Mitochondria respond to Ca2+ already in the submicromolar range: correlation with redox state

Abstract

'The renaissance of mitochondrial calcium transport', the title of a recently published review [1], appropriately characterizes the current interest in the role of mitochondria played in the control of intracellular calcium metabolism. It was beleived that mitochondria sequester Ca 2; in case of calcium intoxication but have no role in the physiological control of calcium metabolism. This view was first challenged by the description of Ca 2; -sensitive mitochondrial dehydrogenases [2]. Thereafter, in the last decade it was revealed that cytoplasmic Summary Rapid formation of high-Ca 2; perimitochondrial cytoplasmic microdomains has been shown to evoke mitochondrial Ca 2; signal and activate mitochondrial dehydrogenases, however, the significance of submicromolar cytoplasmic Ca 2; concentrations in the control of mitochondrial metabolism has not been sufficiently elucidated. Here we studied the mitochondrial response to application of Ca 2; at buffered concentrations in permeabilized rat adrenal glomerulosa cells, in an insulin-producing cell line (INS-1/EK-3) and in an osteosarcoma cell line (143BmA-13). Mitochondrial Ca 2; concentration was measured with the fluorescent dye rhod-2 and, using an in situ calibration method, with the mitochondrially targeted luminescent protein mt-aequorin. In both endocrine cell types, mitochondrial Ca 2; concentration increased in response to elevated cytoplasmic Ca 2; concentration (between 60 and 740 nM) and an increase in mitochondrial Ca 2; concentration could be revealed already at a cytoplasmic Ca 2; concentration step from 60-140 nM. Similar responses were observed in the osteosarcoma cell line, although a clearcut response was first observed at 280 nM extramitochondrial Ca 2; only. As examined in glomerulosa cells, graded increases in cytoplasmic Ca 2; concentration were associated with graded increases in the reduction of mitochondrial pyridine nucleotides, consistent with Ca 2; -dependent activation of mitochondrial dehydrogenases. Our data indicate that in addition to the recognized role of high-Ca 2; cytoplasmic microdomains, also small Ca 2; signals may influence mitochondrial metabolism.

Mitochondrial free [Ca2+] levels and the permeability transition

“…Moreover, aequorin consumption is not a problem to monitor high [Ca 2+ ] M for at least 10 min, as we have shown before using the same aequorin form targeted to the endoplasmic reticulum [13]. In any case, we should also mention that the comparison made by Pitter et al [21], between their in situ calibration and our previous in vitro calibration [22] was made under different conditions of [Mg 2+ ] and temperature, two factors that considerably modify the Ca 2+ -sensitivity of aequorin [15,23], and using a different combination of chelators to prepare the submicromolar Ca 2+ buffers. Thus, in our opinion, confirmation of the different Ca 2+ -sensitivity in situ and in vitro of native aequorin still awaits making the comparison under the same conditions.…”

“…Thus, all the cali-brated [Ca 2+ ] M values obtained in this and previous papers with that aequorin form targeted to mitochondria are clearly valid. We have to remark this point because an in situ calibration of native aequorin reconstituted with native coelenterazine [21] showed important differences with our previously made in vitro calibration of that type of aequorin in the submicromolar [Ca 2+ ] range. This argument, together with the problem of aequorin consumption, has been later used to throw doubts on the validity of all the previous work made with low-Ca 2+ -affinity mitochondrial aequorin [6].…”

Stable Interactions between Mitochondria and Endoplasmic Reticulum Allow Rapid Accumulation of Calcium in a Subpopulation of Mitochondria

“…The emphasis here is on the rapid Ca 2ϩ uptake, but it should be mentioned that a slow Ca 2ϩ accumulation in mitochondria can also occur and it may have important biological functions (7,30,31). Obviously the slow Ca 2ϩ uptake does not require the strict association with the Ca 2ϩ release sites, but rather depends on the bulk increase in [Ca 2ϩ ].…”

Oscillatory control of insulin secretion

“…In addition, Ca 2+ decreases ATP production by depolarizing the mitochondrial inner membrane by its uptake via the uniporter (Magnus and Keizer, 1997;Magnus and Keizer, 1998b;Krippeit-Drews et al, 2000). The situation is further complicated by the fact that Ca 2+ stimulates metabolism by activating mitochondrial dehydrogenases (McCormack et al, 1990;Civelek et al, 1996;Pitter et al, 2002) and that Ca 2+ may regulate glycolytic enzymes (Jung et al, 2000). Metabolic oscillations may indeed reflect intrinsic oscillatory mechanisms in glucose metabolism, but the oscillations are modified both by stimulatory and inhibitory feedback effects of Ca 2+ (Luciani et al, 2006;Bertram et al, 2007b (Henquin, 2000) or after knocking out functional K ATP channels (Nenquin et al, 2004;Szollosi et al, 2007).…”