Enhanced airway contractility following inflammation by cytokines such as tumor necrosis factor alpha (TNFα) or interleukin-13 (IL-13) involves increased intracellular Ca 2+ ([Ca 2+] i) levels in airway smooth muscle (ASM). In ASM, plasma membrane Ca 2+ fluxes form a key component of [Ca 2+] i regulation. There is now growing evidence that the bidirectional plasma membrane Na +/Ca 2+ exchanger (NCX) contributes to ASM [Ca 2+] i regulation. In the present study, we examined NCX expression and function in human ASM cells under normal conditions, and following exposure to TNFα or IL-13. Western blot analysis showed significant expression of the NCX1 isoform, with increased NCX1 levels by both cytokines, effects blunted by inhibitors of nuclear factor NF-κB or mitogen-activated protein kinase. Cytokine-mediated increase in NCX1 involved enhanced transcription followed by protein synthesis. NCX2 and NCX3 remained undetectable even in cytokine-stimulated ASM. In fura-2 loaded human ASM cells, NCX-mediated inward Ca 2+ exchange as well as outward exchange (measured as rates of change in [Ca 2+] i) was elicited by altering extracellular Na + and Ca 2+ levels. Contribution of NCX was verified by measuring [Na +] i using the fluorescent Na + indicator SBFI. NCX-mediated inward exchange was verified by demonstrating prevention of rising [Ca 2+] i or falling [Na +] i in the presence of the NCX inhibitor KBR7943. Inward exchange-mode NCX was increased by both TNFα and IL-13 to a greater extent than outward exchange. NCX siRNA transfection substantially blunted outward exchange and inward exchange modes. Finally, inhibition of NCX expression or function blunted peak [Ca 2+] i and rate of fall of [Ca 2+] i following histamine stimulation. These data suggest that NCX-mediated Ca 2+ fluxes normally exist in human ASM (potentially contributing to rapid Ca 2+ fluxes), and contribute to enhanced [Ca 2+] i regulation in airway inflammation.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)