Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries

The porcine antral follicles, 3–6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca²⁺/Mg²⁺-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r=0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.     

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.     

Symbioramide,a novel Ca2+-ATPase activator from the cultured dinoflagellateSymbiodinium sp.

Summary A novel sphingosine derivative, symbioramide, has been isolated from the laboratory-cultured dinoflagellateSymbiodinium sp. as a sarcoplasmic reticulum (SR) Ca²⁺-ATPase activator, and its structure elucidated to be1 on the basis of spectral and chemical means.Acknowledgments. We thank Ms M. Hamashima and Ms A. Muroyama for their technical assistance.     

Plakorin,a potent Ca2+-ATPase activator from the Okinawan marine spongePlakortis sp.

Summary A new cyclic peroxide, plakorin, which is a potent sarcoplasmic reticulum (SR) Ca^a+-ATPase activator has been isolated from the Okinawan marine spongePlakortis sp., its structure was elucidated on the basis of spectral data.Acknowledgments. We thank Ms M. Hamashima and Ms A. Muroyama for their technical assistance.     

Ca-efflux,from direct membrane injury by CCl4, is elicited by amphiphilic vehicles in vitro

Direct membrane injury by CCl₄, in situations excluding metabolic activation, was evaluated in saponin-permeabilized hepatocytes and in microsomes by measuring immediate Ca²⁺ efflux. A good correlation appears between the Ca²⁺ efflux and the level of CCl₄ in the membrane and also the variations in fluidity. Mixtures of CCl₄ with water-soluble vehicles were used to improve the dispersion of CCl₄ in the medium. The mixtures varied in their ability to elicit the membrane effects of CCl₄. The performance of ethanol and, to a lesser degree, other alcohols, suggests the existence of a water stable structural organization between CCl₄ and these amphiphilic vehicles, facilitating the transfer of CCl₄ to the membrane.     

9-Methyl-7-bromoeudistomin D,a potent inducer of calcium release from sarcoplasmic reticulum of skeletal muscle

Summary The Ca²⁺-releasing action of several derivatives of eudistomin D isolated from a marine tunicate was compared with that of caffeine. It was found that 9-methyl-7-bromoeudistomin D was approximately 1000 times more potent than caffeine in causing Ca²⁺ release from the sarcoplasmic reticulum.The authors thank Ms A. Muroyama of this institute for her technical assistance and Prof. K. L. Rinehart of University of Illinois for his encouragement.     

Sulfhydryl oxidation induces calcium release from fragmented sarcoplasmic reticulum even in the presence of glutathione

Alcian blue and plumbagin induced transient Ca²⁺ release from fragmented sarcoplasmic reticulum. Dithiothreitol (DTT) and glutathione (GSH) partially blocked Ca²⁺ release induced by these oxidizing compounds. Pretreatment of alcian blue and plumbagin with DTT or GSH for more than 1 min was required to abolish the ability of the oxidizing compounds to release Ca²⁺. Mg²⁺ and ruthenium red completely blocked alcian blue-and plumbagin-induced Ca²⁺ release. These results suggest that oxidation of sulfhydryls on Ca²⁺ release channels induces Ca²⁺ release even in the presence of GSH in situ.     

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.     

Calcium binding in chemically skinned fibers of rat myocardium during force development

Summary The amout of bound calcium and force were measured in chemically skinned rat myocardial fibers during isometric contraction at different concentrations of free Ca²⁺. The data obtained suggest that calcium binding by cardiac myofibrils is cooperative and probably depends on mechanical tension.The author expresses his gratitude to Dr V. I. Kapelko for valuable discussion of the experimental results.     

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.     

Calyculin a increases voltage-dependent inward current in,smooth muscle cells isolated from guinea pig taenia coli

The effects of a potent phosphatase inhibitor, calyculin A (CL-A), on inward currents in guinea pig taenia coli smooth muscle cells were examined. CL-A increased the inward current, and this effect of CL-A was inhibited by a protein kinase C inhibitor, H-7, and by nifedipine. Phorbol 12,13-dibutyrate, an activator of protein kinase C, also increased the inward current and this effect was antagonized by H-7. These results suggest that in guinea pig taenia coli smooth muscle cells CL-A may facilitate the opening of thel-type Ca²⁺ channels through the protein kinase C-dependent phosphorylation system.     

Purification and some properties of an amine oxidase from soybean seedlings

Summary An amine oxidase was purified 447-fold from soybean seedlings and some of its properties were investigated. The molecular weight of the enzyme was estimated to be 25,000. It was most active towards putrescine, followed by spermidine and spermine. K_m-values for these substrates were relatively close. The enzyme was strongly inhibited by carbonyl reagents, such as semicarbazide and aminoguanidine.     

Cell-to-cell transmission in the activation of in situ nematocytes in acontia ofCalliactis parasitica

Summary It is reported that Ca²⁺-induced discharge of in situ nematocytes of acontia ofCalliactis parasitica can occur by cell-to-cell transmission along the acontial filament at a speed that averages 9.8·10^–3 cm^–1. The discharge is preceded by protrusion of nematocytes that proceeds along the acontium at a slightly higher speed.     

Sr2+ can become incorporated into an agonist-sensitive,cytoplasmic Ca2+ store in a cell line derived from the equine sweat gland epithelium

We have explored the properties of a Ca²⁺-dependent cell-signalling pathway that becomes active when cultured equine sweat gland cells are stimulated with ATP. The ATP-regulated, Ca²⁺-influx pathway allowed Sr²⁺ to enter the cytoplasm but permitted only a minimal influx of Ba²⁺. Experiments in which cells were repeatedly stimulated with ATP suggested that Sr²⁺, but not Ba²⁺, could become incorporated into the agonist-sensitive, cytoplasmic Ca²⁺ store. Further evidence for this was provided by experiments using ionomycin, a Ca²⁺ ionophore which has no affinity for Sr²⁺.     

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.     

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     

HAb18G/CD147-mediated calcium mobilization and hepatoma metastasis require both C-terminal and N-terminal domains

HAb18G/CD147 is a heavily glycosylated protein containing two immunoglobulin superfamily domains. Our previous studies have indicated that overexpression of HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca²⁺ entry by nitric oxide (NO)/cGMP. In the present study, we investigated the structure-function of HAb18G/CD147 by transfecting truncated HAb18G/CD147 fragments into human 7721 hepatoma cells. The inhibitory effect of HAb18G/CD147 on 8-bromo-cGMP-regulated thapsigargin-induced Ca²⁺ entry was reversed by the expression of either C or N terminus truncated HAb18G/CD147 in T7721C and T7721N cells, respectively. The potential effect of HAb18G/CD147 on metastatic potentials, both adhesion and invasion capacities, of hepatoma cells was abolished in T7721C cells, but not affected in T7721N cells. Release and activation of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were found to be enhanced by the expression of HAb18G/CD147, and this effect was abolished by both truncations. Thapsigargin significantly enhanced release and activation of MMPs (MMP-2 and MMP-9) in non-transfected 7721 cells, and this effect was negatively regulated by SNAP. However, no effects of thapsigargin or SNAP were observed in T7721 cells, and expression of HAb18G/CD147 enhanced secretion and activation of MMPs at a stable and high level. Taken together, these results suggest that both ectodomain and intracellular domains of HAb18G/CD147 are required to mediate the effect of HAb18G/CD147 on the secretion and activation of MMPs and metastasis-related processes in human hepatoma cells by disrupting the regulation of NO/cGMP-sensitive intracellular Ca²⁺ mobilization although each domain may play different roles.Received 1 April 2004; received after revision 15 June 2004; accepted 22 June 2004     

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.