Electric field-directed cell shape changes, displacement, and cytoskeletal reorganization are calcium dependent

C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 10 V/cm. Some cells elongated and aligned perpendicular to the field direction. A preferential positional shift toward the cathode was observed which was inhibited by the calcium channel blocker D-600 and the calmodulin antagonist trifluoperazine. Rhodaminephalloidin labeling of actin filaments revealed a field-induced disorganization of the stress fiber pattern, which was reduced when stimulation was conducted in calcium-depleted buffer or in buffer containing calcium antagonist CoCl2, calcium channel blocker D-600, or calmodulin antagonist trifluoperazine. Treatment with calcium ionophore A23187 had similar effects, except that the presence of D-600 did not reduce the stress fiber disruption. The calcium-sensitive photoprotein aequorin was used to monitor changes in intracellular-free calcium. Electric stimulation caused an increase of calcium to the micromolar range. This increase was inhibited by calcium-depleted buffer or by CoCl2, and was reduced by D-600. A calcium-dependent mechanism is proposed to explain the observed field-directed cell shape changes, preferential orientation, and displacement.     

External electric fields stimulate the electrogenic calcium/sodium exchange in plant protoplasts

External electric fields of low intensity stimulated calcium influx in protoplasts isolated from carrot cell suspension cultures in field intensity dependent and frequency-dependent ways. The field-induced calcium uptake involved a temperature-dependent system that was saturable by external calcium. The induction process appeared mainly cumulative as long as the morphology of the protoplasts did not change (up to 10 min). The stimulation elicited by the electric fields was effective even after switching the field off; the influx increased for 5 min and then slowed down to its initial value 15 min later. During electrostimulation, an additional amount of ATP was accumulated; on removal of the stimulatory field, the extra amount of ATP was consumed, whereas the plasma membrane was hyperpolarized and sodium ions were expelled from the protoplasts. Inhibition of either ATP accumulation or consumption results in the inhibition of both calcium influx and sodium efflux, demonstrating that these processes are coupled. From the data obtained in this work, it may be concluded that the electric field stimulates an ATP synthase like activity; the consumption of the ATP thus formed elicits an electric potential (probably due to the efflux of cations and more specifically sodium) that drives the influx of calcium.     

Effects of ruthenium red, A23187 and D-600 on steroidogenesis in Y-1 cells.

The effects of the calcium antagonists ruthenium red and D-600 and the cation ionophore A23187 on steroidogenesis were investigated. Steroidogenesis triggered by corticotrophin and cyclic AMP was inhibited by each of the agents. Incubation of Y-1 cells with an excess of ethyleneglycol-bis-(beta-amino-ethylether)-N,N'-tetraacetic acid (EGTA) abolished the steroidogenic response to corticotrophin while the response to cyclic AMP was unaffected. The ability of ruthenium red and D-600 (1 . 10(-5) M), and A23187 (6 . 10(-6 M) to inhibit a response which does not require the presence of extracellular calcium (cyclic AMP induced steroidogenesis) suggests that they are altering intracellular calcium. Neither of the calcium antagonists nor the cation ionophore inhibited the steroidogenic response to exogenous pregnenolone, thereby suggesting that the cells were still viable. Only when A23187 was used in the presence of a 15-fold increase in extracellular calcium (4.8 mM) was the response to pregnenolone diminished. The data are interpreted as a further indication that, in intact cells, intracellular calcium plays a role in the steroidogenic pathway.     

Calcium in epithelial cell contraction

Epithelial morphogenesis in many organs involves asymmetric microfilament-mediated cellular contractions. Similar contractions, in terms of ultrastructure and cytochalasin B sensitivity, can be induced in the carcinoma cell line C-4II in culture. This line was used to compare total intracellular calcium levels ([Ca]i) in contracted monolayer fragments and in control cultures, and to determine whether epithelial cell contraction depends on influx of extracellular Ca. [Ca]i, defined as Ca not displaceable by lanthanum, was measured by atomic absorption spectrophotometry. Degrees of contraction were determined from shape changes of monolayer fragments. Detachment from the growth surface initiated cellular contractions and caused an immediate increase in [Ca]i, from 1.0 to 4.0-5.0 micrograms Ca/mg protein in early confluent cultures, and from 0.3 to 1.0-2.0 micrograms Ca/mg protein in crowded cultures. This increase was followed by a gradual decline in [Ca]i, though Ca levels remained higher than in controls and contraction progressed for 30 min. Contraction was inhibited completely by cold (7 degrees C) and by Ca-free medium, and in a dose-dependent manner by papaverine (2.5 x 10(-6) M-2.5 x 10(-4) M), lanthanum (1.0 x 10(-6) M-1.0 x 10(-4) M); and D-600 (1.0-2.0 x 10(- 4) M). The Ca ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 had no effect at 5.0 x 10(-6) M and was inhibitory at higher concentrations. The results provided direct evidence for increased [Ca]i in contracting epithelial cells, and suggest that Ca influx is required for such contraction to take place.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

Clearer demonstration of calcium/calmodulin-dependent events in synaptosomes by use of the differential effects of two calmodulin antagonists, N-(aminohexyl)-5-chloro-1-naphthalenesulfonamide and N-(6-aminohexyl)-1-naphthalenesulfonamide

1. In order to demonstrate more clearly calcium/calmodulin-dependent events, the differential effects of two calmodulin antagonists, W-7 and W-5, on synapsin I phosphorylation and norepinephrine release associated with calcium influx, were investigated using 32Pi in synaptosomes derived from rat cerebral cortex. 2. The calcium ionophore (A23187)-stimulatory effect on synapsin I phosphorylation and norepinephrine release was markedly reduced by W-7 and slightly reduced by W-5; whereas neither the strong nor the weak calmodulin antagonist had an effect on A23187-stimulated synaptosomal uptake of calcium. 3. Preincubation with H-8 reduced both W-5- and W-7-inhibited A23187-stimulated synapsin I phosphorylation by the same amount but did not affect their inhibitory effect nor the ionophore-stimulated norepinephrine release, thereby suggesting that W-5 may serve as an appropriate control for non-calmodulin-mediated effect of both calmodulin antagonists.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Specific high affinity binding of the calcium channel antagonist, [3H]nitrendipine, to rat liver microsomes

Some key properties of the binding of [3H]nitrendipine, an analogue of the 1,4-dihydropyridine, nifedipine, to a plasma membrane enriched microsomal fraction from the rat liver are described. Specific binding was saturable, linear with protein concentration, and reversible. The apparent equilibrium dissociation constant, KD, was 4.20 +/- 0.22 nM and the maximum density of binding, Bmax, was 3.02 +/- 0.17 pmol/mg of protein determined from Scatchard analysis of binding at 10 degrees C. Inhibition of binding was specific for dihydropyridines with competitive inhibition being noted with nifedipine and 4-chloronifedipine, as well as BAY K-8644, a calcium channel agonist. A biphasic displacement curve was recorded for methoxy verapamil (D-600), and a triphasic competition curve with lanthanum (La3+), and diltiazem demonstrated competitive kinetics. The high affinity binding site for nitrendipine in the liver, although having some similar properties to those sites described in skeletal muscle, would appear to be distinctive with respect to its unique sensitivity to D-600 and diltiazem. We speculate that this binding site may represent a Ca2+ channel responsible for regulating Ca2+ influx and hepatic glycogenolysis.     

The mitogenic effect of A23187 in human peripheral lymphocytes.

The mitogenic action of the divalent ionophore A23187 was confirmed and shown to be very sensitive to changes in extracellular calcium ion concentration. At optimal calcium and ionophore concentrations, an increase in [3H]-thymidine incorporation was seen that was similar to that seen after phytohemagglutinin addition. A calcium-dependent stimulation of alpha-aminoisobutyric acid transport was also seen after A23187 addition. Studies with three inhibitors demonstrate a similarity between proliferation induced by phytohemagglutinin and by A23187. Isoproterenol (10(-4) M) and ouabain (10(-7) M) blocked the effects of phytohemagglutinin and A23187. A drug, D-600 that has been shown to block calcium channels in cardiac muscle, inhibited proliferation induced by either phytohemagglutinin or A23187. This concentration of D600 had no effect on either phytohemagglutinin- or A23187-induced 45Ca2+ uptake. Furthermore, the (+) and (-) isomers separated from racemic D600, which have been shown to block sodium and calcium channels respectively in smooth muscle, had equal potency in blocking lymphocyte proliferation.     

influence of Ca channel blockers on the positive inotropic effect of ionophore A23187 in the myocardium

Calcium ionophore A23187 (10(-5) M) increases the force of contraction of the frog atrium up to 27 + 4.8%. The calcium antagonists d-600 (5 X 10- M), Zn2+ (2 X 10(-5) M), Mn2+ (2 X 10(-4) M) decrease the force of contraction 50, 10 and 20%, respectively, and inhibit the positive inotropic effect of ionophore A23187. Inhibition of the ionophore effect by the blockers is determined by the ability of D-600, Zn2+ and Mn2+ to form complexes with the ionophore. Besides, the affinity of these blockers to the ionophore is higher than that of Ca2+. It is assumed that Zn2+, Mn2+ and D-600 possess higher affinity to ionophore A23187 as compared with myocardial Ca-channels. Fenigidin interacts with Ca-channels to a larger degree than with ionophore A23187.     

The mitogenic effect of A23187 in human peripheral lymphocytes

The mitogenic action of the divalent ionophore A23187 was confirmed and shown to be very sensitive to changes in extracellular calcium ion concentration. At optimal calcium and ionophore concentrations, an increase in [³H]-thymidine incorporation was seen that was similar to that seen after phytohemagglutinin addition. A calcium-dependent stimulation of α-aminoisobutyric acid transport was also seen after A23187 addition.Studies with three inhibitors demonstrate a similarity between proliferation induced by phytohemagglutinin and by A23187. Isoproterenol (10^?4 M) and ouabain (10^?7 M) blocked the effects of phytohemagglutinin and A23187. A drug, D-600, that has been shown to block calcium channels in cardiac muscle, inhibited proliferation induced by either phytohemagglutinin or A23187. This concentration of D600 had no effect on either phytohemagglutinin- or A23187-induced ⁴⁵Ca²⁺ uptake. Furthermore, the (+) and (?) isomers separated from racemic D600, which have been shown to block sodium and calcium channels respectively in smooth muscle, had equal potency in blocking lymphocyte proliferation.     

Hypoxic contractile response in isolated human pulmonary artery: role of calcium ion

The mechanism for hypoxic pulmonary vasoconstriction (HPVC) was investigated in human pulmonary arterial strips. Hypoxia in the presence of histamine (10(-6) M) caused marked pulmonary arterial contraction, which was reversed by O2. The hypoxic contraction in the presence of histamine was inhibited by diphenhydramine, but not by cimetidine. The hypoxic histamine-mediated contraction was attenuated but still present in the absence of extracellular Ca2+, or by the inhibitors of voltage-dependent Ca2+ influx. However, it was inhibited significantly by a further depletion of intracellular Ca2+, or by HA 1004, an intracellular calcium antagonist. A low concentration (10(-7) M) of a calcium ionophore, A23187, enhanced the hypoxic contraction in the presence of histamine, whereas procaine completely inhibited it. W-7, a calmodulin inhibitor, significantly decreased the hypoxic histamine-mediated contraction, but 12-O-tetradecanoylphorbol-13-acetate (TPA), a C-kinase promotor, had no effect. The hypoxic contractile response was also observed in the presence of both A23187 and KCl instead of histamine, but the hypoxia-induced contraction with KCl alone was much smaller than that. These results indicate that hypoxia in the presence of certain other vasoactive agents has a potent contractile effect on the human pulmonary artery and that the response is dependent on Ca2+. Enhancement of both Ca2+ influx and Ca2+ release from intracellular storage sites by hypoxia, which interacts with calmodulin, were suggested to be involved in the mechanism of HPVC.     

Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells

Epidermal growth factor (EGF) stimulates the incorporation of 32Pi and [3H]inositol into phosphatidylinositol (5-10-fold) in A-431 cells. EGF also stimulates the incorporation of 32Pi into phosphatidic acid (up to 10-fold). These effects are attributed to an acceleration of the turnover of phosphatidylinositol as a consequence of the binding of EGF to its membrane receptor. The extent of the phosphatidylinositol response to EGF parallels the extent of hormone binding. The phosphatidylinositol response to EGF appears to be dependent on an influx of calcium since (a) external calcium is required for the enhancement of phosphatidylinositol turnover, (2) the accumulation of 45Ca by A-431 cells is stimulated by EGF, (3) blockage of calcium influx with LaCl3 inhibits stimulation of phosphatidylinositol turnover, and (4) calcium influx via ionophore A23187 is sufficient to stimulate phosphatidylinositol turnover. Since the binding, internalization, and degradation of 125I-labeled EGF in A-431 cells are unaffected by the omission of calcium from the medium, external calcium and phosphatidylinositol turnover are not necessary for the internalization and degradation of the EGF-receptor complex.     

HORMONAL CONTROL OF OOCYTE MATURATION IN ARENICOLA MARINA L. (ANNELIDA, POLYCHAETA)

In Arenicola marina (Annelida, Polychaeta) the oocytes are arrested in the first prophase stage of meiosis until spawning. Oocyte maturation is under hormonal control: when incubated in vitro in a brain extract oocytes reach the first metaphase at which they remain arrested until fertilization. The importance of calcium in oocyte maturation has been investigated by using different drugs known to act on membrane calcium permeability and to modify intracellular free calcium concentration. Tetracaine, procaine, D-600, verapamil (Isoptin), propranolol, oxprenolol and lanthanum chloride, calcium deprivation but not ionophore A23187, are all able to induce oocyte maturation. This suggests that the brain hormone may act on the oocyte by regulating, probably increasing, the intracellular free calcium concentration, as it has been proposed for oocytes of other animals. The importance of -SH/-SS- in meiosis reinitiation is suggested by the fact that dithiothreitol and 2, 3-dimercaptopropanol, two disulfide reducing agents, both induce oocyte maturation.     

Effect of calcium entry blockers and adenosine on the relaxation of large and small coronary arteries

The mechanism(s) by which adenosine causes dilation of the vascular smooth muscle is not properly understood. Several mechanisms including the inhibition of calcium influx and intracellular translocation have been suggested for its action. This study is an attempt to further elucidate the site of action of adenosine in relation to calcium by making use of calcium entry blockers. Large (1 +/- 0.2 mm, o.d.) and small (0.5 +/- 0.2 mm, o.d.) branches of bovine left anterior descending coronary artery (LADCA) contracted with 50 mM K+ were used as a model for these studies. Concentration-response curves for various calcium entry blockers were obtained and the order of potency was found to be: D-600 greater than nifedipine greater than verapamil greater than diltiazem greater than lidoflazine for large branches and nifedipine greater than D-600 greater than verapamil greater than lidoflazine greater than diltiazem for small branches of LADCA. The concentration-response relationship for adenosine (10(-6)-10(-4) M) in the presence and absence of these drugs (10(-9)-10(-7) M) was unchanged. 8-phenyltheophylline (2 X 10(-5) M), an adenosine receptor antagonist was without an effect on the relaxations induced by various calcium entry blockers, however, it antagonized the relaxing response to adenosine. Lidoflazine at concentrations of 7 X 10(-7) M and 2 X 10(-7) M potentiated the effect of adenosine in relaxing the large and small LADCA, respectively. In summary, the data show an increased sensitivity of small coronary vessels to nifedipine, D-600 and lidoflazine. The data further suggest a different site of action for adenosine and calcium entry blockers.     

The role of calcium ions in freezing injuries of winter oilseed rape leaves. Effects of calcium channel blockers and mimesis by calcium ionophore

Calcium ionophore A23187 allowing for a calcium ion influx from an apoplast to a cytoplasm, mimicked symptoms of the frost-induced injuries in winter oilseed rape leaves, as estimated by the conductivity method. Both calcium ionophore and freezing treatment induced degradation of phosphatidylcholine. On the other hand lanthanum and gadolinum ions as well as verapamil, the inhibitors of calcium ion channels, decreased the degree of the frost-induced injuries. Lanthanum ions prevented the frost-induced degradation of PC. It is proposed that freezing alters the functioning of calcium ion channels which results in calcium ion influx into a cytosol. This in turn may lead to a degradation of cell membranes.     

Experimental activation of ascidian eggs.

The effects of the ionophore A23187 on the activation of the eggs of Ascidia malaca have been studied. No common external ion in the sea water is found to be essential for the activation but lanthanum and manganese inhibit the response. These observations support the interpretation that activation of these eggs results from changes in free intracellular calcium levels. This has led to the prediction of two other activating treatments, namely high external calcium and addition of theophylline.     

The control of pigment migration in isolated erythrophores of Holocentrus ascensionis (Osbeck). II. The role of calcium

The integumental pigment cells (erythrophores) of the squirrel fish, Holocentrus ascensionis, are specialized for rapid radial transport of the pigment granules contained within their cytoplasm. Pigment granules in isolated denervated erythrophores alternate spontaneously between a centrally aggregated state and a radially dispersed state. In the absence of external calcium, pigment aggregation does not occur spontaneously and cannot be induced by the aggregating agents epinephrine or high concentration of external K+. Pigment aggregation is also impaired in the presence of D600 or papaverine, compounds reported to antagonize calcium influx into the cell. Pigment aggregation can be induced by experimental elevation of the concentration of cytoplasmic free Ca2+, with a Ca-EGTA buffer system in conjunction with ionophore A23187. The threshold concentration of Ca2+ required to produce this effect is 5 X 10(-6) M. These results suggest that cytoplasmic free Ca2+ is involved in mediating pigment aggregation and that some, if not all, the Ca2+ is supplied by influx from the extracellular space.     

Neuritogenesis in mouse NB2a/d1 neuroblastoma cells: triggering by calcium influx and involvement of actin and tubulin dynamics

Ionophore (A23187)-mediated calcium influx induced rapid neurite outgrowth in NB2a/d1 cells. This outgrowth was prevented by colchicine but not by cycloheximide, demonstrating a requirement for microtubule assembly but not de novo synthesis. Cytochalasin B induced rapid, colchicine-sensitive outgrowth, indicating that depolymerization of the submembrane actin network may be sufficient to allow neurite outgrowth under conditions which permitted microtubule assembly. Neurites induced by serum-deprivation or calcium influx were rapidly retracted by colchicine unless cytochalasin B was first added, indicating that the actin network may provide the retractile force which mediates neurite retraction following microtubule depolymerization. We conclude that neurite outgrowth can be initiated in NB2a/d1 cells by calcium influx, and may involve alterations in actin and microtubule dynamics.     

Calcium Antagonists Inhibit Sustained Gibberellic Acid-Induced Growth of Avena (Oat) Stem Segments

The elongation response of Avena sativa (oat) stem segments to gibberellic acid (GA3) is of large magnitude, with high hormonal sensitivity and specificity, but without cell division activity. This system is therefore an excellent model for mechanistic studies on higher plant cell elongation and the action of gibberellin. At millimolar concentrations, the calcium antagonists verapamil, D-600, nicardipine, diltiazem, bepridil, 8-(N,N,-diethylamino)-octyl-3,4,5-trimethoxybenzoate HCl, and lanthanum substantially inhibited the growth of GA3-treated segments but had no effect on the elongation of nonhormone-treated segments. Although verapamil reduced the maximum growth rate and caused premature cessation of growth, even preincubation of the segments with the drug prior to treatment with GA3 failed to inhibit the earliest measured stimulation of growth by the hormone. Inhibition by verapamil was not reversed by increased concentrations of GA3 or calcium. Neither the calcium ionophore A23187 nor agonist BAY K 8644 had any effect on growth. Light microscopic examination of epidermal peels from antagonist-treated internodal tissue revealed no obvious differences from the control except that the cells were not as elongated. Although these results may support a role for calcium ion movement in maintaining the GA3-induced growth of Avena stem segments, they do not support the involvement of calcium ion movement in the hormone-mediated initiation of growth.