Effect of ammonium and other ions on the glucose-dependent active transport of l-histidine in slices of rat-brain cortex

1. The influence of cations on the active transport into cells of rat-brain-cortex slices of l-histidine, an amino acid that is not metabolized by this tissue, has been studied. 2. Like other amino acids, l-histidine accumulated in the cells in the presence of glucose in concentrations up to over double that in the incubation medium. 3. The active transport of l-histidine was highest in a medium containing Ca²⁺ (3mm). The addition of K⁺ (27mm) led to a marked decrease in the intracellular concentration of l-histidine, though the oxygen uptake of the slices was higher. 4. The active l-histidine transport was inhibited by NH₄⁺. The inhibitory effect increased with the NH₄⁺ concentration, being about 25% at 8mm, 65% at 20mm, and 90% at 27 and 50mm. The oxygen uptake of the brain slices was depressed by only 25% by the highest NH₄⁺ concentration used, and less by lower concentrations.     

Rapid stimulation by vasopressin, oxytocin and angiotensin II of glycogen degradation in hepatocyte suspensions

1. The hormonal control of glycogen breakdown was studied in hepatocytes isolated from livers of fed rats. 2. Glucose release was stimulated by [8-arginine]vasopressin (10pm–10nm), oxytocin (1nm–1μm), and angiotensin II (1nm–0.1μm). These responses are all at least as sensitive to hormone as is glucose output in the perfused rat liver. 3. The effect of these three hormones on glucose release was critically dependent on extracellular Ca²⁺, unlike that of glucagon. Half-maximal restoration of the vasopressin response occurred if 0.3mm-Ca²⁺ was added back to the incubation medium. 4. Glycogen breakdown was more than sufficient to account for the glucose released into the medium, in the absence or presence of hormones. Lactate release by hepatocytes was not affected by vasopressin, but was inhibited by glucagon. 5. If Ca²⁺ was omitted from the extracellular medium, vasopressin stimulated glycogenolysis, but not glucose release. 6. The phosphorylase a content of hepatocytes was increased by vasopressin, oxytocin and angiotensin II; minimum effective concentrations were 0.1pm, 0.1nm and 10pm respectively. This response was also dependent on Ca²⁺. 7. These results demonstrate that hepatocytes can respond to low concentrations of vasopressin and angiotensin II, i.e. these effects are likely to be relevant in the intact animal. The role of extracellular Ca²⁺ in the effects of these hormones on hepatic glycogenolysis and glucose release is discussed.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Maturation in liver mitochondria of Ruthenium Red-sensitive calcium-ion-transport activity and the influence of glucagon administration in vivo and in utero

The maturation of Ca²⁺ transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca²⁺ transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-P_i in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca²⁺ influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2–3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca²⁺ influx are observed in the presence of 2mm-P_i; 3–5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2–3h post partum they have reached adult values. The inclusion of 12.5μm-MgATP with the P_i stimulates further the initial rate of Ca²⁺ influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50–60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4–5 days before birth retain the accumulated Ca²⁺ for about 50min in the presence of 2mm-P_i. In the period 2 days before birth to birth, this ability is largely lost, but by 2–3h after birth Ca²⁺ retention is similar to that of adult mitochondria. The presence of 12.5μm-MgATP progressively enhances the Ca²⁺ retention time as development proceeds until 2–3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca²⁺ influx assayed in the presence of 2mm-P_i and the time for which mitochondria retain accumulated Ca²⁺ in the presence of 12.5μm-MgATP and 2mm-P_i. Its administration to neonatal animals leads to an increase in mitochondrial Ca²⁺ retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca²⁺ transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca²⁺ reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver.     

The G Protein-coupled Receptor Family C Group 6 Subtype A (GPRC6A) Receptor Is Involved in Amino Acid-induced Glucagon-like Peptide-1 Secretion from GLUTag Cells

Although amino acids are dietary nutrients that evoke the secretion of glucagon-like peptide 1 (GLP-1) from intestinal L cells, the precise molecular mechanism(s) by which amino acids regulate GLP-1 secretion from intestinal L cells remains unknown. Here, we show that the G protein-coupled receptor (GPCR), family C group 6 subtype A (GPRC6A), is involved in amino acid-induced GLP-1 secretion from the intestinal L cell line GLUTag. Application of l-ornithine caused an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in GLUTag cells. Application of a GPRC6A receptor antagonist, a phospholipase C inhibitor, or an IP₃ receptor antagonist significantly suppressed the l-ornithine-induced [Ca²⁺]_i increase. We found that the increase in [Ca²⁺]_i stimulated by l-ornithine correlated with GLP-1 secretion and that l-ornithine stimulation increased exocytosis in a dose-dependent manner. Furthermore, depletion of endogenous GPRC6A by a specific small interfering RNA (siRNA) inhibited the l-ornithine-induced [Ca²⁺]_i increase and GLP-1 secretion. Taken together, these findings suggest that the GPRC6A receptor functions as an amino acid sensor in GLUTag cells that promotes GLP-1 secretion.     

Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury

Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca²⁺ ([Ca²⁺]_i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca²⁺]_i are linked critically by the ATP-driven plasma membrane Ca²⁺-ATPase (PMCA) important for maintaining low resting [Ca²⁺]_i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca²⁺]_i was measured by fura-2 imaging. An in situ [Ca²⁺]_i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca²⁺ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca²⁺ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca²⁺ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca²⁺ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

Phospholipase Cη2 Activation Redirects Vesicle Trafficking by Regulating F-actin

PI(4,5)P₂ localizes to sites of dense core vesicle exocytosis in neuroendocrine cells and is required for Ca²⁺-triggered vesicle exocytosis, but the impact of local PI(4,5)P₂ hydrolysis on exocytosis is poorly understood. Previously, we reported that Ca²⁺-dependent activation of phospholipase Cη2 (PLCη2) catalyzes PI(4,5)P₂ hydrolysis, which affected vesicle exocytosis by regulating the activities of the lipid-dependent priming factors CAPS (also known as CADPS) and ubiquitous Munc13-2 in PC12 cells. Here we describe an additional role for PLCη2 in vesicle exocytosis as a Ca²⁺-dependent regulator of the actin cytoskeleton. Depolarization of neuroendocrine PC12 cells with 56 or 95 mm KCl buffers increased peak Ca²⁺ levels to ∼400 or ∼800 nm, respectively, but elicited similar numbers of vesicle exocytic events. However, 56 mm K⁺ preferentially elicited the exocytosis of plasma membrane-resident vesicles, whereas 95 mm K⁺ preferentially elicited the exocytosis of cytoplasmic vesicles arriving during stimulation. Depolarization with 95 mm K⁺ but not with 56 mm K⁺ activated PLCη2 to catalyze PI(4,5)P₂ hydrolysis. The decrease in PI(4,5)P₂ promoted F-actin disassembly, which increased exocytosis of newly arriving vesicles. Consistent with its role as a Ca²⁺-dependent regulator of the cortical actin cytoskeleton, PLCη2 localized with F-actin filaments. The results highlight the importance of PI(4,5)P₂ for coordinating cytoskeletal dynamics with vesicle exocytosis and reveal a new role for PLCη2 as a Ca²⁺-dependent regulator of F-actin dynamics and vesicle trafficking.     

Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

Hyperinsulinemia (HI) is elevated plasma insulin at basal glucose. Impaired glucose tolerance is associated with HI, although the exact cause and effect relationship remains poorly defined. We tested the hypothesis that HI can result from an intrinsic response of the β-cell to chronic exposure to excess nutrients, involving a shift in the concentration dependence of glucose-stimulated insulin secretion. INS-1 (832/13) cells were cultured in either a physiological (4 mm) or high (11 mm) glucose concentration with or without concomitant exposure to oleate. Isolated rat islets were also cultured with or without oleate. A clear hypersensitivity to submaximal glucose concentrations was evident in INS-1 cells cultured in excess nutrients such that the 25% of maximal (S_0.25) glucose-stimulated insulin secretion was significantly reduced in cells cultured in 11 mm glucose (S_0.25 = 3.5 mm) and 4 mm glucose with oleate (S_0.25 = 4.5 mm) compared with 4 mm glucose alone (S_0.25 = 5.7 mm). The magnitude of the left shift was linearly correlated with intracellular lipid stores in INS-1 cells (r² = 0.97). We observed no significant differences in the dose responses for glucose stimulation of respiration, NAD(P)H autofluorescence, or Ca²⁺ responses between left- and right-shifted β-cells. However, a left shift in the sensitivity of exocytosis to Ca²⁺ was documented in permeabilized INS-1 cells cultured in 11 versus 4 mm glucose (S_0.25 = 1.1 and 1.7 μm, respectively). Our results suggest that the sensitivity of exocytosis to triggering is modulated by a lipid component, the levels of which are influenced by the culture nutrient environment.     

Light and calcium interactions in chlorella inhibited by sodium chloride

Analysis of NaCl toxicity in Chlorella sorokiniana showed decreased growth rates, increased dry weight per cell, increased intracellular Na⁺ and Cl⁻, more total chlorophyll per cell, a decreased chlorophyll a to chlorophyll b ratio, increased rates of O₂ evolution, and decreased rates of CO₂ fixation when the extracellular concentration of NaCl was increased from zero to 0.3 m. Cultures did not grow at concentrations greater than 0.3 m NaCl unless 10 mm calcium salts were present. Inclusion of that concentration of Ca²⁺ extended the tolerance to 0.5 m NaCl before growth stopped. Increasing the light intensity from 1.2 to 9.4 mw/cm² increased growth rates for cultures in 0.10 to 0.45 m NaCl. At 14 mw/cm² added Ca²⁺ reduced growth rates of cultures in 0.3 m NaCl compared to controls without added Ca²⁺. Maximal growth rates for cultures in NaCl media were achieved by addition of 10 mm CaSO₄ and maintenance of the light intensity at 9.4 mw/cm². The maximal growth rate of the organism was 9.6 doublings/day achieved at 2.7 mw/cm² for control cultures. In 0.3 m NaCl the growth rate was 4.3 doublings/day at 2.7 mw/cm² and 8.2 doublings/day at 9.4 mw/cm² with 10 mm CaSO₄ added.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.     

Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina

Tributyltin in the concentration range 1–4μm failed to stimulate Ca²⁺ transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P_i, despite its promotion of a rapid Cl⁻/OH⁻ exchange. When 2mm-P_i was present, concentrations of tributyltin greater than 1μm inhibited the initial rate of Ca²⁺ transport and induced efflux of the ion from the mitochondria in Cl⁻- or NO₃⁻-containing media. Lower concentrations had little effect. Oligomycin added at up to 10μg/mg of mitochondrial protein had no effect on Ca²⁺ transport. By contrast, approx. 0.3μm-tributyltin completely inhibited respiration supported by α-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca²⁺ transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl⁻/OH⁻ exchange or producing an oligomycin-like effect.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

Directional Ca2+ effect on stimulation of mucin secretion from chicken trachea in vitro

Chicken tracheal mucosa in vitro transported and incorporated radioactive precursors into mucins, which were secreted at a steady rate into the tracheal lumen. Secretion of mucins labelled with ³⁵S and ³H after pulse-labelling of the mucosal layer with Na₂³⁵SO₄ and d-[1-³H]glucosamine as precursors was an energy-dependent process, as it was strongly inhibited by the action of respiratory-chain inhibitors, an uncoupler of oxidative phosphorylation, a metabolic blocker and a temperature shift from 41°C to 5°C. On the other hand, both cholinergic and parasympathomimetic agents considerably increased the secretion of dual-radiolabelled mucins when applied on the submucosal side of the trachea. The effect of Ca²⁺ was directional, since only high submucosal (3.6 or 18mm) or low luminal (zero or 0.18mm) Ca²⁺ massively enhanced the secretion of radiolabelled mucin compared with the mucin output measured under physiological Ca²⁺ conditions (1.8mm). Whereas application of ionophore A23187 on either side of the trachea significantly increased mucin output, its presence in the appropriate tracheal compartment and under appropriate Ca²⁺ conditions further accentuated the output of radiolabelled mucins. Addition of acetylcholine under appropriate conditions also had an additive effect on the Ca²⁺-stimulated secretion of mucins. Ca²⁺ stimulation of mucin secretion appears to be dependent on the metabolic integrity of the mucosal cells. Mucins secreted in response to high submucosal and low luminal [Ca²⁺] appear to consist of a number of different types of glycoproteins, as judged from their ion-exchange-chromatographic behaviour.     

Selenium Modulates Oxidative Stress-Induced Cell Apoptosis in Human Myeloid HL-60 Cells Through Regulation of Calcium Release and Caspase-3 and -9 Activities

Selenium is an essential chemopreventive antioxidant element to oxidative stress, although high concentrations of selenium induce toxic and oxidative effects on the human body. However, the mechanisms behind these effects remain elusive. We investigated toxic effects of different selenium concentrations in human promyelocytic leukemia HL-60 cells by evaluating Ca²⁺ mobilization, cell viability and caspase-3 and -9 activities at different sample times. We found the toxic concentration and toxic time of H₂O₂ as 100 μm and 10 h on cell viability in the cells using four different concentrations of H₂O₂ (1 μm–1 mm) and six different incubation times (30 min, 1, 2, 5, 10, 24 h). Then, we found the therapeutic concentration of selenium to be 200 nm by cells incubated in eight different concentrations of selenium (10 nm–1 mm) for 1 h. We measured Ca²⁺ release, cell viability and caspase-3 and -9 activities in cells incubated with high and low selenium concentrations at 30 min and 1, 2, 5, 10 and 24 h. Selenium (200 nm) elicited mild endoplasmic reticulum stress and mediated cell survival by modulating Ca²⁺ release, the caspases and cell apoptosis, whereas selenium concentrations as high as 1 mm induced severe endoplasmic reticulum stress and caused cell death by activating modulating Ca²⁺ release, the caspases and cell apoptosis. In conclusion, these results explained the molecular mechanisms of the chemoprotective effect of different concentrations of selenium on oxidative stress-induced apoptosis.     

On the origin of the bioelectrical potential gverated by the freshwater clam mantle

Mantles from freshwater clams develop potential differences (PD''s) between the two surfaces when they are bathed in vitro with artificial saline solutions. The magnitude and polarity of the PD is dependent on [Ca²⁺] in the solution bathing the mantle''s shell surface. When the solutions are gassed with 5% CO₂ in oxygen, the PD is in the range 25 to 50 mv, shell side positive. It decreases if [Ca²⁺] in the shell solution is elevated. The concentration dependence is logarithmic with a slope of about -27 mv per 10-fold change in [Ca²⁺], slightly less than predicted by the Nernst equation for a membrane acting as a calcium electrode. Analysis of the electrical behavior both in intact mantles and in isolated epithelia indicates that most of the PD develops across the external membranes of epithelial cells on the shell side. There is no evidence that an active calcium transport system is involved in electrogenesis, and a model based on calcium diffusion across a selectively permeable membrane can explain existent data. If CO₂ is absent, the mantle PD is very small (2–10 mv), but still sensitive to change in external [Ca²⁺]. It is proposed that CO₂ alters intracellular pH, thereby changing the equilibrium between a large store of nonionized calcium and [Ca²⁺] in the cells. A role for carbonic anhydrase in the CO₂ effect is suggested by the action of a specific inhibitor of this enzyme. The diffusion model predicts that increasing ionized calcium should increase the PD as is actually observed. Some implications of this system for the physiology of calcium movement in vivo are discussed.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.