Inflammation alters regional mitochondrial Ca²+ in human airway smooth muscle cells.
Regulation of cytosolic Ca(2+) concentration ([Ca(2+)](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 [Ca(2+)](cyt), helping prevent Ca(2+) 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 Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of [Ca(2+)](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 [Ca(2+)](cyt) and mitochondrial Ca(2+) concentration ([Ca(2+)](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 [Ca(2+)](cyt) and [Ca(2+)](mito), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased [Ca(2+)](mito) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca(2+)](mito) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca(2+)](cyt). TNF-α and IL-13 both increased [Ca(2+)](cyt), which was associated with decreased [Ca(2+)](mito) in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca(2+)](cyt) and [Ca(2+)](mito) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca(2+)](cyt) after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca(2+)](mito) in different parts of ASM cells may serve to locally regulate Ca(2+) 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 [Ca(2+)](cyt) is increased.
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905, USA.
Philippe
P
Delmotte
Binxia
B
Yang
Michael A
MA
Thompson
Christina M
CM
Pabelick
Y S
YS
Prakash
Gary C
GC
Sieck
D000814:Aniline Compounds / Q000494:pharmacology; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002998:Clonazepam / Q000031:analogs & derivatives / Q000494:pharmacology; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007249:Inflammation / Q000188:drug therapy / Q000378:metabolism*; D018793:Interleukin-13 / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D017975:Ruthenium Compounds / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors; D013841:Thiazepines / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014966:Xanthenes / Q000494:pharmacology
D000814:Aniline Compounds; D006575:Heterocyclic Compounds, 3-Ring; D018793:Interleukin-13; C112020:Ru 360; D017975:Ruthenium Compounds; D019831:Sodium-Calcium Exchanger; D013841:Thiazepines; D014409:Tumor Necrosis Factor-alpha; D014966:Xanthenes; C068483:rhod-2; C059715:Fluo-3; D002998:Clonazepam; C079032:CGP 37157; D006632:Histamine; D002118:Calcium