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Chronic in vivo ethanol administration alters the sensitivity of adenylate cyclase coupling in homogenates of rat brain
Neuroscience Letters, 84 (1988) 317 322 Elsevier Scientific Publishers Ireland Ltd.
317
NSL 05084
Chronic in vivo ethanol administration alters the sensitivity of adenylate cyclase coupling in homogenates of rat brain Keith Saffey l, M a r k A. G i l l m a n 2 a n d R i c h a r d C. Cantrill l IM RC Brain Metabolism Research Group and :Departments o["Medical Biochemistry and Pharmacology, Universi O, o["The Witwatersrand Medical School, Johannesburg (South Africa) (Received 28 August 1987; Revised version received 9 October 1987: Accepted 9 October 1987) Key words'." Ethanol; Cyclic AMP; Adenylate cyclase; Noradrenaline; GTP The high activities of adenylate cyclase, phosphodiesterase and protein kinases in the synaptic terminals of the central nervous system makes these enzymes prime candidates for the in vivo actions of ethanol. Adult female rats were fed a liquid diet containing ethanol as 35% of the available calories for 6 days. This resulted in a decrease (22-45%) in the basal activity of adenylate cyclase, as determined by cyclic 3',5'adenosine monophosphate (cAMP) production, in homogenates of all brain areas tested. In these homogenates the ability of guanosine triphosphate and noradrenaline to stimulate basal cyclase activity was severely reduced. These results suggest that ethanol administration causes an uncoupling of the fl-receptor/adenylate cyclase cascade and an interruption of the control of the synthesis of cAMP.
It is proposed that the addictive effects of ethanol may result from modifications in the structure of cerebral membranes in response to changes in membrane fluidity [2, 9]. These changes have been identified as minor changes in the lipid composition of synaptic membranes resultant upon the fluidizing effects of ethanol [5, 8]. Since the activity of membrane proteins may be influenced by their immediate environment, both the structural and functional components of neural membranes could be affected [3]. Thus alterations in the activity of membrane proteins, especially neurotransmitter receptors and receptor-linked enzyme systems involved in the production of 'second messengers' would be predicted. Studies on the effects of ethanol in vivo have shown a decrease in the level of the 'second messenger', cyclic 3',5'-adenosine monophosphate (cAMP), in the brain [12. 15], although the activity offl-adrenergic receptor-coupled adenylate cyclase has been shown to be either increased [10, 14], or unaffected [6] by the presence of ethanol (up to 850 mM). The activity of mouse brain cortical adenylate cyclase can be stimu-
Correspondence." R. Cantrill. Present address: Physiologisch-Chemischeslnstitut, University of Wfirzburg, Koellikerstrasse 2, D-8700 Wfirzburg, F.R.G. 0304-3940/88/$ 03.50 @ 1988 Elsevier Scientific Publishers Ireland Ltd.
318
lated by fl-receptor agonists and guanosine triphosphate (GTP) analogues as seen in the classical fl-receptor stimulation cascade (for reviews see refs. 4 and 7). Since ethanol (in vitro) may stimulate cAMP production at any or all of the following sites: the fl-receptor; the family of guanosine nucleotide binding proteins (separately known as the 'G' [4] or 'N' [7] proteins); and at the catalytic adenylate cyclase unit [11], the reduction in the cAMP levels in the brains of rats following chronic ethanol treatment may be a result of the desensitization of the fl-receptor cascade as described [13]. We have studied the apparent disparity in the in vitro and in vivo effects of ethanol on cAMP levels in rats fed a liquid diet containing ethanol. The stimulation of cAMP production by GTP and noradrenaline was measured in 6 major brain regions; namely the cerebellum, pons-medulla, hypothalamus, midbrain, striatum and cerebral cortex. In all experiments female Sprague-Dawley rats (average weight 200 g) were fed a liquid nutrient diet [1] (Nutrament-T; Bristol-Myers) ad libitum. All animals were kept at constant temperature and humidity with a 12 h light/dark cycle and had free access to the diet. The group of animals receiving ethanol were fed the chocolate flavoured diet containing ethanol as 35% of its calorific content. All animals were fed the diet with an isocaloric quantity of sucrose for 4 days prior to being ascribed to the control or test groups. The control groups of animals received the liquid diet plus sucrose throughout the 144 h experimental period. The ethanol diet was consumed at 80-100% of the control diet intake. Blood samples taken from the tail vein of rats belonging to the ethanol treatment group contained approximately 3.67 mM ethanol (125_+8/lg/mt; n = 3), when measured in an enzymatic assay (Boehringer Mannheim). At the end of the 144 h experimental period, rats were stunned and then killed by cervical dislocation, and their brains were quickly removed and dissected on ice. The 6 brain regions were then homogenized separately in 30 vols. (w/v) of ice-cold 10 mM Tris-HC1 buffer (pH 7.4) containing 2 mM EGTA and assayed immediately. The assay mixture contained in 110 pl: 80 mM Tris-HCl, 2 mM MgCI2, 20 mM theophylline, 0.6 mM EGTA, 50/tl of homogenate and 10/A of either water or (1) GTP (100 /~M); (2) noradrenaline (100 pM); (3) GTP and noradrenaline (100 pM each). The homogenate was prewarmed together with the assay components for 5 min at 22°C before the addition of ATP (1 raM). Incubation was continued at 30°C for a further 5 min before being terminated by heating the tubes to 90°C for 4 rain. cAMP concentrations were determined using a commercially available competitive binding assay using 125I-labelled antibody (New England Nuclear). Protein concentrations were determined using the Coomassie Brilliant Blue method (Biorad) with bovine serum albumin as standard. All estimations were carried out in duplicate and cyclase activity was determined under all experimental conditions allowing for statistical evaluation using paired data. Results from assays containing GTP and/or noradrenaline are expressed as a percentage of the unstimulated value in order to minimize individual variations between cAMP determinations. Basal values for adenylate cyclase activity in the brains of rats fed the control diet
319 TABLE I A C O M P A R I S O N OF T H E BASAL A D E N Y L A T E C Y C L A S E A C T I V I T Y IN D I F F E R E N T B R A I N A R E A S IN C O N T R O L A N D E T H A N O L - F E D RATS The brain areas are labelled (I) cerebellum. (2) pons-medulla, (3) hypothalamus, (4) midbrain, (5) stria-
turn, (6) cortex. Basal activities of adenylate cyclase are expressed as pmol/min/mg prot. + S.D. Control animals received the liquid diet with sucrose throughout the experimental period. Ethanol-fed rats received the same liquid diet as control rats for 4 days after which their diet contained ethanol in place of sucrose. Brain area
Control (5) Ethanol-fed (4) % Decrease
I
2
3
4
5
6
205 +34 141 +21 31
185 +36 145 +26 22
455 + 148 252 +59 45
279 __+51 203 +50 27
409 +56 264 +24 35
212 +44 166 +33 22
containing sucrose ranged from 185 pmol/min/mg prot. in the pons-medulla region to 455 pmol/min/mg prot. in the hypothalamus (Table I). The order of enzyme activity was: pons-medulla < cerebellum < cerebral cortex < midbrain < striatum < hypothalamus. The basal acivity of the enzyme from the brains of rats receiving the ethanol supplemented diet was generally lower than that measured in the same area of the control rat brain. The enzyme activities were reduced by between 22% (ponsmedulla and cortex) and 45% in the hypothalamus. The distribution of activity was similar to that seen in the different regions of the control brain (cerebellum < ponsmedulla < cortex < midbrain < hypothalamus < striatum). In both control and ethanol-fed rats~ highest cyclase activity was detected in the hypothalamus and striatum. Basal enzyme activity was increased in all areas of control rat brain in the presence of G T P except for the striatum (Table II). Noradrenaline alone increased the production of c A M P in the cerebellum, pons-medulla and hypothalamus, but there were variable results in the striatum and no stimulation in the cortex and midbrain. Incubation in the presence of both G T P and noradrenaline significantly increased the production of c A M P in all brain regions except the striatum and stimulation ranged between 49% in the cortex and 124% in the pons-medulla. In contrast, in homogenates of brain areas from ethanol-fed rats, the degree of stimulation elicited by either GTP, noradrenaline or both, was small and highly variable. These results suggest that chronic ethanol intake may lead to membrane modifications which cause an uncoupling of the receptor and guanyl nucleotide binding proteins from the catalytic unit together with a lowering of the basal activity of the unstimulated enzyme. It could be speculated that the catalytic unit, in this instance, is more accessible to inhibitory ' G ' proteins and has reduced interactions with the stimulatory components. The degree of stimulation of the adenylate cyclase system by G T P (42%) in homogenates of control rat brain regions is low in comparison with other published data
320 T A B L E 1I S T I M U L A T I O N O F A D E N Y L A T E C Y C L A S E BY G T P A N D N O R A D R E N A L I N E IN D I F F E R ENT B R A I N A R E A S F R O M C O N T R O L A N D E T H A N O L - F E D RATS The brain areas are labelled (1) cerebellum, (2) pons-medulla, (3) hypothalamus, (4) midbrain, (5) striatum, (6) cortex. Data are presented as % of basal activity using the values presented in Table I, for each of the brain areas studied. Statistical variation was calculated using the Student's t-test for paired data. *P<0.05. Both groups of animals were fed as described in the legend to Table I. Adenylate cyclase was stimulated by the addition of guanosine triphosphate (100/~M, final conc.) ' + GTP'; noradrenaline (100 /~M, final conc.) ' + N A ' : and both together (100 #M each, final conc.) ' + G T P + N A ' . Brain area
Control + G T P (5) + N A (5) + G T P + N A (5)
Ethanol-fed + G T P (4) + N A (4) + G T P + N A (4)
1
2
3
4
5
6
100 135" +22 152" +21 221" -+48
100 172" +27 162" -+25 224* +42
100 167" +25 133" __+13 196" ±40
100 133" + 14 113 -+ 17 164" -+40
100 110 +31 t40 +36 119 +24
100 138" + 12 105 -+ 18 149" +27
100 130 -+22 1 I0 -+15 120 + 15
100 133" +ll 109 __+20 129 _+24
100 121 -+15 96 + 9 126 -+ 18
100 90 +17 99 -+24 116 + 18
100 92 +18 116 +17 101 -t-35
100 100 +17 106 -+13 126 + 14
using mouse cerebral cortex [11], although the basal activity and maximum activity in the presence of both noradrenaline and GTP were increased. The degree of agonist stimulation (36%) is similar to that reported [1 I]. The differences in the basal activities of adenylate cyclase may indicate that the homogenate contained sufficient GTP for partial activation of the cyclase system. Endogenous cAMP levels were measured in ATP-free incubations and subtracted from the values obtained in experiments containing ATP. No attempt was made to control for the action of ATPase enzymes, except for the presence of large quantities of substrate; the high levels of cAMP measured would indicate the presence of sufficient substrate. The regional variations in adenylatecyclase activity and its response to noradrenaline may indicate the relative preponderance of functional adrenergic receptors in those areas. The decrease in the activity of adenylate cyclase and cAMP production in ethanol-fed animals correlates well with the decrease in cAMP levels observed in the brains of ethanol dependent rats [12], although the activity of the mouse cortical membrane adenylate cyclase system was increased when incubated in the presence of ethanol in vitro [ 11]. Adenylate cyclase in cortical slices may be insensitive to nor-
321
adrenaline in rats chronically treated with ethanol [14], and the same intransigence of adenylate cyclase to noradrenaline is seen in the cortical homogenate results presented in Table II. However, the same insensitivity of the cyclase system is seen in the cortical homogenates of control rat brain and is a feature common to all brain areas in the ethanol-fed rats, where the average increase in activity by noradrenaline over that produced by GTP is 9%. The present results could arise from modifications in membrane composition and receptor sensitivity altering the activity of the cAMP generating system; thus reflecting the homeostatic pressures towards the restoration of normal physiological function. It is also possible that the magnitude of the measured effects may be either species dependent or result from the dietary manipulation inherent in these experiments. Feeding rats a liquid diet, such as Nutrament-T, alters their intake of protein and may give rise to increased circulating levels of excitatory amino acids which could alter cerebral neurotransmitter activity. Analysis of the composition of Nutrament-T and proprietary rat chow revealed a 4-fold increase in glutamate concentration which may be either directly or indirectly responsible for alterations in cerebral neurotransmission and the sensitivity of some neurotransmitter receptors (Kurstjens and Cantrill; unpublished data). Basal adenylate cyclase activity can be increased by an average of 68% by feeding the liquid nutrient and many of the receptor-linked alterations can be elicited in chow-fed animals by glutamate supplementation. The authors are grateful for the support of the South African Medical Research Council. 1 Freund, G., Physical dependence on ethanol: conceptual considerations, Drug Alcoholic Depend., 4 (1969) 371 375. 2 Goldstein, D.B. and Chin, J., Interaction of ethanol with biological membranes, Fed. Proc., 40 (1981) 2073 2076. 3 Harris, R.A. and Schroeder, F., Ethanol and the physical properties of brain membranes, Mol. Pharmcol., 20 (1981) 128 137. 4 Helmreich, E.J.M. and Pfeuffer, T., Regulation of signal transduction by fl-adrenergic hormone receptors, Trends Pharmacol. Sci., 6 ([985) 438 443. 5 Johnson, D.A., Lee, N.M., Cooke, R. and Loh, H.H., Ethanol-induced fluidisation of brain lipid bilayers: required presence of cholesterol in membranes for the expression of tolerance, Mol. Pharmacol., 15 ( [ 979) 739 746. 6 Kuriyama, K. and Israel, M., Effects of ethanol administration in cyclic 3'5'-adenosine monophosphate metabolism in the brain, Biochem. Pharmacol., 22 (1973) 2919 2922. 7 Lefkowilz, R.J., Stadel, J.M. and Caron, M.G., Adenylate cyclase-coupled beta-adrenergic receptors: structure and mechanisms of activation and desensitisation, Annu. Rev. Biochem., 52 (1983) [59 186. 8 Littleton, J.M. and John, G., Synaptosomal membrane lipids of mice during continuous exposure to ethanol, J. Pharm. Pharmacol., 29 (1977) 579 580. 9 Lyon, R.C., McComb, J.A., Schreurs, J. and Goldstein, D.B., A relationship between alcohol intoxication and the disordering of brain membranes by a series of short-chain alcohols, J. Pharmacol. Exp. Ther., 218 (1981) 669 675. 10 Rabin, R.A. and Molinoff, P.B., Activation of adenylate cyclase by ethanol in mouse striatal tissue, J. PharmacoI. Exp. Ther., 216 (1981) 129 134. 11 Saito, T., Lee, J.M. and Tabakoff, B., Ethanol's effect on cortical adenylate cyclase activity, J. Neurochem., 44 (1985) 1037 1044.
322 12 Shen, A., Jacobyanski, A., Pathman, D. and Thurman, R.G., Changes in brain cyclic-AMP levels during chronic ethanol treatment and withdrawal in the rat, Eur. J. Pharmacol., 89 (1983) 103 110. 13 Sibley, D.R. and Lefkowitz, R.J., Molecular mechanisms of receptor desensitisation using the/#adrenergic receptor-coupled adenylate cyclase as a model, Nature, 317 (1985) 124-129. 14 Smith, T., Jacobyanski, A., Shen, A., Pathman, D. and Thurman, R.G., Adaptation of the cyclic AMP generating systems in rat cerebral cortical slices during chronic ethanol treatment and withdrawal, Neuropharmacotogy, 20 (1981) 6 7 72. 15 Volicer, L., Mirin, R. and Gold, F., Effect of ethanol on the cyclic AMP system in rat brain, J. Stud. Alcohol, 38 (1977) I I -24.
317
NSL 05084
Chronic in vivo ethanol administration alters the sensitivity of adenylate cyclase coupling in homogenates of rat brain Keith Saffey l, M a r k A. G i l l m a n 2 a n d R i c h a r d C. Cantrill l IM RC Brain Metabolism Research Group and :Departments o["Medical Biochemistry and Pharmacology, Universi O, o["The Witwatersrand Medical School, Johannesburg (South Africa) (Received 28 August 1987; Revised version received 9 October 1987: Accepted 9 October 1987) Key words'." Ethanol; Cyclic AMP; Adenylate cyclase; Noradrenaline; GTP The high activities of adenylate cyclase, phosphodiesterase and protein kinases in the synaptic terminals of the central nervous system makes these enzymes prime candidates for the in vivo actions of ethanol. Adult female rats were fed a liquid diet containing ethanol as 35% of the available calories for 6 days. This resulted in a decrease (22-45%) in the basal activity of adenylate cyclase, as determined by cyclic 3',5'adenosine monophosphate (cAMP) production, in homogenates of all brain areas tested. In these homogenates the ability of guanosine triphosphate and noradrenaline to stimulate basal cyclase activity was severely reduced. These results suggest that ethanol administration causes an uncoupling of the fl-receptor/adenylate cyclase cascade and an interruption of the control of the synthesis of cAMP.
It is proposed that the addictive effects of ethanol may result from modifications in the structure of cerebral membranes in response to changes in membrane fluidity [2, 9]. These changes have been identified as minor changes in the lipid composition of synaptic membranes resultant upon the fluidizing effects of ethanol [5, 8]. Since the activity of membrane proteins may be influenced by their immediate environment, both the structural and functional components of neural membranes could be affected [3]. Thus alterations in the activity of membrane proteins, especially neurotransmitter receptors and receptor-linked enzyme systems involved in the production of 'second messengers' would be predicted. Studies on the effects of ethanol in vivo have shown a decrease in the level of the 'second messenger', cyclic 3',5'-adenosine monophosphate (cAMP), in the brain [12. 15], although the activity offl-adrenergic receptor-coupled adenylate cyclase has been shown to be either increased [10, 14], or unaffected [6] by the presence of ethanol (up to 850 mM). The activity of mouse brain cortical adenylate cyclase can be stimu-
Correspondence." R. Cantrill. Present address: Physiologisch-Chemischeslnstitut, University of Wfirzburg, Koellikerstrasse 2, D-8700 Wfirzburg, F.R.G. 0304-3940/88/$ 03.50 @ 1988 Elsevier Scientific Publishers Ireland Ltd.
318
lated by fl-receptor agonists and guanosine triphosphate (GTP) analogues as seen in the classical fl-receptor stimulation cascade (for reviews see refs. 4 and 7). Since ethanol (in vitro) may stimulate cAMP production at any or all of the following sites: the fl-receptor; the family of guanosine nucleotide binding proteins (separately known as the 'G' [4] or 'N' [7] proteins); and at the catalytic adenylate cyclase unit [11], the reduction in the cAMP levels in the brains of rats following chronic ethanol treatment may be a result of the desensitization of the fl-receptor cascade as described [13]. We have studied the apparent disparity in the in vitro and in vivo effects of ethanol on cAMP levels in rats fed a liquid diet containing ethanol. The stimulation of cAMP production by GTP and noradrenaline was measured in 6 major brain regions; namely the cerebellum, pons-medulla, hypothalamus, midbrain, striatum and cerebral cortex. In all experiments female Sprague-Dawley rats (average weight 200 g) were fed a liquid nutrient diet [1] (Nutrament-T; Bristol-Myers) ad libitum. All animals were kept at constant temperature and humidity with a 12 h light/dark cycle and had free access to the diet. The group of animals receiving ethanol were fed the chocolate flavoured diet containing ethanol as 35% of its calorific content. All animals were fed the diet with an isocaloric quantity of sucrose for 4 days prior to being ascribed to the control or test groups. The control groups of animals received the liquid diet plus sucrose throughout the 144 h experimental period. The ethanol diet was consumed at 80-100% of the control diet intake. Blood samples taken from the tail vein of rats belonging to the ethanol treatment group contained approximately 3.67 mM ethanol (125_+8/lg/mt; n = 3), when measured in an enzymatic assay (Boehringer Mannheim). At the end of the 144 h experimental period, rats were stunned and then killed by cervical dislocation, and their brains were quickly removed and dissected on ice. The 6 brain regions were then homogenized separately in 30 vols. (w/v) of ice-cold 10 mM Tris-HC1 buffer (pH 7.4) containing 2 mM EGTA and assayed immediately. The assay mixture contained in 110 pl: 80 mM Tris-HCl, 2 mM MgCI2, 20 mM theophylline, 0.6 mM EGTA, 50/tl of homogenate and 10/A of either water or (1) GTP (100 /~M); (2) noradrenaline (100 pM); (3) GTP and noradrenaline (100 pM each). The homogenate was prewarmed together with the assay components for 5 min at 22°C before the addition of ATP (1 raM). Incubation was continued at 30°C for a further 5 min before being terminated by heating the tubes to 90°C for 4 rain. cAMP concentrations were determined using a commercially available competitive binding assay using 125I-labelled antibody (New England Nuclear). Protein concentrations were determined using the Coomassie Brilliant Blue method (Biorad) with bovine serum albumin as standard. All estimations were carried out in duplicate and cyclase activity was determined under all experimental conditions allowing for statistical evaluation using paired data. Results from assays containing GTP and/or noradrenaline are expressed as a percentage of the unstimulated value in order to minimize individual variations between cAMP determinations. Basal values for adenylate cyclase activity in the brains of rats fed the control diet
319 TABLE I A C O M P A R I S O N OF T H E BASAL A D E N Y L A T E C Y C L A S E A C T I V I T Y IN D I F F E R E N T B R A I N A R E A S IN C O N T R O L A N D E T H A N O L - F E D RATS The brain areas are labelled (I) cerebellum. (2) pons-medulla, (3) hypothalamus, (4) midbrain, (5) stria-
turn, (6) cortex. Basal activities of adenylate cyclase are expressed as pmol/min/mg prot. + S.D. Control animals received the liquid diet with sucrose throughout the experimental period. Ethanol-fed rats received the same liquid diet as control rats for 4 days after which their diet contained ethanol in place of sucrose. Brain area
Control (5) Ethanol-fed (4) % Decrease
I
2
3
4
5
6
205 +34 141 +21 31
185 +36 145 +26 22
455 + 148 252 +59 45
279 __+51 203 +50 27
409 +56 264 +24 35
212 +44 166 +33 22
containing sucrose ranged from 185 pmol/min/mg prot. in the pons-medulla region to 455 pmol/min/mg prot. in the hypothalamus (Table I). The order of enzyme activity was: pons-medulla < cerebellum < cerebral cortex < midbrain < striatum < hypothalamus. The basal acivity of the enzyme from the brains of rats receiving the ethanol supplemented diet was generally lower than that measured in the same area of the control rat brain. The enzyme activities were reduced by between 22% (ponsmedulla and cortex) and 45% in the hypothalamus. The distribution of activity was similar to that seen in the different regions of the control brain (cerebellum < ponsmedulla < cortex < midbrain < hypothalamus < striatum). In both control and ethanol-fed rats~ highest cyclase activity was detected in the hypothalamus and striatum. Basal enzyme activity was increased in all areas of control rat brain in the presence of G T P except for the striatum (Table II). Noradrenaline alone increased the production of c A M P in the cerebellum, pons-medulla and hypothalamus, but there were variable results in the striatum and no stimulation in the cortex and midbrain. Incubation in the presence of both G T P and noradrenaline significantly increased the production of c A M P in all brain regions except the striatum and stimulation ranged between 49% in the cortex and 124% in the pons-medulla. In contrast, in homogenates of brain areas from ethanol-fed rats, the degree of stimulation elicited by either GTP, noradrenaline or both, was small and highly variable. These results suggest that chronic ethanol intake may lead to membrane modifications which cause an uncoupling of the receptor and guanyl nucleotide binding proteins from the catalytic unit together with a lowering of the basal activity of the unstimulated enzyme. It could be speculated that the catalytic unit, in this instance, is more accessible to inhibitory ' G ' proteins and has reduced interactions with the stimulatory components. The degree of stimulation of the adenylate cyclase system by G T P (42%) in homogenates of control rat brain regions is low in comparison with other published data
320 T A B L E 1I S T I M U L A T I O N O F A D E N Y L A T E C Y C L A S E BY G T P A N D N O R A D R E N A L I N E IN D I F F E R ENT B R A I N A R E A S F R O M C O N T R O L A N D E T H A N O L - F E D RATS The brain areas are labelled (1) cerebellum, (2) pons-medulla, (3) hypothalamus, (4) midbrain, (5) striatum, (6) cortex. Data are presented as % of basal activity using the values presented in Table I, for each of the brain areas studied. Statistical variation was calculated using the Student's t-test for paired data. *P<0.05. Both groups of animals were fed as described in the legend to Table I. Adenylate cyclase was stimulated by the addition of guanosine triphosphate (100/~M, final conc.) ' + GTP'; noradrenaline (100 /~M, final conc.) ' + N A ' : and both together (100 #M each, final conc.) ' + G T P + N A ' . Brain area
Control + G T P (5) + N A (5) + G T P + N A (5)
Ethanol-fed + G T P (4) + N A (4) + G T P + N A (4)
1
2
3
4
5
6
100 135" +22 152" +21 221" -+48
100 172" +27 162" -+25 224* +42
100 167" +25 133" __+13 196" ±40
100 133" + 14 113 -+ 17 164" -+40
100 110 +31 t40 +36 119 +24
100 138" + 12 105 -+ 18 149" +27
100 130 -+22 1 I0 -+15 120 + 15
100 133" +ll 109 __+20 129 _+24
100 121 -+15 96 + 9 126 -+ 18
100 90 +17 99 -+24 116 + 18
100 92 +18 116 +17 101 -t-35
100 100 +17 106 -+13 126 + 14
using mouse cerebral cortex [11], although the basal activity and maximum activity in the presence of both noradrenaline and GTP were increased. The degree of agonist stimulation (36%) is similar to that reported [1 I]. The differences in the basal activities of adenylate cyclase may indicate that the homogenate contained sufficient GTP for partial activation of the cyclase system. Endogenous cAMP levels were measured in ATP-free incubations and subtracted from the values obtained in experiments containing ATP. No attempt was made to control for the action of ATPase enzymes, except for the presence of large quantities of substrate; the high levels of cAMP measured would indicate the presence of sufficient substrate. The regional variations in adenylatecyclase activity and its response to noradrenaline may indicate the relative preponderance of functional adrenergic receptors in those areas. The decrease in the activity of adenylate cyclase and cAMP production in ethanol-fed animals correlates well with the decrease in cAMP levels observed in the brains of ethanol dependent rats [12], although the activity of the mouse cortical membrane adenylate cyclase system was increased when incubated in the presence of ethanol in vitro [ 11]. Adenylate cyclase in cortical slices may be insensitive to nor-
321
adrenaline in rats chronically treated with ethanol [14], and the same intransigence of adenylate cyclase to noradrenaline is seen in the cortical homogenate results presented in Table II. However, the same insensitivity of the cyclase system is seen in the cortical homogenates of control rat brain and is a feature common to all brain areas in the ethanol-fed rats, where the average increase in activity by noradrenaline over that produced by GTP is 9%. The present results could arise from modifications in membrane composition and receptor sensitivity altering the activity of the cAMP generating system; thus reflecting the homeostatic pressures towards the restoration of normal physiological function. It is also possible that the magnitude of the measured effects may be either species dependent or result from the dietary manipulation inherent in these experiments. Feeding rats a liquid diet, such as Nutrament-T, alters their intake of protein and may give rise to increased circulating levels of excitatory amino acids which could alter cerebral neurotransmitter activity. Analysis of the composition of Nutrament-T and proprietary rat chow revealed a 4-fold increase in glutamate concentration which may be either directly or indirectly responsible for alterations in cerebral neurotransmission and the sensitivity of some neurotransmitter receptors (Kurstjens and Cantrill; unpublished data). Basal adenylate cyclase activity can be increased by an average of 68% by feeding the liquid nutrient and many of the receptor-linked alterations can be elicited in chow-fed animals by glutamate supplementation. The authors are grateful for the support of the South African Medical Research Council. 1 Freund, G., Physical dependence on ethanol: conceptual considerations, Drug Alcoholic Depend., 4 (1969) 371 375. 2 Goldstein, D.B. and Chin, J., Interaction of ethanol with biological membranes, Fed. Proc., 40 (1981) 2073 2076. 3 Harris, R.A. and Schroeder, F., Ethanol and the physical properties of brain membranes, Mol. Pharmcol., 20 (1981) 128 137. 4 Helmreich, E.J.M. and Pfeuffer, T., Regulation of signal transduction by fl-adrenergic hormone receptors, Trends Pharmacol. Sci., 6 ([985) 438 443. 5 Johnson, D.A., Lee, N.M., Cooke, R. and Loh, H.H., Ethanol-induced fluidisation of brain lipid bilayers: required presence of cholesterol in membranes for the expression of tolerance, Mol. Pharmacol., 15 ( [ 979) 739 746. 6 Kuriyama, K. and Israel, M., Effects of ethanol administration in cyclic 3'5'-adenosine monophosphate metabolism in the brain, Biochem. Pharmacol., 22 (1973) 2919 2922. 7 Lefkowilz, R.J., Stadel, J.M. and Caron, M.G., Adenylate cyclase-coupled beta-adrenergic receptors: structure and mechanisms of activation and desensitisation, Annu. Rev. Biochem., 52 (1983) [59 186. 8 Littleton, J.M. and John, G., Synaptosomal membrane lipids of mice during continuous exposure to ethanol, J. Pharm. Pharmacol., 29 (1977) 579 580. 9 Lyon, R.C., McComb, J.A., Schreurs, J. and Goldstein, D.B., A relationship between alcohol intoxication and the disordering of brain membranes by a series of short-chain alcohols, J. Pharmacol. Exp. Ther., 218 (1981) 669 675. 10 Rabin, R.A. and Molinoff, P.B., Activation of adenylate cyclase by ethanol in mouse striatal tissue, J. PharmacoI. Exp. Ther., 216 (1981) 129 134. 11 Saito, T., Lee, J.M. and Tabakoff, B., Ethanol's effect on cortical adenylate cyclase activity, J. Neurochem., 44 (1985) 1037 1044.
322 12 Shen, A., Jacobyanski, A., Pathman, D. and Thurman, R.G., Changes in brain cyclic-AMP levels during chronic ethanol treatment and withdrawal in the rat, Eur. J. Pharmacol., 89 (1983) 103 110. 13 Sibley, D.R. and Lefkowitz, R.J., Molecular mechanisms of receptor desensitisation using the/#adrenergic receptor-coupled adenylate cyclase as a model, Nature, 317 (1985) 124-129. 14 Smith, T., Jacobyanski, A., Shen, A., Pathman, D. and Thurman, R.G., Adaptation of the cyclic AMP generating systems in rat cerebral cortical slices during chronic ethanol treatment and withdrawal, Neuropharmacotogy, 20 (1981) 6 7 72. 15 Volicer, L., Mirin, R. and Gold, F., Effect of ethanol on the cyclic AMP system in rat brain, J. Stud. Alcohol, 38 (1977) I I -24.