Insulin-like effect of dichloroacetic acid on hexose transport in Swiss 3T3 cells

Hexose transport in Swiss 3T3 cells was increased by treatment with dichloroacetic acid as well as by treatment with insulin. Neither extra- nor intracellular Ca2+ was found to be involved in their stimulatory action. On the other hand, the removal of intracellular Mg2+ resulted in a loss of the stimulation. These results suggest that dichloroacetic acid stimulates the hexose transport in Mg2+-dependent manner, similar to that of insulin.     

Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe

Effects of extracellular magnesium ions ([Mg²⁺]_o ) on intracellular free Mg²⁺ ([Mg²⁺]_i ) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg²⁺ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg²⁺]_o , [Mg²⁺]_i in yeast cells was 0.91±0.08 mM. Elevation of [Mg²⁺]_o to 1.97 mM induced rapid (within 5 min) increments in [Mg²⁺]_i (2.18±0.11 mM). Lowering [Mg²⁺]_o to 0.06 mM, however, exerted no significant effects on [Mg²⁺]_i (0.93±0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg²⁺]_o used, the subcellular distribution of [Mg²⁺]_i remained hetero geneous, i.e. where the sub-plasma membrane region >cytoplasm >nucleus. [Mg²⁺] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg²⁺]_i when placed in 1.97 mM [Mg²⁺]_o . We conclude that [Mg²⁺]_i in fission yeast is maintained at a physiologic level when [Mg²⁺]_o is low, but intracellular free Mg²⁺ rapidly rises when [Mg²⁺]_o is elevated. Like most eukaryotic cells, yeast may have a Mg²⁺ transport system(s) which functions to maintain gradients of Mg²⁺ from the outside to inside the cell and among its subcellular compartments. Received 18 April 1996; received after revision 4 July 1996; accepted 26 July 1996     

TRPM7 is regulated by halides through its kinase domain

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg²⁺), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg²⁺ ([Mg²⁺]_i) and this is facilitated through the ATP-binding site of the channel’s kinase domain. The synergistic block of TRPM7 by chloride and Mg²⁺ is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg²⁺]_i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.     

Effects of Ca2+ and Mg2+ on motility of sea urchin spermatozoa

Summary Swimming speed of sea urchin spermatozoa, measured by a light scattering technique, did not change with 0-20 mM Ca²⁺ in the medium. The speed was maximum at the normal concentration of Mg²⁺ (49 mM) in sea water.Supported by grants-in-aid from the Ministry of Education, Science and Culture, Japan, and a grant from the Ford Foundation.     

Insulin effect in vitro on human erythrocyte plasma membrane

Summary The effect of porcine insulin has been tested in vitro on human erythrocyte plasma membrane (Na⁺–K⁺) and Mg²⁺-ATPase activities as well as on membrane fluidity. The results indicate that the hormonal treatment significantly inhibits (Na⁺–K⁺)-ATPase activity, and at the same time decreases membrane fluidity.This investigation has been supported by Consiglio Nazionale delle Richerche, Rome, Italy.     

Effects of various inhibitors and 2,4-dinitrophenol on adenosine triphosphatase from Malpighian tubules ofLocusta migratoria L

Summary Both Mg²⁺-ATPase and HCO ₃ ^– -stimulated ATPase activity were inhibited by sodium azide and to a lesser extent ethacrynic acid and amiloride. 1 mM DNP stimulated Mg²⁺-ATPase activity by 22% and HCO ₃ ^– -stimulated ATPase activity by 7%.     

The Ca2+-transport ATPases in smooth muscle

Summary A calmodulin stimulated Ca²⁺-transport ATPase which has many of the characteristics of the erythrocyte type Ca²⁺-transport ATPase has been purified from smooth muscle. In particular, the effect of calmodulin on these transport enzymes is mimiced by partial proteolysis and antibodies against erythrocyte Ca²⁺-transport ATPase also bind to the smooth muscle (Ca²⁺+Mg²⁺)ATPase. A correlation between the distribution of the calmodulin stimulated (Ca²⁺+Mg²⁺)ATPase and (Na⁺+K⁺)ATPase activities in smooth muscle membranes separated by density gradient centrifugation suggests a plasmalemmal distribution of this (Ca²⁺+Mg²⁺)ATPase. A phosphoprotein intermediate in smooth muscle which strongly resembles the corresponding phosphoprotein in sarcoplasmic reticulum of skeletal muscle may indicate the presence in smooth muscle of a similar type of Ca²⁺-transport ATPase.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

Summary The role of Ca²⁺ in secretagogue-induced insulin release is documented not only by the measurements of⁴⁵Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca²⁺, [Ca²⁺]_i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca²⁺]_i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca²⁺ homeostasis by organelles from a rat insulinoma, was investigated with a Ca²⁺ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca²⁺ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca²⁺]_i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca²⁺ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Influence of ethanol on calcium,inositol phospholipids and intracellular signalling mechanisms

Summary Studies have implicated Ca⁺⁺ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca⁺⁺ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca⁺⁺/Mg⁺⁺-ATPase, the high affinity membrane Ca⁺⁺ pump.Current research suggests that a subtype of the voltage-dependent Ca⁺⁺ channel, the dihydropyridine-sensitive Ca⁺⁺ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases⁴⁵Ca⁺⁺-influx and [³H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca⁺⁺/Mg⁺⁺-ATPase activity in neuronal membranes. Changes in Ca⁺⁺ channel and Ca⁺⁺/Mg⁺⁺-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca⁺⁺ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca⁺⁺/Mg⁺⁺-ATPase activity.The increased sensitivity of three Ca⁺⁺-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Effect of free fatty acids and cholesterol in vitro on liver plasma membrane-bound enzymes

Summary The effect of cholesterol and fatty acid treatment in vitro was tested on rat liver plasma membrane-bound enzymes and lipid fluidity. The observed alterations of membrane fluidity affect both (Na⁺–K⁺)-ATPase and Mg²⁺-ATPase activities but not 5-nucleotidase; basal adenylate cyclase as well as its hormonal sensitivity were differentially affected by changes of membrane microenvironment.This investigation was partially supported by the Italian National Research Council.     

Possible involvement of protein kinase C in the stimulation of amino acid transport by phorbol ester,platelet-derived growth factor and A23187 in Swiss 3T3 cells

Summary Stimulation of amino acid transport induced by phorbol-12, 13-dibutyrate, platelet-derived growth factor or A23187 was not observed in cells lacking protein kinase C. On the other hand, stimulation of transport by epidermal growth factor or insulin was not affected. These results suggested that the stimulation of amino acid transport is mediated by at least two separate pathways.This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare of Japan.     

Inhibition ofE. coli ATPase activity by a troponin component,TN-I,and by mitochondrial ATPase inhibitor

Summary The enzymic activity of Mg²⁺-or Ca²⁺-stimulated ATPase fromEscherichia coli was inhibited by one of the troponin components, TN-I, and by mitochondrial ATPase inhibitor (F₁-inhibitor). The inhibitory ability of component TN-I against Mg²⁺-stimulated ATPase activity was lost after digestion of component TN-I with trypsin. The Mg²⁺-stimulated ATPase activity inhibited by component TN-I was completely restored by the addition of another troponin component, TN-C.     

IP<Subscript>3</Subscript> receptor signaling and endothelial barrier function

The endothelium, a monolayer of endothelial cells lining vessel walls, maintains tissue-fluid homeostasis by restricting the passage of the plasma proteins and blood cells into the interstitium. The ion Ca²⁺, a ubiquitous secondary messenger, initiates signal transduction events in endothelial cells that is critical to control of vascular tone and endothelial permeability. The ion Ca²⁺ is stored inside the intracellular organelles and released into the cytosol in response to environmental cues. The inositol 1,4,5-trisphosphate (IP₃) messenger facilitates Ca²⁺ release through IP₃ receptors which are Ca²⁺-selective intracellular channels located within the membrane of the endoplasmic reticulum. Binding of IP₃ to the IP₃Rs initiates assembly of IP₃R clusters, a key event responsible for amplification of Ca²⁺ signals in endothelial cells. This review discusses emerging concepts related to architecture and dynamics of IP₃R clusters, and their specific role in propagation of Ca²⁺ signals in endothelial cells.     

Modulation of 3-hydroxy-3-methyl glutaryl CoA reductase by 2,3-diphosphoglyceric acid

Summary Rat liver microsomal 3-hydroxy-3-methylgularyl CoA (HMG-CoA) reductase was activated by 50% at a concentration of 0.4 mM 2,3-diphosphoglyceric acid (DPG) and by 11-fold at 10 mM DPG. DPG also prevented the inactivation of HMG-CoA reductase by ATP and Mg⁺⁺. Rat liver microsomal HMG-CoA reductase prepared in the presence of 1 mM DPG was significantly more active than when prepared in the absence of DPG. Activation of the enzyme by DPG and protection of the enzyme against inhibition by ATP and Mg⁺⁺ by DPG were also observed with solubilized HMG-CoA reductase.This work was supported by Research Award # 697 G2-1 from the American Heart Association, Greater Los Angeles Affiliate, and by grant # 1R01 HL22672 from the National Institutes of Health. We thank M. Brun and M. Curtis for their excellent technical assistance.     

Experiments on the mechanism of the inhibition of mitochondrial Ca2+ transport by La3+ and ruthenium red

Summary The effects of La³⁺ and ruthenium red on the energy-linked uptake of Ca²⁺ mediated by a synthetic neutral Ca²⁺ ionophore have been investigated in rat liver mitochondria. The results indicate that unspecific surface charge effects do not play a major role in the mechanism of inhibition of mitochondrial Ca²⁺ transport by La³⁺ and ruthenium red.Acknowledgments. The authors are indebted to Prof. W. Simon, ETH Zurich, for having provided samples of the synthetic neutral Ca²⁺ ligand, and to M. Mattenberger for the valuable technical assistence. The work was supported by a grant of the Swiss Nationalfonds (grant No. 3.1720.75).     

Effects of Mn2+ and Mg2+ on the pigeon liver pyruvate kinase

Riassunto Mn²⁺ and Mg²⁺ attivano la piruvato cinasi di fegato di piccione in maniera distinta. In presenza di basse concentrationi di fosfoenolpiruvato Mn⁺ é piú efficace di Mg²⁺ ed é attivatore dell'enzima saturato da Mg²⁺. Piruvato cinasi (EC 2.7.1.40).

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This work was supported by a grant from the Consiglio Nazionale delle Ricerche, Roma, Italia.Silvia Baldi is a fellow of the Italian C.N.R.     

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63 Ag8 cells

Summary Using the suspension cell line P3X63 Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (M_r 40 kD) and Semliki Forest virus core protein (M_r 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0°C. Subsequent resealing of the pores was completed after a 5-min incubation at 37°C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37°C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K⁺, Mg²⁺, and Ca²⁺) and resealing (in the presence of K⁺, Mg²⁺, and Ca²⁺), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion

The role of protein kinase C and Ca²⁺ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca²⁺, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca²⁺ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca²⁺. Increasing the Ca²⁺ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca²⁺ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

The adhesion receptor GPR56 is activated by extracellular matrix collagen III to improve β-cell function

Aims

G-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on β-cell function.

Methods

GPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on β-cell proliferation, apoptosis, intracellular calcium ([Ca²⁺]_i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56^?/? mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56^?/? mice.

Results

Immunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet β-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected β-cells from cytokine-induced apoptosis, triggered increases in [Ca²⁺]_i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56^?/? islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation.

Conclusion

We have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.

     

Glutathione plays different roles in the induction of the cytotoxic effects of inorganic and organic arsenic compounds in cultured BALB/c 3T3 cells

The cytotoxicity of arsenic compounds towards BALB/c 3T3 cells in culture was investigated, together with the role of glutathione (GSH) in the induction of the cytotoxic effects. The rank order of cytotoxicity was as follows: arsenite (As³⁺)>arsenate (As⁵⁺)>dimethylarsinic acid (DMAA)>methylarsonic acid (MAA)>trimethylarsine oxide (TMAO). Arsenobetaine, arsenocholine and the tetramethylarsonium ion were less toxic. Depletion of GSH enhanced the cytotoxic effects of As³⁺, As⁵⁺, MAA and TMAO, while the cytotoxicity of DMAA was markedly reduced by depletion of GSH. These results suggest that GSH plays a role in protecting the cells against the toxic effects of As³⁺, As⁵⁺, MAA and TMAO while it is involved in the induction of the cytotoxic effects of DMAA.