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.     

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.     

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.     

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.     

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.     

Role of myosin-light-chain kinase in biphasic contractile activity of lymphatics isolated from hemorrhagic shock rats

AIM: To elucidate the mechanism by which myosin-light-chain kinase (MLCK) modulates the biphasic contractile activity of lymphatics isolated from the rats subject to hemorrhagic shock (HS). METHODS: Male Wistar rats were randomiz to control group and HS group. In HS group, the rats were subject to HS and then further divided into HS 0 h, 0.5 h, 1 h, 2 h and 3 h subgroups. Thoracic ducts of control and shock rats were isolated and used to determine the protein levels of phosphorylated MLCK (p-MLCK). In addition, thoracic ducts obtained from control, 0.5 h- and 2 h-shocked rats were used to observe the contractile properties of lymphatics by a pressure myograph in vitro . Lymphatic rings were prepared and incubated with ML-7 (a specific inhibitor of MLCK) or substance P (SP, an agonist of MLCK). During the experiment, the contractile frequency (CF), end-diastolic diameter, end-systolic diameter and passive diameter in Ca²⁺-free PSS buffer were measured and used to calculate the lymphatic tonic index (TI), contractile amplitude (CA) and fractional pump flow (FPF) as the indexes of lymphatic contraction activity. RESULTS: The levels of p-MLCK in lymphatics in 0 h- and 0.5 h-shocked rats were significantly increased compared with the control rats, and it was gradually decreased with the development of shock. The values of CF, TI and FPF in 0.5 h-shocked lymphatics were significantly increased at transmural pressure of 1, 3 and 5 cmH₂O compared with those in control group, and significantly blunted by ML-7. SP obviously increased the suppressive effects induced by ML-7 and restored the values of CF, TI and FPF to the levels of HS 0.5 h group. CF, TI and FPF in 2 h-shocked lymphatics significantly declined under different transmural pressure as compared with those in control group, and significantly elevated by SP. Similarly, ML-7 depressed the effects of SP. No significant difference was found in CA between 0.5 h- and 2 h-shocked lymphatics. SP decreased the CA of lymphatics obtained from 2 h-shocked rats and this effect was suppressed by ML-7. However, both agents had no effects on CA of 0.5 h-shocked lymphatics. CONCLUSION: MLCK, as an essential enzyme that influences the contraction of lymphatic smooth muscle cells, involves in the modulation of biphasic changes of lymphatic contractile activity during the process of HS.     

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。     

Estrogen treatment enhances mesenteric lymphatic contractility in rats after hemorrhagic shock

AIM To investigate the effects of 17β-estradiol （E2） treatment on the mesenteric lymphatic microcirculation and isolated lymphatic contractility in rats after hemorrhagic shock, and to explore the relationship between contractility and the difference between intra- and extracellular calcium ion concentrations （［Ca²⁺］） of lymphatic smooth muscle cells （LSMCs）. METHODS Male Wistar rats were divided into sham group, shock group and shock+E2 group. The rats were subjected to hemorrhage ［（40±2） mmHg for 90 min］ and resuscitation with or without subcutaneous injection of E2 （2 mg/kg）. After resuscitation for 3 h, the mesenteric lymphatic microcirculation in vivo was observed. Moreover, the isolated mesenteric microlymphatic rings were prepared for the observations of lymphatic contractility evaluated by the indexes including end-systolic diameter, end-diastolic diameter, contraction frequency （CF） and passive diameter. Meanwhile, the difference between intra- and extracellular ［Ca²⁺］ of LSMCs was recorded during lymphatic contraction. RESULTS Treatment with E2 significantly enhanced the CF, total contractile fraction and lymphatic dynamics index in vivo in the rats after hemorrhagic shock, and increased the CF, the fractional pump flow and the difference between intra- and extracellular ［Ca²⁺］ of LSMCs in isolated lymphatics from the shocked rats （P<0.05）. CONCLUSION Estrogen treatment enhances lymphatic contractility in rats after hemorrhagic shock, which is related to enhancement of difference between intra- and extracellular ［Ca²⁺］ of LSMCs.     

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.     

Changes of lymphatic vessel response to norepinephrine in hemorrhagic shock rats

AIM: To observe the changes of lymphatic vessel response to norepinephrine (NE) in hemorrhagic shock (HS) rats, and to explore the role of lymphatic reactivity in the pathogenesis of shock. METHODS: The lymphatic vessel pressure was observed through intubating into abdomen thoracic duct in 8 rats in sham group and HS group (which was bled from femoral artery until the mean arterial pressure to 40 mmHg). The changes of lymphatic vessel pressure response to NE at different time points were observed by injection of NE (5 μg/kg) through femoral vein. The spontaneous contraction frequency (F), maximal contraction diameter (a), maximal diastolic diameter (b) and static diameter (c) of mesenteric lymphatic (ML) living samples in 8 rats of each group were recorded through microcirculation video systems continuously. The changes of lymphatic fractional contraction index (index I), total contractile activity index (index II) and lymphatic dynamic index (LD-index) (to show the value using △F, △index I, △indexⅡ, △LD-index) were calculated after injection of NE at different time points. RESULTS: The changes of lymphatic boosting pressure response to NE in HS group was started to diminish 30 min after shock, and showed a progressive decreasing trend which significantly reduced than that in sham group at all time points of shock 1 h-3 h. In HS group, the △F, △indexⅡ, △LD-index at shock 1 h, the △F, △index I, △indexⅡ, △LD-index at shock 1.5 h and 2 h were significantly lower than those in sham group, and the △F, △index I, △indexⅡ, △LD-index at all time points were significantly decreased as compared to the values of pre-shock. CONCLUSION: Lymphatic vessel reactivity in shock rats is progressive declined in the process of hemorrhagic shock. The lymphatic vessel hypo-reactivity might play an important role in the pathogenesis of shock.     

Role of Rho kinase in blocking the return of mesenteric lymph to improve vascular calcium sensitivity in hemorrhagic shock rats

AIM: To observe the role of Rho kinase in mesenteric lymph duct ligation or mesenteric lymph drainage to improve vascular calcium sensitivity in the rats subjected to hemorrhagic shock. METHODS: Male Wistar rats were randomly divided into sham group, shock group, shock+ligation (shock plus mesenteric lymph duct ligation) group and shock+drainage (shock plus mesenteric lymph drainage) group. After induction of shock (hypotension at 40 mmHg) for 3 h, the vascular rings of superior mesenteric artery (SMA) were prepared and used to measure the response to gradient calcium ions for determining the calcium sensitivity with a wire myograph system. In shock+ligation group and shock+drainage group, the vascular rings were incubated with Rho kinase agonist angiotensinⅡ or antagonist fasudil before the measurement of the response to gradient calcium ions. RESULTS: The calcium sensitivity of vascular rings in shock group was significantly lower than that in sham group, and that in shock+ligation group and shock+drainage group was significantly higher than that in shock group, but still lower than that in sham group. AngⅡ elevated the contractile activity of the vascular rings in response to gradient calcium ions and the pD₂, and fasudil significantly decreased the response to gradient calcium ions and E_max in shock+ligation group and shock+drainage group. At the same time, fasudil decreased the pD₂ in shock+ligation group. CONCLUSION: Rho kinase plays an important role in blocking shock mesenteric lymph return that improves calcium sensitivity.     

Mechanism of vascular hyporeactivity induced by NO in hemorrhagic shock

AIM: To investigate the role of nitric oxide (NO) in vascular hyporeactivity during prolonged hemorrhagic shock (HS). METHODS: Anesthetized Sprague-Dawley rats (180-220 g) were subjected to HS insult in which they were bled to a mean arterial pressure (MAP) of 40 mmHg (5.33 kPa) and arteriolar reactivity to norepinephrine in spinotrapezius was detected. The constant MAP of 40 mmHg was maintained until vascular hyporeactivity had occurred and then were resuscitated or sacrificed for further analysis. NO synthase (NOS) activity was measured ex vivo by the conversion of [³H]-arginine to [³H]-citrulline in homogenates from heart, lung, liver, spleen, duodunum, skeletal muscle. 24 h survival rates of resuscitated rats were observed with and without administration of aminoguanidine (AG), a selective inducible NOS (iNOS) inhibitor. Mesenteric arteriolar smooth muscle cells (ASMC) were isolated, and the effects of L-arginine (L-Arg) on membrane potential (MP) of ASMC were determined by fluorescent probe and confocal microscopy in the absence and presence of AG. RESULTS: When vascular hyporeactivity occurred, an increase of NOS activity was observed in liver and heart. Resuscitated rats with AG had a higher survival rate compared with that of control. The MP of ASMC was decreased (more negative) immediately following the addition of L-Arg, and the hyperpolarization effects of L-Arg were partially blocked in the presence of AG. CONCLUSION: These results suggest that excessive NO produced in HS is responsible for the occurrence of vascular hyporeactivity in prolonged hemorrhagic shock, and one of the mechanisms of which may be hyperpolarization of ASMC caused by NO.     

Hypertonic NaCl-NaAc improves the microcirculation in rats subjected to hemorrhagic shock

AIM: To study the effects of hypertonic NaCl-NaAc on microcirculation in hemorrhage-shocked rats.METHODS: SD rats were randomized into three groups of 7.5% NaCl(hypertonic saline, HS), 5% NaCl-3.5% NaAc(hypertonic sodium acetate, HSA) and 0.9% NaCl(normal saline, NS). 4 mL/kg HS, HSA or NS was given intravenously following hemorrhagic shock. The microcirculation of spinotrapezius muscle was observed.RESULTS: HS increased mean aortic pressure more significant than HSA. Variables including arteriolar and venular diameter, velocity and volumetric flow rate and open capillaries were increased and erythrocyte aggregation was decreased in 5 min after resuscitation with both HS and HSA solutions.5 min later, variables were deteriorated in HS group.After local treatment, arteriolar and venular diameters were dilated significantly in HSA group.CONCLUSION:HSA had superior effects to HS in improving microcirculation of hemorrhage-shocked rats.     

Caveolae-mediated endocytosis of VE-Cad contributes to vascular hyperpermeability after LPS treatment

AIM: To observe caveolae-mediated endocytosis of vascular endothelial cadherin (VE-Cad) after lipopolysaccharide (LPS) treatment, and its role in vascular hyperpermeability. METHODS: Human vascular endothelial cell line CRL-2922 was used in the experiment. Western blot, co-immunoprecipitation and immunocytochemistry were adopted to observe the protein expression of caveolin-1 (Cav1), a main structural protein of caveolae, after LPS treatment. Caveolae-mediated endocytosis of VE-Cad after LPS treatment, and the effects of caveolae inhibitor on caveolae-mediated endocytosis of VE-Cad, the protein expression of VE-Cad and monolayer cell permeability after LPS treatment were determined. RESULTS: After LPS treatment, the protein expression of Cav1 did not show a significant change, while the phosphorylation (Tyr14) of Cav1 was significantly increased (P<0.05). No co-immunoprecipitation or co-localization of VE-Cad with Cav1 was observed in the normal control, and that was increased time-dependently after LPS treatment (P<0.05). Filipin, an inhibitor of caveolae, at concentration of 5 mg/L significantly reduced the co-immunoprecipitation of VE-Cad with Cav1 (P<0.05), increased the expression of VE-Cad (P<0.05) in the membrane, and improved the monolayer cell permeability at 4 h after LPS treatment (P<0.05). CONCLUSION: Caveolae-mediated endocytosis of VE-Cad contributes to the internalization of VE-Cad and the monolayer cell hyperpermeability after LPS treatment.     

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.     

Mechanism of calcium sensitivity in lymphatic hypo-reactivity in hemorrhagic shock rats

AIM: To observe the changes of lymphatic reactivity to norepinephrine (NE) and calcium sensitivity in vitro in hemorrhagic shock (HS) rats. METHODS: Male Wistar rats were randomly divided into sham group (with only operation), HS group (duplicating HS model, and divided into shock 1 h and shock 2 h subgroups). The thoracic duct rings (n=48 in each group) were prepared for assaying the lymphatic reactivity to NE and calcium sensitivity by lymphatic tension measurement technique in vitro with isolated perfusion system. Meanwhile, the effects of angiotensin Ⅱ (Ang Ⅱ) and insulin (Ins) on lymphatic reactivity were also observed. RESULTS: Compared with sham group, the NE concentration-response curves in HS 1 h and HS 2 h groups, and calcium concentration-response curves in HS 2 h group were obviously shifted to right. The lymphatic reactivity to NE, contraction to calcium, maximum effect(E_max)and avidity index (pD₂) were markedly reduced. In HS group, after incubating with calcium sensitizer Ang Ⅱ, the lymphatic reactivity to NE and calcium sensitivity were significantly increased but reduced in sham group. However, calcium sensitivity inhibitor Ins decreased the lymphatic contractile response to NE and Ca²⁺. CONCLUSION: The lymphatic hypo-reactivity in hemorrhagic shock rats is related to calcium desensitization, indicating a mechanism of lymphatic hypo-contraction.     

Protective effect and mechanism of ulinastatin on rats with hemorrhagic shock

AIM: To investigate the protective effect of ulinastatin on rats with hemorrhagic shock. METHODS: A prospective, controlled animal study was designed. The model of hemorrhagic shock in rats was produced by Chaudry method. After 60 min, rats were resuscitated by transfusion of shed blood and normal saline, but a half of them were treated with ulinastatin. At different time points after reperfusion, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected. RESULTS: The levels of TNF-α, IL-6 and MDA significantly increased and the activity of SOD decreased. In the ulinastatin-treated groups, the blood pressure and heart rate were obviously improved; the levels of TNF-α, IL-6 and MDA significantly decreased and the activity of SOD had little change after hemorrhagic shock and reperfusion. CONCLUSION: Ulinastatin has a protection effect on rats with hemorrhagic shock by suppressing the production of inflammatory factors and reducing oxidative damage.     

Role of nitric oxide in reactivity of isolated lymphatics to substance P in shock rats

AIM: To observe the role of nitric oxide (NO) in the reactivity of isolated lymphatics to substance P (SP),which presents a biphasic change, in the hemorrhagic shock (HS) rats with the technique of lymphatic perfusion in vitro. METHODS: Male Wistar rats were randomly divided into control group (surgical procedure only) and shock group (the rats were further divided into shock 0.5 h and shock 2 h groups after the HS model was established). A segment of lymphatics was pressed and perfused in vitro at transmural pressure of 3 cmH₂O after thoracic ducts were separated from the rats at the corresponding time points in each group. The lymphatics of shock 0.5 h and shock 2 h were incubated with different drugs for changing the activity of No and nitric oxide synthase (NOS), respectively. The end-systolic diameter, end-diastolic diameter, contraction frequency (CF) and passive diameter of isolated lymphatics were measured, while the contraction amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were calculated after stimulated with gradient SP. Different values between pre-and post-administration of SP in CF, CA, TI and FPF were calculated and expressed as ΔCF, ΔTI, ΔCA and ΔFPF for further assessing the reactivity of lymphatics. RESULTS: NO donor L-Arg reduced ΔCF, ΔTI and ΔFPF of 0.5 h-shocked lymphatics treated with different concentrations of SP. The effect of L-Arg was obviously suppressed by a soluble guanylate cyclase inhibitor ODQ. ΔCF, ΔTI and ΔFPF increased strikingly compared with shock 0.5 h+L-Arg group in the presence of SP at certain concentration, and ΔCF and ΔFPF increased remarkably compared with control group. NOS inhibitor L-NAME elevated ΔCF, ΔTI and ΔFPF of 2 h-shocked lymphatics treated with different concentrations of SP and the manifestation of lymphatics exceeded the values of control levels. In the experiment of 2 h-shocked lymphatics treated with L-NAME+phosphodiesterase inhibitor aminophylline (AP), the effect of L-NAME was suppressed significantly, which manifested by the decrease in ΔCF, ΔTI and ΔFPF as compared with the values of shock 2 h+L-NAME group in the presence of SP at the concentrations of 1×10^-8 mol/L and 3×10^-8 mol/L. CONCLUSION: These data indicate that NO involves in the biphasic modulation of shocked lymphatics and the effect might be involved in the action of cyclic guanosine monophosphate.