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Studies on the antidiuretic action of mophine in the rat
EUROPEAN
JOURNAL
OF
PHARMACOLOGY
2 (1968) 301-307.
NORTH-HOLLAND
PUBL.
COMP.,
AMSTERDAM
STUDIES ON THE ANTIDIURETIC ACTION OF MOPHINE IN THE RAT C h a r l e s E. INTURRISI ** a n d J a m e s M. F U J I M O T O Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA Accepted 8 D e c e m b e r ]967
C.E. INTURRISI and J . M . FUJIMOTO, Studies on the antidiuretic action of morphine in the rat, European J. Pharmacol. 2 (1968) 301-307. Morphine sulfate (MS) given s.c. or i.v. produces a d o s e - r e s p o n s e related d e c r e a s e in urine flow in the a l c o h o l - a n e s t h e t i z e d - d i u r e s i n g rat with a bladder fistula. The antidiuretic effect is not seen if the hydrating infusion contains mannitol instead of g l u c o s e - s a l i n e . These r e s u l t s are compatible with the proposal that the acute antidiuretic effect of morphine is mediated by the r e l e a s e of ADH from the neurohypophysis. Tolerance to the antidiuretic effect of MS rapidly develops in rats t r e a t e d on an i n c r e a s ing schedule of MS. After 5 days of MS treatment, tolerance to the antidiuretic effect of MS given i.v. or s.c. is nearly complete and the initial r e s p o n s e to MS b e c o m e s diuretic. At the same time renal sensitivity to P i t r e s s i n is unchanged. The development of tolerance to the antidiuretic effect of MS, seen after one or m o r e doses, may be due to a morphine induced blockade of ADH r e l e a s e . Morphine An t i d i u r e s i s Tolerance
1. I N T R O D U C T I O N T h e a n t i d i u r e t i c a c t i o n of n a r c o t i c a n a l g e s i c s h a s b e e n s t u d i e d in t h e r a t b y m e a s u r i n g t h e e x cretion time after an oral water load (Schnieden a n d B l a c k m o r e , 1951; G i a r m a n a n d C o n d o u r i s , 1954). T h i s m e t h o d h a s a l s o b e e n u s e d to d e m o n s t r a t e t h e d e v e l o p m e n t of t o l e r a n c e t o t h e a n t i d i u r e t i c a c t i o n of l e v o r p h a n o l ( N e w s o m e , T o b i n a n d F u j i m o t o , 1963) a n d m o r p h i n e ( M a r c h a n d a n d F u j i m o t o , 1966; S h i m a i e t a l . , 1965). Y e t , c e r t a i n d i f f i c u l t i e s a r i s e in t h e u s e of t h i s m e t h o d . N a r c o t i c a n a l g e s i c s d e c r e a s e t h e a b s o r p t i o n of w a t e r f r o m the g a s t r o i n t e s t i n a l t r a c t (Schnieden a n d B l a c k m o r e , 1955; F u j i m o t o e t a l . , 1963) a n d c a u s e r e t e n t i o n of u r i n e in t h e b l a d d e r ( R e y n o l d s a n d R a n d a l l , 1957). In t h e p r e s e n t r e p o r t a n a p p r o a c h h a s b e e n adopted which overcomes these difficulties. Ob-
* Supported by USPHS Grant 5 R01 AM 06515. ** Trainee, USPHS R e s e a r c h Training Grant 5 T01 GM 00363. P r e s e n t a d d r e s s : Laboratory of Chemical Pharmacology, National Heart Institute, Bethesda, Maryland.
Pitressin Alcohol-anesthetized Bladder fistula
s e r v a t i o n s a r e made c o n c e r n i n g the c o m p o n e n t s i n v o l v e d in t h e a n t i d i u r e t i c r e s p o n s e a n d t h e d e v e l o p m e n t of t o l e r a n c e . T h e r e s p o n s e to m o r p h i n e a n d P i t r e s s i n i s e v a l u a t e d in t h e s a m e r a t in o r d e r to d e t e r m i n e w h e t h e r t o l e r a n c e t o t h e a n t i d i u r e t i c e f f e c t i s m e d i a t e d by a d i m i n i s h e d r e s p o n s i v e n e s s to a n t i d i u r e t i c h o r m o n e a s s u g g e s t e d by N e w s o m e e t al. (1963) a n d S h i m a i e t a l . (1965).
2. M E T H O D S C e r t a i n f e a t u r e s f o u n d in t h e a n t i d i u r e t i c h o r m o n e b i o a s s a y p r o c e d u r e s of C z a c z k e s , K l e e m a n - a n d K o e n i g (1964) a n d T h o r n , S m i t h a n d S k a d h a u g e (1965) w e r e a d a p t e d f o r u s e in t h e present investigation. Rats were surgically prepared with a bladder fistula. Female rats weighi n g 1 0 0 - 1 3 0 g w e r e a n e s t h e t i z e d w i t h e t h e r , the skin over the s y m p h y s i s pubis s h a v e d and a s m a l l i n c i s i o n made along the linea alba into the p e r i t o n e u m . T h e b l a d d e r w a s d r a w n out, i n c i s e d a n d t h e cut e d g e s s u t u r e d t o t h e s k i n i n c i s i o n s o a s t o e x t e r i o r i z e t h e b l a d d e r a n d a l l o w u r i n e to d r a i n d i r e c t l y f r o m the u r e t e r s to the e x t e r i o r .
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F o u r d a y s l a t e r , t h e a n i m a l s c o u l d be u s e d . On the experimental day, the animal was anesthet i z e d w i t h a 12% e t h a n o l s o l u t i o n g i v e n o r a l l y two t i m e s a t 30 m i n i n t e r v a l s ; e a c h d o s e w a s 3% of b o d y w e i g h t . A t h i r d d o s e , a d m i n i s t e r e d 20 m i n l a t e r , w a s b e t w e e n 2 a n d 3% of b o d y w e i g h t d e p e n d i n g on t h e s t a t e of a n e s t h e s i a . U s u a l l y 15 t o 20 m i n a f t e r t h e l a s t d o s e of e t h a n o l t h e r a t w a s anesthetized. The external jugular vein was cannulated with PE-50 tubing attached via a needle a n d s t o p c o c k to a n i n f u s i o n p u m p . L u b r i c a n t w a s applied around the exteriorized bladder and the r a t w a s p l a c e d on a b o a r d s o t h a t t h e b l a d d e r w a s p o s i t i o n e d o v e r a h o l e in t h e b o a r d . A s m a l l funnel placed under the hole delivered into a 2 m l p i p e t t e . T h e d e l i v e r y t i p of t h e p i p e t t e w a s plugged so that urine could be collected and the v o l u m e r e a d . A s o l u t i o n c o n t a i n i n g 0.3% s o d i u m c h l o r i d e , 1.67% g l u c o s e a n d 1.2% e t h a n o l w a s i n f u s e d i n t o t h e j u g u l a r v e i n a t a r a t e of 0.12 m l / m i n . U r i n e v o l u m e s w e r e r e a d a t 10 m i n i n t e r v a l s . All d r u g s o l u t i o n s w e r e p r e p a r e d in 0.9% s a l i n e a n d i n j e c t e d in v o l u m e s f r o m 0.1 to 0.5 cm3. For the intravenous injection, the infusion s o l u t i o n w a s d i v e r t e d out t h r o u g h a s e c o n d s t o p cock placed between the infusion syringe and the i n j e c t i o n s t o p c o c k . In one s e t of e x p e r i m e n t s , carotid blood pressure was measured with the p r e s s u r e t r a n s d u c e r of a G r a s s p o l y g r a p h . T h e r e s p o n s e of t h e s e r a t s to c h r o n i c t r e a t ment with morphine was also studied. After the 4 day recovery period from the bladder surgery, morphine sulfate was injected subcutaneously t w i c e a d a y , b e g i n n i n g w i t h a n i n i t i a l d o s e of 8 m g / k g a n d i n c r e a s i n g in 8 m g / k g i n c r e m e n t s e a c h d a y f o r 5 d a y s s o t h a t t h e s c h e d u l e w a s 8, 16, 24, 36 a n d 44 m g / k g f o r d a y s 1 t h r o u g h 5. T h e m o r p h i n e w a s i n j e c t e d at 8 a . m . a n d 4 p . m . A f t e r 1, 3 a n d 5 d a y s , r a t s w e r e r e m o v e d f r o m the group, prepared for assay and challenged w i t h t h e i n i t i a l d o s e of 8 m ~ / k g s . c . T h e s e n s i t i v i t y of t h e r e s p o n s e to 10 m i c r o u n i t s (ttU) of P i t r e s s i n w a s a l s o t e s t e d in t h e s a m e a n i m a l . T h i s d o s e of P i t r e s s i n w a s t h e l o w e s t t o w h i c h a c o n s i s t e n t d e c r e a s e in u r i n e flow of 40 to 70% f o r one p e r i o d w a s o b t a i n e d . A n o t h e r g r o u p of r a t s t r e a t e d w i t h t h e a b o v e s c h e d u l e of d a i l y m o r p h i n e i n j e c t i o n s w a s c h a l l e n g e d w i t h 0.4 m g / k g of m o r p h i n e s u l f a t e i.v. S i n c e m a n y of t h e a n t i d i u r e t i c responses l a s t e d f o r s e v e r a l 10 rain c o l l e c t i o n p e r i o d s , t h e r e s p o n s e is e x p r e s s e d in t e r m s of a n u m b e r called the antidiuretic minute time, AMT. The A M T i s a n e x p r e s s i o n , in t e r m s of t i m e , of t h e difference between the urine volume that would h a v e b e e n e x p e c t e d h a d no e f f e c t o c c u r r e d a n d
t h e a c t u a l a m o u n t of u r i n e f o r m e d u n d e r t h e i n f l u e n c e of m o r p h i n e . T h e A M T i s o b t a i n e d a s follows: find the mean control urine volume per 10 m i n f o r t h r e e p r e d r u g p e r i o d s ; d e t e r m i n e t h e actual urine volume excreted during the periods of d r u g e f f e c t w h e n t h e u r i n e v o l u m e s w e r e b e low t h e c o n t r o l m e a n . C a l c u l a t e how l o n g it w o u l d h a v e t a k e n to e x c r e t e t h i s l a t t e r v o l u m e had the excretion continued at the control rate. S u b t r a c t t h i s t i m e f r o m t h e a c t u a l t i m e of a n t i d i u r e s i s to g i v e a d i f f e r e n c e w h i c h i s t h e A M T . F o r e x a m p l e , v o l u m e of u r i n e e x c r e t e d f o r t h r e e p r e d u r g ( c o n t r o l ) p e r i o d s w a s 1.15, 1.20 a n d 1.25 m l / 1 0 m i n p e r i o d o r a m e a n of 1.20 m l / 1 0 m i n . G i v e n 0.8 m g / k g of m o r p h i n e i . v . , t h e e x c r e t i o n w a s b e l o w t h i s m e a n f o r f i v e p e r i o d s : 0.25, 0.15, 0.05, 0 . 0 5 , 0.30 = 0.80 m l t o t a l v o l u m e f o r 5 p e r i o d s o r 50 m i n . A t t h e c o n t r o l r a t e of u r i n e f l o w , 1.2 m l / 1 0 m i n , f i v e p e r i o d s w o u l d h a v e g i v e n a v i r t u a l e x c r e t i o n of 6 m l in 50 m i n . T h e a c t u a l v o l u m e e x c r e t e d w a s 0.8 m l ; t h e r e f o r e , a t t h e c o n t r o l r a t e 6.6 m i n w o u l d h a v e b e e n r e q u i r e d t o e x c r e t e t h i s 0.8 m l . T h u s , t h e A M T i s 50 - 6.6 = 43.4 m i n , w h i c h i s a v i r t u a l t i m e d u r i n g w h i c h n o u r i n e flow o c c u r s .
3. R E S U L T S M o r p h i n e s u l f a t e a t a d o s e of 8 m g / k g s . c . c a u s e d a m a r k e d a n t i d i u r e s i s in t h e a l c o h o l - a n e s t h e t i z e d r a t a s s h o w n in fig. 1. T h i s d o s e of m o r p h i n e w a s w e l l t o l e r a t e d b y t h e r a t s a n d no o b s e r v a b l e c h a n g e s o c c u r r e d in t h e l e v e l of a n e s t h e s i a . In d i f f e r e n t r a t s , a d e c r e a s e in u r i n e 2.0-
MS
MS
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Fig. 1. The antidiuretie r e s p o n s e to morphine (MS) and the diminished r e s p o n s e to a second dose of MS in the rat. MS = morphine sulfate 8 m g / k g s.c.
ANTIDIURETIC ACTION O F MORPHINE IN THE RAT Table 1 Antidiuretic r e s p o n s e to morphine sulfate (MS). Dose mg/kg
Route
No. of animals
Antidiuretic minute time min ± S.E.
0.2 0.4 0.8
i.v. i.v. i.v.
4 4 2
6.7±1.6 17.0±1.6 38.5±1.4
1.0 4.0 8.0
s.c. s.c. s.c.
2 4 8
11.0 ± 1 . 0 34.5±2.4 63.4±6.5
flow of 70 to 90% occurred in the first period after morphine administration and urine excretion was depressed for 7 to 13 periods. For several periods during recovery, the excretion would often exceed the predrug baseline value and then return to a baseline of 1.00 to 1.30 ml per 10 rain period. The data in table 1 indicate that the antidiuretic response to morphine follows a d o s e - r e sponse relationship. To obtain comparable r e sponses, smaller doses a r e needed i.v. than s.c. No difference was observed in the rate of onset of antidiuresis by either route of administration; urine flow fell to about the same degree the f i r s t period after drug injection at all the doses indicated in table 1. An increase in the duration of effect occurred as the dose was increased. In table 2 the data show that morphine was not effective in producing an antidiuresis in the presence of a mannitol-induced diuresis. The experiments were performed by starting the Table 2 Effect of infusion solution on antidiuresis to morphine sulfate (MS). Infusion Antidiuretic minute time solution MS 0.4 mg/kg MS 4 mg/kg MS 8 mg/kg (g/100 i.v. s.c. s.c. ml) glucose (I .67) NaCl (0.30) ethanol (1.20)
17 (4)
35 (4)
63 (8)
mannitol (6.30) ethanol (1.20)
0 (2)
7 (2)
31 (2)
mannitol (10.00) ethanol (1.20)
9 (4)
0 (2)
6 (4)
( ) indicate n u m b e r of animals.
303
various intravenous infusion solutions and giving morphine at the doses and routes indicated. Thus, if the infusion solution contained 6.3% mannitol, a marked reduction in the effect of morphine occurred at all 3 doses of morphine. When the mannitol concentration was increased to 10%, almost a complete absence of antidiuretic effect occurred. P i t r e s s i n at doses from 10 to 40 tiU had no antidiuretic effect under the condition of mannitol infusion. The effect of morphine on blood p r e s s u r e was studied simultaneously with urine flow in six experiments. As seen in table 3, an average drop of 5 mm in blood p r e s s u r e occurred during the f i r s t period after morphine administration (in one case blood p r e s s u r e rose slightly). At the same time the urine volume decreased 65 to 87% of the control volume. During the course of morphine-induced antidiuresis, mean blood p r e s s u r e decreased to the values given in the last column of table 3. Blood p r e s s u r e returned to predrug values at about the same time as urine volume returned to baseline. Morphine at a dose of 0.I mg/kg i.v. produced an 80% decrease in urine volume for one period with no change in blood pressure. Administration of a second dose of morphine often resulted in an antidiuretic response which was much less than seen to the first dose. Fig. 1 gives an example. The AMT for the first injection of morphine was 50, while the second dose gave an AMT of only 13. Thus, a diminished antidiuretic response to repeated morphine administration occurs in a short time. The results of studies on the chronic administration of morphine are given in fig.2 (a,b, c,d). Fig. 2a illustrates the response to 8 mg/kg s.c. of morphine in a rat with no prior morphine treatment. The AMT was 67. Note responses to t h e t e s t d o s e s of 10 a n d 20 ttU of P i t r e s s i n . F i g . 2b s h o w s t h a t a f t e r o n e d a y of t r e a t m e n t , s o m e t o l e r a n c e h a d d e v e l o p e d to m o r p h i n e . T h e A M T w a s r e d u c e d t o 28 b u t P i t r e s s i n s e n s i t i v i t y w a s u n c h a n g e d . A f t e r 3 d a y s of t r e a t m e n t (fig. 2c) t h e A M T w a s r e d u c e d t o 22 a n d P i t r e s s i n s e n s i t i v i t y r e m a i n e d u n c h a n g e d . Note t h a t a f t e r 5 d a y s of t r e a t m e n t (fig. 2d) t h e i n i t i a l r e s p o n s e to m o r p h i n e w a s a n i n c r e a s e in u r i n e flow. T h i s i n c r e a s e w a s t h e n f o l l o w e d b y a d e c r e a s e in u r i n e flow. T h e o v e r a l l A M T w a s 8 a n d P i t r e s s i n s e n sitivity was still unchanged. In a n o t h e r s e t of e x p e r i m e n t s rats were treated on the dosage schedule described prev i o u s l y , b u t a c h a l l e n g e d o s e of 0.4 m g / k g m o r phine sulfate was given. The results are illust r a t e d i n fig. 3 ( a , b , c ) . F i g . 3 a s h o w s t h e a c u t e
C. E. INTURRISI and J. M. F U J I M O T O
304
Table 3 E f f e c t of m o r p h i n e 8 m g / k g s . c . on c a r o t i d blood p r e s s u r e and u r i n e flow in t h e r a t .
1 2 3 4 5 6
Control mean B.P.
Mean B.P. 1st p e r i o d a f t e r MS
C h a n g e in m e a n B.P.
Percent decrease in u r i n e v o l u m e in 1st p e r i o d a f t e r MS
Lowest mean B.P. d u r i n g MS antidiuresis
85 120 77 87 72 120
81 116 80 82 67 105
-4 -4 +3 -4 -5 -15
87 75 77 65 65 75
75 82 73 77 67 95
response of a n u n t r e a t e d r a t t o 0 . 4 m g / k g i . v . o f morphine. The response consisted of a prompt decrease in u r i n e f l o w w h i c h l a s t e d t w o p e r i o d s t o g i v e a n A M T o f 18. T h i s r e s p o n s e w a s o f t e n followed by an overshoot in urine flow. Fig. 3b is an example of the response of a rat treated for 3 days and then challenged with morphine. The immediate response is clearly an increase in urine flow for 3 periods. This increase is then followed by a slow decline; another increase occurs before urine flow is stabilized. A dose of 0.16 mg/kg i.v. in rats treated for 3 days pro-
duced a slight increase in urine flow for 2 periods without the subsequent decline seen at the higher dose. Fig. 3c illustrates the response of rats treated for 5 days with morphine. The chall e n g e d o s e of m o r p h i n e ( 0 . 4 m g / k g i . v . ) p r o d u c e s an even greater initial increase in urine flow t h a n s e e n a f t e r 3 d a y s o f t r e a t m e n t ( f i g . 3b). T h e fall in urine flow which followed was smaller than that seen after 3 days of treatment. When higher challenging doses of morphine of 0.8 and 1.2 mg/kg i.v. were used, an initial increase in urine flow was again followed by a decrease in
201
20-
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Fig. 2. T h e d e v e l o p m e n t of t o l e r a n c e to t h e a n t i d i u r e t i c effect of m o r p h i n e (MS) 8 m g / k g g i v e n s u b c u t a n e o u s l y . MS m o r p h i n e s u l f a t e 8 m g / k g s . c . , P - 1 0 = P i t r e s s i n 10 m i c r o u n i t s i.v., P - 2 0 = P i t r e s s i n 20 m i e r o u n i t s i.v. (a) t h e r e s p o n s e to MS and P - 1 0 , P - 2 0 of an u n t r e a t e d r a t . (b) t h e r e s p o n s e to MS and P - 1 0 of a r a t t r e a t e d f o r 1 day with MS 8 m g / k g s . c . , b.i.d. (c) the r e s p o n s e to MS and P - 1 0 of a r a t t r e a t e d f o r 3 d a y s with MS 8 m g / k g s . c . , b.i.d, i n c r e a s e d 8 m g / k g p e r day so t h a t by day t h r e e t h e d o s e w a s MS 24 m g / k g s . c . See text f o r s c h e d u l e . (d) t h e r e s p o n s e to MS and P - 2 0 of a r a t t r e a t e d f o r 5 d a y s of s c h e d u l e . See text.
ANTIDIURETIC ACTION OF MORPHINE IN THE RAT
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Fig. 3. The development of t o l e r a n c e to the antidiuretic effect of morphine sulfate (MS) 0.4 m g / k g given i n t r a v e nously. MS = morphine sulfate 0.4 m g / k g i.v. (a) the response to MS of an u n t r e a t e d rat. (b) the r e s p o n s e to MS of a rat t r e a t e d for 3 days of schedule. See text. (c) the r e s p o n s e to MS of a rat t r e a t e d for 5 days of schedule. See text. u r i n e flow. T h e s e e x p e r i m e n t s d e m o n s t r a t e t h a t in t h e r a t t r e a t e d f o r s e v e r a l d a y s w i t h m o r phine, another component, a diuretic one, app e a r s in t h e r e s p o n s e . This diuretic response contrasts sharply with the antidiuretic response s e e n in t h e c o n t r o l a n i m a l in w h i c h t h e d e c r e a s e in u r i n e flow i s f o l l o w e d b y a n o v e r s h o o t i n g e f fect. T h e d i u r e t i c c o m p o n e n t a p p e a r s to b e c o m e m o r e p r o m i n e n t a s t h e n u m b e r of t r e a t m e n t d a y s w i t h m o r p h i n e i n c r e a s e . T h i s c o m p o n e n t i s not p a r t i c u l a r l y s e n s i t i v e t o c h a n g e s in t h e c h a l l e n g i n g d o s e of m o r p h i n e i n t h a t g o i n g f o r 0.16 to 1.2 m g / k g i . v . d o e s n o t e n l a r g e o r p r o l o n g t h e r e s p o n s e . On t h e o t h e r h a n d , t h e a n t i d i u r e t i c component is much more dose-dependent as was s h o w n i n t a b l e 1.
4. DISCUSSION The results demonstrate that morphine prod u c e s a n a n t i d i u r e s i s in t h e r a t w h e r e t h e c o m p l i c a t i n g e f f e c t s of m o r p h i n e on t h e g a s t r o i n t e s t i n a l a b s o r p t i o n of w a t e r a n d on t h e r e t e n t i o n of u r i n e in t h e b l a d d e r h a v e b e e n l a r g e l y e l i m i n a t e d . T h e e f f e c t of m o r p h i n e on g a s t r o i n t e s t i n a l a b s o r p t i o n of w a t e r w a s m i n i m i z e d b y i n t r a v e n o u s i n f u s i o n of t h e h y d r a t i n g s o l u t i o n . R e t e n t i o n of u r i n e i n t h e b l a d d e r w a s e l i m i n a t e d b y c r e ating a bladder fistula. Our results are consistent with the mechan i s m s of t h e a n t i d i u r e t i c e f f e c t s of m o r p h i n e g i v e n in t h e l i t e r a t u r e . M o r p h i n e r e l e a s e s a n t i diuretic hormone from the neurohypophysis and
a n t i d i u r e s i s r e s u l t s f r o m a c t i o n of t h e h o r m o n e on t h e k i d n e y (De B o d o , 1944; D u k e , P i c k f o r d a n d W a t t , 1961). L i p s c h i t z a n d S t o k e y (1947) s u g g e s t a d i r e c t e f f e c t of m o r p h i n e on t h e k i d n e y . A n d , o t h e r s r e p o r t e d t h e p o s s i b i l i t y of s o m e c o m b i n a t i o n of e f f e c t s ( G e o r g e a n d W a y , 1959; H a n d l e y a n d K e l l e r , 1950; C r a w f o r d a n d P i n k h a m , 1955). In o u r s t u d y , it d o e s a p p e a r t h a t t h e i n i t i a l s h a r p d r o p in u r i n e v o l u m e s e e n i n t h e f i r s t p e r i o d a f t e r b o t h h i g h a n d low d o s e s of m o r p h i n e m a y b e due to t h e r e l e a s e of ADH a n d i t s s u b s e q u e n t a c t i o n on t h e k i d n e y . At low d o s e s of m o r p h i n e (0ol-0.4 mg/kg i.v.) the effect is rapid, lasts 1-2 p e r i o d s , a n d i s e f f e c t i v e o n l y in t h e p r e s e n c e of a water diuresis. The antidiuretic response is not seen during mannitol infusion. With 4 mg/kg of m o r p h i n e s . c . g i v e n t o a r a t u n d e r g o i n g a m a n n i t o l d i u r e s i s , t h e i n i t i a l r a p i d d e c r e a s e in u r i n e e x c r e t i o n w a s n o t s e e n . T h e r e s p o n s e to Pitressin was also blocked by mannitol. The 8 m g / k g d o s e of m o r p h i n e s . c . , h o w e v e r , did p r o duce some antidiuresis. Thus another mechan i s m m a y b e c o m i n g i n t o p l a y a t h i g h d o s e s of m o r p h i n e ; c h a n g e s in r e n a l h e m o d y n a m i c s l e a d i n g to d e c r e a s e in t h e g l o m e r u l a r f i l t r a t i o n r a t e a n d u r i n e f o r m a t i o n a r e p o s s i b l e . D r o p s in s y s temic blood pressure were observed at larger doses. These interpretations parallel those der i v e d b y G e o r g e a n d W a y (1959). T h e y r e p o r t e d t h a t m o r p h i n e in low d o s e s a c t s to r e l e a s e ADH and that this action is blocked by hypothalamic lesions which produce diabetes insipidus. The e f f e c t of l a r g e d o s e s of m o r p h i n e w e r e n o t blocked by hypothalamic lesions and the morp h i n e a p p e a r e d to a c t b y m e c h a n i s m s o t h e r t h a n
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r e l e a s e of ADH. L a s t l y , t h e u s e of e t h a n o l a s a n anesthetic in our study is compatible with producing a water diuresis since ethanol, unlike m o s t a n e s t h e t i c s d o e s not i t s e l f r e l e a s e ADH ( V a n Dyke a n d A m e s , 1951). The data from rats treated chronically with m o r p h i n e show t h a t t o l e r a n c e d e v e l o p s to t h e a n tidiuretic effect. This tolerance is nearly comp l e t e a f t e r 5 d a y s of t r e a t m e n t w i t h a s c h e d u l e of i n c r e a s i n g d o s e s . T o l e r a n c e to t h e a n t i d i u r e t i c response was measured by administering a chall e n g e d o s e of 8 m g / k g s , c . o r 0.4 m g / k g i . v . of m o r p h i n e a f t e r t h e f i r s t , t h i r d o r f i f t h d a y of treatment. Tolerance occurs without a decrease in s e n s i t i v i t y to P i t r e s s i n ( e x o g e n o u s ADH). T h u s , it i s p o s s i b l e to s e p a r a t e t h e d e v e l o p m e n t of t o l e r a n c e to m o r p h i n e a n t i d i u r e s i s f r o m p o s s i b l e c h a n g e s in s e n s i t i v i t y to ADH. T h i s p o i n t i s o n e of s u b s t a n t i a l i n t e r e s t b e c a u s e of t h e s u g g e s t i o n of N e w s o m e et a l . (1963) a n d S h i m a i et a l . (1965) t h a t t h e d e v e l o p m e n t of t o l e r a n c e t o the antidiuretic effect involves a diminished res p o n s i v e n e s s of t h e r a t to a n t i d i u r e t i c h o r m o n e . Our present data offer convincing evidence that tolerance can occur without a change in sensitivi t y to P i t r e s s i n . P o s s i b l y , t h e c h a n g e s in s e n = sitivity occur with long, sustained tolerance. C e r t a i n l y t h e d u r a t i o n of t r e a t m e n t i s s h o r t in our present experiment. If t h e r e n a l s e n s i t i v i t y to ADH h a s not c h a n g e d , t h e n it i s p o s s i b l e t h a t t h e a b i l i t y of m o r p h i n e t o r e l e a s e ADH f r o m t h e n e u r o h y p o p h ysis has been altered. Our data suggest that with t h e d e v e l o p m e n t of t o l e r a n c e t h e i n i t i a l a n t i d i u r e t i c r e s p o n s e to m o r p h i n e , a s b e s t i l l u s t r a t e d b y t h e 0.4 m g / k g i . v . d o s e , i s a b s e n t o r r e p l a c e d by a diuretic response. This altered response w o u l d b e c o n s o n a n t w i t h l o s s of a b i l i t y of m o r p h i n e to r e l e a s e ADH in r a t s t r e a t e d c h r o n i c a l l y with morphine. In t h e c h r o n i c a l l y - t r e a t e d r a t a n 8 m g / k g s . c . d o s e of m o r p h i n e h a s a b i p h a s i c r e s p o n s e , a n initial increase followed by a decrease in urine flow. N e w s o m e et a l . (1963), H o l m e s , C a r t e r a n d F u j i m o t o (1958) a n d S h i m a i et a l . (1965) o b s e r v e d that prolonged treatmentwith narcotic analgesics l e a d s to p o l y d y p s i a a n d p o l y u r i a a n d a r e v e r s a l of e f f e c t f r o m a n t i d i u r e s i s t o d i u r e s i s in w a t e r l o a d e d r a t s . W e h a v e b e e n a b l e to o b s e r v e b o t h p h a s e s in t h e s a m e a n i m a l in r e s p o n s e to m o r phine. Within 5 days an increase in the diuretic a n d a d e c r e a s e in t h e a n t i d i u r e t i c p h a s e a r e s e e n . If m o r p h i n e i n i t i a l l y r e l e a s e s ADH a n d s u b s e q u e n t l y on c h r o n i c t r e a t m e n t p r o d u c e s a b l o c k a d e of ADH r e l e a s e , t o l e r a n c e c a n b e e x -
p l a i n e d b y m e c h a n i s m s c o n s i s t e n t w i t h m a n y of the known findings. T h e e f f e c t of m o r p h i n e t r e a t m e n t on a d r e n a l ascorbic acid depletion can be sighted as a possible parallel situation. The acute administration of m o r p h i n e s t i m u l a t e s t h e r e l e a s e of A C T H a s measured by adrenal ascorbic acid depletion ( G e o r g e a n d W a y , 1955). W h e n m o r p h i n e i s g i v e n d a i l y f o r 6 d a y s , t h e r e l e a s e of A C T H to a c h a l l e n g e d o s e of m o r p h i n e i s n o t o n l y a b s e n t b u t t h e r e l e a s e of A C T H b y o t h e r d r u g s i s b l o c k e d in morphine-treated animals (Briggs and Munson, 1955). T h e s a m e s o r t of s i t u a t i o n m a y b e p r o d u c e d a c u t e l y b y g i v i n g a c o m b i n a t i o n of m o r p h i n e a n d p e n t o b a r b i t a l w h e r e b l o c k a d e of A C T H release occurs. RE FERENCES Briggs, F.N. and P . L . Munson, 1955, Studies on the m e c h a n i s m of stimulation of ACTH s e c r e t i o n with the aid of morphine as a blocking agent, E n d o c r i nology 57, 205. Crawford, J . D . and B. Pinkham, 1955, The physiology of morphine a n t i d i u r e s i s , J. P h a r m . Exp. Ther. 113, 431. Czaczkes, J . W . , C.R. Kleeman and M. Koenig, 1964, Physiologic studies of antidiuretic h o r m o n e by its d i r e c t m e a s u r e m e n t in human plasma, J. Clin. Invest. 43, 1625. De Bodo, R.C., 1944, The antidiuretic action of m o r phine and its m e c h a n i s m , J. P h a r m . Exp. Ther. 82, Ther. 82, 74. Duke, H.N., M. Pickford and J. A. Watt, 1951, The antidiuretic action of morphine: its site and mode of action on the hypothalamus of the dog. Q . J . Exp. Physiol. 36, 149. Fujimoto, J . M . , R . J . F r e n c h , J . K . G r a h a m and C.E. I n t u r r i s i , 1963, Effects of levorphanol and l e v a l l o r phan on osmolality of s e r u m from water loaded rats, Proc. Soc. Exp. Biol. Med. 114, 193. George, R. and E . L . Way, 1959, Role of the hypot h a l a m u s in p i t u i t a r y - a d r e n a l activation and antid i u r e s i s by morphine, J. P h a r m . Exp. Ther. 125, 111. Giarman, N . J . and G.A. Condouris, 1954, The antidiuretic action of morphine and some of its analogs, Archs. Int. Pharmacodyn. Ther. 97, 28. Handley, C.A. and A. D. Keller, 1950, Changes in r e nal function produced by morphine in normal dogs and dogs with diabetes insipidus, J. P h a r m . Exp. T h e r . 99, 33. Holmes, S., M.K. C a r t e r and J . M . Fujimoto, 1958, Polydipsia and polyuria during chronic a d m i n i s t r a tion of levorphanol to rats, Proc. Soc. Exp. Biol. Med. 99, 319. Lipschitz, W.L. and E. Stokey, 1947, Mechanisms of antidiuresis in the dog and in the rat, Am. J. P h y s iol. 148, 259. Marchand, C.R. and J. M. Fujimoto, 1966, Sodium and potassium excretion in r a t s t r e a t e d chronically with morphine, Proc. Soc. Exp. Biol. Med. 123, 600.
ANTIDIURETIC ACTION OF MORPHINE IN THE RAT N e w s o m e , H . H . , W. Tobin and J . M . F u j i m o t o , 1963, C e r t a i n a s p e c t s of the e f f e c t s of l e v o r p h a n o l on w a t e r m e t a b o l i s m in r a t s , J. P h a r m . Exp. T h e r . 139, 368. Reynolds, A.K. and L . O , Randall, 1957, Morphine and allied d r u g s (University of T o r o n t o P r e s s , Canada) pp. 85-86. Schnieden, H. and E . K . B l a c k m o r e , 1955, Effect of n a l o r p i n e on the a n t i d i u r e t i c action of m o r p h i n e in r a t s and men, Brit. J. P h a r m . C h e m o t h e r . 10, 45.
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Shimai, K., M. Akita, S. T o m i z a w a andH.,Kondo, 1965, On the e o r r e l a t i o n between n e u r o s e e r e t i o n of the h y p o t h a l a m o - h y p o p h y s i a l s y s t e m and w a t e r m e t a b o l i s m in m o r p h i n i z e d r a t s , O k a j i m a s FoI. Anat. Jap. 40, 911. T h o r n , N . A . , M.W. Smith and E. Skadhauge, 1965, The a n t i d i u r e t i c effect of i n t r a v e n o u s and i n t r a e a r o t i d infusion of c a l c i u m c h l o r i d e in h y d r a t e d r a t s , J. E n d o c r . 32, 1(31. Van Dyke, H.B. and R.G. A m e s , 1951, Alcohol d i u r e s i s . Acta Endocr. (Copenhagen) 7, 110.
JOURNAL
OF
PHARMACOLOGY
2 (1968) 301-307.
NORTH-HOLLAND
PUBL.
COMP.,
AMSTERDAM
STUDIES ON THE ANTIDIURETIC ACTION OF MOPHINE IN THE RAT C h a r l e s E. INTURRISI ** a n d J a m e s M. F U J I M O T O Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA Accepted 8 D e c e m b e r ]967
C.E. INTURRISI and J . M . FUJIMOTO, Studies on the antidiuretic action of morphine in the rat, European J. Pharmacol. 2 (1968) 301-307. Morphine sulfate (MS) given s.c. or i.v. produces a d o s e - r e s p o n s e related d e c r e a s e in urine flow in the a l c o h o l - a n e s t h e t i z e d - d i u r e s i n g rat with a bladder fistula. The antidiuretic effect is not seen if the hydrating infusion contains mannitol instead of g l u c o s e - s a l i n e . These r e s u l t s are compatible with the proposal that the acute antidiuretic effect of morphine is mediated by the r e l e a s e of ADH from the neurohypophysis. Tolerance to the antidiuretic effect of MS rapidly develops in rats t r e a t e d on an i n c r e a s ing schedule of MS. After 5 days of MS treatment, tolerance to the antidiuretic effect of MS given i.v. or s.c. is nearly complete and the initial r e s p o n s e to MS b e c o m e s diuretic. At the same time renal sensitivity to P i t r e s s i n is unchanged. The development of tolerance to the antidiuretic effect of MS, seen after one or m o r e doses, may be due to a morphine induced blockade of ADH r e l e a s e . Morphine An t i d i u r e s i s Tolerance
1. I N T R O D U C T I O N T h e a n t i d i u r e t i c a c t i o n of n a r c o t i c a n a l g e s i c s h a s b e e n s t u d i e d in t h e r a t b y m e a s u r i n g t h e e x cretion time after an oral water load (Schnieden a n d B l a c k m o r e , 1951; G i a r m a n a n d C o n d o u r i s , 1954). T h i s m e t h o d h a s a l s o b e e n u s e d to d e m o n s t r a t e t h e d e v e l o p m e n t of t o l e r a n c e t o t h e a n t i d i u r e t i c a c t i o n of l e v o r p h a n o l ( N e w s o m e , T o b i n a n d F u j i m o t o , 1963) a n d m o r p h i n e ( M a r c h a n d a n d F u j i m o t o , 1966; S h i m a i e t a l . , 1965). Y e t , c e r t a i n d i f f i c u l t i e s a r i s e in t h e u s e of t h i s m e t h o d . N a r c o t i c a n a l g e s i c s d e c r e a s e t h e a b s o r p t i o n of w a t e r f r o m the g a s t r o i n t e s t i n a l t r a c t (Schnieden a n d B l a c k m o r e , 1955; F u j i m o t o e t a l . , 1963) a n d c a u s e r e t e n t i o n of u r i n e in t h e b l a d d e r ( R e y n o l d s a n d R a n d a l l , 1957). In t h e p r e s e n t r e p o r t a n a p p r o a c h h a s b e e n adopted which overcomes these difficulties. Ob-
* Supported by USPHS Grant 5 R01 AM 06515. ** Trainee, USPHS R e s e a r c h Training Grant 5 T01 GM 00363. P r e s e n t a d d r e s s : Laboratory of Chemical Pharmacology, National Heart Institute, Bethesda, Maryland.
Pitressin Alcohol-anesthetized Bladder fistula
s e r v a t i o n s a r e made c o n c e r n i n g the c o m p o n e n t s i n v o l v e d in t h e a n t i d i u r e t i c r e s p o n s e a n d t h e d e v e l o p m e n t of t o l e r a n c e . T h e r e s p o n s e to m o r p h i n e a n d P i t r e s s i n i s e v a l u a t e d in t h e s a m e r a t in o r d e r to d e t e r m i n e w h e t h e r t o l e r a n c e t o t h e a n t i d i u r e t i c e f f e c t i s m e d i a t e d by a d i m i n i s h e d r e s p o n s i v e n e s s to a n t i d i u r e t i c h o r m o n e a s s u g g e s t e d by N e w s o m e e t al. (1963) a n d S h i m a i e t a l . (1965).
2. M E T H O D S C e r t a i n f e a t u r e s f o u n d in t h e a n t i d i u r e t i c h o r m o n e b i o a s s a y p r o c e d u r e s of C z a c z k e s , K l e e m a n - a n d K o e n i g (1964) a n d T h o r n , S m i t h a n d S k a d h a u g e (1965) w e r e a d a p t e d f o r u s e in t h e present investigation. Rats were surgically prepared with a bladder fistula. Female rats weighi n g 1 0 0 - 1 3 0 g w e r e a n e s t h e t i z e d w i t h e t h e r , the skin over the s y m p h y s i s pubis s h a v e d and a s m a l l i n c i s i o n made along the linea alba into the p e r i t o n e u m . T h e b l a d d e r w a s d r a w n out, i n c i s e d a n d t h e cut e d g e s s u t u r e d t o t h e s k i n i n c i s i o n s o a s t o e x t e r i o r i z e t h e b l a d d e r a n d a l l o w u r i n e to d r a i n d i r e c t l y f r o m the u r e t e r s to the e x t e r i o r .
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F o u r d a y s l a t e r , t h e a n i m a l s c o u l d be u s e d . On the experimental day, the animal was anesthet i z e d w i t h a 12% e t h a n o l s o l u t i o n g i v e n o r a l l y two t i m e s a t 30 m i n i n t e r v a l s ; e a c h d o s e w a s 3% of b o d y w e i g h t . A t h i r d d o s e , a d m i n i s t e r e d 20 m i n l a t e r , w a s b e t w e e n 2 a n d 3% of b o d y w e i g h t d e p e n d i n g on t h e s t a t e of a n e s t h e s i a . U s u a l l y 15 t o 20 m i n a f t e r t h e l a s t d o s e of e t h a n o l t h e r a t w a s anesthetized. The external jugular vein was cannulated with PE-50 tubing attached via a needle a n d s t o p c o c k to a n i n f u s i o n p u m p . L u b r i c a n t w a s applied around the exteriorized bladder and the r a t w a s p l a c e d on a b o a r d s o t h a t t h e b l a d d e r w a s p o s i t i o n e d o v e r a h o l e in t h e b o a r d . A s m a l l funnel placed under the hole delivered into a 2 m l p i p e t t e . T h e d e l i v e r y t i p of t h e p i p e t t e w a s plugged so that urine could be collected and the v o l u m e r e a d . A s o l u t i o n c o n t a i n i n g 0.3% s o d i u m c h l o r i d e , 1.67% g l u c o s e a n d 1.2% e t h a n o l w a s i n f u s e d i n t o t h e j u g u l a r v e i n a t a r a t e of 0.12 m l / m i n . U r i n e v o l u m e s w e r e r e a d a t 10 m i n i n t e r v a l s . All d r u g s o l u t i o n s w e r e p r e p a r e d in 0.9% s a l i n e a n d i n j e c t e d in v o l u m e s f r o m 0.1 to 0.5 cm3. For the intravenous injection, the infusion s o l u t i o n w a s d i v e r t e d out t h r o u g h a s e c o n d s t o p cock placed between the infusion syringe and the i n j e c t i o n s t o p c o c k . In one s e t of e x p e r i m e n t s , carotid blood pressure was measured with the p r e s s u r e t r a n s d u c e r of a G r a s s p o l y g r a p h . T h e r e s p o n s e of t h e s e r a t s to c h r o n i c t r e a t ment with morphine was also studied. After the 4 day recovery period from the bladder surgery, morphine sulfate was injected subcutaneously t w i c e a d a y , b e g i n n i n g w i t h a n i n i t i a l d o s e of 8 m g / k g a n d i n c r e a s i n g in 8 m g / k g i n c r e m e n t s e a c h d a y f o r 5 d a y s s o t h a t t h e s c h e d u l e w a s 8, 16, 24, 36 a n d 44 m g / k g f o r d a y s 1 t h r o u g h 5. T h e m o r p h i n e w a s i n j e c t e d at 8 a . m . a n d 4 p . m . A f t e r 1, 3 a n d 5 d a y s , r a t s w e r e r e m o v e d f r o m the group, prepared for assay and challenged w i t h t h e i n i t i a l d o s e of 8 m ~ / k g s . c . T h e s e n s i t i v i t y of t h e r e s p o n s e to 10 m i c r o u n i t s (ttU) of P i t r e s s i n w a s a l s o t e s t e d in t h e s a m e a n i m a l . T h i s d o s e of P i t r e s s i n w a s t h e l o w e s t t o w h i c h a c o n s i s t e n t d e c r e a s e in u r i n e flow of 40 to 70% f o r one p e r i o d w a s o b t a i n e d . A n o t h e r g r o u p of r a t s t r e a t e d w i t h t h e a b o v e s c h e d u l e of d a i l y m o r p h i n e i n j e c t i o n s w a s c h a l l e n g e d w i t h 0.4 m g / k g of m o r p h i n e s u l f a t e i.v. S i n c e m a n y of t h e a n t i d i u r e t i c responses l a s t e d f o r s e v e r a l 10 rain c o l l e c t i o n p e r i o d s , t h e r e s p o n s e is e x p r e s s e d in t e r m s of a n u m b e r called the antidiuretic minute time, AMT. The A M T i s a n e x p r e s s i o n , in t e r m s of t i m e , of t h e difference between the urine volume that would h a v e b e e n e x p e c t e d h a d no e f f e c t o c c u r r e d a n d
t h e a c t u a l a m o u n t of u r i n e f o r m e d u n d e r t h e i n f l u e n c e of m o r p h i n e . T h e A M T i s o b t a i n e d a s follows: find the mean control urine volume per 10 m i n f o r t h r e e p r e d r u g p e r i o d s ; d e t e r m i n e t h e actual urine volume excreted during the periods of d r u g e f f e c t w h e n t h e u r i n e v o l u m e s w e r e b e low t h e c o n t r o l m e a n . C a l c u l a t e how l o n g it w o u l d h a v e t a k e n to e x c r e t e t h i s l a t t e r v o l u m e had the excretion continued at the control rate. S u b t r a c t t h i s t i m e f r o m t h e a c t u a l t i m e of a n t i d i u r e s i s to g i v e a d i f f e r e n c e w h i c h i s t h e A M T . F o r e x a m p l e , v o l u m e of u r i n e e x c r e t e d f o r t h r e e p r e d u r g ( c o n t r o l ) p e r i o d s w a s 1.15, 1.20 a n d 1.25 m l / 1 0 m i n p e r i o d o r a m e a n of 1.20 m l / 1 0 m i n . G i v e n 0.8 m g / k g of m o r p h i n e i . v . , t h e e x c r e t i o n w a s b e l o w t h i s m e a n f o r f i v e p e r i o d s : 0.25, 0.15, 0.05, 0 . 0 5 , 0.30 = 0.80 m l t o t a l v o l u m e f o r 5 p e r i o d s o r 50 m i n . A t t h e c o n t r o l r a t e of u r i n e f l o w , 1.2 m l / 1 0 m i n , f i v e p e r i o d s w o u l d h a v e g i v e n a v i r t u a l e x c r e t i o n of 6 m l in 50 m i n . T h e a c t u a l v o l u m e e x c r e t e d w a s 0.8 m l ; t h e r e f o r e , a t t h e c o n t r o l r a t e 6.6 m i n w o u l d h a v e b e e n r e q u i r e d t o e x c r e t e t h i s 0.8 m l . T h u s , t h e A M T i s 50 - 6.6 = 43.4 m i n , w h i c h i s a v i r t u a l t i m e d u r i n g w h i c h n o u r i n e flow o c c u r s .
3. R E S U L T S M o r p h i n e s u l f a t e a t a d o s e of 8 m g / k g s . c . c a u s e d a m a r k e d a n t i d i u r e s i s in t h e a l c o h o l - a n e s t h e t i z e d r a t a s s h o w n in fig. 1. T h i s d o s e of m o r p h i n e w a s w e l l t o l e r a t e d b y t h e r a t s a n d no o b s e r v a b l e c h a n g e s o c c u r r e d in t h e l e v e l of a n e s t h e s i a . In d i f f e r e n t r a t s , a d e c r e a s e in u r i n e 2.0-
MS
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Fig. 1. The antidiuretie r e s p o n s e to morphine (MS) and the diminished r e s p o n s e to a second dose of MS in the rat. MS = morphine sulfate 8 m g / k g s.c.
ANTIDIURETIC ACTION O F MORPHINE IN THE RAT Table 1 Antidiuretic r e s p o n s e to morphine sulfate (MS). Dose mg/kg
Route
No. of animals
Antidiuretic minute time min ± S.E.
0.2 0.4 0.8
i.v. i.v. i.v.
4 4 2
6.7±1.6 17.0±1.6 38.5±1.4
1.0 4.0 8.0
s.c. s.c. s.c.
2 4 8
11.0 ± 1 . 0 34.5±2.4 63.4±6.5
flow of 70 to 90% occurred in the first period after morphine administration and urine excretion was depressed for 7 to 13 periods. For several periods during recovery, the excretion would often exceed the predrug baseline value and then return to a baseline of 1.00 to 1.30 ml per 10 rain period. The data in table 1 indicate that the antidiuretic response to morphine follows a d o s e - r e sponse relationship. To obtain comparable r e sponses, smaller doses a r e needed i.v. than s.c. No difference was observed in the rate of onset of antidiuresis by either route of administration; urine flow fell to about the same degree the f i r s t period after drug injection at all the doses indicated in table 1. An increase in the duration of effect occurred as the dose was increased. In table 2 the data show that morphine was not effective in producing an antidiuresis in the presence of a mannitol-induced diuresis. The experiments were performed by starting the Table 2 Effect of infusion solution on antidiuresis to morphine sulfate (MS). Infusion Antidiuretic minute time solution MS 0.4 mg/kg MS 4 mg/kg MS 8 mg/kg (g/100 i.v. s.c. s.c. ml) glucose (I .67) NaCl (0.30) ethanol (1.20)
17 (4)
35 (4)
63 (8)
mannitol (6.30) ethanol (1.20)
0 (2)
7 (2)
31 (2)
mannitol (10.00) ethanol (1.20)
9 (4)
0 (2)
6 (4)
( ) indicate n u m b e r of animals.
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various intravenous infusion solutions and giving morphine at the doses and routes indicated. Thus, if the infusion solution contained 6.3% mannitol, a marked reduction in the effect of morphine occurred at all 3 doses of morphine. When the mannitol concentration was increased to 10%, almost a complete absence of antidiuretic effect occurred. P i t r e s s i n at doses from 10 to 40 tiU had no antidiuretic effect under the condition of mannitol infusion. The effect of morphine on blood p r e s s u r e was studied simultaneously with urine flow in six experiments. As seen in table 3, an average drop of 5 mm in blood p r e s s u r e occurred during the f i r s t period after morphine administration (in one case blood p r e s s u r e rose slightly). At the same time the urine volume decreased 65 to 87% of the control volume. During the course of morphine-induced antidiuresis, mean blood p r e s s u r e decreased to the values given in the last column of table 3. Blood p r e s s u r e returned to predrug values at about the same time as urine volume returned to baseline. Morphine at a dose of 0.I mg/kg i.v. produced an 80% decrease in urine volume for one period with no change in blood pressure. Administration of a second dose of morphine often resulted in an antidiuretic response which was much less than seen to the first dose. Fig. 1 gives an example. The AMT for the first injection of morphine was 50, while the second dose gave an AMT of only 13. Thus, a diminished antidiuretic response to repeated morphine administration occurs in a short time. The results of studies on the chronic administration of morphine are given in fig.2 (a,b, c,d). Fig. 2a illustrates the response to 8 mg/kg s.c. of morphine in a rat with no prior morphine treatment. The AMT was 67. Note responses to t h e t e s t d o s e s of 10 a n d 20 ttU of P i t r e s s i n . F i g . 2b s h o w s t h a t a f t e r o n e d a y of t r e a t m e n t , s o m e t o l e r a n c e h a d d e v e l o p e d to m o r p h i n e . T h e A M T w a s r e d u c e d t o 28 b u t P i t r e s s i n s e n s i t i v i t y w a s u n c h a n g e d . A f t e r 3 d a y s of t r e a t m e n t (fig. 2c) t h e A M T w a s r e d u c e d t o 22 a n d P i t r e s s i n s e n s i t i v i t y r e m a i n e d u n c h a n g e d . Note t h a t a f t e r 5 d a y s of t r e a t m e n t (fig. 2d) t h e i n i t i a l r e s p o n s e to m o r p h i n e w a s a n i n c r e a s e in u r i n e flow. T h i s i n c r e a s e w a s t h e n f o l l o w e d b y a d e c r e a s e in u r i n e flow. T h e o v e r a l l A M T w a s 8 a n d P i t r e s s i n s e n sitivity was still unchanged. In a n o t h e r s e t of e x p e r i m e n t s rats were treated on the dosage schedule described prev i o u s l y , b u t a c h a l l e n g e d o s e of 0.4 m g / k g m o r phine sulfate was given. The results are illust r a t e d i n fig. 3 ( a , b , c ) . F i g . 3 a s h o w s t h e a c u t e
C. E. INTURRISI and J. M. F U J I M O T O
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Table 3 E f f e c t of m o r p h i n e 8 m g / k g s . c . on c a r o t i d blood p r e s s u r e and u r i n e flow in t h e r a t .
1 2 3 4 5 6
Control mean B.P.
Mean B.P. 1st p e r i o d a f t e r MS
C h a n g e in m e a n B.P.
Percent decrease in u r i n e v o l u m e in 1st p e r i o d a f t e r MS
Lowest mean B.P. d u r i n g MS antidiuresis
85 120 77 87 72 120
81 116 80 82 67 105
-4 -4 +3 -4 -5 -15
87 75 77 65 65 75
75 82 73 77 67 95
response of a n u n t r e a t e d r a t t o 0 . 4 m g / k g i . v . o f morphine. The response consisted of a prompt decrease in u r i n e f l o w w h i c h l a s t e d t w o p e r i o d s t o g i v e a n A M T o f 18. T h i s r e s p o n s e w a s o f t e n followed by an overshoot in urine flow. Fig. 3b is an example of the response of a rat treated for 3 days and then challenged with morphine. The immediate response is clearly an increase in urine flow for 3 periods. This increase is then followed by a slow decline; another increase occurs before urine flow is stabilized. A dose of 0.16 mg/kg i.v. in rats treated for 3 days pro-
duced a slight increase in urine flow for 2 periods without the subsequent decline seen at the higher dose. Fig. 3c illustrates the response of rats treated for 5 days with morphine. The chall e n g e d o s e of m o r p h i n e ( 0 . 4 m g / k g i . v . ) p r o d u c e s an even greater initial increase in urine flow t h a n s e e n a f t e r 3 d a y s o f t r e a t m e n t ( f i g . 3b). T h e fall in urine flow which followed was smaller than that seen after 3 days of treatment. When higher challenging doses of morphine of 0.8 and 1.2 mg/kg i.v. were used, an initial increase in urine flow was again followed by a decrease in
201
20-
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P-20
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MS
1.0
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Fig. 2. T h e d e v e l o p m e n t of t o l e r a n c e to t h e a n t i d i u r e t i c effect of m o r p h i n e (MS) 8 m g / k g g i v e n s u b c u t a n e o u s l y . MS m o r p h i n e s u l f a t e 8 m g / k g s . c . , P - 1 0 = P i t r e s s i n 10 m i c r o u n i t s i.v., P - 2 0 = P i t r e s s i n 20 m i e r o u n i t s i.v. (a) t h e r e s p o n s e to MS and P - 1 0 , P - 2 0 of an u n t r e a t e d r a t . (b) t h e r e s p o n s e to MS and P - 1 0 of a r a t t r e a t e d f o r 1 day with MS 8 m g / k g s . c . , b.i.d. (c) the r e s p o n s e to MS and P - 1 0 of a r a t t r e a t e d f o r 3 d a y s with MS 8 m g / k g s . c . , b.i.d, i n c r e a s e d 8 m g / k g p e r day so t h a t by day t h r e e t h e d o s e w a s MS 24 m g / k g s . c . See text f o r s c h e d u l e . (d) t h e r e s p o n s e to MS and P - 2 0 of a r a t t r e a t e d f o r 5 d a y s of s c h e d u l e . See text.
ANTIDIURETIC ACTION OF MORPHINE IN THE RAT
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Fig. 3. The development of t o l e r a n c e to the antidiuretic effect of morphine sulfate (MS) 0.4 m g / k g given i n t r a v e nously. MS = morphine sulfate 0.4 m g / k g i.v. (a) the response to MS of an u n t r e a t e d rat. (b) the r e s p o n s e to MS of a rat t r e a t e d for 3 days of schedule. See text. (c) the r e s p o n s e to MS of a rat t r e a t e d for 5 days of schedule. See text. u r i n e flow. T h e s e e x p e r i m e n t s d e m o n s t r a t e t h a t in t h e r a t t r e a t e d f o r s e v e r a l d a y s w i t h m o r phine, another component, a diuretic one, app e a r s in t h e r e s p o n s e . This diuretic response contrasts sharply with the antidiuretic response s e e n in t h e c o n t r o l a n i m a l in w h i c h t h e d e c r e a s e in u r i n e flow i s f o l l o w e d b y a n o v e r s h o o t i n g e f fect. T h e d i u r e t i c c o m p o n e n t a p p e a r s to b e c o m e m o r e p r o m i n e n t a s t h e n u m b e r of t r e a t m e n t d a y s w i t h m o r p h i n e i n c r e a s e . T h i s c o m p o n e n t i s not p a r t i c u l a r l y s e n s i t i v e t o c h a n g e s in t h e c h a l l e n g i n g d o s e of m o r p h i n e i n t h a t g o i n g f o r 0.16 to 1.2 m g / k g i . v . d o e s n o t e n l a r g e o r p r o l o n g t h e r e s p o n s e . On t h e o t h e r h a n d , t h e a n t i d i u r e t i c component is much more dose-dependent as was s h o w n i n t a b l e 1.
4. DISCUSSION The results demonstrate that morphine prod u c e s a n a n t i d i u r e s i s in t h e r a t w h e r e t h e c o m p l i c a t i n g e f f e c t s of m o r p h i n e on t h e g a s t r o i n t e s t i n a l a b s o r p t i o n of w a t e r a n d on t h e r e t e n t i o n of u r i n e in t h e b l a d d e r h a v e b e e n l a r g e l y e l i m i n a t e d . T h e e f f e c t of m o r p h i n e on g a s t r o i n t e s t i n a l a b s o r p t i o n of w a t e r w a s m i n i m i z e d b y i n t r a v e n o u s i n f u s i o n of t h e h y d r a t i n g s o l u t i o n . R e t e n t i o n of u r i n e i n t h e b l a d d e r w a s e l i m i n a t e d b y c r e ating a bladder fistula. Our results are consistent with the mechan i s m s of t h e a n t i d i u r e t i c e f f e c t s of m o r p h i n e g i v e n in t h e l i t e r a t u r e . M o r p h i n e r e l e a s e s a n t i diuretic hormone from the neurohypophysis and
a n t i d i u r e s i s r e s u l t s f r o m a c t i o n of t h e h o r m o n e on t h e k i d n e y (De B o d o , 1944; D u k e , P i c k f o r d a n d W a t t , 1961). L i p s c h i t z a n d S t o k e y (1947) s u g g e s t a d i r e c t e f f e c t of m o r p h i n e on t h e k i d n e y . A n d , o t h e r s r e p o r t e d t h e p o s s i b i l i t y of s o m e c o m b i n a t i o n of e f f e c t s ( G e o r g e a n d W a y , 1959; H a n d l e y a n d K e l l e r , 1950; C r a w f o r d a n d P i n k h a m , 1955). In o u r s t u d y , it d o e s a p p e a r t h a t t h e i n i t i a l s h a r p d r o p in u r i n e v o l u m e s e e n i n t h e f i r s t p e r i o d a f t e r b o t h h i g h a n d low d o s e s of m o r p h i n e m a y b e due to t h e r e l e a s e of ADH a n d i t s s u b s e q u e n t a c t i o n on t h e k i d n e y . At low d o s e s of m o r p h i n e (0ol-0.4 mg/kg i.v.) the effect is rapid, lasts 1-2 p e r i o d s , a n d i s e f f e c t i v e o n l y in t h e p r e s e n c e of a water diuresis. The antidiuretic response is not seen during mannitol infusion. With 4 mg/kg of m o r p h i n e s . c . g i v e n t o a r a t u n d e r g o i n g a m a n n i t o l d i u r e s i s , t h e i n i t i a l r a p i d d e c r e a s e in u r i n e e x c r e t i o n w a s n o t s e e n . T h e r e s p o n s e to Pitressin was also blocked by mannitol. The 8 m g / k g d o s e of m o r p h i n e s . c . , h o w e v e r , did p r o duce some antidiuresis. Thus another mechan i s m m a y b e c o m i n g i n t o p l a y a t h i g h d o s e s of m o r p h i n e ; c h a n g e s in r e n a l h e m o d y n a m i c s l e a d i n g to d e c r e a s e in t h e g l o m e r u l a r f i l t r a t i o n r a t e a n d u r i n e f o r m a t i o n a r e p o s s i b l e . D r o p s in s y s temic blood pressure were observed at larger doses. These interpretations parallel those der i v e d b y G e o r g e a n d W a y (1959). T h e y r e p o r t e d t h a t m o r p h i n e in low d o s e s a c t s to r e l e a s e ADH and that this action is blocked by hypothalamic lesions which produce diabetes insipidus. The e f f e c t of l a r g e d o s e s of m o r p h i n e w e r e n o t blocked by hypothalamic lesions and the morp h i n e a p p e a r e d to a c t b y m e c h a n i s m s o t h e r t h a n
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r e l e a s e of ADH. L a s t l y , t h e u s e of e t h a n o l a s a n anesthetic in our study is compatible with producing a water diuresis since ethanol, unlike m o s t a n e s t h e t i c s d o e s not i t s e l f r e l e a s e ADH ( V a n Dyke a n d A m e s , 1951). The data from rats treated chronically with m o r p h i n e show t h a t t o l e r a n c e d e v e l o p s to t h e a n tidiuretic effect. This tolerance is nearly comp l e t e a f t e r 5 d a y s of t r e a t m e n t w i t h a s c h e d u l e of i n c r e a s i n g d o s e s . T o l e r a n c e to t h e a n t i d i u r e t i c response was measured by administering a chall e n g e d o s e of 8 m g / k g s , c . o r 0.4 m g / k g i . v . of m o r p h i n e a f t e r t h e f i r s t , t h i r d o r f i f t h d a y of treatment. Tolerance occurs without a decrease in s e n s i t i v i t y to P i t r e s s i n ( e x o g e n o u s ADH). T h u s , it i s p o s s i b l e to s e p a r a t e t h e d e v e l o p m e n t of t o l e r a n c e to m o r p h i n e a n t i d i u r e s i s f r o m p o s s i b l e c h a n g e s in s e n s i t i v i t y to ADH. T h i s p o i n t i s o n e of s u b s t a n t i a l i n t e r e s t b e c a u s e of t h e s u g g e s t i o n of N e w s o m e et a l . (1963) a n d S h i m a i et a l . (1965) t h a t t h e d e v e l o p m e n t of t o l e r a n c e t o the antidiuretic effect involves a diminished res p o n s i v e n e s s of t h e r a t to a n t i d i u r e t i c h o r m o n e . Our present data offer convincing evidence that tolerance can occur without a change in sensitivi t y to P i t r e s s i n . P o s s i b l y , t h e c h a n g e s in s e n = sitivity occur with long, sustained tolerance. C e r t a i n l y t h e d u r a t i o n of t r e a t m e n t i s s h o r t in our present experiment. If t h e r e n a l s e n s i t i v i t y to ADH h a s not c h a n g e d , t h e n it i s p o s s i b l e t h a t t h e a b i l i t y of m o r p h i n e t o r e l e a s e ADH f r o m t h e n e u r o h y p o p h ysis has been altered. Our data suggest that with t h e d e v e l o p m e n t of t o l e r a n c e t h e i n i t i a l a n t i d i u r e t i c r e s p o n s e to m o r p h i n e , a s b e s t i l l u s t r a t e d b y t h e 0.4 m g / k g i . v . d o s e , i s a b s e n t o r r e p l a c e d by a diuretic response. This altered response w o u l d b e c o n s o n a n t w i t h l o s s of a b i l i t y of m o r p h i n e to r e l e a s e ADH in r a t s t r e a t e d c h r o n i c a l l y with morphine. In t h e c h r o n i c a l l y - t r e a t e d r a t a n 8 m g / k g s . c . d o s e of m o r p h i n e h a s a b i p h a s i c r e s p o n s e , a n initial increase followed by a decrease in urine flow. N e w s o m e et a l . (1963), H o l m e s , C a r t e r a n d F u j i m o t o (1958) a n d S h i m a i et a l . (1965) o b s e r v e d that prolonged treatmentwith narcotic analgesics l e a d s to p o l y d y p s i a a n d p o l y u r i a a n d a r e v e r s a l of e f f e c t f r o m a n t i d i u r e s i s t o d i u r e s i s in w a t e r l o a d e d r a t s . W e h a v e b e e n a b l e to o b s e r v e b o t h p h a s e s in t h e s a m e a n i m a l in r e s p o n s e to m o r phine. Within 5 days an increase in the diuretic a n d a d e c r e a s e in t h e a n t i d i u r e t i c p h a s e a r e s e e n . If m o r p h i n e i n i t i a l l y r e l e a s e s ADH a n d s u b s e q u e n t l y on c h r o n i c t r e a t m e n t p r o d u c e s a b l o c k a d e of ADH r e l e a s e , t o l e r a n c e c a n b e e x -
p l a i n e d b y m e c h a n i s m s c o n s i s t e n t w i t h m a n y of the known findings. T h e e f f e c t of m o r p h i n e t r e a t m e n t on a d r e n a l ascorbic acid depletion can be sighted as a possible parallel situation. The acute administration of m o r p h i n e s t i m u l a t e s t h e r e l e a s e of A C T H a s measured by adrenal ascorbic acid depletion ( G e o r g e a n d W a y , 1955). W h e n m o r p h i n e i s g i v e n d a i l y f o r 6 d a y s , t h e r e l e a s e of A C T H to a c h a l l e n g e d o s e of m o r p h i n e i s n o t o n l y a b s e n t b u t t h e r e l e a s e of A C T H b y o t h e r d r u g s i s b l o c k e d in morphine-treated animals (Briggs and Munson, 1955). T h e s a m e s o r t of s i t u a t i o n m a y b e p r o d u c e d a c u t e l y b y g i v i n g a c o m b i n a t i o n of m o r p h i n e a n d p e n t o b a r b i t a l w h e r e b l o c k a d e of A C T H release occurs. RE FERENCES Briggs, F.N. and P . L . Munson, 1955, Studies on the m e c h a n i s m of stimulation of ACTH s e c r e t i o n with the aid of morphine as a blocking agent, E n d o c r i nology 57, 205. Crawford, J . D . and B. Pinkham, 1955, The physiology of morphine a n t i d i u r e s i s , J. P h a r m . Exp. Ther. 113, 431. Czaczkes, J . W . , C.R. Kleeman and M. Koenig, 1964, Physiologic studies of antidiuretic h o r m o n e by its d i r e c t m e a s u r e m e n t in human plasma, J. Clin. Invest. 43, 1625. De Bodo, R.C., 1944, The antidiuretic action of m o r phine and its m e c h a n i s m , J. P h a r m . Exp. Ther. 82, Ther. 82, 74. Duke, H.N., M. Pickford and J. A. Watt, 1951, The antidiuretic action of morphine: its site and mode of action on the hypothalamus of the dog. Q . J . Exp. Physiol. 36, 149. Fujimoto, J . M . , R . J . F r e n c h , J . K . G r a h a m and C.E. I n t u r r i s i , 1963, Effects of levorphanol and l e v a l l o r phan on osmolality of s e r u m from water loaded rats, Proc. Soc. Exp. Biol. Med. 114, 193. George, R. and E . L . Way, 1959, Role of the hypot h a l a m u s in p i t u i t a r y - a d r e n a l activation and antid i u r e s i s by morphine, J. P h a r m . Exp. Ther. 125, 111. Giarman, N . J . and G.A. Condouris, 1954, The antidiuretic action of morphine and some of its analogs, Archs. Int. Pharmacodyn. Ther. 97, 28. Handley, C.A. and A. D. Keller, 1950, Changes in r e nal function produced by morphine in normal dogs and dogs with diabetes insipidus, J. P h a r m . Exp. T h e r . 99, 33. Holmes, S., M.K. C a r t e r and J . M . Fujimoto, 1958, Polydipsia and polyuria during chronic a d m i n i s t r a tion of levorphanol to rats, Proc. Soc. Exp. Biol. Med. 99, 319. Lipschitz, W.L. and E. Stokey, 1947, Mechanisms of antidiuresis in the dog and in the rat, Am. J. P h y s iol. 148, 259. Marchand, C.R. and J. M. Fujimoto, 1966, Sodium and potassium excretion in r a t s t r e a t e d chronically with morphine, Proc. Soc. Exp. Biol. Med. 123, 600.
ANTIDIURETIC ACTION OF MORPHINE IN THE RAT N e w s o m e , H . H . , W. Tobin and J . M . F u j i m o t o , 1963, C e r t a i n a s p e c t s of the e f f e c t s of l e v o r p h a n o l on w a t e r m e t a b o l i s m in r a t s , J. P h a r m . Exp. T h e r . 139, 368. Reynolds, A.K. and L . O , Randall, 1957, Morphine and allied d r u g s (University of T o r o n t o P r e s s , Canada) pp. 85-86. Schnieden, H. and E . K . B l a c k m o r e , 1955, Effect of n a l o r p i n e on the a n t i d i u r e t i c action of m o r p h i n e in r a t s and men, Brit. J. P h a r m . C h e m o t h e r . 10, 45.
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Shimai, K., M. Akita, S. T o m i z a w a andH.,Kondo, 1965, On the e o r r e l a t i o n between n e u r o s e e r e t i o n of the h y p o t h a l a m o - h y p o p h y s i a l s y s t e m and w a t e r m e t a b o l i s m in m o r p h i n i z e d r a t s , O k a j i m a s FoI. Anat. Jap. 40, 911. T h o r n , N . A . , M.W. Smith and E. Skadhauge, 1965, The a n t i d i u r e t i c effect of i n t r a v e n o u s and i n t r a e a r o t i d infusion of c a l c i u m c h l o r i d e in h y d r a t e d r a t s , J. E n d o c r . 32, 1(31. Van Dyke, H.B. and R.G. A m e s , 1951, Alcohol d i u r e s i s . Acta Endocr. (Copenhagen) 7, 110.