Hydrogen peroxide depolarizes mitochondria and inhibits IP3-evoked Ca2+ release in the endothelium of intact arteries.
ABSTRACT: Hydrogen peroxide (H2O2) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H2O2 regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H2O2 on Ca2+ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca2+ signals and acetylcholine evoked Ca2+ increases were inhibited by H2O2. H2O2 inhibition of acetylcholine-evoked Ca2+ signals was reversed by catalase. H2O2 exerts its inhibition on the IP3 receptor as Ca2+ release evoked by photolysis of caged IP3 was supressed by H2O2. H2O2 suppression of IP3-evoked Ca2+ signalling may be mediated by mitochondria. H2O2 depolarized mitochondria membrane potential. Acetylcholine-evoked Ca2+ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP3-evoked Ca2+ release by H2O2 arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca2+ overload during IP3-linked Ca2+ signals by a H2O2 mediated negative feedback depolarization of the organelle and inhibition of IP3-evoked Ca2+ release.
Project description:BACKGROUND AND PURPOSE:FK506 and rapamycin are modulators of FK-binding proteins (FKBP) that are used to suppress immune function after organ and hematopoietic stem cell transplantations. The drugs share the unwanted side-effect of evoking hypertension that is associated with reduced endothelial function and nitric oxide production. The underlying mechanisms are not understood. FKBP may regulate IP3 receptors (IP3 R) and ryanodine receptors (RyR) to alter Ca2+ signalling in endothelial cells. EXPERIMENTAL APPROACH:We investigated the effects of FK506 and rapamycin on Ca2+ release via IP3 R and RyR in hundreds of endothelial cells, using the indicator Cal-520, in intact mesenteric arteries from male Sprague-Dawley rats. IP3 Rs were activated by acetylcholine or localised photo-uncaging of IP3 , and RyR by caffeine. KEY RESULTS:While FKBPs were present, FKBP modulation with rapamycin did not alter IP3 -evoked Ca2+ release. Conversely, FK506, which modulates FKBP and blocks calcineurin, increased IP3 -evoked Ca2+ release. Inhibition of calcineurin (okadiac acid or cypermethrin) also increased IP3 -evoked Ca2+ release and blocked FK506 effects. When calcineurin was inhibited, FK506 reduced IP3 -evoked Ca2+ release. These findings suggest that IP3 -evoked Ca2+ release is not modulated by FKBP, but by FK506-mediated calcineurin inhibition. The RyR modulators caffeine and ryanodine failed to alter Ca2+ signalling suggesting that RyR is not functional in native endothelium. CONCLUSION AND IMPLICATIONS:The hypertensive effects of the immunosuppressant drugs FK506 and rapamycin, while mediated by endothelial cells, do not appear to be exerted at the documented cellular targets of Ca2+ release and altered FKBP binding to IP3 and RyR.
Project description:Emerging evidence indicates that mitochondria are locally coupled to endoplasmic reticulum (ER) Ca2+ release in myoblasts and to sarcoplasmic reticulum (SR) Ca2+ release in differentiated muscle fibers in order to regulate cytoplasmic calcium dynamics and match metabolism with cell activity. However, the mechanism of the developmental transition from ER to SR coupling remains unclear. We have studied mitochondrial sensing of IP3 receptor (IP3R)- and ryanodine receptor (RyR)-mediated Ca2+ signals in H9c2 myoblasts and differentiating myotubes, as well as the attendant changes in mitochondrial morphology. Mitochondria in myoblasts were largely elongated, luminally connected and relatively few in number, whereas the myotubes were densely packed with globular mitochondria that displayed limited luminal continuity. Vasopressin, an IP3-linked agonist, evoked a large cytoplasmic Ca2+ ([Ca2+]c) increase in myoblasts, whereas it elicited a smaller response in myotubes. Conversely, RyR-mediated Ca2+ release induced by caffeine, was not observed in myoblasts, but triggered a large [Ca2+]c signal in myotubes. Both the IP3R and the RyR-mediated [Ca2+]c rise was closely associated with a mitochondrial matrix Ca2+ ([Ca2+]m) signal. Every myotube that showed a [Ca2+]c spike also displayed a [Ca2+]m response. Addition of IP3 to permeabilized myoblasts and caffeine to permeabilized myotubes also resulted in a rapid [Ca2+]m rise, indicating that Ca2+ was delivered via local coupling of the ER/SR and mitochondria. Thus, as RyRs are expressed during muscle differentiation, the local connection between RyR and mitochondrial Ca2+ uptake sites also appears. When RyR1 was exogenously introduced to myoblasts by overexpression, the [Ca2+]m signal appeared together with the [Ca2+]c signal, however the mitochondrial morphology remained unchanged. Thus, RyR expression alone is sufficient to induce the steps essential for their alignment with mitochondrial Ca2+ uptake sites, whereas the mitochondrial proliferation and reshaping utilize either downstream or alternative pathways.
Project description:The building blocks of intracellular Ca2+ signals evoked by inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ puffs, transient focal increases in Ca2+ concentration that reflect the opening of small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs evoked by photolysis of caged IP3 or activation of endogenous muscarinic receptors with carbachol in human embryonic kidney 293 cells. Ca2+ puffs evoked by carbachol initiated at an estimated 65±7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged IP3 evoked Ca2+ puffs at a similar number of sites (100±35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential stimulation with carbachol or photolysed caged IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites and the sites become more likely to fire as the IP3 concentration increases; there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.
Project description:Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.
Project description:Intracellular Ca2+ stores in rat submandibular acinar cells were characterized using the Ca(2+)-sensitive fluorescent indicator fura 2 and the radiotracer 45Ca2+. Acetylcholine induced a rapid Ca2+ release from a store sensitive to inositol 1,4,5-trisphosphate (IP3) and to thapsigargin (TG). After this store was presumably depleted, ionomycin caused a further increase in cytosolic free Ca2+ concentration ([Ca2+]i), suggesting the presence of an IP3-insensitive Ca2+ release from a store that is more extensive and heterogeneous than the IP3-sensitive one and includes a small mitochondrial component. After both of these stores had been discharged, exposure to monensin caused an additional release of Ca2+ from a third store. This store appears to be associated with secretory granules, since Ca2+ release was significantly reduced when degranulation was induced by isoprenaline. This third store appears to be insensitive to IP3, discharges Ca2+ when the pH gradient across the limiting membrane is collapsed with monensin and only in the presence of both ionomycin and monensin. Ca2+ release from this store is not by Na+/Ca2+ exchange, since simply altering [Na+]i did not cause significant Ca2+ release. In permeabilized cells, IP3 and TG released approx. 35% of 45Ca2+, and ionomycin released an additional 57%, whereas monensin only caused a small additional release, suggesting that only IP3- and ionomycin-sensitive stores are loaded with 45Ca2+ under these conditions. The absence of significant isotope uptake into the ionomycin+monensin-sensitive store may result from a low rate of tracer accumulation or from the lack of Ca2+ pumps in the store. The pattern of response was similar in the presence and absence of mitochondrial inhibitors, indicating that the store is not located in mitochondria. In summary, these results suggest that a substantial IP3-insensitive Ca2+ store is present in secretory granules in rat submandibular acinar cells.
Project description:Cholesterol depletion reversibly abolishes carbachol-evoked Ca(2+) release from inositol (1,4,5)-trisphosphate (IP3)-sensitive stores, without affecting the distribution of IP3 receptors (IP3R) or endoplasmic reticulum, IP3 formation or responses to photolysis of caged IP3. Receptors that stimulate cAMP formation do not alone evoke Ca(2+) signals, but they potentiate those evoked by carbachol. We show that these potentiated signals are entirely unaffected by cholesterol depletion and that, within individual cells, different IP3-sensitive Ca(2+) stores are released by carbachol alone and by carbachol combined with receptors that stimulate cAMP formation. We suggest that muscarinic acetylcholine receptors in lipid rafts deliver IP3 at high concentration to associated IP3R, stimulating them to release Ca(2+). Muscarinic receptors outside rafts are less closely associated with IP3R and provide insufficient local IP3 to activate IP3R directly. These IP3R, probably type 2 IP3R within a discrete Ca(2+) store, are activated only when their sensitivity is increased by cAMP. Sensitization of IP3R by cAMP extends the effective range of signalling by phospholipase C, allowing muscarinic receptors that are otherwise ineffective to recruit additional IP3-sensitive Ca(2+) stores.
Project description:Inositol 1,4,5-trisphosphate (IP3) stimulates Ca2+ release from the endoplasmic reticulum (ER), and the response is potentiated by 3',5'-cyclic AMP (cAMP). We investigated this interaction in HEK293 cells using carbachol and parathyroid hormone (PTH) to stimulate formation of IP3 and cAMP, respectively. PTH alone had no effect on the cytosolic Ca2+ concentration, but it potentiated the Ca2+ signals evoked by carbachol. Surprisingly, however, the intracellular Ca2+ stores that respond to carbachol alone could be both emptied and refilled without affecting the subsequent response to PTH. We provide evidence that PTH unmasks high-affinity IP3 receptors within a discrete Ca2+ store. We conclude that Ca2+ stores within the ER that dynamically exchange Ca2+ with the cytosol maintain a functional independence that allows one store to be released by carbachol and another to be released by carbachol with PTH. Compartmentalization of ER Ca2+ stores adds versatility to IP3-evoked Ca2+ signals.
Project description:Intracellular accumulation of oligomeric forms of ? amyloid (A?) are now believed to play a key role in the earliest phase of Alzheimer's disease (AD) as their rise correlates well with the early symptoms of the disease. Extensive evidence points to impaired neuronal Ca2+ homeostasis as a direct consequence of the intracellular A? oligomers. However, little is known about the downstream effects of the resulting Ca2+ rise on the many intracellular Ca2+-dependent pathways. Here we use multiscale modeling in conjunction with patch-clamp electrophysiology of single inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and fluorescence imaging of whole-cell Ca2+ response, induced by exogenously applied intracellular A?42 oligomers to show that A?42 inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP3R, which is then extended to build a model for the whole-cell Ca2+ signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca2+ release from the endoplasmic reticulum by intracellular A?42 oligomers through G-protein-mediated stimulation of IP3 production. The estimated IP3 concentration as a function of intracellular A?42 content together with the whole-cell model allows us to show that A?42 oligomers impair mitochondrial function through pathological Ca2+ uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and H2O2. We further show that mitochondrial function can be restored by the addition of Ca2+ buffer EGTA, in accordance with the observed abrogation of A?42 cytotoxicity by EGTA in our live cells experiments.
Project description:Extracellular ATP plays important roles in coordinating the activities of astrocytes and neurons, and aberrant signalling is associated with neurodegenerative diseases. In rodents, ATP stimulates opening of Ca2+ -permeable channels formed by P2X receptor subunits in the plasma membrane. It is widely assumed, but not verified, that P2X receptors also evoke Ca2+ signals in human astrocytes. Here, we directly assess this hypothesis. We showed that cultured foetal cortical human astrocytes express mRNA for several P2X receptor subunits (P2X4 , P2X5 , P2X6 ) and G protein-coupled P2Y receptors (P2Y1 , P2Y2 , P2Y6 , P2Y11 ). In these astrocytes, ATP stimulated Ca2+ release from intracellular stores through IP3 receptors and store-operated Ca2+ entry. These responses were entirely mediated by P2Y1 and P2Y2 receptors. Agonists of P2X receptors did not evoke Ca2+ signals, and nor did ATP when Ca2+ release from intracellular stores and store-operated Ca2+ entry were inhibited. We conclude that ATP-evoked Ca2+ signals in cultured human foetal astrocytes are entirely mediated by P2Y1 and P2Y2 receptors, with no contribution from P2X receptors.
Project description:The Ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca2+ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca2+ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca2+ release (but not cADPR- or IP3-evoked Ca2+ release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen 'hits' exhibiting >80% inhibition of NAADP-evoked Ca2+ release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca2+ release without depleting acidic Ca2+ stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca2+ signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP3-dependent Ca2+ signaling with potential therapeutic value.