Effects of mitochondrial ATP-sensitive K⁺ channel on protein kinase C pathway and airway smooth muscle cell proliferation in asthma

Abstract

The effects of ATP-sensitive mitochondrial K⁺ channel (mitoK_ATP) on mitochondrial membrane potential (Δψm), cell proliferation and protein kinase C alpha (PKCα) expression in airway smooth muscle cells (ASMCs) were investigated. Thirty-six Sprague-Dawley (SD) rats were immunized with saline (controls) or ovalbumin (OVA) with alum (asthma models). ASMCs were cultured from the lung of control and asthma rats. ASMCs were treated with diazoxide (the potent activator of mitoK_ATP) or 5-hydroxydencanote (5-HD, the inhibitor of mitoK_ATP). Rhodamine-123 (R-123) was used to detect Δψm. The expression of PKCα protein was examined by using Western blotting, while PKCα mRNA expression was detected by using real-time PCR. The proliferation of ASMCs was measured by MTT assay and cell cycle analysis. In diazoxide-treated normal ASMCs, the R-123 fluorescence intensity, protein and mRNA levels of PKCα, MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls. The ratio of G₀/G₁ cells was decreased (P<0.05) in diazoxide-treated ASMCs from normal rats. However, there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group. In untreated and diazoxide-treated ASMCs of asthmatic rats, the R-123 fluorescence intensity, protein and mRNA levels of PKCα, MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group. Furthermore, in comparison to ASMCs from asthmatic rats, these values were considerably increased in asthmatic group treated with diazoxide (P<0.05). After exposure to 5-HD for 24 h, these values were decreased as compared with asthma control group (P<0.05). In ASMCs of asthma, the signal transduction pathway of PKCα may be involved in cell proliferation, which is induced by the opening of mitoK_ATP and the depolarization of Δψm.

Keywords

asthma / airway smooth muscle cells / ATP-sensitive K⁺ channel / protein kinase C

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