Adenosine and adrenergic agonists modulate neutrophil function by ligating their specific receptors (adenosine A2 and beta-adrenergic) on the neutrophil. When occupied, adenosine A2 and beta-adrenergic receptors stimulate, presumably via G alpha s, an increase in intracellular 3', 5' cyclic adenosine monophosphate (cAMP). cAMP affects cellular functions, in part, via protein kinase-mediated phosphorylation. Therefore, we determined whether inhibition of protein kinase A activity by KT5720 (10 mumol/L) reversed the inhibition of FMLP-stimulated O2- generation by 5'N-ethylcarboxamidoadenosine (NECA), the most potent adenosine A2 agonist, and by isoproterenol a potent beta-adrenergic agonist. KT5720 did not affect O2- generation stimulated by FMLP (125% +/- 13% of control, n = 5). However, KT5720 completely reversed inhibition of O2- generation by dibutyryl cAMP (DbcAMP, 1 mmol/L, from 26% +/- 5% to 84% +/- 25% of control, n = 5, P less than .004), but not by NECA (1 mumol/L, 26% +/- 5% v 33% +/- 7% of control, n = 5) or isoproterenol (10 mumol/L, 20% +/- 8% to 38% +/- 6% of control, n = 5). Nearly identical results were obtained using the less specific protein kinase inhibitor H-7. To determine whether occupancy of adenosine A2 or beta-adrenergic receptors inhibits neutrophil (PMN) activation by uncoupling chemoattractant receptors from G proteins, we determined the effect of NECA and isoproterenol on guanosine triphosphatase (GTPase) activity, a parameter that reflects G protein "activation," of plasma membranes derived from human PMNs. Control GTPase activity was 138.9 pmol/mg protein/min; NECA (1 nmol/L to 1 mumol/L) and isoproterenol (10 nmol/L to 10 mumol/L) alone did not significantly affect GTPase activity. FMLP (0.1 mumol/L) increased GTPase activity by 31.9 +/- .9 pmol/mg/min, an increment that was markedly inhibited to approximately 50% of control by NECA (IC50 = 3 nmol/L, P less than .001, n = 5) and isoproterenol (IC50 = 30 nmol/L, P less than .001, n = 5). Neither cAMP nor dibutyryl cAMP (10 mumol/L and 1 mmol/L) affected resting or stimulated GTPase activity. In addition, neither adenosine nor DbcAMP affected protein phosphorylation in resting or stimulated neutrophils. Our studies are consistent with the hypothesis that ligation of G alpha s-linked receptors uncouples chemoattractant receptors from their signal-transduction mechanisms rather than inhibiting neutrophil function via cAMP-mediated effects.