A calcium ionophore-induced secretion in rabbit lacrimal gland in vivo

Local administration of the calcium ionophore, A-23187 increased basal fluid secretion (non-stimulated) from the cannulated main excretory duct of rabbit lacrimal gland in vivo. A-23187 also facilitated fluid secretion induced by submaximal dose of methacholine (0.1 μg/kg, intraarterially). The stimulatory effect of A-23187 was dependent on the extracellular calcium concentration. Lowering the extracellular calcium by addition of EGTA markedly depressed or abolished the responses to the ionophore while increasing the extracellular calcium with CaCl₂ enhanced it. The results suggest that A-23187 causes increase in cell membrane permeability to extracellular calcium and the rise in intracellular calcium activates the secretory process(es) by an unknown mechanism to produce fluid secretion in the rabbit lacrimal gland.     

The induction of sporulation in the aquatic fungus Blastocladiella emersonii is dependent on extracellular calcium

Induction of sporulation in Blastocladiella emersonii is absolutely dependent on extracellular calcium. Vegetative cells grown in media with or without calcium do not sporulate in media devoid of calcium or in CaCl₂ with EGTA. Calcium channel blockers, CoCl₂ and nifedipine, and ionophore A23187 inhibited the induction of sporulation. The calmodulin antagonists trifluoperazine and chlorpromazine inhibited the sporulation when present in the cultures at least 60 min after induction. So, calcium that is accumulated during growth is not sufficient or is not mobilized to initiate sporulation, and a calcium influx is likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein has been suggested to mediate calcium events in sporulation.     

Indoleamines and calcium channels influence morphogenesis in in vitro cultures of Mimosa pudica L.

The present article reports the interplay of indoleamine neurohormones viz. serotonin, melatonin and calcium channels on shoot organogenesis in Mimosa pudica L. In vitro grown nodal segments were cultured on MS medium with B5 vitamins containing Serotonin (SER) and Melatonin (MEL) at 100 µM and indoleamine inhibitors viz. serotonin to melatonin conversion inhibitor p-chlorophenylalanine (p-CPA) at 40 µM, serotonin reuptake inhibitor (Prozac) 20 µM. In another set of experiment, calcium at 5 mM, calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) 100 µM, and calcium channel blocker varapamil hydrochloride (1 mM) a calcium chelator EGTA (100 µM) were administered to the culture medium. The percentage of shoot multiplication, endogenous MEL and SER were monitored during shoot organogenesis. At 100 µM SER and MEL treatment 60% and 70% explants responded for shoot multiplication respectively. Medium supplemented with either SER or MEL along with calcium (5 mM) 75%–80% explants responded for organogenesis. SER or MEL along with calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) at 100 µM 70% explants responded for shoot multiplication. p-CPA, prozac, verapamil and EGTA, shoot multiplication was reduced and endogenous pools of SER, MEL decreased by 40–70%. The results clearly demonstrated that indoleamines and calcium channels positively influenced shoot organogenesis in M. pudica L.     

Proliferation of human peripheral blood lymphocytes induced by A23187, a streptomyces antibiotic

Incubation of human peripheral blood lymphocyte cultures with streptomyces antibiotic A23187, a divalent cation ionophore, resulted in an increased rate of calcium uptake, enhanced rates of RNA and DNA synthesis, and lymphoblastic transformation. An optimal response was obtained with an initial ionophore concentration of 3–5 μM. The highest rate of thymidine incorporation was detected when the cells were labelled from the 3rd to 4th day of culture. In long-term culture the ionophore was highly toxic to the lymphocytes and optimal response was detected only if the cells were transferred to fresh medium after incubating for some hours with A23187. Both RNA and DNA synthesis, as well as calcium uptake induced by A23187 were completely inhibited if ethyleneglycol-bis-(aminoethylether)tetraacetic acid (EGTA) was present in the culture during the first 6 h of incubation. These findings support the hypothesis that calcium ion has a critical role in the mitogenic response of lymphocytes, and that calcium influx may be an important event in the initiation of proliferation. Possible mechanisms of the effects of A23187 on lymphocytes are discussed.     

The Effect of Calcium Antagonists on Auxin-induced Elongation and on the Expression of Two Auxin-Regulated Genes in Pea Epicotyls

Calcium has been implicated in various regulatory roles in plantcells including auxin-induced cell elongation. Treatment ofpea epicotyl segments with the calcium chelators, EGTA and chlorotetracycline(CTC), the calcium ionophore, A23187 [GenBank] , and channel blocker, D-600,inhibits auxin-induced cell elongation. Depletion of tissuecalcium either by EGTA or EGTA and a calcium ionophore doesnot interfere with the induction of the early auxin induciblemRNAs pIAA4/5 and pIAA6. Similarly, an increase in cytosoliccalcium with calcium and calcium ionophore neither induces thehormonally regulated mRNAs nor interferes with their inductionby auxin. The calcium channel blocker, D-600, is without effecton the auxin-regulated mRNA induction. The results indicatethat calcium is not involved in the rapid induction of IAA4/5and IAA6 genes in pea tissue. However, a possible role for calciumin the translation of these mRNAs, or in the expression of otherauxin-regulated genes, is not excluded. ³Present Address: Department of Biology, Tokyo MetropolitanUniversity, Tokyo, Japan. (Received April 8, 1988; Accepted July 30, 1988)     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Calcium and calcium ionophore A23187 induce high-frequency somatic embryogenesis in cultured tissues of <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr

High-frequency somatic embryogenesis was achieved in Coffea canephora using calcium ionophore A23187, which influences the influx of calcium into a cell. With 100 μM calcium ionophore and 5 mM calcium, 85% and 70% of cultures produced embryogenic tissue, with 105 ± 7 and 95 ± 8 primary embryos from each callus mass respectively. Medium supplemented with 100 μM EGTA (calcium chelator) or 1 mM verapamil (calcium channel blocker) significantly reduced somatic embryogenesis. Calcium imaging studies were done to determine the relationship between morphogenetic response and the cellular calcium levels. The calcium ionophore/calcium treatment was very effective in driving cellular machinery toward embryogenesis. The embryos were regenerated into plantlets when cultured on MS medium supplemented with 5 mM calcium/100 μM calcium ionophore A23187. Somatic embryogenesis-derived plants were successfully transferred to soil and grown to maturity in the field.     

Calcium requirements in the action of cholecystokinin-pancreozymin on pancreatic acinar cell metabolism.

The present study utilized ionophore A23187 to determine the role of Ca²⁺ in pancreatic acinar cell metabolism. The ionophore A23187 in the presence of EGTA increased efflux of Ca²⁺ from the rat pancreatic fragments. Ionophore and CCK-PZ were equally effective in the presence of extracellular Ca²⁺ in stimulating ¹⁴C-labeled protein secretion. The ionophore decreased synthesis of new protein more effectively than CCK-PZ in the presence of extracellular Ca²⁺. The effect of ionophore and CCK-PZ in combination was greater than either agent alone. Phospholipid labeling was not stimulated by A23187 in the presence of extracellular Ca²⁺ in contrast to CCK-PZ. With CCK-PZ, the effect was dependent on the concentration of extracellular Ca²⁺. Protein phosphorylation was stimulated ～ 109% by CCK-PZ and ～ 39% by ionophore. CCK-PZ stimulated protein phosphorylation in the 100,000 g supernatant whereas A23187 was ineffective. Ionophore A23187 inhibited glucose oxidation whereas CCK-PZ stimulated glucose oxidation. These data suggest that more than one kinase system might be involved in metabolic responses to hormonal stimulation of the pancreas viz. a phosphorylase kinase may be directly activated by Ca²⁺ causing protein discharge whereas other kinase system may require binding of the hormone to receptor leading to other events besides protein discharge.     

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.     

Active calcium transport and calcium-dependent membrane phosphorylation in human peripheral blood lymphocytes

The characteristics of the calcium pump were investigated in intact human peripheral blood lymphocytes /PBL/ and in inside-out vesicles prepared from their plasma membranes. Intact PBL were loaded with calcium by a short exposure to A23187 ionophore. After the elimination of the ionophore, calcium-loaded PBL produced an ATP-dependent, external lanthanum sensitive, uphill calcium extrusion. Calcium pump in intact PBL was insensitive to ouabain and /until cellular ATP was provided/ to oligomycin and dinitrophenol. Maximum calcium extrusion rate and the alkali cation sensitivity of the process were similar to those in human red cells. Calcium was partially sequestered by PBL, and this calcium could be released by A23187 ionophore only.Inside-out plasma membrane vesicles prepared from hypotonically lysed PBL showed and ATP + Mg²⁺-dependent uphill calcium uptake. This calcium transport was insensitive to ouabain, oligomycin, or dinitrophenol, while blocked by lanthanum and quercetin. Calmodulin significantly stimulated calcium pumping in EDTA-washed vesicles. ATP-dependent and -independent calcium uptake rates, respectively, showed different calcium concentration dependences.When PBL membrane vesicles were phosphorylated by γ ³²P-ATP, a calcium-induced, hydroxylamine-sensitive incorporation of ³²P was found in 120–150 000 molecular weight proteins. Depending on the way of membrane preparation, the molecular weight of the phosphoprotein was shifted. Similarly to that found in red cell membranes, sensitivity to calmodulin stimulation and partial proteolysis of the calcium pump molecule showed an inverse relationship.     

Calcium and lymphocyte activation.

The role of ionized calcium in the early phases of activation of human peripheral blood lymphocytes was evaluated by stimulating the cells with a calcium ionophore A23187 (Lilly) or with mitogenic lections over a broad range of extracellular calcium concentrations (< 1 to > 1000 μM). A number of biochemical parameters shown previously to be altered during stimulation of these cells by mitogenic lectins were studied including: 1) amino acid transport, 2) phosphatidylinositol turnover, 3) cyclic nucleotide accumulation, and 4) calcium uptake. The ionophore (0.1–0.5 μg/ml) was shown to produce stimulatory effects in all of these systems with the changes closely simulating those produced by the lectins themselves both in regard to time course and magnitude. A23187 also produced 5–10 fold increases in DNA synthesis as measured at 48–72 hr after exposure of the cells to this agent. The responses to A23187 were shown to be almost completely dependent on the presence of ionized calcium. Since mitogenic lectins are known to stimulate calcium uptake and DNA synthesis appears to require extracellular calcium, the early responses to A23187 suggested that calcium was important both during the early and later phases of lymphocyte activation. However, short time course studies of amino acid transport, cyclic AMP accumulation, and phosphatidylinositol turnover in calcium deficient media failed to provide convincing evidence of calcium dependency in lectin stimulation since the three responses were well preserved (<25% inhibition) in “calcium free” medium containing 1–3 mM ethylene bis (ethylene oxynitrilo) tetraacetic acid (EGTA) (an estimated final Ca²⁺ concentration of <1 μM). Greater than 50% inhibition of the lectin response was seen only when the cells were incubated in calcium free, EGTA-containing medium for 30 min prior to stimulation with lectin. Thus despite the striking ability of A23187 complexed with calcium to mimic the action of mitogenic lectins, its effects may involve more than simple transport of calcium into the cell. A23187 may also exert a direct membrane action as suggested by its ability to produce rapid increases in cAMP and the occurrence of cytotoxicity at 5–10 fold higher concentrations (2–4 μg/ml). However, these data do not entirely exclude a mechanism of ionophore action whereby: 1) mobilization of intracellular stores of calcium and 2) diminished intracellular transport of ionized calcium at extracellular concentrations less than or equal to 1 μM combine to provide an effective stimulus for cellular activation.     

Effects of the calcium ionophore A-23187 on the regulation of sugar transport in muscle

The distribution of (¹⁴C)-3-0-methyl-D-glucose and of (⁴⁵Ca) was followed in perifused left atria and intact hemidiaphragms of the rat. The carboxylic calcium ionophore A-23187 affected sugar and Ca²⁺ influx in parallel, with low concentrations inhibiting and higher ones stimulating influx under basal conditions. The stimulation of sugar transport by insulin, high concentrations of adrenaline or ouabain, or by K⁺-free medium was antagonized by the calcium ionophore. Likewise, A-23187 counteracted the depression of sugar transport caused by low concentrations of ouabain or adrenaline. These results support a role of Ca²⁺ in the regulation of sugar transport in muscle. However, increased influx of Ca²⁺ cannot explain all the effects of A-23187. It is suggested that the ionophore may also act by releasing Ca²⁺ from intracellular storage and binding sites.     

Calcium and cyclic nucleotide involvement in exocrine pancreatic enzyme secretion studied with the ionophore A-23187.

The ionophore, A-23187, was an effective pancreatic secretagogue. The response to A-23187 was Ca²⁺-dependent; Mg²⁺ reduced the secretory response to the ionophore. A-23187-stimulated enzyme release was potentiated by dibutyryl cyclic AMP; in the presence of carbachol, output of pancreatic protein paralleled the response to A-23187 alone. The time-course for secretion with A-23187 was similar to that observed with carbachol. The ionophore did not affect basal cyclic AMP levels but did stimulate a rapid Ca²⁺-dependent production of pancreatic cyclic GMP which preceded the onset of the secretory response. A-23187 did not significantly alter basal or carbachol-stimulated ⁴⁵Ca efflux from isotope preloaded glands; yet in Ca²⁺-lowered media, it inhibited (reversed) the secretory response to carbachol, an effect which may have been due to an outward transport by the ionophore of cholinergic-mobilized intracellular Ca²⁺. Like carbachol, A-23187, inhibits the incorporation of amino acid into new protein, the effect being partially dependent on extracellular Ca²⁺. The data suggest that the pancreatic cholinergic receptor acts as a Ca²⁺-ionophore and that extracellular Ca²⁺ is utilized in the synthesis of cyclic GMP.     

On the mechanism of stimulation of H+ transport in gastric mucosa by Ca++ ionophore A23187: I. Pathways for cytosolic calcium increase

The pathways for cytosolic Ca⁺⁺ increase under A23187 stimulation of H⁺ secretion were studied in the isolated gastric mucosa of the toad $\text{Bufo marinus}$. A23187 produced a more potent stimulation of secretion when added to the mucosal side which did not contain calcium. Measurements of ionophore incorporation by fluorometric methods indicated that A23187 incorporates into oxyntic cells intracellularly. The presence of divalent cations inhibited incorporation. This may be the reason for a more potent action when A23187 was added from the mucosal side. With-drawal of calcium from serosal solution largely inhibited the secretory response to A23187 added to the mucosal side. Reintroduction of calcium into the serosal side in the presence of ionophore elicited H⁺ secretion. The results are consistent with an uptake of A23187 from the mucosal side into cellular organelles and basolateral membranes. Calcium entry through the serosal side may be responsible for triggering secretion. Although A23187 likely releases calcium from intracellular stores, its rate of release may not be sufficient to bring about a full stimulation of secretion in serosal-Ca⁺⁺-free conditions.     

Signal transduction in plants: evidence for the involvement of calcium and turnover of inositol phospholipids

Involvement of calcium and turnover of inositol phospholipids in signal transduction was investigated using roots of a variety of corn (Zea mays L., cv. Merit) which require light to develop gravitropic sensitivity. Depletion of calcium in root tips by EGTA plus calcium ionophore A23187 prior to light treatment resulted in the loss of light-dependent gravisensitivity. Replenishment of calcium to depleted roots restored the light-dependent gravisensitivity. Light treatment of dark-grown roots resulted in an increased level of inositol trisphosphate as compared to controls. Furthermore, 5-hydroxytryptamine, which is known to promote the hydrolysis of phosphoinositides, sensitized dark-grown roots to gravity and increased inositol trisphosphate levels. These results support the hypothesis that calcium and inositol phospholipid turnover play a role in signal transduction in plants.     

Erratum

The ionophoretic capabilities of dioleoylphosphatidic acid (DOPA) for transporting calcium across phospholipid bilayers have been investigated. Calcium uptake by large unilamellar vesicles is shown to depend on the presence of DOPA. This uptake is sensitive to the nature and concentration of calcium chelators in the vesicle interior, indicating that accumulation results from DOPA-mediated translocation of calcium across the membrane. Further, it is shown that characteristics of DOPA-mediated Ca²⁺ uptake are similar to those observed for the fungal calcium ionophore, A23187.     

Phorbol ester induces inhibition of arachidonate incorporation into phospholipids in human neutrophils

PMA is known to enhance calcium ionophore A-23187 induced arachidonate release in human neutrophils. Mechanism of enhancement by PMA is not clear. We have found that neutrophils pretreated with PMA showed significant reduction in labeled arachidonate uptake. Decrease in arachidonate uptake following PMA treatment was attributed, at least in part, to inactivation of arachidonoyl CoA synthase and arachidonoyl CoA lysophosphatide acyltransferase, two key enzymes involved in arachidonate incorporation into phospholipids. These results suggest that PMA may induce protein kinase C activation which in turn may cause inactivation of the two enzymes involved in incorporation of arachidonate resulting in greater availability of arachidonate which is liberated by A-23187 for oxygenation and release into extracellular space. Abbreviations: PMA, 4 beta-phorbol 12-myristate 13-acetate; PDD, 4 alpha-phorbol 12,13-didecanoate; TXB2, thromboxane B2; LTB4, leukotriene B4; PC, phosphatidylcholine; LPC, lysophosphatidylcholine; DMSO, dimethylsulfoxide.     

Calcium in the Control of Nitrogenase Activity in Vicia faba L. Root Nodules: Calcium Release from Symbiosomes and the Accompanying Decrease in Their Nitrogenase Activity Is Blocked by Verapamil

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or together with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron-microscopic analysis revealed considerable calcium depletion of symbiosomes in the infected nodule cells treated with EGTA and A23187. Ca²⁺ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca²⁺ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached its initial level in symbiosomes. The experiments on isolated symbiosomes with arsenazo III, a Ca²⁺ indicator, demonstrated that verapamil inhibited Ca²⁺ release from them induced by valinomycin in the presence of K⁺ ions. These data suggest the presence on the peribacteroid membrane of a verapamil-sensitive transporter responsible for Ca²⁺ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of root nodules is discussed.     

Senescence of Rice Leaves XXIV. Involvement of Calcium and Calmodulin in the Regulation of Senescence

Effects of compounds that influenced calcium uptake and calmodulininhibitors on the senescence of detached rice leaves were examined.Chelators, ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraaceticacid (EGTA) and l,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraaceticacid (BAPTA), significantly promoted senescence of detachedrice leaves in the dark and light. The effect of EGTA can bereversed by treating detached rice leaves with calcium. Verapamil,a calcium channel blocker, and lanthanum chloride, a calciumantagonist, promoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. Calcium ionophore A23187 [GenBank] and ruthenium red, believed to raise cytosolic level of Ca²⁺,were quite effective in retarding dark-induced and ABA-promotedsenescence of detached rice leaves. Calmodulin inhibitors, W-7,compound 48/80, chlorpromazine and trifluoperazine, significantlypromoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. It is concluded that cytosoliclevel of Ca²⁺ may regulate senescence of detached rice leavesthrough a calmodulin-dependent mechanism. (Received June 13, 1990; Accepted August 3, 1990)     

Lipochondria and the light response of Aplysia giant neurons

The ultrastructure, absorbance, and elemental content of lipochondria present in the cytoplasm of Aplysia giant neurons have been investigated before and after 30–1,200 sec doses of white light at intensities which produce saturated light responses. The effects of exposure to the calcium ionophore A-23187 and to EGTA were also examined. The lipochondria of nonilluminated neurons are membrane-bound, and contain lipids, protein, Na, K, Mg, Ca, Si, Cl, Br, P, and a pigment which is probably β-carotene. The cytoplasm appeared to have little pigment. When neurons were illuminated for 20 min, 60–70% of the lipochondria showed marked ultrastructural alterations, the most notable being the appearance of membranous material. Earlier changes which occur after 30 sec of illumination include the appearance of paracrystalline arrays and mottling. Less than 10% of lipochondria in nonilluminated neurons have a similar appearance. These effects were greatly enhanced in illuminated neurons exposed to the calcium ionophore or EGTA. In nonilluminated neurons, the ionophore also produced ultrastructural changes. In frozen specimens, the calcium content of the most electron dense lipochondria of illuminated neurons was reduced. Other elements which were counted were also reduced. The lipochondria are the main intracellular site of photopigment. They may also act as an intracellular source for calcium which, as the accompanying paper indicated, may mediate phototransduction in Aplysia neurons.