Effect Of AVP On Vascular Reactivity And Calcium Sensitivity After Hemorrhagic Shock In Rats And Its Relationship To α,δ And ε Isoforms Of Protein Kinase C

The occurrence of vascular hyporeactivity after severe trauma or shock has been shown to have important roles in the incidence, development, and the outcome of shock and interfered with the therapy of shock. Arginine vasopressin (AVP) is a 9-amino-acid neurohypophysial peptide hormone synthesized in the hypothalamus. Our previous studies have demonstrated that hemorrhagic shock could also cause calcium desensitization of blood vessels, which played important roles in vascular hyporeactivity following hemorrhagic shock, and protein kinase C (PKC) and Rho-kinase can regulate the calcium sensitivity of vascular smooth muscle cell (VSMC) by induced the inhibition of myosin light chain phosphatase (MLCP). And recent observations from our laboratory showed that AVP is beneficial to hemorrhagic shock, which may be related to AVP-induce increase of vascular reactivity and calcium sensitivity of vascular smooth muscle cell via the activation of Rho-kinase. However, Rho-kinase antagonist, Fasudil and Y-27632 only partially blocked this effect of AVP, it's suggested that there may be other mechanisms involved in the effect of AVP. PKC was lipid-regulated serine/threonine kinases, included multiple isozymes with distinct tissue distributions and regulatory properties. However, little is known about which isoforms of PKC might be involved in modulating vascular reactivity and calcium sensitivity of vascular smooth muscle after hemorrhagic shock induced by AVP and the precise mechanisms. So with hemorrhagic shock model of rats and hypoxia-treated VSMC, we observed the effects of AVP on vascular reactivity amd calcium sensitivity following hemorrhagic shock in rats and explore its relations withα,δandεisoforms of PKC.Methods:The experiments were conducted in three parts. Part I. The effects of AVP on vascular reactivity following hemorrhagic shock in rats1. In vivo—The hemorrhagic shock (30 mmHg for 2 hours) model of rats was adopted to observe the effects of AVP(0.04,0.1,0.4 U/kg) on the contractile response of SMA to norepinephrine(NE) and pressor effect of NE.2. In isolated SMA—Superior mesenteric artery (SMA) rings from rats were used to observe the effects of AVP(5×10^-11,5×10^-10,5×10^-9 mol/L) on vascular reactivity of SMA with isolated organ perfusion system (The vascular reactivity was observed by measuring the contraction initiated by accumulative NE).3. In VSMC—Primary cultures of VSMCs were obtained from the mesenteric artery of Wistar rats by an explant technique and the third to fifth passage cells were used in the present study. The contractile response of cultured VSMC to NE at different time after 1.5 hours hypoxia and the effects of AVP were observed (The contractile response of VSMC was measured by the ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated bovine serum albumin with transwell).Part II. The effects of AVP on vascular reactivity and calcium sensitivity of vascular smooth muscle and its relationship to PKCα,δandεisoforms1. In isolated SMA—With isolated SMA rings from hemorrhagic shock rats, the effects of AVP on vascular reactivity and calcium sensitivity of SMA from hemorrhagic shock rats and and the effects of PKCα,δ,εinhibitor were observed (The calcium sensitivity of SMA were observed by measuring the contraction initiated by accumulative calcium under depolarizing condition (120 mM K+) with an isolated organ perfusion system). And the myosin light chain (MLC20) phosphorylation of mesenteric artery smooth muscle was detected by Western blotting.2. In VSMC—With hypoxia-treated VSMCs, the contractile response of VSMC to NE after 1.5 h hypoxia and the effects of AVP and PKCα,δ,εinhibitor were observed. And the effect of AVP on the expression of PKC-α,δandεisoforms in the cytosol and particulate fractions of VSMCs, the activity of MLCP and myosin light chain kinase (MLCK) of VSMC were also observed.Part III. The mechanism of AVP induced the PKC isozymes activation 1. In isolated SMA—The effects of V_1a and V₂ receptor inhibitor on AVP improving vascular reactivity and calcium sensitivity of SMA from hemorrhagic shock rats and its relationship to the phosphorylation of MLC₂₀ were observed.2. In VSMC—With hypoxia-treated VSMCs, we observe the effects of V_1a and V₂ receptor inhibitor on AVP regulating the expression of PKC-α,δandεisoforms in the cytosol and particulate fractions of VSMCs. At the same time, the activity of MLCP/MLCK and the contractile response of VSMC to NE were assayed. In addition, the activity of phospholipase C (PLC), phospholipase D (PLD), phospholipase A₂ (PLA₂) and the effects of V_1a and V₂ receptor inhibitor were observed.Results:1. Effects of AVP on vascular reactivity following hemorrhagic shock in ratsIn vivo NE-induced pressor response and vasoconstriction of SMA following hemorrhagic shock was significantly decreased (P<0.01), AVP (0.04, 0.1 and 0.4 U/kg) significantly increased the pressor effect of NE and vasoconstriction of SMA to NE in hemorrhagic shock rats (P<0.05⁰.01). In vitro, AVP (5×10-11, 5×10-10, 5×10-9 mol/L) pretreatment also improved reactivity of SMA rings to NE following hemorrhagic shock and made the cumulative dose-response curve of NE shift to the left, Emax was increased significantly (P<0.01). After hypoxia for 1.5 h, the contractile response of VSMC to NE significant decreased, AVP pretreatment increased the contractile response of VSMCs, its ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated BSA was significantly increased at 45, 60 and 75 min after NE administration (P<0.05^P<0.01). It was suggested that AVP significantly improved the hypoxia or hemorrhagic shock-induced decrease in contractile reactivity of SMA and VSMC.2. Effects of AVP on calcium sensitivity of vascular smooth muscle and its relationship with PKCα,δandεisoforms(1) AVP markedly restored the decreased sensitivity of SMA to NE and Ca2+ following hemorrhagic shock, the concentration-response curve of NE and Ca2+ was shifted to the left and Emax was significantly increased (P<0.01). G? 6976 (the specific PKCαisoform inhibitor), Rottlerin (the specific PKCδisoform inhibitor) and PKCεinhibitor peptide antagonized AVP-induced increase of vascular reactivity and calcium sensitivity of SMA following hemorrhagic shock (P<0.05～P<0.01), in which PKCεinhibitor peptide showed a stronger antagonistic effectiveness than the others. The data suggested that PKCα,δandεisoforms participated in the regulation of AVP on calcium sensitivity and vascular reactivity after shock with different importance.(2) AVP pretreatment significant increased the contractile response of VSMC to NE (P<0.05～P<0.01), the effects of AVP was significantly blocked by G? 6976 and PKCεinhibitor peptide (P<0.01), while partly inhibited by Rottlerin (P<0.05). The expression of particulate PKCαandεincreased in response to hypoxia, with a concomitant decrease in cytosolic fractions. AVP treatment further increased expression of PKCαandεin the particulate fractions (P<0.05^P<0.01), but the PKCαandεlevels in the cytosolic fractions were not significant changes. While PKCδshowed a similar changes in either the particulate or the cytosolic fractions during the process, but there were no statistical differences among the groups. It was suggested that AVP improved vascular reactivity and calcium sensitivity following hemorrhagic shock through translocating PKCαandεisoforms from a cytosol to a particulate and activation.(3) The MLC20 phosphorylation of SMA following hemorrhagic shock were significantly decreased (P<0.01), AVP treatment resulted in an increase in MLC20 phosphorylation (P<0.01), which could be inhibited by G? 6976 and PKCεinhibitor peptide, not Rottlerin. The results suggested that PKCαandεisoforms played an important role in AVP regulating vascular reactivity and calcium sensitivity through MLC20 phosphorylation.(4) Treatment with hypoxia for 1.5 h caused a significant increase in MLCP activity, with a decrease in MLCK activity of VSMC, AVP treatment resulted in an inhibition of MLCP activity (P<0.05), G(o|¨)6976 and PKCεinhibitor peptide (P<0.01), not Rottlerin, abolished AVP-induced decrease of MLCP activity. But there was no significant influence on MLCK activity.. The results of the present study suggested that AVP restores the vascular reactivity and calcium sensitivity of vascular smooth muscle following hemorrhagic shock via an activation of PKCαandεisoforms, and its mechanisms may be related to induce decrease of MLCP activity and MLC20 phosphorylation.3. Mechanism of AVP induced the PKC isozymes activation(1) [d(CH2)5-Tyr2(Me)]AVP, the V_1a receptor inhibitor, significantly antagonized AVP-induced increase of vascular reactivity and calcium sensitivity of SMA following hemorrhagic shock and V₂ receptor inhibitor ([d(CH₂)(d-Ile²Abu⁴)]AVP) also partly inhibit this effect (P<0.05). And V_1a receptor inhibitor also blocked AVP-induced increase of the contractile response of VSMCs to NE (P<0.01), while V₂ receptor inhibitor only slightly inhibited this effect of AVP. The results showed that AVP may restore the decreased vascular reactivity and calcium sensitivity of vascular smooth muscle after hemorrhagic shock through V_1a and V₂ receptor, and V_1a receptor may play more important roles than V₂ receptor.(2) The expression of particulate PKCαandεincreased in response to hypoxia, AVP treatment further increased expression of PKCαandεin the particulate fractions, V_1a receptor inhibitor significantly antagonized this effect of AVP (P<0.05^P<0.01), but V₂ receptor inhibitor had no effect. While PKCδshowed a similar changes, but there were no statistical differences. AVP treatment resulted in an increase in MLC20 phosphorylation of SMA (P<0.01) and decreased in MLCP activity of VSMC (P<0.05), which could be inhibited by V_1a receptor inhibitor, not V₂ receptor inhibitor. MLCK activity of VSMC were significantly decreased after hypoxia (P<0.01), but AVP and V_1a/V₂ receptor inhibitor had no effect on it. It was suggested that AVP regulating the calcium sensitivity was mainly related to V_1a receptor, not V₂ receptor.(3) Treatment with hypoxia for 1.5 h caused a significant increase in PLC and PLD activity, AVP further increased the activity of PLC and PLD (P<0.05). V_1a receptor inhibitor significantly antagonized AVP-induced increased in PLC and PLD activity (P<0.01). The PLA₂ activity had no significant changes among the groups. The results suggested that PLC and PLD took part in the V_1a receptor-mediated effects of AVP.Conclusions:1. AVP can restore the decreased vascular reactivity in hemorrhagic shock rats including the systemic responsiveness and local vascular reactivity, and increase the contractile response of vascular smooth muscle cell to NE.2. AVP restores the vascular reactivity and calcium sensitivity of vascular smooth muscle following hemorrhagic shock via activation of PKCαandεisoforms, and its mechanisms is that AVP firstly induceαandεisoforms of PKC translocation from a cytosol to a particulate and activation, and then inhibits the activity of MLCP and increases MLC20 phosphorylation, followed by improves the calcium sensitivity of VSMC and at last enhances the vascular reactivity.3. V_1a receptor inhibitor antagonized AVP-induced increase expression of PKCαandεin the particulate fractions, decrease in MLCP activity and increase in MLC₂₀ phosphorylation of SMA following hemorrhagic shock. At the same time, V_1a receptor inhibitor also decreased the increased PLC/PLD activity induced by AVP. These data suggested that AVP induced translocation/activation of PKC in vascular smooth muscle through a V_1a receptor dependent mechanism, and PLC or PLD may participate in the signal transduction pathway.