Role of PKC-α and δ isoforms in AVP improved contractile response of VSMC after hypoxia and its relations to MLC20 phosphorylation

AIM:The present study was to investigate the roles of protein kinase C α and δ isoforms (PKC-α, δ) in arginine vasopressin (AVP) improved contractile response of vascular smooth muscle cells (VSMC) to norepinephrine (NE) after hypoxia and its relations to myosin light chain (MLC₂₀) phosphorylation, myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK) activity.METHODS: Primary cultures of VSMC were obtained from the superior mesenteric artery (SMA) of rats by explanting technique and the cells in third to fifth passage were used in the study. The effects of PKC-α and δ antagonists on AVP induced contractile response of VSMC to NE after 1.5 h hypoxia were observed by measuring the ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated bovine serum albumin with transwell, and their effect on the activity of MLCP/MLCK in VSMC was assayed by enzymatic catalysis. At the same time, with the SMA from hemorrhagic shock rats (30 mmHg for 2 h), the effects of PKC α and δ isoforms in the regulation of AVP on MLC₂₀ phosphorylation of SMA after shock were observed by Western blotting.RESULTS: G 6976 (5×10^-6 mol/L, PKC-α isoform inhibitor) significantly antagonized AVP (5×10^-10 mol/L)-induced increase in the contractile response of VSMC to NE after hypoxia, and rottlerin (10^-5 mol/L, PKC-δ isoform inhibitor) also partly inhibited this effect. Hypoxia resulted in a significant increase in MLCP activity, with a decrease in MLCK activity of VSMC, and at the same time, the MLC₂₀ phosphorylation of SMA following hemorrhagic shock was significantly decreased. AVP inhibited the activity of MLCP and increased the phosphorylation of MLC₂₀, which was inhibited by G 6976, while rottlerin treatment only showed a slightly inhibitory effect. AVP and PKC-α, δ inhibitor had no significant influence on MLCK activity.CONCLUSION:AVP up-regulates vascular reactivity and calcium sensitivity of VSMC possibly through inhibiting the activity of MLCP and increasing the phosphorylation of MLC₂₀ by PKC-α isoform.