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.     

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.     

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.     

Role of post-hemorrhagic shock mesenteric lymph in enhancement of vascular permeability

AIM：To investigate the role of post-hemorrhagic shock mesenteric lymph (PHSML) in the enhancement of vascular permeability. METHODS：Eighteen Wistar rats were randomized into sham group, shock group, and shock plus mesenteric lymph drainage (shock + drainage) group. The rats in shock group and shock + drainage group were routinely subjected to hemorrhagic shock and hypotension ［(40±2） mmHg］ was maintained for 90 min, and then the fluid resuscitation was performed. Mesenteric lymph was drained in the rats in shock+drainage group from resuscitation finished to 6 h, for the observation of PHSML drainage on the vascular permeability in multiple tissues of hemorrhagic shock rats. Afterwards, human umbilical vein endothelial cells (HUVECs) were incubated with the PHSML in vitro to observe the effects of PHSML on the morphology and permeability of HUVECs. RESULTS：The degree of blue color and concentrations of Evens blue in the lung, myocardium, kidney, liver, spleen and small intestine were significantly increased in the shocked rats than that in sham group, while the ratios of the dry weight to the wet weight were decreased. The mesenteric lymph drainage reversed these changes. Meanwhile, 4% and 10% of PHSML at 0~3 h and 3~6 h after resuscitation, and lipopolysaccharide (10 mg/L) all caused the damage of HUVECs, decreased the viability and trans-endothelial electrical resistance of HUVECs, and increased the permeability of HUVECs to fluorescein isothiocyanate-labeled albumin. CONCLUSION：PHSML is a vital factor in the enhancement of vascular permeability.     

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.     

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.     

Effects of chronic hypoxia on intracellular calcium concentration in pulmonary arterial smooth muscle cells

AIM:To study the effect of chronic hypoxia (CH) on the intracellular calcium (［²⁺］_i) in pulmonary artery smooth muscle cells (PASMCs) and the role of L-type calcium channel and calcium store. METHODS:The rat chronic hypoxia model was set up and intervene the PASMCs with normal PSS, calcium-free PSS, nifedipine, and heparine respectively. The resting ［Ca²⁺］_i was determined with the Fura-2/AM calcium imaging technique. RESULTS:(1) The ［Ca²⁺］_i in CH group in normal PSS was higher than that in control group in normal PSS (P<0.05). The ［Ca²⁺］_i in CH group in normal PSS was higher than that in calcium-free PSS (P<0.05). (2) No obvious change of ［Ca²⁺］_i before and after application of nifedipine in PASMCs of CH groups was observed. (3) No difference of ［Ca²⁺］_i before and after application of heparine in PASMCs of CH groups was detected. CONCLUSION:Chronic hypoxia increased the ［Ca²⁺］_i in PASMCs. Chronic hypoxia induced increase in ［Ca²⁺］_i may relate to the influx of extracellular calcium, but not due to the L-type calcium channel or the IP3R modulation only.     

Effect of mesenteric lymph reperfusion on multiple organs injury in superior mesenteric artery occlusion shock rats

AIM: To explore the effect of mesenteric lymph reperfusion (MLR) on blood pressure, survival rate and organ injury in superior mesenteric artery occlusion (SMAO) shock rats.METHODS: Male Wistar rats were randomly divided into 4 groups: in sham group, only anesthetized and operation; in MLR group, performed 1 h occlusion of mesenteric lymphatics (ML) followed by 2 h of reperfusion; in SMAO group, performed 1 h occlusion of superior mesenteric artery (SMA) followed by 2 h of reperfusion; in MLR+SMAO group, performed 1 h occlusion of SMA and ML followed by 2 h of reperfusion. Histopathological and function changes of the lung, liver, kidney and myocardium in rats were assessed after 3 h of mean arterial pressure (MAP) was observed continuously in rats. The survival rate of 24 h was recorded.RESULTS: ① The 24 h survival rates of rats in sham, MLR, SMAO group (100%, 83.3%, 66.7%, respectively) were significant higher than those in MLR+SMAO group (0%). ② The changes of MAP in 4 groups were not statistically different before occlusion. The MAP in SMAO group was higher than that in MLR+SMAO and sham groups at multi-time points after clamping. After reperfusion, the change of MAP in MLR and sham groups was not obvious, and the MAP in SMAO group at multi-time points was decreased than that in MLR and sham groups. MAP in MLR+SMAO group was decreased compared with SMAO, MLR, sham group at all time points after reperfusion. ③ The levels of AST, ALT, BUN, Cre, LDH-1 and CK-MB in MLR+SMAO group in serum were higher than those in sham, MLR and SMAO groups, and these indexes in SMAO group were higher than those in sham and MLR groups. The morphologic observation showed that the structures in kidney, lung, liver and myocardium were normal in sham and MLR groups, however, inflammation, hemorrhage, congestion and necrosis were found in MLR+SMAO group, but only mild histopathological injury was found in SMAO group.CONCLUSION: Our data suggest that the MLR aggravates the multiple organs injury in SMAO shock, therefore, intestinal lymph pathway may play an important role in the pathogenesis of SMAO shock.     

Neuropeptide Y induced redistribution of intracellular free calcium in rat cardiomyocytes

AIM: To investigate the effect of neuropeptide Y (NPY) on intracellular free calcium(［Ca²⁺］_i) and Ca²⁺ sarcoplasmic reticulum of cardiomyocytes in rats.METHODS: Cardiomyocytes of neonatal Sprague-Dawley rats were incubated with NPY at concentration of 100 nmol/L for 24 h. Fluorescent indicator Fluo-4 AM was used to detect ［Ca²⁺］_i and Fluo-5N AM was used to detect Ca²⁺ in sarcoplasmic reticulum (SR). Calcium image was recorded by laser scanning confocal microscope. The SR Ca²⁺ load was estimated by caffeine-induced Ca2+ transient (CCT). RESULTS: 24 h after incubation with NPY, compared with control group, the concentration of ［Ca²⁺］_i was significantly elevated (P<0.05), and the concentration of free Ca2+ in SR (［Ca²⁺］_SR) was significantly decreased (P<0.05), and the peak of CCT was attenuated.CONCLUSION: Stimulation with NPY for 24 h causes redistribution of free calcium in rat cardiomyocytes, namely the elevation in ［Ca²⁺］_i and decline in ［Ca²⁺］_SR.     

Role of hydrogen sulfide in alleviation of liver injury by mesenteric lymph drainage in hemorrhagic shock rats

AIM：To investigate the role of hydrogen sulfide (H2S) in alleviation of liver injury by mesenteric lymph drainage in hemorrhagic shock rats. METHODS：A hemorrhagic shock model was established in male Wistar rats. DL-propargylglycine (PPG), an inhibitor of cystathionine γ-lyase (CSE) which is a synthase of H2S, or sodium hydrosulfide (NaHS), a donor of H2S, was administered to the hemorrhagic shock rats with mesenteric lymph drainage. The rats were randomly divided into sham, shock, shock+drainage, shock+drainage+PPG (45 mg/kg, ip, 0.5 h before hemorrhage) and shock+drainage+NaHS (28 μmol/kg, ip, 0.5 h before hemorrhage) groups. Fluid resuscitation was performed 1 h after hypotension, and then mesenteric lymph was drained in the rats of shock+drainage, shock+drainage+PPG and shock+drainage+NaHS groups for 3 h. The hepatic histomorphology was observed. The biochemical indexes of hepatic function in plasma, and H2S, CSE, Toll-like receptor 4 (TLR4), interleukin (IL)-10, IL-12 and tumor necrosis factor α (TNF-α) in hepatic homogenate were also examined. RESULTS：The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bile acid (TBA) in plasma, and H2S, CSE, TLR4, IL-10, IL-12 and TNF-α in hepatic homogenate in shock group were significantly higher than those in sham group. Mesenteric lymph drainage obviously reduced these indexes in shock rats, except for TLR4. PPG further decreased these indexes except for CSE, while NaHS increased these indexes except for TBA and CSE. Morphological observation showed that liver injury appeared in the rats from shock and shock+drainage+NaHS groups, and there was nearly normal hepatic structure in the rats from sham, shock+drainage and shock+drainage+PPG groups. CONCLUSION：The mechanism of mesenteric lymph drainage alleviating liver injury in hemorrhagic shock rats is related to reducing the production of H2S and alleviating the H2S-mediated inflammation.     

Mechanism of mesenteric lymph duct ligation against the renal insufficiency in hemorrhagic shock rats

AIM: To observe the effect of mesenteric lymph duct ligation on free radical and inflammatory mediator in serious hemorrhagic shock rats at different periods, and explore the mechanism of intestinal lymphatic pathway on renal insufficiency. METHODS: 78 male Wistar rats were divided into the sham group, shock group, and ligation group. The model of serious hemorrhagic shock was established in shock group, ligation group, and mesenteric lymph was blocked by ligating mesenteric lymph duct in ligation group after resuscitating. All rats were executed and kidneys were taken out for making homogenate of 10 percent to determine levels of MDA, SOD, NO, NOS, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and myeloperoxidase (MPO) at time points after shock 90 min, after transfusion and resuscitate 0 h, 1 h, 3 h, 6 h, 12 h and 24 h. The expression of inducible nitric oxide synthase (iNOS) mRNA in kindey was detected by RT-PCR. RESULTS: The contents of MDA, NO, NOS, TNF-α, IL-6, MPO and iNOS expressions in renal homogenate of shock group were increased after transfusion and resuscitation, and were higher at 6 h and 12 h, and was significantly higher than that in sham group. The acvitity of SOD was significantly lower than that in sham group (P<0.01, P<0.05). The contents of MDA, NO, NOS, TNF-α, IL-6, MPO and iNOS expression in renal homogenate of ligation group after transfusion and resuscitation 6 h, 12 h and 24 h were significantly lower than those in shock group at same points, and the SOD activity was higher (P<0.01, P<0.05). CONCLUSION: The results demonstrate that the ligation of mesenteric lymph duct can antagonise the development of renal failure in serious hemorrhagic shock rats, and its mechanism might relate to reduce the PMN sequestration, decrease the levels of TNF-α and IL-6, inhibit NO production and expression of iNOS mRNA, suppress the release of free radical and consumption of SOD.     

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.     

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.     

Role of intracellular free Ca2+ concentration in the regulation of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells

AIM: To investigate the role of intracellular free Ca²⁺ concentration (［Ca²⁺］_i) in the regulation of calcium-activated chloride (Cl_Ca) channels in pulmonary artery smooth muscle cells (PASMCs) of rats under normoxic, acute and chronic hypoxic conditions. METHODS: Acute hypoxia-induced contraction was observed in rat pulmonary artery by using routine blood vascular perfusion in vitro. The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe ［Ca²⁺］_i of rat PASMCs in normal and chronic hypoxic condition. The influences of Cl_Ca channels on PASMCs proliferation were assessed by MTT assay. RESULTS: (1) The Cl_Ca channel blockers niflumic acid (NFA) and indaryloxyacetic acid (IAA-94) produced inhibitory effects on acute hypoxia-evoked contractions in pulmonary artery. (2) Under chronic hypoxic condition, ［Ca²⁺］_i was increased. In normoxic condition, ［Ca²⁺］_i was (123.63±18.98) nmol/L, and in hypoxic condition, ［Ca²⁺］_i was (281.75±16.48)nmol/L (P<0.01). (3) In normoxic condition, ［Ca²⁺］_i had no significant change and no effect on Cl_Ca channels was observed (P>0.05). (4) Chronic hypoxic increased ［Ca²⁺］_i which opened Cl_Ca channels. The NFA and IAA-94 blocked them and decreased ［Ca²⁺］_i from (281.75±16.48)nmol/L to (117.66±15.36)nmol/L (P<0.01). (5) MTT assay showed that in chronic hypoxic condition NFA and IAA-94 decreased the value of absorbing light degree (A value) from 0.459±0.058 to 0.224±0.025 (P<0.01). CONCLUSION: Hypoxia increased ［Ca²⁺］_i which opened Cl_Ca channels and had a positive-feedback to ［Ca²⁺］_i. This may play an important role in hypoxic pulmonary hypertension. In chronic hypoxic condition, Cl_Ca channel may play a role in the regulation of PASMCs proliferation.     

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.     

Trichosanthin-induced apoptosis in melanoma B-16 cell line

AIM: To investigate the antitumor effect and mechanism of trichosanthin (TCS) on melanoma B-16 cells.METHODS: (1) The injury of B-16 cells by trichosanthin was observed with SCGE and hoechst33258 staining method.(2) LCSM and specificity fluorescent probe Fluo-3/AM,H₂DCF-DA,DAF-FM diacetate were applied to analyze the dynamic changes of Ca²⁺,ROS and NO in single cell cultured with TCS.Simultaneously,the relationship between ROS,NO and increase of Ca²⁺ was also revealed.RESULTS: (1)When treated with TCS (50 mg/L) for 3 h and 6 h,neither cytotoxicity assay nor SCGE showed the differences compared with control group.After 12 h incubation,specificity phenomena of DNA injury-comet tail appeared in SCGE and chromatin condensation even apoptotic body formation were seen by Hoechst33258 staining.（2） TCS (50 mg/L) evoked rapid enhancement of the production of Ca²⁺,ROS and NO in the cell and the differences between TCS and control group had statistical significance (P<0.01).Augment of ROS and NO was involved in the increase of Ca²⁺.CONCLUSION: Trichosanthin has relatively strong cytotoxicity on melanoma B-16 cells and contributes to apoptosis as well.Trichosanthin-induced apoptosis is related to the increase of Ca²⁺,NO and ROS in the cells.     

Mesenteric lymph drainage alleviates spleen injury in hemorrhagic shock rats

AIM：To observe the effects of post-shock mesenteric lymph (PSML) drainage on histopathology, apoptosis, cell cycle and proliferation of the spleen in rats with hemorrhagic shock. METHODS：Eighteen Wistar rats were randomly divided into sham, shock and shock+drainage groups (n=6 in each group). The hemorrhagic shock model was established in the shock and shock+drainage groups. Fluid resuscitation for 30 min was performed 1.5 h after hypotension, and PSML was drained in the rats in shock+drainage group from 1 h after hypotension to 3 h after resuscitation finished. The fixed spleen tissue was harvested from each rat for histological observation with HE staining. The apoptosis of splenocytes was observed by Hoechst 33258 staining. The expression of Bcl-2 and Bax proteins was detected by immunohistochemical staining. The cell cycle and the expression of p53 protein were measured by flow cytometry, and the proliferation index (PI) was calculated. RESULTS：Compared with sham group, splenic tissue injury appeared in the shocked rats. The apoptotic cells and the expression of Bax and p53 in shock group were increased, while Bcl-2 expression was decreased. The percentage of G2/M cells in shock group was decreased. Compared with shock group, the splenic tissue damage in shock+drainage group was significantly attenuated. Moreover, the number of apoptotic cells, the percentage of G0/G1 cells, and the expression of Bax and p53 were obviously decreased, and the G2/M cells, Bcl-2 protein expression and PI were significantly increased in shock+drainage group. CONCLUSION: PSML drainage alleviates splenic injury in hemorrhagic shock rats, which may be related to reducing the apoptosis of splenocytes.     

Changes of ［Ca2+］i and the activity of ACE in alveolar macrophages of rabbits with high-fat diet

AIM: To investigate the effects of high-fat diet on the level of intracellular free calcium (［Ca²⁺］_i) and the activity of angiotensinⅠconverting enzyme (ACE) in alveolar macrophages (AMs) of rabbits.The association between asthma and high-fat diet was also observed.METHODS: Twelve male New Zealand rabbits were medially divided into normal diet group and 1.2% high-cholesterol diet group randomly.8 weeks later,bronchial alveolar lavage was performed in vitro.［Ca²⁺］_i was determined by Fluo-2/am.The activity of ACE was detected with ultraviolet method.RESULTS: The levels of ［Ca²⁺］_i in AMs greatly increased (P＜0.01).The activity of ACE both in BALF and in culture supernatants of AMs was all greatly increased compared with normal diet group (P＜0.01).In hypercholesterolemic group the number of macrophages in BALF showed a positive correlation with the content of cholesterol in serum,such as the level of ［Ca²⁺］_i in AMs and the activity of ACE in the culture supernatants of AMs (all P＜0.05).CONCLUSION: The results suggest that AMs of rabbits may be activated by hyperlipoidemia and become ease to be stimulated.     

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.