UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Central and peripheral components of the vasoactive actions of vasopressin and adrenergic amines King, Kathryn Anne 1987

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1987_A1 K56.pdf [ 7.31MB ]
Metadata
JSON: 831-1.0097408.json
JSON-LD: 831-1.0097408-ld.json
RDF/XML (Pretty): 831-1.0097408-rdf.xml
RDF/JSON: 831-1.0097408-rdf.json
Turtle: 831-1.0097408-turtle.txt
N-Triples: 831-1.0097408-rdf-ntriples.txt
Original Record: 831-1.0097408-source.json
Full Text
831-1.0097408-fulltext.txt
Citation
831-1.0097408.ris

Full Text

CENTRAL AND PERIPHERAL COMPONENTS OF THE VASOACTIVE ACTIONS OF VASOPRESSIN AND ADRENERGIC AMINES By KATHRYN ANNE KING B.Sc , University of Victoria, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1987 ©Kathryn Anne King, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmacology & Therapeutics The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1 Y 3 Date 1 8 August 1987 - i i -ABSTRACT Three major systems par t i c ipa te in the control of the peripheral c i r c u l a t i o n : the renin-angiotensin, the arginine vasopressin (AVP) and the sympathetic nervous systems. These studies examined the ro les of the AVP and the sympathetic nervous systems in the regulat ion of blood pressure at both the central and the peripheral l e v e l . Anatomical studies have revealed that hypothalamic neurons containing AVP extend to the nucleus tractus s o l i t a r i u s (NTS) in the medulla. Since the NTS. i s the primary s i t e of termination of the afferent neurons of the baroreceptor re f lex arc, i t suggests that AVP may be involved in central cardiovascular regula t ion. The ef fect of central AVP on mean a r te r i a l pressure (MAP) and sympathetic nerve a c t i v i t y , estimated from plasma catecholamine l eve l s , was invest igated. The in jec t ion of AVP into the fourth cerebroventr ic le and NTS of conscious, unrestrained rats increased MAP and plasma noradrenaline and adrenaline l eve l s , suggesting that AVP may act cen t ra l l y at the NTS to modulate sympathoadrenal outf low. However, the in jec t ion of a se lec t ive vascular antagonist of AVP, d(CH 2) 5Tyr(Me)AVP, into the fourth ven t r i c le or NTS did not af fect MAP or plasma catecholamine l eve l s , e i ther in normotensive r a t s , in rats subjected to hypotensive s t ress , or in neurogenical ly-stressed r a t s . This suggests that endogenously-released AVP may not have a tonic inf luence on central cardiovascular regu la t ion. (cont'd) The ro le of AVP in the control of MAP, cardiac output (CO) and i t s d i s t r i bu t ion was invest igated in anesthet ized, surg ica l l y -s t ressed ra t s . The i . v . in jec t ion of d(CH2)5Tyr(Me)AVP decreased MAP and to ta l peripheral resistance (TPR), did not a l te r CO, and increased the d is t r i bu t ion of blood flow (BF) to the stomach and sk in . The vascular ro le of AVP was found to be greater in the absence of inf luence from the renin-angiotensin and the sympathetic nervous systems. Af ter blockade of the renin-angiotensin system by the infusion of sara las in the AVP antagonist increased BF to the skin and muscle, while af ter blockade of the a-adrenergic system with the infusion of phentolamine, the AVP antagonist markedly increased BF to the muscle. Thus, the amount of vasoconstr ict ion produced by AVP in d i f ferent vascular beds was found to depend on the endogenous vasomotor tone from the renin-angiotensin and a-adrenergic systems. Cross-c i rcu la t ion studies were conducted to concurrently observe the peripheral and central e f fects of a-agonists in two anesthetized ra t s , designated rat A and B, respect ive ly . The i . v . in jec t ion of c lonid ine into rat A was found to increase MAP and decrease HR in rat A, and reduce MAP and HR in rat B. Since the st imulat ion of peripheral a-adrenoceptors in rat A by c lonid ine increased MAP, i t suggests that the ef fects of peripheral post- junct ional o^-adrenoceptors predominate over those of peripheral pre- junct ional c^-adrenoceptors. In contrast , the i . v . in jec t ion of the o^-agonis t , methoxamine, in rat A increased MAP and decreased HR in rat A, (cont'd) and increased both MAP and HR in rat B. This suggests that central (^-adrenoceptors may mediate responses in the opposite d i rec t ion to those produced by o^-adrenoceptors. To ver i f y the resu l ts of the c ross -c i r cu la t i on studies in animals free of the inf luence of surgery and anesthesia, and to determine whether the responses to a-agonists were mediated by changes in sympathoadrenal outf low, c lonid ine and a more se lec t ive c^-agonis t , B-HT 920, were injected cen t ra l l y in conscious ra t s . The i . e . v . in jec t ion of c lonid ine (1 ug) s i g n i f i c a n t l y decreased MAP and HR and s l i g h t l y decreased plasma noradrenaline and adrenaline l e v e l s ; however, contrary to expectat ions, the i . c . v . in jec t ion of B-HT 920 (1, 10 yg) increased MAP, decreased HR and s l i g h t l y increased plasma noradrenaline and adrenaline l eve l s . To determine whether the responses to central in jec t ion of c lon id ine or B-HT 920 were due to the st imulat ion of ©^-adrenoceptors, i . c . v . in ject ions of these drugs were given af ter pretreatment with rauwolseine, a se lec t i ve (^-antagonist . The i . c . v . in jec t ion of rauwolscine in conscious rats increased MAP and plasma noradrenaline and adrenaline l eve l s , suggesting that central ag-adrenoceptors may mediate tonic i nh ib i t i on of the cardiovascular system. However, i . c . v . in ject ions of c lonid ine or B-HT 920 produced the same responses in the absence or presence of rauwolscine. Further studies with d i f ferent a-adrenergic agonists and antagonists with various s e l e c t i v i t i e s are necessary before we can explain the d i f f e ren t i a l e f fects of central c lonid ine and B-HT 920. - V -TABLE OF. CONTENTS CHAPTER Page 1 INTRODUCTION 1 1.1 General overview 1 1.2 The vasopressin system 2 1.2.1 Structure of AVP 2 1.2.2 Synthesis of AVP 2 1.2.3 Release of AVP 4 1.2.4 Physio logical ro le of AVP 11 1.2.5 Role of AVP in hemorrhage, dehydration and surgical s t ress 16 1.2.6 Role of AVP in hypertension. 18 1.3 The sympathetic nervous system 21 1.3.1 Structure of the sympathetic nervous system 21 1.3.2 C lass i f i ca t i on of adrenoceptors 24 1.3.3 Peripheral a-adrenoceptors 26 1.3.4 Central a-adrenoceptors 29 1.4 Aims .of the studies 31 1.4.1 Role of AVP in cardiovascular regulat ion 31 1.4.2 Role of the a-adrenergic system in cardiovascular regulat ion 34 2 METHODS - 37 2.1 Central AVP in conscious rats 37 2.1.1 Surgical preparation 37 2.1.1.1 Implantation of in t racerebroventr icu lar cannulae 37 2.1.1.2 Implantation of vascular cannulae 38 2.1.2 Experimental protocol 38 2.1.3 Catecholamine analysis by HPLC 41 2.1.3.1 Extract ion of plasma samples 41 2.1.3.2 HPLC with electrochemical detection 42 2.1.4 S t a t i s t i c a l analysis 42 (cont'd) - vi -CHAPTER Page 2.2 Central AVP in hypotensive rats 43 2.2.1 Experimental protocol 43 2.2.2 S t a t i s t i c a l analysis 43 2.3 Micro in ject ion of AVP into the NTS 43 2.3.1 Experimental protocol 43 2.3.2 H is to log ica l technique 45 2.3.3 S t a t i s t i c a l analysis 45 2.4 Central AVP in neurogenical ly-stressed rats 45 2.4.1 Experimental protocol 45 2.4.2 S t a t i s t i c a l analysis 46 2.5 Vascular ro le of AVP 46 2.5.1 Surgical preparation 46 2.5.2 Microsphere technique 46 2.5.3 Experimental protocol 48 2.5.4 Calculat ions , 49 2.5.5 S t a t i s t i c a l analysis 49 2.6 Central and peripheral actions of a-agonists 50 2.6.1 Cross-c i rcu la ted rat preparation 50 2.6.2 Blood flow to the brain v ia the l e f t carot id artery 52 2.6.3 Microsphere technique 53 2.6.4 Determination of c i rcu la to ry leakage 53 2.6.5 Ef fects of c lon id ine and methoxamine 53 2.6.6 Calcu lat ions 54 2.6.7 S t a t i s t i c a l analysis 54 2.7 Central ct2-agonists in conscious rats 54 2.7.1 Experimental protocol 54 2.7.2 S t a t i s t i c a l analysis 56 2.8 Drugs 56 (cont'd) CHAPTER Page 3 RESULTS 57 3.1 Central AVP in conscious rats 57 3.1.1 Central administrat ion of AVP 57 3.1.2 Central administrat ion of AVP antagonist 57 3.1.3 Peripheral administrat ion of AVP and AVP antagonist 62 3.2 Central AVP in hypotensive rats 62 3.3 Micro in ject ion of AVP into the nucleus t ractus s o l i t a r i u s 65 3.4 Central AVP in neurogenical ly-stressed rats 65 3.5 Vascular ro le of AVP 70 3.5.1 Ef fect of antagonism of pressor systems, on MAP, CO and TPR 70 3.5.2 Ef fect of AVP antagonist on MAP, CO, and TPR 70 3.5.3 Ef fect of AVP antagonist on the d i s t r i bu t i on of blood flow 74 3.6 Central and peripheral actions of a-agonists 78 3.6.1 Blood flow to the brains of c ross-c i rcu la ted rats 78 3.6.2 Blood flow to the brain v ia the l e f t carot id artery 78 3.6.3 Ci rcu la tory leakage 78 3.6.4 Cross-c i rcu la ted rat preparation 82 3.7 Central (^adrenergic agonists in conscious rat 82 4 DISCUSSION 93 4.1 Central AVP in conscious rats 93 4.2 Central AVP in hypotensive rats 97 4.3 Micro in ject ion of AVP into the NTS 98 4.4 Central AVP in neurogenical ly-stressed rats 101 4.5 Vascular ro le of AVP 102 4.6 Central and peripheral actions of a-agonists 105 (cont'd) - v i i i -CHAPTER Page 4.7 Central a2~agonists in conscious rats 109 4 .8 General conclusions 113 4 . 8 . 1 Role of AVP in cardiovascular regulat ion 113 4 . 8 . 2 Role of the a-adrenergic system in cardiovascular regulat ion 114 5 REFERENCES • - ix -LIST-OF TABLES Table Page 1 Control values of MAP, CO and TPR in Groups I, I I , and III 71 2 Effects of AVP antagonist on MAP, CO and TPR in Groups I, II 73 and III 3 Cpm in rats A and B expressed as a % of cpm detected in rat A 81 in 1 min 4 Control values of MAP, HR and plasma noradrenaline and adrenaline 87 concentration in Groups I, I I , III and IV pr ior to drug administrat ion - X -LIST OF - FIGURES F ig . Page 1 The structures of arginine vasopressin (AVP) and lysine vasopressin 3 2 The biosynthet ic pathway for noradrenaline and adrenaline 22 3 Vascular connections between rats A and B in the c ross -c i r cu la t i on 51 preparation 4 The ef fects of i . e . v . in ject ions of AVP on MAP and plasma 58 noradrenaline and adrenaline concentrations in ra ts from Group I and Group II 5 The ef fects of i . e . v . in ject ions of a r t i f i c i a l CSF on MAP and 59 plasma noradrenaline and adrenaline levels in rats from Group III and Group IV 6 The effect of i . e . v . in jec t ion of AVP antagonist on MAP and plasma 60 noradrenaline and adrenaline concentrations in rats from Group V 7 The effect of pretreatment of the fourth ven t r i c le with AVP 61 antagonist on MAP and plasma noradrenaline and adrenaline leve ls fo l lowing i . e . v . in ject ions of AVP in rats from Group VI 8 The ef fect of i . v . in ject ions of AVP and AVP antagonist on MAP and 63 plasma noradrenaline and adrenaline concentrations in rats from Group VII , Group VIII and Group IX 9 The ef fect of i . e . v . in ject ions of a r t i f i c i a l CSF (Group I) and AVP 64 antagonist (Group II) on MAP and plasma noradrenaline and adrenaline leve ls in rats receiv ing i . v . infusions of ni t roprusside 10 The ef fect of in jec t ion into the NTS of 2 ul a r t i f i c i a l CSF, 66 2 ng AVP, 10 ng AVP, or 10 ng AVP antagonist on MAP and HR 11 The ef fect of in jec t ion into the NTS of 2 yl a r t i f i c i a l CSF, 67 2 ng AVP, 10 ng AVP, or 10 ng AVP antagonist on plasma noradrenaline and adrenaline concentration 12 The ef fect of in jec t ion into the NTS of a r t i f i c i a l CSF (Group I) 68 and AVP antagonist (Group II) of rats subjected to neurogenic stress on MAP and HR 13 The ef fect of in jec t ion into the NTS of a r t i f i c i a l CSF (Group I) 69 and AVP antagonist (Group II) of rats subjected to neurogenic stress on plasma noradrenaline and adrenaline concentration - x i -F i g . Page 14 Effect of AVP antagonist on MAP, CO and TPR in anesthet ized, 72 su rg i ca l l y stressed r a t s : intact and sal ine-pretreated (Group I ) , saralasin-pretreated (Group II) and phentolamine-pretreated (Group III) 15 Effect of AVP antagonist on regional d i s t r i bu t ion of BF in rats 75 from group I (sal ine-pretreated) 16 Effect of AVP antagonist on regional d i s t r i bu t ion of BF in rats 76 from group II (saralasin-pretreated) 17 Effect of AVP antagonist on regional d i s t r i bu t ion of BF in rats 77 from group III (phentolamine-pretreated) 18 BF to the le f t and r ight brain hemispheres and brainstem before 79 and after l i ga t ion of the subclavian ar ter ies in ra ts A and B of c ross-c i rcu la ted rat preparations 19 BF to the le f t and r ight brain hemispheres and brainstem in the 80 presence and absence of funct ional subclavian ar ter ies in s ingle rats 20 MAP and HR responses of rat A and rat B to i . v . in jec t ion 83 of c lonidine (25 yg/kg) in rat A 21 Representative recordings of MAP and HR responses in rat A 84 and rat B fo l lowing i . v . in jec t ion of c lonidine (25 yg/kg) in rat A 22 MAP and HR responses of rat A and rat B to i . v . in jec t ion 85 of methoxamine (25 yg/kg) in rat A 23 Representative recordings of MAP and HR responses of rat A 86 and rat B fo l lowing i . v . in ject ion of methoxamine (25 yg/kg) in rat A 24 The ef fect of i . c . v . in jec t ion of B-HT 920 (Group I) and c lonid ine 88 (Group II) on MAP and HR 25 The ef fect of i . c . v . in jec t ion of B-HT 920 (Group I) and c lonid ine 89 (Group II) on plasma noradrenaline and adrenaline levels 26 The ef fect of i . c . v . in jec t ion of 10 yg rauwolscine followed by 91 1 yg B-HT 920 (Group III) and 10 yg rauwolscine followed by 1 yg c lonid ine (Group IV) on MAP and HR 27 The ef fect of i . c . v . in jec t ion of 10 yg rauwolscine followed by 92 1 yg B-HT 920 (Group III) and 10 yg rauwolscine followed by 1 yg clonidine (Group IV) on plasma noradrenaline and adrenaline levels - x i i -ACKNOWLEDGEMENTS I am grateful to both the B.C. and Canadian Heart Foundations, without whose f i nanc ia l support th is work would not have been poss ib le . I would l i ke to express my appreciat ion to Dr. M.C. Sut ter , Dr. M.J.A. Walker, Dr. V.W. Yong, Dr. M.J . Cur t is and Carol ine Bruce for the i r support and encouragement. I am also grateful to Wee for providing me with space to wr i te . I would l i ke to thank Dr. R. Wall for his expert advice regarding the HPLC ana lys is , and for his endless patiencel I would also l i ke to thank Fedrick Wong and Maureen Murphy for the i r assistance in several of the s tudies. I would l i k e to thank Ron for his patient support. I thank Reza for a l l his help, and for his sense of lunacy and his exceptional ins igh t . I am very grateful to my supervisor, Dr. C.C.Y. Pang for her careful guidance and encouragement, and espec ia l l y for her enthusiasm for science. - x i i i -LIST OF ABBREVIATIONS arginine vasopressin AVP adenosine triphosphate ATP blood flow BF cardiac output CO central nervous system CNS cerebrospinal f l u i d CSF counts per minute cpm deoxycort icosterone/sal t DOC/salt 3,4-dihydroxybenzylamine DHBA heart rate HR high performance l i qu id chromatography HPLC hour(s) h internat ional units IU int racerebroventr icular i . c . v . in t raper i toneal i . p . intravenous i . v . mean a r te r ia l pressure MAP minute(s) min nucleus tractus s o l i t a r i u s NTS paraventr icular nucleus PVN polyethylene PE second(s) sec spontaneously hypertensive rat (s) SHR standard deviat ion SD standard error of the mean SEM to ta l peripheral resistance TPR 1 INTRODUCTION 1.1 General overview The c i r cu la t i on of the blood was f i r s t described by Wil l iam Harvey in his t rea t i se "Exc i ta t i o anatomica de motu cordis et sanguinis in animalibus" in 1628. Since the time of Harvey, i t has been establ ished that the peripheral c i r cu la t i on funct ions to provide t issues with nutr ients and oxygen, remove waste mater ia ls , maintain normal t issue f l u i d volume, regulate body temperature and f a c i l i t a t e food absorpt ion. In ef fect the primary ro le of the c i r cu la t i on i s to maintain homeostasis. A number of mechanisms regulate the flow of blood to the t i ssues . Local control of blood flow or autoregulation occurs at the level of the blood vesse l . Autoregulation is the tendency for blood flow to remain constant in sp i te of changes in a r te r i a l pressure, and i t is achieved by non-neural mechanisms. It may involve a myogenic mechanism, the a b i l i t y of the a r te r i o le to d i l a te in response to a reduction in a r te r i a l pressure and to contract in response to an increase in pressure, and/or a metabolic mechanism, the accumulation of metabolites by t issues which cause vasodi la t ion during periods of decreased f low. In addit ion to these non-neural regulatory systems, several hormonal systems including the renin-angiotensin-aldosterone and vasopressin systems have been implicated in the control of the c i r c u l a t i o n . s Perhaps the greatest inf luence on the peripheral c i r c u l a t i o n , however, i s exerted by the sympathetic nervous system. The sympathetic nervous system, v ia the neurotransmitter noradrenaline released from sympathetic nerve endings, causes contract ion of vascular smooth muscles and therefore contr ibutes to the maintenance of blood pressure. - 2 -1.2 The vasopressin system 1.2.1 Structure of AVP. The discovery that extracts of the posterior pituitary gland have an effect on blood vessels was first made by Oliver and Schafer in 1895. Krogh (1929) later observed that local application of posterior pituitary extracts to capillary beds in vivo in the webbed feet of frogs and in the ear of dogs caused the vessels to constrict. The structure of the vasoactive substance present in pituitary extracts was postulated by du Vigneud and his group in 1953 and was subsequently synthesized in 1954, an unprecedented accomplishment at this time. Vasopressin was found to be a nonapeptide containing two cysteine residues at positions one and six which participate in a disulfide linkage so that the first six amino acids form a ring, which is essential for biological activity (Fig. 1). Two major naturally occurring forms exist. Lysine vasopressin, which occurs in the pig and hippopotamus, contains a lysine residue in position eight, and arginine vasopressin (AVP), the form present in other mammals, contains an arginine residue at position eight. 1.2.2 Synthesis of AVP. The hypothalamohypophyseal complex, the neurosecretory system for AVP, is comprised of magnocellular or neurosecretory cells which have their perikarya in the supraoptic and paraventricular nuclei of the hypothalamus. The magnocellular neurons form the floor and ventrolateral walls of the third ventricle, and their axons project through the pars tuberalis to terminate on capillaries in the neurohypophysis. The secretory process for AVP consists of four phases: synthesis, packaging into secretory granules,transport of the granules to the release site, and release. AVP and its binding protein neurophysin II are synthesized within the cell bodies of the magnocellular neurons in the form of a precursor molecule, and packaged into membrane-bound secretory granules (Brownstein et al . 1983). The granules are transported along the - 3 -C y s — T y r — P h e — G l u — A s p — C y s — P r o — A r g — G l y ( N H 2 J 1 2 3 4 5 6 7 8 9 8-Arginine Vasopressin (ADH, AVP; mammals) Lys-8-Lysine Vasopressin (lypressin, LVP; swine) F i g . 1. The structures of arginine vasopressin (AVP) and lys ine vasopressin. - 4 -axons to the axon terminals where they are stored (Russell et a l . 1981). AVP i s released from the nerve endings into the systemic c i r cu la t i on by a 2+ Ca -dependent exocytosis in response to membrane depolar izat ion e l i c i t e d by e l e c t r i c a l impulses generated within the supraoptic and paraventr icular nuclei (Thorn 1978). Sachs et a l . (1971) have shown that only 10-20 percent of the to ta l pool of the hormone stored within the neural lobe can be readi ly and rapid ly released, and once th is occurs AVP secret ion continues in response to st imulat ion but at a slower rate. The complete process of synthesis, transport and storage i s completed in 1-2 h in the rat (Pickering and McPherson 1977). Once secreted into the blood stream, AVP is subject to metabolic clearance by the kidney and l i v e r with a h a l f - l i f e of 1-8 min in the rat (Lauson 1974; Gazis and Sawyer 1978), 4-8 min in the dog (Gazis and Sawyer 1978), and 17-35 min in humans (Robertson et a l . 1973; Baumann and Dingman 1976; Robertson 1977). 1.2.3 Release of AVP. AVP appears to be released in response to two types of physio logical s t i m u l i : osmotic and non-osmotic. The concept of an osmoreceptor, or c e l l which can respond to changes in i t s own volume and accordingly inf luence AVP re lease, was f i r s t postulated by Verney in 1947. This proposal was based on experiments in which the in t racarot id in jec t ion of hypertonic solut ions of solutes which are not readi ly permeant to c e l l s , such as sodium ch lo r ide , sucrose and sodium su l fa te , e l i c i t e d an immediate f a l l in urine flow in dogs, while the administrat ion of hypertonic solut ions of solutes which read i ly penetrate c e l l s , such as glucose or urea, were less e f f ec t i ve . Subsequent studies involving administrat ion of various hypertonic solut ions c l ea r l y demonstrated that an increase in the osmotic pressure of the ex t race l l u la r f l u i d stimulates ant id iu res is (Jewell and Verney 1957; Eriksson et a l . 1971; Athar and Robertson 1974; Robertson et a l . 1977) and AVP release (Thrasher et a l . 1980a). In v i t ro experiments - 5 -using rat hypothalamo-neurohypophysial explants also demonstrated that an increase in osmolal i ty induced' with hypertonic solut ions of sa l ine or mannitol caused AVP re lease, and that reduction of osmolal i ty reduced AVP release (Sladek and Knigge 1977). A l te rna t i ve l y , Andersson (1971) proposed that AVP release i s regulated by a sodium-sensit ive mechanism, or "sodium receptor" , present in the wal ls of the th i rd ven t r i c l e , which responds to an increase in the sodium concentration of the cerebrospinal f l u i d (CSF). The most convincing evidence for a sodium receptor i s provided by the observations that in t racerebroventr icu lar ( i . c . v . ) in jec t ion of hypertonic sucrose so lu t ion , which would be expected to increase the osmolal i ty but not the sodium concentration of the CSF, did not produce an t i d iu res i s ; yet hypertonic sodium chlor ide so lu t ion , which would increase both the osmolal i ty and sodium concentration of the CSF, did produce an t id iu res is (Olsson 1969). The i . c . v . administrat ion of iso ton ic or hypertonic saccharide solut ions into the th i rd ven t r i c l e , which would be expected to decrease CSF sodium concentration by a d i l u t i ona l e f f ec t , inh ib i ted AVP release in normovolemic goats, supporting the concept of a sodium receptor rather than .an osmoreceptor (Eriksson 1974). Moreover, infusions of isotonic or hypertonic saccharide solut ions into a la te ra l ven t r i c le were shown to i nh ib i t the an t id iu res is due to in t racaro t id infusion of hypertonic sa l ine solut ion (Olsson 1973). McKinley et a l . (1978) showed that the in t racaro t id infusion of hypertonic urea solut ion in sheep caused a much greater increase in CSF sodium concentration than did infusion of hypertonic sa l ine or sucrose so lu t ions , and yet was much less e f fec t i ve in st imulat ing an t id iu res i s . This would suggest that an osmoreceptor rather than a sodium receptor inf luences AVP re lease, and that the receptor i s located outside the blood-brain barr ier since c i r cu la t i ng hypertonic urea solut ion should - 6 -provide an osmotic stimulus to a receptor within the blood brain bar r ie r which i t does not rapid ly penetrate. However, they also found that the i . c . v . administrat ion of hypertonic sa l ine in a r t i f i c i a l CSF produced a greater an t id iu re t i c ef fect than the i . c . v . in jec t ion of equiosmolar sucrose solut ions which produced an ident ica l e levat ion of CSF osmolal i ty but decreased sodium concentrat ion. As a resu l t , they proposed that a dual osmoreceptor-sodium receptor system mediates AVP re lease, involving both osmoreceptors located outside the blood-brain bar r ie r and sodium receptors wi thin the blood-brain bar r ie r . More recent studies by Thrasher et a l . (1980a,b) appear to have c l a r i f i e d the issue of osmoreceptors versus sodium receptors. The i . v . infusion of hypertonic solut ions of sa l i ne , sucrose, glucose or urea in conscious dogs was shown to increase both the osmolal i ty and the sodium concentration of the CSF, and yet only hypertonic sa l ine and sucrose solut ions increased plasma AVP concentration suggesting that the elevat ion of the sodium concentration of the CSF i s not a stimulus fo r AVP release (Thrasher et a l . 1980a). It i s also un l i ke ly that AVP release is mediated by a sodium receptor outside the blood-brain bar r ie r since hypertonic sucrose solut ion was equally as e f fec t i ve as hypertonic sa l ine solut ion in causing AVP release, but s i g n i f i c a n t l y decreased plasma sodium concentrat ion. These observations rule out the existence of a sodium receptor e i ther within or outside the blood-brain bar r ie r . The i . v . infusion of hypertonic sodium chlor ide or sucrose solut ions increased both the osmolal i ty of the CSF and the plasma AVP concentrat ion, observations consistent with the existence of osmoreceptors within the blood-brain bar r ie r . On the other hand, the i . v . infusion of hypertonic glucose or urea solut ions also elevated both the osmolal i ty and the sodium concentration of the CSF, but did not cause AVP release. While these substances readi ly - 7 -penetrate most c e l l s , they penetrate the blood-brain bar r ie r very s lowly, and should provide an osmotic stimulus within the barr ier by causing osmosis of water across the ba r r ie r . The observation that these solut ions do not cause AVP release after i . v . infusion suggests that the osmoreceptors are not located within the blood-brain ba r r ie r . The circumventr icular organs, such as the subfornical organ or the organum vasculosum of the lamina terminal is were proposed as a possible anatomical s i t e of these osmoreceptors (Thrasher et a l . 1980b), although i t has also been suggested that neurons in the supraoptic nucleus may detect osmotic s t imul i (Leng et a l . 1982). Non-osmotic s t imul i of AVP release include changes in blood pressure or blood volume, hypoxia, nausea, hormones, surgery and other s t ress- re la ted s t i m u l i . It i s f a i r l y c lear that the carot id sinus baroreceptors inf luence the release of AVP. Carotid occlusion has been shown to cause AVP release or an t id iu res is (Share and Levy 1962; Share 1965; Clark and Rocha e S i l v a 1967; Schr ier and Berl 1972). Stimulat ion of a r t e r i a l baroreceptors by increasing carot id sinus pressure has been shown to reduce plasma AVP concentration in dogs (Thames and Schmid 1981). Receptors with vagal a f ferents , possib ly a t r i a l receptors, have been shown to i nh ib i t AVP release in anesthetized dogs with s ino-aor t i c denervation (Thames and Schmid 1979), as well as in conscious dogs with aor t ic or s ino-aor t i c denervation (Bishop et a l . 1984). I t has also been reported that when carot id sinus pressure was held constant at 50 mmHg or 135 mmHg, vagal cold block increased plasma AVP l e v e l s , but when carot id sinus pressure was increased to 200 mmHg, plasma AVP levels decreased with vagal cold block (Thames and Schmid 1981). These studies suggest that there may be an in teract ion between baroreceptors and a t r i a l receptors in the regulat ion of AVP re lease. - 8 -The l e f t a t r i a l receptors have been proposed to regulate AVP release (Gauer and Henry 1963; Perlmutt 1964; Schr ier and Berl 1972). Ear ly studies showed that the elevat ion of l e f t a t r i a l pressure produced by i n f l a t i n g a small balloon in the l e f t atrium caused d iu res is which was abolished by b i l a t e ra l vagotomy (Henry et a l . 1956). A number of other workers showed that l e f t a t r i a l d istension reduced plasma AVP levels measured by bioassay (Share 1965; Shu'ayb et a l . 1965; Johnson et a l . 1969). More loca l i zed st imulat ion of l e f t a t r i a l receptors achieved by i n f l a t i n g a small balloon at the junct ion of the pulmonary vein and l e f t atrium was shown to cause d iures is in dogs (Ledsome and Linden 1968). Vagotomy was shown to cause an t id iu res is which was abolished by acute hypophysectomy (Schrier and Berl 1972). In addi t ion, pacing-induced a t r i a l tachycardia, which was shown to increase l e f t a t r i a l pressure, caused d iures is in dogs which was abolished by acute hypophysectomy or vagotomy (Boykin et a l . 1975). In contrast to these observations, Kappagoda et a l . (1975) did not observe a change in bioassayable AVP levels af ter l e f t a t r i a l d istension in dogs. These ear ly studies were subject to the l im i ta t ion that plasma AVP levels were e i ther not measured or the bioassay procedure used to measure AVP was not sens i t ive enough to detect small changes in AVP l eve l s . More recent studies using radioimmunoassay techniques showed that AVP leve ls decreased af ter l e f t a t r i a l d istension (Johnson et a l . 1969;, de Torrente et a l . 1975). Left a t r i a l d istension also reduced plasma AVP concentration in anesthetized dogs (Ledsome et a l . 1983) and in conscious dogs (Fater et a l . 1982; Schultz et a l . 1982), but not af ter vagal cold block, or af ter cardiac denervation, respect ive ly . Stimulation of l e f t a t r i a l receptors by i n f l a t i on of a balloon placed at the pulmonary ve in - l e f t a t r i a l junct ion was also shown to decrease plasma AVP leve ls in anesthetized dogs (Wilson and Ledsome 1983). Direct st imulat ion of cardiac receptors with vagal afferents - 9 -by in jec t ion of veratrum a lka lo ids has been shown to i nh ib i t AVP re lease, although i t is not c lear whether the response was mediated by l e f t a t r i a l or vent r icu lar receptors (Thames et a l . 1980). These studies provide evidence to suggest that the st imulat ion of l e f t a t r i a l receptors i nh ib i t s AVP re lease. Small reductions (<10 percent) in blood volume have been shown to increase the release of AVP, an ef fect which may be mediated by l e f t a t r i a l receptors (Share 1968; Claybaugh and Share 1973). I t has been suggested that th i s pathway may mediate AVP release in response to non-hypotensive hemorrhage (Share 1968; Henry et a l . 1968; Claybaugh and Share 1973; Share 1974). Moderate or severe hemorrhage has been shown to be an extremely potent stimulus for the release of AVP (Ginsburg and Hel le r 1953; Weinstein et a l . 1960; Rocha e S i l v a and Rosenberg 1969; Szczepanska-Sadowska 1972; Claybaugh and Share 1973; Cousineau et a l . 1973; Arnauld et a l . 1977; Weitzman et a l . 1978; Bierman et a l . 1979; Laycock et a l . 1979; Pul lan et a l . 1980; Fyhrquist et a l . 1981). I t i s not c lear i f l e f t a t r i a l receptors play an important ro le in the mediation of large elevat ions of plasma AVP concentrations in response to more hypotensive hemorrhage. Vagotomy has been shown to s i g n i f i c a n t l y reduce the increase in plasma AVP concentration which occurs in response to hypotensive hemorrhage (Clark and Rocha e S i l v a 1967; Share 1968). A logarithmic re la t ionsh ip was found to ex is t between blood volume and plasma AVP concentration in anesthetized dogs in which elevat ion of blood volume resul ted in a reduction in plasma AVP levels while reduction of blood volume led to an increase in plasma AVP concentration (Ledsome et a l . 1985). However, i t was also found that the plasma AVP concentration was more highly correlated with mean a r te r i a l pressure than with mean l e f t a t r i a l pressure, suggesting that the l e f t a t r i a l receptors are un l i ke ly to provide the major - 1 0 -stimulus for AVP release during large changes in blood volume. Subsequent studies in anesthetized rabbits showed that 10 percent hemorrhage caused s ign i f i can t elevat ion of plasma AVP concentration and reduction of mean a r te r i a l pressure and r ight a t r i a l pressure, even af ter sect ion of the ao r t i c , vagus and carot id sinus afferent nerves, suggesting that another receptor type may stimulate AVP release during hemorrhage (Rankin et a l . 1986). Whether th is f ind ing i s unique to the rabbit or extends to other species i s not c lea r . These studies suggest that both a r t e r i a l baroreceptors and a t r i a l receptors do par t i c ipa te in the non-osmotic regulat ion of AVP re lease; however the re la t i ve ro les of the two receptor types in various pathophysiological condit ions has yet to be e luc idated. Other pathways have also been implicated in the non-osmotic release of AVP, including chemoreceptors (Share and Levy 1966), the cerebral emetic center (Robertson 1977) and a cerebral pain center (Hayward and Jennings 1973). Hypoxia has been shown to cause AVP release in anesthetized dogs (Fors l ing and Ullmann 1977), f e ta l sheep (Alexander et a l . 1972), and conscious rats (Bhatia et a l . 1977) but not in man (Fors l ing and Ullmann 1977) . Pain pathways may also inf luence AVP re lease. A group of pat ients report ing to a surgical emergency department with pain had s i g n i f i c a n t l y higher leve ls of plasma AVP than a control group of pat ients , although there was no di f ference in plasma osmolal i ty between the two groups (Kendler et a l . 1978). Emesis (Robertson 1977) and motion sickness (Fela l et a l . 1978) are powerful s t imul i of AVP re lease. Although the mechanisms through which AVP release i s stimulated during hypoxia, nausea and pain remain unclear, i t i s possible that sympathetic st imulat ion or a l te ra t ion of baroreceptor tone may par t i c ipa te in the mediation of AVP release under these condit ions (Schrier et a l . 1979). - 11 -Anesthesia has been implicated in the release of AVP, although recent evidence suggests that the large amount of AVP released i s more l i k e l y to be associated with surgical stress since the induction of anesthesia was found to cause l i t t l e change in plasma AVP l eve l s , but subsequent surgery released large amounts of AVP (Moran et a l . 1964; Moran and Zimmerman 1967; Ukai et a l . 1968; Bonjour and Malvin 1970; Oyama et a l . 1971; Ph i l b in et a l . 1976; Ishihara et a l . 1978; Ph i lb in and Coggins 1978; Wu et a l . 1980). Moreover, inadequate anesthesia, indicated by the return of corneal or limb re f lexes , was reported to increase plasma AVP concentrations (Ukai 1971). The l a t t e r observation i s also consistent with the involvement of a pain pathway in the release of AVP. Hormones may also modulate AVP re lease. While Bonjour and Malvin (1970) suggested that angiotensin I I , infused intravenously or i n t r a - a r t e r i a l l y , increased bioassayable plasma AVP levels in anesthetized dogs, la te r studies showed that such angiotensin II infusions neither al tered water excretion in anesthetized animals undergoing water d iu res is nor increased bioassayable AVP (Claybaugh et a l . 1972). However, both i . c . v . infusion of angiotensin II (Ke i l et a l . 1975) and appl icat ion of angiotensin II to iso la ted p i t u i t a r y glands (Gagnon et a l . 1975) were reported to cause AVP re lease, suggesting that brain rather than plasma angiotensin II may modulate AVP re lease. Central ly-administered PGE2 has been shown to release AVP (Yamamoto et a l . 1978). TSH, thyroid hormones (Skowsky and Fisher 1977), estrogen, progesterone and androgens (Skowsky and Swan 1977) have a l l been suggested to modulate AVP re lease. 1.2.4 Phys io log ica l ro le of AVP. Although i t was the pressor action of AVP which was f i r s t observed, the conventional physio logica l ro le of AVP i s that of an an t id iu re t i c hormone. The par t i c ipa t ion of AVP in the renal conservation of water was f i r s t noted by Verney in 1947. AVP interacts with - 12 -receptors on the basolateral surface of the co r t i ca l and medullary segments of the co l l ec t i ng duct, increasing the permeabil i ty of these segments to water and thereby f a c i l i t a t i n g water reabsorpt ion. The renal AVP receptors, designated V 2 receptors, act through the adenylate cyclase system (Michel 1 et a l . 1979). Many studies have attempted to assess the vascular ef fects of AVP. Krogh (1929) reported that in vivo appl icat ion of poster ior p i t u i t a ry extracts to the c a p i l l a r y bed in the webbed feet of frogs and the ear of dogs caused vasoconst r ic t ion, and that hypophysectomy increased blood flow to these vesse ls . More recent studies have shown that administrat ion of crude p i t u i t a r y extracts or P i t ress in increased systemic a r te r i a l pressure and peripheral vascular resistance in rats (Bisset and Lewis 1962; A l tu ra et a l . 1965), rabbi ts (Friedman and Pauls 1952), cats (Barer 1961; Friedman and Pauls 1952), dogs (Car l i e r et a l . 1960; Co r l i ss et a l . 1968) and humans (Davis et a l . 1957). Although i t was c lear that AVP could cons t r i c t a r t e r i o l e s , i t was general ly believed that blood vessels were r e l a t i v e l y insens i t i ve to the hormone (Sawyer 1961; Mellander and Johansson 1968; Saameli 1968). However, A l tu ra and A l tura (1977) showed that AVP was in fact more potent even than angiotensin II in cons t r i c t ing rat terminal a r t e r i o l e s , mesenteric resistance vessels and aor t ic s t r i p s . I t should be noted that vasoconstr ic t ion could be e l i c i t e d with physio logical leve ls of AVP at 1 0 " 1 3 to 1 0 " 1 2 M (Al tura 1973). Vascular responses to AVP appeared to be dependent on the species and the vascular bed. AVP was found to cause potent coronary vasoconstr ict ion in the dog heart-lung preparation (Bodo 1927), in anesthetized dogs (Wegria et a l . 1940) and in conscious dogs (Essex et a l . 1940). The infusion of AVP in anesthetized cats caused cons t r i c t ion in splenic and in tes t ina l vascular beds but d i l a ta t i on in hepatic a r t e r i a l beds (Cohen et a l . 1970). The g r a c i l i s muscle i s more - 13 -sens i t i ve than the mesenteric beds to the vasoconstr ictor ef fect of AVP, and these in turn are more sens i t i ve than the renal bed in anesthetized dogs (Schmid et a l . 1974). AVP also appears to have l i t t l e ef fect on iso la ted veins (Sutter 1965; Stamm 1972), although in human umbi l ical ve in , high concentrations of AVP were shown to cause re laxat ion (Somlyo et a l . 1966). As w e l l , in vivo experiments have shown that AVP has l i t t l e ef fect on mean c i r cu la to ry f i l l i n g pressure, an index of to ta l body venous tone (Pang and Tabrizchi 1986). The AVP receptors present on smooth muscles (Michel 1 et a l . 1979), as well as those present on hepatocytes which mediate hepatic glycogenolysis (Gopalnakrishnan et a l . 1986), have been c l a s s i f i e d V} receptors, and are believed to use phosphoinosit ides and calcium as second messengers. I t has been reported that subpressor doses of AVP can enhance the blood pressure response to noradrenaline and adrenaline in ca ts , rats and dogs (Bartelstone and Nasmyth 1965). AVP has also been shown to potent iate the vasoconstr ictor ef fects of noradrenaline, angiotensin II and potassium in v i t ro in iso lated rat mesenteric ar ter ies (Karmazyn et a l . 1978). The response of mesenteric artery to AVP was diminished in the presence of indomethacin, suggesting that prostaglandins may play a ro le in mediating the vascular ef fect of AVP (Manku and Horrobin 1977). Based on his observations of the vascular ef fects of p i t u i t a ry ex t rac ts , Krogh (1929) proposed that poster ior p i t u i t a r y hormones might have a tonic const r ic to r inf luence on peripheral blood vessels and therefore par t i c ipa te in the regulat ion of blood f low. Many studies have attempted to determine whether AVP has a physio logical ro le in the maintenance of blood pressure. To produce a 10 mmHg increase in blood pressure in intact conscious dogs (Cowley et a l . 1974; Montani et a l . 1980) and rats (Mohring et a l . 1979; Morton et a l . 1982), AVP was infused at a rate of - 14 -1-2 ng/min/kg, a dose corresponding to a 30-80 pg/ml increase in plasma AVP concentrat ion. In humans, infusions of AVP which increase plasma AVP levels by 50-500 pg/ml were shown to have l i t t l e ef fect on blood pressure (Padf ield et a l . 1976; Mohring et a l . 1980); the normal physio logical plasma concentration of AVP in humans i s in the range of 1-5 pg/ml (Robertson 1977). Bussien et a l . (1984) reported that in jec t ion of the se lec t ive AVP pressor antagonist d(CH2)gTyr(Me)AVP in healthy, normally hydrated subjects did not af fect blood pressure, heart rate or skin blood f low, even af ter inac t iva t ion of the renin-angiotensin system with cap top r i l . Since plasma leve ls of at least 50 pg/ml are required to elevate blood pressure -a concentration 10-100 times both the normal phys io log ica l level and the level required to achieve maximal an t id iu res is - and AVP pressor antagonists have l i t t l e ef fect on blood pressure under normal condi t ions, the vascular ef fects of AVP have been considered to be of l i t t l e physio logical s ign i f i cance . However, low concentrations of AVP have been reported to increase blood pressure after baroreceptor denervation in dogs (Cowley et a l . 1974) and in patients with autonomic insu f f i c iency (Mohring et a l . 1980). It was also shown that AVP antagonist decreased a r te r i a l pressure in conscious, water-replete rats only af ter s inoaor t ic denervation and gangl ionic blockade or adrenalectomy ( I r iuch i j ima 1983). This led I r iuch i j ima to suggest that the AVP system may be recru i ted to maintain blood pressure only af ter f a i l u r e of the sympathoadrenal system. It has been suggested that AVP may increase the s e n s i t i v i t y of the baroreceptor r e f l ex , since AVP produces a greater reduction in heart rate in response to equivalent increases in blood pressure than other pressor agents (Heyndrickx et a l . 1976; Mohring et a l . 1981; Lumbers and Potter 1982). On the other hand, Bratt leboro r a t s , which lack AVP, d isp lay a decreased s e n s i t i v i t y of the baroreceptor re f lex system which can be restored by the - 15 -administrat ion of AVP (Imai et a l . 1983). I.v. infusions of AVP produce only small elevat ions of blood pressure in animals with in tact baroreceptors (Cowley et a l . 1974; Montani et a l . 1980). Af ter baroreceptor i nac t i va t i on , in fusion of physio logical doses of AVP was shown to s i g n i f i c a n t l y increase blood pressure in anesthetized dogs (Rocha e S i l v a and Rosenberg 1969) and in conscious dogs (Cowley et a l . 1974; Cowley et a l . 1983). Moreover, Montani et a l . (1980) found that small increases in plasma AVP concentrations in conscious dogs increased to ta l peripheral res is tance, but not blood pressure, due to a reduction in cardiac output; af ter baroreceptor denervation, ident ica l AVP leve ls led to an increase in blood pressure since cardiac output was not reduced. Evidence suggests that th is in teract ion between AVP and the baroreceptor re f lex occurs within the central nervous system (CNS). L iard et a l . (1981) showed that infusion of AVP into a vertebral artery caused greater re f lex bradycardia than i . v . infusion of the same dose of AVP. Infusion of AVP in rabbi ts was shown to i nh ib i t lumbar sympathetic efferent nerve a c t i v i t y but did not change aor t ic baroreceptor afferent a c t i v i t y suggesting that the sens i t i za t ion occurs cen t ra l l y (Guo et a l . 1986). Based on les ion s tud ies, Undesser et a l . (1985) proposed the area postrema as a s i t e at which AVP acts to enhance baroref lex a c t i v i t y . It i s possible that AVP may also l o c a l l y sens i t i ze baroreceptors. Inject ion of AVP into the iso la ted carot id sinus in anesthetized rabbits produced a small increase in the a c t i v i t y of carot id sinus nerves (Holmes and Ledsome 1984). I t has also been shown that while AVP did not inf luence the rest ing a c t i v i t y of s ing le f i b res from aor t ic baroreceptors in anesthetized rabb i ts , or from l e f t vent r icu la r receptors in anesthetized ca ts , i t s i g n i f i c a n t l y enhanced s ingle f i b re a c t i v i t y during elevat ion of a r t e r i a l pressure and l e f t vent r icu lar end-d ias to l ic pressure in rabbi ts and cats respect ive ly (Abboud et a l • 1986). Therefore AVP may - 16 -f a c i l i t a t e the baroreceptor re f lex by both central and peripheral act ions. This extremely e f fec t i ve buffering of the pressor ef fect of AVP may explain why AVP, although i t i s a potent vasopressor agent, does not normally appear to increase blood pressure in animals with in tact baroreceptors. In addit ion to i t s an t id iu re t i c and vascular act ions, AVP has a number of other e f fec ts . AVP has been found to be e f fec t i ve in the management of hemophilia and von Wil lebrand's disease, since i t can increase leve ls of factor VIII poss ib ly by inducing i t s release from vascular endothelium. The secret ion of adrenocort icotropic hormone i s stimulated by AVP which reaches the anter ior p i t u i t a ry v ia the hypophyseal portal c i r cu la t i on (Zimmerman et a l . 1977). An area which has generated much in terest recent ly i s the possib le ro le of AVP as a central neurotransmitter. Central AVP may f a c i l i t a t e memory consol idat ion (de Wied 1976; Kovacs et a l . 1979; Weingartner et a l . 1981). Central AVP may par t i c ipa te in the regulat ion of body temperature since a number of studies have shown that central AVP has ant ipyre t ic propert ies (Cooper et a l . 1979; Veale et a l . 1981). I t has also been reported that central AVP may inf luence the e x c i t a b i l i t y of neurons since i t can cause convulsions in conscious rats (Abood et a l . 1980; Kasting et a l . 1980). In add i t ion , central AVP may inf luence spontaneous water intake (Szczepanska-Sadowska 1982), regulate the permeabil i ty of the brain to water (Raichle and Grubb 1978), and control CSF absorption by the choroid plexus (Schultz et a l . 1977). 1.2.5 Role of AVP in hemorrhage, dehydration and surg ical s t ress . Since the leve ls of AVP detected in volume-depleted states such as hemorrhage, dehydration and in s t ress- re la ted condit ions such as surgical stress are much greater than required for maximal an t i d i u res i s , a number of studies have examined the ro le of AVP in blood pressure regulat ion under these condi t ions. The amount of AVP secreted in response to hypotensive - 17 -hemorrhage was found to be su f f i c i en t to cause a pressor response in anesthetized dogs in which the baroreceptor ref lexes were suppressed (Rocha e S i l v a and Rosenberg 1969). Infusion of pathophysiological amounts of AVP (equivalent to levels of plasma AVP induced by condit ions such as surgery or hemorrhage) increased blood pressure in conscious dogs (Szczepanska-Sadowska 1973). Blood loss of 1 percent of body weight did not af fect blood pressure in control r a t s , but decreased blood pressure in Bratt leboro rats with diabetes ins ip idus (Laycock et a l . 1979). Dogs with diabetes ins ip idus have also been shown to be more suscept ible to hemorrhage than normal dogs, an ef fect that can be reversed by the administrat ion of P i t r ess i n (Frieden and Ke l l e r 1954). An AVP pressor antagonist administered to anesthetized rats subjected .to surgical stress (Pang 1983a) or hypotensive hemorrhage (Pang 1983b) was found to decrease blood pressure through a reduction in to ta l peripheral res is tance, and increase the d is t r i bu t ion of blood flow to the skin and stomach. Schwartz and Reid (1981) found that AVP par t ic ipates in the maintenance of blood pressure af ter non-hypotensive hemorrhage in conscious dogs, since the i . v . administrat ion of a se lec t ive antagonist of the pressor ef fect of AVP decreased blood pressure in dogs. A se lec t ive vascular antagonist of AVP was also found to prevent the restorat ion of blood pressure af ter hemorrhage in animals subjected to baroreceptor denervation and inact iva t ion of the renin-angiotensin system, leading the authors to conclude that AVP released in hemorrhage can funct ion as a rapid and potent system to maintain blood pressure (Cowley et a l . 1980). Dehydration resu l t ing from r e s t r i c t i o n of water intake has been shown to increase plasma AVP levels to 10-30 pg/ml, an increase su f f i c i en t to produce s ign i f i can t hemodynamic ef fects (Andrews and Brenner 1981; Aisenbrey et a l . 1981). The in jec t ion of a vascular antagonist of AVP in conscious dehydrated rats (Aisenbrey et a l . 1981) and dogs (Schwartz and - 18 -Reid 1983) s i g n i f i c a n t l y decreased blood pressure. In contrast , i t has been shown that the in jec t ion of an AVP pressor antagonist in conscious dehydrated rats did not decrease blood pressure even af ter blockade of the renin-angiotensin system (Fejes-Toth et a l . 1985). Rascher et a l . (1985) found that an AVP vascular antagonist decreased to ta l peripheral resistance but did not decrease blood pressure in conscious dehydrated rats unless they were also subjected to s ino-aor t i c denervation. Furthermore, the administrat ion of AVP antiserum in anesthetized rats with renal f a i l u r e induced by glycerol (a condit ion in which plasma volume is reduced and plasma osmolal i ty increased) was found to s i g n i f i c a n t l y decrease blood pressure (Hofbauer et a l . 1977). It may be argued that while an AVP antagonist may not decrease blood pressure under some condi t ions, i t does decrease peripheral res is tance, suggesting that endogenous AVP has vascular act ions. In summary, in pathophysiological condit ions such as hemorrhage, renal f a i l u r e and poss ib ly dehydration, AVP may par t i c ipa te in the short-term maintenance of blood pressure and peripheral res is tance. 1.2.6 Role of AVP in hypertension. Since AVP may be involved in cardiovascular regulat ion and par t ic ipates in the regulat ion of f l u i d volume through i t s an t id iu re t i c ac t ions, i t i s tempting to speculate that i t may have a ro le in hypertension. The ro le of AVP in a number of animal models of hypertension has been examined. Both an increase in plasma AVP level and s e n s i t i v i t y to the pressor ef fect of AVP were reported in the spontaneously hypertensive rat (SHR), suggesting that AVP may play a ro le in the development of th is genetic model of hypertension (Hoffman et a l . 1977). However, a se lec t ive antagonist of the pressor but not the an t id iu re t i c ef fect of AVP produced only a small reduction in blood pressure in ten-week old SHR. It has also been reported that spec i f i c AVP antiserum decreased blood pressure in stroke-prone SHR with well-developed hypertension (Mohring - 19 -et a l . 1979). AVP is ce r ta in l y not essent ia l for the development of spontaneous hypertension since Ganten et a l . (1983) have developed a s t ra in of SHR which also develop hypothalamic diabetes ins ip idus . Therefore i t i s possible that AVP may contr ibute to th i s form of hypertension only in i t s la te r stages. Increases in both plasma AVP levels (Matsuguchi et a l . 1981; Share et a l . 1982) and-urinary excretion of AVP (Share et a l . 1982) were observed in Dahl sa l t - sens i t i ve rats fed a high sa l t d ie t . Although enhanced pressor responsiveness was also observed (Matsuguchi et a l . 1981), suggesting that AVP plays a ro le in th is model of hypertension, in jec t ion of an AVP pressor antagonist did not decrease blood pressure (Matsuguchi et a l . 1981; Share et a l . 1982). F a i r l y convincing evidence ex is ts to suggest that AVP plays a c ruc ia l ro le in the development of another form of sa l t - re la ted hypertension. Friedman et a l . (1960) f i r s t suggested that AVP may contr ibute to the pathogenesis of deoxycort icosterone/sal t (DOC/salt) hypertension based on studies in which the administrat ion of exogenous AVP accelerated the development of hypertension, and neurohypophyseal denervation prevented i t . This type of hypertension cannot be induced in Bratt leboro rats with diabetes ins ip idus , except af ter treatment with AVP (Berecek et a l . 1982). Both plasma leve ls (Mohring et a l . 1977; Crofton et a l . 1980) and renal excret ion of AVP (Crofton et a l . 1979, 1980; Marchetti et a l . 1980) were increased in DOC/salt hypertensive ra t s . Furthermore, in rats subjected to the administrat ion of e i ther a spec i f i c AVP antiserum (Mohring et a l . 1977) or an AVP pressor antagonist (Crofton et a l . 1979; Mento et a l . 1982) a s ign i f i can t decrease in blood pressure was observed. Although plasma AVP levels have been reported to be elevated in one kidney-one c l i p renal hypertensive rats (Pullan et a l . 1980), i t i s un l i ke ly - 20 -that AVP i s essent ia l for the development of t h i s type of hypertension, since i t can be produced in Bratt leboro rats (Johnston et a l . 1981). Only 50 percent of rats subjected to two kidney-one c l i p renal hypertension displayed elevated plasma AVP levels or a reduction in blood pressure in response to AVP antiserum (Mohring et a l . 1978). An in tact neurohypophyseal system is not c r i t i c a l to the development of two kidney-one c l i p renal hypertension (Johnston et a l . 1981) suggesting that AVP does not play a s ign i f i can t ro le in th is type of hypertension. S ign i f i can t increases in plasma concentration and urinary excret ion of AVP were observed in rats which had 70 percent of the renal mass removed and were given normal sa l ine to drink (Lee-Kwon et a l . 1981). However, i t i s un l i ke ly that the pressor act ion of AVP i s so le ly responsible for the resu l t ing hypertension since a pressor antagonist did not decrease blood pressure in these rats (Lee-Kwon et a l . 1981). I t i s possible however, that AVP contr ibutes to the hypertension through i t s an t id iu re t i c e f fec t , since pa r t i a l nephrectomy-salt hypertension i s a volume-dependent model of hypertension. The ro le of AVP in human essent ia l hypertension i s con t rovers ia l . Plasma AVP concentrations have been reported to be decreased (Padf ie ld et a l . 1976; Shimamoto et a l . 1979) or elevated (Cowley et a l . 1981). Elevated plasma AVP levels have been observed in pat ients with malignant hypertension (Padf ie ld et a l . 1981). A small increase in s e n s i t i v i t y to the pressor ef fect of AVP was reported in pat ients with mild to moderate hypertension, but the level was un l i ke ly su f f i c i en t to account for the elevated blood pressure of these pat ients (Padf ie ld et a l . 1976). While AVP leve ls have been shown to be elevated in a number of hypertensive animal models, the resu l t ing leve ls appear to be i nsu f f i c i en t to account for the increase in blood pressure, unless appreciable enhancement of the s e n s i t i v i t y to the pressor ef fect of AVP also occurs. An - 21 -increased s e n s i t i v i t y to AVP occurring in combination with elevated plasma AVP l eve l s , as observed in DOCA-salt hypertensive ra t s , SHR and Dahl s a l t - s e n s i t i v e ra t s , may contr ibute to elevated blood pressure in these animals. It has also been suggested that the an t id iu re t i c action of AVP may contr ibute to the development of some forms of hypertension, espec ia l l y those involv ing volume expansion (Share and Crofton 1984). 1.3 The sympathetic nervous system 1.3.1 Structure of the sympathetic nervous system. In ject ion of extracts of the adrenal gland were reported by Ol iver and Schafer in 1895 to increase blood pressure in animals. The act ive component, which was subse-quently named adrenal ine, was soon iso lated and iden t i f i ed (Aldr ich .1901; Takamine 1901; F i g . 2 ) . The primary homologue of adrenal ine, d l -nor-adren-a l ine (arterenol) was a r t i f i c i a l l y synthesized in 1904 by S t o l z , who observed that i t was as e f fec t ive as adrenaline in increasing blood pressure in an i -mals ( F i g . 2) . The observation that adrenal extracts produced responses s im i la r to st imulat ion of sympathetic nerves led to the suggestion that adrenaline was released from sympathetic nerves ( E l l i o t t 1904). However i t was suggested by Barger and Dale in 1910 that noradrenaline produced responses which were more s im i la r to sympathetic nerve st imulat ion than those produced by adrenal ine. Nerve, f i b res which release adrena l ine- l ike substances were la te r c l a s s i f i e d as adrenergic (Dale 1933). There were various ind icat ions that the transmitter released by the sympathetic nerves was not adrenal ine. Through experiments involv ing st imulat ion of the hepat-ic nerve, Cannon and U r i d i l (1921) found evidence of a substance with sympa-thomimetic actions which was not adrenal ine. Cannon and Bacq (1931) through another ser ies of experiments concluded that sympathetic nerve st imulat ion led to the appearance of a substance other than adrenal ine, which they named sympathin. I t was not un t i l 1946 that von Euler establ ished that the t rans-mit ter released by adrenergic nerves was noradrenaline. - 22 L-Tyrosine H O < f >— C H 2 — C H — N H 2 C O O H Tyrosine hydroxylase H O , - D O P A H 0 \ / ~ C H ^ - C H - N H 2 C O O H l - D O P A decarboxylase H Q . Dopamine HO<( 7— C H 2 — C H 2 — N H 2 Dopamine p-hydroxylase HC Noradrenal ine H O < f > - C H ( O H ) - C H 2 - N H 2 Phenylethanoiamine W-mcthyltransferase H Q Adrenal ine H O < f 7— C H ( O H ) — C H 2 — N H — C H j F i g . 2. The biosynthet ic pathway for noradrenaline and adrenal ine. -23-The general organization and d i s t r i bu t ion of the sympathetic nervous system was described by Langley in 1921. The sympathetic efferent pathway i s comprised of a chain of at least two types of neurons: preganglionic neurons located in the CNS and postganglionic neurons located in the gangl ia . Ear ly experiments by Kibjakow (1933) and Feldberg and Gaddum (1933) led to the i den t i f i ca t i on of the transmit ter released by sympathetic preganglionic f i b res as acety lcho l ine. Burn and Rand (1959) then establ ished that acety lcho l ine , through a n i co t i n i c e f fec t , caused the release of noradrenaline from the sympathetic nerve terminal and noradrenaline and adrenaline from the chromaffin c e l l s of the adrenal medulla which are anatomically re lated to autonomic gangl ia . Noradrenaline i s synthesized from the amino acid tyrosine within sympathetic nerve endings according to the pathway proposed by Blaschko in 1939 (F i g . 2 ) . The synthetic pathway continues to the synthesis of adrenaline only in the chromaffin c e l l s . Noradrenaline ex is ts in three states within the neuron: f ree noradrenaline in cytoplasm, f ree noradrenaline wi thin membrane-bound storage v e s i c l e s , and bound noradrenaline wi thin storage ves ic les (Burnstock and Costa 1975). 2+ Noradrenaline i s taken up into the ves ic les by an ATP and Mg -dependent process where i t i s stored with ATP and ac id ic proteins ca l led chromogranins. Noradrenaline i s released from the ves ic les by a 2+ Ca -dependent exocytosis in response to nerve impulses and i t subsequently acts on receptors on the ef fector organ. The ef fects of released noradrenaline are terminated pr imar i ly by reuptake into the axons (uptake-^) or the ef fector c e l l s (uptake2). Noradrenaline may also be subject to enzymatic catabolism by catechol-O-methyltransferase and monoamine oxidase, although enzymatic degradation is much less important for terminating the ef fects of noradrenaline than reuptake. - 24 -1.3.2 C l a s s i f i c a t i o n of adrenoceptors. In 1905, Langley proposed that the ef fector c e l l s contained two d i f ferent "recept ive substances" with which the released transmit ter combined to produce ei ther inh ib i to ry or exci tatory e f fec ts . Cannon and Rosenblueth (1933) proposed a somewhat d i f ferent concept of the receptor when they suggested that the transmit ter sympathin combined with e i ther of two receptive substances, E or I, wi thin the ef fector t issues which conferred e i ther exc i ta tory or inh ib i to ry propert ies on the t ransmit ter , respect ive ly . This theory was eventual ly rejected af ter evidence in support of Langley 1s o r ig ina l proposal became ava i lab le . Although unaware of i t s s ign i f icance at the time, Dale (1906) made one of the f i r s t observations in support of the receptor theory when he blocked the pressor ef fect of adrenaline in the spinal cat with ergotoxine and provided the f i r s t demonstration of receptor antagonism. A lqu is t (1948) compared the re la t i ve potencies of a ser ies of sympathomimetic amines in a number of d i f ferent t i ssues . This work resulted in the c l a s s i f i c a t i o n of adrenoceptors into two d i s t i nc t i ve types: a-receptors which are highly sens i t i ve to noradrenaline and adrenaline but are r e l a t i v e l y insens i t i ve to isopropylnoradrenal ine, and e-receptors at which the order of potency of the amines was isopropylnoradrenaline > adrenaline > noradrenaline. The a-adrenoceptors were found to be located pr imar i ly in the vasculature, while the B-adrenoceptors were found in the heart, vasculature and other smooth muscles. The 6-adrenoceptors were eventual ly subc lass i f ied into and &2 types, based on the d i f ferent s e n s i t i v i t i e s of a number of t issues to various 3-adrenergic agonists (Lands et a l . 1967). It also gradual ly became apparent that the a-adrenoceptors did not comprise a d iscrete populat ion. Brown and G i l l e s p i e (1957) observed that the a-adrenoceptor antagonists dibenamine and phenoxybenzamine potentiated the overflow of noradrenaline e l i c i t e d by nerve st imulat ion in the --25 -blood-perfused cat spleen. At the time they at t r ibuted the increased overflow of transmitter to occupation of the post- junct ional receptors by the antagonist, resu l t ing in higher leve ls of unbound transmit ter present in the neuroeffector junct ion. While phenoxybenzamine inh ib i t s the metabolism of noradrenaline released by nerve s t imula t ion, and i nh ib i t s i t s neuronal uptake, i t was shown that these ef fects alone could not account for the enhanced overflow of noradrenaline caused by phenoxybenzamine and other a-adrenergic antagonists such as phentolamine (Langer 1970; Langer and Vogt 1971; Starke et a l . 1971; Enero et a l . 1972). Based on these f ind ings , the hypothesis that a-receptors were present on the adrenergic nerve terminals was postulated. I t was proposed that these pre- junct ional a-receptors when act ivated by noradrenaline present in the neuroeffector junct ion decreased the subsequent release of noradrenaline through a negative feedback mechanism (Langer et a l . 1971; Farnebo and Hamberger 1971; Enero et a l . 1972; Starke 1972; Rand et a l . 1973; Langer 1973, 1974). Phenoxybenzamine was then found to be 30 times more potent in blocking post- junct ional a-receptors than pre- junct ional receptors mediating noradrenaline re lease, suggesting that these two types of receptors were not ident ica l pharmacologically (Dubocovich and Langer 1974; Langer 1974). Langer (1974) suggested that the a-adrenoceptors be subdivided into two classes based on anatomic loca t ion : post- junct ional receptors were designated a^-adrenoceptors, while those located pre- junc t iona l ly on the nerve terminal were ca l led ag-adrenoceptors. The receptor c l a s s i f i c a t i o n scheme of Langer (1974) assumed that post- junct ional a-receptors were a homogeneous populat ion. However, i t was observed in cats and rats that vasoconstr ic t ion mediated by the post- junct ional receptors could not be completely abolished by prazosin, an a-^-adrenergic antagonist, suggesting that perhaps a sub-population of -26 -a-receptors was present post - junc t iona l ly (Bentley et a l .1977). Moulds and Jauernig (1977) also reported a s im i la r f ind ing in vivo in human a r te r i es . The existence of two classes of a-receptors at the post- junct ional s i t e was f i n a l l y demonstrated by Drew and Whiting (1979) in the anesthetized cat and pithed ra t . They found that prazosin, a so-ca l led a^-adrenergic antagonist, or yohimbine, a se lec t i ve a2~adrenergic antagonist, could not completely antagonize the pressor response to phenylephrine, although the response was abolished i f both antagonists were given together. I t was then establ ished that both a-^  and a2-adrenoceptors are present pos t - junc t iona l l y . As a resu l t , Langer's o r ig ina l proposal to designate the post- junct ional receptors a-j-adrenoceptors and the pre- junct ional receptors a2-adrenoceptors led to confusion. Therefore the terms pre- and post- junct ional presently refer only to anatomical locat ion of receptors, while the terms a^- and a2~adrenoceptor designate the pharmacological s e l e c t i v i t y based on the preference of the receptors for cer ta in agonists and antagonists. a-j-Adrenoceptors were designated as those s i tes where methoxamine or phenylephrine showed greater potency than c lon id ine , and a 2-adrenoceptors as those s i tes where the reverse order of potency was observed (Berthelsen and Pett inger 1977). 1.3.3 Peripheral a-adrenoceptors. Pre- junct ional a2~adrenoceptors have been demonstrated in a number of t i ssues , including the rabbi t iso la ted pulmonary artery (Starke 1981), rabbi t ear artery (Drew 1979) and autoperfused hindlimb (Steppeler et a l . 1978), rat heart (Drew 1979; P ich ler and Kobinger 1978), anococcygeus muscle (Leighton et a l . 1979; Doxey 1979) and vas deferens (Doxey et a l . 1977; Drew 1977), cat hindlimb (Pich ler and Kobinger 1978), dog heart (Lokhandwala et a l . 1977) and mouse vas deferens (Marshall at a l . 1978). It has been c l e a r l y demonstrated that the st imulat ion of pre- junct ional a ?-adrenoceptors decreases the amount of -.11 -noradrenaline released per impulse. No mechanism has yet been demonstrated which could account for these e f fec ts . In addi t ion, the physio logical s ign i f i cance of th is ef fect has been questionned (Drew 1980; Holman and Surprenant 1980; F i tzGera ld et a l . 1981; Kalsner 1982, 1983; Kalsner and Qui 11 en 1984). This modulation of noradrenaline release from the nerve terminal could possib ly play a ro le in cardiovascular regula t ion. Prazos in , a se lec t ive antagonist of a-j-adrenoceptors, causes vasodi la tat ion and a decrease in blood pressure but l i t t l e re f lex tachycardia (Lund-Johanssen 1974; Safar et a l . 1974) or renin release (Massingham and Hayden 1975; Graham et a l . 1976). Since the act iva t ion of peripheral pre- junct ional ag-adrenoceptors i nh ib i t s noradrenaline released by st imulat ion of the post-gangl ionic sympathetic nerves, i t has been suggested that the i n a b i l i t y of prazosin to block these receptors may account for the lack of re f lex tachycardia and renin release observed in response to th i s drug (Stokes and Oates 1978). I t has also been suggested that pre- junct ional (^-adrenoceptors in the heart may contr ibute to the bradycardia induced by drugs such as c lon id ine , since c lonid ine was shown to decrease the amount of noradrenaline overflowing into the coronary sinus blood in dogs (Cavero and Roach 1980), and also to i nh ib i t tachycardia in response to sympathetic st imulat ion in pithed rats (de Jonge et a l . 1981). However the physio logica l ro le of these cardiac c^-adrenoceptors remains unclear. Post- junct ional a-adrenoceptors which mediate the ef fects of the sympathetic nervous system in vascular smooth muscle were a l l considered to be of the c l a s s i c a l a-^-adrenoceptor type un t i l i t was rea l ized that receptors with the pharmacological s e l e c t i v i t y of pre- junct ional o^-adrenoceptors were also present at post- junct ional s i tes (Drew and Whiting 1979). The subdiv is ion of vascular post- junct ional a-adrenoceptors into ay- and a2-adrenoceptors has been demonstrated in rats (Timmermans - 28 -et a l . 1979; Drew and Whiting 1979), rabbi ts (Hamilton and Reid 1982), dogs (Langer et a l . 1981), cats (Timmermans 1981) and humans (van Brummelen et a l . 1982; Flavahan et a l . 1987). The a^/ot 2 receptor ra t io at post- junct ional vascular s i tes was determined by examining blood pressure responses to se lec t ive ay- and o^-agonists in decapitated cats (Kobinger and P ich le r 1982) and pithed rats (Kobinger and P ich le r 1981) and i t was suggested that both subtypes are of equal importance. Both in v i t ro studies in dog saphenous vein (de Mey and Vanhoutte 1981; Vanhoutte 1982) and in vivo studies (Greenway 1979; Stevens and Moulds 1982; Pang and Tabrizchi 1986) suggest that o^-adrenoceptors. are present in ve ins. I t has been suggested that the post- junct ional o^-adrenoceptors may be located ex t ra - junc t iona l l y , and that they are pr imar i ly stimulated by c i r cu l a t i ng Catecholamines rather than noradrenaline released from the nerve terminal (Langer 1981; Wi l f fe r t et a l . 1982). Although the re la t i ve a f f i n i t y of the post- junct ional c i 2 - r e c e P t o r s f ° r various agonists and antagonists is ident ica l to that of the pre- junct ional q^-receptors, the post- junct ional receptors were shown to mediate both vasoconstr ict ion (Kobinger and P ich le r 1980a,b; Timmermans and Van Zwieten 1980a,b; E l l i o t t and Reid 1983; Van Meel et a l . 1983; Hicks et a l . 1984; Pang and Tabrizchi 1986) and venoconstr ict ion (Schumann and Lues 1983; Shoji et a l . 1983; Kalkman et a l . 1984; Steen et a l . 1984; Pang and Tabrizchi 1986) in response to sympathetic nerve st imulat ion or the administrat ion of se lec t ive adrenergic agonists. In contrast , while the (^-adrenoceptors were found to mediate vasoconstr ict ion and maintain peripheral resistance and blood pressure in r a t s , they have not been found to contr ibute to the maintenance of mean c i rcu la to ry f i l l i n g pressure, an index of to ta l body venous tone (Pang and Tabr izchi 1986). - 29 -1.3.4 Central a-adrenoceptors. Central a-adrenoceptors have been shown to be pr imar i ly a.^ in nature by both binding studies (U'Prichard et a l . 1977; Perry and U'Prichard 1981; Ja r ro t t et a l . 1979) and pharmacological studies (Timmermans et a l . 1981; Kobinger and P ich le r 1982). Central a^-adrenoceptors have been shown to be pharmacologically s im i la r to both pre- junct ional a2~receptors in the rat vas deferens (Doxey et a l . 1983) and post- junct ional a2~adrenoceptors on vascular smooth muscle (Kobinger and Pich ler 1980b). I t remains unclear whether these central a 2 -adrenoceptors are located on the nerve terminals of adrenergic nerves (pre-synaptic) or on the c e l l bodies or dendrites of post-synaptic neurons (post-synapt ic) . A presynaptic locat ion for central ag-adrenoceptors i s supported by the observation that c lon id ine decreases noradrenaline turnover in the CNS (Anden et a l . 1970; Fu l l e r et a l . 1977), which could be at t r ibuted to i nh ib i t i on of noradrenaline release resu l t ing from st imulat ion of pre-synaptic a2-adrenoceptors. However, i t has been shown that c lon id ine was able to produce a response even af ter depletion of central noradrenaline with reserpine and inh ib i t i on of noradrenaline synthesis with a -methyl-p- tyrosine in cats (Hausler 1974) and dogs (Kobinger and P ich le r 1975), and af ter destruct ion of central adrenergic neurons with 6-hydroxydopamine (Warnke and Hoefke 1977; Kubo and Misu 1981). There was also no change in the binding charac te r i s t i cs of these receptors af ter catecholamine deplet ion (Greenberg et a l . 1976; Langer et a l . 1983). In e f fec t , c lon id ine could s t i l l produce a response even in the absence of neuronal noradrenaline, which would not be possible i f the ef fect resulted from a reduction in noradrenaline release from the nerve terminal . Therefore st imulat ion of central pre-synaptic a2~adrenoceptors does not provide a sa t i s fac to ry explanation for the ef fects of c lon id ine . A l t e rna t i ve l y , the central a2~adrenoceptors may be located - 30 -pos t -synapt ica l l y . This would be consistent with the observation that c lon id ine i s s t i l l e f fec t i ve af ter central catecholamine deplet ion. The decrease in noradrenaline turnover could be explained by an inh ib i to ry neuronal feedback mechanism in which the post-synaptic neuron would exert an inh ib i to ry influence on the pre-synaptic neuron through a neuronal project ion (Hausler 1982). Hausler (1982) has suggested that the o^-adrenoceptors may be located on non-catecholamine neurons which release a transmit ter other than noradrenaline. o^-Adrenoceptors located post -synapt ica l ly on such neurons could mediate decreases in MAP and HR i f these neurons had a tonic inh ib i to ry inf luence on the cardiovascular system. On the other hand, pre-synaptic (^-adrenoceptors on non-catecholamine neurons could mediate a reduction in MAP and HR i f the transmit ter released normally stimulated the cardiovascular system. C lea r l y , the anatomical s i t e of central c^-adrenoceptors has yet to be def ined. Within the CNS, o^-adrenoceptors are present in brain stem areas such as the nucleus tractus s o l i t a r i u s (NTS), the vasomotor center and the nucleus motoris dorsal is vagus (Chalmers 1975). A decrease in blood pressure and heart rate was shown to occur in response to the st imulat ion of central o^-adrenoceptors by in t rac is te rna l in jec t ion of c lonid ine in cats (Kobinger 1967) and dogs (Onesti et a l . 1971), in jec t ion of c lonid ine into a vertebral artery in cats (Sat t le r and van Zwieten 1967) and dogs (Constantine and McShane 1968). S imi lar responses were observed af ter the in jec t ion of noradrenaline or a-methyl noradrenal ine, a se lec t ive o^-adrenergic agonist (de Jonge and Nijkamp 1976; Kubo and Misu 1981) or c lonidine (Kubo and Misu 1981) into the NTS. Clonidine was also shown to reduce or abol ish spontaneous e l e c t r i c a l discharges in preganglionic and postganglionic sympathetic nerve f ibers in cats (Hukuhara et a l . 1968) and rats and dogs (Schmitt et a l . 196 7; Klupp - 31 -et a l . 1970). I t i s also possible that central c^-adrenoceptors may f a c i l i t a t e the baroreceptor r e f l ex , since the re f lex bradycardia in response to i . v . in jec t ion of angiotensin II in dogs was increased by in t rac is te rna l but not i . v . in jec t ion of c lonid ine (Kobinger and Walland 1971). The experiments were performed on animals subjected to cardiac sympathetic blockade with t o l i p r o l o l , suggesting that th is ef fect i s vagally-mediated. Central (^-adrenoceptors have also been shown to enhance baroref lex a c t i v i t y by decreasing sympathetic outflow to the periphery. The in jec t ion of the o^-antagonist yohimbine into a vertebral artery in bi lateral ly-vagotomized dogs was shown to i nh ib i t the re f lex bradycardia e l i c i t e d by i . v . in jec t ion of noradrenaline or st imulat ion of the carot id sinus nerve (Huchet et a l . 1983). Thus, central a 2-adrenoceptors appear to decrease blood pressure and heart rate through an increase in vagal a c t i v i t y and a reduction in sympathetic nervous a c t i v i t y ; the mechanism by which st imulat ion of these receptors brings about such ef fects remains to be e luc idated. 1.4 Aims of the studies This thes is w i l l focus on the ro les of the vasopressin and the sympathetic nervous systems in the regulat ion of blood pressure, at both the central and the peripheral l e v e l . 1.4.1 Role of AVP in cardiovascular regu la t ion. A possible new ro le for AVP as a neurotransmitter in the CNS has recent ly come to l igh t as a resu l t of neuroanatomical studies which have revealed that neurons containing AVP or ig ina t ing in the paraventr icular nucleus (PVN) of the hypothalamus extend to a number of areas in the ventro latera l medulla. Studies employing retrograde d is t r i bu t ion of horseradish peroxidase (Saper et a l . 1976) and dyes (Swanson and Sawchenko 1980) and immunohistochemical techniques (Swanson 1977) have shown that these AVP-containing neurons - 32 -terminate on catecholamine-containing neurons of the A-^  group at the NTS. Ascending catecholaminergic f i b res of the A-^  group have also been shown to terminate predominantly in the region of AVP-containing neurons in the PVN (Swanson and Sawchenko 1980). Therefore a descending-ascending neuronal connection is present between the PVN and NTS. Moreover, neurons which project from the medulla to the PVN can i nh ib i t the release of AVP (Blessing et a l . 1982), although a more recent study has shown that st imulat ion of the A^ c e l l group in the medulla enhances the a c t i v i t y of AVP-secreting neurons in the PVN (Day et a l . 1984). The discharge rate of PVN neurons has also been shown to be al tered by st imulat ion of the buffer nerves (Calaresu and C i r i e l l o 1980; Yamashita et a l . 1984). AVP project ions to the NTS have been found to terminate in axosomatic and axodendrit ic contacts, which suggests that an in teract ion with neurons occurs rather than the release of hormone into the blood (Sofroniew 1980). Immuno-electron microscopic studies have shown that the AVP present in PVN neurons i s contained within ves ic les (Bui js and Swaab 1979; Voorn and Bui js 1983). Binding studies have also revealed spec i f i c binding s i tes for AVP on neural membranes in the NTS (Dorsa et a l . 1983; Brinton et a l . 1984). This evidence suggests that AVP may funct ion as a neurotransmitter in the CNS. Since the NTS is the primary s i t e of termination of the afferent neurons of the baroreceptor re f lex arc (Cot t le 1964; C r i l l and Reis 1968), i t i s possible that AVP may be involved in the neurogenic control of the c i r c u l a t i o n . A number of studies have invest igated th i s p o s s i b i l i t y . Nashold et a l • (1961) showed that in jec t ion of AVP into a la te ra l cerebral ven t r i c le in cats elevated blood pressure. I .c .v. in ject ions of AVP have since been shown to increase both blood pressure and heart rate in both anesthetized (Pittman et a l . 1982) and conscious rats (Zerbe et a l . 1983). In contrast , - 33 -other studies have shown that i . c . v . in jec t ions of AVP decrease blood pressure and heart rate in anesthetized ra t s , yet increase blood pressure and heart rate in conscious rats (Zerbe and Feuerstein 1985). Inject ions of AVP d i rec t l y into the NTS of anesthetized rats (Matsuguchi et a l . 1982; Va l le jo et a l . 1984) and conscious rabbits (Martin et a l . 1983, 1985) have also been shown to increase blood pressure, impl icat ing the NTS as a s i t e of action of the central pressor ef fect of AVP. The pressor responses to central in jec t ion of AVP were abolished by gangl ionic blockade (Matsuguchi et a l . 1982), suggesting that they were mediated by the sympathetic nervous system. The purpose of our experiments was f i r s t of a l l to examine more d i r ec t l y whether the responses to central AVP are mediated by an increase in sympathetic nerve a c t i v i t y . The ef fect of i . c . v . in jec t ion of AVP on blood pressure and plasma noradrenaline and adrenaline concentrations was examined in conscious ra t s . Plasma levels of noradrenaline and adrenaline have been shown to provide a r e l i a b l e estimate of sympathetic nerve a c t i v i t y (Yamaguchi and Kopin 1979; Goldstein et a l . 1983; Es ler et a l . 1985; Hubbard et a l . 1986). As w e l l , in order to assess the ro le of central AVP, the ef fect of central in jec t ion of a se lec t ive AVP pressor antagonist was invest igated. A second ser ies of experiments was car r ied out to assess the central ro le of endogenously-released AVP on blood pressure and catecholamine release in hypotensive ra ts . In these experiments, AVP antagonist was injected cen t ra l l y into conscious rats subjected to infusion of n i t ropruss ide. In a f i n a l ser ies of experiments, the possible ro le of the NTS as a s i t e of action of central AVP was examined by in jec t ing AVP and AVP antagonist d i r e c t l y into the NTS in conscious rats and monitoring blood pressure, heart rate and plasma catecholamine responses. In order to determine i f endogenously-released AVP in the region of the NTS par t ic ipates - 34 -in cardiovascular regulat ion during neurogenic s t ress , AVP antagonist was injected into the NTS of rats exposed to a 150 watt heat lamp. This model of stress has been shown to reproducibly e l i c i t an increase in blood pressure in a l l the animals tested (Chan et a l . 1985). Since anesthetics may in ter fere with the responses to central in jec t ions of AVP, a l l studies were car r ied out in conscious, unrestrained ra ts . Another object ive of our study was to invest igate the vascular ro le of AVP. Surgery has been reported to cause the release of large amounts of AVP (Moran et a l . 1964; Bonjour and Malvin 1970; Ishihara et a l . 1978). Since AVP has been shown to be a potent pressor agent (Al tura and A l tu ra 1977), i t i s log ica l to speculate that AVP released during surgery may contr ibute to the maintenance of blood pressure. AVP released by surgery has indeed been shown to par t i c ipa te in the control of blood pressure and peripheral vascular resistance (McNeil l and Pang 1982; Pang 1983a). However, the vascular ro le of AVP may have been underestimated in the presence of the buffer ing re f lex systems, such as the sympathetic nervous and the renin-angiotensin systems. It has been shown that af ter antagonism of the sympathetic nervous and renin-angiotensin systems, the amount of AVP released (Burnier et a l . 1983a; Waeber et a l . 1983; Dipette et a l . 1984) and i t s ef fect on blood pressure are increased (Cowley et a l . 1980; Andrews and Brenner 1981; Burnier et a l . 1983a; I r iuch i j ima 1983; McNeil l 1983). Therefore we invest igated the inf luence of endogenous AVP on cardiac output and i t s d i s t r i bu t ion in pentobarbi ta l -anesthet ized, surg ica l l y -s t ressed rats in the presence and absence of inf luence from the renin-angiotensin or the a-adrenergic systems. 1.4.2 Role of the a-adrenergic system in cardiovascular regu la t ion. The mechanism of antihypertensive action of a2-agonists such as c lon id ine , guanfacine and a-methydopa has general ly been at t r ibuted to the st imulat ion --35 -of central c^-adrenoceptors leading to a reduction in sympathetic nerve a c t i v i t y , blood pressure and heart rate (Kobinger 1981; Kobinger and P ich le r 1982). However, c lon id ine has also been shown to stimulate o^-adrenoceptors at peripheral pre- junct ional s i t e s , resu l t ing in less tachycardia in response to e l e c t r i c a l st imulat ion of sympathetic outflow in pithed rats (Kobinger and P ich ler 1980a,b). Therefore i t i s possible that peripheral pre- junct ional o^-adrenoceptors in the heart and vasculature and central o^-adrenoceptors may contr ibute to the bradycardic and hypotensive ef fects of c lon id ine . Kobinger and P ich le r (1980a,b) also reported that th i s drug stimulates c^-adrenoceptors at peripheral post- junct ional s i t e s , resu l t ing in vasoconstr ic t ion and an increase in blood pressure. In addi t ion, c lonid ine i s an c^-adrenergic agonist with par t ia l a-^-agonistic propert ies (Timmermans and van Zwieten 1980a) which may also contr ibute to i t s pharmacological e f fec t . Therefore the central r e l a t i ve to the peripheral ef fects of c^-adrenergic agonists are not yet c l ea r l y estab l ished. In order to completely separate the central from the peripheral ef fects of adrenergic agonists we developed a c ross -c i r cu la t i on technique in anesthetized ra ts . The c i r cu la t ions of two rats were joined so that peripheral blood from one rat supplied the brain of a second rat and then returned to the body of the f i r s t ra t , and vice versa for blood from the second ra t . A drug could then be injected intravenously in one ra t , which would d isp lay the peripheral ef fects of the drug, and the drug would be del ivered to only the brain of the second, rat which would exhib i t the central e f fects of the drug. Using th is technique, we separated the central from the peripheral component of the cardiovascular e f fect of c lon id ine . The actions of methoxamine, a se lec t ive ctj-adrenergic agonist, were also invest igated in th is preparation to provide a contrast to c lon id ine , since - 36 -methoxamine increases blood pressure by an action on peripheral o^-adrenoceptors but, unl ike c lon id ine , has not been shown to decrease blood pressure or heart rate by central act ions. A disadvantage of the c ross -c i r cu la t i on experiments was the requirement for anesthesia and surgery, which could inf luence neurotransmitter and hormone release and the responses to drugs. Due to the surgical stress to which these animals were subjected, i t was not feas ib le to examine the ef fects of these drugs on catecholamine re lease. Therefore in order to determine f i r s t l y whether i t was possible to extend the resu l ts of the c ross -c i r cu la t i on studies to conscious animals, and secondly whether the sympathetic nervous system was involved in mediating the responses to c lon id ine , the ef fects of i . c . v . in jec t ion of c lon id ine on blood pressure, heart rate and plasma catecholamine concentrations were examined in conscious ra t s . Since i t i s also unclear whether a l l c^-adrenergic agonists have s im i la r act ions, the ef fects of i . c . v . in jec t ion of a more se lec t ive o^-adrenergic agonist (Kobinger and P ich le r 1980a,b), B-HT 920, were also invest igated. The a b i l i t y of a se lec t i ve ag-adrenergic antagonist, rauwolscine, to block the responses to central in jec t ion of c lon id ine and B-HT 920 were also invest igated to determine whether the responses to these drugs were a resu l t of ^ -adrenoceptor ac t i va t ion . - 37 -2 METHODS 2.1 Central AVP in conscious rats 2.1.1 Surgical preparation 2.1.1.1 Implantation of in t racerebroventr icu lar cannulae. Male Wistar rats (280-320 g, Charles River Canada, Inc.) were anesthetized with sodium pentobarbital (65 mg/kg, i .p . ) and mounted in a stereotaxic device (David Kopf) to allow implantation of a cannula into the fourth cerebroventr ic le . The inc iso r bar was placed 5 mm above the ear bar.. A 2-3 cm inc is ion was made through the skin and subcutaneous t i s sues . The t i ssue was retracted with hemostats at each corner and the sku l l was allowed to dry. The guide cannula posi t ion was marked by measuring from the bregma using the stereotaxic manipulator. Stereotaxic coordinates used in the posi t ioning of the cannula were 10.5 mm poster ior to the bregma and 7.8 mm ventral to the dura (Pel legr ino et a l . 1979). Using a d r i l l (Dremel, Model 280) with a dental d r i l l b i t , three holes were d r i l l e d just through the sku l l at s i tes around the cannula posi t ion and s ta in less steel screws were inserted in the holes. The hole for the cannula was then d r i l l e d and a s ta in less steel cannula (23-gauge, 15 mm long) was lowered into p lace. The cannula was surrounded with gelfoam and held in place with dental cement (P las t i c Products Co.) su f f i c i en t to s t a b i l i z e the cannula and cover the 3 screws. The stereotaxic manipulator, with the guide cannula holder, was removed once the cement was hardened. Patency was maintained using a plug consist ing of 15 mm of a 30-gauge needle fused to a short length of a crimped 19-gauge needle. When necessary, the inc is ion was closed with a suture. Animals were i nd i v idua l l y housed and allowed to recover for 7 days. - 38 -2.1.1.2 Implantation of vascular canriulae. On the day of the experiment, rats were b r i e f l y anesthetized with 1.5% halothane and the femoral artery and femoral vein were cannulated with polyethylene (PE) 50 tubing. The tubing was f i l l e d with normal sa l ine containing heparin (25 Ill/ml) to prevent c l o t t i n g . The cannulae were held in place with three s i l k t i e s . A l l cannulae were burrowed subcutaneously, ex ter ior ized and secured at the back of the neck. The cannulae were f lushed and heat-sealed by melting the end with a flame and squeezing with the f ingers . The animals were allowed to recover for at least 4 h before the experimental protocol was begun in order to avoid the inf luence of anesthesia on the experimental r esu l t s . 2.1.2 Experimental p ro toco l . An in jec t ion device, consis t ing of an in jec t ion needle (30-gauge, 15 mm) fused to a 19-gauge needle connected v ia PE 50 tubing to a Hamilton 10 ul syr inge, was inserted into the vent r icu lar cannula for i . c . v . in ject ions of drugs. The femoral a r t e r i a l cannula was connected to a pressure transducer (Gould, Model P23ID) with a length of PE 90 tubing. Conscious and unrestrained rats were placed in a small cage and allowed to accl imat ize for at least twenty min pr ior to the s tar t of the experiment. Mean a r te r i a l pressure (MAP) was continuously monitored using a Grass polygraph (Model 7D). MAP values were recorded immediately pr ior to the removal of each blood sample. A control blood sample (1 ml) was then obtained from the femoral a r t e r i a l cannula of each rat from the various groups for the determination of plasma concentrations of noradrenaline and adrenaline by high performance l i qu id chromatography (HPLC). A l l blood samples obtained for the assay were 1 ml in volume and a l l blood removed was replaced with the in jec t ion of an ident ica l volume of normal sa l ine so lu t ion . Nine groups of animals were used in the study. A l l animals were prepared with i . c . v . cannulae for central in jec t ions of drugs, except for - 39 -three groups which were prepared with the femoral a r t e r i a l and venous cannulae only and which received i . v . in jec t ions of drugs, i Five min af ter the control blood sample was obtained, rats in Group I, (n = 10) were given an i . c . v . in jec t ion of 23 ng/kg of AVP in 1 ul a r t i f i c i a l CSF over 5 sec followed by the removal of a second blood sample af ter 1 min. Ten min la ter a second i . c . v . in jec t ion of 73 ng/kg of AVP in 3 ul of a r t i f i c i a l CSF was given, followed by the removal of a f i n a l blood sample 1 min l a te r . Two d i f ferent doses of AVP were given to determine whether a re la t ionsh ip existed between dose and response. The two doses were given in sucession to achieve a cumulative dose of AVP. Animals in Group II (n = 8) received the same treatment as those in Group I except that the responses were examined at a d i f ferent time in order to assess the time course of the response to central AVP. A blood sample was withdrawn 10 min rather than 1 min af ter i . c . v . in jec t ion of the low dose of AVP. Af ter 5 min, the high dose of AVP was in jec ted; 10 min la ter a f i n a l blood sample was obtained. Groups III (n - 7) and IV (n = 6) served as controls in which the ef fects of the veh ic le , a r t i f i c i a l CSF, on MAP and catecholamine concentration were assessed 1 and 10 min af ter i . c . v . i n jec t i ons , respect ive ly . Following the protocol described above, 1 and 3 ul of a r t i f i c i a l CSF were injected in both groups. To determine whether endogenously released AVP plays a ro le in the modulation of sympathetic nervous a c t i v i t y , Group V (n = 5) was given i . c . v . in ject ions of a se lec t ive antagonist of the pressor ef fect of AVP, [ l-(B-mercapto-e,s-cyclopentamethylenepropionic ac id ) , 2-(0-methyl)- tyrosine] arginine vasopressin (d(CH 2) 5Tyr(Me)AVP) (Kruszynski et a l . 1980; Pang and Leighton 1981; Manning and Sawyer 1982). Two doses, 0.5 ug/kg in 1 ul and 1.5 ug/kg in 3 ul a r t i f i c i a l CSF, were given according to the protocol - 40 -described for Group I I . Since the antagonist i s known to have a long duration of action (Pang and Leighton 1981), blood was sampled 10 min af ter each in jec t ion only. In order to determine whether the receptors involved in mediating the pressor response to central administrat ion of AVP are ident ica l to vascular AVP receptors present per iphera l l y , the response to i . c . v . in jec t ion of AVP was observed in Group VI (n = 11) af ter pretreatment of the rats with the AVP antagonist. Five min after the f i r s t blood sample was obtained, 2.0 ug/kg of AVP antagonist (the to ta l cumulative dose used in the previous study) was injected into the fourth ven t r i c le and a blood sample obtained 1 min l a te r . Ten min after AVP antagonist was given, 96 ng/kg AVP was in jec ted, and a f i na l blood sample was removed 1 min l a te r . To ensure that the reponses observed fo l lowing central in ject ions of drugs were not due to d i f fus ion of drugs into the peripheral c i r c u l a t i o n , AVP and AVP antagonist were in jected intravenously in 3 fur ther groups. Group VII (n = 8) received doses of AVP ident ica l to those given to Group I except that the drug was dissolved in a volume of 0.1 ml normal sa l i ne . Blood samples were obtained 1 min af ter i . v . in jec t ion of each dose of AVP. Group VIII (n = 5) also received the same doses of AVP but blood samples were removed 10 min after i . v . in ject ions of AVP. Group IX (n = 5) was given the same dose of AVP antagonist ( in 0.1 ml of normal sa l ine) as Group V. Blood samples were obtained 10 min af ter i . v . in jec t ions of AVP antagonist. Blood samples were immediately placed on i c e , centr i fuged and the plasma removed and stored at , -80°C un t i l assayed for noradrenaline and adrenaline content. On completion of the experiments, rats were anesthetized with sodium pentobarbital (30 mg/kg, i . v . ) . Methylene blue (2 ul) was injected through the i . c . v . cannula to permit la te r ve r i f i ca t i on - 41 -of the posi t ion of the cannula in the fourth ven t r i c l e . Rats were then perfused t ranscard ia l l y to allow in s i t u f i xa t i on of the brain t i s sue . The thorax was cut open, the r ight atrium was snipped to allow blood to flow out, and 60 ml of normal sa l ine followed by 60 ml of 10% buffered neutral formalin (BDH) was injected into the l e f t ven t r i c l e . The brain was removed, stored in 10% neutral formalin for 10 days and la ter sectioned in a f reezing microtome (American Optical Corp. , model 880) for v isual inspection of the cannula pos i t i on . Data obtained from animals in which the cannula was not present in the fourth ven t r i c le were discarded. 2.1.3 Catecholamine analysis by HPLC 2.1.3.1 Extract ion of plasma samples. The plasma samples were obtained and stored at -80°C as described in the previous sec t ion . The extract ion procedure used was a modif icat ion of the method of Davis et a l . (1981). Polypropylene microcentri fuge tubes (Western S c i e n t i f i c Ltd.) were prepared with 20 mg alumina, 250 ul 1.5 M Tr is buffer (pH 8 .7 ) , and 25 ul 10% EDTA. To each tube, 0.5 ml plasma and 100 ul of internal standard, 10 pg/ul 3,4-dihydroxybenzylamine (DHBA), were added. For each assay, 2 spiked samples were also prepared with 0.5 ml 0.1 M sodium phosphate buffer and 50 ul each of 100 pg/ul noradrenaline, adrenaline and DHBA so lu t ions . The tubes were then shaken to mix the contents and placed on a rec iprocal shaker for 5 min. The tubes were spun in an. Eppendorf centr i fuge (Model 3200) for 30 sec, the supernatant removed by aspi rat ion and the alumina washed twice with doub le -d i s t i l l ed water. In order to extract the catecholamines from the alumina, 100 ul 0.1 M HCIO^ was added and the tubes were again agitated for 5 min. The tubes were then centri fuged and the supernatant drawn into a 1 ml disposable syringe and ejected through a disposable f i l t e r (M i l l i po re , 0.45 um pore s ize) into a f resh tube. The f i l t r a t e was immediately stored at -80°C and assayed for catecholamine content by HPLC the same day. - 42 -2.1.3.2 HPLC with electrochemical detect ion. The samples were assayed fo r noradrenaline and adrenaline content by reverse-phase ion pa i r HPLC with electrochemical detection (Davis et a l . 1981). The HPLC system consisted of a l i qu id chromatograph (Waters Associates, Model 590) and a 12.5 cm x 4.6 mm 5 urn column packed with ODS Hypers i l . The mobile phase was composed of a 0.1 M KH 2 P0 4 buffer (pH 3.77) with 50 ml methanol, 100 mg sodium octyl sulphate and 60 mg EDTA added to each l i t r e of buffer. The flow rate was 1.2 ml/min. The electrochemical detection system consisted of a carbon paste detector electrode (Bioanaly t ica l Systems Inc . , Model TL-3) packed with a graphite:nujol paste (B ioanaly t ica l Systems Inc., CP-0). The electrode potent ia l was maintained at +0.60V versus a Ag-AgCl reference electrode (Bioanaly t ica l Systems Inc., Model RE-1). Peak areas were integrated using an Apple l i e computer. The recoveries averaged 75-80 . Plasma catecholamine concentrations were calculated using the equation: r . . , . (Catecholamine/DHBA) , •- r . , , Catecholamine = j ' sample x Catecholamine . , (ng/ml) (Catecholamine/DHBA) ' , . (ng/ml) stanaan 3 standard 3 ' The coe f f i c ien ts of var ia t ion fo r 10 rep l ica te analyses (run on the same day) of a human plasma sample spiked with standard catecholamine solut ions were 8 fo r noradrenaline and 14 for adrenal ine. 2.1.4 S t a t i s t i c a l ana lys is . A l l values reported represent mean ± SD. Analysis of variance with repeated measures was used to assess the s ign i f i cance of di f ferences between drug responses and control values of MAP and plasma noradrenaline and adrenaline concentrations within each group. Since plasma catecholamine concentrations were not normal ly-d is t r ibuted, the analysis was performed af ter logarithmic transformation of these values. Where s ign i f i can t di f ferences were found, Tukey's mult ip le range test was used to compare group means. A probab i l i t y of error of less than 0.05 was preselected as the c r i t e r i on fo r s t a t i s t i c a l s ign i f i cance . - 43 -2.2 Central AVP in hypotensive rats 2.2.1 Experimental p ro toco l . Male Wistar rats were prepared with i . c . v . cannulae and femoral a r te r i a l and venous cannulae as described previously in sect ion 2 .1 .1 . MAP, recorded from the femoral ar tery , and heart rate (HR) of conscious, unrestrained rats were monitored continuously using a Grass polygraph and tachograph (Model 7P4G). To assess the inf luence of endogenously released central AVP in hypotensive animals, AVP antagonist was injected cen t ra l l y in rats made hypotensive by continuous infusion of n i t ropruss ide. Control blood samples were obtained at the beginning of the experiment for la ter plasma catecholamine ana lys is . Rats received a continuous i . v . infusion of n i t roprusside (0.083 mg/kg/min), and a second blood sample was removed 10 min l a te r . Rats in Groups I (n = 8) and II (n = 8) were then given i . c . v . in ject ions of 4.0 pi a r t i f i c i a l CSF alone or with 2.0 pg/kg AVP antagonist, respect ive ly . A f i na l blood sample was obtained 10 min l a te r . The plasma was separated, stored and analyzed as described in Section 2 .1 .3 . Rats were t ranscard ia l l y perfused and the cannula pos i t ion ve r i f i ed h i s t o l o g i c a l l y as previously described in Section 2 .1 .2 . 2.2.2 S t a t i s t i c a l ana lys is . Analysis of variance with repeated measures and Duncan's mult ip le range test were used to assess s t a t i s t i c a l s ign i f i cance . A probab i l i t y of error of less than 0.05 was preselected as the c r i t e r i on for s t a t i s t i c a l s ign i f i cance . A l l values represent mean ± SEM. 2.3 Micro in ject ion of AVP into the NTS 2.3.1 Experimental p ro toco l . Male Wistar rats were prepared by implanting cannulae cen t ra l l y 7 days pr io r to the experiment as described previously except that commercially avai lab le cannulae (P las t i c Products Co.) were used, and they were placed at the NTS rather than the fourth ven t r i c l e . The stereotaxic coordinates used were 11.6 mm poster ior to the •> 4 4 -bregma and 7.8mm ventral to the dura, in the mid l ine. The guide cannula (26-gauge, 20 mm long) was lowered in the dorso-ventral plane to a posi t ion of 0.5 mm above the NTS. A dummy cannula (33-gauge, 20 mm long) was inserted into the cannula to maintain patency. Femoral a r t e r i a l and venous cannulae were also inserted on the day of the experiment at least 4 h p r io r to the experiment. Conscious unrestrained rats were placed in a cage and MAP and HR were continuously monitored. An internal cannula (33-gauge) connected by PE tubing to a 1 ul Hamilton syringe and preloaded with drug or vehic le was inserted into the guide cannula. The internal cannula was 0.5 mm longer that, the guide cannula so that when inserted into the guide, i t was posit ioned at the level of the NTS. Af ter al lowing at least 20 min for each rat to acc l imat ize , a control blood sample was then obtained. F i f teen min l a t e r , the appropriate drug or i t s veh ic le , a r t i f i c i a l CSF, was injected into the NTS of four groups of r a t s . Each animal received one in jec t ion consis t ing of a volume of 0.2 ul given over 10 sec. Animals in Group I (n = 10) received in ject ions of 0.2 ul a r t i f i c i a l CSF. Rats in Group II (n = 8) were given 2 ng AVP, the minimally e f fec t i ve pressor dose. Group III (n = 10) received 10 ng AVP. Group IV (n = 8) rats were injected with 10 ng AVP antagonist. This dose has been shown to block the pressor ef fect of vascular responses to central in ject ions of 1-30 ng AVP (Val le jo et a l . 1984; Berecek et a l . 1984). MAP and HR were recorded at the time of maximal pressor response, within 1 min of drug i n j ec t i on , and a second blood sample was taken immediately afterward. On completion of the experiment the rats were t ranscard ia l l y perfused and the brain removed and stored in 30% sucrose formalin for at least 10 days before sect ioning and s ta in ing . - 45 -2.3.2 H is to log ica l technique. The f ixed brain t issue was sectioned on the f reezing microtome into 50 u sect ions. Sections were t ransferred to gelat in-coated glass s l ides and dried 0.5-1 h at 30-60°C. The sections were hydrated to water through the fo l lowing ser ies of so lu t ions : xylene, xylene, 100% ethanol, 70% ethanol, 35% ethanol and d i s t i l l e d water. They _ were stained with cresyl v io le t fo r 0.5-1 min and r insed quickly in d i s t i l l e d water, 70% ethanol and 95% ethanol. The sections were then placed — in d i f f e ren t i a to r , dehydrated in 50:50 absolute ethanol:xylene and cleared in 2 changes of xylene. The cresy l v i o le t working solut ion was made by combining 12 ml of cresyl v io le t solut ion (0.02 g cresyl v i o le t + 15 ml d i s t i l l e d water) with 100 ml of buffer (94 ml 0.1 M acet ic acid + 6 ml 0.1 M sodium acetate, pH 3.5) . The d i f f e ren t ia to r consisted of 90 ml 95% ethanol , — 10 ml chloroform and 3 drops g lac ia l acet ic ac id . Coversl ips were f ixed to the s l ides with mounting medium (Permount) and the s l ides were allowed to dry. The placement of the cannula at the NTS was confirmed by examining the cannula track marks with reference to a stereotaxic at las (Pel legr ino et a l . 1979). Data obtained from animals in which the cannula was not present at the NTS were re jected. 2.3.3 S t a t i s t i c a l ana lys is . Analysis of variance and Duncan's mul t ip le range test was used to assess s t a t i s t i c a l s ign i f i cance . A p robab i l i t y of error of less than 0.05 was preselected as the c r i t e r i on for s t a t i s t i c a l s ign i f i cance . A l l resu l ts are presented as mean ± SEM. 2.4 Central AVP in neurogenical ly-stressed rats 2.4.1 Experimental p ro toco l . Male Wistar rats were prepared with cannulae at the NTS, and femoral a r t e r i a l and venous cannulae as in the previous study. MAP and HR of conscious, unrestrained rats were monitored continuously. To assess the ro le of endogenous AVP in central cardiovascular regulat ion during neurogenic s t ress , AVP antagonist was - 46 -in jected into the NTS in rats subjected to a 150 watt heat lamp. A control blood sample was obtained for la ter plasma catecholamine analysis af ter the animals were allowed 20 min to become accustomed to the cage. Ten min l a te r , the heat lamp was turned on, and af ter ten min of exposure to the lamp, a second blood sample was obtained. Af ter 1 min, rats in Groups I (n = 5) and II (n = 5) were given in ject ions into the NTS of 0.2 ul a r t i f i c i a l CSF and 10 ng AVP antagonist, respect ive ly . A f i n a l blood sample was obtained 5 min la te r . The plasma samples were prepared as described previously , and the rats were t ranscard ia l l y perfused as described in 2.1.2 and the cannula posi t ion ver i fed h i s t o l o g i c a l l y as described in 2 .3 .2 . 2.4.2 S t a t i s t i c a l ana lys is . Analys is of variance with repeated measures and Duncan's mult ip le range test were used to assess s t a t i s t i c a l s ign i f i cance . A probab i l i t y of error of less than 0.05 was preselected as the c r i t e r i on for s t a t i s t i c a l s ign i f i cance . A l l values represent mean ± SEM. 2.5 Vascular ro le of AVP 2.5.1 Surgical preparat ion. Male Sprague-Dawley rats (375-450 g) were anesthetized with sodium pentobarbital (60 mg/kg, i .p . ) and subjected to a standard laparotomy with a 5 cm midline i n c i s i o n . A PE 50 cannula was then inserted into the l e f t ven t r i c le v ia the r ight carot id artery with the help of the a r te r i a l pressure t rac ing , for the in jec t ion of microspheres. PE 50 cannulae were also introduced into the femoral vein for the infusion of drugs, and into the abdominal aorta through the caudal and i l i a c ar ter ies for blood pressure recordings and the withdrawal of blood during the in jec t ion of microspheres, respect ive ly . 2.5.2 Microsphere technique. Cardiac output (CO) and the d is t r i bu t ion of blood flow (BF) were determined by the reference sample method (Malik et a l . 1976) using rad ioact ive ly-1abel led microspheres (15 um diameter, New England Nuclear). It has been shown that these microspheres - 47 -are trapped within one c i r cu la t i on af ter in jec t ion in rats (Nishiyama et a l . 1976). Beginning 10 sec before the in jec t ion of microspheres, blood was withdrawn with an infusion-withdrawal pump (Harvard Apparatus) from the i l i a c a r te r i a l cannula at 0.35 ml/min for 1 min into a heparinized syr inge. A 200 ul sample of a vigorously vortexed precounted microsphere suspension 57 (containing 20,000-30,000 microspheres, labe l led with e i ther Co or 113 Sn) was injected and flushed with 200 y l of normal sa l ine for 10 sec into the l e f t ven t r i c l e . I t has been shown that three repeated in ject ions of 20,000 microspheres in rats gave reproducible d i s t r i bu t ion with no systemic hemodynamic changes; only a cumulative in jec t ion of over 100,000 microspheres caused reductions of oxygen consumption, CO and a r te r i a l pressure (Tsuchiya et a l . 1977). F i c o l l 70 (10%, Pharmacia Fine Chemicals AB, Sweden) and Tween 80 (0.05%) were used to suspend the microspheres (Foster and Frydman 1978). To avoid the p o s s i b i l i t y of a var ia t ion in the d is t r i bu t ion between microspheres label led with ^ C o and ^ S n and to avoid var ia t ions re lated to di f ferences in the counting e f f i c i enc ies of the 57 1 two d i f ferent isotopes, in hal f the experiments Co was given before 113 Sn. In the remaining experiments, the order in which the isotopes were administered was reversed. At the end of the experiments, whole rats were completely d issected. A l l organs were removed, weighed and loaded into v ia l s for counting, bones were charred at 300°C overnight and large organs were cut into small pieces and loaded into several v i a l s to a level less than 3.0 cm from the base. In rare instances ( less than 5%) where BF to the le f t and r ight kidney d i f fered by more than 20%, the experiment was rejected as i t was assumed that the mixing of the microspheres was not adequate. Blood samples, t i ssue samples, test tubes, and syringes used for the in jec t ion of microspheres and the co l l ec t i on of blood were counted for rad ioac t i v i t y using a Beckman 8000 Gamma Counter with a 3 inch Nal c rys ta l -•48 -at energy set t ings of 95-165 and 320-460 keV for 5 7 C o and 1 1 3 S n , respect ive ly . At these energy se t t ings , the sp i l l ove r of Co into the Sn channel i s neg l ig ib le (0.03%) and therefore no correct ion was made for Co s p i l l o v e r . The sp i l l ove r of Sn into the Co channel was 16.7% and correct ion of Co counts was done by subtract ing Sn sp i l l ove r from Co counts. 2.5.3 Experimental p ro toco l . CO and i t s d i s t r i bu t i on were determined by the in jec t ion of rad ioac t i ve ly - labe l led microspheres into the l e f t ven t r i c le p r io r to and fo l lowing the administrat ion of AVP antagonist. Rats were divided into 3 groups (n = 8) . In Group I, normal sa l ine was infused (0.08 ml/min/kg) 30 min af ter surg ical preparation of the rats and the infusion was continued un t i l the end of the experiment. The f i r s t in jec t ion of microspheres was made 10 min af ter the s tar t of sa l ine in fus ion . Ten min af ter the in jec t ion of the f i r s t set of microspheres, AVP antagonist was in jected into the l e f t ven t r i c l e . A dose of 4 ug/kg of the antagonist has been reported to block pressor responses to i . v . infusions of supramaximal doses of AVP in rats (Pang and Leighton 1981). Ten min af ter the in jec t ion of AVP antagonist, microspheres labe l led with a d i f ferent isotope were in jected into the l e f t ven t r i c l e . Group II rats were subjected to i . v . infusion of sara las in (10 ug/min/kg) at 0.08 ml/min/kg instead of normal sa l i ne . The infusion of sara las in was continued during the in jec t ion of the microspheres and the AVP antagonist. Prel iminary studies showed that a continuous infusion of sara las in at t h i s rate for 10 min completely blocked the pressor responses to i . v . in jec t ions of angiotensin I I . The pressor ef fect of angiotensin II was tested in a l l rats p r io r to the infusion of sara las in and af ter the second in jec t ion of microspheres. In a l l cases, sara las in completely blocked pressor responses to i . v . in jec t ions of angiotensin I I . Group III was subjected to i . v . infusion of phentolamine (0.5 mg/kg/min) at 0.08 ml/min/kg started 10 min pr io r to the f i r s t - 49 -in jec t ion of microspheres and continued during the in jec t ion of AVP antagonist and the second set of microspheres. Prel iminary studies showed that a 10 min infusion of phentolamine at th is rate completely blocked the pressor responses to i . v . in jec t ion of methoxamine (Sigma, 250 ng/kg), B-HT 933 (1 mg/kg) and c lonid ine (5 mg/kg). MAP recordings obtained during the f i r s t and second in ject ions of microspheres were used to indicate MAP during control and drug treatment per iods, respect ive ly . 2.5.4 Ca lcu la t ions . Total peripheral resistance (TPR, mmHg min/ml) was calculated by d iv id ing MAP (mmHg) by CO (ml/min). CO and the d is t r i bu t ion of BF were calculated as fo l lows: CO (ml/min) = r a t e °^ withdrawal of blood (ml/min) x to ta l in jected cpm cpm in withdrawn blood Tissue BF (ml/min) = r a t e °^ withdrawal of blood (ml/min) x t issue cpm cpm in withdrawn blood Total amount of rad ioac t i v i t y (cpm) injected was obtained by subtract ing the amount of rad ioac t i v i t y l e f t in the tube, in jec t ing syr inge, and f lushing syringe from the amount of r ad ioac t i v i t y o r i g i n a l l y present in the tube. Rad ioact iv i ty (cpm) in blood was obtained by adding the amount of rad ioac t i v i t y in the blood sample, cannulae and syringe used for co l l ec t i ng blood. 2.5.5 S t a t i s t i c a l ana lys is . Analysis of variance with repeated measures was used to compare data obtained during the f i r s t and second determinations of CO. Analysis of var iance, complete random design, was used to compare data from d i f ferent groups of r a t s . Duncan's mul t ip le range test was used to compare group means of MAP, CO and TPR. Tukey's mult ip le range test was used to compare group means of t i ssue BF where a more s t r i c t test was desired for mul t ip le comparisons of data. A probab i l i t y of error of less than 0.05 was preselected as the c r i t e r i on for s t a t i s t i c a l s ign i f i cance . 50 -2.6 Central and peripheral actions of ot-agonists 2.6.1 Cross-c i rcu la ted rat preparat ion. Two male Sprague-Dawley rats of ident ica l weight (350-400 g, n = 8 pairs) were anesthetized with pentobarbital (65 mg/kg, i . p . ) , and a PE 50 cannula was inserted into a femoral artery and femoral vein of each ra t . The MAP and HR of each rat were monitored continuously. The d i s ta l end of the l e f t common carot id artery of each rat was cannulated with PE 50 tubing, while the proximal end of the same artery was cannulated with PE 90 tubing. The d i s ta l and proximal ends of both l e f t and r ight jugular veins were cannulated with PE 90 and PE 50 tubing, respect ive ly . A l l tubing was f i l l e d with heparinized normal sa l ine (25 I l l /ml) . The cannulae from corresponding vessels of the two rats were then jo ined , al lowing peripheral a r t e r i a l blood from the body of each rat to supply the head of the other ra t , and venous blood to drain back into the peripheral c i r cu la t i on of the f i r s t rat ( F i g . 3 ) . The trachea was cannulated with a s ta in less -s tee l cannula to allow a r t i f i c i a l resp i ra t i on . A PE 50 cannula was inserted v ia the r ight common carot id artery into the l e f t ven t r i c le of one ra t , designated rat A. The r ight common carot id artery of the other r a t , designated rat B, was l i ga ted . Prel iminary studies have shown that l i ga t ion of the r ight common carot id artery of rats did not a l te r MAP and HR responses to various vasoactive drugs. Microspheres labe l led with ^ C o or ^ S n were injected into the l e f t ven t r i c le of rat A in order to determine the d i s t r i bu t ion of BF from rat A to rat B. Each rat was then a r t i f i c i a l l y vent i la ted with O2, the chest was opened along the sternum, and the l e f t and r ight subclavian ar ter ies were l i ga ted . This abolished the vertebral a r t e r i a l blood supply to the head of each ra t , rendering the brain of each rat dependent on the peripheral blood supply from the other ra t . Methoxamine (n = 6 pairs) or c lonid ine (n = 4 - 51 -F i g . 3 . V a s c u l a r c o n n e c t i o n s between r a t s A and B i n t h e c r o s s - c i r c u l a t i o n p r e p a r a t i o n , c a = common c a r o t i d a r t e r y , j v = e x t e r n a l j u g u l a r v e i n . - 5 2 -pairs) was injected intravenously into rat A. Afterward, microspheres labe l led with a d i f ferent isotope were injected into the l e f t ven t r i c le of rat A to determine BF from rat A to rat B af ter l i ga t ion of the subclavian a r te r i es . 2.6.2 Blood flow to the brain v ia the l e f t carot id ar tery . In the c ross-c i rcu la ted rat preparation the c ross -c i r cu la t i on of blood from the l e f t carot id artery of one rat to another was ve r i f i ed using rad ioac t i ve ly - labe l led microspheres. Since th is enta i led placing a cannula through the r ight carot id artery into the l e f t ven t r i c le for the in jec t ion of microspheres, the r ight carot id artery could not be used in the c ross - c i r cu l a t i on . To ensure that the l e f t carot id artery alone could provide su f f i c i en t BF to the bra in , experiments were carr ied out to determine BF to the brain v ia the l e f t carot id ar tery. A polyethylene occluder was placed loosely around each subclavian artery of halothane-anesthetized rats and ex ter io r ized at the back of the neck. These occluders, s im i la r to those designed by Johnston et a l . (1983), consisted of a length of PE 10 tubing one end of which had been f la red with a soldering i ron . A polythene suture was threaded through the tubing, around the artery and back through the f l a r e at the bottom of the tubing, where i t was melted down s l i g h t l y with a solder ing iron to secure i t . Af ter the rats recovered for 1 week they were anesthetized with sodium pentobarbital (65 mg/kg, n = 6) and cannulae were inserted into the femoral artery and v ia the r ight carot id artery into the l e f t ven t r i c l e . Radioact ively l abe l l ed ' microspheres ( C o ^ or ^ S n ) were injected into the l e f t ven t r i c l e . The subclavian ar ter ies were l igated by pu l l i ng on the suture while holding the polyethylene guide, and heat-seal ing both the suture and the guide. Microspheres labe l led with a d i f fe rent isotope were then in jected into the l e f t ven t r i c l e . - 53 -2.6.3 Microsphere technique. BF was determined using the microsphere technique as described in sect ion 2 .5 .2 . During the in jec t ion of microspheres, blood was withdrawn from the femoral a r te r i a l cannula rather than an i l i a c a r te r i a l cannula. Blood samples, l e f t and r ight brain hemispheres (cerebrum and cerebellum), and brain stem were counted for rad ioac t i v i t y using a Picker Gamma Counter (Model 644-055 Dual Channel/Dual Window Analyzer) . 2.6.4 Determination of c i r cu la to ry leakage. Since internal jugular venous drainage from the head to the body of each rat was s t i l l func t iona l , the extent of c i rcu la to ry leakage from the head of rat B to i t s peripheral c i r cu la t i on was assessed. Radioactive solut ions were obtained by leaching 57 the Co microspheres (approximately 50,000 microspheres in 300 ul of normal sa l ine) with 150 ul of concentrated n i t r i c ac id . The radioact ive solut ion was neutral ized to pH 6 with sodium bicarbonate and suspended in 0.5 ml of 10% F i c o l l 70 so lu t ion . This suspension (150 ul) was then in jected intravenously into rat A in c ross-c i rcu la ted rat preparations (n = 4 p a i r s ) , and blood (0.5 ml) was removed from both rats A and B at 1, 3, 6, 9 and 15 min af ter the in jec t ion of the radioact ive suspension for measurement of r ad ioac t i v i t y . 2.6.5 Ef fects of c lon id ine and methoxamine. The c ross -c i r cu la t i on preparation was used to invest igate both the central and peripheral e f fects of c lon id ine (n = 4) and methoxamine (n = 6) . Maximum MAP and HR responses to the i . v . in jec t ion in rat A of e i ther c lon id ine (25 ug/kg in 100 ul of normal sal ine) or methoxamine (25 ug/kg in 100 ul of normal sal ine) were observed in both rats A and B. In a l l cases MAP and HR responses were observed within 2 min in rat A and between 2 and 3 min in rat B. - 54 -2.6.6 Calcu la t ions. Tissue BF was calculated as shown in sect ion 2 .5 .4 . C i rcu la tory leakage was calculated as fo l lows: % leakage = c ' 3- n i ™ blood sample from rat B at any time x ^ n n ^ cpm in blood of rat A, 1 min af ter in jec t ion of ^ C o 2.6.7 S t a t i s t i c a l ana lys i s . Results were expressed as mean ± SEM. Analysis of variance and Duncan's mul t ip le range test were used to assess s t a t i s t i c a l s ign i f i cance . A p robab i l i t y of error of less than 0.05 was preselected as the c r i t e r i on for s t a t i s t i c a l s ign i f i cance . 2.7 Central ag -agonists in conscious rats 2.7.1 Experimental p ro toco l . Male Sprague-Dawley rats (280-320 g) were prepared with i . c . v . cannulae as described in sect ion 2 .1 .1 , except that commercially avai lab le cannulae (P las t i c Products Co.) were used. The guide cannula (22-gauge) and the dummy cannula (28-gauge) were both cut to a length of 20 mm. Af ter recovering 7 days, rats were prepared with femoral a r t e r i a l and venous cannulae as previously described. Conscious unrestrained rats were placed in a cage and MAP and HR were continuously monitored. An internal cannula (28-gauge, 20 mm long), connected with PE tubing to a 5 ul Hamilton syringe and preloaded with drug or vehic le was inserted into the guide cannula. The experiments were conducted over 2 days. On the f i r s t day, MAP and HR responses to i . c . v . doses (0.01, 0 .1 , 1.0, 10.0 ug) of a-adrenergic agonists were observed. Rats in Group I (n = 8) were given c lon id ine , while rats in Group II (n = 8) received 6-a l ly l -2-amino-5,6 ,7 ,8- te t rahydro-4H-thiazolo-[4,5-d]-azepine (B-HT 920), a more se lec t ive c^-adrenergic agonist (Kobinger and P ich ler 1980b, 1982). A l l drugs were dissolved in normal sa l ine and injected in a volume of 2 y l . Doses were administered at least 1 h apart. At the end of the day, the vascular cannulae were flushed with heparin and resealed, and the dummy cannula was reinserted into the guide cannula. - 55 -On the second day, MAP and HR were again monitored. Af ter rats were given 20 min to acc l imat ize , a control blood sample was obtained for la ter analysis of catecholamine concentrat ion. Twenty min l a t e r , 1 pg of the same drug given the previous day was injected cen t ra l l y . MAP and HR were recorded once the response was f u l l y developed, within 2-5 min af ter i n jec t i on , and a second blood sample was taken immediately afterward. Af ter 30 min, 10 pg of the same drug was in jec ted , MAP and HR were recorded and a f i n a l blood sample was obtained. Each animal received in jec t ions of a s ingle drug only. In order to determine whether the responses to i . c . v . in jec t ion of c lon id ine and B-HT 920 were mediated by c * 2 ~ a d r e n o c e P t o r s > these drugs were also in jected in rats pretreated cen t ra l l y with the se lec t ive o^-antagon is t , rauwolscine. Twenty min after the control blood sample was obtained, rats in Groups III (n = 8) and IV (n = 8) received i . c . v . in jec t ions of 10 pg rauwolscine in 4 pi normal s a l i n e . MAP and HR responses were recorded and a second blood sample was obtained within 2-5 min. Af ter 30 min, rats in Group III and IV received i . c . v . in ject ions of 1 pg c lonid ine and 1 pg B-HT 920, respec t ive ly , in 1 pi normal sa l i ne . By test ing for blockade of (^-adrenoceptors with various agonists, i t has been found that rauwolscine e f fec t i ve l y blocks pressor responses to B-HT 920 for at least 40 min fo l lowing i . v . administrat ion in rats (Pang et a l . , personal observat ion). MAP and HR responses were recorded and a f i n a l blood sample was obtained within 2-5 min of i n j ec t i on . On completion of the experiments, rats were t ranscard ia l l y perfused and the brain removed and sectioned to ve r i f y the cannula pos i t i on . Blood samples were assayed by HPLC for plasma catecholamine concentrat ions. - 56 -2.7.2 S t a t i s t i c a l ana lys is . Results were expressed as mean ± SEM. Analysis of variance and Duncan's mult ip le range test were used to assess s t a t i s t i c a l s ign i f i cance . A probab i l i t y of er ror of less than 0.05 was preselected as the c r i t e r i on for s t a t i s t i c a l s ign i f i cance . 2.8 Drugs A l l drug solut ions were made fresh da i l y except fo r AVP and the AVP antagonist which were made up f resh ly each day from stock so lu t ions. For the central AVP studies, AVP (Calbiochem) and AVP antagonist (the g i f t of Dr. M. Manning, Department of Biochemistry, Medical College of Ohio) were dissolved in a r t i f i c i a l CSF. The a r t i f i c i a l CSF consisted of 1.24 x IO -''' M NaCl; 5.0 x 10" 3 KC1; 2.6 x 10~ 2 M NaHC03; 1.25 x 10" 3 M KH 2 P0 4 ; 2.0 x 10~3 M MgS04 7H 20; 2.65 x 10~ 3 M C a C l 2 . The AVP, AVP antagonist, sara las in (the g i f t of Dr. K. 0. E l l i s , Norwich-Eaton Pharmaceuticals) and angiotensin II (Ciba-Geigy Canada) which were injected intravenously were dissolved in normal sa l i ne . The c lonid ine HCl (Boehringer Ingelheim Canada L td . ) , methoxamine HCl (Burroughs Wellcome), B-HT 920 HCl (Boehringer Ingelheim Canada Ltd.) and rauwolscine HCl (Carl Roth) used in the c ross -c i r cu la t i on and CNS studies were also dissolved in normal sa l i ne . - 57 -3 RESULTS 3.1 Central AVP in conscious rats 3.1.1 Central administrat ion of AVP. One min af ter the i . c . v . in jec t ion of a low dose of AVP in Group I, there were s ign i f i can t increases in MAP and the plasma leve ls of adrenaline but not noradrenaline (F i g . 4a). There were increases in MAP and plasma noradrenaline and adrenaline concentrations 1 min af ter the i . c . v . in jec t ion of the high dose of AVP (F ig . 4a). Ten min af ter the i . c . v . in ject ions of both high and low doses of AVP in Group I I , MAP and plasma noradrenaline and adrenaline leve ls were increased (F i g . 4b). The increase in MAP began within 0.5-1.0 min af ter the in jec t ion of AVP and reached a maximum within 1 min. Catecholamine measurements were therefore made once the change in MAP reached a steady s ta te . Heart rate did not change s i g n i f i c a n t l y . There was no change in MAP or plasma catecholamine leve ls 1 min af ter i . c . v . in jec t ions of 1 and 3 y l of the vehic le in Group III (F ig . 5a). However, small but s ign i f i can t reductions in plasma adrenaline concentration were observed 10 min af ter i . c . v . in jec t ions of both volumes of a r t i f i c i a l CSF (F ig . 5b). 3 .1.2 Central administrat ion of AVP antagonist. There were no changes in MAP or plasma concentrations of noradrenaline or adrenaline af ter i . c . v . in ject ions of both doses of AVP antagonist in Group V (F ig . 6 ) . In order to determine i f the receptors involved in mediating the pressor response to central AVP were ident ica l to peripheral V-^  receptors, a further set of experiments was carr ied out on Group VI. As in Group V, the i . c . v . in jec t ion of AVP antagonist did not a l te r MAP or plasma leve ls of noradrenaline or adrenaline (F ig . 7) . However, AVP antagonist prevented both the pressor response and the increase in catecholamine levels fol lowing the central in jec t ion of AVP (F ig . 7) . 58 • C O N T R O L S 2 3 n g / k g A V P | 7 3 n g / k g A V P M A P N A A —r- * F i g . 4. The ef fects of i . c . v . in jec t ions of AVP on MAP and plasma noradrenaline and adrenaline concentrations in rats from Group I (a) and Group II (b). Responses in Groups I (n = 10) and II (n = 8) were measured at 1 and 10 min, respec t ive ly , after i . c . v . * in ject ions of AVP. Values represent mean ± SD. S ign i f i can t l y d i f ferent from control (P < 0.05). - 59 -• CONTROL 0 1 ul VEHICLE • 3 ul VEHICLE The e f f e c t s o f i . c . v . i n j e c t i o n s o f a r t i f i c i a l CSF on MAP and p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e l e v e l s i n r a t s f r om Group I I I (a) and Group IV ( b ) . R e s p o n s e s i n Groups I I I (n = 7) and IV (n = 6) were measured a t 1 and 10 m i n , r e s p e c t i v e l y , a f t e r i . c . v . i n j e c t i o n s o f a r t i f i c i a l C S F . V a l u e s r e p r e s e n t mean * SD. S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (P < 0 . 0 5 ) . - 60 -• CONTROL 0 0.5 pg/kg AVP ANTAGONIST • 1.5 pg/kg AVP ANTAGONIST X £ E 14 o r 12 0 iooU MAP 2 r-oi c NA F i g . 6 . The e f f e c t o f i . c . v . i n j e c t i o n o f AVP a n t a g o n i s t on MAP and p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e c o n c e n t r a t i o n s i n r a t s f r o m Group V * (n = 5 ) . V a l u e s r e p r e s e n t mean ± SD. S i g n i f i c a n t l y d i f f e r e n t f r om c o n t r o l (P < 0 . 0 5 ) . - 61 -• CONTROL 0 2.0 ug/kg AVP ANTAGONIST • 96 ng/kg AVP 140 1 0 O ^ -MAP NA F i g . 7 . The e f f e c t o f p r e t r e a t m e n t o f t h e f o u r t h v e n t r i c l e w i t h AVP a n t a -g o n i s t on MAP and p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e l e v e l s ... (mean * SD) f o l l o w i n g i . c . v . i n j e c t i o n s o f AVP i n r a t s f r om Group VI (n = 1 1 ) . S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (P < 0 . 0 5 ) . - 62 -3.1.3 Peripheral administrat ion of AVP and AVP antagonist. An increase in MAP and reductions in plasma concentrations of noradrenaline and adrenaline were observed 1 min af ter i . v . in ject ions of AVP in Group VII (F ig . 8a). In Group VI I I , MAP and plasma catecholamine leve ls measured 10 min af ter i . v . in ject ions of AVP were not d i f ferent from control values (F ig . 8b). The i . v . in jec t ion of AVP antagonist in Group IX caused a reduction in MAP but no change in plasma noradrenaline or adrenaline concentrations (F ig . 8c) . 3.2 Central AVP in hypotensive rats The continuous i . v . infusion of sodium ni t roprusside in rats in Group I, which served as cont ro ls , led to a s ign i f i can t reduction of MAP and s ign i f i can t increases in plasma noradrenaline and adrenaline concentration af ter 10 min. Ten min af ter the i . c . v . in jec t ion of the vehic le in Group I during the continuous infusion of n i t ropruss ide, there was a s l i g h t , but not s ign i f i can t fur ther reduction of MAP and a further increase in plasma noradrenaline and adrenaline concentrations (F i g . 9) . Only the noradrenaline concentration was s i g n i f i c a n t l y increased from the level observed 10 min af ter the s tar t of the ni t roprusside in fus ion . Rats in Group II showed ident ica l responses to those in Group I. Ten min after the i n i t i a t i o n of the ni t roprusside in fus ion , MAP was s i g n i f i c a n t l y reduced from control and plasma noradrenaline and adrenaline leve ls were s i g n i f i c a n t l y increased. Ten min af ter the in jec t ion of the AVP antagonist during the infusion of n i t ropruss ide, MAP was s l i g h t l y reduced further and plasma noradrenaline and adrenaline leve ls o increased fur ther , although only the increase in plasma noradrenaline concentration was s t a t i s t i c a l l y s ign i f i can t (F ig . 9) . - 63 -• C O N T R O L S 23 ng/kg AVP | 73 ng/kg AVP • 0 0.5 (jg kg AVP ANTAGONIST ED 1.5 yg/kg AVP A N T A G O N I S T MAP NA A F i g . 8 . The e f f e c t o f i . v . i n j e c t i o n s o f AVP and AVP a n t a g o n i s t on MAP and p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e c o n c e n t r a t i o n s i n r a t s f r o m Group VI I ( a ) , Group V I I I (b) and Group IX ( c ) . Responses i n Groups V I I (n = 8) and V I I I (n = 5) were measured a t 1 and 10 m i n , r e s p e c t i v e l y , a f t e r i . v . i n j e c t i o n s o f A V P . R e s p o n s e s i n Group IX (n = 5) were o b t a i n e d 10 min a f t e r i . v . i n j e c t i o n s o f AVP a n t a g o n i s t . V a l u e s r e p r e s e n t mean ± S D . . S i g n i f i c a n t l y d i f f e r e n t f r om c o n t r o l (P < 0 . 0 5 ) . - 64a -F i g . 9. The ef fect of i . c . v . in ject ions of a r t i f i c i a l CSF (Group I) and AVP antagonist (Group II) on MAP and plasma noradrenaline and adrena-l ine levels in ra ts receiv ing i . v . infusions of n i t ropruss ide. Open columns represent control values, shaded columns represent values af ter 10 min of ni t roprusside infusion and so l id columns represent values 10 min after in jec t ion of vehic le or AVP antagon-i s t during continuous ni t roprusside in fus ion. Values represent mean * SEM. a S i g n i f i c a n t l y d i f fe rent from control value (P < 0.05). ^S ign i f i can t l y d i f ferent from value reported after infusion of ni t roprusside (P < 0.05). - 65 -3.3 Micro in ject ion of AVP into the nucleus t ractus s o l i t a r i u s The ef fects of the in jec t ion of AVP and the AVP antagonist on MAP are shown in F i g . 10. Inject ion of both 2 and 10 ng AVP into the NTS in Groups II and III s i g n i f i c a n t l y elevated MAP by 4 and 15 mmHg, respect ive ly . The r i se in MAP began within 15-20 sec of in jec t ion and the maximal response was reached within 1 min of i n j ec t i on . The in jec t ion of the vehic le (0.2 y l ) in Group I and the AVP antagonist in Group IV had no ef fect on MAP. None of the treatments s i g n i f i c a n t l y changed HR (F ig . 10). The in jec t ion of the 10 ng dose of AVP s i g n i f i c a n t l y elevated plasma noradrenaline concentration (F ig . 11). The administrat ion of the lower dose of AVP, veh ic le , or AVP antagonist did not s i g n i f i c a n t l y change plasma noradrenaline l eve l s . Plasma adrenaline concentration was s i g n i f i c a n t l y increased after the in jec t ion of 10 ng AVP into the NTS in rats (F ig . 11). There was no s ign i f i can t change in plasma adrenaline leve ls af ter the in jec t ion of the lower dose of AVP, AVP antagonist or veh ic le . 3.4 Central AVP in neurogenical ly-stressed rats Rats in Group I, which served as cont ro ls , had a s l i g h t l y increased MAP, no change in HR and s l i g h t l y increased plasma noradrenaline and adrenaline leve ls af ter a 10 min exposure to the heat lamp (F ig . 12,13). None of these changes were s i g n i f i c a n t l y d i f ferent from pretreatment l eve l s . MAP, HR and plasma noradrenaline and adrenaline concentrations were further increased 5 min af ter in jec t ion of the vehic le during continuous neurogenic s t ress , although th is fur ther increase was s ign i f i can t only in the case of plasma adrenaline concentrat ion. Af ter in jec t ion of the vehic le MAP was, however, s i g n i f i c a n t l y greater than the control l e v e l . Rats in Group II showed s imi la r responses to neurogenic stress (F ig . 12,13). Af ter 10 min exposure to the heat lamp, MAP of rats in Group II was s i g n i f i c a n t l y increased, while HR and plasma noradrenaline and adrenaline concentrations (a) .(b) (c) (d) 450 (a) -(b)- . (c) (d) 10. The ef fect of in jec t ion into the NTS of (a.) 2 ul a r t i f i c i a l CSF (n = 10); (b) 2 ng AVP (n = 8 ) ; (c) 10 ng AVP (n = 10); (d) 10 ng AVP antagonist (n =8) on MAP and HR. Open columns represent control values, shaded columns represent treatment values. Values are mean * SEM. * S i g n i f i c a n t l y d i f ferent from control value (P < 0.05). - 67 -. 7 5 - 1 .60 -3 .45 -(a) (b) (c) (d) .75 ~\ .60 -(a) (b) (c) (d) . F i g . 11. The ef fect of in jec t ion into the NTS of (a) 2 y l a r t i f i c i a l CSF (n = .10 ) ; Cp) 2 ng AVP (n = 8); (c) 10 ng AVP (n = 10); (d) 10 ng AVP antagonist (n = 8) on plasma noradrenaline and adrenaline concentrat ion. Open columns represent control values, shaded columns represent treatment values. Values are mean •* SEM. * S i g n i f i c a n t l y d i f ferent from control value (P < 0.05). - 68a -F i g . 12. The ef fect of in jec t ion into the NTS of a r t i f i c i a l CSF (Group I) and AVP antagonist (Group II) of rats subjected to neurogenic stress on MAP and HR. Open columns represent control va lues; shaded and so l i d columns represent values obtained 10 min after i n i t i a t i o n of neurogenic s t ress , and 5 min after the in ject ion of vehic le or AVP antagonist, respec t ive ly . Values represent mean ^ SEM. a S i g n i f i c a n t l y d i f ferent from, control value (P < 0.05). ^S ign i f i can t l y d i f ferent from value obtained 10 min after i n i t i a t i o n of neurogenic stress (P < 0.05). - 68 -Noradrenaline (ng/ml) Adrenaline (ng/ml) CTS 73 O CD O PJ _1_ 0) 00 PJ _L 0) J CO - 69a -F i g . 13. Plasma noradrenaline and adrenaline levels af ter in jec t ion into the NTS of a r t i f i c i a l CSF (Group I) and AVP antagonist (Group II) of rats subjected to neurogenic s t ress . Open columns represent control va lues; shaded and so l i d columns represent values obtained 10 min after i n i t i a t i o n of neurogenic s t ress , and 5 min after in jec t ion of vehic le or AVP antagonist, respec t ive ly . Values represent mean * SEM. a S i g n i f i c a n t l y d i f ferent from control value (P < 0.05). ^S ign i f i can t l y d i f ferent from value obtained 10 min after i n i t i a t i o n of neurogenic stress (P < 0.05). - 70 -were s l i g h t l y but not s i g n i f i c a n t l y elevated. Five min af ter the i . c . v . in jec t ion of the AVP antagonist in rats subjected to neurogenic s t ress , MAP, HR and plasma adrenaline levels showed further s ign i f i can t elevations and plasma noradrenaline concentration was s l i g h t l y but not s i g n i f i c a n t l y increased. Therefore, the AVP antagonist did not prevent the increase in MAP and plasma adrenaline leve ls caused by neurogenic s t ress . 3.5 Vascular ro le of AVP 3.5.1 Ef fect of antagonism of pressor systems on MAP, CO and TPR. Table 1 shows control values of MAP pr ior to and fo l lowing the infusion of sa l ine or drugs in the various groups of ra t s . A 10 min infusion of sa l ine did not a l te r MAP in rats from Group I. Infusions of sara las in and phentolamine decreased MAP in Groups II and I I I , respec t ive ly . However, a comparison of resu l ts between groups I and II during infusions of sa l ine and sa ra las in , respect ive ly , shows that the infusion of sara las in in Group II did not cause any s ign i f i can t change in MAP, CO, or TPR from the corresponding values in Group I (Table 1) . Therefore, unl ike comparisons of MAP within the same animal, we were unable to detect a s ign i f i can t decrease of MAP during the infusion of sara las in in Group II compared to control MAP in Group I which received infusion of sa l i ne . Comparisons of values between Groups I and III show that MAP and CO in Group III were s i g n i f i c a n t l y lower than the corresponding values in Group I. There was no di f ference in TPR between Groups I and I I I . 3..5.2 Ef fect of AVP antagonist on MAP, CO, and TPR. The in jec t ion of AVP antagonist decreased MAP and TPR but not CO in the three groups of rats (F ig . 14). Table 2 shows the MAP, TPR and CO responses normalized as a percentage of control to allow comparisons of the ef fects of the AVP antagonist in the d i f ferent groups. The antagonist caused a greater decrease of percent control of MAP in Group II than in Group I. Although - 71 -Table 1. Control values of MAP, CO and TPR in Groups I, I I , and III Groups I II III MAP (mmHg)a 103 ± 17 106 ± 14 95 ± 13 MAP (mmHg)b 103 ± 17 91 ± 17 c 79 ± i o c d CO (ml/min) b 105 ± 26 90 ± 20 69 ± 20 d TPR (mmHg min/ml) b 1.04 ± 0.29 1.03 ± 0.15 1.20 ± 0.37 A l l values represent mean ± SD. n = 8 in each group. a Denotes MAP readings pr io r to drug (sal ine) in fus ion . Denotes readings of MAP, CO or TPR during the f i r s t determination of CO, at 10 min fo l lowing the infusions of sa l i ne , sara las in or phentolamine in Groups I, II and I I I , respect ive ly . c S i gn i f i can t l y d i f ferent from MAP pr io r to the infusions of drug or sa l ine within the same group (p < 0.05). d S i g n i f i c a n t l y d i f ferent from corresponding values in Group I (p < 0.05). - 72 -150 n 100 8 so A '_! 1.5 1 £ f • l.o 4 X E e - 0.5 -\ Q_ X x GROUPS GROUPS GROUPS Fig. 14. Effect of AVP antagonist on MAP, CO and TPR in anesthetized, surgically stressed rats: intact and saline-pretreated (Group I), saralasin-pretreated (Group II) and phentolamine-pretreated (Group III). CO was determined twice in each group of rats. AVP anta-gonist was given to each group between the first and second deter-* minations of CO. Significantly different from values obtained from the first determination (P < 0.05). - 73 -Table 2. Ef fects of AVP antagonist on MAP, CO and TPR in Groups I, II and III Groups I II III % of control MAP 87 ± 12 75 ± I 0 a 71 ± 13 a % of control CO 105 ± 17 106 ± 22 118 ± 30 % of control TPR 85 ± 1 8 73 ± 15 61 ± 21 a A l l values represent mean ± SD. n = 8 in each group. Values of MAP, CO and TPR obtained during the second determination of CO fol lowing the administrat ion of AVP antagonist were expressed as a % of the corresponding values obtained during the f i r s t determination of CO in the various groups of ra t s : Group I (sa l ine- t rea ted) ; Group II (sara las in - t rea ted) ; Group III (phentolamine-treated). a S i g n i f i c a n t l y d i f ferent from Group I (p < 0.05). the AVP antagonist caused a greater decrease in control TPR in Group II than in Group I, the decrease was not s t a t i s t i c a l l y s i g n i f i c a n t . The AVP antagonist caused s i g n i f i c a n t l y greater decreases of percent control of MAP and TPR in Group III than in Group I. The AVP antagonist did not cause any d i f f e ren t i a l e f fects in the percent of control CO. The resu l ts show that the AVP antagonist exerts greater depressor ef fects in rats with antagonism of the renin-angiotensin or a-adrenergic systems than in in tact r a t s . 3.5.3 Ef fect of AVP antagonist on the d i s t r i bu t ion of blood f low. In in tact rats (Group I ) , the AVP antagonist increased BF to the stomach and skin (F ig . 15) but did not a l te r BF to any other organs. This indicates that AVP has the greatest vasoconstr ictor inf luence in the area of the stomach and sk in . During the infusion of sara las in (Group I I ) , AVP antagonist increased BF to muscles and skin and decreased BF to the lungs and l i v e r (F ig . 16). Therefore in the absence of inf luence from angiotensin I I , AVP has the greatest vasoconstr ictor ef fects in the vascular beds in the skin and muscles. During a continuous infusion of phentolamine (Group I I I ) , AVP antagonist markedly increased muscle BF (note change of scale) and decreased BF to the l i v e r , i n tes t ine , kidneys, and testes (F ig . 17). Thus, in the absence of the a-adrenergic system, AVP has the greatest inf luence on BF to the muscle. - 75 -25 n F i g . 15. Effect of AVP antagonist on regional d i s t r i bu t ion of BF in rats from Group I (sa l ine-pret reated) . Whole rats were dissected for the determination of BF. A l l values represent BF to ent i re organs. Glands include thyro id , parathyroid, sa l i va ry and adrenal glands. BF was determined twice in each ra t . AVP antagonist was given between the f i r s t and second determinations of BF. * S i g n i -f i c a n t l y d i f ferent from BF obtained from the f i r s t determination (P < 0.05). - 76 -30 25 -e 20 -5 o _l Lu Q O O. _l CD 15 10 O CC z «c =3 u» —I X rift r^l m*a r^ h 3C ac o UJ <_i o UJ o O _ l o. W1 UJ I— _ j o o co F i g . 16. Effect of AVP antagonist on regional d i s t r i bu t ion of BF in rats from Group II (sara las in-pret reated) . Whole rats were dissected for the determination of BF. A l l values represent BF to ent i re organs. Glands include thyro id , parathyroid, sa l i va ry and adrenal glands. BF was determined twice in each r a t . AVP antagonist was given between the f i r s t and second determinations of BF. S i g n i -f i c a n t l y d i f ferent from BF obtained from the f i r s t determination (P < 0.05). - 77 50 | 40 E — 30 H o H 1 20 -I Q O O 10 m 1. < < z 2: o r> _ J C J o LU C J C J -o CO >-2 : CO r*Th r*Tih co CO Q z _ J o z o CO F i g . 17 . E f f e c t o f AVP a n t a g o n i s t on r e g i o n a l d i s t r i b u t i o n o f BF i n r a t s f r o m Group I I I ( p h e n t o l a m i n e - p r e t r e a t e d ) . Whole r a t s were d i s s e c t e d f o r t he d e t e r m i n a t i o n o f B F . A l l v a l u e s r e p r e s e n t BF t o e n t i r e o r g a n s . G l a n d s i n c l u d e t h y r o i d , p a r a t h y r o i d , s a l i v a r y and a d r e n a l g l a n d s . BF was d e t e r m i n e d t w i c e i n e a c h r a t . AVP a n t a g o n i s t was g i v e n between t h e f i r s t and second d e t e r m i n a t i o n s o f B F . S i g n i f i c a n t l y d i f f e r e n t f r om BF o b t a i n e d f r o m t h e f i r s t d e t e r m i n a t i o n (P < 0 . 0 5 ) . - 78 -3.6 Central and peripheral actions of a-agonists 3.6.1 Blood flow to the brains of c ross-c i rcu la ted ra t s . P r io r to l i ga t i on of the subclavian a r te r i es , peripheral blood from rat A supplied the l e f t and r ight brain hemispheres and brainstem of rat A (F i g . 18a) and only the l e f t and r ight hemispheres but not the brainstem of rat B (F ig . 18b). Af ter l i ga t ion of the subclavian ar ter ies peripheral blood from rat A supplied neg l ig ib le BF to the brain of rat A but instead a l l BF was diverted to both brain hemispheres and brainstem of rat B. BF to a l l parts of the brain of rat A was s i g n i f i c a n t l y decreased while BF to a l l parts of the brain of rat B was s i g n i f i c a n t l y increased fo l lowing l i ga t ion of the subclavian a r t e r i es . 3.6.2 Blood flow to the brain v ia the l e f t carot id ar tery . The BF to the brain before and af ter l i ga t ion of the subclavian ar ter ies in a s ingle rat i s shown in F i g . 19. In the control cond i t ion, BF to the. brain was supplied by the l e f t common carot id ar ter ies and both subclavian a r te r i es . Af ter l i ga t ion of both subclavian a r te r i es , BF to the brain was supplied only by the l e f t common carot id a r te r i es . Our resu l ts show that af ter l i ga t ion of the subclavian ar ter ies BF to the l e f t and r ight brain hemispheres was s l i g h t l y although not s i g n i f i c a n t l y increased while BF to the brainstem was s l i g h t l y but not s i g n i f i c a n t l y reduced. 3.6.3 C i rcu la tory leakage. The amount of rad ioac t i v i t y detected in the blood samples removed from rats A and B, expressed as a percentage of the rad ioac t i v i t y o r i g i n a l l y present in rat A (measured 1 min af ter 57 in jec t ion of Co), i s shown in Table 3. Leakage was less than 10% at 3 min, and reached a maximum of 18% at 6 min. Rad ioac t iv i ty in blood obtained from rat A was found to gradual ly decrease with t ime, probably pr imar i ly as a resu l t of t i ssue uptake. - 79 -u- e CO " \ a) RAT A i -i — . 6 E • * L. BRAIN b) RAT B • 8 — . 6-e Lu E CO \ J. •'' . 2 1 ^ 1 I L. BRAIN CONTROL Q LIGATION ^ MEAN + SE. N=8 R. BRAIN 4 R. BRAIN BRAINSTEM BRAINSTEM F i g . 18. BF to the le f t and r ight brain hemispheres and brainstem before and af ter l i ga t ion of the subclavian ar ter ies in rats A (a) and B (b) of c ross-c i rcu la ted rat preparations. *S i gn i f i c a n t l y d i f ferent from pre- l iga t ion BF (P < 0.05). - 80 -CONTROL Q LIGATION MEAN + SE N=6 l - SH .E 1 . 5-L. BRAIN R. BRAIN BRAINSTEM F i g . 1 9 . BF t o t h e l e f t and r i g h t b r a i n h e m i s p h e r e s and b r a i n s t e m i n t h e p r e s e n c e and absence o f f u n c t i o n a l s u b c l a v i a n a r t e r i e s i n s i n g l e r a t s . - 81 -Table 3. Cpm in rats A and B expressed as a of cpm detected in rat A in 1 min (n = 4) . Values represent mean ± SEM. Time (min) 1 3 6 9 15 Rat A 100 74 ± 9 47 ± 5 43 ± 5 35 ± 5 Rat B 5 ± 2 8 ± 3 18 ± 4 10 ± 2 13 ± 3 - 82 -3.6.4 Cross-c i rcu lated rat preparat ion. The in jec t ion of c lonid ine in rat A caused a s ign i f i can t increase in MAP, and a s l i gh t decrease (not s ign i f i can t ) in HR in rat A, and a s ign i f i can t decrease in MAP and a s l igh t reduction (not s ign i f i can t ) in HR in rat B ( F i g . 20). MAP and HR tracings from a typ ica l experiment with c lonid ine are shown in F i g . 21. The response of rat A to c lonid ine became evident within 5-10 sec and lasted about 2 min, while the response to c lonid ine became apparent in rat B within 10-30 sec and lasted 3-5 min. Conversely, in jec t ion of methoxamine in rat A s i g n i f i -cant ly increased MAP and decreased HR in rat A, and, s i g n i f i c a n t l y increased MAP and s l i g h t l y but not s i g n i f i c a n t l y increased HR in rat B (F i g . 22). MAP and HR tracings from a typ ica l experiment with methoxamine are shown in F i g . 23. The response of rat A to methoxamine was apparent within 5-10 sec of i n jec t i on , and lasted 2-3 min, while the response of rat B was evident within 10-30 sec and persisted 4-6 min. 3.7 Central o^adrenergic agonists in conscious rats The control values of MAP and HR pr io r to the administrat ion of the various doses of the a-agonists are l i s t ed in Table 4. The i . c . v . in ject ion of B-HT 920 in rats in Group I on the f i r s t day was found to increase MAP and decrease HR in a dose-dependent manner (F i g . 24). On the second day of the experiment, the in jec t ion of 1 and 10 yg B-HT 920 caused changes in MAP and HR s im i la r to those on day 1 ( resul ts not shown). The ef fects of 1 and 10 yg B-HT 920 on plasma catecholamine levels are shown in F i g . 25. While the accompanying changes in plasma catecholamine levels did not reach s ta t -i s t i c a l s ign i f i cance , the 10 yg dose of B-HT 920 appeared to increase the plasma concentration of noradrenaline, while the 1 yg dose had l i t t l e ef fect on noradrenaline concentrat ion. Both doses of B-HT 920 had a tendency to increase the plasma levels of adrenaline. In contrast , the i . c . v . in ject ion of c lonid ine in Group II on the f i r s t day led to a dose-dependent decrease - 83 -4 0 0 -CONTROL . [ ] CLONIDINE 2 0 0 - 1 MEAN + SE N=4 1 5 0 -cn CL. I < ICO -3; E so-1 I RAT A RAT B c E. Q : U I 2 0 0 -T H • RAT A I I RAT B F i g . 20. MAP and HR r e s p o n s e s o f r a t A (a) and r a t B (b) t o i . v . i n j e c t i o n o f c l o n i d i n e (25 u g / k g ) i n r a t A . * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (P < 0 . 0 5 ) . - 84 -200 r 200 • r  CLONIDINE 1 min F i g . 2 1 . R e p r e s e n t a t i v e r e c o r d i n g s o f MAP and HR r e s p o n s e s i n r a t A (a) and r a t B (b) f o l l o w i n g i . v . i n j e c t i o n o f c l o n i d i n e (25 y g / k g ) i n r a t A . - 85 -CONTROL Q METHOXAMINE ^ MEAN + SE T * 1 I N=6 T | 1 RAT A RA1 B 6 O O - 1 5 0 0 -— 4 0 0 -RAT A RAT B F i g . ?.?.. MAP and HR r e s p o n s e s o f r a t A (a ) and r a t B (b) t o i . v . i n j e c t i o n o f methoxamine (25 u g / k g ) i n r a t A . * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (P < 0 . 0 5 ) . - 86 -200 r F i g . 23. R e p r e s e n t a t i v e r e c o r d i n g s o f MAP and HR r e s p o n s e s o f r a t A (a) and r a t B (b) f o l l o w i n g i . v . i n j e c t i o n o f methoxamine (25 p g / k g ) i n r a t A . - 87 -Table 4. Control values of MAP, HR and plasma noradrenaline and adrenaline concentration in Groups I, I I , III and IV pr ior to drug administrat ion. Drug Dose MAP HR Noradrenaline Adrenaline (yg) (mmHg) (beats/min) (ng/ml) (ng/ml) I: B-HT 920 0.01 104 ± 1 386 11 — — 0.1 105 ± 2 398 ± 10 — 1.0 86 ± 4 385 9 0.39 ± 0.05 0.24 ± 0.07 10.0 101 ± 5 381 ± 11 0.39 0.05 0.24 ± 0.07 I I : Clonidine 0.01 105 ± 3 398 ± 14 — — 0,1 102 ± 2 389 ± 9 — — 1.0 104 ± 2 388 ± 7 0.20 ± 0.06 0.14 ± 0.06 10.0 104 ± 4 384 ± 8 0.20 0.06 1.14 ± 0.06 I I I : Rauwolscine 10.0 99 ± 3 358 ± 13 0.17 0.05 0.65 0.25 B-HT 920 1.0 104 ± 3 376 ± 11 0.17 0.05 0.65 ± 0.25 IV: Rauwolscine 10.0 106 ± 2 354 12 0.16 0.04 0.53 ± 0.15 Clonidine 1.0 103 ± 2 364 11 0.16 0.04 0.53 ± 0.15 GROUP I GROUP II F i g . 24. The ef fect of i . c . v . in jec t ion of B-HT 920 (Group I) and c lonid ine (Group II) on MAP and HR. Q = 0.01 jig, [jjff] = 0.1 yg, ^ = 1.0 yg, ^ | • = 10.0 yg. Values represent change from con t ro l . * S i g n i f i c a n t l y d i f ferent from control (P < 0.05), - 89 -~ 0 . 3 -i 0 . 3 i - 0 . 3 J GROUP I GROUP I I F i g . 25. The e f f e c t o f i . c . v . - i n j e c t i o n o f B-HT 920 (Group I ) and c l o n i d i n e (Group I I ) on p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e l e v e l s . ^ = 1 .0 p g , H= 1 0 . 0 p g . V a l u e s r e p r e s e n t change f r om c o n t r o l . * S i g n i f i c a n t l y d i f f e r e n t f r om c o n t r o l (P < 0 . 0 5 ) . - 90 -in both MAP and HR in the dose range of 0.01-1 pg and a s ign i f i can t elevat ion of MAP and reduction of HR af ter the 10 pg dose (F ig . 24). The i . c . v . in jec t ion of 1 and 10 pg of c lon id ine on the second day also caused s im i la r changes in MAP and HR (resul ts not shown). Plasma noradrenaline and adrenaline leve ls decreased in response to both 1 and 10 pg of c lon id ine (F ig . 25). However, only the noradrenaline level af ter the in jec t ion of 10 pg of c lon id ine was s i g n i f i c a n t l y d i f ferent from the pretreatment value. The i . c . v . in jec t ion of the se lec t ive c^-antagonis t , rauwolscine, in rats in Group III increased MAP, HR, and both plasma noradrenaline and adrenaline concentrations (F ig . 26), although these changes were not s i g n i f i c a n t l y d i f ferent from the pretreatment values shown in Table 4. The subsequent i . c . v . in jec t ion of B-HT 920 in these animals increased MAP s i g n i f i c a n t l y and decreased HR ( F i g . 26) compared to control values obtained after the treatment with rauwolscine (Table 4 ) . The in jec t ion of B-HT 920 also increased plasma noradrenaline and adrenaline concentrations ( F i g . 27), although these changes were not s i g n i f i c a n t l y d i f fe rent from control values or rauwolscine treatment values (Table 4 ) . The i . c . v . in ject ion of rauwolscine in rats in Group IV s i g n i f i c a n t l y increased MAP, s l i g h t l y but not s i g n i f i c a n t l y decreased HR, and s l i g h t l y but not s i g n i f i c a n t l y increased plasma noradrenaline and adrenaline leve ls (F igs . 26,27). Although the ef fect of rauwolscine on HR in rats in Group IV was d i f ferent from that in Group I I I , these changes were small and not s i g n i f i c a n t l y d i f ferent from pretreatment control values shown in Table 4. The i . c . v . in jec t ion of c lonid ine af ter pretreatment with rauwolscine s i g n i f i c a n t l y decreased MAP, s l i g h t l y but not s i g n i f i c a n t l y decreased HR and plasma adrenaline l eve l s , and s l i g h t l y increased plasma noradrenaline concentration (F igs. 26,27), compared to control values or rauwolscine treatment values (Table 4 ) . 91 c n CL. 0) CD c ra JZ o 15 - i 10 5 0 -5 -10 -15 J JL 1 20 -, = io J3 OJ C n c o •10 -•20 J T GROUP I I I GROUP IV F i g . 26. The e f f e c t o f i . c . v . i n j e c t i o n o f 10 ug r a u w o l s c i n e f o l l o w e d by 1 pg B-HT 920 (Group I I I ) and 10 pg r a u w o l s c i n e f o l l o w e d by 1 pg c l o n i d i n e (Group IV) on MAP and HR. V a l u e s r e p r e s e n t change f r o m c o n t r o l . Open co lumns r e p r e s e n t v a l u e s a f t e r i n j e c t i o n o f r a u w o l s c i n e ; shaded co lumns r e p r e s e n t v a l u e s a f t e r i n j e c t i o n o f B-HT 920 and c l o n i d i n e i n Groups I I I and I V , r e s p e c t i v e l y . S i g n i f i c a n t l y d i f f e r e n t f r om c o n t r o l (P < 0 .05) , r- 0.3 H E cn 0.2 -^ o.i H O) s_ •o n3 J -O cn c: 0 -o . i H -0.2 -0.3 92 -1 MEL cn 0.3 -i 0.2 -<y 0.1 S--0.1 -g, "0.2 c 5 -0.3 J GROUP III GROUP IV F i g . 27. The ef fect of i . c . v . in jec t ion of 10 yg rauwolscine followed by 1 yg B-HT 920 (Group III) and 10 yg rauwolscine followed by 1 yg c lonid ine (Group IV) on plasma noradrenaline and adrenaline l eve l s . Values represent change from con t ro l . Open columns represent values after in jec t ion of rauwolscine; shaded columns represent values after in jec t ion of B-HT 920 and clonidine in Groups III and IV, respec t ive ly . *S ign i f i can t l y d i f ferent from control (P < 0.05). - 93 -4 DISCUSSION 4.1 Central AVP in conscious rats Neuroanatomical studies have provided evidence to suggest that AVP may play a ro le in the modulation of the cardiovascular r e f l ex . Moreover, AVP has been shown to increase catecholamine turnover in the NTS (Tanaka et a l . 1977). Therefore, i t i s possible that AVP modulates cardiovascular ref lexes by af fect ing sympathoadrenal outflow. In th is study, we determined whether central administrat ion of AVP a l te rs MAP and plasma leve ls of noradrenaline and adrenaline in conscious ra ts . Numerous studies have shown that plasma concentrations of catecholamines provide a r e l i a b l e estimate of sympathetic nerve a c t i v i t y (Yamaguchi and Kopin 1979; Goldstein et a l . 1983; Esler et a l . 1985; Hubbard et a l . 1986). The resu l ts show that i . c . v . in jec t ion of both a high and a low dose of AVP produced increases in MAP and plasma concentrations of noradrenaline and adrenaline 1 and 10 min af ter drug administrat ions. The level of noradrenaline detected 1 min af ter the in jec t ion of a low dose (23 ng/kg) of AVP was however, not s i g n i f i c a n t l y d i f ferent from the control noradrenaline l e v e l . The release of adrenaline was found to be greater 1 min af ter i . c . v . in jec t ion of AVP than i t was af ter 10 min. Yamaguchi and Kopin (1979) reported that sustained sympathetic st imulat ion of pithed rats resulted in a rapid ( less than 1 min) increase of adrenaline level but slow elevat ion of noradrenaline level which peaked af ter 4 min of s t imula t ion. Therefore, i t i s not surpr is ing that the r i se in adrenaline level was found in th is study to be higher af ter 1 min, while the increase in noradrenaline level was greater 10 min after the i . c . v . in jec t ion of AVP. Since the increase in blood pressure was associated with increased plasma catecholamine l eve l s , the resu l ts suggest that the pressor ef fect was mediated by increased - 94 -sympathetic nerve a c t i v i t y . It i s possible that th i s act ivat ion of sympathoadrenal outflow by central AVP resu l ts from an inh ib i t i on of baroreceptor re f lexes . Our resu l ts from conscious rats are consistent with those of Matsuguchi et a l . (1982) who found that microin ject ions of AVP in the area of the NTS of anaesthetized rats increased MAP and HR, and that these responses could be abolished by gangl ionic blockade. We have found that i . c . v . in ject ions of a second higher dose of AVP did not produce a greater response than that obtained with the f i r s t dose. However, the doses of AVP injected were very high, so that i t i s possible that the f i r s t dose given had produced the maximal response. I t i s also possib le that tolerance may have developed to a high local concentration of AVP, since Matsuguchi et a l . (1982) have reported the development of tolerance to the pressor ef fects of consecutive in jec t ions of AVP cen t ra l l y . In prel iminary experiments, lower doses (1-4 ng/kg) of AVP were injected into the fourth ven t r i c l e , but no changes in blood pressure or plasma catecholamine concentrations were observed. Anatomical studies have revealed that AVP-containing neurons terminate on somata and dendrites in the area of the NTS (Sofroniew et a l . 1981). Therefore i t i s l i k e l y that the local concentrations of AVP may be much higher than those normally detected in the CSF. This may be the reason why i . c . v . in ject ions of low doses of AVP did not produce any response. There was no change in MAP or plasma catecholamine leve ls in Group I I I , in which the responses were observed 1 min af ter in jec t ions of a r t i f i c i a l CSF. However, in Group IV, s l i gh t but s ign i f i can t reductions in plasma adrenaline leve ls were observed 10 min after i . c . v . in jec t ions of the veh ic le . The reason for th is i s obscure at present. - 95 -I t was found that in ject ions of AVP antagonist into the fourth ven t r i c le did not af fect MAP or plasma concentrations of catecholamines. If one assumes that both AVP and the AVP antagonist have s imi la r d i f fus ion proper t ies, the resu l ts suggest that endogenously released AVP does not have a tonic inf luence on cardiovascular re f lexes . To ru le out the p o s s i b i l i t y that the receptors involved in the mediation of AVP responses cen t ra l l y are not ident ica l to those present per iphera l l y , we pretreated rats with AVP antagonist pr ior to the i . c . v . in jec t ion of AVP. We have found that the i . c . v . in jec t ion of AVP after central administrat ion of AVP antagonist did not produce any change in MAP or plasma noradrenaline or adrenaline concentrat ions. This indicates that the receptors involved were synonymous with peripheral AVP receptors. I t i s controvers ia l whether a d i f fus ion bar r ie r for AVP ex is ts between the blood and CSF. Several studies have shown the existence of a blood-CSF barr ier to AVP (Ang and Jenkins 1982; Vorherr et a l . 1968; Stark et a l . 1985). However, Schmid et a l . (1984) have reported that access of plasma AVP to the CNS is poss ib le . To ensure that the observed responses to central AVP in jec t ions were not secondary to the d i f fus ion of the drug into the peripheral c i r c u l a t i o n , the ef fects of i . v . in jec t ions of AVP and AVP antagonist were also examined. One min af ter i . v . in jec t ion of AVP, MAP was s i g n i f i c a n t l y increased, while plasma noradrenaline and adrenaline leve ls were reduced. The increase in MAP can be at t r ibuted to the d i rec t pressor ef fect of AVP on the vasculature, which leads to a re f lex reduction in sympathetic nerve a c t i v i t y , as re f lec ted by the decreased catecholamine l eve l s . Ten min af ter the i . v . in jec t ion of AVP, the pressor ef fect of AVP and plasma noradrenaline and adrenaline concentrations had returned to basal l eve ls . I.v. in jec t ions of AVP antagonist did not af fect plasma catecholamine l e v e l s , although they did reduce MAP. The dose (2 yg/kg) of - 96 -antagonist used should have been su f f i c ien t to produce blockade of peripheral pressor receptors for AVP (Pang and Leighton 1981). Since the responses to i . v . in ject ions of AVP and AVP antagonist were d i s t i nc t from those to central i n jec t ions , the ef fect of central drug administrat ion cannot be at t r ibuted to d i f fus ion of the drug into the peripheral c i r c u l a t i o n . Each rat was subjected to the removal of 1 ml blood three times with approximately 10 to 15 min in terva ls between successive samplings. The blood removed was replaced with an equal volume of normal sa l i ne . Results from our control experiments involving i . c . v . in ject ions of a r t i f i c i a l CSF have shown that th is procedure neither decreased MAP nor elevated the plasma leve ls of noradrenaline or adrenal ine. Thus, the elevat ions of MAP and catecholamine release fo l lowing i . c . v . in ject ions of AVP should not have been the resu l t of blood l oss . However, i t i s possible that the plasma vasopressin level may have been elevated by the removal of blood since hemorrhage has been shown to be a potent stimulus for vasopressin release (Pang 1983b; Rocha e S i l v a and Rosenberg 1969; Szczepanska-Sadowska et a l . 1983). Indeed, even a non-hypotensive hemorrhage has been reported to increase the release of AVP (Claybaugh and Share 1972; Szczepanska-Sadowska 1972). The i . v . in jec t ion of AVP antagonist was found in th is study to cause a decrease in MAP but no change in plasma leve ls of noradrenaline and adrenal ine. Therefore, the decrease in "MAP fo l lowing the i . v . in jec t ion of AVP antagonist was l i k e l y to have been due to the antagonism of pressor ef fects of endogenously-released AVP. This study does not conclus ively iden t i f y the brainstem s i te of the cardiovascular actions of AVP. AVP-containing neurons project from the PVN to a number of brainstem s i t e s , including the nucleus motoris dorsal i s - 97 -vagus, and the nucleus in t ramedio la tera l is of the spinal cord (Sofroniew 1980). I t i s possib le that AVP mediates i t s cardiovascular ef fects through any of these s i t e s . In summary, i t was found that in jec t ion of AVP into the fourth ven t r i c le of conscious and unrestrained rats produced increases in MAP which could be at t r ibuted to increased plasma catecholamine l eve l s , and therefore increased sympathetic nerve a c t i v i t y . Thus, central AVP act ivates sympathoadrenal outf low, possib ly v ia an i nh ib i t i on of baroreceptor re f l exes . Since centra l ly-administered AVP antagonist did not inf luence MAP or plasma noradrenaline or adrenaline l eve l s , i t i s un l i ke ly that AVP exerts a tonic inf luence on the central cardiovascular re f lex system. 4.2 Central AVP in hypotensive rats The previous study showed that the central in jec t ion of AVP increased plasma noradrenaline and adrenaline leve ls as well as MAP, suggesting that the blood pressure response to central AVP was mediated by an increase in sympathetic nerve a c t i v i t y . I t would be expected that i f AVP played an act ive ro le in central cardiovascular regu la t ion, the central in jec t ion of AVP antagonist would produce ef fects on the cardiovascular system in opposit ion to those produced by AVP. The resu l ts of the previous study indicated that the central in jec t ion of AVP antagonist did not af fect MAP or plasma noradrenaline or adrenaline l eve l s , suggesting that AVP does not have a tonic inf luence on the cardiovascular system. However, i t i s possible that under condit ions in which the sympathetic nervous system is ac t ivated, the central AVP system may par t i c ipa te in the modulation of sympathetic nerve a c t i v i t y . In th is study the physio logical ro le of the central AVP system under condit ions of hypotensive stress was determined by cen t ra l l y administering a pressor antagonist of AVP in rats made hypotensive with continuous infusions of n i t ropruss ide. - 98 -The r e s u l t s show t h a t f o l l o w i n g t h e i n f u s i o n o f n i t r o p r u s s i d e i n t h e c o n t r o l g r o u p , MAP d e c r e a s e d and p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e l e v e l s i n c r e a s e d . T h i s was f o l l o w e d by a f u r t h e r s l i g h t r e d u c t i o n o f MAP and f u r t h e r i n c r e a s e s i n p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e c o n c e n t r a t i o n a f t e r t h e c o n t r o l i . c . v . i n j e c t i o n o f a r t i f i c i a l CSF and t h e c o n t i n u e d i n f u s i o n o f n i t r o p r u s s i d e . The second d e c r e a s e i n MAP and i n c r e a s e i n p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e c o n c e n t r a t i o n s was l i k e l y t h e r e s u l t o f c o n t i n u e d i n f u s i o n o f n i t r o p r u s s i d e f o r 10 m i n , s i n c e t h e p r e v i o u s s t u d y shows t h a t i . c . v . i n j e c t i o n o f a r t i f i c i a l CSF a l o n e does no t a f f e c t e i t h e r MAP o r p l a s m a c a t e c h o l a m i n e l e v e l s . The g roup o f r a t s w h i c h r e c e i v e d i . c . v . i n j e c t i o n s o f AVP a n t a g o n i s t showed a p a t t e r n o f r e s p o n s e i d e n t i c a l t o t h a t o f t h e c o n t r o l g r o u p . The AVP a n t a g o n i s t n e i t h e r d e c r e a s e d MAP no r p r e v e n t e d t h e i n c r e a s e i n p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e l e v e l s p r o d u c e d by n i t r o p r u s s i d e . T h e r e f o r e t h e r e s u l t s f r o m b o t h s t u d i e s show t h a t t h e c e n t r a l i n j e c t i o n o f AVP a n t a g o n i s t does no t r e d u c e MAP o r s y m p a t h e t i c n e r v e a c t i v i t y e i t h e r under normal c o n d i t i o n s o r d u r i n g s y m p a t h o a d r e n a l a c t i v a t i o n . T h i s s u g g e s t s t h a t e n d o g e n o u s l y - r e l e a s e d AVP does no t have a t o n i c i n f l u e n c e on c e n t r a l c a r d i o v a s c u l a r r e g u l a t i o n . 4.3 M i c r o i n j e c t i o n o f AVP i n t o t h e NTS T h i s s t u d y shows t h a t t h e m i c r o i n j e c t i o n o f 2 ng AVP i n t o t h e NTS o f c o n s c i o u s r a t s i n c r e a s e d MAP but no t p l a s m a n o r a d r e n a l i n e o r a d r e n a l i n e c o n c e n t r a t i o n s . The i n j e c t i o n o f a l a r g e r dose (10 ng) o f AVP s i g n i f i c a n t l y e l e v a t e d b o t h MAP and p l a s m a c a t e c h o l a m i n e l e v e l s . A d m i n i s t r a t i o n o f v e h i c l e a l o n e had no e f f e c t on MAP o r p l a s m a c a t e c h o l a m i n e l e v e l s i n d i c a t i n g t h a t t h e p r e s s o r r e s p o n s e was no t c a u s e d by t h e v e h i c l e . These r e s u l t s a r e c o n s i s t e n t w i t h t h e t h e o r y t h a t AVP a c t s i n t h e NTS t o i n c r e a s e b l o o d p r e s s u r e and s y m p a t h e t i c n e r v e a c t i v i t y . A l t h o u g h t h e l o w e r dose o f AVP a l s o i n c r e a s e d MAP, i t d i d no t i n c r e a s e p l a s m a c a t e c h o l a m i n e l e v e l s . T h i s - 99 -was l i k e l y to be a resu l t of the large v a r i a b i l i t y associated with the assay. Continuous sympathetic st imulat ion of pithed rats has been reported to produce a rapid (<1 min) increase in plasma adrenaline concentrat ion, but a slower (>4 min to peak levels) e levat ion of plasma noradrenaline leve ls (Yamaguchi and Kopin 1979). Since blood samples were obtained within 1 min after in jec t ion of the lower dose of AVP, we were able to detect a small but i ns ign i f i can t increase in plasma adrenaline but not noradrenaline concentrat ion. Homozygous Bratt leboro rats which lack hypothalamic AVP also showed pressor responses to central in jec t ion of AVP (Pittman et a l . 1982). Therefore th is suggests that the a b i l i t y of central AVP to increase MAP may not necessar i ly be ind ica t ive of a funct ional central AVP pathway. In th is study, each rat was subjected to the removal of 1 ml blood only twice, with an in terval of at least 15 min between the removal of each sample, and the f l u i d volume was replaced with 1 ml normal sa l ine as in the previous s tud ies. The resu l ts of control experiments in which rats received an in jec t ion of a r t i f i c i a l CSF (Group I) showed there was neither a reduction of MAP nor elevat ion of plasma catecholamine leve ls af ter th is treatment. As w e l l , in the previous studies three 1 ml blood samples were removed from conscious rats without af fect ing MAP or plasma catecholamine concentrat ions. Therefore the elevat ion of MAP and plasma catecholamine leve ls which occurred af ter central in jec t ion of AVP cannot be at t r ibuted to the loss of blood. I t is un l i ke ly that the ef fects of AVP within the NTS can be accounted for by d i f fus ion of drug into the peripheral c i r c u l a t i o n , since most evidence suggests that the blood-brain barr ier l im i t s the access of phys io log ica l l y s ign i f i can t amounts of AVP from the CSF to the blood (Ermisch et a l . 1985; Ang and Jenkins 1982; Vorherr et a l . 1968). Moreover, the doses of drugs injected were very small and thus, were - 100 -un l i ke ly to have much d i rect ef fect in the peripheral c i r c u l a t i o n . In add i t ion , the f i r s t study showed that the systemic administrat ion of AVP produced responses d i f ferent from those to the central in jec t ion of AVP. HR was not s i g n i f i c a n t l y affected by the in jec t ion of AVP or the veh ic le . The central in jec t ion of AVP was reported to increase HR (Matsuguchi et a l . 1982). However, these experiments were performed on anaesthetized rats in which ref lexes were depressed. Since our studies were carr ied out in conscious animals, pressor responses to AVP in jec t ion could resu l t in a re f lex reduction in HR which may have concealed any d i rec t increase in HR produced by central AVP in jec t i on . Zerbe et a l . (1983) reported that in conscious rats the response to lower doses of i . c . v . AVP was characterized by tachycardia, while at higher doses bradycardia was predominant. Therefore, i t was possible that the higher dose of AVP produced a greater elevat ion of MAP, hence st imulat ing the baroreceptors to a greater extent, causing re f lex bradycardia. The in jec t ion of the AVP antagonist into the NTS did not af fect MAP, HR or plasma noradrenaline or adrenaline concentrat ions. These resu l ts are consistent with those of the previous two studies in which the ef fects of i . c . v . in jec t ion of AVP antagonist were invest igated, and together they suggest that AVP does not have a tonic inf luence on sympathoadrenal outf low. In add i t ion , the resu l ts from th i s study allow us to ident i fy the NTS as one s i t e of action of the central cardiovascular ef fects of AVP. However, we cannot exclude the p o s s i b i l i t y that exogenously-administered AVP or AVP antagonist stimulates or b locks, respec t ive ly , extra-synapt ic receptors while endogenously released AVP stimulates synaptic receptors that may be r e l a t i v e l y inaccess ib le . - 101 -4.4 Central AVP in neurogenical ly-stressed rats The previous studies showed that the i . c . v . in jec t ion of AVP antagonist alone did not af fect MAP, HR or plasma catecholamine l eve l s , even in conscious rats subjected to hypotensive s t ress . These resu l ts suggest that central AVP does not have a tonic inf luence on the cardiovascular system. Another study showed that the NTS could be a possible s i t e of the actions of central AVP. The in jec t ion of the AVP antagonist into the NTS in th is study was also without e f fec t . A f i n a l study was carr ied out to determine whether AVP acting at the NTS s p e c i f i c a l l y has a tonic inf luence on the cardiovascular system under condit ions of neurogenic stress induced by exposing the rats to a 150 watt heat lamp. The resu l ts indicate that af ter 10 min of exposure to the heat lamp, the MAP and plasma noradrenaline and adrenaline leve ls of control rats in Group I increased s l i g h t l y , but HR was not a f fected. This was followed by further increases in MAP and plasma adrenaline concentration which were s i g n i f i c a n t l y d i f ferent from con t ro l , and smaller increases in HR and plasma noradrenaline concentration af ter the i . c . v . in jec t ion of a r t i f i c i a l CSF into the NTS under condit ions of neurogenic s t ress . Rats in Group II showed responses s im i la r to those of the control group, both 10 min af ter the i n i t i a t i o n of neurogenic stress and 5 min af ter the in jec t ion of AVP antagonist into the NTS. As observed af ter the i . c . v . in jec t ion of AVP antagonist in rats subjected to hypotensive s t ress , AVP antagonist did not prevent the elevat ions of MAP, HR and plasma noradrenaline and adrenaline concentrations which occurred in response to neurogenic s t ress , and in fac t MAP, HR and plasma adrenaline concentration a l l s i g n i f i c a n t l y increased further af ter the in jec t ion of AVP antagonist. Therefore a l l of our resu l ts suggest that endogenously-released AVP does not act t o n i c a l l y to regulate the cardiovascular system. - 102 -4.5 Vascular ro le of AVP I t has been shown that large amounts of AVP are released fo l lowing surgery in d i f fe rent species of animals (Moran et a l . 1964; Bonjour and Malvin 1970; Ishihara et a l . 1978). The in jec t ion of AVP antagonist in halothane-anaesthetized, surg ica l l y -s t ressed rats has been shown to decrease MAP by the reduction of TPR (Pang 1983a). I t is expected that the depressor response fo l lowing the in jec t ion of AVP antagonist would resu l t in re f lex act ivat ion of other endogenous pressor systems, thereby masking the vascular ef fects of the antagonism of AVP. This study invest igates the vascular ef fects of endogenously released AVP in the presence and absence of inf luence from other endogenous pressor systems, namely the renin-angiotensin or the a-adrenergic systems. The in jec t ion of the AVP antagonist decreased MAP and TPR but did not a l te r CO in a l l groups of ra ts . The AVP antagonist caused a s i g n i f i c a n t l y greater decrease of % control MAP in Groups II and III than in Group I. As we l l , the AVP antagonist caused a s l i g h t , but not s i g n i f i c a n t l y greater, decrease of % control TPR in Group I I , and a s i g n i f i c a n t l y greater decrease of % control TPR in Group III than in Group I. The resu l ts show that in the absence of vasoconstr ictor inf luences from ei ther the renin-angiotensin or the a-adrenergic systems, endogenously-released AVP has a greater inf luence on the control of MAP and vascular resistance in anaesthetized, surg ica l l y -s t ressed ra ts . The administrat ion of AVP antagonist in pentobarbi ta l-anaesthet ized, surg ica l l y -s t ressed and in tact rats in Group I increased BF to the stomach and skin but not the other organs. The resu l ts are s im i la r to those previously reported using halothane-anaesthetized surg ica l l y -s t ressed rats (Pang 1983a). - 103 -Surgery has been shown to increase plasma renin a c t i v i t y (McKenzie et a l . 1967). I t has been shown previously in halothane-anaesthetized, su rg ica l l y -s t ressed rats that the infusion of sara las in caused a decrease of MAP and TPR but no change in CO (Pang 1983a). In th is study, the infusion of sara las in in Group II also resul ted in a decrease of MAP within the same group of animals. Although the values of MAP in Group II rats subjected to the infusion of sara las in were less than the MAP values in Group I given sa l ine in fus ion , the decrease was not s t a t i s t i c a l l y s i g n i f i c a n t . This was probably due to the d i f f i c u l t y associated with the detection of small di f ferences between animals due to b io log ica l va r ia t ions . L ikewise, the infusion of sara las in was found to decrease TPR in a previous study (Pang 1983a) and not in th is one because comparisons in the previous study were made within the same animal while in th is one, they were made between d i f ferent animals. The administrat ion of the AVP antagonist in Group II caused an increase of BF to the skin and muscle and a decrease of BF to the lungs and l i v e r . Therefore, in the absence of vasomotor tone from the renin-angiotensin system, AVP plays the greatest vasoconstr ictor inf luence in the areas of the muscle and skin and the least in the lungs and l i v e r . It should be emphasized that the depressor response to the infusion of sara las in would be expected to act ivate the sympathetic nervous system. Therefore one should not expect to obtain the same ef fects from the in jec t ion of the AVP antagonist in groups I and II which have d i f fe rent endogenous vasomotor tones. The in fus ion of phentolamine in Group III decreased MAP by the reduction of CO but not TPR. This i s consistent with resu l ts from a previous study (Tabrizchi and Pang 1987). The decrease of CO was probably a resu l t of reduced venous return due to the blockade of post- junct ional a-adrenoceptors in veins. I t has been shown that i . v . infusions of - 104 -noradrenaline and B-HT 920 (a se lec t ive o^-agonist) but not methoxamine (a se lec t i ve a^-agonist) into conscious rats caused a dose-dependent increase in MAP and mean c i rcu la to ry f i l l i n g pressure, an index of to ta l body venous tone, suggesting that c^-adrenoceptors in veins play a ro le in the control of venous tone. Therefore, i t i s quite possible that phentolamine, by blocking venous o^-adrenoceptors and decreasing venous tone, caused a reduction in venous return and CO. The administrat ion of the AVP antagonist during the infusion of phentolamine markedly increased BF to the muscle and decreased BF to the l i v e r , i n tes t i ne , kidneys and tes tes . Therefore, in the absence of inf luence from the a-adrenergic system, AVP plays the greatest vasoconstr ictor inf luence in the muscle and the least in the l i v e r , i n tes t i ne , kidneys and tes tes . I t has been shown that the administrat ion of phentolamine in rats increased plasma renin a c t i v i t y (Burnier et a l . 1983b). Renin release i s known to be increased by a reduction of MAP or renal a r te r i a l pressure (Keeton and Campbell 1981). I t has been shown that endogenously released angiotensin II played the greatest vasoconstr ictor inf luence in areas of the kidneys and skin (Pang 1983a). Increased vasomotor tone from the renin-angiotensin system in rats from Group III may have overcome the inf luence of AVP antagonist on skin BF (compared to the ef fect of the AVP antagonist in rats from Groups I and I I ) . Control skin BF in rats from Group III during the infusion of phentolamine was indeed very low, 3.3 ± 0.8 verses 10.6 ± 3.8 and 8.6 ± 4.0 ml/min (mean ± SD) in Groups I and I I , respect ive ly . The resu l ts were not expressed as changes in vascular conductance (or res is tance) . The ca lcu la t ion of conductance (BF/MAP) involves the d i v i s ion of a l l BF readings by MAP obtained from an eas i l y accessible s i t e ( e . g . , femoral a r te ry ) . This method of ca lcu la t ion of a r t e r i a l conductance is based on the assumption that the same MAP can be recorded from any ar tery. - 105 -I t has been reported that unl ike the case in larger animals ( e . g . , dogs and ca ts ) , large or medium sized ar ter ies can of fer resistance to BF in ra t s . In the ra t , an a r te r i a l pressure di f ference of about 5-6 mm Hg ex is ts between the carot id artery and the femoral artery (Pang and Chan 1985). Therefore, ca lcu la t ion of a r t e r i a l conductance of d i f ferent vascular beds using MAP obtained in the femoral artery could resu l t in higher conductance values in many organs ( e . g . , b ra in , heart, e tc . ) than the true conductance which can only be obtained by d iv id ing BF by MAP obtained at the par t i cu la r vascular bed in question. In summary, th is study shows that endogenously-released AVP plays a s ign i f i can t ro le in the control of MAP and vascular res is tance. Moreover, AVP plays a greater pressor ro le in the absence of inf luence from the renin-angiotensin or the sympathetic nervous systems. The extent of vasoconstr ictor inf luence exerted by AVP in d i f ferent vascular beds varies depending on the endogenous vasomotor tone from the angiotensin II and/or a-adrenergic systems. In the absence of vasomotor tone from the renin-angiotensin system, AVP has the greatest vasoconstr ictor inf luence in the vascular beds of the skin and muscle. In the absence of inf luence from the sympathetic nervous system, AVP has the greatest vasoconstr ictor inf luence in the vascular beds of the muscle. 4.6 Central and peripheral actions of a-agonists Most c ross -c i r cu la t i on techniques developed in the past have involved connecting both l e f t and r igh t common carot id ar ter ies of two animals (Bickerton and Buckley 1961; Takahashi and Bunag 1980). I t i s possible to connect only one common carot id artery from each ra t . Our studies using s ingle rats have shown that in the absence of funct ional subclavian a r te r i es , the l e f t common carot id artery alone could provide su f f i c ien t BF to the bra in . This i s to be expected since rats have extensive co l l a t e ra l - 106 -c i r cu la t i on supplying the c i r c l e of W i l l i s ( P u l s i n e l l i and B r i e r l y 1979). Although none of the changes in BF to the d i f ferent areas of the brain were s i g n i f i c a n t , i t i s in terest ing to note that af ter l i ga t i on of the subclavian a r te r i es , BF to the l e f t and r ight hemispheres was increased, while that to the brainstem was reduced. This change in the d i s t r i bu t ion of blood to the brain suggests that the brainstem is highly dependent on blood supplied by the subclavian a r te r i es . There was s im i la r BF to both brain hemispheres, ind icat ing that the l e f t common carot id artery supplied both hemispheres. A c ross -c i r cu la t i on preparation was developed in which the l e f t common carot id ar ter ies and both the l e f t and r ight external jugular veins of two rats were connected. The subclavian ar ter ies of both rats were l igated to occlude blood supply to the vertebral a r te r i es . Unlike previous preparat ions, in which one animal served as a donor and one as a rec ip ien t , th is technique involved complete c ross -c i r cu la t i on of blood between ra t s , so that each rat acted both as a donor and a rec ip ien t . In contrast to the preparation of Takahashi and Bunag (1980), no p e r i s t a l t i c pump was required to i n i t i a t e or maintain BF. Results from microsphere experiments ve r i f i ed that peripheral blood from one rat supplied the brain of another rat but not i t s own bra in . This c ross -c i r cu la t i on preparation makes i t possible to completely separate the central from the peripheral cardiovascular ef fects of drugs. Thus, one can administer a drug intravenously into one rat to observe the peripheral e f fects in the same ra t , , and the central ef fects in another ra t . This preparation was used to separate the ef fects of methoxamine and c lonid ine into central and peripheral components. The i . v . administrat ion of these drugs to one rat e l i c i t e d completely d i f ferent responses from the two ra t s . The resu l ts show that c lon id ine reduced MAP and HR by a central ac t ion , while i t s peripheral action resul ted in a pressor response and a - 107 -r e f l e x b r a d y c a r d i a . These o b s e r v a t i o n s c o r r e l a t e w e l l w i t h t h e e s t a b l i s h e d v i e w t h a t c l o n i d i n e s t i m u l a t e s c e n t r a l o ^ - a d r e n o c e p t o r s w h i c h m e d i a t e a r e d u c t i o n o f s y m p a t h e t i c n e r v e a c t i v i t y and MAP. I t i s p o s s i b l e t h a t a s i g n i f i c a n t r e d u c t i o n i n HR was no t o b s e r v e d i n t h e second r a t b e c a u s e i t was masked by a r e f l e x i n c r e a s e i n HR i n r e s p o n s e t o t h e c e n t r a l d e p r e s s o r a c t i o n o f c l o n i d i n e . S i n c e t h e r e s p o n s e t o s t i m u l a t i o n o f p e r i p h e r a l o ^ - a d r e n o c e p t o r s was c h a r a c t e r i z e d by i n c r e a s e d MAP. and d e c r e a s e d HR, i t s u g g e s t s t h a t t he e f f e c t s o f p e r i p h e r a l p o s t - j u n c t i o n a l o ^ - a d r e n o c e p t o r s p r e d o m i n a t e o v e r t h o s e o f p r e - j u n c t i o n a l c ^ - a d r e n o c e p t o r s . C l o n i d i n e may s t i m u l a t e p r e - j u n c t i o n a l o ^ - a d r e n o c e p t o r s i n t h e p e r i p h e r y , bu t t h i s e f f e c t may be o f l i t t l e i m p o r t a n c e s i n c e t h e o v e r a l l e f f e c t o f s t i m u l a t i o n o f p e r i p h e r a l o ^ - a d r e n o c e p t o r s can be a t t r i b u t e d p r i m a r i l y t o a c t i v a t i o n o f p o s t - j u n c t i o n a l ( ^ " a d r e n o c e p t o r s . In c o n t r a s t , methoxamine i n c r e a s e d MAP by b o t h c e n t r a l and p e r i p h e r a l a c t i o n s . T h i s f i n d i n g c o r r e l a t e s w e l l w i t h t h e o b s e r v a t i o n t h a t low doses o f p h e n y l e p h r i n e a d m i n i s t e r e d c e n t r a l l y i n c r e a s e b l o o d p r e s s u r e and s p l a n c h n i c s y m p a t h e t i c n e r v e a c t i v i t y ( S c h m i t t and F e n a r d 1971). In a d d i t i o n , b i n d i n g s t u d i e s have r e v e a l e d s i t e s f o r 3 3 H-WB 4101 and H - p r a z o s i n w i t h i n t he CNS w h i c h meet t h e c r i t e r i a f o r a - | - a d r e n o c e p t o r s ( U ' P r i c h a r d e t a l . 1978; U ! P r i c h a r d and S n y d e r 1979). Our r e s u l t s w i t h me thoxam ine , a s e l e c t i v e a ^ - a g o n i s t , were no t n e c e s s a r i l y i n c o n f l i c t w i t h t h e r e s u l t s o f T a y l o r and Page (1951) who o b s e r v e d d e p r e s s o r r e s p o n s e s i n t h e r e c i p i e n t a f t e r a d m i n i s t r a t i o n o f n o r a d r e n a l i n e and a d r e n a l i n e , w h i c h may s t i m u l a t e o ^ - a d r e n o c e p t o r s i n a d d i t i o n t o a ^ - a d r e n o c e p t o r s . However o u r r e s u l t s c o n f l i c t w i t h o b s e r v a t i o n s t h a t i n j e c t i o n o f p h e n y l e p h r i n e , a n o t h e r a - j - a g o n i s t , i n t o t h e head c i r c u l a t i o n o f a r e c i p i e n t r a t d e c r e a s e d i t s b l o o d p r e s s u r e and s y m p a t h e t i c n e r v e a c t i v i t y ( T a k a h a s h i and Bunag 1980). I t s h o u l d be no ted t h a t t he a u t h o r s d i d no t remove t h e v e r t e b r a l a r t e r i a l b l o o d s u p p l y t o t h e b r a i n o f t h e - 108 -rec ip ient ra t . BF from the donor rat was found to be d is t r ibuted mainly to the cerebrum of the rec ip ient ra t ; BF to the brainstem of the rec ip ient rat was neg l i g i b l e . Therefore, the responses to phenylephrine which they observed were due to ef fects of the drug on the brain hemispheres. In our c ross -c i r cu la t i on preparat ion, the determination of BF from rat A to the brains of the two rats revealed that before l i ga t ion of the subclavian a r t e r i es , most BF was d is t r ibuted to the brain of rat A, while the l e f t and r ight hemispheres of rat B received some BF but the brainstem did not. Thus the subclavian ar ter ies alone were found to supply a l l brain areas in rat A. Af ter l i ga t ion of the subclavian ar ter ies the brain of rat A did not receive any BF, while BF to a l l areas of the brain of rat B was subs tan t ia l l y increased. Therefore, the resu l ts show that l i ga t i on of the subclavian ar ter ies i s essent ia l to achieve c ross -c i r cu la t i on between the two rats v ia the carot id a r te r i es . Since the venous connections between the head and body of the rats in th is preparation were not completely severed, the p o s s i b i l i t y remained of c i r cu la to ry leakage from the head to the body of rat B. To determine the 57 extent of c i r cu la to ry leakage, Co isotope was injected into rat A and the rad ioac t i v i t y in the blood of both rats measured at spec i f i c t imes. Leakage was less than 10% unt i l af ter 3 min. We observed drug responses within 5-10 s of drug in jec t ion in rat A, and within 10-30 s in rat B. Drug responses were measured within 2 min in rat A and between 2-3 min in rat B. Within 2-3 min, drug responses in rat A had disappeared while responses in rat B had reached a maximum and c i rcu la to ry leakage in rat B was s t i l l minimal. Therefore, i t i s un l i ke ly that the drug response observed in rat B was due to a peripheral action of the drug. However, i t i s possible that i f drugs with a long duration of action were to be invest igated using th is - 109 -technique, that the drug responses observed in rat B could be a consequence of c i r cu la to ry leakage from the head to the body of B. Using th is c ross -c i r cu la t ion preparation we found that c lonid ine acted per iphera l ly to increase MAP, while i t s central act ion reduced MAP. Methoxamine on the other hand increased MAP by a peripheral act ion, while i t s central act ion increased MAP and HR. 4.7 Central o^-agonists in conscious rats The resul ts of th is study show that the i . c . v . in jec t ion of doses of 0.01 to 1 yg of c lonid ine caused dose-dependent reductions in MAP and HR. The i . c . v . in jec t ion of 1 yg was accompanied by reductions in the levels of plasma noradrenaline and adrenal ine. These changes, however, were not found to be s i gn i f i can t , but th is may have been the resul t of the large v a r i a b i l i t y of the assay. These resul ts are consistent with those of the c ross -c i r cu la t i on study, in which c lonid ine was shown to decrease MAP and HR by a central act ion in anesthetized ra ts . The 10 ug dose of c lonid ine was l i k e l y a s u f f i c i e n t l y high dose to leak into the peripheral c i r c u l a t i o n , where i t stimulated post- junct ional o^-adrenoceptors, causing vasoconstr ict ion and masking the central ly-mediated decrease in sympathoadrenal a c t i v i t y . The s ign i f i can t decrease in HR af ter the i . c . v . in jec t ion of 10 yg c lonid ine may have been the consequence of a ref lex response to the increase in MAP as well as an enhancement of the baroreceptor ref lex by a central act ion of c lonid ine (Huchet et a l . 1983). The s ign i f i can t decrease in plasma noradrenaline level and small and non-s igni f icant decrease in adrenaline concentration with 10 yg clonidine may represent a baroreceptor reflex-mediated reduction in sympathetic nerve a c t i v i t y which pa ra l l e l s the decrease in HR. The responses to the i . c . v . in jec t ion of the more se lect ive oto -agonis t B-HT 920 were unexpected. Rather than decreasing MAP, the - 110 -i . c . v . in jec t ion of B-HT 920 increased MAP and decreased HR in a dose-dependent fashion. In contrast to the responses to the i . c . v . in jec t ion of c lon id ine , the responses to a l l 4 doses of B-HT 920 were in the same d i r ec t i on . I f a high dose of B-HT 920 were to leak out of the CNS into the peripheral c i r c u l a t i o n , i t would be expected to cause an ef fect addi t ive to i t s central ac t ion. While plasma noradrenaline concentration did not appear to change af ter the i . c . v . in jec t ion of 1 yg B-HT 920, the plasma adrenaline concentration appeared to increase, and both noradrenaline and adrenaline concentrations were increased af ter the in jec t ion of 10 yg B-HT 920, although none of these changes were s t a t i s t i c a l l y s i g n i f i c a n t . These resu l ts are opposite to our expectat ions. Our hypothesis was that as a more se lec t ive agonist at a 2 ~ t n a n a t aj-adrenoceptors, B-HT 920 should produce a greater reduction of MAP and sympathetic nerve a c t i v i t y af ter i . c . v . administrat ion than c lon id ine . Paradox ica l ly , the i . c . v . in jec t ion of B-HT 920 produced MAP and plasma catecholamine responses in the opposite d i rec t ion to those observed af ter the i . c . v . in jec t ion of c lon id ine . These resu l ts are not consistent with e a r l i e r reports that the in t rac is te rna l in jec t ion of B-HT 920 in anesthetized cats decreased MAP and HR (P ich ler and Kobinger 1981). I t i s not c lear whether di f ferences in the s i t e of i n j ec t i on , in the species or the use of anesthesia in the study by P ich ler and Kobinger (1981) could account for the di f ference in response. These resu l ts suggest that e i ther the central ly-mediated inh ib i to ry ef fects of c lonid ine are not mediated by central (^-adrenoceptors, or that the central pressor ef fects of B-HT 920 are mediated by receptors other than (^-adrenoceptors, or that the act ivat ion of other receptors by B-HT 920 masks the ef fect of act ivat ion of c^-adrenoceptors. In order to determine whether the response to centra l ly-administered B-HT 920 or that to c lonid ine was mediated by c^-adrenoceptors, i . c . v . - I l l -in jec t ions of these drugs were given af ter the i . c . v . in jec t ion of the se lec t i ve o^-antagonist , rauwolscine (Tabrizchi and Pang 1987). The i . c . v in jec t ion of rauwolscine alone was found to increase MAP. The ef fect of central rauwolscine on HR was less c lea r . Rauwolscine increased HR in Group III but decreased i t in Group IV. However, i t should be noted that neither change was of s t a t i s t i c a l s ign i f i cance , and that the amount of change was small (about 10 beats/min when control values of HR were above 350 beats/min). Furthermore, rauwolscine may have central as well as re f lex ef fects on HR which may act in opposite d i rec t ions . The increase in MAP was accompanied by increases in both plasma noradrenaline and adrenaline concentrat ions, although again, due to the large v a r i a b i l i t y in the assay, these changes were not s i g n i f i c a n t . I f i t i s assumed that these rauwolscine-sensi t ive receptors are (^-adrenoceptors, the resu l ts suggest that these receptors may have a tonic inh ib i to ry inf luence on sympathetic nerve a c t i v i t y and MAP. The i . c . v . in jec t ion of B-HT 920 af ter the central administrat ion of rauwolcine produced the same responses as in i t s absence: an increase in MAP and plasma noradrenaline and adrenaline concentrat ions, and a decrease in HR. This suggests that the ef fects of i . c . v . in jec t ion of B-HT 920 were not mediated by central ag-adrenoceptors. However, the ef fects of central in jec t ions of c lonid ine were also not abolished by rauwolscine. MAP, HR and plasma adrenaline concentration were also decreased by c lonid ine af ter pretreatment with rauwolscine, although the magnitude of the reduction in plasma noradrenaline level was not as great. The resu l ts suggest that the dose of rauwolscine used was not su f f i c i en t to achieve complete blockade of central a^-adrenoceptors. However, the ra t io of doses of rauwolscine to B-HT 920 used in th is study was higher than that required to completely block the peripheral pressor actions of B-HT 920 (Tabrizchi and Pang 1987). - 112 -Due to s o l u b i l i t y problems, i t was not possible to make a more concentrated solut ion of rauwolscine. As w e l l , i t was not possible to in jec t a larger volume of the drug since a 4 pi volume was already used to carry the 10 pg dose. Thus, i t i s s t i l l possible that neither the ef fect of B-HT 920 nor that of c lon id ine was mediated by central o^-adrenoceptors. Clonidine has been shown to have par t ia l agonist ic propert ies on (^-adrenoceptors (Timmermans and van Zwieten 1980a). However, the observation that methoxamine increased MAP and HR cen t ra l l y in c ross -c i r cu la t i on studies would suggest that st imulat ion of central o^-adrenoceptors leads to ac t iva t ion rather than depression of the cardiovascular system. Therefore the central e f fects of c lon id ine cannot read i l y be at t r ibuted to i t s a c t i v i t y on a^-adrenoceptors. Although B-HT 920 has been shown to be a se lec t i ve c^ -agon is t , i t acted d i f f e ren t l y than c lon id ine , suggesting that i t may st imulate another type of receptor within the CNS which leads to act ivat ion of the sympathetic nervous system. I t has been reported that B-HT 958, an analog of B-HT 920, stimulates central dopamine receptors (Brown and Harland 1986). I t was shown that i . v . or i . c . v . in ject ions of idazoxan, a se lec t ive o^-antagonist , could not prevent the reductions in MAP, HR and plasma noradrenaline concentration produced by the i . v . in jec t ion of B-HT 958, although these ef fects were abolished by the i . c . v . administrat ion of the dopamine antagonist, s u l p i r i d e . However, since the responses to B-HT 958 were in the opposite d i rec t ion to those observed af ter central in jec t ion of B-HT 920, i t i s un l i ke ly that the pressor e f fects of B-HT 920 were mediated by dopamine receptors. These studies produced some in terest ing and rather unexpected resu l t s . Further studies using se lec t ive c^-antagonists which can ensure blockade of central a ? -adrenoceptors, and other a ? -agon is ts with varying - 113 -s e l e c t i v i t i e s for c^-adrenoceptors are required before an explanation for these paradoxical resu l ts can be obtained. 4.8 General conclusions 4.8.1 Role of AVP in cardiovascular regu la t ion . These studies demonstrated that AVP can increase MAP and plasma catecholamine leve ls through an action in the CNS. A possible s i t e for th i s action of AVP may be the NTS, the primary s i t e of termination of the afferent neurons of the baroreceptor re f lex arc, since the micro in ject ion of small amounts of AVP into th is s i t e could evoke responses s im i la r to those observed af ter i . c . v . in jec t ion of AVP. However, the in jec t ion of AVP antagonist alone into e i ther the fourth vent r i c le or the NTS was without e f fec t . The in jec t ion of AVP antagonist into the fourth ven t r i c le or the NTS of rats subjected to hypotensive stress or neurogenic s t ress , respec t ive ly , also did not af fect MAP, HR or plasma catecholamine l eve l s . This suggests that endogenously-released central AVP does not have a tonic inf luence on the cardiovascular system under e i ther normal condit ions or condit ions in which the sympathoadrenal system may be act ivated. Therefore the a b i l i t y of cen t ra l l y - in jec ted AVP to elevate MAP and sympathetic nerve a c t i v i t y may be of l i t t l e physio logica l s ign i f i cance . The in jec t ion of AVP antagonist in control rats decreased MAP and TPR. This suggests that at the peripheral l e v e l , endogenously-released AVP plays a s ign i f i can t ro le in the control of MAP and vascular resistance in su rg ica l l y -s t ressed ra t s . This ro le was found to be greater in the absence of inf luence from the renin-angiotensin or sympathetic nervous systems, since the in jec t ion of AVP antagonist in rats subjected to blockade of the renin-angiotensin or the a-adrenergic systems caused a greater reduction in MAP and TPR than in control r a t s . Therefore the AVP system may have a greater ro le in the maintenance of peripheral resistance and MAP in the event of f a i l u r e of the renin-angiotensin and sympathetic nervous systems. - 114 -The amount o f v a s o c o n s t r i c t i o n p r o d u c e d by AVP i n d i f f e r e n t v a s c u l a r beds a p p e a r s t o depend on t h e endogenous v a s o m o t o r t o n e f r o m t h e r e n i n - a n g i o t e n s i n and t h e a - a d r e n e r g i c s y s t e m s . In t h e absence o f t h e r e n i n - a n g i o t e n s i n s y s t e m , AVP a n t a g o n i s t i n c r e a s e d BF t o s k i n and m u s c l e , s u g g e s t i n g t h a t i n t h e absence o f t o n e f r o m t h i s s y s t e m , AVP has t h e g r e a t e s t i n f l u e n c e i n t h e m u s c l e and s k i n . D u r i n g b l o c k a d e o f t h e a - a d r e n e r g i c s y s t e m , t h e AVP a n t a g o n i s t i n c r e a s e d BF t o t h e m u s c l e , s u g g e s t i n g t h a t i n t h e a b s e n c e o f t h i s s y s t e m , AVP has t h e g r e a t e s t v a s o c o n s t r i c t o r e f f e c t on m u s c l e . 4 . 8 . 2 R o l e o f t h e a - a d r e n e r g i c s y s t e m i n c a r d i o v a s c u l a r r e g u l a t i o n . The r e s u l t s f r o m c r o s s - c i r c u l a t i o n s t u d i e s and s t u d i e s w h i c h i n v o l v e d c e n t r a l i n j e c t i o n s o f c l o n i d i n e s u g g e s t e d t h a t c e n t r a l a 2 ~ a d r e n o c e p t o r s m e d i a t e a r e d u c t i o n o f MAP and HR, o b s e r v a t i o n s c o n s i s t e n t w i t h t h e a c c e p t e d v i e w s i n t h e l i t e r a t u r e . M o r e o v e r , c e n t r a l a 2 ~ a d r e n o c e p t o r s may e x e r t a t o n i c i n h i b i t o r y i n f l u e n c e o v e r t h e c a r d i o v a s c u l a r s y s t e m , s i n c e t h e i . c . v . i n j e c t i o n o f r a u w o l s c i n e was shown t o i n c r e a s e MAP and p l a s m a n o r a d r e n a l i n e and a d r e n a l i n e c o n c e n t r a t i o n s . The r e s u l t s f r o m t h e c r o s s - c i r c u l a t i o n s t u d y a l s o s u g g e s t t h a t t h e e f f e c t s o f p e r i p h e r a l p o s t - j u n c t i o n a l a g - a d r e n o c e p t o r s p r e d o m i n a t e o v e r t h o s e o f p e r i p h e r a l p r e - j u n c t i o n a l a 2 ~ a d r e n o c e p t o r s , s i n c e t h e s t i m u l a t i o n o f p e r i p h e r a l a 2 ~ a d r e n o c e p t o r s by c l o n i d i n e r e s u l t e d i n an i n c r e a s e i n MAP and a r e d u c t i o n o f HR. U n t i l a n t a g o n i s t s w h i c h p r e f e r e n t i a l l y b l o c k e i t h e r t h e p o s t - j u n c t i o n a l o r t h e p r e - j u n c t i o n a l a 2 - a d r e n o c e p t o r s become a v a i l a b l e , t h e r e l a t i v e c o n t r i b u t i o n o f t h e s e two t y p e s o f r e c e p t o r s t o s y m p a t h o a d r e n a l a c t i v i t y and hemodynamics c a n n o t be d e t e r m i n e d . The c r o s s - c i r c u l a t i o n s t u d i e s a l s o showed t h a t methoxamine i n c r e a s e d MAP and p o s s i b l y HR by a c e n t r a l a c t i o n , s u g g e s t i n g t h a t c e n t r a l a - ^ - a d r e n o c e p t o r s may m e d i a t e r e s p o n s e s i n t h e o p p o s i t e d i r e c t i o n t o t h o s e - 115 -produced by cxg-adrenoceptors. However, i t i s not c lear whether these receptors have a physio logical ro le in cardiovascular regula t ion. F i n a l l y , the observation of a paradoxical elevat ion of MAP in response to the i . c . v . in jec t ion of B-HT 920 ra ises new questions about the ro le of central o^-adrenoceptors. Since the central in jec t ion of c lonid ine produced the expected decrease in MAP and HR, i t suggests that the response to B-HT 920 was mediated by receptors other than ©^-adrenoceptors. Unfortunately, neither the response to c lonid ine nor that to B-HT 920 was abolished by ©^-adrenoceptor blockade with rauwolscine. Therefore, we cannot de f i n i t e l y conclude that the response to c lon id ine was due to the act ivat ion of rauwolscine-sensi t ive ©^-adrenoceptors and that the pressor response to B-HT 920 was not. Further studies with d i f fe rent a-antagonists and o^-agonists with various s e l e c t i v i t i e s are necessary before we can explain the d i f f e ren t i a l ef fects of central c lonid ine and B-HT 920. - 116 -5 REFERENCES ABBOUD, F .M. , AYLWARD, P . E . , FLORAS, O.S. and GUPTA, B.N. Sens i t i za t ion of aor t ic and cardiac baroreceptors by arginine vasopressin in mammals. J . Phys io l . (Lond.) 377:251-265, 1986. ABOOD, L . G . , KNAPP,. R., MITCHELL, T . , BOOTH, H. and SCHWAB, L. Chemical requirements of vasopressins for barrel rotat ion convulsions and reversal by oxytocin. J . Neurosci. Res. 5:191-199, 1980. AISENBREY, G.A. , HANDELMAN, W.A., ARNOLD, P. and MANNING, M. Vascular ef fects of arginine vasopressin during f l u i d deprivat ion in the ra t . J . C l i n . Invest. 67:961-968, 1981. ALDRICH, T.B. A prel iminary report on the act ive p r inc ip le of the suprarenal gland. Am. J . Phys io l . 5^:457-461, 1901. ALEXANDER, D.P. , FORSLING, M.L., MARTIN, M.S. , NIXON, D.A., RATCLIFFE, J . G . , REDSTONE, D. and TURNBRIDGE, D. The ef fect of maternal hypoxia on fe ta l p i t u i t a ry hormone release in the sheep. B i o l . Neonate 21:219-228, 1972. ALQUIST, R.P. A study of the adrenotropic receptors. Am. J . Phys io l . 153:586-600, 1948. ALTURA, B.M. Select ive microvascular const r ic tor actions of some neurohypophyseal peptides. Eur. J . Pharmacol. 24:49-60, 1973. ALTURA, B.M. and ALTURA, B.T. Vascular smooth muscle and neurohypophyseal hormones. Fed. Proc. 36 :1.853-1860, 1977. ALTURA, B.M., HERSHEY, S.G. and ZWEIFACH, B.W. Ef fects of a synthetic analogue of vasopressin on vascular smooth muscle. Proc. Soc. Exp. B i o l . Med. 119:258-261, 1965. ANDEN, N .E . , CORRODI, H. , FUXE, K., HOKFELT, B. , HOKFELT, T . , RYDIN, C. and SVENSSON, T. Evidence for a central noradrenaline receptor st imulat ion by c lon id ine . L i fe S c i . 2:513-523, 1970. ANDERSSON, B. Thi rs t - and brain control of water balance. Am. S c i . 59:408-415, 1971. ANDREWS J R . , C .E . and BRENNER, B.M. Relat ive contr ibut ions of arginine vasopressin and angiotensin II to maintenance of systemic a r te r i a l pressure in the anesthetized water- deprived ra t . C i r c . Res. 48:254-258, 1981. ANG, V.T.Y. and JENKINS, J . S . Blood- cerebrospinal f l u i d bar r ie r to argin ine-vasopressin, desmopressin, and desglycinamide arg in ine-vasopressin in the dog. J . Endocr inol . 93:319-325, 1982. - 117 -ARNAULD, E . , CZERNICHOW, P . , FUMOUX, F. and VINCENT, J .D . The ef fect of hypertension and hypovolaemia on the l ibera t ion of vasopressin during haemorrhage in the unanaesthetized monkey. Pfluegers Arch. 371:193-200, 1977. ATHAR, S. and ROBERTSON, G.L. Osmotic control of vasopressin secret ion in man. C l i n . Res. 22:335A, 1974. BARER, G.R. A comparison of the c i r cu la to ry ef fects of angiotensin, vasopressin and adrenaline in the anaesthetized cat . J . Phys io l . (Lond.) 156:49-66, 1961. BARGER, G. and DALE, H.H. Chemical structure and sympathomimetic action of amines. J . Phys io l . (Lond.) 41:19-59, 1910. BARTELSTONE, H.J . and NASMYTH, P.A. Vasopressin potent iat ion of catecholamine actions in dog, ra t , cat and rat aor t ic s t r i p . Am. J . Phys io l . 208:754-762, 1965. BAUMAN, G. and DINGMAN, J . F . D i s t r i bu t i on , blood transport and degradation of an t id iu re t i c hormone in man. J . C l i n . Invest. 57:1109-1116, 1976. BENTLEY, S .M. , DREW, G.M. and WHITING, S.B. Evidence for two d i s t i nc t types of postsynaptic alpha- adrenoceptor. Br. J . Pharmacol. 61:116P-117P, 1977. BERECEK, K .H . , MURRAY, R.D., GROSS, F. and BRODY, M.J. Vasopressin and vascular r eac t i v i t y in the development of DOCA hypertension in rats with hereditary diabetes ins ip idus . Hypertension 4_:3-12, 1982. BERECEK, K .H . , OPLE, H. , JONES, R.S.G. and HOFBAUER, K.G. Micro in ject ion of vasopressin into the locus coeruleus of conscious ra t s . Am. J . Phys io l . 247:H675-H681, 1984. BERTHELSEN, S. and PETTINGER, W.A. A funct ional basis for c l a s s i f i c a t i o n of a - adrenergic receptors. L i fe S c i . 21:595-606, 1977. — BHATIA, B. , SUBRAMANIAN, S. and SIDDIQUI, H.H. S ign i f icance of the changes in urine output on acute exposure to hypoxia. In: Respiratory Adaptations, Cap i l l a ry .Exchange and Reflex Mechanisms. EcC P a i n t a l , A . S . , G i l l - Kumar, P.) Univers i ty of Deh l i , Deh l i . 1977), pp. 244-252. BICKERTON R.K. and BUCKLEY, J . P . Evidence for a central mechanism in angiotensin induced hypertension. Proc. Soc. Exp. B i o l . Med. 106:834-836, 1961. BIERMAN, U. , FORSLING, M.L., ELLENDORFF, F. and MCDONALD, A.A. The cardiovascular responses of the. chron ica l l y catheter ized pig fetus to infused lys ine vasopressin and to hemorrhage. J . Phys io l . (Lond.) 296:28P, 1979. - 118 -BISHOP, V . S . , THAMES, M.D. and SCHMID, P.G. Ef fects of b i l a t e ra l vagal cold block on vasopressin in conscious dogs. Am. J . Phys io l . 246:R566-R569, 1984. BISSET, G.W. and LEWIS, G.P. A spectrum of pharmacological ac t i v i t y in some b io l og i ca l l y act ive peptides. Br. J . Pharmacol. 19:168-182, 1962. BLASCHKO, H. The spec i f i c action of I- dopa decarboxylase. J . Phys io l . (Lond.) 96:50-51P, 1939. BLESSING, W.W., SVED, A .F . and REIS, D .J . Elevated plasma vasopressin contr ibutes to fulminating hypertension produced by funct ional impairment of A l catecholamine neurons in rabbit medulla. C l i n . S c i . 63:289s-292s, 1982. BODO, R. The ef fect of the "heart- ton ics" and other drugs upon the heart- tone and coronary c i r c u l a t i o n . J . Phys io l . (Lond.) 64:365-387, 1927-28. BONJOUR, J . P . and MALVIN, R.L. Plasma concentrations of ADH in conscious and anesthetized dogs. Am. J . Phys io l . 218:1128-1132, 1970. BOYKIN, J . , CADNOPAPHORNCHAI, P . , MCDONALD, K.M. and SCHRIER, R.W. Mechanism of d iu re t i c response associated with a t r i a l tachycardia. Am. J . Phys io l . 229:1486-1491, 1975. BRINTON, R . E . , GEE, K.W., WAMSLEY, J . K . , DAVIS, T .P . and YAMAMURA, I. Regional d i s t r i bu t ion of putat ive vasopressin receptors in rat brain and p i tu i t a ry by quant i tat ive autoradiography. Proc. Na t l . Acad. S c i . (USA) 81:7248-7252, 1984. BROWN, G.L. and GILLESPIE, J . S . The output of sympathetic transmitter from the spleen of the cat . J . Phys io l . (Lond.) 138:81-102, 1957. BROWN, M.J. and HARLAND, D. B- HT 958 lowers blood pressure and heart rate in the rat through st imulat ion of dopamine receptors. Br. J . Pharmacol. 87:361-370, 1986. BROWNSTEIN, M.J. Biosynthesis of vasopressin and oxytocin. Ann. Rev. Phys io l . 45:129-135, 1983. BUIJS, R.M. and SWAAB, D.F. Immuno- electron microscopical demonstration of vasopressin and oxytocin synapses in the l imbic system of the ra t . Ce l l Tissue Res. 204:355-365, 1979. BURN, J . H . and RAND, M.J. Sympathetic postganglionic mechanism. Nature (Lond.) 184:163-165, 1959. BURNIER, M., BIOLLAZ, J . , BRUNNER, D.B. and BRUNNER, H.R. Blood pressure maintenance in awake dehydrated ra t s : ren in , vasopressin, and sympathetic a c t i v i t y . Am. J . Phys io l . 245:H203-H209, 1983a. - 119 -BURNIER, M., BIOLLAZ, J . , BRUNNER, D.B., GAVRAS, H. and BRUNNER, H.R. Alpha and beta adrenoceptor blockade in normotensive and deoxycorticosterone (DOC)- hypertensive ra t s ; plasma vasopressin and vasopressin pressor e f fec t . J . Pharmacol. Exp. Ther. 224: . 222-227, 1983b. BURNSTOCK, G. and COSTA, M. Adrenergic Neurons: the i r organizat ion,  funct ion and development in the peripheral nervous system. Chapman and H a l l , London, (197S), pp. 1-50. BUSSIEN, J . P . , WAEBER, B. , NUSSBERGER, J . , SCHALLER, M.D., GAVRAS, H. , HOFBAUER, K. and BRUNNER, H.R. Does vasopressin sustain blood pressure of normally hydrated healthy volunteers? Am. J . Phys io l . 246:H143-H147, 1984. CALARESU, F.R. and CIRIELLO, J . Project ions to the hypothalamus from buffer nerves and nucleus tractus s o l i t a r i u s in the cat . Am. J . Phys io l . 239:R130-R136, 1980. CANNON, W.B. and URIDIL, J . E . Studies on the condit ions of a c t i v i t y in endocrine glands. VI I I . Some ef fects on the denervated heart of st imulat ing the nerves of the l i v e r . Am. J . Phys io l . 58:353-364, 1921. CANNON, W.B. and BACQ, Z.M. Studies on the condit ions of a c t i v i t y in endocrine organs. XXVI. A hormone produced by sympathetic action on smooth muscle. Am. J . Phys io l . 96:392-412, 1931. CANNON, W.B. and ROSENBLUETH, A. Studies on condit ions of a c t i v i t y in endocrine organs. XXIX. Sympathin E and sympathin I. Am. J . Phys io l . 104:557-574, 1933. CARLIER, J . G . , LEJEUNE, G. and BARAC, G. Ef fets hemodynamiques compares d'une vasopressine synthetique et de la p i t ress ine chez le chien. Arch. Int. Pharmacodyn. 125:287-303, 1960. CAVERO, I. and ROACH, A.G. Ef fects of c lonid ine on canine cardiac neuroeffector structures cont ro l l ing heart ra te . Br. J . Pharmacol. 70:269-276, 1980. CHALMERS, J . P . Brain amines and models of experimental hypertension. C i r c . Res. 36:469-480, 1975. CHAN, T . C . K . , WALL, R.A. and SUTTER, M.C. Chronic ethanol consumption, s t ress , and hypertension. Hypertension 7:519-524, 1985. CLARK, B . J . and ROCHA E SILVA J R . , M. An afferent pathway for the se lec t ive release of vasopressin in response to carot id occlusion and hemorrhage in the cat . J . Phys io l . (Lond.) 191:529-542, 1967. CLAYBAUGH, J . R . and SHARE, L. Role of the ren in- angiotensin system in the vasopressin response to hemorrhage. Endocrinology 90:453-460, 1972. - 120 -CLAYBAUGH, J .R . and SHARE, L. Vasopressin, ren in , and cardiovascular responses to continuous slow hemorrhage. Am. J . Phys io l . 224:519-523, 1973. CLAYBAUGH, J . R . , SHARE, L. and SHIMUZI, K. The i n a b i l i t y of infusions of angiotensin to elevate the plasma vasopressin concentration in the anesthetized dog. Endocrinology 90: 1647-1652, 1972. COHEN, M.M., SITAR, D.S. , MCNEILL, J . R . and GREENWAY, C V . Vasopressin and angiotensin on resistance vessels of spleen, in test ine and l i v e r . Am. J . Phys io l . 218:1704-1706, 1970. CONSTANTINE, J.W. and MCSHANE, W.K. Analysis of the cardiovascular e f fects of 2-(2,6-dichlorophenylamino)-2-imidazol ine hydrochloride (Catapres). Eur. J . Pharmacol. 4:109-123, 1968. COOPER, K . E . , KASTING, N.W., LEDERIS, K. and VEALE, W.L. Evidence supporting a ro le for endogenous vasopressin in natural suppression of fever in the sheep. J . Phys io l . (Lond.) 295:33-45, 1979. CORLISS, R . J . , MCKENNA, O.H. , SIALER, S . , O'BRIEN, G.S. and ROWE, G.G. Systemic and coronary hemodynamic ef fects of vasopressin. Am. J . Med. S c i . 256:293-299, 1968. COTTLE, M.K. Degeneration studies of primary afferents of ix th and xth cran ia l nerves in the cat . J . Comp. Neurol. 122:329-345, 1964. COUSINEAU, D., GAGNON, D.J . and SIROIS, P. Changes in plasma leve ls of vasopressin and renin in response to hemorrhage in dogs. Br. J . Pharmacol. 47:315-324, 1973. COWLEY J R . , A.W., MONOS, E. and GUYTON, A .C. Interact ion of vasopressin and the baroreceptor re f lex in the regulat ion of a r t e r i a l pressure in the dog. C i r c . Res. 34:505-514, 1974. COWLEY J R . , A.W., SWITZER, S . J . and GUINN, M.M. Evidence and quant i f ica t ion of the vasopressin a r te r i a l pressure control system in the dog. C i r c . Res. 46:58-67, 1980. COWLEY J R . , A.W., CUSHMAN, W.C., QUILLEN J R . , E.W., SKELTON, M.M. and LANGFORD, H.G. Vasopressin elevat ion in essent ia l hypertension and increased responsiveness to sodium intake. Hypertension 3 (Supp l . l ) :93 - l00 , 1981. COWLEY J R . , A.W., QUILLEN J R . , E.W. and SKELTON, M.M. Role of vasopressin in cardiovascular regu la t ion. Fed. Proc. 42:3170-3176, 1983. CRILL, W.E. and REIS, D .J . D is t r ibu t ion of carot id sinus and depressor nerves in cat brain stem. Am. J . Phys io l . 214:269-276, 1968. - 121 -CROFTON, J . T . , SHARE, L . , SHADE, R . E . , LEE-KWON, W . J . , MANNING, M. and SAWYER, W.H. The importance of vasopressin in the development and maintenance of DOC-salt hypertension in the ra t . Hypertension 1^:31-38, 1979. CROFTON, J . T . , SHARE, L . , WANG, B.C. and SHADE, R.E. Pressor responsiveness to vasopressin in the rat with DOC-salt hypertension. Hypertension 2_:424-431, 1980. DALE, H.H. On some physio logical actions of ergot. J . Phys io l . (Lond.) 34:163-206, 1906. DALE, H.H. Nomenclature of f ib res in the autonomic system and the i r e f f ec t s . J . Phys io l . (Lond.) 80:10-11P, 1933. DAVIS, G . C , KISSINGER, P.T. and SHOUP, R. E. Strategies for deter-mination of serum or plasma norepinephrine by reverse-phase l i qu id chromotography. Anal . Chem. 63:156-159, 1981. DAVIS J R . , W.D., GORLIN, R., REICHMAN, S. and STORAASLI, J . P . Ef fect of p i t u i t r i n in reducing portal pressure in the human being. New Engl . J . Med. 256:108-111, 1957. DAY, T .A . , FERGUSON, A.V. and RENAUD, L .P . F a c i l i t a t o r y inf luence of noradrenergic afferents on the e x c i t a b i l i t y of rat paraventr icular nucleus neurosecretory c e l l s . J . Phys io l . (Lond.) 355:237-249, 1984. DE JONGE, W. and NIJKAMP, F .P . Cent ra l ly induced hypotension and bradycardia af ter administration of a-methylnoradrenaline into the area of the nucleus tractus s o l i t a r i i of the ra t . Br. J . Pharmacol. 58:593-598, 1976. DE JONGE, A . , TIMMERMANS, P.B.M.W.M. and VAN ZWIETEN, P.A. Par t i c ipa t ion of cardiac presynaptic a2~adrenoceptors in the bradycardiac ef fects of c lon id ine and analogues. Naunyn Schmiedeberg's Arch. Pharmacol. 317:8-12, 1981. DE MEY, J . G . and VANHOUTTE, P.M. Uneven d i s t r i bu t ion of post junct ional alpha^- and alpha2~like adrenoceptors in canine a r t e r i a l and venous smooth muscle. C i r c . Res. 48:875-884, 1981. DE TORRENTE, A . , ROBERTSON, G . L . , MCDONALD, K.M. and SCHRIER, R.W. Mechanism of d iu re t i c response to increased l e f t a t r i a l pressure in the anesthetized dog. Kidney Int. 8:355-361, 1975. DE WIED, D. Behavioural ef fects of i n t raven t r i cu la r l y administered vasopressin and vasopressin fragments. L i f e S c i . 19:685-690, 1976. DIPETTE, D., GAVRAS, I., NORTH, W., DIPETTE, P. and GAVRAS, H. Vasopressin response to hyperosmotic st imulus: blood pressure ef fect in normal subjects and patients with impaired sympathetic system. C l i n . Exp. Hypertens. 6:851-861, 1984. - 122 -DORSA, D . M . , MAJUMDAR, L . A . F . , PETRACCA, M . , B A S K I N , D . G . and CORNETT, L . E . C h a r a c t e r i z a t i o n and l o c a l i z a t i o n o f 3 H - a r g i n i n e 8 - v a s o p r e s s i n b i n d i n g t o r a t k i d n e y and b r a i n t i s s u e . P e p t i d e s 4 :699-706, 1983. DOXEY, J . P r e - and p o s t s y n a p t i c e f f e c t s o f a - a g o n i s t s i n t h e a n n o c o c c y g e u s m u s c l e o f t h e p i t h e d r a t . E u r . J . P h a r m a c o l . 54:185-189, 1979. DOXEY, J . C . , SMITH, C . F . C . and WALKER, J . M . S e l e c t i v i t y o f b l o c k i n g a g e n t s f o r p r e - and p o s t s y n a p t i c a - a d r e n o c e p t o r s . B r . J . P h a r m a c o l . 60:91-96, 1977. DOXEY, J . C , GADIE , B . , LANE, A . C . and TULLOCH, I . F . E v i d e n c e f o r p h a r m a c o l o g i c a l s i m i l a r i t y between a 2 - a d r e n o c e p t o r s i n t h e vas d e f e r e n s and c e n t r a l n e r v o u s s y s t e m o f t h e r a t . B r . J . P h a r m a c o l . 80:155-161, 1983. DREW, G . M . P h a r m a c o l o g i c a l c h a r a c t e r i z a t i o n o f t h e p r e s y n a p t i c a - a d r e n o c e p t o r i n t h e r a t vas d e f e r e n s . E u r . J . P h a r m a c o l . 42:123-130, 1977. DREW, G . M . P r e s y n a p t i c a - a d r e n o c e p t o r s : t h e i r p h a r m a c o l o g i c a l c h a r a c t e r i z a t i o n and f u n c t i o n a l s i g n i f i c a n c e . I n : P r e s y n a p t i c  R e c e p t o r s , A d v a n c e s i n t h e B i o s c i e n c e s , V o l . 18. (Eds~I L a n g e r , S . Z . , S t a r k e , KT, and D u b o c o v i c h , M . L . ) Pergamon P r e s s , O x f o r d . (1979), pp . 5 9 - 6 5 . DREW, G . M . P r e s y n a p t i c m o d u l a t i o n o f h e a r t r a t e r e s p o n s e s t o c a r d i a c n e r v e s t i m u l a t i o n i n p i t h e d r a t s . J . C a r d i o v a s c . P h a r m a c o l . 2_:843-856, 1980. DREW, G . M . and WHITING, S . B . E v i d e n c e f o r two d i s t i n c t t y p e s o f p o s t s y n a p t i c a - a d r e n o c e p t o r i n v a s c u l a r smooth m u s c l e i n v i v o . B r . J . P h a r m a c o l . 67:207-215, 1979. DU VIGNEUD, H . , LAWLER, H . C . and POPENOE, E . A . E n z y m a t i c c l e a v a g e o f g l y c i n a m i d e f r o m v a s o p r e s s i n and a p r o p o s e d s t r u c t u r e f o r t h i s p r e s s o r - a n t i d i u r e t i c hormone o f t h e p o s t e r i o r p i t u i t a r y . J . Am. Chem. S o c . 75:4880-4881, 1953. DU VIGNEUD, H . , G I S H , D . T . and KATSOYANNIS, P . G . A s y n t h e t i c p r e p a r a t i o n p o s s e s s i n g b i o l o g i c a l p r o p e r t i e s a s s o c i a t e d w i t h a r g i n i n e - v a s o p r e s s i n . J . Am. Chem. S o c . _75:4751-4752, 1954. DUBOCOVICH, M . L . and LANGER, S . Z . N e g a t i v e f e e d b a c k r e g u l a t i o n o f n o r a d r e n a l i n e r e l e a s e by n e r v e s t i m u l a t i o n i n t h e p e r f u s e d c a t ' s s p l e e n : d i f f e r e n c e s i n p o t e n c y o f phenoxybenzamine i n b l o c k i n g t h e p r e - and p o s t - s y n a p t i c a d r e n e r g i c r e c e p t o r s . J . P h y s i o l . ( L o n d . ) 237:505-519, 1974. E L L I O T T , H . L . and R E I D , J . L . E v i d e n c e f o r p o s t j u n c t i o n a l v a s c u l a r a 2 - a d r e n o c e p t o r s i n p e r i p h e r a l v a s c u l a r r e g u l a t i o n i n man. C l i n . S c i . 65:237-241, 1983. - 123 -ELLIOTT, T.R. On the action of adrenal in. J . Phys io l . (Lond.) 32:401-467, 1904. ENERO, M.A., LANGER, S . Z . , ROTHLIN, R.P. and STEFANO, F . J . E . The ro le of the alpha adrenoceptor in regulat ing noradrenaline overflow by nerve s t imula t ion. Br. J . Pharmacol. 44:672-688, 1972. ERIKSSON, L. Ef fect of lowered CSF sodium concentration on the central control of f l u i d balance. Acta. Phys io l . Scand. 91:61-68, 1974. — ERIKSSON, L . , FERNANDEZ,. 0. and OLSSON, K. Differences in an t id iu re t i c response to in t racaro t id infusions of various hypertonic solut ions in the conscious goat. Ac ta . Phys io l . Scand. 83:554-562, 1971. ERMISCH, A . , BARTH, T . , RUHLE, H . J . , SKOPKOVA, J . , HRBAS, P. and LANDGRAF, R. On the blood-brain bar r ie r to peptides: accumulation of labe l led vasopressin, desglyNH^-vasopressin and oxytocin by brain regions. Endocrinologia experimental i s 19:29-37, 1985. ESLER, M.D., HASKING, G . J . , WILLETT, I .R .P . , LEONARD, W. and JENNINGS, G.L. Noradrenaline release and sympathetic nervous system a c t i v i t y . Journal of Hypertension 3^ : 117-129, 1985. ESSEX, H .E . , WEGRIA, G . E . , HERRICK, J . F . and MANN, F.C. The ef fect of cer ta in drugs on the coronary blood flow of the trained dog. Am. Heart J . 19:554-565, 1940. FARNEBO, L-0. and HAMBERGER, B. Drug-induced changes in the release of 3H-noradrenal ine from f i e l d stimulated rat i r i s . Br. J . Pharmacol. 43:97-106, 1971. FATER, D . C , SCHULTZ, H.D., SUNDET, W.D., MAPES, J . S . and GOETZ, K.L. Ef fects of l e f t a t r i a l stretch in cardiac denervated and intact conscious dogs. Am. J . Phys io l . 242:H1056-1064, 1982. FEJES-TOTH, G . , NARAY-FEJES-TOTH, A. and RATGE, D. Evidence against ro le of an t id iu re t i c hormone in support of blood pressure during dehydration. Am. J . Phys io l . 249:H42-H48, 1985. FELAL, I.; GOTTSMAN, M., EVERSMANN, T . , JEHLE, W. and UHLICH, E. Influence of various stress s i tuat ions on vasopressin secret ion in man. Acta. Endocr inol . [Suppl.] (Copenh) 215:122-123, 1978. FELDBERG, W. and GADDUM, J . H . The chemical t ransmitter at synapses in a sympathetic gangl ion. J . Phys io l . (Lond.) 80:12-13P, 1933. FITZGERALD, G.A. , WATKINS, J . and DOLLERY, C T . Regulation of norepinephrine release by peripheral a2~receptor s t imula t ion. C l i n . Pharmac. Ther. 29:160-167, 1981. - 124 -FLAVAHAN, N.A., COOKE, J . P . , SHEPHERD, J .T . and VANHOUTTE, P.M. Human post junct ional alpha-1 and alpha-2 adrenoceptors: d i f f e ren t i a l d i s t r i bu t ion in ar ter ies of the l imbs. J . Pharm. Exp. Ther. 241:361-365, 1987. FORSLING, M.L. and ULLMANN, E.A. Non-osmotic st imulat ion of vasopressin re lease. In: Neurohypophys i s . Int. Conf. Key Biscayne, F l o r i da . Karger, BaseT (1977), pp. 128-135. FOSTER, D.O. and FRYDMAN, M.L. Comparison of microspheres and 8 6 R b as t racers of the d is t r i bu t ion of cardiac output in rats indicates i n v a l i d i t y of 8 6 Rb + -based measurements. Can. J . Phys io l . Pharmacol. 56:97-109, 1978. FRIEDEN, J . and KELLER, A.D. Decreased resistance to hemorrhage in neurohypophysectomized dogs. C i r c . Res. 2_:214-220, 1954. FRIEDMAN, S.M. and PAULS, H. The ef fect of p i t ress in infusion on blood pressure of the rabb i t , ca t , and ra t . Am. Heart J . 44:131-142, 1952. FRIEDMAN, S .M. , FRIEDMAN, C L . and NAKASHIMA, M. Accelerated appearance of DCA hypertension in rats treated with p i t r e s s i n . Endocrinology 67:752-759, 1960. FULLER, R.W., SNODDY, H.D. and MARSHALL, U.S. Lowering of rat brain 3-methoxy-4-hydroxyphenylethylene glycol suphate (MOPEG sulphate) concentration by 2,6-dichlorobenzyl idene aminoguanidine. J . Pharm. Pharmacol. 29:375-376, 1977. FYHRQUIST, F . , TIKKANEN, I. and LINKOLA, J . Plasma,vasopressin concentration and renin in the ra t : e f fect of dehydration and hemorrhage. Acta. Phys io l . Scand. 113:507-510, 1981. GAGNON, D . J . , SIROIS, P. and BOUCHER, P . J . St imulat ion by angiotensin II of the release of vasopressin from incubated rat neurohypophysis: possible involvement of c y c l i c AMP. C l i n . Exp. Pharmacol. Phys io l . ^:305-313, 1975. GANTEN, U. , RASCHER, W. and LANG, R.E. Development of a new s t ra in of spontaneously hypertensive rats homozygous for hypothalamic diabetes ins ip idus . Hypertension 5(Suppl.1):119-128, 1983. GAUER, O.H. and HENRY, J . P . C i rcu la tory basis of f l u i d volume con t ro l . Phys io l . Rev. 43:423-481, 1963. GAZIS, D. and SAWYER, W.H. El iminat ion of infused arginine-vasopressin and i t s long-act ing deaminated analogue in ra ts . J . Endocr inol . 78:179-186, 1978. GINSBURG, M. and HELLER, H. An t id iu re t i c a c t i v i t y in blood obtained from various parts of the cardiovascular system. J . Endocrinol . 9:274-282, 1953. - 125 -GOLDSTEIN, D.S. , McCARTY, R., POLINSKY, R . J . and KOPIN, I . J . Relat ionship between plasma norepinephrine and sympathetic neural a c t i v i t y . Hypertension 5_:552-559, 1983. GOPALAKRISHNAN, V . , TRIGGLE, C .R . , SULAKHE, P.V. and MCNEILL, J .R . Character izat ion of a s p e c i f i c , high a f f i n i t y [^H]arginine° vasopressin-binding s i t e on l i v e r microsomes from d i f ferent s t ra ins of rat and the ro le of magnesium. Endocrinology 118:990-997, 1986. GRAHAM, R.M., MUIR, M.R. and HAYES, J .M . D i f fe r ing ef fects of the vasodi lator drugs, prazosin and diazoxide on plasma renin a c t i v i t y in the dog. C l i n . Exp. Pharmacol. Phys io l . 3^:173-177, 1976. GREENBERG, D.A., U'PRICHARD, D.C. and SNYDER, S.H. Alpha-noradrenergic receptor binding in mammalian bra in : d i f f e ren t i a l label ing of agonist and antagonist s ta tes . L i f e S c i . 19:69-76, 1976. GREENWAY, C V . Ef fects of sodium n i t ropruss ide, isosorbide d i n i t r a t e , isoproterenol , phentolamine and prazosin on hepatic venous responses to sympathetic nerve st imulat ion in the ' ca t . J . Pharmacol. Exp. Ther. 209:56-61, 1979. GUO, G .B . , SCHMID, P.G. and ABBOUD, F.M. Si tes at which vasopressin f a c i l i t a t e s the a r te r i a l baroreflex in rabb i ts . Am. J . Phys io l . 251:644-655, 1986. HAMILTON, C A . and REID, J . L . A postsynaptic locat ion of alpha-2-adrenoceptors in vascular smooth muscle: in vivo studies in the conscious rabb i t . Cardiovasc. Res. 16:11-15, 1982. HAUSLER, G. Clonidine-induced inh ib i t i on of sympathetic nerve a c t i v i t y : no ind icat ion for a central presynaptic or an ind i rec t sympathomimetic mode of ac t ion . Naunyn-Schmiedeberg's Arch. Pharmacol. 286:97-111, 1974. HAUSLER, G. Central a-adrenoceptors involved in cardiovascular regula t ion. J . Cardiovasc. Pharmacol. £:S72-S76, 1982: HAYWARD, J . N . and JENNINGS, D.P. Influence of sleep-waking and nociceptor-induced behaviour on the a c t i v i t y of supraoptic neurons in the hypothalamus of the monkey. Brain Res. 57^:461-466, 1973. HENRY, J . P . , GAUER, O.H. and REEVES, J . L . Evidence of the a t r i a l locat ion of receptors inf luencing urine f low. C i r c . Res. 4:85-94, 1956. HENRY, J . P . , GUPTA, P .D. , MEEHAN, J . P . , SINCLAIR, R. and SHARE, L. The ro le of afferents from the low-pressure system in the release of an t id iu re t i c hormone during non-hypotensive hemorrhage. Can. J . Phys io l . Pharmacol. 46:286-295, 1968. - 126 -HEYNDRICKX, G.R. , BOETTCHER, D.H. and VATNER, S .F . Ef fects of angiotensin, vasopressin, and methoxamine on cardiac funct ion and blood flow d is t r ibu t ion in conscious dogs. Am. J . Phys io l . 231:1579-1587, 1976. HICKS, P . E . , MEDGETT, I.C. and LANGER, S.Z. Postsynaptic alpha-2 adrenergic receptor-mediated vasoconstr ict ion in SHR t a i l ar ter ies in v i t r o . Hypertension 6^:112-118, 1984. HOFBAUER, K .G . , KONRADS, A . , BAUEREISS, K., MOHRING, B. , MOHRING, J . and GROSS, F. Vasopressin and renin in glycerol- induced acute renal f a i l u r e in the ra t . C i r c . Res. 41:424-428, 1977. HOFFMAN, W.E., PHILLIPS, M.I. and SCHMID, P.G. Central angiotensin II-induced responses in spontaneously hypertensive ra t s . Am. J . Phys io l . 232:H426-H433, 1977. HOLMAN, M.E. and SURPRENANT, A. An electrophysiooogical analysis of the ef fects of noradrenaline and a-receptor antagonists on neuromuscular transmission in mammalian muscular a r te r i es . Br. J . Pharmacol. 71:651-661, 1980. HOLMES, A . E . and LEDSOME, J .R . Ef fect of norepinephrine and vasopressin on carot id sinus baroreceptor a c t i v i t y in the anesthetized rabb i t . Exper ient ia 40:825-827, 1984. HUBBARD, J .W. , BUCHHOLTZ, R.A. , KEETON, T.K., and NATHAN, M.A. Plasma norepinephrine re f l ec ts pharmacological a l tera t ions of sympathetic a c t i v i t y in the conscious cat . J . Autonom. Nerv. Syst . 15:93-100, 1986. ~ " HUCHET, A .M . , DOURSOUT, M.F. , OSTERMANN, G . , CHELLY, J . and SCHMITT, H. Possible ro le of cq-and ©^-adrenoceptors in the modulation of the sympathetic component of the baroref lex. Neuropharmacology 22:1243-1248, 1983. HUKUHARA J R . , T . , OTSUKA, Y . , TAKEDA, R. and SAKAI, F. Die zentralen wirkungen des 2-(2,6-dichlorophenylamino)-2- imidazol in-hydrochlor ide. Arzneim. Forsch. 18:1147-1153, 1968. IMAI, Y . , NOLAN, P .L . and JOHNSTON, C . I . Endogenous vasopressin modulates the baroreflex s e n s i t i v i t y in r a t s . C l i n . Exp. Pharmacol. Phys io l . 10:289-292, 1983. IRIUCHIJIMA, J . Conditions for secret ion of vasopressin in pressor amounts in water-replete ra t s . Jpn. J . Phys io l . 33:887-894, 1983. ISHIHARA, H. , ISHIDA, K., OYAMA, T . , KUDO, T. and KUDO, M. Ef fects of general anaesthesia and surgery on renal function and plasma ADH leve l s . Can. Anaesth. Soc. J . 25:312-318, 1978. JARROTT, B. , LOUIS, W.H. and SUMMERS. R . J . The charac te r i s t i cs of (3H)-clonidine binding to an a-adrenoceptor in membranes from guinea-pig kidney. Br. J . Pharmacol. 65:663-670, 1979. - 127 -JEWELL, P.A. and VERNEY, E.B. An experimental attempt to determine the s i t e of the neurohypophyseal osmoreceptors in the dog. Ph i l os . Trans. Roy. Soc. London Ser. B 240:197-324, 1957. JOHNSON, J . A . , MOORE, W.W. and SEGAR, W.E. Small changes in l e f t a t r i a l pressure and plasma an t id iu re t i c hormone t i t e r s in dogs. Am. J . Phys io l . 217:210-214, 1969. JOHNSTON, C . I . , NEWMAN, M. and WOODS, R. Role of vasopressin in cardiovascular homeostasis and hypertension. C l i n . S c i . 61:129s-139s, 1981. JOHNSTON, K.M. , MACLEOD, B.A. and WALKER, M.J.A. Responses to l i ga t ion of a coronary artery in conscious rats and the actions of antiarrhythmics. Can. J . Phys io l . Pharmacol. 61:1340-1353, 1983. KALKMAN, H.O., THOOLEN, M . J . M . C , TIMMERMANS, P.B.M.W.M. and VAN ZWIETEN, P.A. The inf luence of a\- and (^-adrenoceptor agonists on cardiac output in' rats and ca ts . J . Pharm. Pharmacol. 36:265-268, 1984. KALSNER, S. The presynaptic receptor controversy. Trends in Pharmacological Sciences 3-1:11-16, 1982. KALSNER, S. Evidence that transmitter release in sympathetic nerves is not set by feedback v ia presynaptic receptors. Can. J . Phys io l . Pharmacol. 61:1197-1201, 1983. KALSNER, S. and QUILLAN, M. A hypothesis to explain the presynaptic ef fects of adrenoceptor antagonists. Br. J . Pharmacol. 82:515-522, 1984. KAPPAGODA, C T . , LINDEN, R . J . , SNOW, H.M. and WHITAKER, E.M. Ef fect of destruct ion of the poster ior p i t u i t a ry on the d iures is from le f t a t r i a l receptors. J . Phys io l . (Lond.) 244:757-770, 1975. KARMAZYN, M.M., MANKU, S. and HORROBIN, D.F. Changes of vascular r eac t i v i t y induced by low vasopressin concentration in teract ion w i t h C o r t i s o l a n d l i t h i u m possible i n v o l v e m e n t of p r o s t a g l a n d i n s . Endocrinology 102:1230-1236, 1978. KASTING, N.W., VEALE, W.L. and COOPER, K.E. Convulsive and hypothermic ef fects of vasopressin in the brain of the ra t . Can. J . Phys io l . Pharmacol. 58:316-319, 1980. KEETON, T.K. and CAMPBELL, W.B. The pharmacologic a l te ra t ion of renin re lease. Pharmacol. Rev. 31:81-227, 1981. KEIL, L . C , SUMMY-LONG, J . and SEVERS, W.B. Release of vasopressin by angiotensin I I . Endocrinology 96:1063-1065, 1975. KENDLER, K . S . , WEITZMAN, E.A. and FISHER. D.A. The ef fect of pain on plasma arginine vasopressin concentrations in man. C l i n . Endocr inol . (Oxf) 8:89-94, 1978. - 128 -KIBJAKOW, A.W. Uber humorale Ubertragung den Erregung von einem neuron auf das andere. Arch. i . d. ges. Phys io l . 232:432-443, 1933. , KLUPP, H. , KNAPPEN, F . , OTSUKA, Y . , STRELLER, J . and TEICHMANN, H. Ef fects of c lonid ine on central sympathetic tone. Eur. J . Pharmacol. 10:225-229, 1970. KOBINGER, W. Uber den Wirkungsmechanismus einer neuen antihypertensiven substanz mit imidazol inst ruktur . Naunyn Schmiedeberg's Arch. Pharmacol. 258:48-58, 1967. KOBINGER, W. The ro le of a-adrenoceptors in central nervous and peripheral vascular regu la t ion. Jpn. J . Pharmacol. 31(Suppl.):13P-2QP, 1981. KOBINGER, W. and PICHLER, L. The central modulatory ef fect of c lonid ine on the cardiodepressor re f lex af ter suppression of synthesis and storage of noradrenaline. Eur. J . Pharmacol. 30:56-62, 1975. KOBINGER, W. and PICHLER, L. Invest igat ion into d i f ferent types of post- and presynaptic a-adrenoceptors at cardiovascular s i tes in ra ts . Eur. J . Pharmacol. 65:393-402, 1980a. KOBINGER, W. and PICHLER, L. Relat ion between central sympathoinhibitory and peripheral pre- and postsynaptic a-adrenoceptors as evaluated by d i f ferent c l on id i ne - l i ke substances in ra ts . Naunyn-Schmiedeberg1s Arch. Pharmacol. 315:21-27, 1980b. KOBINGER, W. and PICHLER, L. Alphai and a lpha 2 adrenoceptor subtypes: s e l e c t i v i t y of various agonists and re l a t i ve d is t r ibu t ion of receptors as determined in ra t s . Eur. J . ' Pharmacol. 73:313-321, 1981. KOBINGER, W. and PICHLER, L. a-Adrenoceptor subtypes in cardiovascular regula t ion. J . Cardiovasc. Pharmacol. 4:S81-S85, 1982. KOBINGER, W. and WALLAND, A. Involvement of adrenergic receptors in central vagus a c t i v i t y . Eur. J . Pharmacol. 16:120-122, 1971. KOVACS, G . L . , BOHUS, B. , VERSTEGG, D.H.G., DE KLOET, E.R. and DE WIED, D. Ef fects of oxytocin and vasopressin on memory consol idat ion: s i tes of action and catecholaminergic corre lates af ter local micro in ject ion into l imbic-midbrain s t ructures. Brain Res. 175:303-314, 1979. KROGH, A. The reactions of c a p i l l a r i e s to d i rect s t imula t ion. In: The Anatomy and Physiology of C a p i l l a r i e s . Yale Univers i ty Press, Hafner N.Y. ( 1 9 2 9 ) , p p . 1 8 2 - 2 0 8 . - 129 -KRUSZYNSKI, M., LAMMEK, B. , MANNING, M., SETO, J . , HALDAR, J . and SAWYER, W.H. [l-(B-mercapto-B,B-cyclopentamethylenepropionic ac id ) , 2-(0-methyl) tyrosine]arginine-vasopressin and [ l-(B-mercapto-B,B-cyc1opentamethy1enepropi oni c acid) ] argin ine-vasopressin, two highly potent antagonists of the vasopresssor response to argin ine-vasopressin. J . Med. Chem. 23:364-368, 1980. KUBO, T. and MISU, Y. Pharmacological character izat ion of the a-adrenoceptors responsible for a decrease of blood pressure in the nucleus tractus s o l i t a r i i of the ra t . Naunyn-Schmiedeberg1s Arch. Pharmacol. 317:120-125, 1981. LANDS, A . M . , ARNOLD, A . , MCAULIFF, J . P . , LUDUENA, F .P . and BROWN J R . , T.G. D i f fe ren t ia t ion of . receptor systems act ivated by sympathomimetic amines. Nature 214:597-598, 1967. LANGER, S.Z. The metabolism of ^H-noradrenaline released by e l e c t r i c a l st imulat ion from the iso la ted n i c t i t a t i n g membrane of the cat and from the vas deferens of the ra t . J . Phys io l . (Lond.) 208:515-546, 1970. LANGER, S.Z. The regulat ion of t ransmitter release e l i c i t e d by nerve st imulat ion through a presynpatic feedback mechanism. In: Front iers in Catecholamine Research. (Ed. Usdin, E. and Snyder, ST) Pergammon Press, New York. (T973), pp. 543-549. LANGER, S.Z. Presynaptic regulat ion of catecholamine re lease. Biochem. Pharmac. 23:1793-1900, 1974. LANGER, S.Z. Presynaptic regulat ion of the release of catecholamines. Pharmacol. Rev. 32:337-362, 1981. LANGER, S.Z. and VOGT, M. Noradrenaline release from iso la ted muscle of the n i c t i t a t i n g membrane of the cat . J . Phys io l . (Lond.) 214:159-171, 1971. LANGER, S . Z . , ADLER, E . , ENERO, M.A. and STEFANO, F . J . E . The ro le of the alpha receptor in regulat ing noradrenaline overflow by nerve s t imulat ion. XXV Int. Cong. Phys io l . Sciences (1971), p. 335. LANGER, S . Z . , SHEPPERSON, M.B. and MASSINHAM, R. Pre ferent ia l noradrenergic innervation of alpha-adreneric receptors in vascular smooth muscle. Hypertension 3(Suppl. 1):1112—1118, 1981. LANGER, S . Z . , PIMOULE, C. and SCATTON, B. [3H]-RX781094, a preferent ia l a2-adrenoceptor antagonist rad io l igand, labels a2~adrenoceptors in the rat brain cortex. Br. J . Pharmacol. 78(Suppl . ) : l09P, 1983. LANGLEY, J . N . On the react ion of c e l l s and nerve endings to cer ta in poisons; ch i e f l y as regards the react ion of s t r ia ted muscle to n icot ine and to curare. J . Phys io l . (Lond.) 33:374-413, 1905. LANGLEY, J . N . The Autonomic Nervous System, Part I. Heffer, Cambridge. (1921). : - 130 -LAUSON, H.D. Metabolism of the neurohypophysial hormones. In: Handbook of Physiology Endocrinology Sect. 7, Vo l . IV, P t . l . (Ed. Greep, R.O., Astwood, E.B.) AirTi Phys io l . Soc . , Washington, D.C. (1974), pp. 287-393,. LAYCOCK, J . F . , PENN, W., SHIRLEY, D.G. and WALTER, S . J . The ro le of vasopressin in blood pressure regulat ion immediately fol lowing acute hemorrhage in the ra t . J . Phys io l . (Lond.) 296:267-275, 1979. LEDSOME, J .R . and LINDEN, R . J . The ro le of the l e f t a t r i a l receptors in the d iu re t i c response to l e f t a t r i a l d is tens ion . J . Phys io l . (Lond.) 198:487-503, 1968. LEDSOME, J . R . , NGSEE, J . and WILSON, N. Plasma vasopressin concentration in the anaesthetized dog before, during and af ter a t r i a l d is tens ion. J . Phys io l . (Lond.) 338:413-422, 1983. LEDSOME, J . R . , WILSON, N. and COURNEYA, C.A. Plasma vasopressin during increases and decreases in blood volume in anaesthetized dogs. Can. J . Phys io l . Pharmacol. 63:224-229, 1985. LEE-KWON, W . J . , SHARE, L . , CROFTON, J . T . and SHADE, R.E. Vasopressin in the rat with par t ia l nephrectomy-salt hypertension. C l i n . Exp. Hypertens. _3:281-297, 1981. LEIGHTON, J . , BUTZ, K.R. and PARMETER, L .L . Ef fect of a-adrenergic agonists and antagonists on neurotransmission in the rat anococcygeus muscle. Eur. J . Pharmacol. 58:27-38, 1979. LENG, G . , MASON, W.T. and DYER, R.G. The supraoptic nucleus as an osmoreceptor. Neuroendocrinology 34:75-82, 1982. LIARD, J . F . , DERIAZ, 0 . , TSCHOPP, M. and SCHOUN, J . Cardiovascular e f fects of vasopressin infused into the vertebral c i r cu la t i on of conscious dogs. C l i n . S c i . 61:345-347, 1981. LOKHANDWALA, M.F. , COATS, J . T . and BUCKLEY, J . P . Ef fects of several catecholamines on sympathetic transmission to the myocardium: ro le of presynaptic a-adrenoceptors. Eur. J . Pharmacol. 42:257-265, 1977. ~~ LUMBERS, E.R. and POTTER, E.K. The ef fect of vasoactive peptides on the carot id cardiac baroref lex. C l i n . Exp. Pharmacol. Phys io l . Suppl. 7:45-49, 1982. LUND-JOHANSEN, P. Hemodynamic changes at rest and during exercise in lon-term prazosin therapy of essent ia l hypertension. In: Prazosin: Evaluation of a new antihypertensive agent. (Ed. Cotton, D.W.K.) Exerpta Medica, Amsterdam, ( i y / 4 ) , p p . 4 B - b J . MALIK, A . B . , KAPLAN, J . E . and SABA, T.M. Reference sample method for cardiac output and regional blood flow determinations in the ra t . J . Appl . Phys io l . 40:472-475, 1976. - 131 -MANKU, M.S. and HORROBIN, D.F. Indomethacin i nh ib i t s response to a l l vasoconstr ictors in the rat mesenteric vascular bed. Restoration of response by prostaglandin Ep. Prostaglandins 12:369-376, 1977. ~~ MANNING, M. and SAWYER, W.H. Antagonists of vasopressor and an t id iu re t i c responses to arginine vasopressin. Ann. Int. Med. 96:520-522, 1982. MARCHETTI, J . , THIBONNIER, M., GONZALES, M.F. , CORVOL, P. and MENARD, J . Dynamic study of an t id iu re t i c hormone during benign mineralocort icoid hypertension. Acta Endocr inol . 95:444-453, 1980. MARSHALL, I., NASMYTH, P .A . , NICHOLL, C.G. and SHEPPERSON, N.B. a-Adrenoceptors in the mouse vas deferens and the i r e f fects on response to e l e c t r i c a l s t imula t ion. Br. J . Pharmacol. 62:147-151, 1978. ~~ MARTIN, S . , MALKINSON, T . J . , VEALE, W.L. and PITTMAN, Q . J . Central ef fect of arginine vasopressin on blood pressure in the rabb i t . Can. J . Phys io l . Pharmacol. ( J l ^ A x v i i - A x v i i i , 1983. MARTIN, S . , MALKINSON, T . J . , VEALE, W.L. and PITTMAN, Q . J . The act ion of cen t ra l l y administered arginine vasopressin on blood pressure in the conscious rabb i t . Brain Res. 348:137-145, 1985. MASSINGHAM, R. and HAYDEN, M.L. A comparison of the ef fects of prazosin and hydralazine on blood pressure, heart ra te , and plasma renin a c t i v i t y in conscious renal hypertensive dogs. Eur. J . Pharmacol. 30:121-124, 1975. MATSUGUCHI, H. , SCHMID, P . G . , VAN ORDEN, D. and MARK, A .L . Does vasopressin contr ibute to sal t - induced hypertension in the Dahl s t ra in? Hypertension 3:174-181, 1981. MATSUGUCHI, H. , SHARABI, F .M . , GORDON, F . J . , JOHNSON, A.K. and SCHMID, P.G. Blood pressure and heart rate responses to microin ject ion of vasopressin into the nucleus t ractus s o l i t a r i u s region of the ra t . Neuropharmacology 21:687-693, 1982. MCKENZIE, J . K . , RYAN, J.W. and LEE, M.R. Ef fect of laparotomy on plasma renin a c t i v i t y in the rabb i t . Nature (London) 215:542-543, 1967. MCKINLEY, M . J . , DENTON, D.A. and WEISINGER, R.S. Sensors for an t id iu res is and t h i r s t - osmoreceptors or CSF sodium detectors? Brain Res. 141:89-103, 1978. MCNEILL, J . R . . Redundant nature of the vasopressin and renin-angiotensin systems in the control of mesenteric resistance vessels of the conscious fasted cat . Can. J . Phys io l . Pharmacol. 61:770-773, 1983. - 132 -MCNEILL, J .R . and PANG, C.C.Y. Ef fect of pentobarbital anaesthesia and surgery on the control of a r t e r i a l pressure and mesenteric resistance in cats : ro le of vasopressin and angiotensin. Can. J . Phys io l . Pharmacol. 60:363-368, 1982. MELLANDER, S. and JOHANSSON, B. Control of res is tance, exchange, and capacitance functions in the peripheral c i r c u l a t i o n . Pharmacol. Rev. 20:117-196, 1968. MENTO, P . F . , WANG, H.H. and SAWYER, W.H. Relat ive contr ibut ions of arginine vasopressin (AVP) and the sympathetic nervous system in maintaining DOC-salt hypertension in ra t s . Fed. Proc. 41:1230, 1982. — MICHELL, R .H . , KIRK, C . J . and BILLAH, M.M. Hormonal st imulat ion of phosphatidyl i nos i to l breakdown with par t i cu la r reference to the hepatic ef fects of vasopressin. Biochem. Soc. Trans. 7:861-865, 1979. MOHRING, J . , MOHRING, B. , PETRI, M. and HAACK, D. Vasopressor ro le of ADH in the pathogenesis of malignant DOC hypertension. Am. J . Phys io l . 232:F260-F269, 1977. MOHRING, J . , MOHRING, B. , PETRI, M. and HAACK, D. Plasma vasopressin concentrations and ef fects of vasopressin antiserum on blood pressure in rats with malignant two-kidney Goldblatt hypertension. C i r c . Res. 42:17-22, 1978. MOHRING, J . , . KINTZ, J . and SCHOUN, J . Studies on the ro le of vasopressin in blood pressure control of spontaneously hypertensive rats with establ ished hypertension (SHR, stroke-prone s t r a i n ) . J . Cardiovasc. Pharmacol. 1^593-608, 1979. MOHRING, J . , GLANZER, K. , MACIEL J R . , J . A . , DUSING, R., KRAMER, H . J . , ARBOGAST, R. and KOCH-WESER, J . Greatly enhanced pressor response to an t id iu re t i c hormone in patients with impaired cardiovascular ref lexes due to id iopath ic or thostat ic hypotension. J . Cardiovasc. Pharmacol. 21:367-376, 1980. MOHRING, J . , KINTZ, J.,.SCHOUN, J . and MCNEILL, J .R . Pressor responsiveness and cardiovascular re f lex a c t i v i t y in spontaneously hypertensive and normotensive rats during vasopressin in fus ion . J . Cardiovasc. Pharmacol. 3:948-957, 1981. MONTANI, J . P . , LIARD, J . F . , SCHOUN, J . and MOHRING, J . Hemodynamic ef fects of exogenous and endogenous . vasopressin at low plasma concentration in conscious dogs. C i r c . Res. 47:346-355, 1980. MORAN, W.H. and ZIMMERMAN, B. Mechanism of an t id iu re t i c hormone control of importance to the surgical pat ient . Surgery 62:639-644, 1967. — MORAN, W.H., MILTENBERGER, F.W., SHU'AYB, W.A. and ZIMMERMAN, B. The re la t ionsh ip of an t id iu re t i c hormone secret ion to surg ical s t ress . Surgery 56:99-108, 1964. - 133 -MORTON, J . J . , GARCIA DEL RIO, C. and HUGHES, M.J. Ef fect of acute vasopressin infusion on blood pressure and plasma angiotensin II in normotensive and DOCA-salt hypertensive ra t s . C l i n . S c i . 62:143-149, 1982. MOUILLE, P . , HUCHET, A . M . , CHELLY, J . , LUCET, B. , DOURSOUT, M.F. and SCHMITT, H. Pharmacological propert ies of AR-C239, 2- [2-[4(0-methoxyphenyl)-piperazine- l -Yl ] -ethyl ]4,4-dimethyl- l ,3(2H-4 H) isoquinol inedione, a new a-adrenoceptor blocking drug. J . Cardiovasc. Pharmacol. 2^:175-191, 1980. MOULDS, R . J . and JAUERNIG, A.R. Mechanism of prazosin co l lapse . Lancet j . :200-20l , 1977. NASHOLD, B . S . , MANNARINO, E.M. and WUNDERLICH, M. Pressor-depressor blood pressure response in the cat af ter in t ravent r icu lar administrat ion of drugs. Nature 193:1297-1298, 1961. NISHIYAMA, K., NISHIYAMA, A. and FROHLICH, E.D. Regional blood flow in normotensive and spontaneously hypertensive ra t s . Am. J . Phys io l . 230:691-198, 1976. OLIVER, G. and SCHAFER, E.A. On the physio logical action of extracts of p i t u i t a r y body and cer ta in other glandular organs. J . Phys io l . (Lond.) 18:277-279, 1895a. OLIVER, G. and SCHAFER, E.A. Phys io log ica l e f fects of extracts of the suprarenal capsules. J . Phys io l . (Lond.) 18:230-279, 1895b. OLSSON, K. Studies on central regulat ion of secret ion of an t id iu re t i c hormone (ADH) in the goat. Acta. Phys io l . Scand. 78:465-474, 1969. OLSSON, K. Further evidence for the importance of CSF Na + concentration in central control of f l u i d balance. Acta. Phys io l . Scand. 88:183-188, 1973. ONESTI, G . , SCHWARTZ, A . B . , KIM, K . E . , PAZ-MARTINEZ, V. and SWARTZ, C H . Antihypertensive ef fect of c lon id ine . C i r c . Res.> 28(Supp1.2):53-69, 1971. OYAMA, T . , KIMURA, K. and SATO, K. The ef fect of anesthesia and surgery on plasma an t id iu re t i c hormone. Med. J . Osaka Univ. 21:113-120, 1971. PADFIELD, P . L . , LEVER, A . F . , BROWN, J . J . , MORTON, J . J . and ROBERTSON, J . I . S . Changes of vasopressin in hypertension: cause or ef fect? Lancet 1255-1257, 1976. PADFIELD, P . L . , BROWN, J . J . , LEVER, A . F . , MORTON, J . J . and ROBERTSON, J . I . S . Does vasopressin play a ro le in the pathogenesis of hypertension? C l i n . S c i . 61:141s-143s, 1981. - 134 -PANG, C.C.Y. Vasopressin and angiotensin in the control of a r t e r i a l pressure and regional blood flow in anaesthetized, surg ica l l y -s t ressed ra t s . Can. J . Phys io l . Pharmacol. 61:1494-1500, 1983a. PANG, C.C.Y. Ef fect of vasopressin antagonist and sara las in on regional blood flow fo l lowing hemorrhage. Am. J . Phys io l . 245:H749-H755, 1983b. PANG, C.C.Y. and CHAN, T.C.K. D i f fe ren t ia l i n t r a -a r t e r i a l pressure recordings from d i f ferent ar ter ies in the ra t . J . Pharmacol. Methods 13:325-330, 1985. PANG, C.C.Y. and LEIGHTON, K.M. Prolonged inh ib i t i on of pressor response to vasopressin by a potent spec i f i c antagonist, [ l-(B-mercapto-B,B-cyclopentamethylenepropionic acid)2-(0-methyl)-tyros ine]arg in ine-vasopressin. Can. J . Phys io l . Pharmacol. 59:1008-1012, 1981. PANG, C.C.Y. and TABRIZCHI, R. The ef fects of noradrenaline, B-HT 920, methoxamine, angiotensin II and vasopressin on mean c i r cu la to ry f i l l i n g pressure in conscious ra ts . Br. J . Pharmacol. 89:389-394, 1986. PELLEGRINO, L . J . , PELLEGRINO, A .S . and CUSHMAN, A . J . A Stereotaxic  At las of the Rat Bra in . Plenum Press, New York (1979T: PERLMUTT, J . H . Ef fect of vagotomy on renal funct ion during water d iu res i s . Proc. Soc. Exp. B i o l . Med. 116:270-273, 1964. PERRY, B.D. and U'PRICHARD, D.C. ( 3H)-Rauwolscine (a-yohimbine): a spec i f i c antagonist radiol igand for brain (^-adrenoceptors. Eur. J . Pharmacol. 76:461-464, 1981. PHILBIN, D.M. and COGGINS, C H . Plasma an t id iu re t i c hormone leve ls in cardiac surgical pat ients during morphine and halothane anesthesia. Anesthesiology 49:95-98, 1978. PHILBIN, D.M., WILSON, N .E . , S0K0L0SKI, J . and COGGINS, C. Radioimmunoassay of an t id iu re t i c hormone during anesthesia. Can. Anaesth. Soc. J . 23:290-295, 1976. PICHLER, L. and KOBINGER, W. Presynaptic a c t i v i t y at peripheral adrenergic s i tes and blood pressure ef fect of a-adrenoceptor st imulat ing drugs. Eur. J . Pharmacol. 52:287-295, 1978. PICHLER, L. and KOBINGER, W. Centra l ly mediated cardiovascular ef fects of B-HT 920 (6-a l ly l -2-amino-5,6,7,8- tet rahydro-4H-th iazolo- [4,5-d] -azepine d ihydrochlor ide) , a hypotensive agent of the "c lon id ine type". J . Cardiovasc. Pharmacol. 3:269-277, 1981. - 135 -PICKERING, B.T. and MCPHERSON, M.A. Progress in the study of biosynthesis and transport in the neurohypophysial system. In: Neurohypophysis. Int. Conf. Key Biscayne, F l o r i d a . Karger, Base l . (197/), pp. 30-42. PITTMAN, Q . J . , LAWRENCE, D. and MCLEAN, L. Central e f fects of arginine vasopressin on blood pressure in r a t s . Endocrinology 110:1058-1059, 1982. PULLAN, P .T . , JOHNSTON, C . I . , ANDERSON, W.P. and KORNER, P . I . Plasma vasopressin in blood pressure homeostasis and in experimental renal hypertension. Am. J . Phys io l . 239:H81-H87, 1980. PULSINELLI, W.A. and BRIERLEY, J . B . A new model of b i l a t e ra l hemispheric ischemia in the unanesthetized ra t . Stroke 10:267-272, 1979. — RAICHLE, M.E. and GRUBB, R.L. Regulation of brain water permeabil i ty by cent ra l ly - re leased vasopressin. Brain Res. 143:191-194, 1978. RAND, M . J . , STORY, D.F. , ALLEN, G . S . , GLOVER, A .B . and MCCULLOCH, M.W. Pulse- to-pulse modulation of noradrenaline release through a prejunct ional a-receptor auto- inh ib i tory mechanism. In: Front iers  in Catecholamine Research. (Ed. Usdin, E. and Snyder^ S.) Pergammon Press, New York. (1973), pp. 579-581. RANKIN, A . J . , KELPIN, B .G. , COURNEYA, C . A . , WILSON, N. and LEDSOME, J . R . Plasma vasopressin in response to haemorrhage in the anaesthetized rabb i t . Can. J . Phys io l . Pharmacol. 64:904-908, 1986. — RASCHER, W., MEFFLE, H. and GROSS, F. Hemodynamic ef fects of arginine vasopressin in conscious water-deprived ra t s . Am. J . Phys io l . 249:H29-H33, 1985. ROBERTSON, G.L. The regulat ion of vasopressin funct ion in health and disease. In: Recent Progress in Hormone Research. 23:333-388, 1977. — ROBERTSON, G . L . , MAHR, E .A . , ATHAR, S. and SINHA, T. Development and c l i n i c a l appl icat ion of a new method for the radioimmunoassay of arginine vasopressin in human plasma. J . C l i n . Invest. 52:2340-2352, 1973. ROBERTSON, G . L . , ATHAR, S. and SHELTON, R.L. Osmotic control of vasopressin funct ion. In: Disturbances in Body F lu id Osmolality (Ed. Andreo l i , T . E . , Grantham^ J . J . , Rector, F.C.) IW. Phys io l . S o c , Washington, D.C. (1977), pp. 125-248. ROCHA E SILVA J R . , M. and ROSENBERG, M. The release of vasopressin in response to haemorrhage and i t s ro le in the mechanism of blood pressure regu la t ion. J . Phys io l . (Lond.) 202:535-557, 1969. - 136 -RUSSELL, J . T . , BROWNSTEIN, M.J. and GAINER, H. Time course of appearance and release of [ " "Sjcysteine labe l led neurophysins and peptides in the neurohypophysis. Brain Res. 205:299-311, 1981. SAAMELI, K. Neurohypophyseal hormones and s im i la r peptides. In: Handbook of Experimental Pharmacology, Vo l . 23. (Ed. Berde, B.) Spr inger-Ver lag, Heidelberg. (1968), pp. 545-612. SACHS, H.L. , SHARE, L . , OSINCHAK, J . and CARPI, A. Capacity of the neurohypophysis to release vasopressin. Endocrinology. 81:755-770, 1971. SAFAR, M.E. , WEISS, Y.A. and LONDON, G.L. Short-term hemodynamic studies with prazosin. Prazosin: Evaluation of a new  antihypertensi ve agent. (Ed"! Cotton, D.W.K.) Exerpta Medica, Amsterdam. (1974), pp.64-70. SAPER, C . B . , LOEWY, A . D . , SWANSON, L.W. and COWAN, W.M. Direct hypothlamoautonomic connections. Brain Res. 117:305-313, 1976. SATTLER, R.W. and VAN ZWIETEN, P.A. Acute hypotensive action of 2-(2,6-dichlorophenylamino)-2-imidazol ine hydrochloride (St 155) af ter infusion into the ca t ' s vertebral ar tery. Eur. J . Pharmacol. 2_:9-13, 1967. SAWYER, W.H. Neurohypophysial hormones. Pharmacol. Rev. 13:225-277, 1961. — SCHMID, P . G . , ABBOUD, F .M. , WENDLING, M.G., RAMBERG, E . S . , MARK, A . L . , HEISTAD, D.D. and ECKSTEIN, J.W. Regional vascular ef fects of vasopressin: plasma leve ls and c i rcu la to ry response. Am. J . Phys io l . 227:998-1004, 1974. SCHMID, P . G . , SHARABI, F .M. , GUO, G .B . , ABBOUD, F.M. and THAMES, M.D. Vasopressin and oxytocin in the neural control of the c i r c u l a t i o n . Fed. Proc. 43:97-102, 1984. SCHMITT, H. and FENARD, S. Ef fets des substances sympathomimetiques sur les centres vasomoteurs. Arch. Int. Pharmacodyn. 190:229-240, 1971. . SCHMITT, H. , SCHMITT, H. , BOISSIER, J . R . and GUIDICELLI, J . F . Centra l ly mediated decrease in sympathetic tone induced by 2-(2,6-dichlorophenylamino)-2-imidazol ine (St 155, Catapresan). Eur. J . Pharmacol. 2_:147-148, 1967. SCHRIER, R.W. and BERL, T. Mechanisms of the an t id iu re t i c ef fect associated with interrupt ion of parasympathetic pathways. J . C l i n . Invest. 51:2613-2620, 1972. SCHRIER, R.W., BERL, T. and ANDERSON, R . J . Osmotic and nonosmotic control of vasopressin re lease. Am. J . Phys io l . 236:F321-F332, 1979. - 137 -SCHULTZ, H.D., FATER, D.C. , SUNDET, W.D., GEER, P.G. and GOETZ, K.L. Reflexes e l i c i t e d by acute stretch of a t r i a l vs. pulmonary receptors in conscious dogs. Am. J . Phys io l . 242:1065-1076, 1982. SCHULTZ, W . J . , BROWNFIELD, M.C. and KOZLOWSKI, G.P. The hypothalamo-choroidal t rac t I I . U l t ras t ruc tura l response of the choroid plexus to vasopressin. Ce l l T i s s . Res. 178:129-141, 1977. SCHUMANN, H-J . and LUES, I. Postjunctional a-adrenoceptors in the iso la ted saphenous vein of the rabb i t : character izat ion and inf luence of angiotensin. Naunyn-Schmiedeberg's Arch. Pharmacol. 323:328-334, 1983. SCHWARTZ, J . and REID, I.A. Ef fect of vasopressin blockade on blood pressure regulat ion during hemorrhage in conscious dogs. Endocrinology 109:1778-1780, 1981. SCHWARTZ, J . and REID, I.A. Role of vasopressin in blood pressure regulat ion in conscious water-deprived dogs. Am. J . Phys io l . 244:R74-R77, 1983. SHARE, L. Ef fects of carot id occlusion and l e f t a t r i a l d is tent ion on plasma vasopressin t i t e r . Am. J . Phys io l . 208:219-223, 1965. SHARE, L. Control of plasma ADH t i t e r in hemorrhage: ro le of a t r i a l and a r te r i a l baroreceptors. Am. J . Phys io l . 215:1385-1389, 1968. SHARE, L. Blood pressure, blood volume, and the release of vasopressin. In: Handbook of Physiology Endocrinology Sect. 7,  Vo l . IV, P t . 1. (Ed. Knob i l , E . , Sawyer, W.H.) Am. Phys io l . S o c , Washington, D.C. (1974), pp. 243-255,. SHARE, L. and CROFTON, J . T . The ro le of vasopressin in hypertension. Fed. Proc. 43:103-106, 1984. SHARE, L. and LEVY, M.N. Cardiovascular receptors and blood t i t e r of an t id iu re t i c hormone. Am. J . Phys io l . 203:425-428, 1962. SHARE, L. and LEVY, M. Ef fect of carot id chemoreceptor st imulat ion on plasma an t id iu re t i c hormone t i t e r . Am. 0. Phys io l . 210:157-161, 1966. SHARE, L . , CROFTON, J . T . , R0CKH0LD, R.W. and RAPP, J . P . Vasopressin secret ion and responses to captopr i l and a vasopressin antagonist in the Dahl r a t . Proc. Fourth Int . Symp. Rats Spontaneous Hypertens. Related Studies. (Ed. Rascher, W., Clough, D. and Ganten, D.) Schattauer Ver lag, New York. (1982), 574-576. SHIMAM0T0, K., AND0, T . , NAKHASHI, Y . , NAKAO, T . , TANAKA, S . , SAKUMA, M. and MIYAHARA, M. Plasma and urinary ADH levels in patient with essent ia l hypertension. Jpn. C i r c . J . 43:43-47, 1979. SHOJI, T . , TSURU, H. and SHIGEI, T. A regional di f ference in the d i s t r i bu t ion of postsynaptic alpha-adrenoceptor subtypes in canine veins. Naunyn-Schmiedeberg1s Arch. Pharmacol. 324:246-255, 1983. - 138 -SHU'AYB, W.A., MORAN, W.H. and ZIMMERMAN, B. Studies of the mechanism of an t id iu re t i c hormone secret ion and post-commissurotomy d i l u t i ona l syndrome. Ann. Surg. 162:690-699, 1965. SKOWSKY, W.R. and FISHER, D.A. Arginine vasopressin secret ion on thyroidectomized sheep. Endocrinology 100:1022-1026, 1977. SKOWSKY, W.R. and SWAN, L. Ef fects of androgens and estrogens on arginine vasopressin in the rat and the human. Abstract 370, 59th Meeting of Endocrine Society, 1977. SLADEK, C P . and KNIGGE, K.M. Chol inergic st imulat ion of vasopressin release from the rat hypothalamo-neurohypophysial system in organ cu l tu re . Endocrinology 101:411-420, 1977. SOFRONIEW, M.V., WEINDL, A . , SCHRELL, U. and WETZSTEIN, R. Immunohistochemistry of vasopressin, oxytoc in, and neurophysin in the hypothalamus and extrahypothalamic regions of the human and primate bra in . Acta histochemica, Suppl. Band XXIV:S79-95, 1981. SOMLYO, A . V . , WOO, C Y . and SOMLYO, A . P . Response of nerve-free vessels to vasoactive amines and polypeptides. Am. J . Phys io l . 208:748-753, 1966. STAMM, W. The inf luence of carbon dioxide on the react ion of iso la ted veins to vasoconstr ictor substances. Agents Actions 2:261-269, 1972. STARK, R . I . , DANIEL, S . S . , HUSAIN, H.K., TROPPER, P . J . and LAMB, L .S . Cerebrospinal f l u i d and plasma vasopressin in the fe ta l lamb: basal concentration and the ef fect of hypoxia. Endocrinology 116:65-72, 1985. STARKE, K. Influence of ex t race l lu la r noradrenaline on the st imulat ion evoked secret ion of noradrenaline from sympathetic nerves: evidence for an a-receptor-mediated feeback inh ib i t i on of noradrenaline re lease. Naunyn Schmiedebergs Arch. Pharmacol. 275:11-23, 1972. STARKE, K. a-Adrenoceptor subc lass i f i ca t i on . Rev. Phys io l . Biochem. Pharmacol. 88:199-228, 1981. STARKE, K., MONTEL, H. and SCHUMANN, H .J . Influence of cocaine and phenoxybenzamine on noradrenaline uptake and re lease. Naunyn Schmiedebergs Arch. Pharmacol. 270:210-214, 1971. STEEN, S . , SKARBY, T .V . , NORGREN, L. and ANDERSSEN, K.E. Pharmacological character izat ion of post junct ional alpha-adrenoceptors in iso lated human omental a r ter ies and veins. Acta Phys io l . Scand. 129:109-116, 1984. STEPPELER, A . , TANAKA, T. and STARKE, K.A. A comparison of pre- and postsynaptic a-adrenergic ef fects of phenylephrine and tramazoline on blood vessels of the rabbit in v ivo . Naunyn Schmiedeberg's Arch. Pharmacol. 304:223-230, 1978. - 139 -STEVENS, M.J. and MOULDS, R.F.W. Are the pre- and postsynaptic a-adrenoceptors in human vascular smooth muscle a typ t ica l? J . Cardiovasc. Pharmacol. 4:129-133, 1982. STOKES, G.S. and OATES, H.F. Prazos in: new alpha-adrenergic blocking agent in treatment of hypertension. Cardiovasc. Med. 3:41-57, 1978. STOLZ, F. Veber adrenalin und alkylaminoaceto-brenzcatechin. Ber . , deutsch. chem. Gese l lsch. 37:4149-4154, 1904. SUTTER, M.C. The pharmacology of iso la ted ve ins. Br. J . Pharmacol. 24:742-751, 1965. SWANSON, L.W. Immunohistochemical evidence for a neurophysin-containing autonomic pathway a r i s ing in the paraventr icular nucleus of the hypothalamus of the rat and monkey. Brain Res. 128:356-363, 1977. SWANSON, L.W. and SAWCHENKO, P .E. Paraventr icular nucleus: a s i t e for the integrat ion of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31:410-417, 1980. SZCZEPANSKA-SADOWSKA, E. The a c t i v i t y of the hypothalamo-hypophysial an t i d iu re t i c system in concious dogs. I. The inf luence of iso-osmotic blood volume changes. Pfluegers Arch. 335:139-146, 1972. SZCZEPANSKA-SADOWSKA, E. Hemodynamic ef fect of a moderate increase of the plasma vasopressin level in conscious dogs. Pfluegers Arch. 338:313-322, 1973. SZCZEPANSKA-SADOWSKA, E . , SOBOCINSKA, J . and SADOWSKI, B. Central dipsogenic ef fect of vasopressin. Am. J . Phys io l . 242:R372-R379, 1982. SZCZEPANSKA-SADOWSKA, E . , GRAY, D. and SIMON-OPPERMANN, C. Vasopressin in blood and th i rd ven t r i c le CSF during dehydration, t h i r s t , and hemorrhage. Am. J . Phys io l . 245:R549-R555, 1983. TABRIZCHI, R. and PANG, C.C.Y. Comparative ef fects of rauwolscine, prazosin and phentolamine on blood pressure and cardiac output in anesthetized ra t s . Can. J . Phys io l . Pharmacol. 1987; ( in press) . TAKAHASHI, H. and BUNAG, R.D. Augmentation of cen t ra l l y induced alpha-adrenergic vasodepression in spontaneously hypertensive ra t s . Hypertension 2^:198-206, 1980. TAKAMINE, J . Adrenal ine; the act ive p r inc ip le of the suprarenal glands and i t s mode of preparat ion. Am. J . Pharm. 73:523-531, 1901. ~~ TANAKA, M., DE KLOET, E .R. , DE WIED, D. and VERSTEEG, D.H.G. Arginine°-vasopressin af fects catecholamine metabolism in spec i f i c brain nuc le i . L i f e S c i . 20:1799-1808, 1977. - 140 -TAYLOR, R.D. and PAGE, I.H. Peripheral vasomotor e f fects of adrenaline and noradrenaline acting upon the iso la ted perfused central nervous system. C i rcu la t ion :563-575, 1951. THAMES, M.D. and SCHMID, P.G. Cardiopulmonary receptors with vagal afferents t o n i c a l l y i nh ib i t ADH release in the dog. Am. J . Phys io l . 237:H299-H304, 1979. THAMES, M.D. and SCHMID, P.G. Interaction between carot id and cardiopulmonary baroreflexes in control of plasma ADH. Am. J . Phys io l . 241:H431-H434, 1981. THAMES, M.D., PETERSON, M.G. and SCHMID, P.G. St imulat ion of cardiac receptors with veratrum a lka lo ids i nh ib i t s ADH secret ion. Am. J . Phys io l . 239:H784-H788, 1980. THORN, N.A. Introductory remarks about the ant idiuret ic-hormone-releasing neuron system. In: Osmotic and - Volume Regulat ion. (Ed. Barker Jorgens, C. and Skadhuage, E.) Munksgaard, Copenhagen. (1978), pp. 225-228. THRASHER, T . N . , BROWN, C . J . , KEIL, L .C. and RAMSAY, D.J . Thi rs t and vasopressin release in the dog: an osmoreceptor or sodium receptor mechanism? Am. J . Phys io l . 238:R333-R339, 1980a. THRASHER, T . N . , JONES, R .G . , KEIL, L . C . , BROWN, C . J . and RAMSAY, D .J . Drinking and vasopressin release during vent r icu lar infusions of hypertonic so lu t ions. Am. J . Phys io l . 238:R340-R345, 1980b. TIMMERMANS, P.B.M.W.M. Calcium antagonism and (^-adrenoceptor ac t i va t i on . Naunyn Schmiedeberg's Arch. Pharmacol. 316:57, 1981. TIMMERMANS, P.B.M.W.M. and VAN ZWIETEN, P.A. Postsynaptic c ^ - and ©^-adrenoceptors in the c i r cu la to ry system of the pithed ra t : se lec t ive st imulat ion of the o^-type by B-HT 933. Eur. J . Pharmacol. 63:199-202, 1980a. TIMMERMANS, P.B.M.W.M. and VAN ZWIETEN, P.A. Vasoconstr ict ion mediated by postsynaptic ©^-adrenoceptor s t imulat ion. Naunyn-Schmiedeberg's Arch. Pharmacol. 313:17-20, 1980b. TIMMERMANS, P.B.M.W.M., KWA, H.Y. and VAN ZWIETEN, P.A. Possib le subdiv is ion of postsynaptic a-adrenoceptors mediating pressor responses in the pithed ra t . Naunyn Schmiedeberg1 s Arch. Pharmacol. 310:189-193, 1979. TIMMERMANS, P.B.M.W.M., SCHOOP, A .M.C. , KWA, H.Y. and VAN ZWIETEN, P.A. Character izat ion of a-adrenoceptors par t i c ipa t ing in the central hypotensive and sedative ef fects of c lon id ine using yohimbine, rauwolscine and corynanthine. Eur. J . Pharmacol. 70:7-15, 1981. TSUCHIYA, M., WALSH, G.M. and FROHLICH, E.D. Systemic hemodynamic ef fects of microspheres in conscious ra t s . Am. J . Phys io l . 235:H617-H621, 1977. - 141 -U'PRICHARD, D.C. and SNYDER, S.H. D is t inc t a-noradrenergic receptors d i f fe ren t ia ted by binding and physio logical re la t ionsh ips . L i f e S c i . 24:79-88, 1979. U'PRICHARD, D . C , GREENBERG, D.A. and SYNDER, S.H. Binding charac te r i s t i cs of a .radio- labeled agonist and antagonist at central nervous system alpha noradrenergic receptors. Mol. Pharmacol. 13:454-473, 1977. U'PRICHARD, D . C , CHARNESS, M.E. , ROBERTSON, D. and SYNDER, S.H. Prazosin: d i f f e ren t i a l a f f i n i t i e s for two populations of a-noradrenergic receptor binding s i t e s . Eur. J . Pharmacol. 50:87-89, 1978. UKAI, M. An t id iu re t i c hormone responses to surgical and experimental v iscera l s t imula t ion. Med. J . Osaka Univ. 21:121-129, 1971. UKAI, M., MORAN, W.H. AND ZIMMERMAN, B. The ro le of v iscera l afferent pathways on vasopressin secret ion and urinary excretory patterns during surg ical s t ress . Ann. Surgery 168:16-28, 1968. UNDESSER, K . P . , HASSER, E .M. , HAYWOOD, J . R . , JOHNSON, A.K. and BISHOP, V .S . Interact ions of vasopressin with the area postrema in a r te r i a l baroreflex funct ion in conscious rabb i ts . C i r c . Res. 56:410-417, 1985. VALLEJO, M., CARTER, D.A. and LIGHTMAN, S .L . Haemodynamic ef fects of arginine-vasopressin microinject ions into the nucleus t ractus s o l i t a r i u s : a comparative study of vasopressin, a se lec t i ve vasopressin receptor agonist and antagonist, and oxytocin. Neurosci. Le t t . 52:247-252, 1984. VAN BRUMMELEN, P . , VERMEY, P . L . , TIMMERMANS, P.B.M.W.M. and VAN ZWIETEN, P.A. Prel iminary evidence for a postsynaptic alpha2 adrenoceptor in the vasculature of the human forearm. Br. J . C l i n . Pharmacol. 15:134-135, 1982. VAN MEEL, J . C . A . , TIMMERMANS, P.B.M.W.M. and VAN ZWIETEN, P.A. <*]-and <x2-adrenoceptor st imulat ion in the iso la ted perfused hindquarters of the ra t : an in v i t ro model. J . Cardiovasc. Pharmacol. 5:S80-S85, 1983. VANHOUTTE, P.M. Heterogeneity of post junct ional vascular a-adrenoceptors and handling of calcium. J . Cardiovasc. Pharmacol. 4:91-96, 1982. VEALE, W.L., KASTING, N.W. and COOPER, K .E . Arginine vasopressin and endogenous an t ipy res is : evidence and s ign i f i cance . Fed. Proc. 40:2750-2753, 1981. VERNEY, E.B. The an t id iu re t i c hormone and the factors which determine i t s re lease. Proc. R. Soc. Lond. (B i o l . ) 135:25-106, 1947. - 142 -VON EULER, U.S. A spec i f i c sympathomimetic ergone in adrenergic nerve f ib res (sympathin) and i t s re la t ion to adrenaline and noradrenaline. Acta. Phys io l . Scand. 12^:73-97, 1946. VOORN, P. and BUIJS, R.M. An immuno-electronmicroscopical study comparing vasopressin, oxytocin, substance P and enkephalin containing nerve terminals in the nucleus of the s o l i t a r y t rac t of the ra t . Brain Res. 270:169-173, 1983. VORHERR, H. , BRADBURY, M.W.B., HOGHOUGHI, M. and KLEEMAN, C.R. An t id iu re t i c hormone in cerebrospinal f l u i d during endogenous and exogenous changes in i t s blood l e v e l . Endocrinology 83:246-250, 1968. — WAEBER, B. , NUSSBERGER, J . and BRUNNER, H.R. Blood pressure dependency on vasopressin and angiotensin II in prazosin-treated conscious normotensive ra t s . J . Pharmacol. Exp. Ther. 225: 442-446, 1983. WARNKE, E. and HOEFKE, W. Influence of central pretreatment with 6-hydroxydopamine on the hypotensive ef fect of c lon id ine . Arzneim. Forsch. 27:2311-2313, 1977. WEGRIA, G . E . , ESSEX, H .E . , HERRICK, J . F . and MANN, F.C. The simultaneous action of cer ta in drugs on the blood pressure and on the flow in the r ight and l e f t coronary a r te r i es . Am. Heart J . 20:557-572, 1940. WEINGARTNER, H. , GOLD, P . , BALLENGER, J . C , SMALLBER, S . A . , SUMMERS, R., RUBINOW, D.R., POST, R.M. and GOODWIN, F.K. Ef fects of vasopressin on human memory funct ions. Science 211:601-693, 1981. WEINSTEIN, H. , BERNE, R.M. and SACHS, H. Vasopressin in blood: ef fect of hemorrhage. J . Endocr inol . 66^:712-718, 1960. WEITZMAN, R . E . , GLATZ, T.H. and FISHER, D.A. The ef fect of hemorrhage and hypertonic sa l ine upon plasma oxytocin and arginine vasopressin in conscious dogs. Endocrinology 103:2154-2160, 1978. WILFFERT, B. , TIMMERMANS, P.B.M.W.M. and VAN ZWIETEN, P.A. Extrasynaptic locat ion of alpha-2 and non-innervated beta-2 adrenoceptors in the vascular system of the pithed normotensive ra t . J . Pharmacol. Exp. Ther. 221:762-768, 1982. WILSON, N. and LEDSOME, J .R . Distension of the pulmonary ve in -a t r i a l junct ions and plasma vasopressin in the chloralose anaesthetized dog. Can. J . Phys io l . Pharmacol. 61:905-910, 1983. WU, W., ZBUZEK. V.K. and BELLEVUE, C. Vasopressin release during cardiac operat ion. J . Thorac. Cardiovasc. Surg. _79:83-90, 1980. YAMAGUCHI, I. and KOPIN, I . J . Plasma catecholamine and blood pressure responses to sympathetic st imulat ion in pithed ra t s . Am. J . Phys io l . 237:H305-H310, 1979. - 143 -YAMAMOTO, M., SHARE, L. and SHAD, R.E. Ef fect of ven t r i cu lo -c is te rna l perfusion with angiotensin II and indomethacin on the plasma vasopressin concentrat ion. Neuroendocrinology 25 :^ 166-173, 1978. YAMASHITA, H. , KANNAN, H. , INENAGA, K. and KOIZUMI, K. The ro le of cardiovascular and muscle afferent systems in control of body water balance. J . Auton. Nerv. Syst . 10:305-316, 1984. ZERBE, R.L. and FEUERSTEIN, G. Cardiovascular ef fects of cen t ra l l y administered vasopressin in conscious and anesthetized ra t s . Neuropeptides 6:471-484, 1985. ZERBE, R .L . , KIRTLAND, S . , FADEN, A . I . and FEUERSTEIN, G. Central cardiovascular ef fects of mammalian neurohypophyseal peptides in conscious ra ts . Peptides 4-:627-630, 1983. ZIMMERMAN, E.A. and DEFENDI, R. Hypothalamic pathways containing oxytocin, vasopressin and associated neurophysins. In: Neurohypophysis. Int. Conf. Key Biscayne, F l o r i d a . (Ed. Moses, M., Share, L.) Karger, Base l . (1977), pp. 9-21. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0097408/manifest

Comment

Related Items