The α1-blocker dapiprazole inhibits diuresis but not drinking and feeding induced by U-50,488H

0361-9230/92$5.00 + .OO Copyright0 1992Pergamon Press Ltd.

Vol. 29, pp. 401-405, 1992 Printed in the USA. All rights reserved.

Brain Research Buliefin,

The cII-Blocker Dapiprazole Inhibits Diuresis but not Drinking and Feeding Induced by U-50,488H I

PAOLO

NENCINI,*’

PACIFIC0

VALERIt

AND

GIUSEPPE

PIMPINELLAT

*Institute of Medical Pharmacology and fInstitute of Pharmacology and Pharmacognosy, University of Rome “La Sapienza”, Italy

Received

1 May 199 1; Accepted

3 1 January

1992

NENCINI. P., P. VALERI AND G. PIMPINELLA. The cu,-blocker dapiprazole inhibils diuresis but not drinking and feeding induced by U50,488H. BRAIN RES BULL 29(3/4)401-405, 1992.-To further explore the interaction between opiates and catecholamines in the control of water balance, we studied the effects of the a,-adrenoceptor antagonist dapipmzole on the modifications in drinking and diuresis produced by U-50,488H (a selective K-opiate agonist), morphine, naloxone, and amphetamine in rats. Because animals were maintained in a free-feeding paradigm and water intake is also controlled by feeding (prandial drinking), food intake was also measured. At doses administered (3-6 mg/kg, IP), dapiprazole had no effect on basal food and water intake or on diuresis. Nor did it modify changes in feeding and drinking produced by U-50,488H, morphine, naloxone, and amphetamine. It did, however, antagonize the diuretic effect of both U-50,488H and amphetamine. In addition, suppression of diuresis was obtained by combining doses of dapiprazole and morphine or naloxone that were devoid of antidiuretic effects when administered independently. A further experiment showed that diuresis produced by water load was also prevented by dapiprazole. n,-Adrenoceptors thus appear to play a role in the regulation of water balance in a condition of free access to water, inhibiting diuresis without affecting drinking. Dapiprazole Rats

U-50.488H

Morphine

Naloxone

Amphetamine

SlNCE the discovery that opiates affect water balance (5) and ingestive behavior (6). the attempt to integrate opioidergic mechanisms in the framework of the catecholaminergic control of these functions has led to much research. In line with this concept, we observed that chronic administration of an indirectly acting catecholaminergic agent, that is, amphetamine, is associated with sensitization to the ingestive response to both morphine and U-50.488H ( 17,I8). Besides dopaminergic mechanisms (19). noradrenaline may play an important role in the interaction between amphetamine and opiates because aadrenergic mechanisms appear to be involved in feeding ( I3,14) and drinking (16) responses to opiates. A role for the adrenergic system in the opioidergic regulation of water balance is also suggested by the finding that adrenal demedullation reduces plasma adrenaline levels and diuretic response to K-opioid agonists (1,x). Because most of the studies on the interaction between opioid and adrenergic mechanisms on ingestive behavior primarily focused on the role of a,-adrenoceptors (l3,14), the possibility that cu,-adrenoceptors are also involved in these effects deserves further attention. Among a,-adrenoceptor antagonists. our interest was focused on dapiprazole, a trazodone derivative, which

Diuresis

Drinking

Feeding

in an in vitro binding study using brain membranes has shown a higher selectivity for cu,-adrenoceptors than for (Ye, Dz, 5-hydroxytryptamine (5-HT, , 5-HT& and opioid receptors (22). Despite the lack of direct interaction with opioid receptors, dapiprazole has been reported to modify the pharmacological effects of opiates, as evidenced by the prevention of a naloxone-precipitated withdrawal syndrome in morphineaependent mice (2 I). We therefore examined the effects ofdapiprazole on changes in eating, drinking, and diuretic responses induced by morphine, U-50,488H, and naloxone in rats. As a positive control for catecholaminergic specificity of the actions of dapiprazole, we also evaluated its influence on eating, drinking, and diuretic responses to amphetamine. METHODS Animals Seventy-two Sprague-Dawley male rats (Morini, San Polo D’Enza, RE, Italy) were used. At the beginning of the study, they had an average weight of 280 g. Two weeks after their arrival at the laboratory colony, they were housed singly in metabolic

’ To whom requests for reprints should be addressed.

401

402

NENCINI.

VALERI

AND

PIMPINELLA

T.Sr

2.6

a0

6-

6’

FIG. 1. Two hour food intake. The influence of IP injection of water (m) or of dapiprazole 3 mg/kg (KI)or 6 mg/kg (0) on the effects produced by IP administration of morphine (MORPH) (upper left panel), U-50,488H (U50) (upper right panel), amphetamine (AMPH) (lower left panel), and naloxone (NAL) (lower right panel) on 2-h food intake. Figures that follow drug acronyms indicate doses in mg/kg. Each bar represents the mean + SEM of six data. *p < 0.05 vs. controls (i.e., the group that received two water injections), Duncan’s test.

cages (Tecniplast, Buggiate, VA, Italy) at 2 1“C with an alternating 12 L: 12 D cycle. Animals had free access to food and water. Food was made available in the form of a powder by grinding chows (Standard Diet 4RF21, Charles River). We avoid dispensing chows to minimize food spillage due to stereotyped gnawing. To allow rats to adapt to the new environment, manipulation during the first week was restricted to daily handling for weight recording. Independent Measures During the experimental procedures described below, food and water intake and urine output were measured by weighing (approx 0.1 g) food receptacles, water bottles, and urine cylinders before and 2,5, and 24 h after drug administration. This schedule of measures was chosen because in previous studies we observed significant effects of both amphetamine and opiates at 2 and 5 h (17,18). In addition, 24-h measure of food and water intake and urine output allowed to determine the reversibility of these effects. A layer of mineral oil was put into the cylinders to prevent urine evaporation. Experimental Procedure The experiment was performed according to a 4 X 3 design, in which groups treated with two different doses of dapiprazole or three different doses of the drug to be tested (i.e., morphine.

U-50,488H. amphetamine, or naloxone) were given alone or in combination, whereas the 12th group received water only. A total of 24 rats were injected daily first IP and 15 min afterward SC with water: food and water intakes were measured at 2 and 5 h. When responses were stable (no more than 10% variation between 2 consecutive days), the experiments to test the interaction between dapiprazole and each of the other four drugs began. Tests were performed on Tuesday and Friday; on the other 5 days of the week, animals were injected with water as described. On the first test day, animals were assigned to 12 groups (2 rats per group); each group received an IP injection of water or dapiprazole (3 or 6 mg/kg, respectively) 15 min before a SC injection of water or morphine (0.5, 1.O, or 2.0 mg/kg, respectively). Food and water intake, as well as urine output, were measured at 2 and 5 h as described. On subsequent test days, naloxone (2.0, 4.0, or 8.0 mg/kg, respectively), U50,488H (2.0, 4.0, or 8.0 mg/kg, respectively), or amphetamine (0.5. I .O, or 2.0 mg/kg, respectively) were substituted for morphine; each animal was maintained in the same treatment group. This block of experiments was replicated twice in such a way that each group included six rats. To minimize bias linked to administration of different drugs to the same animal. in each replication of the block of experiments the order of treatments was randomly changed.

WATER

to

BALANCE

AND

a,-ADRENOCEPTORS

1

403

I-

u602

MU0

NM2

WA6

FIG. 2. Two-hour urine output. The influence of IP injection of water (m) or of dapiprazole 3 mg/kg (N) or 6 mg/kg (0) on the effects produced by IP administration of amphetamine (AMPH) (upper left panel), U-50,488H (U50) (upper right panel), morphine (MORPH) (lower left panel), and naloxone (NAL) (lower right panel) on 2-h urine output. Figures that follow drug acronyms indicate doses in mg/kg. Each bar represents the mean + SEM of data obtained from six rats. *p < 0.05 vs. controls (i.e., the group that received two water injections); tp < 0.05 vs. the matched dapiprazoleuntreated group, Duncan’s test.

Water Load In a previous pilot experiment, we obtained evidence of an antidiuretic effect for dapiprazole. This study was further extended by assigning 24 rats to 3 different groups, receiving an IP injection of water (1 ml/kg) or dapiprazole at doses of 3 or 6 mg/kg, respectively. Twenty minutes later, all animals received by gavage 25 ml/kg distilled water. The urine output at 2 h was measured as described; urine sodium and potassium concentrations were measured by flame photometry (Flame Photometer, Mod. 602, Biotechnical). Animals were then anesthetized with ethylether and killed and the urine remaining in the bladder was measured.

Data Anal_vsis The data were represented as mean fSEM and analyzed by analysis of variance (ANOVA). Group comparisons were subsequently performed with Duncan’s multiple-range tests.

acetamide methane sulfonate) by Upjohn Co. (Kalamazoo, MI), and morphine sulfate and naloxone HCl by SIFAC (Milan, Italy). RESULTS

Food and Water Intake Because modifications of feeding and drinking were evident at 2 h, only data collected at that time are reported here. Both dapiprazole doses (3 and 6 mg/Kg) were devoid of effects on food intake (Fig. 1). As expected, both morphine and U-50,488H significantly increased food intake and these changes were not affected by dapiprazole pretreatment. Neither did dapiprazole modify the inhibition on food intake produced by administration of amphetamine or naloxone. Naloxone was the only drug that significantly affected drinking. It decreased water intake at 2 h and this effect was not modified by dapiprazole pretreatment (data not shown).

Urine Output Drugs d, l-Amphetamine

was obtained by BDH Laboratory Chemicals Division (Poole, England) and dapiprazole was kindly provided by Angelini (Rome, Italy), U-50,488H (tran- f -3,4-dichloro-~-methyl-N-[2-(i-py~olidinyl)-cyclohexyl)]-benzene-

Figure 2 shows that, as expected, U-50,488H produced a significant and dose-related increase in urine output at 2 h. This diuretic effect was still present at 5 h (data not shown). Dapiprazole did not significantly affect basal diuresis, but did antagonize U-50,488H-induced diuresis. The dose of 3 mg/kg significantly

404

NENCINI.

TABLE

VALERI

AND

PIMPINELLA

I

EFFECTS OF DAP (3 OR 6 mg/kg IP) ON URINE Nat AND K’ CONCENTRATIONS 2 h AFTER WITH DISTILLED WATER

VOLUME AND ON HYDRATATION

DAP (mg/kg IP)

Urine voided (ml) Urine remaining in bladder Na’ (mEq/l) K’ (mEq/l)

Il.3 (ml)

*

0.7

0.33 f 0. I 9.5 + 3.4 24.0 -t 4.3

3.7

f

1.1*

0.1’ t 0.03* 2.5 35.4

f 0.6? + 5.7

3.8

t

1.3+

0.0I t o.o* 4.1 33.3

+ 0.9t i 6.8

Results are expressed as mean + SEM (n = 8 per group) * /, < 0.01 and t/l < 0.05 vs. DAP 0. Dunnett’s test.

reduced the U-50.488H diuretic effect and apparently no further reduction was obtained with the higher dose (6 mg/kg). The lower dose of dapiprazole also fully suppressed the increase in urine output produced by amphetamine. Finally. a significant reduction in urine output in comparison to controls was observed at 2 h. when dapiprazole was given in combination with morphine or naloxone-two drugs that did not affect diuresis when given alone. Dapiprazole was also able to reduce significantly diuresis produced by water load (Table 1). This effect was not due to urine retention because the residual volume of urine into the bladder was also reduced by dapiprazole. Reduction of diuresis was associated with a decrease in Na+ excretion and a slight increase in K+ excretion. DISCUSSION

The present study shows that dapiprazole affects water balance, inhibiting diuresis stimulated by water load and by unrelated pharmacological agents, such as U-50,488H and amphetamine. In addition, an almost complete suppression of diuresis was obtained by combining doses of dapiprazole and morphine or naloxone that were devoid of antidiuretic effects when administered independently. Although preliminary, these results allow speculation on the antidiuretic effects ofdapiprazole. The reduction in urine output, already observed in conditions of hydratation ( 15). was certainly not due to a retentive action of dapiprazole because the residual volume of urine into the bladder was also reduced by dapiprazole in the water load experiment. As the complete inhibition produced by this drug on the increased urine output caused by a sympathomimetic drug. such as amphetamine. indirectly suggests, presumably dapiprarole acts on water and sodium excretion through an antiadrenergic mechanism. This may consist in a block of cu,-adrenoceptors because prazosin, another selective oc,-antagonist, also has antidiuretic effects in unanesthetized dogs and mice (4). Because dapiprazole does not bind to opiate receptors (22). the block of cu,-adrenoceptors is also likely to be involved in its inhibition of U-50,488H-mediated diuresis. A large body of ev-

idence suggests that the diuretic effect of U-50,488H depends upon the inhibition of vasopressin release (IO,1 I). However. it is unlikely that dapiprazole’s antidiuretic action is due to the release of vasopressin because corynanthine. a selective al-antagonist (20). does not interfere with vasopressin release (3). Recently. diuresis produced by K-opiate agonists was seen to be suppressed by demedullation of adrenal glands, a condition in which circulating epinephrine was reduced by as much as 92% (I .2). In contrast, kidney denervation does not alter K-opiatemediated diuresis (9). As a whole, these findings suggest that, whether or not sympathetic innervation of the kidneys is functionally intact. the diuretic action of K-Opiate agonists requires the presence of circulating catecholamines. Our results appear to be consistent with this finding and suggest that a,-receptors are responsible for the facilitation of U-50.488H-mediated diuresis produced by adrenal catecholamines. Potentiation by dapiprazole of the antidiuretic action of either morphine or naloxone may also be the result of the interaction between cu,-adrenergic and K-opioid mechanisms, Although the inhibition of diuresis produced by morphine is considered mediated by the activation of p-receptors (7.8). this drug is also active at K-IXceptors. Dapiprazole may therefore shift the balance of morphine actions toward antidiuresis. The influence that dapiprazole exerted on the effects produced bv opiates on urine output did not extend to opioidergic control of ingestive behavior. Neither did it antagonize the anorectic effects of amphetamine. Because norepinephrine has been claimed to play a role in both the prophagic effects of p-opiate agonists ( 13) and the anorectic effects of amphetamine ( 12). our results suggest that this noradrenergic control is not mediated by a,-adrenoceptors. In conclusion, the use of a selective a,-antagonist such as dapiprazole elucidates further the opioidergic control of water balance and ingestive behavior, providing evidence that oc,-adrenoceptors are involved in the diuretic but not ingestive effects of LJ-50.488H. This finding may be of clinical interest because diuresis is a troublesome side effect that prevents the use of Kopiate analgesics.

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