Inflammation alters regional mitochondrial Ca²⁺ in human airway smooth muscle cells

Regulation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca²⁺]_cyt, helping prevent Ca²⁺ overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca²⁺ buffering by mitochondria and mitochondrial Ca²⁺ transport mechanisms in the regulation of [Ca²⁺]_cyt in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca²⁺]_cyt and mitochondrial Ca²⁺ concentration ([Ca²⁺]_mito) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca²⁺]_cyt and [Ca²⁺]_mito, with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (1 μM CGP- 37157) increased [Ca²⁺]_mito responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca²⁺]_mito responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca²⁺]_cyt. TNF-α and IL-13 both increased [Ca²⁺]_cyt, which was associated with decreased [Ca²⁺]_mito in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca²⁺]_cyt and [Ca²⁺]_mito were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca²⁺]_cyt after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca²⁺]_mito in different parts of ASM cells may serve to locally regulate Ca²⁺ fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca²⁺]_cyt is increased.