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J. Biol. Chem., Vol. 281, Issue 13, 8409-8416, March 31, 2006

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Cyclic GMP-dependent Protein Kinase I Inhibits Thrombin Receptor-mediated Calcium Mobilization in Vascular Smooth Muscle Cells^*

From the Molecular Cardiology Research Institute, Tufts-New England Medical Center and Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

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 and PKGI, 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 or PKGI 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 or PKGI, 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- and PKGI-specific antibodies showed PKGI 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 levels by RNA interference (RNAi) led to a significantly greater TRAP-stimulated rise in [Ca²⁺]_i as compared with control RNAi-treated Co403 cells. In the presence of 8-Br-cGMP, the suppression of PKGI expression by RNAi led to the complete loss of cGMP-mediated inhibition of [Ca²⁺]_i. Adenoviral overexpression of PKGI in Co403 cells was unable to alter TRAP-stimulated Ca²⁺ mobilization either before or after suppression of PKGI expression by RNAi. These results support that PKGI is the principal cGMP-dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/cyclic GMP pathway.

Received for publication, November 29, 2005 , and in revised form, January 30, 2006.

^* This work was supported in part by National Institutes of Health Grant HL55309 (to M. E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Tufts University School of Medicine, New England Medical Center, Molecular Cardiology Research Inst., 750 Washington St., Box 80, Boston, MA 02111. Tel.: 617-636-9370; Fax: 617-636-1444; E-mail: mmendelsohn{at}tufts-nemc.org.

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