Dynamic cytosolic Ca2+ and force responses to muscarinic stimulation in airway smooth muscle.

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca2+]cyt (Ca2+ sensitivity) was assessed as the ratio of maximum force to maximum [Ca2+]cyt. However, this static assessment of Ca2+ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca2+]cyt and isometric force responses to three concentrations of ACh (1, 2.6, and 10 µM). Both maximum [Ca2+]cyt and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca2+ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca2+]cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca2+]cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca2+]cyt and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca2+]cyt, the extent of regulatory myosin light-chain (rMLC20) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca2+]cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC20 phosphorylation.

Sodium-calcium exchange in intracellular calcium handling of human airway smooth muscle.

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.