Cyclic GMP-dependent protein kinase Iα inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells

Vascular smooth muscle contractile state is regulated by intracellular calcium levels. Nitric oxide causes vascular relaxation by stimulating production of cyclic GMP, which activates type I cGMP- dependent protein kinase ( PKGI) in vascular smooth muscle cells ( VSMC), inhibiting agonist- induced intracellular Ca2+ mobilization ([Ca2+] (i)). The relative roles of the two PKGI isozymes, PKGI alpha and PKGI beta, in cyclic GMP- mediated inhibition of [Ca2+] (i) in VSMCs are unclear. Here we have investigated the ability of PKGI isoforms to inhibit [Ca2+](i) in response to VSMC activation. Stable Chinese hamster ovary cell lines expressing PKGI alpha or PKGI beta were created, and the ability of PKGI isoforms to inhibit [Ca2+](i) in response to thrombin receptor stimulation was examined. In Chinese hamster ovary cells stably expressing PKGI alpha or PKGI beta, 8- Br- cGMP activation suppressed [ Ca2+](i) by thrombin receptor activation peptide ( TRAP) by 98 +/- 1 versus 42 +/- 5%, respectively ( p < 0.002). Immunoblotting studies of cultured human VSMC cells from multiple sites using PKGI alpha- and PKGI beta- specific antibodies showed PKGI alpha is the predominant VSMC PKGI isoform. [Ca2+](i) following thrombin receptor stimulation was examined in the absence or presence of cyclic GMP in human coronary VSMC cells ( Co403). 8- Br- cGMP significantly inhibited TRAP- induced [Ca2+](i) in Co403, causing a 4- fold increase in the EC50 for [Ca2+](i). In the absence of 8- Br- cGMP, suppression of PKGI alpha levels by RNA interference ( RNAi) led to a significantly greater TRAP- stimulated rise in [Ca-i(2+]) as compared with control RNAi- treated Co403 cells. In the presence of 8- Br- cGMP, the suppression of PKGI alpha expression by RNAi led to the complete loss of cGMP- mediated inhibition of [Ca2+](i). Adenoviral overexpression of PKGI beta in Co403 cells was unable to alter TRAP- stimulated Ca2+ mobilization either before or after suppression of PKGI alpha expression by RNAi. These results support that PKGI alpha is the principal cGMP- dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/ cyclic GMP pathway.