Divalent Cation Influx Research Articles

Ba 2+ current oscillations modulated by cyclic AMP and phorbol esters in ras-transformed fibroblasts

An oscillatory influx of divalent cations was measured as Ba 2+ inward currents (Ba 2+ current oscillations) by voltage-clamp recording in v-Ki- ras-transformed NIH 3T3 (DT) fibroblasts after activation with bradykinin or serum. Application of forskolin or dibutyryl cyclic AMP onto DT cells initiated Ba 2+ current oscillations. Increasing intracellular cyclic AMP reduced the amplitude but increased the frequency of the Ba 2+ current oscillations. Activation of protein kinase C by phorbol esters terminated Ba 2+ current oscillations. No inhibition of Ba 2+ current oscillations by phorbol esters was observed in down-regulated cells that had been pretreated with phorbol esters for 24 hrs. The results suggest that Ba 2+ current oscillations are regulated by intracellular second messengers.

Mn and Cd transport by the Na-Ca exchanger of ferret red blood cells

Ca influx via the Na-Ca exchanger into ferret red blood cells is easily measured from a Na-free solution; the intracellular Na concentration is normally approximately 150 mM in ferret red blood cells. We have found that Mn and Cd competitively inhibit Ca influx. Mn influx and Cd influx were a saturable function of the divalent cation concentration, consistent with a carrier mechanism. Indeed, the Km (approximately 10 microM) and the Vmax (usually 1-3 mmol.l packed cells-1.h-1) were similar for Ca, Cd, and Mn. Extracellular Na inhibited divalent cation influx, and intracellular Na stimulated influx. These results are consistent with Na-Cd and Na-Mn influx pathways in ferret red blood cells. Ca (1 mM) almost completely inhibited Mn influx and Cd influx, whereas 1 mM Mg inhibited 5-15%. These results strongly support the notion that Mn and Cd are alternative substrates for Ca on the ferret red cell Na-Ca exchanger. The similarity in the behavior of all three divalent cation places important constraints on kinetic and structural models of the exchanger.

Agonist-activated cobalt uptake identifies divalent cation—Permeable kainate receptors on neurons and glial cells

Activation of kainate receptors causes Co 2+ influx into neurons, type-2 astrocytes, and O-2A progenitor cells. Agonist-activated Co 2+ uptake can be performed using cultured cells or fresh tissue slices. Based on the pattern of response to kainate, glutamate, and quisqualate, three functionally different kainate-activated ion channels (K1, K2, and K3) can be discriminated. Co 2+ uptake through the K1 receptor was only activated by kainate. Both kainate and glutamate activated Co 2+ uptake through the K2 receptor. Co 2+ uptake through the K3 receptor was activated by all three ligands: kainate, glutamate, and quisqualate. Co 2+ uptake occurred through a nonselective cation entry pathway permeable to Co 2+, Ca 2+, and Mn 2+. The agonist-dependent activation of divalent cation influx through different kainate receptors could be correlated with expression of certain kainate receptor subunit combinations. These results are indicative of kainate receptors that may contribute to excitatory amino acid-mediated neurotoxicity.

Translocation of Ca2+ across lipid bilayer membrane due to defects induced by teleocidin

The effect of defects in a dipalmitoylphosphatidylcholine (DPPC) membrane on Ca2+ permeability across the membrane was studied. Addition of teleocidin to a suspension of DPPC vesicles encapsulating Quin 2 increased the fluorescence intensity of Quin 2. Change of fluorescence intensity was significant below the phase-transition temperature of the membrane, and increased according to the kind of divalent metal ions in the medium in the order of Mg2+<Ba2+<Ca2+. It was confirmed that DPPC vesicles did not change the vesicular structure upon binding teleocidin to the membrane. Therefore, the fluorescence increase below the phase-transition temperature was ascribed to the influx of divalent cations into DPPC vesicles through cracks formed in the membrane upon distribution of teleocidin. By contrast, 12-0-tetradecanoylphorbol-13-acetate (TPA) did not change the fluorescence intensity of Quin 2 significantly. It should be noted that teleocidin, which located at the membrane surface, yielded more significant defects across the lipid membrane than TPA, which was incorporated into the hydrophobic core of the membrane.

Effect of membrane potential on divalent cation transport catalyzed by the “electroneutral” ionophores A23187 and ionomycin

Depolarization of plasma membrane potential has a potent inhibitory effect on divalent cation influx catalyzed by the carboxylic ionophores ionomycin and A23187. This effect is observed in different cell models and does not depend on either inhibition of Ca2+-activated cation channels or activation of Ca2+ extrusion mechanisms as suggested previously. A dependence of divalent cation influx on the magnitude of membrane potential is observed also in artificial liposomes. The inhibition of ionophore-dependent divalent cation transport by membrane potential depolarization can be modified varying the ionophore concentration and the external pH. These findings suggest that both neutral and positively charged ionophore-cation complexes can cross the plasma membrane and that their contribution to the overall transport process can be varied according to the experimental conditions.

Chromatin solubilization in rapidly growing hepatomas

The presence of substantial amounts of DNA in the cytosol fractions prepared from rapidly growing transplanted rat hepatomas was confirmed. Agarose gel electrophoresis of this DNA from the Morris hepatomas 7288CTC, 7777, and 7800 suggested that it corresponds primarily to nucleosome monomers with smaller amounts of nucleosome dimers and trimers. Despite the evidence for solubilization of chromatin in rapidly growing hepatomas the potential DNA endonuclease activity was less in nuclei from these tumors than in nuclei from more slowly growing hepatomas or host livers. It was suggested that focal necrosis in rapidly growing solid tumors may be associated with the influx of divalent cations and the activation of DNA endonuclease activity.

Modulation of platelet-activating-factor-induced calcium influx and intracellular calcium release in platelets by phorbol esters.

The mechanisms by which platelet-activating factor (PAF) and thrombin increase intracellular calcium were examined. Platelets were loaded with the calcium-sensitive fluorescent probe Quin 2 and then were suspended in buffer containing 0.5 mM-Mn2+ in order to quantify simultaneously calcium release from intracellular stores and divalent cation influx. Pretreating platelets with agents which activate protein kinase C [the phorbol ester phorbol myristate acetate (PMA) or the diacylglycerol 1-oleoyl-2-acetylglycerol (OAG)] inhibited increased intracellular calcium by PAF and thrombin in a dose-related manner. That protein kinase C regulates intracellular calcium by phosphorylating proteins in two distinct pathways was suggested by two observations. PAF-induced calcium release was more sensitive to inhibition by PMA and OAG than was manganese influx and the kinetics of recovery from inhibition were different for the two pathways. Both PMA and OAG aggregated Quin 2-loaded platelets without eliciting measurable increases in intracellular calcium. In contrast, prostacyclin, which increases intracellular cyclic AMP, inhibited calcium release and influx in parallel, suggesting that this agent acts at a step common to both pathways.

Transmembrane cation movements during infection of Lactobacillus lactis by bacteriophage LL-H.

Phage LL-H-induced cation (K+, Na+, Mg2+, Ca2+, Cd2+) movements in Lactobacillus lactis bacteria have been studied. The effects of the m.o.i. and external cation concentration have been quantified. LL-H-induced effluxes showed cation specificity: K+ but practically no Mg2+ was lost during LL-H infection at low and moderate m.o.i. (up to about 100). Simultaneously to K+ efflux, divalent cation influxes were observed. These were dependent on the m.o.i. and on concentrations of external divalent cations and were concomitant with phage DNA transport, as concluded from the timing of the first phage-promoted biochemical changes in host cell metabolism and from electron microscopical observations. Host energy was not mobilized with phage-induced divalent cation influx. Several features of divalent cation influxes support the view that divalent cations have to be cotransported into the cell as counterions of LL-H DNA. Phage DNA associated with divalent cations may be the basic feature of the divalent cation dependence of LL-H infection.

The effect of calcium channel blockers on blood platelet function, especially calcium uptake

The effects of organic and inorganic calcium antagonists on washed platelets from rat and human been studied. Platelet aggregation was assessed by turbidimetry. Endogenous serotonin release was measured on the same sample by means of electrochemically treated carbon fiber electrodes. The organic calcium antagonist, nitrendipine, and the inorganic calcium channel blockers (Co 2+, Mn 2+, Cd 2+, La 3+) drastically inhibited rat and human platelet aggregation induced by thrombin, ADP or adrenaline in teh presence of 0.32 mM Ca 2+. In our conditions, the thrombin-induced release of endogenous serotonin was found to be external Ca 2+-dependent and completely inhibited by 20 μM nitrendipine or 1 mM Cd 2+. In addition, Ba 2+ or Sr 2+ ions can be substituted for Ca 2+ to bring about platelet aggregation as well as endogenous serotonin secretion. In Ba 2+ or Sr 2+-containing media, rat platelet aggregation and/or serotonin secretion can be inhibited by either nitrendipine or Cd 2+. Finally, we have also studied the thrombin- and external Ca 2+-dependence of radiolabeled calcium uptake by rat platelets. We found that the thrombin-induced 45Ca uptake was inhibited by either 18 μM nitrendipine or 1 mM Cd 2+. These results provide strong evidence for the existence of an influx of divalent cations (Ca 2+, Sr 2+, Ba 2+) triggering platelet function. They also suggest, although they do not prove, that the translocation of these cations occurs through an agonist-operated channel as proposed by Hallam and Rink (FEBS Lett. 186 (1986) 175–179).

Mechanism and characteristics of platelet activation by haematin.

The mechanism as well as some characteristics of haematin-induced human platelet aggregation were investigated. Haematin-induced platelet aggregation required the presence of devalent cations; Mg2+, and to a lesser extent, Co2+, were just as effective as Ca2+ in supporting the aggregation. Mono- and trivalent cations were ineffective. Verapamil inhibited the aggregation. The aggregation was accompanied by thromboxane formation which could be abolished by aspirin. The release of adenine nucleotides was only slightly inhibited by aspirin. The rate of aggregation and the ultrastructure of the aggregated platelets were comparable between control and aspirin-treated samples. It is concluded therefore that haematin-induced aggregation is not dependent on platelet prosta-glandin synthesis. Haematin induced binding of fibrinogen to platelets, and failed to aggregate thrombasthenic platelets. These findings indicate that haematin may induce platelet aggregation by promoting influx of divalent cations in association with increased fibrinogen binding and release of adenine nucleotides.

Phorbol 12-myristate 13-acetate, ionomycin or ouabain, and raised extracellular magnesium induce proliferation of chicken heart mesenchymal cells.

Cultured chicken heart mesenchymal cells are proliferatively quiescent at low densities in medium containing plasma at 10%. Mitogenic hormones like epidermal growth factor and insulin-like growth factors cause these cells to proliferate very actively, as does infection with avian sarcoma viruses, erythroblastosis virus, or myelocytomatosis virus. We have found that the combination of phorbol 12-myristate 13-acetate (PMA), ionomycin or ouabain, and raised extracellular magnesium, likewise, causes these cells to proliferate very actively. Although these agents have no significant effect when acting singly, the combination of PMA at 100 ng/ml and 0.5 microM ionomycin induces a 6-fold increase in cell number at 4 days, and the combination of PMA, ionomycin, and 5.6 mM magnesium induces 12-fold multiplication. Likewise, PMA plus 1 microM ouabain induces 3-fold multiplication, whereas the combination of PMA, ouabain, and magnesium induces 6-fold multiplication. The tumor promoter PMA, like diacylglycerol released by breakdown of plasma membrane phosphatidylinositol diphosphate, is known to activate the serine- and threonine-specific intracellular enzyme kinase C. The divalent cation ionophore ionomycin is known to carry calcium into cells down an electrochemical gradient, and the Na+,K+-ATPase inhibitor ouabain appears to elevate intracellular calcium by means of a sodium-mediated exchange mechanism. Magnesium, like calcium, is known to enter cells passively down an electrochemical gradient and to be involved in the regulation of many key intracellular reactions. Our findings with PMA, ionotropes, and magnesium support a hypothesis that diacylglycerol-mediated activation of kinase C plus cellular divalent cation influx and/or mobilization, caused by the action of mitogenic hormones or the protein products of onc genes, are key events in the initiation of cell replication.

The effect of α-latrotoxin on the neurosecretory PC12 cell line: Studies on toxin binding and stimulation of transmitter release

α-Latrotoxin of black widow spider venom was found to bind with high affinity (K A = 1.8·10 9M −1) to specific sites present in discrete number (∼6300/cell, ∼12/aμn2) at the surface membrane of PC12 cells. This binding correlated with (and therefore, probably caused) the secretory response produced by the toxin. Binding was enhanced (∼ 2-fold) in the presence of mM concentrations of various divalent cations (Ca 2+, Mn 2+ and Co 2+) while Ba 2+ and Sr 2+ had a smaller effect and Mg 2+ was inactive. Hypertonicity, concanavalin A and trypsin pretreatment of the cells blocked the binding interaction. The α-latrotoxin-induced stimulation of 3H-dopamine release was massive and occurred very rapidly when cells were exposed to the toxin in a Ca 2+-containing Krebs-Ringer medium, whereas it occurred at a much slower rate in a Ca 2+-free, Mg 2+-containing Ringer. Introduction of Ca 2+ into the latter medium resulted in a shift of the release rate from slow to fast. In contrast, in divalent cation-free medium the response was abolished. The toxin-induced secretory response was unaffected by Na 2+ and Ca 2+ channel blockers (tetrodotoxin and D600) as well as by calmodulin inhibitors (calmidazolium and trifluoperazine). The effects of Ca 2+ and Mg 2+ were found to be concentration-dependent, with half maximal responses occurring at approximately 0.3 and l.5mM for the two divalent cations, respectively. Other divalent cations could substitute for Ca 2+ and Mg 2+, the relative efficacy being Sr 2+ > Ca 2+ ⩾> Ba 2+ > Mn 2+ > Mg 2+ > Co 2+. Moreover, the response occurring at suboptimal concentration of Ca 2+ (0.4 mM) was potentiated by the concomitant addition of either Mg 2+, Mn 2+ or Co 2+. The effect(s) of divalent cations in supporting the α-latrotoxin-induced release response seem(s) to occur primarily at step(s) beyond toxin binding because (a) the stimulatory effects of the various cations on release were not matched by parallel effects on binding (b) Ca 2+ maintained its ability to stimulate fast release even when toxin binding had occurred in a Ca 2+-free medium. Delays in the release responses were observed when cells were exposed to αLTx in Na 2+-free, glucosamine or methylamine-based media, or depolarized with high K 2+ (in the presence of D600) before toxin treatment. Moreover, in these two conditions the ability of Mg 2+ to support the αLTx response was considerably decreased. Taken together with previous data of the literature these results appear consistent with the hypothesis that αLTx acts by activating a ‘non-conventional’ cation channel of low ionic specificity in the plasma membrane. This would result in a stimulated influx of various divalent cations, not only Ca 2+ but also Mg 2+ and the others investigated, which would then be able to stimulate the release response, although with different efficacy.

Elementary currents through Ca2+ channels in guinea pig myocytes.

Elementary Ca2+ and Ba2+ currents were recorded from cell-attached membrane patches of ventricular myocytes from adult guinea pig hearts using the improved patch-clamp technique (Hamill et al. 1981). High concentrations of Ba2+ or Ca2+ (50 or 90 mM) were used in the pipettes to increase the signal-to-noise ratio. All data were derived from elementary current analyses in patches containing only one channel. 1) In response to voltage steps, channel openings occurred singly or in bursts of closely spaced unitary current pulses separated by wider shut intervals. During depolarizations of small amplitude from the resting potential, channel openings occurred almost randomly, whereas during larger depolarizations the events were grouped preferentially at the beginning. 2) Channel openings became more probable with increased depolarization; simultaneously, unitary current amplitudes declined in an ohmic manner. Elementary current amplitudes were slightly larger, when 50 mM Ba2+ replaced 50 mM Ca2+ in the pipettes (slope conductances 9 and 10 pS, respectively), but more than doubled, when Ba2+ was increased to 90 mM (slope conductance 18 pS). Clear outward currents through Ca2+ channels were not observed under these conditions. 3) Peak amplitudes of reconstructed mean currents doubled when 50 mM Ba2+ replaced 50 mM Ca2+ and were larger still when 90 mM Ba2+ was used in the pipettes. The current-voltage relations of the reconstructed mean currents showed a positive shift along the voltage axis as Ba2+ was increased or substituted equimolarly by Ca2+. correspondingly, the open state probability-voltage relations (activation curves) showed a parallel shift as Ba2+ was increased, which was less pronounced when Ba2+ was replaced equimolarly by Ca2+. 4) Determination of Ca2+ channel inactivation using 90 mM Ba2+ in the pipettes indicated an overlap with channel activation in a limited voltage range, resulting in a steady-state "window" current. Inactivation can occur without divalent cation influx. 5) Formation of an inside-out patch resulted in a fast rundown of elementary Ca2+ channel currents. 6) Channel openings were often grouped in bursts. The lifetimes of the open state, the bursts, and the closed states were estimated for Ba2+ and Ca2+ as permeating ions. At least two exponentials were needed to fit the histogram of the lifetimes of all closed states. The lifetimes of the individual openings and bursts were mono-exponentially distributed. The kinetics of the Ca+ channel depended on the voltage and the permeating ion. During +30 mV depolarizations, no significant effect on the permeating ion on channel gating could be detected.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

The roles of extracellular and intracellular calcium in lysosomal enzyme release and superoxide anion generation by human neutrophils

The relative importance of intracellular and extracellular calcium in the stimulation of human neutrophils was studied by use of chelators and calcium antagonists. In the first series of experiments, EGTA was used to see if extracellular calcium was an absolute requirement for neutrophil activation. Stimulated neutrophils secreted lysosomal enzymes and generated superoxide anion radicals in the presence or absence of 5 mM EGTA or Mg-EGTA. Seven different stimuli were employed: the chemotactic peptide N-formylmethionylleucylphenylalanine (10 −7 M), calcium ionophore A23187 (10 −5 M), concanavalin A (30 μg/ml), serum-treated zymosan (2 mg/ml), zymosan-treated serum serum (10%), an immune complex (150 μg/ml) and phorbol myristate acetate (1 μg/ml). Release of the lysosomal enzymes β-glucuronidase and lysozyme and generation of superoxide anions in response to these stimuli were readily demonstrable in the presence of EGTA. Each response of neutrophils was variably, but not completely, reduced in the presence of the chelator; EGTA did not completely block the responses elicited by any of the stimuli. Consequently, it is unlikely that an influx of divalent cations from the extracellular medium is obligatory for stimulus-response coupling in human neutrophils. The role of intracellular calcium was then studied using calcium antagonists. Inhibition of neutrophil responses was readily obtained using TMB-8, an antagonist of the mobilization of intracellular calcium, and trifluoperazine and W-7, inhibitors of calmodulin. These results suggest that intracellular, but not extracellular, calcium plays an obligatory role in the responses of neutrophils to a variety of soluble and insoluble stimuli.

The role of calcium in secretion of inflammatory mediators from neutrophils

We have found that cytochalasin B-treated human PMN respond to surface stimulation in the presence of saturating concentrations of EGTA or Mg · EGTA. It is extremely unlikely that extracellular divalent cations or, by implication, an influx of divalent cations is an obligate requirement for stimulation of PMN. An obligate requirement for mobilization of intracellular calcium and action of calmodulin was suggested by the effects of TMB-8 and stelazine.

Requirement for magnesium influx in activation of alveolar macrophages mediated by ionophore A23187

NORMAL antibacterial activity in the lung is largely dependent on alveolar macrophage function1. Products of antigen-stimulated, sensitised lymphocytes can activate alveolar macrophages, which then exhibit greater adherence, phagocytosis and nonspecific bactericidal capacity than normal2. Although the molecular basis of this activation is unknown, influx or intracellular redistribution of divalent cations (Ca2+ and Mg2+) has been implicated in other cell types3–10. We have therefore examined the effect on alveolar macrophage function of exposure to the ionophore A23187, which selectively binds divalent cations at neutral pH and passively transfers them across biological membranes11,12.

Modification by temperature of the response of isolated aorta to stimulatory agents and transmural stimulation.

Contractile responses of helically-cut strips of the rabbit aorta to drugs, ions and transmural electrical stimulation were compared at different temperatures of the bathing medium, the response at 37 degrees C being taken as control. The dose-response curve of norepinephrine was moved to the right and downward by lowering the temperature from 37 to 25 degrees C and by raising temperature to 40 degrees C. Responses to transmural neural stimulation at frequencies of 5 and 20/sec were attenuated at 25 degrees C, the attenuation being greater in the response at the lower frequency. Concentrations of exogenous norepinephrine needed to produce the same magnitude of contraction as that with transmural stimulation were markedly increased by lowering the temperature to 25 degrees C. Contractile responses to norepinephrine (2 X 10(-6) M), histamine (2 X 10(-5) M) and angiotensin II (10(-7) M) were attenuated by 32-44% at 25 degrees C, whereas the responses to K+ (25 mM) and Ba++ (2 mM) were dependent on temperatures between 25 and 37 degrees C and were attenuated by 69 and 92%, respectively, at 25 degrees C. Contractures induced by Ca++ in K+-depolarized preparations exposed to Ca++-free media and also by Ba++ in preparations exposed to Ca++-free media varied directly by raising temperatures. Interference with the influx of divalent cations, such as Ca++ and Ba++, may be involved in the cold inhibition of aortic contractility.