Evidence that opiate receptors mediate suppression of hypertonic saline-induced drinking in the mouse by narcotic antagonists

Life Sciences, Vol. 26, pp. 1543-1550 Printed in the U.S.A.

Pergamon Press

EVIDENCE THAT OPIATE RECEPTORS MEDIATE SUPPRESSION OF HYPERTONIC SALINE-INDUCED DRINKING IN THE MOUSE BY NARCOTIC ANTAGONISTS David R. Brown and Stephen G. Holtzman Department of Pharmacology Emory University School of Medicine Atlanta, Georgia 30322 (Received in final form March 6, 1980) S~mmry The effects of naloxone, its dextro-stereolsomer, and five other narcotic antagonists were determined on water intake induced by intracellular dehydration in the mouse. The intraperitoneal administration of a 2M sodium chloride solution served as the model for intracellular dehydration, l_-Naloxone (0.01-I0 mg/kg) reduced drinking in a dose-dependent fashion with an EDs0 of 0.55 mg/kg. In contrast, d_-naloxone failed to suppress water consumption at doses up to i0 mg/kg. The other narcotic antagonists tested --- naltrexone, diprenorphine, levallorphan, oxilorphan, and nalorphine --- also produced dosedependent decreases in water consumption. The order of potency of these narcotic antagonists in suppressing water intake was highly correlated with their orders of potency in other procedures involving the opiate receptor. The stereoselectivlty and order of potency suggest that the suppressant effects of the narcotic antagonists on drinking induced by hypertonic saline administration in the mouse are mediated through an opiate receptor-dependent mechanism. The specific opiate antagonist, naloxone, has been considered to possess little intrinsic activity in m o s t procedures (1,2). However, the administra~ tion of naloxone has been associated with a variety of physiological and behavioral effects, such as alterations in the release of pituitary hormones (3-5), increased sexual behavior (6-8), and heightened sensitivity to painful stimuli (9-11). These effects appear to be a manifestation of the blockade of opiate receptors by naloxone, and the resultant disruption of the activity of endogenous opiate (endorphin) systems. Thus, naloxone has become a useful pharmacological tool in efforts to elucidate the physiological roles of the endorphins (12,13). Recent evidence suggests that endorphin pathways mediate some aspects of ingestive behavior. Beta-endorphin, administered intrahypothalamically (14) or intracerebroventricularly (15), stimulates food intake in satiated or mildly food-deprlved rats. Genetlcally-obese rats and mice display exceptionally high levels of 8-endorphin in the pituitary and plasma, and low doses of naloxone partially a t t e n u a t e the excessive food consumption characteristic of both rodent strains (16). Moreover, naloxone has been shown to decrease food consumption in normal, food-deprived rats (17-23) and mice (17,18). 0024-3205/80/181543-08502.00/0 Copyright (c) 1980 Pergamon Press Ltd

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Water intake in rats and mice is also reduced by low doses of naloxone, and appears to be more sensitive to the suppressive effects of naloxone than is food intake (17). In the rat, naloxone produces a dose-related decrease of water consumption induced by periods of water deprivation (17,18,20-22,24-28) or by the administration of hypertonic saline, an intracellular thirst stimulus (18,24,26). Water intake in the mouse following periods of water deprivation is also attenuated by naloxone (17,18), but the suppressant effects of naloxone on post-deprivational water intake in the mouse appear to be of a smaller magnltude than those observed in the rat. However, naloxone has been found to suppress water consumption following hypertonic saline administration in the mouse, and this effect seems to be more pronounced in the mouse compared to the rat

(18).

This study was undertaken to expand upon preliminary findings (18) of a suppressant effect of naloxone on water consumption induced by hypertonlc saline injection in mice. We now report that the suppression of hypertonic salineelicited drinking in the mouse by naloxone is stereoselective. Furthermore, other narcotic antagonists also suppress hypertonic sallne-lnduced drinking in mice, and in so doing, exhibit an order of potency that corresponds with their relative potencies in other procedures involving the opiate receptor. Methods Subjects. The subjects were male CF-I mice (Charles River Breeding Laboratories, Wilmington, Mass.) weighing between 20 - 40 g at the start of the experiment. All mice were housed in groups of 3 - 5 per cage in a large colony room with an average temperature of 72 ° F and under continuous illumination between 0700 and 1900 hrs. Water and food (Rodent Laboratory Chow No. 5001, Ralston Purina Co., St. Louis, Mo.) were freely available in home cages. Water Intake. Subjects were weighed and injected s.c. with drug or isotonic saline 30 mln prior to the testing of water intake. Fifteen minutes before the start of the water intake tests, each mouse received 0.25 ml of a 2 molar sodium chloride solution, i.p., and was placed into an individual plastic cage. Water intake was measured for 30 min from modified i0 ml Mohr pipets described previously (17). Food was absent during the testing period. At the end of the session, mice were returned to home cages where they again had free access to food and water. Water intake was determined to the nearest 0.i ml; mice that drank less than i0 ml of water per kg of body weight after an injection of isotonic saline were eliminated from the study. This criterion led to the exclusion of approximately 40% of the mice initially available for this study. All experiments were conducted between 0900 - 1500 hrs, 3 times weekly. Each subJect underwent 2 - 3 trial sessions with isotonic saline prior to the start of actual drug testing. Dr u6s. The following drugs were used in this investigation: diprenorphine hydrochloride, naltrexone hydrochloride, and l__-naloxone hydrochloride (supplied by Dr. Robert Willette, National Institute on Drug Abuse), d_-naloxone hydrochloride (a gift from Dr. Arthur Jacobson, National Institute of Arthritis, Metabolism, and Digestive Diseases), nalorphine hydrochlorlde (donated by Merck Chemical Division, Rahway, N.J.), levallorphan tartrate (kindly provided by Hoffmann-LaRoche, Inc., Nutley, N.J.), and oxilorphan tartrate ( a gift from Bristol Laboratories, Syracuse, N.Y.). Oxilorphan tartrate was dissolved in distilled water, and the remaining drugs were dissolved in 0.9% saline. In each series of experiments, various doses of drug (0.01-I0 mg/kg) and isotonic saline were administered subcutaneously in random order in an injection volume of 5.0 ml per kg of body weight. All drug doses are expressed in terms of the free base. Sodium chloride (Fisher Scientific Co., Reagent grade) was dlssolv-

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ed in distilled water (w/w) to yield a 2.0 molar solution. The hypertonic saline solution was administered intraperitoneally in a volume of 0.25 ml, corresponding to a 5mM saline load. Data Analysis. Water intake data were converted to ml of water consumed per kg of body weight. In order to facilitate comparisons among the different experimental groups, the data were further normalized to a percentage of the saline control value for each animal in each series of experiments. The transformed data were evaluated by an analysis of variance for randomized block designs, and comparisons of treatment means to those of saline controls were made by two-tailed Dunnett (29) and paired ~-tests. The dose of each drug required to produce a 50% suppression of water intake (EDb0) was calculated from the treatment means by probit analysis and least-squares regression procedures (30). The rank order of potency of narcotic antagonists in suppressing water intake compared to potencies of the antagonists in other procedures was evaluated by the Spearman rank-order correlation (31). A E value of 0.05 was selected as the upper limit for statistical significance. Results The average water intake was similar among all experimental groups in control experiments of each drug series: administration of hypertonic saline (i.p.) following 15 min pretreatment with isotonic saline (s.c.) (F-1.37 for 7,97 df, p > 0.05). The mean volumes of water consmned by each experimental group under saline control conditions are presented in the legends of Figs. 1 and 2. The administration of l_.-naloxone (0.01-i0 mg/kg) reduced water intake stimulated by hypertonic saline treatment in a dose-related fashion (Fig. i). Water intake was reduced to only 60% of the saline baseline value (p < 0.01) by as little as 0.i mg/kg of naloxone, and 10 mg/kg reduced water intake to 20X of the saline control (Fig. i). In contrast, d_-naloxone was ineffective in decreasing water consumption even at i0 mg/kg, the highest dose tested (Fig. 1). The other narcotic antagonists examined in this study---naltrexone, diprenorphine, levallorphan, oxilorphan, and nalorphine---also decreased water intake after hypertonic saline administration in a dose-dependent manner (Fig. 2). Naltrexone was the most potent compound tested. A dose of only 0.01 mg/kg reduced water intake to 75% of saline baseline (p < 0.05), and i0 mg/kg further reduced water intake to 19% of the saline control value. Levallorphan, although less potent than either naltrexone or diprenorphine, appeared to exert the greatest suppressant effect on water consumption among the drugs tested. Levallorphan produced a 96% reduction in water intake at a dose of 10 mg/kg. Nalorphine, the least potent drug tested, exhibited a biphasic effect on hypertonic saline-lnduced water intake. In doses of 0.01-1.0 mg/kg, nalorphine tended to increase water consumption, an effect that reached a maximum of 134% of saline control at 0.1 mg/kg. However, there was considerable interanimal variability at these low doses of nalorphine which is reflected by relatively large standard errors. At higher doses---3.0-30 mg/kg---nalorphine reduced water consumption in a dose-related manner. Ten and 30 mg/kg of nalorphine significantly (p < 0.01) decreased water intake to 46 and 34% of saline baseline. The dose of each antagonist that decreased hypertonic sallne-induced drinking to 50% of control (i.e. ED50) is presented in Table i. All drugs, at the doses employed, did not appear to affect the general health or overt behavior of any experimental subject. Discussion The results of this study indicate that naloxone and other narcotic

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Opiate Receptors and Drinking in Mice

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140 120 0 0

-÷

I00 80 60

Z

m

40 LIJ I-20 0

l-Nal A d-Naioxone

I

I

I

Sal

0.01

0.1

I

1.0

I

I0

DRUG DOSE (mg/kg) FIG.____~I Stereoselectivity of the suppression of hypertonic salineinduced drinking in mice by naloxone. Each point represents the mean and S.E. of 9 and 13 observations in groups receiving d_- and l_-naloxone, respectively. The absolute volumes of water consumed in tests with isotonic saline (points above Sal) are 30.2 ± 3.1 and 31.6 ± 3.3 ml/kg of body weight for d-- and l._-naloxone-treated groups, respectively.

antagonists attenuate drinking in the mouse induced by the administration of hyper tonic saline, an intracellular thirst stimulus. This effect of narcotic antagonists appears to be mediated through opiate receptors. First, the suppressant effects of naloxone on drinking were stereoselective, l-Naloxone decreased water intake after hypertonic saline with an ED50 of 0.55 mg/~g (Table i), whereas d-naloxone, its inactive enantiomer (35,36), had no significant effect on water Tntake at doses of up to i0 mg/kg. Second, the other narcotic antagonists evaluated in this study exhibited an order of potency in suppressing water intake that is highly correlated with

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Opiate Receptors and Drinking in Mice

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160

140

•,--.

120

so so z ,=, 4o 2o o

I

Sal

|

0.01

I

0.1

i,

I

1.0

I

I,

I0

I

!

I00

DRUG DOSE(mg/kg). FIG. 2 Dose-related suppression of hypertonic sallne-induced water intake in mice by narcotic antagonists. Each point represents the mean and S.E. of 10-16 observations; the actual number of subjects used in each experimental group are given in Table i. The absolute volumes of water consumed in tests with isotonic saline (points above Sal) are: 34.2 ± 5.4, 25.6 ± 2.7, 21.6 ± 2.8, 30.2 ± 2.7, and 31.5 ± 2.3 ml/kg of body weight for groups treated with naltrexone, diprenorphine, levallorphan, oxilorphan, and nalorphine, respectively.

the potencies of these compounds in displacing 3B-naloxone from guinea pig ileal and rat brain binding sites, and for the precipitation of morphine withdrawal jumping in dependent mice (Table I). The high correlation between narcotic antagonist potencies in the present drinking paradigm, and potencies in other procedures involving opiate receptors lends strong support to the hypothesis that the suppressant effects of the narcotic antagonists on water consumption are similarly mediated through an interaction with opiate receptors. Although a number of mice initially assigned to this study were eliminated due to low control values, the fact that 60~ of mice tested did drink reliably

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TABLE i COMPARISONS OF NARCOTIC ANTAGONIST POTENCIES IN VARIOUS PROCEDURES a

ED50 for

Guinea Pig

Ra~ Brain

Precipitation of MorphineWithdrawal

Ileum c

Homogenate d

Jumping e

OPIATE RECEPTOR AFFINITY

DRUG

(n) b

Naltrexone (11) Diprenorphine

(16) Levallorphan

(i0) Naloxone (13) Oxilorphan g

Wa~er Intake Suppression (mg/kg)

Potency

0.13 (0.06-0.28) f

i

2

1.5

1

0.21

2

i

1.5

2

3

3

3

4

4

4

4

3

5

5

5

0.90

0,98

0.94

(0.06-0.74) 0.45

(0.29-0.69) 0.55 (0.20-1.48) 0.83

(I0)

(0.22-3.09)

Nalorphlne (11-15)

11.22 (8.12-15.49)

5

6

Spearman c o r r e l a t i o n c o e f f i c i e n t h, rs -

apotencies are expressed in rank o r d e r . bNumber of s u b j e c t s . CPotency in d i s p l a c i n g 3H-naloxone from guinea pig ileum (32). dpotency in d i s p l a c i n g 3H-naloxone from r a t brain homogenates in presence of 100mM sodium (32). epotency in producing morphlne-withdrawal

Jumping in the mouse (33,34).

fStandard error of the estimate on regression line. gData were unavailable for opiate receptor affinity of oxilorphan. hcorrelations were made between rank potency for suppressing hypertonic salineinduced drinking and each of the other three parameters. All correlations were significant at p < 0.05.

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and to similar degrees supports the contention that a representative sample was employed in the present study. Furthermore, it does not appear that suppression of drinking behavior by the narcotic antagonists is the result of increasing pain sensitivity or the generation of non-speciflc behaviors following hypertonic saline injections: no differences in the degree of discomfort or in other overt behaviors could be observed in mice under control or experimental conditions. The suppressant effects of naloxone on drinking induced by hypertonic saline administration have now been established in both the rat (24) and the mouse. These effects appear to be greater in magnitude than the suppressant effects of naloxone on water consumption induced by periods of water deprivation in both species (17, 20,21,23-25,27,28). Water deprivation produces a general depletion of both intraand extracellular fluid compartments (37), and naloxone may exert its suppressant activity through a thirst pathway responsive to imbalances in one or both specific fluid compartments. The effects of naloxone on drinking elicited by mediators of extracellular thirst have yet to be determined. Nevertheless, it is clear that intracellular dehydration in both rats and mice induces drinking which is highly susceptible to the suppressant actions of naloxone. The involvement of endorphln pathways in the regulation of water intake remains inferential at this point. However, the present results argue strongly for the participation of an opiate receptor-dependent mechanism in the suppressant effects of the narcotic antagonists on drinking behavior.

Ackn0wledgements 1.

This i n v e s t i g a t i o n was s u p p o r t e d i n p a r t by USPHS Grants TO1 GMO0179, DA00541, and Research Scientist Development Award K02 DA00008 to S.G.H. References

1.

2. 3. 4. 5. 6. 7. 8. 9. i0. 11. 12. 13. 14. 15. 16.

H. BLUMBERG and H. B. DAYTON, in Narcotic Antagonists (eds. M.C. Braude, L.S. Harris, E.L. May, J.P. Smith, a n d J.E. Villarreal), pp. 33-43, Raven Press, New York (1974). D. R. JASINSKI, W. R. MARTIN, and C. A. HAERTZEN, J. Pharmacol. Exp. Ther., 157, 420-426 (1967). M. S. BLANK, A. E. PANERAI, and H. G. FRIESEN, Science, 203, 1129-1131 (1979). T. MURAKI, H. NAKADATE, Y. TOKUNAGA, R. KATO, and T. MAKINO, Neuroendocrinology, 28, 241-247 (1979). J. F. BRUNI, D. VAN VUGT, S. MARSHALL, and J. MEITES, Life Scl., 21, 461466 (1977). B. M. MYERS and M. J. BAUM, Pharmacol. Biochem. Behav., i_~0, 615-618 (1979). J. H. MENDELSON, J. ELLINGBOE, J. C. KEUHNLE, and N. K. MELLO, PsychqneuroendocrinoloLv, !, 231-236 (1979). G. L. GESSA, E. PAGLIETTI, and B. PELLEGRINI QUAKANTOTTI, Science, 20_~4, 203205 (1979). J. J. CARMODY, P. R. CARROLL, and D. MORGANS, Life Sci___...__.~2~4, ., 1149-1152 (1979). J. J. JACOB, E. C. TREMBLAY, and M.-C. COLOMBEL, Psycho~harmacologica, 37, 217-233 (1974). P. GREVERT and A. GOLDSTEIN, PsychopharmacoloKy, 53, 111-113 (1974). A. BEAUMONT and J. HUGHES, Ann. Rev. Pharmacol. Toxlcol., 19, 245-267 (1979). A. GOLDSTEIN, Science, 19__~3,1081-i086 (1976>. L. GRANDISON and A. GUIDOTTI, Ne_uropharmacology, 16, 533-536 (1977). N. J. KENNEY, L. D. McKAY, S. C. WOODS, and R. H. WILLIAMS, Soc. Neuroscl. Abstr., ~, 176, (1978). D. L. MARGULES, B. MOISSET, M. J. LEWIS, H. SHIBD'YA, and C. B. PERT, Science, 202, 988-991 (1978).

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17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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D. R. BROWN and S. G. HOLTZMAN, Pharmacol. Biochem. Behav., ii, in press. D. R. BROWN and S. G. HOLTZMAN, Fedn. Proc., 38, 856 (1979). B. BRANDS, J. A. THORNHILL, M. HIRST, and C. W. GOWDEY, Life Sci., 24, 17731778 (1979). H. FRENK and G. H. ROGERS, Behav. Neural B i o l . , 26, 23-40 (1979). S. G. HOLTZMAN, L i f e S c i . , 2~4, 219-226 (1979). G. H. ROGERS~ H. FRENK, A. N. TAYLOR, and J. C. LIEBESKIND, Proc. West. Pharmacol. Sot., 21, 457-460 (1978). S. G. HOLTZMAN, J. Pharmacol. Exp. Ther., 189, 51-60 (1974). D. R. BROWN and S. G. HOLTZMAN, Life Sci______.___~in ., press. J . M . STAPLETON, N. L. OSTROWSKI, V. J. MERRIMAN, M. D. LIND, and L. D. REID, Bull. Psychonom. Soc., 13, 237-239 (1979). J . M . STAPLETON, M. D. LIND, J. R. QUINAN, T. L. FOLEY, M. F. WU, and L. D. REID, Soc. Neurosci. Abstr., ~, 663 (1979). R. P. MAICKEL, M. C. BRAUDE, and J. E. ZABIK, Neuropharmacology, 16, 863-866 (1977). S. G. HOLTZMAN, Life Sci., 16, 1465-1470 (1975). C. W. DUNNETT, J. Am. Star. Assoc., 50, 1096-1121 (1955). J. T. LITCHFIELD, JR. and F. WILCOXON, J. Pharmacol. Ex~. Ther., 96, 99-113 (1949). S. SIEGEL, NonparametrlcSta=istics for the Behavloral ScienGe~, pp. 202-213, McGraw-Hill, New York (1956). I. CREESE and S. H. SNYDER, J. Pharmacol. Exp. Ther., 19__~4,205-219 (1975). A. COWAN, J. Pharm. Pharmacol., 28, 177-182 (1976). A. W. PIRCIO and J. A. GYLYS, J. Pharmacol. Exp~ Ther. , 193, 23-34 (1975). I. IIJIMA, J.-I. MINAMIKAWA, A. E. JACOBSON, A. BROSSI, K. C. RICE, and W. A. KLEE, J. Med. Chem., 21, 398-400 (1978). R. J. GAYTON, L. A. LAMBERT, and P. B. BRADLEY, Neuropharmacolo~y, 17, 549551 (1978). J. T. FITZSIMONS, Physiol. Rev., 52, 468-561 (1972).