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.     

Receptor and signaling mechanisms involved in zacopride-induced potentiation of IK1

AIM: To investigate the receptor and signaling mechanisms involved in the potentiation of inward rectifier K⁺ current (I_K1) induced by zacopride, a potent 5-HT₃ receptor antagonist and 5-HT₄ receptor agonist. METHODS: The whole-cell patch clamp technique was used to record I_K1. 5-HT₄-receptor antagonist RS23597-190, 5-HT₃-receptor agonist m-chlorophenylbiguanide (m-CPBG), PKA inhibitor KT5720, PKC inhibitor GF109203X and PKG inhibitor KT5823 were applied respectively to determine the regulatory mechanism of I_K1. RESULTS: In the presence of RS23597-190 at concentration of 10 μmol/L which inhibited I_K1, zacopride at concentration of 1 μmol/L still increased I_K1 with the mean increment of 32.5% in inward current (at -100 mV, P<0.05). The I_K1 increment induced by zacopride was not inhibited by m-CPBG at concentration of 10 μmol/L (P>0.05). Furthermore, PKA inhibitor KT5720 at concentration of 5 μmol/L reversed the effect of zacopride (P<0.05), while PKC inhibitor GF109203X and PKG inhibitor KT5823 both at concentration of 5 μmol/L didn't influence the effect (P>0.05). CONCLUSION: Zacopride potentiates I_K1 via a PKA-mediated signaling pathway, which is independent on 5-HT₄ and 5-HT₃ receptors.     

Vasonatrin peptide attenuates injuries of dopaminergic neurons induced by 1-methyl-4-phenylpyridinium

AIM：To investigate the neuroprotective effects of vasonatrin peptide (VNP) on the injury of dopaminergic neurons induced by 1-methyl-4-phenylpyridinium (MPP+). METHODS：Cultured dopaminergic neurons from the mouse ventral mesencephalon were exposed to MPP+, and the effects of VNP on the neurotoxicity of MPP+ were eva-luated by cell viability analysis and immunofluorescence staining. Various kinds of agonists and antagonists were used to clarify the mechanism underlying the effects of VNP. RESULTS：MPP+ caused injuries in the dopaminergic neurons. VNP significantly increased the viability, axon number and axon length of the dopaminergic neurons. The MPP+-induced depolymerization of β-tubulin Ⅲ was also attenuated by the treatment with VNP. In addition, VNP significantly increased the intracellular levels of cGMP. These effects of VNP were mimicked by 8-Br-cGMP (a cell-permeable analog of cGMP), whereas inhibited by HS-142-1 ［the antagonist of the particulate guanylyl cyclase-coupled natriuretic peptide receptors (NPR)］, or KT-5823 ［a cGMP-dependent protein kinase (PKG) inhibitor］. CONCLUSION：VNP attenuates the neurotoxicity of MPP+ via guanylyl cyclase-coupled NPR/cGMP/PKG pathway, indicating that VNP might be a new effective reagent in the treatment of neural degeneration of dopaminergic neurons in Parkinson disease.     

Effect of monocyte-secreted VEGF induced by electrical burn serum on monocyte-endothelial cell adhesion

AIM: To observe the level of vascular endothelial growth factor (VEGF) secreted by monocytes cultured with electrical burn serum, and to explore the effect of VEGF on monocyte-endothelial cell adhesion.METHODS: The electrical burn serum of the rat was prepared. The normal serum from the rats without treating electric current was also collected for control. The contents of VEGF and its soluble receptor sFlt-1 in electrical burn group were determined by double-antibody sandwich ELISA. THP-1 cells were randomly divided into normal serum group and electrical burn serum group. The contents of VEGF and sFlt-1 in the culture supernatants were measured by double-antibody sandwich ELISA. THP-1 cells were also randomly divided into another 4 groups:normal serum group, electrical burn serum group, normal serum+inhibitor group and electrical burn serum+inhibitor group. THP-1 cells, which were incubated with the serum for 3 h and 6 h, were labeled with calcein-AM and then were added into the well with monolayer of endothelial cell line EA.hy926 to detect monocyte-endothelial cell adhesion.RESULTS: The levels of serum VEGF of the rats with electrical burns were significantly increased, the levels of serum sFlt-1 were significantly decreased as compared with the controls. The levels of VEGF secreted by THP-1 cells cultured with electrical burn serum were significantly increased, the levels of sFlt-1 were decreased correspondingly. Electrical burn serum enhanced monocyte-endothelial cell adhesion, sFlt-1 inhibited the adhesion between monocytes and endothelial cells.CONCLUSION: The monocytes exposed to the electrical burn serum secrete VEGF, which enhance the adhesion between monocytes and endothelial cells. Blockage of VEGF activity may effectively inhibit monocyte-endothelial cell adhesion.     

Effects of Ginsenosides on LPS-induced TF and PAI-1 expression in vascular endothelial cells

AIM: To study the effect of ginsenosides on lipopolysaccharide-induced expression of tissue factor (TF) and plasminogen activator inhibitor type-1 (PAI-1) in vascular endothelial cells (EC), and to investigate the mechanism of ginsenosides in the healthy protection and treatment of cardiovascular diseases. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured by trypsin digestion method. PAI-1 was measured in the conditioned medium of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA), whereas TF activity was measured in the lysates of these cells by a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. RESULTS: Treatment of HUVEC with LPS resulted in a significant increase in PAI-1 antigen and TF activity. Ginsenosides inhibited this LPS-induced upregulation of PAI-1 protein and TF activity in HUVEC. These effects were also confirmed on the level of specific PAI-1 and TF mRNA expression by Northern blotting. CONCLUSION: Ginsenosides counteract endothelial cell activation by inhibition LPS-induced PAI-1 and TF expression in these cells. This ability of ginsenosides might explain its efficacy in the healthy protection and the treatment of cardiovascular diseases.     

The effect of intercellular adhesion molecule-1 monoclonal antibody on microcirculation of burn shock in rats

AIM: To detect the effect of intercellular adhesion molecule-1(ICAM-1) monoclonal antibody on microcirculation disorder in burn shock of Wistar rats. METHODS: The blood flow velocity and diameter of venule were measured with RBC tracking correlator and IV550 model video microscaler in burn shock models of rats. The number of leukocytes adhered on venule wall was calculated under microscope. The animal survival time was observed. RESULTS: ICAM-1monoclonal antibody could attenuate the falling of mean arterial pressure, significantly reduce the number of leukocytes adhered on venule wall, and obviously prolong the animal mean survival time, but less than 24h.CONCLUSION: ICAM-1 monoclonal antibody can decrease the number of adhered leukocytes to endothelial cells, attenuate the tether of leukocytes to venule and improve microcirculation and protect tissue cells in burn shock of rats. However, a comprehensive therapy should be taken in severe shock.     

VEGF gene expression in norepinephrine/ burn serum-induced rat astrocytes

AIM: To investigate the changes of gene expression of vascular endothelial growth factor (VEGF) in norepinephrine/ burn serum-induced astrocytes. METHODS: Immunofluorescence staining was used to show the distribution of VEGF in astrocytes after 24 h using norepinephrine/burn serum stimulation. Western blotting was used to detect protein expression of VEGF. Real time PCR was used to investigate expression of VEGF mRNA. RESULTS: ① Green fluorescence of protein expression of VEGF in astrocytes was increased when treated with high dose norepinephrine (50 μmol/L). Green fluorescence of protein expression of VEGF in astrocytes was increased distinctness after burn serum stimulation. Green fluorescence protein expression of VEGF in astrocytes was increased significantly when high dose norepinephrine combined with burn serum stimulation was added. ② VEGF protein expression in burn serum stimulating group was increased, and VEGF protein expression was significantly increased when burn serum was added during moderate (20 μmol/L), high dose norepinephrine stimulation. ③ Expression of VEGF mRNA was increased in burn serum-treated astrocytes. Expression of VEGF mRNA was increased significantly when norepinephrine-stimulated astrocytes exposed to barn serum, and as norepinephrine dose increases gradually. CONCLUSION: Norepinephrine and burn serum play an important role in inducing VEGF protein expression in astrocytes, suggesting that stress reaction of postburn is an important cause in inducing brain edema by excreting VEGF in astrocytes.     

In vivo screening and characterization of peptides specifically binding to vasculature endothelial cells of liver in mice with septic shock

AIM: To screen peptides specifically binding to vasculature of liver in mice with septic shock. METHODS: Peptide display libraries were injected into septic shock mice by tail vein injection. After circulating for 10 min, the phages from mouse livers were recovered, amplified and purified following a routine protocol, and then re-injected for the next round screening. After screening for 4 rounds in vivo, the phage libraries were injected into normal mice by tail vein injection and the phages were recovered by serum collection to subtract the peptides binding with endothelial cells of normal mice. Then the phage clones were sequenced and further analyzed by bioinformatics. The interested phage clones were reinfused to the mice with septic shock, and appraised by titering the phage distribution in vivo and immunohistochemical staining of the mouse liver. RESULTS: After 4 rounds of biopanning, the phage recovery rates increased step by step, indicating that the phage library was successfully enriched in the livers of mice with septic shock. Fifty percent (5/10) of the analyzed sequences were identical, and with a sequence of LTTWAPA. In vivo titering and immunohistochemical staining displayed that this sequence selectively targets liver of mice with septic shock. CONCLUSION: The peptide of LTTWAPA specifically binds to vascular endothelial cells of liver in septic shock mice, which may have important significance for the therapy of septic shock.     

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.     

Effects of resveratrol on the L-type calcium current by protein kinase G pathway in isolated guinea pig ventricular myocytes

AIM: To investigate the effects of resveratrol on the L-type calcium current in isolated guinea pig ventricular myocytes. METHODS: The whole cell patch clamp method was used. RESULTS: （1）Resveratrol (1, 50, 100 μmol/L) reduced the I_Ca-L by 18.31%±3.15%, 56.20%±2.50% and 84.51%±4.01% in a concentration-dependent manner (n=5, P<0.05). But it has no change on I-V shape of I_Ca-L. (2) 8Br-cGMP (100 μmol/L), an activator of protein kinase G(PKG), deduced the density of I_Ca-L by 10.50%±1.11%. Applying resveratrol and 8Br-cGMP simultaneously decreased the I_Ca-L significantly by 87.58%±3.49％ (n=6, P<0.05). (3) 5 μmol/L H8, a PKG inhibitor, inhibited the decrease in I_Ca-L caused by resveratrol. CONCLUSION: Resveratrol inhibits I_Ca-L in guinea pig ventricular myocytes, and this inhibitory effect involves the PKG pathway.     

Effects of nicotine on the release of t-PA and PAI-1 in cultured human umbilical vein endothelial cells

AIM: To study the effect of nicotine on the release of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in human umbilical vein endothelial cells (HUVECs).METHODS: HUVECs were cultured in 24-well tissue-culture plates and randomly divided into control group and nicotine treatment groups. In nicotine treatment groups, HUVECs were either incubated with nicotine at the concentrations of 0.1, 1, 10 and 100 μmol/L for 12 h, or exposed to 100 μmol/L nicotine for 0 h, 4 h, 6 h, 8 h, 12 h and 24 h. The cells in control group were exposed to the same volume of PBS instead of nicotine at corresponding time points. The supernatants were collected for determining the concentrations of t-PA and PAI-1 by ELISA. RESULTS: (1) Compared with control group, the protein levels of PAI-1 in 100 μmol/L nicotine treatment group increased significantly (P<0.01). No significant difference of t-PA protein among the groups was observed (P>0.05). (2) After stimulated with 100 μmol/L nicotine for 4 h, 6 h, 8 h, 12 h and 24 h, the protein levels of PAI-1 in the cells increased over time and reached the peak at 12 h, which was significantly higher than those in the control cells (P<0.05). However, no significant difference of t-PA protein in these cells was found as compared to the controls (P>0.05).CONCLUSION: Nicotine inhibits the fibrinolytic activity in HUVECs in vitro, thereby damaging the vascular endothelial cells.     

Effects of cholecystokinin octapeptide on changes in rabbit thoracic aortic reactivities induced by lipopolysaccharides in vitro

AIM and METHODS: To elucidate the mechanism of anti-endotoxic shock of cholecystokinin octapeptide(CCK-8), the effects of CCK-8 on changes in rabbit thoracic aortic reactivities induced by lipopolysaccharides(LPS) in vitro were studied, and the ultrastructure of the endothelial cells was observed under scanning electron microscope. RESULTS: Incubation of thoracic aortic rings(TARs) with LPS(100 mg/L) resulted in an time-dependent impairment of the endothelium-dependent relaxations to acetylcholine(incubation for 3, 7, 14 h), a reduction of contractive response to phenylphrine(incubation for 14 h) and ultrastructural injury in endothelial cells(incubation for 7 h), all of which were alleviated by concomitant incubation with CCK-8(1 mg/L). In contrast, neither the vascular contractions nor the relaxations were affected by CCK-8 (1 mg/L) alone. CONCLUSION: CCK-8 improved the vascular reactivities in the presence of LPS, which may be one of the anti-endotoxic shock mechanisms of CCK.     

Influence of complement bypass-activation on IL-8 expression in endothelial cells

AIM:To study the induction of IL-8 expression by bypass-activated complement in human umbilical vein endothelial cells (HUVECs) and regulatory effect of nuclear factor-kappa B on the expression of IL-8. METHODS:In vitro, zymosan-activated human serum(ZAHS) directly challenged the HUVECs monolayers. Following techniques were used in the experiment: ① RIA for measurement of IL-8,ISH for measurement of their mRNA.② EMSA for measurement of nuclear factor-kappa B(NF-κB). RESULTS:①After HUVECs monolayers were stimulated with ZAHS, the level of IL-8 increased significantly at 4 h. ②The NF-κB activity began upregulated within 30 min after ZAHS stimulation, maximal NF-κB activity was observed at 120 min. Pretreatment of endothelial monolayers with PDTC (20 μmol/L) significantly inhibited the secretion of IL-8 (P＜0.05). CONCLUSION:Bypass-activated complement directly challenged HUVECs to secret IL-8. Cytoplasma to nuclear translocation of NF-κB was necessary for this response.     

Effects of insulin and glucose on secretion of tissue-type plasminogen activator and its inhibitor-1 in hypoxic vascular endothelial cells

AIM: To study the effects of insulin and glucose on tissue-type plamingen activator (tPA) and its inhibitor-1 (PAI-1) secretion in cultured human endothelial cells. METHODS: Human endothelial cell line ECV-304 was cultured with glucose and/or insulin at different concentrations with or without hypoxic exposure. RESULTS: The tPA, PAI-1 secretion and ratio of tPA/PAI-1 increased in endothelial cells during hypoxia. Insulin and glucose increased the tPA and PAI-1 secretion in endothelial cells exposed to hypoxia, and increase in tPA/PAI-1 ratio was also observed at 4 h and 8 h. CONCLUSION: Hypoxia stimulates the release of tPA and PAI-1. Insulin and glucose also stimulate the tPA and PAI-1 secretion during hypoxia.