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.     

Acute60Co-gamma irradiation of rats decreases the inhibitory effect of succinate on the lipid peroxidation of liver mitochondria

Summary Succinate inhibits NADPH-dependent lipid peroxidation of liver mitochondria. This effect of succinate decreased 12 h after whole-body⁶⁰Co-gamma irradiation, depending on the dose of irradiation.     

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.     

Further evidence for the dissociation of digoxin-like immunoreactivity from Na+, K+-ATPase inhibitory activity

Summary The effects of adrenalectomy or nephrectomy, carried out one hour previously, on the levels of endogenous digitalis-like factors were determined in rat plasma. Factors were assayed by digoxin-like immunoreactivity and direct Na⁺, K⁺-ATPase inhibitory activity. Digoxin-like immunoreactivity significantly decreased one hour after bilateral ablation of adrenals, while Na⁺, K⁺-ATPase inhibitory activity remained unaltered. There were no changes in either activity one hour after bilateral nephrectomy. These results suggest that digoxin-like immunoreactivity may be derived from the adrenal gland or under adrenal control and the major substances detected by digoxin-like immunoreactivity and direct Na⁺, K⁺-ATPase inhibitory activity may be different.     

Decreased rates of Ca2+-dependent heat production in slow- and fast-twitch muscles from the dystrophic (mdx) mouse

Using a newly developed microcalorimetric approach to assess the rate of energy expenditure for intracellular [Ca²⁺] homeostasis in isolated muscles at rest, we found this was lower inmdx than in control mouse muscles, by 62% and 29% in soleus and extensor digitorum longus, respectively. Differences in total and Ca²⁺-dependent rates of specific heat production betweenmdx and control were enhanced during sustained, KCl-induced stimulation of energy dissipation. These results suggest that the low sacroplasmic energy status of dystrophic muscles is not due to any excessive energy expenditure for intracellular [Ca²⁺] homeostasis.     

Purification from human plasma of endogenous dodium transport inhibitor(s)

Summary Na⁺, K⁺-ATPase inhibitors extracted from plasma of healthy human subjects displaced³H-ouabain binding to human erythrocytes and inhibited the Na⁺ efflux catalyzed by the Na⁺, K⁺-pump and unexpectedly the Na⁺, K⁺-cotransport system without alteration of the Na⁺, Na⁺-exchange or the Na⁺ passive permeability. This suggests the presence in healthy human plasma of endogenous factors with ouabain-like and furosemide-like activities.Acknowledgments. We are indebted to Dr M. A. Devynck for her advice on chemical measurements and to Dr R. P. Garay for his help with flux measurements     

Altered K+ movement in liver mitochondria from alloxan diabetic rats

Potassium movements were monitored in liver mitochondria from control and alloxan diabetic rats with a cationic electrode. There was net accumulation of K+ after Ca2+ addition to the mitochondria with the diabetic but not with the control.     

The interaction of organic cations with the mitochondrial membrane

Summary Weak and strong organic bases behave in an opposite manner in respect to several mitochondrial functions. The former induce a catalytic exchange with K⁺ in valinomycon-treated, respiratory-inhibited mitochondria, and act as uncouplers in respiring mitochondria. The latter induce a stoicheometric exchange with K⁺ and are actively taken up by respiring mitochondria.     

Peroxovanadate inhibits Ca2+ release from mitochondria

Mitochondria contain a specific Ca²⁺ release pathway which operates when oxidized mitochondrial pyridine nucleotides are hydrolyzed. NAD⁺ hydrolysis and therefore Ca²⁺ release is possible when some vicinal thiols are cross-linked. Here we report that the thiol oxidant peroxovanadate inhibits the specific Ca²⁺ release pathway. In mitochondria, peroxovanadate causes a complete loss of reduced glutathione, which is not accompanied by formation of glutathione disulfide, and a partial loss of protein thiols. In model reactions, peroxovanadate oxidizes reduced glutathione predominantly to the sulfonate derivative, but does not react with glutathione disulfide. When the vicinal thiols relevant for Ca²⁺ release are cross-linked, Ca²⁺ release is no longer inhibited by peroxovanadate. Conversely, pretreatment of mitochondria with peroxovanadate makes them insensitive to compounds promoting the disulfide state. These results suggest that peroxovanadate inhibits the prooxidant-induced Ca²⁺ release from mitochondria by (i) depleting mitochondria of reduced glutathione and (ii) oxidizing the vicinal thiols relevant for Ca²⁺ release to a state higher than disulfide, presumably the sulfonate state. The findings provide further insight into the regulation of Ca²⁺ release from intact mitochondria, and may be relevant for a better understanding of the action of peroxovanadate in cells, where the compound can be insulin mimetic. Received 28 March 2002; received after revision 8 May 2002; accepted 15 May 2002     

The effect of cyclic AMP on Na+ and K+ transport systems in mouse macrophages

Summary Exogenous cyclic AMP (cAMP) inhibits the Na⁺, K⁺-cotransport system and stimulates the Na⁺, K⁺-pump and Na⁺, Ca²⁺ exchange in mouse macrophages. These effects are enhanced by inhibition of phosphodiesterase with methylisobutylxanthine (MIX). MIX alone showed little or no effect. A similar response was observed after stimulation of endogenous production of cAMP by isoproterenol.     

Die Abhängigkeit der Ca++-Aufnahme isolierter Mitochondrien des Herzmuskels von der Na+-und K+-Konzentration als mögliche Ursache der inotropen Digitaliswirkung

Summary In isolated mitochondria of heart muscle from rabbits and oxen there is, under suitable conditions, an accumulation of Ca⁺⁺, which is significantly enhanced by elevating the K⁺/Na⁺ quotient of the incubation medium. K-strophanthine (10^–5–10^–7) does not influence the accumulation of Ca⁺⁺ by the mitochondria of heart muscle. Therefore the intracellular increase in exchangeable Ca⁺⁺ observed after digitalis-glycosides could be explained by a decrease of the intracellular K⁺/Na⁺ quotient, which is caused by inhibition of the membrane ATPase and diminishes the capacity for Ca⁺⁺ accumulation in mitochondria.     

Dual modes of 5-(N-ethyl-N-isopropyl)amiloride modulation of apical dipeptide uptake in the human small intestinal epithelial cell line Caco-2

Selective pharmacological Na⁺/H⁺ exchange (NHE) inhibitors were used to identify functional NHE isoforms in human small intestinal enterocytes (Caco-2) and to distinguish between direct and indirect effects on transport via the intestinal di/tripeptide transporter hPepT1. The relative potencies of these inhibitors to inhibit ²²Na⁺ influx identifies NHE3 and NHE1 as the apical and basolateral NHE isoforms. The Na⁺-dependent (NHE3-sensitive) component of apical dipeptide ([¹⁴C] Gly-Sar) uptake was inhibited by the selective NHE inhibitors with the same order of potency observed for inhibition of apical ²²Na⁺ uptake. However, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) also reduced [¹⁴C]Gly-Sar uptake in the absence of Na⁺ and this inhibition was concentration and pH (maximal at pH 5.5) dependent. NHE3 inhibition by S1611 and S3226 modulates dipeptide uptake indirectly by reducing the transapical driving force (H⁺ electrochemical gradient). EIPA (at 100 μM) has similar effects, but at higher concentrations (>200 μM) also has direct inhibitory effects on hPepT1.Received 28 February 2005; received after revision 20 April 2005; accepted 20 May 2005     

Lack of Na+,K+-ATPase expression in intercalated cells may be compensated by Na+-ATPase: A study on MDCK – C11 cells

The lack of Na⁺,K⁺-ATPase expression in intercalated cells (IC) is an intriguing condition due to its fundamental role in cellular homeostasis. In order to better understand this question we compared the activities of Na⁺,K⁺-ATPase and Na⁺-ATPase in two MDCK cell clones: the C11, with IC characteristics, and the C7, with principal cells (PC) characteristics. The Na⁺,K⁺-ATPase activity found in C11 cells is far lower than in C7 cells and the expression of its β-subunit is similar in both cells. On the other hand, a subset of C11 without α-subunit expression has been found. In C11 cells the Na⁺-ATPase activity is higher than that of the Na⁺,K⁺-ATPase, and it is increased by medium alkalinization, suggesting that it could account for the cellular Na⁺-homeostasis. Although further studies are necessary for a better understanding of these findings, the presence of Na⁺-ATPase may explain the adequate survival of cells that lack Na⁺,K⁺-ATPase. Received 09 July 2008; received after revision 03 August 2008; accepted 12 August 2008     

Suppressed collagenolytic activity in polymorphonuclear leucocytes from diabetic humans

Summary Extracts of polymorphonuclear leucocytes (PMNL) from diabetic human exhibited less collagenolytic activity than extracts from normoglycemic control subjects. Partially purified control extracts produced ^A and ^B collagen breakdown products of the type generated by mammalian collagenase; the diabetic preparation produced decreased amounts of the same products. The diabetic PMNLs may synthesize abnormally low levels of collagenase or contain inactive forms of this enzyme.Supported by a grant (No. DE-03987) from the National Institute of Dental Research (N.I.H.), USA. This study forms part of the Ph.D. thesis of G.A. Nicoll.Acknowledgments. The authors wish to thank Ms Salema Karim and Mr F.R. Singh for excellent technical assistance.     

Regulation of mitochondrial aconitase by phosphorylation in diabetic rat heart

Mitochondrial dysfunction and protein kinase C (PKC) activation are consistently found in diabetic cardiomyopathy but their relationship remains unclear. This study identified mitochondrial aconitase as a downstream target of PKC activation using immunoblotting and mass spectrometry, and then characterized phosphorylation-induced changes in its activity in hearts from type 1 diabetic rats. PKCβ₂ co-immunoprecipitated with phosphorylated aconitase from mitochondria isolated from diabetic hearts. Augmented phosphorylation of mitochondrial aconitase in diabetic hearts was found to be associated with an increase in its reverse activity (isocitrate to aconitate), while the rate of the forward activity was unchanged. Similar results were obtained on phosphorylation of mitochondrial aconitase by PKCβ₂ in vitro. These results demonstrate the regulation of mitochondrial aconitase activity by PKC-dependent phosphorylation. This may influence the activity of the tricarboxylic acid cycle, and contribute to impaired mitochondrial function and energy metabolism in diabetic hearts. Received 31 October 2008; received after revision 17 December 2008; accepted 2 January 2009     

Zinc ions block the second but not the first phasic response to repetitive application of carbachol and histamine in guinea-pig taenia caeci

In the presence of Zn²⁺ (0.3 mM), carbachol (10^–6 M) or histamine (10^–5 M) induced the phasic response in guinea-pig taenia caeci while the tonic response was markedly inhibited. However, when the muscles were kept in Zn²⁺-containing medium following the first stimulation with either carbachol or histamine, neither application of carbachol nor of histamine elicited another phasic contraction. Caffeine (25 mM) did not induce contraction in the presence of Zn²⁺. After the washing out of caffeine in the presence of Zn²⁺, however, the muscle did then develop the phasic response on the application of carbachol or histamine. In conclusion, Zn²⁺ did not affect the carbachol or histamine-induced Ca²⁺ release from the storage sites. However, when Zn²⁺ was continuously present, Ca²⁺ was not supplied to the storage sites. Furthermore, carbachol and histamine mobilized a common cellular Ca²⁺ store, but they activated Ca²⁺ release channels different from the ones activated by caffeine in the Ca²⁺ storage sites.     

Studies of total and protein-bound plasma Mg++ in wild and hatchery-reared coho salmon smolts in freshwater and in seawater

Summary Total plasma Mg⁺⁺ and Ca⁺⁺, Mg⁺⁺ in erythrocytes as well as protein-bound plasma Mg⁺⁺ were investigated in wild and hatchery-reared smolts. The proportion of plasma Mg⁺⁺ which was bound to plasma protein did not change significantly during entry into seawater, even though the in vitro addition of exogenous Mg⁺⁺ to the plasma showed that additional binding was possible.     

Protective effect of Shigyaku-to,a traditional Chinese herbal medicine,on the infection of herpes simplex virus type 1 (HSV-1) in mice

The antiviral activity of Shigyaku-to (TJS-109), a traditional Chinese herbal medicine, was investigated in mice infected with herpes simplex virus type 1 (HSV-1). TJS-109 is a combination of the medicinal plant extracts fromZingiberis siccatum rhizoma,Aconiti tuber andGlycyrrhizae radix in a specific proportion. Mice infected with a 10 LD₅₀ dose of HSV-1 were treated with TJS-109 orally at doses of 1.25 to 20 mg/kg 2 days before, and 1 and 4 days after the infection. The treated groups had 80% (1.25 mg/kg), 40% (5 mg/kg) and 23% (20 mg/kg) mortality rates 25 days after the infection as compared with a 100% mortality rate in control mice treated with saline. When HSV-1 infected mice (recipients) received CD8⁺T cell fractions derived from spleens of mice treated with TJS-109 (donors), 70% of recipients survived, as compared with 0% survivors in the groups of mice treated with saline, B cell fractions, CD4⁺ T cell fractions or macrophage-enriched fractions prepared from the same donors. TJS-109 did not show any virucidal activities against HSV-1 or any virostatic activities on the growth of HSV-1 in Vero cells. These results suggest that TJS-109 protected mice exposed to lethal amounts of HSV-1 through the activation of CD8⁺ T cells.     

Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, ³²P-poly(Glu-Tyr)_4:1 and ³²P-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria.Received 17 May 2004; received after revision 20 July 2004; accepted 26 July 2004     

Chromate reduction inStreptomyces

Summary Streptomyces species 3M grew in peptone yeast extract medium with 1000 g/ml K₂Cr₂O₇. Incubation of the chromate with different cell fractions in the presence of NADH and NADPH resulted in a decrease of Cr⁶⁺ in the reaction mixture. The level of Cr⁶⁺ was reduced by 82.7% by a particulate cell fraction obtained by centrifugation at 105,000×g for 1 h, in the presence of NADH. The reducing enzyme was associated with this cell fraction. The enzyme was constitutive and reduced Cr⁶⁺ to Cr³⁺.