Several models of control networks suggest that the cytosolic calcium concentration ([Ca 2+ ] c ) regulates both the utilization of ATP in the contractile process, as well as the mitochondrial production of ATP, by increasing the mitochondrial matrix free-calcium concentration ([Ca 2+ ] m ) through a mechanism that activates the citrate cycle dehydrogenases in response to specific cell demands [1,2].Indeed, under pathological conditions, such as those observed during ischemia-reperfusion (I ⁄ R), mitochondrial calcium overload might cause a series of vicious cycles, leading to the transition from reversible to irreversible myocardial injury [3,4] Mitochondrial calcium overload has been implicated in the irreversible damage of reperfused heart. Accordingly, we studied the effect of an oxygen-bridged dinuclear ruthenium amine complex (Ru 360 ), which is a selective and potent mitochondrial calcium uniporter blocker, on mitochondrial dysfunction and on the matrix free-calcium concentration in mitochondria isolated from reperfused rat hearts. The perfusion of Ru 360 maintained oxidative phosphorylation and prevented opening of the mitochondrial permeability transition pore in mitochondria isolated from reperfused hearts. We found that Ru 360 perfusion only partially inhibited the mitochondrial calcium uniporter, maintaining the mitochondrial matrix free-calcium concentration at basal levels, despite high concentrations of cytosolic calcium. Additionally, we observed that perfused Ru 360 neither inhibited Ca 2+ cycling in the sarcoplasmic reticulum nor blocked ryanodine receptors, implying that the inhibition of ryanodine receptors cannot explain the protective effect of Ru 360 in isolated hearts. We conclude that the maintenance of postischemic myocardial function correlates with an incomplete inhibition of the mitochondrial calcium uniporter. Thus, the chemical inhibition by this molecule could be an approach used to prevent heart injury during reperfusion.Abbreviations Dw, mitochondrial membrane potential; [Ca 2+ ] c , cytosolic calcium concentration; [Ca 2+ ] m , mitochondrial matrix free-calcium concentration;

Thapsigargin-sensitive sarco/endoplasmic reticulum Ca(2+) pumps (SERCAs) are involved in maintaining and replenishing agonist-sensitive internal stores. Although it has been assumed that release channels act independently of SERCA pumps, there are data suggesting the opposite. Our aim was to study the relationship between SERCA pumps and the release channels in smooth muscle cells. To this end, we have rapidly blocked SERCA pumps with thapsigargin, to avoid depletion of the internal Ca(2+) stores, and induced Ca(2+) release with either caffeine, to open ryanodine receptors, or acetylcholine, to open inositol 1,4,5-trisphosphate receptors. Blocking SERCA pumps produced smaller and slower agonist-induced [Ca(2+)](i) responses. We determined the Ca(2+) level of the internal stores both indirectly, measuring the frequency of spontaneous transient outward currents, and directly, using Mag-Fura-2, and demonstrated that the inhibition of SERCA pumps did not produce a reduction of the sarco/endoplasmic reticulum Ca(2+) levels to explain the decrease in the agonist-induced Ca(2+) responses. It appears that SERCA pumps are involved in sustaining agonist-induced Ca(2+) release by a mechanism that involves the modulation of Ca(2+) availability in the lumen of the internal stores.

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We have combined patch clamp recording with simultaneous [Ca2+]i measurements in single LNCaP cells (a human prostate cancer cell line), to study the activation of Ca2+‐permeable channels by two different inducers of apoptosis, ionomycin and serum deprivation. In perforated patch recording, LNCaP cells had a membrane potential of ‐40 mV and a resting [Ca2+]i of 90 nM. Application of ionomycin at levels that induced apoptosis in these cells (10 μM) produced a biphasic increase in [Ca2+]i. The first rise in [Ca2+]i was due to release of Ca2+ from internal stores and it was associated with a membrane hyperpolarization to ‐77 mV. The latter was probably due to the activation of high conductance, Ca2+‐ and voltage‐dependent K+ channels (maxi‐K). Conversely, the second rise in [Ca2+]i was always preceded by and strictly associated with membrane depolarization and required external Ca2+. Serum deprivation, another inducer of apoptosis, unmasked a voltage‐independent Ca2+ permeability as well. A lower concentration of ionomycin (1 μM) did not induce apoptosis, and neither depolarized LNCaP cells nor produced the biphasic increase in [Ca2+]i. However, the first increment in [Ca2+]i due to release from internal Ca2+ stores was evident at this concentration of ionomycin. Simultaneous recordings of [Ca2+]i and ion channel activity in the cell attached configuration of patch clamp revealed a Ca2+‐permeable, Ca2+‐independent, non‐selective cation channel of 23 pS conductance. This channel was activated only during the second increment in [Ca2+]i induced by ionomycin. The absence of serum activated the 23 pS channel as well, albeit at a lower frequency than with ionomycin. Thus, the 23 pS channel can be activated by two unrelated inducers of apoptosis and it could be another Ca2+ influx mechanism in programmed cell death of LNCaP cells.

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