Human Airway Smooth Muscle Cell Ca2+ Dynamics in Asthma and Health

Intracellular Ca2+ homeostasis and handling were investigated in passaged human airway smooth muscle, hASM, cells from asthma and normal donors. Temporal changes in fluorescence of Ca2+-sensitive indicator fura-2 loaded into quiescent sub-confluent hASM cells were monitored using epifluorescence video microscopy. Spontaneous amplitude changes in basal fluorescence of temporal waveforms, or Ca2+ oscillations, were measured. Also, spectral analysis using the FFT transform generated a Ca2+ oscillation dominant frequency (CODF) variable. Neither amplitude nor CODF were significantly different in asthma compared to normal hASM cell donors. However, there was a significant difference (P<0.0001) between CODF in airflow obstruction (AFO), defined as FEV1/FVC<70% and FEV1< 80%, and non-AFO donors, making CODF a strong phenotypic predictor of AFO. hASM cell Ca2+ handling was investigated by Ca2+ uncaging using confocal microscopy and by bradykinin stimulation using epifluorescence microscopy. Basal Ca2+ level, Ca2+ handling exponential decay rate constants (K), SERCA activity and expression, and SOCE after a SR Ca2+-store depletion event, all demonstrated that Ca2+ handling was not significantly different between hASM cells from asthma or normal donors. There was no correlation between FEV1 and K, however there was an emerging correlation between FEV1/FVC and K for bradykinin. The postulate that Ca2+ homeostasis and handling are intrinsically dysfunctional in hASM cells from asthma compared to normal donors is ergo not supported by these data. Caffeine was found to decrease basal Ca2+ and inhibit Ca2+ oscillations in hASM cells. Future work using freshly dispersed hASM cells is required to understand in vivo Ca2+ dynamics using the methods described in this thesis. Since CODF correlates with FEV1, pattern recognition of Ca2+ oscillation frequency spectra has the potential to help define clinical asthma phenotypes. Inevitably, a post-genomic approach to comparative protein expression in asthma and normal hASM cell donors will accelerate understanding of Ca2+ dynamics.