Ca2+ homeostasis in the agonist-sensitive internal store: functional interactions between mitochondria and the ER measured In situ in intact cells.
Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals.
Landolfi
B
B
Istituto di Fisiologia Generale, Università degli Studi di Bari, Via Amendola 165/A, I-70126 Bari, Italy.
Curci
S
S
Debellis
L
L
Pozzan
T
T
Hofer
A M
AM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology*; D007295:Inositol Phosphates / Q000378:metabolism; D008928:Mitochondria / Q000502:physiology*; D009840:Oligomycins / Q000494:pharmacology; D012402:Rotenone / Q000494:pharmacology
D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D009840:Oligomycins; D012402:Rotenone; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004533:Egtazic Acid; D000968:Antimycin A; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2