ACELL May 45/5

Monteith, Gregory R., and Mordecai P. Blaustein. Heterogeneity of mitochondrial matrix free Ca21: resolution of Ca21 dynamics in individual mitochondria in situ. Am. J. Physiol. 276 (Cell Physiol. 45): C1193–C1204, 1999.—The role of mitochondria in Ca21 homeostasis is controversial. We employed the Ca21-sensitive dye rhod 2 with novel, high temporal and spatial resolution imaging to evaluate changes in the matrix free Ca21 concentration of individual mitochondria ([Ca]m) in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 μM serotonin (5-HT) evoked modest cytosolic Ca21 transients [cytosolic free Ca21 concentration ([Ca]cyt) ,500 nM; measured with fura 2] and triggered contractions in short-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca]m) in only 4% of cells. This revealed heterogeneity in the responses of individual mitochondria, all of which stained with MitoTracker Green FM. In contrast, stimulation with 100 μM ATP evoked large cytosolic Ca21 transients (.1,000 nM) and induced pronounced, reversible elevation of [Ca]m (measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca21 uptake usually lagged behind the cytosolic Ca21 transient peak by 3–5 s, and [Ca]m declined more slowly than did bulk [Ca]cyt. The uptake delay may prevent mitochondria from interfering with rapid signaling events while enhancing the mitochondrial response to large, long-duration elevations of [Ca]cyt. The responses of arterial myocytes to modest physiological stimulation do not, however, depend on such marked changes in [Ca]m.