Akt-eNOS-NO signal pathway mediates regulatory effect of Ang-1 and Ang-2 on vascular hyperreactivity in early hemorrhagic shock rats

AIM:To observe the role of endothelial nitric oxide synthase(eNOS) in the regulatory effect of angiopoietin-1(Ang-1) and angiopoietin-2(Ang-2) on the biphasic change of vascular reactivity after hemorrhagic shock in rats. METHODS:The protein expression of eNOS was measured in the superior mesenteric artery(SMA) after hemorrhagic shock by Western blotting. The effect of eNOS inhibitor on the vascular reactivity of SMA treated with Ang-1 and Ang-2 in the early(hyperreactivity) and late(hyporeactivity) periods of hypoxia were observed via an isolated organ perfusion system. The protein levels of eNOS in the hypoxic mixture of vascular endothelial cells(VECs) and vascular smooth muscle cells(VSMCs), and the concentration of nitric oxide(NO) in the medium supernatant of the mixture cells treated with Ang-1, Ang-2 and the inhibitors of Tie-2, Akt, p38 MAPK and ERK were measured. RESULTS:The protein expression of eNOS in SMA was low in normal control group, and increased significantly after hemorrhagic shock, which was 1.84, 3.55, 4.75, 5.96 and 6.33 folds of the normal control level in shock 10 min, 30 min, 1 h, 2 h and 4 h groups, respectively(P<0.01). Inhibitor of eNOS decreased the vascular hyperreactivity in hypoxia 10 min group, in which the E_max of norepinephrine(NE) was decreased from 13.479 mN to 9.043 mN(P<0.05). It also repressed the maintenance effect of Ang-1 on vascular reactivity in hypoxia 10 min group, in wihich the E_max of NE was decreased from 15.283 mN to 11.219 mN(P<0.01). The effect of Ang-2 on the vascular hyperreactivity in hypoxia 10 min group, the vascular hyporeactivity in hypoxia 4 h group, or the effect of Ang-1 or Ang-2 on the vascular reactivity in hypoxia 4 h group did not change. The protein expression of eNOS was increased 10 min after hypoxia as compared with the normal control, which was decreased by Ang-2 and the inhibitors of Tie-2 and Akt(P<0.01), but was not decreased by p38 MAPK and ERK inhibitors. The concentration of NO in the medium supernatant was increased 10 min after hypoxia, and was significantly decreased by Ang-2 and the inhibitors of Tie-2, Akt and eNOS, while the inhibitors of p38 MAPK and ERK had no influence on it. CONCLUSION:Ang-1 and Ang-2 regulate the vascular hyperreactivity in the early hemorrhagic shock rats through Akt-eNOS-NO pathway.     

Role of A3 adenosine receptor on vascular reactivity of superior mesenteric artery in hemorrhagic shock in rat

AIM: To explore whether A3 adenosine receptor plays a role in the modulation of vascular reactivity after hemorrhagic shock in rat, and to find out the prospective drug target to restore the decreased vascular reactivity following hemorrhagic shock. METHODS: The hemorrhagic shock (40 mmHg) model was established in rat, and the reactivity of superior mesenteric artery (SMA) to norepinephrine (NE) was observed. A3AR expression at protein level and mRNA level were measured by Western blotting and RT-PCR respectively. RESULTS: The vascular reactivity of SMA to NE after hemorrhagic shock (40 mmHg) was decreased significantly in a biphasic response manner. The expression of A3AR mRNA in SMA after hemorrhagic shock decreased without significant difference. The expression of A3AR protein has a slight increase without statistical difference after 30 min of hemorrhagic shock and then has a significant decrease (especially at 2 h and 4 h after hemorrhagic shock). The usage of IB-MECA, a selective A3AR agonist, significantly increased the responsiveness of SMA to NE in hemorrhagic shock in rat. MRS1523, the selective A3AR antagonist, significantly abolished the restoration of the vascular reactivity to NE by IB-MECA in hemorrhagic shock in rat. CONCLUSION: A3AR plays a role in the modulation of vascular responsiveness to NE in hemorrhagic shock in rat, and the selective agonist of A3AR could restore the reactivity of SMA to NE in hemorrhagic shock in rat.     

菜薹BrNAP克隆及其在采后叶片衰老中的功能分析

在菜薹(Brassica rapa var. parachinensis)中克隆Br NAP1并分析其功能。Br NAP1编码区全长813 bp,编码270个氨基酸,具有NAP转录因子特有的保守结构域,属于NAP亚家族成员。Br NAP1表达量与叶片衰老程度呈正相关且受ABA诱导表达上调。亚细胞定位试验表明Br NAP1定位于细胞核。互补试验显示Br NAP1能使拟南芥atnap滞绿表型回复至野生型,过表达则能引起采后叶片早衰。双荧光素酶试验表明Br NAP1能够激活Br SAG113表达。这些说明Br NAP1是菜薹采后叶片衰老的正调控基因。     

Effect of ischemic preconditioning on vascular reactivity and calcium sensitivity in hemorrhagic shock rats

AIM: To investigate the effect of ischemic preconditioning (IPC) on vascular reactivity and calcium sensitivity during hemorrhagic shock. METHODS: Appropriate method of IPC was selected by observing the effect of different strategies of IPC on the survival time and the survival rate in hemorrhagic shock rats. The effect of IPC on the pressor effect of norepinephrine (NE, 3 μg/kg) and the contractile response of superior mesenteric artery (SMA) to NE and calcium in vivo and in vitro were observed. RESULTS: Among 3 strategies of IPC, 3 cycles of abdominal aorta occlusion for 1 min and loosing for 5 min increased the survival time and 24 h survival rate significantly, which was superior to the other two IPC methods. In vivo, IPC significantly increased the pressor response to NE and the contractile response of SMA to NE (P<0.01). In vitro, IPC significantly improved the reactivity of SMA to NE and Ca²⁺. The E_max values of SMA to NE and Ca²⁺ in IPC group were significantly higher than that in shock control group (P<0.01). CONCLUSION: Ischemic preconditioning reverses Shock-induced vascular hyporeactivity via improving calcium sensitivity of the vasculatures.     

Pinacidil pretreatment increases vascular reactivity and calcium sensitivity after hemorrhagic shock

AIM: To observe the protective effects of protein kinase Cα(PKCα) and protein kinase Cε(PKCε) activated by pinacidil pretreatment on vascular reactivity and calcium sensitivity after hemorrhagic shock in rats. METHODS: The changes of the pressor effect(the change of mean arterial pressure) and vasoconstriction response(the changes of diameter) of superior mesenteric artery(SMA) to norepinephrine(NE) were observed. The vascular reactivity and calcium sensitivity of the first class arborization of SMA induced by pinacidil pretreatment with different volume and at different time points before shock were determined. The effects of PKCα and PKCε antagonists on the protection of pinacidil pretreatment, and the effects of pinacidil pretreatment on the translocation of PKCα and PKCε were also measured. RESULTS: (1) The pressor effect and vasoconstriction response of SMA to NE, and the vascular reactivity and calcium sensitivity of the first class arborization of SMA in 2 h shock group were significantly decreased as compared to those in normal controls(P<0.01). Pinacidil(25 μg/kg) pretreated at 30 min before shock attenuated the above changes.(2) The inhibitors of PKCα and PKCε suppressed the protective effects of pinacidil pretreatment(25 μg/kg pinacidil pretreated at 30 min before shock) on the vascular reactivity and calcium sensitivity. The E_max of NE was decreased by 42.9% and 62.9%, respectively(P<0.01). The E_max of Ca²⁺ was decreased by 31.1% and 56.1%, respectively(P<0.01). Pinacidil(25 μg/kg) pretreated at 30 min before shock increased the protein expression of PKCα and PKCε on the membrane, and decreased the protein expression in the cytoplasm as compared to those in 2 h shock group(P<0.01). CONCLUSION: Pinacidil pretreatment activates PKCα and PKCε, and induces the increasing effects of vascular reactivity and calcium sensitivity after hemorrhagic shock in rats.     

The signal transducers involved in the regulation of calcium sensitivity of vascular smooth muscle in hemorrhagic shock

AIM: To observe the regulatory effects of Rho-kinase, PKC and PKG on calcium sensitivity of vascular smooth muscle in hemorrhagic shock in rats. METHODS: The superior mesenteric artery (SMA) from hemorrhagic shock model of rat was adopted to assay the calcium sensitivity via observing the contraction initiated by Ca2+ under depolarizing conditions (120 mmol/L K+) with isolated organ perfusion system. Rho-kinase agonist Ang-Ⅱ and inhibitor fasudil, PKC agonist PMA and inhibitor staurosporine, PKG agonist 8Br-cGMP and inhibitor KT-5823 were used as tool agents to study the regulatory effect of Rho-kinase, PKC and PKG on the calcium sensitivity of SMA following shock. RESULTS: Ang-Ⅱ, PMA and KT-5823 improved the calcium sensitivity of SMA and made the cumulative dose-response curve of SMA to Ca2+ shift to the left, their Emax of Ca2+ (at 3×10-2 mol/L) was 0.630 g/mg, 0.595 g/mg and 0.624 g/mg, respectively, which were all higher than that in shock control (0.377 g/mg) (P<0.05, P<0.01). Fasudil, staurosporine and 8Br-cGMP delimitated the calcium sensitivity of SMA and made the cumulative dose-response curve of Ca2+ shift to the right, their Emax at 3×10-2 mol/L of Ca2+ was 0.242 g/mg, 0.230 g/mg and 0.256 g/mg, respectively, which were all lower than that in shock control (0.377 g/mg) (P<0.05, P<0.01). CONCLUSION: Rho-kinase, PKC, PKG play important roles in the regulation of calcium sensitivity of vascular smooth muscle in hemorrhagic shock.     

Role of ZIPK in the regulatory effects of PKCα and PKCε on calcium sensitivity during hemorrhagic shock in rats

AIM: To observe the role of zipper-interacting protein kinase (ZIPK) in the regulatory effects of protein kinase Cα (PKCα) and protein kinase Cε (PKCε) on calcium sensitivity during hemorrhagic shock(HS) in rats. METHODS: The skinned first class arborization of superior mesenteric artery (SMA) from HS rats were adopted to observe the influence of inhibitor of ZIPK on the effects of PKCα and PKCε agonists on calcium sensitivity after shock via measuring the contraction initiated by Ca2+ with isolated organ perfusion system, hypoxic vascualr smooth muscle cells (VSMCs) were adopted to measure the protein expression and activity of ZIPK after applying PKCα and PKCε agonists following hypoxia via Western blotting. RESULTS: (1) The calcium sensitivity of SMA was decreased after 2 h shock, and increased by agonists of PKCα and PKCε. Emax of Ca2+ was increased from 47.2％to 66.5％ (P<0.01) and 66.3％ (P<0.01) of normal control respectively as compared with 2 h shock group. The increasing effects of PKCα and PKCε agonists on calcium sensitivity of SMA after 2 h shock were weakened by the inhibitor of ZIPK. The cumulative dose-response curve of Ca2+ was shifted to the right, the Emax of Ca2+ was decreased to 42.6％ and 47.5％ of normal control (P<0.01), respectively. (2) The protein expression and activity of ZIPK in VSMCs were decreased after 2 h hypoxia, and were increased by the agonists of PKCα and PKCε following 2 h hypoxia. CONCLUSION: PKCα and PKCε regulate the calcium sensitization probably through changing the protein expression and activity of ZIPK following HS in rats.     

Changes of RyR-mediated Ca2+ release in VSMCs is involved in the development of vascular hyporeactivity in hemorrhagic shock rats

AIM: In order to investigate the mechanisms involved in the vascular hyporeactivity after hemorrhagic shock, the changes of Ca²⁺ release from calcium store in vascular smooth muscle cells (VSMCs) with hypoxia were observed and the role of Ca²⁺ release from calcium store in the occurrence of vascular hyporeactivity to norepinephrine (NE) after hemorrhagic shock in rats was further explored.METHODS: A hemorrhagic shock model (40 mmHg for 2 h) in rats and a VSMCs hypoxic model were established. The changes of intracellular Ca²⁺ concentration in VSMCs were evaluated by fura3-AM and the role of IP₃R and RyR mediated Ca²⁺ release from calcium store was further observed. The role of IP₃R and RyR mediated Ca²⁺ release from Ca²⁺ store in the development of vascular hyporeactivity was measured with an isolated organ perfusion system. RESULTS: In the absence of extracellular Ca²⁺, NE upregulated by mobilizing Ca²⁺ release through calcium store. Compared to the normal control, the VSMCs had a slight increase when treated with hypoxia and NE-induced intracellular down-regulated, both without significant difference. Compared to the normal control cells, there was a significant change of Ca²⁺ release from calcium store in hypoxia-treated VSMCs, characterized by the significant increase in triggered by RyR-sensitive Ca²⁺ releasing activator caffeine. However, the increase in triggered by IP₃R-mediated Ca²⁺ release agonist adenophostin A (10^-5 mol/L) and ATP-Na₂ (10^-4 mol/L) had no significant difference in hypoxic VSMCs. Furthermore, the vascular reactivity to NE decreased in abdominal aorta in hemorrhagic shock (40 mmHg, 2 h) rats. The activation of IP₃R mediated Ca²⁺ release with ATP-Na₂ (10^-4 mol/L) did not improve the vascular reactivity to NE, while inhibition of IP₃R mediated Ca²⁺ release with heparin (10⁴ U/L) significantly antagonized the vascular reactivity to NE in hemorrhagic shock rats. In addition, in normal K-H solution (with about 2.2 mmol/L) and Ca²⁺-free K-H solution, RyR antagonist ryanodine (10^-5 mol/L) partly restored the vascular reactivity to NE in hemorrhagic shock rats, while RyR agonist caffeine(10^-3 mol/L) further decreased the vascular reactivity. CONCLUSION: The over-activation of RyR-mediated Ca²⁺ release from calcium store is partly involved in the development of vascular hyporeactivity after hemorrhagic shock in rats.     

Neuregulin-1 induces expression of angiogenic factors in coronary artery smooth muscle cells

ATM: To investigate the effects of neuregulin-1 (NRG-1) on the expression of angiogenic factors in human coronary artery smooth muscle cells (HCASMCs). METHODS: HCASMCs were cultured in vitro, and the cells at the 3rd passage were collected to assess the expression and phosphorylation of ErbB by Western blot. After HCASMCs were cultured under normal condition, with hypoxia and serum deprivation, or with NRG-1 treatment, the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) was determined by Western blot. RESULTS: The expression of ErbB2, ErbB3 and ErbB4 was observed in the HCASMCs, and the phosphorylation of these receptors was increased by NRG-1 treatment. Compared with control group, the expression of VEGF and Ang-1 in the HCASMCs was significantly increased in hypoxia and serum deprivation group (P<0.05﹚,while no difference in the expression of Ang-2 between the 2 groups was found.Compared with hypoxia and serum deprivation group,the expression of VEGF and Ang-1 in the H CASM Cs treated with NRG-1 was furtherincreased﹙P<0.05﹚,and no difference in the expression of Ang-2 between the 2 groups was observed.CONCLUSION: HCASMCs express ErbB2, ErbB3 and ErbB4, and the phosphorylation of the receptors is increased by NRG-1. Hypoxia, serum deprivation and NRG-1 treatment induce the increased expression of VEGF and Ang-1 significantly.     

Mechanism of RhoA on vascular reactivity following hemorrhagic shock in rats

AIM: To observe the mechanisms of RhoA on vascular reactivity following hemorrhagic shock (HS) in rats. METHODS: The superior mesenteric artery (SMA) in rats subjected to hemorrhagic shock was adopted to assay the vascular reactivity via observing the contraction initiated by norepinephrine (NE) with isolated organ perfusion system. Meanwhile, the effects of Rho kinase, myosin light chain phosphatase (MLCP), myosin light chain kinase (MLCK) on RhoA regulating vascular reactivity were observed. The effects of RhoA agonist U-46619 and inhibitor C3 enzyme on the activities of Rho kianse, MLCP, MLCK and phosphorylation of MLC20 in the vascular smooth muscle cells (VSMC) with hypoxia were also measured. RESULTS: As compared to control group, the cumulative dose-response curves of SMA to NE at 2 h after shock shifted to the right, the maximal contractions (Emax) of NE was significantly decreased. RhoA agonist U-46619 increased the vascular reactivity in the late period of shock. C3 enzyme abolished U-46619 induced the increase in the contractile response of SMA to NE. Rho kinase inhibitor Y-27632 decreased U-46619-induced the increase in the vascular reactivity, MLCP inhibitor calyculin further promoted the increase in the vascular reactivity. However, MLCK inhibitor had no effect on the U-46619-induced change of vascular reactivity. After hypoxia, the activities of Rho kinase and MLCK, and the level of MLC20 phosphorylation were decreased, MLCP activity was increased. RhoA agonist U-46619 increased the activity of Rho kinase and phosphorylation of MLC20, decreased the activity of MLCP, but had no effects on MLCK activity. CONCLUSION: RhoA plays an important role in the regulation of vascular reactivity following shock. The mechanism is closely related to regulating the activities of Rho kinase and MLCP, and increasing the phosphorylation of MLC20 in VSMC.     

Blockage of mesenteric lymph return promotes the vascular reactivity and calcium sensitivity in hemorrhagic shock rats

AIM: To observe the effects of mesenteric lymph duct ligation and mesenteric lymph drainage on the vascular reactivity and calcium sensitivity in hemorrhagic shock (HS) rats, and to investigate the role of mesenteric lymph on the vascular hyporeactivity during shock. METHODS: Seventy-two male Wistar rats were randomly divided into sham group (only operation), shock (duplicating HS model) group, shock+ligation group (duplicating HS model and mesenteric lymph duct ligation) and shock+drainage group (duplicating HS model and mesenteric lymph drainage). The changes of mean artery pressure (MAP) after injection of norepinephrine (NE, 3 μg/kg) at different time points were recorded. After hypotension (40 mmHg) for 3 h, the vascular ring of superior mesenteric artery (SMA) was made for determining the vascular reactivity and sensitivity to calcium by observing the contraction initiated by NE and Ca²⁺ under depolarizing conditions (120 mmol/L K⁺) in the isolated organ perfusion system. Meanwhile, the effects of angiotensin Ⅱ (AngⅡ) and insulin (Ins) on the vascular reactivity were also observed. RESULTS: Compared to sham group, the △MAP in shock group was increased significantly at 0 h and 0.5 h after shock, and that was decreased markedly at 1.5 h, 2 h, 2.5 h and 3 h after shock, respectively, and that in shock+ligation group and shock+drainage group was increased at 0 h, 0.5 h and 1 h after shock, decreased at 2.5 h and 3 h after shock, respectively. The △MAP in shock+ligation group and shock+drainage group was higher than that in shock group at 0.5 h after shock and all the time points followed. The SMA reactivity to NE and sensibility to Ca²⁺ in shock group, shock+ligation group and shock+drainage group were lower markedly than those in sham group. The vascular reactivity and calcium sensitivity in shock+ligation and shock+drainage groups were higher than those in shock group. The vascular reactivity and calcium sensitivity in shock group, shock+ligation group and shock+drainage group were lower than those in sham group, and those in shock+ligation and shock+drainage groups were increased as compared to shock group, respectively. CONCLUSION: Blockage of mesenteric lymphatic return with the methods of mesenteric lymph duct ligation and mesenteric lymph drainage promotes the vascular reactivity of HS rats. The mechanism may be related to improving the calcium sensitivity in the vasculature.     

Construction of lentiviral vector carrying the Ang-1 gene and its expression in the rMSCs

AIM: To construct lentiviral vector carrying the angiopoietin-1 (Ang-1) gene,and make it express Ang-1 in the rat mesenchymal stem cells (rMSCs).METHODS: The cDNA encoding the CDS of Ang-1 gene was obtained from the placenta of the adult Fisher 344 rats with RT-PCR.After digestion with restrication endonuclease,the Ang-1 gene was recombined to construct the transfer plasmid PNL-Ang1-IRES2-EGFP.The three-plasmid system of lentiviral vector was consisted of PNL-Ang1-IRES2-EGFP,the packaging plasmid HELPER,and the envelope plasmid VSVG,which were co-transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles.The rMSCs were infected by obtained lentiviral particles.The insertion of Ang-1 gene was detected by PCR,the mRNA expression of Ang-1 in rMSCs was detected with RT-PCR,the protein expression of Ang-1 was observed with immunocytochemistry and Western blotting methods.RESULTS: The result of sequencing showed that the cloned Ang-1 gene was consistent with the sequence reported in GenBank.After digestion with restrication endonuclease,the 1 512 bp fragment of Ang-1 gene and the 10.5 kb vector fragment of PNL-IRES2-EGFP were observed with gel electrophoresis.The insertion of Ang-1 gene in viral genome was confirmed.The EGFP expression was observed with the fluorescent microscope.In infected rMSCs,the mRNA and protein expressions of Ang-1 were confirmed.CONCLUSION: Lentiviral vector carrying Ang-1 gene has been successfully constructed.The infected rMSCs are able to express the Ang-1 mRNA and Ang-1 abundantly.This will facilitate the following exploratory development of Ang-1 gene-modified rMSCs.     

Effects of perindopril at different doses on cardiac function and ACE2/Ang-(1-9)/Ang-(1-7) axis of ischemic cardiac dysfunction rabbits

AIM: To investigate the different dose of perindopril on cardiac function in the rabbits with ischemic cardiac dysfunction. METHODS: Male rabbits weighing 2.5~3.0 kg(n=30) were randomly divided into 3 groups(n=10):high dose perindopril group(HD group), low dose perindopril group(LD group) and cardiac dysfunction group(CD group). The Left anterior descending coronary artery of the rabbits was ligatured for model preparation. In HD group, the rabbits were treated with perindopril split normal saline solution(1 g/L)2 mL·kg^-1·d^-1. In LD group, the rabbits were treated with perindopril split normal saline solution(0.33 g/L)2 mL·kg^-1·d^-1. In CD group, the rabbits were treated with normal saline solution 2 mL·kg^-1·d^-1. Four weeks after treatment, the cardiac function was measured via echocardiography, the mRNA expression of angiotensin-converting enzyme 2(ACE2) and angiotensin type 2 receptor(AT2R) was analyzed by real-time PCR, serum angiotensin(Ang)-(1-9) and Ang-(1-7) levels were detected by ELISA. RESULTS: Compared with CD group, the cardiac function of the 2 groups treated with perindopril was significantly improved(P<0.01), and more improvement in HD group was observed than LD group(P<0.05). The serum angiotensin(Ang)-(1-9) and Ang-(1-7) level and the mRNA expression of ACE2 and AT2R in the 2 groups treated with perindopril were significantly improved(P<0.01). Compared with LD group, the mRNA expression of ACE2 and AT2R and the serum levels of Ang-(1-9) in HD group were significant improved(P<0.05), while no difference of serum Ang-(1-7) level was observed. Correlation analysis revealed that the improvement of the cardiac function was associated with serum Ang-(1-9) level, mRNA expression of ACE2 and AT2R(P<0.01), but has no significant correlation with serum Ang-(1-7) level. CONCLUSION: High dose of perindopril may improve more cardiac function in ischemic cardiac dysfunction model in rabbits. The mechanism may relate to increasing serum Ang-(1-7) level to activate AT2R.     

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.     

Signal pathway of Cx40/43 regulates vascular contractile reactivity of superior mesenteric artery under hypoxic condition

AIM: To investigate the effect of connexin 40/43(Cx40/43) on cyclic adenosine monophosphate-protein kinase A (cAMP-PKA), cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) and diacylglycerol-protein kinase C (DG-PKC) signal pathways and the regulatory role on the vascular contractile reactivity. METHODS: Rat superior mesenteric arteries (SMA) were isolated. The vascular rings of SMA were prepared and treated with Cx40/43 antisense oligodeoxyribonucleotide (Cx40/43AODN). The changes of the concentration of cAMP, cGMP and DG, the activity of PKA, PKG and PKC, the contractile response of hypoxia treated SMA rings were observed. RESULTS: Cx40AODN decreased the concentrations of cAMP, cGMP and the activities of PKA and PKG, increased the level of DA, the activity of PKC and the endothelium-dependent contractile response in SMA. Cx43AODN increased the concentrations of cAMP, cGMP and the activities of PKA and PKG, decreased the level of DA, the activity of PKC and the endothelium-dependent contractile response in SMA. CONCLUSION: Cx40/43 regulates endothelium-dependent vasoconstrictor reactivity of SMA after hemorrhagic shock, which may be related to cAMP-PKA, cGMP-PKG and DG-PKC signal pathways.     

Regulatory mechanism of Cx40 and Cx43 on endothelium-dependent vascular contractile reactivity in rat superior mesenteric artery

AIM: To investigate the regulatory mechanism of Cx40 and Cx43 on endothelium-dependent vascular contractile reactivity and calcium sensitivity following hemorrhagic shock in rats. METHODS: The rat superior mesenteric arteries (SMAs) were isolated and vascular rings were prepared. Transfection of Cx40 and Cx43 antisense oligodeoxyribonucleotide (Cx40 and Cx43AODN) was conducted to block the expressions of Cx40 and Cx43 in SMAs. The changes of contractile response, calcium sensitivity, the activity of myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK), 20kD myosin light chain (MLC₂₀) phosphorylation in hypoxia treated SMA were observed. RESULTS: Cx40AODN decreased the activity of MLCP, increased the MLC₂₀ phosphorylation, by which Cx40AODN improved the calcium sensitivity and the contractile response of SMA. Cx43AODN increased the activity of MLCP, reduced the MLC₂₀ phosphorylation, by which Cx43AODN depressed the calcium sensitivity and the contractile response of SMA. No effect of Cx40 and Cx43AODN on the MLCK′s activity of SMA was observed. CONCLUSION: The mechanism that Cx40 and Cx43 regulates endothelium-dependent vaso-contractile responses following hemorrhagic shock may be mainly through regulating the activity of MLCP and MLC₂₀ phosphorylation in vascular smooth muscle cells.     

Effects of angiotensinⅡ and angiotensin-(1-7) on expression of(pro) renin receptor in rat vascular smooth muscle cells

AIM: To observe the effects of angiotensinⅡ (AngⅡ) and angiotensin-(1-7) on the expression of (pro)renin receptor in rat vascular smooth muscle cells.METHODS: The cultured VMSCs were randomly divided into control group, AngⅡ group, Ang-(1-7) group, AngⅡ+losartan (an AT₁ receptor antagonist) group, AngⅡ+PD123319(an AT₂ receptor antagonist) group and CGP42112A (an AT₂ receptor agonist)group. The expression of (P)RR at protein and mRNA levels was detected by Western blotting and real-time PCR,respectively.RESULTS: Compared with control group, AngⅡ distinctly increased and Ang-(1-7) decreased the expression of (P)RR mRNA and protein in VMSCs in a dose-dependent manner (P<0.01). Compared with AngⅡ group, losartan did not inhibit the expression of (P)RR mRNA and protein in VMSCs induced by AngⅡ (P>0.05), but PD123319 did (P<0.01). CGP42112A also induced the expression of (P)RR protein and mRNA in VMSCs (P<0.01).CONCLUSION: AngⅡ induces the expression of (P)RR in VMSCs by AT₂. However, Ang-(1-7) inhibits the expression of (P)RR in VMSCs.     

中甘21号

AIM: To observe the pathological role of 3-nitrotynosine (3-NT) on Escherichia coli LPS-induced vascular hyporeactivity in rats and the therapeutic effect of antioxidants. METHODS: Forty male SD rats weighting from 200 g to 250 g were randomly divided into four groups: the control group (n=10); LPS shock group (n=10); uric acid-treated group (n=10); melatonin-treated group (n=10). 6 h after LPS shock, phenylephrine (0.5-2.5 μg·kg-1) was applied intravenously to all groups and the percentage increase in MAP was detected, respectively. The concentration-response curve of aorta rings from all groups rats were obtained by cumulative addition of phenylephrine (PE), and PE Emax, EC50 were calculated. The concentrations of plasma malondialdehyde (MDA), nitrate/nitrite and 3-NT were assayed in all groups 6 h after LPS shock. RESULTS: The MAP level induced by PE significantly decreased to 54.60% in LPS shock rats compared with the control (P<0.05). However, PE induced MAP level increased 37.70% and 43.05% in uric acid and melatonin treated rats, respectively, compared with the LPS shock rats (P<0.05). The maximum response and EC50 to PE were significant reduced in LPS shock rats ［Emax, 35.30%±9.80%; EC50, (15.70±4.50)nmol/L］ compared with control group ［Emax, 100%; EC50, (4.71±2.04)nmol/L, P<0.05］; but the reactivity of aorta to PE was improved obviously in uric acid and melatonin treated groups (P<0.05). The plasma concentration of MDA, nitrate/nitrite and 3-NT were much lower in uric acid and melatonin groups than those in LPS shock group (P<0.05). CONCLUSIONS: 3-NT is an important pathological factor on vascular hyporeactivity in LPS shock. Antioxidants effectively improve α-adrenergic receptor-mediated vascular reactivity in LPS shock rats partially by removing lipid peroxidative production, reducing nitric oxide and 3-NT biosynthesis in LPS shock. These results suggest that antioxidants have potential beneficial therapeutic effect for septic shock patients.