Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells

Abstract

Intracellular Ca2+ ([Ca2+]i) regulation in smooth muscle involves multiple mechanisms such as second messengers and ion channels. Intra- and inter-cellular heterogeneities in these mechanisms are likely, and will be reflected by heterogeneities in [Ca2+]i. In the present study, real-time confocal imaging was used to examine intracellular and intercellular heterogeneity in spontaneous Ca2+ sparks and acetylcholine-induced [Ca2+]i oscillations in porcine tracheal smooth muscle (TSM) cells. Ca2+ sparks were highly localized to multiple (2-5) foci in a cell. Individual sparks displayed relatively constant rise times (14.5 +/- 0.3% variance) and amplitudes (11.1 +/- 0.2% variance), but across regions these attributes varied. The incidence of sparks was often coupled across adjacent regions (r2 = 0.93 +/- 0.04). Spark frequency was increased approximately 350% by ryanodine and caffeine, suggesting that they represent unitary Ca2+ release through ryanodine receptor (RyR) channels. In TSM cells, acetylcholine induced [Ca2+]i oscillations that initiated from foci with the highest spark frequency. Results using beta-escin-permeabilized TSM cells indicated that [Ca2+]i oscillations also represent Ca2+ release through RyR channels. [Ca2+]i oscillations displayed intracellular heterogeneity in amplitude (30 +/- 4% variance) and intercellular heterogeneities in amplitude (100-800 nM) and frequency (5-35 per minute). Within a region, the amplitude and frequency of [Ca2+]i oscillations were correlated to both acetylcholine concentration (r = -0.79 +/- 0.04 for amplitude and 0.77 +/- 0.05 for frequency) and basal [Ca2+]i level (r = -0.94 +/- 0.02 for amplitude and 0.84 +/- 0.03 for frequency). Compared with TSM cells, acetylcholine-induced [Ca2+]i oscillations in bronchial cells were slower and lower in amplitude. We conclude that intracellular and intercellular heterogeneity in [Ca2+]i levels in airway smooth muscle reflects heterogeneities in Ca2+ regulatory mechanisms.