Cytoplasmic and mitochondrial Ca levels in brown adipocytes.
OBJECTIVE
We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline- and forskolin-induced Ca(2+) mobilizations in cold-acclimated rats.


METHODS
A confocal laser-scanning microscope of brown adipocytes from warm- or cold-acclimated rats was employed using probes rhodamine 123 which is a mitochondria-specific cationic dye, and the cytoplasmic and mitochondrial Ca(2+) probes fluo-3 and rhod-2. X-ray microanalysis was also studied.


RESULTS
The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold-acclimated cells than warm-acclimated cells. Cytoplasmic and mitochondrial Ca(2+) in cold-acclimated brown adipocytes double-loaded with fluo-3 and rhod-2 were measured. Noradrenaline induced the rise in cytoplasmic Ca(2+) ([Ca(2+)](cyto)) followed by mitochondrial Ca(2+) ([Ca(2+)](mito)), the effect being transformed into an increase in [Ca(2+)](cyto) whereas a decrease in [Ca(2+)](mito) by antimycin A or carbonyl cyanide m-chlorophenylhydrazone (CCCP). Antimycin A induced small Ca(2+) release from mitochondria. CCCP induced Ca(2+) release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca(2+)](cyto) followed by [Ca(2+)](mito) in the cells. The Ca measured by X-ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold-acclimated cells in comparison to warm-acclimated cells.


CONCLUSIONS
These results suggest that noradrenaline and forskolin evoked an elevation in [Ca(2+)](cyto) followed by [Ca(2+)](mito), in which H(+) gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca(2+) signalling in cold-acclimated brown adipocytes.
Nakagaki
I
I
Department of Physiology, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan.
Sasaki
S
S
Yahata
T
T
Takasaki
H
H
Hori
S
S
D017667:Adipocytes / Q000378:metabolism / Q000648:ultrastructure; D002001:Adipose Tissue, Brown / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002118:Calcium / Q000032:analysis*; D003080:Cold Temperature; D005576:Colforsin / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004577:Electron Probe Microanalysis / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D020112:Rhodamine 123 / Q000032:analysis; D015398:Signal Transduction / Q000502:physiology; D046509:Tissue Culture Techniques / Q000379:methods
D005456:Fluorescent Dyes; D005576:Colforsin; D020112:Rhodamine 123; D000968:Antimycin A; D002118:Calcium; D009638:Norepinephrine