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.     

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     

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.     

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.     

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     

The ATP synthase (F0−F1) complex in oxidative phosphorylation

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P_i. The bacterial and mitochondrial H⁺-ATP synthases both consist of a membranous sector, F₀, which forms a H⁺-channel, and an extramembranous sector, F₁, which is responsible for catalysis. When detached from the membrane, the purified F₁ sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H⁺-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

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     

Effect of monensin and diabetes on asialoglycoprotein degradation in rat hepatocytes

We have studied the effects of two modulations — streptozotocin-induced diabetes in vivo, and the presence of the carboxylic proton ionophore monensin in vitro — on the degradation of³H-asialoorosomucoid ligand in isolated rat hepatocytes.The ligand was internalized by means of a synchronous wave procedure. Diabetes was associated with a marked decrease in the amount of total degraded radioactive ligand compared to that in normal cells (3.6% and 37.3% of internalized ligand respectively, at 60 min), together with increased secretion of degradation products into the incubation medium (87% and 46.3% of the total degraded ligand was secreted by diabetic and normal cells, respectively). Monensin induced similar effects in normal cells, but had no apparent effect in diabetic cells.     

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.     

Magnesium enhances human pancreatic elastase digestion of125I-labeled elastin

Summary The effect of some divalent cations, especially Mg⁺⁺, on elastinolysis by porcine or human pancreatic elastase has been determined using¹²⁵Iodine-labeled elastin as substrate. Elastin degradation was significantly increased in the presence of 10^–3 M Mg⁺⁺. If elastin was pre-incubated with 0.5 (w/v) Triton, there was a further increase in elastinolysis to 2.6 times the original rate.     

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.     

Lipid characterization and14C-acetate metabolism in catfish taste epithelium

Summary The catfish,Ictalurus punctatus is an important model system for the study of the biochemical mechanisms of taste reception. A detailed lipid analysis of epithelial tissue from the taste organ (barbel) of the catfish has been performed. Polar lipids account for 62±1% of the total, neutrals for 38±1%. Phosphatidyl-cholines, serines and ethanolamines are the major constitutents of the polar fraction. Plasmalogen concentration is high relative to that of non-neural tissues. [¹⁴C]-Acetate is incorporated into cell lipid fractions after incubation of barbel tissue at 37°C for 60 min. Percentage amounts of most lipids change with time during this in vitro incubation. The phospholipids are the most metabolically active fractions. This work yields information for continuing reconstitution experiments and indicates that the taste epithelium of this important model system is a metabolically active tissue capable of supporting lipid turnover/synthesis.This work was supported in part by NIH Research Grant No. NS-23622, NS-22620, and by the Veterans Administration.     

Na+ mechanism of δ-opioid receptor induced protection from anoxic K+ leakage in the cortex

Activation of δ-opioid receptors (DOR) attenuates anoxic K⁺ leakage and protects cortical neurons from anoxic insults by inhibiting Na⁺ influx. It is unknown, however, which pathway(s) that mediates the Na⁺ influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na⁺ influxes mediated by voltage-gated Na⁺ channels; (2) DOR activation inhibited Na⁺ influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K⁺ derangement; and (3) DOR activation had little effect on Na⁺/Ca²⁺ exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K⁺ derangement by restricting Na⁺ influx mediated by Na⁺ channels and NMDA receptors, and that non-NMDA receptors and Na⁺/Ca²⁺ exchangers, although involved in anoxic K⁺ derangement in certain degrees, are less likely the targets of DOR signal. Received 26 November 2008; received after revision 26 December 2008; accepted 13 January 2009     

Allometry of mammalian cellular oxygen consumption

In the 1930s, Max Kleiber and Samuel Brody established that the interspecies correlation between mammalian body mass and metabolic rate (αM^0.75) cannot be explained (solely) by whole body surface area (αM^0.66) to volume ratios. Metabolic considerations must also be taken into account. Decreases in the proportion of visceral organ mass to whole body mass can account for some of the whole body metabolic differences. However, superimposed upon these anatomical differences, the metabolism of tissues and cells has been demonstrated to decrease with increasing body mass. These decreases in oxygen consumption rates (with increasing body mass) in cells and tissues can be explained by a decrease in ATP turnover and mitochondrial density and an increase in mitochondrial functional efficiency (decrease in proton leak). The majority of the proton leak differences reflect differences in mitochondrial inner membrane surface area. Indeed, liver metabolism correlates directly with liver mitochondrial inner membrane surface area. Apart from being a significant contributor (~25 %) to basal metabolism, mitochondrial proton leak is a major factor determining the differences in basal metabolism between mammals of different body mass. Received 31 May 2000; received after revision 2 October 2000; accepted 14 November 2000     

Dissection of cytotoxic and helper T cell responses

Cytotoxic (CD8⁺) and helper (CD4⁺) T cells play a crucial role in resolving infections by intracellular pathogens. The development of technologies to visualize antigen-specific T cell responses in mice and men over the past decade has allowed a dissection of the formation of adaptive T cell immunity. This review gives a brief overview of the currently used detection techniques and possible future additions. Furthermore, we discuss our current understanding of the formation of antigen-specific T cell responses, with particular attention to the similarities and differences in CD4⁺ and CD8⁺ T cell responses, the functional heterogeneity within responder T cell pools and the regulation of CD8⁺ T cell responses by dendritic cells and CD4⁺ helper T cells. Received 16 June 2005; received after revision 2 August 2005; accepted 15 August 2005     

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.     

Mechanism of age-dependent involution in embryonic chick notochords

To study the possible mechanism of the age-dependent involution of the notochord, isolated mesenchymefree notochords of chick embryos were cultured in vitro and compared with their counterparts in vivo. Two different aspects were evaluated: (1) DNA synthesis measured by [³H]thymidine incorporation and visualized by autoradiography and (2) cell death quantified by counting the number of pyknotic nuclei. The results demonstrate that [³H]thymidine uptake by notochords shows an age-dependent decrease in vitro as well as in vivo. The number of [³H]thymidine-labelled notochord cells, however, is higher in vitro than in vivo. At the same time, there is an age-dependent increase in pyknosis in the notochord in vivo and in vitro. So, during the aging process, the number of both pyknotic nuclei and of [³H]thymidine-labelled nuclei suggest a high turnover of notochord cells in vitro. From these results, we can conclude that the process of involution in aging notochord seems to be controlled by a programmed intrinsic process, which might be influenced partially by the microenvironment in vivo.     

Role of calcium in the histamine release induced by D-galactosamine from rat mast cells

Summary Rat peritoneal mast cells were isolated and purified by differential centrifugation in Ficoll. Cells pooled from three to four rats were suspended at approximately 10⁶ cells/ml in a buffered salt solution and incubated for 1 h at 37°C in 300 l volumes in the absence or presence (9×10^–4 M) of calcium chloride. Addition of D-galactosamine hydrochloride (DGM; 2.8×10^–4 M) caused (in addition to basal release) a mean ±SEM percent histamine release of 15.7±5.2 in the presence of Ca⁺⁺ and 19±4.9 in the absence of Ca⁺⁺ (p>0.05). It is suggested that D-galactosamine does not require extracellular Ca⁺⁺ for the release of histamine from the rat mast cell.A preliminary analysis of these results was presented at the International Symposium on calcium entry blockers and tissue protection, Rome, 15–16 March 1984.     

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⁺.