[Role of protein kinase C in protecting rats against pulmonary ischemia reperfusion injury through opening of mitochondrial ATP sensitive potassium channel].

OBJECTIVE: To investigate the effect of opening of mitochondrial ATP-sensitive potassium channel on inducing lung protection against ischemia reperfusion injury (I/R) and the role of PKC in the protective effect. METHODS: Fifty-six rats were divided into six groups, including sham-operation group, I/R group, DE (diazoxide) +I/R group, 5-HD (5-hydroxydecanoic acid) +DE+I/R group, CHE (chelerythrine) +DE+I/R group and 5-HD +PMA (phorbol 12-myristate 13-acetate)+ I/R group. Pulmonary I/R injury rat models were established by 45 min of left hilar clipping followed by 180 min of reperfusion. Morphological changes in lung tissues were detected by HE staining. The wet-to-dry weight ratio of lung tissues was evaluated. Cytochrome C expression in lung tissues was assessed by immunohistochemical staining. TUNEL was used to determine apoptosis. Protein kinase C (PKC) activity in lung tissues was assessed by PepTag non-radioactive assay. RESULTS: Compared with I/R group, the lung tissue morphology of the rats in the DE+I/R group was preserved well and the wet-to-dry weight ratio, expression of cytochrome C and apoptosis index decreased significantly (P < 0.05). The PKC activity in the lung tissues of the rats in the DE+ I/R group increased dramatically. Both pretreatment with 5-HD and CHE blocked the protective effect induced by DE preconditioning. There were no differences between 5-HD+PMA+I/R group and I/R group in the above indicators except for PKC activation. These results showed that blocking of mitochondrial ATP-sensitive potassium channel by 5-HD did not protect lung from ischemia reperfusion injury even though PKC were activated. CONCLUSION: Opening of mitochondrial ATP-sensitive potassium channel plays an essential protective role in pulmonary ischemia reperfusion injury. The pulmonary protection appears to be dependent on PKC activation.

[Effects of histamine on endothelial nitric oxide synthase expression in pulmonary artery endothelial cells].

All three nitric oxide synthase (NOS) isoforms are found in the lungs. It has been demonstrated that eNOS-derived NO plays an important role in modulating pulmonary vascular tone and inhibiting pulmonary vascular remodeling. Histamine induces pulmonary vasoconstriction by activating H(1)-receptor on the smooth muscle cells and vasodilation by stimulating H(2)-receptor. It remains unclear whether histamine also modulates the pulmonary vascular tone by regulating eNOS gene expression and NO production in pulmonary artery endothelial cells. Therefore, the present study was performed on cultured primary porcine pulmonary artery endothelial cells (PAECs) to investigate the effects of histamine on eNOS gene expression, and to explore the role of CaMK II in eNOS gene expression. After treatment with different concentrations histamine for different times, the levels of eNOS mRNA and protein were measured by RT-PCR and Western blot, respectively. The results showed that histamine upregulated eNOS mRNA and protein levels in a concentration- and time-dependent manner. Incubation with 10 micromol/L histamine for 24 h could increase eNOS mRNA and protein level to 160.8+/-12.2% (P<0.05) and 136.2+/-11.2% (P<0.05), respectively, of the control values. These up-regulation effects were prevented by selective CaMK II inhibitor, KN-93 (10 micromol/L). To investigate whether or not histamine increases eNOS expression by upregulating eNOS gene transcription, PAECs were transiently transfected with 1.6-kb fragment of the human eNOS promoter driving a luciferase reporter gene. The results suggested that eNOS gene promoter activity was enhanced to 148.2+/-33.7% (P<0.05) of the control after PAECs were incubated with 10 micromol/L histamine for 24 h. The nitrite and nitrate content in culture media measured by colorimetric method after incubation with 10 micromol/L histamine for 24 h indicated that the NO production in PAECs was increased. These results suggest that histamine up-regulates eNOS gene transcription and enhances NO production in PAECs by a signaling pathway involving CaMK II, which might be one of the mechanisms of histamine modulating pulmonary vascular tone.

[Effects of hypoxia on endothelial nitric oxide synthase gene expression in pulmonary artery endothelial cells and the role of protein kinase C isoforms].

OBJECTIVE: To explore the regulation of eNOS gene expression in pulmonary arterial endothelial cells (PAECs) by protein kinase C (PKC) and its isoforms during hypoxia. METHODS: Primary cultured porcine PAECs were exposed to 5%O(2) for 2, 6, 12, 24, 48 hours. The eNOS mRNA level was measured by RT-PCR. Western blot technology was used to detect the contents of eNOS protein and the 8 PKC isoforms. After addition of selective PKC inhibitors, bisindolylmaleimideI (BIMI, 1 micro mol/L) or Gö6983 (1 micro mol/L), PAECs were exposed to 5%O(2) for 24 hours, then the expression of eNOS mRNA was detected by RT-PCR. Promoter activity of eNOS gene was determined by luciferase reporter gene assay. PAECs were transfected transiently with 1.6 kb fragment of the human eNOS promoter driving a luciferaes reporter gene, then exposed to 5%O(2). 24 h later, the activity of luciferase and beta-galactosidase was examined and the relative luciferase activity, representing the eNOS promotor activity, was calculated. After addition of actinomycine D (5 micro g/ml) and exposure to 5%O(2) or normoxia for 6, 12, 24 hours, and eNOS mRNA in PAECs was measured by RT-PCR. RESULTS: After exposed to hypoxia for 24 hours, the expression of eNOS mRNA and protein level increased by 171% +/- 18% (P < 0.05) and 166% +/- 21% (P < 0.01) respectively. These up-regulation effects were prevented by BIM I and Gö6983. Further experiments showed that among 8 isoforms of PKC detected in this study, only nPKCepsilon protein expression was changed in PAECs after exposure to hypoxia for 24 h. After exposure to hypoxia nPKCepsilon was translocated from cytosol to cell membrane, showing the activation of nPKCepsilon during hypoxia. Reporter gene assay showed that hypoxia enhanced eNOS promoter activity up to 2.3 +/- 0.7 fold. In addition, hypoxia did not change the stability of eNOS mRNA. CONCLUSION: Hypoxia may up-regulate eNOS expression in PAECs by transcriptional mechanism through nPKCepsilon signaling pathway. Higher levels of mRNA observed during hypoxia are due to increased transcription, not to increased stability of mRNA.

[Influence of hypoxia-reoxygenation on nitric oxide synthesis III gene expression in cultured cerebral arterial endothelial cell].

OBJECTIVE: To investigate the change of nitric oxide synthase (NOS) III gene expression in cultured cerebral arterial endothelial cells during hypoxia and reoxygenation. METHODS: (1) The cells were divided into six groups: control, hypoxia for 1 hour, reoxygenation for 2, 6, 12, 24 hours after hypoxia for 1 hour. (2) The expression of NOSIII mRNA was detected semiquantitatively by reverse transcription-polymerase chain reaction (RT-PCR). (3) Immunocytochemistry was used to detect the expression of NOSIII protein. RESULTS: (1) The gene and protein expression of NOSIII was increased during hypoxia for 1 hour. (2) The gene and protein expression of NOSIII was decreased during reoxygenation for 2, 6, 12 hours after hypoxia for 1 hour, especially at 6 hours after reoxygenation. After cells were reoxygenation for 24 hours, the expression was restored to the normal level. CONCLUSION: The experiment showed that hypoxia could increase the levels of NOSIII gene and protein expression and reoxygenation inhibited the increment of this gene expression.