The PKC Isoforms Responsible For The Regulation Of The Vascular Reactivity And Calcium Sensitivity Following Hemorrhagic Shock And Its Regulatory Mechanism Through MLC₂₀ Phosphorylation Signal Transduction Pathway

After severe trauma or shock, including hemorrhagic, endotoxic and septic shock, vascular reactivity to vasoconstrictors and vasodilators is greatly reduced. Our previous studies showed that the vascular hyporeactivity is not only caused by the functional disorder of the K+ and Ca2+ cannels in vascular smooth muscle cell (VSMC), the hyperpolarization of VSMC membrane, but also by the calcium desensitization (the decrease of force/Ca2+ ratio) in VSMC. Our previous study showed that calcium desensitization is one of the important mechanisms responsible for the occurrence of vascular hyporeactivity, PKC take part in the regulation of vascular reactivity and calcium sensitization following hemorrhagic shock (HS). Protein kinase C (PKC) is a widely distributed protein serine/threonine kinase with broad substrate specificity, it comprises of a family of at least 12 isozymes, which are classified into three main groups: conventional, novel and atypical PKC. Conventional PKC are the Ca2+-dependent isozymesα,β?,β??,γ. Novel PKC are the Ca2+-independent isoformsδ,ε,η,θ,μ. The atypical isozymes are PKCζ,ι,λ. Among these numerous isoforms, only PKCα,ε,δandζare identified as the contraction-related PKC isoforms in aorta and superior mesenteric artery of rats, While which isoform plays an important role in the regulation of calcium desensitivity and vascular hyporeactivity following hemorrhagic shock and what is the precise mechanism is still unknown. So with hemorrhagic shock model of rats and hypoxia-treated VSMCs, we investigated the PKC isoforms responsible for the regulation of the vascular reactivity and calcium sensitivity following hemorrhagic shock and its calcium-independent regulatory mechanism through the 20kDa myosin light chain (MLC20) phosphorylation signal transduction pathway, and the induction effects by ischemia precondition and pinacidil pretreatment. The experiments were conducted in three parts:①To identify the PKC isoforms responsible for the regulation of the vascular reactivity and calcium sensitivity following hemorrhagic shock.②To investigate the regulatory mechanism of the related PKC isoforms on vascular reactivity and calcium sensitivity.③To observe the induction effects of ischemia precondition and pinacidil pretreatment on the related PKC isoforms, and their protective effects on vascular reactivity and calcium sensitivity after hemorrhagic shock.Methods:Part I. To identify the PKC isoforms responsible for the regulation of the vascular reactivity and calcium sensitivity following hemorrhagic shock.1. The first class arborizations of superior mesenteric artery (SMA) from normal and hemorrhagic shock rats (40mmHg) at different time after shock (immediately, 30min, 1h, 2h and 4h) were adopted to assay the vascular reactivity and calcium sensitivity via observing the contraction initiated by norepinephrine (NE) and Ca2+ with isolated organ perfusion system.2. The first class arborizations of SMA from hemorrhagic shock rats (40mmHg, 2h) were adopted to observe the effects of agonists and antagonists of PKCα,ε,δandζon the vascular reactivity and calcium sensitivity following HS.3. The SMAs from normal and hemorrhagic shock rats (40mmHg) at different time after shock (immediately, 30min, 1h, 2h and 4h) were adopted to determine the mRNA, protein expression and distribution of the related PKC isoforms using RT-PCR and western blot technique.Part II. To investigate the regulatory mechanism of the related PKC isoforms on vascular reactivity and calcium sensitivity through the MLC20 phosphorylation signal transduction pathway.1. The first class arborizations of SMA from hemorrhagic shock rats (40mmHg, 2h) were adopted to observe the influence of inhibitors of PKC–potentiated phosphatase inhibitor of 17 kDa (CPI-17), integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK) (using their neutralizing antibodies after permeabilization) on the effects of the related PKC isoforms agonists on calcium sensitivity after shock.2. Two hours hypoxia-treated vascular smooth muscle cells (VSMCs) were adopted to measure the protein expression of CPI-17, ILK and ZIPK, the phosphorylation of CPI-17, and the activity of ILK and ZIPK after applying the agonists of the related PKC isoforms following hypoxia via western blot and substrate phosphorylation.3. Two hours hypoxia-treated VSMCs were adopted to observe the direct effect of CPI-17, ILK and ZIPK, and their relationship to PKC isoforms via co-immunoprecipitation and immunoblot analysis.4. Two hours hypoxia-treated VSMCs were adopted to determine the myosin light chain phosphatase (MLCP) activity, and SMA from hemorrhagic shock rats (40mmHg, 2h) were adopted to determine the MLC20 phosphorylation after applying the agonists of the related PKC isoforms and the inhibitors of CPI-17, ILK and ZIPK via substrate phosphorylation and western blot.Part III. To observe the induction effects of ischemia precondition and pinacidil pretreatment on the related PKC isoforms, and their protective effects on vascular reactivity and calcium sensitivity after hemorrhagic shock.1. The NE-induced pressor response and vasoconstriction response, the vascular reactivity and calcium sensitivity of the first class arborization of SMA after ischemia precondition with different hemorrhage (2.5%, 5%, 10%), and pinacidil pretreatment with different dosage (12μg/kg body weight, 25μg/kg body weight, 50μg/kg body weight) implemented at different time before hemorrhagic shock (30min, 1h, 2h and 3h) were measured to determine the optimal ischemia precondition and pinacidil pretreatment which induce the protection against vascular hyporeactivity and calcium desensitization after hemorrhagic shock2. The SMAs tissue and the first class arborization artery rings of SMA from hemorrhagic shock (40 mmHg, 2 h), the optimal ischemia preconditioned and pinacidil pretreatmented rats were used to observe the effects of the related PKC isoforms antagonists on the protection of vascular reactivity and calcium sensitivity after hemorrhagic shock, and the effects of ischemia precondition and pinacidil pretreatment on the protein translocation of the related PKC isoforms. Results:1. The PKC isoforms responsible for the regulation of the vascular reactivity and calcium sensitivity following HS(1) The contractile response of SMA to NE and Ca2+ were increased at the early stage of shock (P<0.01), and decreased since 30min after shock (P<0.01), and continued to decrease at the late stage of shock (P<0.01).(2) PKCαagonist thymelea toxin, PKCεagonist carbachol, and PKC nonselective agonist PMA significantly restored the contractile response to NE and Ca2+ as compared with shock group (P<0.01 or P<0.05), PKCαantagonist, G?-6976, and PKCεantagonist, PKCεpseudosubstrate inhibition peptide, further decreased the contractility to NE and Ca2+ as compared with shock group or permeabilized shock group (P<0.01 or P<0.05). While PKCζantagonist did not change the contractility to NE and Ca2+ after shock, and PKCδantagonist did not inhibit PMA induced increase of contractile response of SMA to NE and Ca2+.(3) The mRNA expression of PKCαand PKCεexhibited a time-dependent increase following HS as compared with normal control (P<0.01). The protein expression of PKCαand PKCεincreased in membrane fraction and decrease in cytosolic fraction following HS as compared with normal control (P<0.01), and exhibited a time-dependent translocation from cytoplasm to membrane fraction following HS as compared with normal control.2. The regulatory mechanism of the related PKC isoforms on vascular reactivity and calcium sensitivity through the MLC20 phosphorylation signal transduction pathway.(1) The contractile response of SMA to Ca2+ decreased after 2h shock was restored by PKCαagonist, thymelea toxin, and PKCεagonist, carbachol. The neutralizing antibodies of CPI-17, ILK and ZIPK abolished the increase of calcium sensitivity induced by the agonists of PKCαandε(P<0.01).(2) The protein expression of CPI-17, ILK and ZIPK after 2h hypoxia was decreased obviously as compared with normal control, and restored by PKCαagonist, thymelea toxin, and PKCεagonist, carbachol (P<0.01). The CPI-17 phosphorylation and the activity of ILK and ZIPK were also decreased in VSMC after 2h hypoxia as compared with normal control, and restored by PKCαagonist, thymelea toxin, and PKCεagonist, carbachol (P<0.01).(3) ILK and ZIPK, especially ILK was present in the immunoprecipitates of PKCα, while CPI-17 was not, ILK and ZIPK were both present in the immunoprecipitates of PKCε, while CPI-17 was not either. ILK and ZIPK were both presented in the immunoprecipitates of CPI-17, ZIPK was presented in the immunoprecipitates of ILK.(4) MLCP activity was significantly increased after 2h hypoxia as compared with the normal control (P<0.01), MLC20 phosphorylation was significantly decreased after 2h shock as compared with normal control (P<0.01). The agonists of PKCαand PKCεdecreased the MLCP activity and increased the MLC20 phosphorylation after 2h hypoxia or shock (P<0.01). The neutralizing antibodies of CPI-17, ILK and ZIPK obviously reversed the effects of the PKCαandεagonists on MLCP activity and MLC20 phosphorylation (P<0.01).3. The induction effects of ischemia precondition and pinacidil pretreatment on the related PKC isoforms, and their protective effects on vascular reactivity and calcium sensitivity after hemorrhagic shock.(1) Ischemia precondition, especially 5% hemorrhage precondition, and pinacidil pretreatment, especially 25μg/kg pinacidil pretreatment, implemented at 30min before shock, improved the decreased NE-induced pressor response and vasoconstriction response, and improved the decreased vascular reactivity and calcium sensitivity after hemorrhagic shock (P<0.01).(2) The inhibitor of PKCα, G?-6976, and the inhibitor of PKCε, PKCεpseudosubstrate inhibitory peptidee, could significantly suppress the protection effects of ischemia precondition (5% hemorrhage precondition implemented at 30min before shock) and pinacidil pretreatment (25μg/kg pinacidil pretreatment also implemented at 30min before shock) on the vascular reactivity and calcium sensitivity after hemorrhagic shock (P<0.01).The ischemia precondition and pinacidil pretreatment could further promote shock induced PKCαand PKCεtranslocation from cytosolic to membrane fraction (P<0.01). Conclusions:1. PKCαand PKCεmay be the main isoforms of PKC responsible for the regulation of the vascular reactivity and calcium sensitivity following hemorrhagic shock, and they may be the important endogenous protective molecule of vascular reactivity.2. The regulatory effects of PKCαand PKCεon vascular reactivity and calcium sensitivity may be through altering the protein expression and activity of CPI-17, ILK and ZIPK. PKCαandεmay directly act on ILK and ZIPK. ILK and ZIPK may directly alter or via CPI-17 to alter MLCP activity and MLC20 phosphorylation to regulate the vascular calcium sensitivity.3. Ischemia precondition and pinacidil pretreatment may induce the translocation and activation of PKCαand PKCε, leading to the protection against vascular hyporeactivity and calcium desensitization after hemorrhagic shock.