Endogenous ghrelin and its synthetic counterpart hexarelin are peptide GH secretagogues (GHS) that exert a positive ionotropic effect in the cardiovascular system. The mechanism by which GHS modulate cardiac electrophysiology properties to alter myocyte contraction is poorly understood. In the present study, we examined whether GHS regulates the transient outward potassium current (I(to)) as well as the putative intracellular signaling cascade responsible for such regulation. GHS and experimental agents were applied locally onto freshly isolated adult Sprague-Dawley rat ventricular myocytes and action potential morphology and I(to) was recorded using nystatin-perforated whole-cell patch-clamp recording technique. Under current clamp, ghrelin and hexarelin (10 nm) significantly prolonged action potential duration. Under voltage clamp, hexarelin and ghrelin inhibited I(to) in a concentration-dependent manner. This inhibition was abolished in the presence of the GHS receptor (GHS-R) antagonist [D-Lys(3)]GH-releasing peptide-6 (10 microm) and GHS-R1a-specific antagonist BIM28163 (1 microm). GHS-induced I(to) inhibition was totally reversed by the phospholipase C inhibitor U73122 (5 microm) and protein kinase C inhibitors GO6983 (1 microm) and calphostin C (0.1 microm) but not by the cAMP antagonist Rp-cAMP (100 microm) or the PKA inhibitor H89 (1 microm). We conclude that hexarelin and ghrelin activate phospholipase C and protein kinase C signaling cascade through the stimulation of the GHS-R, resulting in a decrease in the I(to) current and subsequent prolongation of action potential duration.