Differential effects of prostaglandins on macrophage activation induced by calcium ionophore A23187 or IFN-gamma.

Calcium ionophore A23187 can mimic IFN-gamma-induced macrophage activation for intracellular Leishmania killing and secretion of L-arginine-derived nitrite. Because the effects of ionophore are not restricted to calcium mobilization but also involve alterations of phospholipid metabolism, we have examined the role of PGE2 in the activation process. Macrophages exposed to A23187 or IFN-gamma in the presence of LPS and FCS secreted significant amounts of PGE2 independently of the presence of L-arginine in the incubation medium. The addition of the cyclooxygenase inhibitor indomethacin or omission of FCS abrogated PGE2 secretion but had little effect on nitrite production or intracellular killing. The addition of exogenous PGE2, of agents increasing PGE2 production such as arachidonic acid and colchicine, or of an analogue of cAMP, dibutyryl cAMP inhibited A23187 + LPS-induced activation whereas that mediated by IFN-gamma + LPS remained unimpaired. Our results indicate that PGE2 can modulate activation induced by A23187 but not by IFN-gamma, probably by a process involving cAMP. Conceivably, ionophore can mimic IFN-gamma for the induction of activation but lacks the capacity to help maintain the activated state because of its inability to desensitize macrophages to negative regulation by PGE2, as suggested previously for IFN-gamma-dependent activation.     

Calcium ionophore A23187 does not stimulate lipopolysaccharide nonresponsive C3H/HeJ peritoneal macrophages to produce interleukin 1

C3H/HeJ mice are hyporesponsive to the biologic effects of bacterial lipopolysaccharide (LPS). The defect in the strain of mice is believed to be due to the expression of a mutant allele designated Lpsd at the chromosome four locus. The molecular basis of this hyporesponsiveness is not known, but it may result from some defective membrane signal transductions. To examine this possibility, we compared the abilities of interleukin 1 (IL-1) production by C3H/HeJ macrophages with those by C3H/He macrophages (LPS responsive) after stimulation with the calcium ionophore A23187 or phorbol myristate acetate (PMA). A23187 induced IL-1 production by C3H/He macrophages, but it did not induce IL-1 production by C3H/HeJ macrophages and neither did LPS. However, it had the ability to increase intracellular free Ca2+ in C3H/HeJ macrophages as well as in C3H/He macrophages, this being examined by the changes in cytosolic Ca2+ in the macrophages by using Quin 2. In contrast, PMA was able to induce IL-1 production by both C3H/He and C3H/HeJ macrophages without increasing intracellular Ca2+. Since polymyxin B did not inhibit A23187- or PMA-induced IL-1 production by C3H/He macrophages, these results are not due to the little amount of LPS in culture medium, but due to their own characteristics. A calmodulin antagonist W-7 effectively inhibited A23187-induced IL-1 production by C3H/He macrophages. However, it hardly inhibited LPS-induced IL-1 production except at high concentration, and it caused no inhibition of the PMA-stimulated one. These results suggest that the blocking sites expressed phenotypically by the Lpsd are shared by LPS- and A23187-stimulated cellular processes, although the actions of LPS and A23187 are different from each other. In addition to the direct study with LPS or lipid A, A23187 should provide another useful approach to clarify the molecular mechanisms of Lpsd defect in C3H/HeJ macrophages.     

Macrophage activation for intracellular killing as induced by calcium ionophore. Correlation with biologic and biochemical events

Changes in the concentration of cytosolic Ca2+ are known to affect various macrophage functions; in particular, exposure in vitro to the Ca2+ ionophore A23187 primes macrophages for tumor cell killing. In the present report, it is shown that treatment with this ionophore similarly mimics IFN-gamma as a priming signal for induction of microbicidal activity. Incubation of mouse bone marrow-derived macrophages with 10(-7) to 10(-6) M A23187 (in the presence of Ca2+) led to intracellular killing of the protozoan parasite Leishmania enriettii within 24 h, provided LPS (1 ng/ml) was also present; no microbicidal activity was observed using either compound alone. A 4-h exposure to the ionophore in the presence of Ca2+ (priming phase) was sufficient to induce leishmanicidal activity upon reincubation with LPS, here acting as a necessary second signal. Addition of EGTA during the priming phase blocked intracellular killing upon subsequent LPS treatment; microbicidal activity could be restored by excess Ca2+, but not Mg2+, suggesting that changes in the concentration of cytosolic Ca2+ are sufficient to mediate the molecular events that lead to acquisition of microbicidal potential. Ionophore-induced leishmanicidal activity was paralleled by a stimulation of the hexosemonophosphate shunt pathway and production of nitrites, which are biochemical correlates of the activated state. In addition, sequential exposure to A23187 and LPS markedly stimulated macrophages to release TNF and PGE2, two agents thought to act as modulators of macrophage activation.     

The role of calcium in macrophage chemotaxis to the phorbol ester tumor promoter TPA

The significance of the macrophage in the inflammatory response that occurs concurrently with phorbol ester induced tumor promotion has not yet been determined. Biologically active phorbol ester tumor promoters modify several functional responses of macrophages including chemotaxis, cytotoxicity, secretion and prostaglandin synthesis and release. The present study examines calcium metabolism as a possible underlying biochemical mechanism through which 12-0-tetradecanoyl-phorbol-13-acetate (TPA) exerts its effects on macrophage chemotaxis. The chemotaxis of mouse resident peritoneal macrophages was evaluated in the presence of pharmacological agents known to alter cellular calcium metabolism. The calcium ionophore A23187 in microM concentrations enhanced macrophage chemotaxis to TPA by approximately 41%. This enhancement was dependent on the presence of extracellular calcium. TPA-induced chemotaxis was also enhanced by the histological dye ruthenium red (RR), an agent known to modify mitochondrial calcium fluxes and calcium-dependent neuronal transmission. Ruthenium red (0.1 and 1.0 microM) produced a maximal stimulation of macrophage chemotaxis to TPA of approximately 62%. An intracellular calcium antagonist, 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) inhibited macrophage chemotaxis to TPA in a dose related fashion (1.0 to 100 microM). Varying extracellular calcium concentrations (0-3.6 mM) had no effect on macrophage chemotaxis in response to TPA. In drug combination studies neither A23187 nor RR was able to overcome the inhibitory effects of TMB-8 on macrophage chemotaxis to TPA. These results indicate that intracellular calcium metabolism may be playing a significant role in modulating TPA's effect on macrophage chemotaxis, while extracellular calcium may be of little import. A possible mode of TPA's effect on the macrophage via mobilization of calcium from cellular storage sites is discussed.     

Effect of vitamin E on production of macrophage migration inhibitory factor (MIF) by macrophages

Macrophage migration inhibitory factor (MIF), a putative cytokine involved in inflammatory and immune responses, was identified in rat peritoneal macrophages by Western blot analysis and its secretion into culture medium by enzyme-linked immunosorbent assay. To clarify the possibility of vitamin E as an immune modulator, we investigated the effect of vitamin E on MIF production in macrophages in response to calcium ionophore A23187 and lipopolysaccharide (LPS). Intraperitoneal injections of vitamin E (5 mg per rat) for 6 successive days resulted in a significant increase of alpha-tocopherol content in peritoneal macrophages. Alpha-tocopherol content of macrophages in vitamin E-treated rats was 478.3 +/- 90.7 ng/10(6) cells, whereas in control rats it was 1.5 +/- 0.5 ng/10(6) cells. For the control macrophages, total MIF content of the medium (2.5 x 10(6) cells/18 ml) without stimulation was 40.7 +/- 3.6 ng after 14 h culture, whereas stimulation with calcium ionophore A23187 (400 nM) and LPS (5.0 microg/ml) induced the elevation of MIF content to 65.9 +/- 7.5 ng and 74.3 +/- 10.4 ng, respectively (p < 0.05, n = 3). On the other hand, vitamin E-enriched macrophages without stimulation showed less MIF content (14.0 +/- 4.2 ng) than the control (p < 0.05, n = 3). Similarly, the increase of MIF of vitamin E-treated macrophages was significantly suppressed after stimulation with calcium ionophore A23187 or LPS, compared with the control macrophages (p < 0.01, n = 3). From analysis of intracellular MIF content by Western blot, we found no alteration of intracellular MIF content of vitamin E-macrophages, in contrast to the decreased content of control stimulated-macrophages, showing that vitamin E suppressed MIF secretion into the culture medium. Taken together, these results indicate that vitamin E may contribute to the regulation of inflammatory and immune responses through regulation of MIF secretion, possibly by modulating macrophage-membrane architecture.     

Superoxide responses of immune complex-stimulated rat alveolar macrophages. Intracellular calcium and priming

ATP is known to induce calcium transients in rat and human neutrophils and to "prime" these cells for enhanced oxygen radical responses after stimulation with chemotactic peptide, FMLP, or immune complexes. Calcium ionophores are also well known for their ability to prime phagocytic cells. In the current studies, nonelicited rat alveolar macrophages were analyzed for the ability of ATP as well as FMLP, C5a, platelet-activating factor and calcium ionophore (A23187) to modify levels of intracellular calcium and to enhance superoxide anion (O2-) production in response to immune complexes. Although none of these agents induced a O2- response under the conditions employed, all, except FMLP and C5a (human, recombinant) increased intracellular calcium, although the temporal features of the increases varied with the agent. In contrast to the inability of FMLP and C5a to cause intracellular calcium increases in macrophages, these same peptides caused dose-dependent intracellular calcium increases in rat neutrophils, whether the cells were derived from the blood or from the peritoneal cavity. On the basis of the effects of EGTA, the calcium increases in alveolar macrophages were caused by intracellular release of calcium in addition to some influx of extracellular calcium. Although ATP caused a dose-related increase in the level of intracellular calcium in alveolar macrophages, the cells were not "primed" for enhanced O2- responses to immune complexes. In contrast, platelet-activating factor and A23187, each of which induced increased intracellular levels of calcium, were able to prime macrophages for enhanced O2- responses. C5a and FMLP neither increased intracellular calcium levels nor primed macrophages for enhanced O2- responses to immune complexes. It is not clear if the inability of ATP to prime alveolar macrophages is caused entirely by insufficient increases in intracellular calcium or if ATP is unable to bring about additional changes that are relevant to the priming phenomenon.     

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.     

Involvement of intracellular Ca2+ in the regulation of bovine leukemia virus expression

The calcium ionophore A23187, which was used to increase the intracellular calcium concentration ([Ca2+]i), was analyzed for effects on bovine leukemia virus (BLV) expression in two BLV infected cell lines. To clarify the role of intracellular free calcium in this response, [Ca2+]i was measured during ionophore treatment with the fluorescent calcium indicator Fura-2. Elevation of intracellular calcium under these conditions caused an enhancement of BLV gp51 and p24 synthesis as well as an activation of the BLV long terminal repeat (LTR) in a dose-dependent manner. Furthermore, it was observed that elevated levels of intracellular calcium following A23187 stimulation lead to activation of NF-kappaB. Based on inhibitor studies, we hypothesize that the effect of A23187 on BLV expression appears to be mediated by PKC.     

Regulation of interleukin 1 production by mouse peritoneal macrophages. Effects of arachidonic acid metabolites, cyclic nucleotides, and interferons

The effects of prostaglandin E2 (PGE2), cyclic nucleotides, leukotriene B4 (LTB4), and interferons on interleukin 1 (IL 1) production by lipopolysaccharide (LPS)-stimulated C3H/HeNCrl mouse peritoneal macrophages were studied. IL 1 production was inhibited by PGE2, the adenosine 3':5'-monophosphate analog dibutyryl cAMP, the cAMP agonist isoproterenol, and the phosphodiesterase inhibitor isobutylmethylxanthine. These agents were more inhibitory when added early in the latent phase of IL 1 synthesis following stimulation with LPS rather than just prior to release of IL 1 into the medium. Production of both the intracellular and extracellular forms of IL 1 was blocked by PGE2 and cAMP. Suppression of LPS-induced IL 1 production by PGE2 was prevented by leukocyte alpha-interferon. Moreover, alpha-interferon augmented LPS-induced IL 1 production but did not stimulate IL 1 production in the absence of LPS. Immune gamma-interferon markedly inhibited LPS-stimulated IL 1 production. The lipoxygenase inhibitor eicosa-5,8,11,14-tetraynoic acid suppressed, whereas 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline augmented, LPS-induced IL 1 production. The opposing effects of these agents suggested that lipoxygenase metabolites do not act as inducers of IL 1 production. Purified LTB4 did not stimulate base-line or augment LPS-induced IL 1 production (both intracellular and extracellular forms). Moreover, calcium ionophore A23187 (a lipoxygenase activator) did not stimulate IL 1 production, alone or in combination with LTB4. Thus, net IL 1 production by macrophages may be regulated by a balance between the effects of PGE2, cAMP, alpha-interferon, and gamma-interferon, but not LTB4.     

The role of endoplasmic reticular Ca2+ stores in cell viability and tumor necrosis factor-α production of the murine macrophage RAW 264.7 cell line

Thapsigargin (TG), an endoplasmic reticular (ER) Ca(2+)-ATPase inhibitor, can increase the intracellular calcium concentration and then deplete the TG-sensitive intracellular Ca(2+) pool. In this study, we investigated the effects of TG on cell viability and tumor necrosis factor-alpha (TNF-alpha) production in the murine macrophage RAW 264.7 cell line. We found that treatment with TG (10-800 nM) induced apoptosis in RAW 264.7 cells in a dose-dependent manner (IC(50), 200 nM). Lipopolysaccharide (LPS, 1 microg/ml) markedly potentiated low concentrations of TG (10-75 nM) in inducing apoptosis (IC(50), 20 nM) as revealed by the DNA ladder. Polymycin B (an LPS receptor antagonist) inhibited the cytotoxic effect induced by LPS plus TG. Although TG, A23187 and ionomycin all definitely increased intracellular Ca(2+) concentrations, neither A23187 nor ionomycin mimicked TG in inducing apoptotic events in LPS-activated RAW 264.7 cells. Moreover, the production of TNF-alpha induced by LPS was profoundly potentiated by TG but not by A23187 or by ionomycin. We conclude from these combined results that TG-sensitive ER Ca(2+) stores play a pivotal role in modulating cell viability and TNF-alpha production. The mutual potentiation between the LPS receptor signaling pathway and the depletion of ER Ca(2+) stores implies the existence of cross-talk between these multiregulatory mechanisms in this murine macrophage RAW 264.7 cell line.     

Mannheimia haemolytica leukotoxin-induced increase in leukotriene B4 production by bovine neutrophils is mediated by a sustained and excessive increase in intracellular calcium concentration

Isolated bovine neutrophils were used to study the relationship between the duration and magnitude of the Mannheimia haemolytica leukotoxin-induced increase in intracellular calcium concentration and leukotriene B4 synthesis. In contrast to recombinant human C5a, which caused a transient, small increase in intracellular calcium concentration and no effects on leukotriene B4 synthesis, exposure of neutrophils to leukotoxin resulted in a rapid, sustained, large increase in intracellular calcium concentration, followed by leukotriene B4 synthesis. This leukotoxin-induced response was similar to those produced by the calcium ionophore, A23187, and phorbol myristate acetate, which also caused significant leukotriene B4 production. Manipulation of the duration and magnitude of leukotoxin- and A23187-induced intracellular calcium concentration increase confirmed that a high and sustained intracellular calcium concentration was necessary to stimulate production of leukotriene B4, which is believed to play an important role in the pathogenesis of pulmonary M. haemolytica infection.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity

Exposure of cultured Chinese hamster ovary (CHO) cells to hydrogen peroxide results in the production of extensive DNA breakage which can be prevented by the intracellular calcium chelator Quin 2. This effect occurs at Quin 2 AM concentrations as low as 0.1 microM and is maximal at 1 microM. Addition of the extracellular calcium chelator, EGTA, does not affect the level of DNA breakage generated by H2O2. Quin 2 also significantly reduces cellular toxicity caused by the oxidant. Experiments with spin-trapping techniques demonstrate that Quin 2 does not affect the formation of hydroxyl radicals generated by the action of Fe2+ on H2O2. Quin 2 at high concentrations, similar to those reached within the cell, actually enhanced generation of hydroxyl radical in the absence of other iron chelators under our experimental conditions. These results suggest that H2O2 or H2O2-derived radicals do not directly induce DNA strand breakage in intact mammalian cells; rather, these radicals may disturb intracellular Ca2+ homeostasis which results in secondary reactions ultimately leading to DNA strand breakage. In addition to strand breakage, membrane and protein oxidation probably contribute to the cytotoxic effect of H2O2.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

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.     

Effects of bacterial lipopolysaccharide on the hydrolysis of phosphatidylinositol-4,5-bisphosphate in murine peritoneal macrophages

LPS and lipid A initiated enhanced hydrolysis of PIP2 in macrophages. When murine peritoneal macrophages were labeled with [2-3H]myoinositol and stimulated with either LPS or lipid A, a rapid (within 10 sec) rise in Ins(1,4,5)P3 was observed. The breakdown pattern of Ins(1,4,5)P3 was complex; this included breakdown of Ins(1,4,5)P3 and formation of Ins(1,3,4,5)P4 (approximately 10 to 30 sec), and ultimately formation of Ins(1,3,4)P3 (approximately 60 sec). Within 10 sec after treatment, LPS caused an average increase of about fourfold to fivefold in Ins(1,4,5)P3, which declined over 5 min. When the total isomers of InsP3 were measured, levels rose about twofold in response to LPS or to lipid A and remained elevated for as long as 5 min. Lipid A, in the concentration range of 0.1 to 10 micrograms/ml, induced elevated intracellular levels of Ca2+ as quantified by fluorescence with Quin 2 or with Fura 2. When single, adherent Fura 2-loaded macrophages were treated with lipid A, basal levels of calcium rose over 10 sec from approximately 55 nM to almost 600 nM. LPS, paradoxically, did not cause such substantial increases in intracellular calcium (i.e., increases of approximately 26 nM) when judged by Fura 2 fluorescence. LPS treatment led to enhanced phosphorylation of a characteristic set of proteins, similar to those induced by stimulating protein kinase C (PKC) with phorbol myristate acetate as previously reported. The enhanced phosphorylation of pp28, pp33, and pp67 in macrophages was evident by 15 min and optimal by 30 min. Taken together, these observations indicate that LPS and lipid A cause increased breakdown of phosphatidylinositol 4,5-bisphosphate, which led to enhanced intracellular levels of calcium and also to enhanced protein phosphorylation, presumably mediated by PKC. The data thus suggest that one major intracellular signal transduction mechanism, initiated by LPS and lipid A in macrophages, is the rapid breakdown of PIP2.     

Participation of the microtubular-microfilamentous system on intracellular Ca2+ transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[14C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca2+. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca2+, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca2+ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca2+. In addition, an increase in cytosolic Ca2+ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca2+, suggesting that Ca2+ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca2+ increase induced by A23187 in a Ca2+-free medium. On the other hand, in a Ca2+-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca2+ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca2+ uptake. These observations indicate that intracellular Ca2+ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca2+ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

Intracellular calcium does not appear to be essential for arachidonic acid release from stimulated macrophages as shown by studies with Quin-2

The present investigation was undertaken to study the potential role of intracellular calcium on the release of arachidonic acid from mouse peritoneal macrophages activated by inflammatory stimuli. The intracellular calcium concentration, as measured using fluorescent probe Quin-2, was 112 +/- 8.4 nM. The chelation of intracellular calcium with Quin-2 did not affect the release of arachidonic acid from macrophages upon stimulation with phorbol myristate acetate, opsonized zymosan or calcium ionophore A23187. However, the removal of calcium from the extracellular medium resulted in a 30-50% decrease in arachidonic acid release from phorbol myristate acetate- and zymosan-stimulated macrophages and also the stimulation of arachidonic acid release from calcium ionophore-stimulated cells were nullified. These studies indicated the existence of calcium-dependent and independent mechanisms modulating the release of arachidonic acid from macrophages subjected to inflammatory stimuli.     

Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca2+ concentration ([Ca2+]i) and induces production of leukotriene C4 (LTC4). This model was used to examine the role of Ca2+ in LTC4 formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2 x 10(-7) M), selectively prevented the stimulatory effect of the antigen on LTC4 production whereas the response to calcium ionophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 micrograms/ml) did not overcome the inhibitory action of HC. An elevated [Ca2+]i is known to be essential for the activation of both 5-lipoxygenase and phospholipase A2. The stimulatory effect of the antigen on LTC4 production was abolished when the cells were incubated in Ca2+-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca2+. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca2+ concentration of 150 microM and 40 microM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca2+]i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of 45Ca2+ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca2+]i, and this may be associated with the inhibitory action of HC on LTC4 formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.     

Prostaglandin synthesis and release by subpopulations of rat alveolar macrophages

Alveolar macrophages are the primary phagocytic cell of lung, but are also capable of a variety of other functions, which include initiating or modulating inflammatory and immune responses through the production of soluble mediators. One such group of mediators is the eicosanoids. Further, recent data indicate that alveolar macrophages are not functionally homogeneous, but are heterogeneous with several subpopulations that differ both morphologically and functionally. Considering the apparent importance of prostaglandin synthesis and release in inflammatory and immune responses, the current study was undertaken to determine whether alveolar macrophage subpopulations differ in their ability to synthesize and release prostaglandin (PG) E, PGI2, and thromboxane A2 after stimulation by calcium ionophore A23187, zymosan, or aggregated IgG. Alveolar macrophages were harvested by bronchoalveolar lavage and were separated into 18 density-defined fractions. Density-defined alveolar macrophages (DD-AM) showed marked heterogeneity in prostaglandin synthesis and release. Maximal PGE synthesis and release was seen as a single peak after calcium ionophore A23187 and zymosan stimulation. In contrast, two peaks in PGE synthesis were seen after aggregated IgG stimulation. PGI2 synthesis was seen as a single peak generated by different DD-AM after calcium ionophore A23187 and zymosan. In contrast, aggregated IgG stimulation of subpopulations exhibited uniform synthesis and release of PGI2. Thromboxane A2 synthesis and release was maximal from a broad range of various DD-AM after calcium ionophore A23187, zymosan, and aggregated IgG stimulation. The results demonstrate that DD-AM are heterogeneous in ability to synthesize and release prostaglandins which is dependent on the stimuli. Therefore, specific subpopulations of alveolar macrophages may be central to the control of the pulmonary inflammatory response through specific eicosanoid synthesis and release.