Physiological roles of A1 and A2A adenosine receptors : studies using genetically modified mice

Abstract

Since adenosine receptors were identified and cloned, their roles in animals and humans have been extensively studied. Because pharmacological tools have limitations, we have used mice in which adenosine receptors have been deleted to further study their roles under physiological and pathophysiological conditions. Caffeine exerts its effects mainly via blockade of A1 and A2A adenosine receptors. In this thesis, the physiological roles of these receptors have been explored using in vivo and ex vivo models of heart physiology in mice with genetically modified expression of A1R and A2AR. The possible role of A1R in cardiac protection due to by preconditioning was also studied, and the action of caffeine explored.

When A1R and/or A2AR genes were knocked out or down little compensatory adaptation was observed in the other adenosine receptors or related genes and proteins, which facilitates the use of genetically modified mice to study the roles of the specific receptors.

We show a critically important role of A1R in the cardiac protection following in vivo ischemic preconditioning and in tolerance to ischemia. The cardioprotective effect induced by ischemic preconditioning was A1R gene dose-dependent in vivo: the protective effect was enhanced by overexpression of A1R but eliminated by knockout of A1R; a reduced protection was seen in partially A1R-deleted hearts. The tolerance to ischemia was significantly reduced in isolated A1R knockout hearts.

We also examined physiological roles of adenosine receptors. Compared to WT, HR increased in male A1R KO mice, but deceased in A2AR KO mice. The HR difference was eliminated by administration of beta-blocker, indicating that the modulatory effect of alteration of A1R and A2AR on HR is exerted via sympathetic tone. Interestingly, the HR of A1R-A2AR dKO and A1R-A2AR dHz mice was lower than that of WT mice, suggesting that the contribution of A1R and A2AR to HR modulation is not equal. Injection of caffeine in a low dose increased HR in WT mice. Its stimulatory effect was reduced in mice lacking A1R and eliminated in mice lacking the A2AR, suggesting both A1R and A2AR contribute to HR stimulation by caffeine, where A2AR is more critical than A1R. High dose caffeine decreased HR in all mouse genotypes, which clearly indicates that the depressant effect of caffeine is A1R and A2AR independent. Long-term caffeine treatment and partial deletion of A1R and A2AR genes decreased HR. Both A1R and A2AR were involved in modulation of body temperature and oxygen consumption, an effect which was sex-dependent, and altered by caffeine. Locomotor activity was slightly higher in female (but not male) A1R KO mice, and lower in mice lacking A2AR than in their WT controls, suggesting that A2AR may be more important than A1R in regulating locomotion. Caffeine stimulated locomotion dose-dependently. This effect was eliminated in mice lacking A2AR and markedly reduced in the other genotypes. Long-term caffeine treatment increased locomotion in a sex-dependent manner, but not in mice lacking A2AR, indicating that caffeine modulates locomotion mainly via blockade of the A2AR. Deleting one of the copies of both the A1R and A2AR genes increased activity, thus resembling long-term caffeine treatment. The response to caffeine was blunted in A1R-A2AR dHz mice, again resembling long-term caffeine use. Hence in some respects A1R-A2AR dHz mice are similar to long-term caffeine treated mice.

In summary, A1R plays a critical role in cardiac protection. Physiologically both A1R and A2AR contribute to modulation of HR, body temperature, locomotor activity, and oxygen consumption in a sex-dependent manner. Caffeine modulates these effects by partially blocking A1R and A2AR, and A2AR may be more important than A1R. Deletion of one copy of both A1R and A2AR genes mimics the long-term caffeine effect, at least in some aspects. Both A1R and A2AR contribute to the development of tolerance to caffeine. Thus, adenosine plays both physiological and pathophysiological roles.