Cyclic GMP-dependent protein kinase Ialpha inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells. J Biol Chem 2006 Mar 31;281(13):8409-16
Date
02/01/2006Pubmed ID
16446362DOI
10.1074/jbc.M512770200Scopus ID
2-s2.0-33646853170 (requires institutional sign-in at Scopus site) 20 CitationsAbstract
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.
Author List
Christensen EN, Mendelsohn MEMESH terms used to index this publication - Major topics in bold
8-Bromo Cyclic Adenosine MonophosphateAdenoviridae
Animals
Blotting, Western
CHO Cells
Calcium
Cell Culture Techniques
Cell Line, Transformed
Cell Transformation, Viral
Coronary Vessels
Cricetinae
Cyclic GMP-Dependent Protein Kinases
Enzyme Activation
Enzyme Inhibitors
Humans
Immunohistochemistry
Isoenzymes
Muscle, Smooth, Vascular
RNA Interference
RNA, Small Interfering
Receptors, Thrombin