Heterologous sensitization of adenylate cyclase by Gj-coupled receptors

Prolonged stimulation of G_i-coupled receptors often sensitizes adenylate cyclase to subsequent activation by forskolin (FSK) or Gscoupled receptors. To identify mechanisms of heterologous sensitization, we characterized sensitization induced by recombinant dopamine (DA) D2 receptors expressed in C6 glioma and HEK293 cells. Cells were treated with D2 receptor agonists, followed by determination of isoproterenol (ISO)- or FSK-stimulated cAMP accumulation. Pretreatment with DA or other agonists for 2 hr induced heterologous sensitization that was blocked by the D2 antagonist spiperone, but not by the AR antagonist propranolol. Sensitization was evident after 30 min treatment with agonist, maximal by 2 hr, and persisted for at least 2 hr after washout. Sensitization was observed in intact cells, but not in membranes prepared from cells that had been pretreated with DA. The EC50 for sensitization by DA in HEK cells was 100 nM, -20-fold higher than the EC50 for DA inhibition of adenylate cyclase. In Cg cells, sensitization of ISOstimulated activity was manifested as a 140% increase in maximal response, with no chance in potency. In contrast, the potency for FSKstimulated activity was increased 4-fold, with no apparent change in maximal response. Overnight treatment with pertussis toxin (25 ng/ml) did not affect ISO or FSK activation of adenylate cyclase, but prevented D2-mediated sensitization in both cell lines, indicating an involvement of G/G,. Heterologous sensitization was not inhibited by colchicine, amiloride, or by nonspecific activators (cAMP analogs) or an inhibitor (H7) of protein kinase A. VA Merit Review and PHS MH45372.