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Effects of plasma factor(s) on vascular smooth muscle function and its role in the etiology of essential… Pillai, Gnanaranjitham 1989

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EFFECTS OF PLASMA FACTOR(S) ON VASCULAR SMOOTH MUSCLE FUNCTION AND ITS ROLE IN THE ETIOLOGY OF ESSENTIAL HYPERTENSION By GNANARANJITHAM PILLA1 M.B.B .S . , Universi ty of Ceylon, Colombo, Sr i Lanka, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics) We accept th is thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1989 ^Gnanaranjitham P i l l a i , 1989 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 P h a r m a c o l o g y and T h e r a p e u t i c s . The University of British Columbia Vancouver, Canada n a t p 21 A p r i l 1989 . DE-6 (2/88) - i i -ABSTRACT Some recent invest igat ions suggest that there ex is ts a c i r cu la t i ng factor in the plasma from pat ients with high blood pressure that sens i t i zes the vascular smooth muscle (Cappuccio et a l . 1986). The present invest igat ion was to determine i f any c i r cu la t i ng plasma factor could be detected in the hypertensive patients which a l te rs the vascular smooth muscle functions of iso la ted blood vessels from the ra t . Human plasma was obtained from normotensive and hypertensive subjects who were not on medication. Vascular s t r i ps were prepared from aortae and portal veins of normotensive wistar rats (Wt. 250 ± 50g) and placed in physio logical so lut ion in muscle baths. Cont rac t i le a c t i v i t i e s of aor t i c or portal vein s t r i ps in response to agonists (noradrenaline (NA) or potassium (K +) in the absence or presence of plasma from hypertensive pat ients, normotensive people and spontaneous con t rac t i l e a c t i v i t y of portal vein in the presence of normotensive plasma, hypertensive plasma or human plasma proteins (albumin, gamma g lobu l i n , alpha (B ) and beta (a) g lobu l i n , alpha (p) g lobul in and immunoglobulin IgG obtained commercially) were determined. As well as, possib le mechanisms involved in the a l te ra t ion of con t rac t i l e response of the vascular smooth muscles in response to the various plasma proteins were examined using Ca antagonists and receptor antagonists and ouabain in order to determine i f the changes were dependent on spec i f i c membrane recep-to rs , in f lux of Ca through Ca channels or the membrane receptors of the vascular smooth muscle c e l l s . The resu l ts show that the aor t ic s t r i ps exposed to hypertensive plasma developed greater force to NA but less force to K + compared to the aor t ic s t r ips exposed to normotensive plasma. When the portal veins were exposed to hypertensive plasma i t s spontaneous a c t i v i t y was completely lost at the end of 20 min, but the portal veins exposed to normotensive plasma retained a very small percentage of t h i s spontaneous a c t i v i t y . NA did not produce any response in the portal veins exposed to hypertensive or normotensive plasma but response to K + in the presence of normotensive plasma was greater than that of hypertensive plasma. Increasing concentration of normotensive or hypertensive plasma increased the sponta-neous a c t i v i t y of the portal vein up to 50 concentration of the plasma. Further addi t ion of e i ther of the splasma inh ib i ted the spontaneous a c t i v i t y . The responses obtained in the presence of hypertensive plasma was s i g n i f i -cant ly greater than the corresponding concentration of normotensive plasma. The plasma f rac t ions albumin and gamma globul in both increased the sponta-neous a c t i v i t y of the portal ve in . In contrast the e-globul in alone or e- and a -g lobu l in together inh ib i ted the spontaneous a c t i v i t y . Immunoglobu-l i n IgG which i s about 70 of the gamma g lobul in f rac t i on also increased the spontaneous a c t i v i t y of the portal ve in. Phentolamine an e-adrenoceptor antagonist, blocked the increased sponta-neous ac t i v i t y produced by albumin in the portal ve in. Depolarizing the portal vein with ouabain did not have any inf luence on the spontaneous a c t i v i t y produced by albumin. Albumin s t i l l increased the spontaneous a c t i v i t y of porta l veins denervated by 6-hydroxy dopamine. Depolar iz ing the portal vein with ouabain completely blocked the increase in the spontaneous a c t i v i t y produced by gamma g lobu l in . Blocking the 6-adrenoreceptors, chol inerg ic receptors, histamine receptors, serotonin receptors or angiotensin receptors did not block the increased spontaneous - iv -a c t i v i t y produced by gamma g lobu l in . Blocking the calcium channel by vera-pamil also blocked the increased spontaneous a c t i v i t y produced by gamma g lobu l in . The studies with aor t ic s t r i ps exposed to normotensive and hypertensive plasma to NA or K + support the idea that there ex is ts a vascular sens i -t i z i n g agent in the hypertensive plasma. The studies of spontaneous a c t i v i t y of portal vein in the presence of plasma also indicate that there is a vascular sens i t i z ing agent in the hypertensive plasma. The studies with plasma f rac t ions on the spontaneous a c t i v i t y of the portal veins suggest that the vascular sens i t i z ing agent may or ig inate in the IgG f rac t i on of the gamma g lobu l in . The increased vascular tone produced by gamma globul in seems dependent on the membrane potent ia l of the vascular t issue and the in f lux of Ca through the Ca channels. - V -TABLE OF CONTENTS CHAPTER TITLE Page 1 INTRODUCTION 1 1.1 General 1 1.2 Def in i t i on of Essent ia l Hypertension 2 1.3 Essent ia l Hypertension: Scope of the Problem 4 1.3.1 Prevalence and consequences. 4 1.3.2 Risks of elevated blood pressure. 5 1.3.3 Hypertension: Risks and benef i ts of treatments. 6 1.4 Review of H is to r i a l Background in Essent ia l Hypertention 8 1.4.1 Genetic fac to rs . 8 1.4.2 Elevated sympathetic nerve a c t i v i t y . 9 1.4.3 Cations. 10 1.4.4 A l te ra t ions in vascular smooth muscles. 11 1.4.5 C i rcu la t ing plasma or serum fac to rs . 18 1.5 Research Objectives 20 1.5.1 P o s s i b i l i t y of vascular sens i t i z ing agent in plasma of hypertensive pat ients . 21 1.5.2 Ef fects of human plasma proteins on vascular response. 21 1.5.3 Mechanism of action of plasma prote ins. 22 2 MATERIALS AND METHODS 23 2.1 General 23 2.2 Mater ia ls 23 2.3 Methods 24 2.3.1 Tissue preparations. 24 2.3.2 Recordings. 25 2.4 Experimental Protocols 25 2.4.1 Determine i f there ex is ts a vascular sens i t i z ing agent 25 in plasma. 2.4.2 Determine ef fect of plasma f rac t ions on spontaneous a c t i v i t y . 28 - v i -CHAPTER TITLE Page 2.4.3 Determine the mechanism of action of plasma prote ins: 30 2.4.4 S t a t i s t i c a l ana lys is . 37 3 RESULTS, OBSERVATIONS AND STATISTICAL ANALYSIS 38 3.1 Vascular Sens i t i z ing Agent in Hypertensive Plasma 38 3.1.1 S e n s i t i v i t y of aor t ic and portal vein s t r ips to agonists. 38 3.1.2 Cont ract i le ac t i v i t yo f porta l vein to agonist. 39 3.2 Spontaneous A c t i v i t y of Porta l Vein 48 3.2.1 Spontaneous a c t i v i t y obtained with heated and unheated normotensive plasma and serum. 48 3.2.2 Spontaneous a c t i v i t y produced with unheated normotensive and hypertensive plasma. 54 3.3 Plasma Fract ions(s). That Sens i t i ze the Vascular Tissue 58 3.3.1 Albumin. 58 3.3.2 Gamma g lobu l in . 58 3.3.3 Alpha (a) g lobu l in . 58 3.3.4 Alpha (a) and beta (e) g lobu l in . 68 3.3.5 Immunoglobulin IgG. 68 3.4 Mechanism of Act ion of Plasma Proteins 68 3.4.1 Albumin. 68 3.4.2 Gamma g lobu l in . 76 3.4.3 Endothelium. 92 4 DISCUSSION 96 4.1 General 96 4.2 Our Investigations 98 4.2.1 Response to K + and NA in aor t ic s t r i ps and portal veins. 98 4.2.2 Spontaneous a c t i v i t y of portal vein with plasma. 103 4.2.3 Spontaneous a c t i v i t y of portal vein with plasma f rac t ions . 105 5 CONCLUSION 113 6 REFERENCES 115 - v i i -LIST OF TABLES TABLE TITLE Page Table 1 NA Dose-response re la t ionsh ip in aor t ic s t r i ps exposed to hypertensive (n = 6) or normotensive plasma (n = 6 ) . 40 Table 2 NA Dose-response re la t ionsh ip in aor t i c s t r i ps (n = 6) at d i f ferent pH of the Krebs and with no bubbling with carbogen. 42 Table 3 K Dose-response re la t ionsh ip in aor t i c s t r i ps (n = 6) exposed to hypertensive (n = 6) or normotensive plasma (n = 6 ) . 44 Table 4 K Dose-response re la t ionsh ip in aor t ic s t r i ps (n = 6) at d i f ferent pH of the Krebs and with no bubbling with carbogen. 46 Table 5 Dose-response re la t ionsh ip in portal vein s t r i p (n = 6) exposed to hypertensive (n = 6) or normotensive plasma (n = 6) . 50 Table 6 Spontaneous a c t i v i t y of portal vein in heated and unheated normotensive plasma and unheated normotensive serum (n = 6) . 52 Table 7 Spontaneous a c t i v i t y of the portal vein in normotensive (n = 6) or hypertensive plasma (n = 3) . 55 Table 8 Spontaneous a c t i v i t y of portal vein with albumin (n = 6 ) . 59 Table 9 Spontaneous a c t i v i t y of portal vein with gamma globul in (n = 6 ) . 61 Table 10 Spontaneous a c t i v i t y of por ta l vein with a -g lobu l in (n = 6) . 66 Table 11 Spontaneous a c t i v i t y of portal vein with a and B-globul in (n = 6) . 69 Table 12 Changes in spontaneous a c t i v i t y induced by gamma globul in or IgG (n = 6 ) , % Max (integrated) response, amplitude and frequency of spontaneous a c t i v i t y are shown. 70 Table 13 Spontaneous a c t i v i t y of porta l vein in response to albumin in the presence and absence of phentolamine, ouabain and 6-0HDA respect ive ly (n = 6) . 74 Table 14 Spontaneous a c t i v i t y of the portal vein in response to gamma g lobul in in the presence and absence of ouabain 10~^ M (n = 6 ) . 79 - v i i i -TABLE TITLE Page Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Spontaneous a c t i v i t y of portal vein in response to gamma g lobul in in the presence and absence of phentolamine 10~ 7 M (n = 6 ) . 81 Spontaneous a c t i v i t y of the portal vein in the response to gamma globul in in the presence and absence of atropine IO" 5 M (n = 6) . 83 Spontaneous a c t i v i t y of portal vein in response to gamma g lobul in in the presence and absence of ketanserin I O - " M (n = 6) . 85 Spontaneous a c t i v i t y of the portal vein in response to gamma globul in in the presence and absence of ch lo r -pheniramine I O - 8 M (n = 6 ) . 87 Spontaneous a c t i v i t y of the portal vein in response to Y -g lobu l in in the presence of sa ra las in . 89 Spontaneous a c t i v i t y of portal vein in response to gamma globul in in the presence and absence of verapamil 10~ 8 M (n = 6 ) . 93 Spontaneous a c t i v i t y of portal vein with gamma globul in using intact/damaged endothelium (n = 6) . 94 - ix -LIST OF FIGURES FIGURE TITLE Page F i g . 1 NA dose-response curves in aor t i c s t r i ps (n = 6) exposed to normotensive and hypertensive plasma. 41 F i g . 2 NA dose-response curves in aor t i c s t r ips (n = 6) at d i f fe rent pH and in the presence and absence of bubbling with carbogen. 43 F i g . 3 K dose-response curves in aor t i c s t r ips (n = 6)exposed to normotensive and hypertensive plasma. 45 F i g . 4 K dose-response curves in aor t i c s t r ips (n = 6) at d i f fe rent pH and in the presence and absence of bubbling with carbogen. 47 F i g . 5 Tracing of NA (M) dose-response curves in por ta l vein exposed to normotensive plasma (B), hypertensive plasma (D) and Krebs (A and C) . 49 F i g . 6 K dose-response curves in portal veins (n = 6) exposed to normotensive and hypertensive plasma. 51 F i g . 7 % maximum of spontaneous a c t i v i t y of portal veins (n = 6) with increasing concentration of unheated and heated normotensive plasma and normotensive serum. 53 F i g . 8 % maximum of spontaneous a c t i v i t y of porta l vein (n = 3) with increasing concentration of normotensive plasma and hypertensive plasma. 56 F i g . 9 Tracing of spontaneous a c t i v i t y and integrated ac t i v i t y of porta l veins in the presence of increasing volume of Krebs solut ion (A), normotensive (B) and hypertensive (C) plasma. 57 F i g . 10 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) with albumin and cont ro ls . 60 F i g . 11 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) with gamma globul in and cont ro ls . 62 F i g . 12 Tracing of spontaneous a c t i v i t y and integrated ac t i v i t y of portal vein in the presence of Krebs solut ion (A) and gamma globul in (B). 63 F i g . 13 Amplitude (maximum spike tension in grams) of spontaneous a c t i v i t y of portal vein (n = 5) with gamma g lobu l i n . 64 - X -FIGURE TITLE Page F i g . 14 Frequency (no. of spikes/min) of Spontaneous a c t i v i t y of porta l vein (n = 5) with gamma g lobu l in . 65 F i g . 15 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) with a -g lobu l i n , a - and e-g lobu l in . 67 F i g . 16 % maximum of spontaneous ac t i v i t y of portal vein (n = 6) with gamma g lobu l in , IgG. 71 F i g . 17 Amplitude of Spontaneous a c t i v i t y of portal vein (n = 6) with gamma g lobu l in , IgG. 72 F i g . 18 Frequency of Spontaneous a c t i v i t y of portal vein (n = 6 ) , with gamma g lobu l in , IgG. 73 F i g . 19 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) in response to albumin in the presence of phentolamine 10" 7 M. 75 F i g . 20 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) in response to albumin af ter depolar izat ion with ouabain 10~ 4 M. 77 F i g . 21 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) in response to albumin af ter chemical denervation with 6-0HDA. 78 F i g . 22 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) in response to gamma g lobu l in in the presence of ouabain 10" 4 M. 80 F i g . 23 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) in response to gamma globul in in the presence of phento-lamine 10~7 M. 82 F i g . 24 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) in response gamma globul in in the presence of atropine 10~ 5 M. 85 F i g . 25 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) in response to gamma globul in in the presence of ketanserin 10~ 6 M. 86 F i g . 26 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) in response to gamma globul in in the presence of ch lo r -pheniramine 10~° M. 88 - x i -FIGURE TITLE Page F i g . 27 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) in response to gamma g lobu l in in the presence of sara las in IO" 9 M. 90 F i g . 28 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) in response to gamma globul in in the presence of verapamil 10~ 8 M. 91 F i g . 29 % maximum of spontaneous a c t i v i t y of porta l vein in response to gamma globul in in the presence of endothelium and the absence of endothelium. 95 - x i i -ACKNOWLEDGMENTS I wish to express my deepest grat i tude to Professor Morley C. Sutter for his in terest and valuable guidance and suggestions. I would also l i ke to express my sincere thanks to Drs. C.C.Y. Pang, B.R. Sastry and R. Vrba for t he i r many helpful suggestions and encouragements. I am grateful to Dr. J . M. Wright of the Hypertensive C l i n i c , Univers i ty Hospital at The Univers i ty of B r i t i s h Columbia and the volunteers for providing human plasma. Special thanks are to Ms. Su L in Lim for her valuable technical help, Ms. Margaret Wong for her sec re t r i a l assistance and to other colleagues for providing a pleasant and cord ia l atmosphere to work. Also my special thanks and love are to my two l i t t l e sons Ray (2 y r s . ) and Rex (6 y r s . ) for t he i r unassuming cooperation during the las t two years. F i n a l l y B.C. Heart Foundation's pa r t ia l f i nanc ia l assistance is grate-f u l l y acknowledged. - x i i i -Dedicated to my mother ( la te) Mrs. Ponnammah Chellappah father ( late) Mr. Ehamparam Chellappah - 1 -1 INTRODUCTION 1.1 General Essent ia l hypertension is a condit ion in which the cause for the elevated blood pressure is not known. A generalized increase in peripheral a r te r i o l a r resistance to blood flow is a charac te r i s t i c manifestation of essent ia l hypertension. It is perhaps not appropriate to c a l l i t a disease as i t usual ly does not have any symptoms; the symptoms appear only when there are secondary changes in various systems. The major consequential complications are stroke, coronary heart d isease, cardiac f a i l u r e and peripheral a r t e r i a l d isease. The problems of essent ia l hypertension facing both the society and the medical profession are overwhelming. At least 20 percent of the adult population (U.S.) suffer from some degree of high blood pressure condit ion (Kane! et a l . 1984). Abundant s t a t i s t i c a l evidence ex is ts demonstrating an inverse cor re la t ion between the level of a r t e r i a l pressure and the average expected length of human l i f e . Data from the Framingham of fspr ing study (Kanel 1986) indicate that the incidence of hypertension from the th i rd to f i f t h decade increases th ree- fo ld in men and e igh t - fo ld in women. Aside from age e f fec ts , hypertension i s more common in blacks. Essent ia l hyper-tension is pa r t i cu l a r l y common in persons with a family h is tory of hyper-tension and in persons with such a pred ispos i t ion , there i s substant ial evidence incr iminat ing high NaCl, low potassium and low calcium intakes as r i sk factors (Dustan 1983; Tobian 1983). Various drugs such as e-adrenergic b lockers, d i u r e t i c s , C a + + antagon-i s t s and vasodi lators have been used for the management of essent ia l hyper-tens ion. These treatments provide only a temporary r e l i e f , as they do not - 2 -el iminate the causative factors which produce high blood pressure. As soon as treatment is discont inued, the hypertension general ly returns, sometimes within days. In order to provide a permanent cure for essent ia l hypertension, the primary factor (s) that " i n i t i a t e ( s ) " the essent ia l hypertension should be determined. Although much progress has been made and several e t i o l og i ca l fac tors are proposed, i t i s not c lear whether these are the primary factors or secondary changes. This research focuses on the i den t i f i ca t i on of possible c i r cu la t i ng factors which may increase the con t rac t i l e a c t i v i t y of vascular smooth muscles leading to an increase in vascular tone and blood pressure. 1.2 Def in i t i on of Essent ia l Hypertension As much as 90% of a l l hypertension is of unknown cause, and are therefore c l a s s i f i e d as "essen t ia l " or "primary" hypertension; and the rest (10%) with i den t i f i ab le causes are c l a s s i f i e d as "secondary" hypertension. " E s s e n t i a l " , apparently derived from an erroneous t rans la t ion of the German word "essen t i a l e " , which means id iopath ic , may be mistakenly interpreted to in fer an essent ia l need for higher pressure to push blood through vessels narrowed by age. Essent ia l hypertension i s not a disease but i s a physio logical d i s tu r -bance in which the causative factor for the elevated blood pressure is not i d e n t i f i a b l e . The fundamental hemodynamic a l te ra t ion in hypertension, essent ia l or secondary, is an increased resistance to the outflow of blood in the systemic a r t e r i a l bed, resu l t ing in an increase in peripheral r e s i s -tance and ra ised d i a s t o l i c blood pressure. Invar iably, there is a concomit-ant e levat ion of the sys to l i c blood pressure, and usual ly to an extent that the pulse pressure is also increased. Elevat ion of s y s t o l i c pressure alone, - 3 -with normal d i a s t o l i c pressure, is not considered to be a re f l ec t i on of increased peripheral resistance and thus, i s not an indicator of essent ia l hypertension. Since the elevat ion of a r t e r i a l pressure is the only measur-able change in essent ia l hypertension, a r t e r i a l pressure has been used to c l a s s i f y essent ia l hypertension. According to World Health Organization (W.H.O. 1980) the c l i n i c a l c r i t e r i a for normotension i s blood pressure (BP) < 140/90, borderl ine hypertension, BP > 140/90 and de f in i te hyperten-s ion , BP > 160/95. Although the term "hypertensive disease" is used quite often as synonymous with essent ia l hypertension, i t should properly be res t r i c ted to designate the as yet unident i f ied physio logical disturbance (or disturbances) cha rac te r i s t i c of t h i s d isease, and which leads u l t imate ly to elevat ion of d i a s t o l i c and sys to l i c blood pressures, anatomical changes in the vascular t r ee , and funct ional impairment of the involved t i s sues . The extent to which the blood pressure i s elevated in hypertensive subjects i s determined by the interplay of many dynamic fac to rs , among which are the degree of anatomical narrowing of the vascular bed, and var ia t ions in the factors such as levels of c i r cu la t i ng or local vasoconstr ictor and d i l a to r substances. The to ta l vascular peripheral resistance can be increased by diminution in the c ross-sect iona l s ize of the resistance vascular vesse ls . Since s t ructura l vascular occlusive changes do not appear un t i l the hypertensive process has been present for some t ime, s t ruc tura l a l te ra t ions cannot be the causative factor of essent ia l hypertension. Furthermore, vascular changes may not be extensive enough in hypertensive disease to so le ly account for increased peripheral resistance (Laher and Tr iggle 1984; Pang and Scott 1985). - 4 -The extent of blood pressure elevat ion as well as i t s f luc tuat ion i s related to the factors that normally control the level of blood pressure, namely, cardiac output, peripheral res is tance, e l a s t i c i t y of the central a r te r i es , and volume and v i scos i t y of the blood. These factors can form a pseudostable but dynamic balance maintaining a kind of an equi l ibr ium with various metabolic, neuronal and c e l l membrane a c t i v i t i e s , and th i s e q u i l i -brium may be slowly sh i f ted with time by an e t i o l og i ca l agent or the primary disturbing factor in essent ia l hypertension. Despite years of intensive inves t iga t ion , the primary e t i o l og i ca l agent in hypertensive disease is s t i l l unknown and evaluation of i t s ef fect on the level of blood pressure can be only a matter of speculat ion. I t is true that cer ta in disease processes are often associated with increased blood pressure; b i l a t e r a l renal disease such as d i f fuse glomerulonephrit is and atrophic pye lonephr i t is ; congenital defects such as coarctat ion of aorta and po lycyst ic renal disease; disease of the endocrine glands such as tumors of the p i tu i t a ry and adrenals; and the spec i f i c hypertensive diseases of pregnancy. The mechanism of hyper-tension in these pat ients is jus t as obscure as in those patients in whom the hypertension i s not associated with recognizable s t ructura l d isease. 1.3 Essent ia l Hypertension: Scope of the Problem 1.3.1 Prevalence and consequences. High blood pressure i s one of the major r i sk factors for premature death and d i s a b i l i t y because of the large number of people af fected. A survey conducted in 1983, by the NHANES-II (National Center for Health S t a t i s t i c s National Health and Examination Surveys) e s t i - mated that over 57 m i l l i on Americans or about 25% of a l l U.S. population are at increased r isk of morbidity and premature morta l i ty associated with high blood pressure. Of th is more than 90% belongs to essent ia l hypertension. - 5 -Hypertension is the leading cause of st roke, myocardial i n fa r c t i on , peripheral vascular diseases e t c . Because of i t s high incidence in general , coronary heart disease is the most common sequela (Kane! 1986). In persons with de f i n i t i ve hypertension (> 160/95 mmHg) myocardial in fa rc t ion occurred at more than twice the rate of normotensive persons (< 140/90). The sever i ty of myocardial in fa rc t ion that went unrecognized increased with the sever i ty of hypertension. Hypertension is the most important contr ibutor to the occurrence of cerebrovascular diseases. I t i s often asserted that mild hypertension leads to atherothrombotic brain i n fa rc t i on , whereas severe hypertension resu l ts in int racerebral haemorrhage. However, the Framingham study data do not support t h i s contention. The proport ion of strokes due to int racerebral hemorrhage was not d i f fe rent at d i f fe rent sever i t ies of hyper-tens ion, whereas the proportion of strokes due to brain in fa rc t ion increased with sever i ty of hypertension (Kanel 1986). 1.3.2 Risks of elevated blood pressure. The r i s ks of elevated blood pressure have been determined mainly from large scale epidemiological surveys, such as the Framingham study (Kanel et a l . 1975), Pooling Project (Pooling Project Research Group 1978), Actuar ia l data (Spence et a l . 1980) and many others conducted in the U.S. These groups have studied the r i sk leve ls for various groups of race, sex, and age. The Framingham study (Kanel 1970; Kanel et a l . 1984) was conducted in the U.S. s ta r t ing in 1948, and observations have been made every 2 years upon 5200 men and women of age 30 to 62 at entry. The pooling project is one in which the data from the Framingham study have been combined with those from four other studies to examine the r i s ks for major coronary diseases. Overa l l , more than 7000 white men aged 40 to 59 with c l i n i c a l evidence of heart diseases were followed for 40 years by invest igators in f i ve locat ions in the U.S. Since the data from - 6 -these separate studies were considered comparable, they have been pooled (Pooling Project Research Group, 1978). Actuar ia l data comes from the 1979 Bui ld and Blood study conducted by Soc ie t ies of Actuar ies, U.S.A. (1980) conducted on a large population (4.5 mi l l ion) of large ly white and upper middle c l a s s , and the subjects were followed for up to 20 years. The resul ts from a l l these studies indicate that the higher the blood pressure, the higher the morbidity and mor ta l i ty , and there is apparently no c r i t i c a l hypertensive l e v e l . In summary, prevalence of hypertension and the r i sks vary among the race, sex and age groups. Blacks tend to have higher leve ls of blood pressure and suffer more overa l l morta l i ty at a l l levels than whites. Likewise, men in general have higher morbidity and morta l i ty rates than women. The r isk of hypertension tends to increase with advancing age. Data from the VA Cooper-at ive study (1972) estimates that 62.8% of those 60 years or older with DBP between 90 and 114 mm Hg, developed major cardiovascular complications during an average 5 years on placebo. 1.3.3 Hypertension: Risks and benefi ts of treatments. The Framingham study indicates that the lower the blood pressure, the less cardiovascular disease i s seen in the general population that is not on antihypertensive therapy. However, that fac t cannot be used as evidence in support of the benefi ts of therapy. In moderate to severe hypertension antihypertensive drug therapy reduced cardiovascular morbidity by up to 75% compared to placebo (Helgeland 1980). The benefi ts of t reat ing mild hypertension are con t rovers ia l . Besides drug therapy, nonpharmacological treatment may also be valuable. Several measures including stopping smoking, meditat ion, weight reduction (Andrew 1982) and dietary measures such as sodium - 7 -r e s t r i c t i o n (Beard 1982; MacGregor et a l . 1982) have received at tent ion with varying degrees of success. As with drugs, here, patients compliance i s an important fac to r . The drug treatments avai lab le in the current p rac t i ce , such as B-b"lockers, d i u re t i cs , C a + + channel blockers and vasodi lators only lower the blood pressure but they do not have any ef fect on the primary factor which causes the elevat ion of blood pressure. Often the high blood pressure returns as soon as the drug treatment i s discont inued. Hypertension is a well establ ished coronary r i sk factor and there has been expectation that lowering the elevated blood pressure by means of drugs may be an e f fec t i ve preventive measure against coronary heart disease. The Oslo Hypertensive study and other randomized contro l led hypertension drug t r i a l s with placebo or untreated control groups have f a i l ed to show a de f i n i t i ve preventive ef fect on the incidence of coronary heart disease (Helgeland 1980). Aust ra l ian t r i a l s (Management report 1982) also found the same incidence of myocardial in fa rc t ion incidence between treated pat ients and placebo-treated cont ro ls . The lack of ef fect on coronary heart disease is in contrast to the preventive e f fec t on stroke in a l l adequately con t ro l -led drug studies (Helgeland 1980; Leren et a l . 1975). However, as long as coronary heart disease remains uninfluenced, there is l i t t l e impact of the antihypertensive treatment on to ta l cardiovascular disease because coronary heart disease is the more prevalent disease compared to stroke. The i n a b i l i t y of drug treatment of hypertension to inf luence the impact of coronary heart disease is c l ea r l y a major health problem and a challenge to the medical profession (Leren et a l . 1986). Better understanding of the causat ive/predisposing factor of essent ia l hypertension and mechanism of essent ia l hypertension should lead to the - 8 -prevention of essent ia l hypertension and vascular d iseases. This would be a better way of treatment than t reat ing the manifestations of essent ia l hyper-tension. 1.4 Review of H is to r i ca l Background in Essent ia l Hypertension For the las t several decades, the et io logy of essent ia l hypertension has been under intense invest igat ions but no s ingle causative factor or e t i o -log ica l agent has been i d e n t i f i e d . The f a i l u r e has been a consequence of i n a b i l i t y to detect a "prehypertensive" fac to r . Even when studies were done among adolescents and "border l ine" hypertensive pat ients , the i n i t i a t i n g factors may have been obscured by the adaptational changes invoked by raised blood pressure by the time hypertension is detected. The background of some of the invest igat ions are b r i e f l y reviewed in the fo l lowing sect ions. The pressure required to move blood through the c i rcu la to ry system is provided by the pumping act ion of the heart which provides cardiac output and the tone of ar ter ies (peripheral res is tance) . Cardiac output is i n f l u -enced by various factors including heart ra te , cardiac c o n t r a c t i l i t y , vascular compliance, vascular resistance and f l u i d volume. Peripheral resistance is regulated by mult ip le fac to rs , such as, (a) local factors (myogenic and metabol ic), (b) nervous (c) humoral vasoconstr ictor and vaso-d i l a t o r agent and s t ructura l fac to rs . Hypertension in general has been ascribed to abnormalit ies in every one of these pos i t i ve fac to rs , and essen-t i a l hypertension in par t i cu la r to those which ra ise peripheral res is tance. 1.4.1 Genetic fac to rs . The Framingham study of fami l ies and other studies of twins have provided estimates of the genetic contr ibut ion to the v a r i a b i - l i t y of blood pressure (Havlik et a l . 1982). The Tecumseh study (Longini et a l . 1984) suggests that a shared gene explains about 25% of the var iance, a much larger contr ibut ion than the environmental factor (5%), but - 9 -less than the age and sex (30% and 40%), leaving another 30% to 40% unexplained. It i s not c lear whether s ingle gene or polygenic inheritance is involved. 1.4.2 Elevated sympathetic nerve a c t i v i t y . Elevated sympathetic nerve a c t i v i t y has been postulated to be a causative factor for the development of essent ia l hypertension. Moreover, sympathetic a c t i v i t y large ly mediates the cardiovascular e f fects of various psychogenic factors which have been incriminated in human hypertension. Plasma noradrenaline and adrenaline levels were elevated in some hyper-tens ives, pa r t i cu l a r l y those with high plasma renin leve ls which were, in turn, thought to re f l ec t the higher levels of sympathetic nerve ac t i v i t y (Engelman et a l . 1970). Some reports of high plasma noradrenaline (NA) levels in hypertensives were l i k e l y in error because of the f a i l u r e to recognize the upward d r i f t in these leve ls with age and the use of younger normotensives as controls for the hypertensives (Lake et a l . 1977). High plasma NA levels may not however re f l ec t higher sympathetic nervous system tone since plasma levels of a hormone re f l ec t the balance between i t s entry and removal from the c i r c u l a t i o n . The higher plasma leve ls in e lder l y subjects were found to re f l ec t normal rates of NA production but not higher rates of entry of NA into the c i r cu la t i on (Hoeldtke et a l . 1985). The removal or clearance of NA was reported to be slower in some patients with essent ia l hypertension (Esler et a l . 1981). Baroreceptor dysfunction has been suggested for the increase in blood pressure by various invest igators (Falkner et a l . 1981). These re f lexes , when activated by a r i se in blood pressure,normally reduce heart rate and blood pressure by vagal st imulat ion and sympathetic i nh i b i t i on . The re f lex is reset within hours when hypertension i s induced so that , despite higher - 10 -pressure, the afferent neuronal ac t i v i t y is s im i la r to that seen in normo-tensives (Falkner et a l . 1981). Populations of adolescents with a combina-t ion of borderl ine hypertension plus a strong family h is tory of essent ia l hypertension were found to show a pronounced blood pressure and heart rate response to mental stress (Falkner et a l . 1981). Not only were t he i r base l ine values higher but also the absolute increase during stress was greater than in cont ro ls . Add i t i ona l l y , there was a cha rac te r i s t i c pattern of response noted in the hypertensive adolescents. The normal damping ef fect in the heart rate and blood pressure responses observed in controls with continued stress was not present in hypertensives. These observations would suggest l im i ta t ion or withdrawal of normal feedback or adaptive mechanisms, perhaps as a resu l t of reduced s e n s i t i v i t y of baroreceptors. Falkner et a l . (1981) showed that adolescents with borderl ine hyperten-sion have a greater r i sk of progression to sustained hypertension than previously reported. Character is t ics of adolescents who do develop high blood pressure include a uniformly strong family h istory of essent ia l hypertension, higher body weight and rest ing heart rate and abnormal card io-vascular response to mental s t ress . These f indings are consistent with the concept of a dysregulatory neurogenic component in essent ia l hypertension. 1.4.3 Cat ions. There i s no compelling evidence that too l i t t l e or too much dietary sodium, potassium, calcium or magnesium is responsible for the genesis of essent ia l hypertension, or that changes in the intake of any of these cations w i l l cons is tent ly lower elevated blood pressure to normal l e v e l . This i s not to suggest that a l terat ions in cer ta in aspects. of metabolism or intake may not be important in a cer ta in hypertensive sub population (MacGregor et a l . 1982; Kawaski et a l . 1978; McCarron et a l . 1985). - 11 -Because of the long-standing be l ie f in a putative ro le for high sa l t intake in hypertension, the experimental, c l i n i c a l and epidemiologic studies on sodium and blood pressure, number in the thousands. Yet, only two f i rm conclusions emerge. The f i r s t i s that i n t r ace l l u l a r sodium concentration in white and red blood c e l l s (and presumably vascular smooth muscle c e l l s ) is s i g n i f i c a n t l y higher in hypertensive animals and humans than in normotensive controls (Beard et a l . 1982). The second is that there is a s izab le subgroup of pat ients with essent ia l hypertension whose blood pressure i s s a l t - s e n s i -t i ve (Kawasaki et a l . 1978). There is even less agreement on ef fects of a l te ra t ion in the intake of other cat ions, potassium, calcium and magnesium on blood pressure. Inter-pretat ion of data in th i s area is complicated by the lags in methodology in the determination of cy toso l i c ion ic concentrat ions. For example, sodium and calcium compete for reabsorption in the proximal tubules, so that in theory, increasing the f i l t e r e d load of e i ther one could cause increased excret ion of the other (Walser 1961). I t was speculated that increased calcium intake could lower blood pressure by causing a subtle d iu res is of sodium (Walser 1961). Dietary sources of calcium and magnesium are also r i ch in potassium. Potassium loading was shown to cause sodium d iu res is (Young et a l . 1976). The increase in i n t r ace l l u l a r sodium concentration in hypertension was suggested to be a factor in trapping f ree calcium within the c e l l , which then increases vascular tone (Young et a l . 1976). 1.4.4 A l tera t ions in vascular smooth muscles. The increased resistance in essent ia l hypertensive patients may be caused by a combination of s t ructura l and funct ional changes in the vasculature. In normal subjects, vascular resistance is maintained by the autonomic nervous system, hormones with e i ther pressor or inh ib i to ry a c t i v i t y , e l ec t ro l y te , blood - 12 -volume and local metabolic fac to rs . In hypertensive subjects, there i s a l te ra t ion in one or more of these control systems resu l t ing in increased cont rac t i le a c t i v i t y of vascular smooth muscles and elevated peripheral resistance (Kaplan 1986). Many invest igators have attempted to use hypertensive and normotensive animals to corre la te a l te ra t ions in vascular smooth muscle function with development of hypertension (Sutter et a l . 1977; F i t z p a t r i c et a l . 1980; winquistel et a l . 1983; Devynck et a l . 1981; Greenberg et a l . 1981; Laher et a l . 1984; Lipe et a l . 1985). 1.4.4.1 Structural changes. The c l a s s i c experiments by Folkow (1956) provided quant i tat ive data concerning the structure of the vasculature in hypertension by showing that under condit ions of hyperthermia, when vacu-lature appeared f u l l y re laxed, the forearm resistance was raised in essent ia l hypertension. Autopsy reports showed increased media/lumen ra t i o of micro c i r cu la t i on in essent ia l hypertensive patients (Short 1966). These f indings have been confirmed (Aalkjaer et a l . 1987) by d i rec t measurements of vascular structures in iso lated subcutaneous resistance vessels . This observed i n -crease in media/lumen ra t i o could be secondary to the elevat ion of a r t e r i a l pressure and may not be the primary cause of i t s e levat ion. One of the c learest indicat ions that the pressure can d i r e c t l y inf luence vascular growth is seen in the hypertrophic response of veins after they have been grafted in connection to coronary bypass surgery (Spray 1977). Ce l l u l a r mechanisms responsible for hypertrophy may simply be mechanical s t re tch ing. For example, in f i b rob las t s , mitosis has been found to be increased i f these are grown on " s n a i l s " which undergo repe t i t i ve stretching (Leung et al.1977; Cur t is et a l . 1978). Also embryonic chick tendons which are cultured in tac t , - 13 -increases both protein and DNA synthesis i f they are held under load (Slack et a l . 1984). This suggests that the stretch may be an i n i t i a t o r of vascular growth. Although i t is c lear that increased blood pressure can cause s t ructura l changes in vascular smooth muscles, the resu l ts from various studies were inconclusive with respect to whether inherent di f ferences in the structure of vascular smooth muscles of hypertensive subjects could i n i t i a t e the increase in blood pressure. I t has been shown that small mesenteric ar ter ies from Wistar Kyoto (WKY) rats when grafted into mesenteric ar ter ies of spon-taneously hypertensive rats (SHR) responded with a structural ly-mediated decrease in lumen or increase in media (Pang 1985), suggesting that the changes could have been caused by a plasma factor or secondary to the e leva-t ion of a r t e r i a l pressure in SHR. In another study, the blood pressure of the of fspr ings of human hypertensives were found to be s l i g h t l y higher than that of the cont ro ls . Subcutaneous small ar ter ies dissected out from these of fspr ings of hypertensives showed th icker media than those of the cont ro ls , but in a manner which corresponded to the s l i g h t l y higher pressure of these groups (Aalkjaer et a l . 1987). Lee (1986) provided evidence that s t ructura l a l te ra t ions of the blood vessels observed in hypertensive animals could be primary changes. He showed that primary changes are seen only in some vascular t issues namely, carot id and main renal ar tery, whereas those in aorta and superior mesenteric ar ter ies are secondary adaptive changes. As well in the muscular ar ter ies and also some a r t e r i o l es , vascular changes are primary in nature as these changes are present in the prehypertensive phase and blood pressure normalization with ant i-hypertensive treatment had no ef fect in preventing these changes from taking place. - 14 -Although much research work has been carr ied out with respect to s t ruc -tura l changes, i t i s not c lear whether s t ructura l changes in vascular smooth muscle may i n i t i a t e the elevat ion of blood pressure. 1.4.4.2 Functional changes. There i s considerable evidence in the l i t e ra tu re point ing to fundamental defects in the vascular smooth of the SHR. Despite close to 20 years of research, the s ign i f icance of these defects remain unknown, and the question of whether changes in the propert ies of vascular smooth muscles are involved in the i n i t i a t i o n of the increase in blood pressure remains unanswered. A number of studies u t i l i z i n g isolated perfused hind quarters of SHR have demonstrated an elevated s e n s i t i v i t y and an increased maximum response to adrenaline when compared with the response in normotensive control ra ts . However, studies with iso lated vascular t issues are not consistent . Thoracic aorta from the SHR were shown to have reduced con t rac t i l e response to NA in comparison to aorta from normotensive animals (Spector et a l . 1969; Shibata et a l . 1973; Antanaicco et a l .1980). Sutter et a l . (1977), Pang and Sutter (1980) showed that while portal veins had increased maximum response to NA compared to normotensive Wistar and Wistar Kyoto (WKY) control ra ts , aor t ic s t r i ps from SHR showed the opposite response to NA. In contrast , Pegram et a l . (1981) showed that there was no di f ference in s e n s i t i v i t y to NA between the portal veins of SHR and wistar Kyoto control r a t s . The anter ior mesenteric portal vein possesses spontaneous action potent ia ls and slow wave e l e c t r i c a l a c t i v i t y associated with contract ions (Rhodes et a l . 1971; Co l l i ns et a l . 1972). Anter ior mesenteric portal veins also depends on external C a + + for i t s spontaneous myogenic and agonist induced contract ions (Sutter et a l . 1977; Co l l i ns et a l . 1972). In these respects the peripheral resistance vessels seems to be more analogous to the mesenteric portal veins - 15 -than aortae (Rhodes et a l . 1971; Ljung 1970; Sutter et a l . 1977). Thus the portal vein may be a better model than the aorta to study the con t rac t i l e response of vascular smooth muscles independent of the ef fect of high blood pressure. Numerous studies indicate that the occurrence of spontaneous con t rac t i l e a c t i v i t y is increased in vascular preparations from hypertensive animals (Winquist et a l . 1983). These spontaneous contractions can be e i ther tonic or phasic, and they are independent of ex t r i ns i c s t i m u l i . The spontaneous a c t i v i t y presumably re f lec ts al tered membrane propert ies thereby increasing the e x c i t a b i l i t y of muscle c e l l . Greenberg et a l . (1981) observed that the frequency and amplitude of spontaneous phasic contract ion of the portal vein was greater in SHR, compared with the portal vein of WKY ra t s . Normotensive WKY rats parabiosed to SHR were found to develop hypertension. As well the frequency of phasic contractions in portal veins was increased in these parabiosed WKY rats compared with that in control ra ts . This suggests that a c i r cu la t i ng fac tor may be responsible for a l te ra t ions of c e l l membrane propert ies and phasic con t rac t i l e ac t i v i t y of vascular smooth muscle in SHR. Since there were var iable re la t ionsh ips of al tered membrane funct ion with blood pressure in d i f ferent populations, i t i s d i f f i c u l t to draw a unifying hypothesis to corre late c e l l membrane funct ion to hypertension. ++ Many invest igators suggest that a change in the handling of act ivator Ca by the smooth muscle during exc i ta t ion contract ion coupling may explain some of the al tered functions associated with hypertension (Folkow et a l . 1977; Pang and Sutter 1981; Webb and Bohr 1981). Zsoter et a l . (1977) found more rapid e f f lux of calcium from aortae of SHR. However, the use of aor t ic s t r ips as a model of vascular smooth muscle may be questioned since the i r responses are r e l a t i ve l y independent of - 16 -external calcium (Sutter 1977), whereas resistance vessels are markedly dependent on external calcium (Sutter et a l . 1977). Sutter (1985) used calcium antagonists as a probe to ident i fy the di f ference in calcium t rans -port or permeabil i ty s i tes in blood vessels from SHR. In his invest igat ions he found no di f ference in the ef fect of calcium antagonists on aor t ic or portal vein s t r i ps from SHR compared to WKY or Wistar cont ro ls . This was s im i la r to those of Harris et a l . (1984), who also found no di f ference in the ef fect of calcium antagonists n i fed ip ine , n i t rendipine and n iso ld ip ine on portal vein s t r i ps from SHR compared to cont ro ls . This does not exclude the p o s s i b i l i t y of increased calcium permeabil i ty in SHR because the calcium ion movement is not only dependent on spec i f i c channels blocked by calcium antagonist but also on passive permeabi l i ty . I t i s not known whether the passive permeabil i ty is al tered in blood vessels from hypertensive animals. I t has been shown that to ta l [Ca ] is higher than normal in the wal ls of a r te r ies from SHR and DOCA sa l t hypertensive rats (Jones et a l . 1975; Massingham et a l . 1973; Bahal la et a l . 1978). Studies with radioact ive Ca indicate an increase of exchangeable [Ca ] within the c e l l (Postnov I | et a l .1974), suggesting inadequate membrane control over Ca in the ++ cytoplasm. Two abnormalit ies of Ca handling have been demonstrated in various types of c e l l s in SHR. The f i r s t one is defect ive calcium binding to the wall of the c e l l membrane leading to an act iva t ion of potent ia l operated channels and to an increase of Ca in f lux into the c e l l (Devynck et a l . 1981; Devynck et a l .1981; Robinson 1984). The second abnormality ++ concerns the ATP - dependent Ca accumulation in membrane ves ic les (Robinson 1984), suggesting the existence of an impaired in teract ion of calmodulin with Ca -ATPase in the plasma membrane (Rhodes et a l . 1971). These a l tera t ions lead to a reduction of calcium extrusion across the plasma - 17 -membrane. In both cases the net resu l t is an increase in the steady state concentration of i n t r ace l l u l a r calcium. However, none of these f indings indicate what caused these abnormalit ies of C a + + handl ing. Most of the studies done on ionic permeabil i ty are ind i rec t and done on red blood c e l l s (RBC), leucocytes or p l a t e l e t s . Chan et a l . (1983) found that passive permeabil i ty to calcium at 4°C was increased in RBC from SHR but not from DOCA-salt hypertensive ra ts . These resul ts d i f f e r s l i g h t l y from those of Devynck et a l . (1981) who found only a trend towards increased permeabi l i ty . There has been increasing number of reports of a l tered c e l l u l a r ca t ion ic transport in blood c e l l s from hypertensive patients (Parker et a l . 1983; Swales et a l . 1983; Friedman et a l . 1986; Hi l ton et a l . 1986) suggesting that s im i la r changes may have occured in vascular smooth muscles. Increased RBC sodium content in essent ia l hypertensive pat ients has been reported by the majority of workers (Parker et a l . 1983; Friedman et a l . 1983). This suggests a disturbance in the re la t ionsh ip between sodium in f l ux , sodium pump a c t i v i t y and i n t r ace l l u l a r sodium. Many groups have observed enhanced sodium and potassium in f lux in RBC from hypertensive pat ients using a var iety of methods (Parker et a l . 1983; Hi l ton et a l . 1986; Birks et a l . 1982). In some cases, a r i se in c e l l sodium with a f a l l or no change in the rate constant for sodium ef f lux suggests that the pump is not responding adequately. This has not been cons is tent ly observed in leucocytes (Parker et a l . 1983; Blaustein (1977) hypothesized that an + ++ increased Na -Ca exchange across the c e l l membrane may be responsible for an elevat ion of i n t r ace l l u l a r f ree Ca in primary hypertension. This increase in i n t r ace l l u l a r calcium resul ts in an increased c o n t r a c t i l i t y of the a r te r i a l smooth muscle c e l l s , u l t imately causing vasoconstr ict ion and - 18 -hypertension. Zidek et a l . (1986) demonstrated a large f luc tua t ion in in t ra -ery th rocy t ic calcium a c t i v i t i e s in both normotensives and hyperten-s ives . However, the a l te ra t ion of i n t r ace l l u l a r calcium correlated more to the presence of hypertension than to a family h istory of hypertension. Mean erythrocyte calcium a c t i v i t i e s observed in hypertensive pat ients were s i g n i -f i c a n t l y higher than those in normotensives. I t has been reported that there was an increase in cy toso l i c calcium concentration (Bruschi et a l . 1985; Folsom et a l . 1986; Zidek et a l . 1986) in RBC, leucocytes, p la te le ts and vascular smooth muscles of hypertensive pat ients . Lindner et a l . (1987) measured the calcium content of p la te le ts from normotensive and hypertensive humans and found that free i n t r a c e l l u l a r calcium was increased in "hyper-tensive" p la te le t s , which also corre la tes with the d i a s t o l i c blood pressure. This f inding is in agreement with Bruschi et a l . (1985) who also reported that the cy toso l i c Ca concentration was increased in p la te le ts of pat ients with essent ia l hypertension and th i s increase also d i r ec t l y corre-lated with the increase in the blood pressure. What causes the increase in i n t r a c e l l u l a r calcium in essent ia l hypertension is not c lea r . An a l ternat ive explanation at t r ibutes disturbances of ion transport to an i n t r i n s i c abnormality in the physicochemical structure of the c e l l membrane associated with essent ia l hypertension (Heagerty et a l . 1982). Since the cation transport systems that are al tered in the hypertensive population are independent of each other and of c e l l membrane permeabil i ty (Post et a l . 1967) i t is necessary to postulate that e i ther mul t ip le abnormalit ies or global disturbances of several pathways contribute to the development of high blood pressure. 1.4.5 C i rcu la t ing plasma or serum fac to rs . Recently, i t has been suggested that c i r cu la t i ng factor (s) may be responsible for the development - 19 -of essent ia l hypertension. Serum or plasma from hypertensive animals were reported to sens i t i ze vascular t issue to pressor agents (Wright 1981; Michelakis et a l .1975; Cappuccio et a l . 1986). The administrat ion of low molecular weight protein obtained from hypertensive human urine induced hypertension in experimental animals (Sen et a l . 1977). Administrat ion of serum from hypertensive humans to experimental animals enhanced the pressor responses of the rec ip ient animal to vasoactive substances, such as NA, angiotensin II and tyramine (Greenberg et a l . 1975). A r t e r i a l s t r ips from normotensive subjects exposed to human hypertensive plasma showed increased s e n s i t i v i t y to NA (Cappuccio et a l . 1986). Parabiosis of WKY with SHR showed an increased c o n t r a c t i l i t y , r e a c t i v i t y , and protein content, a decrease in e x t e n s i b i l i t y and hypertrophy of portal vein obtained from the WKY member of WKY-SHR. These changes are s im i la r to those which occurred in the portal vein of SHR, in the absence of parabios is , and are also correlated with the r i se in a r te r i a l pressure in the WKY in the absence of an elevated porta l venous pressure (Greenberg et a l .1981). Hamyln et a l . (1982) suggested that a c i r cu la t i ng inh ib i to r of + + Na + K -ATPase i s associated with essent ia l hypertension. They hypo-+ + thesized that the increase in c i r cu la t i ng Na + K -ATPase inh ib i to r i s responsible for the increased peripheral res is tance. This was demonstrated by the k ine t i c Na + + K +-ATPase assay, which showed a highly s ign i f i can t cor re la t ion between the leve ls of the plasma inh ib i to r of Na + + K +-ATPase a c t i v i t y and mean a r te r i a l pressure. Lindner et a l . (1987) reported that when p la te le ts from normotensives were incubated with the plasma from hypertensives the cy toso l i c f ree calcium was increased. In contrast there was no increase in the cy toso l ic free ++ Ca of normotensive p la te le ts when incubated with normotensive plasma - 20 -from another subject. In addit ion when the hypertensive p la te le ts were incubated with normotensive plasma the cy toso l ic calcium was reduced to normal l e v e l . This indicates that the changes in cy toso l i c calcium content are revers ib le . Reducing blood pressure of hypertensive patients was found to reduce cy toso l i c calcium of p la te le ts but did not reduce the a b i l i t y of the plasma to increase the cy toso l i c calcium of normotensive p la te le t s . Lindner et a l . (1987) concluded that the plasma from pat ients with essent ia l hypertension contain a substance that ra ises the i n t r ace l l u l a r f ree C a + + concentration of normotensive p la te le t s . 1.5 Research Objectives Ava i lab le information suggests that there ex is ts a " fac tor " or " fac to rs" that could " i n i t i a t e " the complex chain of a c t i v i t i e s leading to the increase of peripheral vascular resistance and consequently essent ia l hypertension. It is conceivable that the factor or factors could be c i r cu la t i ng type in the plasma. Wright (1981) suggested that there was a vascular sens i t i z ing agent in the plasma of SHR. Aor t ic s t r i ps from normotensive rats when exposed to plasma from (SHR) showed an increase in responsiveness to NA and K + and th is was not related to low or high level of calcium in the plasma. Cappuccio et a l . (1986) showed that ar ter ies from normotensive humans when exposed to plasma from essent ia l hypertensives showed an increase in s e n s i t i v i t y to NA. These observations suggest that there is a vascular sens i t i z ing agent in the plasma of ind iv iduals with essent ia l hypertension. However the research of c i r cu la t i ng plasma factor (s) s t i l l leaves many unanswered questions. The main object ive of th i s research is to attempt to character ize the in v i t ro vascular ef fects of the c i r cu la t i ng vascular sens i t i z ing fac to r , to determine i f the plasma factor a l te rs vascular - 21 -response and to elucidate the possible mechanism by which the factor a l te rs vascular response. To accomplish the object ives, the invest igat ions were carr ied out in several steps. 1.5.1 P o s s i b i l i t y of vascular sens i t i z ing agent in plasma of  hypertensive pat ients . Plasmas from human essent ia l hypertensives and normotensives were used to incubate aor t ic and portal veins s t r i ps from normotensive (Wistar) ra ts . Both these t issues were tested for the i r response to NA or K + during exposure to plasma from normotensive or hypertensive pat ients . As w e l l , the spontaneous a c t i v i t y of the portal vein in the presence of d i f fe rent concentrations of normotensive and hypertensive plasma was also examined as an index of vascular tone. In th i s ser ies of experiments the spontaneous a c t i v i t y of portal vein was measured in the presence of unheated, heated normotensive plasma and unheated normotensive serum in order to examine whether the plasma s e n s i t i -zing fac tor is heat sens i t ive and/or whether i t i s present only in serum and or plasma f rac t ions . 1.5.2 Ef fects of human plasma proteins on vascular response. I t was found in sect ion 1.5.1 that both normotensive and hypertensive plasmas cause an increase in the spontaneous ac t i v i t y of rat portal ve in. In th is ser ies of experiments human plasma prote ins, namely, albumin, gamma g lobu l in , a-g lobul in and e-globul in were used to determine which human plasma protein f rac t ion from normotensive people af fects vascular response. Since agonist may bind to plasma proteins, i t i s d i f f i c u l t to determine the concentration of an agonist that produces a measured response. To avoid cor re la t ing agonist concentration to vascular response, the spontaneous ac t i v i t y of rat porta l vein was used as an index of vascular con t rac t i l e a c t i v i t y to various doses of plasma prote ins. - 22 -1.5.3 Mechanism of action of plasma prote ins . The myogenic spontaneous ac t i v i t y of the portal vein depends on external calcium. It is possible that the plasma protein(s) may increase spontaneous a c t i v i t y by increasing the in f lux of calcium v ia the act ivat ion of receptor mediated calcium channels, d i rec t action on the plasma membrane, ac t iva t ing the potent ia l dependent calcium channels or act ivat ion of an unknown receptor. To examine whether the increase in the spontaneous a c t i v i t y of portal vein induced by the plasma proteins from normotensive plasmas are mediated by the act iva t ion of membrane receptors, the spontaneous a c t i v i t y of the portal vein was determined in the absence and presence of spec i f i c receptor antagonists, namely, phentolamine (a-adrenoceptor antagonist) , atropine (muscarinic antagonist) , ketanserin (5HT2 antagonist) , chlorpheniramine (H-p histamine antagonist) and sara las in (angiotensin antagonist) . In addi t ion, ouabain was used to depolarise the vascular smooth muscles in order to see whether the increase in the spontaneous a c t i v i t y produced by the plasma f rac t ions was dependent on c e l l membrane po ten t ia l . In other ser ies of experiments, the vascular smooth muscles were chemical ly denervated with 6 - hydroxy dopamine (6-OHDA) to f ind out whether the increase in the spon-taneous a c t i v i t y produced by the plasma f rac t ions were due to the release of NA from nerve terminals in the vascular smooth muscle. A cumulative dose of tyramine was also added to the chemical ly denervated portal vein to determine i f there was releasable NA after denervation. A ser ies of experiments were done in the portal veins in which the endothelium was removed to study whether the an endothelium dependent factor(s) was involved in increased spontaneous ac t i v i t y produced by the plasma prote ins. F i n a l l y , the C a + + antagonist verapamil was used to examine i f the inh ib i t i on of C a + + in f lux af fects the increase in the spontaneous ac t i v i t y produced by the plasma prote ins. - 23 -2 MATERIALS AND METHODS 2.1 General In order to invest igate the plasma factor in essent ia l hypertension the fol lowing materials and methods were used. 2.2 Mater ia ls Mater ia ls used in the experimental invest igat ions were: (a) Tissues: Portal vein and aor t ic s t r i ps were obtained from normo-tensive male Wistar ra ts . (b) Plasma: Plasma was obtained from age and sex matched essent ia l hypertensive and normotensive humans from the Hypertensive C l i n i c , U.B.C. Health Sciences Hospital and volunteers from Univers i ty of B r i t i s h Columbia who were not under drug treatment. From each donor, a 20 ml of blood sample was drawn into a heparinised tube and centri fuged immediately at 10,000 RPM for 10 min (Beckman, Model 0-21 Centr i fuge). The plasma was used on the same day or frozen immediately and used on the next day. For preparation of serum, 20 ml of blood was drawn into nonheparinised tubes and allowed to c lo t in the re f r igera tor overnight, then centri fuged on the next day and the supernatant used on the same day. (c) Krebs So lu t ion: Composition (mM) of the Krebs Solut ion was: NaCl, 118; KC1, 4 .7 ; C a C l 2 , 2 .5; KH 2 P0 4 , 1.2; NaHC03, 2 .5; Glucose, 11; EDTA, 0.226; MgCl 2 .6H 2 0, 1.2. (d) Plasma proteins: The plasma proteins were purchased from Sigma Chemical Co. The fo l lowing plasma proteins were dissolved in Krebs solut ions a few minutes before addit ion to the t issue bath. (1) Human albumin (2) Human gamma-globulin - 24 -(3) Human alpha ( a ) and beta (e) g lobul in (4) Human a -g lobu l i n (5) Human IgG 2.3 Methods 2.3.1 Tissue preparat ions. Male Wistar rats weighing about 250g were k i l l e d by a blow on the head. Four d i f ferent vascular s t r i ps were prepared. 2.3.1.1 Surgical preparation - 1 (Portal Ve in) . The abdominal cav i ty was opened and the mesenteric portal vein was separated from the connective t i ssues . A thread was t ied at the lower end and a loop was made on the thread. Another long thread was attached to the end located c loser to the l i v e r . A l l fa t ty t issues around the vein were removed and a longi tudinal s l i t was made along the vein (Pang and Sutter 1981). The vein was then removed and placed in Krebs solut ion bubbled with carbogen (95% 0 2 and 5% C 0 2 ) . 2.3.1.2 Surg ica l preparation - 2 (Aort ic S t r i p ) . The thoracic cavi ty of the rat was opened and the thoracic aorta between the diaphragm and the or ig in at the heart was removed and placed in Krebs solut ion bubbling with carbogen. The fa t and connective t issue around the aorta was removed and a he l i ca l s t r i p was cut from the aorta (Furchgott and Bhadrakam 1953) by cut t ing at 15 degrees re l a t i ve to the c i r c u l a r axis of the ar tery . A long thread was t ied at one end and a loop with the thread was made at the other end. The length of the s t r i ps were about 5 to 7 mm. 2.3.1.3 Surgical preparation - 3 (Portal Vein) . The abdominal cav i ty was opened and the mesenteric portal vein was separated from the connective t i ssue . The vein was removed and placed in Krebs solut ion bubbled with carbogen. A needle threaded with s i l k thread was passed - 25 -i through the wall of the vein at each end and a loop was made at each end. A long thread for attachment to the force transducer was t ied to the loop at one end. The vein was handled ca re fu l l y so that the endothelium of the vein was kept undamaged. 2.3.1.4 Surgical preparation - 4 (Portal Ve in) . The abdominal cav i ty was opened and mesenteric portal vein was prepared as in surg ical preparation in Section 2 .3 .1 .1 . A longi tudinal s l i t was made along the portal ve in . Endothelium was separated off gently using a small forcep through the s l i t . The vein was removed and placed in Krebs solut ion bubbled with carbogen. 2.3.2 Recordings. The s t r i ps were mounted in organ baths for isometric recording with Grass, Model FT-03 force displacement transducers. Contrac- t ions were recorded isomet r ica l ly at 0.5g rest ing tension on a Grass poly- graph (Model 7P1). Force s ignals from portal vein and aor t ic s t r i ps were integrated e l ec t ron i ca l l y by an integrator (Grass, Model 7P10) over 1 min in terval on a separate channel. The spontaneous a c t i v i t y of the portal vein was measured as an integrated response, maximum force in grams or maximum rate as number of spikes/min. 2.4 Experimental Protocols Since bubbling the plasma with carbogen produced foaming of the plasma, a l l the s t r i ps incubated with plasma or proteins were not bubbled with carbogen. 2.4.1 Determine i f there ex is ts a vascular sens i t i z ing agent in plasma. 2.4.1.1 Responsiveness to noradrenaline (NA) and potassium (K + ^ of t issues exposed to normotensive or hypertensive plasma. A ser ies of s ix experiments were carr ied out using the above method (protocol) with plasma obtained from s ix normotensive and s ix hypertensive persons. Two rats were - 26 -used for the preparation of one set of portal vein and aor t ic s t r i p s . The porta l veins and aor t ic s t r ips were prepared as described in 2.3.1.1 and 2.3.1.2 respect ive ly . A portal vein and an aor t ic s t r i p from the same animal were attached to d i f ferent force displacement transducers (Grass, Model FT03) and mounted in the same t issue bath. The t issues were exposed to Krebs solut ion being bubbled with carbogen at 37° C. Af ter equ i l ib ra t ing for 1 hr, the t i ssue baths were f i l l e d with 5 ml of Krebs solut ion and cumulative doses of noradrenaline (NA) were added un t i l plateau responses were obtained. The t issues were washed twice and allowed to recover completely. The pH of the normotensive and hypertensive plasma was measured with a pH meter (Fisher Accumet Se lec t i ve Ion Analyzer, Model 750) and was found to be in the range of 7.8-8.0. One bath was then f i l l e d with 5 ml of normotensive plasma and another t issue bath was f i l l e d with 5 ml of hypertensive plasma. Bubbling with carbogen was stopped and 2.5 mM C a + + was added to baths. Af ter 20 minutes, a cumulative dose-response to NA was again obtained. On completion, a l l t issues were washed and allowed to recover completely in Krebs solut ion bubbled with carbogen. The pH of the Krebs solut ion was altered to that of plasma (pH 7.8-8.0) , and 5 ml of t h i s solut ion was added to both baths. Bubbling with carbogen was again stopped. Af ter 20 minutes, a cumulative dose-response re la t ionsh ip to NA was constructed. The t issues were washed again and allowed to recover in Krebs solut ion bubbled with carbogen and the same procedures as above were repeated with potassium ( K + ) . 2.4.1.2 Ef fect of unheated and heated normotensive plasma and  serum on spontaneous a c t i v i t y . Heated plasma was used to f ind out whether the factor(s) which af fect the spontaneous a c t i v i t y is (are) heat sens i t ive or not. The heated plasma was prepared by wanning normotensive plasma to 57° C for 45 min. The plasma was then allowed to cool to room temperature before use. - 27 -A ser ies of s ix experiments were carr ied out. In each experiment a portal vein was prepared as described in Section 2.3.1.1 and mounted into a t i ssue bath containing Krebs solut ion bubbled with carbogen at 37° C. Since the minimum volume of Krebs needed in the t issue bath to measure the spontaneous a c t i v i t y was 2.5 ml, bath was f i l l e d with 3 ml of f resh Krebs solut ion after equl ibrat ion of the s t r ips for 1 hr. The bubbling with carbogen was stopped. A f te r recording the spontaneous a c t i v i t y for 3 min, 1 ml of the Krebs solut ion was removed from the bath and 1 ml of f resh Krebs solut ion was added to the bath and the spontaneous a c t i v i t y was again recorded for 3 min. Then 0.5 ml of f resh Krebs solut ion was repeatedly added into bath un t i l a to ta l volume of 5.5 ml of was present in the baths. The spontaneous a c t i v i t y was recorded for 3 min. af ter each addit ion of fresh Krebs so lu t ion . At the end, the t issues were washed and the bubbling with carbogen was resumed and the t issues were allowed to recover to i t s o r ig ina l spontaneous a c t i v i t y . Then a se r i a l addit ion of unheated plasma was done to get a dose response as described before. The s t r ips were then allowed to recover in Krebs solut ion bubbled with carbogen to allow comparison of the ef fects of se r i a l addit ion of heated plasma. Af ter recovery in Krebs so lu t i on , the e f fec ts of se r i a l addit ion unheated serum was determined. The procedure with Krebs was used as time control for a l l the experiments done with plasma and plasma proteins to see whether increasing volume of solut ion in the t issue bath or withdrawal of bubbling has any ef fect on the spontaneous a c t i v i t y of the portal ve in . 2.4.1.3 Ef fect of • hypertensive and normotensive plasma on  spontaneous a c t i v i t y : A portal vein was prepared in each of the three experiments as described before (Section 2 .3 .1 .1 ) . In order to compare the ef fects of hypertensive and normotensive plasmas on the spontaneous ac t i v i t y - 28 -of the portal ve ins, the same procedure as in control (Section 2.4.1.2) was repeated but af ter removing 1 ml of krebs from the bath, 1 ml of e i ther normotensive or hypertensive plasma and subsequently 0.5 ml of e i ther hypertensive plasma or normotensive plasma was added to the bath. The pH of the normotensive and hypertensive plasma was found to be in the range of 7.8 - 8. I t was suggested that perhaps the a l te ra t ion seen in the spontaneous a c t i v i t y of the portal vein produced by these plasmas may have been due to the a lka l ine pH of these plasmas and/or no bubbling during the experiments. To provide controls the pH of the Krebs was matched to that of plasma (7 .8-8 .0) , and the same procedure as in control (Section 2.4.1.2) was repeated with the Krebs solut ion pH matched to that of plasma. 2.4.2 Determine ef fect of plasma f ract ions on spontaneous a c t i v i t y . The responses to various f rac t ions from normotensive human plasma were studied using the fol lowing protocols. The plasma f rac t ions studied included albumin, gamma g lobu l in , a- and e-g lobu l in , a -g lobu l in and the immunoglobulin f rac t i on IgG. Two controls were done for each of the plasma proteins: the f i r s t with Krebs at the normal pH of 7.4 and the other with the Krebs pH matched to that of plasma prote in . This was done in order to check whether the di f ference in spontaneous a c t i v i t y was due to the plasma protein and not due to pH change. 2.4.2.1 Albumin. Six experiments were car r ied out. In each, one portal vein was prepared as before (Section 2.3.1.1) and the volume control for albumin was done as in volume control for plasma (Section 2 .4 .1 .2 ) , but a f ter removing 1 ml of Krebs from the bath, 0.5 ml of f resh Krebs was added to the bath and the spontaneous ac t i v i t y was recorded for 3 min each time un t i l a to ta l volume of 5.5 ml was present in the bath. The pH of the Krebs - 29 -was matched to that of the Krebs solut ion containing albumin and the same procedure as above was repeated with th i s pH matched Krebs so lu t ion . F i n a l l y albumin solut ion was prepared as (Section 2.2.d) and the same procedure as above was repeated with Krebs containing albumin (30 mg/ml). 2.4.2.2 Gamma g lobu l in . Portal veins (n = 6) were prepared as before (Section 2.3.1.1) and se r i a l addit ion of Krebs solut ion was carr ied out as described in (Section 2 .4 .2 .1 ) . The gamma globul in solut ion was prepared (Section 2.2.d) and the pH was measured. The procedure as in Section 2.4.2.1 was repeated but instead of Krebs solut ion 0.5 ml of gamma globul in (7 mg/ml) in Krebs was added. F i n a l l y , se r i a l addit ion of 0.5 ml of Krebs solut ion with pH matched to that of gamma globul in solut ion was added to the bath. 2 .4.2.3 a and e -g lobul in: To study how a - and e-globulins a l te r the spontaneous a c t i v i t y of the portal vein portal veins (n = 6) were prepared as before (Section 2 .3 .1 .1 ) . Ser ia l addit ion of Krebs solut ion was carr ied out as described in Sect ion 2 .4 .2 .1 . The a - and e-globul in solut ion was prepared (2.2.d) and the pH was measured. The same procedure as in sect ion 2.4.2.1 was repeated with the addit ion of 0.5 ml of a - and e-globul in (10.4 mg/ml) in Krebs instead of 0.5 ml of Krebs. The pH of the a - and e-globul in was found to be in the same range as the gamma globul in (pH 7.6-7.8) and the ef fects of pH in the Krebs solut ion containing a - and e-globul ins was not studied. 2.4.2.4 g -g lobu l i n: To study how a -g lobu l i n a l te rs the spontaneous a c t i v i t y of the portal ve in , portal veins (n = 6) were prepared as before (Section 2 .3 .1 .1 ) . Se r ia l addit ion of Krebs solut ion was carr ied out as described in Section 2 .4 .2 .1 . The a -g lobu l i n solut ion was prepared (Section 2.2.d) and the pH was measured. The same procedure as above was - 30 -repeated with 0.5 ml of a -g lobul in (5.2 mg/ml) in Krebs. The pH of the a -g lobu l in was found to be in the same range as the gamma globul in (pH 7.6-7.8) and therefore the e f fec ts of pH was not fur ther examined. 2.4.2.5 Immunoglobulin f rac t ion IgG and gamma g lobu l i n . The immuno- g lobul in f rac t ion IgG is the major component of the immunoglobulins in the intravascular system. To compare the ef fects of IgG and gamma globul in on the spontaneous a c t i v i t y of the portal vein IgG solut ion as well as gamma globul in solut ion was prepared by d isso lv ing in Krebs solut ion (Section 2 .2 .d ) . Portal veins were prepared as before (Section 2.3.1.1) and the experiment was set up as described in Section 2.4.1.2 except that the t issue baths were s t i r red by a magnetic s t i r r e r . The t issue baths were f i l l e d with 5 ml of Krebs and the bubbling with carbogen was stopped and s t i r r ed using a magnetic s t i r r e r . The spontaneous a c t i v i t y was recorded for 3 min and at the end of 3 min, a cumulative dose-response curve to gamma globul in was obtained. This was used to compare with cumulative dose-response curve to IgG. The maximum concentration of gamma globul in used was 5 mg/ml. The t issues were washed with f resh Krebs solut ion and the s t i r r i n g was stopped; the bubbling with carbogen was resumed. When the t issues recovered to t he i r o r ig ina l spontaneous a c t i v i t y , a cumulative dose-response re la t ionships as above was repeated with IgG. The maximum concentration used with IgG was 5 mg/ml. 2.4.3 Determine the mechanism of action of plasma prote ins: 2.4.3.1 Albumin: The fo l lowing ser ies of experiments were performed to study the mechanism by which albumin a l te rs the spontaneous a c t i v i t y of the portal ve in . 2.4.3.1.1 g-adrenoreceptors: This ser ies of experiments were performed to study whether a-adrenoreceptors have any ro le in the increase in the spontaneous ac t i v i t y produced by albumin. Prel iminary invest igat ion indicates that phentolamine an a-antagonist at 10" M completely blocked the response to noradrenaline in the rat portal ve in. Phentolamine was therefore used to examine whether the blocking of the a-adrenoreceptors has any inf luence on albumin induced increase in the spontaneous a c t i v i t y of the por ta l . Porta l veins (n = 6) were prepared as before (Section 2.3.1.1) and the procedure in control Section 2.4.2.5 was repeated but a cumulative dose-response to albumin was obtained instead of IgG. The maximum concentration of albumin used was 20 mg/ml. The to ta l volume of f l u i d added was 200 p i . This i s used as the control for the dose response to albumin in the presence of phentolamine. When the t issues recovered to t he i r o r ig ina l spontaneous ac t i v i t y in Krebs so lu t ion , the t issues were washed with Krebs solut ion containing phentolamine and the baths were f i l l e d with 5 ml of t h i s so lu t ion . Af ter 5 min, a cumulative dose-response to albumin was repeated. 2.4.3.1.2 Membrane po ten t ia l : The fol lowing ser ies of experiments were performed to study whether the increase in the spontaneous a c t i v i t y produced by albumin in the portal vein is dependent on the membrane poten- t i a l . Ouabain is a Na + - K + ATPase inh ib i to r which can depolarize the plasma membrane by blocking the Na + - K + pump. Prel iminary invest iga- t ions indicated that ouabain (10~ 4 M) produced an i n i t i a l contract ion, but with time the t issues relaxed and returned to the base l ine in 45 min to 1 hr. At t h i s time the spontaneous a c t i v i t y was completely abol ished. I t was shown by other invest igators (Mathews and Sutter 1967) that ouabain 10-5 M produced s imi la r changes in the rabbi t porta l vein and i n t r ace l l u l a r recording showed that ouabain at th is concentration progressively reduce the membrane potent ia l and produced depo lar isa t ion . - 32 -Portal veins (n = 6) were prepared (Section 2.3.1.1) and the procedure described in Section 2.4.2.5 was repeated. A cumulative dose-response re la t ionsh ip to albumin was obtained. Afterwards the t issues were washed with Krebs and allowed to recover completely. The t issues were then washed _4 with Krebs solut ion containing 10 M ouabain. The t issues were exposed to t h i s so lut ion for 1 hr, washing with the same solut ion in between. When the t issues relaxed completely and returned to the base l i n e , a second cumulative dose-response re la t ionsh ip to albumin was obtained. 2.4.3.1.3 Albumin release of NA from nerve terminals: The fo l lowing ser ies of experiments were carr ied out to study whether the increase in the spontaneous a c t i v i t y produced by albumin was due to NA released by the albumin from the nerve terminals. I t has been shown that portal veins can be chemical ly denervated by 6-hydroxy dopamine (Apr igl iano et a l . 1976). In t h i s ser ies of experiments the portal veins were chemically denervated with 6 - OH dopamine. Portal veins (n = 6) were prepared (Section 2.3.1.1) and the procedure as in Section 2.4.2.5 was done. A dose-response curve to albumin was constructed. The maximum concentration of albumin used was 18 mg/ml. The t issues were washed, the s t i r r i n g was stopped, and bubbling with carbogen was res tar ted . When the t issues recovered to t he i r o r ig ina l spontaneous a c t i v i t y , a cumulative dose-response curve with tyramine was obtained. In order to f ind out the concentration of tyramine which caused maximum release of NA from the nerve terminals of the portal ve in. The t issues were washed in Krebs solut ion and allowed to recover. The pH of the unbuffered Krebs so lut ion (NaHC02 and NaH^PO^ omitted) was reduced to 4.9 by addit ion of approximately 20 pM of glutathione in order to reduce the oxidation of 6-hydroxy dopamine. A concentration of 300 pg/ml of 6-hydroxy dopamine was - 33 -dissolved in the pH-reduced so lu t ion . The baths were f i l l e d with 5 ml of th is solut ion and the t issues were exposed to th i s so lut ion for two 10 min periods with an interval of 1/2 hr between exposure (Apr igl iano et a l . 1976). The t issues were washed with normal Krebs solut ion in between and were allowed to recover to the base l ine for 2 hr in Krebs so lu t ion . A dose-response to tyramine was again constructed. Af ter the t issues had recovered from tyramine, another cumulative dose-response curve to albumin was constructed. 2.4.3.2 Gamma g lobu l in : The fol lowing ser ies of experiments were performed to study the mechanism by which gamma globul in a l te rs the spontan-eous a c t i v i t y of the portal ve in . 2.4.3.2.1 Membrane po ten t ia l : The ser ies of experiment was to f ind out whether the increase in the spontaneous a c t i v i t y of the portal vein obtained with gamma globul in was dependent on the membrane po ten t ia l . Portal veins (n = 6) were prepared (Section 2.3.1.1) and the procedure as in Section 2.4.2.1 was repeated but a cumulative dose-response curve to gamma globul in was constructed by addit ion of 0.5 ml samples of gamma globul in (7 mg/ml) in Krebs. The t issues were treated with ouabain Krebs ( 1 0 - 4 M) as in Section 2 .4 .3 .1 .2 . When the t issues were completely relaxed and returned to the base l i n e , a 0.5 ml sample of ouabain ( I O - 4 M) Krebs was s e r i a l l y added to the baths un t i l i t reached a volume of 5.5 ml. Af ter washing the t issues with ouabain Krebs solut ion a second dose-response curve to gamma globul in (7 mg of gamma globul in dissolved in 1 ml of ouabain ( 1 0 - 4 M) Krebs) was constructed. 2 .4 .3 .2 .2 a-Adrenoceptors: The fo l lowing ser ies of experiments were performed to study whether a-adrenoreceptors have a ro le in the increase in the spontaneous ac t i v i t y caused by gamma g lobu l in . Portal - 34 -veins (n = 6) were prepared (Section 2.3.1) and the same procedure as in Section 2.4.2.1 was repeated but dose-response curve to gamma globul in (as in sect ion 2.4.3.2.1) was carr ied out. The t issues were then exposed to 3 ml of Krebs containing phentolamine (10~7 M) for 5 min. A 0.5 ml sample of phentolamine Krebs solut ion was s e r i a l l y added to the baths un t i l the bath solut ion reached 5.5 ml volume. Another dose-response curve to gamma globul in (7 mg of gamma globul in dissolved in 1 ml phentolamine Krebs solut ion) was constructed. 2 .4.3.2.3 Chol inergic receptors: Prel iminary invest igat ions indicated that the maximum response produced by acety lchol ine (10~ 4 M) in the portal vein was completely blocked by atropine (10~5 M). In t h i s _5 ser ies of experiments, atropine (10 M) was used to block the chol inerg ic receptors to see i f t h i s also blocked the increase in the spontaneous a c t i v i t y produced by gamma g lobu l i n . Porta l veins (n = 6) were prepared (Section 2 .3 .1 .1 ) . A dose-response curve to gamma g lobul in as in sect ion 2.4.3.2.1 was constructed. The t issues _5 were washed with atropine (10 M) Krebs solut ion and f i l l e d with 3 ml atropine Krebs. A 0.5 ml sample of atropine Krebs solut ion was added un t i l the bath volume reached 5.5 ml. Another dose-response curve to gamma globul in (7 mg of gamma globul in d issolved in 1 ml of atropine Krebs) was constructed. 2.4.3.2.4 Histamine receptors: Prel iminary invest igat ion indicated the maximum cont rac t i l e response obtained by histamine (10 M) _ Q in the portal vein was blocked by chlorpheniramine (10 M). In t h i s _o ser ies of experiments chlorpheniramine (10 M) was used to block the histamine H^  receptors of the portal vein to see i f th is also blocked the increase in the spontaneous ac t i v i t y produced by gamma g lobu l in . - 35 -Portal veins (n = 6) were prepared (Section 2 .3 .1 .1 ) . Dose-response curve to gamma globul in (as in sect ion 2.4.3.2.1) was constructed. The baths were f i l l e d with 3 ml of chlorpheniramine Krebs and 0.5 ml sample of —8 chlorpheniramine (10 M) Krebs was s e r i a l l y added un t i l the bath volume reached 5.5 ml. Another dose-response curve to gamma globul in (7 mg of gamma globul in dissolved in 1 ml of chlorpheniramine Krebs) was constructed. 2 .4.3.2.5 Serotonin receptors: Prel iminary invest igat ion in —ft the rat portal vein indicated that ketanserin (10 M) blocked more than 75% of the cont rac t i le response produced by serotonin (10~ M). In t h i s ser ies of experiments ketanserin (10 - ^ M) was used to antagonise serotonin (5HT2) receptors. Por ta l veins (n = 6) were prepared (Section 2 .3 .1 .1 ) . Dose-response curve to gamma globul in as in section 2.4.3.2.1 was constructed. The baths were f i l l e d with 3 ml of ketanserin Krebs and 0.5 ml sample of ketanserin ft (10 M) Krebs was s e r i a l l y added un t i l the bath volume reached 5.5 ml. Another dose-response curve to gamma globul in (7 mg of gamma globul in dissolved in 1 ml of ketanserin Krebs) was constructed. 2.4.3.2.6 Angiotensin receptors: This ser ies of experiments were performed to study whether angiotensin receptors play a ro le in the increase in the spontaneous a c t i v i t y produced by gamma g lobu l in , and whether the increase in ac t i v i t y is due to the contamination of gamma globul in with a -g lobu l in (angiotensinogen, rennin substrate is an a -g lobu l i n ) . The prel iminary invest igat ion indicated that the maximum response produced by -9 -9 angiotensin (10 M) was blocked by sara las in (10 M) which is a competitive antagonist of angiotensin. Por ta l veins (n = 6) were prepared (Section 2 .3 .1 .1 ) . Dose-response curve to gamma globul in as in section 2.4.3.2.1 was constructed. The baths -9 were f i l l e d with 3 ml of sara las in (10 M) Krebs and 0.5 ml sample of - 36 -sara las in Krebs was s e r i a l l y added unt i l the bath volume reached 5.5 ml . Another dose-response curve to gamma globul in (7 mg of gamma globul in dissolved in 1 ml of sara las in Krebs) was constructed. 2.4.3.2.7 Calcium channels: This ser ies of experiments were performed to study whether the in f lux of C a + + through the voltage sens i t i ve C a + + channels play a ro le in the gamma globul in induced increase in the spontaneous a c t i v i t y of the portal ve in . The prel iminary invest igat ions indicated that the antagonist verapamil (10 M) blocked about 75% of the spontaneous a c t i v i t y of the portal ve in. Portal veins (n = 6) were prepared (Section 2.3.1.1) and the same procedure as in Section 2.4.2.5 was carr ied out. Dose-response to gamma globul in was constructed and th i s was used as a control for subsequent —8 experiment in the presence of verapamil (10 M). The t issues were washed with verapamil Krebs and the baths were f i l l e d with 5 ml of verapamil Krebs. Af ter exposing the t issues in t h i s so lut ion for 5 min another dose-response curve to gamma globul in was constructed. 2.4.3.2.8 Endothelium dependent fac to rs : This ser ies of experiments were performed to study whether the increase in the spontaneous a c t i v i t y of the portal vein was due to the release of endothelium dependent factor(s) from the endothelium of portal vein by gamma g lobu l in . In th is ser ies of experiments s ix portal veins were prepared as in Section 2.3.1.3 without damaging the endothelium and s ix portal veins were prepared as in Sect ion 2.3.1.4 with scraping off the endothelium to see whether there is any di f ference in the spontaneous ac t i v i t y produced by gamma globul in in these two preparat ions. Dose-response curve to gamma globul in (procedure as described in Section 2.4.2.5) was constructed. The presence and absence of - 37 -the endothelium was confirmed at the end of experiment with acety lchol ine which did not produce any s ign i f i can t re laxat ion in the portal veins in which the endothelium was scraped off but produced s ign i f i can t re laxat ion in veins with intact endothelium. 2.4.4 S t a t i s t i c a l ana lys is . Analys is of variance (ANOVA) with repeated measures was used to compare various dose-response curves. Duncan's mult ip le range test was used to compare the group means. In a l l cases, a p robab i l i t y of error of less than 0.05 was pre-selected as the c r i t e r i o n for s t a t i s t i c a l s ign i f i cance . Results are presented as mean ± SEM. - 38 -3 RESULTS, OBSERVATIONS AND STATISTICAL ANALYSIS 3.1 Vascular Sens i t i z ing Agent in Hypertensive Plasma 3.1.1 Sens i t i v i t y of aor t ic and portal vein s t r ips to agonists. When portal veins were exposed to normotensive plasma the spontaneous a c t i v i t y was gradual ly i nh ib i ted . In aor t ic s t r i p s , the base l ine tension shi f ted upwards but with time i t came down. When a cumulative dose-response curve + was attempted with NA or K , i t was found that these agents did not produce any measurable response in e i ther portal vein or aor t ic s t r i p s . A dose-response curve to Ca was done in the presence of NA (10 M) or K (100 mM) in the t issues exposed to normotensive plasma. Ca caused a dose dependent increase in response in the aor t ic s t r ips in the presence + + of NA or K and portal vein in the presence of K but not in the portal veins in the presence of NA. The dose range of Ca used in t h i s p re l im-inary invest igat ion was 0.5 mM-3.0 mM. The maximum amount of Ca needed in the plasma to produce maximum response to NA or K + was found to be 2.5 mM Ca .•. Further addit ion of Ca did not increase the response in e i ther of these t i ssues . Portal veins exposed to plasma with extra 2.5 mM C a + + produced a very l i t t l e spontaneous ac t i v i t y compared to the complete inh ib i t i on of spontaneous a c t i v i t y in the portal vein exposed to plasma without extra 2.5 mM Ca . The maximum response to each agonist in Krebs solut ion expressed as 100% a dose-response curve was constructed (F ig . 1-4). The maximum force produced by aor t ic s t r i p in Krebs solut ion at pH 7.4 was 0.62 ± 0.08g. Addit ion of normotensive plasma (pH 7.8-8.0) s i g n i f i c a n t l y decreased the response to NA. Maximum response to NA was s i g n i f i c a n t l y reduced (by 77%) to 0.13 ± 0.03g in normotensive plasma. Hypertensive plasma (pH 7.8-8.0) also caused a s ign i f i can t reduction (45%) of the maximum response to NA in - 39 -aor t ic s t r i ps in Krebs solut ion (pH 7.4) from 0.64 ± 0.06g to 0.36 ± 0.05g (Table 1 ana F i g . 1) . The maximum response to NA obtained in the aor t ic s t r i ps exposed to Krebs, with i t s pH matched to that of plasma (pH 7.8-8) was 0.56 ± 0.07g, which represents 18% reduction from that obtained in t issues exposed to Krebs solut ion (pH 7.4) (Table 2 and F i g . 2 ) . Therefore, the aor t ic s t r ips exposed to hypertensive plasma maintain the i r response to NA better than the aor t ic s t r i ps exposed to normotensive plasma. This di f ference could not be en t i re l y at t r ibuted to the a lka l ine pH of the plasma or to the hypoxic condit ion under which the experiment was carr ied out. The % maximum response obtained in the aor t ic s t r ips exposed to the hypertensive plasma was s i g n i f i c a n t l y greater than that in normotensive plasma. Figure 3 and Table 3 show that the maximum response (0.51 * O.Ollg) to K + in the aor t ic s t r ips exposed to normotensive plasma was reduced from the corresponding response (1.3 ± 0.56g) in Krebs solut ion (pH 7.4). The + maximum response to K in the aor t ic s t r ips exposed to hypertensive plasma was reduced (by 46%) to 0.38 ± 0.7g from the control response 1.24 ± 0.08g in Krebs solut ion (pH 7.4) . The maximum response 1.34 * ,63g obtained in the aor t ic s t r ips exposed to Krebs solut ion with i t s pH matched to that of plasma (pH 7.8-8) (F ig . 4 and Table 4) was not s i g n i f i c a n t l y d i f ferent from that in Krebs solut ion (pH 7.4) (1.4 ± 0.63g). Therefore the aor t ic s t r i ps exposed to hypertensive plasma were less sens i t ive to K + than the aor t i c s t r ips exposed to normotensive plasma. The maximum response to K + in Krebs at pH 7.8-8 was s i g n i f i c a n t l y greater than the maximum responses to K + in the aor t ic s t r ips exposed to hypertensive or normotensive plasma. 3.1.2 Cont rac t i le a c t i v i t y of portal vein to agonist. F i g . 5 shows a typ ica l t rac ing of portal vein exposed to normotensive or hypertensive plasma. Addit ion of 2.5 mM C a + + increased the spontaneous ac t i v i t y of the - 40 -[NA] M CONT 1 NORMO CONT 2 HYPER i o - 8 12.2 ± 4.1 3.5 ± 0.8 6.9 + 1.8 3.2 ± 0.3 i o - 7 17.6 ± 5.2 5.2 ± 1.3 12.2 ± 5.4 8.8 ± 4.9 i o - 6 59.2 ± 6.3 8.4 ± 2.7 53.9 ± 7.5 33.4 ± 8.4 IO" 5 91.7 ± 2.0 14 .0 ± 1.8 91.9 ± 2.6 51.5 ± 7.8 IO" 4 99.7 ± 0.3 22.5 ± 3.9 100 ± 0 56.7 ± 6.7 TABLE 1 NA Dose response re la t ionsh ip in aor t i c s t r i ps exposed to hyperten-s ive (n = 6) or normotensive plasma (n = 6) . CONT 1 = Control for normotensive plasma, Krebs pH 7.4; NORMO = Normotensive plasma; CONT 2 = Control fo r hypertensive plasma, Krebs pH 7.4; HYPER = Hypertensive plasma. Results are expressed as % maximum response ± SEM. - 41 -120 [ N A ] ( M ) O C . 1 • N O R A C . 2 A H Y P FIG. 1 NA dose-response curves in aor t i c s t r i ps (n = 6) exposed to normotensive and hypertensive plasma. C . l = Control for Normotensive plasma, ph 7.4; NOR.= Normotensive plasma; C.2 = Control for Hypertensive plasma, pH 7.4; HYP = Hypertensive plasma. Each point and v e r t i c a l bar represents mean ± SEfo. Curve a and curve b - s i g n i f i c a n t l y d i f fe rent from C . l and C.2 (P < 0.05). Curve b - s i g n i f i c a n t l y d i f fe rent from curve a (P < 0.0b). - 42 -[NA] M CONT EXPT 10-8 10.5 ± 2.5 7.4 ± 1.2 I O - 7 13.6 ± 3.2 11.7 ± 2.3 i o - 6 35.3 ± 5.7 25.3 ± 4.7 I O " 5 79.1 ± 4.0 52.9 ± 8.9 i o - 4 100 ± 0 82.4 ± 6.8 TAbLE 2 NA Dose-response r e l a t i o n s h i p i n a o r t i c s t r i p s (n = 6) at d i f f e r e n t pH of the Krebs ana with no bubbling with carbogen. CONT = Bubbled Krebs ph 7.4; EXPT = Krebs pH matched to plasma with no bubbling with carbogen. R e s u l t s are expressed as % maximum response ± SEM. - 43 -FIG. 2 NA dose-response curves in aor t ic s t r ips (n = 6) at d i f fe rent pH and in the presence ana absence of bubbling with carbogen. CON = Bubbling Krebs pH 7.4; EXP = Krebs pH matched to plasma with no bubbling with carbogen. Each point and ve r t i ca l bar represents mean ^ SEM. - s i g n i f i c a n t l y d i f fe ren t from CON (P < 0.05). - 44 -fK] mM CONT1 NORMO CONT 2 HYPER 20 82.3 + 4.1 28.5 + 4.4 84.0 ± 2.4 30.5 ± 5.9 40 96.1 ± 2.1 66.0 ± 13.6 79.4 ± 14 51.1 ± 5.8 60 98.8 + 1.3 75.0 ± 12.6 100 ± 0 55.7 + 7.9 80 99.5 ± 0.3 76.1 ± 11.8 99.1 ± 0.3 53.0 ± 8.4 TABLE 3 K Dose-response re la t ionsh ip in aor t ic s t r i ps ( n = b ) exposed to hypertensive (n = 6) or normotensive plasma ( n = 6 ) . CONT 1 = Control for . normotensive plasma, Krebs pH 7.4; NORMO = Normotensive plasma; CONT 2 = Control for hypertensive plasma, Krebs pH 7.4; HYPER = Hypertensive plasma. Results are expressed as % maximum response ± SEM. - 45 -120 Ul 0 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 [ K ] ( m M ) O C . 1 • N O R A C . 2 A H Y P FIG. 3 K dose-response curves in aor t ic s t r i ps (n = 6)exposed to normo-tensive and hypertensive plasma. C l = Control for Normotensive plasma, Ph 7.4; NOR = Normotensive plasma; C.2 = Control for Hypertensive plasma, pH 7 .4; HYP = Hypertensive plasma. Each point and v e r t i c a l bar represents mean ± SEM. Curve a^  - s i g n i f i c a n t l y d i f fe ren t from C l and C.2 (P < 0.05). Curve a - s i gn i f i can ta l y d i f fe ren t from curve b (P < 0.05) . - 46 -[K] mM CONT EXPT 20 77.2 ± 6.9 49.4 + 16 40 93.4 ± 2.9 87.3 ± 14 60 97.4 ± 1.7 98.8 + 12 80 99.6 ± 0.2 106.7 + 12 TABLE 4 K Dose-response re la t i onsh ip in aor t ic s t r ips (n = 6) at d i f fe rent pH of the Krebs and with no bubbling with carbogen. CONT = Bubbled Krebs pH 7.4; EXPT = Krebs ph matched to plasma with no bubbling with carbogen. Results are expressed as % maximum response ± SEM. - 47 -120 [ K ] ( m M ) O C O N • E X P FIG. 4 K dose-response curves in aor t i c s t r i ps (n = 6) at d i f ferent pH and in the presence and absence of bubbling with carbogen. CON = Bubbling Krebs pH 7.4; EXP = Krebs pH matched to plasma with no bubbling with carbogen. Each point and ve r t i ca l bar represents mean ± SEM. - 48 -portal vein exposed to normotensive plasma but not to hypertensive plasma. NA neither produced a contract ion nor increased the spontaneous ac t i v i t y in the presence of normotensive or hypertensive plasma with addi t ional C a + + (F ig . 5 ) . Table 5 and F i g . 6 shows the tension developed in response to K + in the portal vein s t r ips in the presence and absence of normotensive or hypertensive plasma. The maximum tension obtained in the portal vein to K + in Krebs solut ion (pH 7.4) was 0.73 ± 0.12g. Normotensive plasma s i gn i f i can t l y + + decreased the response to K . The maximum response to K in the presence of normotensive plasma was 0.29 ± 0.06g. Maximum response to K + in the presence of hypertensive plasma was 0.19 0.03g, which is s i g n i f i c a n t l y less than the tension 0.73 ± O . l l g obtained in Krebs solut ion (pH 7.4). The maximum response obtained in the presence of hypertensive plasma was s i g n i f i c a n t l y less than that of normotensive plasma. 3.2 Spontaneous A c t i v i t y of Portal Vein 3.2.1 Spontaneous ac t i v i t y obtained with heated and unheated normotensive  plasma and serum. The spontaneous ac t i v i t y of the portal vein was s i g n i f i -cant ly increased when the vein was exposed to increasing concentrations of normotensive plasma, heated plasma or serum (Table 6 and F i g . 7). The maximum response was obtained when the volume of unheated plasma, heated plasma and serum was 50% , 45% and 50% respect ive ly . The maximum force developed during spontaneous a c t i v i t y of the portal vein in the absence of plasma or serum was 0.08 * 0.03g. The unheated plasma, heated plasma or unheated serum increased the force to 0.25 ± 0.04g, 0.26 ± 0.04g and 0.24 ± 0.03g respect ive ly . Af ter th is maximum response was at ta ined, further addit ion of the plasma or serum reduced the spontaneous a c t i v i t y . The force developed in the presence of 65% volume of unheated plasma, heated plasma and unheated serum were 0.14 ± 0.02g, 0.14 ± 0.03g and 0.15 ± 0.02g respect ive ly . The force - 49 -i i i y 1 1 PL A S M A 10 ' ' , 0 ' ' I0"« io? » 4 ADDED . FIG. 5 Tracing of NA (M) dose-response curves in portal vein exposed to normotensive plasma (B) , hypertensive plasma (D) and Krebs (A and C) . - 50 -[K] mM CONT1 NORMO CONT 2 HYPER 20 54.7 ± 5.4 12.3 ± 2.6 57.8 ±5.8 10.7 ± 4.7 40 91.2 ± 4.3 27.6 ± 4.3 93.1 ± 1.8 15.1 ± 4.2 60 99.3 ± 0.5 34.8 ± 3.3 99.7 ± 0.3 21.7 ± 4.9 80 97.5 ± 1.1 36.4 ± 3.5 91.6 ± 2.9 25.1 ± 5.0 TABLE 5 Dose-response r e l a t i o n s h i p i n p o r t a l vein s t r i p s (n = 6) exposed to hypertensive ( n = 6) or normotensive plasma ( n = 6 ) . CONT 1 = Control f o r normotensive plasma, Krebs at pH 7.4; NORMO = Normo-t e n s i v e plasma;" CONT 2 = Contr o l f o r hypertensive plasma, Krebs at pH 7.4; HYPER = Hypertensive plasma. Results are expressed as % maximum response ± SEM. - 51 -FIG, b K dose-response curves in por ta l veins (n = b) exposed to normoten-sive and hypertensive plasma. C . l = Control for Normotensive plasma, Krebs; NOR = Normotensive plasma; C.2 = Control for Hypertensive plasma, Krebs; hYP = Hypertensive plasma. Each point and ve r t i ca l bar represents mean ± SEM. Curves a and b - s i g n i f i c a n t l y d i f fe rent from curves C . l and C.2 (P < 0.05). Curve b - s i g n i f i c a n t l y d i f fe ren t from curve a (P < 0.05) . - 52 -% Vol PLASMA HEATED SERUM 0 21.6 ± 0.8 41.6 ± 6.4 26.9 ± 4.2 33 71.3 ± 4.4 91.2 ± 7.9 83.9 ± 3.8 44 94.5 ± 2.8 95.3 ± 9.7 95.7 ± 3.8 50 94.5 ± 1.9 84.8 ± 9.3 96.2 ± 1.5 55 85.3 ± 3.4 72.5 ± 8.2 84.3 ± 1.9 60 75.3 ± 5.6 67.7 ± 7.8 72.9 ± 2.3 64 59.4 ± 3.5 55.1 ± 6.8 62.9 ± 2.1 TABLE 6 Spontaneous a c t i v i t y of porta l vein in heatea and unheated normo-tensive plasma and unheated normotensive serum (n = 6). % VOL = % Volume added; PLASMA = Unheated normotensive plasma; HEATED = Heated normotensive plasma; SERUM = Unheated normotensive serum. Resul ts are expressed as % maximum response ± SEM. - 53 -2 0 0 UJ * tn z o CL in 0 I i i i i i i i O 10 2 0 3 0 4 0 5 0 6 0 7 0 8 0 % V O L U M E A D D E D O P L A • H E A A S E R FIG. 7 % maximum of spontaneous a c t i v i t y of portal veins (n = 6) with increasing concentrat ion of unheated and heated normotensive plasma and normotensive serum. PLA = Unheated normotensive plasma; HEA = heated normotensive plasma; SER = Normotensive serum. Each point and v e r t i c a l bar represents mean ± SEM. - 54 -developed in the portal vein at any given concentration of unheated plasma, heated plasma or unheated serum was not s i g n i f i c a n t l y d i f ferent from each other. 3.2.2 Spontaneous ac t i v i t y produced with unheated normotensive and  hypertensive plasma. The maximum force developed during spontaneous a c t i v i t y of the portal vein in the control Krebs (pH 7.4) p r io r to the addit ion of normotensive or hypertensive plasma was 0.08 * 0.03g. The spontaneous a c t i v i t y increased with addit ion of small volume of normotensive or hyper-tensive plasma (Table 7 and F i g . 8, 9 ) . The maximum response obtained with normotensive plasma was taken as 100% and the % maximum response was ca lcu -lated for each concentration of hypertensive and normotensive plasma. Both normotensive and hypertensive plasmas increased the spontaneous a c t i v i t y . The base l ine tension was shi f ted with hypertensive plasma, but i t came down with time. This sh i f t was also seen with some normotensive plasma. The maximum response obtained with the hypertensive plasma was 60% more than the maximum response obtained for the normotensive plasma. At 50% volume of plasma, the spontaneous ac t i v i t y with the normotensive plasma was taken as 100% (0.20 ± 0.03g) while the spontaneous a c t i v i t y of the hypertensive plasma was about 161.2 ± 7% (0.33 ± 0.07g) (n = 3) (Table 7). When the concentra-t ion of normotensive or hypertensive plasmas were increased, the spontaneous ac t i v i t y decreased. At the 65% plasma volume the force developed with norm-otensive plasma was 0.14 ± 0.03g and hypertensive plasma was 0.21 ± 0.05g. The response at 60% volume of hypertensive plasma was not s i g n i f i c a n t l y d i f fe rent from the maximum response obtained with 50% volume of normotensive plasma. These changes were not due to the change in f l u i d volume, tempera-ture or pH of the t issue bath s ince, the spontaneous a c t i v i t y of the portal vein was not al tered (Table 7, F i g . 8,9) by Krebs under the ident ica l condi-t ions as with plasma. - 55 -% VOL NORMO HYPER CONT 1 CONT 2 0 33.2 ± 5.3 46.7 ± 7.5 25.9 ± 5.0 27.5 ± 3.8 33 i 78.8 ± 9.3 127.9 ± 4.4 23.6 + 6.5 31.3 ± 4.6 44 91.8 ± 3.5 154.9 ± 9.3 24.7 ± 7.0 28.5 ± 1.2 50 100 ± 0 161.6 ± 6.9 27.6 ± 9.2 26.9 ± 0.8 55 88.4 ± 3.9 140.6 ± 11 30.8 ± 6.1 27.5 ± 0.9 60 77.4 ± 7.9 105.7 ± 7.4 29.2 ± 7.6 28.1 ± 3.2 64 66.1 ± 4.2 98.2 ± 7.8 30.3 ± 5.4 27.6 ± 1.9 TABLE 7 Spontaneous a c t i v i t y of the por ta l vein in normotensive (n = 6) or hypertensive plasma (n = 3 ) . VOL = Volume added; NORMO = Normo-tensive plasma; HYPER = Hypertensive Plasma; CONT 1 = Krebs pH 7.4; CONT 2 = Krebs pH matched to that of plasma. Results are expressed as maximum response ± SEM. - 56 -FIG. 8 % maximum of spontaneous a c t i v i t y of portal vein (n = 3) with increasing concentrat ion of normotensive plasma ana hypertensive plasma. NOR = Normotensive plasma; HYP = hypertensive plasma; C . l = Krebs pH 7.4; C.2 = Krebs pH matched to that of plasma. Each point and ve r t i ca l bar represents mean ± SEM. * - s i g n i f i -cant ly d i f fe rent from NOR, C . l , C.2 (P < 0.05). - 57 -FIG. 9 Tracing of spontaneous a c t i v i t y and integrated a c t i v i t y of porta l veins in the presence of increasing volume of Krebs solut ion (A), normotensive (B) ana hypertensive (C) plasma. The arrow indicates the % volume of plasma in the bath (B and C) or equivalent volume of f resh Krebs (A). - 58 -3.3 Plasma Fract ion(s) That Sens i t i ze The Vascular Tissue 3.3.1 Albumin. Table 8 and Figure 10 show that the spontaneous a c t i -v i t y of the portal vein increased with increasing concentration of albumin and reached a plateau at 16mg/ml concentrat ion, which is about 40% of the concentration o rd ina r i l y found in the human plasma. The maximum force develpoed during spontaneous a c t i v i t y of the portal vein was increased from 0.09 ± 0.02g to 0.24 ± 0.04g in the presence of albumin. The increase in spontaneous ac t i v i t y of the portal vein was neither due to a l te ra t ion of the pH of the albumin solut ion nor due to the increase in the volume of the f l u i d in the t issue bath (Table 8, F i g . 10). 3.3.2 Gamma g lobu l in . The maximum force developed during spontaneous a c t i v i t y of the portal vein increased from the control value of 0.07 ± O.Olg with increasing concentration of gamma globul in (Table 9, F i g . 11, 12) and reached a plateau (0.15 ± 0.06g) at 4.2 - 4.5 mg/ml concentration of gamma g lobu l in . This is approximately 47-52% of the concentration of gamma globu-l i n normally present in the human plasma. Gamma g lobul in increased both amplitude and frequency of the spontaneous ac t i v i t y (Table 9, F i g . 13, 14). The increase in spontaneous a c t i v i t y of the portal vein was neither due to a l te ra t ion of the pH of the gamma globul in nor due to the increase in the volume of the f l u i d in the t issue bath (Table 9, F i g . 11). 3.3.3 Alpha (a) g lobu l in . The spontaneous a c t i v i t y of the portal vein tended to increase i n i t i a l l y with the addit ion of a -g lobu l in (Table 10, F i g . 15) but s t a t i s t i c a l l y there was no s ign i f i can t di f ference between the maximum force developed during spontaneous a c t i v i t y in the presence of Krebs (0.17 -± 0.03g) and the maximum force developed in the presence of a -g lobu l in . Further addit ion of a -g lobul in decreased the spontaneous a c t i v i t y . At a 3.3 - 59 -[ALB] ALBUMIN % MAX CONT 1 % MAX CONT 2 % MAX 0.0 32.5 + 5.2 24.1 ± 4.3 31.7 ± 5.4 1.0 58.4 ± 7.5 26.3 ± 4.5 31.9 ± 6.3 12.8 83.3 ± 6.9 28.3 ± 4.7 29.4 ± 3.9 15.0 91.1 ± 2.7 30.9 ± 4.6 30.1 + 3.9 16.6 96.2 + 2.2 32.3 + 4.5 30.8 ± 4.4 18.0 90.9 ± 4.3 33.2 ± 4.5 29.9 ± 4.5 19.0 89.5 ± 4.9 33.6 ± 4.5 20.6 ± 4.4 TABLE 8 Spontaneous a c t i v i t y of portal vein with albumin (n = 6) . [ALB] = Concentration of albumin mg/ml. % MAX = % Maximum response; % VOL = % Volume added; CONT 1 = Krebs pH 7.4; CONT-2 = Krebs pH matched to albumin pH (7.4 - 7 .6) . Results are expressed as % maximum response ± SEM. Note: In both con t ro ls , instead of albumin Krebs, equivalent volumes of Krebs which corresponds to each dose of albumin was added to the bath. - 60 -UJ U) z O CL if) UJ ft X < 2 0 0 100 -0 o 10 [ A L B U M I N ] A L B • C . 1 2 0 ( M G / M L ) A C . 2 3 0 FIG. 10 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) with albumin and con t ro ls . ALB = Albumin Krebs; C l = Krebs pH 7.4 alone; C.2 = Krebs ph matched to albumin and no bubbling with carbogen, ( in C l and C.2 equivalent volume of fresh Krebs so lut ion was added to the bath instead of albumin Krebs s o l u t i o n ) . Each point and ve r t i ca l bar represents mean ± SEM. - s i g n i f i c a n t l y d i f f e ren t from C l and C.2 (P < 0.05) . - 61 -[GLOB] GAMMA % MAX GAMMA AMP'(g) GAMMA FREQ CONT 1 % MAX CONT 2 % MAX 0.0 39.8 ± 4.8 0.4 ± 0.01 2.7 ± 0.6 24.1 ± 4.3 31.7 ± 5.4 1.4 52.8 ± 3.6 0.6 ± 0.01 2.3 + 0.8 26.3 + 4.5 31.9 ± 6.3 2.2 60.4 ± 4.6 0.6 ± 0.01 3.4 + 0.6 28.3 ± 4.7 29.4 ± 3.9 3.0 73.3 + 5.5 0.7 ± 0.01 3.9 ± 0.7 30.9 ± 4.6 30.1 ± 3.9 3.5 84.5 ± 4.4 0.8 ± 0.01 3.9 ± 0.9 32.3 ± 4.5 30.8 ± 4.4 3.8 91.7 ± 2.7 0.8 ± 0.01 4.2 ± 0.5 33.2 ± 4.5 29.9 ± 4.5 4.2 97.5 ± 1.2 0.8 ± 0.01 4.4 ± 0.7 33.6 ± 4 . 5 20.6 ± 4.4 4.4 91.7 ± 3.3 0.8 ± 0.01 4.3 ± 0.7 34.9 ± 5.4 39.0 ± 4.5 TABLE 9 Spontaneous a c t i v i t y of portal vein with gamma globul in ( n = 6 ) . Table shows amplitude frequency and two cont ro ls . [GLOB] = Concen-t ra t i on of gamma g lobu l in in" mg/ml; % MAX = % Maximum response ± SEM; AMP = Amplitude of spontaneous ac t i v i t y ± SEM (gram tension) . FREQ = Frequency of spontaneous a c t i v i t y ± SEM (no ot spikes/min); % VOL = % Volume added; CONT 1 = Krebs - pH 7.4; CONT 2 = Krebs pH matched to gamma g lobu l in (pH 7.7 - 7.8). Note: In both con t ro ls , instead of gamma globul in Krebs, equivalent volume of Krebs which corresponds to each dose of gamma globul in was added to the bath. - 62 -HI (f) z o n (/) HI ft X < 2 0 0 1 0 0 -0 1 2 3 4 [ G A M M A G L O B ] ( M G / M L O G A M • C . 1 A C . 2 FIG. 11 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) with gamma g lobu l in and con t ro l s . GAM = Gamma g lobul in Krebs; - C . l = Krebs pH 7.4; C.2 = Krebs matched to g lobul in and -no bubbl ing, ( in C . l ana C.2 equivalent volume of f resh Krebs was added instead of gamma g lobul in Krebs ^ s o l u t i o n ) . Each point and v e r t i c a l bar represents mean ± SEM. - s i g n i f i c a n t l y d i f fe ren t from C . l and C.2 (P < 0.05). - 63 -F I G . 12 Tracing of spontaneous a c t i v i t y and integrated a c t i v i t y of por ta l vein in the presence of Krebs so lu t ion (A) ana gamma g lobu l in (B) . The arrow ina icate O.b ml of gamma globul in Krebs (7 mg/ml) added (B) or equivalent volume of f resh Krebs added (A). - 64 -Z o or z HI H 1.00 0 .90 0 .80 0 .70 0 .60 -0 .50 -0.40« 0 .30 0 .20 0 .10 0 .00 0 1 2 3 4 5 [ G A M M A G L O B ] ( M G / M L ) F I G . 13 Amplitude (maximum spike tension in grams) of spontaneous a c t i v i t y of por ta l vein (n = 5) with gamma-globulin. Each point and v e r t i c a l bar represents mean ± SEM. - 65 -Frequency (no. of spikes/mi n) of Spontaneous a c t i v i t y of por ta l vein (n= 5) with gamma g lobu l i n . Each point and v e r t i c a l bar represents mean ± SEM. - 66 -[a -Gl ] % MAX 0.0 80.9 ± 5.5 1.1 87.8 ± 5.9 1.7 96.6 ± 1.9 2.2 82.6 ± 4.9 2.6 65.4 ± 6.3 2.8 49.5 ± 6.9 3.1 34.2 ± 5.4 3.3 26.2 ± 5.7 TABLE 10 Spontaneous a c t i v i t y of por ta l vein with a -g lobu l in (n = 6 ) . L a - G l J = Concentration of a -g lobu l in in mg/ml; % MAX = % maximum response ± SEM. - 67 -ID (/) Z O a w ID CC X < 200 1 0 0 b O A L N ] ( M G / M L ) • A & B FIG. 15 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) with a - g l o b u l i n , a- ana e -g lobu l i n . AL = a - g l o b u l i n ; A & B = a- and 3 -g lobu l in . Each point and v e r t i c a l bar represents mean ± SEM. - 68 -mg/ml concentration of a -g lobu l i n , which is approximately 50% of the concen-t ra t ion in the human plasma, the maximum force developed during spontaneous a c t i v i t y of the portal vein was 0.06 ± 0.02g. 3.3.4 Alpha (a) and beta (e) g lobu l in . The spontaneous ac t i v i t y of the portal vein was inh ib i ted with the addit ion of a-and e-globul in together (Table 11, F i g . 15). At a concentration of 6.6 mg/ml, which is approximately 50% concentration present in the human plasma, the maximum force developed during spontaneous ac t i v i t y of the portal vein was decreased to 0.05 * O.Olg from 0.16 ± 0.03g. 3.3.5 Immunoglobulin IgG. Control values of maximum force developed during spontaneous a c t i v i t y in portal vein before exposure to IgG or gamma globul in were 0.11 * O.Olg and 0.11 * 0.02g respect ive ly . IgG increased the spontaneous a c t i v i t y (both amplitude and frequency) of the portal vein (Table 12, F i g . 16, 17, 18). The maximum force obtained in the presence of IgG (0.16 ± 0.01 g) was not s i g n i f i c a n t l y d i f ferent from that in the presence of gamma globul in (0.19 0.02g) The IgG produced maximum response at the concentration of 4.5-5 mg/ml. This is approximately 50% of the concentration of IgG present in the human plasma. 3.4 Mechanism of Act ion of Plasma Proteins The maximum spontaneous a c t i v i t y obtained by the control (plasma proteins without antagonist) was taken as 100% response and the % maximum response was calculated with each concentration of albumin or gamma g lobu l in . 3.4.1 Albumin. Albumin increased maximum force developed during spontaneous a c t i v i t y of the portal vein from 0.09 •* 0.02g to 0.25 * 0.03g. The increased spontaneous a c t i v i t y obtained with albumin was blocked when the porta l veins were exposed to 10~^ M phentolamine (Table 13, F i g . 19). Af ter the exposure to ouabain the spontaneous ac t i v i t y was abol ished. Subsequent - 69 -to & G] % MAX 0.0 84.2 ± 7.7 2.2 93.9 ± 3.5 3.4 84.9 ± 6.3 4.4 66.7 ± 7.1 5.2 48.3 f 6.0 5.6 35.4 ± 4.4 6.2 26.1 ± 4.5 6.6 22.2 ± 3.8 TABLE 11 Spontaneous a c t i v i t y of p o r t a l v ein w i t h o and e - g l o b u l i n (n = 6). [a & ej = C o n c e n t r a t i o n of a - ana e-globulin i n mg/ml; % MAX = maximum response ± SEM. - 70 -CON'C GAMMA % MAX IgG % MAX GAMMA AMP IgG AMP GAMMA FREQ IgG FREQ 0.0 58.4 ± 4.4 59.3 ± 2.6 0.55 ± 0.08 0.61 ± 0.07 2.3 + 0.4 2.0 ± 0.3 2.0 64.1 ± 3.4 70.2 ± 2.7 0.61 ± 0.07 0.63 ± 0.07 2.4 ± 0.4 2.3 ± 0.4 2.5 77.3 ± 4.7 74.9 ± 4.1 0.68 ± 0.08 0.71 ± 0.08 2.7 ± 0.3 2.9 ± 0.3 3.0 89.1 ± 1.3 82.4 ± 3.4 0.75 ± 0.10 0.70 ± 0.07 2.9 ± 0.3 2.9 + 0.4 3.5 89.3 ± 1.3 83.7 ± 3.9 0.72 ± 0.09 0.72 ± 0.08 3.2 ± 0.4 3.1 ± 0 . 4 4.0 94.7 ± 1.4 83.1 ± 3.7 0.74 ± 0.08 0.74 ± 0.06 3.3 ± 0.4 3.3 ± 0.4 4.5 93.9 ± 1.9 85.9 + 1.5 0.77 ± 0.07 0^75 ± 0.07 3.4 ± 0.4 3.4 ± 0.4 5.0 98.3 ± 0.9 85.2 ± 3.4 0.77 ± 0.08 0.74 ± 0.08 4.1 ± 0.4 3.6 ± 0.4 TABLE 12 Changes in spontaneous a c t i v i t y induced by gamma g lobu l in or IgG (n= 6), %Max ( integrated) response, amplitude ana frequency of spontaneous a c t i v i t y are shown. C0NC = Concentration of IgG or gamma g lobu l in in mg/ml; % MAX = % Maximum response ± SEM; GAMMA = Gamma g lobu l i n ; AMP = Amplitude of spontaneous a c t i v i t y ± SEM (gram tens ion) ; FREQ = Number of spikes/min ± SEM. - 71 -2 0 0 HI if) Z o n if) HI DC X < 100 0 ~ 1 2 3 4 5 C O N C E N T R A T I O N M G / M L O G A M • I g G FIG. 16 % maximum of spontaneous a c t i v i t y of .portal vein (n = 6) with gamma g lobu l i n , IgG. GAM = Gamma g lobu l in , IgG = igG. Each point and v e r t i c a l bar represents mean ± SEM. - 72 -1 . 0 0 -0 . 9 0 -z 0 . 8 0 -o - 0 . 7 0 . : 0 . 6 0 r * z 0 . 5 0 . : Ul 0 . 4 0 -I— 0 . 3 0 Oi 0 . 2 0 0 . 1 0 0 . 0 0 o 1 4 C O N C E N T R A T I O N M G / M L O G L O • I n G FIG. 17 Amplitude of Spontaneous a c t i v i t y of portal vein (n = 6) with gamma g l obu l i n , IgG. GAM = Gamma g lobu l in , IgG = IgG. Each point and v e r t i c a l bar represents mean ± SEM. - 73 -Z U) UJ * D. 0) LL o o z C O N C E T R A T I O N G L O B • I g G FIG. 18 Frequency of Spontaneous a c t i v i t y of portal vein (n = 6 ) , with gamma g lobu l i n , IgG. GAM = Gamma g lobu l i n , IgG = IgG. Each point and v e r t i c a l bar represents mean ± SEM. - 74 -[ALB] CONT 1 PHEN OUAB CONT 2 6 OH DA 0 56.1 ± 8.7 67.7 ± 10.4 30.7 ± 4.8 55.5 ± 7.2 49.8 ± 7.4 10 64.8 ± 9.7 66.8 ± 16.1 63.1 ± 8.4 78.5 ± 4.9 81.1 ± 13.6 12 75.5 ± 5.9 59.4 ± 9.1 89.2 ± 13.9 91.6 ± 1.6 121.7 ± 19.6 14 83.9 ± 3.9 60.7 ± 9.4 82.2 ± 18.4 96.4 ± 2.8 138.0 ± 21.9 16. 97.1 ± 1.6 64.3 ± 8.2 82.2 ± 10.9 96.4 ± 2.8 142.8 ± 23.4 18 92.2 ± 3.2 66.0 ± 9.1 74.8 ± 10.4 89.2 ± 4.0 139.2 + 22.8 20 85.3 ± 4.1 67.9 ± 8.3 71.7 ± 10.3 TABLE 13 Spontaneous a c t i v i t y of p o r t a l v e i n in response to albumin i n the presence and absence of phentolamine, ouabain and 6-OHDA r e s p e c t i v e l y (n = 6 ) . [ALB] = Concentration i n mg/ml; CONT 1 = Albumin alone; PHEN = Albumin with phentolamine 10"'; OUAB = Albumin w i t h Ouabain 10~ 4M; CONT 2. = Albumin alone c o n t r o l f o r 6-OHDA; 6-0HDA = Albumin a f t e r denervation wit h 6-0HDA. Re s u l t s are expressed as % maximum response ± SEM. - 75 -2 0 0 UJ co Z O o. co UJ cr x < ^° 1 0 0 -0 1 0 2 0 3 0 o A L [ A L B U M I N ] ( M G / M L ) B • P H E -FIG. 19 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) in response to albumin in the presence of phentolamine 10 M. Alb = Albumin alone; PHE = Albumin in the presence of phentolamine. Each point and v e r t i c a l bar represents mean ± SEto. is s i g n i f i c a n t l y d i f f e ren t from PHE LP < 0.05) . - 7 6 -addit ion of albumin restarted the spontaneous a c t i v i t y and the maximum force developed was 0.13 ± O.Olg (Table 13, F i g . 20) . The increase in the spon-taneous a c t i v i t y produced by albumin in the presence or absence of ouabain was not s i g n i f i c a n t l y d i f ferent from each other. Albumin increased the maximum force developed during spontaneous a c t i v i t y of the portal vein from 0.11 ^ 0.03g to 0.20 ± 0.06g before the vein was denervated. The maximum force developed during spontaneous a c t i v i t y of the portal vein chemically denervated by 6-hydroxydopamine was 0.10 ± 0.03g, which was not s i g n i f i c a n t l y d i f fe rent from the force developed in the portal vein without denervation. Albumin increased the spontaneous ac t i v i t y of the denervated portal vein from 0.10 ± 0.03g to 0.28 ± 0.08g, t h i s i s not s i g n i f i c a n t l y d i f ferent from the spontaneous ac t i v i t y produced in the vein by albumin before denervation. Tyramine did not produce any s ign i f i can t response in these chemical ly denervated portal veins (Table 13, F i g . 21). 3.4.2 Gamma g lobu l i n . Gamma globul in increased the maximum force developed during spontaneous a c t i v i t y from 0.08 O.Olg to 0.14 * 0.03g, which was completely blocked by ouabain (Table 14, F i g . 22). Gamma globul in s t i l l increased the maximum force developed during spontaneous ac t i v i t y of the portal vein in the presence of phentolamine (from 0.05 ± O.Olg to 0.15 * 0.04g, Table 15, F i g . 23), atropine (from 0.12 ± 0.02g to 0.19 * 0.04g, Table 16, F i g . 24), ketanserin (from 0.05 ± O.Olg to 0.21 ± 0.03g, Table 17, F i g . 25) Chlorpheniramine (from 0.10 ± O.Olg to 0.20 ± 0.03g, Table 18, F i g . 26) and sara las in (from 0.08 ± 0.02g to 0.13 ± O.Olg, Table 19, F i g . 27) the spontaneous a c t i v i t y produced by gamma globul in in the presence of these blockers were not s i g n i f i c a n t l y d i f ferent from the corresponding control values in the absence of the blockers. The maximum force developed during spontaneous a c t i v i t y of the portal vein when exposed - 77 -FIG. 20 % maximum of spontaneous a c t i v i t y of por ta l vein (n = 6) in response to albumin a f te r depo lar iza t ion with ouabain 10" 4 M. ALB = Albumin alone; OUA = Albumin af ter af ter depolar izat ion with ouabain l O - 4 M. Each point and ve r t i ca l bar represents mean ± SEM. - 78 -200 l±i co Z O 10 20 [ A L B U M I N ] ( M G / M L ) o A L B • 6 O H F I G . 21 % maximum of spontaneous a c t i v i t y of p o r t a l v e i n (n = 6) i n response to albumin a f t e r chemical denervation w i t h 6-OHDA. ALB = Albumin alone; 60h = Albumin - a f t e r d e n e r v a t i o n . Each point and v e r t i c a l bar represents mean ± SEto. - 7 9 -[GLOB] OUAB CONT 1 CONT 2 0.0 42.3 ± 2.1 41.5 ± 5.2 14.2 ± 2.4 1.4 56.2 ± 2.5 55.1 ± 3.4 24.5 ± 5.6 2.2 63. 4=-± 4.8 61.9 ± 4.4 25.5 ± 4.4 3.0 78.8 ± 4.9 73.8 ± 4.8 27.1 ± 5.3 3.5 87.7 ± 4.8 83.7 ± 3.7 26.9 ± 5.8 3.8 90.0 ± 1.2 92.3 ± 2.9 27.5 ± 6.0 4U2 96.7 ± 2.1 96.1 ± 2.4 27.3 ± 6.1 4.4 87.6 ± 5.7 91.6 ± 5.7 26.8 ± 6.3 TABLE 14 Spontaneous a c t i v i t y of the porta l vein in response to gamma lobu l in in the presence ana absence of ouabain 1 0 " 4 M ( n = 6 ) . GLOB] = Concentration of gamma g lobu l in in mg/ml; OUAB = Gamma g lobu l in with . ouabain; CONT 1 = Gamma g lobu l in alone; CONT 2 = Ouabain without gamma g lobu l i n . Results are expressed as * maximum response ± SEM. - 80 -2 0 0 111 (/) Z o Q . CO 0 1 2 3 4 5 [ G A M M A G L O B ] ( M G / M L ) O O U A • C . 1 A C . 2 FIG. 22 % maximum of spontaneous a c t i v i t y of porta l ve in . (n = b) in response to gamma g lobu l in in the presence of ouabain 10~ 4 M. C . l = Gamma g lobu l in alone; OUA = Gamma g lobu l in in the presence of ouabain; C.2 | Ouabain Krebs alone (in_ C.2 equivalent volume of ouabain - - (10 M) Krebs was added instead of gamma g lobu l in Krebs). Each point and v e r t i c a l bar represents mean ± SEM. is s i g n i f i c a n t l y d i f f e ren t from OUA (P < 0.05) - 81 -[GLOB] PHENTOL CONT 1 CONT 2 0.0 32.0 ± 3.8 49.3 + 2.1 17.4 ± 3.1 1.4 42.7 ± 7.1 56.2 ± 2.5 22.9 +4 .1 2.2 44.9 + 5.7 69.4 + 4.8 22.9 ± 4.2 3.0 59.5 ± 8.9 78.8 ± 4.9 27.0 ± 7.2 3.5 75.3 + 7.3 87.7 + 4.8 24.9 ± 7.6 3.8 83.5 ± 6.3 98.0 ± 1,2 24.9 ± 7.6 4.2 90.3 ± 8.6 96.7 ± 2.1 23.7 ± 7.9 4.4 102.3 ± 11 87.6 ± 5.7 23.1 ± 8.1 TABLE 15 Spontaneous a c t i v i t y of p o r t a l v e i n i n response to gamma g l o b u l i n i n the presence ana absence of phentolamine 10"^ M (n = b ) . [GLOBj = Concentration of gamma g l o b u l i n mg/ml; PHENTOL = Gamma g l o b u l i n w i t h phentolamine; CONT 1 = Gamma g l o b u l i n without phento-lamine; CONT 'I = Phentolamine without gamma g l o b u l i n . Results are expressed as % maximum response ± SEM. - 82 -FIG. 23 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) in response to gamma g lobu l in in the presence of phentolamine l u ~ 7 M. C . l = Gamma g lobul in alone; PHE = Gamma globul in in the presence of phentolamine Krebs; C.2 = Phentolamine Krebs alone, ( in C.2 equivalent volume of phentolamine ( 1 0 _ / M) Krebs was added instead of gamma g lobul in Krebs s o l u t i o n ) . Each point and ve r t i ca l bar represents mean ± SEM. - 83 -[GLOB] ATRO CONT 1 CONT 2 0.0 66.0 ± 10.8 44.3 ± 4.6 63.2 ± 9.6 1.4 73.6 ± 12.3 52.9 ± 6.5 58.5 ± 6.1 2.2 82.8 + 15.2 71.9 + 6.9 53.1 ± 7.4 3.0 102.6 ± 16.9 88.3 ± 4.7 56.6 ± 9.1 3.5 109.1 ± 16.0 94.5 ± 2.1 61.3 ± 9.5 3.8 111.5 ± 15.6 92.9 ± 2.2 61.3 ± 9.5 4.2 109.7 ± 14.7 93.6 ± 3.3 62.3 ±10.2 4.4 107.3 ± 15.5 88.7 ± 4.3 59.3 ± 8.9 TABLE 16 Spontaneous a c t i v i t y of the porta l vein in the response to gamma g lobu l in in the presence and absence of atropine 10 M (n = 6 ) . [GLOB] = Concentration of gamma globul in in mg/ml; ATRO = Gamma g lobu l in with atropine; CONT 1 = Gamma globul in alone; CONT 2 = Atropine alone without gamma g lobu l in . Results are expressed as % maximum response ± SEM. - 84 -2 0 0 UJ to z o CL CO 111 CC X < 1 0 0 0 1 2 3 4 [ G A M M A G L O B ] ( M G / M L ) o A T R • C . 1 A C . 2 FIG. 24 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) in response gamma g lobul in in the presence of atropine 1CT^ M. C . l = Gamma g lobu l in alone; ATR = gamma globul in in the presence of atropine; CONT 2 = Atropine Krebs alone, ( in C.2 equivalent volume of atropine (10 M) Krebs was added instead of gamma globul in Krebs). Each point and v e r t i c a l bar represents mean ± SEM. - 85 -[GLOB] KETAN CONT 1 CONT 2 0.0 27.2 ± 4.1 44.3 ± 4.1 32.7 ± 4.3 1.4 38.8 ± 4.9 52.9 ± 6.5 31.9 + 5.1 2.2 51.7 ± 7.4 71.2 ± 6.9 29.5 ± 4.5 3.0 64.5 ± 8.7 88.3 ± 4.7 31.3 ± 5.5 3.5 90.2 ± 12.2 94.5 ± 2.1 37.9 ± 5.7 3.8 103.8 ± 13.8 92.9 ± 2.2 38.4 ± 5.9 4.2 116.4 ± 15.4 93.6 ± 3.3 39.2 ± 6.6 4.4 117.1 ± 13.9 88.7 ± 4.3 38.0 ± 9.0 TABLE 17 Spontaneous a c t i v i t y of portal vein in response to gamma globul in in the _presence and absence of ketanserin 10~ b M (n = 6 ) . [GLOB] = Concentration of gamma g lobul in mg/ml; KETAN = gamma g lobu l in with ketanser in; CONT 1 = gamma globul in alone; CONT 2 = Ketenserin alone without gamma g lobu l in . Results are expressed as % maximum response ± SEM. - 86 -2 0 0 UJ CO Z O fl CO LU ft X < 1 0 0 -[ G A M M A G L O B ] ( M G / M L ) O K E T • ' G . 1 A C . 2 FIG. 25 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) in response to gamma g lobu l in in the presence of ketanserin 10~ b M. C . l = Gamma g lobul in alone; KET = Gamma globul in in the presence of ketanser in; C.2 = Ketanserin Krebs alone, ( in C.2 equivalent volume of ketanserin (10 M) Krebs was added instead of gamma globul in Krebs). Each point and ve r t i ca l bar represents mean ± SEM. - 87 -[GLOB] CH.LOR CONT 1 CONT 2 0.0 57.8 ± 4.0 51.0 ± 4.9 49.9 ± 4.2 1.4 72.8 ± 8.8 65.4 ± 6.3 44.9 ± 4.7 2.2 73.9 ± 5.8 76.2 ± 6.3 50.5 ± 3.5 3.0 87.8 + 7.6 87.7 ± 5.5 54.2 + 4.2 3.5 101.4 ± 10.3 96.2 + 2.4 54.9 ± 4.5 3.8 104.4 ± 12.2 84.7 ± 3.6 55.7 ± 4.6 4.2 94.4 ± 10.3 82.9 ± 6.1 56.7 + 3.8 4.4 90.5 ± 9.7 80.5 ± 5.3 55.5 ± 4.6 TABLE 18 Spontaneous a c t i v i t y of the portal vein in response to gamma g lobu l in in the_ presence and absence of chlorpheniramine 1 0 - 8 M (n = 6 ) . [6L0B] = Concentration of gamma - g lobul in in mg/ml; CHLOR = Gamma g lobu l in with chlorpheniramine; C0NT1 = Gamma g l i b u l i n alone;C0NT2 = Chlorpheniramine without gamma g lobu l in . Resul ts are expressed as % maximum response ± SEM. - 88 FIG. 26 % maximum of spontaneous a c t i v i t y of p o r t a l vein (n = 6) i n response to gamma g l o b u l i n i n the presence of chlorpheniramine 10 -^ M. C . l = Gamma g l o b u l i n alone; CHL ="Gamma g l o b u l i n in the presence of chlopheniramine; C.2 ^ c h l o r p h e n i r a m i n e Krebs alone, ( i n C.2 eq u i v a l e n t volume of chlorpheniramine (10~° M) Krebs was added i n s t e a d of gamma g l o b u l i n Krebs). Each point and v e r t i c a l bar represents mean ± SEM. - 89 -[GLOB] SARA CONT 1 CONT 2 0.0 49.4 ± 7.1 50.4 ± 4.4 49.9 ± 4.2 1.4 68.8 ± 11.7 65.4 ± 6.3 46.6 ± 4.7 2.2 67.3 ± 9.9 78.5 ± 6.4 46.1 ± 3.6 3.0 74.6 ± 7.1 87.7 ± 5.5 45.1 ± 4.2 3.5 81.1 ± 5.5 96.3 ± 2.3 44.9 ± 4.5 3.8 84.4 + 10.2 83.7 ± 3.3 42.7 ± 4.6 4.2 80.8 ± 6.7 82.9 ± 6.4 40.7 ± 3.8 4.4 79.5 ± 4.8 80.5 ± 5.3 41.6 ± 4.6 TABLE 19 Spontaneous a c t i v i t y of portal vein in response to gamma globul in in the presence and absence of sara las in 10 - ^ M (n = fa). [GLOB] = Concentration of gamma g lobul in in mg/ml; SARA = gamma g lobu l in with sa ra las in ; CONT 1 = gamma globul in without s a r a l a s i n ; CONT 2 = Sara las in with out gamma g lobu l in . Results are expressed as a % maximum response ± SEM. - 90 -2 0 0 1 0 0 -O o 1 2 ' [ G A M M A G L O B ] S A R • C . l ( M G / M L ) A C . 2 FIG. 27 % maximum of spontaneous a c t i v i t y of portal vein (n = 6) in response to gamma g lobu l in in the presence of sara las in I O - 9 M. C . l = Gamma g lobu l in alone; SAR = Spontaneous a c t i v i t y of portal vein in the presence of s a r a l a s i n ; C.2 = Sara las in Krebs alone, ( in C.2 equiva-lent volume of sara las in (10~ 9 M) Krebs was added instead of gamma g lobu l in Krebs) . Each point and v e r t i c a l bar represents mean ± SEM. - 91 -2 0 0 LU CO z o CL CO 0 I i i i i i 0 1 2 3 4 5 6 [ G A M M A G L O B ] ( M G / M L ) O G L O • V E R FIG. 28 % maximum of spontaneous a c t i v i t y of porta l vein (n = 6) in response to gamma g lobu l in in the presence of verapamil 1 0 - 8 M. GLO = Gamma g lobu l in alone; VER = Gamma g lobul in in the presence of verapamil. Each point and v e r t i c a l bar represents mean ± SEM. * is S i g n i f i -can t l y d i f fe rent from GLO (p < 0.05) . to verapamil (10~ M) was reduced 0.08 * O.Olg from 0 . 1 0 * O.Olg. In the absence of verapamil the gamma globul in increased the force from 0.10 ± O.Olg to 0.15 * 0.02g. The gamma globul in did not increase the spontaneous a c t i v i t y in the presence of verapamil (Table 20, F i g . 28). 3.4.3 Endothelium. The maximum force developed during spontaneous a c t i v i t y of the portal vein s t r i ps with and without endothelium was 0.06 * O.Olg and 0.05 * 0.03g respect ive ly . Table 21 and F i g . 29 shows the maximum response obtained in the portal vein with endothelium taken as 100% response. The maximum force obtained by the gamma globul in in the portal vein with endothe- lium (0.14 ± 0.02g) was not s i g n i f i c a n t l y d i f ferent from the maximum force obtained by gamma globul in without endothelium (0.12 ± 0.03g). The presence of endothelium was tested with acety lcho l ine. Aety lchol ine did not produce any re laxat ion in the portal vein in which the endothelium was removed but i t produced s ign i f i can t re laxat ion in the portal veins with intact endothelium. Therefore the increase in the spontaneous a c t i v i t y produced by the gamma globul in was not dependent on the endothelium. - 93 -[GLOB] CONT VERA 0.0 66.9 ± 6.3 53.8 ± 8.1 2.0 73.5 ± 7.2 50.2 + 7.8 2.5 81.9 ± 8.3 50.9 ± 6.9 3.0 87.1 ± 5.1 51.9 ± - 4 . 9 3.5 96.3 ± 2.6 53.5 ± 4.7 4.0 90.0 ± 2.4 52,2 ± 5.9 4.5 91.2 ± 3.1 51.7 ± 6.5 5.0 91.7 ± 3.2 45.2 ± 5.9 TABLE 20 Spontaneous a c t i v i t y of portal vein in response to gamma globul in the presence and absence of verapamil I 0 _ b M (n = 6) . LGL0B] Concentration of gamma g lobul in mg/ml; CONT = gamma globul in VERA = gamma globul in with 10 - ^ M verapamil. Results are expressed as % maximum response ± SEM. - 94 -[GLOB] INTACT DAMAG 0.0 66.9 ± 6.3 46.6 ± 9.6 2.0 73.5 ± 7.2 54.6 ± 10.8 2.5 81.9 ± 8.3 63.5 ± 11.3 3.0 87.1 ±"5.1 75.6 ± 12.2 3.5 96.3 ± 2.6 80.6 ± 10.5 4.0 90.0 ± 2.4 83.6 ± 10.9 4.5 91.2 ± 3.1 82.0 ± 11.9 5.0 91.7 ± 3.2 79.6 ± 14.0 TABLE 21 Spontaneous a c t i v i t y of portal vein with gamma globul in using intact/damaged endothelium (n = 6 ) . [GLOB] = Concentration of gamma g lobu l in in mg/ml; INTACT = Intact endothelium; DAMAG = Damaged endothelium. Results are expressed as % maximum response ± SEM. - 95 -FIG. 29 % maximum of spontaneous a c t i v i t y of portal vein in response to gamma g lobul in in the presence of endothelium and the absence of endothelium. INT = Gamma g lobul in in the presence of endothelium; DAM = Gamma g lobul in in t the absence of endothelium. Each point and v e r t i c a l bar represents mean ± SEM. - 96 -4 DISCUSSION 4.1 General I t i s well accepted that the primary abnormality in human essent ia l hypertension is the increase in peripheral vascular res is tance. Since the vascular smooth muscle is the regulator of the to ta l peripheral res is tance, the con t rac t i l e state of the vascular smooth muscle regulates the a r te r i a l blood pressure and organ blood f low. Current evidence suggests that the cont rac t i le apparatus of vascular smooth muscle is composed of th in and th ick f i laments, and the force generated between these two f i laments provides the mechanism of the c e l l shortening (Hartshorn et a l . 1980; Webb et a l . 1981; Page et a l . 1965). The molecular events that i n i t i a t e the in teract ion between these f i laments depend upon the sarcoplasmic concentration of a c t i -vator free calcium which is regulated by the c e l l membrane and at subcel lu lar s i t e s . Changes in the e l e c t r i c a l a c t i v i t y of the c e l l membrane and in te r -act ion of the pharmacological agents with membrane receptors cause ei ther a decrease or increase in the sarcoplasmic calcium concentration thus changing the con t rac t i l e state of the vascular smooth muscle c e l l (Kwan 1985). A l te ra t ions in the c e l l u l a r mechanisms that regulate the i n t r ace l l u l a r f ree calcium may contr ibute to the abnormal vascular funct ion in pathological s ta te . It is known that the con t rac t i l e state of the vascular smooth muscle i s al tered in essent ia l hypertension; however i t i s not c lear what causes th i s vascular smooth muscle abnormality. Whether or not the increased peripheral resistance i s due to s t ructura l or funct ional changes in the vascular smooth muscle is s t i l l con t rovers ia l . Evidence incr iminat ing s t ructura l changes in the vascular smooth muscle as primary changes is less than convincing (Aalkjaer et a l . 1987; Leung et a l . 1977; Cur t is et a l . 1978; Slack et a l . 1984). The st ructura l changes - 97 -observed (Curt is et a l . 1978; Slack et a l . 1984) may be due to secondary adaptive changes to the elevated blood pressure. Lee (1986) studied s t ructura l changes in d i f ferent models of hypertension and concluded that a l l models of hypertension shared common changes in the endothelium, media and advent i t ia layers of the vessel wa l l . However, changes in the endothe-lium and advent i t ia are general ly secondary to changes to the elevated blood pressure. In contrast , hyperplasia of medial smooth muscle c e l l s is a primary change related to the development of hypertension; whereas hyper-trophy of the smooth muscle c e l l is a secondary adaptive change. This conclusion was based on the observation that the medial hyperplasia was seen in the prehypertensive s ta te . Even though th i s change was seen in the prehypertensive s ta te , i t i s not c lear i f the hyperplasia is produced by an unknown agent(s) or f ac to r ( s ) , which i n i t i a t e s the chain of processes which u l t imate ly leads to increased peripheral res is tance. The funct ional changes in essent ia l hypertension are as controvers ia l as s t ructura l changes. I t was shown that aor t ic s t r ips from SHR produced decreased contract ion with NA compared to t he i r control normotensive rats (Spray et a l . 1977; Spector et a l . 1969; Antanaicco et a l . 1980). A number of studies of the iso lated perfused mesenteric preparations from SHR indicate that the reac t i v i t y and c o n t r a c t i l i t y are raised and the threshold agonist s e n s i t i v i t y is increased in vessels from the SHR compared to vessels from normotensive control rats (Sutter et a l . 1977). I t was suggested by F i t zpa t r i ck et a l . (1980) that the vascular smooth muscles of SHR possess an i n t r i n s i c myogenic tone, possibly due to increased permeabil i ty to Ca ; and the decreased responsiveness of SHR s t r i ps in the presence of C a + + i s only an apparent hyporesponsiveness which may be at t r ibutab le to the pre-ex is t ing tone. When the aor t ic s t r ips from SHR were exposed to Ca free - 98 -medium, the response produced by the aor t ic s t r i ps from SHR and WKY rats to agonist plus Ca were s im i l a r . Some of the di f ference between the resul ts from in vivo and in v i t ro studies may be related to neuronal inf luences i nd i rec t l y af fect ing the sens i t i v i t y of the t issues to NA, or due to the inf luence of s t ructura l adaptations in the vascular smooth muscles. Aorta is exposed to high blood pressure, yet i t seldom contr ibutes to increased peripheral res is tance. Therefore the aorta is a poor model for the r e s i s -tance blood vessels . There is ample evidence to show that the s e n s i t i v i t y of the vascular smooth muscle to C a + + i s al tered in essent ia l hypertension. I t was shown (Sutter et a l . 1977) that portal vein s t r i ps from SHR retained t he i r c o n t r a c t i l i t y in low Ca better than those from the normotensive ra ts . This may be due to a l tera t ion in the Ca permeabil i ty of the vascular smooth muscle. There i s not much d i rect evidence to show that the hyperten-sive vascular smooth muscle, per se, handles Ca d i f f e ren t l y . Sut ter (1985) studied the ionic permeabil i ty of vascular smooth muscles from SHR using C a + + channel blockers and did not f ind any di f ference between the C a + + handling of the vascular smooth muscles of SHR, WKY and Wistar ra ts . The only evidence for the increase in C a + + permeabil i ty came from the studies of the blood c e l l s (Chan et a l . 1983; Devynck et a l . 1981; Parker et a l . 1983). 4.2 Our Invest igat ions + 4.2.1 Response to K and NA in aort ic s t r ips and portal ve ins. In t h i s present study, the aor t ic s t r i ps and portal veins exposed to normotensive plasma did not produce any measurable contract ion with NA ( 1 0 - 4 M) or K + (100 mM). NA act ivates vascular smooth muscle contract ion not only by st imulat ing Ca entry through the voltage - 99 -sensi t ive calcium channels (VOC) but also by st imulat ion of C a + + entry both through receptor operated channels of the c e l l membrane (ROC) and also through the release of internal calcium bound within the c e l l s (Lipe et a l .1985). Potassium is thought to produce vascular smooth muscle contraction p r i n c i p a l l y by st imulat ion of the entry of C a + + into the c e l l through the voltage sens i t ive calcium channels of the c e l l membrane (Lipe et a l . 1985). The portal vein is dependent on the in f lux of external calcium for the i n i t i a t i o n and maintenance of contract ion produced by K + and NA (Sutter 1976). The absence of the response to K + and NA observed in th i s study may be due to less avai lab le ex t race l lu la r calcium in the plasma (plasma [Ca + + ] 1.4 mM), or to reduced a v a i l a b i l i t y of K + and NA for act ivat ion due to binding of these agonists to plasma prote ins. NA response was not dependent on external calcium for the i n i t i a t i o n of response in aor t ic s t r i ps (Sutter 1976), the rapid phasic con t rac t i l e response was due to the release of i n t r ace l l u l a r C a + + . Even in zero ++ ++ external Ca aor t ic s t r i ps produce 25-55% of the control (2.5 mM Ca ) response to NA (Sutter 1976). Therefore th i s absence of response to NA seen in the aor t ic s t r i ps exposed to plasma can not be due to the low C a + + present in the plasma. When the portal veins were exposed to normotensive plasma during the prel iminary invest igat ions, the spontaneous a c t i v i t y of the portal vein was reduced gradual ly and i t was almost abolished at the end of 20 min. The spontaneous a c t i v i t y of the portal vein is myogenic and is dependent on ++ external Ca . The loss of spontaneous ac t i v i t y observed may not be due to the di f ference in the C a + + content of the plasma and Krebs since spontaneous a c t i v i t y was seen at reduced (0.8 mM) external C a + + concentra-t ion (Sutter 1976). - 100 -++ + Addi t ion of 2.5 mM Ca allowed a measurable response to K and NA in the aor t ic s t r i ps exposed to normotensive plasma but not in the portal veins. The spontaneous ac t i v i t y was increased by the addit ion of 2.5 mM C a + + to normotensive plasma but i t was s i g n i f i c a n t l y less than the level seen with Krebs ( C a + + 2.5 mM). The response to K + and NA did not increase with further increases in Ca . The addit ion of Ca may have produced an increase in the avai lable ex t race l l u la r C a + + . The amount of external calcium af ter addit ion of 2.5 mM Ca may have been s t i l l too low + to produce contract ion with K and NA in the portal vein because the portal vein depends exc lus ive ly on external calcium for the contract ions produced by K + and NA (Co l l i ns et a l .1972). The maximum response to NA produced in the aor t ic s t r i ps exposed to hypertensive plasma was s i g n i f i c a n t l y higher than in aor t ic s t r ips exposed to normotensive plasma (F ig . 1) . This increase is not due to the added calcium in the plasma because the same amount of calcium was added to both plasmas. This increase also is not due to the a lka l ine pH of the plasma or hypoxic condit ion under which the experiment was carr ied out (F ig . 2) since controls were pH matched and not bubbled with carbogen. This indicates that the hypertensive plasma must be acting d i f f e ren t l y from the normotensive plasma. The mechanism by which the hypertensive plasma increases the responsiveness of the aor t ic s t r ips to NA was not examined in the study. Possible mechanisms include a d i rec t e f fect on plasma membrane permeabi l i ty, ion ic f luxes , or the agonist receptor in teract ion or d i rect or ind i rec t e f fec t on i n t r ace l l u l a r calcium. However, the a l te ra t ion of i n t r ace l l u l a r C a + + could be due to the in f lux of ex t race l lu la r calcium which provides an increase in the i n t r ace l l u l a r calcium store. This calcium could be released by the agonist NA or and by the act ivat ion of i nos i to l phosphate pathway resu l t ing in an increase in the i n t r a c e l l u l a r C a + + level (Heagerty 1986). - 101 -+ The maximum response to K observed in the aor t ic s t r ips exposed to hypertensive plasma was less than the controls (aor t ic s t r ips exposed to Krebs pH 7.4); but the maximum response to K + observed in the aor t i c s t r i ps exposed to normotensive plasma was s i g n i f i c a n t l y higher than that of the hypertensive plasma (F ig . 3 ) . Potassium produces vascular smooth muscle contract ion mainly by st imulat ion of the entry of external calcium in to the c e l l through the voltage sensi t ive calcium channels of the c e l l membrane (Lipe et a l . 1985). The decreased response to K + seen in the aor t i c s t r i ps exposed to hypertensive plasma may be due to the decrease in the entry of ex t race l l u la r calcium into the c e l l . The mechanism by which the hypertensive plasma reduces the response to K + in the aor t ic t issue i s not known. I t was observed in p la te le ts that the normotensive p la te le ts exposed to ++ hypertensive plasma had increased cy toso l i c Ca ; normotensive p la te le t s exposed to normotensive plasma did not show any di f ference (Linder et a l .1987). When calcium is infused intravenously into hypertensive pat ients and normotensive people at the same ra te , serum calcium rose almost l i nea r l y in a l l subjects, although at a lower rate in hypertensive subjects; serum increment over base l ine was s i g n i f i c a n t l y lower in hypertensives than in controls at the end of in fus ion. There was no s ign i f i can t di f ference in the calcium excret ion in both groups. S t razzu l lo et a l . (1986) suggested that the decrease in the serum C a + + in hypertensives was due to an increase ++ in the net entry of Ca into the c e l l u l a r compartment. In our s tud ies , the increased responsiveness to NA seen in the aor t i c s t r i ps exposed to hypertensive plasma compared to normotensive plasma may also be due to increase in the i n t r ace l l u l a r C a + + in the aor t ic s t r i ps exposed to hypertensive plasma. The mechanism by which the hypertensive - 102 -plasma increases the i n t r ace l l u l a r C a + + is not c lear at t h i s stage. This ++ could be due to the act ivat ion of i n t r ace l l u l a r Ca releasing process or increase of the calcium permeabil i ty of the aor t ic t issue and increasing the i n t r a c e l l u l a r C a + + stores which could be released by NA. Portal veins are very sens i t ive to ex t race l l u la r calcium concentrat ion, spontaneous contract ions and response to a l l agonists i s los t within 10 min of p lacing the t issue in calcium free solut ion (Cuthbert et a l . 1964; Axelsson et a l . 1967). When the portal veins were exposed to hypertensive and normotensive plasma in the presence of 2.5 mM C a + + , the spontaneous a c t i v i t y was gradual ly abolished in the veins exposed to hypertensive plasma, however the veins exposed to normotensive plasma retained a l i t t l e spontaneous ac t i v i t y (F ig . 5, 6 ) . NA did not produce any response in the portal veins exposed to normotensive or to hypertensive plasma (F ig . 5) but portal veins in the presence of normotensive plasma showed an increased response to K + compared to veins exposed to hypertensive plasma (F ig . 6) . Spontaneous a c t i v i t y was more sens i t ive to external calcium than the response to NA. The myogenic a c t i v i t y was general ly absent at 0.4 mM Ca and was general ly present at 0.8 mM Ca but the spontaneous ac t i v i t y was about 10% of that with C a + + 2.5 mM (Sutter 1976). Even though contract ion produced by both K and NA i s dependent on ex t race l l u la r calcium in the portal ve ins , i t is not c lear why NA did not produce at least an increase in the spontaneous ac t i v i t y of the portal vein. NA usual ly induces spontaneous a c t i v i t y at Ca concentration as low as 0.2 mM Ca (Sutter 1976). I t was shown by other invest igators that the portal veins exposed to C a + + f ree Krebs solut ion for 15 min did not e l i c i t any response to NA or K + (Co l l ins et a l . 1972). This dependence on ex t race l l u la r Ca may be because a l l of the act ivator calcium came from external f l u i d and there i s - 103 -no t i gh t l y bound store of C a + + which can be released by agonist or perhaps t i g h t l y bound Ca may act as a source of act ivator Ca but the presence of ex t race l lu la r C a + + i s required for i t s release (Co l l i ns et a l . 1972). The absence of response to NA in the portal veins exposed to normotensive and hypertensive plasma may be also due to the decrease in the ex t race l l u la r Ca . I t i s not c lear how these plasmas reduce the ex t race l lu la r calcium. The reason for the di f ference seen in the portal vein and aor t ic s t r i ps exposed to the plasmas (normotensive and hypertensive plasma) are unknown. Based on the prel iminary observations of the spontaneous a c t i v i t y of the porta l veins exposed to the normotensive and hypertensive plasmas, a cumula-t i ve dose response to normotensive and hypertensive plasma was done to see how the spontaneous ac t i v i t y of the portal vein was altered with each of these plasmas. The spontaneous ac t i v i t y of the portal vein i s myogenic, i t depends on ++ the membrane propert ies of the vascular smooth muscle c e l l s . At low Ca concentrat ions, the myogenic contract ions of the portal veins are more depressed than those induced by NA (Sutter 1976). The myogenic tone is due ++ to the in f lux of Ca from the external medium, th i s myogenic tone i s depressed by reduction of C a + + in the immediate chemical environment (Sutter 1976). 4.2.2 Spontaneous ac t i v i t y of portal vein with plasma. When the portal veins were exposed to e i ther normotensive or hypertensive plasma, the spontaneous ac t i v i t y increased at very low concentrations of the plasma (both plasmas). Normotensive or hypertensive plasma showed maximum sponta-neous ac t i v i t y at 50% volume of plasma. However, the maximum response observed with the hypertensive plasma was more than that with the normoten-sive plasma. Since the spontaneous ac t i v i t y of the portal vein is dependent - 104 -++ on the in f lux of Ca from the external medium,the increased spontaneous a c t i v i t y produced by these plasmas also may be due to the in f lux of C a + + in to the c e l l . The in f lux of Ca into the c e l l s exposed to hypertensive plasma must be more than the c e l l s exposed to normotensive plasma to produce th is di f ference in the spontaneous ac t i v i t y (F ig . 8 and 9 ) . Further increase in the concentration of the plasma (both plasmas) decreases the spontaneous a c t i v i t y . Even though both these plasmas eventual ly decrease the spontaneous a c t i v i t y , at 60% volume of the plasma the spontaneous a c t i v i t y observed with the hypertensive plasma was s t i l l s i g n i f i c a n t l y higher than the normotensive plasma. This increase in spontaneous a c t i v i t y with low concentration and decrease in the spontaneous ac t i v i t y with higher concentration of plasma indicates that both normotensive and hypertensive plasma may have st imulatory and inh ib i tory factors in the plasma and the amount of these factor(s) may vary. Since the spontaneous a c t i v i t y at a l l concentrations of the hyperten-s ive plasma was more than that of normotensive plasma i t i s probable that the st imulatory factor may be of more importance in the hypertensive plasma. This st imulatory factor (s) may be increasing the C a + + permeabil i ty of the plasma membranes to Ca and increase the spontaneous rhythmic ac t i v i t y of the vascular smooth muscle c e l l s and the tone of the vascular smooth muscle. Since the portal vein i s c losest to the peripheral resistance vessels (Ljung 1970; Rhodes and Sutter 1971), and increased spontaneous a c t i v i t y also means increased e x c i t a b i l i t y , i t i s possible that th is putative stimulatory factor may also increase the vascular tone and the peripheral res is tance. The spontaneous ac t i v i t y of the portal vein was elevated by increasing concentrations of normotensive plasma, normotensive heated plasma or normo-tensive serum up to 50% volume. The maximum responses to a l l three were not s i g n i f i c a n t l y d i f f e ren t . They also decreased spontaneous a c t i v i t y at higher - 105 -concentrations in a s imi la r manner (F i g . 7). This indicates that there i s no s ign i f i can t di f ference in the behavior of the t issues exposed to plasma, serum and heated plasma. Therefore the vascular sens i t i z ing factor (s) in the plasma was not heat sens i t ive and i s present in serum f rac t ions . 4.2.3 Spontaneous a c t i v i t y of portal vein with plasma f rac t i ons . Albumin and globul ins are the major proteins present in the serum or plasma f rac t i ons . Albumin is the major plasma protein in the plasma and i t i s about 55-65% of the to ta l plasma protein (Phys ic ian 's hand book). The spontaneous a c t i v i t y of the portal vein was increased in a dose dependent manner with albumin (F ig . 10). The maximum response was obtained at 40% concentration of the albumin normally present in the normotensive plasma (16 mg/ml). This increase was not due to the change in the temperature of the bath due to the added volume of f l u i d , increase in the volume of the f l u i d in the t i ssue bath or due to the a lka l ine pH of albumin (pH of albumin solut ion 7.4-7.6) . The ident ica l condit ions without albumin did not produce an increase in the spontaneous ac t i v i t y (F ig . 10). The increase in the spontaneous ac t i v i t y produced by albumin was not dependent on the membrane potent ia l of the vascular smooth muscle c e l l s . Albumin s t i l l increased the spontaneous a c t i v i t y of the portal vein depolarized by ouabain ( F i g . 20); but the increase in the spontaneous ac t i v i t y produced by albumin was completely blocked by phentolamine 10"^ M (F ig . 19). This indicated that albumin i s not dependent on the membrane potent ia l for i t s ac t ion , but i t s act ion dependent on a-adrenoreceptors. Mathews and Sutter (1967) demonstrated that NA produced the same or more response in the portal vein depolarized by ouabain or K + compared to portal vein without depolar izat ion. I t was suggested that the stimulant drugs are able to contract the anter ior mesenteric portal vein both v ia act ion potent ia ls and by mechanisms - 106 -r e l a t i v e l y independent of the e l e c t r i c a l events at the c e l l membrane. Since albumin behaved in a s imi la r manner as NA, spontaneous ac t i v i t y produced by albumin was completely blocked by phentolamine, albumin could be re leasing NA from the nerve terminals or acting l i k e NA. Albumin also st imulates the spontaneous a c t i v i t y of a chemical ly denervated portal vein (by 6-OHDA) (F i g . 21) which indicates that albumin i s behaving l i ke NA. The increase in the spontaneous a c t i v i t y produced by albumin in the denervated portal vein was more than that without denervation. The gamma globul in f rac t ion of the plasma protein is about 35-45% of the to ta l plasma prote ins. The- g lobul in f rac t ion i s again subdivided into a -g lobu l in (14.2-18.1%), e-globul in (14.2-18.1%) and gamma globul in (16.6-26.5%) (Phys ic ian 's hand book). The gamma globul in component contains a l l the immunoglobulins. IgG is the major immunoglobulin in the serum forming about 70% of the to ta l immunoglobulin, and i s evenly d is t r ibuted within the intravascular and extravascular pool . IgM represents about 10% of the to ta l immunoglobulin and is largely confined to the intravascular pool . Other immunoglobulins, IgE, IgA and IgD are present in very low concentrat ions. Gamma globul in also caused a dose dependent increase in the spontaneous a c t i v i t y of the portal vein (F i g . 11-14). The maximum spontaneous a c t i v i t y was obtained with the gamma globul in at concentration of 4.2-5 mg/ml, which is about 40% of gamma globul in normally present in the normotensive plasma. The increase in the spontaneous ac t i v i t y produced by gamma globul in seems dependent on the membrane potent ia l of the portal ve in . Depolar izing the membrane by ouabain blocked the increase produced by gamma globul in ( F i g . 22). The increase in spontaneous ac t i v i t y produced by gamma globul in -7 -5 was not blocked by phentolamine 10 M, atropine 10 M, chlorpheniramine - 107 -10~ 8 M, ketanserin 10~ b M or sara las in 10~ 9 M. This indicates that the a c t i v i t y of gamma g lobul in is not effected by a -adrenergic, cho l i ne rg i c , serotoninergic or histaminergic transmit ters or the renin angiotensin system ( F i g . 23-27). The increase in the spontaneous a c t i v i t y of the portal vein _o produced by gamma globul in was blocked by verapamil 10 M (F ig . 28). This suggests that the increases in the spontaneous a c t i v i t y produced by ++ gamma g lobul in was dependent on the in f lux of Ca through the voltage dependent C a + + channels. The probable mechanism by which the gamma globu-l i n increases the in f lux of C a + + through the voltage dependent C a + + channels may be by depolar iz ing the plasma membrane and ac t iva t ing the potent ia l dependent C a + + channels to open. a - and e-globul in together i nh ib i t the spontaneous a c t i v i t y of the portal vein (F ig . 15). a -g lobu l in by i t s e l f i nh ib i t s the spontaneous a c t i v i t y ( F i g . 15). I t is not known at th i s point whether e-globul in by i t s e l f has an inh ib i to ry ac t ion . I t was not tested alone because of the i n a b i l i t y to purchase e-globul in alone. Since both the inh ib i to ry and stimulatory ef fects are present in the g lobul in f rac t ion of the plasma pro te in , g lobul in may play an important ro le in the regulat ion of the vascular tone. These stimu-latory and inh ib i to ry e f fec ts of the g lobul in together may be responsible for the maintenance of the vascular tone. Once th is normal balance is disturbed e i ther by the increase in the st imulatory e f fec t or decrease in the inh ib i to ry ef fect of g lobu l in , the vascular tone w i l l be increased. The gamma g lobu l in contains a l l the major immunoglobulins present in the serum. The immunoglobulin IgG which i s the major immunoglobulin in the serum (Burton et a l . 1986) also increased the spontaneous ac t i v i t y of the portal vein (F ig . 16). I t was suggested that the IgE mediates release of histamine and other mast c e l l products and these may produce coronary artery spasm in - 108 -animals (Shimokawa et a l . 1983), but the increase in spontaneous a c t i v i t y produced by gamma globul in is not mediated by the release of histamine ( F i g . 26). The maximum response produced by IgG was not s i g n i f i c a n t l y d i f fe rent from that of gamma globul in (F ig . 16-18). This indicates that IgG may be the immunoglobulin in the gamma globul in which increased the spontaneous a c t i v i t y of the portal ve in. I t i s possible that IgG may be the st imulatory fac tor present in the normotensive plasma which increased the spontaneous a c t i v i t y of the portal ve in. The increase in the spontaneous a c t i v i t y produced by gamma globul in was not dependent on the presence of intact endothelium (F ig . 29). Recent invest igat ions (Yanagisawa et a l . 1988) have described a protease sens i t i ve vasoconstr ictor a c t i v i t y in the supernatant of cul tured endothel ial c e l l s . This a c t i v i t y is dependent on the presence of ex t race l l u la r calcium and is not affected by blocking the action of a-adrenergic, cho l inerg ic , seroto-nergic or histaminergic neurotransmitters (Yanagisawa et a l . 1988). The production of th i s peptidergic substance by endothel ial c e l l i s influenced by NA, anoxia and neuropeptide Y (Daly et a l . 1987). The vasoconstr ictor peptide endothelin was iso lated by Yanagisawa et a l . (1988). They showed that endothelin induced vasoconstr ic t ion depended on the presence of ex t ra -c e l l u l a r calcium and was inh ib i ted by low dose of n icard ip ine, suggesting that the endothelin act ion was c lose ly associated with C a + + in f lux through the dihydropyridine sensi t ive C a + + channels (Daly et a l . 1987). Even though the gamma globul in induced spontaneous a c t i v i t y is blocked by vera-pamil which also blocks the dihydropyridine sens i t ive C a + + channels, i t i s not releasing the vasoconstr ictor endothelin from the endothelium, because the increase in the spontaneous a c t i v i t y is not dependent on the endothelium ( F i g . 29). There is no s ign i f i can t di f ference between the response produced - 109 -by the gamma g lobul in in the portal vein with and without endothelium. The hypoxic condit ion under which the gamma globul in dose response was done cannot be accounted for the observed response. The same condit ions without gamma globul in did not increase the spontaneous ac t i v i t y (F ig . 11). The act ion of the gamma globul in on the portal vein is due to the d i rec t act ion on the vascular smooth muscles. The IgG may be acting d i r ec t l y on the vascular smooth muscles and increasing the permeabil i ty of the vascular „ ++ smooth muscles to Ca . Increases in IgG in plasma and serum of hypertensive patients were reported in c l i n i c a l studies (Kristensen 1978; Kristensen et a l . 1983; Olsen et a l . 1973; Ebringer et a l . 1971; Gudbrandsson et a l . 1981). Furthermore i t was demonstrated that IgG was increased in treated as well as untreated hypertensive patients (Kristensen 1978; Ebringer et a l . 1970), IgG level also correlated pos i t i ve l y to blood pressure in untreatd as well as insuf-f i c i e n t l y treated patients (Kristensen 1978). Ebringer and Doyle (1970) suggested that ra ised levels of IgG in severe hypertension were secondary to the raised blood pressure. Others (Ebringer and Doyle 1970; Olsen 1972; Kristensen 1978) suggested a possible primary immunological disturbance. We also demonstrated that the stimulatory factor (present in the normotensive plasma) which increased the spontaneous a c t i v i t y of the portal vein is present in IgG. Perhaps, the greater in spontaneous ac t i v i t y caused by the hypertensive plasma could be due to the increased amount of IgG present in the hypertensive plasma. The pr inc ipa l disturbance in organ or t issue re jec t ion , which resul ts in the destruct ion of organ or t issue graft within a short t ime, is rap id ly progressive ischemia. Rosenberg et a l . (1971) reported that during the hyperacute re jec t ion , within 8 min. the perfusion sinks to 1/60 of i n i t i a l - 110 -volume and the vascular resistance increases f i f t y f o l d . Hobbs (1972) observed conspicuous al ternat ing vasoconstr ict ion and vasodi la tat ion a f f l i c -t ing the g ra f t , but not the host 's own vesse ls . These rap id ly increasing contract ions apparently are symptoms of vasospastic c r i s i s (Hobbs 1972; Demster 1970). The vasoconstr ict ion of hyperacute re ject ion is character ized by i t s fulminating onset and resistance to a l l therapeutic agents used; i t s b ru ta l i t y is unique among a l l defensive react ion in immunology (Hobbs 1972). Recently calcium channel blockade has been shown to prevent renal ischemic damage (Wagner et a l . 1987; Agatstein et a l . 1987; Foeghet et a l . 1985). IgG also produces increase in vascular tone and the mechanism by which i t increases the vascular tone is also calcium channel re la ted . The IgG may play a ro le in producing vasoconstr ic t ion during hyperacute t issue re jec t ion . IgG is a 150 kD protein with 4-chain domain structure consist ing of two l ight and two heavy chains. Each heavy chain is folded into two domains in the Fab arm (antigen binding arm) forms a region of extended polypeptide chain in the hinge and is then folded into two domains in the Fc region (complement f i x i ng region). The l ight chain forms two domains associated only with Fab arm. Each domain has a molecular weight varying from 12 kD leading to a molecular weight of 50 kD for Fc and Fab fragments (Burton et a l . 1986). I t i s not c lear whether the intact IgG molecule is necessary to increase the permeabil i ty of the vascular smooth muscle to C a + + or the fragments i t s e l f have th i s property. Zidek et a l . (1988) obtained a sub-stance with molecular mass 1-1.5 kD from essent ia l hypertensive subjects and showed that th i s f rac t ion when injected intravenously into normotensive rats increased blood pressure. The same f rac t ion s i g n i f i c a n t l y accelerated neutrophil Ca uptake by permeabilized neutrophi ls after addit ion of ex t race l l u la r C a + + . As C a + + transport has been observed in permeabilized - I l l -c e l l s , the question ar ises as to how far the ef fects are comparable with condit ions in v ivo . This small molecular f rac t ion extracted from hyperten-sive plasma could be the fragments of IgG molecule fragmented during the ge l l f i l t r a t i o n process, (during separation of the plasma f r ac t i on ) . Wright et a l . (1986) and McCumbee et a l . (1985) observed an increase 45 ++ uptake of Ca in aor t ic t issues when incubated with small molecular weight f rac t ions extracted from SHR red blood c e l l s . Intravenous in jec t ion of th i s f rac t ion into normotensive rats increased the blood pressure (McCumbee et a l . 1985). This small molecular weight peptide did not have any ef fect on rest ing tension; but i t s i g n i f i c a n t l y enhanced K + and NA induced con t rac t i l e response in the aor t ic s t r i p s . The con t rac t i l e ef fects of t h i s peptide were abolished by removal of ex t race l lu la r calcium or addi t ion of calcium channel antagonists, verapamil and n i fed ip ine . There was no s ign i f i can t di f ference in the rate or magnitude of re laxat ion to verapamil or n i fed ip ine in K + stimulated aor t ic rings with or without peptide incubation (Huang et a l . 1988). The above invest igators suggested that the mechanism of action of the peptide is Ca channel related and also that peptide i t s e l f played no d i rec t ro le in the opening of the C a + + channels (Huang et a l . 1988). They also showed that th i s peptide has s imi la r e f fec ts to Bay K8644, a calcium channel agonist . The mechanism of act ion of the peptide as well as the gamma globul in are calcium channel re la ted . It is not known whether the small molecular weight peptide which was extracted from the RBC of SHR are the fragments of IgG adsorbed to the RBC or a d i f ferent peptide. The blood pressure in the hypertensives may be related to the amount or the potency of the vascular sens i t i z ing agent in the plasma. Whether the nature of the IgG molecule/submolecules or only the amount of IgG is - 112 -increased in essent ia l hypertension is not known at th i s point . IgG is formed as a resu l t of prolonged antigenic st imulat ion or as a secondary immune response. The increase in the amount of the IgG could be due to the a l te ra t ion in the mechanism of the immune response. The increase in the potency of IgG in essent ia l hypertension may be due to some s t ruc tura l a l te ra t ion in the IgG molecule. Ei ther of these could produce an increase in the C a + + permeabil i ty of the vascular smooth muscle. Separation of d i f ferent plasma f rac t ions from essent ia l hypertensive plasma and studying the nature and structure of the f rac t ions which produce the increase in the vascular s e n s i t i v i t y would answer some of these questions. - 113 -5 CONCLUSION Based on the invest igat ive study and the discussion, the fol lowing conclusions are made. I t i s evident from the responsiveness obtained from the aor t ic s t r ips exposed to hypertensive plasma to NA and the increased spontaneous a c t i v i t y of the portal vein produced by the hypertensive plasma compared to normoten-sive plasma that the plasma of essent ia l hypertensives has ef fects d i f fe rent from that of normotensives. Observations of the spontaneous ac t i v i t y of portal vein exposed to the normotensive or the hypertensive plasma again c l e a r l y indicate that both these plasmas contain a vascular sens i t i z ing agent but the a c t i v i t y of t h i s agent is s i g n i f i c a n t l y more in the hypertensive plasma (F ig . 5, 6). However, i t i s not c lear whether the absolute amount of t h i s agent is increased in the hypertensive subjects or the nature of the agent in the hypertensives i s changed. The spontaneous ac t i v i t y of the portal vein depends on the in f lux of external C a + + . Increased spontaneous ac t i v i t y of the portal vein produced by gamma globul in is dependent d i r ec t l y on the membrane potent ial of the vascular smooth muscle as well as the inf lux of the C a + + through the calcium channels, but the increased spontaneous ac t i v i t y produced by albumin i s dependent on the in f lux of C a + + i nd i r ec t l y through the act ivat ion of a-adrenoreceptors. A vascular sens i t i z ing agent may or ig inate in the gamma globul in f rac t ion of the plasma prote ins. The vascular tone i s al tered by the st imulatory ef fect of the gamma globul in and the inh ib i tory ef fect of a- and 6 -g lobul ins . When the st imulat ing ac t i v i t y of the gamma globul in increases the balance between the inh ib i to ry and the stimulatory ef fect - 114 -could be disturbed and the vascular tone increased. I t is not c lear whether or how th is stimulatory ef fect produced by the gamma globul in is increased in essent ia l hypertension. Also the study provides further evidence that the plasma factor (agent) which increases the vascular s e n s i t i v i t y is present in the IgG f rac t ion of the immunoglobulin, which i s a major component (> 70%) of the gamma g lobu l in . The IgG is produced as an immune response and i t is not known whether there i s an abnormality in the immune response in essent ia l hypertensives. The increase in the vascular tone produced by the plasma factor (agent) in essent ia l hypertension i s Ca and potent ia l dependent. The increase in the vascular tone produced by the globul in f rac t ion is antagonized by verapamil, but the mechanism by which gamma globul in increases the in f lux of C a + + through the calcium channels is not known. Monitoring th is f rac t ion in people with essent ia l hypertension as well as ear ly hypertensives might be a d iagnos t i ca l l y valuable measure at ear ly stages of development of hypertension. 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