Angiotensin II reduces voluntary alcohol intake in the rat

Pharmacology Btochemlstry & Behawor, Vol 29, pp 479-482 ©Pergamon Press plc, 1988 Pnnted m the U S A

0091-3057/88 $3 00 + 00

Angiotensin II Reduces Voluntary Alcohol Intake in the Rat L A. GRUPP, M KILLIAN,* E P E R L A N S K I

A N D R. B. S T E W A R T

D e p a r t m e n t o f Pharmacology, University o f Toronto, Toronto, Ontario, Canada M 5 S 1A8 Neurobtology Department, Addt~ tion Research F o u n d a t u m o f Ontario, Toronto, Ontario, Canada M 5 S 2S1 *Rudolf M a g n u s Instttute, Utrecht, The Netherlands R e c e i v e d 11 D e c e m b e r 1986 GRUPP, L A , M KILLIAN, E PERLANSKI AND R B STEWART Angtoten~m H redm e~ voluntary alcohol mtal,e m the rat PHARMACOL BIOCHEM BEHAV 29(3) 479-482, 1988 --The voluntary retake of alcohol has been shown to be attenuated by a variety of mampulat~ons which increase activity in the renln-anglotensm system In the present study we examined the effects of peripheral rejections of the peptlde anglotensm II on alcohol dnnklng The peptlde produced a dose-dependent decrease m alcohol retake wzth 20/xg/kg hawng httle effect, 200/xg/kg reducing intake by approximately 50% and 1 mg/kg wrtually abohshlng all alcohol drinking Thxs decrease was not due to a peptlde reduced motor deficit, or state of sickness, and could also not be accounted for by the increased water intake, or by a change m pharmacokmetlcs and taste function These data provide direct evidence that anglotensm II can modulate voluntary alcohol dnnkmg The posslbdlty that the level of anglotensm II serves as a satiety signal m alcohol dnnkmg is discussed Alcohol drinking

Anglotensm II

Remn-anglotensm system

O V E R the past few years, our laboratory has demonstrated that manipulations which are known to change acuvlty m the renIn-anglotensln (r-a) system wdl markedly alter the voluntary intake of alcohol For example, when r-a activity ~s elevated by the use of a low salt diet and diuretic [6,7], by the administration of certain drugs [4,15], or by constricting one renal artery (Two-Kidney, One-Chp procedure) [3], alcohol drinking is reduced On the other hand, when r-a activity ms suppressed as is the case in the genetically selected saltsensitive line of Dahl rats [5], by the use of a high salt diet [4], or if the anglotensln " s i g n a l " from the periphery is decreased by leslonlng the anglotensln-receptor-rich area postrema [16], voluntary alcohol drinking is enhanced The following experiments examine the involvement of the bloactive component of the r-a system, angiotensln II, on voluntary alcohol dnnking These experiments demonstrate that subcutaneous rejections of angIotensln II produce a potent and dose-dependent attenuation of voluntary alcohol drinking METHOD

Subjects The subjects were 35 naive male Wlstar rats weighing 270-300 g at the start of the experiment. They were individually housed m cages equipped with a water bottle and food hopper and kept on a reverse 12-hr/12-hr light/dark cycle with lights off at 7 00 a m

Procedure The ammals were randomly assigned to three groups, one designated to receive 20/xg/kg (n= 12) of angiotensmn II (AID (asparagme-1 vahne-5 anglotensin II-Hypertensin, Clba),

479

Satiety

one to receive 1 mg/kg anglotensln II (n= 12) and the control group to receive the sahne vehicle (n= l l ) The agents were administered by the subcutaneous route A hmlted access drinking procedure was used [13,14]. Each day during the dark cycle, the ammals were removed from their home cages, weighed, injected and then placed for 1 hr in separate "drinking" cages which had two graduated tubes, one containing alcohol, the other containing water No food was available in the drinking cage, and positions of the two fluids were alternated dally After the hour had elapsed, the amounts consumed of both fluids were recorded and the ammals were then returned to their home cages Since the half-hfe of AII in the orculation of the rat is less than one minute, a 1 hr session should be sufficient to be able to observe all of the effects of the peptlde on intake For 14 days the alcohol concentration was 3% w/v and was then increased to 6% w/v for the next 14 days For a further 14 days the alcohol concentration remained at 6%, bat the group that was previously treated with 20/xg/kg AII now received 200 /xg/kg AII The doses of the other two groups remained unchanged Following this, and m order to probe for any group differences m taste sensitivity, the drug Injections continued and a 14% w/v glucose solution was substituted for the alcohol during the one hour drinking period This test was carried out for 4 consecutive days Finally, at the end of the experiment all rats were first injected with their respective doses of AII and then with an lntraperitoneal dose of 2 5 g/kg alcohol (12 5%) Blood samples were taken from the cut tip of the tall at 20 mm intervals during the first hour after the Injection and thereafter at hourly intervals for the next 5 hours These blood samples were used to examine the effect of AII on the disposition and metabolism of alcohol

480

G R U P P , K I L L I A N , P E R L A N S K I A N D STEgC,~RT

One w a y analyses of varmnce were used to analyze group differences m c o n s u m p t i o n of alcohol and w a t e r Post hoc multiple c o m p a r i s o n s were done with the D u n c a n ' s test

1.= 3% Alcohol

A 15

Water

RESULTS

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3 % A It ohol- Wate~ Chol( ('

Figure IA shows the m e a n alcohol and w a t e r intake across the 14 days of choice b e t w e e n 3% alcohol and w a t e r S~gnificant differences in the intake o f alcohol, F(2,32)= I 1 5. p < 0 01, and of water, F(2,32)=4 44. p < 0 02, were found a m o n g the three groups of ammals The group r e c e w i n g 20 /xg/kg A I I did not differ s~gmficantly in its intake of e~ther alcohol or w a t e r from the control group H o w e v e r , the group receiving 1 mg/kg A I I showed a v e r y slgmficant r e d u c u o n in alcohol intake along w=th a slgmficant elevation in w a t e r intake c o m p a r e d to the control group

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W h e n the concentration of alcohol was increased to 6% (Fig 1B), slgmficant group differences in the intake of alcohol, F(2,32)= 19 2 , p < 0 01, persisted, with the 1 mg/kg group continuing to s h o w a dramatic suppression in alcohol retake Again, the 20/xg/kg group did not show a change m intake c o m p a r e d to control Group d~fferences in water retake, on the other hand, were marginally s~gntficant, F(2,32)=2 85, p < 0 07, largely because the e n h a n c e d water intake o f the 1 mg/kg group seen was shghtly l o w e r than ~ts level during the first 14 days of the experament (~ e , from a p p r o x i m a t e l y 3 ml to 2 ml) D u r m g the final 14 days of the e x p e r i m e n t , the group of ammals that previously r e c e i v e d 20/zg/kg A l l , now began to receive a dose of 200/xg/kg A l l The other two groups still r e c e i v e d their respective agents and a choice b e t w e e n 6% alcohol and w a t e r continued to be offered Figure 1C shows that the 200 p.g/kg dose s~gnlficantly reduced the c o n s u m p tion of alcohol c o m p a r e d to control, whale the 1 mg/kg dose continued to suppress alcohol drinking, F(2,32)=41 99, p < 0 01 G r o u p differences m w a t e r mtake w e r e also obtained, F(2,32)=8 01, p < 0 01, with both the 200/zg/kg and 1 mg/kg groups showing an e n h a n c e d intake relatwe to control

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The control, 200/zg/kg and 1 mg/kg groups drank similar amounts of the glucose solution across the four day testing period ( m e a n = 4 4 4 ml/kg, 41 2 ml/kg and 35 3 ml/kg, respectively) and statistical analysis indicated no significant group differences, F(2,32)=2 6, n s Although m e a n w a t e r retake for these groups was minimal (l 2 ml/kg, 0 85 ml/kg and 3 9 ml/kg, respectively) the 1 mg/kg group did c o n s u m e slgmficantly more than the o t h e r two groups, F(2,32)=4 0, p < 0 01 Drug Handhng

Figure 2 shows the m e a n blood alcohol levels for the three groups at the eight samphng times following the 2 5 g/kg injection of alcohol The last four points on the descending portion of the c u r v e s w e r e used to calculate the slopes which r e p r e s e n t the rate o f alcohol m e t a b o h s m Statistical analysis showed a significant difference in m e t a b o h s m a m o n g the three groups, F(2,31)=3 29, p < 0 . 0 5 , and post hoc tests indicated that the 1 mg/kg group had a faster rate than control This accelerated rate o f metabolism, h o w e v e r , could not ac-

Control

A II A II 2 0 0 / z g 1 mg

Control

A II A II 200 ~ug 1 rng

FIG 1 (A) Mean 3% (w/v) alcohol and water retake for the two anglotensm II groups and the control group across the first 14 days of the hmlted access procedure Bars represent +_standard error of the mean (B) Mean 6% (w/v) alcohol and water retake for the two anglotensm II groups and the control group across the next 14 days of the limited access procedure Bars represent +standard error of the mean (C) Mean 6% (w/v) alcohol and water retake for the two anglotensm II groups and the control group across the next 14 days of the hmlted access procedure The group that received 20/~g/kg anglotensm II now received 200 #g/kg anglotensm lI Bars represent _+standard error of the mean

count for the reduced alcohol drinking in this group because an accelerated rate would be e x p e c t e d to permit a greater but not a lesser alcohol retake Extension of the linear portion of the curves back to the ordinate yields an estimate o f the concentration at time zero from which the v o l u m e of distribution is calculated There were no significant group differ-

ANGIOTENSIN REDUCES ALCOHOL DRINKING

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ences m volume of distribution, F(2,31)=0.55, n s Finally a two-way analysis of variance of the blood alcohol levels measured at the first three intervals following the alcohol lnjecUon did not show any group differences in absorption, F(2,31)=0 44, n s , or group×interval mteracUon, F(2,31)= 0 25, n s A slgnLficant effect of interval, F(2,31)=13 28, p < 0 01, reflected the increasing blood alcohol levels during the absorption phase These findings suggest that A l l did not alter alcohol retake through a change m drug dlsposlUon or metabolism DISCUSSION This experiment has shown that anglotensm II can significantly reduce voluntary alcohol drinking m a dosedependent fashion. A 20 p.g/kg dose was meffectwe, a 200 /~g/kg dose produced a 50% reductson m retake and a 1 mg/kg dose almost completely abolished all alcohol retake The effect of AII on alcohol intake lasted at least 1 hr because there was no obwous recovery within that period of t~me A timeresp~,h~e study would dehneate the duration of action of the peptlde on retake At the same time that alcohol drinking was suppressed, water intake was elevated From casual observauons the effect on water intake was over within the first 15-30 mm following the rejection Since anglotensm II is a dlpsogen, th~s increase in water mtake Is a reflection of the peptidereduced increase in thirst. Th~s thirst, however, appears to be hnked specifically to water rather than a general increase m the motwatlon for both available fluids It is not merely that ammals are " t h i r s ti e r " for water and indifferent to alco-

hol Rather they are "thtrstler" for water whde at the same time avoid the alcohol. Furthermore, this suppression of alcohol dnnkmg cannot be the result of a regulatory compensaUon secondary to the enhanced water intake because regulatory considerations are not relevant in a hm~ted access procedure where animals are not weight reduced, have full access to food and water in their home cages and are m the dnnkmg cage for only one hour every day Finally, the fact that anglotensm II stimulated consummatory behawor directed to water and did not change glucose retake rules out the possibility that the pept~de-mduced decrease m alcohol retake was related to a non-specific decrement in the ammals' ability to approach or consume the avadable fluids Taken together these findings suggest that the effect of anglotensm II on alcohol intake is independent of its effects on water intake. It is possible that anglotensln II reduced alcohol intake by inducing a general state of malaise or by altering taste function in such a way as to reduce palatability To examine this, all groups continued to receive their respective injections of either anglotensln II or vehicle and were run m the limited access procedure where they were offered a choice between a highly palatable glucose solution and water No significant group differences In glucose consumption were found suggesting that if the animals that received anglotensm II were feeling unwell, it was not reflected in the avidity with which they consumed the sweet solution. Furthermore, ff anglotensln II reduced alcohol retake through a change in taste funcUon, It would have to be a specific change m the acceptability of the taste of alcohol rather than a general effect on all taste sensauons Whde anglotensm II ~s a potent pressor agent, it ~s unlikely that these pressor effects per se mediated the reducUon in alcohol drinking because previous work was shown that alcohol retake can be enhanced in certam types of hypertension For example, Grupp et al [4,5] have shown that Dahl salt-sensitive hypertensive rats. or Wistar rats rendered hypertensive by the chromc feeding of a high salt diet show an elevated and not a reduced alcohol intake It ~s also unhkely that anglotensm II exerted its effect on alcohol drinking by altering the dlsposmon or metabolism of alcohol An alcohol disappearance curve showed no group differences m the absorption or dlstnbuUon of alcohol Furthermore, the group receiving 200/xg/kg anglotensln II did not differ slgmficantly in its rate of alcohol metabolism from control The 1 mg/kg group had a significant accelerated rate of metabolism, however, even this cannot readdy account for the abthty of this dose to reduce alcohol drinking since an accelerated rate would, if anything, permit a greater not a lesser alcohol intake Taken together, these findings indicate that a change in drug handhng cannot account for the effect of anglotensm II on alcohol retake In the present experiment ang~otensm II does not appear to alter alcohol intake because of motor lmpmrment, general malaise, taste factors or pharmacokmetlcs, and appears to exert its effect independently of changes in water intake Anglotensm II is known to play a role in the control of fluid and electrolyte balance, blood pressure and thirst, but the suggesUon that this pepUde in particular or the renmanglotensm system in general might also be involved in regulating alcohol intake is new and somewhat unusual relative to the biological functions normally associated with this pepttde It may therefore be appropriate to outhne, even if somewhat speculatively, how this pepUde might be involved in voluntary alcohol intake Recently, it has been suggested

482

GRUPP, KILLIAN, PERLANSKI

t h a t a n g i o t e n s m II m a y act as a satiety signal in c o n t r o l l i n g a l c o h o l i n t a k e [2] T h i s w a s b a s e d o n t w o sets o f findings First, it h a s b e e n d e m o n s t r a t e d in a n u m b e r o f d~fferent labo r a t o r i e s t h a t a l c o h o l i n g e s t i o n c a n e l e v a t e a n g i o t e n s l n II levels [8, 10--12, 17] S e c o n d , m a n i p u l a t i o n s w h i c h are k n o w n to e l e v a t e a n g l o t e n s l n II levels r e s u l t m a r e d u c t i o n in v o l u n t a r y a l c o h o l d r i n k i n g [2, 3, 6, 7] P u t t i n g t h e s e t w o sets o f findings t o g e t h e r led to the s u g g e s t i o n t h a t the c e s s a t i o n of alcohol d r i n k i n g m i g h t o c c u r w h e n o r c u l a t i n g a n g l o t e n s m II e x c e e d s a "cratical'" level t h a t signals satiety T h e following p r o c e s s is s u g g e s t e d B e f o r e alcohol d r i n k i n g c o m m e n c e s , the level o f a n g i o t e n s l n II is b e l o w this " s a t i e t y " level O n c e d r i n k i n g starts, a n d b e c a u s e a l c o h o l ~tself e l e v a t e s a n g l o t e n s i n II, the circulating level o f a n g l o t e n s l n II would a p p r o a c h this satiety level A l c o h o l d r i n k i n g w o u l d c e a s e w h e n e n o u g h alcohol h a s b e e n c o n s u m e d to c a u s e t h e circ u l a t i n g levels o f a n g l o t e n s l n II to e x c e e d the satiety level In the p r e s e n t e x p e r i m e n t a n g l o t e n s l n II was a d m i n i s t e r e d by Injection p r i o r to alcohol d r i n k i n g so that the circ u l a t i n g levels o f a n g l o t e n s l n II w o u l d be e l e v a t e d If this b r o u g h t the a n i m a l s c l o s e r to the satiety level prior to a n y a l c o h o l intake, o n c e alcohol d r i n k i n g b e g a n , less could b e c o n s u m e d b e f o r e t h e satiety level w o u l d h a v e b e e n surpassed T h u s , a n g l o t e n s l n II injections m a y h a v e r e d u c e d alcohol intake by bringing the animals closer to this satiety level b e y o n d w h i c h n o m o r e alcohol would b e c o n s u m e d F o r the c o n t r o l a n i m a l s not r e c e i v i n g a n g l o t e n s m II, the satiety level w o u l d be r e a c h e d only a f t e r sufficient a n d larger q u a n h t l e s o f alcohol were c o n s u m e d S u c h c o n s i d e r a t i o n s a w a i t empirical vahdat~on b y m e a s u r e m e n t s o f p l a s m a a n g l o t e n s l n II levels d u r i n g the 1 hr a c c e s s p e r i o d This raises t h r e e o t h e r p o i n t s First, t h a t a role for a n g l o t e n s l n II as a satiety signal for s t o p p i n g alcohol retake d o e s not imply t h a t a n g l o t e n s m II ~s r e d u c i n g alcohol i n t a k e b y d~mlnishlng t h e p o s l t w e l y r e i n f o r c i n g p r o p e r h e s o f alco-

AND STEWART

hol S e c o n d , t h a t this role for a n g l o t e n s i n II in alcohol drinklng d o e s not c o m m e n t on or e l u c i d a t e t h o s e factors t h a t are r e s p o n s i b l e for the r u i n a t i o n o f alcohol d r i n k i n g Finally, the n o t i o n t h a t a n g l o t e n s m II d o e s act as a satiety signal r e q u i r e s t h a t t h e levels o f this peptsde for a c t i v a t i n g the satiety c u t - o f f be e s t a b l i s h e d F u t u r e e x p e r i m e n t s will b e d i r e c t e d t o w a r d s d e t e r m i n i n g th~s t h r e s h o l d T h e e f f e c t i v e n e s s o f a n g s o t e n s i n II ( a s p - l , val-5 a n g i o t e n s i n II H y p e r t e n s l n . C~ba) in r e d u c i n g alcohol mtake raises the intriguing possibility t h a t an a n a l o g or p e r h a p s e v e n a f r a g m e n t o f this p e p t l d e m i g h t be d e v e l o p e d w h i c h c a n a n t a g o n i z e v o l u n t a r y alcohol d r i n k i n g w i t h o u t the a t t e n d a n t cardiac a n d thirst side-effects o f H y p e r t e n s l n In fact, f r a g m e n t s o f a n g l o t e n s l n II are usually less p o t e n t t h a n the p a r e n t p e p t l d e w~th r e s p e c t to thew effects on b l o o d p r e s s u r e (e g , [9]) a n d t h w s t (e g , [I]) Yet, if a f r a g m e n t could be ~dentlfied t h a t stdl m a i n t a i n e d b~ologlcal activity V l S a VlS alcohol intake, s u c h a n a g e n t could t h e o r e h c a l l y be v e r y useful in the t r e a t m e n t o f h u m a n a l c o h o l a b u s e In s u m m a r y , we f o u n d t h a t the s u b c u t a n e o u s a d m i n i s t r a tion o f a n g l o t e n s l n II m a r k e d l y r e d u c e s v o l u n t a r y alcohol intake in a d o s e d e p e n d e n t m a n n e r This r e d u c t i o n d o e s not a p p e a r to be related to an a n g ~ o t e n s l n - l n d u c e d state of malaise, g e n e r a l m o t o r deficit, c h a n g e m taste f u n c h o n or c h a n g e m drug h a n d l i n g a n d is i n d e p e n d e n t o f c h a n g e s m w a t e r retake T h e s e results p r o w d e n e w a n d direct e v i d e n c e for the i n v o l v e m e n t o f the r e n l n - a n g l o t e n s l n s y s t e m in the c o n t r o l of alcohol d r i n k i n g

ACKNOWLEDGEMENTS The authors w~sh to thank Mrs V Cabral for preparing the manuscript and Ms Cathenne Nash, C~ba-Ge~gy Canada Ltd for the generous supply of Hypertensm Th~s research was supported by the Add~chon Research Foundation of Ontario

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10 Lmkola, J , F Fyhrqmst. M M N~emmen, T H Weberand K Tonttl Remn-aldosterone axis m ethanol intoxication and hangover E u r J Chn l m e ~ t 6. 191-194, 1976 11 Lmkola, J , F Fyhrqmst and R Yhkahn Renm, aldosterone and cortlsol during ethanol mtox~cahon and hangover A¢ta P h w t o l Sc and 106: 75-82, 1979 12 Lmkola, J , 1 Tlkkanen F Fyhrqulst and M Rusl Renm, water drinking salt preference m alcohol preferring and alcohol avoiding rats Pharma~ ol B:o~ hem Bchav 12: 293-296, 1980 13 Lmseman, M A Alcohol consumption m free-feeding rats Procedural, genetic and pharmacokmet~c factors Psvt hopharma~ olog~ (Berhn) 92. 254-261, 1987 14 MacDonall, J S and H Marcucella Increasing the rate of ethanol consumptmn m food and water satmted rats Pharma~ ol Bto~hem heha~ 10: 211-216, 1979 15 Spmosa, G , E PerlanskL R B Stewart and L A Grupp Ang~otensm converting enzyme mh~bltors Ammal expenment~ suggest a new approach to the treatment of alcohol abuse m man A h ohoh~m Chn E~p Red, m press 16 Stewart, R B , L A Grupp and E Perlansk~ Area postrema and alcohol Effects of area postrema les~ons on ethanol selfadministration, pharmacokmehcs and ethanol-reduced condltmned taste aversmn Sot Neuros~ t Ab~tr 12: 285, 1986 17 Wright, J W , S L Morseth, R H Abholdand J W Harding Elevations m plasma angJotensm II with prolonged ethanol treatment m rats Pharma~ olB:o~ hem B e h a v 24. 813-818, 1986