Angiotensin II antagonists block ethanol effects on the aerial righting reflex

Alcohol, Vol. 13, No. 3,287-289, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0741-8329/96 $15.00 + .00

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Angiotensin II Antagonists Block Ethanol Effects on the Aerial Righting Reflex 1 HARRY

A. TRACY,

JR., MATTHEW

J. W A Y N E R 2 A N D

DEBORAH

L. A R M S T R O N G

Division o f Life Sciences, The University o f Texas at San Antonio, San Antonio, T X 78249-0662 R e c e i v e d 17 J u l y 1995; A c c e p t e d 3 N o v e m b e r 1995 TRACY, JR., H., M. J. WAYNER AND D. L. ARMSTRONG. A ngiotensin llantagonists block ethanol effects on the aerial righting reflex. ALCOHOL 13(3) 287-289, 1996. - T h e purpose of the present study was to determine the effects of an angiotensin II (AID AT~ antagonist, losartan 10, 15, and 20 mg/kg IP, and the AII AT 2 antagonist, PD 123319, 20 mg/kg IP on ethanol (EtOH) intoxication as measured by the aerial righting reflex in male rats. EtOH (25070), 2.0 g/kg, was administered by stomach tube under mild metaphane anesthesia and the aerial righting reflex was determined at 30-min intervals for 3.5 h. The AII antagonists were administered IP 2 h before the EtOH. There were six groups of 10 rats each: EtOH alone, 10, 15, or 20 mg/kg losartan plus ethanol, 20 mg/kg losartan plus 20 mg/kg PD 123319 plus EtOH, and 20 mg/kg Iosartan alone. Data were analyzed by a two-way ANOVA with repeated measures on one factor, time. Results show a clear intoxicating effect of ethanol on the aerial righting reflex that was blocked significantly by losartan in a dose-dependent way. Losartan alone had no observable effect. The administration of both antagonists, losartan and PD 123319 injected IP in two different sites, completely blocked the EtOH effect on the aerial righting reflex. The involvement of AII in the mediation of EtOH intoxication effects on the aerial righting reflex supports results of our previous studies on th e effects of EtOH on open field behavior, AII impairment of the retention of an inhibitory shock avoidance response, and A I | inhibition of hippocampal granule cell long-term potentiation, all of which can be blocked by losartan. Ethanol intoxication

PD 123319

Angiotensin II

AT~ receptor

H I P P O C A M P A L dentate granule cell long-term potentiation (LTP) induction is inhibited by ethanol in relatively low doses. The inhibition is mediated by angiotensin II (AII) and the AT~ receptor because it can be blocked by losartan, a specific AT~ antagonist (6). L T P is a type o f frequency-dependent synaptic plasticity involved in learning and the m e m o r y process (5). A I I administered directly to the dentate gyrus of the hippocampus impairs the 24-h retention o f an inhibitory shock avoidance response and the impairment can be blocked by losartan (3). We have previously shown that the intoxicating effects of low dose ethanol directly observable in the rat in an open field can be effectively blocked by losartan (7). The purpose of the present study was to examine the intoxicating effects of a low dose o f ethanol on the aerial righting reflex in male rats and to determine if the effects can be blocked by losartan and P D 123319, an A T 2 antagonist. It has been previously reported that ethanol impairs the aerial righting reflex (4). Also the

Aerial righting reflex

Losartan

aerial righting reflex involves both learning and m o t o r coordination components (1,2). Present results support the hypothesis that A I I is involved in both cognitive and m o t o r functions and that both mechanisms are impaired by ethanol, and the impairment can be blocked by pretreatment with combined losartan and P D 123319. METHOD

Animals Male Harlan Sprague-Dawley rats, 235-335 g in weight, were used. Animals were housed in separate cages in the animal facility and were maintained on a 12-h light/dark cycle, with lights on between 0600 and 1800 h, and at a constant 21 ± 1 °C ambient temperature. Cages contained chipped corn cob as bedding and all animals were given food and water ad lib. Animals were food deprived for 24 h prior to testing.

t These results were presented at the Fourth International Behavioral Neuroscience Society Conference held in Santiago de Compostela, Spain, May 18-21, 1995. 2 Requests for reprints should be addressed to Matthew J. Wayner, Division of Life Sciences, The University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, TX 78249-0662. 287

288

TRACY, JR., WAYNER AND ARMSTRONG RESULTS AND DISCUSSION

Drug Administration Rats were mildly sedated with metaphane prior to drug administration. Ethanol 25°70 v/v was administered by gavage (PO) at a dose of 2 g/kg body weight. Losartan was administered IP at a doses of 10, 15, and 20 mg/kg body weight and PD 123319, 20 mg/kg IP, 2 h prior to administration of ethanol. The losartan-only group was injected 2.5 h prior to testing. Ethanol, losartan, and PD 123319 were prepared the afternoon before a test day and were stored in a refrigerator in light-tight sealed containers.

Experimental Design Rats were divided into groups of 10 animals each. One group received losartan only, 20 mg/kg IP, and served as a control group. A second group received EtOH only, 2 g/kg PO body weight. A third group received EtOH, 2 g/kg PO, following pretreatment with losartan, 10 mg/kg IP. The fourth and fifth groups received EtOH, 2 g/kg PO, following pretreatment with losartan 15 and 20 mg/kg IP, respectively. A sixth group was administered losartan, 20 mg/kg IP, and PD 123319, 20 mg/kg IP, in two separate injections 2 h prior to administration of ethanol, at 2 g/kg PO.

Aerial Righting Reflex The aerial righting reflex test was conducted in a small room at the animal facility. The room contained a freezer, a fume hood, a large cabinet, and a laboratory bench with numerous items, all of which served as visual cues for the rats during the test. A meter stick was suspended above a 2-in. mat of foam rubber utilized to cushion the animal's fall. The meter stick was used to measure the minimum height above the foam rubber mat at which the rat could successfully right itself. Animals were tested at 30-min intervals, starting at 30 min following administration of EtOH, or at 2.5 h following administration of losartan alone, and PD 123319 and losartan 2 h prior to EtOH. The rat was raised in an inverted position to a height of approximately 45 cm above the foam rubber mat and always facing in the same direction to maintain the same spatial orientation of the visual cues. The rat was suspended by placing the right hand just beneath the front legs and the left hand above the hips. The hands were extended laterally with slight pressure to keep the animal in a horizontal position. When the animal was at 45 cm and was not moving, it was dropped by quickly pulling the two hands outward, so that no rotational movement was applied to the animal as it fell. If the animal could successfully right itself, determined by landing on all four paws, then the procedure was repeated at a lower height. The height was decreased until the animal could no longer successfully right itself. The last previous successful righting height was recorded as the height of the aerial righting reflex. This procedure was repeated three times for each animal at each time tested and these three measurements were averaged to obtain the final height above ground for that test. Measures of the aerial righting reflex were recorded for a total of 210 min following EtOH administration.

Data are summarized in Fig. 1 where the means and SEMs for each group are presented for each 30 min when the aerial righting reflex was measured following the administration of the ethanol. An ANOVA revealed a significant between subjects effect, F(5, 54) = 53.5, p < 0.0001; a significant within subjects effect, F(6, 324) = 55.8, p < 0.0001; and a significant interaction, F(30, 324) = 12.7, p < 0.0001. The differences between the ethanol plus pretreatment with losartan group, 20 mg/kg, and the ethanol plus pretreatment with losartan, 20 mg/kg, and PD 123319 group, 20 mg/kg, were significant, F(1, 18) = 5.21, p < 0.05. Differences between the ethanol plus pretreatment with losartan and PD 123319 group and the losartan-only group were not significant, F(I, 18) = 2.92. A Newman-Keuls analysis of the differences between the means at each 30-rain interval substantiates the observation that obvious differences are significant. These results demonstrate clearly that ethanol has an intoxicating effect on the aerial righting reflex in male rats and that it can be effectively blocked by pretreatment with losartan, in a dose-dependent way, and completely blocked by pretreatment with losartan and PD 123319. Once again, these data support our previous results that demonstrated a newly discovered inhibitory role for AII not only in dentate granule cell LTP but also in the mediation of an alcohol-related acutely impaired physiological function. Because the aerial righting reflex involves both learning and motor coordination components, it seems reasonable that both the ATI and AT 2 AII receptors would be involved. Experiments in progress are examining dose effects for different treatments with losartan and PD 123319 alone. ACKNOWLEDGEMENTS This research was supported by Grant #4038 from The Council for Tobacco Research-USA, Inc. We appreciate the help of Marianne Van Wagner in typing the manuscript and Barbara Smith who did the data entry and statistical analysis.

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Statistical A nalysis Data were analyzed by a two-factor ANOVA with repeated measures on one factor, time. Means and SEMs were calculated for each group for every 30 min throughout the experiment. Post hoc comparisons between the group means were made by means of the Newman-Keuls test.

FIG. 1. Mean aerial righting reflex in cm and SEMs for six groups, presented as a function of time in minutes following administration of 25% ethanol alone, 2.0 g/kg PO, and losartan pretreatment, 10, 15, and 20 mg/kg IP. Two other groups were pretreated before the ethanol was administered with 20 mg/kg IP losartan and PD 123319, 20 mg/kg IP and losartan only, respectively.N = 10 in each group.

ETHANOL

AND AERIAL RIGHTING

REFLEX

289

REFERENCES 1. Brindley, G. S. How does an animal that is dropped in a nonupright posture know the angle through which it must turn in the air so that its feet point to the ground? J. Physiol. 180:20-21P; 1965. 2. Brindley, G. S. Ideal and real experiments to test the memory hypothesis of righting in free fall. J. Physiol. 184:72-73P; 1966. 3. Lee, E. H. Y., Ma, Y. L.; Wayner, M. J.; Armstrong, D. L. Impaired retention by angiotensin II mediated by the ATt receptor. Peptides 16:1069-1071; 1995. 4. Mattucci-Schiavone, L.; Ferko, A. P. An inhalation procedure to produce tolerance to the behavioral effects of ethanol. Physiol. Behav. 36:643-646; 1986.

5. Rison, R. A.; Stanton, P. K. Long-term potentiation and Nmethyl-D-aspartate receptors: Foundations of memory and neurologic disease? Neurosci. Biobehav. Rev. 19(4):533-552, 1995. 6. Wayner, M. J.; Armstrong, D. L.; Polan-Curtain, J. L.; Denny, J. B. Role of angiotensin II and AT~ receptors in hippocampal LTP. Pharmacol. Biochem. Behav. 45:455-464; 1993. 7. Wayner, M. J.; Polan-Curtain, J. L.; Chiu, S.-C.; Armstrong, D. L. Losartan reduces ethanol intoxication in the rat. Alcohol 11:343-346; 1994.