The calcium ATPase of sarcoplasmic reticulum is inhibited by one Ca2+ ion

Inhibition by calcium of the steady-state turnover of the calcium ATPase from sarcoplasmic reticulum of rabbit muscle follows a Hill slope of 0.8 +/- 0.2 (pH 7.0, 0.1 M KCl, varying [Mg2+] and 2 microM A23187 ionophore). It is concluded that dissociation of the two Ca2+ ions from E-P.Ca2 is sequential and that the inhibition arises from the binding of one Ca2+ to A-P.Ca1.     

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.     

The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium

There has been some dispute in the literature as to the sensitivity of the zinc indicator FluoZin-3 to calcium, with suggestions that physiological levels of calcium and magnesium effectively occlude the response of the probe to zinc. In this communication we demonstrate that calcium concentrations as high as 10mM do not prevent FluoZin-3 from detecting zinc elevations as low as 100pM. Moreover, the inclusion of a few muM Ca-EDTA does not prevent FluoZin-3 from responding to increases in zinc concentration but does extend the dynamic range of the probe by reducing contaminating zinc levels and allowing the probe to respond to multiple zinc additions. In addition, we have derived a mathematical model to account for the kinetics of FluoZin-3 response to zinc in the presence of an additional zinc and calcium chelator.     

Growth factor-stimulated protein synthesis is inhibited by sodium orthovanadate.

The study of intracellular signaling pathways has been aided by the use of sodium orthovanadate, a cell-permeable inhibitor of tyrosine phosphatases. However, long-term addition of sodium orthovanadate is often cytotoxic. In this study we demonstrate that the growth factor-mediated increase in the rate of protein synthesis was inhibited by sodium orthovanadate. This effect of sodium orthovanadate was dose-dependent, with an IC50 of 40 microM and maximal inhibition obtained at 100 microM. As a consequence, the fetal bovine serum-mediated induction of the immediate-early genes, c-Fos and MKP-1, at the protein level was inhibited by orthovanadate. Orthovanadate's ability to attenuate protein synthesis was partially reversible, and was no longer evident when the agent was added 6 h after addition of growth factor to cells. Analysis of several elements of signaling pathways which are known to regulate protein synthesis in a positive manner (p42/p44 MAPK, AKT and p70 S6K stimulation, and hyperphosphorylation of PHAS-I) were not inhibited but rather were stimulated by orthovanadate. Thus, sodium orthovanadate is a potent inhibitor of growth factor-stimulated protein synthesis independent of p42/p44 MAPK or PI3K-p70 S6K activation.     

Calcium-dependent proteolysis in neural tissue is activated at physiologic intracellular Ca2+ levels and inhibited by some anticonvulsants

The level of calcium-activated neutral protease (CANP) activity in the brain and nerve cord of the crayfish Procambarus clarkii was assayed by measuring the degradation of casein yellow by tissue homogenates. When care was taken to maintain the ionic strength of all incubation media at 0.15M and to buffer the Ca²⁺activity of the media with 5mM EGTA, CANP was found to be very sensitive to Ca²⁺; maximal activity was achieved at 1 × 10^?3M Ca²⁺, with 50% of this maximum present at the physiologic intracellular Ca²⁺activity of 1 × 10^?7M. We found that the anticonvulsant agent phenytoin was without effect on CANP activity while pentobarbital and a relatively new anticonvulsant agent, valproic acid (an eight-carbon branched fatty acid), significantly inhibited CANP activity in a dose-dependent manner. The inhibitory effect of valproic acid was shared by a straight-chain eight-carbon fatty acid, caprylic acid. These findings demonstrate that inhibition of CANP activity is not limited to agents with a specific molecular structure. They also suggest that CANP plays a role in the normal turnover of proteins that control various cellular functions.     

Identification of the major positional isomer of pegylated interferon alpha-2b

Interferons display a wide range of antiviral, antiproliferative, and immunomodulatory activities on a variety of cell types and have been used to treat many diseases including hairy-cell leukemia and hepatitis B and C and have also been applied to other therapeutic areas. To improve the pharmacological properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with average molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated proteins with differing sites of PEG attachment. To identify the major positional isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were conducted on a subtilisin-digested N-acetylated peptide of the major positional isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-34, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a novel interferon-polymer attachment site as a histidine-linked interferon conjugate. We show that the major component of PEG-IFN is pegylated in the imidazole side chain of histidine-34. Chemical shift data suggest that pegylation occurs mainly at the N(delta)(1) position in the imidazole side chain of this residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the current experimental conditions at pH 6.5 with an electrophilic derivative of PEG, succinimidyl carbonate PEG. The reversibility of the histidine modification was examined. The PEG-imidazole adduct in the intact protein, PEG-IFN(His-34), is labile but much more stable than in the peptide, PEG-IFN(His-34)dig. Apparently, the tertiary structure of the intact protein protects the His(34)-imidazole ring from depegylation.     

Quantitative microchemical imaging of calcium in Na-K pump inhibited heart cells

Quantitative electron probe X-ray imaging techniques have been utilized to determine simultaneously the element content within a single cultured embryonic chick heart cell and its intracellular compartments as well as the average elemental content of several heart cells within a population. These features of microchemical imaging have permitted establishment of data regarding: (1) the heterogeneity of calcium accumulation in mitochondrial, cytoplasmic and nuclear compartments under conditions which elevate total cell calcium without producing irreversible cell injury; and (2) the variability of calcium accumulation from cell to cell within the population sampled. The results indicate that during Na-K pump inhibition (K-free HT-BSS, 10(-4) M ouabain, 60 min) elevation of mitochondrial calcium, measured in situ by electron probe X-ray microanalysis, to levels more than 100 times greater than in the basal state, may not cause irreversible mitochondrial uncoupling and cell death.     

Hormone-stimulated calcium release is inhibited by cytoskeleton-disrupting toxins in AR4-2J cells

We have studied the role of the actin cytoskeleton in bombesin-induced inositol 1,4,5-trisphosphate (IP(3))-production and Ca(2+)release in the pancreatic acinar tumour cell line AR4-2J. Intracellular and extracellular free Ca(2+)concentrations were measured in cell suspensions, using Fura-2. Disruption of the actin cytoskeleton by pretreatment of the cells with latrunculin B (10 microM), cytochalasin D (10 microM) or toxin B from Clostridium difficile (20 ng/ml) for 5-29 h led to inhibition of both, bombesin-stimulated IP(3)-production and Ca(2+)release. The toxins had no effect on binding of bombesin to its receptor, on Ca(2+)uptake into intracellular stores and on resting Ca(2+)levels. Ca(2+)mobilization from intracellular stores, induced by thapsigargin (100 nM) or IP(3)(1 microM) was not impaired by latrunculin B. In latrunculin B-pretreated cells inhibition of both, bombesin-stimulated IP(3)- production and Ca(2+)release was partly suspended in the presence of aluminum fluoride, an activator of G-proteins. Aluminum fluoride had no effect on basal IP(3)and Ca(2+)levels of control and toxin-pretreated cells. We conclude that disruption of the actin cytoskeleton impairs coupling of the bombesin receptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP(3)-production and Ca(2+)release.     

Voltage-activated sodium current is inhibited by capsaicin in rat atrial myocytes

The effects of capsaicin, the active principle of hot pepper genus Capsicum, were studied on voltage-activated, tetrodotoxin-sensitive Na+ currents in isolated rat atrial cells using the patch clamp technique in the whole-cell configuration. 0.4 and 4 microM of capsaicin produced a significant tonic block on voltage-activated Na+ current (I(Na)) evoked by a depolarizing step to -40 mV from a holding potential of -100 mV (49 +/- 7% n = 11, P < 0.05 and 72 +/- 13% n = 4, P < 0.05 respectively). We didn't observe any use-dependent block of capsaicin in our experimental conditions. Capsaicin slowed the time decay of inactivation of I(Na), and increased the time constant of the recovery of inactivation. Capsaicin and tetrodotoxin (TTX) depressed contractility of isolated electrically driven left rat atria, being the depression of maximal velocity of force development (dF/dt(max)) with respect to control values of 19 +/- 3% at 1 microM of capsaicin and 22 +/- 2% at 1 microM of TTX. These results show an inhibitory effect of capsaicin on I(Na) in isolated atrial cells that may modify the electrical and contractile function of the rat heart.     

Synaptosomal calcium influx is activated by sodium fluoride

Neuronal calcium channels can be modulated by changes in membrane potential or by activation of channel-associated receptors. The latter may be modulated by guanine nucleotide binding proteins. NaF, which activates guanine nucleotide binding proteins, caused a large stimulation of 45Ca2+ uptake by synaptosomes prepared from rat brain. Stimulation of 45Ca2+ influx by NaF (i) was apparent in media containing either 5 mM-K+ or 50 mM-K+, (ii) was slower than the fast-phase of voltage-dependent 45Ca2+ influx but continued for a longer period of time than did depolarization-induced 45Ca2+ influx, and (iii) was not mimicked or modified by a number of drugs, including ouabain, dinitrophenol, sodium azide or sodium vanadate. These results are consistent with the hypothesis that NaF activates a guanine nucleotide binding protein associated with receptor-coupled calcium channels, resulting in stimulation of calcium influx.     

Glutathione reductases from a variety of sources are inhibited by physiological levels of glutathione.

1. Glutathione reductase from human platelets, bovine intestinal mucosa, yeast and E. coli were inhibited in vitro by physiological levels of reduced glutathione with IC50s of 6.61 mM, 2.92 mM, 2.40 mM and 12.11 mM, respectively. 2. A steady-state kinetic examination revealed that glutathione inhibited the NADPH oxidation (at constant [glutathione-disulphide]) catalysed by the eucaryotic enzymes uncompetitively, whereas the E. coli enzyme appeared unaffected by glutathione concentrations of up to 10 mM. 3. With respect to glutathione inhibition of glutathione-disulphide reduction (at constant [NADPH]), the human enzyme was inhibited uncompetitively; the bovine and yeast enzymes displayed apparent mixed hyperbolic inhibition; the E. coli enzyme was inhibited competitively.     

The ubiquitin-protein ligase activity of Hdm2 is inhibited by nucleic acids

The proto-oncoprotein Hdm2 is a member of the RING finger-type family of ubiquitin-protein ligases E3. The RING finger domain is assumed to mediate the specific interaction of an E3 with its cognate ubiquitin-conjugating enzyme E2, which catalyzes the covalent attachment of ubiquitin to substrate proteins. In addition, the RING finger domain of Hdm2 is involved in Hdm2 homooligomer formation and has the capacity to bind to RNA in a sequence-specific manner. Here we report that interaction with nucleic acids interferes with both Hdm2/Hdm2 complex formation and auto-ubiquitination of Hdm2 in vitro. Furthermore, although binding of Hdm2 to the tumor suppressor p53 is not inhibited by nucleic acids, Hdm2-mediated ubiquitination of p53 is significantly decreased. Taken together, these results provide the first example of an E3 whose activity can be regulated by direct interaction with nucleic acids.     

The ATP-sensitive potassium channel in pancreatic B-cells is inhibited in physiological bicarbonate buffer

The effects of bicarbonate buffer (HCO3-/CO2) on the activity of the two K+ channels proposed by some to control the pancreatic B-cell membrane response to glucose were studied. Single K+-channel records from membrane patches of cultured B-cells dissociated from adult rat islets exposed to a glucose- and bicarbonate-free medium (Na-Hepes in place of bicarbonate) exhibit the activity of both the ATP-sensitive as well as the [Ca2+]i-activated K+ channels. However, in the presence of bicarbonate-buffered Krebs solution, the activity of the ATP-sensitive K+ channel is inhibited leaving the activity of the K+ channel activated by intracellular [Ca2+]i unaffected. In the absence of bicarbonate (Hepes/NaOH in place of bicarbonate), lowering the external pH from 7.4 to 7.0 also has differential effects on the two K+ channels. While the K+ channel sensitive to ATP is inhibited, the K+ channel activated by a rise in [Ca2+]i is not affected. To determine whether the response of the B-cell in culture to bicarbonate is also present when the B-cell is functioning within the islet syncytium, the effects of bicarbonate removal on membrane potential of B-cells from intact mouse islets were compared. These studies showed that glucose-evoked electrical activity is also blocked in bicarbonate-free Krebs solution. Furthermore, in the absence of bicarbonate and presence of glucose (11 mM), electrical activity was recovered by lowering the pHo from 7.4 to 7.0. The ATP-sensitive K+-channel activity is greatly reduced by physiologically buffered solutions in pancreatic B-cells in culture. The most likely explanation for the bicarbonate effects is that they are mediated by cytosolic pH changes. Removal of bicarbonate (keeping the external pH at 7.4 with Hepes/NaOH as buffer) would increase the pHi. Since the activity of the [Ca2+]i-dependent K+ channels is not affected by the removal of the bicarbonate buffer, our patch-clamp data in cultured B-cells indicate an involvement of [Ca2+]i-activated K+ channels in the control of the membrane potential. For the B-cell in the islet, we propose that the burst pattern of electrical activity (Ca2+ entry) is controlled, at least in part, by the [Ca2+]i-activated K+ channel.     

The Bcl-2 regulator FKBP38-calmodulin-Ca2+ is inhibited by Hsp90

FKBP38 is a negative effector of the anti-apoptotic Bcl-2 protein in neuroblastoma cells. The interaction with Bcl-2 and the enzyme activity of FKBP38 depend on prior binding of calmodulin-Ca(2+) (CaM-Ca(2+)) at high Ca(2+) concentrations. The FKBP38 protein structure contains three tetratricopeptide repeat (TPR) motifs corresponding to the Hsp90 interaction sites of other immunophilins. In this study we show that the TPR domain of FKBP38 interacts with the C-terminal domain of Hsp90, but only if the FKBP38-CaM-Ca(2+) complex is preformed. Hence, FKBP38 is the first example of a TPR-containing immunophilin that interacts cofactor-dependently with Hsp90. In the ternary Hsp90-FKBP38-CaM-Ca(2+) complex the active site of FKBP38 is blocked, thus preventing interactions with Bcl-2. The dual control of the active site cleft of FKBP38 by CaM-Ca(2+) and Hsp90 highlights the importance of the enzyme activity of the FKBP38-CaM-Ca(2+) complex in the regulation of programmed cell death.