The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3'':5''-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.

1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.     

Oligomers of prostaglandin B1 inhibit arachidonic acid mobilization in human neutrophils and endothelial cells

The previous paper (Biochim. Biophys. Acta 1006 (1989) 272-277) has demonstrated that oligomers of prostaglandin B1 are effective in vitro inhibitors of a wide range of both cell-derived and extracellular phospholipases A2. The present study has investigated the effects of prostaglandin oligomers on agonist-stimulated phospholipase activity on intact human cells. PGBx, an oligomer (n = 6) or PGB1, and PGB-trimer inhibit as much as 95% of the A23187-stimulated release of arachidonic acid from human neutrophils. The effect is dose-dependent, with an IC50 of 4-5 microM; near maximal inhibition is obtained with as little as 1 min of preincubation with PGB-trimer. Consistent with its role as a phospholipase A2 inhibitor, PGB-trimer also inhibits the A23187-stimulated incorporation of [3H]acetate into platelet-activating factor. PGBx and PGB-trimer also inhibit the release of arachidonic acid from human umbilical vein endothelial cells stimulated with histamine, thrombin, or ionophore A23187; inhibition of the basal or unstimulated turnover of both arachidonic acid and oleic acid is also observed. Inhibition by PGB-trimer can be blocked by simultaneous addition of 50 microM albumin; cells preincubated with PGB-trimer are not affected by albumin. Furthermore, removal of exogenous PGB-trimer prior to challenge with A23187 does not reverse the inhibition of either endothelial cells and neutrophils. Thus, prostaglandin B1 oligomers are taken up by human neutrophils and vascular endothelial cells and serve as potent inhibitors of arachidonic acid mobilization. One mechanism for the pharmacological effects of PGBx may be inhibition of cell-associated and extracellular phospholipase A2.     

Regulation of leukotriene synthesis by arachidonic acid in human polymorphonuclear leukocyte adhesive interactions is dependent on the presence of albumin

We have previously demonstrated that the pretreatment of polymorphonuclear leukocytes (PMNs) with the chemotherapeutic drug, Suramin, increases both cell attachment and inhibits calcium ionophore A23187-stimulated leukotriene (LT) synthesis. Here, we examined the effects of extracellular arachidonic acid (AA) and albumin on attachment and LT synthesis in the interaction of PMNs with both collagen-coated surfaces and human umbilical vein endothelial cell (HUVEC) monolayers. Suramin decreased the release of radiolabelled AA and 5-lipoxygenase metabolites by [(14)C-AA]-prelabelled PMNs stimulated with A23187, with and without human serum albumin (HSA) in the culture medium. Addition of 1 microM AA together with calcium ionophore stimulated the release of endogenous AA to the same level as control and Suramin-pretreated cells, but attachment was unaffected and LT synthesis was still inhibited with Suramin treatment. Using 24 microM AA, regulation of LT synthesis was dependent on the presence of HSA in the medium. Without HSA, 24 microM AA induced detachment of PMNs and increased LT synthesis in Suramin-treated cells above the control level. In the presence of HSA, 24 microM AA did not influence PMN attachment or abolish Suramin-induced inhibition of LT synthesis. These results suggest that tight attachment of PMNs to a solid surface leads to decreased LT synthesis during subsequent stimulation of the cells by A23187 in the presence or absence of exogenous substrate.     

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.     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.     

Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore

In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O2-)release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca2+ activated, phospholipid-dependent protein kinase, designated "protein kinase C." PMA enhanced O2- release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca2+. A similar increase was produced by A23187; omission of extracellular Ca2+ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O2- release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2-.     

Influence of extracellular calcium on cell permeabilization and growth regulation by the lymphokine leukoregulin

Permeablization of human K562 leukemia cells was measured in the presence and absence of extracellular ionic calcium to examine the relationship of ionic calcium to increased membrane permeability and the inhibition of cell proliferation by this lymphokine. In the absence of extracellular calcium, the ability of leukoregulin to permeabilize the cell membrane is diminished but is fully restored by addition of 1 mM extracellular Ca++ as shown flow cytometrically by loss of intracellular fluorescein. Membrane permeability is also increased by calcium ionophore A23187 but permeablization is completely blocked in calcium-free medium despite the intramembrane presence of the calcium ionophore. Membrane permeablization by the lectin phytohemagglutinin, in contrast, is independent of extracellular calcium. A similar divergence in cell proliferation activity of the three modulators of calcium flux and membrane permeability occurs in the absence of extracellular calcium. Leukoregulin inhibition of cell proliferation is abolished, inhibition by calcium ionophore A23817 is greatly reduced, and inhibition by phytohemagglutinin is unchanged. Leukoregulin permeabilized K562 cells isolated by fluorescence activated cell sorting resume proliferation after 72 h. In contrast cells permeablized by calcium ionophore A23187 or phytohemagglutinin fail to resume proliferation by 7 days. The membrane permeablizing action of leukoregulin is, therefore, partially dependent upon extracellular calcium. It is also effected through a mechanism other than calcium ionophore transport or lectin type transmembrane signaling, and is accompanied by a reversible inhibition of cell proliferation.     

Role of phospholipid hydrolysis in the mechanism of toxic cell death by calcium and ionophore A23187

Manganese chloride inhibits the hydrolysis of arachidonate-containing phospholipids stimulated in 3T3 mouse fibroblasts by ionophore A23187 in the presence of extracellular calcium. The inhibition is reduced by increasing extracellular calcium concentrations. Stimulation by A23187 of this phospholipid hydrolysis and cell killing are inhibited at similiar concentrations by (i) manganese chloride or (ii) reduced extracellular calcium. These results indicate an important role for the phospholipid hydrolysis in the mechanism of cell killing by A23187 plus calcium. Analysis of the rates of the two processes indicates that phospholipid hydrolysis triggers cell killing, but it is not itself the membrane permeabilizing step.     

Calcium as a regulator of juvenile hormone biosynthesis and release in the cockroach Diploptera punctata

The role of calcium in the modulation of juvenile hormone (JH) biosynthesis and release by the corpora allata (CA) of Diploptera punctata was examined using an in vitro radiochemical assay. JH production showed a dose dependence on extracellular calcium in the incubation medium. Rates of JH release were maximal between calcium concentrations of 3–5 mM and were almost totally inhibited in its absence. Upon return to medium containing 5 mM calcium, CA exhibited a rapid increase in JH release, although rates of release remained slightly below normal.Blockers of voltage-dependent calcium channels (verapamil, nifedipine), at physiological doses, were able to modulate JH production whereas non-specific calcium channel blockers such as lanthanum effectively inhibited JH release. The calcium ionophore A23187 caused a rapid and irreversible decline in JH release. The calcium dose-response for A23187 showed 50% inhibition of JH release at about 1 mM calcium and maximal inhibition (93%) at 6 mM calcium. Treatment with lanthanum or A23187 did not result in an accumulation within the CA of either JH or methyl farnesoate and accordingly, these compounds appeared to reduce overall JH biosynthesis rather than inhibiting release. Inhibition of JH release by A23187 was dramatically attenuated by coincubation with cobalt, although cobalt alone was found to stimulate JH release significantly. Intracellular calcium levels thus appear to be important in the regulation of JH biosynthesis and release.     

Acrosomal status in fresh and capacitated human ejaculated sperm

The acrosomal status of human sperm was evaluated by immunofluorescence utilizing a specific monoclonal antibody that recognizes target antigen(s) localized in the acrosomal cap region. Spontaneous acrosomal loss was first examined in sperm preparations used for successful in vitro fertilization of human eggs. In these sperm populations, less than 20% of the sperm underwent degenerative or spontaneous acrosomal loss following 24 h of incubation. The correlation of acrosomal loss with changes in motility and viability suggested that sperm senescence was not necessarily coupled to acrosomal loss. Chemical induction of acrosomal loss by calcium ionophore A23187 and lysophosphatidylcholine (LPC) was characterized. Maximal ionophore induction (10 microM A23187 in media containing calcium) was observed in cells exposed to capacitating conditions in vitro; sperm exposed to noncapacitating conditions did not readily acquire the ability to respond to ionophore. The reaction induced by ionophore was slow (60 min), and at least 30% of the cells were always resistant to induction. In contrast, LPC induced rapid, synchronous acrosomal loss in either freshly ejaculated or capacitated sperm in the presence or absence of extracellular calcium, suggesting that this loss was not a physiologic reaction. These studies may provide a basis for evaluating capacitation and ultimately fertility potential in the human male.     

Intracellular or extracellular calcium can be used to trigger C5a-stimulated enzyme secretion from rabbit neutrophils

The ability of C5a to stimulate lysosomal enzyme release and 45Ca2+ efflux from rabbit neutrophils was studied. C5a stimulated beta-glucuronidase release from cytochalasin B-treated neutrophils either in the presence or absence of extracellular calcium. Depletion of cell calcium by pretreatment with the calcium ionophore A23187 blocked both the ability of C5a to elicit enzyme release in the absence of extracellular calcium and its ability to stimulate 45Ca2+ efflux. Both actions were dose-dependent over the same concentration range (10(-8)-10(-6) M ionophore A23187). In contrast, ionophore pretreatment had no effect on C5a-stimulated enzyme release in the presence of extracellular calcium. These results suggest that (a) release of cell calcium is required for enzyme secretion in the absence of extracellular calcium, and (b) C5a can trigger near-maximal enzyme release by using calcium from either of two sources: the extracellular space or an intracellular site.     

Changes in cellular levels of cyclic AMP in rat mast cells during secretion of histamine induced by immunoglobulin E decapeptide and ACTH(1–24) peptide: Comparison with immunological and ionophore triggers

The role of cyclic AMP in the secretory mechanism of mast cells has been investigated by comparing the time course of changes in cellular levels of this cyclic nucleotide with the kinetics of secretion induced by basic peptides, antigen, anti-IgE and calcium ionophore. ACTH(1–24) peptide and a synthetic decapeptide representative of the sequence 497–506 within the Cε4 domain of human IgE induced a transient rise in cyclic AMP which reached approx. 150% of the resting levels by 10 s. Peptide-induced secretion of histamine was also rapid, reaching a maximum after 5–10 s. Immunological triggering of mast cells with antigen and anti-IgE raised levels of cyclic AMP to 150% of resting levels within 15 s, accompanying secretion of histamine which reached a maximum after 30 s. A relatively slower release of histamine induced by the calcium ionophore A23187 was paralleled by a significant reduction in cyclic AMP to 50% of the resting levels after 300 s. These data suggest a relationship between the accumulation of cyclic AMP in mast cells and secretion of histamine mediated by the Cε4 decapeptide and the ACTH(1–24) peptide as well as by IgE-dependent mechanisms. However, the simultaneous increase in cyclic AMP and secretion of histamine suggests that the two events may not be causally related.     

Amino acid transport in isolated rat thymocytes. Effects of divalent cations and ethanol.

In dispersed rat thymocytes neither basal alpha-aminoisobutyric acid influx nor influx stimulated by insulin, prostaglandin theophylline, or butyryl adenosine 3':5'-monophosphate (cyclic AMP) depended on extracellular calcium or magnesium. The divalent cation ionophore A23187 inhibited both basal and stimulated alpha-aminoisobutyric acid influx. The extent to which influx was inhibited depended on ionophore concentration, extracellular calcium concentration, and time but did not depend on extracellular magnesium. Significant inhibition could be detected at an ionophore concentration of 1 muM and maximal inhibition occurred with 6 muM A23187. A23187 increased cellular uptake of calcium and there was good agred calcium uptake and that for ionophore inhibition of alpha-aminoisobutyric acid influx. Incubating cells with A23187 and then adding ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid completely reversed ionophore-stimulated cellular calcum uptake but did not reverse inhibition of alpha-aminoisobutyric acid influx. Thus, A23187 produces irreversible inhibition of alpha-aminoisobutyric acid transport in dispersed rat thymocytes. Ethanol abolished insulin-stimulated alpha-aminoisobutyric acid influx but did not alter basal influx or that stimulated by prostaglandin E1, theophylline, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate. Inhibition could be detected with 0.2% (v/v) ethanol and insulin-stimulated alpha-aminoisobutyric influx was abolished with 1% ethanol. The effect of ethanol occurred immediately and could be reversed completely. This ability of ethanol to inhibit selectively insulin-stimulated alpha-aminoisobutyric acid influx indicates that the mechanism through which insulin stimulates alpha-aminoisobutyric acid influx is functionally distinct from the stimulation produced by cyclic AMP.     

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.     

Intracellular calcium as a modulator of transepithelial permeability to water in frog urinary bladder

The divalent cation ionophore A 23187 was used to evaluate the action of intracellular calcium on net transepithelial water movement across the isolated frog urinary bladder. Incubation with the ionophore increases the net basal water flux in a dose-dependent fashion but independent of the extracellular calcium concentration. Bladders pretreated with A 23187 and exposed thereafter to an increase in calcium concentration exhibit a water permeability that under certain conditions can be comparable to that achieved with antidiuretic hormone (ADH). Lowering the serosal calcium at the peak of the hydrosmotic responses to both ADH and A 23187 inhibited the maintenance of the net water flux. The action of a supramaximal dose of ADH is blunted in bladders pretreated with A 23187, while the hydrosmotic effects of a submaximal dose are enhanced when the ionophore is added together with the hormone. The results show that an increase in transepithelial water movement can be triggered by calcium and that serosal calcium is needed to sustain the response. This hydrosmotic response may be dependent upon the rate at which intracellular calcium concentrations change and on the absolute concentration attained. It is suggested that calcium is involved in the action of ADH on water permeability and may act as a modulator of the hydrosmotic response.     

Inhibition of Tyrosinase Activity and Protein Synthesis in Melanoma Cells by Calcium lonophore A23187

Calcium ionophore A23187 lowers basal levels of tyrosinase and inhibits the MSH-induced increase in tyrosinase in Cloudman S-91 mouse melanoma cell cultures. lonophore at a concentration of 10^–6 g/ml causes a 50% reduction in basal levels of tyrosinase and inhibits the MSH stimulated level of enzyme. lonophore A23187 also inhibits the PGEi mediated stimulation of tyrosinase, as well as the rise in enzyme activity observed in cells exposed to either theophylline (1 mM) or dbcAMP (10^–4M). lonophore does not affect basal levels of cyclic AMP nor the elevated levels produced by either MSH or PGEi, suggesting then, that the antagonistic activity of A23187 is localized to a point in the pathway of tyrosinase activation distal to the formation of cAMP. lonophore causes a rapid and marked (> 50%) inhibition of cellular protein synthesis and it is possible that this calcium mobilizing compound may exert its inhibitory effects on tyrosinase activity by causing a general reduction in cellular translation. Since the inhibition of protein synthesis occurs in cells exposed to ionophore in either the presence or absence of calcium in the medium, it seems, likely that the ionophore may exert its effects by causing the release of calcium from intracellular sites.     

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.     

Histamine release from human basophils by synthetic block co-polymers composed of polyoxyethylene and polyoxypropylene and synergy with immunologic and non-immunologic stimuli

Co-polymers composed of polyoxyethylene and polyoxypropylene have been shown previously to trigger histamine release from mouse peritoneal mast cells; this property quantitatively is directly related to the ionophorous ability of these compounds to cause a functional exchange of intracellular K+ for extracellular Na+ across the cell membrane. We investigated the effect of an inflammatory copolymer, T130R2, on human basophils. The data demonstrate that T130R2 can cause calcium-dependent histamine release from human basophils in vitro. Further, at concentrations that do not cause histamine release, this co-polymer markedly augments release by suboptimal concentrations of the lectin Con A or anti-IgE antibody and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate but not the calcium ionophore A23187. Thus, these co-polymers induce mediator release from cells of both rodents and humans. In both instances it is likely that calcium-dependent cell triggering is the result of an influx of sodium ions with concomitant depolarization of the transmembrane potential. In common with the calcium ionophore A23187, the co-polymer T130R2 has the ability to synergize with stimuli which trigger the IgE receptor as well as those which directly activate the cellular calcium- and phospholipid-dependent protein kinase.     

Evidence for a role of NA+/H+ exchange in platelets activated with calcium-ionophore A 23187

We have investigated the release of protons from human platelets and platelet aggregation induced by the calcium ionophore, A 23187. Addition of the ionophore to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.2 mM amiloride, a potent inhibitor of Na+/H+ countertransport, the amount of protons liberated was decreased by 50% and was further reduced to about 10% by 1 mM amiloride. Similar inhibition of H+-release was observed after decreasing Na+ in the incubation medium. Both results suggest that increasing internal Ca2+ by the ionophore induces Na+/H+ exchange in human platelets. Platelet aggregation could be induced by adding the ionophore to the platelet suspension. This aggregation was inhibited by amiloride, at least when induced by low ionophore concentrations. The results suggest that stimulation of Na+/H+ exchange, and the concomitant increase in intraplatelet pH, are important mechanisms in platelet activation.     

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.