Inhibition Of Mitochondrial Ca2 Research Articles

Abstract 5142: Direct Monitoring of Mitochondrial Calcium Levels in Cultured Cardiomyocytes Using a Novel Fluorescent Indicator Protein, GCaMP2-mt

Background: An opening of the mitochondrial permeability transition pore (MPTP), which leads to loss of mitochondrial membrane potential (ΔΨ m ), is the earliest event that commits the cell to death. Mitochondrial matrix calcium ([Ca 2+ ] m ) is considered a critical regulator of MPTP, but direct monitoring of [Ca 2+ ] m has been difficult with previously reported sensors due to limited signal intensity or low targeting efficiency. We developed a novel fluorescent indicator for [Ca 2+ ] m , GCaMP2-mt, by adding the mitochondrial targeting sequence of cytochrome oxidase to a high signal-to-noise Ca 2+ sensor protein GCaMP2, and monitored the dynamic changes in oxidant-induced cardiomyocyte death. Methods and Results: GCaMP2-mt was transduced in neonatal rat cardiomyocytes using a recombinant adenovirus. We confirmed that GCaMP2-mt colocalized well with tetramethylrhodamine ethyl-ester, a fluorescent indicator of ΔΨ m . We monitored oxidant-induced response of [Ca 2+ ] m and ΔΨ m using time-lapse confocal microscopy. After H 2 O 2 exposure, [Ca 2+ ] m remained unchanged for about 20 min, followed by kinetically-distinct two-step increase; the first increase lasted for about 5 min, and the second increase was slower and persisted until cells underwent irreversible ΔΨ m loss. Importantly, there was a cellular heterogeneity in the second increase, and the level of [Ca 2+ ] m overload was variable. However, ΔΨ m loss occurred in an all-or-none manner depending on cellular [Ca 2+ ] m level, with a clear cut-off value. A Ca 2+ chelator BAPTA suppressed both the first and the second increase of [Ca 2+ ] m , and protected the cells against ΔΨ m loss. In contrast, ruthenium red, an inhibitor of mitochondrial Ca 2+ uniporter, suppressed only the second increase of [Ca 2+ ] m , but not the first increase. Despite the suppressed [Ca 2+ ] m overload, ruthenium red was unable to protect against ΔΨ m loss; it caused earlier increase in cytosolic Ca 2+ ([Ca 2+ ] c ) level which might have accelerated the MPTP opening, as measured by an indicator of [Ca 2+ ] c , Fluo-4. These findings support the central role of [Ca 2+ ] m as well as [Ca 2+ ] c overload in the MPTP opening. Conclusion: Direct monitoring of [Ca 2+ ] m using GCaMP2-mt provides deeper insight into the mechanism of cardiomyocyte death.

Mitochondrial Ca2+ and Apoptosis in Multiple Myeloma

Multiple myeloma is a hematological cancer of plasma cells. Proteasome inhibitors, such as Bortezomib (BZ, Velcade/PS‐341) are used to treat myeloma because they induce apoptosis via activation of ER stress. We showed that ER stress results in a transient increase in cytosolic calcium ([Ca2+]c), and subsequent initiation of apoptosis. Since inhibition of mitochondrial Ca2+ ([Ca2+]m) uptake with ruthenium red blocked apoptotic initiation and activation of caspase activity, we analyzed [Ca2+]m in the myeloma line 8226 using Rhod‐2. Although BZ elicits a transient increase in [Ca2+]c, [Ca2+]m was found to rapidy (< 1 min) decrease. Following this initial decrease, two distinct responses were identified; either [Ca2+]m returned to then exceeded baseline, or [Ca2+]m remained below baseline levels. Our results indicate that there are two discrete sub‐populations of myeloma cells with respect to response to BZ which is consistent with the observed efficacy of BZ on <70% of the myeloma population. We propose that the population in which [Ca2+]m remains low is refractory to BZ induced apoptosis. Current studies will test this hypothesis, and aim to further characterize these two sub‐populations of myeloma cells. These data will allow us to identify targets for initiating apoptosis in the BZ refractory population with the goal of increasing the efficacy and specificity of drug treatment.

Extracellular ATP Mediates The Intercellular Ca2+ Wave Induced By Mechanical Stimulation In Human Salivary Gland Cells

Intracellular Ca2+ signaling has a central role in regulation of salivary gland cell function. Coordination of Ca2+ signaling between cells contributes to synchronized and effective secretion of saliva. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca2+ waves (ICW) and their propagation in human salivary gland (HSG) cells were investigated using fura-2 fluorescence imaging. While not well understood, mechanical stimulation of a single cell in a cluster with a micropipette induces ICW. The Ca2+ signal is propagated from the stimulated cell to the 7-9th tier of cells or ∼120 μm. The following findings indicate that ICW propagation in HSG cells uses an extracellular and ATP-dependent pathway. The purinergic receptor antagonist suramin significantly decreased ICW propagation. Extracellular ATP or UTP abolished ICW suggestive of receptor desensitization. Gap junction intercellular communication is not involved in ICW in HSG cells because the gap junction inhibitor oleamide did not inhibit ICW. Furthermore, HSG cells showed poor dye coupling upon microinjection of Lucifer Yellow. The Ca2+ transients observed within each cell are dependent on Ca2+ release from the ER as thapsigargin abolished the ICW. The phospholipase C inhibitor U73122 also blocks ICW indicating that these transients are IP3-dependent. Furthermore, store operated Ca2+ entry (SOCE) modulates the amplitude of Ca2+ signal since removal of extracellular Ca2+ or a SOCE inhibitor SK&F 96365 decreased the amplitude of Ca2+ signal. Inhibition of mitochondrial Ca2+ uptake with FCCP/oligomycin or ruthenium red showed similar effects on the amplitude. These results indicate that propagation of this ICW utilizes extracellular ATP, likely through the P2U(P2Y2) receptor in HSG cells. The major Ca2+ mobilization mechanisms are IP3-dependent ER Ca2+ release and SOCE. Finally, mitochondrial energy metabolism and Ca2+ uptake modulated this ICW propagation.

Mitochondria Act Within InsP3R Clusters To Maintain Ca2+ Release In Smooth Muscle

Many smooth muscle activities including contraction, transcription, growth and apoptosis are regulated by transient inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c). InsP3 binds to receptors (InsP3R) present on the sarcoplasmic reticulum to evoke Ca2+ release. InsP3R exist in clusters and Ca2+ released from one receptor may activate nearby InsP3R within this cluster in a CICR-like process to evoke a ‘puff’. Ca2+ released may also diffuse to adjacent clusters to trigger further Ca2+ release and generate a Ca2+ rise throughout the cell. Mitochondrial Ca2+ uptake limits a negative feedback process operative on InsP3R to maintain Ca2+ release. Inhibition of mitochondrial Ca2+ uptake decreases InsP3-mediated Ca2+ waves by ≥50%. We addressed whether mitochondria act to maintain release by operating within or between InsP3R clusters. Ca2+ puffs were evoked by localized photolysis of InsP3 in single voltage-clamped colonic smooth muscle cells in which [Ca2+]c and ΔΨM were measured simultaneously. EGTA, a slow Ca2+ buffer, was used to functionally uncouple puff sites to prevent the formation of Ca2+ waves. EGTA was used at a concentration ([300 μM]) which does not affect the magnitude or kinetics of Ca2+ puffs. InsP3-evoked Ca2+ puffs had amplitudes of 0.5-5.0 F/F0 and durations of ∼200 ms at half-maximum amplitude. Puffs were abolished by the InsP3R inhibitor 2-APB. The protonophore CCCP and the mitochondrial inhibitor rotenone, each used with oligomycin, depolarized the mitochondrial membrane potential (ΔΨM) and prevented mitochondrial Ca2+ uptake. Depolarizing ΔΨM with CCCP attenuated Ca2+ puffs by ∼65% while rotenone inhibited them by ∼60%. These results indicate mitochondrial Ca2+ uptake occurs quickly enough to influence InsP3R communication at the intra-cluster level. Supported by the Wellcome Trust and British Heart Foundation.

Inhibition of Autophagy Promotes Bortezomib-Mediated Cell Death in Myeloma Cells

The 26S proteasome is a key regulator of proteins controlling many important cellular functions, including cell cycle progression, differentiation, gene transcription and apoptosis. Proteasome inhibition is a new therapeutic strategy that has shown promise in the treatment of B cell malignancies, primarily multiple myeloma. We and others have demonstrated that proteasome inhibitors induce endoplasmic reticulum (ER) stress and activate an unfolded protein response (UPR) in transformed cells. Our previous work demonstrated that bortezomib induces an endoplasmic reticulum (ER) stress response ultimately leading to calcium-dependent apoptotic cell death. Co-treatment of myeloma cells with the mitochondrial uniporter inhibitor ruthenium red (RuR) abrogated bortezomib mediated cell death, indicating that the cytotoxic effects of proteasomal inhibition requires dysregulation of intracellular Ca2+. Intracellular Ca2+ has also been implicated in the cellular stress response known as autophagy or “self-eating”. Macroautophagy (hereafter referred to as autophagy) is induced by various cellular stresses including nutrient deprivation, metabolic insufficiency, interruption of growth factor signaling, elevated ROS, accumulation of intracellular Ca2+, and the UPR. The biochemical events linking the cellular stress response with the induction of autophagy, and the relationship between autophagy and apoptosis is not well understood.In this study, we investigate the role of the Ca2+ dependent serine protease, calpain, as a mediator of the conversion from autophagic cell survival to accelerated cell death in the ER stress response. We demonstrate that the proteasome inhibitor, bortezomib, initiates autophagy in myeloma cells, and protection from bortezomib-mediated cell death by mitochondrial Ca2+ inhibitors is associated with a promotion and stabilization of the autophagosome. This response can be reversed, and indeed, accelerated, leading to enhanced cell death, by blockade of calpain activity. Inhibition of calpain activity with the tri-peptide zLLY-FMK (Calpain Inhibitor IV, (CiIV) or the non-peptide inhibitor, PD150606, demonstrated a significant increase in the cytotoxic activity of bortezomib. Similarly, elimination of the small catalytic subunit, CAPNS1, using siRNA, enhanced bortezomib-mediated cell death, and prevented autophagosome-lysosomal progression. Furthermore, inhibition of calpain by clinically approved HIV protease inhibitors including Nelfinavir, Ritonavir, Saquinavir, and Indinavir sulfate, significantly increased the cytotoxic activity of bortezomib in vitro. We suggest that disregulation of Ca2+ by bortezomib-mediated ER stress activates the autophagic survival response. Inhibition of mitochondrial Ca2+ uptake by the uniporter inhibitor RuR promotes autophagy, and confers resistance to bortezomib. Conversely, inhibition of the Ca2+-dependent serine protease, calpain, prevents autophagolysosome maturation, and subverts the survival response to cell death. These data are likely to have important clinical implications for the treatment of refractory myeloma and other B cell malignancies.

Mechanisms of Prolonged Presynaptic Ca2+Signaling and Glutamate Release Induced by TRPV1 Activation in Rat Sensory Neurons

Transient receptor potential vanilloid receptor 1 (TRPV1)-mediated release of neuroactive peptides and neurotransmitters from the peripheral and central terminals of primary sensory neurons can critically contribute to nociceptive processing at the periphery and in the CNS. However, the mechanisms that link TRPV1 activation with Ca2+ signaling at the release sites and neurosecretion are poorly understood. Here we demonstrate that a brief stimulation of the receptor using either capsaicin or the endogenous TRPV1 agonist N-arachidonoyl-dopamine induces a prolonged elevation of presynaptic [Ca2+](i) and a concomitant enhancement of glutamate release at sensory synapses. Initiation of this response required Ca2+ entry, primarily via TRPV1. The sustained phase of the response was independent of extracellular Ca2+ and was prevented by inhibitors of mitochondrial Ca2+ uptake and release mechanisms. Measurements using a mitochondria-targeted Ca2+ indicator, mtPericam, revealed that TRPV1 activation elicits a long-lasting Ca2+ elevation in presynaptic mitochondria. The concentration of TRPV1 agonist determined the duration of mitochondrial and cytosolic Ca2+ signals in presynaptic boutons and, consequently, the period of enhanced glutamate release and action potential firing by postsynaptic neurons. These data suggest that mitochondria control vanilloid-induced neurotransmission by translating the strength of presynaptic TRPV1 stimulation into duration of the postsynaptic response.

Mechanisms of Mitochondrial Complex 1 Inhibition by Intermittent Hypoxia

We previously reported that intermittent hypoxia (IH) inhibits the mitochondrial complex I but not the complex III. IH has been shown to activate NADPH oxidases. The present study tests the hypothesis that activation of NADPH oxidases and the resulting increase in reactive oxygen species (ROS) mediate complex I inhibition by IH. To test this possibility, experiments were performed on rat PC12 cells exposed to IH or normoxia. IH increased NADPH oxidase activity in a stimulus‐dependent manner and the effects could be reversed following re‐oxygenation for 3h. Apocynin (1mM), an inhibitor of NADPH oxidase as well as ROS scavengers prevented IH‐evoked inhibition of complex I. ROS are known to mobilize intracellular Ca2+. 2APB (75μM; inhibitor of IP3 receptors) but not nifedipine (3μM; L‐type calcium channel blocker) restored IH‐evoked complex I inhibition. Furthermore, ruthenium red (20μM; inhibitor of mitochondrial Ca2+ uniporter) or CGP37157 (10μM; inhibitor of mitochondrial Na+/Ca2+ exchanger) prevented complex I inhibition by IH. Taken together these observations suggest that ROS generated by NADPH oxidases mobilize Ca2+ flux from cytosol to mitochondria during IH resulting in inhibition of complex I. (Supported by NIH HL‐25830).

Target Cell-Specific Involvement of Presynaptic Mitochondria in Post-Tetanic Potentiation at Hippocampal Mossy Fiber Synapses

Previous studies indicate that boutons from the same axon exhibit distinct Ca2+ dynamics depending on the postsynaptic targets. Mossy fibers of hippocampal granule cells innervate synaptic targets via morphologically distinct boutons. We investigated mitochondrial involvement in the generation of post-tetanic residual Ca2+ (Ca(res)) at large and small en passant mossy fiber boutons (MFBs). Mitochondria limited the [Ca2+]i build-up during high-frequency stimulation (HFS) at large MFBs, but not at small MFBs. The amount of Ca(res), quantified as a time integral of residual [Ca2+]i, was significantly larger at large MFBs than at small MFBs, and that at large MFBs was substantially attenuated by inhibitors of mitochondrial Ca2+ uniporter and mitochondrial Na+/Ca2+ exchanger (mitoNCX). In contrast, blockers of mitoNCX had no effect on the Ca(res) at small MFBs. Post-tetanic Ca(res) has been proposed as a mechanism for post-tetanic potentiation (PTP). We examined mitochondrial involvement in PTP at mossy fiber synapses on hilar mossy cells (MF-->MC synapse) and on hilar interneurons (MF-->HI synapse), which are presumably innervated via large and small MFBs, respectively. Consistent with the differential contribution of mitochondria to Ca(res) at large and small MFBs, mitoNCX blockers significantly reduced the PTP at the MF-->MC synapse, but not at the MF-->HI synapse. In contrast, protein kinase C (PKC) inhibitors significantly reduced the PTP at MF-->HI synapse, but not at the MF-->MC synapse. These results indicate that mitochondria- and PKC-dependent PTP are expressed at distinct hilar mossy fiber synapses depending on postsynaptic targets.

The mitochondrial membrane potential and Ca2+ oscillations in smooth muscle

Ca2+ uptake by mitochondria might both modulate the cytosolic Ca2+ concentration ([Ca2+]c) and depolarize the mitochondrial membrane potential (delta Psi m) to limit ATP production. To investigate how physiological Ca2+ signaling might affect energy production, delta Psi m was examined during Ca2+ oscillations in smooth muscle cells. In single, voltage-clamped smooth muscle cells, inhibition of mitochondrial Ca2+ accumulation inhibited inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-evoked Ca2+ release and prolonged the time required for restoration of [Ca2+]c following activation of plasmalemmal Ca2+ currents (ICa). Ca2+ could be released from mitochondria immediately (within 15 seconds) after a [Ca2+]c rise evoked by Ins(1,4,5)P3 or ICa. Despite this evidence of mitochondrial Ca2+ accumulation, no change in delta Psi m was observed during single or repetitive [Ca2+]c oscillations evoked by these conditions. Occasionally, spontaneous, repetitive, persistent Ca 2+ oscillations were observed. In these cases, mitochondria displayed stochastic delta Psi m depolarizations, which were independent both of events in neighboring mitochondria and of the timing of the [Ca 2+]c oscillations themselves. Such delta Psi m depolarizations could be mimicked by increased exposure to either fluorescence excitation light or the delta Psi m-sensitive dye tetramethylrhodamine ethyl ester (TMRE) and were inhibited by antioxidants (ascorbic acid, catalase, Trolox and TEMPO) or the mitochondrial permeability transition pore (mPTP)-inhibitor cyclosporin A (CsA). Individual mitochondria within smooth muscle cells might depolarize during repetitive Ca2+ oscillations or during oxidative stress but not during the course of single [Ca2+]c transients evoked by Ca2+ influx or store release.

Role of mitochondria in spontaneous rhythmic activity and intracellular calcium waves in the guinea pig gallbladder smooth muscle

Mitochondrial Ca(2+) handling has been implicated in spontaneous rhythmic activity in smooth muscle and interstitial cells of Cajal. In this investigation we evaluated the effect of mitochondrial inhibitors on spontaneous action potentials (APs), Ca(2+) flashes, and Ca(2+) waves in gallbladder smooth muscle (GBSM). Disruption of the mitochondrial membrane potential with carbonyl cyanide 3-chlorophenylhydrazone, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, rotenone, and antimycin A significantly reduced or eliminated APs, Ca(2+) flashes, and Ca(2+) waves in GBSM. Blockade of ATP production with oligomycin did not alter APs or Ca(2+) flashes but significantly reduced Ca(2+) wave frequency. Inhibition of mitochondrial Ca(2+) uptake and Ca(2+) release with Ru360 and CGP-37157, respectively, reduced the frequency of Ca(2+) flashes and Ca(2+) waves in GBSM. Similar to oligomycin, cyclosporin A did not alter AP and Ca(2+) flash frequency but significantly reduced Ca(2+) wave activity. These data suggest that mitochondrial Ca(2+) handling is necessary for the generation of spontaneous electrical activity and may therefore play an important role in gallbladder tone and motility.

Abstract 274: Physical Coupling Supports Local Ca2+ Coupling Between SR Subdomains and the Mitochondria in Heart Muscle

Propagation of ryanodine receptor (RyR) dependent SR Ca2+ release to the mitochondria is locally supported by high [Ca2+] microdomains at close contacts between the organelles. The objective of this study was to clarify if the close associations of SR and mitochondria underlying the local Ca2+ communication were secured via a physical coupling. Crude and Percoll-purified mitochondrial fractions of rat heart homogenates were tested for the presence of mitochondria-associated SR fragments capable of RyR-dependent Ca2+ mobilization and Ca2+ delivery to the mitochondria. SR and mitochondrial marker proteins were analyzed by Western blot or visualized in situ by immunofluorescent labeling and imaged using confocal microscopy. Mitochondrial matrix [Ca2+] ([Ca2+]m) was monitored using fluorescence imaging of rhod-2-loaded adherent individual vesicles. Ample presence of SR markers (calsequestrin, SERCA2a, phospholamban) was detected in the crude mitochondria, whereas after percoll purification they were barely detectable in the ‘heavy’ fraction but remained present in the ‘light’ fraction. ‘Immunomitochemistry’ revealed various sizes of SR particles in the crude fraction, a large portion of which was colocalized with mitochondria, while showed less SR markers in the percoll-purified heavy fraction. Fluorescence Ca2+ imaging in the adherent particles revealed well-detectable [Ca2+]m responses to caffeine stimulation that was prevented by Ca2+ predepletion of the SR using the SERCA inhibitor thapsigargin (Tg), or by inhibitors of mitochondrial Ca2+ uptake. Because the Ca2+ released from SR was diluted in the large volume of the incubation medium without causing a change in the bulk [Ca2+], only local Ca2+ regulation could mediate the Ca2+ transfer to the mitochondria. Despite the hardly detectable presence of SR, the percoll-purified ‘heavy’ mitochondrial fraction still displayed a caffeine-induced [Ca2+]m rise that was abolished by Tg-pretreatment. Our data suggest that the SR-mitochondrial associations where the local Ca2+ coupling is established, are supported by physical links between the two organelles and the mitochondria bound SR surface represents a relatively small fraction of the total SR.

Intracellular organelles and calcium homeostasis in rods and cones.

The role of intracellular organelles in Ca2+ homeostasis was studied in salamander rod and cone photoreceptors under conditions that simulate photoreceptor activation by darkness and light. Sustained depolarization evoked a Ca2+ gradient between the cell body and ellipsoid regions of the inner segment (IS). The standing pattern of calcium fluxes was created by interactions between the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Pharmacological experiments suggested that mitochondria modulate both baseline [Ca2+]i in hyperpolarized cells as well as kinetics of Ca2+ entry via L type Ca2+ channels in cell bodies and ellipsoids of depolarized rods and cones. Inhibition of mitochondrial Ca2+ sequestration by antimycin/oligomycin caused a three-fold reduction in the amount of Ca2+ accumulated into intracellular organelles in both cell bodies and ellipsoids. A further 50% decrease in intracellular Ca2+ content within cell bodies, but not ellipsoids, was observed after suppression of SERCA-mediated Ca2+ uptake into the ER. Inhibition of Ca2+ sequestration into the endoplasmic reticulum by thapsigargin or cyclopiazonic acid decreased the magnitude and kinetics of depolarization-evoked Ca2+ signals in cell bodies of rods and cones and decreased the amount of Ca2+ accumulated into internal stores. These results suggest that steady-state [Ca2+]i in photoreceptors is regulated in a region-specific manner, with the ER contribution predominant in the cell body and mitochondrial buffering [Ca2+] the ellipsoid. Local [Ca2+]i levels are set by interactions between the plasma membrane Ca2+ channels and transporters, ER and mitochondria. Mitochondria are likely to play an essential role in temporal and spatial buffering of photoreceptor Ca2+.

Involvement of L-type calcium channels and mitochondria in post-tetanic potentiation: Is it a general rule for different types of synapses?

Our experiments and studies of a few other authors demonstrated that L-type calcium channels and mitochondria are involved in the induction of post-tetanic potentiation (PTP) in a number of preparations (Aplysia central nervous system, hippocampal cell cultures, crayfish neuromuscular junctions, etc.). We extend this conclusion on cortical synapses by the demonstration that inhibitors of mitochondrial Ca2+ uptake and release suppress PTP in rat neocortical cell cultures.

Mitochondrial Nitric Oxide Mediates Decreased Vulnerability of Hippocampal Neurons from Immature Animals to NMDA

Mitochondrial membrane potential (DeltaPsim)-dependent Ca2+ uptake plays a central role in neurodegeneration after NMDA receptor activation. NMDA-induced DeltaPsim dissipation increases during postnatal development, coincident with increasing vulnerability to NMDA. NMDA receptor activation also produces nitric oxide (NO), which can inhibit mitochondrial respiration, dissipating DeltaPsim. Because DeltaPsim dissipation reduces mitochondrial Ca2+ uptake, we hypothesized that NO mediates the NMDA-induced DeltaPsim dissipation in immature neurons, underlying their decreased vulnerability to excitotoxicity. Using hippocampal neurons cultured from 5- and 19-d-old rats, we measured NMDA-induced changes in [Ca2+]cytosol, DeltaPsim, NO, and [Ca2+]mito. In postnatal day 5 (P5) neurons, NMDA mildly dissipated DeltaPsim in a NO synthase (NOS)-dependent manner and increased NO. The NMDA-induced NO increase was abolished with carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone and regulated by [Ca2+]mito. Mitochondrial Ca2+ uptake inhibition prevented the NO increase, whereas inhibition of mitochondrial Ca2+ extrusion increased it. Consistent with this mitochondrial regulation, NOS and cytochrome oxidase immunoreactivity demonstrated mitochondrial localization of NOS. Furthermore, NOS blockade increased mitochondrial Ca2+ uptake during NMDA. Finally, at physiologic O2 tensions (3% O2), NMDA had little effect on survival of P5 neurons, but NOS blockade during NMDA markedly worsened survival, demonstrating marked neuroprotection by mitochondrial NO. In P19 neurons, NMDA dissipated DeltaPsim in an NO-insensitive manner. NMDA-induced NO production was not regulated by DeltaPsim, and NOS immunoreactivity was cytosolic, without mitochondrial localization. NOS blockade also protected P19 neurons from NMDA. These data demonstrate that mitochondrial NOS mediates much of the decreased vulnerability to NMDA in immature hippocampal neurons and that cytosolic NOS contributes to NMDA toxicity in mature neurons.

Growth hormone promotes Ca(2+)-induced Ca2+ release in insulin-secreting cells by ryanodine receptor tyrosine phosphorylation.

Elevation in cytoplasmic free Ca2+ concentration ([Ca2+]i) is a common mechanism in signaling events. An increased [Ca2+]i induced by GH, has been observed in relation to different cellular events. Little is known about the mechanism underlying the GH effect on Ca2+ handling. We have studied the molecular mechanisms underlying GH-induced rise in [Ca2+]i in BRIN-BD11 insulin-secreting cells. GH (500 ng/ml, 22 nm) induced a sustained increase in [Ca2+]i. The effect of GH on [Ca2+]i was prevented in the absence of extracellular Ca2+ and was inhibited by the ATP-sensitive K(+)-channel opener diazoxide and the voltage-dependent Ca(2+)-channel inhibitor nifedipine. However, GH failed to induce any changes in Ca2+ current and membrane potential, evaluated by patch-clamp recordings and by using voltage-sensitive dyes. When the intracellular Ca2+ pools had been depleted using the Ca(2+)-ATPase inhibitor thapsigargin, the effect of GH was inhibited. In addition, GH-stimulated rise in [Ca2+]i was completely abolished by ruthenium red, an inhibitor of mitochondrial Ca2+ transport, and caffeine. GH induced tyrosine phosphorylation of ryanodine receptors. The effect of GH on [Ca2+]i was completely blocked by the tyrosine kinase inhibitors genistein and lavendustin A. Interestingly, treatment of the cells with GH significantly enhanced K(+)-induced rise in [Ca2+]i. Hence, GH-stimulated rise in [Ca2+]i is dependent on extracellular Ca2+ and is mediated by Ca(2+)-induced Ca2+ release. This process is mediated by tyrosine phosphorylation of ryanodine receptors and may play a crucial role in physiological Ca2+ handling in insulin-secreting cells.

Modulation of [Ca2+]i Signaling Dynamics and Metabolism by Perinuclear Mitochondria in Mouse Parotid Acinar Cells

Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke[Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion.

Inhibition of mitochondrial Ca2+ uptake affects phasic release from motor terminals differently depending on external [Ca2+

We investigated how inhibition of mitochondrial Ca2+ uptake affects stimulation-induced increases in cytosolic [Ca2+] and phasic and asynchronous transmitter release in lizard motor terminals in 2 and 0.5 mM bath [Ca2+]. Lowering bath [Ca2+] reduced the rate of rise, but not the final amplitude, of the increase in mitochondrial [Ca2+] during 50-Hz stimulation. The amplitude of the stimulation-induced increase in cytosolic [Ca2+] was reduced in low-bath [Ca2+] and increased when mitochondrial Ca2+ uptake was inhibited by depolarizing mitochondria. In 2 mM Ca2+, end-plate potentials (epps) depressed by 53% after 10 s of 50-Hz stimulation, and this depression increased to 80% after mitochondrial depolarization. In contrast, in 0.5 mM Ca2+ the same stimulation pattern increased epps by approximately 3.4-fold, and this increase was even greater (transiently) after mitochondrial depolarization. In both 2 and 0.5 mM [Ca2+], mitochondrial depolarization increased asynchronous release during the 50-Hz train and increased the total vesicular release (phasic and asynchronous) measured by destaining of the styryl dye FM2-10. These results suggest that by limiting the stimulation-induced increase in cytosolic [Ca2+], mitochondrial Ca2+ uptake maintains a high ratio of phasic to asynchronous release, thus helping to sustain neuromuscular transmission during repetitive stimulation. Interestingly, the quantal content of the epp reached during 50-Hz stimulation stabilized at a similar level ( approximately 20 quanta) in both 2 and 0.5 mM Ca2+. A similar convergence was measured in oligomycin, which inhibits mitochondrial ATP synthesis without depolarizing mitochondria, but quantal contents fell to <20 when mitochondria were depolarized in 2 mM Ca2+.

Mitochondrial Ca2+ uptake prevents desynchronization of quantal release and minimizes depletion during repetitive stimulation of mouse motor nerve terminals.

We investigated how inhibition of mitochondrial Ca2+ uptake affects transmitter release from mouse motor terminals during brief trains of action potentials (500 at 50 Hz) in physiological bath [Ca2+]. When mitochondrial Ca2+ uptake was inhibited by depolarizing mitochondria with antimycin A1 or carbonyl cyanide m-chlorophenyl-hydrazone, the stimulation-induced increase in cytosolic [Ca2+] was greater (> 10 microM, compared to < or = 1 microM in control solution), the quantal content of the endplate potential (EPP) depressed more rapidly (approximately 84 % depression compared to approximately 8 % in controls), and asynchronous release during the stimulus train reached higher frequencies (peak rates of approximately 6000 s-1 compared to approximately 75 s-1 in controls). These effects of mitochondrial depolarization were not accompanied by a significant change in EPP quantal content or the rate of asynchronous release during 1 Hz stimulation, and were not seen in oligomycin, which blocks mitochondrial ATP synthesis without depolarizing mitochondria. Inhibition of endoplasmic reticular Ca2+ uptake with cyclopiazonic acid also had little effect on stimulation-induced changes in cytosolic [Ca2+] or EPP amplitude. We hypothesize that the high rate of asynchronous release evoked by stimulation during mitochondrial depolarization was produced by the elevation of cytosolic [Ca2+], and contributed to the accelerated depression of phasic release by reducing the availability of releasable vesicles. During mitochondrial depolarization, the post-tetanic potentiation of the EPP observed under control conditions was replaced by a post-tetanic depression with a slow time course of recovery. Thus, mitochondrial Ca2+ uptake is essential for sustaining phasic release, and thus neuromuscular transmission, during and following tetanic stimulation.

The inhibition of mitochondrial Ca2+ uniporter influences the propagation of Ca2+ release

The inhibition of mitochondrial Ca2+ uniporter influences the propagation of Ca2+ release

Swelling-induced Ca2+ release from intracellular calcium stores in rat submandibular gland acinar cells.

The effects of osmotically-induced cell swelling on cytoplasmic free Ca2+ concentration ([Ca2+]i) were studied in acinar cells from rat submandibular gland using microspectrofluorimetry. Video-imaging techniques were also used to measure cell volume. Hypotonic stress (78% control tonicity) caused rapid cell swelling reaching a maximum relative volume of 1.78 +/- 0.05 (n = 5) compared to control. This swelling was followed by regulatory volume decrease, since relative cell volume decreased significantly to 1.61 +/- 0.08 (n = 5) after 10 min exposure to hypotonic medium. Osmotically induced cell swelling evoked by medium of either 78% or 66% tonicity caused a biphasic increase of [Ca2+]i. The rapid phase of this increase in [Ca2+]i was due to release of Ca2 + from intracellular stores, since it was also observed in cells bathed in Ca2+-free solution. The peak increase of [Ca2+]i induced by cell swelling was 3.40 +/- 0.49 (Fura-2 F340/F380 fluorescence ratio, n = 11) and 3.17 +/- 0.43 (n = 17) in the presence and the absence of extracellular Ca2+, respectively, corresponding to an absolute [Ca2+]i of around 1 microm. We found that around two-thirds of cells tested still showed some swelling-induced Ca2+ release (SICR) even after maximal concentrations (10(-5) M - 10(-4) M) of carbachol had been applied to empty agonist-sensitive intracellular Ca2+ stores. This result was confirmed and extended using thapsigargin to deplete intracellular Ca2+ pools. Hypotonic shock still raised [Ca2+]i in cells pretreated with thapsigargin, confirming that at least some SICR occurred from agonist-insensitive stores. Furthermore, SICR was largely inhibited by pretreatment of cells with carbonyl cyanide m-cholorophenyl hydrazone (CCCP) or ruthenium red, inhibitors of mitochondrial Ca2+ uptake. Our results suggest that the increase in [Ca2+]i, which underlies regulatory volume decrease in submandibular acinar cells, results from release of Ca2+ from both agonist-sensitive and mitochondrial Ca2+ stores.