During cell activation, mitochondria play an important role in Ca²⁺ homoeostasis due to the presence of a fast and specific Ca²⁺ channel in its inner membrane, the mitochondrial Ca²⁺ uniporter. This channel allows mitochondria to buffer local cytosolic [Ca²⁺] changes and controls the intramitochondrial Ca²⁺ levels, thus modulating a variety of phenomena from respiratory rate to apoptosis. We have described recently that SB202190, an inhibitor of p38 MAPK (mitogen-activated protein kinase), strongly activated the uniporter. We show in the present study that a series of natural plant flavonoids, widely distributed in foods, produced also a strong stimulation of the mitochondrial Ca²⁺ uniporter. This effect was of the same magnitude as that induced by SB202190 (an approx. 20-fold increase in the mitochondrial Ca²⁺ uptake rate), developed without measurable delay and was rapidly reversible. In intact cells, the mitochondrial Ca²⁺ peak induced by histamine was also largely increased by the flavonoids. Stimulation of the uniporter by either flavonoids or SB202190 did not require ATP, suggesting a direct effect on the uniporter or an associated protein which is not mediated by protein phosphorylation. The most active compound, kaempferol, increased the rate of mitochondrial Ca²⁺ uptake by 85±15% (mean±S.E.M., n=4) and the histamine-induced mitochondrial Ca²⁺ peak by 139±19% (mean±S.E.M., n=5) at a concentration of 1 μM. Given that flavonoids can reach this concentration range in plasma after ingestion of flavonoid-rich food, these compounds could be modulating the uniporter under physiological conditions.

Background and purpose: The thiourea derivative KB‐R7943, originally developed as inhibitor of the plasma membrane Na⁺/Ca²⁺exchanger, has been shown to protect against myocardial ischemia‐reperfusion injury. We have studied here its effects on mitochondrial Ca²⁺fluxes.Experimental approach.: [Ca²⁺] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura‐2, tetramethylrhodamine ethyl ester) probes in HeLa cells.Key results: KB‐R7943 was also a potent inhibitor of the mitochondrial Ca²⁺uniporter (MCU). In permeabilized HeLa cells, KB‐R7943 inhibited mitochondrial Ca²⁺uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB‐R7943 reduced by 80% the mitochondrial [Ca²⁺] peak induced by histamine. KB‐R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na⁺/Ca²⁺exchanger. KB‐R7943 inhibited histamine‐induced ER‐Ca²⁺release in intact cells, but not in cells loaded with a Ca²⁺‐chelator to damp cytosolic [Ca²⁺] changes. Therefore, inhibition of ER‐Ca²⁺‐release by KB‐R7943 was probably due to the increased feedback Ca²⁺‐inhibition of inositol 1,4,5‐trisphosphate receptors after MCU block. This mechanism also explains why KB‐R7943 reversibly blocked histamine‐induced cytosolic [Ca²⁺] oscillations in the same range of concentrations required to inhibit MCU.Conclusions and Implications: Inhibition of MCU by KB‐R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca²⁺‐overload during ischemia‐reperfusion. In addition, the effects of KB‐R7943 on Ca²⁺homeostasis provide new evidence for the role of mitochondria modulating Ca²⁺‐release and regenerative Ca²⁺‐oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.British Journal of Pharmacology(2007)151, 647–654; doi:10.1038/sj.bjp.0707260

There is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in non-excitable cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+ release and in that way controls cytosolic Ca2+ oscillations.

The secretory granules constitute one of the less well‐known compartments in terms of Ca²⁺ dynamics. They contain large amounts of total Ca²⁺, but the free intragranular [Ca²⁺] ([Ca²⁺]_SG), the mechanisms for Ca²⁺ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca²⁺]_SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50–100 μm Ca²⁺. Ca²⁺ accumulation into this compartment takes place mainly through an ATP‐dependent mechanism, namely, a thapsigargin‐sensitive Ca²⁺‐ATPase. In addition, fast Ca²⁺ release was observed in permeabilized cells after addition of inositol 1,4,5‐trisphosphate (InsP₃) or caffeine, suggesting the presence of InsP₃ and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP₃‐producing agonist histamine or with caffeine also induced Ca²⁺ release from the vesicles, whereas acetylcholine or high‐[K⁺] depolarization induced biphasic changes in vesicular [Ca²⁺], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca²⁺ during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca²⁺, and this strongly supports the hypothesis that granular Ca²⁺ may contribute to its own secretion.

The recent availability of activators of the mitochondrial Ca²⁺ uniporter allows direct testing of the influence of mitochondrial Ca²⁺ uptake on the overall Ca²⁺ homeostasis of the cell. We show here that activation of mitochondrial Ca²⁺ uptake by 4,4′,4″‐(4‐propyl‐[1H]‐pyrazole‐1,3,5‐triyl)trisphenol (PPT) or kaempferol stimulates histamine‐induced Ca²⁺ release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na⁺–Ca²⁺ exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca²⁺ uptake and release from mitochondria control the ability of local Ca²⁺ microdomains to produce feedback inhibition of inositol 1,4,5‐trisphosphate receptors (InsP₃Rs). In addition, the ability of mitochondria to control Ca²⁺ release from the ER allows them to modulate cytosolic Ca²⁺ oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca²⁺ oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca²⁺ oscillations are exquisitely sensitive to the rates of mitochondrial Ca²⁺ uptake and release, which precisely control the size of the local Ca²⁺ microdomains around InsP₃Rs and thus the ability to produce feedback activation or inhibition of Ca²⁺ release.