In rat pinealocytes, alpha 1-adrenergic receptor activation increases intracellular pH (pHi) through Ca2+/protein kinase-C-dependent activation of the Na+/H+ antiporter. Using a series of amiloride analogs, norepinephrine stimulation of cGMP accumulation is also found to be pHi dependent. In this study, we examined the postreceptor mechanisms involved in the amiloride effects on cyclic nucleotide accumulation using agents that simulate alpha 1-adrenoceptor activation. Four amiloride analogs, with a 500-fold difference in their inhibitory potency of the Na+/H+ antiporter, were used. 5-(N,N-Hexamethylene)amiloride (HA), the most active inhibitor of the Na+/H+ antiporter, had a stimulatory effect on isoproterenol (ISO)-stimulated cAMP, while its effect on cGMP was inhibitory. The other three amiloride derivatives had no effect on the ISO-stimulated cAMP or cGMP responses. All four amilorides (at 10 microM) had no effect on the phenylephrine potentiation of cAMP responses in beta-adrenergically stimulated cells, while they inhibited the potentiation of cGMP accumulation according to their inhibitory potency on the Na+/H+ antiporter. Using depolarizing concentrations of K+, it was found that HA was additive to the submaximal potentiation by K+ on ISO-stimulated cAMP, while its effect on cGMP was inhibitory. Amiloride hydrochloride dihydrate, the amiloride that is least potent in its inhibitory action on the Na+/H+ antiporter, had no effect on the K+ potentiation of either cAMP or cGMP. Using 4 beta-phorbol 12-myristate 13-acetate in cells treated with 10 mM K+ and ISO, it was found that HA was additive to phorbol 12-myristate 13-acetate and K+ potentiation of the cAMP response, while its effect on the cGMP response was inhibitory. Amiloride hydrochloride had no effect on either the cAMP or cGMP response. It can be concluded from these studies that 1) HA has a stimulatory effect on the beta-adrenoceptor-Gs-adenylate cyclase pathway that is independent of inhibition of the Na+/H+ antiporter; 2) postreceptor mechanisms are involved in HA's effects on cAMP and cGMP accumulation; and 3) the action of HA on cGMP is likely to be related to its effect on the Na+/H+ antiporter.