Inhibition of mitochondrial calcium uptake rather than efflux impedes calcium release by inositol-1,4,5-trisphosphate-sensitive receptors.
Mitochondria modulate cellular Ca2+ signals by accumulating the ion via a uniporter and releasing it via Na+- or H+-exchange. In smooth muscle, inhibition of mitochondrial Ca2+ uptake inhibits Ca2+ release from the sarcoplasmic reticulum (SR) via inositol-1,4,5-trisphosphate-sensitive receptors (IP(3)R). At least two mechanisms may explain this effect. First, localised uptake of Ca2+ by mitochondria may prevent negative feedback by cytosolic Ca2+ on IP(3)R activity, or secondly localised provision of Ca2+ by mitochondrial efflux may maintain IP(3)R function or SR Ca2+ content. To distinguish between these possibilities the role of mitochondrial Ca2+ efflux on IP(3)R function was examined. IP(3) was liberated in freshly isolated single colonic smooth muscle cells and mitochondrial Na+-Ca2+ exchanger inhibited with CGP-37157 (10microM). Mitochondria accumulated Ca2+ during IP(3)-evoked [Ca2+](c) rises and released the ion back to the cytosol (within approximately 15s) when mitochondrial Ca2+ efflux was active. When mitochondrial Ca2+ efflux was inhibited by CGP-37157, an extensive and sustained loading of mitochondria with Ca2+ occurred after IP(3)-evoked Ca2+ release. IP(3)-evoked [Ca2+](c) rises were initially unaffected, then only slowly inhibited by CGP-37157. IP(3)R activity was required for inhibition to occur; incubation with CGP-37157 for the same duration without IP(3) release did not inhibit IP(3)R. CGP-37157 directly inhibited voltage-gated Ca2+ channel activity, however SR Ca2+ content was unaltered by the drug. Thus, the gradual decline of IP(3)R function that followed mitochondrial Na+-Ca2+ exchanger inhibition resulted from a gradual overload of mitochondria with Ca2+, leading to a reduced capacity for Ca2+ uptake. Localised uptake of Ca2+ by mitochondria, rather than mitochondrial Ca2+ efflux, appears critical for maintaining IP(3)R activity.
Chalmers
Susan
S
Strathclyde Institute of Pharmacy & Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, UK.
McCarron
John G
JG
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D002998:Clonazepam / Q000031:analogs & derivatives / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D025461:Feedback, Physiological; D006168:Guinea Pigs; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects; D008297:Male; D008856:Microscopy, Fluorescence; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013841:Thiazepines / Q000494:pharmacology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D013841:Thiazepines; D002998:Clonazepam; C079032:CGP 37157; D002118:Calcium