Inhaled nitric oxide (NO) and other cyclic guanosine (cGMP) or adenosine (cAMP) monophosphate-dependent pulmonary vasodilators are often utilized in combination for the treatment of the persistent pulmonary hypertension of the newborn syndrome (PPHN). There is in vitro evidence to indicate that NO downregulate the pulmonary vascular response to cGMP-dependent agonists raising concern as to whether a synergistic effect is observed when employing a combined strategy in newborns. Hypothesizing that a synergistic effect is absent, we evaluated newborn and juvenile rat pulmonary arteries to determine the individual and combined vasodilatory effect of cGMP- and cAMP-dependent agonists. In precontracted near-resistance pulmonary arteries, the addition of sildenafil reduced vasorelaxation response to NO donor S-nitroso-N-acetyl penicillamine (SNAP). A similar decrease in SNAP-induced vasodilation was observed in arteries pre-treated with BAY 41-2272 (10^-9 M), a soluble guanylate cyclase stimulator, cGMP and its downstream protein kinase activator. cGMP also reduced the vasorelaxant response to the cAMP-dependent forskolin. Inhibition of endogenous vascular NO generation enhanced SNAP-induced relaxation. The present data suggest that the mechanism involved in the cGMP desensitization to other relaxant agonists involves downregulation of the small heat shock protein HSP20 and is evident in rat pulmonary and systemic vascular smooth muscle cells. In newborn rats with chronic hypoxia-induced pulmonary hypertension, the combination of sildenafil and inhaled NO resulted in a lesser reduction in pulmonary vascular resistance, when compared with their individual effect. These data suggest that clinical exposure to one cGMP-dependent pulmonary vasodilator may affect the response to other cGMP- or cAMP-mediated agonists.