Role of Mitochondria in SR Calcium Buffering in Human Airway Smooth Muscle

Abstract

Sarcoplasmic reticulum (SR) Ca2+ release and reuptake are important mechanisms in airway smooth muscle (ASM) intracellular Ca2+ [Ca2+]i regulation. In other tissues, there is increasing evidence for a role of mitochondria in [Ca2+]i buffering, especially following SR Ca2+ release. Mitochondrial buffering may also influence Ca2+ influx. The purpose of the present study was to examine the role of mitochondria in [Ca2+]i buffering in human ASM. Cells were loaded with fura‐2 and exposed to 10 µM histamine in "0" Ca2+ HBSS containing 1mM lanthanum and 1µM nifedipine (to block Ca2+ influx/efflux) and in the additional presence of 1µM RU360 (mitochondrial Ca2+ uniporter inhibitor) or 1µM CGP37157 (CGP; mitochondrial Na+/Ca2+ exchange inhibitor). Compared to controls, peak [Ca2+]i levels were increased by 30% in the presence of CGP, while RU360 had not effect. Both CGP and RU360 significantly increased store‐operated Ca2+ entry. In cells co‐loaded with fura‐2 and rhod‐2 (mitochondrial Ca2+ indicator), exposure to 5 µM caffeine showed instantaneous SR Ca2+ release followed by an increase in [Ca2+]mito. Exposure to 1 µM ACh resulted in ACh‐induced [Ca2+]i oscillations that were reflected by mitochondria (but with a time delay). In ASM cells co‐labeled for mitochondria (MitoTracker) and SR (fluorescent ryanodine) migration of mitochondria towards SR was observed upon exposure to ACh or cyclopiazonic acid. This migration was completely inhibited by phalloidin (actin inhibitor). These data demonstrate an important role of mitochondria in [Ca2+]i buffering in ASM.Supported by NIH grant RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.