title: Cyclic GMP-dependent protein kinase Ialpha inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells.
abstract: 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, PKGIalpha and PKGIbeta, 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 PKGIalpha or PKGIbeta 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 PKGIalpha or PKGIbeta, 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 PKGIalpha- and PKGIbeta-specific antibodies showed PKGIalpha 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 PKGIalpha 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 PKGIalpha expression by RNAi led to the complete loss of cGMP-mediated inhibition of [Ca2+]i. Adenoviral overexpression of PKGIbeta in Co403 cells was unable to alter TRAP-stimulated Ca2+ mobilization either before or after suppression of PKGIalpha expression by RNAi. These results support that PKGIalpha is the principal cGMP-dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/cyclic GMP pathway.
authors.affiliation: Molecular Cardiology Research Institute, Tufts-New England Medical Center and Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: Eva N
authors.initials: EN
authors.lastname: Christensen
authors.forename: Michael E
authors.initials: ME
authors.lastname: Mendelsohn
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003331:Coronary Vessels / Q000166:cytology; D006224:Cricetinae; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D018179:Receptors, Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D034741:RNA, Small Interfering; D018179:Receptors, Thrombin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017869:Cyclic GMP-Dependent Protein Kinases; D002118:Calcium