Cigarette smoke extract activates PKC isoforms and down-regulates the expressions of potassium channels BK(Ca) and Kv1.5 in rat bronchial smooth muscle cells.

Large-conductance calcium-activated potassium channel (BK(Ca)) and voltage-gated potassium channel Kv1.5 play an important role in the pathogenesis of bronchial hyperresponsiveness (BHR). It is known that cigarette smoke can induce BHR, however, the role of BK(Ca) and Kv1.5 expression in it remains to be further elucidated. The purpose of the present study was to investigate the direct effects of cigarette smoke extract (CSE) on BK(Ca) and Kv1.5 expression, and the role of protein kinase C (PKC) isoforms activation in primary cultured rat bronchial smooth muscle cells (BSMCs). Primarily cultured rat BSMCs were treated with 5% CSE, the expression and translocation of PKC isoforms were measured by Western blot, and the mRNA and protein levels of BK(Ca) and Kv1.5 alpha-subunits were determined by semi-quantitative RT-PCR and Western blot, respectively. The results showed that 5% CSE induced the translocation of PKCepsilon, PKCeta, PKCtheta from soluble fraction to particulate fraction, and reduced mRNA and protein expressions of BK(Ca) and Kv1.5 alpha-subunits. The decreased expression of potassium channels was partly restored by PKC inhibitor, BIM or Goe6983. In summary, CSE may activate PKC isoforms epsilon, eta, theta, thereby down-regulate the expressions of BK(Ca) and Kv1.5 in BSMCs.

[Effect of exercise stress on cigarette smoking induced downregulation of BKca and Kv1.5 expression in pulmonary arterial smooth muscle cells of rats].

OBJECTIVE: To investigate the effect of exercise stress on chronic cigarette smoking induced downregulation of large conductance calcium-activated potassium channel (BKca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) expression in pulmonary arterial smooth muscle cells of rats. METHODS: Rats were divided into three groups: the normal control group, the smoking control group and the smoking + exercise group. The plasma cortisol level, the potassium channel expression and the pathological changes in lung tissue were determined with HE staining, the immunohistochemistry and the in-situ hybridization. RESULTS: (1) In the smoking + exercise group, the plasma cortisol level was determined immediately after exercise [(1528.7 +/- 469.7) ng/L] and was higher than that determined before exercise [(672.4 +/- 235.7) ng/L] (P < 0.01); (2) The HE staining showed that the chronic pulmonary inflammatory response in the smoking control group was severe while it was mild in the smoking + exercise group; (3) The mRNA and protein expression (OD value) of BKca in the smoking control group (mRNA: 0.2206 +/- 0.0415 for big artery and 0.3935 +/- 0.1378 for small artery; protein: 0.2634 +/- 0.1219 for big artery and 0.0995 +/- 0.0851 for small artery) were less than those in the normal control group. The mRNA expression of BKca in the smoking + exercise group (OD value) (0.5022 +/- 0.1134 for big artery and 0.6408 +/- 0.2135 for small artery) was higher than that in the smoking control group; (4) The mRNA and protein expression of Kv1.5 in the smoking control group (OD value) (mRNA: 0.9354 +/- 0.3290 for big artery and 0.5012 +/- 0.1170 for small artery; protein: 1.1112 +/- 0.3310 for big artery and 0.4736 +/- 0.1250 for small artery) were less than those in the normal control group. The protein expression of Kv1.5 in the smoking + exercise group (0.7445 +/- 0.2690) in small artery was higher than that in the smoking control group. CONCLUSION: Proper exercise stress can decrease inhibition effect of the chronic smoking on the expression of potassium channel BKca and Kv1.5, which perhaps partly results from exercise induced increase of cortisol secretion.

Passive sensitization increases histamine-stimulated calcium signaling and NF-kappaB transcription activity in bronchial epithelial cells.

AIM: To find out if the two aspects of asthma (chronic airway inflammation and bronchial hyperresponsiveness) are related to hypersensitivity of calcium signaling in bronchial epithelial cells. METHODS: Porcine bronchial epithelial cells (PBEC) were divided into sensitized (S) and non-sensitized (N) groups. In group S, the cells were preincubated with serum from ovalbumin sensitized guinea pigs. In group N, the cells were preincubated with serum from nonsensitized guinea pigs. Single cell calcium imaging and ELISA-based NF-kappaB activity were used to evaluate the histamine-stimulated intracellular free calcium level and NF-kappaB activity, respectively. RESULTS: First, 0.1 micromol/L histamine could induce [Ca(2+)](i) oscillations in PBEC of group S, but not in group N. Second, 1 micromol/L histamine could induce [Ca(2+)](i) oscillations of PBEC in both group S and group N. The [Ca(2+)](i) oscillation frequency of PBEC was significantly higher in group S than in group N, though the [Ca(2+)](i) oscillation amplitude showed no difference between the two groups. Finally, when 10 micromol/L histamine was used to stimulate PBEC, a transient initial increase followed by a sustained elevation (FSE) of [Ca(2+)](i) was observed in PBEC in both groups. The amplitude of the FSE of [Ca(2+)](i) in PBEC was significantly higher in group S than in group N. The subsequent NF-kappaB activity was in accordance to the calcium oscillation frequency evoked by histamine, but not to the amplitude. CONCLUSION: It was suggested that the increased sensitivity of calcium signaling in bronchial epithelial cells might contribute to the exorbitant inflammation or increased susceptibility in asthmatic airway epithelial cells.

Effect of chronic cigarette smoking on large-conductance calcium-activated potassium channel and Kv1.5 expression in bronchial smooth muscle cells of rats.

To investigate the role of potassium channels in the pathogenesis of airway hyperresponsiveness induced by cigarette smoking, the alteration in expression of large-conductance calcium-activated potassium channel (BKca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) in bronchial smooth muscle cells were investigated in chronic cigarette smoking rats. Airway responsiveness was determined, hematoxylin and eosin staining, immuno-histochemistry, in-situ hybridization and western blot techniques were used. The results showed: (1) Chronic cigarette smoking down-regulated the protein synthesis and mRNA expression of BKca and Kv1.5 in bronchial and bronchiolar smooth muscles. (2) BKca decreased more markedly than Kv1.5 in bronchi, but there was no difference between them in bronchioli. (3) No changes in the expression of these two potassium channel proteins were found in extracted cell membrane protein from lung tissue. The results suggest that chronic cigarette smoking can down-regulate the levels of BKca and Kv1.5 in rat bronchial smooth muscle cells in vivo, which might contribute to the mechanism of airway hyperresponsiveness induced by cigarette smoking.

[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.