Sodium-Calcium Exchange in Intracellular Calcium Handling of Human Airway Smooth Muscle

Abstract

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.