Cytoplasmic Free Ca2 Research Articles

Apolipoprotein CIII links islet insulin resistance to β-cell failure in diabetes

Insulin resistance and β-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and β-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.

Protein kinase A signaling and calcium ions are major players in PAF mediated toxicity against Aspergillus niger

The Penicillium chrysogenum antifungal protein PAF is toxic against potentially pathogenic Ascomycetes. We used the highly sensitive aequorin-expressing model Aspergillus niger to identify a defined change in cytoplasmic free Ca2+ dynamics in response to PAF. This Ca2+ signature depended on an intact positively charged lysine-rich PAF motif. By combining Ca2+ measurements in A. niger mutants with deregulated cAMP/protein kinase A (PKA) signaling, we proved the interconnection of Ca2+ perturbation and cAMP/PKA signaling in the mechanistic function of PAF. A deep understanding of the mode of action of PAF is an invaluable prerequisite for its future application as new antifungal drug.

6]-shogaol induces Ca²⁺ signals by activating the TRPV1 channels in the rat insulinoma INS-1E cells.

[6]-shogaol is a vanilloid compound present in steamed ginger (Zingiber officinale), a commonly used spice. Pancreatic beta-cells respond to nutrients like glucose, amino acids and fatty acids, by an increase in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺](i)), which mediates diverse cellular processes in these cells. Some vanilloid compounds activate the transient receptor potential vanilloid receptor type 1 (TRPV1) channel. We investigated whether [6]-shogaol could trigger Ca²⁺ signals in the beta-cell. [Ca²⁺](i) was measured from single INS-1E cells by microscope-based fluorometry using fura-2 as the Ca²⁺ indicator. In fura-2 loaded single rat insulinoma INS-1E cells, a widely used model of beta-cell, [6]-shogaol increased [Ca²⁺](i) in a concentration-dependent manner. [Ca²⁺](i) increase by [6]-shogaol was completely blocked when Ca²⁺ was omitted from the extracellular medium. Capsazepine, an inhibitor of the TRPV1 ion channel completely inhibited the [6]-shogaol-induced [Ca²⁺](i) increase. [Ca²⁺](i) increase obtained by 1 µM [6]-shogaol was greater than that obtained by 10 mM glucose. Moreover, a sub-stimulatory concentration of [6]-shogaol (300 nM), significantly enhanced the glucose-induced [Ca²⁺](i) increase in these cells. We conclude that [6]-shogaol induces Ca²⁺ signals in the beta-cell by activating the TRPV1 channels, and it sensitizes the beta-cells to stimulation by glucose.

ARA290 Improves Insulin Release and Glucose Tolerance in Type 2 Diabetic Goto-Kakizaki Rats.

Effects of ARA290 on glucose homeostasis were studied in type 2 diabetic Goto-Kakizaki (GK) rats. In GK rats receiving ARA290 daily for up to 4 wks, plasma glucose concentrations were lower after 3 and 4 wks, and hemoglobin A1c (Hb A1c) was reduced by ~20% without changes in whole body and hepatic insulin sensitivity. Glucose-stimulated insulin secretion was increased in islets from ARA290-treated rats. Additionally, in response to glucose, carbachol and KCl, islet cytoplasmic free Ca2+ concentrations, [Ca2+]i, were higher and the frequency of [Ca2+]i oscillations enhanced compared with placebo. ARA290 also improved stimulus-secretion coupling for glucose in GK rat islets, as shown by an improved glucose oxidation rate, ATP production and acutely enhanced glucose-stimulated insulin secretion. ARA290 also exerted an effect distal to the ATP-sensitive potassium (KATP) channel on the insulin exocytotic pathway, since the insulin response was improved following islet depolarization by KCl when KATP channels were kept open by diazoxide. Finally, inhibition of protein kinase A completely abolished effects of ARA290 on insulin secretion. In conclusion, ARA290 improved glucose tolerance without affecting hematocrit in diabetic GK rats. This effect appears to be due to improved γ-cell glucose metabolism and [Ca2+]i handling, and thereby enhanced glucose-induced insulin release.

Inhibitory motor neurons of the esophageal myenteric plexus are mechanosensitive.

Mechanosensitivity of enteric neurons has been reported in the small intestine and colon, but not in the esophagus. Our earlier in vivo studies show that mechanical stretch of the esophagus in the axial direction induces neurally mediated relaxation of the lower esophageal sphincter, possibly through mechanosensitive motor neurons. However, this novel notion that the motor neurons are mechanosensitive has not been examined in isolated esophageal myenteric motor neurons. The goal of our present study was to examine the mechanosensitivity of esophageal motor neurons in primary culture and elucidate the underlying molecular mechanisms. Immmunocytochemical analysis revealed that >95% cells were positive for the neuronal marker protein gene product 9.5 and that 66% of these cells costained with protein gene product 9.5 and neuronal nitric oxide (NO) synthase. Hypotonic solution induced an increase in the cytoplasm volume in all cells that was independent of extracellular Ca(2+). Hypotonic solution and mechanical stretch induced cytoplasmic free Ca(2+) signaling in ~65% of neurons in the presence, but not absence, of extracellular Ca(2+). Neurons grown on the elastic membrane responded to mechanical stretch by an increase in neuronal size and Ca(2+) signaling simultaneously. Hypotonic stretch-induced cytoplasmic free Ca(2+) signaling was not affected by extracellular Mg(2+), 5-nitro-2-(3-phenylpropylamino)benzoic acid, and nifedipine but was attenuated by 2-aminoethoxydiphenyl borate, Gd(3+), and Grammostola mechanotoxin 4, blockers of the stretch-activated ion channels. In ~57% of the neurons, hypotonic stretch also induced Ca(2+)-dependent cytoplasmic NO production, which was abolished by Grammostola mechanotoxin 4. These results prove that the esophageal inhibitory motor neurons possess a mechanosensitive property and also provide novel insights into the stretch-activated ion channel-Ca(2+)-NO signaling pathway in these neurons.

Enhanced Membrane Permeability of <i>Saccharomyces ce</i><i>revisiae</i> Induced by Dielectric Barrier Discharge Plasma at Atmospheric Pressure

In this study, we investigated the effect of DBD plasma at atmospheric pressure on membrane permeability, potential membrane and cytoplasmic free calcium concentration inSaccharomyces cerevisiae. S. cerevisiaecells were treated with DBD plasma for 0, 1, 2, 3, 4 and 5 min and then subjected to various analyses before the commencement of fermentation or at the middle (9 h) and end (21 h) stages of fermentation. The results show that compared to non-treated cells, cells treated with plasma for 2-5 min show significant increase in membrane potential, while cells treated for 2-4 min show significant increase in membrane permeability prior to fermentation. Plasma-treated cells also show increased cytoplasmic free Ca2+that depends on plasma treatment time, with the highest increase, 35%, observed for 5-min treatment time. However, at the middle (9 h) and end (21 h) stages of fermentation, these changes become insignificant compared to the control.

β-Cell Ca(2+) dynamics and function are compromised in aging.

Defects in pancreatic β-cell function and survival are key components in type 2 diabetes (T2D). An age-dependent deterioration in β-cell function has also been observed, but little is known about the molecular mechanisms behind this phenomenon. Our previous studies indicate that the regulation of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) may be critical and that this is dependent on the proper function of the mitochondria. The [Ca(2+)]i dynamics of the pancreatic β-cell are driven by an interplay between glucose-induced influx of extracellular Ca(2+) via voltage-dependent Ca(2+) channels and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-mediated liberation of Ca(2+) from intracellular stores. Our previous workhas indicated a direct relationship between disruption of Ins(1,4,5)P3-mediated Ca(2+) regulation and loss of β-cell function, including disturbed [Ca(2+)]i dynamics and compromised insulin secretion. To investigate these processes in aging we used three mouse models, a premature aging mitochondrial mutator mouse, a mature aging phenotype (C57BL/6) and an aging-resistant phenotype (129). Our data suggest that age-dependent impairment in mitochondrial function leads to modest changes in [Ca(2+)]i dynamics in mouse β-cells, particularly in the pattern of [Ca(2+)]i oscillations. These changes are driven by modifications in both PLC/Ins(1,4,5)P3-mediated Ca(2+) mobilization from intracellular stores and decreased β-cell Ca(2+) influx over the plasma membrane. Our findings underscore an important concept, namely that even relatively small, time-dependent changes in β-cell signal-transduction result in compromised insulin release and in a diabetic phenotype.

BLX-1002 restores glucose sensitivity and enhances insulin secretion stimulated by GLP-1 and sulfonylurea in type 2 diabetic pancreatic islets.

BLX‐1002 is a novel thiazolidinedione with no peroxisome proliferator‐activated receptor (PPAR) activity that has been shown to improve glycemia in type 2 diabetes without weight gain. We previously found that BLX‐1002 selectively augments glucose‐sensitive (but not basal) insulin secretion in normal mouse β‐cells. We have now extended these observations to other insulin secretagogues and to diabetic rat islets. To this end, dynamics of insulin secretion stimulated by glucose, GLP‐1, and the sulfonylurea tolbutamide were examined in pancreatic islets from nondiabetic Wistar and type 2 diabetic Goto‐Kakizaki rats ex vivo. BLX‐1002 restored normal glucose‐sensitive insulin secretion in otherwise “glucose‐blind” islets from GK rats, but did not affect basal or glucose‐stimulated secretion in normal Wistar rat islets. The stimulatory effect of BLX‐1002 on insulin secretion at high glucose required Ca2+ and involved phosphatidylinositol 3‐kinase (PI3K) activity. Consistent with its effects on insulin secretion, BLX‐1002 also augmented insulin secretion and cytoplasmic‐free Ca2+ concentrations ([Ca2+]i) stimulated by high glucose, GLP‐1, and tolbutamide in islets from GK, but not Wistar, rats. The inactive analog BLX‐1237 had no effects. In conclusion, our findings suggest that BLX‐1002 potentiates insulin secretion by different stimuli in diabetic β‐cells only, in a Ca2+‐dependent manner and involving PI3K.

Calcium involved in the poly(γ-glutamic acid)-mediated promotion of Chinese cabbage nitrogen metabolism

Plant growth can reportedly be promoted by poly(γ-glutamic acid) (γ-PGA). However, the underlying mechanism is unknown. To reveal the mechanism of γ-PGA, we designed an experiment that investigated the effect of γ-PGA on the nitrogen metabolism of Chinese cabbage hydroponic cultured at different calcium (Ca) levels and varied exogenous Ca2+ inhibitors. The results showed that nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase, and glutamate dehydrogenase activities in leaves and roots were obviously enhanced by γ-PGA at the normal Ca2+ level (4.0 mM). Meanwhile, γ-PGA increased the content of total nitrogen, soluble protein, and soluble amino acids in leaves. However, the promotional effect of γ-PGA on fresh weight weakened when Ca2+ was inadequate. Moreover, γ-PGA not only induced the influx of extracellular Ca2+ and Ca2+ in organelles into cytoplasm, but also increased the Ca2+-ATPase level to modify Ca2+ homeostasis in plant cells. In addition, exogenous Ca2+ inhibitors significantly suppressed the γ-PGA-mediated promotion of cytoplasmic free Ca2+ level, calmodulin (CaM) content, GS and glutamate dehydrogenase activities. In summary, γ-PGA accelerated the nitrogen metabolism of plants through the Ca2+/CaM signaling pathway, thereby improving the growth of the plant.

Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator of sarcoplasmic reticulum Ca(2+) release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca(2+) ([Ca(2+)]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70Hz, 350ms tetani given at 2s (high-intensity fatigue, HIF) or 10s (low-intensity fatigue, LIF) intervals, while force and [Ca(2+)]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution by transmission electron microscopy. At fatigue, tetanic [Ca(2+)]i was reduced to 70±4% and 54±4% of the initial in HIF (P<0.01, n=9) and LIF (P<0.01, n=5) fibres, respectively. At fatigue, the mean inter- and intramyofibrillar glycogen content was 60-75% lower than in rested control fibres (P<0.05), whereas subsarcolemmal glycogen was similar to control. Individual fibres showed a good correlation between the fatigue-induced decrease in tetanic [Ca(2+)]i and the reduction in intermyofibrillar (P=0.051) and intramyofibrillar (P=0.0008) glycogen. In conclusion, the fatigue-induced decrease in tetanic [Ca(2+)]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca(2+)]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca(2+) release critically depends on energy supply from the intramyofibrillar glycogen pool.

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure

We demonstrate that ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. We find that Ca2+ is sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca2+ leads to cell contraction. Furthermore, endogenous Ca2+ fluxes correlate with cell contraction in the amnioserosa during closure, whereas the chelation of Ca2+ slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca2+ and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. We identify two channel subunits, ripped pocket and dtrpA1 (TrpA1), that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. Our results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo.

Role of Transient Receptor Potential Melastatin–like Subtype 5 Channel in Insulin Secretion From Rat β-Cells

Several studies have reported that the transient receptor potential melastatin-like subtype 5 (TRPM5) channel, a Ca(2+)-activated monovalent cation channel, is involved in the stimulus-secretion coupling in the mouse pancreatic β-cells. We have studied the role of the TRPM5 channel in regulating insulin secretion and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in the rat β-cells by using triphenylphosphine oxide, a selective inhibitor of the channel. Insulin secretion from islets from Sprague-Dawley rats was measured in batch incubations. Cytoplasmic free Ca(2+) concentration was measured from single β-cells by fura-2-based microfluorometry. Triphenylphosphine oxide did not alter insulin secretion and [Ca(2+)](i) response triggered by KCl or fructose. It inhibited insulin secretion in response to glucose, L-arginine, and glucagon-like peptide 1. It also inhibited glucose-induced insulin secretion by mechanisms that are independent of the adenosine triphosphate-sensitive potassium channels and [Ca(2+)](i) increase. Our results suggest that in the rat islets, TRPM5 is involved in mediating insulin secretion by glucose and l-arginine and in potentiating the glucose-induced insulin secretion by glucagon-like peptide 1.

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure

We demonstrate that ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. We find that Ca(2+) is sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca(2+) leads to cell contraction. Furthermore, endogenous Ca(2+) fluxes correlate with cell contraction in the amnioserosa during closure, whereas the chelation of Ca(2+) slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca(2+) and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. We identify two channel subunits, ripped pocket and dtrpA1 (TrpA1), that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. Our results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo.

Role of store-operated Ca2+ channels in ethanol-induced intracellular Ca2+ increase in HepG2 cells

To investigate the mechanism of ethanol-induced calcium overload in hepatocytes and the related role of store-operated calcium channels (SOCs). HepG2 cells were treated an ethanol concentration gradient with or without intervention treatment with the extracellular calcium chelator EGTA or the SOCs inhibitor 2-aminoethoxydiphenyl borate (2-APB). Effects on cell viability were assessed by the CCK8 assay. Effects on leakage of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by automatic biochemical analyzer measurements of the culture supernatants. Effects on cytoplasmic free Ca2+ concentration ([Ca2+]i) were accessed by detecting fluorescence intensity of the calcium indicator Fluo-3/AM with a flow cytometer. Effects on mRNA and protein expression levels of SOCs, stromal interacting factor 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) were evaluated by qPCR and western blotting. The ethanol treatment produced dose-dependent reduction in cell viability (r = -0.985, P less than 0.01) and increases in leakage of ALT (F = 15.286, P less than 0.01) and AST (F = 39.674, P less than 0.01). Compared to untreated controls, the ethanol treatments of 25, 50, 100, 200 and 400 mM induced significant increases in [Ca2+]i level (1.25+/-0.36, 1.31+/-0.15, 1.41+/-0.18, 2.29+/-0.25, 2.58+/-0.19; F = 15.286, P less than 0.01). Both intervention treatments, EGTA and 2-APB, significantly reduced the 200 mM ethanol treatment-induced [Ca2+]i increase (2.32+/-0.08 reduced to 1.79+/-0.15 (t = 7.201, P less than 0.01) and 1.86+/-0.09 (t = 8.183, P less than 0.01) respectively). EGTA and 2-APB also increased the ethanol-treated cells' viability and reduced the ALT and AST leakage. The 200 mM ethanol treatment stimulated both gene and protein expression of STIM1 and Orai1, and the up-regulation effect lasted at least 72 h after treatment. Ethanol-induced dysregulation of SOCs may be an important molecular mechanism of ethanol-induced [Ca2+]i rise in hepatocytes and the related liver cell injury.

Autocrine/Paracrine Function of Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) for Glucose Homeostasis in Pancreatic β-Cells and Adipocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger for mobilizing Ca(2+) from intracellular stores in various cell types. Extracellular application of NAADP has been shown to elicit intracellular Ca(2+) signals, indicating that it is readily transported into cells. However, little is known about the functional role of this NAADP uptake system. Here, we show that NAADP is effectively transported into selected cell types involved in glucose homeostasis, such as adipocytes and pancreatic β-cells, but not the acinar cells, in a high glucose-dependent manner. NAADP uptake was inhibitable by Ned-19, a NAADP mimic; dipyridamole, a nucleoside inhibitor; or NaN3, a metabolic inhibitor or under Ca(2+)-free conditions. Furthermore, NAADP was found to be released from pancreatic islets upon stimulation by high glucose. Consistently, administration of NAADP to type 2 diabetic mice improved glucose tolerance. We propose that NAADP is functioning as an autocrine/paracrine hormone important in glucose homeostasis. NAADP is thus a potential antidiabetic agent with therapeutic relevance.

Gene Expression Profiles of Peripheral Blood Mononuclear Cells Reveal Transcriptional Signatures as Novel Biomarkers of Cardiac Remodeling in Rats With Aldosteronism and Hypertensive Heart Disease

In searching for a noninvasive surrogate tissue having mimicry with the prooxidant/-proinflammatory hypertensive heart disease (HHD) phenotype, we turned to peripheral blood mononuclear cells (PBMC). We tested whether iterations in [Ca2+]i, [Zn2+]i and oxidative stress in cardiomyocytes and PBMC would complement each other eliciting similar shifts in gene expression profiles in these tissues demonstrable during preclinical (wk 1) and pathologic (wk 4) stages of aldosterone/salt treatment (ALDOST). Inappropriate neurohormonal activation contributes to pathologic remodeling of myocardium in HHD associated with aldosteronism. In rats receiving chronic ALDOST, evidence of reparative fibrosis replacing necrotic cardiomyocytes and coronary vasculopathy appears at wk 4 associated with the induction of oxidative stress by mitochondria that overwhelms endogenous, largely Zn2+-based, antioxidant defenses. Biomarker-guided prediction of risk prior to the appearance of cardiac pathology would prove invaluable. In PBMC and cardiomyocytes, quantitation of cytoplasmic free Ca2+ and Zn2+, H2O2 and 8-iosprostane levels, as well as isolation of RNA and gene expression, together with statistical and clustering analyses, and confirmation of genes by in situ hybridization and RT-PCR, were performed. Compared to controls, at wk 1 and 4 ALDOST, we found comparable: increments in [Ca2+]i, [Zn2+]i and 8-isoprotane coupled to increased H2O2 production in cardiac mitochondria and PBMC, together with the common networks of expression profiles dominated by genes involved in oxidative stress, inflammation and repair. These included three central Ingenuity pathway-linked genes: p38MAPK, a stress-responsive protein; NFκB, a redox-sensitive transcription factor and a proinflammatory cascade it regulates; and TGF-β1, a fibrogenic cytokine involved in tissue repair. Significant overlapping demonstrated in the molecular mimicry of PBMC and cardiomyocytes during preclinical and pathologic stages of ALDOST implicates that transcriptional signatures of PBMC may serve as early noninvasive and novel sentinels predictive of impending pathologic remodeling in HHD.

Effect of temperature decrease on hemocyte apoptosis of the white shrimp Litopenaeus vannamei

This study investigated the effect of continuous temperature decrease on hemocyte apoptosis of the white shrimp Litopenaeus vannamei. In the stress group, water temperature decreased from 26 to 17 °C at a rate of 1 °C/h. Shrimp kept at 26 ± 0.5 °C were used as control group. Total hemocyte count (THC), reactive oxygen species (ROS) production, cytoplasmic free-Ca2+ (CF-Ca2+) concentration, mitochondrial membrane potential (MMP), apoptotic cell ratio, and caspase-3 activity of L. vannamei hemocytes were determined when water temperature decreased to 23, 20, and 17 °C, respectively. Increased ROS production in hemocytes was observed when water temperature decreased to 20 and 17 °C. Decreased THC and cellular MMP, increased CF-Ca2+ concentration, apoptotic cell ratio, and caspase-3 activity were shown when water temperature decreased to 17 °C. These results indicate that water temperature decrease can induce oxidative stress on shrimp hemocytes and then cause mitochondria and caspase-3 mediated hemocyte apoptosis and THC reduction, when water temperature decreased to an unconformable level.

Patch-Clamp Electrophysiology of Intracellular Ca2+ Channels

The modulation of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) is a universal intracellular signaling pathway that regulates numerous cellular physiological processes. Ubiquitous intracellular Ca(2+)-release channels localized to the endoplasmic/sarcoplasmic reticulum-inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels-play a central role in [Ca(2+)]i signaling in all animal cells. Despite their intracellular localization, electrophysiological studies of the single-channel permeation and gating properties of these Ca(2+)-release channels using the powerful patch-clamp approach have been possible by application of this technique to isolated nuclei because the channels are present in membranes of the nuclear envelope. Here we provide a concise description of how nuclear patch-clamp experiments have been used to study single-channel properties of different InsP3R channels in the outer nuclear membrane. We compare this with other methods for studying intracellular Ca(2+) release. We also briefly describe application of the technique to InsP3R channels in the inner nuclear membrane and to channels in the outer nuclear membrane of HEK293 cells expressing recombinant RyR.

RhoGEF-mediated vasoconstriction in hypertension

Arterial hypertension is a common health problem that is a major risk factor for many diseases, including myocardial infarction and stroke.1, 2 The pathogenesis of arterial hypertension and the basic mechanisms of blood pressure control are still insufficiently understood. An increase in the concentration of cytoplasmic free Ca2+ is thought to be the major trigger for contraction in smooth muscle (Figure 1).3 This increase in intracellular Ca2+([Ca2+]i) induces Ca2+ binding to calmodulin (CaM), and the Ca2+–CaM complex activates myosin light chain kinase to phosphorylate the myosin light chain (MLC) of myosin II. The degree of MLC phosphorylation is the determining factor in smooth muscle contraction: MLC phosphorylation promotes smooth muscle contraction, whereas MLC dephosphorylation, following a reduction in [Ca2+]i, results in muscle relaxation.

Metabolome Response to Glucose in the β-Cell Line INS-1 832/13

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is triggered by metabolism of the sugar to increase ATP/ADP ratio that blocks the KATP channel leading to membrane depolarization and insulin exocytosis. Other metabolic pathways believed to augment insulin secretion have yet to be fully elucidated. To study metabolic changes during GSIS, liquid chromatography with mass spectrometry was used to determine levels of 87 metabolites temporally following a change in glucose from 3 to 10 mM glucose and in response to increasing concentrations of glucose in the INS-1 832/13 β-cell line. U-[(13)C]Glucose was used to probe flux in specific metabolic pathways. Results include a rapid increase in ATP/ADP, anaplerotic tricarboxylic acid cycle flux, and increases in the malonyl CoA pathway, support prevailing theories of GSIS. Novel findings include that aspartate used for anaplerosis does not derive from the glucose fuel added to stimulate insulin secretion, glucose flux into glycerol-3-phosphate, and esterification of long chain CoAs resulting in rapid consumption of long chain CoAs and de novo generation of phosphatidic acid and diacylglycerol. Further, novel metabolites with potential roles in GSIS such as 5-aminoimidazole-4-carboxamide ribotide (ZMP), GDP-mannose, and farnesyl pyrophosphate were found to be rapidly altered following glucose exposure.