Multiple mechanisms for desensitization of A2a adenosine receptor-mediated cAMP elevation in rat pheochromocytoma...

To understand the regulation of A2a adenosine receptor (A2a-R) response, we examined the molecular mechanisms underlying the desensitization of A2a response in rat pheochromocytoma PC12 cells, which possess an A2a-R identical with the A2a receptor we recently cloned from rat brain. Prolonged exposure of PC12 cells to adenosine agonists significantly inhibited the response of the cells to subsequent stimulation with an A2a-selective adenosine agonist (CGS21680). No significant change in the number of binding sites and affinity for CGS21680 was observed in desensitized cells, nor did we find any significant change in the transcript level of A2a-R in cells pretreated with adenosine agonists. However, the basal adenylyl cyclase activity and the cyclase activities stimulated by adenosine agonists, by GTP gamma S, and by forskolin were reduced in desensitized cells. Prolonged exposure of PC12 cells to dibutyryl-cAMP did not significantly change either the basal or the adenosine agonist-evoked adenylyl cyclase activity. Therefore, elevation of cellular cAMP content is by itself not sufficient to produce the observed reductions of adenylyl cyclase activity with A2a desensitization. Inhibition of adenylyl cyclase activity in desensitized cells occurred after short-term (30 min) incubation with CGS21680 and could be blocked by the adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine. Gs alpha protein levels did not significantly change after a 30-min exposure to CGS21680. In contrast, long-term exposure (12-20 hr) of PC12 cells to adenosine agonists resulted in a slight further reduction of adenylyl cyclase activity and a consistent decline in the Gs alpha protein level. In addition, long-term incubation with adenosine agonists or with forskolin-enhanced phosphodiesterase (PDE) activity in the cytosolic and membrane fractions by 57 +/- 9% and 53 +/- 18%, respectively. Hydrolysis of cAMP was significantly faster in agonist-desensitized cells than in control cells. PDE might therefore play an important role in desensitization of the A2a response in PC12 cells. Polymerase chain reaction-based analysis of the mRNA for A2a-R and A2b-R indicated that both A2a-R and A2b-R were present in PC12 cells; the A2b response was also diminished in A2a-desensitized cells. Our data suggest that inhibition of adenylyl cyclase after short-term agonist treatment, down-regulation of Gs alpha protein level after long-term agonist treatment, and activation of PDE after long-term agonist treatment account for desensitization of the A2a-mediated response in PC12 cells.