cGMP signals mainly through cAMP kinase in permeabilized murine aorta

Abstract

GMP affects vascular tone by multiple mechanisms, including inhibition of the Rho/Rho kinase-mediated Ca2+sensitization, a process identified as Ca2+desensitization. Ca2+desensitization is mediated probably by both cGMP- and cAMP-dependent protein kinases (cGKI and PKA). We investigate to which extent Ca2+desensitization is initiated by cGKI and PKA. cGMP/cAMP-induced relaxation was studied at constant [Ca2+] in permeabilized aortas from wild-type and cGKI-deficient mice. [Ca2+] increased aortic tone in the absence and presence of 50 μM GTPγS with EC50values of 160 and 30 nM, respectively. In the absence of GTPγS, the EC50for [Ca2+] was shifted rightward from 0.16 μM to 0.43 and 0.82 μM by 1 and 300 μM 8-bromo-cGMP (8-Br-cGMP), and to 8 μM by 10 μM Y-27632. Contractions induced by 300 nM [Ca2+] were relaxed by 8-Br-cGMP with an EC50of 2.6 μM. Surprisingly, [Ca2+]-induced contractions were also relaxed by 8-Br-cGMP in aortas from cGKI−/−mice (EC50of 19 μM). Western blot analysis of the vasodilator-stimulated phosphoprotein indicated “cross”-activation of PKA by 1 mM 8-Br-cGMP in aortic smooth muscle cells from cGKI−/−mice. Indeed, the PKA inhibitor peptide (PKI 5–24) completely abolished the relaxant effect of 8-Br-cGMP in muscles from cGKI−/−mice and to 65% in wild-type aortas. The thromboxane analogue U-46619 induced contraction at constant [Ca2+], which was only partially relaxed by 8-Br-cGMP but completely relaxed by Y-27632. The effect of 8-Br-cGMP on U-46619-induced contraction was attenuated by PKI 5–24. These results show that cGKI has only a small inhibitory effect on Ca2+sensitization in murine aortas.