Reduction of responses to angiotensin II by antidepressant drugs

8jp ELSEVIER

European Journal of Pharmacology264 (1994)295-300

Reduction of responses to angiotensin II by antidepressant drugs Paul R. Gard *, Anne Mandy, Jane M. Whiting, David P.D. Nickels, Amanda J.L.S. Meakin Department of Pharmacy, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, UK

Received 28 April 1994;revised MS received 26 July 1994; accepted 2 August 1994

Abstract

The effects of the antidepressant drugs desipramine, fluoxetine and tranylcypromine and the non-antidepressant control cocaine on angiotensin II function were determined in vivo by use of angiotensin-induced drinking in rats and in vitro using contractile responses of the rat uterus. The results of the drinking studies showed that the three antidepressants, but not cocaine, reduced the dipsogenic effects of angiotensin II. In vitro, all of the drugs reduced the effects of not only angiotensin but also acetylcholine and oxytocin on the uterus. The inhibition appeared to be non-competitive in all cases. These results indicate that the antidepressant drugs reduced the activity of angiotensin II, albeit non-selectively, and suggest that the previously reported effects of antidepressants on isoprenaline-induced drinking in rats reflect an action on angiotensin activity rather than a reduction of/3-adrenoceptor activity as previously suggested. Keywords: Angiotensin II; Antidepressive agent; Drinking behavior

1. Introduction

Goldstein et al. (1985) reported that subacute (4-day) coadministration of antidepressant drugs and an a 2adrenoceptor antagonist caused a decrease in isoprenaline-induced drinking in rats. A more rapid effect of an antidepressant has been demonstrated using salbutamol-induced drinking where the drinking behaviour was decreased 21 h, but not 4 h, after a single dose of desipramine (Gard and Kerr, 1990). This drinking paradigm is believed to be mediated, at least in part, by central/3-adrenoceptors and it has been proposed that the decreased isoprenaline-induced drinking is a behavioural demonstration of the central fl-adrenoceptor down-regulation that follows chronic administration of antidepressant drugs (Goldstein et al., 1985). The rapid rise in fluid intake and simultaneous inhibition of urine output following administration of /3-adrenoceptor agonists to rats was first reported by Lehr et al. (1967) and was subsequently shown to be mediated by fl2-adrenoceptors (Katovich and Fregly, 1978), the stimulation of which results in the release of

* Corresponding author. Tel. 44-273-642084,fax 44-273-679333. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(94)00481-1

renin and the production of angiotensin II, the ultimate dipsogen (Gutman et al., 1971). The timing of the drinking response to fl-adrenoceptor stimulation in the rat is independent of any hypotensive effect (Lehr et al., 1967) suggesting that the renin release is a direct consequence of the fl-adrenoceptor stimulation; a proposal supported by the finding that renin release from isolated renal cell preparations can be enhanced by catecholamines (Weinberger et al., 1975). The site of the fl-adrenoceptors in question is unclear although they are probably central as direct injections of minute doses of isoprenaline to the lateral ventricles (Goldstein et al., 1985), the lateral septal area (Giardino and Fisher, 1971) and the hippocampus (Mountford, 1969) all produce the drinking response. The angiotensin receptors via which the drinking behaviour is mediated are believed to be in the circumventricular organs, probably in the region of the medial preoptic nucleus (Phillips, 1987). The aim of the present study was to investigate further the effects of antidepressant drugs on drinking behaviour in rats by determining the effects of three dissimilar antidepressant drugs, and one non-antidepressant control, on responses to angiotensin II in vivo and in vitro using angiotensin-induced drinking in the

296

P.R. Gard et al. / European Journal of Pharmacology 264 (1994) 295-300

rat and angiotensin-induced contractions of the rat isolated uterus. The drugs selected for study were desipramine, fluoxetine and tranylcypromine which are proven antidepressant drugs from dissimilar chemical groups and cocaine which was selected as a non-antidepressant, psychostimulant control. Desipramine and fluoxetine are antidepressants which inhibit the neuronal reuptake of noradrenaline and 5-hydroxytryptamine respectively, whilst cocaine inhibits neuronal reuptake of noradrenaline but does not possess antidepressant properties in clinical use. Tranylcypromine is neither an inhibitor of noradrenaline reuptake nor of 5-hydroxytryptamine reuptake but it increases intrasynaptic noradrenaline and 5-hydroxytryptamine by inhibition of monoamine oxidase.

2. Materials and methods 2.1. Drinking studies

Male Wistar rats (226 + 24 g) were housed in groups of four under controlled lighting conditions (lights on: 07.00-21.00 h) at an average temperature of 21.6°C and relative humidity of 59%. Food and water was allowed ad libitum throughout. Angiotensin- and saline-induced (baseline) drinking were determined following administration of either antidepressant drug, cocaine or vehicle control. For determination of angiotensin-induced drinking, rats were placed into individual cages containing specialized water dispensers. The dispensers allowed the animal free access to the water bottle nozzle and were fitted with a removable receptacle for the collection of any spillage. Angiotensin II (1.5 mg kg -1 s.c.) was administered and the fluid intake was monitored for 3 h by weighing the water bottle together with the receptacle at 30 min intervals, assuming the density of water to be 1.0 g ml-1. Baseline drinking was determined by monitoring water intake for 3 h after subcutaneous administration of saline (0.9% w/v; 1 ml kg-1). To study the effects of pretreatment with antidepressant drugs or cocaine, the rats received a single intraperitoneal injection of either desipramine, fluoxetine, tranylcypromine, or cocaine (all 10 mg kg -1) or saline control, 1 h before determination of either angiotensin-induced or baseline drinking. All experiments were commenced between 09.00 and 10.00 h at an ambient temperature of 17-21°C. The group size for the angiotensin-induced drinking was 8 in all cases; for the saline-induced drinking the sample size for the control group was 8 and for each of the treatment groups was 4.

2.2. In vitro studies

Sexually mature female Wistar rats, housed as previously described for males, were killed by cervical dislocation, the stage of oestrous cycle determined by microscopic examination of vaginal cytology, and the uterine horns removed. Each uterine horn was divided longitudinally and bathed in de Jalon's physiological salt solution, under 1 g of tension, at 28°C and gassed with 95% 0 2 / 5 % CO 2. Contractile responses to geometrically ascending doses of acetylcholine (4 x 10 -8 to 9 x 10 -5 M), angiotensin II (5 x 10 -11 to 2 X 10 -6 M) and oxytocin (6 x 10 -11 to 2 X 10 -7 M) were recorded isotonically in the absence or presence of desipramine (1 and 10/xM), fluoxetine (1 and 10 ~M), tranylcypromine (1 /zM) or cocaine (1 and 10 /xM). Acetylcholine and angiotensin were added using a 3min dose cycle; the contact times were 30 s for acetylcholine and 60 s for angiotensin. Oxytocin was added using a 5-min dose cycle with 2 min contact time. The contractile responses to potassium chloride (17 mM) were also determined either in the presence or absence of desipramine (1 and 10 ~M) and fluoxetine (1 and 10 ~M). 2.3. Data analysis

Data from the drinking experiments are expressed as mean cumulative fluid intake (+S.E.M.). Results were compared using 2-way analysis of variance. Data from the studies in vitro are expressed as the percentage of the maximal mean control response and are plotted as log10 molar concent rat i on- response curves using the mean results (±S.E.M.). The group size was 5-12. Treatment groups were compared with the appropriate control group using 2-way analysis of variance. 2.4. Drugs and chemicals

Angiotensin II (acetate salt), desipramine (hydrochloride salt), tranylcypromine (trans-2-phenylcyclopropylamine, hydrochloride salt), oxytocin (acetate salt), acetylcholine (chloride salt) and cocaine hydrochloride were all obtained from commercial sources. Fluoxetine hydrochloride ( L Y l l 0 1 4 0 ) w a s obtained from Eli Lilly and company, Indianapolis, USA. For experiments in vivo drugs were diluted in water prepared by reverse osmosis, except for angiotensin which was dissolved in saline. For the experiments in vitro drugs were dissolved in de Jalon's solution. De Jalon's solution contains 0.154 M NaCI, 2.775 mM D-glucose, 0.595 mM NaHCO 2, 5.633 mM KCI and 0.551 mM CaC1. All drugs were prepared immediately prior to use.

P.R. Gard et aL / European Journal of Pharmacology 264 (1994) 295-300

297

2-

E

"o

"= 0 r.

m

E 1

0 3(I

60

911

120

1 5 1 1 1811 Time afler saline

311 1rain)

60

911

1211

1 5 1 1 1811

Fig. 1. Effects of a single dose of fluoxetine (*), tranylcypromine ( • ) , desipramine (e), cocaine ( • ) , all 10 m g k g - i , or vehicle control ( • ) on cumulative baseline (saline-induced) fluid intake in the rat. Values represent the m e a n of 4 - 8 replicates + S.E.M. Significant differences from vehicle control: *** P < 0.001.

3. Results 3.1. Drinking studies The results presented in Fig. 1 show that neither fluoxetine nor tranylcypromine had any significant ef-

fect on baseline (saline-induced) drinking; however, baseline drinking was significantly reduced by desipramine (P < 0.001) and significantly increased by cocaine (P < 0.0011. All of the antidepressant drugs studied caused a significant decrease in angiotensin-induced fluid intake

4

-~2 I

"=

m t._

0

6-

-=43" 2" I

0.7. o

311

60

90

1211 150 180 ' ' 3'0 " " 6'11 "l'ime after a n g i o t e n s i n (mill)

'

'

911 ' "

"

1211 ~ '

'

1 5' 1 1' ' 1811 '

Fig. 2. Effects of a single dose of fluoxetine (*), tranylcypromine ( • ) , desipramine (e), cocaine ( • ) , all 10 mg k g - t , or vehicle control ( • ) on cumulative angiotensin-induced fluid intake in the rat (1.5 mg kg -1 s.c.). Values represent the m e a n of 8 replicates + S.E.M. Significant differences from vehicle control: * P < 0.05, ** P < 0.01 and *** P < 0.001.

P.R. Gard et al. / European Journal of Pharmacology 264 (1994) 295-300

298

150-

( P < 0.01 in all cases; Fig. 2). Cocaine, conversely, caused a significant increase in angiotensin-induced drinking ( P < 0.05; Fig. 2).

100 to

3.2. In vitro studies 50

The majority of the animals used were found to be in either oestrus or the metaphase of the oestrous cycle; a small number were found to be in dioestrus. None of the animals was in prooestrus. The contractile responses to acetylcholine, angiotensin and oxytocin in the control group were not significantly affected by the stage of oestrous cycle. The graphs presented in Fig. 3 show that desipramine 1 0 / z M significantly reduced the contractile responses to angiotensin as did fluoxetine at both 10 /xM and 1/zM; tranylcypromine 1/~M and cocaine 10 /xM and 1 /zM. There was no significant effect of desipramine 1/zM. The contractile responses to acetylcholine were also significantly reduced by desipramine 10/xM ( P < 0.01); and by fluoxetine 10/zM ( P < 0.01); tranylcypromine 1 /zM ( P < 0.001) and cocaine at both 10 izM and 1/~M ( P < 0.001 in each case). There were no significant effects of desipramine 1 /zM or fluoxetine 1 /xM. The minimum group size for the control tissues was 9 and for the treated tissues was 7. The contractile responses to oxytocin were completely abolished by fluoxetine at 1 0 / x M (control n = 12; fluoxetine n = 6; P < 0.001) whilst at 1 tzM slight responses were recorded at the higher concentrations

f.

N

-5:.~.~:

0-

I

N N

~

50"

I 0 O- ~

***

N 5 I)-

O" 0

-9 I,og

-S

Molar

-7

Concentration

-6 Oxylocin

Fig. 4. Effects of desipramine ( A ) and tranylcypromine ( ~ ) on oxytocin-induced contractions of the rat uterus. Values represent the m e a n of at least 9 replicates +S.E.M. Significant differences from control (1:1): *** P < 0.001.

of oxytocin (control n = 14; fluoxetine n = 6; P < 0.001). Cocaine similarly significantly reduced the responses to oxytocin at both concentrations (control n = 12; cocaine 10 /xM n = 7; P < 0.05 and cocaine 1 /xM n = 7; P < 0.025) and as with the effects of cocaine on angiotensin (presented in Fig. 3) there were

150

c loo

~e

o

50

"~ E

0

v iv v

v iv v

v v

-8 I,og

-7 Moll|r

150 100

50

i

0 -10

-9

-6 - I 0 Concenlralion

-9 -8 A n g i o t e n s i n !1

-7

-6

Fig. 3. Effects of fluoxetine (o: 1/~M; , : 10/zM), tranylcypromine ( ~ : 10/zM), desipramine (zx: 1 p,M; A: 10 ~ M ) and cocaine (o: 1/.~M; e: 10 /.~M) on angiotensin II-induced contractions of rat uterus. Values represent the m e a n s of 5 - 1 4 replicates + S.E.M. Significant differences from control (12): * P < 0.05, ** P < 0.01 and *** P < 0.001 respectively.

P.R. Gard et al. / European Journal of Pharmacology 264 (1994) 295-300

no quantitative differences between the effects of the two doses of cocaine studied. Tranylcypromine 1 /zM and desipramine at 10 /zM also significantly reduced the responses to oxytocin (Fig. 4), unlike desipramine 1 /zM which had no significant effect (control n = 12; desipramine n = 6). Compared to control (n = 11), neither desipramine 1/~M (n = 5) or 10/zM (n = 6) nor fluoxetine (1/zM, n = 4) had any significant effect on the contractile responses induced by potassium chloride; fluoxetine 10 /zM (n = 5), however, caused a significant reduction (P < 0.01).

4. Discussion

The results of the drinking studies show that the three antidepressant drugs investigated all reduced the dipsogenic effects of angiotensin II. These results therefore question the proposal that the reduction of isoprenaline-induced drinking by antidepressants is a behavioural demonstration of central /3-adrenoceptor down-regulation (Goldstein et al., 1985), and suggest that it is due to the antagonism of angiotensin II, possibly in the circumventricular organs. Such results in themselves are, however, inconclusive as there are many factors which may influence fluid intake in rats, for example sedative drugs decrease motor activity and thus might be expected to reduce drinking behaviour; angiotensin-induced drinking can be reduced by 5-hydroxytryptamine (5-HT), 5-HT 1 and 5-HT 2 receptor antagonists (Dourish et al., 1992); and, perhaps more relevant in the light of the known anticholinergic actions of the tricyclic antidepressants, muscarinic antagonists are known to decrease fluid intake (Block and Fisher, 1970), although atropine has no effect on angiotensin-induced drinking in rats (Giardino and Fisher, 1971). In the case of tranylcypromine and fluoxetine the results from the studies of their effects on baseline drinking indicate that they had no effect on drinking itself, but only on angiotensin-induced drinking, which would suggest that their actions are not due to nonspecific sedative effects or interaction with the cholinergic mechanisms controlling drinking. Desipramine, however, reduced both angiotensin-induced and baseline drinking which could be due to antagonism of both endogenous and exogenous angiotensin or a nonspecific effect such as sedation, although the latter suggestion is unlikely as there are no reports of desipramine-induced sedation in the rat, but rather it has been shown to reduce the hypoactivity induced by salbutamol (Przegalinski et al., 1983). Cocaine increased both baseline and angiotensin-induced drinking which could be due to an effect on the neuronal control of drinking or may reflect a psychostimulant action.

299

In vitro all of the antidepressant drugs tested reduced the contractile responses to angiotensin II, and for fluoxetine and desipramine the effect was dose related. Cocaine also inhibited the angiotensin-induced uterine contractions. In all cases the shapes of the log concentration-response curves suggest that the antagonism of the angiotensin II was insurmountable and non-competitive in nature. These results, when considered in conjunction with those from the drinking studies, imply that the three antidepressant drugs studied are all able to antagonize the effects of angiotensin II at concentrations equivalent to or less than the concentrations required to elicit an anticholinergic effect (see later) and comparable to the therapeutic plasma concentrations in humans (e.g. up to 0.7 ~M, 3.0/.~M and 0.5 /zM for desipramine, fluoxetine and tranylcypromine respectively; Ziegler et al., 1978; McEvoy, 1993). It is unclear whether the reduction of angiotensin activity by these drugs is of clinical importance, but the finding is particularly interesting in the light of evidence that reduction of angiotensin function in humans may have an antidepressant effect; for example it has been reported that the angiotensin converting enzyme inhibitor captopril, which decreases the production of angiotensin II from angiotensin I, has a mood elevating effect in depressed patients (Zubenko and Nixon, 1984; Deicken, 1986; Germain a n d Chouinard, 1988, 1989). Another angiotensin converting enzyme inhibitor, enalapril, has similarly been seen to produce mood elevation in normal volunteers (Cohen et al., 1984). Captopril also exhibits antidepressant-like activity in the forced swim test in mice (Giardina and Ebert, 1989), and it is equipotent with imipramine in the reversal of the learned helplessness paradigm in rats (Martin et al., 1990). In the case of cocaine there is an apparent disparity between the results obtained in vivo and those obtained in vitro. This indicates some action other than antagonism of angiotensin II receptors, for example a non-specific membrane stabilizing effect, may be involved in its actions. Such a non-specific action would also explain its inhibition of the uterine contractions induced by acetylcholine and oxytocin. The three antidepressants studied also reduced the responses to both acetylcholine and oxytocin which may point to a non-specific action similar to that of cocaine. The results of the studies of the effects of desipramine and fluoxetine (1 /zM) on the contractile responses to potassium chloride, however, suggest that, at least in the cases of these two antidepressants, the inhibition of the agonist-induced uterine contractions is not due to a non-specific action nor due to physiological antagonism, although this may not be so for the higher concentration of fluoxetine. Given the dissimilar chemical structures of the three antidepressant drugs studied it is unlikely that they are producing their effects

300

P.R. Gard et al. / European Journal of Pharmacology 264 (1994) 295-300

on the uterus by interacting directly with the three membrane-bound receptors concerned, i.e. muscarinic M 3 (Varol et al., 1988), angiotensin (AT 1) and oxytocin, although it could be speculated that they are acting on post-receptor events. All of these receptors utilize the inositol triphosphate/diacylglycerol second messengers. In human platelets it has been shown that a wide variety of antidepressants, including desipramine, are able to inhibit thrombin-induced inositol triphosphosphate/diacylglycerol production, although the mechanism of this effect is unknown (Rehavi et al., 1993). The current study used antidepressant concentrations 10-100-fold less than the above mentioned study; however, the possibility remains that the current results from the rat uterus reflect an effect of these antidepressant drugs on this inositol triphosphate/diacylglycerol second messenger which would account for the apparent non-competitive nature of the antagonism. In conclusion, this study shows that the dissimilar antidepressant drugs desipramine, fluoxetine and tranylcypromine are all able to inhibit the actions of angiotensin II in vivo and in vitro at concentrations comparable to the therapeutic plasma concentrations in humans. It is therefore likely that the previously reported effects of antidepressants on isoprenaline-induced drinking in rats reflect an action of these drugs on angiotensin activity rather than/3-adrenoceptor activity. The nature of the inhibition of angiotensin, however, suggests that the antidepressants are not acting selectively at the angiotensin receptor.

Acknowledgements Fluoxetine hydrochloride (LYl10140)was a kind gift from Eli Lilly and Company, Indianapolis, USA. The work wa s supported by Royal Society Research Grant No. 11641.

References Block, M.L. and A.E. Fisher, 1970, Anticholinergic central blockade of salt aroused and deprivation induced drinking, Physiol. Behav. 5, 525. Cohen, L., G. Anderson, R.F. White, G. Griffing and J. Melby, 1984, Enalapril and hypertension, Am. J. Psychiatry 141, 1012. Deicken, R.F., 1986, Captopril treatment of depression, Biol. Psychiatry 21, 1425.

Dourish, C.T., J. Francis and J.A. Duggan, 1992, Multiple angiotensin receptors and angiotensin-5HT interactions in the control of drinking in the rat, Appetite 19, 174. Gard, P.R. and K.P. Kerr, 1990, Behavioural evidence of beta 2adrenoceptor 'down-regulation' within 21 h of a single dose of desipramine, Eur. J. Pharmacoi. 183, 2054. Germain, L. and G. Chouinard, 1988, Treatment of recurrent unipolar major depression with captopril, Biol. Psychiatry 23, 637. Germain, L. and G. Chouinard, 1989, Captoprii treatment of major depression with serial measurements of blood cortisol concentrations, Biol. Psychiatry 25, 489. Giardina, W.J. and D.M. Ebert, 1989, Positive effects of captopril in the behavioral despair swim test, Biol. Psychiatry 25, 697. Giardina, A.R. and A.E. Fisher, 1971, Effect of atropine on drinking induced by carbachol, angiotensin and isoproterenol, Physiol. Behav. 7, 653. Goldstein, J.M., L.C. Krlobloch-Litwin and J.B. Malik, 1985, Behavioural evidence for fl-adrenoceptor subsensitivity after subacute antidepressant/a2-adrenoceptor antagonist treatment, Naunyn-Schmied. Arch. Pharmacol. 329, 355. Gutman, Y., F. Bcnzakein and P. Liunch, 1971, Polydipsia induced by isoprenaline and by lithium: relation to kidneys and renin, Eur. J. Pharmacol. 16, 380. Katovich, M.J. and M.J. Fregly, 1978, Mediation of isoproterenol induced thirst in rats by beta2-adrenergic receptors, Can. J. Physiol. Pharmacol. 56, 465. Lehr, D., J. Mallow and M. Krukowski, 1967, Copious drinking and simultaneous inhibition of urine flow elicited by beta-adrenergic stimulation and contrary effect of alpha-adrenergic stimulation, J. Pharmacol. Exp. Ther. 158, 150. Martin, P., J. Massol and A.J. Puech, 1990, Captopril as an antidepressant? Effects on the learned helplessness paradigm in rats, Biol. Psychiatry 27, 968. McEvoy, G.K. (ed.), 1993, American Hospital Formulary Services: Drug Information 1993 (American Society of Hospital Pharmacists, Bethesda, MD) p. 1301. Mountford, D., 1969, Alterations in drinking following isoproterenol stimulation of the hippocampus, Physiologist 12, 309. Phillips, M.I., 1987, Functions of angiotensin in the central nervous system, Annu. Rev. Physiol. 49, 413. Przegalinski, E., L. Baran and J. Siwanowicz, 1983, The effect of chronic treatment with antidepressant drugs on salbutamol-induced hypoactivity in rats, Psychopharmacology 80, 355. Rehavi, M., Z. Jerushalemi, A. Aviv, N. Laor, E. Podliszewski, L. Karp, S. Shavit and R. Weizman, 1993, Interaction between antidepressants and phosphoinositide signal transduction system in human platelets, Biol. Psychiatry 33, 40. Varol, F.G., M. Hadjiconstantinou, F.P. Zuspan and N.H. Neff, 1988, Muscarinic receptor mediated phosphoinositide hydrolysis in rat uterus, FASEB J. 2, A613. Weinberger, M.H., W. Aoi and D.P. Henry, 1975, Direct effect of beta-adrenergic stimulation on renin release by the rat kidney slice in vitro, Circ. Res. 37, 318. Ziegler, V.E., L.T. Wylie and J.T. Biggs, 1978, Intrapatient variability of serial steady-state plasma tricyclic antidepressant concentrations, J. Pharm. Sci. 67, 554. Zubenko, G.S. and R.A. Nixon, 1984, Mood-elevating effect of captopril in depressed patients, Am. J. Psychiatry 141, 110.