Protamine does not affect the formation of cGMP or cAMP in pig vascular smooth muscle cells in response to vasodilators

Objectives: Protamine has recently been shown to have a direct vasodilator action in isolated vascular tissue. As one possible mechanism for this action, it has been hypothesized that protamine might increase the response of vascular smooth muscle to the endothelium-derived relaxing factor, nitric oxide. In this study, we tested this hypothesis and examined the effect of protamine on other guanosine 3'5'-cyclic monophosphate (cGMP)- and adenosine 3'5'-cyclic monophosphate (cAMP)-dependent processes. Design: Prospective, repeated measures analysis of concentration-response curves. Setting: Anesthesia research laboratory in an academic medical center. Subjects: Cultured coronary artery smooth muscle cells from pig heart. Interventions: Sodium nitroprusside was used to mimic the action of the endothelium-derived relaxing factor by stimulating the soluble guanylyl cyclase and increasing intracellular cGMP. Atrial natriuretic peptide was used to stimulate the particulate guanylyl cyclase. Isoproterenol and forskolin were used to increase intracellular cAMP. The responses to these agents were determined in the presence and absence of protamine. Measurements and Main Results: In cultured vascular smooth muscle cells, sodium nitroprusside increased cGMP, the second messenger for endothelium-derived relaxing factor, in a concentration-dependent manner. In cells treated with protamine (32 to 250 μg/mL), we could detect no effect of protamine on basal intracellular levels of cGMP until a concentration of 250 μg/mL of protamine was used. At this concentration, protamine increased basal cGMP concentrations from 4.2 ± 0.3 to 9.0 ± 0.6 pmol/mg protein (p < .001). The response of intracellular cGMP to sodium nitroprusside in cells treated with 250 μg/mL or other concentrations of protamine was not different from control. Likewise, we could detect no effect of protamine on intracellular cGMP stimulated with the atrial natriuretic peptide or on cAMP stimulated with the β-adrenergic receptor agonist, isoproterenol, or with forskolin. Conclusions: These experiments show that protamine does not alter the responses of the intracellular second messengers, cGMP and cAMP, to the vasodilators sodium nitroprusside, atrial natriuretic peptide, isoproterenol, and forskolin. These results do not support the hypothesis that protamine sensitizes vascular smooth muscle cells to the endothelium-derived relaxing factor, nitric oxide.