Further studies on alterations in magnesium binding during cold storage of erythrocytes

Free intracellular Mg2+ concentration ([Mg2+]i) was measured in cold-stored human erythrocytes by the method of null-point titration with ionophore A23187. [Mg2+]i was 311 +/- 41 microM (mean +/- S.D.) for cells stored 0-10 days, increasing to 458 +/- 64 microM for cells stored 22-48 days. The values for stored cells were higher than those previously determined by a 31P-NMR method (Bock et al. (1985) Blood 65, 1526-1530); however, the null-point method requires extensive washing of the cells, which we have found to increase NMR-measured [Mg2+]i. The null-point values still represent a small fraction of total cell Mg2+, and confirm that binding of Mg2+ to ligands other than ATP and 2,3-bisphosphoglycerate must increase during storage. As an initial test of whether this may imply suboptimal availability of Mg2+ for cell preservation, we used A23187 to prepare erythrocytes with altered Mg2+ content, then removed ionophore and stored the cells in plasma-free medium for up to 2 weeks. Higher Mg2+ content had a very significant positive correlation (P less than 0.0001) with higher cell ATP concentrations. Storage did not significantly affect basal or Na+-stimulated efflux of Mg2+ from Mg2+-loaded red cells.     

Ethyl Pyruvate Inhibits HMGB1 Phosphorylation and Release by Chelating Calcium

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca²⁺ and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K⁺-induced transient [Ca²⁺]_i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca²⁺ as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca²⁺ indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187-induced intracellular Ca²⁺ surge in BV2 cells and, under this condition, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187-induced activations of Ca²⁺-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca²⁺, and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.     

Stimulation and inhibition of secretion in Paramecium: role of divalent cations

The effects of Ca2+ and Mg2+ on exocytosis in Paramecium tetraurelia cells were examined with light microscopy, freeze fracture (FEM) and transmission electron microscopy (TEM) of thin-sectioned embedded cells. Picric acid-Ca2+-induced secretion in wild type (wt) cells was captured by "quick" fixation with OsO4, and TEM demonstrated membrane fusion occurring before trichocyst matrix (tmx) expansion. Cells stimulated with picric acid in the presence of high extracellular Mg2+ showed very few sites of membrane fusion and no tmx expansion, suggesting that Ca2+ is required for both membrane fusion and tmx expansion. Further information was obtained by comparing secretory responses of wt cells with a temperature-sensitive secretory mutant, nd 9. These cells when grown at the permissive temperature (18 degrees C) possess normal rosettes at the secretory site and secrete in response to picric acid-Ca2+, but when grown at 27 degrees C they lack rosettes and do not secrete (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). Quantitation of picric acid-Ca2+-induced secretion revealed that: (a) the number of tmx secreted by wt and nd 9 cells was independent of their cultural growth phase, (b) wt cells secreted the same number of tmx when grown either at 18 or 27 degrees C, and (c) nd 9 18 degrees C cells secreted the same number of tmx as wt 18 or 27 degrees C cells. Wild type and nd 9 cells had the same frequencies of occupied and unoccupied secretory sites as determined by quantitative analysis of freeze-fracture replicas. After stimulation with divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+, wt cells showed a significant reduction in the frequency of occupied sites. FEM and TEM studies revealed that {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-Ca2+ induced tmx expansion and normal fusion of the plasma and trichocyst membranes in wt and nd 9 18 degrees C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27 degrees C cells. The lack of membrane fusion in nd 9 27 degrees C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.     

The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187

Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.     

Calcium control of ciliary reversal in ionophore-treated and ATP- reactivated comb plates of ctenophores

Previous work showed that ctenophore larvae swim backwards in high-KCl seawater, due to a 180 degrees reversal in the direction of effective stroke of their ciliary comb plates (Tamm, S. L., and S. Tamm, 1981, J. Cell Biol., 89: 495-509). Ion substitution and blocking experiments indicated that this response is Ca2+ dependent, but comb plate cells are innervated and presumably under nervous control. To determine whether Ca2+ is directly involved in activating the ciliary reversal mechanism and/or is required for synaptic triggering of the response, we (a) determined the effects of ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+ on the beat direction of isolated nerve-free comb plates dissociated from larvae by hypotonic, divalent cation-free medium, and (b) used permeabilized ATP- reactivated models of comb plates to test motile responses to known concentrations of free Ca2+. We found that 5 microM {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and 10 mM Ca2+ induced dissociated comb plate cells to beat in the reverse direction and to swim counterclockwise in circular paths instead of in the normal clockwise direction. Detergent/glycerol-extracted comb plates beat actively in the presence of ATP, and reactivation was reversibly inhibited by micromolar concentrations of vanadate. Free Ca2+ concentrations greater than 10(-6)M caused reversal in direction of the effective stroke but no significant increase in beat frequency. These results show that ciliary reversal in ctenophores, like that in protozoa, is activated by an increase in intracellular free Ca2+ ions. This allows the unique experimental advantages of ctenophore comb plate cilia to be used for future studies on the site and mechanism of action of Ca2+ in the regulation of ciliary motion.     

Gap junction dynamics: reversible effects of divalent cations

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode''s solutions containing a Ca++ ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.     

Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. II. Contrasting effects of local anesthetics and a calcium ionophore

In the previous study, lymphocyte surface molecules were separated into two subsets depending on whether capping was associated was associated with redistribution of cytoplasmic myosin. In the present study, the effects of the local anesthetic chlorpromazine and of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 were compared. Both drugs affected the surface redistribution of immunoglobulin (Ig), Fc receptors, and the TL antigen- -molecules that appear to cap by association with microfilaments--but had no effect on the Thy.1 (theta) and H2 antigens--molecules that cap slowly, apparently unlinked to microfilament function. The capping of Ig, Fc receptor, and TL was inhibited while that of H2 and theta was not. Both drugs reversed the Ig Fc receptor, and TL caps but not the H2 and theta caps. In the former group, the reversal of caps was accompanied by a parallel reversal of the myosin segregated to the cap area. The appearance of myosin after drug treatment varied: chlorpromazine resulted in a diffuse pattern similar to that of normal lymphocytes, whereas {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 produced an array of aggregates and coarse filaments. The results are compatible with the view that two mechanisms for capping exist in the lymphocyte. The Ca2+ ionophore may affect capping of microfilament-dependent caps by producing a systemic activation of contractile proteins while chlorpromazine may act by disrupting a Ca2+-dependent link between surface complexes and the contractile proteins.     

A trypanosome-soluble factor induces IP3 formation,intracellular Ca2+ mobilization and microfilament rearrangement in host cells

Lysosomes are recruited to the invasion site during host cell entry by Trypanosoma cruzi, an unusual process suggestive of the triggering of signal transduction mechanisms. Previous studies showed that trypomastigotes, but not the noninfective epimastigotes, contain a proteolytically generated trypomastigote factor (PGTF) that induces intracellular free Ca2+ transients in several mammalian cell types. Using confocal time-lapse imaging of normal rat kidney (NRK) fibroblasts loaded with the Ca(2+)-sensitive dye fluo-3, we show that the initial intracellular free Ca(2+) concentration ([Ca2+]i) transient detected a few seconds after exposure to trypomastigote extracts is a result of Ca2+ release from intracellular stores. Removal of Ca2+ from the extracellular medium or inhibition of Ca2+ channels with NiCl2 did not affect the response to PGTF, while depletion of intracellular stores with thapsigargin abolished it. [Ca2+]i transients induced by PGTF were shown to be coupled to the activity of phospholipase C (PLC), since the specific inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 completely blocked the response, while its inactive analogue {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 had no effect. In addition, polyphosphoinositide hydrolysis and inositol 1,4,5-trisphosphate (IP3) were detected upon cell stimulation with PGTF, suggesting the participation of IP3-sensitive intracellular Ca2+ channels. An immediate effect of the signaling induced by PGTF and live trypomastigotes was a rapid and transient reorganization of host cell microfilaments. The redistribution of F-actin appeared to be a direct consequence of increased [Ca2+]i, since thrombin and the Ca2+ ionophore ionomycin produced a similar effect, with a time course that corresponded to the kinetics of the elevation in [Ca2+]i. These observations support the hypothesis that PGTF-induced disassembly of the cortical actin cytoskeleton may play a role in T. cruzi invasion, by facilitating lysosome access to the invasion site. Taken together, our findings suggest that the proteolytically generated trypomastigote factor PGTF is a novel agonist that acts through the PLC/phosphoinositide signaling pathway of mammalian cells.     

Regulation of mitochondrial K+/H+ antiport activity by hydrogen ions

The effect of matrix pH (pHi) on the activity of the mitochondrial K+/H+ antiport has been studied using the fluorescence of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to monitor pHi and passive swelling in K+ acetate to follow antiport activity. Heart mitochondria suspended in hypotonic K+ acetate in the absence of respiration show an initial delta pH of -0.4 (interior acid) that decays slowly. Addition of A23187 to deplete matrix Mg2+ results in a further acid shift in pHi followed by equilibration of delta pH. This equilibration appears to depend on K+/H+ antiport and is slow at acid pHi but very rapid when the matrix is alkaline. Swelling of Mg(2+)-depleted mitochondria in K+ acetate is multiphasic with a slow initial rate, a period of maximum swelling, and a final period in which the rate declines. At constant external pH (pH0), the initial rate of swelling is faster with increasing pHi and the time to the onset of the maximum swelling rate decreases. The maximum swelling rate is initiated at pHi 7.4 when pH0 is 7.8 and at pHi 7.1 when pH0 is 7.4. The maximum rate of swelling increases linearly with increasing pH0 in the range from 7.0 to 8.2. This rate also shows a linear relationship to the value of pHi at the time the maximum rate is attained. Dixon plots of the reciprocal of the maximum swelling rate vs [H+]0 suggest that external [H+] is a noncompetitive inhibitor of K+ entry on the antiport. It is concluded that K+/H+ antiport in Mg(2+)-depleted heart mitochondria can be regulated by matrix [H+] (see Beavis, A. D., and Garlid, K. D. (1990) J. Biol. Chem. 265, 2538-2545), but that this antiport is also sensitive to external [H+] or to delta pH when it acts in the direction of K+ uptake.     

Modulation of the activity of cytosolic phospholipase A2�� (cPLA2��) by cellular sphingolipids and inhibition of cPLA2�� by sphingomyelin

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2α (cPLA2α) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2α inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2α. Phosphatidylcholine vesicles containing SM reduced cPLA2α activity. Furthermore, SM disturbed the binding of cPLA2α to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2α-dependent release of AA by inhibiting the binding of cPLA2α to glycerophospholipids.     

Diffusion rates of cell surface antigens of mouse-human heterokaryons. III. Regulation of lateral diffusion rates by calcium ions

In mouse-human heterokaryons, the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membrane is enhanced by treatment of parent cells with ouabain. Ouabain treatment is ineffective if the medium lacks calcium ion, or if Verapamil, a blocker of calcium channels, is present. The divalent ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 also enhances lateral diffusion of MHC antigens, to the same extent as ouabain, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 is effective only if calcium is present in the medium. Thus it appears that increased levels of cell calcium release constraints to lateral diffusion of MHC antigens.     

Regulation of intracellular Mg2+ by superoxide in amnion cells.

Changes of intracellular free Mg2+ concentration ([Mg2+]i) in human amnion cells induced by superoxide anion were determined using a highly Mg(2+)-sensitive fluorescent dye Mg(2+)-fura2 or Mg(2+)-indol. Superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, induced decrease of [Mg2+]i. The decrease was significantly inhibited by an anion channel blocker, 4,4'diisothiocyano-2,2' disulfonic acid stilbene (DIDS). Superoxide dismutase (SOD), injected into cells by cell fusion, also inhibited the change of [Mg2+]i, but catalase did not. Superoxide anion induced prompt increase of intracellular pH (pHi) as well as decrease of [Mg2+]i and subsequently activated the increase of intracellular free Ca2+ ([Ca2+]i) and the release of arachidonate. In contrast to superoxide anion, NH4Cl which induces increase of pHi in amnion cells increased [Mg2+]i. The elevation of basal level of [Mg2+]i by Mg(2+)-ionophore inhibited the change of [Ca2+]i and the release of arachidonate induced by superoxide anion. These results suggest that superoxide anion, transported through anion channels into cells, decreases [Mg2+]i directly, not due to a pH-effect and that the decrease of [Mg2+]i may regulate biological functions of the cells via increase of [Ca2+]i.     

Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.

alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin''s effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.     

Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A₂α (cPLA₂α) or calcium-independent PLA₂. Surprisingly, release of arachidonic acid and LDH from cPLA₂α-deficient fibroblasts was inhibited by the cPLA₂α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca²⁺ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and H₂O₂. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.     

Inhibitors of arachidonic acid metabolism eliminate the increase in cytosolic free calcium induced by the mitogen concanavalin A in rat thymocytes

Using inhibitors of arachidonic acid (AA) metabolism, the possible involvement of AA products in the generation of [Ca2+]i and the pHi rise induced by the mitogen concanavalin A (Con A) in rat thymocytes has been studied. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) and the phospholipase A2 inhibitor bromophenacyl bromide (10 microM) eliminated the [Ca2+]i signal induced by Con A; the cyclooxygenase blocker indomethacin also inhibited it. However, neither NDGA nor indomethacin suppressed the pHi rise stimulated by Con A. Exogenous AA induced an increase in [Ca2+]i but not in the pHi. These results indicate that AA metabolites, probably of the lipoxygenase pathway, take part in the generation of the [Ca2+]i response to the mitogen. In contrast, they appear not to be involved in the pHi rise evoked by Con A.     

Lipoxygenase inhibitors suppress intracellular calcium rise induced by ionomycin in rat thymocytes

The lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and 15S-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE) have been found to suppress the rise in free cytoplasmic Ca2+ concentration [( Ca2+]i) induced by the Ca2+ ionophores ionomycin and A23187 in rat thymocytes. Bromophenacyl bromide (BPB), a phospholipase A2 (PLA2) inhibitor, produced a much weaker inhibitory effect, and indomethacin, a cyclo-oxygenase inhibitor, practically did not influence the [Ca2+]i response to ionomycin. These findings implicate the involvement of LO product(s) in the [Ca2+]i rise triggered by the Ca2+ ionophores. The contribution of the NDGA-sensitive component to the ionomycin-induced [Ca2+]i rise was significant in the ionomycin concentration range of 0.1 nM to 0.1 microM whereas at higher doses of the ionophore it gradually diminished. By contrast, the [Ca2+]i rise induced by exogenous arachidonic acid (AA) or melittin, a PLA2 activator, was not suppressed but potentiated by NDGA. Ionomycin and exogenous AA also elicited opposite changes in thymocyte cytoplasmic pH (pHi): the former elevated the pHi while the latter induced a pronounced acidification of the cytoplasm. This difference in the pHi responses may account for the different sensitivity of ionomycin- and AA-elicited [Ca2+]i signal to LO inhibitors.     

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.     

Trifluoperazine,a calmodulin antagonist,inhibits muscle cell fusion

We investigated the effect of trifluoperazine (TFP), a calmodulin antagonist, on the fusion of chick skeletal myoblasts in culture. TFP was found to inhibit myoblast fusion. This effect occurs at concentrations that have been reported to inhibit Ca2+-calmodulin in vitro, and is reversed upon removal of TFP. In addition, other calmodulin antagonists, including chlorpromazine, N-(6-aminohexyl)-5- chloro-1-naphthalene-sulfonamide (W7), and N-(6-aminohexyl)-1- naphthalene-sulfonamide (W5), inhibit fusion at doses that correspond closely to the antagonistic effects of these drugs on calmodulin. The expression of surface acetylcholine receptor, a characteristic aspect of muscle differentiation, is not impaired in TFP-arrested myoblasts. Myoblasts inhibited from fusion by 10 microM TFP display impaired alignment. In the presence of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, the fusion block by 10 microM TFP is partially reversed and myoblast alignment is restored. The presence and distribution of calmodulin in both prefusional myoblasts and fused muscle cells was established by immunofluorescence. We observed an apparent redistribution of calmodulin staining that is temporally correlated with the onset of myoblast fusion. Our findings suggest a possible role for calmodulin in the regulation of myoblast fusion.