期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 281, 期 13, 页码 8409-8416出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M512770200
关键词
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资金
- NHLBI NIH HHS [HL535309] Funding Source: Medline
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.
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