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The effects of morphine on the hypothalamo-neurohypophyseal system Ngsee, Johnny Kuan 1979

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9 1 THE EFFECTS OF MORPHINE ON THE HYPOTHALAMO-NEUROHYPOPHYSEAL SYSTEM by JOHNNY KUAN NGSEE B.Sc. , the U n i v e r s i t y o f B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT.OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1979 (c) Johnny Kuan Ngsee, 1979 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I further agree that permission for extensive copying of th i s thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t i on of th is thes is fo r f inanc ia l gain sha l l not be allowed without my written permission. Department of Physiology The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date October 11, 1979 i i ABSTRACT The acute a d m i n i s t r a t i o n of an a n a l g e s i c dose (1mg/kg) of morphine s u l f a t e to conscious and hydrated r a t s produced a pronounced a n t i d i u r e t i c response. T h i s was accompanied by an i n c r e a s e i n u r i n e o s m o l a l i t y and a decrease i n f r e e water c l e a r a n c e i n the normal and B r a t t l e b o r o r a t s heterozygous f o r the d i a b e t e s i n s i p i d u s t r a i t * The response was comparable t o t h a t of an exogenous dose of v a s o p r e s s i n . A n t i d i u r e s i s was a l s o observed i n the homozygous D . I . . r a t s . Since these r a t s are i n c a p a b l e of s y n t h e s i z i n g v a s o p r e s s i n , the a n t i d i u r e s i s must be mediated by other mechanism(s). When a r t e r i a l blood pressure was monitored, i t was found that the mean a r t e r i a l pressure decreased s h a r p l y immediately a f t e r the morphine i n j e c t i o n i n a l l animals. T h i s can account f o r the a n t i d i u r e s i s i n the homozygous D.I. r a t s . In the normal and heterozygous D.I. animals, i t i s q u i t e p o s s i b l e that both morphine-mediated r e l e a s e of v a s o p r e s s i n and hypotension are r e s p o n s i b l e f o r the decrease i n u r i n a r y flow. In a d d i t i o n , hypotension per se may act as a s t i m u l u s f o r v a s o p r e s s i n r e l e a s e . To study the c h r o n i c e f f e c t s of morphine, r a t s were rendered t o l e r a n t and p h y s i c a l l y dependent by two means: m u l t i p l e i n j e c t i o n s and p e l l e t i m p l a n t a t i o n of morphine s u l f a t e . In c o n t r a s t to the a n t i d i u r e t i c e f f e c t s of acute morphine a d m i n i s t r a t i o n , c h r o n i c treatment r e s u l t e d i n p o l y u r i a . Using a v a s o p r e s s i n radioimmunoassay (RIA), i t was found t h a t r a t s implanted with a morphine i i i p e l l e t f o r 3 days had a s i g n i f i c a n t l y lower neurohypophyseal s t o r e of v a s o p r e s s i n (744 . 3 ± 27.9 ng, n=6) as compared to the placebo p e l l e t implanted c o n t r o l s (1024. 1 ± 66.0 ng, n=6). This d e p l e t i o n was r e p l e n i s h e d as the animals developed t o l e r a n c e to the drug.. abrupt withdrawal of the drug from p h y s i c a l l y dependent animals a l s o produced a s i g n i f i c a n t d e p l e t i o n of the neurohypophyseal v a s o p r e s s i n s t o r e s - from 902.4 ± 37.0 ng (n=6) to 638. 3 ± 36.0 ng (n=6). In c o n t r a s t to r a t s implanted with morphine p e l l e t s , no s i g n i f i c a n t changes i n the neurohypophyseal v a s o p r e s s i n s t o r e s were observed i n those i n j e c t e d d a i l y with morphine f o r 2 weeks..Withdrawal from the drug i n these animals a l s o d i d not produce any d e t e c t a b l e changes i n the v a s o p r e s s i n s t o r e s . A withdrawal symptom, r e d u c t i o n i n body weight, monitored a f t e r 24 hr of abstinence suggested that the degree of p h y s i c a l dependence i n these animals i s very l i g h t . 3H-naloxone b i n d i n g performed on whole b r a i n homogenate of r a t s i n j e c t e d with morphine f o r 2 weeks rev e a l e d no s i g n i f i c a n t changes i n the number of bin d i n g s i t a s (q). The a f f i n i t y constant (Kd) was augmented from a c o n t r o l value of 5.33 nM to 22. 37 nM i n the morphine-injected r a t s . The changes i n g and Kd suggested the presence of morphine i n the whole b r a i n homogenates. Moreover, the Kd was r e s t o r e d to the c o n t r o l value i n animals withdrawn from the drug f o r 24 hr. Vasopressin or o x y t o c i n i i d not have any d i r e c t e f f e c t on the 3H-naloxone bi n d i n g . Thus, i t i s u n l i k e l y t h a t the f a c i l i t a t i o n of i v morphine t o l e r a n c e by the neurohypophyseal peptides i s mediated by t h e i r d i r e c t a c t i o n on the o p i a t e r e c e p t o r . V TABLE OF CONTENTS Abstract i i List of Tables . v i i List of Figures v i i i List of Abbreviations v ................................ . x Acknowledgements. ................................. . x i I n t r o d u c t i o n ........................................ 1 I. E f f e c t s of Acute Morphine A d m i n i s t r a t i o n ...... 1 I I . E f f e c t s of Chronic Morphine A d m i n i s t r a t i o n ... 5 A. I n d u c t i o n of Tolerance and P h y s i c a l Dependence ............................... 6 B. P h y s i o l o g i c a l Changes Associated With Chronic Morphine A d m i n i s t r a t i o n ......... 7 C. Tolerance t o and P h y s i c a l Dependence on the Endogenous Opiates ............. 10 D. Biochemical Mechanisms of Tolerance 10 E. Studies L i n k i n g AVP and Memory to Morphine Tolerance and Dependence ........ 12 I I I . O b j e c t i v e s of the Current Study ............. 17 Methods 19 I . Acute Morphine Experiments 19 I I . Chronic Morphine Experiments ................. 21 I I I . . Extract ion o f the Neurohypophyseal Peptides . 22 IV. Opiate Receptor Binding ...................... 23 V. Radioimmunoassay (RIA) of Vasop r e s s i n ......... 24 v i . VI. Radioimmunoassay (RIA) of Oxytocin ............ 31 R e s u l t s 32 I . Acute E f f e c t s Of Morphine ...................... 32 I I . Chronic E f f e c t s Of Morphine ................... 39 A. .The Neurohypophyseal Stores Of AVP ...... 43 B. Neurohypophyseal Sto r e s of Oxytocin ..... 51 C. Serum O s m o l a l i t y and Sodium ............. 52 D. Opiate Receptor Binding .................. 55 D i s c u s s i o n .......................................... 59 I . Acute E f f e c t s of Morphine A d m i n i s t r a t i o n ...... 59 I I . C h r o n i c E f f e c t s of Morphine A d m i n i s t r a t i o n ... 65 Conclusion .......................................... 75 B i b l i o g r a p h y ......................................... 77 v i i LIST OF TABLES Table I, E f f e c t s of a S i n g l e Dose of Morphine on Mean A r t e r i a l Pressure, Urine Flow And Urine O s m o l a l i t y 37 Table I I . The Hypothalamic and Neurohypophyseal Sto r e s of AVP i n the M u l t i p l e I n j e c t i o n S e r i e s 46 Table I I I . . Serum O s m o l a l i t y and Serum Sodium i n the P e l l e t Implantation S e r i e s 54 Table IV..Number of 3H-naloxone B i n d i n g S i t e s and Binding A f f i n i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 LIST OF FIGURES Figure 1. . Chromatography of 1 2 5 I - A V P on CM Sephadax C-25 .................................... 26 F i g u r e 2. C r o s s - r e a c t i v i t y (a) of the Antiserum GP-13 28 Fig u r e 3. C r o s s - r e a c t i v i t y (b) of the Antiserum GP-13 29 Fig u r e 4..Renal and C a r d i o v a s c u l a r E f f e c t s of Morphine on a Normal Long-Evans Rat ............ 33 F i g u r e 5.,Renal and C a r d i o v a s c u l a r E f f e c t s of Morphine on a Heterozygous D.I r a t 35 Figure 6. .Renal and C a r d i o v a s c u l a r E f f e c t s of Morphine on a Homozygous D.I r a t ............... 36 F i g u r e 7. Changes i n Body Weight During Chronic i . p. i n j e c t i o n of Morphine .................... 40 F i g u r e 8. .Changes i n Body Weight, Water Intake and Urine Output r a t s Implanted With Morphine P e l l e t s 41 F i g u r e 9. .Changes i n Body Weight During Withdrawal i n the M u l t i p l e I n j e c t i o n S e r i e s ................ 44 Fig u r e 10. Changes i n Body Weight During Withdrawal i n the P e l l e t Implantation S e r i e s .............. 45 F i g u r e ^..Neurohypophyseal Stores of AVP i n the P e l l e t Implantation S e r i e s 48 Fig u r e 12.. Hypothalamic Stores of AVP i n the P e l l e t Implantation S e r i e s ....................... 50 F i g u r e 13. _Neurohypophyseal Stores of Oxytocin i n the P a l l e t Implantation S e r i e s .................. 53 F i g u r e 14. . S t e r e o s p e c i f i c Binding of 3H-naloxone .... 56 Figure 15. ,Scatchard P l o t of S t e o r e o s p e c i f i c 3H-naloxone B i n d i n g ................................. 57 X LIST OF ABBREVIATIONS ACTH a d r e n o c o r t i c o t r o p h i c hormone ADH a n t i d i u r e t i c hormone AVP a r g i n i n e v a s o p r e s s i n AVT a r g i n i n e v a s o t o c i n CEzO f r e e water c l e a r a n c e C i c u r i e CNS c e n t r a l nervous system Cos osmolar cl e a r a n c e CSF c e r e b r o s p i n a l f l u i d D.I. d i a b e t e s i n s i p i d u s fmol fentomole g gram GFR glomerular f i l t r a t i o n r a t e hr hour i . p . . i n t r a p e r i t o n e a l kg kilogram LVP l y s i n e v a s o p r e s s i n M molar M. A. P. mean a r t e r i a l pressure mCi m i l l i c u r i e mEq m i l l i e q u i v a l e n t mg m i l l i g r a m min minute ml m i l l i l i t e r mM m i l l i m o l a r mmol m i l l i m o l e mOsm m i l l i o s m o l e ng nanogram nM nanomolar pM picomolar Pos plasma o s m o l a l i t y RBF r e n a l blood flow RIA radioimmunoassay u l m i c r o l i t e r V u r i n a r y flow r a t e ACKNOWLEDGEMENTS I t i s my pleasure to thank, my s u p e r v i s o r , Dr..N. Wilson, f o r her i n v a l u a b l e a d v i c e and i n t e r e s t i n t h i s p r o j e c t . I would a l s o l i k e to thank Dr. ,R.,Keeler and Mr. B. T i b e r i i s f o r the many h e l p f u l d i s c u s s i o n s . 1 I N T R O D U C T I O N I. E f f e c t s of Acute Morphine A d m i n i s t r a t i o n The acute a d m i n i s t r a t i o n of morphine and other o p i a t e a g o n i s t s has been known to ex e r t a v a r i e t y of b e h a v i o r a l , c a r d i o v a s c u l a r , g a s t r o i n t e s t i n a l and endocrine e f f e c t s (1,2). In man, morphine produces a f e e l i n g of drowsiness, euphoria, p e r i p h e r a l v a s o d i l a t i o n and a marked decrease i n the m o t i l i t y of the g a s t r o i n t e s t i n a l system (2,3). In a d d i t i o n , i t produces a s t a t e of c a t a t o n i a and waxy f l e x i b i l i t y i n experimental animals (4) . C l i n i c a l l y , i t i s used e x t e n s i v e l y i n the a l l e v i a t i o n of p a i n . In c o n t r a s t t o most non- n a r c o t i c a n a l g e s i c s , morphine has a r e l a t i v e l y s e l e c t i v e e f f e c t i n d i m i n i s h i n g the s u b j e c t i v e aspect of pain as w e l l as r a i s i n g the pain t h r e s h o l d (5). I t has s e v e r a l u n d e s i r a b l e s i d e e f f e c t s , however, most notably the development of t o l e r a n c e and p h y s i c a l dependence, and the depression of r e s p i r a t i o n . S e v e r a l endocrine systems are known to be a l t e r e d by p a i n f u l or s t r e s s f u l s t i m u l i . . T h e ; c l a s s i c example i s the a c t i v a t i o n of the A C T H - c o r t i s o l a x i s i n response to s t r e s s (6). A l t e r e d endocrine s t a t e s are a l s o known to be a s s o c i a t e d with c e r t a i n changes i n p s y c h o l o g i c a l s t a t u s or mood, such as a n x i e t y and depr e s s i o n . Hence, i t i s c o n c e i v a b l e t h a t p s y c h o a c t i v e drugs which a l t e r the mood or the p e r c e p t i o n of p a i n f u l s t i m u l i by the animal would l e a d t o corresponding changes i n endocrine s t a t u s . These changes, however, are not 2 p r e d i c t a b l e at present. For example, morphine would be expected t o have an i n h i b i t o r y e f f e c t on ACTH s e c r e t i o n i n view of i t s a b i l i t y t o block the p e r c e p t i o n of pain and s t r e s s . Yet experimental evidence has shown t h a t morphine s t i m u l a t e d the r e l e a s e of ACTH (7-9). . S e v e r a l other endocrine systems were shown to be a f f e c t e d by morphine. Both growth hormone (7,10,11) and p r o l a c t i n (10,12-14) s e c r e t i o n were s t i m u l a t e d by the acute a d m i n i s t r a t i o n of the drug.. I t had an i n h i b i t o r y e f f e c t on t h e . r e l e a s e of the p i t u i t a r y gonadotrophins, FSH and LH (15)., Chronic treatment with the drug r e s u l t e d i n the a l t e r a t i o n of t e s t i c u l a r f u n c t i o n i n the male (15). I r r e g u l a r menstrual c y c l e has a l s o been r e p o r t e d i n human female drug a d d i c t s (16). Morphine has a l s o been shown t o be a potent a n t i d i u r e t i c agent (17-20). At doses as low as 0.4 mg/kg, morphine produced a d e t e c t a b l e a n t i d i u r e t i c e f f e c t on water-loaded and ethanol a n e s t h e t i z e d r a t s (17). f S e v e r a l s t u d i e s (17-20) have a s c r i b e d t h i s a n t i d i u r e t i c e f f e c t to the r e l e a s e of AVP, as proposed i n i t i a l l y by de Bodo (21). A n t i d i u r e s i s , f o r example, was shown to be a b o l i s h e d by neurohypophysectomy (21). This i m p l i c a t e d the involvement of the hypothalamo-neurohypophyseal system.. P r i o r a d m i n i s t r a t i o n of an o p i a t e a n t a g o n i s t , such as na l o r p h i n e , was shown to prevent the onset of a n t i d i u r e s i s (19). Th i s suggested t h a t the a c t i o n of morphine i s mediated through o p i a t e r e c e p t o r s . C a u t i o n , however, must be. e x e r c i s e d i n the i n t e r p r e t a t i o n of these s t u d i e s . 3 F a c t o r s a f f e c t i n g u r i n a r y output have, by f a r , been poorly c o n t r o l l e d and have not been c o n c l u s i v e l y e l i m i n a t e d . These f a c t o r s i n c l u d e d the p o t e n t i a t i o n o f the a n t i d i u r e t i c a c t i o n of AVP, c a r d i o v a s c u l a r changes and a d i r e c t e f f e c t of the drug on r e n a l f u n c t i o n . Although a n t i d i u r e s i s i s the predominant response, the a d m i n i s t r a t i o n of morphine has been shown to cause d i u r e s i s i n some cases. In p a t i e n t s with pulmonary edema, morphine produced a p e r i o d o f d i u r e s i s (22) . D i u r e s i s was a l s o observed when morphine was given to a hydropenic r a t (23). However, the same dose gi v e n to a water-loaded r a t produced a n t i d i u r e s i s . . T h u s , the e f f e c t s of morphine appeared to be dependent upon the p h y s i o l o g i c a l s t a t u s and the s t a t e of h y d r a t i o n of the animal. A n t i d i u r e s i s may be brought on by a number of f a c t o r s . For example, changes i n the c a r d i o v a s c u l a r system can cause a sharp decrease i n u r i n a r y flow. T h i s i s h i g h l y probable i n view of the powerful c a r d i o v a s c u l a r e f f e c t s of morphine (24-27). In conscious human s u b j e c t s , a 15 mg dose of morphine was shown to induce p e r i p h e r a l venous and a r t e r i o l a r v a s o d i l a t i o n (24).. T h i s , i n t u r n , l e d to a s i g n i f i c a n t decrease i n systemic blood pressure. Renal f u n c t i o n can be d r a s t i c a l l y a l t e r e d by changes i n systemic blood pressure (28-29). Thus, hypotension may c o n c e i v a b l y be r e s p o n s i b l e f o r the decrease i n u r i n a r y flow. In a d d i t i o n , i t may also serve as a s t i m u l u s f o r the r e l e a s e of AVP (30). Most s t u d i e s have i n d i c a t e d t h a t the 4 c a r d i o v a s c u l a r e f f e c t s of morphine are mediated c e n t r a l l y (24-27). T h i s , however, does not exclude the p o s s i b i l i t y of a p e r i p h e r a l a c t i o n . Green et a l . (31) have shown t h a t morphine i n c r e a s e d the h e p a t i c venous r e s i s t a n c e . T h i s i n turn l e d to an i n c r e a s e i n the r a t e of a s c i t e s f l u i d formation and i n p o o l i n g of f l u i d s i n the p e r i t o n e a l c a v i t y ^ The r e s u l t i n g decrease i n plasma volume can d i r e c t l y a l t e r r e n a l f u n c t i o n as well as s t i m u l a t e the r e l e a s e of v a s o a c t i v e peptides (30). In the r a t , the c a r d i o v a s c u l a r response to morphine appeared to be dependent upon the s t a t e of consciousness of the animal. Hypertension and t a c h y c a r d i a were observed i n the conscious, unanesthetized r a t (25,26). The same dose given to a c h l o r a l hydrate a n e s t h e t i z e d r a t produced the opposite e f f e c t s , namely hypotension and b r a d y c a r d i a . L a s t l y , a n t i d i u r e s i s may a l s o be e x p l a i n e d by a d i r e c t e f f e c t of the drug on r e n a l f u n c t i o n . A r e d u c t i o n i n RBF and GFR was observed by Handley and K e l l e r (32) i n normal and hypophysectomized dogs a f t e r the a d m i n i s t r a t i o n of morphine. Contrary to the r e s u l t s presented by de Bodo (21) , they found t h a t a n t i d i u r e s i s was not a b o l i s h e d by hypophysectomy (32). T h i s has l e d them to conclude t h a t a n t i d i u r e s i s was caused by the r e d u c t i o n of RBF and GFR. However, the v a l i d i t y of t h e i r experiment i s q u e s t i o n a b l e i n view of the abnormally low RBF and GFR i n t h e i r experimental animals. 5 I I . E f f e c t s of Chronic Morphine A d m i n i s t r a t i o n Prolonged treatment with morphine or o p i a t e a g o n i s t s has been known to induce t o l e r a n c e and p h y s i c a l dependence ( 3 3 ) . . Tolerance can be d e f i n e d as the development of i n s e n s i t i v i t y to the drug upon repeated exposure. Thus, i n c r e a s i n g l y l a r g e r doses must be given to o b t a i n the e f f e c t s observed with the o r i g i n a l dose. Tolerance i s o f t e n assessed by comparing the a n a l g e s i c potency o f the drug before and a f t e r i t s repeated use. P h y s i c a l dependence, on the other hand, r e f e r s to an a l t e r e d p h y s i o l o g i c a l s t a t e whereby the continued a d m i n i s t r a t i o n of the drug i s necessary t o prevent the onset of a s t e r e o t y p e d withdrawal or abstinence syndrome. The withdrawal syndrome encompasses a c o n s t e l l a t i o n of b e h a v i o r a l and p h y s i o l o g i c a l s i g n s . , In the r a t , t h i s i n c l u d e d h y p e r a c t i v i t y , jumping, wat dog shaking, t e e t h c h a t t e r i n g , v o c a l i z a t i o n , a g r e s s i v e behavior, body weight l o s s and e l e v a t i o n of body temperature^ jAssessment of the degree of dependence i s based mainly on the i n c i d e n c e of occurrence and/or i n t e n s i t y of each symptom. Q u a n t i t a t i v e assessment of the degree of t o l e r a n c e and p h y s i c a l dependence i s o f t e n a complicated task. This i s due to the f a c t t h a t v a r i o u s s i g n s of t o l e r a n c e and p r e c i p i t a t e d withdrawal do not i n c r e a s e concomitantly with i n c r e a s i n g t o l e r a n c e or dependence. Writhing and wet dog shaking, f o r example, were shown to be the predominant abstinence s i g n s i n moderately dependent animals ( 3 4 ) . These s i g n s decreased or even 6 disappeared with i n c r e a s i n g dependence, and were r e p l a c e d by more vigorous motor e x c i t a t i o n such as jumping ( 3 4 ) . Moreover, q u a n t i f i c a t i o n based only on one s p e c i f i c s i g n may not be adequate, e s p e c i a l l y i n s t u d i e s i n v o l v i n g pre-treatment with another drug. Such pre-treatment may c o n c e i v a b l y i n t e r f e r e with the n e u r a l mechanism u n d e r l y i n g one s p e c i f i c s i g n without a f f e c t i n g the b a s i c mechanism r e s p o n s i b l e f o r t o l e r a n c e and p h y s i c a l dependence. . hs. Indu ction of Tolerance and P h y s i c a l Dependence Animals can be made t o l e r a n t and p h y s i c a l l y dependent by v a r i o u s means. E a r l y s t u d i e s r e l i e d mainly on the d a i l y i n j e c t i o n of morphine ( 3 5 ) . One major disadvantage with t h i s procedure, other than the f a c t t h a t i t i s l a b o r i o u s , i s the time r e q u i r e d to induce a reasonable degree of t o l e r a n c e and p h y s i c a l dependence. This may be due to the f l u c t u a t i o n i n the c i r c u l a t i n g l e v e l of the drug between i n j e c t i o n s . The dosage and the time i n t e r v a l between i n j e c t i o n s are c r u c i a l i n these s t u d i e s . The animal may go through a pe r i o d of withdrawal i f the dosage per i n j e c t i o n i s low or the time i n t e r v a l between i n j e c t i o n s too l o n g , which i n t u r n may a f f e c t the measured response v a r i a b l e . Water i n t a k e and u r i n e e x c r e t i o n measurements, f o r example, are o f t e n i n f l u e n c e d by the dosage and i n j e c t i o n schedule used, because such measurements are o f t e n made over a time period much longer than that r e q u i r e d f o r the c l e a r a n c e of the drug. Rats p r e - t r e a t e d with morphine were shown to consume food and water v o r a c i o u s l y s h o r t l y a f t e r a m o r p h i n e ; i n j e c t i o n . . 7 Moreover, food and water i n t a k e decreased s i g n i f i c a n t l y as the e f f e c t of the drug wore o f f (36). These changes would not have been det e c t e d were the measurements made over 24 hr. T h e r e f o r e , the i d e a l method to study the c h r o n i c e f f e c t of a drug i s to d e l i v e r the drug on a continuous and steady b a s i s . Morphine base p e l l e t s designed by Gibson and Tingstad (37) have been used e x t e n s i v e l y i n s t u d i e s r e q u i r i n g t o l e r a n t and p h y s i c a l l y dependent animals. Maximal t o l e r a n c e and dependence can be induced with t h i s method over a very s h o r t p e r i o d of time (38) as compared to t h a t of the m u l t i p l e i n j e c t i o n of morphine. B_. P h y s i o l o g i c a l Changes A s s o c i a t e d With Chronic Morphine A d m i n i s t r a t i o n For most animals, the development of t o l e r a n c e and p h y s i c a l dependence seem to occur simultaneously. Changes i n s e v e r a l p h y s i o l o g i c a l parameters have been observed i n the p h y s i c a l l y dependent s t a t e s (39,40). R e s p i r a t o r y r a t e , f o r example, was depressed i n c h r o n i c drug users (40). T h i s has been shown to be p r i m a r i l y due to the d e p r e s s i o n of the r e s p i r a t o r y c e n t e r and the decrease i n s e n s i t i v i t y of the c e n t e r to CO2 (41).. Blood pressure, pulse r a t e and body temperature were a l s o p e r s i s t e n t l y e l e v a t e d i n p h y s i c a l l y dependent animals.. Moreover, t o l e r a n t and p h y s i c a l l y dependent animals o f t e n respond d i f f e r e n t l y to a s i n g l e morphine i n j e c t i o n . Whereas a n t i d i u r e s i s (17-21) and i n h i b i t i o n of d r i n k i n g (42) were o f t e n seen a f t e r morphine a d m i n i s t r a t i o n to a naive animal, p o l y u r i a and p o l y d i p s i a are the predominant 8 responses encountered i n a p h y s i c a l l y dependent animal (36,42-44). Thus, t h e r e appeared to be a complete r e v e r s a l of some of the e f f e c t s of the drug with repeated use. Studies on the e f f e c t s of a drug on water balance are f r e q u e n t l y complicated by the c l o s e l i n k between water intake:and urine e x c r e t i o n . I t i s d i f f i c u l t to examine the two processes independently. A drug capable of i n d u c i n g e x c e s s i v e water l o s s w i l l i n v a r i a b l y cause p o l y d i p s i a , and v i c e v e r s a . Hence, i t i s not c l e a r whether d r i n k i n g i n response t o morphine was due t o the m o d i f i c a t i o n of the animal's p e r c e p t i o n of t h i r s t or a s a l t and water imbalance caused by the drug. The i n h i b i t i o n of d r i n k i n g a f t e r the a d m i n i s t r a t i o n of morphine t o a naive animal may have been due to the s e d a t i v e and e u p h o r i c e f f e c t s of the drug; the animal may have been t e m p o r a r i l y i n c a p a c i t a t e d or i t s s e n s a t i o n of t h i r s t may have been masked by the euphoric e f f e c t of morphine. On the other hand, the p o l y d i p s i a observed i n t o l e r a n t and dependent animals may p o s s i b l y be due to water imbalance caused by the e x c e s s i v e water l o s s . There i s evidence to suggest t h a t morphine has an i n h i b i t o r y e f f e c t on AVP r e l e a s e i n a t o l e r a n t animal (17)..Thus, the r e s u l t i n g water d i u r e s i s may have caused a temporary s t a t e of dehydration and s t i m u l a t e d water consumption. S a l t and water metabolism may f u r t h e r be complicated by the apparent n e p h r o t o x i c e f f e c t of the n a r c o t i c drugs. S e v e r a l r e p o r t s have c i t e d the c o e x i s t e n c e of r e n a l d isease and n a r c o t i c a d d i c t i o n (45-47). The 9 s t a t i s t i c a l evidence a v a i l a b l e thus f a r i s c o n t r o v e r s i a l , and the e x i s t e n c e of a s p e c i f i c h e r o i n - a s s o c i a t e d nephropathy i s a t best c i r c u m s t a n t i a l . There i s no unequivocal proof t h a t h e r o i n or any of the o p i a t e s are capable of cau s i n g r e n a l f a i l u r e . V a r i o u s e t i o l o g i c a l agents have been i m p l i c a t e d by re s e a r c h e r s to e x p l a i n the onset of r e n a l d i s e a s e i n drug a d d i c t s (45-50) . These range from h e r o i n t o the u b i q u i t o u s v i r a l p a r t i c l e . B i c h t e r et a l . (48) have suggested t h a t a d u l t e r a n t s or substances used to d i l u t e the h e r o i n can act as antigens and promote the d e p o s i t i o n of immunoglobulins on the glomerular basement membrane.. Some a d u l t e r a n t s (49) as we l l as immunoglobulins (50) have been detected i n kidneys of drug a d d i c t s with r e n a l d i s e a s e . Bhabdomyolysis or s k e l e t a l muscle n e c r o s i s i s another w e l l known cause of acute r e n a l f a i l u r e found i n drug a d d i c t s (51). I t was o r i g i n a l l y d e s c r i b e d i n p a t i e n t s with crush syndrome (52). Since acute r e n a l f a i l u r e i s not a common f e a t u r e of rhabdomyolysis, i t i s l i k e l y t h a t other f a c t o r s are e s s e n t i a l t o b r i n g about the r e n a l disturbance..Imbalance i n calcium and phosphate metabolism are among two of the most l i k e l y c a n d i d a t e s (53,54). The a c t u a l mechanisms r e s p o n s i b l e f o r rhabdomyolysis f o l l o w i n g h e r o i n a d d i c t i o n remain t o be proven. 10 - Tolerance to and P h y s i c a l Dependence on the Endogenous Opiates The recent d i s c o v e r y of endogenous pe p t i d e s with o p i a t e - l i k e a c t i v i t y (55) posed an i n t r i g u i n g q u e s t i o n to drug a d d i c t i o n r e s e a r c h e r s . These p e p t i d e s have been shown to a c t c o m p e t i t i v e l y with morphine on the o p i a t e r e c e p t o r (56). There were high e x p e c t a t i o n s t h a t these: o p i o i d peptides may be used i n place of morphine as an a n a l g e s i c , without the impending development of t o l e r a n c e and p h y s i c a l dependence. However, t o l e r a n c e and dependence were shown to be e a s i l y evoked by the repeated a d m i n i s t r a t i o n of the endogenous o p i o i d p e p t i d e s , B-endorphin and the enkephalins (57,58). The p o s s i b l e advantage of t o l e r a n c e and dependence on these endogenous pep t i d e s i s not c l e a r . I t i s p o s s i b l e t h a t s h i f t s i n the l e v e l of r e s p o n s i v e n e s s to these peptides may serve as a mechanism to r e g u l a t e t h e i r a c t i o n s on s p e c i f i c n e u r a l c i r c u i t s . The development of t o l e r a n c e and dependence may thus be viewed as an adaptive response t o the e x c e s s i v e i n f l u e n c e of the o p i o i d peptides or exogenous o p i a t e s . Rs. B i o c h e m i c a l Mechanisms of Tolerance S e v e r a l hypotheses have been proposed to e x p l a i n the development of morphine t o l e r a n c e and p h y s i c a l dependence (59). Lampert et a l . (57) and C o l l i e r et a l . . (60) have suggested t h a t t o l e r a n c e and dependence may be due to the a l t e r a t i o n of adenyl c y c l a s e a c t i v i t y of the t a r g e t c e l l . Morphine and endogenous o p i o i d p e p t i d e s have 11 been shown to i n h i b i t adenyl c y c l a s e a c t i v i t y i n c e l l s possessing o p i a t e r e c e p t o r s (57,61). Prolonged exposure t o the drug r e s u l t e d i n an enhanced s y n t h e s i s of adenyl c y c l a s e (62). Thus, the c e l l s adapt to the i n h i b i t o r y e f f e c t of morphine by i n c r e a s i n g the s y n t h e s i s of the enzyme. T h i s allows them to maintain a normal l e v e l of c e l l u l a r c y c l i c AMP and to appear t o l e r a n t t o the i n h i b i t o r y e f f e c t of morphine on adenyl cyclase.„At t h i s stage, the c e l l s are dependent upon the continued presence of morphine to maintain a normal l e v e l of c y c l i c AMP. The abrupt withdrawal of the drug would r e s u l t i n the d i s i n h i b i t i o n of adenyl c y c l a s e and s y n t h e s i s of an abnormally high l e v e l of c y c l i c AMP. This has been a s s o c i a t e d with neuronal h y p e r e x c i t a b i l i t y (62) and may account f o r the high l e v e l of a c t i v i t y i n the CNS during withdrawal* .Tolerance and dependence can, t h e r e f o r e , be regarded as normal processes which help t o r e g u l a t e the adenyl c y c l a s e coupled s y n a p t i c communication. S e v e r a l other b i o c h e m i c a l changes were noted i n dependent animals. Turnover r a t e s of s e v e r a l n e u r o t r a n s m i t t e r s were a l t e r e d i n p h y s i c a l l y dependent animals (63,64). T h i s has been thought to be r e s p o n s i b l e f o r the b e h a v i o r a l changes observed d u r i n g p e r i o d s of i n t o x i c a t i o n and withdrawal* E l e v a t e d l e v e l s of b r a i n enkephalin has a l s o been observed i n morphine dependent r a t s (65). K o s t e r l i t z and Hughes (66) proposed t h a t t o l e r a n c e and dependence might be e x p l a i n e d by the r e d u c t i o n of neuronal enkephalin r e l e a s e as the normal 12 f u n c t i o n s of these p e p t i d e s were r e p l a c e d by the exogenous o p i a t e , morphine. Malfroy et a l . , (67) on the other hand, suggested t h a t the i n c r e a s e d l e v e l s of a h i g h - a f f i n i t y enkaphalia-degrading peptidase serves as a mechanism of t o l e r a n c e and dependence. Both thesa hypotheses imply t h a t symptoms of withdrawal are due to the l a c k of a c t i o n of the e n k e p h a l i n - c o n t a i n i n g neurons on t h e i r r e s p e c t i v e t a r g e t c e l l s . Changes i n r e c e p t o r a f f i n i t y or number of b i n d i n g s i t e s can c o n c e i v a b l y a l t e r the r e s p o n s i v e n e s s to a drug (68). In most bi o c h e m i c a l pathways, feedback c o n t r o l i s exerted on the i n i t i a l step of a c y c l e . The b i n d i n g of a drug molecule to the r e c e p t o r i s often considered the i n i t i a l step of drug a c t i o n . Hence, a l t e r a t i o n s i n the l i g a n d - r e c a p t o r b i n d i n g or the c o n c e n t r a t i o n of the r e c e p t o r can not be excluded as a p r i n c i p a l s i t e of c o n t r o l . Enhancement of the number of o p i a t e b i n d i n g s i t e s has been rep o r t e d by Pert and Snyder (69) i n morphine-treated mice. , E. S t u d i e s l i n k i n g AVP and Memory to Morphine-Tolerance and Dependence Aside from the b i o c h e m i c a l changes, morphine t o l e r a n c e and dependence have been l i n k e d t o l e a r n i n g and memory processes of the animal (70,71). Memory storage, according t o the c u r r e n t dogma (72), c o n s i s t s of a t l e a s t two stages. The f i r s t stage i n v o l v e s a l t e r a t i o n s i n e l e c t r i c a l events and/or metabolic processes s h o r t l y a f t e r the l e a r n i n g experience. Storage a t t h i s stage i s 13 temporary and i s o f t e n r e f e r r e d t o as short-term memory. Long-term memory i s considered more permanent i n nature and i n v o l v e s a process of i n f o r m a t i o n c o n s o l i d a t i o n . T h i s i s accompanied by metabolic changes, and growth of axon c o l l a t e r a l s and n e u r a l c o n n e c t i v i t y (73). A number of s t u d i e s have shown that AVP and i t s analogues have a f a c i l i t a t i n g e f f e c t on memory c o n s o l i d a t i o n (74-76). In view o f t h i s , i t i s probable t h a t the peptide a l s o plays a r o l e i n the development of morphine t o l e r a n c e and dependence. K r i v o y e t a l . (77) have shown t h a t c h r o n i c treatment of mice with a b i o l o g i c a l l y i n e r t AVP analogue, d e s g l y c i n a m i d e 9 - i y s i n e 8 v a s o p r e s s i n (DG-LVP), f a c i l i t a t e d the development of t o l e r a n c e . In a d d i t i o n , h e r e d i t a r y D.I. ( B r a t t l e b o r o s t r a i n ) r a t s not only e x h i b i t e d memory d e f i c i e n c y but al s o impairment i n the development of morphine t o l e r a n c e as compared to t h e i r normal or heterozygous l i t t e r m a t e s (78). The bi o c h e m i c a l mechanism by which AVP and i t s analogues f a c i l i t a t e memory c o n s o l i d a t i o n i s as yet unknown. The a b i l i t y of DG-LVP to p r o t e c t the animal a g a i n s t puromycin-induced amnesia (79) suggested t h a t AVP a f f e c t s memory processes through the a l t e r a t i o n of p r o t e i n metabolism i n the CNS. F a c i l i t a t i o n of morphine:tolerance may a l s o be mediated by a p o s s i b l e a c t i o n of AVP on the o p i a t e r e c e p t o r s . I n t e r a c t i o n s between endocrine systems, and between endocrine systems and the CNS have been w e l l documented (80-82). I t i s of i n t e r e s t to s p e c u l a t e t h a t a hormone - AVP i n t h i s case - may modulate or e x e r t some 14 i n f l u e n c e on the o p i a t e r e c e p t o r s . . A l t e r n a t i v e l y , AVP may e x e r t i t s e f f e c t on the o p i a t e r e c e p t o r s through the r e l e a s e of ACTH. The s e c r e t i o n of ACTH i s known to be p o t e n t i a t e d by AVP (83,84). Furthermore, ACTH and i t s analogues have been shown by T e r e n i u s et a l . (85) to i n t e r f e r e with the b i n d i n g of the o p i a t e s t o the r e c e p t o r . As with AVP, the other neurohypophyseal hormone, o x y t o c i n , a l s o appeared to f a c i l i t a t e the development of morphine t o l e r a n c e and p h y s i c a l dependence (86) Moreover, t h i s a c t i v i t y was shown to r e s i d e predominantly on the C-terminal t r i p e p t i d e , p r o - l e u - g l y - ( N H 2 ) . Tocinamide, the N-terminal r i n g s t r u c t u r e of o x y t o c i n , was found to be i n a c t i v e (86). I t i s q u e s t i o n a b l e whether o x y t o c i n and i t s by-products play a s i g n i f i c a n t r o l e i n the B r a t t l e b o r o r a t s . Although these r a t s e x h i b i t e d an impairment i n the development of morphine t o l e r a n c e (77), they are capable of s y n t h e s i z i n g o x y t o c i n . Most s t u d i e s on the p h y s i o l o g i c a l r o l e of the neurohypophyseal peptide thus f a r have been d i r e c t e d at t h e i r e x t r a n e u r o n a l a c t i o n s . In order to understand t h e i r a c t i o n s on the CNS, knowledge of t h e i r presence: and the route by which these p e p t i d e s reach the CNS are important. AVP and o x y t o c i n have been found i n v a r i o u s hypothalamic n u c l e i (87,88).,It i s not c l e a r whether these p e p t i d e s are s y n t h e s i z e d i n a l l the n u c l e i i n which they were detected or are s e l e c t i v e l y taken up by the hypothalamic neurons a f t e r being s y n t h e s i z e d elsewhere.. N e v e r t h e l e s s , t h e i r presence i n these n u c l e i suggests a p o s s i b l e neuronal 15 connection and/or f u n c t i o n . Neural pathways c o n t a i n i n g AVP and/or o x y t o c i n have been shown by B u i j s (89) to spread out from the p a r a v e n t r i c u l a r nucleus towards v a r i o u s areas of the b r a i n . . These areas i n c l u d e d the hippocampus, amygdala and v a r i o u s medullary n u c l e i . Some of these pathways have been thought to be the anatomical b a s i s f o r the b e h a v i o r a l e f f e c t s of the p e p t i d e s (86,90). The neurohypophyseal p e p t i d e s have been shown to be present i n both axons and d e n d r i t e s of the hypothalamic and extrahypothalamic f i b e r s (89)., There i s evidence that these two peptides may f u n c t i o n as n e u r o t r a n s m i t t e r s i n these neurons. Iontophoresis of AVP has been shown to exert an i n h i b i t o r y e f f e c t on a l a r g e percentage of neurons i n the s u p r a o p t i c nucleus (91). The: other neurohypophyseal hormone, o x y t o c i n , has an i n h i b i t o r y e f f e c t on p a r a v e n t r i c u l a r nucleus neurons (92). . The g e n e r a l i d e a s emerging from these s t u d i e s are t h a t AVP and o x y t o c i n act as neuromodulators and g e n e r a l r e g u l a t o r s of neuronal membrane p r o p e r t i e s . In c o n t r a s t to the c l a s s i c a l n e u r o t r a n s m i t t e r s , t h e i r a c t i o n s appear to be of longer d u r a t i o n . Aside from d i r e c t neuronal c o n t a c t , the neurohypophyseal hormones may be t r a n s p o r t e d throughout the CNS v i a the c e r e b r o s p i n a l f l u i d (CSF) . There i s morphological evidence of connections between the neurosecretory c e l l s and the i n f u n d i b u l a r r e c e s s of the t h i r d v e n t r i c l e (93). T h i s suggested t h a t AVP and o x y t o c i n may be taken up and/or secreted i n t o the CSF from neuron 16 t e r m i n a l s . Immune-reactive AVP has been det e c t e d by Dogterom e t a l . (94) i n dog, human and r a t CSF. I t s presence i n the CSF has been i m p l i c a t e d i n the c o n d i t i o n e d avoidance behavior of the r a t (95). Removal of AVP i n the CSF by the i n t r a c e r e b r o v e n t r i c u l a r a d m i n i s t r a t i o n of A V P - s p e c i f i c a n t i b o d i e s (96) r e s u l t e d i n a marked r e d u c t i o n of avoidance behavior. There i s no doubt t h a t the endogenous o p i o i d peptides play a s i g n i f i c a n t r o l e i n modulating various neuroendocrine f u n c t i o n s (97).. As i n most endocrine systems, proper f u n c t i o n i n g r e q u i r e s some form of feedback c o n t r o l . With regards to the hypothalamo-neurohypophyseal system, the e l a b o r a t i o n of AVP i n CNS s t r u c t u r e s may act as a mechanism t o prevent the e x c e s s i v e i n f l u e n c e of the endogenous o p i o i d p e p t i d e s . S t i m u l a t i o n of AVP r e l e a s e i n t o the systemic c i r c u l a t i o n by the presence of excess endogenous o p i o i d peptides or exogenous o p i a t e s may cause an unnecessary imbalance i n the animal's s a l t and water metabolism. One method o f d i m i n i s h i n g such s t i m u l a t i v e i n f l u e n c e i s t o promote the development of t a r g e t c e l l t o l e r a n c e . Thus, the r e l e a s e of AVP e i t h e r as a ne u r o t r a n s m i t t e r or i n t o the CSF may serve to f a c i l i t a t e the development of t o l e r a n c e and prevent the h y p e r s e c r e t i o n of AVP i n t o the systemic c i r c u l a t i o n . 17 I I I . O b j e c t i v e s - of the Cu r r e n t Study The present experiments were designed to examine the e f f e c t s of both acute and c h r o n i c morphine treatment on the hypothalamo-neurohypophyseal system. I t was apparent from the l i t e r a t u r e t h a t the morphine-induced a n t i d i u r e t i c response was governed by at l e a s t two f a c t o r s : the s t a t e of h y d r a t i o n and l e v e l of consciousness of the animal.. In t h i s study, the acute e f f e c t s of morphine were examined using unanesthetized and hydrated r a t s . The r e l e a s e of AVP i s known to be i n h i b i t e d i n hydrated animals or by a decrease i n plasma o s m o l a l i t y (98,99). I f the morphine-induced a n t i d i u r e s i s i s mediated by AVP s e c r e t i o n from the neurohypophysis, then the drug must overcome the i n h i b i t o r y e f f e c t of plasma hy p o o s m o l a l i t y . To f u r t h e r e l u c i d a t e the r o l e of AVP i n the i n d u c t i o n of a n t i d i u r e s i s , the responses of B r a t t l e b o r o r a t s (100) with f a m i l i a l hypothalamic D.I. were compared t o t h a t of t h e i r normal and heterozygous l i t t e r r a a t e s . The homozygous D.I. r a t s have served as e x c e l l e n t c o n t r o l s i n many s t u d i e s i n v o l v i n g AVP r e l e a s e and a c t i o n s . Trauma and t e c h n i c a l d i f f i c u l t i e s o f t e n encountered i n hypophysectomy can thus be avoided with the use of these animals. The presence of an a n t i d i u r e t i c response i n the normal r a t s coupled to the f a i l u r e of the homozygous D*I. r a t s to respond t o morphine would c o n c l u s i v e l y prove t h a t a n t i d i u r e s i s i s AVP mediated. The e f f e c t s of c h r o n i c morphine treatment on the 18 hypothalamic and neurohypophyseal AVP s t o r e s were a l s o s t u d i e d . . Changes i n AVP storage were determined by radioimmunoassay (RI A) . Attempts were made to c o r r e l a t e these changes with the animal's d a i l y water i n t a k e and urine o u t p u t . . P o l y u r i a o f t e n encountered i n p h y s i c a l l y dependent animals may be due to the d e p l e t i o n of the neurohypophyseal AVP s t o r e s . Changes i n neurohypophyseal AVP s t o r e s may a l s o provide some i n s i g h t t o the mechanism fo morphine t o l e r a n c e . . Studies on the f a c i l i t a t i o n of morphine t o l e r a n c e by the neurohypophyseal peptides (77,78) have been based e x c l u s i v e l y on the exogenous a p p l i c a t i o n of these peptides. There i s no study thus f a r on the p o s s i b l e e f f e c t s of c h r o n i c morphine a d m i n i s t r a t i o n on the hypothalamo-neurohypophyseal system.. The measurement of the hypothalamic and neurohypophyseal s t o r e s o f AVP might shed some l i g h t on the p o s s i b l e i n t e r a c t i o n s between the neurohypophyseal peptides and morphine. 19 METHODS 1^  Acute -Morphine Experiments Male B r a t t l e b o r o r a t s , homozygous and heterozygous f o r the hypothalamic diabetes i n s i p i d u s (D.I.) t r a i t , were s t u d i e d . Normal male r a t s from the Long-Evans s t r a i n , the s t r a i n from which the B r a t t l e b o r o r a t s were: d e r i v e d , were used as c o n t r o l * The r a t s , weighing 200-300 g, were a n e s t h e t i z e d with e t h e r . The r i g h t c a r o t i d a r t e r y was exposed by a midline i n c i s i o n and cannulated with a h e p a r i n - f i l l e d PE-50 tubing (Clay-Adams). The cannula was passed subcutaneously t o the back o f the neck and brought out through a small i n c i s i o n . The c a r o t i d blood pressure was monitored with a pressure transducer (Statham) and recorded on a u.v. r e c o r d e r (S.E. Lab. L t d . ) . For intravenous i n f u s i o n * the l a t e r a l t a i l v e i n was cannulated using a PE-10 t u b i n g . Urine samples were c o l l e c t e d by c a t h e t e r i z i n g the u r i n a r y bladder* The bladder was exposed by a sm a l l midline i n c i s i o n * A small opening was made i n the apex and a f l a r e - t i p p e d c a t h e t e r was i n s e r t e d . The c a t h e t e r was held i n p l a c e with a suture and care was taken t o minimize the amount of dead space i n the bladder. The bladder was then gently guided back i n t o the abdominal space with the t i p of the c a t h e t e r p r o t r u d i n g out the abdominal i n c i s i o n . A f t e r s u r g e r y , 2.5 mg of Mepivacaine (Winthrop) was i n j e c t e d subcutaneously around the i n c i s i o n s i t e s . T h i s l o c a l a n e s t h e t i c was used i n order to l e s s e n the 20 s u r g i c a l pain which may s t i m u l a t e the r e l e a s e of AVP. The animal was then allowed t o recover f o r 2 hours before commencing the experiment. A f t e r t h i s recovery p e r i o d , the r a t was hydrated with a s o l u t i o n of 0.3% NaCI and 1.6% glucose, i n f u s e d i n t r a v e n o u s l y at a r a t e of 51 ul/min. The i n f u s i o n s o l u t i o n was s i m i l a r t o t h a t used by I n t u r r i s i and Fujimoto (17) with the exception t h a t e t h a n o l was omitted and a slower i n f u s i o n r a t e was chosen* Drine samples were c o l l e c t e d at 10 min i n t e r v a l s . Morphine s u l f a t e (BDH Chemicals) was d i s s o l v e d i n 0.9% NaCI and i n j e c t e d i n t o the t a i l v e i n at volumes of 20-30 u l to give a dose o f 1 mg/kg body weight. Morphine i n j e c t i o n s were given only a f t e r the establishment of a constant u r i n e flow r a t e f o r at l e a s t two c o n t r o l periods* The r e s u l t s were expressed as the percentage r e d u c t i o n i n the urin e e x c r e t i o n r a t e a f t e r morphine i n j e c t i o n . The u r i n e o s m o l a l i t y (Oos) was measured by f r e e z i n g p o i n t depression (Osmette, P r e c i s i o n System). Osmolar c l e a r a n c e (Cos) and f r e e water c l e a r a n c e (CH2O) were c a l c u l a t e d using the f o l l o w i n g formulae: Cos = Uos x V / Pos CH 20 = V - Cos Where V i s the u r i n e e x c r e t i o n r a t e and Pos the plasma o s m o l a l i t y . 21 i i i Chronic Morphine Experiments Male Wistar r a t s , weighing 200-250 g, were housed i n d i v i d u a l l y i n metabolic cages. Food and water were given ad l i b . The room was maintained at 22° C with a 12 hr l i g h t - d a r k c y c l e ( l i g h t on from 0700-1900 h r ) . . The r a t s were rendered p h y s i c a l l y dependent on morphine by two means: m u l t i p l e i n j e c t i o n and p e l l e t i m p l a n t a t i o n . For the m u l t i p l e i n j e c t i o n method, 24 r a t s were d i v i d e d i n t o 3 equal groups. Two groups were i n j e c t e d i n t r a p e r i t o n e a l l y (i.p,*) with i n c r e a s i n g doses of morphine s u l f a t e twice d a i l y f o r 14 days (101). The c o n t r o l r a t s were i n j e c t e d with an equal volume of 0.9% NaCI. On the f o u r t e e n t h day, the c o n t r o l group and one morphine-treated group were s a c r i f i c e d one hour a f t e r t h e i r d a i l y i n j e c t i o n * . D a i l y morphine i n j e c t i o n s were withheld from the second morphine-treated group f o r 24 hr before s a c r i f i c e . . This group w i l l be r e f e r r e d t o as the withdrawal group. For the p e l l e t i m p l a n t a t i o n method, morphine p e l l e t s c o n t a i n i n g 75 mg morphine s u l f a t e ( A l l e n & Hanbury's); were prepared a c c o r d i n g t o the method of Gibson and T i n g s t a d (37). T h i r t y - s i x r a t s were d i v i d e d i n t o 6 equal groups and placed i n d i v i d u a l l y i n metabolic cages. A f t e r two days of housing, f o u r groups were implanted with morphine p e l l e t s i n the d o r s a l subcutaneous space under l i g h t e t h e r a n e s t h e s i a . The remaining two groups served as c o n t r o l s and were implanted with placebo p e l l e t s composed of the i n e r t i n g r e d i e n t s . . S t u d i e s (33,34) 22 have shown t h a t the absorption of morphine from each p e l l e t r a p i d l y decreased a f t e r a few days and tended to p l a t e a u a f t e r a week. Hence, a second p e l l e t was implanted 3 days a f t e r the f i r s t p e l l e t i n order to maintain a constant d e l i v e r y of the drug. To examine the changes i n the neurohypophyseal s t o r e s of AVP, a morphine-implanted group was s a c r i f i c e d a f t e r 1, 3 and 5 days of treatment. One of the c o n t r o l groups was a l s o s a c r i f i c e d on day 5. Withdrawal. was p r e c i p i t a t e d on one morphine-implanted group on day 5 by s u r g i c a l l y removing the implanted p e l l e t s . The implanted placebo p e l l e t s were a l s o removed from the remaining c o n t r o l group* Both groups were s a c r i f i c e d 3 days a f t e r the e x c i s i o n of the p e l l e t s . A l t e r a t i o n i n body weight during the withdrawal p e r i o d was used as an index of p h y s i c a l dependence (102). I I I . E x t r a c t i o n of the Neurohypophyseal Peptides On p r e v i o u s l y designated days, the t r e a t e d r a t s were s a c r i f i c e d by d e c a p i t a t i o n . Trunk blood was c o l l e c t e d i n t o a t e s t tube and allowed t o c l o t f o r 1 h r . The c l o t was removed by low speed c e n t r i f u g a t i o n (1400 x g f o r 10 min) and the serum s t o r e d a t -20° C. Serum o s m o l a l i t y was measured by f r e e z i n g p o i n t d e p r e s s i o n (Osmette), and serum sodium by flame photometry (Instrumentation Lab., model 143). A f t e r the d e c a p i t a t i o n , the neurohypophysis was q u i c k l y separated from the adenohypophysis and f r o z e n at -20° C. The hypothalamus, according to the boundaries set by Bie and Thorn (103), was removed and f r o z e n at -20° C. The t i s s u e s (neurohypophysis and hypothalamus) were then 23 suspended i n 6-10 volumes of i c e c o l d 0.2 M a c e t i c a c i d (pH 2.85) and homogenized i n a Potter-Ehlvejehm assembly at 300 RPM f o r 30 sec. The homogenate was c e n t r i f u g e d at 6000 x g f o r 30 min. The p e l l e t was d i s c a r d e d and the supernatant l y o p h i l i z e d . The l y o p h i l i z e d powder was s t o r e d at -20 ° C. . H i Opiate - Receptor B i n d i n g B r a i n s from r a t s i n j e c t e d c h r o n i c a l l y with s a l i n e or morphine were q u i c k l y removed a f t e r d e c a p i t a t i o n . . S i n c e the cerebellum has been shown to be devoid of r e c e p t o r a c t i v i t y (104), i t was removed and discarded. Each b r a i n was homogenized i n 10 ml of i c e c o l d 50 mM T r i s - H C l b u f f e r (pH 7.4) f o r 30 sec. The homogenate was c e n t r i f u g e d at 12,000 x g f o r 10 min. The p e l l e t was re-suspended i n 10 ml of T r i s - H C l b u f f e r and c e n t r i f u g e d a second time. The f i n a l p e l l e t was suspended i n 5 ml of T r i s - H C l b u f f e r . T h i s suspension had an average p r o t e i n c o n c e n t r a t i o n of 16.33 ± 0.16 mg/ml (n=22) as estimated by the Folin-Lowry method (105) . A r e c e p t o r - b i n d i n g assay s i m i l a r to t h a t d e s c r i b e d by Pert and Snyder (106) was used. A 100 u l a l i q u o t of the b r a i n homogenate, c o n t a i n i n g approximately 1.5 mg p r o t e i n , was incubated with 2.33 nM of 3H-naloxone (New England Nuclear, at 17.15 Ci/mmol) at 4° C f o r 60 min. N o n - s p e c i f i c b i n d i n g was determined by adding 115 nM of l e v a l l o r p h a n (Hoffman-LaRoche) to tubes c o n t a i n i n g the b r a i n homogenate and 3H-naloxone. A l l assays were performed i n t r i p l i c a t e . A f t e r the 60 min i n c u b a t i o n 24 p e r i o d , the samples were f i l t e r e d through Whatman GF/B g l a s s - f i b e r f i l t e r s (24 mm diameter) and washed twice with 5 ml volunes of i c e c o l d T r i s - H C l b u f f e r . The f i l t e r s were p a r t i a l l y d r i e d under i n f r a - r e d lamp and the p r o t e i n d i g e s t e d overnight with 1 ml P r o t o s o l (New England Nuclear)..10 ml of Omnifluor (New England Nuclear) was added to each s c i n t i l l a t i o n v i a l and the r a d i o a c t i v i t y was counted i n a Beckman (LS-233) s c i n t i l l a t i o n counter. S t e r e o s p e c i f i c b i n d i n g was determined by s u b t r a c t i n g the b i n d i n g which occurred i n the presence of the o p i a t e a n t a g o n i s t ^ l e v a l l o r p h a n , from that which occurred i n i t s absence* The n o n - s p e c i f i c b i n d i n g i n the presence of excess i e v a l l o r p h a n was not a l t e r e d by adding 100 mM NaCI, which i s known to enhance the b i n d i n g of o p i a t e a n t a g o n i s t s (107). T h i s showed t h a t the amount of l e v a l l o r p h a n used was adequate to bind to a l l o p i a t e r e c e p t o r s i n the tube. V. Radioimmunoassay jRIA). of Vasopressin The assay was performed a c c o r d i n g to the method de s c r i b e d by Burget and Wilson (108). AVP (Spectrum Med. Ind. ) was i o d i n a t e d by a m o d i f i c a t i o n (109) of the chloramine-T method d e s c r i b e d by Greenwood e t a l . r (110). 10 ug of s y n t h e t i c AVP was d i s s o l v e d i n 10 / i l of 0.05 M a c e t i c a c i d (pH 3.0) and 15 u l of 0.5 M phosphate b u f f e r (pH 7.4). 10 u l of 1 mg/ml chloramine-T (Eastman) and 1.5 mCi of 1 2 . 5j (Amersham) were added to the s o l u t i o n . The reagents were allowed t o r e a c t f o r 50 sec. The r e a c t i o n was stopped by the a d d i t i o n of 100 u l of 25% BSA (Pentex). 25 In addition, 200 u l of Bio-Rad anion exchanger, AG1-X10 at 250 mg/ml, was added to help bind the unreacted 1 2 5 1 a s well as the complex polyiodides. The ion exchange beads were then removed by low-speed centrifugation. The resul t i n g supernatant, containing the iodinated AVP, was separated and p u r i f i e d on a CM Sephadex C-25 column (9 x 800 mm) equilibrated with 0.6 M sodium acetate buffer (pH 4.85).. The eluti o n p r o f i l e of the iodination mixture on CM Sephadex C-25 i s shown in f i g . 1. Iodinated BSA was the f i r s t derivative to be eluted from the column and i t showed no binding to the antiserum (GP-13).. This was followed by several small radioactive peaks corresponding to unreaoted 1 2 5 1 a n d complex polyiodides. The next radioactive derivative to be eluted was mono-iodinated AVP, followed by di-iodinated AVP. 25a FIGURE 1. Chromatography of i 2 5 I - A V P on CM Sephadex C-25 column (9 x 800 mm).. The column was e q u i l i b r a t e d with 0.6 M a c e t a t e b u f f e r (pH 4.85) at a flow r a t e of 18 ml/hr. Each f r a c t i o n c o n s i s t e d of 70 drops (or 5 ml). The order of e l u t i o n was i o d i n a t e d BS A (a), unreacted i 2 s j a n Q the complex p o l y i o d i d e s (b), mono-iodinated AVP (c) and d i - i o d i n a t e d AVP (d). 2 6 27 The amount of mono- and d i - i o d i n a t e d AVP obtained was dependent on the r e a c t i o n time..A longer r e a c t i o n time (>45 sec) favored the production of more d i - i o d i n a t e d AVP. Standardized p o s t e r i o r p i t u i t a r y e x t r a c t , s u p p l i e d by Dr. Ri . E. Weitzman (Harbor Gen. H o s p i t a l , Torrance, C a l i f . ) at 2.1 IO/ml, was used i n the p r e p a r a t i o n of the standards. The HIA b u f f e r was 0.15 M sodium phosphate (pH 7.2) with 0.25% normal r a b b i t serum. The antiserum used (GP-13) was obtained from guinea pigs immunized a g a i n s t s y n t h e t i c LVP coupled to BSA by the c a r b o d i i m i d e r e a c t i o n (111). In the assay, the antiserum (GP-13) was used at a f i n a l d i l u t i o n of 1:90,000. T h i s antiserum showed no c r o s s - r e a c t i v i t y with o x y t o c i n (112) and AVT ( f i g . 2). Although the antiserum was r a i s e d a g a i n s t LVP, i t e x h i b i t e d a g r e a t e r preference f o r AVP. I t c r o s s - r e a c t e d with A VP at a c o n c e n t r a t i o n of 13 pM whereas LVP c r o s s - r e a c t e d at 100 pM. An AVP analogue, desamino-dicarba AVP, was also t e s t e d and i t c r o s s - r e a c t e d at 45 pM ( f i g . 3). Hence, the 3P-13 antiserum appeared to c o n s i s t of at l e a s t two p o p u l a t i o n s of a n t i b o d i e s ; one s p e c i f i c a g a i n s t the N-terminus and the other a g a i n s t the region bounded by the d i s u l f i d e b r i d g e . 27a FIGURE 2. I n h i b i t i o n of bindin g of i 2 5 I - A V P t o antiserum GP-13 by the neurohypophyseal p e p t i d e s . Oxytocin (o) and a r g i n i n e v a s o t o c i n (o) showed no i n h i b i t i o n of b i n d i n g up to a c o n c e n t r a t i o n of 500 pM/L. The antiserum (GP-13) showed g r e a t e r preference f o r AVP (•) than f o r LVP (•) . 28 28a FIGURE 3. Comparison of e f f e c t i v e n e s s of an AVP analogue and l e u - e n k e p h a l i n i n the displacement of l 2 s I - A V P from the antiserum, GP-13.. The AVP analogue, desamino-dicarba AVP (o) showed l e s s e r c r o s s - r e a c t i v i t y as compared to AVP (•). The endogenous o p i o i d p e p t i d e , l e u -enkephalin (A) d i d not depress the b i n d i n g of 1 2 S I - A V P . B O U N D / F R E E o o 30 The l i m i t of d e t e c t i o n of the standard curve was a r b i t r a r i l y d e f i n e d as 80% of the i n i t i a l or maximal binding* T h i s i s more than the three standard d e v i a t i o n s of i n i t i a l b i n d i n g o f t e n used by other authors. Osing t h i s c o n d i t i o n , the l i m i t of d e t e c t i o n was about 13 fmol of AVP (13.6 ± 1.9 fmol, n=15), and 50% displacement of * 2 5I-AVP occurred a t 52 fmol (52.1 ± 3.6, n=15). The i n t r a - a s s a y v a r i a t i o n at 50 fmol l e v e l was 3 . 2 % (n=18). The i n t e r - a s s a y v a r i a t i o n u s ing pooled dog plasma was 13.9% (n=15) . The l y o p h i l i z e d t i s s u e s to be assayed were d i s s o l v e d i n the RIA b u f f e r . A 1:500 d i l u t i o n was made f o r the p o s t e r i o r p i t u i t a r y e x t r a c t and a 1:10 d i l u t i o n f o r the hypothalamic e x t r a c t . The f i n a l volume of each RIA tube was 1.0 ml. The assay was incubated a t 4° C f o r 3 days. Separation of bound and f r e e * 2 5I-AVP was accomplished by using dextran-coated c h a r c o a l (2.5 mg/ml dextran T-70, 25 mg/ml N o r i t A c h a r c o a l i n 0.15 M phosphate b u f f e r , pH 7.2). The r e s u l t s were expressed as the amount of i 2 S I - A V P bound d i v i d e d by the t o t a l amount added versus the l o g a r i t h m of u n l a b e l l e d AVP present.. L i n e a r i z a t i o n was done by expressing the response v a r i a b l e i n l o g i t (113). 31 ILs. Radioimmunoassay (RIA) of Oxytocin A c e t i c a c i d e x t r a c t s of the neurohypophysis from the p e l l e t i m p l a n t a t i o n s e r i e s were a l s o assayed f o r o x y t o c i n . The RIA procedure has been d e s c r i b e d p r e v i o u s l y (114). The antiserum used, SP-4, has been shown not to c r o s s - r e a c t with a r g i n i n e and l y s i n e v a s o p r e s s i n s , v a s o t o c i n , a n g i o t e n s i n I, c a l c i t o n i n or epinephrine (114). The l i m i t of d e t e c t i o n of t h i s assay was about 25 fmol of o x y t o c i n . The i n t r a - and i n t e r - a s s a y v a r i a t i o n s were s i m i l a r to that of the AVP radioimmunoassay. 32 RESULTS ia. Acute E f f e c t s Of Morphine The animals were. hydrated by i n f u s i n g i n t r a v e n o u s l y a hypotonic g l u c o s e - s a l i n e s o l u t i o n . T h i s was i n f u s e d slowly to prevent any abrupt changes i n plasma o s m o l a l i t y . Urine e x c r e t i o n r a t e was observed t o i n c r e a s e g r a d u a l l y . Once a steady ur i n e flow r a t e was obtained f o r at l e a s t two 10 min c o l l e c t i o n p e r i o d s , morphine s u l f a t e was i n j e c t e d i n t o the t a i l v ein cannula. A moderately low dose, 1 mg/kg, was chosen. This dose r e s u l t e d i n a marked a n t i d i u r e s i s i n the hydrated normal and D.I. (Brattleboro) r a t s . An example of the response of a normal Long-Evans r a t i s shown i n f i g . 4. The a n t i d i u r e t i c response occurred almost immediately a f t e r the morphine i n j e c t i o n . . I t g e n e r a l l y l a s t e d f o r 10-20 min and there was a gradual recovery to the p r e - i n j e c t i o n e x c r e t i o n r a t e . .A rebound phenomenon was observed i n s e v e r a l r a t s d u r i n g the recovery phase. The r a t e of u r i n e e x c r e t i o n was seen to i n c r e a s e beyond the p r e - i n j e c t i o n l e v e l before r e t u r n i n g back t o b a s e l i n e . 32a FIGUEE 4. An example of the r e n a l and c a r d i o v a s c u l a r response of a normal Long-Evans r a t to a s i n g l e dose (1mg/Kg) of morphine s u l f a t e . The drug was i n j e c t e d (MS) a f t e r e s t a b l i s h i n g a steady u r i n a r y flow f o r at l e a s t two 10 min c o l l e c t i o n p e r i o d . U r i n a r y flow was c o r r e c t e d t o 100 g body weight. The dashed l i n e i n d i c a t e s changes i n u r i n e o s m o l a l i t y (Uos). Changes i n mean a r t e r i a l pressure (M.A.P.) were monitored through an i n d w e l l i n g c a r o t i d cannula. Corresponding changes i n osmolar c l e a r a n c e (Cos) and f r e e water c l e a r a n c e (CEUO) are shown i n the bottom graph. 34 The responses of heterozygous and homozygous D.I. r a t s , shown r e s p e c t i v e l y i n f i g . 5 and f i g . 6, were comparable t o t h a t of the normal Long-Evans r a t s . Morphine a l s o produced a marked r e d u c t i o n i n the r a t e of urine e x c r e t i o n i n these animals. The p e r i o d of a n t i d i u r e s i s was b r i e f (10-20 min) with subsequent r e t u r n to pre - i n j e c t i o n l e v e l . . As with the normal r a t s , a rebound phenomenon was observed i n s e v e r a l r a t s . In a l l three groups of r a t s , 1 mg/kg of morphine s u l f a t e produced a mild but n o t i c e a b l e s e d a t i v e e f f e c t . The animals remained immobile or c a t a t o n i c f o r 5-15 min a f t e r the i n j e c t i o n . Most animals, however, appeared to have recovered from the s e d a t i v e e f f e c t of the drug and were f u l l y c o nscious w i t h i n 20 min. Table I summarizes the e f f e c t s of morphine on urine e x c r e t i o n , u r i n e o s m o l a l i t y and a r t e r i a l blood pressure* At 1 mg/kg, morphine caused over 50% (n = 6) r e d u c t i o n i n u r i n e flow i n the normal r a t . The same dose caused a 46% (n=6) r e d u c t i o n i n the heterozygous D.I. group.. Although t h i s r e d u c t i o n was not as great as that observed i n the normal group, the d i f f e r e n c e was not s i g n i f i c a n t . Homozygous D.I. r a t s a l s o showed an a n t i d i u r e t i c response. A 25% (n=6) r e d u c t i o n i n u r i n e e x c r e t i o n was observed a f t e r the morphine i n j e c t i o n . T h i s was s i g n i f i c a n t l y l e s s than that of the normal and heterozygous D.I. groups. 34a FIGURE 5. An example of the r e n a l and c a r d i o v a s c u l a r e f f e c t s of morphine s u l f a t e (1mg/Kg) on a heterozygous D.I. (B r a t t l e b o r o ) r a t . Legend i s the same as t h a t i n f i g * 4. The response to a t e s t dose of AVP (at 50 ug) was a l s o i n c l u d e d f o r comparison. 1 0 min period 35a FIGURE 6. An example of the r e n a l and c a r d i o v a s c u l a r e f f e c t s of morphine s u l f a t e (1mg/Kg) and AVP (10ug) on a homozygous D.I. (B r a t t l e b o r o ) r a t . Legend i s the same as t h a t i n f i g . 4. 36 36a TABLE I. Changes i n mean a r t e r i a l pressure, urine flow rate and urine osmolality in Long-Evans (LE), heterozygous D.I. (HE-Dl) and homozygous D.I. (HO-DI) rats after a 1 mg/Kg dose of morphine sulfate. The values represent the mean ± SEM and each group comprises of 6 animals. * Values i n the HO-DI are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) as compared to the LE group. 3 7 GROUP MEAN ARTERIAL PRESSURE (mmHg) URINE FLOW RATE URINE OSMOLALITY (ul/min) (mOsm/Kg) ^Reduction %Reduct ion Xlncrease LE 8.4 ± 1.2 51.4 ± 9. 1 54. 1 + 20.7 HE-DI 11.3 ± 1. 5 45.9 ± 9.9 64. 8 ± 18.5 HO-DI 16.7 ± 1. 0 25. 1 ± 7.7 9.0 ± 8*3 38 When blood pressure was measured simultaneously, an i n t e r e s t i n g phenomenon was observed* Contrary to the r e p o r t s given by Gomes e t a l . . ( 2 5 , 2 6 ) , a decrease i n a r t e r i a l blood pressure was observed. In the normal Long-Evans group, t h i s amounted to an average of about 8% (n=6) r e d u c t i o n (Table I) . Both B r a t t l e b o r o groups gave g r e a t e r r e d u c t i o n s , with the a r t e r i a l pressure reduced by as much as 16% (n=6) i n the homozygous D.I..group. The decrease observed i n the heterozygous D.I. r a t s (11.3 ± 1.5%, n=6) , however, was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of the normal r a t s . Heart r a t e was monitored i n some experiments and b r a d y c a r d i a was c o n s i s t e n t l y observed a f t e r morphine a d m i n i s t r a t i o n . In the homozygous D.I. group, the heart r a t e decreased from a p r e - i n j e c t i o n l e v e l of 445 + 7 beats/min (n=6) to 320 ± 18 beats/min (n=6) . S i m i l a r changes were observed i n the normal Long-Evans and heterozygous D.I. groups. The r e d u c t i o n i n the rate of urine e x c r e t i o n was accompanied by a s l i g h t i n c r e a s e i n the urine o s m o l a l i t y i n both the normal Long-Evans and the heterozygous D.I. .groups. The homozygous D.I. group, on the other hand, produced only a small and r e l a t i v e l y i n s i g n i f i c a n t i n c r e a s e i n u r i n e o s m o l a l i t y (Table I ) . A l l three groups of r a t s were capable of i n c r e a s i n g u r i n e o s m o l a l i t y i n response t o a t e s t dose of s y n t h e t i c AVP, as shown i n f i g . 5 and 6. Thus, a n t i d i u r e s i s can not be a t t r i b u t e d to the development of r e n a l i n s e n s i t i v i t y t o AVP. 39 I I . C hronic E f f e c t s Of Morphine Development of p h y s i c a l dependence on morphine has been known to occur with repeated use. As shown i n f i g . . 7 , r a t s i n j e c t e d d a i l y with morphine did not gain weight as r a p i d l y as the s a l i n e i n j e c t e d c o n t r o l s . T h i s was s i g n i f i c a n t l y d i f f e r e n t , as determined by Student's t - t e s t , by day 7. Water intake and u r i n e output were not monitored i n t h i s s e r i e s , thus the s t a t e of f l u i d balance of these animals was not known. Body weight gain was a l s o attenuated i n r a t s implanted with morphine p e l l e t s as compared to animals implanted with placebo p e l l e t s ( f i g - 8) . D a i l y water in t a k e o f the morphine-pellet implanted animals was h i g h l y v a r i a b l e d u r i n g the treatment p e r i o d . There was a n o t i c e a b l e decrease i n water i n t a k e immediately a f t e r morphine i m p l a n t a t i o n ( f i g . 8), which presumably i s due to the n o n - s p e c i f i c b e h a v i o r a l d i s r u p t i o n by morphine* D a i l y water i n t a k e , however, r e t u r n e d t o l e v e l s comparable to t h a t o f p l a c e b o - p e l l e t implanted groups on the second day of treatment. Thus, the animals appeared t o have developed t o l e r a n c e to the s e d a t i v e e f f e c t of. the drug. 39a FIGURE 7. Changes i n body weight of rats injected daily with saline (A, n=8) or increasing dosage of morphine sulfate ( A , n=14). The values represent means + SEM of the two groups of ra t s . * Values are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) as determined by Student's t - t e s t . 40a FIGURE 8. Changes i n body weight, water intake and urine output of rats implanted with placebo (•-•) and morphine (a~m,) p e l l e t s . Each animal was implanted with either a placebo or morphine p e l l e t on day 0 and day 3. Both d a i l y water intake and urine output were corrected to 100 g body weight. Withdrawal was precipitated by the excision of the implanted p e l l e t s on day 5. * Values are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) as determined by Student's t - t e s t . 42 As with water i n t a k e , d a i l y urine output was reduced on the f i r s t day of morphine-pellet i m p l a n t a t i o n . Changes i n water i n t a k e and urine e x c r e t i o n were c l o s e l y r e l a t e d throughout the treatment p e r i o d with o n l y one exception.. Urine e x c r e t i o n was s i g n i f i c a n t l y i n c r e a s e d i n the m o r p h i n e - p e l l e t implanted group on day 5, while d a i l y water i n t a k e was s i g n i f i c a n t l y reduced. T h i s change may account f o r the sudden decrease i n body weight observed on day 5. A l s o , i n c o n t r a s t to the a n t i d i u r e t i c e f f e c t of a s i n g l e dose of morphine, the drug appeared to promote u r i n e f o r m a t i o n when given c h r o n i c a l l y . E x c r e t i o n of a dark brown u r i n e was observed i n a few r a t s a f t e r morphine p e l l e t s i m p l a n t a t i o n . Attempts to c h a r a c t e r i z e the nature of the pigmentation through Sephadex G-75 were u n s u c c e s s f u l . There i s reason, however, to b e l i e v e t h a t such pigmentation i s due t o a combination of hemoglobin and myoglobin, as w i l l be d i s c u s s e d l a t e r . Body weight l o s s i s one of the p h y s i o l o g i c a l parameters o f t e n used i n the q u a n t i t a t i v e assessment of p h y s i c a l dependence (103). When morphine was withheld from r a t s i n j e c t e d d a i l y with morphine, the r e s u l t i n g r e d u c t i o n i n body weight was s m a l l ( f i g . 9) . Approximately 355 (n=8) r e d u c t i o n i n body weight was observed 24 hr a f t e r the l a s t i n j e c t i o n . . T h i s i m p l i e s that the degree of dependence of these animals i s very l i g h t . In the morphine-pellet implanted r a t s , however, the s u r g i c a l removal of the implanted p e l l e t s r e s u l t e d i n a dramatic decrease i n body weight. As shown i n f i g . 10, the body weight decreased by 43 as much as 10% 24 hr a f t e r the e x c i s i o n of the p e l l e t s . No s i g n i f i c a n t changes i n body weight were observed i n the p l a c e b o - p e l l e t implanted animals. D a i l y water i n t a k e was depressed s l i g h t l y on the f i r s t withdrawal day but r e t u r n e d to c o n t r o l l e v e l s by the second day. P o l y u r i a was q u i t e marked throughout the withdrawal p e r i o d . T h i s temporary s t a t e of p o l y u r i a accompanied by the t r a n s i e n t decrease i n water i n t a k e can le a d to a negative f l u i d balance and may account f o r the s u b s t a n t i a l decrease i n body weight. The Neurohypophyseal Stores Of AVP Table I I shows the the neurohypophyseal s t o r e s of AVP a f t e r 14 days of morphine s u l f a t e i n j e c t i o n . The average amount of AVP store d i n the neurohypophysis of r a t s i n j e c t e d with morphine f o r 14 days was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of s a l i n e - i n j e c t e d c o n t r o l (423 ± 60 ng vs. 401 ± 32 ng) . . A 24 hr withdrawal from the drug a l s o d i d not appear t o i n f l u e n c e the l e v e l s of AVP i n the neurohypophysis (449 ± 55 ng) . The AVP content i n the hypothalamus of the morphine-injected (45.5 ± 3 . 7 ng) and withdrawn r a t s (34.4 ± 5 . 2 ng) were both lower than that of s a l i n e - i n j e c t e d c o n t r o l (50.4 ± 8.4 ng) . T h i s d i f f e r e n c e , however, was a l s o not s i g n i f i c a n t . 43a FIGURE 9 . Changes i n body weight during withdrawal i n the m u l t i p l e i n j e c t i o n s e r i e s . The r a t s (n=7) were i n j e c t e d with an i n c r e a s i n g dose of morphine s u l f a t e f o r IU days p r i o r t o the withdrawal of the drug., 44 Hours Af ter the Last I n j e c t i o n 44a FIGURE 10. Changes i n body weight during withdrawal i n the p e l l e t i m p l a n t a t i o n s e r i e s . The r a t s (n=6) were implanted with e i t h e r placebo (•) or morphine p e l l e t s (o) f o r f i v e days. * Values are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) as determined by Student's t - t e s t . 45 Hours A f t e r Pellet Exc i s ion 45a TABLE I I . Hypothalamic and neurohypophyseal s t o r e s of AVP i n the m u l t i p l e i n j e c t i o n s e r i e s . The c o n t r o l group was i n j e c t e d with s a l i n e . a n d the morphine-treated group with morphine s u l f a t e f o r 14 days. Morphine was withheld from the morphine-withdrawn group f o r 24 hr a f t e r the 14 day treatment p e r i o d . Each group c o n s i s t e d of 8 r a t s . No s i g n i f i c a n t d i f f e r e n c e was found between the t h r e e treatments. HYPOTHALAMUS NEUROHYPOPHYSIS GROUP (ng AVP) (ng AVP) CONTROL MORPHINE WITHDRAWN 50.44 ± 8.43 45. 50 ± 3.67 34. 43 ± 5.15 401 ± 32 423 ± 60 449 ± 55 47 Neurohypophyseal AVP s t o r e s of the morphine p e l l e t i m p l a n t a t i o n s e r i e s are shown i n f i g . 11. Morphine treatment r e s u l t e d i n a s i g n i f i c a n t d e p l e t i o n of the neurohypophyseal AVP s t o r e s . T h i s was p a r t i c u l a r l y n o t i c e a b l e on day 3 , i n which the amount of AVP s t o r e d was reduced from a c o n t r o l value of 1024 ± 66 ng (n=6) to 744 ± 28 ng (n= 6 ) . Continued treatment with the drug d i d not cause any f u r t h e r d e p l e t i o n of the neurohypophyseal AVP s t o r e s . In f a c t , i t appeared t h a t the neurohypophyseal s t o r e s of the hormone, which were deplet e d d u r i n g the f i r s t t h r e e days of treatment, were being r e p l e n i s h e d by the f o u r t h and f i f t h day. The amount of RVP stored on the f i f t h day of treatment was not s i g n i f i c a n t l y d i f f e r e n t from t h a t of the p l a c e b o - p e l l e t implanted c o n t r o l . I f the AVP s t o r e d i n the gland i s maintained near the normal l e v e l with the continuous a p p l i c a t i o n of morphine a f t e r the f i f t h day, i t may e x p l a i n the l a c k o f d e p l e t i o n of AVP i n the neurohypophysis of r a t s given d a i l y i * p . i n j e c t i o n of morphine on the f o u r t e e n t h day. P o s s i b l e mechanisms by which the AVP s t o r e s were r e p l e n i s h e d w i l l be d i s c u s s e d l a t e r . 47a FIGURE 11.. Neurohypophyseal s t o r e s of AVP i n r a t s implanted with placebo p e l l e t s (C) and morphine p e l l e t s f o r one (1), three (3) or f i v e (5) days.. S t i p p l e d bars r e p r e s e n t neurohypophyseal AVP i n r a t s implanted with morphine p e l l e t s f o r 5 days and allowed to withdraw from the drug f o r 3 days t h e r e a f t e r (W). The c o n t r o l group (Wc) was implanted with placebo p e l l e t s and subjected to the same treatment as the withdrawal group (W). . * Values are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) as determined by Student's t - t e s t . 48 W sz a o a 3 <D C \ CL > < CO c 1200 800+ 400+ C 1 3 5 Wc W Morphine Treatment 49 When morphine withdrawal was p r e c i p i t a t e d on the f i f t h day by the s u r g i c a l removal of the implanted p e l l e t s , a marked r e d u c t i o n i n the neurohypophyseal fcVP s t o r e s was observed ( f i g 11)..The l e v e l s decreased from a pre-withdrawal value of 1012 ± 70 ng (n=6) to 638 ± 36 ng (n=6) 72 hr a f t e r the e x c i s i o n of the pellets..Removal of the implanted p l a c e b o - p e l l e t s did not r e s u l t i n any s i g n i f i c a n t change i n the neurohypophyseal AVP s t o r e s (902 ± 37 ng, n=6) as compared to the p l a c e b o - p e l l e t implanted c o n t r o l s * D e p l e t i o n of the neurohypophyseal AVP s t o r e , t h e r e f o r e , can only be observed d u r i n g the e a r l y stages of morphine treatment. As the animal develops t o l e r a n c e to and p h y s i c a l dependence on the drug, the neurohypophyseal s t o r e s of AVP returned t o pre-treatment l e v e l s . . Abrupt withdrawal of the drug from p h y s i c a l l y dependent animals again l e d to a d e p l e t i o n of the AVP s t o r e s . During morphine treatment, no changes i n the hypothalamic AVP s t o r e s were observed i n the p e l l e t i m p l a n t a t i o n s e r i e s ( f i g - 12). During withdrawal, however, a s i g n i f i c a n t r e d u c t i o n i n AVP s t o r e s was seen. The hypothalamic AVP s t o r e s were reduced from the pre-withdrawal value of 53 ± 5 ng (n=6) to 33 ± 5 ng (n=6) 72 hr a f t e r the e x c i s i o n of the implanted p e l l e t s . T h i s change was s i g n i f i c a n t as determined by the Student's t - t e s t (p<0.03) . 49a FIGURE 12.. Hypothalamic AVP s t o r e s i n r a t s implanted with placebo p e l l e t s (C) or morphine p e l l e t s (1, 3 and 5). S t i p p l e d bars r e p r e s e n t hypothalamic s t o r e s of AVP i n the withdrawal group (W) and i t s placebo implanted c o n t r o l (He),.* Values are s i g n i f i c a n t l y d i f f e r e n t (p<0. 05) as determined by Student's t - t e s t . 50 3 E o a O) c 8 0 4 0 + J u l C 1 3 5 W c M o r p h i n e T r e a t m e n t w 51 The c o n t r o l neurohypophyseal AVP s t o r e s i n the morphine-pellet implanted s e r i e s ( f i g . 11) was much higher than t h a t observed i n the i . p . . i n j e c t e d s e r i e s (Table I I ) . This d i s c r e p a n c y can n e i t h e r be e x p l a i n e d by v a r i a t i o n s i n the e x t r a c t i o n procedure nor by v a r i a t i o n s i n the RIA of AVP. I t may be due to the d i f f e r e n c e s i n the age of the animals used. Young male Wistar r a t s (193.5 ± 2.5 g, n = 8) were used i n the i . p . i n j e c t i o n s e r i e s , whereas a d u l t male Wistar r a t s (266.0 ± 2.5 g, n=6) were used i n the morphine-pellet i m p l a n t a t i o n s e r i e s . Seasonal v a r i a t i o n s may a l s o p l a y a r o l e i n a l t e r i n g the neurohypophyseal s t o r e s of AVP. The i . p . i n j e c t i o n s e r i e s was performed i n the month of December, whereas the p e l l e t i m p l a n t a t i o n s e r i e s was performed i n May. B.,Neurohypophyseal Stores of Oxytocin The neurohypophyseal o x y t o c i n s t o r e s of the morphine-pellet implanted s e r i e s are shown i n F i g . 13. Unlike the neurohypophyseal AVP s t o r e s , o x y t o c i n s t o r e s i n t h e . r a t s implanted with morphine p e l l e t s were u n a l t e r e d i n the f i r s t t h r e e days of treatment. However, a s l i g h t i n c r e a s e i n the neurohypophyseal o x y t o c i n s t o r e s from a c o n t r o l value of 1051 ± 52 ng (n=6) to 1353 ± 50 ng (n=6) was observed on the f i f t h day. T h i s d i f f e r e n c e was s i g n i f i c a n t as determined by Student's t - t e s t (p<0.01). This change a l s o c o i n c i d e d with the replenishment of the depleted AVP s t o r e s . The abrupt withdrawal from the drug i n i t i a t e d on the f i f t h day of treatment r e s u l t e d i n a s i g n i f i c a n t r e d u c t i o n i n the neurohypophyseal o x y t o c i n 52 s t o r e s . The oxytocin s t o r e s f e l l from a pre-withdrawal value o f 1328 ± 54 ng (n=6) to 901 ± 38 ng (n=6) 72 hr a f t e r the e x c i s i o n o f the p e l l e t s . C. Serum O s m o l a l i t y and Sodium No s i g n i f i c a n t changes i n serum o s m o l a l i t y and serum sodium were det e c t e d between the morphine and placebo p e l l e t implanted animals (Table V). There was a s l i g h t i n c r e a s e i n serum o s m o l a l i t y i n the morphine withdrawn group (303.5 ± 2.6 mOsm/kg, n=6) as compared to i t s placebo implanted c o n t r o l (296.8 ± 2.4 mOsm/kg, n=6) . T h i s d i f f e r e n c e , however, was not s i g n i f i c a n t as determined by Student's t - t e s t . 52a FIGURE 13. _ Neurohypophyseal s t o r e s of oxytocin i n r a t s implanted with placebo p e l l e t s (C) or morphine p e l l e t s (1,3 and 5 ) . . S t i p p l e d bars r e p r e s e n t the withdrawal group (W) and i t s placebo implanted c o n t r o l (Wc). * Values are s i g n i f i c a n t l y d i f f e r e n t (p<0.05) as determined by Student's t - t e s t . 53 a o a. >« 3 c \ c o o 1600 t 1 2 0 0 2 8 0 0 4 -2 400-f >» X o c n c X 5 Wc W 53a TABLE III.„ Serum o s m o l a l i t y and sodium of r a t s i n the p e l l e t i m p l a n t a t i o n s e r i e s . Each group c o n s i s t e d of 6 r a t s . The groups were c l a s s i f i e d as f o l l o w s : C r a t s implanted with placebo p e l l e t s f o r 5 days; 1 r a t s implanted with morphine p e l l e t f o r 1 day; 3 r a t s implanted with morphine p e l l e t s f o r 3 days; 5 r a t s implanted with morphine p e l l e t s f o r 5 days; Wc r a t s implanted with placebo p e l l e t s f o r 5 days; p e l l e t s were ex c i s e d on the f i f t h day and the animals allowed t o recover f o r 3 days; W r a t s implanted with morphine p e l l e t s f o r 5 days; p e l l e t s were ex c i s e d on the f i f t h day and the animals allowed t o recover f o r 3 days; No s i g n i f i c a n t d i f f e r e n c e was found amongst a l l groups. 54 SERUM OSMOLALITY SERUM SODIUM GROUP (mOsm/Kg) (mEq/L) C 293.7 ± 2.0 144.8 ± 0.9 1 295. 2 ± 3. 4 142.7 ± 1. 1 3 297.0 ± 1 . 8 146.0 ± 2. 8 5 292.7 ± 3.2 143.8 ± 1.0 Wc 29 6.8 ± 2.4 14 5.8 ± 1.7 W 303.5 ± 2.6 147.7 ± 1 . 1 55 P i Opiate Receptor Binding The b i n d i n g of 3H-naloxone to whole b r a i n homogenate was depressed with c h r o n i c i . p . i n j e c t i o n of morphine ( f i g . 14). Morphine abstinence f o r 24 hr r e s u l t e d i n near complete recovery of the 3H-naloxone binding* Since the decrease i n r e c e p t o r b i n d i n g can be e x p l a i n e d by e i t h e r a change i n the number of b i n d i n g s i t e s or b i n d i n g a f f i n i t y , a Scatchard (115) a n a l y s i s of the 3H-naloxone b i n d i n g was performed ( f i g . .15). The a f f i n i t y constant (Kd) f o r 3H-naloxone was found to be 3.2 and 5.3 nM f o r the c o n t r o l and withdrawal groups, r e s p e c t i v e l y . . T h i s was i n c r e a s e d four to seven f o l d (about 22.4 nM) i n the morphine t r e a t e d group (Table V I ) . A small and r e l a t i v e l y i n s i g n i f i c a n t i n c r e a s e i n the number of r e c e p t o r b i n d i n g s i t e s (q) was a l s o observed in the morphine-treated group (Table VI) . The l a c k of change i n the number of r e c e p t o r b i n d i n g s i t e s coupled t o the decrease i n the a f f i n i t y of the r e c e p t o r f o r 3H-naloxone i n the morphine-treated r a t s suggest the presence of a competitive l i g a n d , such as morphine, i n the b r a i n homogenate. Repeated washing of the r e c e p t o r p r e p a r a t i o n from the morphine-injected r a t s r e s u l t e d i n an enhancement of the 3H-naloxone b i n d i n g . E f f e c t s of the neurohypophyseal hormones, AVP and o x y t o c i n , on the r e c e p t o r b i n d i n g assay were a l s o t e s t e d . No s p e c i f i c or n o n - s p e c i f i c i n t e r f e r e n c e were det e c t e d at c o n c e n t r a t i o n s of 20 pM to 2 nM. 55a FIGURE 14,, S t e r e o s p e c i f i c b i n d i n g of 3H-naloxone to whole b r a i n homogenates from r a t s i n j e c t e d d a i l y with s a l i n e (©) or morphine s u l f a t e (A) f o r 14 days. The t h i r d group (A) r e p r e s e n t s bin d i n g to whole b r a i n homogenates from r a t s withdrawn from the drug f o r 24 hr. . 56 C o n c e n t r a t i o n of 3H - N a l o x o n e (ViM) 56a FIGURE 15., Double r e c i p r o c a l (Scatchard) p l o t of s t e r e o s p e c i f i c 3H-naloxone b i n d i n g t o whole b r a i n homogenates from r a t s i n the m u l t i p l e i n j e c t i o n s e r i e s . Legend i s the same as that i n f i g . 14. 5 7 1 / 3 H - Na loxone C o n c e n t r a t i o n (nM" ' ) 57a TABLE IV. Number of bin d i n g s i t e s (q) and binding a f f i n i t y (Kd) as determined from Scatchard a n a l y s i s of 3H-naloxone b i n d i n g t o whole brain homogenates from r a t s i n j e c t e d with morphine. GROUP BINDING SITES (pmol) BINDING AFFINITY (nM) CONTROL 0. 370 5. 32 MORPHINE 0.588 22.37 WITHDRAWN 0.213 3.21 59 DISCUSSION The present study d e a l t with two aspects of morphine e f f e c t s on AVP f u n c t i o n - the immediate sytemic e f f e c t s of a s i n g l e dose a d m i n i s t r a t i o n (acute experiments) and the e f f e c t s of continuous a d m i n i s t r a t i o n of the drug (chronic experiments). These two a s p e c t s w i l l be d e a l t with s e p a r a t e l y i n the d i s c u s s i o n * I. Acute E f f e c t s of Morphine Ad m i n i s t r a t i o n One of the approaches i n s t u d y i n g the a n t i d i u r e t i c a c t i o n of a drug i s to measure the a b i l i t y of the drug t o reduce the r a t e of u r i n a r y flow i n a water hydrated animal. In t h i s type of study, i t i s important to e s t a b l i s h a constant s t a t e of h y d r a t i o n and water d i u r e s i s . I f the a c t i o n of the drug i s d i r e c t e d at the kidneys, then f a c t o r s a f f e c t i n g u r i n e flow r a t e , such as endogenous r e l e a s e of AVP, must be c o n t r o l l e d . T h i s i s the b a s i s f o r using e t h a n o l to suppress the endogenous r e l e a s e of AVP i n the a n t i d i u r e t i c hormone (ADH) bioassay (116) . The study r e p o r t e d by I n t u r r i s i and Fujimoto (132) on the a n t i d i u r e t i c a c t i o n of morphine u t i l i z e d t h i s f e a t u r e of the ADH bioassay* One major problem i n using ethanol-hydrated animal p r e p a r a t i o n s i s t h a t a n t i d i u r e s i s mediated through the r e l e a s e of AVP may not be detected. The reason being t h a t the drug i n question has to counteract the s u p p r e s s i v e e f f e c t of both ethanol and low plasma o s m o l a l i t y on AVP r e l e a s e . In view of the p o s s i b i l i t y t h a t morphine-induced a n t i d i u r e s i s i s mediated 60 by the r e l e a s e of AVP, ethanol was excluded from the i n f u s i o n s o l u t i o n i n these experiments. T h i s a l s o has an added p h y s i o l o g i c a l advantage i n that the animals were f u l l y c o nscious. The i n h i b i t i o n of endogenous AVP r e l e a s e was e v i d e n t by the maintenance of a s t a b l e high u r i n a r y flow r a t e and low u r i n e o s m o l a l i t y . The r e s u l t s from the acute experiments c l e a r l y e s t a b l i s h e d t h a t morphine at 1 mg/kg, induced a s u b s t a n t i a l r e d u c t i o n i n u r i n a r y flow i n the hydrated r a t (Table I) . T h i s agreed w e l l with s t u d i e s by s e v e r a l i n v e s t i g a t o r s on the a n t i d i u r e t i c a c t i o n of morphine (17-21). The same dose a l s o produced a s i g n i f i c a n t r e d u c t i o n i n the mean a r t e r i a l pressure (Table I ) . . In terms of the r e n a l and c a r d i o v a s c u l a r responses t o morphine, both heterozygous D.I. and normal Long-Evans r a t s may be c o n s i d e r e d i d e n t i c a l . However, the responses of the homozygous D.I. r a t were s t a t i s t i c a l l y d i f f e r e n t from those of heterozygous D.I. and normal Long-Evans r a t s . The r e d u c t i o n i n u r i n a r y flow was accompanied by an i n c r e a s e i n u r i n e o s m o l a l i t y . The c o n c e n t r a t i n g a b i l i t y o f the kidney i n a l l three groups improved a f t e r the a d m i n i s t r a t i o n of morphine. A s u b s t a n t i a l d e c r e a s e . i n f r e e water c l e a r a n c e was a l s o observed. In some cases, f r e e water c l e a r a n c e r e v e r s e d t o negative values, suggesting net water r e a b s o r p t i o n . I t i s d i f f i c u l t t o a c c u r a t e l y determine the u n d e r l y i n g mechanism f o r t h i s decrease as f r e e water c l e a r a n c e i s a f f e c t e d by a multitude of 61 p h y s i o l o g i c a l parameters. N e v e r t h e l e s s , these changes suggested the involvement of AVP i n the heterozygous D.I. and normal r a t s . The r e l e a s e of AVP has been known t o be i n f l u e n c e d by a number of f a c t o r s . These i n c l u d e changes i n blood o s m o l a l i t y , volume, and pressure (30). . I t s r e l e a s e i s a l s o a f f e c t e d by v a r i o u s hormones, water and e l e c t r o l y t e balance, and emotional s t r e s s (117). i t has been widely accepted t h a t the morphinerinduced a n t i d i u r e s i s i s due t o the d i r e c t s t i m u l a t i o n of the hypothalamo-neurohypophyseal system (17-21). In order to c o n c l u s i v e l y prove t h i s , a l l f a c t o r s a f f e c t i n g the r e l e a s e of AVP must be adequately c o n t r o l l e d . In view of the powerful c a r d i o v a s c u l a r e f f e c t of morphine (25-27), the s e c r e t i o n of AVP may not be a d i r e c t r e s u l t of the s t i m u l a t i o n of the hypothalamo-neurohypophyseal system by the drug. Recent evidence i n d i c a t e s t h a t blood pressure changes as sm a l l as 5% could s i g n i f i c a n t l y i n c r e a s e the plasma AVP l e v e l s (30). As much as 8 and 11% r e d u c t i o n i n mean a r t e r i a l pressure (Table I) was observed a f t e r morphine a d m i n i s t r a t i o n i n the normal Long-Evans and heterozygous D.I. r a t s , r e s p e c t i v e l y . ,Hence, hypotension can not be r u l e d out as a p o s s i b l e mechanism by which the drug e f f e c t s the r e l e a s e o f AVP. I f the endogenous r e l e a s e of AVP i s the s o l e mechanism r e s p o n s i b l e f o r the a n t i d i u r e t i c a c t i o n of morphine, one would not expect an a n t i d i u r e t i c response from animals i n c a p a b l e of s y n t h e s i z i n g AVP. For example, 62 t h i s was shown to be the case f o r chlorpropamide-induced a n t i d i u r e s i s (118). Chlorpropamide, at doses e f f e c t i v e i n causing a n t i d i u r e s i s i n normal r a t s was i n e f f e c t i v e i n r a t s with h e r e d i t a r y hypothalamic dia b e t e s i n s i p i d u s (118). Morphine s u l f a t e , on the other hand, was e f f e c t i v e i n r e d u c i n g the u r i n a r y flow i n the homozygous D.I. r a t s (Table I ) . Hence, mechanisms other than AVP must be i n v o l v e d i n the i n d u c t i o n of a n t i d i u r e s i s by morphine* The a n t i d i u r e s i s observed i n the homozygous D.I. r a t s was s i g n i f i c a n t l y l e s s than t h a t of the normal and heterozygous D.I. r a t s . This d i f f e r e n c e may be due to the l a c k o f AVP i n the homozygous D.I. r a t s , l e a d i n g to a decreased a b i l i t y of the kidneys to reabsorb water. Compared to the normal and heterozygous D.I. r a t s , there was h a r d l y any change in the u r i n e o s m o l a l i t y of the homozygous D.I,, r a t during the a n t i d i u r e t i c p e r i o d . T h i s again can be a t t r i b u t e d to the l a c k of AVP i n these animals. A n t i d i u r e s i s can be brought on by s e v e r a l means., For example, a l t e r a t i o n s i n r e n a l f u n c t i o n can be induced by v a r i o u s v a s o a c t i v e agents. The r e s u l t i n g changes i n f i l t r a t e r e a b s o r p t i o n , amount of f i l t r a t e formed, or both, can l e a l t o a marked r e d u c t i o n i n u r i n a r y flow.. A n t i d i u r e s i s and a n t i n a t r i u r e s i s have been shown t o be induced by changes i n the p h y s i c a l f o r c e s r e s p o n s i b l e f o r t u b u l a r f l u i d r e a b s o r p t i o n (119) as w e l l as by v a s o a c t i v e agents such as a n g i o t e n s i n (120,121), epinephrine (121), and AVP (122). Changes i n 3FR, caused by e i t h e r an 6 3 a l t e r a t i o n i n glomerular membrane p e r m e a b i l i t y or r e n a l v a s c u l a r tone (123) can a l s o cause a s u b s t a n t i a l r e d u c t i o n i n u r i n a r y flow. Since the homozygous D.I. r a t s are devoid of AVP, only AVP-independent mechanisms may be used to e x p l a i n the a n t i d i u r e s i s observed i n these animals. In view of the marked decrease i n mean a r t e r i a l pressure observed a f t e r morphine a d m i n i s t r a t i o n , i t i s h i g h l y probable that n e u r a l and hormonal compensatory mechanisms, s i m i l a r to those i n v o l v e d i n hemorrhagic hypotension (124,125) are a c t i v a t e d . An i n c r e a s e i n sympathetic nervous tone and c i r c u l a t i n g catecholamines has been shown to d r a m a t i c a l l y a l t e r r e n a l f u n c t i o n (126, 1 27) .. Reduction i n r e n a l blood flow, and r e d u c t i o n i n s a l t and water e x c r e t i o n have been demonstrated during p e r i o d s of heightened sympathetic discharge (126). An i n c r e a s e i n c i r c u l a t i n g catecholamine l e v e l s (mainly epinephrine and norepinephrine) can cause a decrease i n the t o t a l r e n a l blood flow and an i n c r e a s e i n f i l t r a t i o n f r a c t i o n (127). In a d d i t i o n , hypotension may a l s o serve as a stimulus f o r the r e l e a s e of other v a s o a c t i v e peptides (120-122).„Activation of the r e n i n - a n g i o t e n s i n system, f o r example, was shown to r e s u l t i n a decrease i n GFR and RBF, a n t i d i u r e s i s and a n t i n a t r i u r e s i s (128) . I t i s t h e r e f o r e l i k e l y t h a t a n t i d i u r e s i s i n the homozygous D.I. r a t i s due to changes i n the c a r d i o v a s c u l a r system and r e n a l hemodynamics brought on by n e u r a l and hormonal mechanisms other than AVP. Morphine appeared to exert a more: potent 6 4 hypotensive e f f e c t on the homozygous D.I. r a t s as compared to the normal and heterozygous D.I. r a t s (Table I ) . T h i s suggested t h a t the l a c k of AVP may have i n t e r f e r e d with the c a r d i o v a s c u l a r compensatory mechanisms i n these animals* Under normal c o n d i t i o n s , AVP has been shown to be i n v o l v e d i n the maintenance of the mesenteric v a s c u l a r tone (129). Hypophysectomy was shown to cause d i l a t a t i o n of the mesenteric r e s i s t a n c e v e s s e l s as w e l l as to a b o l i s h the i n t e s t i n a l v a s o c o n s t r i c t o r response to volume d e p l e t i o n (130). In a d d i t i o n , r e n a l blood v e s s e l s appeared normally t o be under the v a s o c o n s t r i c t o r i n f l u e n c e of AVP (131). The g r e a t e r s e n s i t i v i t y t o AVP i n the diabetes i n s i p i d u s animals has been shown to be p a r t i a l l y due to the v a s o c o n s t r i c t o r e f f e c t of the hormone (132). Hence, i n the normal r a t , the v a s o c o n s t r i c t o r e f f e c t of AVP may a s s i s t i n the r e s t o r a t i o n of a normal systemic blood pressure. The r a t e of c l e a r a n c e of morphine i s slow and i t s d u r a t i o n of a c t i o n i s g e n e r a l l y i n the order of 4-5 hr (133). Uptake of the drug by the CNS can be detected as long as 60 minutes a f t e r a s i n g l e i . v . i n j e c t i o n (134,135)..Hence, the presence of the drug should provide a continuous s t i m u l u s f o r AVP s e c r e t i o n and a p r o t r a c t e d a n t i d i u r e t i c p e r i o d . However, the a n t i d i u r e t i c p e r i o d observed only l a s t e d f o r 10 to 20 minutes. T h i s could e i t h e r mean t h a t the drug was c l e a r e d r a p i d l y t o below the s t i m u l u s t h r e s h o l d or the animal has developed r a p i d t o l e r a n c e or t a c h y p h y l a x i s to the drug. When a second dose 65 o f morphine was administered 30 min a f t e r the i n i t i a l dose, the r e s u l t i n g a n t i d i u r e s i s observed was much reduced. T h e r e f o r e , the s h o r t a n t i d i u r e s i s p e r i o d observed a f t e r the i n i t i a l dose i s l i k e l y due to the development of ta c h y p h y l a x i s * . Morphine t a c h y p h y l a x i s was a l s o observed by I n t u r r i s i and Fujimoto (17). They hypothesized t h a t morphine has an i n h i b i t o r y e f f e c t on AVP re l e a s e a f t e r one or more doses. The mechanism u n d e r l y i n g the development of t a c h y p h y l a x i s i s not known a t present. The c h r o n i c experiments i n the c u r r e n t study were designed to examine the long term e f f e c t s of morphine a d m i n i s t r a t i o n on the hypothalamo-neurohypophyseal system.„ Changes i n the neurohypophyseal s t o r e s of AVP might shed some l i g h t on the e f f e c t s of morphine on the r e l e a s e of AVP. II • C h r o n i c E f f e c t s of Morphine A d m i n i s t r a t i o n Chronic osmotic s t i m u l i or dehydration has been known to d e p l e t e the neurohypophyseal s t o r e s of both AVP and o x y t o c i n (136,137). Since the acute a d m i n i s t r a t i o n of morphine causes the r e l e a s e of AVP, i t would be of i n t e r e s t to examine the adaptive response of the neurohypophysis upon c h r o n i c a d m i n i s t r a t i o n . . Eats implanted with morphine p e l l e t s showed p r o g r e s s i v e d e p l e t i o n of neurohypophyseal AVP i n t h e . f i r s t t h r e e days of treatment. The neurohypophyseal s t o r e s subsequently recovered to the pre-treatment l e v e l s on the f i f t h day. These changes may r e f l e c t the development of t o l e r a n c e to the s t i m u l a t o r y e f f e c t s of the drug. Although t a c h y p h y l a x i s can be demonstrated a f t e r a s i n g l e dose 66 i n j e c t i o n o f morphine, the development of t o l e r a n c e i s reported t o be maximal a f t e r 3 days (33)..Hence, the d e p l e t i o n of AVP i n the f i r s t 3 days of treatment might be due to the d i r e c t s t i m u l a t i o n of AVP r e l e a s e or by morphine-induced a l t e r a t i o n of the a f f e r e n t s i g n a l s t o the hypothalamo-neurohypophyseal system. Presumably, the amount of hormone r e l e a s e d during t h i s 3 day p e r i o d exceeded the b i o s y n t h e t i c r a t e * With the development of t o l e r a n c e , the amount of hormone r e l e a s e d p r o g r e s s i v e l y decreases, thus a l l o w i n g b i o s y n t h e s i s to overcome the r a t e of r e l e a s e and r e p l e n i s h the neurohypophyseal s t o r e s of the hormone. In r a t s given d a i l y i . p . . i n j e c t i o n s of morphine, the neurohypophyseal AVP s t o r e s d i d not show any s i g n i f i c a n t d i f f e r e n c e compared to those of s a l i n e - i n j e c t e d c o n t r o l s . T h i s may be due to the f a c t t h at the animals are t o l e r a n t t o the s t i m u l a t o r y e f f e c t s of morphine at the time of s a c r i f i c e . I t i s q u i t e p o s s i b l e t h a t the l a c k of d i f f e r e n c e be due to an adaptive change i n AVP s y n t h e s i s i n response to c h r o n i c s t i m u l a t i o n . I t i s d i f f i c u l t to compare the values obtained i n the m u l t i p l e i n j e c t i o n s e r i e s with those of the p e l l e t i m p l a n t a t i o n s e r i e s . F i r s t , the degree of dependence on morphine i n these two s e r i e s i s d i f f e r e n t as i s e v i d e n t by the changes i n body weight during withdrawal..A 10% body weight l o s s was encountered i n the p e l l e t i m p l a n t a t i o n s e r i e s a f t e r 24 hr of drug abs t i n e n c e . The morphine i n j e c t e d r a t s , on the other hand, only e x h i b i t e d a 3% 67 decrease i n body weight over the same p e r i o d of withdrawal. Secondly, the mode of d e l i v e r y of the drug i s d i f f e r e n t . In the m u l t i p l e i n j e c t i o n s e r i e s , morphine was given twice d a i l y at 0800 and 1800 hr. Since morphine i s c l e a r e d i n 4-5 hr, the animals may go through a b r i e f p e r i o d of withdrawal between i n j e c t i o n s . On the other hand, morphine was d e l i v e r e d at a reasonably continuous r a t e i n the p e l l e t i m p l a n t a t i o n s e r i e s . Thus, these animals are not as l i k e l y to have encountered withdrawal periods throughout the s e r i e s . T h i r d l y , the d u r a t i o n of exposure t o the drug v a r i e s between the two s e r i e s . The r a t s were only exposed to morphine f o r 5 days i n the p e l l e t i m p l a n t a t i o n s e r i e s , whereas r a t s were given morphine f o r 14 days i n the m u l t i p l e i n j e c t i o n s e r i e s . Hence, the neurohypophyseal s t o r e s of AVP are not comparable between the two s e r i e s . . I t i s of i n t e r e s t , however, t h a t the neurohypophyseal AVP of the s a l i n e i n j e c t e d r a t s (423 ± 60 ng, n=8) was much lower than t h a t of the p l a c e b o - p e l l e t i n p l a n t e d r a t s (1024 ± 66 ng, n=6) . T h i s i s p a r t i a l l y due to the d i f f e r e n c e s ' i n the age of the animals used. Young r a t s were used i n the m u l t i p l e i n j e c t i o n s e r i e s , whereas adult r a t s were used i n the p e l l e t i m p l a n t a t i o n s e r i e s . A d u l t r a t s have been shown to have s i g n i f i c a n t l y more AVP store d i n the neurohypophysis than young r a t s (137). I t i s a l s o p o s s i b l e that the s t r e s s caused by the d a i l y h a n d l i n g and i n j e c t i o n s may have c o n t r i b u t e d t o the d e p l e t i o n of AVP s t o r e s i n the m u l t i p l e i n j e c t i o n s e r i e s . S t r e s s f u l and p a i n f u l s t i m u l i have long 68 been known to s t i m u l a t e the r e l e a s e of AVP (117).,Animals i n the p e l l e t i m p l a n t a t i o n s e r i e s were not sub j e c t e d to s t r e s s f u l s t i m u l i and, consequently, a be t t e r r e f l e c t i o n of the c h r o n i c e f f e c t s of the drug. D e p l e t i o n of neurohypophyseal AVP s t o r e s i n the morphine-pellet implanted r a t s can be a t t r i b u t e d t o a decreased b i o s y n t h e t i c r a t e or an i n c r e a s e d r a t e of r e l e a s e * Morphine has been known to a f f e c t the s y n t h e s i s of p r o t e i n s (138). Furthermore, t h i s has been a t t r i b u t e d to the decrease i n food i n t a k e r e s u l t i n g from the s e d a t i v e e f f e c t of the drug (138). Ho wever, s t a r v a t i o n per se i s not a l i k e l y cause f o r the d e p l e t i o n of the neurohypophyseal AVP s t o r e s . I t has been shown to have no e f f e c t on AVP s t o r e s d e s p i t e the presence of a s l i g h t decrease i n water i n t a k e (139). T h i s , however, does not r u l e out the p o s s i b i l i t y of a d i r e c t e f f e c t of morphine on the b i o s y n t h e s i s or t r a n s p o r t of AVP. Increased r e l e a s e , such as that o c c u r r i n g during dehydration (139), can a l s o deplete the neurohypophyseal s t o r e s of AVP. T h i s may p a r t i a l l y e x p l a i n the d e p l e t i o n observed a f t e r the f i r s t day of p e l l e t i m p l a n t a t i o n . Water i n t a k e i n r a t s implanted with morphine p e l l e t s decreased s i g n i f i c a n t l y a f t e r the f i r s t day ( F i g . 10). As water i n t a k e recovered t o c o n t r o l l e v e l s by the second day, dehydration can be r u l e d out as a f a c t o r f o r the d e p l e t i o n of neurohypophyseal AVP s t o r e s observed on the t h i r d day of treatment. Since AVP was shown to be r e l e a s e d with the acute a d m i n i s t r a t i o n of morphine (17-21) , the continuous 69 a p p l i c a t i o n of the drug should d e p l e t e the s t o r e s of the hormone. A c h r o n i c a l l y e l e v a t e d c i r c u l a t i n g RVP l e v e l would tend t o promote water r e t e n t i o n and development of a syndrome of i n a p p r o p r i a t e s e c r e t i o n of a n t i d i u r e t i c hormone (SIADH). The two most prominent symptoms of SIADH are reduced serum o s m o l a l i t y and d i l u t i o n a l hyponatremia (140). Serum de t e r m i n a t i o n s performed on r a t s t r e a t e d with morphine r e v e a l e d no changes i n e i t h e r serum o s m o l a l i t y or sodium (Table V). T h i s r a i s e s the p o s s i b i l i t y t h a t the hormone may not be r e l e a s e d i n t o the systemic c i r c u l a t i o n i f the d e p l e t i o n of AVP i s due t o an i n c r e a s e d r a t e of r e l e a s e * In c o n t r a s t to AVP, o x y t o c i n s t o r e s i n the neurohypophysis were unalte r e d by morphine-pellet i m p l a n t a t i o n . Although the neurosecretory neurons producing these two hormones are a n a t o m i c a l l y c l o s e l y a s s o c i a t e d , t h e r e i s evidence t h a t these hormones can be r e l e a s e d independently of each other (141). Thus, s t i m u l i causing AVP r e l e a s e may not n e c e s s a r i l y a f f e c t the r e l e a s e of oxytocin* A l s o , morphine and i t s analogues have been shown to i n h i b i t the s u c k l i n g - i n d u c e d o x y t o c i n r e l e a s e (140). T h i s may account f o r the l a c k of d e p l e t i o n of o x y t o c i n s t o r e s a f t e r 3 days of morphine t r e a t m e n t . . I t may a l s o e x p l a i n the s l i g h t e l e v a t i o n i n o x y t o c i n s t o r e s observed on the f i f t h day of treatment. Abrupt withdrawal of morphine from the p h y s i c a l l y dependent animals r e s u l t e d i n the d e p l e t i o n of both AVP and o x y t o c i n s t o r e s . Changes i n AVP storage may 70 p a r t i a l l y be due to the sudden changes i n water and e l e c t r o l y t e balance. Diarrhea (142), and a decrease i n water i n t a k e (40) during withdrawal can d r a s t i c a l l y reduce the body's s t o r e s of s a l t and water. These changes, however, were not observed i n the c u r r e n t study ( f i g . 10) . Aside from the t r a n s i e n t decrease i n water i n t a k e observed on the f i r s t day of withdrawal, no s i g n i f i c a n t changes were observed i n the morphine or placebo p e l l e t implanted group on the second and t h i r d day of withdrawal. There was, however, a n o t i c e a b l e i n c r e a s e i n d a i l y u r i n e output throughout the 3 days o f withdrawal. Because of the small number of animals i n each group (n=6), t h i s i n c r e a s e was not s i g n i f i c a n t as determined by Student's t - t e s t . N e v e r t h e l e s s , t h i s change i n urine e x c r e t i o n c o u l d have a l t e r e d the animal's s a l t and water balance. In a d d i t i o n , i t may account f o r the dramatic decrease i n body weight observed d u r i n g withdrawal. The g e n e r a l i z e d i n c r e a s e i n CNS a c t i v i t y i s e q u a l l y l i k e l y to p o t e n t i a t e the r e l e a s e and subsequent d e p l e t i o n of the two hormones. The presence of various withdrawal s i g n s , such as jumping, wet dog shaking, and t e e t h c h a t t e r i n g , i s no doubt r e l a t e d to the h y p e r a c t i v i t y of the CNS. I t i s c l e a r t h a t both acute and c h r o n i c a d m i n i s t r a t i o n of morphine a f f e c t s the f u n c t i o n i n g of the hypothalmo-neurohypophyseal system., The d i s c o v e r y of opiate r e c e p t o r s i n the neurohypophysis (143) r a i s e d the p o s s i b i l i t y t h a t endorphin and other endogenous o p i o i d peptides may r e g u l a t e the neurohypophysis. A d m i n i s t r a t i o n 71 of B-endorphin has been shown to s t i m u l a t e the r e l e a s e of AVP i n the r a b b i t (144). The presence of enkephalin i n n e r v a t i o n i n the neurohypophysis and the f a c t t h a t both AVP and enkephalin content were depleted during dehydration (145) f u r t h e r strengthen the concept of the c o n t r o l of AVP s e c r e t i o n by these p e p t i d e s . Aside from the i n f l u e n c e of the endorphins and enkephalins on the neurohypophysis, the neurohypophyseal hormones may i n t u r n c o n t r o l the a c t i o n s of these endogenous o p i a t e s . Krivoy e t a l . (79) have repo r t e d that the neurohypophyseal hormones can f a c i l i t a t e the development of t o l e r a n c e t o and p h y s i c a l dependence on o p i a t e a g o n i s t s * A number of t h e o r i e s have been proposed to e x p l a i n the mechanism of t o l e r a n c e and p h y s i c a l dependence (59). One of these t h e o r i e s i s the change i n a d enylate c y c l a s e a c t i v i t y (60). Acute a d m i n i s t r a t i o n of morphine was shown to cause a t r a n s i e n t suppression of the adenylate c y c l a s e a c t i v i t y . An enhanced formation of c y c l i c AMP was found dur i n g the development of t o l e r a n c e and p h y s i c a l dependence. Since AVP has been shown t o exert i t s a c t i o n on t a r g e t c e l l s by a c c e l e r a t i n g the formation of c y c l i c AMP (146), the p o s s i b i l i t y e x i s t s that i t may be one of the compensating mechanisms i n the maintenance of a s t a b l e c e l l u l a r l e v e l of the c y c l i c n u c l e o t i d e . A l t e r a t i o n i n o p i a t e r e c e p t o r b i n d i n g has a l s o been proposed as a mechanism of t o l e r a n c e and p h y s i c a l dependence (68). Whole b r a i n homogenates from r a t s i n j e c t e d d a i l y with morphine f o r 14 days showed a decrease 72 i n 3H-naloxone b i n d i n g (Table V I ) . In c o n t r a s t to the f i n d i n g s by Pert and Snyder (69), no s i g n i f i c a n t changes i n the number of b i n d i n g s i t e s were detected. Scatchard a n a l y s i s r e v e a l e d t h a t the changes are l i k e l y due to the presence of a c o m p e t i t i v e a g o n i s t . The repeated washing of the homogenate caused a p r o g r e s s i v e i n c r e a s e i n the r e c e p t o r a f f i n i t y . T h i s suggested the i n t e r f e r e n c e of 3H-naloxone b i n d i n g by the i n j e c t e d morphine. Also, changes i n r e c e p t o r a f f i n i t y are probably caused by the presence of minute q u a n t i t i e s of morphine i n the whole b r a i n homogenate. D i r e c t i n t e r f e r e n c e of o p i a t e r e c e p t o r b i n d i n g by the neurohypophyseal hormones can be r u l e d out as a p o s s i b l e mechanism of t o l e r a n c e and dependence. Both oxyt o c i n and AVP were found to have no e f f e c t on the 3H-naloxone b i n d i n g . The temporal r e l a t i o n s h i p between op i a t e r e c e p t o r b i n d i n g and t o l e r a n c e to the a n a l g e s i c e f f e c t of morphine appears r a t h e r weak. Pert and Snyder (69) found t h a t enhancement of o p i a t e receptor b i n d i n g appeared 2 hr a f t e r morphine treatment, and remained a t the same l e v e l f o r 4 days. T o l e r a n c e , on the other hand, has been shown to i n c r e a s e p r o g r e s s i v e l y and reaches a peak on the t h i r d day o f treatment (35). Hence, the appearance of maximal enhancement of o p i a t e r e c e p t o r b i n d i n g occured long before the development of maximal t o l e r a n c e • and p h y s i c a l dependence. However, the l a c k of c o r r e l a t i o n i n o p i a t e r e c e p t o r b i n d i n g and t o l e r a n c e development does not n e c e s s a r i l y r u l e out the p o s s i b l e involvement of the 73 o p i a t e r e c e p t o r s . . I t i s h i g h l y probable t h a t changes i n r e c e p t o r a f f i n i t y or b i n d i n g s i t e s occur r e g i o n a l l y i n the b r a i n . Such a l t e r a t i o n s may only occur i n b r a i n s t r u c t u r e s r e s p o n s i b l e f o r parameters or b e h a v i o r a l s i g n s measured i n the assessment of t o l e r a n c e and p h y s i c a l dependence. Hence, they may only be detected by examining the binding i n these s p e c i f i c s t r u c t u r e s of the b r a i n * Crude p r e p a r a t i o n s , such as whole b r a i n homogenate, may not be able t o d e t e c t such small changes. One i n t e r e s t i n g phenomenon encountered i n the p e l l e t i m p l a n t a t i o n s e r i e s i s the a c t i o n of morphine on the kidneys. Dark brown u r i n e was observed from a few r a t s 24 hr a f t e r the i m p l a n t a t i o n of a morphine p e l l e t . The e x c r e t i o n of dark brown u r i n e r e s u l t i n g from n a r c o t i c use has been r e p o r t e d i n human drug a d d i c t s (51). The pigmentation has been c h a r a c t e r i z e d as a combination of hemoglobin and myoglobin (48)., T h i s symptom i s q u i t e s i m i l a r to the crush syndrome d e s c r i b e d by S c h r e i b e r (52).. Regional s k e l e t a l n e c r o s i s or rhabdomyolysis were found i n some p a t i e n t s (51) and i s l i k e l y the cause of the myoglobinuria.. Rhabdomyolysis may be caused by the prolonged i m m o b i l i z a t i o n as the animal i s under the s e d a t i v e e f f e c t of morphine.. The p o s s i b i l i t y t h a t rhabdomyolysis i s caused by a d u l t e r a n t s present i n the h e r o i n used by the drug a d d i c t , as proposed by R i c h t e r et a l . (48), may be excluded. The morphine used i n the present study i s devoid of any a d u l t e r a n t s . The l i k e l i h o o d that rhabdomyolysis be due to the i n e r t b i n d e r s used i n 74 the p r o c e s s i n g of the p e l l e t s may a l s o be. excluded. C o n t r o l animals were implanted with p e l l e t s made e x c l u s i v e l y of such m a t e r i a l s , yet myoglobinuria was never observed i n these animals. I t i s s t i l l q u e s t i o n a b l e whether t h e c h r o n i c use of n a r c o t i c drugs can i n some unknown way p r e d i s p o s e s the onset of r e n a l d i s e a s e . The c u r r e n t o b s e r v a t i o n supported s t u d i e s (48-51) on a p o s s i b l e l i n k between r e n a l disease and heroin a d d i c t i o n . I t does not; however, c o n c l u s i v e l y r u l e out the p o s s i b i l i t y t h a t the onset of r e n a l d i s e a s e be due to other c o m p l i c a t i o n s a s s o c i a t e d with a d d i c t i o n . I t would be of i n t e r e s t though to determine whether the r e n a l disease improves o r s t a b i l i z e s with the withdrawal of the drug. 75 C O N C L U S I O N I t i s c l e a r t h a t morphine administered a c u t e l y has an a n t i d i u r e t i c e f f e c t . The r e l e a s e of AVP i s at l e a s t p a r t i a l l y r e s p o n s i b l e f o r the a n t i d i u r e s i s . The a c t u a l s t i m u l u s f o r AVP r e l e a s e , however, remains q u e s t i o n a b l e . I t i s p o s s i b l e t h a t morphine a c t s d i r e c t l y on the hypothalamic n e u r o s e c r e t o r y c e l l s . However, the hypotensive e f f e c t of the drug can a l s o exert a s u b s t a n t i a l i n f l u e n c e on the r e l e a s e of the hormone. The a p p l i c a t i o n of morphine d i r e c t l y to an i n v i t r o c u l t u r e of the hypothalamo-neurohypophyseal complex may provide some c l u e t o the d i r e c t e f f e c t of the drug on AVP r e l e a s e . I t i s e q u a l l y l i k e l y t h a t a p o r t i o n of the a n t i d i u r e s i s observed i s mediated by the e f f e c t s of the. r e s u l t i n g hypotension on r e n a l f u n c t i o n . .. In view of the c a r d i o v a s c u l a r e f f e c t s o f morphine, i t would be of i n t e r e s t to examine i t s e f f e c t s on the r e n a l v a s c u l a t u r e and the output of v a r i o u s u r i n a r y c o n s t i t u e n t s . When given c h r o n i c a l l y , morphine de p l e t e d the neurohypophyseal s t o r e s of AVP. Th i s d e p l e t i o n , however, was t r a n s i e n t and only l a s t e d f o r 3 days. The cause of t h i s d e p l e t i o n i s p a r t i a l l y due to dehydration i n i t i a t e d by a decrease i n food and water i n t a k e . . P o t e n t i a t i o n of AVP r e l e a s e due t o the d i r e c t a c t i o n of the drug on the hypothalamo-neurohypophyseal system i s an e q u a l l y l i k e l y p o s s i b i l i t y . The subsequent development of t o l e r a n c e and p h y s i c a l dependence on morphine presumably al l o w s the animal to adapt t o the e f f e c t s of the drug by e i t h e r 7 6 decreasing the r a t e of s e c r e t i o n or i n c r e a s i n g the b i o s y n t h e t i c r a t e . D e p l e t i o n observed when the drug was abr u p t l y withdrawn from p h y s i c a l l y dependent animals can be a t t r i b u t e d t o the s t i m u l a t o r y s f f e c t of s a l t and water l o s s on AVP r e l e a s e as well as to the g e n e r a l i z e d h y p e r a c t i v i t y of the CNS. The a c t u a l cause of AVP d e p l e t i o n d u r i n g morphine treatment and withdrawal remains u n s e t t l e d . Simultaneous measurements of the b i o s y n t h e t i c and s e c r e t i o n r a t e s of the peptide may shed some l i g h t to t h i s problem,* Furthermore, i f the d e p l e t i o n was due to the augmented r e l e a s e of AVP, i t would be of i n t e r e s t to determine the e v e n t u a l f a t e of the hormone. There i s a p o s s i b i l i t y t h a t i t may be e l a b o r a t e d not only t o the systemic c i r c u l a t i o n but a l s o to v a r i o u s CNS s t r u c t u r e s and the CSF. Enhancement of o p i a t e r e c e p t o r b i n d i n g with morphine treatment as repor t e d by Pert and Snyder (69) was not d u p l i c a t e d i n t h i s study. I t was concluded t h a t the minute q u a n t i t i e s of the drug l e f t i n the whole b r a i n homogenates s e v e r e l y a f f e c t e d the b i n d i n g of 3H-naloxone to the r e c e p t o r . 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