Nrurofi~urt~~o~oloy~, Vol. lg. pp. X7 I to X76 Pergamon Press Ltd 1979. Prmted in Great Britain
EFFECTS OF ETHANOL, ARECOLINE, ATROPINE AND NICOTINE, ALONE AND IN VARIOUS COMBINATIONS, ON RAT CEREBELLAR ‘CYCLIC GUANOSINE 3’.5’-MONOPHOSPHATE R. A. DODSON* and W. E. JOHN~N Colleges
of Pharmacy. Washington
Idaho State University, Pocatello, ID 83209, U.S.A. and State University, Pullman, WA 99163, U.S.A. (Accepted
9 May 1979)
Summary-Rat cerebellar cyclic guanosine 3’S’-monophosphate (cGMP) concentrations were determined by radioimmune methods after sacrifice with focused microwave fixation in animals pretreated with ethanol, arecoline, atropine and nicotine alone and in various combinations. Although arecoline and nicotine treatments resulted in large cerebellar increases in cGMP, they failed to antagonize ethanol’s depressive effects. Atropine treatment did not augment ethanol-induced decreases in cGMP
content. These data suggest that ethanol’s depressant actions on cerebellar cGMP are independent of cholinergic mechanisms.
Volicer and Hurter (1977) recently reported that acute and chronic ethyl alcohol administration caused a dose-dependent decrease in cyclic adenosine 3’,5’-monophosphate (CAMP) and cyclic guanosine 3’,5’-monophosphate (cGMP) in the cerebellum, cerebral cortex and the pons-medulla in the rat. These authors postulated that the levels of these cyclic nucleotides were decreased as the result of the effects of ethanol on the release of norepinephrine and acetylcholine from their respective central neurons. More recently, Hunt, Redos, Dalton and Catravas (1977) confirmed the observations of Volicer and Hurter. In addition, these investigators conducted experiments concerned with the effects of ethanol on guanylate cyclase and cGMP phosphodiesterase. Under optimal enzymatic conditions, ethanol had no direct effect on either enzyme in vitro. Currently, many investigators believe that cGMP is associated with central muscarinic receptors (Lee, Kuo and Greengard, 1972; Dinnendahl and Stock, 1975; Kebabian, Steiner and Greengard, 1975; Hanley and Iverson, 1978). Arecoline is a cholinomimetic derived from the betel nut (Areca catechh) which readily penetrates the blood-brain barrier and affects central muscarinic receptors. Dinnendahl and Stock (1975) were able to demonstrate an in uiuo dosedependent increase in cGMP concentrations in mouse cerebellum in response to arecoline. The elevations were 3-fold at doses of 1&20mg/kg (s.c.), of this cholinomimetic. The effect revealed a maximum between 3 and 5 min after the injection. Nicotine, a potent cholinomimetic which is also * To whom reprint Key cGMP.
words:
requests should ethanol, arecoline,
be addressed. atropine, nicotine,
able to penetrate the blood-brain barrier, has numerous pharmacological actions which are complex and often unpredictable. Its best characterized sites of action are the autonomic ganglia (both parasympathetic and sympathetic) and the neuromuscular junction. The excitatory effects on ganglia are rapid in onset, followed by a persistent depolarization (i.e. blockade) even at small doses. Nicotine can markedly stimulate the central nervous system (CNS) in laboratory animals and man at doses of 1:Omg/kg or greater. Such doses produce tremors. With larger doses, the tremor is followed by convulsions due to stimulation of the motor cortex. These central actions are known to be blocked by a variety of agents (e.g. CNS depressants, antiparkinson agents, anticonvulsants, curariform compounds, and adrenergic blocking drugs). The present available literature concerning the effects of nicotine on tissue levels of cGMP contains little information. If ethanol causes depression of central cGMP via inhibition of the release of acetylcholine, or through more direct action on the cholinergic receptor (as many investigators believe), certain pharmacological manipulations should help clarify the basic mechanism. In either case cholinomimetic agonists (e.g. muscarinic and nicotinic agents) could be predicted to antagonize the ethanol-induced decreases in cGMP levels. Further, the presence of an antimuscarinic agent (e.g. atropine) would be expected to augment ethanol-induced decreases in the cGMP content. The purpose of the present study was to examine the effects of ethanol, arecoline, atropine, and nicotine alone and in various combinations on rat cerebellar cGMP in an attempt to clarify the mechanism(s) whereby ethanol lowers cerebellar cGMP in rat brain. 871
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R. A. Donso~ and W. E. METHODS
Each individual experiment employed four or more male Sprague-Dawley (CAW:CFE) rats weighing 15&250 g obtained from Charles River Breeding Labs (Wilmington, MA). Ethanol (USP) was diluted with isotonic saline, in a concentration of, not more than 20% (w/v). The volume administered orally @.o.) was 3.0 ml /lo0 g of .body weight. Control animals (0.9% NaCI) were also administered the same volume via the oral route. A dose of 6.Og/kg, of ethanol was administered to previously (24 hr) fasted rats, followed at 15, 30, 45 and 6b min by the removal of 0.5 ml of blood via cardiac puncture. The blood was deproteinized with 6.25% (w/v) trichloroacetic acid (Fisher Scientific Co.,) and centrifuged at 3000 rpm for 5 min. The blood ethanol concentration was determined using Ethanol Determination Kits 3331-UV (Sigma Chemical Corp., St Louis, MO) and a Beckman DB-G grating Spectrophotometer (Beckman Instruments, Irving, CA). Rat cerebellar cGMP concentrations were determined after sacrifice with 6 set of focused microwave fixation on the dorsal side of the head using a modified method of Stavinoha. Weintraub and’ Modak (1973) and Guidotti, Cheney, Trabucchi, Doteuchi and Wang (1974). The brains wer’e excised and the cerebellum removed as described by Glowinki and Iversen (1966). This was followed by homogenization with Tris-HCL-EDTA buffer, pH 7.5, to yield a 10% (w/v) homogenate. The homogenates were boiled for 3 min and centrifuged for IO-15 min at 3000 rpm. The supernatants were assayed for their cGMP content in duplicate with the use of tritium radiolabelled radioimmune kits (Cyclic-GMP RIA Kit, TRK. 500, Amersham/Searle, Corp.) that employed the method of Steiner, Paghara, Chase and Kipnis (1972). Protein determinations employed the method of Lowry, Rosebrough, Farr and Randall, 1951) with minor modifications. Protein determinations were used to express the cGMP content of cerebellum in relationship to its protein content. Arecoline HBr was given in varying doses of 1.0, 5.0, 10.0. 20.0 and 30.0mg/kg, intraperitoneally (ip.) 5 min before killing the rats with microwave irradiation. Nicotine was given in doses of 0.25, 0.50, 1.00 and 5.00 mg/kg (i.p.). Each animal was sacrificed 5 min after this drug administration. Saline injection, 2.Oml (i.p.) per 200g of body weight, was administered to each animal. Guided by the preliminary investigations and the published observations of Dinnendahl and Stock (1975) further investigations were undertaken using the following treatments: (1) Saline, 3.0 ml (p.o.)/lOO g of body weight, 25 min before the intraperitoneal injection of saline, 2.0 ml/200 g of body weight, followed 5 min later by lethal microwave exposure; (2) Ethanol 6.0 g/kg, (p.o.), 30 min prior to microwave radiation; (3) Ethanol, 6.0g/kg, (p.0.) 25 min prior to either arecohne, 20 mg/kg. (ip.). or nicotine 1.0 mg/kg, (ip.), fol-
JOHNSON
lowed 5 min later by microwave sacrifice; (4) atropine SO4 5.0 mgkg, (ip.) 30 min prior to microwave sacrifice and (5) ethanol, 6.0 g/kg, (p.o.), and atropine SO+ 5.0mg/kg, (i.p.), 30min prior to microwave sacrifice. Statistical evaluation used included means, SEM and Student’s unparied t-test. Drugs
Drugs used in the study were arecoline HBr (Regis Chemical Company, Morton Grove, IL), atropine SO, (Mallinkrodt Chemical Works, St Louis, MO), nicotine (Pfactz Bauer, Inc.. Flushing. NY) and ethanol, 9555%, USP (Gold Shield, Commercial Solvent Corp., Terre Haute. IN.). RESULTS
A summary of the preliminary dose-response curves are presented in Figures 1 and 2. Increasing doses of arecoline at 10, 20 and 30mg/Icg, (i.p.), resulted in significant (P < 0.05) dose-dependent increases in cerebellar cGMP (Fig. 1). Of the arecoline doses used. 20mg/kg. (ip.), produced the largest increase in cGMP levels, 1900/, of controls. Nicotine also significantly (P < 0.05) increased cGMI? concentrations in a dose-related fashion. Of the nicotine doses used, 5 mgkg, (ip.), resulted in the largest positive change in cGMP content, 496”,/, of controls. Log dose comparisons between arecoline and nicotine are shown in Figure 2, which indicated that. on a weight basis, nicotine was approximately five times more potent than arecoline in producing cerebellar cGMP increases in rat. Blood ethanol levels in fasted rats resulting from doses of 6 mg/kg, (p.o.), are presented in Figure 3. Ethanol blood levels rose rapidly, peaked at 30min. then declined towards control levels 15 min later. Figure 4 summarizes the effects of treatments with ethanol alone and in combination with atropine, arecoline, or nicotine on rat brain cerebellar levels of cGMP. Atropine. 5 mg/kg, (i.p.), produced a slight nonsignificant rise in cGMP content. Oral saline control values were statistically the same as intraperitoneal saline controls. However, ethanol, 6 g/kg, (p.0.). alone reduced cGMP cerebellum levels by a significant (P < 0.05) 57% of controls. Oral plus intraperitoneal saline injections (i.e. dual route controls) resulted in a large significant (P < 0.05) increase in cGMP cerebellar content compared to either intraperitoneal saline (62%) or oral saline (1039/,). Administration of ethanol, 6 g/kg, (p.0.). in animals treated with arecoline. 20mg/kg. (i.p.), or nicotine, 1 mg/kg. (i.p.), or atropine, 5 mg/kg, (i.p.) caused significant (P < 0.05) decreases in cerebellar cGMP concentrations of 637, (arecolineeethanol). 72% (nicotine-ethanol). and 78% (atropineeethanol), respectively as compared to the oral plus intraperitoneal controls. There was no difference between the ethanol-treated grdups, either alone or in the various combinations.
Ethanol and cholinergic agents on cyclic GMP
Fig. I. Dose-response
873
of arecoline and nicotine on cerebellum cGMP content in rats. Each graph represents the mean & SEM of four or more animals.
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DISCUSSION
The results obtained by increasing doses of arecoline (Fig. I) agree well with previously reported observations in mice (Dinnendahl and Stock, 1975), and rat brain corpus striatum (Hanley and Iversen, 1978). Arecoline, a direct acting muscarinic agonist, can readily penetrate the blood-brain barrier and interact with central muscarinic receptors, causing an increase in cGMP levels of the cerebellum. Nicotine is a direct acting cholinomimetic agonist. Of the doses of nicotine used, 5 mg/kg, (i.p.), caused the largest increase in cerebellar cGMP. However, this dose is near the reported LD,, values (Chen, Rose and Robbins, 1938) so I mg/kg, (i.p.), of nicotine was selected for subsequent experiments. This smaller dose markedly elevated cGMP concentration in the cerebellum of treated rats (Fig. 1). This in uiuo observation has not been previously reported. Log-dose comparisons (Fig. 2) of elevations of cGMP caused by arecoline and nicotine resulted in parallel curves. This suggests similar mechanism(s) of action for these drugs. Any attempt to construct welldefined conclusions without further investigations would be premature; however, the ability of two different cholinergic agonists to increase cGMP levels in the cerebellum adds further support to the current hypothesis that cGMP is implicated in central as well as peripheral cholinergic transmission. Volicer and Hurter (1977) observed a large significant decrease (62%) in central cGMP with acute ethanol intoxication (6 g/kg, p.o.) in rats, but these investigators did not report blood ethanol levels. However, Hunt et al. (1977) did include ethanol blood determinations in rats treated with an acute dose (6 g/kg, p.0.) of ethanol. In addition, these workers confirmed the earlier findings of Volicer and Hurter (1977) that ethanol significantly reduced cerebellar cGMP levels. The blood ethanol levels reported here (Fig. 3) agree well with those reported by Hunt et al. (1977). In this present work, ethanol (6 g/kg, p.o.) resulted in significant decreases in cGMP in the rat cerebellum (Fig. 4). This observation agrees with the reports of Volicer and Hurter (1977) and Hunt et al. (1977) and more recently with those of Klosowicz, Pelikan and Volicer (1979). Atropine administration (5 g/kg, i.p.) has previously been reported to increase cGMP levels in the mouse cerebellum (Dinnendahl and Stock, 1975) and in certain in vitro preparations (Ferrendelli, Steiner, McDougal and Kipnis, 1970). Although atropine (5 mg/kg, i.p.) in this study did cause a slight increase in cGMP, the change was not significant (Fig. 4). This accords with the findings of certain other workers (Lee et al., 1972; Kuo, Lee, Procerfina, Walton, Donnelly and Greengard, 1972). Oral plus intraperitoneal saline injections caused large increases in cerebellar cGMP content when compared to either separate oral or intraperitoneal saline injections. This finding is most likely due to
agents
on cyclic GMP
875
the additional stress (e.g. handling and immobilization) associated with the two different, closely-spaced administrations in the same animal. Previously, Dinnendahl and Gumulka (1977) reported similar results in mice subjected to stressful conditions (e.g. swimming and immobilization). More recently, Klosowicz et al. (1979) demonstrated similar findings in rats subjected to various stressful manipulations (e.g. handling, ice bath and hot plate). When ethanol was administered to rats previously treated with arecoline, nicotine or atropine, a significant reduction in cerebellar cGMP content resulted (Fig. 4). These responses were not significantly different from those .of animals subjected to ethanol alone. Thus, it appears that neither a muscarinic nor a nicotinic agonist is capable of antagonizing the depressive effects of ethyl alcohol on cerebellar cGMP in rats. Furthermore, the presence of atropine did not produce a synergistic effect with ethanol. These data show that the depressant actions of ethanol on the cGMP content in the rat cerebellum are independent of cholinergic receptor mechanisms. REFERENCES
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tide-dependent protein kinases. X. An assay method for the measurement of guanosine 3’,5’-monophosphate in various biological materials and a study of agents regulating its levels in heart and brain. J. hiol. Chem. 247: 16-22. Lee, T. P., Kuo, J. F. and Greengard, P. (1972), Role of muscarinic cholinergic receptors in regulation of cGMP content in mammalier brain, heart muscle and intestinal smooth muscle. Proc. nutn Acad. Sci., i1.S.A. 69: 3287-3291. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin reagent. .I. biol. Chem. 193: 265-215.
Stavinoha. W. B., Weintraub, S. T. and Modak, A. T. (1973). The use of microwave heating to inactivate cholinesterase in the rat brain prior to analysis for acetylcholine. J. Neurochem. 20: 361-371. Steiner, A. L.. Pagliara, A. S., Chase, L. R. and Kipnis. D. M. (1972). Radioimmunoas~y for cyclic nucleotides II. Adenosine 3’S’-monophosphate and guanosine 3’S’-monophosphate in mammalian tissues and body fluid. J. hiol. Chem. 247: 1114-I 120. Volicer. L. and Hurter. R. P. (1977). Effects of acute and chronic ethanol administration and withdrawal on adenosine 3’,5’-monophosphate levels in the rat brain. J. Pharmac. exp. Ther. 200: 298-304.