Capacitative calcium entry mechanism in porcine oocytes

The presence of the capacitative Ca(2+) entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca(2+)-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca(2+) entry. A similar divalent cation influx could also be detected with the Mn(2+)-quench technique after inositol 1,4,5-triphosphate-induced Ca(2+) release. In both cases, lanthanum, the Ca(2+) permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca(2+) influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca(2+) entry mechanism might help in refilling the intracellular stores after the release of Ca(2+) from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca(2+) entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca(2+) entry mechanism and thus contributes to Ca(2+) influx.     

Divalent cation influx and calcium homeostasis in germinal vesicle mouse oocytes

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca²⁺) influx and spontaneous Ca²⁺ oscillations. The oscillations cease during maturation but Ca²⁺ influx continues, as the oocytes’ internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca²⁺ influx has not been completely determined. GV and matured oocytes are known to express three Ca²⁺ channels, Ca_V3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca²⁺ homeostasis, suggesting a complex regulation of Ca²⁺ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca_V3.2 and TRPM7 channels contributed the majority of Ca²⁺ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca²⁺ entry. Sr²⁺ influx was promoted by Ca_V3.2 channels, as Sr²⁺ oscillations were negligible in Ca_V3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn²⁺ entry relied on expression of Ca_V3.2 and TRPM7 channels, but Ni²⁺ entry depended on the latter. Ca_V3.2 and TRPV3 channels combined to fill the Ca²⁺ stores, although Ca_V3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca²⁺ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies.     

The plasma membrane potential of human neutrophils. Role of ion channels and the sodium/potassium pump

Calcium-depleted human neutrophils are depolarised when suspended in calcium-free media containing sodium ions, and are repolarised by extracellular replenishment of Ca2+. The depolarisation is due to a high inward sodium current, which is blocked by calcium and by several other divalent cations, but not by barium. Addition of calcium results in a rise in the cytosolic concentration from approx. 20 nM to the resting level of approx. 130 nM. Calcium influx is strongly accelerated by a voltage-gated calcium channel. This channel might be responsible for the depolarising Na+ current in the absence of divalent cations. In the polarised state the neutrophil membrane has a high intrinsic permeability to K+, which may be low or absent in the depolarised state. Generation of membrane potential from the depolarised state is mainly due to the electrogenic sodium/potassium pump. However, the resting potential of about -75 mV is maintained primarily by the K+ conductance, and only to a small extent by the sodium/potassium pump.     

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.     

Barium permeability of neuronal nicotinic receptor alpha 7 expressed in Xenopus oocytes.

The rat alpha 7 neuronal nicotinic acetylcholine receptor was expressed and studied in Xenopus oocytes. The magnitude and reversal potential of instantaneous whole cell currents were examined in solutions containing varying concentrations of either calcium or barium, and in the presence or absence of the intracellular calcium chelator BAPTA. In external barium, application of nicotine elicits an inwardly rectifying response; in calcium the response is larger and has a linear IV relation. Pretreatment of oocytes with BAPTA-AM could not prevent activation of calcium-dependent chloride channels in external Ringer containing calcium. Using an extended GHK equation, the permeability ratio PBa/PNa of the alpha 7 receptor was determined to be about 17. Our results suggest that alpha 7 nicotinic receptors are highly permeable to divalent cations.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Protective effects of manganese, cobalt, nickel, and barium against a calcium paradox in the isolated frog heart

The effect of inorganic slow channel blockers on the calcium paradox in the frog heart was examined. Addition of the divalent cations of manganese, cobalt, nickel, or barium during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that in the control paradox in the absence of divalent cations, there is an efflux of calcium from myocardial cells during calcium depletion and a massive influx of calcium during the following reperfusion, leading to a calcium overload. Divalent cations protected frog myocardial cells, when present in the calcium-free perfusion medium, by reducing both calcium efflux during calcium depletion and the massive calcium influx during reperfusion. The effectiveness of the added divalent cations showed a strong dependence upon their ionic radius. The most potent inhibitors of the calcium paradox in the frog heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanism involved in the protective effect of manganese, cobalt, nickel, and barium.     

Target-cell contact activates a highly selective capacitative calcium entry pathway in cytotoxic T lymphocytes

Calcium influx is critical for T cell activation. Evidence has been presented that T cell receptor-stimulated calcium influx in helper T lymphocytes occurs via channels activated as a consequence of depletion of intracellular calcium stores, a mechanism known as capacitative Ca(2+) entry (CCE). However, two key questions have not been addressed. First, the mechanism of calcium influx in cytotoxic T cells has not been examined. While the T cell receptor-mediated early signals in helper and cytotoxic T cells are similar, the physiology of the cells is strikingly different, raising the possibility that the mechanism of calcium influx is also different. Second, contact of T cells with antigen-presenting cells or targets involves a host of intercellular interactions in addition to those between antigen-MHC and the T cell receptor. The possibility that calcium influx pathways in addition to those activated via the T cell receptor may be activated by contact with relevant cells has not been addressed. We have used imaging techniques to show that target-cell-stimulated calcium influx in CTLs occurs primarily through CCE. We investigated the permeability of the CTL influx pathway for divalent cations, and compared it to the permeability of CCE in Jurkat human leukemic T cells. CCE in CTLs shows a similar ability to discriminate between calcium, barium, and strontium as CCE in Jurkat human leukemic T lymphocytes, where CCE is likely to mediated by Ca(2+) release-activated Ca(2+) current (CRAC) channels, suggesting that CRAC channels also underlie CCE in CTLs. These results are the first determination of the mechanism of calcium influx in cytotoxic T cells and the first demonstration that cell contact-mediated calcium signals in T cells occur via depletion-activated channels.     

The Staphylococcal Pore-forming Leukotoxins Open Ca2+ Channels in the Membrane of Human Polymorphonuclear Neutrophils

The ability of leukotoxins secreted by Staphylococcus aureus to modify the permeability of the membrane of human polymorphonuclear neutrophils has been studied by spectrofluorometry and appropriate fluorescent probes. This family of bicomponent leukotoxins is constituted by, at least, three pairs of proteins: LukS-PV/LukF-PV, HlgA/HlgB, HlgC/HlgB. After binding of both components to the membrane, each pair induces influxes of divalent cations and ethidium in polymorphonuclear neutrophils, although with different intensities. The influx of divalent cations appears sooner than the influx of ethidium. The pathway for divalent cations is not permeable to monovalent cations (Na⁺, K⁺, ethidium⁺) and is blocked by Ca²⁺ channel inhibitors that do not block the fluxes of ethidium and monovalent cations. It is concluded that the leukotoxins bind to a receptor linked to a divalent cation-selective channel or to the channel itself which is activated. Then, the leukotoxins open a second pathway by insertion into the membrane and subsequent formation of aspecific pores allowing an influx of ethidium. Received: 8 May 1997/Revised: 22 December 1997     

Somatostatin: a metabolic regulator

Somatostatin, the hypothalamic release-inhibiting factor, has been found to stimulate gluconeogenesis in rat kidney cortical slices. Stimulation by somatostatin was linear and dose-dependent. Other bioactive peptides such as cholecystokinin, gastrointestinal peptide, secretin, neurotensin, vasoactive intestinal peptide, pancreatic polypeptide, beta endorphin and substance P did not affect the renal gluconeogenic activity. Somatostatin-induced gluconeogenesis was blocked by phentolamine (alpha adrenergic antagonist) and prazosin (alpha1 adrenergic antagonist) but not by propranolol (beta adrenergic antagonist) and yohimbine (alpha2 adrenergic antagonist) suggesting that the effect is via alpha1 adrenergic stimuli. Studies on the involvement of Ca2+ revealed that tissue depletion and omission of Ca2+ from the reaction mixture would abolish the stimulatory effect of somatostatin. Furthermore, somatostatin enhanced the uptake of 45calcium in renal cortical slices which could be blocked by lanthanum, an inhibitor of Ca2+ influx. It is proposed that the stimulatory effect of somatostatin on renal gluconeogenesis is mediated by alpha1 adrenergic receptors, or those which functionally resemble alpha1 receptors and that the increased influx of Ca2+ may be the causative factor for carrying out the stimulus.     

Influx of calcium, strontium, and barium in presynaptic nerve endings

Depolarization-induced (potassium-stimulated) influx of 45Ca, 85Sr, and 133Ba was measured in synaptosomes prepared from rat brain. There are two phases of divalent cation entry, "fast" and "slow;" each phase is mediated by channels with distinctive characteristics. The fast channels inactivate (within 1 s) and are blocked by low concentrations (less than 1 micro M) of La. The slow channels do not inactivate (within 10 s), and are blocked by high concentrations (greater than 50 micro M) of La. Divalent cation influx through both channels saturates with increasing concentrations of permeant divalent cation; in addition, each permeant divalent cation species competitively blocks the influx of other permeant species. These results are consistent with the presence of "binding sites" for divalent cations in the fast and slow channels. The Ca:Sr:Ba permeability ratio, determined by measuring the influx of all three species in triple-label experiments, was 6:3:2 for the fast channel and 6:3:1 for the slow channel. A simple model for ion selectivity, based on the presence of a binding site in the channel, could account well for slow and, to some extent, for fast, channel selectivity data.     

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.     

A calcium requirement for electric field-induced cell shape changes and preferential orientation

C3H/10T1/2 mouse embryo fibroblasts stimulated by a steady electric field (10 V/cm) for 30 min exhibited lamellar retraction on the sides facing the electrodes. Some cells elongated and preferentially oriented with their long axis perpendicular to the field direction. Depletion of external calcium or blockage of calcium influx with lanthanum or the calcium channel blocker D-600 resulted in a reduction of the field-induced response. When external calcium was elevated stepwise from 0 to 10 mM, the field-induced response increased correspondingly. Electric stimulation in the presence of the calcium ionophore A23187 resulted in an increase of spindle-shaped cells with no preferential orientation. This response was blocked by calcium depletion and lanthanum, but not by D-600. The anticalmodulin drug W-13 inhibited the field-induced responses observed in normal buffer as well as in the presence of A23187. Some cell death resulted from prolonged electric field exposure, and the mortality was reduced by calcium depletion, lanthanum or D-600, but was not affected by W-13. We postulate that local calcium influx through channels opened by the electric field produces areas of high intracellular calcium which stimulate the cytoskeletal network to induce lamellar retraction. Prolonged field-induced calcium influx may eventually overcome the cell's mitochondrial calcium-buffer system, leading to necrotic calcification.     

Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na(+)-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (<60 μM), but not at normal calcium (1.2 mM); this mutual inhibition is consistent with dicarboxylate transport. The inhibition varied progressively at intermediate levels of extracellular calcium. In addition to changing the relative magnitude and interaction of citrate and succinate transport, decreasing calcium also increased the affinity of the transport process for various other dicarboxylates. Also, the affinity for succinate, at low concentrations of substrate, was increased by calcium removal. In contrast, in oocytes expressing NaDC1, calcium did not have a similar effect on transport, indicating that NaDC1 could not likely account for the findings in OK cells. In summary, extracellular calcium regulates constitutive citrate and succinate transport in OK proximal tubule cells, probably via a novel transport process that is not NaDC1. The calcium effect on citrate transport parallels in vivo studies that demonstrate the regulation of urinary citrate excretion with urinary calcium excretion, a process that may be important in decreasing urinary calcium stone formation.     

Passive calcium influx by plasma membrane vesicles isolated from rat liver

Passive Ca2+ influx independent of ATP addition to the incubation medium, took place in plasma membrane vesicles isolated from rat liver. The rate of Ca2+ influx was found to depend on the concentration of added Ca2+, and on the incubation temperature, and was inhibited by La3+, Hg2+ and by p-chloromercuribenzoate. Influx was not blocked by calcium channel blockers, or affected by a range of uncouplers. Addition of the Ca2+ ionophore A23187 to vesicles that had taken up the ion induced a rapid efflux of Ca2+ especially when EGTA also was added to the incubation medium. A number of divalent cations inhibited Ca2+ influx. The vesicles could be frozen and stored overnight with little loss in activity. The kinetics of Ca2+ influx could be related to that which occurs in the unstimulated perfused rat liver. The data suggest that the plasma membrane vesicle preparation may be useful for further studies on the basal liver cell Ca2+ influx system in vitro.     

Transient potassium current in native Xenopus oocytes

Depolarization of follicle-enclosed oocytes of Xenopus laevis obtained from some donors elicits, in addition to other responses, a fast transient outward current. After holding the membrane potential at -100 mV this response begins to be activated by depolarizations to around -30 mV, and increases progressively as the voltage is raised further. A striking characteristic is that the current recovers only slowly (several seconds) from inactivation following a depolarizing pulse. Because of its outward direction and insensitivity to removal of extracellular chloride or addition of tetrodotoxin, the current probably arises largely through a flux of potassium ions. The current was abolished after treatment of oocytes with collagenase to remove enveloping cells, and although it was blocked by barium and zinc ions, tetraethylammonium was relatively ineffective. In addition, the potassium current was unaffected by 5 mM manganese, suggesting that it does not arise as a consequence of an influx of calcium into the oocyte.     

Role of intracellular calcium in Pasteurella haemolytica leukotoxin-induced bovine neutrophil leukotriene B4 production and plasma membrane damage

Isolated neutrophils were used to study the intracellular calcium ([Ca2+]i) dependency of Pasteurella haemolytica leukotoxin-induced production of leukotriene B4 and plasma membrane damage. Exposure of neutrophils to leukotoxin caused a rapid and concentration-dependent increase in [Ca2+]i, followed by simultaneous plasma membrane damage and production of leukotriene B4. Removal of extracellular Ca2+, replacement of Ca2+ with other divalent cations, or exposure to high concentration of verapamil, an inhibitor of voltage-dependent calcium channels, inhibited leukotoxin-induced increases in [Ca2+]i, leukotriene B4 production, and membrane damage, thus indicating that influx of extracellular Ca2+ is necessary to produce these leukotoxin-induced neutrophil responses.     

Monovalent cation conductance in Xenopus laevis oocytes expressing hCAT-3

hCAT-3 (human cationic amino acid transporter type three) was investigated with both the two-electrode voltage clamp method and tracer experiments. Oocytes expressing hCAT-3 displayed less negative membrane potentials and larger voltage-dependent currents than native or water-injected oocytes did. Ion substitution experiments in hCAT-3-expressing oocytes revealed a large conductance for Na+ and K+. In the presence of L-Arg, voltage-dependent inward and outward currents were observed. At symmetrical (inside/outside) concentrations of L-Arg, the conductance of the transporter increased monoexponentially with the L-Arg concentrations; the calculated Vmax and KM values amounted to 8.3 microS and 0.36 mM, respectively. The time constants of influx and efflux of [3H]L-Arg, at symmetrically inside/outside L-Arg concentrations (1 mM), amounted to 79 and 77 min, respectively. The flux data and electrophysiological experiments suggest that the transport of L-Arg through hCAT-3 is symmetric, when the steady state of L-Arg flux has been reached. It is concluded that hCAT-3 is a passive transport system that conducts monovalent cations including L-Arg. The particular role of hCAT-3 in the diverse tissues remains to be elucidated.     

High calcium content in Streptomyces spores and its release as an early event during spore germination.

The metal ion content of spores of five Streptomyces species was studied. A general feature of this study was the finding of a very high calcium content (1.1 to 2.1% of the dry weight). Accumulation of calcium occurred preferentially during the sporulation process. Spore calcium was located in the integument fraction, and more than 95% of the calcium was removed from intact spores by ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. Several divalent cations (Mg2+, Mn2+, Zn2+, and Fe2+) which induced darkening of spores and loss of heat resistance also caused the release of calcium from spores. In addition, darkening of spores was blocked by metabolic inhibitors, whereas calcium excretion was not affected. Two different categories of events in the initiation of germination may be differentiated; first, calcium release from spores which is not energy dependent and is a consequence of triggering of germination by some divalent cations, and second, some other events including loss of heat resistance, loss of spore refractility, and a decrease in absorbance, with at least one energy-dependent step.     

Human melanoma cells express a novel integrin receptor for laminin

This study sought to determine whether human melanoma cells express integrin-related receptors that mediate their adhesion to laminin. We found that antibodies against the integrin beta 1 chain blocked cell attachment to laminin-coated surfaces. Furthermore, immunofluorescence staining demonstrated beta 1 complexes in vinculin-positive focal adhesion plaques on the basal surface of cells attached to laminin substrates. Chromatography of detergent extracts of 125I-surface-labeled cells on laminin-Sepharose columns recovered two major laminin-binding proteins (100 and 130 kDa, reduced) that bound with high affinity to the columns and were eluted with EDTA. Both proteins were specifically immunoprecipitated from column fractions with monoclonal and polyclonal antibodies to the integrin beta 1 subunit, indicating that they form a noncovalent heterodimer complex. The alpha-like subunit is composed of a 30-kDa light chain that is joined by a disulfide bond to the 100-kDa heavy chain. This complex was not recovered from columns of fibronectin- or collagen type I- or IV-Sepharose. Laminin-binding by the alpha beta 1 complex was independent of Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg-like sequences, but required the presence of divalent cations. The 100-kDa alpha-like subunit was electrophoretically and immunochemically distinct from the other known alpha subunits, alpha 1-alpha 6. The results indicate that human melanoma cells express a novel laminin-specific integrin beta 1 complex which may mediate the cells' interactions with this ligand.