Mitochondrial Ca 2+ in airway smooth muscle

There is increasing evidence in different cell types for mitochondrial regulation of intracellular Ca 2+ ([Ca 2+ ] i ). In addition to modulation of energy levels, mitochondrial Ca 2+ ([Ca 2+ ] mito ) may also play a role. We examined the role of mitochondria in airway smooth muscle (ASM). In ASM cells, immunostaining for sarcoplasmic reticulum (SR) and plasma membrane proteins and fluorescent mitochondrial markers showed significant co‐localization of mitochondria with both IP 3 and RyR channels of the SR as well as with the plasma membrane. In ASM cells co‐loaded with the fluorescent Ca 2+ fluorescent indicators fluo‐3 ([Ca 2+ ] i ) and rhod‐2 ([Ca 2+ ] mito ), 1 μM ACh induced [Ca 2+ ] i oscillations as well as [Ca 2+ ] mito oscillations that were phase delayed and reduced in amplitude and frequency compared to [Ca 2+ ] i oscillations. Similarly, there was a delayed and damped [Ca 2+ ] mito response with 5 mM caffeine. These [Ca 2+ ] mito responses were inhibited by Ru‐360, a blocker of the mitochondrial Ca 2+ uniporter. Inhibition by Ru‐360 also decreased the extent of Ca 2+ entry following SR Ca 2+ depletion by cyclopiazonic acid (i.e. store‐operated Ca 2+ entry). In cells loaded with the mitochondrial membrane potential dye JC‐1, ACh increased mitochondrial membrane potential but to a lesser extent than the uncoupler of oxidative phosphorylation CCCP. These data demonstrate that mitochondria act as a Ca 2+ buffer with increased [Ca 2+ ] I , facilitated by close proximity between mitochondria, SR and plasma membrane. Alterations in membrane potential suggest that agonists such as ACh influence cellular energy levels thereby potentially influencing cellular metabolic reserve. Supported by NIH grants GM56686, HL74309, Foundation for Anesthesia Education and Research (FAER), and Department of Anesthesiology, Mayo Clinic