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

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 ~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 β-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.