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Up-regulation of rat cortical adenosine receptors following chronic administration of theophylline
European Journal of Pharmacology, 82 (1982) 113-114
113
Elsevier Biomedical Press
Rapid communication U P - R E G U L A T I O N OF RAT CORTICAL A D E N O S I N E R E C E P T O R S F O L L O W I N G C H R O N I C A D M I N I S T R A T I O N OF T H E O P H Y L L I N E * T.F. M U R R A Y
College of Pharmacy 6510, Washington State University, Pullman, WA 99164, U.S.A. Received I July 1982, accepted 5 July 1982
Adenosine functions as a putative endogenous neuromodulator or cotransmitter in both the peripheral and central nervous system (Stone, 1981). The methylxanthines, caffeine and theophylline, have been shown to exert their central stimulant effects through a competitive antagonism of adenosine (AI) receptors in the brain (Snyder et al., 1981). Caffeine tolerance has been demonstrated in man (Colton et al., 1967), and recently tolerance development to the stimulating effect of theophylline on the mouse nigrostriatal system has been reported (Watanabe et al., 1982). This latter tolerance was proposed to be a functional rather than a metabolic tolerance as the concentrations of theophylline in the forebrains of tolerant and nontolerant animals did not differ significantly (Watanabe et al., 1982). One neurochemical mechanism of functional tolerance is an up-regulation or increase in the density of the receptors for a given neurotransmitter or drug. The up-regulation can occur in response to the receptors reduced access to the endogenous transmitter substance, or it can occur as a result of the presence of an antagonist for that receptor which in effect reduces it access to its endogenous ligand. Therefore, the present investigation was undertaken to investigate the regulation of adenosine A ~-receptors following chronic treatment of rats with theophylline, an adenosine receptor antagonist. N6-Cyclohexyl-[3H]adenosine ([3H]CHA) (New England Nuclear, spec. act. 11.5 C i / m m o l ) specific binding to brain membranes was performed as * Supported in part by a PMA Foundation Research Starter Grant. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
previously described (Murray and Cheney, 1982). Specific binding was defined as the excess over blanks determined in the presence of 10 /~M Lphenylisopropyladenosine (L-PIA). Saturation isotherms of specific [3H]CHA binding were performed in the presence of a 0.3-10.5 nM concentration range. After 120 min of incubation at 23°C, the entire 1 ml incubation vol was filtered through Whatman G F / B glass-fiber filters. Filters were then washed three times with 5 ml each of ice cold TRIS-HC1 buffer (50 mM, pH 7.7). Radioactivity was counted after 18 h of elution in a Beckman liquid scintillation counter. Male Sprague-Dawley rats weighing 220-250 g were injected twice daily with either 25 m g / k g i.p. theophylline or saline vehicle. After 14 consecutive days of this chronic regimen, the theophylline dose was increased to 45 m g / k g twice daily for seven more days (i.e. total treatment regimen -- 21 days). On day 24 groups of 4 rats each were sacrificed and [3H]CHA binding was determined that day in freshly prepared membranes from the control and chronic theophylline cortices and hippocampi. As shown in fig. l, the chronic administration of theophylline for 21 days resulted in a significant increase in the maximum number of [3H]CHA binding sites (27.9% increase) in cortical membranes, while the affinity of [3H]CHA for its recognition site remained unchanged (Control K D = 1.06 riM; theophylline K D = 1.11 nM). There was also a trend for the Bmax to increase in hippocampal membranes (12.1% increase), but this change did not reach the level of significance in 2 separate experiments (data not shown). There were not any significant effects on the chronic theophylline treated animals weights (X =
114
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300
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100
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100
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300
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500
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Fig. 1. Scatchard analysis of saturation isotherms of specific [3H]CHA binding to cortical membranes of control (A) and theophylline-treated ( I ) (21 days) rats. Results are from a single experiment performed in triplicate, which was repeated twice with results varying less than 10%. Theophylline-treated cortical Bm~× = 659 f m o l / m g protein; control injected cortical B..... - 508 f m o l / m g protein (P<0.05). Theophylline-treated cortical K o = 1.11 nM; control injected cortical K D = 1.06 nM. Ordinate: bound/free ( f m o l / m g protein per nM; abscissa: [3H]CHA bound (fmol/mg protein).
348g) as compared to the controls (X= 355g) following the chronic regimen. Furthermore, the percentage of the homogenates in the two groups that was comprised of protein was identical. From these results one may infer that adenosine Al-receptors, as labeled by [3H]CHA, in the rat cortex are responsive to fluctuations in their access to adenosine in a manner analogous to many other neurotransmitters and hormones. This suggests that recognition sites for endogenous cotransmitters and neuromodulators may be subject to the same types of regulation and plasticity as neurotransmitter receptors. The role of the presently observed upregulation of cortical adenosine Al-receptors and previous reports of tolerance development to theophylline remains to be established.
Acknowledgements The author gratefully acknowledges the helpful suggestions and discussions of Dr. E. Costa, and the excellent secretarial assistance of Ms. Julie M. Kent.
References Colton, T., R.E. Gosselin and R.P. Smith, 1967, The tolerance of coffee drinkers to caffeine, Clin. Pharmacol. Ther. 9, 31. Murray, T.F. and D . L Cheney, 1982, Neuronal location of N6-cyclohexyl [3H]adenosine binding sites in rat and guinea-pig brain, Neuropharmacol. (in press). Snyder, S.H., J.J. Katims, Z. Annau, R.F. Brans and J.W. Daly, 1981, Adenosine receptors and behavioral actions of methylxanthines, Proc. Natn. Acad. Sci. 78, 3260. Stone, T.W., 1981, Physiological roles for adenosine and adenosine 5'-triphosphate in the nervous system, Neuroscience 6, 523. Watanabe, H., M. Ikeda and K. Watanabe, 1982, Development of tolerance to dopaminergic stimulation effect of theophylline in mice with unilateral striatal 6-hydroxydopamine lesions, European J. Pharmacol. 79, 125.
113
Elsevier Biomedical Press
Rapid communication U P - R E G U L A T I O N OF RAT CORTICAL A D E N O S I N E R E C E P T O R S F O L L O W I N G C H R O N I C A D M I N I S T R A T I O N OF T H E O P H Y L L I N E * T.F. M U R R A Y
College of Pharmacy 6510, Washington State University, Pullman, WA 99164, U.S.A. Received I July 1982, accepted 5 July 1982
Adenosine functions as a putative endogenous neuromodulator or cotransmitter in both the peripheral and central nervous system (Stone, 1981). The methylxanthines, caffeine and theophylline, have been shown to exert their central stimulant effects through a competitive antagonism of adenosine (AI) receptors in the brain (Snyder et al., 1981). Caffeine tolerance has been demonstrated in man (Colton et al., 1967), and recently tolerance development to the stimulating effect of theophylline on the mouse nigrostriatal system has been reported (Watanabe et al., 1982). This latter tolerance was proposed to be a functional rather than a metabolic tolerance as the concentrations of theophylline in the forebrains of tolerant and nontolerant animals did not differ significantly (Watanabe et al., 1982). One neurochemical mechanism of functional tolerance is an up-regulation or increase in the density of the receptors for a given neurotransmitter or drug. The up-regulation can occur in response to the receptors reduced access to the endogenous transmitter substance, or it can occur as a result of the presence of an antagonist for that receptor which in effect reduces it access to its endogenous ligand. Therefore, the present investigation was undertaken to investigate the regulation of adenosine A ~-receptors following chronic treatment of rats with theophylline, an adenosine receptor antagonist. N6-Cyclohexyl-[3H]adenosine ([3H]CHA) (New England Nuclear, spec. act. 11.5 C i / m m o l ) specific binding to brain membranes was performed as * Supported in part by a PMA Foundation Research Starter Grant. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
previously described (Murray and Cheney, 1982). Specific binding was defined as the excess over blanks determined in the presence of 10 /~M Lphenylisopropyladenosine (L-PIA). Saturation isotherms of specific [3H]CHA binding were performed in the presence of a 0.3-10.5 nM concentration range. After 120 min of incubation at 23°C, the entire 1 ml incubation vol was filtered through Whatman G F / B glass-fiber filters. Filters were then washed three times with 5 ml each of ice cold TRIS-HC1 buffer (50 mM, pH 7.7). Radioactivity was counted after 18 h of elution in a Beckman liquid scintillation counter. Male Sprague-Dawley rats weighing 220-250 g were injected twice daily with either 25 m g / k g i.p. theophylline or saline vehicle. After 14 consecutive days of this chronic regimen, the theophylline dose was increased to 45 m g / k g twice daily for seven more days (i.e. total treatment regimen -- 21 days). On day 24 groups of 4 rats each were sacrificed and [3H]CHA binding was determined that day in freshly prepared membranes from the control and chronic theophylline cortices and hippocampi. As shown in fig. l, the chronic administration of theophylline for 21 days resulted in a significant increase in the maximum number of [3H]CHA binding sites (27.9% increase) in cortical membranes, while the affinity of [3H]CHA for its recognition site remained unchanged (Control K D = 1.06 riM; theophylline K D = 1.11 nM). There was also a trend for the Bmax to increase in hippocampal membranes (12.1% increase), but this change did not reach the level of significance in 2 separate experiments (data not shown). There were not any significant effects on the chronic theophylline treated animals weights (X =
114
600
500
400
300
200
100
I
100
200
300
400
500
600
700
Fig. 1. Scatchard analysis of saturation isotherms of specific [3H]CHA binding to cortical membranes of control (A) and theophylline-treated ( I ) (21 days) rats. Results are from a single experiment performed in triplicate, which was repeated twice with results varying less than 10%. Theophylline-treated cortical Bm~× = 659 f m o l / m g protein; control injected cortical B..... - 508 f m o l / m g protein (P<0.05). Theophylline-treated cortical K o = 1.11 nM; control injected cortical K D = 1.06 nM. Ordinate: bound/free ( f m o l / m g protein per nM; abscissa: [3H]CHA bound (fmol/mg protein).
348g) as compared to the controls (X= 355g) following the chronic regimen. Furthermore, the percentage of the homogenates in the two groups that was comprised of protein was identical. From these results one may infer that adenosine Al-receptors, as labeled by [3H]CHA, in the rat cortex are responsive to fluctuations in their access to adenosine in a manner analogous to many other neurotransmitters and hormones. This suggests that recognition sites for endogenous cotransmitters and neuromodulators may be subject to the same types of regulation and plasticity as neurotransmitter receptors. The role of the presently observed upregulation of cortical adenosine Al-receptors and previous reports of tolerance development to theophylline remains to be established.
Acknowledgements The author gratefully acknowledges the helpful suggestions and discussions of Dr. E. Costa, and the excellent secretarial assistance of Ms. Julie M. Kent.
References Colton, T., R.E. Gosselin and R.P. Smith, 1967, The tolerance of coffee drinkers to caffeine, Clin. Pharmacol. Ther. 9, 31. Murray, T.F. and D . L Cheney, 1982, Neuronal location of N6-cyclohexyl [3H]adenosine binding sites in rat and guinea-pig brain, Neuropharmacol. (in press). Snyder, S.H., J.J. Katims, Z. Annau, R.F. Brans and J.W. Daly, 1981, Adenosine receptors and behavioral actions of methylxanthines, Proc. Natn. Acad. Sci. 78, 3260. Stone, T.W., 1981, Physiological roles for adenosine and adenosine 5'-triphosphate in the nervous system, Neuroscience 6, 523. Watanabe, H., M. Ikeda and K. Watanabe, 1982, Development of tolerance to dopaminergic stimulation effect of theophylline in mice with unilateral striatal 6-hydroxydopamine lesions, European J. Pharmacol. 79, 125.