The involvement of nitric oxide (NO) in human GH-releasing hormone (hGHRH)-induced GH secretion was studied with freshly dissociated male rat pituitary cells. The cells were packed in a column of Bio-Gel-P2 and continuously perifused at 37 C. Hemoglobin (Hb; 10 microM), which is known to strongly bind NO, potentiated 0.01, 0.1, and 1 nM hGHRH-induced GH secretion by 73%, 52%, and 39%, respectively, without affecting the basal secretion of GH. As reported previously, 1-nM or higher concentrations of hGHRH elicit an increase in GH secretion during the application of hGHRH (on-response) and also a transient increase after the cessation of hGHRH application (off-response). It was found that Hb potentiated only the off-response in 1 nM hGHRH-induced GH secretion, and the same concentration of Hb had no effect on 10 nM hGHRH-induced GH secretion. N-Methyl-L-arginine (MeArg; 500 microM), a competitive inhibitor of NO synthase, also potentiated both the on- and off-responses of 1 nM hGHRH-induced GH secretion by 39% without affecting basal GH secretion. Since cAMP is thought to be an intracellular messenger of hGHRH action, the effects of Hb and MeArg on 1 mM (Bu)2AMP-induced GH secretion were examined. Their actions were found to be greater than those in hGHRH-induced GH secretion. Excess K+ (15 and 50 mM)-induced GH secretion, which does not involve cAMP, however, was not affected by either Hb or MeArg. In contrast, 3 mM sodium nitroprusside, which releases NO, suppressed the 1 nM hGHRH-induced off-response by 18%. The same concentration of sodium nitroprusside had no effect on excess K(+)-induced GH secretion. The effect of 8-bromo-cGMP on hGHRH-induced GH secretion was also examined, since NO is thought to exert its action through cGMP by activating guanylate cyclase in neural tissue. The application of 8-bromo-cGMP, however, did not affect 1 nM hGHRH-induced GH secretion. These observations suggest that hGHRH stimulates the synthesis of NO at least partly through cAMP, thereby partially inhibiting hGHRH-induced GH secretion.