Basal Cytosolic Calcium Research Articles

Polycystin-2 Mediated Calcium Signalling in the Dictyostelium Model for Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, PKD1) and Polycystin-2 (PC2, PKD2) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism Dictyostelium discoideum by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca2+ responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca2+ signalling also impacted other downstream Ca2+-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that Dictyostelium is a useful model to study Polycystin-2 mediated calcium signalling.

CaV1.3 enhanced store operated calcium promotes resistance to androgen deprivation in prostate cancer

Androgen deprivation therapy (ADT) is the main treatment for advanced prostate cancer (PCa) but resistance results in progression to terminal castrate resistant PCa (CRPC), where there is an unmet therapeutic need. Aberrant intracellular calcium (Cai2+) is known to promote neoplastic transformation and treatment resistance. There is growing evidence that voltage gated calcium channel (VGCC) expression is increased in cancer, particularly CACNA1D/CaV1.3 in CRPC. The aim of this study was to investigate if increased CaV1.3 drives resistance to ADT and determine its associated impact on Cai2+ and cancer biology. Bioinformatic analysis revealed that CACNA1D gene expression is increased in ADT treated PCa patients. This was corroborated in both in vivo LNCaP xenograft mouse and in vitro PCa cell line models, which demonstrated a significant increase in CaV1.3 protein expression following ADT with bicalutamide. Expression was found to be of a shortened 170kDa CaV1.3 isoform associated with plasma and intracellular membranes, which failed to induce calcium influx following membrane depolarisation. Instead, under ADT CaV1.3 mediated a rise in basal cytosolic calcium and an increase in store operated calcium entry (SOCE). This mechanism was found to promote the proliferation and survival of ADT resistant CRPC cells. Overall, this study demonstrates for the first time in PCa that under ADT specific CaV1.3 isoforms promote an upregulation of SOCE which contributes to treatment resistance and CRPC biology. Thus, this novel oncochannel represents a target for therapeutic development to improve PCa patient outcomes.

ATT has positive effects on pulmonary hypertension hallmarks

Pulmonary hypertension (PH) is a multifactorial disease characterized by pulmonary arteries (PA) hyperreactivity and remodelling involving an imbalance of contractile and proliferative agents such as 5-hydroxytryptamine (5-HT). Mitochondria and associated reactive oxygen species (ROS) production were shown to contribute to these phenomena. OP2 Drugs recently demonstrated that Anethole Trithione (ATT), the active ingredient of its principal lead OP2113, specifically inhibits mitochondrial ROS (mtROS) production from complex I in its reverse mode. In this context, this study aims to assess the effects of ATT on PH hallmarks. Male rats were either exposed to normoxia or chronic hypoxia (10% O2) during 3 weeks to induce PH (CH-PH). The effect of ATT on PA reactivity was determined ex vivo by isometric tension measurements of arterial rings. PA contraction was induced by 5-HT, prostaglandin F2α (PGF2α), phenylephrine (Phe) or endothelin-1 (ET-1) whereas relaxation was induced by carbachol (Cch) on preconstricted vessels with PGF2α. The effect of ATT on calcium responses induced by 5-HT was assessed in rat PA smooth muscle cells using confocal imaging. Proliferative responses induced by 5-HT were evaluated by ELISA BrdU on human PASMC in presence or absence of ATT. In rats with CH-PH, ATT reduced contractile hyperreactivity induced by 5-HT and PGF2α. In control rats, ATT treatment also decreased contraction to 5-HT and Phe, but not contraction to ET-1 and PGF2α as well as relaxation to Cch. Interestingly, acute ATT exposure did not modify basal cytosolic calcium signal in control rat PASMC. However, the number of 5-HT responding cells was reduced in presence of ATT whereas neither maximal peak of cytosolic calcium responses nor the global calcium variations were affected. Finally, 5-HT-induced proliferation was reduced by ATT in control human PASMC. In conclusion, ATT improves vascular hyperreactivity of PA from rats with CH-PH whereas in control condition ATT altered 5-HT-induced contraction in rat PA, calcium signalling to 5-HT in rat PASMC and 5-HT-induced human PASMC proliferation. Although mechanisms underlying ATT influence on PH hallmarks need to be further elucidated, these Results suggest that OP2113 may be used to develop new PH therapies.

Examining the effects of the microglial PLCG2 P522R mutation by transplantation of human stem cell‐derived microglia in chimeric AD mice

AbstractBackgroundGenetic studies have uncovered several Alzheimer’s Disease (AD) risk genes that are highly expressed in microglia, suggesting that immune dysregulation plays a critical role in the development and progression of AD. In line with this, a recently discovered polymorphism (P522R) in the microglia‐expressed gene Phospholipase C‐Gamma 2 (PLCG2) was shown to be protective for AD. Interestingly, PLCG2 codes for a protein that regulates the release of calcium from ER stores and is also a component of the downstream signaling pathway of TREM2, another microglial AD risk gene. This underscores the need to improve our understanding of microglial calcium signaling, the effects of AD risk genes on this pathway, and the impacts of PLCG2 and calcium signaling on human microglial function.MethodsCRISPR gene editing was used to produce isogenic iPSCs that express the AD protective mutation in PLCG2. Several in vitro assays were then performed to examine calcium signaling pathways and phagocytosis. PLCG2‐P522R and WT HPCs were also transplanted directly into xenotransplantation‐compatible mouse pups (MITRG‐5xfAD mice), where they readily differentiated into human microglia, exhibiting typical markers and morphology.. Xenotransplanted human microglia (x‐MG) were then examined by confocal microscopy and isolated from the brain for single‐cell RNA sequencing.ResultsThus far our in vitro studies suggest that the P522R coding mutation increases both basal cytosolic calcium concentrations and amyloid phagocytosis. Additional functional experiments are further examining the interactions between PLCG2 mutations and calcium signaling during the uptake of AD‐relevant substrates. In addition, single cell RNA sequencing studies of chimeric mice reveal that the P522R mutation leads to an increased percentage of human microglia that have adopted a disease‐associated microglial (DAM) phenotype in vivo.ConclusionsBy combining iPSC‐microglia, CRISPR gene editing, and chimeric AD mice these studies have begun to uncover the impact of PLCG2 mutations on human microglial function and reveal that the protective P522R mutation increases Ab phagocytosis and promotes the adoption of a DAM‐like microglial response.

The 2β Splice Variation Alters the Structure and Function of the Stromal Interaction Molecule Coiled-Coil Domains.

Stromal interaction molecule (STIM)-1 and -2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels after oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil (CC) domains couple to PM Orai1 proteins and gate these Ca2+ release-activated Ca2+ (CRAC) channels, which facilitate store-operated Ca2+ entry (SOCE). Unlike STIM1 and STIM2, which are SOCE activators, the STIM2β splice variant contains an 8-residue insert located within the conserved CCs which inhibits SOCE. It remains unclear if the 2β insert further depotentiates weak STIM2 coupling to Orai1 or independently causes structural perturbations which prevent SOCE. Here, we use far-UV circular dichroism, light scattering, exposed hydrophobicity analysis, solution small angle X-ray scattering, and a chimeric STIM1/STIM2β functional assessment to provide insights into the molecular mechanism by which the 2β insert precludes SOCE activation. We find that the 2β insert reduces the overall α-helicity and enhances the exposed hydrophobicity of the STIM2 CC domains in the absence of a global conformational change. Remarkably, incorporation of the 2β insert into the STIM1 context not only affects the secondary structure and hydrophobicity as observed for STIM2, but also eliminates the more robust SOCE response mediated by STIM1. Collectively, our data show that the 2β insert directly precludes Orai1 channel activation by inducing structural perturbations in the STIM CC region.

Elevated basal cytosolic calcium of endothelial cells influences the post-surgical outcome in diabetic CTS.

Background Type 2 diabetes mellitus (T2DM)-induced neuropathy and ischemia-reperfusion post-surgery prolong carpal tunnel syndrome (CTS) pathology, but the effect of T2DM on the prognostic outcome of carpal tunnel (CT) release surgery needs to be investigated. Materials and methods A total of 64 individuals with CTS underwent CT release surgery. HbA1c levels identified their diabetic status. The individual prognostic outcomes were measured by nerve conduction velocity (NCV), amplitude, and latency. Measurement of [Ca2+]c and reactive oxygen species (ROS) from isolated endothelial cells (ECs) revealed the oxidative burden of the normal and diabetic CTS phenotypes. Results CTS individuals with HbA1c > 7 showed decreased NCV (≈22m/s) and amplitude (≈4.2mV) with increased latency (≈6ms), compared to groups with HbA1c ≤ 7. Further to CT release surgery, the reversal of the nerve conduction to normalcy was greatly influenced by the diabetic profile of the individuals. Our results showed elevated basal [Ca2+]c and corresponding high cytosolic ROS in the ECs isolated from individuals with HbA1c > 7 compared to the diabetic and healthy control groups. Conclusion The individuals with diabetic index showed suboptimal neuronal performance pre- and post-CT release surgery. Oxidative stress mediated by high [Ca2+]c and ROS of ECs dissipates to adjoining cells worsening the pathology of the untreated CTS.

CREB decreases astrocytic excitability by modifying subcellular calcium fluxes via the sigma-1 receptor.

Astrocytic excitability relies on cytosolic calcium increases as a key mechanism, whereby astrocytes contribute to synaptic transmission and hence learning and memory. While it is a cornerstone of neurosciences that experiences are remembered, because transmitters activate gene expression in neurons, long-term adaptive astrocyte plasticity has not been described. Here, we investigated whether the transcription factor CREB mediates adaptive plasticity-like phenomena in astrocytes. We found that activation of CREB-dependent transcription reduced the calcium responses induced by ATP, noradrenaline, or endothelin-1. As to the mechanism, expression of VP16-CREB, a constitutively active CREB mutant, had no effect on basal cytosolic calcium levels, extracellular calcium entry, or calcium mobilization from lysosomal-related acidic stores. Rather, VP16-CREB upregulated sigma-1 receptor expression thereby increasing the release of calcium from the endoplasmic reticulum and its uptake by mitochondria. Sigma-1 receptor was also upregulated in vivo upon VP16-CREB expression in astrocytes. We conclude that CREB decreases astrocyte responsiveness by increasing calcium signalling at the endoplasmic reticulum-mitochondria interface, which might be an astrocyte-based form of long-term depression.

Studies on the resolution of subcellular free calcium concentrations: a technological advance. Focus on “Detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells”

One of the most striking facts about the elements is their unequal distribution and occurrence in nature . —C. R. Hammond free calcium is highly controlled in biological tissues, and much like the physical chemistry of elements ([5][1]), its concentration is unequal in distribution (for recent

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.

REDUCED NO BIOAVAILABILITY, OXIDATIVE STRESS AND ALTERATION OF CALCIUM HOMEOSTASIS IN VASCULAR ENDOTHELIUM FROM DIABETIC MICE

Endothelial dysfunction is a major risk factor for vascular complications associated with diabetes. The purpose of this study was to evaluate the possible link between oxidative stress and alteration in calcium homeostasis in dysfunctional endothelial cells from db/db diabetic mice, 20 or 45 week old. Impaired flow‐induced dilation in mesenteric arteries and acetylcholine‐induced relaxation in aorta due to a decreased nitric oxide (NO) bioavailability were already shown in this model. For this study, mice aortic endothelial cells (MAEC) were isolated from vessel explants. Oxidative stress and Ca2+ homeostasis in MAEC were simultaneously recorded with DHE and Fura‐2 probes using an inverted epi‐fluorescence microscope. Compared to control mice, oxidative stress in MAEC from db/db mice increased with age leading to a progressive alteration of Ca2+ homeostasis, characterized by a rise in basal cytosolic calcium and a decrease in amplitude of acetylcholine‐induced calcium peak. Interestingly, a NO donor reduced oxidative stress and improved calcium signaling in endothelial cells from db/db mice. These results suggest that reduced NO bioavailability contributes to oxidative stress and abnormal calcium homeostasis in endothelial cells during progression of diabetes.

The Need for Calcium Channels in Cell Proliferation

Both increases in the basal cytosolic calcium concentration ([Ca2+]cyt) and [Ca2+]cyt transients play a major role in cell cycle progression, cell proliferation and division. Calcium influx and release from endoplasmic reticulum are the major routes involved in the variations in [Ca2+]cyt and past studies have clearly shown that calcium influx controls cell growth and proliferation in several cell types. Furthermore, various studies in the past thirty years have demonstrated that calcium channel expression levels, as well as cell specific expression, were determinant in these physiological processes. Cell proliferation is directly linked to cell cycle progression, and again, it became evident that calcium channel expression interferes here. It is also clear that calcium influx and cell proliferation relationship can be uncoupled in transformed and cancer cells, resulting in an external calcium-independent proliferation. Other divalent cations such as iron and zinc involved in cell proliferation permeating some calcium channels may interfere in this cellular process. This patent review is focused on transient receptor potential and ORAI channels, and, as calcium influx regulates several other transduction pathways, we assume that specific signalization complexes are needed to trigger activation of proliferation and cell division in mammalian cells.

Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia

We investigated whether Kif3a in osteoblasts has a direct role in regulating postnatal bone formation. We conditionally deleted Kif3a in osteoblasts by crossing osteocalcin (Oc; also known as Bglap)-Cre with Kif3a(flox/null) mice. Conditional Kif3a-null mice (Kif3a(Oc-cKO)) had a 75% reduction in Kif3a transcripts in bone and osteoblasts. Conditional deletion of Kif3a resulted in the reduction of primary cilia number by 51% and length by 27% in osteoblasts. Kif3a(Oc-cKO) mice developed osteopenia by 6 weeks of age unlike Kif3a(flox/+) control mice, as evidenced by reductions in femoral bone mineral density (22%), trabecular bone volume (42%) and cortical thickness (17%). By contrast, Oc-Cre;Kif3a(flox/+) and Kif3a(flox/null) heterozygous mice exhibited no skeletal abnormalities. Loss of bone mass in Kif3a(Oc-cKO) mice was associated with impaired osteoblast function in vivo, as reflected by a 54% reduction in mineral apposition rate and decreased expression of Runx2, osterix (also known as Sp7 transcription factor 7; Sp7), osteocalcin and Dmp1 compared with controls. Immortalized osteoblasts from Kif3a(Oc-cKO) mice exhibited increased cell proliferation, impaired osteoblastic differentiation, and enhanced adipogenesis in vitro. Osteoblasts derived from Kif3a(Oc-cKO) mice also had lower basal cytosolic calcium levels and impaired intracellular calcium responses to fluid flow shear stress. Sonic hedgehog-mediated Gli2 expression and Wnt3a-mediated β-catenin and Axin2 expression were also attenuated in Kif3a(Oc-cKO) bone and osteoblast cultures. These data indicate that selective deletion of Kif3a in osteoblasts disrupts primary cilia formation and/or function and impairs osteoblast-mediated bone formation through multiple pathways including intracellular calcium, hedgehog and Wnt signaling.

Transient Receptor Potential Channel 1 (TRPC1) Reduces Calcium Permeability in Heteromeric Channel Complexes

Specific biological roles of the classical transient receptor potential channel 1 (TRPC1) are still largely elusive. To investigate the function of TRPC1 proteins in cell physiology, we studied heterologously expressed TRPC1 channels and found that recombinant TRPC1 subunits do not form functional homomeric channels. Instead, by electrophysiological analysis TRPC1 was shown to form functional heteromeric, receptor-operated channel complexes with TRPC3, -4, -5, -6, and -7 indicating that TRPC1 proteins can co-assemble with all members of the TRPC subfamily. In all TRPC1-containing heteromers, TRPC1 subunits significantly decreased calcium permeation. The exchange of select amino acids in the putative pore-forming region of TRPC1 further reduced calcium permeability, suggesting that TRPC1 subunits contribute to the channel pore. In immortalized immature gonadotropin-releasing hormone neurons endogenously expressing TRPC1, -2, -5, and -6, down-regulation of TRPC1 resulted in increased calcium permeability and elevated basal cytosolic calcium concentrations. We did not observe any involvement of TRPC1 in store-operated cation influx. Notably, TRPC1 suppressed the migration of gonadotropin-releasing hormone neurons without affecting cell proliferation. Conversely, in TRPC1 knockdown neurons, specific migratory properties like distance covered, locomotion speed, and directionality were increased. These findings suggest a novel regulatory mechanism relying on the expression of TRPC1 and the subsequent formation of heteromeric TRPC channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration.

Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia

We investigated whether Kif3a in osteoblasts has a direct role in regulating postnatal bone formation. We conditionally deleted Kif3a in osteoblasts by crossing osteocalcin (Oc; also known as Bglap)-Cre with Kif3a(flox/null) mice. Conditional Kif3a-null mice (Kif3a(Oc-cKO)) had a 75% reduction in Kif3a transcripts in bone and osteoblasts. Conditional deletion of Kif3a resulted in the reduction of primary cilia number by 51% and length by 27% in osteoblasts. Kif3a(Oc-cKO) mice developed osteopenia by 6 weeks of age unlike Kif3a(flox/+) control mice, as evidenced by reductions in femoral bone mineral density (22%), trabecular bone volume (42%) and cortical thickness (17%). By contrast, Oc-Cre;Kif3a(flox/+) and Kif3a(flox/null) heterozygous mice exhibited no skeletal abnormalities. Loss of bone mass in Kif3a(Oc-cKO) mice was associated with impaired osteoblast function in vivo, as reflected by a 54% reduction in mineral apposition rate and decreased expression of Runx2, osterix (also known as Sp7 transcription factor 7; Sp7), osteocalcin and Dmp1 compared with controls. Immortalized osteoblasts from Kif3a(Oc-cKO) mice exhibited increased cell proliferation, impaired osteoblastic differentiation, and enhanced adipogenesis in vitro. Osteoblasts derived from Kif3a(Oc-cKO) mice also had lower basal cytosolic calcium levels and impaired intracellular calcium responses to fluid flow shear stress. Sonic hedgehog-mediated Gli2 expression and Wnt3a-mediated β-catenin and Axin2 expression were also attenuated in Kif3a(Oc-cKO) bone and osteoblast cultures. These data indicate that selective deletion of Kif3a in osteoblasts disrupts primary cilia formation and/or function and impairs osteoblast-mediated bone formation through multiple pathways including intracellular calcium, hedgehog and Wnt signaling.

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca 2+] cyt) and [Ca 2+] cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G 1 phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca 2+ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.

Tri-phasic modulation of ACh- and NA-maintained calcium plateau by high potassium in isolated mouse submandibular granular convoluted tubular cells

ObjectivesThe fact that submandibular glands secrete a large amount of potassium ion upon nerve stimulation has long been recognized, but a physiological role for such high potassium in the saliva has never been systematically investigated. In the present work, high potassium effect has been investigated in the freshly isolated mouse submandibular granular convoluted tubules. DesignIsolated intact mouse submandibular granular convoluted tubules were loaded with Fura-2, and cytosolic calcium concentration in individual tubular cells was measured by microscopic fluorospectrometry. ResultsIt was found that high potassium had no effect on basal cytosolic calcium concentration, but had a tri-phasic modulation of the calcium plateau maintained by continued stimulation by acetylcholine (ACh) or noradrenaline (NA): a minor initial transient depression, followed by steady increase, completed by a robust calcium rebound spike upon removal of high potassium. The phase of steady increase was blocked without major effect on the plateau by KB-R 7943, a sodium/calcium exchange (NCX) inhibitor. ConclusionThese data together suggest that high potassium in saliva bathing the mouse submandibular granular convoluted tubular cells has a potent feedback effect on ACh and NA stimulation, and sodium/calcium exchange is likely to play a major role in this process. Such positive feedback actions of high potassium may suggest a role for enhancing ACh- or NA-stimulated protein factor secretion from the granulated convoluted tubular cells.

Bax inhibitor 1, a modulator of calcium homeostasis, confers affective resilience

The endoplasmic reticulum (ER) is a critical site for intracellular calcium storage as well as protein synthesis, folding, and trafficking. Disruption of these processes is gaining support for contributing to heritable vulnerability of certain diseases. Here, we investigated Bax inhibitor 1 (BI-1), an anti-apoptotic protein that primarily resides in the ER and associates with B-cell lymphoma 2 (Bcl-2) and Bcl-XL, as an affective resiliency factor through its modulation of calcium homeostasis. We found that transgenic (TG) mice with BI-1 reinforced expression, via the neuronal specific enolase promoter, showed protection against the learned helplessness (LH) paradigm, an animal model to test stress coping. TG mice were also protected against anhedonia following both serotonin and catecholamine depletion as measured in two different models, the female urine sniffing test and the saccharine preference test. In addition, we used primary mouse cortical cultures to explore the ability of BI-1 to influence calcium homeostasis under basal conditions and also following challenge with thapsigargin (THPS), an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) that disrupts calcium homeostasis. TG neurons showed decreased basal cytosolic calcium levels and decreased Ca2+ cytosolic accumulation following challenge with THPS as compared to WT neuronal cultures. Together, these data suggest that BI-1, through its actions on calcium homeostasis, may confer affective resiliency in multiple animal models of depression and anhedonia.

PONM17 Augmentation of the heat shock response in an in vitro model of sporadic inclusion body myositis

Sporadic inclusion body myositis (IBM), the commonest myopathy acquired by those aged over 50 years, remains without proven treatment. We developed an in vitro disease model, using primary satellite cells,...

Dual inhibition of sodium-mediated proton and calcium efflux triggers non-apoptotic cell death in malignant gliomas

Malignant glioma cells maintain an elevated intracellular pH (pH i) within hypoxic–ischemic tumor microenvironments through persistent activation of sodium–proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH i of malignant gliomas by inhibiting isoform 1 of sodium–proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium–calcium exchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na +-dependent calcium efflux by isoform 1.1 of the sodium–calcium exchanger (NCX1.1) , which increases [Ca 2+] i and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na +] i) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calcium levels ([Ca 2+] i) also are increased 5-fold. 2′, 4′-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forward mode of NCX1.1 at ≤ 1 μM, while dually inhibiting both NHE1 and NCX1.1 at ≥ 20 μM. DCB (1 μM) was not cytotoxic to glioma cells, while DCB (20 μM) further increased basal elevated levels of [Ca 2+] i in glioma cells that was followed by cell demise. Cariporide and SEA0400 are more selective inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase activation.

FAD mutations in amyloid precursor protein do not directly perturb intracellular calcium homeostasis.

Disturbances in intracellular calcium homeostasis are likely prominent and causative factors leading to neuronal cell death in Alzheimer's disease (AD). Familial AD (FAD) is early-onset and exhibits autosomal dominant inheritance. FAD-linked mutations have been found in the genes encoding the presenilins and amyloid precursor protein (APP). Several studies have shown that mutated presenilin proteins can directly affect calcium release from intracellular stores independently of Aβ production. Although less well established, there is also evidence that APP may directly modulate intracellular calcium homeostasis. Here, we directly examined whether overexpression of FAD-linked APP mutants alters intracellular calcium dynamics. In contrast to previous studies, we found that overexpression of mutant APP has no effects on basal cytosolic calcium, ER calcium store size or agonist-induced calcium release and subsequent entry. Thus, we conclude that mutated APP associated with FAD has no direct effect on intracellular calcium homeostasis independently of Aβ production.