Calcium signalling in sarcoplasmic reticulum, cytoplasm and mitochondria during activation of rabbit aorta myocytes.
This study investigated the relationship between cytoplasmic, mitochondrial, and sarcoplasmic reticulum (SR) [Ca(2+)] in rabbit aorta smooth muscle cells, following cell activation. Smooth muscle cells were loaded with the Ca(2+)-sensitive fluorescent indicator Mag-Fura-2-AM, and then either permeabilized by exposure to saponin, or dialyzed with a patch pipette in the whole-cell configuration to remove cytoplasmic indicator. When the intracellular solution contained millimolar EGTA or BAPTA, activation of SR Ca(2+)release through IP(3)or ryanodine receptors induced a decrease in the [Ca(2+)] reported by Mag-Fura-2. However, when EGTA was present at < or =100 microM, the same stimuli caused an increase in the [Ca(2+)] reported by Mag-Fura-2. The increase in [Ca(2+)] caused by phenylephrine or caffeine was delayed, and prolonged, with respect to the cytoplasmic Ca(2+)transient. Evidence is presented that this Mag-Fura-2 signal reflected a rise in mitochondrial [Ca(2+)]. Agents that inhibit mitochondrial function, such as FCCP or cyanide in combination with oligomycin B, converted the increase in organelle Mag-Fura-2 fluorescence to a decrease, while also prolonging the cytoplasmic Ca(2+)transient. There was considerable similarity between the localization of Mag-Fura-2 fluorescence and the mitochondria-selective indicator tetramethylrhodamine ethyl ester. Thus, we propose that there is close functional integration between the SR and mitochondria in aorta smooth muscle cells, with mitochondria taking up Ca(2+)from the cytoplasm following cell activation.
Gurney
A M
AM
Department of Physiology and Pharmacology, University of Strathclyde, Strathclyde Institute for Biomedical Sciences, Glasgow, UK.
Drummond
R M
RM
Fay
F S
FS
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D003956:Dialysis; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives* / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008297:Male; D008931:Mitochondria, Muscle / Q000187:drug effects / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D011817:Rabbits; D012503:Saponins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*
D005456:Fluorescent Dyes; D012503:Saponins; C072323:Mag-Fura-2-acetoxymethyl ester; D010656:Phenylephrine; D002110:Caffeine; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D016257:Fura-2