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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

Author: YangGuangMing
Tutor: LiuLiangMing
School: Third Military Medical University
Course: Surgery
Keywords: Hemorrhagic shock Arginine vasopressin Vascular hyporeactivity Calcium sensitivity PKCα PKCδ PKCε V1a receptors V2 receptor Phospholipase C Phospholipase D Phospholipase A2
CLC: R459.7
Type: PhD thesis
Year: 2008
Downloads: 103
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Abstract


Severe trauma / shock, as well as clinical many severe vascular response to vasoactive medications are weakened or even reaction phenomenon (vascular hyporeactivity), which seriously affect the treatment of these diseases. Arginine vasopressin (AVP) is a the hypothalamus synthetic nonapeptide neurohypophysis hormones, the chamber's past studies have demonstrated that the vascular smooth muscle cells in the presence of calcium desensitization, calcium desensitization in shock vascular hyporesponsiveness occurred after shock from important role, AVP of vascular hyporeactivity after shock and calcium desensitization recovery, but the exact mechanism has not been fully elucidated. Our previous studies have shown that protein kinase C (PKC) and Rho kinase is an important regulatory shock vascular reactivity and vascular smooth muscle cells calcium sensitivity kinase molecules; Rho kinase involved in the anti-shock effect of AVP, but we found Rho kinase inhibitor Fasudil and Y-27632 pretreatment only partially antagonized AVP adjust shock the role of vascular reactivity and calcium sensitivity, suggesting that there may be other mechanisms involved in the role of AVP. PKC is a class of serine / threonine protein kinase family, there are a variety of tissue distribution and function of different subtypes of multiple isozymes. AVP whether by one or several PKC isoforms play restore vascular smooth muscle cell calcium sensitivity and vascular reactivity role specific regulation mechanism is unclear. Accordingly, we used a rat model of hemorrhagic shock and hypoxic injury in vitro cultured vascular smooth muscle cells (VSMC) model to study the AVP shock vascular reactivity and vascular smooth muscle cells calcium sensitivity regulation and regulatory molecules from the calcium sensitivity PKC pathway starting to study its mechanism. Mainly includes three parts: ① further clarify the AVP recovery hemorrhagic shock vascular hyporeactivity; ② AVP adjustment shock, vascular reactivity and calcium sensitivity PKCα, δ, ε subtypes; ③ AVP activation of PKC mechanism AVP activation of PKC isoforms V 1a V 2 receptor relationship, PLC, PLD and PLA2 in the AVP signal transduction pathway. Main experimental methods: the first part, to further clarify the role of AVP on vascular reactivity in hemorrhagic shock recovery 1. Overall experimental hemorrhagic shock rat model (30 mmHg, 2 h, the same below) were observed in rats with hemorrhagic shock AVP (0.04,0.1,0.4 U / kg) induced vasoconstriction of the superior mesenteric artery (SMA) reaction and boost effects of norepinephrine (NE). Vitro vascular ring experiments take hemorrhagic shock rat mesenteric artery, using isolated vascular ring tension measurement techniques to observe the AVP (5 × 10 -11 5 × 10 - 10 , 5 × 10 -9 mol / L) the impact of the the shock rats SMA vascular reactivity (reaction the concentration gradient NE contraction reflect vascular ring vascular reactivity). 3. VSMC experiments attached method to take the rat SMA vascular smooth muscle cells in primary culture, passage to generation 3-5 inoculated to establish VSMC dual chamber Transwell chamber culture model, observed hypoxia after 1.5 h of VSMC NE contraction reaction changes AVP on hypoxic VSMC contraction reactivity (by observing the fluorescence-labeled bovine serum albumin permeability variation in the Transwell chamber reflects the contractile response of VSMC). The second part, AVP improve hemorrhagic shock vascular reactivity and vascular smooth muscle cells calcium sensitivity of PKCα in δ and ε isoforms. Vascular ring experiment, take hemorrhagic shock in rats SMA observation AVP treatment ring of shock, vascular reactivity and calcium sensitivity changes, and the specificity of PKCα in, δ, ε subtype inhibitor pretreatment on AVP role (to depolarization of state [120 mmol / LK < / sup>] vascular rings reflect the calcium sensitivity of the contractile response of the gradient of concentration of Ca 2 ). At the same time take the mesenteric artery detection of vascular smooth muscle myosin light chain (MLC 20 ) phosphorylation changes, analysis of PKCα, δ and ε isoforms in AVP regulating of hemorrhagic shock vascular reactivity and calcium sensitivity the role and with MLC 20 relationship between. 2. VSMC experiments using cultured rat mesenteric artery of VSMC observed in the various PKC isoforms (α, δ, ε) inhibitor pretreatment hypoxia VSMC, AVP VSMC contraction reactive; while observing the AVP hypoxia the VSMC cytoplasm and cell membrane components of PKCα in δ and ε isoforms expression studies AVP through which one or several PKC isoforms to regulate shock vascular reactivity. AVP and PKC isoforms inhibitors of hypoxia VSMC myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activity, and to explore AVP restore calcium sensitivity of vascular reactivity and vascular smooth muscle cells MLCK / MLCP relationship, and PKC isoforms in which the role. The third part of the AVP mechanism of activation of PKC. Vascular ring experiments take hemorrhagic shock in rats SMA the observed V 1a , the V 2 receptor antagonist pretreatment AVP improve shock vascular reactivity and calcium sensitivity detection the mesentery artery smooth muscle MLC 20 phosphorylation changes the AVP induced PKC activation and V of 1a and V 2 receptor relationship and its mechanisms. 2. VSMC experiments, including: using cultured rat mesenteric VSMC the observed V 1a V 2 receptor antagonists on the AVP regulation of PKCα in, δ, ε Asia expression, while observing the the VSMC contractile response MLCK / MLCP activity changes, analyze the relationship between receptor the AVP induced PKC activation and V 1a and V 2 mechanisms; ② observation of hypoxia after vascular smooth muscle cells three kinds phospholipase (PLC, PLD, PLA 2 ) of the activity as well as AVP of its role and further observation V 1a , V 2 receptor antagonist pretreatment regulate PLC, PLD, PLA 2 activity of AVP explore PLC, PLD, PLA 2 whether to participate in a shock the AVP induced PKC activation regulates calcium sensitivity of the signal transduction process. Main results: First, the recovery effect of AVP on hemorrhagic shock vascular reactivity after hemorrhagic shock the rat pressor response to NE and mesenteric arterial contractile response of NE-induced significantly lower (P lt; 0.01), three doses of AVP (0.04,0.1,0.4 U / kg) treatment of hemorrhagic shock in rats increased the pressor response to NE and superior mesenteric arteries to NE contractile response (P lt; 0.05 ~ 0.01); isolated vascular ring experiment, AVP (5 × 10 -11 5 × 10 -10 , 5 × 10 -9 mol / L) treatment can significantly restore the shock-induced vascular reactivity decreased SMA to NE dose - response curve was significantly shifted left, Emax increased (P lt; 0.01); hypoxia treatment significantly reduced the contractile response of VSMC to NE, AVP may go up high VSMC contractile response to NE cumulative penetration of the fluorescent markers respectively to the NE after 45 min, 60 min, 75 min was significantly higher than hypoxia 1.5 h group (P lt; 0.05 ~~ P lt; 0.01). The results suggest that, AVP of severe hemorrhagic shock / hypoxia treatment of the whole animal, away from the body of blood vessels and vascular smooth muscle cells in response to NE have significantly improved. Second, AVP 1. AVP (5 × 10 -10 mol / L) may improve the effect of PKCα in δ and ε subtypes of hemorrhagic shock vascular reactivity and vascular smooth muscle cells calcium sensitivity relationship significantly increased the shock rat vascular ring to NE reactivity and calcium sensitivity of the NE and Ca2 dose - response curve significantly to the left, Emax increased (P lt; 0.01). The specificity of PKCα in δ and ε subtypes inhibitors or inhibitory peptide could antagonize AVP shock after vascular reactivity and calcium sensitivity recovery role, which PKCε subtype inhibitory peptides showed strong antagonistic. The results of preliminary evidence of PKCα in δ and ε subtypes are involved in AVP of shock after vascular reactivity and calcium sensitivity of improvement, but their role is not the same intensity. 1.5 h 2. Hypoxia can significantly reduce the the VSMC contractile response to NE, AVP significantly increased hypoxia VSMC contractile response to NE sex (P lt; 0.05 P lt; 0.01) specific inhibitor of PKCα subtype G? 6976 and PKCε inhibitory peptide isoforms pretreatment could significantly inhibit the AVP to restore the role of hypoxia VSMC contractile responses (P lt; 0.01); PKCδ subtypes of specific antagonists Rottlerin pretreatment partially inhibited the AVP role (P lt; 0.05). PKCα the VMSC membrane components and ε isoforms expression was significantly increased after hypoxia, protein expression decreased and cytoplasmic components. AVP treatment further increased the expression of membrane PKCα and ε subtypes, compared with the hypoxia group there is a significant difference (P lt; 0.05 P lt; 0.01); However, AVP of cytoplasmic PKCα and ε The expression levels had no significant effect. While the cytoplasm and membrane PKCδ subtypes show a similar trend, but no significant difference. AVP may by promoting the the VSMC cytoplasm of PKC to the plasma membrane translocation and activation of hemorrhagic shock rat vascular smooth muscle cells to regulate calcium sensitivity and vascular reactivity major isoforms PKCα and ε subtypes. . Shock rat mesenteric artery vascular smooth muscle MLC 20 phosphorylation level was significantly lower than that in normal control group (P lt; 0.01), AVP pretreatment could significantly elevated MLC 20 phosphorylation levels (P lt; 0.01), PKCα and ε subtypes significant inhibition (P lt; 0.01), while PKCδ inhibitor of AVP-induced MLC 20 increased phosphorylation levels The subtypes inhibitors had no significant inhibition. The results suggest that, AVP may improve vascular reactivity and calcium sensitivity after shock, PKCα and ε subtypes in which play an important role through the regulation of vascular smooth muscle MLC 20 phosphorylation levels. 4. Hypoxia VSMC MLCP activity was significantly higher in MLCK activity was significantly lower (P lt; 0.01), AVP treatment inhibit hypoxia VSMC MLCP activity increased (P lt; 0.05) MLCK activity increased slightly but compared with the hypoxia group no significant difference; inhibitor of PKCα in subtypes G? 6976 and PKCε inhibitory peptide isoforms pretreatment could antagonize caused by AVP MLCP activity reduction (P lt; 0.05), the PKCδ subtypes the inhibitor Rottlerin preprocessing to reduce somewhat elevated AVP-induced MLCP activity, but there was no significant difference (P gt; 0.05); three inhibitors had no significant effect on cell MLCK activity. The results suggest that AVP activate PKC, mainly by inhibiting MLCP activity increased MLC 20 phosphorylation levels increased VSMC calcium sensitivity play a role to improve the shock vascular reactivity. SMA to NE, AVP mechanism of activation of PKC. Vasopressin V 1a receptor antagonist [d (CH2) 5-Tyr2 (Me)] AVP pretreatment can significantly inhibit AVP-induced and the the elevated Ca2 reaction (P lt; 0.01), the NE and Ca2 dose - response curve significantly shifted to the right, Emax reduce (P lt; 0.01); V 2 receptor antagonist [ d (CH2) (d-Ile2Abu4)] AVP pretreatment also partially inhibited AVP role (P lt; of 0.05 P lt; 0.01). Also shows a similar trend in the contractile response of the change in hypoxic VSMC, V 1a receptor antagonist significantly antagonized AVP elevated the role of hypoxia in VSMC contractile response (P LT; 0.01) V 2 receptor antagonist pretreatment group cell reactivity also slightly reduced, but no significant difference. The the mechanism its V of the results show, AVP recovery of hemorrhagic shock animals vascular reactivity and calcium sensitivity 1a and V 2 receptor, which V 1a receptor may play a more important role. 2 protein expression in hypoxic the VMSC cytomembrane PKCα and ε subtypes significantly increased, AVP further increased expression of membrane PKCα and ε, V 1a receptor antagonist pretreatment can antagonized AVP-induced membrane PKCα and ε subtype of protein expression increased (P lt; 0.05 P lt; 0.01), V 2 receptor antagonists. a significant role. PKCδ protein subtypes cytoplasmic components and membrane components show a similar trend, but no statistically significant difference among the groups. V 1a receptor antagonist pretreatment could antagonize caused by AVP of hypoxic VSMC MLCP activity reduction and shock of vascular smooth muscle MLC 20 increased phosphorylation levels (P lt ; 0.05 to 0.01), while the V 2 receptor antagonist had no significant role; the AVP their V 1a , the V 2 receptor antagonists of MLCK activity hypoxic cells had no significant effect. The results suggest that AVP through activation of PKC activation and regulation of vascular reactivity and vascular smooth muscle cells in calcium sensitivity may be through V 1a receptor-mediated V 2 by The body in this signal transduction pathway may not play a major role. PLC and PLD activity increased hypoxia AVP processing PLC and PLD activity can be increased further, significantly higher than the hypoxia group (P lt; 0.05). V 1a receptor antagonist pretreatment significantly antagonized AVP-induced PLC and PLD activity increased role (P lt; 0.01), V 2 receptor antagonist had no significant role. Groups PLA 2 activity had no significant difference. The results suggest that PLC and PLD in shock after AVP through V 1a receptor-mediated PKC activation process. Conclusion: 1. Vascular reactivity after hemorrhagic shock significantly reduce systemic and local vascular reactivity after shock, and the contractile response of vascular smooth muscle cells, AVP has a better recovery effect. 2. AVP by activating PKCα and ε subtypes to regulate calcium sensitivity and vascular reactivity in hemorrhagic shock rat vascular smooth muscle cells, the mechanism AVP first of all to promote the activity of PKC to the plasma membrane in the cytoplasm of vascular smooth muscle cells translocation and activation, thereby inhibiting the activity of MLCP, MLC 20 phosphorylation levels, and finally increase the calcium sensitivity of vascular smooth muscle cells, increased vascular reactivity. 3. V 1a receptor antagonist pretreatment inhibited the AVP-induced membrane PKCα and ε protein expression increased MCLP activity decreased the MLC 20 phosphorylation level l high and increased vascular reactivity, also inhibit AVP-induced PLC and PLD activity increased. Suggesting that AVP-induced PKC activation mechanism may be related to V 1a receptor -PLC/PLD ways.

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