Expanding the neuron's calcium signaling repertoire: intracellular calcium release via voltage-induced PLC and IP3R activation.
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ABSTRACT:
SUBMITTER: Ryglewski S
PROVIDER: S-EPMC1808487 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cβ (PLC-β) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-β activity led to greater release of endoplasmic reticulum Ca2+ via the inositol trisphosphate receptor (IP3R), increased mitochondrial Ca2+ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-β-IP3R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca2+ into endolysosomes was an intermediary in the regulation of lifespan by IP3Rs. Manipulations that either lowered PLC-β/IP3R abundance or attenuated endolysosomal Ca2+ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-β-IP3R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca2+ overload.
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Project description:Improving health of the rapidly growing aging population is a critical medical, social, and economic goal. Identification of genes that modulate healthspan, the period of mid-life vigor that precedes significant functional decline, will be an essential part of the effort to design anti-aging therapies. Because locomotory decline in humans is a major contributor to frailty and loss of independence and because slowing of movement is a conserved feature of aging across phyla, we screened for genetic interventions that extend locomotory healthspan of Caenorhabditis elegans. From a group of 54 genes previously noted to encode secreted proteins similar in sequence to extracellular domains of insulin receptor, we identified two genes for which RNAi knockdown delayed age-associated locomotory decline, conferring a high performance in advanced age phenotype (Hpa). Unexpectedly, we found that hpa-1 and hpa-2 act through the EGF pathway, rather than the insulin signaling pathway, to control systemic healthspan benefits without detectable developmental consequences. Further analysis revealed a potent role of EGF signaling, acting via downstream phospholipase C-gammaplc-3 and inositol-3-phosphate receptor itr-1, to promote healthy aging associated with low lipofuscin levels, enhanced physical performance, and extended lifespan. This study identifies HPA-1 and HPA-2 as novel negative regulators of EGF signaling and constitutes the first report of EGF signaling as a major pathway for healthy aging. Our data raise the possibility that EGF family members should be investigated for similar activities in higher organisms.
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Project description:Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localisation and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localisation is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we demonstrated that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs displayed reduced 1,4,5-inositol trisphosphate receptor (IP3R)-mediated ER calcium release downstream of Phospholipase C activation. Collectively, our data indicate that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the level of the ER, in addition to its well-known role in regulating apoptosis through the mitochondria.
| S-EPMC11294475 | biostudies-literature
Project description:BackgroundPolycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca(2+)) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells.MethodsWe investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca(2+) signaling. Plasma membrane (PM) Ca(2+) permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy.ResultsWe demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca(2+) signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca(2+) oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca(2+) permeability. Intracellular Ca(2+) buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca(2+)/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM.ConclusionsThese novel findings demonstrate intracellular Ca(2+)-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.
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Project description:The engagement of antigen receptors on lymphocytes leads to the activation of phospholipase C-γ, the mobilization of intracellular calcium and the activation of the NFAT transcription factor. The coupling of antigen receptors to the activation of NFAT is modulated by numerous cellular effectors including phospho-inositide 3-kinase (PI3K), which is activated following receptor cross-linking. The activation of PI3K has both positive and negative effects on the receptor-mediated activation of NFAT. An increase in the level and activity of Akt2, a target of activated PI3K, potently inhibits the subsequent activation of NFAT. In contrast, an elevation in Akt1 has no effect on signaling. Signaling pathways operating both upstream and downstream of inositol 1,4,5-trisphosphate (IP3)-stimulated calcium release from intracellular stores are unaffected by Akt2. An increase in the level of Akt2 has no significant effect on the initial amplitude, but substantially reduces the duration of calcium mobilization. The ability of Akt2 to inhibit prolonged calcium mobilization is abrogated by the administration of a cell permeable peptide that blocks the interaction between Bcl-2 and the IP3 receptor. Thus, Akt2 is a negative regulator of NFAT activation through its ability to inhibit calcium mobilization from the ER.
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Project description:We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization.
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Project description:The paper describes a novel cellular mechanism for rapid calcium-dependent nitric oxide (NO) release. This release occurs due to NO liberation from S-nitrosothiols. We have analysed the changes of NO concentration in acutely isolated pancreatic acinar cells. Supramaximal acetylcholine (ACh) stimulation induced a Ca(2+)-dependent increase in the fluorescence in the majority of cells loaded with the NO probe DAF-FM via a patch pipette. The ACh-induced NO signals were insensitive to inhibitors of calmodulin and protein kinase C but were inhibited by calpain antagonists. The initial part of the NO signals induced by 10 muM ACh showed little sensitivity to inhibition of NO synthase (NOS); however, cell pretreatment with NO donors (increasing cellular S-nitrosothiol contents) substantially enhanced the initial component of NO responses. Pancreatic acinar cells were able to generate fast calcium-dependent NO responses when stimulated with physiological or supramaximal doses of secretagogues. Importantly, the source of this NO is the already available S-nitrosothiol store rather than de novo synthesis by NOS. A similar mechanism of NO release was found in dorsal root ganglia neurons.
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Project description:Arteriolar smooth muscle cells (SMCs) and capillary pericytes dynamically regulate blood flow in the central nervous system in the face of fluctuating perfusion pressures. Pressure-induced depolarization and Ca2+ elevation provide a mechanism for regulation of SMC contraction, but whether pericytes participate in pressure-induced changes in blood flow remains unknown. Here, utilizing a pressurized whole-retina preparation, we found that increases in intraluminal pressure in the physiological range induce contraction of both dynamically contractile pericytes in the arteriole-proximate transition zone and distal pericytes of the capillary bed. We found that the contractile response to pressure elevation was slower in distal pericytes than in transition zone pericytes and arteriolar SMCs. Pressure-evoked elevation of cytosolic Ca2+ and contractile responses in SMCs were dependent on voltage-dependent Ca2+ channel (VDCC) activity. In contrast, Ca2+ elevation and contractile responses were partially dependent on VDCC activity in transition zone pericytes and independent of VDCC activity in distal pericytes. In both transition zone and distal pericytes, membrane potential at low inlet pressure (20 mmHg) was approximately -40 mV and was depolarized to approximately -30 mV by an increase in pressure to 80 mmHg. The magnitude of whole-cell VDCC currents in freshly isolated pericytes was approximately half that measured in isolated SMCs. Collectively, these results indicate a loss of VDCC involvement in pressure-induced constriction along the arteriole-capillary continuum. They further suggest that alternative mechanisms and kinetics of Ca2+ elevation, contractility, and blood flow regulation exist in central nervous system capillary networks, distinguishing them from neighboring arterioles.
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Project description:Hypochlorhydria during Helicobacter pylori infection inhibits gastric Sonic Hedgehog (Shh) expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through intracellular calcium (Ca2(+)(i))-dependent protein kinase C (PKC) or cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation.We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1, a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, ethylene glycol-bis(?-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) + 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), PKC-overexpressing adenoviruses, and PKC inhibitors were used to modulate Ca(2+)(i)-release, PKC activity, and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H(+)/K(+)-?-cholera-toxin-overexpressing mice.Mice that expressed secreted hedgehog-interacting protein-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression also was repressed in the hyperchlorhydric H(+)/K(+)-?-cholera-toxin model with increased cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca(2+)(i) release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin-, and carbachol-mediated release of Ca(2+)(i) induced Shh expression. Ca(2+)-chelation with BAPTA + EGTA reduced Shh expression. Overexpression of PKC-?, -?, and -? (but not PKC-?) induced an Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene.Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca(2+)(i)-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin.
| S-EPMC2997213 | biostudies-literature
Project description:As documented in our previous study, notoginsenoside R1 (NGR1) can inhibit neuron apoptosis and the expression of endoplasmic reticulum (ER) stress-associated pro-apoptotic proteins in hypoxic-ischemic encephalopathy. Recent evidence indicates that the Phospholipase C (PLC)/inositol 1,4,5-trisphosphate receptor (IP3R) is important for the regulation of Ca2+ release in the ER. Ca2+ imbalance can stimulate ER stress, CAMKII, and cell apoptosis. The purpose of this study was to further investigate the neuroprotective effect of NGR1 and elucidate how NGR1 regulates ER stress and cell apoptosis in the oxygen-glucose deprivation/reoxygenation (OGD/R) model. Cells were exposed to NGR1 or the PLC activator m-3M3FBS. Then, IP3R- and IP3-induced Ca2+ release (IICR) and activation of the ER stress and CaMKII signal pathway were measured. The results showed that NGR1 inhibited IICR and strengthened the binding of GRP78 with PERK and IRE1. NGR1 also alleviated the activation of the CaMKII pathway. Pretreatment with m-3M3FBS attenuated the neuroprotective effect of NGR1; IICR was activated, activation of the ER stress and CaMKII pathway was increased, and more cells were injured. These results indicate that NGR1 may suppress activation of the PLC/IP3R pathway, subsequently inhibiting ER Ca2+ release, ER stress, and CaMKII and resulting in suppressed cell apoptosis.
| S-EPMC5701215 | biostudies-literature