Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle

Abstract

Following smooth muscle excitation and contraction, depletion of intracellular Ca2+stores activates capacitative Ca2+entry (CCE) to replenish stores and sustain cytoplasmic Ca2+(Ca2+i) elevations. The objectives of the present study were to characterize CCE and the Ca2+idynamics underlying human colonic smooth muscle contraction by using tension recordings, fluorescent Ca2+-indicator dyes, and patch-clamp electrophysiology. The neurotransmitter acetylcholine (ACh) contracted tissue strips and, in freshly isolated colonic smooth muscle cells (SMCs), caused elevation of Ca2+ias well as activation of nonselective cation currents. To deplete Ca2+istores, the sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid were added to a Ca2+-free bathing solution. Under these conditions, addition of extracellular Ca2+(3 mM) elicited increased tension that was inhibited by the cation channel blockers SKF-96365 (10 μM) and lanthanum (100 μM), suggestive of CCE. In a separate series of experiments on isolated SMCs, SERCA inhibition generated a gradual and sustained inward current. When combined with high-speed Ca2+-imaging techniques, the CCE-evoked rise of Ca2+iwas associated with inward currents carrying Ca2+that were inhibited by SKF-96365. Regional specializations in Ca2+influx and handling during CCE were observed. Distinct “hotspot” regions of Ca2+rise and plateau were evident in 70% of cells, a feature not previously recognized in smooth muscle. We propose that store-operated Ca2+entry occurs in hotspots contributing to localized Ca2+elevations in human colonic smooth muscle.