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Astrocytes display context-specific diversity in their functions and respond to noxious stimuli between brain regions. Astrocytic mitochondria have emerged as key players in governing astrocytic functional heterogeneity, given their ability to dynamically adapt their morphology to regional demands on ATP generation and Ca2+ buffering functions. Although there is reciprocal regulation between mitochondrial dynamics and mitochondrial Ca2+ signaling in astrocytes, the extent of this regulation in astrocytes from different brain regions remains unexplored. Brain-wide, experimentally induced mitochondrial DNA (mtDNA) loss in astrocytes showed that mtDNA integrity is critical for astrocyte function, however, possible diverse responses to this noxious stimulus between brain areas were not reported in these experiments. To selectively damage mtDNA in astrocytes in a brain-region-specific manner, we developed a novel adeno-associated virus (AAV)-based tool, Mito-PstI expressing the restriction enzyme PstI, specifically in astrocytic mitochondria. Here, we applied Mito-PstI to two brain regions, the dorsolateral striatum and dentate gyrus, and we show that Mito-PstI induces astrocytic mtDNA loss in vivo, but with remarkable brain-region-dependent differences on mitochondrial dynamics, Ca2+ fluxes, and astrocytic and microglial reactivity. Thus, AAV-Mito-PstI is a novel tool to explore the relationship between astrocytic mitochondrial network dynamics and astrocytic mitochondrial Ca2+ signaling in a brain-region-selective manner. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine and has been implicated in pain regulation. Neuronal activity in the spinal dorsal horn contributes to nociceptive transmission. However, it is not fully understood how TNFα affects the activity of spinal dorsal horn neurons. In the present study, we used calcium imaging to characterize the mechanism by which TNFα regulates calcium influx in the cultured spinal dorsal horn neurons. We observed that TNFα incubation caused an increase in fluorescent intensity of Fura-2, a specific intracellular calcium indicator, in the cultured spinal dorsal horn neurons, and such effect was significantly inhibited by co-incubation with R7050, a selective TNFα receptor antagonist, which suggests that TNFα can enhance calcium influx and increase neuronal activity via activating TNFα receptors in the spinal dorsal horn. Using double immunofluorescence staining, we showed that TNFα receptors were co-expressed with a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor subunit GluA1 in the spinal dorsal horn neurons. We further observed that treatment with 1-naphthyl acetyl spermine (NASPM), a specific calcium-permeable AMPA receptor blocker, completely blocked the effect of TNFα incubation on calcium influx in the cultured neurons. Moreover, lipopolysaccharide (LPS)-induced calcium influx was inhibited by co-incubation with R7050. Together, our results suggest that TNFα in the spinal dorsal horn can increase calcium-indicated neuronal activity through the interaction between its receptor and calcium-permeable AMPA receptors. [ABSTRACT FROM AUTHOR]

Background: Hypoxia is a pathological phenomenon of the central nervous system that occurs during ischemia and stroke, leading to brain cell damage. Astrocytes play an important role in maintaining homeostasis in the brain microenvironment under normal as well as in pathological conditions. 3-hydroxymorphinan (3-HM) is an active metabolite of dextromethorphan (DM) that is widely used in anti-cough remedies and is more potent and safer than DM in the treatment of neurological disorders. However, previous research has not examined whether 3-HM has a protective effect in ischemic condition. Objective: In this study, we aimed to confirm the effect of 3-HM on hypoxia-induced astrocyte death. We also investigated the protective mechanism of 3-HM and its potential applicability in ischemia brain injury. Result: For the in vitro hypoxic conditions, CoCl2, a hypoxia-mimetic agent, was added to mouse primary astrocytes. 200 μM of CoCl2 showed cytotoxicity, whereas pre-incubation with 3-HM attenuated cell death by CoCl2. Moreover, pre-treatment with 3-HM dramatically reduced oxidative stress and mitochondrial damage by CoCl2. Further, 3-HM significantly reduced intracellular calcium influx induced by extracellular glutamate and finally modulated the glutamate released from the astrocytes. Conclusion: Taken together, these results suggest that 3-HM has a potential to regulate glutamate release from the astrocytic cell death in hypoxic condition and could be used as a promising preventive or therapeutic agent for ischemic brain injury. [ABSTRACT FROM AUTHOR]

Bisphenol F (BPF) is a replacement to bisphenol A, which has been extensively used in industrial manufacturing. Its wide detection in various human samples raises increasing concern on its safety. Currently, whether a low dose of BPF compromises cardiac function is still unknown. This study provides the first evidence that low-dose BPF can induce cardiac hypertrophy by using cardiomyocytes derived from human embryonic stem cells (hES). Non-cytotoxic BPF increased cytosolic Ca 2+ influx ([Ca2+ ]c), which was most remarkable at low dose (7 ng/ml) rather than at higher doses. Significant changes in the morphological parameters of mitochondria and significant decreases in ATP production were induced by 7 ng/ml BPF, representing a classic hypertrophic cardiomyocyte. After eliminating the direct effects on mitochondrial fission-related DRP1 by administration of the DRP1 inhibitor Mdivi-1, we examined the changes in [Ca 2+ ]c levels induced by BPF, which enhanced the calcineurin (Cn) activity and induced the abnormal mitochondrial fission via the CnAβ-DRP1 signaling pathway. BPF triggered excessive Ca 2+ influx by disrupting the L-type Ca 2+channel in cardiomyocytes. The interaction between ERβ and CnAβ cooperatively involved in the BPF-induced Ca 2+ influx, which resulted in the abnormal mitochondrial fission and compromised the cardiac function. Our findings provide a feasible molecular mechanism for explaining low-dose BPF-induced cardiac hypertrophy in vitro, preliminarily suggesting that BPF may not be as safe as assumed in humans. [ABSTRACT FROM AUTHOR]

According to the Environmental Protection Agency in Taiwan, the common carp (Cyprinus carpio) is one species of fish for acute toxic test. It has been found to be extremely sensitive to the toxicity of Cd2+; Furthermore, the goldfish (Carassius auratus) has a higher resistance than common carp upon Cd2+ exposure, but both fish are the same family. The aim of the study was to compare the physiological and histo-pathological responses between goldfish and common carp under exposure to sublethal concentrations of Cd2+ in order to understand the reasons behind the Cd2+-resistance. Results showed that metallothionein (MT) protein levels in visceral tissues were exceptionally increased and elevated at an earlier time in goldfish than in common carp. Meanwhile, the amount of Cd2+ accumulation in goldfish was higher than common carp after Cd2+ exposure. The histo-pathological results revealed that the density of gill mucus cells and the thickness of gill epithelium in common carp were raised earlier than in goldfish, but the histo-pathological findings resemble each other. According to the data, we suggested the efficient response of MT proteins may contribute to goldfish with a higher Cd2+ tolerance. [ABSTRACT FROM AUTHOR]

Nanosecond pulsed electric fields (nsPEFs) induce changes in the plasma membrane (PM), including PM permeabilization (termed nanoporation), allowing free passage of ions into the cell and, in certain cases, cell death. Recent studies from our laboratory show that the composition of the PM is a critical determinant of PM nanoporation. Thus, we hypothesized that the biological response to nsPEF exposure could be influenced by lipid microdomains, including caveolae, which are specialized invaginations of the PM that are enriched in cholesterol and contain aggregates of important cell signaling proteins, such as caveolin-1 (Cav1). Caveolae play a significant role in cellular signal transduction, including control of calcium influx and cell death by interaction of Cav1 with regulatory signaling proteins. Present results show that depletion of Cav1 increased the influx of calcium, while Cav1 overexpression produced the opposite effect. Additionally, Cav1 is known to bind and sequester important cell signaling proteins within caveolae, rendering the binding partners inactive. Imaging of the PM after nsPEF exposure showed localized depletion of PM Cav1 and results of co-immunoprecipitation studies showed dissociation of two critical Cav1 binding partners (transient receptor potential cation channel subfamily C1 (TRPC1) and inositol trisphosphate receptor (IP3R)) after exposure to nsPEFs. Release of TRPC1 and IP3R from Cav1 would activate downstream signaling cascades, including store-operated calcium entry, which could explain the influx in calcium after nsPEF exposure. Results of the current study establish a significant relationship between Cav1 and the activation of cell signaling pathways in response to nsPEFs. [ABSTRACT FROM AUTHOR]

Excitability in astroglia is controlled by Ca2+ fluxes from intracellular organelles, mostly from the endoplasmic reticulum (ER). Astrocytic ER possesses inositol 1,4,5-trisphosphate receptors (InsP3R) that can be activated upon stimulation through a vast number of metabotropic G-protein-coupled receptors. By contrast, the role of Ca2+-gated Ca2+ release channels is less explored in astroglia. Here we address this process by monitoring Ca2+ dynamics directly in the cytosol and the ER of astroglial cells. Cultured astrocytes exhibited spontaneous and high-K-evoked cytosolic Ca2+ transients, both of them reversibly abolished by external Ca2+ removal, addition of plasma membrane channel blockers or ER Ca2+ depletion with SERCA inhibitors. Resting astrocyte [Ca2+]ER averaged 400 μM and maximal stimulation with ATP provoked a complete and reversible ER discharge. Direct monitoring of Ca2+ in the lumen of ER showed that high-K induced a Ca2+ release from the ER, and its amplitude was proportional to the [K]. Furthermore, by combining the low affinity GAP3 indicator targeted to the ER with the high affinity cytosolic Rhod-2, we simultaneously imaged ER- and cytosolic-Ca2+ signals, in astrocytes in culture and in situ. Plasma membrane Ca2+ entry triggered a fast ER Ca2+ release coordinated with an increase in cytosolic Ca2+. Thus, we identify a Ca2+-induced Ca2+-release (CICR) mechanism that is likely to participate in spontaneous astroglial oscillations, providing a graded amplification of the cytosolic Ca2+ signal. [ABSTRACT FROM AUTHOR]

The mechanical response of the heart to myocardial stretch has been understood since the work of muscle physiologists more than 100 years ago, whereby an increase in ventricular chamber filling during diastole increases the subsequent force of contraction. The stretch-induced increase in contraction is biphasic. There is an abrupt increase in the force that coincides with the stretch (the rapid response), which is then followed by a slower response that develops over several minutes (the slow force response, or SFR). The SFR is associated with a progressive increase in the magnitude of the Ca2+ transient, the event that initiates myocyte cross-bridge cycling and force development. However, the mechanisms underlying the stretch-dependent increase in the Ca2+ transient are still debated. This review outlines recent literature on the SFR and summarizes the different stretch-activated Ca2+ entry pathways. The SFR might result from a combination of several different cellular mechanisms initiated in response to activation of different cellular stretch sensors. [ABSTRACT FROM AUTHOR]

Chronic exposure to inorganic arsenic (iAs), a common drinking water and food contaminant, has been associated with an increased risk of type 2 diabetes in population studies worldwide. Several mechanisms underlying the diabetogenic effects of iAs have been proposed through laboratory investigations. We have previously shown that exposure to arsenite (iAs(III)) or its methylated trivalent metabolites, methylarsonite (MAs(III)) and dimethylarsinite (DMAs(III)), inhibits glucose-stimulated insulin secretion (GSIS) in pancreatic islets, without significant effects on insulin expression or insulin content. The goal of the present study was to determine if iAs(III) and/or its metabolites inhibit Ca2+ influx, an essential mechanism that regulates the release of insulin from β cells in response to glucose. We found that in vitro exposures for 48 h to non-cytotoxic concentrations of iAs(III), MAs(III), and DMAs(III) impaired Ca2+ influx in isolated murine pancreatic islets stimulated with glucose. MAs(III) and DMAs(III) were more potent inhibitors of Ca2+ influx than iAs(III). These arsenicals also inhibited Ca2+ influx and GSIS in islets treated with depolarizing levels of potassium chloride in the absence of glucose. Treatment with Bay K8644, a Cav1.2 channel agonist, did not restore insulin secretion in arsenical-exposed islets. Tolbutamide, a KATP channel blocker, prevented inhibition of insulin secretion in MAs(III)- and DMAs(III)-exposed islets, but only marginally in islets exposed to iAs(III). Our findings suggest that iAs(III), MAs(III), and DMAs(III) inhibit glucose-stimulated Ca2+ influx in pancreatic islets, possibly by interfering with KATP and/or Cav1.2 channel function. Notably, the mechanisms underlying inhibition of GSIS by iAs(III) may differ from those of its trivalent methylated metabolites. [ABSTRACT FROM AUTHOR]

Benzo[a]pyrene (B[a]P) is an ubiquitous environmental pollutant that is generated during combustion of fossil fuels. We examine the effect of noradrenaline (NA) on B[a]P-induced neurotoxicity in brain tumor cell lines like neuroblastoma (Neuro2a) and glioma (C6). We pre-treated tumor cells with NA for 6 h, followed by addition of B[a]P for additional 24 h. Cell viability was measured using trypan blue dye-exclusion assay and comet assay was performed to measure DNA damage. Cell cycle status was analyzed using flow cytometry and oxidative DNA damage (8-oxodG) production was examined by immunostaining. The intracellular Ca2+ concentration was analyzed using Fura-2AM. Our results showed viability of Neuro2a and C6 cells declined (24% and 20%) in B[a]P-treated groups. However, pre-treating with NA increased viability of cells by reducing percentage of cell death in both. Furthermore, B[a]P-induced deregulation of cell cycle (G2/M and S phase cell arrest) was significantly restored by pre-treatment with NA in Neuro2a cells as compared to C6 cells. We further observed increased 8-oxodG production in B[a]P-treated cells; however, NA pre-treatment significantly (p < 0.05) reduced the 8-oxodG production in Neuro2a, while C6 cells were less affected possibly due to better protective machinery. B[a]P-induced intracellular Ca2+ influx was significantly reduced in both the cell lines due to co-treatment of NA possibly by reducing Ca2+ influx. NA protects brain tumor cells against B[a]P-induced neurotoxicity may be by decreasing percentage of G2 cell arrest, oxidative DNA damage, and reducing intracellular Ca2+ influx. These findings suggested that NA may be considered as a natural potential protective agent against B[a]P-induced neurotoxicity. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this review article is to discuss the potential of T-type voltage gated calcium channels (VGCCs) as drug targets in breast cancer.Summary: Breast cancer, attributable to the different molecular subtypes, has a crucial need for therapeutic strategies to counter the mortality rate. VGCCs play an important role in regulating cytosolic free calcium levels which regulate cellular processes like tumorigenesis and cancer progression. In the last decade, T-type VGCCs have been investigated in breast cancer proliferation. Calcium channel blockers, in general, have shown an anti-proliferative and cytotoxic effects. T-type VGCC antagonists have shown growth inhibition owing to the inhibition of CaV3.2 isoform. CaV3.1 isoform has been indicated as a tumour-suppressor gene candidate and is reported to support anti-proliferative and apoptotic activity in breast cancer. The distribution of T-type VGCC isoforms in different breast cancer molecular subtypes is diverse and needs to be further investigated. The role of T-type VGCCs in breast cancer migration, metastasis and more importantly in epithelial to mesenchymal transition (EMT) is yet to be elucidated. In addition, interlaced therapy, using a combination of chemotherapy drugs and T-type VGCC blockers, presents a promising therapeutic approach for breast cancer but more validation and clinical trials are needed. Also, for investigating the potential of T-type VGCC blocker therapy, there is a need for isoform-specific agonists/antagonists to define and discover roles of T-type VGCC specific isoforms.Conclusion: Our article provides a review of the role of T-type VGCCs in breast cancer and also discusses future of the research in this area so that it can be ascertained whether there is any potential of T-type VGCCs as drug targets in breast cancer. [ABSTRACT FROM AUTHOR]

We have previously reported that cationic poly-arginine and arginine-rich cell-penetrating peptides display high-level neuroprotection and reduce calcium influx following in vitro excitotoxicity, as well as reduce brain injury in animal stroke models. Using the neuroprotective peptides poly-arginine R12 (R12) and the NR2B9c peptide fused to the arginine-rich carrier peptide TAT (TAT-NR2B9c; also known as NA-1), we investigated the mechanisms whereby poly-arginine and arginine-rich peptides reduce glutamate-induced excitotoxic calcium influx. Using cell surface biotin protein labeling and western blot analysis, we demonstrated that R12 and TAT-NR2B9c significantly reduced cortical neuronal cell surface expression of the NMDA receptor subunit NR2B. Chemical endocytic inhibitors used individually or in combination prior to glutamate excitotoxicity did not significantly affect R12 peptide neuroprotective efficacy. Similarly, pretreatment of neurons with enzymes to degrade anionic cell surface proteoglycans, heparan sulfate proteoglycan (HSPG), and chondroitin sulfate proteoglycan (CSPG), as well as sialic acid residues, did not significantly affect peptide neuroprotective efficacy. While the exact mechanisms responsible for R12 peptide-mediated NMDA receptor NR2B subunit cell surface downregulation were not identified, an endocytic process could not be ruled out. The study supports our hypothesis that arginine-rich peptides reduce excitotoxic calcium influx by reducing the levels of cell surface ion channels. [ABSTRACT FROM AUTHOR]

The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca entry and processes outgrowth in cortical neurons, and pharmacological activation of L-type Ca channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders. [ABSTRACT FROM AUTHOR]

The increasing number of osteoporosis patients is a pressing issue worldwide. Osteoporosis frequently causes fragility fractures, limiting activities of daily life and increasing mortality. Many osteoporosis patients take numerous medicines due to other health issues; thus, it would be preferable if a single medicine could ameliorate osteoporosis and other conditions. Here, we screened 96 randomly selected drugs targeting various diseases for their ability to inhibit differentiation of osteoclasts, which play a pivotal role in development of osteoporosis, and identified methotrexate (MTX), as a potential inhibitor. MTX is currently used to treat sarcomas or leukemic malignancies or auto-inflammatory diseases such as rheumatoid arthritis (RA) through its anti-proliferative and immunosuppressive activities; however, a direct effect on osteoclast differentiation has not been shown. Here, we report that osteoclast formation and expression of osteoclastic genes such as NFATc1 and DC-STAMP, which are induced by the cytokine RANKL, are significantly inhibited by MTX. We found that RANKL-dependent calcium (Ca) influx into osteoclast progenitors was significantly inhibited by MTX. RA patients often develop osteoporosis, and osteoclasts are reportedly required for joint destruction; thus, MTX treatment could have a beneficial effect on RA patients exhibiting high osteoclast activity by preventing both osteoporosis and joint destruction. [ABSTRACT FROM AUTHOR]

Pancreatic β-cells are particularly susceptible to fatty-acid-induced endoplasmic reticulum (ER) stress and apoptosis. To understand how β-cells sense fatty acid stimuli and translate into a long-term adaptive response, we investigated whether palmitic acid (PA) regulates early growth response-1 (Egr-1), an immediate-early transcription factor, which is induced by many environmental stimuli and implicated in cell proliferation, differentiation, and apoptosis. We found that Egr-1 was rapidly and transiently induced by PA in MIN6 insulinoma cells, which was accompanied by calcium influx and ERK1/2 phosphorylation. Calcium chelation and MEK1/2 inhibition blocked PA-induced Egr-1 upregulation, suggesting that PA induces Egr-1 expression through a calcium influx-MEK1/2-ERK1/2 cascade. Knockdown of Egr-1 increased PA-induced caspase-3 activation and ER stress markers and decreased PA-induced Akt phosphorylation and insulin secretion and signaling. Akt replenishment and insulin supplementation rescued PA-induced apoptosis in Egr-1 knockdown cells. These results suggest that the absence of Egr-1 loses its ability to couple the short-term insulin/Akt pathway to long-term survival adaptation. Finally, Egr-1-deficient mouse islets are more susceptible to ex vivo stimuli of apoptosis. In human pancreatic tissues, EGR1 expression correlated with expression of ER stress markers and anti-apoptotic gene. In conclusion, Egr-1 is induced by PA and further attempts to rescue β-cells from ER stress and apoptosis through improving insulin/Akt signaling. Our study underscores Egr-1 as a critical early sensor in pancreatic β-cells to translate fatty acid stimuli into a cellular adaptation mechanism. Key Message: [ABSTRACT FROM AUTHOR]

Hybrid necrosis is a well-known reproductive isolation mechanism in plant species, and an autoimmune response is generally considered to trigger hybrid necrosis through epistatic interaction between disease resistance-related genes in hybrids. In common wheat, the complementary Ne1 and Ne2 genes control hybrid necrosis, defined as type I necrosis. Two other types of hybrid necrosis (type II and type III) have been observed in interspecific hybrids between tetraploid wheat and Aegilops tauschii. Another type of hybrid necrosis, defined here as type IV necrosis, has been reported in F hybrids between Triticum urartu and some accessions of Triticum monococcum ssp. aegilopoides. In types I, III and IV, cell death occurs gradually starting in older tissues, whereas type II necrosis symptoms occur only under low temperature. To compare comprehensive gene expression patterns of hybrids showing growth abnormalities, transcriptome analysis of type I and type IV necrosis was performed using a wheat 38k oligo-DNA microarray. Defense-related genes including many WRKY transcription factor genes were dramatically up-regulated in plants showing type I and type IV necrosis, similarly to other known hybrid abnormalities, suggesting an association with an autoimmune response. Reactive oxygen species generation and necrotic cell death were effectively inhibited by ZnCl treatment in types I, III and IV necrosis, suggesting a significant association of Ca influx in upstream signaling of necrotic cell death in wheat hybrid necrosis. [ABSTRACT FROM AUTHOR]

The adenosine 5′-triphosphate (ATP)-gated P2X7 receptor is a membrane-bound, non-selective cation channel, expressed in a variety of cell types. The P2X7 senses high extracellular ATP concentrations and seems to be implicated in a wide range of cellular functions as well as pathophysiological processes, including immune responses and inflammation, release of gliotransmitters and cytokines, cancer cell growth or development of neurodegenerative diseases. In the present study, we identified natural compounds and analogues that can block or sensitize the ATP (1 mM)-induced Ca response using a HEK293 cell line stably expressing human P2X7 and fluorometric imaging plate reader technology. For instance, teniposide potently blocked the human P2X7 at sub-miromolar concentrations, but not human P2X4 or rat P2X2. A marked block of ATP-induced Ca entry and Yo-Pro-1 uptake was also observed in human A375 melanoma cells and mouse microglial cells, both expressing P2X7. On the other hand, agelasine (AGL) and garcinolic acid (GA) facilitated the P2X7 response to ATP in all three cell populations. GA also enhanced the YO-PRO-1 uptake, whereas AGL did not affect the ATP-stimulated intracellular accumulation of this dye. According to the pathophysiological role of P2X7 in various diseases, selective modulators may have potential for further development, e.g. as neuroprotective or antineoplastic drugs. [ABSTRACT FROM AUTHOR]

Calcium influx is required for the pituitary adenylyl cyclase activating polypeptide (PACAP)-induced increase in guinea pig cardiac neuron excitability, noted as a change from a phasic to multiple action potential firing pattern. Intracellular recordings indicated that pretreatment with the nonselective cationic channel inhibitors, 2-aminoethoxydiphenylborate (2-APB), 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96365), and flufenamic acid (FFA) reduced the 20-nM PACAP-induced excitability increase. Additional experiments tested whether 2-APB, FFA, and SKF 96365 could suppress the increase in excitability by PACAP once it had developed. The increased action potential firing remained following application of 2-APB but was diminished by FFA. SKF 96365 transiently depressed the PACAP-induced excitability increase. A decrease and recovery of action potential amplitude paralleled the excitability shift. Since semiquantitative PCR indicated that cardiac neurons express TRPC subunit transcripts, we hypothesize that PACAP activates calcium-permeable, nonselective cationic channels, which possibly are members of the TRPC family. Our results are consistent with calcium influx being required for the initiation of the PACAP-induced increase in excitability, but suggest that it may not be required to sustain the peptide effect. The present results also demonstrate that nonselective cationic channel inhibitors could have other actions, which might contribute to the inhibition of the PACAP-induced excitability increase. [ABSTRACT FROM AUTHOR]

Our previous studies have demonstrated that ginsenoside Rd (GSRd), one of the principal ingredients of Pana notoginseng, has neuroprotective effects against ischemic stroke. However, the possible mechanism(s) underlying the neuroprotection of GSRd is/are still largely unknown. In this study, we treated glutamate-injured cultured rat hippocampal neurons with different concentrations of GSRd, and then examined the changes in neuronal apoptosis and intracellular free Ca concentration. Our MTT assay showed that GSRd significantly increased the survival of neurons injured by glutamate in a dose-dependent manner. Consistently, TUNEL and Caspase-3 staining showed that GSRd attenuated glutamate-induced cell death. Furthermore, calcium imaging assay revealed that GSRd significantly attenuated the glutamate-induced increase of intracellular free Ca and also inhibited NMDA-triggered Ca influx. Thus, the present study demonstrates that GSRd protects the cultured hippocampal neurons against glutamate-induced excitotoxicity, and that this neuroprotective effect may result from the inhibitory effects of GSRd on Ca influx. [ABSTRACT FROM AUTHOR]

The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca] ([Ca]) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca] are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca release, and Ca entry, as well as those that maintain cellular Ca homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca] signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca] in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca signaling mechanisms that determine salivary gland fluid secretion. [ABSTRACT FROM AUTHOR]

The history of the discovery of the transient receptor potential (TRP) cation channel superfamily began in 1969 with Cosens and Manning's isolation of the Drosophila transient receptor potential mutant, in which the photoreceptor response decays during continuous illumination. Early studies from Minke found that the elementary light response was unaffected in trp mutants, and he attributed the defect to an intermediate stage of phototransduction. Montell and Rubin cloned the trp gene in 1989: they recognised it as a transmembrane protein, but also concluded that it did not encode the light-sensitive channels. In 1991, Minke and Selinger proposed that TRP represented a Ca transporter required for refilling intracellular InsP-sensitive Ca stores, in turn required for activation of the light-sensitive channels. Also in 1991, after developing a photoreceptor patch clamp preparation, I showed that the light-sensitive channels themselves were highly permeable to Ca, questioning the need for such a dedicated Ca transporter. In 1992, in collaboration with Minke, I resolved this paradox by showing there were two classes of light-sensitive channels, one highly Ca permeable and eliminated in trp mutants. This represented the first and compelling evidence that TRP represented a light-sensitive channel and was supported by the cloning of the second light-sensitive channel, TRPL, by Kelly's lab. Three years later, in 1995, the labs of Montell and Birnbaumer independently cloned TRPC1, the first of 29 vertebrate TRP isoforms distributed amongst seven subfamilies. [ABSTRACT FROM AUTHOR]

Skeletal muscle contraction is basically controlled by Ca release and its reuptake into the sarcoplasmic reticulum. However, the long-term maintenance of muscle function requires an additional Ca influx from extracellular. Several mechanisms seem to contribute to the latter process, such as store-operated Ca entry, stretch-activated Ca influx and resting Ca influx. Candidate channels that may control Ca influx into muscle fibers are the STIM proteins, Orai, and the members of the transient receptor potential (TRP) family of cation channels. Here we show that TRPV4, an osmo-sensitive cation channel of the vanilloid subfamily of TRP channels is functionally expressed in mouse skeletal muscle. Western blot analysis showed the presence of TRPV4-specific bands at about 85 and 100 kDa in all tested muscles. The bands were absent when muscle proteins from TRPV4 deficient mice were analyzed. Using the manganese quench technique, we studied the resting influx of divalent cations into isolated wild-type muscle fibers. The specific TRPV4-channel activator 4α-phorbol-12,13-didecanoate (4α-PDD) stimulated resting influx by about 60% only in wild-type fibers. Electrical stimulation of soleus muscles did not reveal changes in isometric twitch contractions upon application of 4α-PDD, but tetanic contractions (at 120 Hz) were slightly increased by about 15%. When soleus muscles were stimulated with a fatigue protocol, muscle fatigue was significantly attenuated in the presence of 4α-PDD. The latter effect was not observed with muscles from TRPV4 mice. We conclude that TRPV4 is functionally expressed in mouse skeletal muscle and that TRPV4 activation modulates resting Ca influx and muscle fatigue. [ABSTRACT FROM AUTHOR]

Prostate cancer (PC) is a major health problem in adult males. TRPM8, a cationic TRP channel activated by cooling and menthol is upregulated in PC. However, the precise role of TRPM8 in PC is still unclear. Some studies hypothesized that TRPM8-mediated transmembrane Ca fluxes play a key role in cellular proliferation of PC cells. In contrast, other findings suggest that high TRPM8 levels may reduce the metastatic potential of PC cells. A detailed understanding of the response of TRPM8 channels to pharmacological modulators of their activity is relevant when considering potential therapies, targeting this ion channel to treat PC. We characterized the pharmacological and functional properties of native TRPM8 channels in four human prostate cell lines, PNT1A, LNCaP, DU145, and PC3, commonly used as experimental models of PC. PNT1A is a non-tumoral prostate cell line while the other three correspond to different stages of PC. Here, we show that cold- and agonist-evoked [Ca] responses in PC cells are much less sensitive to well-characterized agonists (menthol and icilin) and antagonists (BCTC, clotrimazole, and DD01050) of TRPM8 channels, compared to TRPM8 channels in other tissues, suggesting a different molecular composition and/or spatial organization. In addition, the forced overexpression of human TRPM8 facilitated the trafficking of TRPM8 channels residing in the endoplasmic reticulum to the plasma membrane, leading to a marked potentiation in the efficacy of the different blockers. These results predict that blockers of canonical TRPM8 channels may be less effective in halting proliferation of PC cells than expected. [ABSTRACT FROM AUTHOR]

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca entry into the cells via L-type voltage-gated Ca channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca level ([Ca]) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca] elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca] response of the cells, suggesting Ca influx occurs only via VGCCs. Lowering extracellular K concentration from 5 to 2 mM or pulsing cells in Na-free medium suppressed the pulse-induced rise in [Ca] in the majority of cells. Thus, both membrane potential and Na entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca influx. However, activation of voltage-gated Na channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca]. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na transport across the plasma membrane. Whether Na transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined. [ABSTRACT FROM AUTHOR]

The effect of blueberry ( Vaccinium angustifolium) root methanolic extract (BRM) on the A23187 plus phorbol myristate acetate (PMA)-induced degranulation in human basophilic KU812F cells was investigated. The total phenolic content (TPC) was 170±1.9 mg gallic acid equivalents (GAE)/g BRM. BRM reduced levels of histamine and β-hexosaminidase released from stimulated KU812F cells. Intracellular calcium concentration ([Ca] i) were determined by spectrofluorometric analysis using Fura 2-AM was found to be reduced by BRM with dose-dependent manner. Moreover, Western blot analysis revealed that protein kinase C (PKC) translocation was inhibited by BRM with dose-dependent manner. These results indicated that BRM inhibited histamine and β-hexosaminidase through the suppression of Ca influx and PKC translocation. Therefore, we suggest that BRM is potent inhibitor of degranulation in mast cells and basophils, and may be useful in preventing the allergic reactions. [ABSTRACT FROM AUTHOR]

The transient receptor potential mucolipins (TRPMLs) are the most recently discovered subfamily of TRP ion channel proteins. Positional cloning approach has identified two mutations in the TRPML3 ( Mcoln3) gene that cause the varitint–waddler mouse phenotypes. Short for variable tint (diluted coat color), the varitint–waddler consists two phenotypes Va and Va J . The mutation associated with the Va phenotype is an alanine to proline substitution at position 419 (A419P) within the predicted fifth transmembrane (TM5) domain of TRPML3. The second Va J mouse phenotype arose spontaneously from an isoleucine to threonine substitution at position 362 (I362T) that is proximal to the predicted TM3 domain in addition to the existing A419P mutation on TM5. Mice with the Va and Va J mutations exhibit a spectrum of disease phenotypes from diluted coat color to auditory and vestibular problems, depending on which alleles are present. It has been over 5 years since the discovery of these TRPML3 mutations, and it was just recently that the nature of these mutations has been characterized. In this review, we discuss the molecular and cell physiological effects of the two distinct TRPML3 mutations. We reveal the effects of proline substitution on transmembrane domain structure and channel function and discuss how the Va mutation confers its cytotoxicity, while the Va J mutation results in an apparent rescue phenotype. Finally, we briefly tackle molecular strategies that have been employed to neutralize the cytotoxic effect and constitutive channel activity of the Va mutation. [ABSTRACT FROM AUTHOR]

Here, we describe novel mechanisms limiting a toxic cytosolic Ca2+ rise during adenosine 5′-triphosphate (ATP) depletion. We studied the effect of ATP depletion on Ca2+ signalling in mouse pancreatic acinar cells. Measurements of ATP in isolated cells after adenovirus-mediated expression of firefly luciferase revealed that the cytosolic ATP concentration fell from approximately 1 mM to near zero after treatment with oligomycin plus iodoacetate. ATP depletion resulted in the inhibition of Ca2+ extrusion, which was accompanied by a remarkably synchronous inhibition of store-operated Ca2+ influx. Alternative inhibition of Ca2+ extrusion by carboxyeosin had a much smaller effect on Ca2+ influx. The coordinated metabolic inhibition of Ca2+ influx and extrusion suggests the existence of a common ATP-dependent master regulator of both processes. ATP-depletion also suppressed acetylcholine (ACh)-induced Ca2+ oscillations, which was due to the inhibition of Ca2+ release from internal stores. This could be particularly important for limiting Ca2+ toxicity during periods of hypoxia. In contrast, metabolic control of Ca2+ influx and Ca2+ release from internal stores spectacularly failed to prevent large toxic Ca2+ responses induced by bile acids—activators of acute pancreatitis (a frequent and often fatal disease of the exocrine pancreas). The bile acids taurolithocholic acid 3-sulphate (TLC-S), taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC) were used in our experiments. Neither Ca2+ release from internal stores nor Ca2+ influx triggered by bile acids were inhibited by ATP depletion, emphasising the danger of these pathological mechanisms. [ABSTRACT FROM AUTHOR]

To further understand the significance of bone as a target tissues of lead toxicity, as well as a reservoir of systemic lead, it is necessary to define the effect of lead on the calcium release activated calcium influx (CRACI) in primary cultures of human osteoblast-like cells (OLC). Pb2+ inhibited the immediate CRACI dose-dependent manner. Influx of Pb2+ into human OLC was increased dose-dependent manner. The present study demonstrates that the interference of Pb2+ with CRACI of human OLC is at least twofold: (1) the initiation of CRACI, i.e., the measurable influx of Ca2+ upon Ca2+ readdition, is partially inhibited by Pb2+ and (2) the influx of Pb2+ was enhanced after CRACI had been induced. [ABSTRACT FROM AUTHOR]

We investigated the influence of the polyunsaturated docosahexaenoic acid (22:6 n-3; DHA) on the constitutive expression of choline acetyltransferase (ChAT) in native and induced expression in differentiated cholinergic cells NG108-15 grown in serum-free medium. Elimination of serum-derived trophic support resulted in growth arrest and a strong decrease of ChAT activity. In either conditions, DHA largely rescued general indicators of cell growth and function, and partially prevented the decrease of ChAT activity. However, the maximal effect on general cell state in native and differentiated cells, and ChAT activity in native cells, was reached at or below 10 μmol/l of DHA. In contrast, maximal induction of ChAT activity in differentiated cells required about six times higher concentrations of DHA. These data thus demonstrate stimulatory effect of DHA on ChAT activity that is independent of its general cell protective properties. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: The role of gamma-aminobutyric acid (GABA) and A-type GABA receptors (GABAARs) in modulating islet endocrine function has been actively investigated since the identification of GABA and GABAARs in the pancreatic islets. However, the reported effects of GABAAR activation on insulin secretion from islet beta cells have been controversial. Methods: This study examined the hypothesis that the effect of GABA on beta cell insulin secretion is dependent on glucose concentration. Results: Perforated patch-clamp recordings in INS-1 cells demonstrated that GABA, at concentrations ranging from 1 to 1,000 μmol/l, induced a transmembrane current (IGABA) which was sensitive to the GABAAR antagonist bicuculline. The current-voltage relationship revealed that IGABA reversed at -42±2.2 mV, independently of glucose concentration. Nevertheless, the glucose concentration critically controlled the membrane potential (VM), i.e., at low glucose (0 or 2.8 mmol/l) the endogenous VM of INS-1 cells was below the IGABA reversal potential and at high glucose (16.7 or 28 mmol/l), the endogenous VM of INS-1 cells was above the IGABA reversal potential. Therefore, GABA dose-dependently induced membrane depolarisation at a low glucose concentration, but hyperpolarisation at a high glucose concentration. Consistent with electrophysiological findings, insulin secretion assays demonstrated that at 2.8 mmol/l glucose, GABA increased insulin secretion in a dose-dependent fashion (p<0.05, n=7). This enhancement was blocked by bicuculline (p<0.05, n=4). In contrast, in the presence of 28 mmol/l glucose, GABA suppressed the secretion of insulin (p<0.05, n=5). Conclusions/interpretation: These findings indicate that activation of GABAARs in beta cells regulates insulin secretion in concert with changes in glucose levels. [ABSTRACT FROM AUTHOR]

TRPM2 channels play an important role in the activation process of neutrophil granulocytes. One mechanism of TRPM2 channel gating is the binding of intracellular ADP ribose (ADPR) to the Nudix box domain in the C-terminal tail of TRPM2. Intracellular Ca2+, although not an activator of TRPM2 by its own, significantly enhances TRPM2 gating by ADPR. Stimulation of neutrophil granulocytes with the chemoattractant peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) induces release of Ca2+ ions from intracellular stores which in cooperation with endogenous ADPR levels enable Ca2+ influx through TRPM2. Stimulation of the ectoenzyme CD38, a membrane-associated glycohydrolase with ADPR as main product, and uptake of ADPR into the cell may contribute to the effects of fMLP. Inhibition of ADPR production, of uptake and of binding to TRPM2 are all potential pharmacological principles by which a modulation of neutrophil function may become possible in future. [ABSTRACT FROM AUTHOR]

We studied the effects of adrenergic agents on the five main cell types of the rat anterior pituitary by monitoring the changes of the cytosolic free [Ca2+] ([Ca2+]i) in single cells that were identified by multiple sequential primary immunocytochemistry at the end of the Ca2+ measurements. Adrenaline (100 nM) increased [Ca2+]i in 30% of the cells. Responses were most prominent in somatotrophs and corticotrophs (40–65% of the cells responded) whereas the other three cell types, lactotrophs, thyrotrophs and gonadotrophs, gave poorer responses. Selective agonists and antagonists revealed the presence of both α1- and β-adrenergic receptors. α1-Receptors dominated in corticotrophs, β-receptors in somatotrophs. The α1-adrenergic responses increased with culture of the cells. The β-adrenergic responses were mediated by cAMP and consisted of stimulation of Ca2+ entry through L-type voltage-gated channels. Stimulation of α1-receptors released Ca2+ from intracellular stores in corticotrophs and induced cAMP-independent Ca2+ entry in somatotrophs. The effects of α1-agonists were additive with those of the releasing factors growth hormone-releasing hormone (GHRH) and corticotropin releasing factor (CRF) whereas those of the β-agonists were not. Our results suggest that direct effects of plasma catecholamines on AP cells may contribute to the hormonal response to stress. [ABSTRACT FROM AUTHOR]

We have compared the effects of pituitary adenylate cyclase-activating polypeptide (PACAP-27) on the five main cell types of rat anterior pituitary in primary culture by monitoring changes in cytosolic Ca2+ concentration ([Ca2+]i) in single fura-2-loaded cells. Cells were typed by multiple sequential primary immunocytochemistry at the end of the Ca2+ measurements. PACAP-27 increased [Ca2+]i by three different mechanisms, each one dominant in a given cell type. These involved Ca2+ entry or release from the stores and mediation through different second messenger pathways: (1) stimulation of Ca2+ entry mediated by cAMP was the main mechanism in somatotrophs; (2) Ca2+ release from the intracellular Ca2+ stores mediated by phospholipase C (PLC) was the dominant modality in gonadotrophs; (3) stimulation of Ca2+ entry not mediated by cAMP was the main mechanism in lactotrophs. A minor fraction of somatotrophs (11%) may also use mechanism 3. Corticotrophs and thyrotrophs exhibited weak responses to PACAP (<10% of the cells responded), which in all cases were mediated by mechanism 1. Mechanism 3 represents a novel effect of PACAP which cannot be explained by interaction with the conventional PACAP receptor families. [ABSTRACT FROM AUTHOR]

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 µM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 µM) and cibacron blue 3GA (3 µM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 µM; by 47%), by the N-type calcium channel blocker ω-conotoxin GVIA (100 nM; by 35%), and by the α2-adrenoceptor agonist UK-14,304 (1 µM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by ω-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 µM) caused a marked increase in intraaxonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and ω-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 µM) induced a large depolarisation and action potential firing. UDP (100 µM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and – probably – the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by α2-adrenocep-tors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to α2-adrenoceptor modulation. [ABSTRACT FROM AUTHOR]

The characteristics of the increased calcium (Ca) influx observed in metabolically depleted red blood cells (RBCs) of hypertensive patients were investigated. Twenty-four normotensives, 16 untreated essential hypertensives, and 10 essential hypertensives under sufficient blood pressure control by 50-100 mg/day atenolol were studied. Free intracellular concentrations of Ca, sodium (Na), and potassium (K) were assessed using ion-selective electrodes in freeze-thawed RBCs, which were metabolically depleted by 30 m M desoxy-glucose at 37°C for 48 h. In the treated hypertensives values for Ca and K at 24 and 48 h were not different from values for the normotensives, whereas elevated Ca was found in RBCs of untreated hypertensives. Na in treated hypertensives was significantly increased at 0 and 48 h, thus, being similar to values for untreated hypertensives. Additionally, RBCs of six normals were stressed in a glass/teflon potter. Before metabolic depletion electrolytes were not affected by this procedure, while Ca at 24 and 48 h of metabolic depletion increased to significantly higher values for the hypertensive patients as compared to the controls. These results suggest that the altered Ca metabolism in the RBCs of hypertensives may reflect a secondary phenomenon due to the mechanical damage to RBCs by the elevated blood pressure. [ABSTRACT FROM AUTHOR]

We have analysed Ca waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca] responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC (200 nM). Upon receptor activation, [Ca] signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca] rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca release is higher than other regions of the cell. Removal of extracellular Ca or blockade of Ca channels by inorganic cations (Cd and Ni) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca] response. In the absence of Ca release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca entry is not operative. Furthermore, our experiments provide evidence for local Ca oscillations in cells which can support both wave propagation as well as spatially discrete Ca signalling. [ABSTRACT FROM AUTHOR]

The noncontractile aortic cell line A7r5 was chosen to study the effect of the vasoconstrictor peptide vasopressin on transmembrane Ca movements, using conventional whole-cell patch recording techniques. Conditions in which previously characterised vasoconstrictor-modulated currents were suppressed revealed a tiny inward current component (−18±2 pA, n=50, at -61 mV in 110 mM CaCl). The vasopressin-activated inward current was absent when Ca was absent from the extracellular solution, and the current amplitude increased with [Ca] (0.01-110 mM), with an apparent dissociation constant for Ca of 9.7 mM. It was highly selective for Ca over monovalent cations (permeability ratio Ca/Cs greater than 17). It was not voltage gated, except that the current/potential characteristic showed some inwards rectification. Amplitudes of the evoked inward currents had the same order of magnitude in Sr and Ca, whereas they were much smaller in Mn, suggesting that this pathway is highly permeable to Sr but poorly permeable to Mn. Inward currents evoked in Ca were inhibited by other cations with the following order of potency: La>Cd>Co∼Ni∼Mn. The channel producing this current corresponds most probably to the ionic pathway originally called the receptor-operated calcium channel, which produces a long-lasting, constrictor-induced plateau of increased intracellular free calcium concentration in smooth muscle. [ABSTRACT FROM AUTHOR]

Cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate (ATP) were investigated in primary cultures of rabbit proximal convoluted tubule cells. Extracellular ATP (50 μM) induced a biphasic increase of [Ca] measured with the calcium probe fura-2. In the early phase, the mobilization of intracellular pools resulted in a transient increase of [Ca] from 106±11 nM ( n=36) to 1059±115% ( n=29) of the resting level within 10 s. In the presence of external calcium, [Ca] then decreased within 3 min to a sustained level (398±38%, n=8). Measurements of fura-2 quenching by external manganese revealed that this phase was the result of an increased Ca uptake, blocked by lanthanum (10 μM) and verapamil (100 μM) but not by the nifedipin (25 μM). Internal calcium store depletion by ATP induced an increased calcium influx through lanthanum- and verapamil-sensitive, nifedipininsensitive calcium channels, located on the apical membrane of the cells. As indicated byRb efflux measurements, ATP activated a potassium efflux that was blocked by barium and Leiurus quinquestriatus hebraeus (LQH) venom (containing charybdotoxin) indicating the involvement of Ca-sensitive K channels. Moreover, in the presence of the LQH venom, the internal calcium stores were not replenished after being depleted by ATP. Our results indicate that an ATPevoked hyperpolarization of the plasma membrane leads to increased Ca influx, which facilitates the replenishment of the internal stores. [ABSTRACT FROM AUTHOR]

A pharmacological approach was employed in order to visualize a Ca-dependent component of the extracellularly recorded nerve terminal impulse in the secretory regions of the sympathetic postganglionic nerves in the rat tail artery. Application of potassiumchannel-blocking agents within the recording electrode caused the nerve terminal impulse to acquire a delayed negative deflection, which we have termed the late negative component (LNC) of the nerve terminal impulse. The time course and the latency of the LNC differed from that of the postjunctional transmitter-induced excitatory junction current, and the LNC persisted when the excitatory junction current was blocked by adenosine [α, βmethylene] triphosphate, and was resistant to the α-antagonist prazosin and the α-antagonist yohimbine. Probably, therefore, the LNC was exclusively prejunctional in origin. For the following reasons it seems likely that the LNC, at least in part, was caused by influx of Ca into the secretory regions of these nerves: (a) the LNC occurred only when potassium-blocking agents were present within the recording electrode; (b) the LNC amplitude increased with the Ca concentration inside the recording electrode and was reduced by the removal of Ca; (c) the LNC was enhanced by replacing Ca in the medium inside the recording electrode with Ba; (d) the LNC was depressed by the inorganic Ca-channel blocker cadmium or the Ca-channel-blocking peptide ω-conotoxin added within the recording electrode only, or by addition of cadmium or cobalt (but not the organic Ca-channel blocker nifedipine) inside and outside the recording electrode. In our opinion, these lines of evidence indicate that the LNC represents at least in part a Ca-dependent component of the nerve terminal impulse in the secretory regions of these nerves. The extent to which the LNC can be used as a quantitative measure of nerve-impulse-induced influx of Ca into the nerve terminals remains to be established. [ABSTRACT FROM AUTHOR]

In the ventral horn of the lumbar spinal cord of cats anaesthetised with pentobarbitone sodium, microelectrophoretically administered (–)-baclofen, but not (+)-baclofen, reversibly reduced the duration of the orthodromic action potential of muscle group Ia afferent terminations, but not those of muscle group I afferent myelinated fibres. The presumably submicromolar concentrations are already known to reversibly reduce excitatory transmitter release from muscle group Ia afferent terminations. Action potential durations were estimated from threshold recovery curves after an orthodromic impulse using an extracellular microstimulation technique. Both of these presynaptic effects of (–)-baclofen were blocked by baclofen antagonists, and neither appeared to be reduced by the potassium channel blocking agents tetraethylammonium and 4-aminopyridine. Tetraethylammonium and 4-aminopyridine also did not significantly modify the reduction by (–)-baclofen of monosynaptic field potentials in the lumbar cord of rats anaesthetised with pentobarbitone sodium. In the cat the maximum reduction by (–)-baclofen of termination action potentials was considerably less than that produced by cadmium ions, which, unlike (–)-baclofen, also reduced the action potential duration of group I myelinated fibres. These findings are consistent with a reduction by (–)-baclofen of the influx of calcium through voltage-activated channels in the membrane of group Ia terminations, a proposal which also accounts for the reduction by (–)-baclofen of the release of GABA at axo-axonic depolarizing synapses on these terminations. The results are discussed in relation to the mode of action of (–)-baclofen and the different sensitivities of transmitter release at various central synapses. [ABSTRACT FROM AUTHOR]

Glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylate (PDC, 20 μM) elevated basal and N-methyl-D-aspartate (NMDA, 100 μM)-induced extracellular glutamate accumulation, while it did not augment kainate (100 μM)-induced glutamate accumulation in cultured cerebellar granule neurons. However, pretreatment with PDC for 1 h significantly reduced NMDA-induced glutamate accumulation, but did not affect kainate-induced response. Pretreatment with glutamate (5 μM) for 1 h also reduced NMDA-induced glutamate accumulation, but did not kainate-induced response. Upon a brief application (3–10 min), PDC did neither induce elevation of intracellular calcium concentration ([Ca2+]i) nor modulate NMDA-induced [Ca2+]i elevation. Pretreatment with PDC for 1 h reduced NMDA-induced [Ca2+]i elevation, but it did not reduce kainate-induced [Ca2+]i elevation. These results suggest that glutamate concentration in synaptic clefts of neuronal cells is increased by prolonged exposure (1 h) of the cells to PDC, and the accumulated glutamate subsequently induces selective desensitization of NMDA receptor. [ABSTRACT FROM AUTHOR]

In order to study the fine structural effect of calcium influx on secretory activity of rat anterior pituitary cells, small pieces of anterior pituitary were incubated in Krebs' medium containing the calcium ionophore A23187 (0.15 mM) and were examined electron microscopically. Marked changes were present in all types of secretory cells incubated for 3, 12 and 20 min in the medium containing calcium and A23187. Secretory granules tended to accumulate in the peripheral cytoplasm of the secretory cells, and more numerous images of granule release by exocytosis were observed in somatotroph (STH cell), luteotroph (LTH cell), thyrotroph (TSH cell), corticotroph (ACTH cell), type 1 gonadotroph (Type 1 GTH cell), and type 2 gonadotroph (Type 2 GTH cell). In addition to the increase in the number of exocytoses of single granules, the simultaneous extrusion of multiple granules, 'multigranular exocytosis', was often observed in all kinds of secretory cells, especially the ACTH-cells. Large numbers of granule cores were often located in large vacuole-like or channel-like structures, irregular in shape and size, which were open to the intercellular or pericapillary space. Some parts of the membrane of the vacuole-like or channel-like structures were coated. These observations are interpreted to suggest that the calcium influx stimulates the extrusion of the secretory granules by single or multigranular exocytosis. [ABSTRACT FROM AUTHOR]

In contrast to insect species, light-activated influx of divalent ions into Limulus ventral photoreceptors has proven difficult to demonstrate. We used the quench of the fluorescent indicator dye, fura-2, to measure Mn2+ influx. Limulus ventral photoreceptors were injected with fura-2 and excited at 360 nm. When the photoreceptors were bathed in 1 mmol · l−1 Mn2+, an approximately 1% per 10 s decline in the fura-2 fluorescence during intervals between 50-ms flashes was taken as a measure of Mn2+ entry in darkness. Fluorescence decline during 10-s flashes was used to monitor Mn2+ entry during the photoresponse. During the 10-s flashes we observed a small rapid decline of the fura-2 fluorescence even in the absence of Mn2+. This reflected a contamination of the fluorescence signal arising from light-induced release of intracellular calcium stores. A subsequent slower decline in fluorescence during the 10-s flash, amounting to approximately 9% per 10 s, was only observed in the presence of extracellular Mn2+ and was attributed to Mn2+ influx. This light-activated influx was not through voltage-gated calcium channels since it persisted under voltage clamp, was not stimulated by depolarizing current injections, nor blocked by NiCl2. Depletion of internal calcium stores by cyclopiazonic acid treatment did not accelerate Mn2+ influx. [ABSTRACT FROM AUTHOR]

The proposal that aluminium (Al) toxicity in plants is caused by either inhibition of Ca influx or by displacement of Ca from the cell wall, was examined. For this study the giant alga Chara corallina Klein ex Will. em. R.D. Wood was selected because it shows a similar sensitivity to Al as in roots of higher plants and, more importantly, it is possible to use the large single internodal cells to make accurate and unambiguous measurements of Ca influx and Ca binding in cell walls. Growth of Chara was inhibited by Al at concentrations comparable to those required to inhibit growth of roots, and with a similar speed of onset and pH dependence. At Al concentrations which inhibited growth, influx of calcium (Ca) was only slightly sensitive to Al. The maximum inhibition of Ca influx at 0.1 mol·m Al at pH 4.4 was less than 50%. At the same concentration, lanthanum (La) inhibited influx of Ca by 90% but inhibition of growth was similar for both La and Al. Removal of Ca from the external solution did not inhibit growth for more than 8 h whereas inhibition of growth by Al was apparent after only 2.5 h. Ca influx was more sensitive to Al when stimulated by addition of high concentrations of potassium (K) or by action potentials generated by electrical stimulation. Other membrane-related activities such as sodium influx, rubidium influx and membrane potential difference and conductance, were not strongly affected by Al even at high concentrations. In isolated cell walls equilibrated in 0.5 mol·m Ca at pH 4.4, 0.1 mol·m Al displaced more than 80% of the bound Ca with a half-time of 25 min. From the poor correlation between inhibition of growth and reduction in Ca influx, it was concluded that Al toxicity was not caused by limitation of the Ca supply. Short-term changes in other membrane-related activities induced by Al also appeared to be too small to explain the toxicity. However the strong displacement, and probable replacement, of cell wall ca by Al may be sufficient to disrupt normal cell development. [ABSTRACT FROM AUTHOR]

This paper deals with the effect of calcium binding in the cell wall on the measured Ca influx in Chara corallina Klein ex Will. esk. R.D. Wood. Calcium in the cell wall was in the range 687-1197 (μmol · m compared to the sap which contained only 144-256 μmol · m. In dilute culture solutions the calcium content of the cell wall was relatively independent of external calcium at concentrations above about 0.1 mol · m. The half-times for exchange of calcium from Ca-labelled cell walls varied from 45 min at 0.05 mol · m to less than 2 min at 2 mol · m. The effectiveness of other cations in displacing calcium from cell walls was in the order La > Zn > Co > Ni > Mg. Rinsing of Ca-labelled cell walls in 2 mol · m LaCl for 20 min removed more than 99% of the bound Ca. However, the residual Ca activity in isolated cell walls following La rinsing was similar to that in whole cells. It is concluded that in whole cells Ca influx cannot normally be distinguished from extracellular binding of calcium. Methods are described for the measurement of Ca fluxes in charophyte cells by isolation of intracellular Ca after the uptake period using techniques which avoid contamination from the large amount of tracer bound in the cell wall. At an external calcium concentration of 1 mol · m, the plasmalemma influx was approx. 0.2 nmol · m · s of which about half entered the vacuole and half was effluxed back into the external solution. The cytoplasm filled with calcium with a half-time of 40-50 min with an 'apparent' pool size of 50 mmol · m. After 2 h the net flux to the cell was almost the same as the vacuolar flux. The fluxes reported are an order of magnitude lower than previously reported calcium fluxes in plants. [ABSTRACT FROM AUTHOR]

GM1 ganglioside was previously shown to function as a specific regulator of excitatory opioid activity in dorsal root ganglion neurons and F11 hybrid cells, as seen in its facilitation of opioid-induced activation of adenylyl cyclase and its ability to dramatically reduce the threshold opioid concentration required to prolong the action potential duration. The elevated levels of GM1 resulting from chronic opioid exposure of F11 cells were postulated to cause the ensuing opioid excitatory supersensitivity. We now show that GM1 promotes opioid (DADLE)-induced activation of adenylyl cyclase in NG108-15 cells which possess the δ-type of receptor. In keeping with previous studies of other systems, this can be envisioned as conformational interaction of GM1 with the receptor that results in uncoupling of the receptor from Gi and facilitated coupling to Gs. This would also account for the observation that DADLE-induced attenuation of forskolin-stimulated adenylyl cyclase was reversed by GM1, provided the cells were not pretreated with pertussis toxin. When the cells were so pretreated, GM1 evoked an unexpected attenuation of forskolin-stimulated adenylyl cyclase attributed to GM1-promoted influx of calcium which was postulated to inhibit a calcium-sensitive form of adenylyl cyclase. This is concordant with several studies showing GM1 to be a potent modulator of calcium flux. Pertussis toxin in these experiments exerted dual effects, one being to promote interaction of the δ-opioid receptor with Gs through inactivation of Gi, and the other to enhance the GM1-promoted influx of calcium by inactivation of Go; the latter is postulated to function as constitutive inhibitor of the relevant calcium channel. NG108-15 cells thus provide an interesting example of competitive interaction between two GM1-regulated systems involving enhancement of both opioid receptor excitatory activity and calcium influx. [ABSTRACT FROM AUTHOR]

The effects of Mg on the glutamate-, kainate-, N-methyl- d-aspartate- and quisqualate-induced influx ofCa were studied in cultured cerebellar granule cells. The N-methyl- d-aspartate- and quisqualate-evoked influx was totally and the kainate- and glutamate-evoked influx partially blocked in 1.3 mM extracellular Mg. The increase in influx induced by kainate, quisqualate and glutamate was maximal at 0.1 mM Mg, whereas N-methyl- d-aspartate was most effective in totally Mg-free media. d-2-Amino-5-phosphonovalerate blocked partially and phencyclidine completely the enhancement of Ca influx by 1 mM quisqualate in 0.1-mM Mg medium. The effect of 10 μM quisqualate was also significantly inhibited by antagonists specific for different glutamate receptor subtypes, including N-methyl- d-aspartate, (RS)α-amino-3-hydroxy-5-methyl-4-isozazolepropionate and metabotropic recptors. This evidences a heterogeneous action of quisqualate, mediated by different glutamate receptor subtypes in 0.1 mM Mg medium. The efficacy of quisqualate in inducing influx of Ca and the selectivity of antagonists for different receptors are also modified by extracellular Mg. [ABSTRACT FROM AUTHOR]

Diabetes-induced changes in the calcium influx and contractile responses of aortic rings to various drugs were investigated in streptozotocin-treated rats. Diabetes is associated with calcium influx into the aortic rings (1.5-and 2.5-fold, respectively, after either KCl or noradrenaline stimulation compared with normal). The maximum KCl-induced contraction of the arorta in diabetic rats was reduced by 38%, but the EC of KCl remained unchanged. The pA of nifedipine for inhibiting the contractile response of aorta to KCl decreased one order of magnitude in the diabetic rats (8.26 vs 9.03 for non-diabetic rats). It is concluded that diabetes reduces the sensitivity of aortic tissue to nifedipine and may affect the stimulation-contraction coupling of vascular smooth muscle in such a way that a higher influx of calcium results after stimulation and that this may be responsible for diabetes-induced vascular complications. [ABSTRACT FROM AUTHOR]

We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca transport pathway and an unsaturable Ca flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289-296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3-5-fold higher Ca influx than control liposomes (without protein). Ca uptake into PrL was dependent on the [protein] in PrL and steady state [Ca] in PrL was in equilibrium with external [Ca]. These data demonstrate that a passive, protein-mediated Ca transport has been reconstituted from BLMV into PrL. Ca influx into liposomes did not saturate with increasing [Ca] in the assay medium. In contrast, PrL displayed saturable Ca influx and exhibited a single Ca flux component with an apparent K=242 ± 50.9 μ m and V=13.5 ± 1.14 nmoles Ca/mg protein/ minute. The K of Ca-transport in PrL was similar to that of the high affinity Ca influx component in BLMV while the V was about 4-fold higher. The unsaturable Ca flux component was not detected in PrL. Ca influx in PrL was inhibited by divalent cations in the order of efficacy, Zn>Mn>Co=Ni, and appeared to be more sensitive to lower concentrations of Zn than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N′-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40-50% decrease in V of Ca transport without an alteration in K. These data strongly suggest that the high affinity, passive Ca transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca influx in the rat parotid gland basolateral plasma membrane. [ABSTRACT FROM AUTHOR]