Caffeine, a member of the methylxanthine class of drugs, has been called “the most widely used of all psychoactive drugs” (Fredholm, Chen et al. 2005), largely because of its stimulant properties. The mechanisms by which caffeine produces these stimulatory effects are therefore of considerable cultural and clinical interest.
Pharmacology suggests that caffeine stimulates by blocking the A1 adenosine receptor
Although at high doses caffeine can inhibit phosphodiesterase activity (Butcher and Sutherland 1962), dose-response studies indicated that caffeine produces its stimulant effects by blocking adenosine receptors (Snyder, Katims et al. 1981). Even in these early studies it was apparent that there were at least 2 kinds of adenosine receptors, called at that time A1 and A2, and careful comparison of the dose-response relationship between binding to A1 receptors and stimulatory effects led to the conclusion that it seemed “probable that stimulant effects of methylxanthines involve blockade of A1-receptors” (Snyder, Katims et al. 1981). Similarly, A1 receptor agonists led to an electrophysiological state very similar to that observed during sleep (Benington, Kodali et al. 1995), while A1 receptor antagonists reduced sleep (Virus, Ticho et al. 1990). Furthermore, administration of anti-sense constructs directed against the A1 receptor into the basal forebrain decreased the time spent in non-REM sleep (Thakkar, Winston et al. 2003) and produced other effects similar to those produced by caffeine. Therefore it has been generally assumed that the effects of caffeine in increasing wakefulness are due in large part to antagonism of A1 receptors.
Genetic studies indicate that caffeine stimulates by blocking the A2a, not A1, adenosine receptor
Although at high doses caffeine can inhibit phosphodiesterase activity (Butcher and Sutherland 1962), dose-response studies indicated that caffeine produces its stimulant effects by blocking adenosine receptors (Snyder, Katims et al. 1981). Even in these early studies it was apparent that there were at least 2 kinds of adenosine receptors, called at that time A1 and A2, and careful comparison of the dose-response relationship between binding to A1 receptors and stimulatory effects led to the conclusion that it seemed “probable that stimulant effects of methylxanthines involve blockade of A1-receptors” (Snyder, Katims et al. 1981). Similarly, A1 receptor agonists led to an electrophysiological state very similar to that observed during sleep (Benington, Kodali et al. 1995), while A1 receptor antagonists reduced sleep (Virus, Ticho et al. 1990). Furthermore, administration of anti-sense constructs directed against the A1 receptor into the basal forebrain decreased the time spent in non-REM sleep (Thakkar, Winston et al. 2003) and produced other effects similar to those produced by caffeine. Therefore it has been generally assumed that the effects of caffeine in increasing wakefulness are due in large part to an antagonism of A1 receptors.
Genetic studies indicate that caffeine stimulates by blocking the A2a, not A1, adenosine receptor
On the other hand, genetic ablation of the A1 receptor does not lead to any detectable effect on sleep or sleep regulation (Stenberg, Litonius et al. 2003). Of particular interest, although an A1 receptor antagonist reduced sleep in wild-type mice, it had no effect in A1 knockout mice (Stenberg, Litonius et al. 2003). These important studies do not rule out a role for the A1 receptor in sleep regulation in wild-type individuals, since functional compensation (e.g., by the A2a receptor) cannot be completely ruled out. Furthermore, the results with the A1 receptor antagonist do support a potentially important role for the A1 receptor in mediating at least some stimulatory effects of pharmacological agents, since a specific A1 antagonist inhibited sleep in wild-type mice but not in A1 knockout mice (the latter result being the evidence that the drug did indeed function as a specific antagonist to the A1 receptor). Of particular importance, these studies indicate a correlation between the extent to which a drug acts through the A1 receptor to inhibit sleep, and the extent to which that drug will fail to inhibit sleep in A1 knockout mice.
This latter observation is particularly important in the interpretation of the recent study by Huang et al. (2005), in which A1 receptor knockout mice were used to test the long-standing hypothesis that caffeine increases wakefulness by antagonizing the A1 receptor. In this study, sleep was monitored using EEG and ECG; vigilance states were classified into three stages: wakefulness, REM sleep, and non-REM sleep. As expected, administration of caffeine robustly increased wakefulness in wild-type mice. Surprisingly, however, the same dose of caffeine produced essentially the same degree of wakefulness in A1 receptor knockout mice. In contrast, the same dose of caffeine produced no effect on wakefulness in mice in which the A2a receptor had been genetically ablated.
Pharmacology and genetics can be reconciled, but genetics is a better approach in understanding pharmacology
Although original pharmacological studies led to the conclusion that the stimulant effects of methylxanthines (including caffeine) “involve [a] blockade of the A1 receptor” (Snyder, Katims et al. 1981), newer genetic studies have now clearly indicated that the A1 receptor plays little or no role in mediating the stimulant effects of caffeine (Huang, Qu et al. 2005). Seemingly these two approaches would appear to be in conflict. However, this is not necessarily the case. Pharmacological data overwhelmingly indicate a role for the A1 receptor in regulating arousal state, so the fact that A1 knockout mice have normal regulation of sleep is probably due to a functional compensation, most likely by A2a receptors. Nevertheless, the possibility of functional compensation probably does not compromise the conclusion that A2a, but not A1, receptors primarily mediate stimulatory effects of caffeine (Huang, Qu et al. 2005). The credibility of this conclusion is based mainly on two observations. First, as described above, a specific A1 antagonist failed to increase wakefulness in A1 knockout mice (Stenberg, Litonius et al. 2003); thus, if there is functional compensation, it does not appear to entail the induction of A1-like pharmacological targets. Second, the stimulatory effect of caffeine was completely blocked in A2a knockout mice; if A1 receptors played a significant role in mediating effects of caffeine, one would expect to observe at least some stimulatory effect in the A2a knockout mice, indeed one might expect to observe if anything an accentuated effect due to compensation by the A1 receptor system. Therefore with respect to determining the physiological function of the A1 receptor, pharmacological strategies have proved useful, although ironically with respect to elucidating the pharmacology of the A1 receptor, pharmacological strategies have been misleading and genetic studies have been more definitive.
References
Benington, J. H., S. K. Kodali and H. C. Heller. Stimulation of A1 adenosine receptors mimics the electroencephalographic effects of sleep deprivation. Brain Res 1995 692(1-2): 79-85.
Butcher, R. W. and E. W. Sutherland. Adenosine 3',5'-phosphate in biological materials. I. Purification and properties of cyclic 3',5'-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3',5'-phosphate in human urine. J Biol Chem 1962 237: 1244-50.
Fredholm, B. B., J. F. Chen, S. A. Masino and J. M. Vaugeois. Actions of adenosine at its receptors in the CNS: insights from knockouts and drugs. Annu Rev Pharmacol Toxicol 2005 45: 385-412.
Huang, Z. L., W. M. Qu, N. Eguchi, J. F. Chen, M. A. Schwarzschild, B. B. Fredholm, Y. Urade and O. Hayaishi. Adenosine A(2A), but not A(1), receptors mediate the arousal effect of caffeine. Nat Neurosci 2005.
Snyder, S. H., J. J. Katims, Z. Annau, R. F. Bruns and J. W. Daly. Adenosine receptors and behavioral actions of methylxanthines. Proc Natl Acad Sci U S A 1981 78(5): 3260-4.
Stenberg, D., E. Litonius, L. Halldner, B. Johansson, B. B. Fredholm and T. Porkka-Heiskanen. Sleep and its homeostatic regulation in mice lacking the adenosine A1 receptor. J Sleep Res 2003 12(4): 283-90.
Thakkar, M. M., S. Winston and R. W. McCarley. A1 receptor and adenosinergic homeostatic regulation of sleep-wakefulness: effects of antisense to the A1 receptor in the cholinergic basal forebrain. J Neurosci 2003 23(10): 4278-87.
Virus, R. M., S. Ticho, M. Pilditch and M. Radulovacki. A comparison of the effects of caffeine, 8-cyclopentyltheophylline, and alloxazine on sleep in rats. Possible roles of central nervous system adenosine receptors. Neuropsychopharmacology 1990 3(4): 243-9.
