Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism.

This study investigated the role of intracellular free Ca2+ concentration ([Ca2+]i) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), caused a time- and concentration-dependent decrease in the viability of MIN6 cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic of apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+]i by other mechanisms did not induce apoptosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellular Ca2+ stores were completely depleted in cells treated by SERCA inhibitors, but not by other agents that increase [Ca2+]i. The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ influx or by clamping [Ca2+]i with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+]i per se. However, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic acid and nordihydroguaiaretic acid partially prevented MIN6 cell apoptosis, while exposure of cells to the product of lipoxygenase, 12-hydroxy-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, inhibition of cyclooxygenase with indomethacin did not abolish thapsigargin-induced apoptosis in MIN6 cells. Our findings indicate that thapsigargin causes apoptosis in MIN6 cells by depleting intracellular Ca2+ stores and leading to release of intermediate metabolites of arachidonic acid metabolism.

Adaptation to hyperglycemia enhances insulin secretion in glucokinase mutant mice.

The present study was undertaken to test the hypothesis that exposure to high glucose concentrations enhances insulin secretion in pancreatic islets from glucokinase-deficient mice. Insulin secretion and intracellular calcium ([Ca2+]i) were measured as the glucose concentration was increased from 2 to 26 mmol/l in islets from heterozygous glucokinase (GK)-deficient mice (GK+/-) and their wild-type littermates (GK+/+). Results obtained in islets incubated in 11.6 or 30 mmol/l glucose for 48-96 h were compared. GK+/- islets that had been incubated in 30 mmol/l glucose showed improved although not normal insulin secretory and [Ca2+]i responses to the standard glucose challenge as well as an enhanced ability to sense small amplitude glucose oscillations. These effects were associated with increased glucokinase activity and protein. In contrast, exposure of GK+/+ islets to 30 mmol/l glucose increased their basal insulin secretion but reduced their incremental secretory responses to glucose and their ability to detect small amplitude glucose oscillations. Thus exposure of GK+/- islets to 30 mmol/l glucose for 48-96 h enhanced their ability to sense and respond to a glucose stimulus, whereas similar exposure of GK+/+ islets induced evidence of beta-cell dysfunction. These findings provide a mechanistic framework for understanding why glucokinase diabetes results in mild hyperglycemia that tends not to increase over time. In addition, the absence of one allele of the glucokinase gene appears to protect against glucose-induced beta-cell dysfunction (glucose toxicity).

Assessment of Fura-2 for measurements of cytosolic free calcium.

Fura-2 has become the most popular fluorescent probe with which to monitor dynamic changes in cytosolic free calcium in intact living cells. In this paper, we describe many of the currently recognized limitations to the use of Fura-2 in living cells and certain approaches which can circumvent some of these problems. Many of these problems are cell type specific, and include: (a) incomplete hydrolysis of Fura-2 acetoxymethyl ester bonds by cytosolic esterases, and the potential presence of either esterase resistant methyl ester complexes on the Fura-2/AM molecule or other as yet unidentified contaminants in commercial preparations of Fura-2/AM; (b) sequestration of Fura-2 in non-cytoplasmic compartments (i.e. cytoplasmic organelles); (c) dye loss (either active or passive) from labeled cells; (d) quenching of Fura-2 fluorescence by heavy metals; (e) photobleaching and photochemical formation of fluorescent non-Ca2+ sensitive Fura-2 species; (f) shifts in the absorption and emission spectra, as well as the Kd for Ca2+ of Fura-2 as a function of either polarity, viscosity, ionic strength or temperature of the probe environment; and (g) accurate calibration of the Fura-2 signal inside cells. Solutions to these problems include: (a) labeling of cells with Fura-2 pentapotassium salt (by scrape loading, microinjection or ATP permeabilization) to circumvent the problems of ester hydrolysis; (b) labeling of cells at low temperatures or after a 4 degrees C pre-chill to prevent intracellular organelle sequestration; (c) performance of experiments at lower than physiological temperatures (i.e. 15-33 degrees C) and use of ratio quantitation to remedy inaccuracies caused by dye leakage; (d) addition of N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to chelate heavy metals; (e) use of low levels of excitation energy and high sensitivity detectors to minimize photobleaching or formation of fluorescent non-Ca2+ sensitive forms of Fura-2; and (f) the use of 340 nm and 365 nm (instead of 340 nm and 380 nm) for ratio imaging, which diminishes the potential contributions of artifacts of polarity, viscosity and ionic strength on calculated calcium concentrations, provides a measure of dye leakage from the cells, rate of Fura-2 photobleaching, and can be used to perform in situ calibration of Fura-2 fluorescence in intact cells; however, use of this wavelength pair diminishes the dynamic range of the ratio and thus makes it more sensitive to noise involved in photon detection. Failure to consider these potential problems may result in erroneous estimates of cytosolic free calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning, expression, and chromosomal mapping of a novel human CC-chemokine receptor (CCR10) that displays high-affinity binding for MCP-1 and MCP-3.

Chemokines mediate their chemotactic, proinflammatory effects by binding to and activating a variety of specific receptors belonging to the G protein-coupled superfamily of seven-transmembrane serpentine receptors. We report the cloning, chromosomal localization, expression, and ligand binding of a novel CC chemokine receptor, CCR10. CCR10 is expressed primarily in placenta and fetal liver, and binds two of the CC chemokines, monocyte chemoattractant protein (MCP)-1 and MCP-3, with highest affinity. The KD for MCP-3 binding was 1 nM, and MCP-1 competed for MCP-3 binding with an IC50 of 1.2 nM. The CC chemokines MCP-4 and RANTES competed for MCP-3 binding with IC50 values of 7.5 and 5.4 nM, respectively. The chromosomal location of CCR10 was determined to coincide with the CC chemokine receptor cluster on chromosome 3 (3p21.31-3p21.32). These results indicate that CCR10 is a novel CC chemokine receptor with a unique expression pattern that would be consistent with a role in placental immunity or hematopoiesis.

Glucagon-like peptide-1 stimulates insulin secretion by a Ca2+-independent mechanism in Zucker diabetic fatty rat islets of Langerhans.

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca2+ concentration ([Ca2+]i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca2+]i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 micromol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca2+]i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca2+ influx through voltage-dependent Ca2+ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca2+-independent signaling pathways.

Deposition pattern of inorganic particles at the alveolar level in the lungs of rats and mice.

The early pathogenesis of particle-induced lung disease is likely to be determined in large part by the initial pattern of dust deposition at the alveolar level. We have studied the deposition pattern of 5 aerosolized dusts (chrysotile and crocidolite asbestos, fiber glass, alpha-quartz, and ash from Mt. St. Helens) in the lungs of rats. Mice were exposed to chrysotile asbestos. Quantitative electron microscopy was carried out on tissues fixed by vascular perfusion. Immediately after a brief exposure, significantly (p less than or equal to 0.01) greater numbers of particulates had deposited on alveolar duct bifurcations when compared with the number of particles on duct surfaces adjacent to the bifurcations. Few particles were counted at midpoints between bifurcations, and particles rarely were observed within alveoli. Our data show that regardless of mineral nature, shape, or concentration, inhaled particulates small enough to pass through the conducting airways are deposited primarily at alveolar duct bifurcations. We proposed that the alveolar deposition patterns observed are the result of air-flow characteristics that cause enhanced deposition of particles at alveolar duct bifurcations intersecting the flow. This is similar to deposition patterns that occur at bifurcations of conducting airways.

Epidermal growth factor and other mitogens induce binding of a protein complex to the c-fos serum response element in human astrocytoma and other cells.

Rapid stimulation of c-fos transcription by many agonists requires the serum response element (SRE), which binds at least two distinct nuclear proteins, p67SRF and p62TCF. Using nuclear protein extracts from 1321-N1 human astrocytoma cells, we investigated ligand-induced changes in binding of these proteins to SRE probes. In these cells c-fos mRNA expression can be induced by epidermal growth factor (EGF) through protein kinase C-independent pathways and by phorbol esters through protein kinase C. We detected two DNA-protein complexes that formed specifically with the SRE (bands 1 and 2). Band 2 formation was increased 4-6 min after stimulation with EGF as well as serum and phorbol esters; this peaked at 10-30 min and returned to basal levels by 60 min. Induction of band 2 formation preceded the onset and peak accumulation of c-fos mRNA (15 and 30 min after EGF stimulation, respectively) and its return to basal levels (by 1-2 h). Band 2 formation was also increased A431 cells stimulated with EGF and in HeLa and Swiss-3T3 cells stimulated with serum. We found that band 1 contained p67SRF bound to the SRE; band 2 contained p67SRF and a second protein. Gel shift analyses using [35S]methionine-labeled p67SRF and nonradioactive DNA probes suggested that hormone treatment most likely modified the second protein component of band 2. Transient transfection of 1321-N1 cells with plasmids containing point mutations that prevented band 2 formation in vitro also abolished induction of c-fos transcription in vivo as assayed by RNase protection analysis. Thus, hormone-stimulated formation of the protein-DNA complex represented by band 2 may be involved in the activation of c-fos transcription.

Purinergic-independent calcium signaling mediates recovery from hepatocellular swelling: implications for volume regulation.

Swelling of hepatocytes and other epithelia activates volume-sensitive ion channels that facilitate fluid and electrolyte efflux to restore cell volume, but the responsible signaling pathways are incompletely defined. Previous work in model HTC rat hepatoma cells has indicated that swelling elicits ATP release, which stimulates P2 receptors and activates Cl(-) channels, and that this mechanism is essential for hepatocellular volume recovery. Since P2 receptors are generally coupled to Ca(2+) signaling pathways, we determined whether hepatocellular swelling affected cytosolic [Ca(2+)], and if this involved a purinergic mechanism. Exposure of HTC cells to hypotonic media evoked an increase in cytosolic [Ca(2+)], which was followed by activation of K(+) and Cl(-) currents. Maneuvers that interfered with swelling-induced increases in cytosolic [Ca(2+)], including extracellular Ca(2+) removal and intracellular Ca(2+) store depletion with thapsigargin, inhibited activation of membrane currents and volume recovery. However, the swelling-induced increases in cytosolic [Ca(2+)] were unaffected by either extracellular ATP depletion with apyrase or blockade of P2 receptors with suramin. These findings indicate that swelling elicits an increase in hepatocellular Ca(2+), which is essential for ion channel activation and volume recovery, but that this increase does not stem from activation of volume-sensitive P2 receptors. Collectively, these observations imply that regulatory responses to hepatocellular swelling involve a dual requirement for a purinergic-independent Ca(2+) signaling cascade and a Ca(2+)-independent purinergic signaling pathway.

Insulin and other growth factors induce binding of the ternary complex and a novel protein complex to the c-fos serum response element.

Rapid, transient induction of c-fos transcription follows treatment of cells with insulin and other growth factors. This is mediated through the serum response element (SRE), which binds the serum response factor (SRF), as well as accessory factors such as p62 ternary complex factor. Using a gel shift assay we found that formation of the ternary complex increased transiently within 2 min of insulin or phorbol ester treatment of several insulin-sensitive cell lines. However, mutations that prevented formation of this ternary complex did not inhibit insulin- or phorbol ester-stimulated induction of c-fos transcription in these cell lines. We also identified a novel SRF-containing multiprotein complex that forms on the SRE within 2 min following insulin, phorbol ester, or other growth factor treatment. Formation of this novel complex, called band 3, occurred rapidly and transiently, with a time course parallel to the induction of c-fos transcription. Band 3 also formed with gamma-actin and zif268/3 SRE probes. Methylation and carboxy ethylation interference analysis, as well as extensive SRE mutagenesis, suggest that only the SRF directly contacts the SRE in forming band 3. Formation of this novel complex appears to involve protein-protein interactions between SRF and other nuclear protein(s) that may play a role in growth factor stimulation of c-fos transcription.

Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells.

Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-derived growth factor and angiotensin II cause increases in cytosolic free calcium by different mechanisms in vascular smooth muscle cells.

Platelet-derived growth factor (PDGF) and angiotensin II (AII) are thought to mediate their biological effects in vascular smooth muscle cells (VSMCs) by causing alterations in cytosolic free calcium ([ Ca2+]i). In this study we examine the pathways by which PDGF and AII alter [Ca2+]i in VSMCs. Addition of PDGF resulted in a rapid, transient, concentration-dependent increase in [Ca2+]i; this rise in [Ca2+]i was blocked completely by preincubation of cells with ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) or CoCl2, by the voltage-sensitive Ca2+-channel antagonists verapamil or nifedipine, by 12-O-tetradecanoylphorbol-13-acetate (TPA), or by pertussis toxin. AII also caused an increase in [Ca2+]i; however, AII-stimulated alterations in [Ca2+]i displayed different kinetics compared with those caused by PDGF. Pretreatment of cells with 8-(diethylamine)-octyl-3,4,5-trimethyoxybenzoate hydrochloride (TMB-8), almost totally inhibited AII-induced increases in [Ca2+]i. EGTA or CoCl2 only slightly diminished AII-stimulated increases in [Ca2+]i. Nifedipine, verapamil, TPA, and pertussis toxin pretreatment were without effect on AII-induced increases in [Ca2+]i. PDGF and AII both stimulated increases in total inositol phosphate accumulation, although the one-half maximal concentration (ED50) for alterations in [Ca2+]i and phosphoinisitide hydrolysis differed by a factor of 10 for PDGF (3 X 10(-10) M for Ca2+ vs. 2.5 X 10(-9) M for phosphoinositide hydrolysis), but they were essentially identical for AII (7.5 X 10(-9) M for Ca2+ vs. 5.0 X 10(-9) M for phosphoinositide hydrolysis). PDGF stimulated mitogenesis (as measured by [3H]-thymidine incorporation into DNA) in VSMCs with an ED50 similar to that for PDGF-induced alterations in phosphoinositide hydrolysis. PDGF-stimulated mitogenesis was blocked by pretreatment of cells with voltage-sensitive Ca2+ channel blockers, TPA, or pertussis toxin. These results suggest that PDGF and AII cause alterations in [Ca2+]i in VSMCs by at least quantitatively distinct mechanisms. PDGF binding activates a pertussis-toxin-sensitive Ca2+ influx into cells via voltage-sensitive Ca2+ channels (blocked by EGTA, verapamil, and nifedipine), as well as stimulating phosphoinositide hydrolysis leading to release of Ca2+ from intracellular stores. AII-induced alterations in [Ca2+]i are mainly the result of phosphoinositide hydrolysis and consequent entry of Ca2+ into the cytoplasm from intracellular stores. Our data also suggest that changes in [Ca2+]i caused by PDGF are required for PDGF-stimulated mitogenesis.

Deposition and translocation of inhaled silica in rats. Quantification of particle distribution, macrophage participation, and function.

Chronic exposure to silica dust causes fibrotic lung disease. Using brief exposures, we have attempted to define the initial patterns of dust deposition, anatomical compartments through which silica is translocated, and the participation of pulmonary macrophages in clearing the inhaled dust. To accomplish this, particle distribution and translocation at the alveolar level were studied in rats exposed to aerosolized alpha-quartz. Animals were exposed to 109 mg. per cu. m. of crystalline silica in inhalation chambers for 3 hours and sacrificed at varying times after exposure. The lungs were fixed by vascular perfusion through the right ventricle and tissue blocks were prepared for transmission and scanning electron microscopy. Lungs of additional animals were lavaged to recover populations of pulmonary macrophages for in vitro studies. Scanning electron microscopy in concert with back-scattered electron imaging showed that 24 hours postexposure there was a significant decrease in the number of silica particles per unit area of alveolar duct surface when compared with lung tissue from animals sacrificed immediately after exposure. Transmission electron microscopy revealed that silica crystals had been translocated to alveolar type I cells, interstitium, and macrophages. The percentage of silica-containing macrophages on alveolar surfaces increased from 36 +/- 2 per cent (mean +/- S.E.) immediately after exposure to 66 +/- 2 per cent during the 24 hours following exposure. This high percentage of macrophage participation was maintained through a 24-day postexposure period and then returned to 25 +/- 2 per cent 42 days after exposure. The percentage of silica-containing macrophages recovered by lavage were remarkably similar to those studied in situ: 24 +/- 4 per cent immediately postexposure, 62 +/- 3 per cent from 12 hours through 24 days, and 28 +/- 4 per cent 42 days postexposure. Although the percentage of macrophages with silica remained steady, the amount of silica per cell decreased during this 12-hour to 24-day period. Metabolic and viability studies of lavaged macrophages in vitro showed no differences between sham and silica-exposed animals. We propose that the events reported here represent normal, steady state clearance of a subpathogenic dose of potentially toxic particulates.

Use of backscattered electron imaging to quantify the distribution of inhaled crystalline silica.

Inhalation of crystalline silica causes fibrotic pulmonary disease. The lung pathology of silicosis is well characterized and predictable, but the initial patterns of particle deposition and translocation are unknown. Scanning electron microscopy and backscattered electron imaging were utilized to quantify silica particle distribution in the distal air spaces of rats following a three-hour exposure to silica dust at a concentration of 100mg/m3. Lungs were perfused through the vasculature with 2% Karnovsky's fixative at a pressure of 15cm of water for 30 minutes. Blocks of tissue were dissected from five predetermined regions of the left lung and critical point dried. Mounted blocks were further dissected to reveal terminal bronchioles and their attached alveolar ducts. The tissue was sputter-coated with gold. Silica particles were visualized on the alveolar duct surfaces, then counted using negative backscattered electron imaging. The precise area of alveolar duct surfaces was calculated by using a standard magnification of 10,000X and a grid of 64cm2 over the viewing cathode ray tube. Thus, quantitation of silica particles could be expressed as number of particles per square micron. Our data show that there were fewer particles on the alveolar duct surfaces and in alveolar spaces of animals 8 hours after exposure when compared with animals 3 hours post-exposure.

Thapsigargin inhibits the glucose-induced decrease of intracellular Ca2+ in mouse islets of Langerhans.

Stimulation of pancreatic islets of Langerhans with glucose results in changes in intracellular Ca2+ concentration ([Ca2+]i). With the use of mouse islets loaded with fura 2, the earliest glucose-induced alteration of [Ca2+]i was a pronounced decline in [Ca2+]i. This effect (phase 0) was evident 1 min after increasing extracellular glucose from 2 to 12 mM and was sustained for 3-5 min. Phase 0 was also observed when glucose was increased from 5 to 12 mM, indicating that it was not an experimental artifact resulting from substrate depletion. The [Ca2+]i-lowering effect of glucose was mimicked by D-glyceraldehyde but not by 2-deoxyglucose, pyruvate, glyburide, or 30 mM extracellular KCl. Mannoheptulose inhibited phase 0, whereas diazoxide, sodium azide, calmidazolium, or increasing extracellular [Ca2+] to 10 mM were all without effect. After the elevation of islet [Ca2+]i with 5 microM glyburide, 12 mM glucose caused a considerable transient decrease in [Ca2+]i. Under similar conditions, 5 mM caffeine attenuated phase 0, whereas 1 microM thapsigargin, a specific inhibitor of the sarcoplasmic and endoplasmic reticulum family of Ca(2+)-adenosinetriphosphatases (SERCA), almost completely inhibited any glucose-induced reduction of [Ca2+]i. These observations suggest that glucose causes an elevation of beta-cell SERCA activity triggered by factors generated during the cytosolic stages of glycolysis.

Voltage-dependent intracellular calcium release from mouse islets stimulated by glucose.

Glucose-activated beta-cell insulin secretion depends upon elevation of intracellular calcium concentration, [Ca2+]i, which is thought to arise from Ca2+ influx through voltage-dependent calcium channels. Using fura-2-loaded mouse islets, we demonstrate, in fact, that the major component of the glucose-activated [Ca2+]i rise represents voltage-dependent intracellular Ca2+ release. Furthermore, the Ca2+ release pool possesses a novel pharmacology in that it is caffeine-sensitive but ryanodine-insensitive. In the absence of external Ca2+, glucose still caused intracellular Ca2+ release, an effect blockable by tetrodotoxin. However, depolarization of the islet with KCl in low Ca(2+)-containing solutions induced intracellular Ca2+ release, which was resistant to tetrodotoxin. We conclude that glucose release of intracellular Ca2+ is dependent upon depolarization alone, possibly through increasing inositol 1,4,5-trisphosphate production.

Activation of potassium and chloride channels by tumor necrosis factor alpha. Role in liver cell death.

Despite abundant evidence for changes in mitochondrial membrane permeability in tumor necrosis factor (TNF)-mediated cell death, the role of plasma membrane ion channels in this process remains unclear. These studies examine the influence of TNF on ion channel opening and death in a model rat liver cell line (HTC). TNF (25 ng/ml) elicited a 2- and 5-fold increase in K(+) and Cl(-) currents, respectively, in HTC cells. These increases occurred within 5-10 min after TNF exposure and were inhibited either by K(+) or Cl(-) substitution or by K(+) channel blockers (Ba(2+), quinine, 0.1 mm each) or Cl(-) channel blockers (10 microm 5-nitro-2-(3-phenylpropylamino)benzoic acid and 0.1 mm N-phenylanthranilic acid), respectively. TNF-mediated increases in K(+) and Cl(-) currents were each inhibited by intracellular Ca(2+) chelation (5 mm EGTA), ATP depletion (4 units/ml apyrase), and the protein kinase C (PKC) inhibitors chelerythrine (10 micrometer) or PKC 19-36 peptide (1 micrometer). In contrast, currents were not attenuated by the calmodulin kinase II 281-309 peptide (10 micrometer), an inhibitor of calmodulin kinase II. In the presence of actinomycin D (1 micrometer), each of the above ion channel blockers significantly delayed the progression to TNF-mediated cell death. Collectively, these data suggest that activation of K(+) and Cl(-) channels is an early response to TNF signaling and that channel opening is Ca(2+)- and PKC-dependent. Our findings further suggest that K(+) and Cl(-) channels participate in pathways leading to TNF-mediated cell death and thus represent potential therapeutic targets to attenuate liver injury from TNF.

Endoplasmic reticulum calcium store regulates membrane potential in mouse islet beta-cells.

Glucose stimulation of islet beta-cell insulin secretion is initiated by membrane depolarization and an elevation in intracellular free calcium concentration ([Ca2+]i) from a combination of influx through depolarization-activated Ca2+ channels and intracellular Ca2+ store release. Prevention of Ca2+ store refilling with thapsigargin produced a sustained depolarization, leading to enhanced Ca2+ influx and an elevation in [Ca2+]i in 12 mM glucose. Depletion of intracellular Ca2+ stores by external EGTA reduced [Ca2+]i and also caused a long-lasting depolarization. In single beta-cells, external EGTA activated an inward current, the voltage range and kinetic properties of which differed from those of voltage-dependent Ca2+ channels. A novel pathway thus exists in beta-cells by which depletion of endoplasmic reticulum Ca2+ stores results in the activation of an inward current that, by inducing depolarization, facilitates Ca2+ influx through voltage-gated Ca2+ channels. The physiological relevance of this pathway in the control of beta-cell function is indicated by the stimulation of insulin secretion by thapsigargin.

Defective glucose-dependent endoplasmic reticulum Ca2+ sequestration in diabetic mouse islets of Langerhans.

Non-insulin-dependent diabetes mellitus (NIDDM) is a metabolic disease associated with abnormal insulin secretion, the underlying mechanisms of which are unknown. Glucose-dependent signal transduction pathways were investigated in pancreatic islets derived from the db/db mouse, an animal model of NIDDM. After stimulation with glucose (4-12 mM), the changes in intracellular Ca2+ concentration ([Ca2+]i) were different; unlike control islets, db/db islets lacked an initial reduction of [Ca2+]i and the subsequent [Ca2+]i oscillations following stimulation with 12 mM glucose. The severity of these defects in Ca2+ signaling correlated with the age-dependent development of hyperglycemia. Similarly defective glucose-induced Ca2+ signaling were reproduced in control islets by pre-exposure to thapsigargin, a selective inhibitor of endoplasmic reticulum (ER) Ca(2+)-ATPase. Estimation of ATPase activities from rates of ATP hydrolysis and by immunoblot hybridization with an antiserum directed against the sarco/endoplasmic reticulum Ca(2+)-ATPase both demonstrated that the ER Ca(2+)-ATPase was almost entirely absent from db/db islets. The effects of inhibition of ER Ca(2+)-ATPase on insulin secretion were also examined; a 4-day exposure of control islets to 1 microM thapsigargin resulted in basal and glucose-stimulated insulin secretion levels similar to those found in db/db islets. These results suggest that aberrant ER Ca2+ sequestration underlies the impaired glucose responses in the db/db mouse and may play a role in defective insulin secretion associated with NIDDM.

Dependence on NADH produced during glycolysis for beta-cell glucose signaling.

An increase in cytosolic ATP following glucose metabolism by pancreatic beta-cells is the key signal initiating insulin secretion by causing blockade of ATP-dependent K+ channels (KATP). This induces membrane depolarization, leading to an elevation in cytosolic Ca2+ ([Ca2+]i) and insulin secretion. In this report we identify the critical metabolic step by which glucose initiates changes in beta-cell KATP channel activity, membrane potential, and [Ca2+]i. The signal stems from the glycolytic production of NADH during the oxidation of glyceraldehyde 3-phosphate, which is subsequently processed into ATP by mitochondria via the operation of discrete shuttle systems.