Intracellular sodium modulates mitochondrial calcium signaling in vascular endothelial cells

We have investigated the role of extramitochondrial Na(+) for the regulation of mitochondrial Ca(2+) concentration ([Ca(2+)](m)) in permeabilized single vascular endothelial cells. [Ca(2+)](m) was measured by loading the cells with the membrane-permeant Ca(2+) indicator fluo-3/AM and subsequent removal of cytoplasmic fluo-3 by surface membrane permeabilization with digitonin. An elevation of extramitochondrial Ca(2+) resulted in a dose-dependent increase in the rate of Ca(2+) accumulation into mitochondria (k(0.5) = 3 microm) via the mitochondrial Ca(2+) uniporter. In the presence of 10 mm extramitochondrial Na(+) ([Na(+)](em)), repetitive application of brief pulses of high Ca(2+) (2-10 microm) to simulate cytoplasmic [Ca(2+)] oscillations caused transient increases of [Ca(2+)](m) characterized by a fast rising phase that was followed by a slow decay. Removal of extramitochondrial Na(+) or inhibition of mitochondrial Na(+)/Ca(2+) exchange with clonazepam blocked mitochondrial Ca(2+) efflux and resulted in a net accumulation of Ca(2+) by the mitochondria. Half-maximal activation of mitochondrial Na(+)/Ca(2+) exchange occurred at [Na(+)](em) = 4.4 mm, which is well within the physiological range of cytoplasmic [Na(+)]. This study provides evidence that Ca(2+) efflux from the mitochondria in vascular endothelial cells occurs solely via Na(+)/Ca(2+) exchange and emphasizes the important role of intracellular Na(+) for mitochondrial Ca(2+) regulation.     

Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis

Pituitary gonadotropes transduce hormonal input into cytoplasmic calcium ([Ca(2+)](cyt)) oscillations that drive rhythmic exocytosis of gonadotropins. Using Calcium Green-1 and rhod-2 as optical measures of cytoplasmic and mitochondrial free Ca(2+), we show that mitochondria sequester Ca(2+) and tune the frequency of [Ca(2+)](cyt) oscillations in rat gonadotropes. Mitochondria accumulated Ca(2+) rapidly and in phase with elevations of [Ca(2+)](cyt) after GnRH stimulation or membrane depolarization. Inhibiting mitochondrial Ca(2+) uptake by the protonophore CCCP reduced the frequency of GnRH-induced [Ca(2+)](cyt) oscillations or, occasionally, stopped them. Much of the Ca(2+) that entered mitochondria is bound by intramitochondrial Ca(2+) buffering systems. The mitochondrial Ca(2+) binding ratio may be dynamic because [Ca(2+)](mit) appeared to reach a plateau as mitochondrial Ca(2+) accumulation continued. Entry of Ca(2+) into mitochondria was associated with a small drop in the mitochondrial membrane potential. Ca(2+) was extruded from mitochondria more slowly than it entered, and much of this efflux could be blocked by CGP-37157, a selective inhibitor of mitochondrial Na(+)-Ca(2+) exchange. Plasma membrane capacitance changes in response to depolarizing voltage trains were increased when CCCP was added, showing that mitochondria lower the local [Ca(2+)](cyt) near sites that trigger exocytosis. Thus, we demonstrate a central role for mitochondria in a significant physiological response.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.     

Geldanamycin derivative ameliorates high fat diet-induced renal failure in diabetes

Diabetic nephropathy is a serious complication of longstanding diabetes and its pathogenesis remains unclear. Oxidative stress may play a critical role in the pathogenesis and progression of diabetic nephropathy. Our previous studies have demonstrated that polyunsaturated fatty acids (PUFA) induce peroxynitrite generation in primary human kidney mesangial cells and heat shock protein 90β1 (hsp90β1) is indispensable for the PUFA action. Here we investigated the effects of high fat diet (HFD) on kidney function and structure of db/db mice, a widely used rodent model of type 2 diabetes. Our results indicated that HFD dramatically increased the 24 h-urine output and worsened albuminuria in db/db mice. Discontinuation of HFD reversed the exacerbated albuminuria but not the increased urine output. Prolonged HFD feeding resulted in early death of db/db mice, which was associated with oliguria and anuria. Treatment with the geldanamycin derivative, 17-(dimethylaminoehtylamino)-17-demethoxygeldanamycin (17-DMAG), an hsp90 inhibitor, preserved kidney function, and ameliorated glomerular and tubular damage by HFD. 17-DMAG also significantly extended survival of the animals and protected them from the high mortality associated with renal failure. The benefit effect of 17-DMAG on renal function and structure was associated with a decreased level of kidney nitrotyrosine and a diminished kidney mitochondrial Ca(2+) efflux in HFD-fed db/db mice. These results suggest that hsp90β1 is a potential target for the treatment of nephropathy and renal failure in diabetes.     

Inflammation alters regional mitochondrial Ca²⁺ in human airway smooth muscle cells

Regulation of cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca(2+)](cyt), helping prevent Ca(2+) overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of [Ca(2+)](cyt) in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca(2+)](cyt) and mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca(2+)](cyt) and [Ca(2+)](mito), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased [Ca(2+)](mito) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca(2+)](mito) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca(2+)](cyt). TNF-α and IL-13 both increased [Ca(2+)](cyt), which was associated with decreased [Ca(2+)](mito) in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca(2+)](cyt) and [Ca(2+)](mito) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca(2+)](cyt) after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca(2+)](mito) in different parts of ASM cells may serve to locally regulate Ca(2+) fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca(2+)](cyt) is increased.     

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains

The mitochondrial Ca(2+) uniporter has low affinity for Ca(2+), therefore it has been assumed that submicromolar Ca(2+) signals cannot induce mitochondrial Ca(2+) uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca(2+) diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca(2+) microdomains (HCMDs) capable of activating mitochondrial Ca(2+) uptake. The possibility of mitochondrial Ca(2+) uptake at low submicromolar [Ca(2+)](c) has not yet been generally accepted. Earlier we found in permeabilized glomerulosa, luteal and pancreatic beta cells that [Ca(2+)](m) increased when [Ca(2+)](c) was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca(2+) response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca(2+) response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K(+) (inducing Ca(2+) influx), no correlation was found between the mitochondrial Ca(2+) signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca(2+) uptake may occur both with and without the formation of HCMDs within the same cell.     

Ca2+]i signaling between mitochondria and endoplasmic reticulum in neurons is regulated by microtubules. From mitochondrial permeability transition pore to Ca2+-induced Ca2+ release

The positioning and dynamics of organelles depend on membrane-cytoskeleton interactions. Mitochondria relocate along microtubules (MT), but it is not clear whether MT have direct effects on mitochondrial function. Using two-photon microscopy and the mitochondrial fluorescent dyes rhodamine 123 and Rhod-2, we showed that Taxol and nocodazole, which correspondingly stabilize and disrupt MT, decreased potential and Ca(2+) in the mitochondria of brain stem pre-Botzinger complex neurons. Without changing basal cytoplasmic Ca(2+) ([Ca(2+)](i)), Taxol promoted the generation of [Ca(2+)](i) spikes in dendrites. These spikes were abolished after blockade of Ca(2+) influx and after depletion of internal Ca(2+) stores, indicating the involvement of Ca(2+)-induced Ca(2+) release. Nocodazole decreased mitochondrial potential and [Ca(2+)](m) and produced a long lasting increase in [Ca(2+)](i). MT-acting drugs depolarized single immobilized mitochondria and released previously stored Ca(2+). All of these effects were inhibited by pretreatment with blockers of mitochondrial permeability transition pore (mPTP), cyclosporin A, and 2-aminoethoxydiphenyl borate. Induction of mPTP by Taxol and nocodazole was confirmed by using a calcein/Co(2+) imaging technique. Electron and optical microscopy revealed tubulin bound to mitochondria. Mitochondria, MT, and endoplasmic reticulum (ER) showed strong co-localization, the degree of which decreased after MT were disrupted. We propose that changes in the structure of MT by Taxol and nocodazole promote the induction of mPTP. Subsequent Ca(2+) efflux stimulates the Ca(2+) release from the ER that drives spontaneous [Ca(2+)](i) transients. Thus, close positioning of mitochondria to the ER as determined by MT can be essential for the local [Ca](i) signaling in neurons.     

Mitochondrial Ca2+ uptake is important over low [Ca2+]i range in arterial smooth muscle

Mitochondrial Ca(2+) uptake is usually thought to occur only when intracellular Ca(2+) concentration ([Ca(2+)](i)) is high. We investigated whether mitochondrial Ca(2+) removal participates in shaping [Ca(2+)](i) signals in arterial smooth muscle over a low [Ca(2+)](i) range. [Ca(2+)](i) was measured using fura 2-loaded, voltage-clamped cells from rat femoral arteries. Both diazoxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized the mitochondria. Diazoxide application increased resting [Ca(2+)](i), suggesting that Ca(2+) is sequestered in mitochondria. Over a low [Ca(2+)](i) range, diazoxide and CCCP slowed Ca(2+) removal rate, determined after a brief depolarization. When [Ca(2+)](i) was measured during sustained depolarization to -30 mV, CCCP application increased [Ca(2+)](i). When Ca(2+) transients were repeatedly evoked by caffeine applications, CCCP application elevated resting [Ca(2+)](i). Caffeine-induced Ca(2+) transients were compared before and after CCCP application using the half decay time, or time required to reduce increase in [Ca(2+)](i) by 50% (t((1/2))). CCCP treatment significantly increased t((1/2)). These results suggest that Ca(2+) removal to mitochondria in arterial smooth muscle cells may be important at a low [Ca(2+)](i).     

Mitochondria recycle Ca(2+) to the endoplasmic reticulum and prevent the depletion of neighboring endoplasmic reticulum regions

To study Ca(2+) fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in approximately 20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 +/- 5 microm, indicating that Ca(2+) transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca(2+)](ER) averaged 371 +/- 21 microm at rest and decreased to 133 +/- 14 microm and 59 +/- 5 microm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca(2+) uptake was prevented by oligomycin and rotenone or when Ca(2+) efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca(2+) taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca(2+) than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca(2+) uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca(2+) release and recycle a substantial portion of the captured Ca(2+) back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca(2+) signals and the filling state of neighboring ER regions.     

Mitochondrial Ca2+ uptake requires sustained Ca2+ release from the endoplasmic reticulum

We analyzed the role of inositol 1,4,5-trisphosphate-induced Ca(2+) release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca(2+) uptake and (ii) in maintaining a sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)). For this purpose, we expressed in HeLa cells aequorin-based Ca(2+)-sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H(1) receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. Glutamate induced a transient [Ca(2+)](c) rise and drop in ER luminal [Ca(2+)] ([Ca(2+)](er)), and then the ER refilled with [Ca(2+)](c) at resting values. With histamine, [Ca(2+)](c) after the initial peak stabilized at a sustained plateau, and [Ca(2+)](er) decreased to a low steady-state value. In mitochondria, histamine evoked a much larger mitochondrial Ca(2+) response than glutamate ( approximately 15 versus approximately 65 microm). Protein kinase C inhibition, partly relieving mGluR1a desensitization, reestablished both the [Ca(2+)](c) plateau and the sustained ER Ca(2+) release and markedly increased the mitochondrial Ca(2+) response. Conversely, mitochondrial Ca(2+) uptake evoked by histamine was drastically reduced by very transient ( approximately 2-s) agonist applications. These data indicate that efficient mitochondrial Ca(2+) uptake depends on the preservation of high Ca(2+) microdomains at the mouth of ER Ca(2+) release sites close to mitochondria. This in turn depends on continuous Ca(2+) release balanced by Ca(2+) reuptake into the ER and maintained by Ca(2+) influx from the extracellular space.     

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria

Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local [Ca(2+)] control that operates between IP(3)receptor Ca(2+)release channels (IP(3)R) and mitochondrial Ca(2+)uptake sites, and displays functional similarities to synaptic transmission. Activation of IP(3)R by IP(3)is known to evoke quantal Ca(2+)mobilization that is associated with incremental elevations of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)). Here we report that activation of IP(3)R by adenophostin-A (AP) yields non-quantal Ca(2+)mobilization in mast cells. We also show that the AP-induced continuous Ca(2+)release causes relatively small [Ca(2+)](m)responses, in particular, the sustained phase of Ca(2+)release is not sensed by the mitochondria. Inhibition of ER Ca(2+)pumps by thapsigargin slightly increases IP(3)-induced [Ca(2+)](m)responses, but augments AP-induced [Ca(2+)](m)responses in a large extent. In adherent permeabilized cells exposed to elevated [Ca(2+)], ER Ca(2+)uptake fails to affect global cytosolic [Ca(2+)], but attenuates [Ca(2+)](m)responses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca(2+)pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca(2+)uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca(2+)release through the IP(3)R or during gradual Ca(2+)influx to the junction between ER and mitochondria.     

Mode of mitochondrial Ca2+ clearance and its influence on secretory responses in stimulated chromaffin cells

To study the role of mitochondrial Ca(2+) clearance in stimulated cells, changes in free Ca(2+) concentration in the cytosol, [Ca(2+)](c) and that in mitochondria, [Ca(2+)](m) along with secretory responses were observed using chromaffin cells co-loaded with Fura-2 and Rhod-2 in the perfused rat adrenal medulla. When the cells were stimulated with 40 mM K(+) in the perfusate, the duration of [Ca(2+)](m) response markedly increased with prolongation of the stimulation period, exhibiting a mean half-decay time of 21 min with 30s stimulation, whereas its amplitude was not altered with stimulations of 10-30s. A computer simulation analysis showed that such a mode of [Ca(2+)](m) response can be produced if excess Ca(2+) taken up by mitochondria precipitates as calcium phosphate (Pi) salt. In the presence of 5 microM rotenone plus 10 microM oligomycin, a decrease in the duration of [Ca(2+)](m) response and a slight but significant increase (24%) in the secretory response to 30s stimulation with 40 mM K(+) were observed. Simulation analyses suggested that this effect of rotenone may be due to reduction in mitochondrial Ca(2+) uptake induced by rotenone-elicited partial depolarization of the mitochondrial membrane potential. In chromaffin cells transsynaptically stimulated through the splanchnic nerve, the intensity of NAD(P)H autofluorescence changed with time courses similar to those of [Ca(2+)](m) responses. The temporal profiles of those two responses were prolonged in a similar manner by application of an inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, CGP37157. Thus, due to the unique Ca(2+) buffering mechanism, [Ca(2+)](m) responses associated with massive mitochondrial Ca(2+) uptake may occur within a limited concentration range in which Ca(2+)-sensitive dehydrogenases are activated to control the mitochondrial redox state in stimulated chromaffin cells.     

Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells.

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca(2+)-dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca(2+)/calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca(2+)](i), this late stage of eNOS activation is strictly conditional on the initial VEGF-induced Ca(2+)-dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90.     

Mitochondrial Ca2+ and the heart

It is now well established that mitochondria accumulate Ca(2+) ions during cytosolic Ca(2+) ([Ca(2+)](i)) elevations in a variety of cell types including cardiomyocytes. Elevations in intramitochondrial Ca(2+) ([Ca(2+)](m)) activate several key enzymes in the mitochondrial matrix to enhance ATP production, alter the spatial and temporal profile of intracellular Ca(2+) signaling, and play an important role in the initiation of cell death pathways. Moreover, mitochondrial Ca(2+) uptake stimulates nitric oxide (NO) production by mitochondria, which modulates oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and in turn provides negative feedback for the regulation of mitochondrial Ca(2+) accumulation. Controversy remains, however, whether in cardiac myocytes mitochondrial Ca(2+) transport mechanisms allow beat-to-beat transmission of fast cytosolic [Ca(2+)](i) oscillations into oscillatory changes in mitochondrial matrix [Ca(2+)](m). This review critically summarizes the recent experimental work in this field.     

Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.     

Role of mitochondria in Ca(2+) homeostasis of mouse pancreatic acinar cells

The effects of mitochondrial Ca(2+) uptake on cytosolic Ca(2+) concentration ([Ca(2+)](c)) were investigated in mouse pancreatic acinar cells using cytosolic and/or mitochondrial Ca(2+) indicators. When calcium stores of the endoplasmic reticulum (ER) were emptied by prolonged incubation with thapsigargin (Tg) and acetylcholine (ACh), small amounts of calcium could be released into the cytosol (Delta[Ca(2+)](c)=46 +/- 6 nM, n=13) by applying mitochondrial inhibitors (combination of rotenone (R) and oligomycin (O)). However, applications of R/O, soon after the peak of Tg/Ach-induced Ca(2+) transient, produced a larger cytosolic calcium elevation (Delta[Ca(2+)](c)=84 +/- 6 nM, n=9), this corresponds to an increase in the total mitochondrial calcium concentration ([Ca(2+)](m)) by approximately 0.4 mM. In cells pre-treated with R/O or Ru360 (a specific blocker of mitochondrial Ca(2+) uniporter), the decay time-constant of the Tg/ACh-induced Ca(2+) response was prolonged by approximately 40 and 80%, respectively. Tests with the mitochondrial Ca(2+) indicator rhod-2 revealed large increases in [Ca(2+)](m) in response to Tg/ACh applications; this mitochondrial uptake was blocked by Ru360. In cells pre-treated with Ru360, 10nM ACh elicited large global increases in [Ca(2+)](c), compared to control cells in which ACh-induced Ca(2+) signals were localised in the apical region. We conclude that mitochondria are active elements of cellular Ca(2+) homeostasis in pancreatic acinar cells and directly modulate both local and global calcium signals induced by agonists.