Prostaglandin release but not superoxide production by rat Kupffer cells stimulated in vitro depends on Na+/H+ exchange

The release of the prostaglandins E2 and D2, induced by zymosan and phorbol ester in cultured rat Kupffer cells, was found to depend on the extracellular concentration of Na+. Eicosanoid formation following the administration of the Ca2+ ionophore A23187 or of arachidonic acid, however, did not require the presence of sodium ions in the medium. A half-maximal rate of prostaglandin release by zymosan-treated Kupffer cells was obtained between 4 mM and 5 mM Na+; and a Na+ concentration of greater than or equal to 30 mM was required to maximally stimulate prostaglandin E2 and D2 formation in the cultured liver macrophages. In contrast, the superoxide production following the administration of zymosan or of phorbol ester was quite independent of extracellular Na+. The zymosan and phorbol-ester-stimulated release of prostaglandins E2 and D2 was inhibited by amiloride. Artificial intracellular alkalization enhanced the prostanoid production of unstimulated and of zymosan-stimulated cells whereas artificial intracellular acidification inhibited the zymosan-elicited prostaglandin synthesis. In contrast, the superoxide formation was independent of the pH changes. The data presented here suggest that the prostaglandin production elicited by zymosan or phorbol ester in cultured rat Kupffer cells requires an activated Na+/H+ exchange.     

Ca2+ is mobilized by hydroxyl radical but not by superoxide in RTH-149 cells: the oxidative switching-on of Ca2+ signaling

Differential effects of superoxide and hydroxyl radical on intracellular calcium were investigated in trout hepatoma cells (RTH-149). [Ca2+]i variations were recorded using confocal imaging, fluo-3 loading, and exposure to various mixtures consisting of hypoxanthine/xanthine oxidase (HX/XO), and of sub-stimulatory concentrations of H2O2 and Cu2+ . No [Ca2+]i variation was found with HX/XO, a slight [Ca2+]i rise with a mixture of Cu2+ and HX/XO, a sustained rise with Cu2+ and H2O2, and the highest rise with Cu2+, H2O2 and HX/XO. Fluorimetric assay using dihydrorhodamine 123 revealed a correlation between the oxidizing power of a mixture and its effect on [Ca2+]i. The [Ca2+]i rise induced by Cu2+, H2O2 and HX/XO, was partially reduced in Ca2+ free medium or in the presence of SOD, converted into Ca2+ transient by verapamil, and almost abolished by the PLC inhibitor U73122 or in the presence of the hydroxyl radical quencher TEMPOL. Data indicate that Ca2+ is mobilized by hydroxyl radical but not by superoxide. The mechanism consists of PLC activation causing intracellular Ca2+ release, while Ca2+ entry potentiates Ca2+ release thus leading to sustained [Ca2+]i rise. A role of hydroxyl radicals in the oxidative switching-on of Ca2+ signaling is discussed.     

Ca2+ flux as an initial event in phagocytosis by rat Kupffer cells

Ca2+ uptake by monolayer cultures of rat Kupffer cells was strongly stimulated immediately after contact with phagocytosable material, e.g. zymosan particles. The intensity of the luminol-mediated chemiluminescence following zymosan addition was dependent on extracellular Ca2+; addition of the Ca2+-ionophore A 23187 did not further increase the zymosan-elicited response. The superoxide-mediated chemiluminescence was not inhibited by indomethacin but could be suppressed by compounds known to inhibit Ca2+-calmodulin-dependent reactions. Analysis of their efficiency, however, suggests that the O-2 production is mediated by Ca2+ rather than Ca2+-calmodulin and that the inhibitors exert another, e.g. membrane-directed influence.     

Platelet-activating factor increases inositol phosphate production and cytosolic free Ca2+ concentrations in cultured rat Kupffer cells

Platelet-activating factor (PAF) stimulates glycogenolysis in perfused livers but not in isolated hepatocytes [(1984) J. Biol. Chem. 259, 8685-8688]. PAF-induced glycogenolysis in liver is associated closely with a pronounced constriction of the hepatic vasculature [(1986) J. Biol. Chem. 261, 644-649]. These and other observations suggest that PAF stimulates glycogenolysis in liver indirectly by interactions with cells other than hepatocytes. We have evaluated effects of PAF on hepatic Kupffer cells, which regulate flow through the hepatic sinusoids. Application of PAF to [3H]inositol-labeled Kupffer cells produced dose-dependent increases in [3H]inositol phosphates with an EC50 value of 4 x 10(-10) M. Increases in inositol phosphate production in response to PAF were inhibited by a specific PAF receptor antagonist, SRI 63-675 (2 x 10(-7) M), and stimulus of protein kinase C, phorbol 12-myristate 13-acetate (1 x 10(-7) M). Measurements of cytosolic free Ca2+ concentrations ([Ca2+]i) in single Kupffer cells loaded with Fura-2 demonstrated that application of PAF (2 x 10(-9) M) resulted in significant increases in [Ca2+]i. These observations lead us to propose that interactions of PAF with Kupffer cells may result in the hemodynamic and metabolic responses to PAF in liver.     

Differential inhibition of prostaglandin and superoxide production by dexamethasone in primary cultures of rat Kupffer cells

Dexamethasone inhibited the stimulus-induced prostaglandin E2 formation by rat Kupffer cells in primary culture, e.g. after treatment with zymosan, phorbol ester, calcium ionophore A23187, platelet-activating factor or lipopolysaccharide. Prostaglandin E2 production from added free arachidonic acid was not influenced by the hormone. The time course, as well as the partial inhibition of the hormone effect by actinomycin D and cycloheximide, point to the hormone-induced formation of a protein which regulates phospholipase A2. The hormone did not affect the phagocytotic activity of the Kupffer cells. The quantity of [3H]arachidonic acid incorporated into phospholipids was also not altered by dexamethasone. After stimulation with zymosan, [3H]arachidonic acid was liberated from phosphatidylcholine only. Superoxide generation by rat Kupffer cells was induced by zymosan, phorbol ester and, to a much smaller extent, by platelet-activating factor. A23187 and lipopolysaccharide were without effect. In contrast to prostaglandin formation, the generation of superoxide was not influenced by dexamethasone. These results indicate that in cultured rat Kupffer cells prostaglandin formation and superoxide generation are independently triggered processes.     

Inactivation by Ca2+ of Ca2+-stimulated ATPase from rat red cells

Summary The effect of Ca²⁺ on the stability of the Ca²⁺-stimulated ATPase has been investigated. Our results showed that the preincubation of the rat red cell membranes in presence of Ca²⁺ causes an irreversible inhibition of the enzyme. The same effect was obtained with Ba²⁺ instead of Ca²⁺. Once initiated the inactivation of the enzyme could be halted by the addition of ethylene glycol bis (B-amino ethyl ether) N,N-tetra acitic acid (EGTA), but inactivation was irreversible. The presence of ATP in the preincubation with Ca²⁺ prevented the inactivation but calmodulin did not.     

Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent.

We investigated the hypothesis that extracellular Na+ is required for the rapid mobilization of Ca2+ by rat parotid cells after adrenergic stimulation. When Na+ salts in the media were osmotically replaced with either choline chloride (+atropine) or sucrose, efflux of 45Ca2+ from preloaded cells, caused by 10 microM-(-)-adrenaline, was unchanged. Similarly adrenaline stimulated 45Ca2+ uptake into cells under nonsteady-state conditions in the presence or absence of Na+. Monensin, a Na+ ionophore, was able to elicit a modest increase in 45Ca2+ efflux, compared with controls. Studies of net 45Ca2+ flux, performed under near-steady-state conditions, showed that adrenaline caused net 45Ca2+ accumulation, whereas monensin caused net 45Ca2+ release. The effect of monensin required the presence of Na+ in the incubation medium. Both 1 mM-LaCl3 and 0.1 mM-D-600 prevented adrenaline-stimulated 45Ca2+ uptake into cells, but had no effect on monensin-induced changes. We conclude that (1) the rapid mobilization of Ca2+ by adrenergic agonists seen in rat parotid cells does not require a Na+out greater than Na+in gradient and (2) the nature of the monensin effect is quite different from the adrenergic-agonist-induced response.     

Capsaicin inhibits platelet-activating factor-induced cytosolic Ca2+ rise and superoxide production

Platelet-activating factor (PAF) is an important participant in the inflammatory process. We studied the regulation of PAF activity by capsaicin in human promyelocytic leukemia HL-60 cells. Capsaicin inhibited PAF-induced superoxide production in a concentration-dependent manner. In addition to PAF, the fMLP- and extracellular ATP-induced superoxide productions were inhibited by capsaicin, whereas PMA-induced superoxide production was not affected. In the PAF-stimulated cytosolic Ca2+ increase, capsaicin inhibited in particular the sustained portion of the raised Ca2+ level without attenuation of the peak height. In the absence of extracellular Ca2+, the PAF-induced Ca2+ elevation was not inhibited by capsaicin because capsaicin only inhibited the Ca2+ influx from the extracellular space. In addition, capsaicin did not affect PAF-induced inositol 1,4,5-trisphosphate production, suggesting that phospholipase C activation by PAF is not affected by capsaicin. Store-operated Ca2+ entry (SOCE) induced by thapsigargin was inhibited by capsaicin in a concentration-dependent manner. This capsaicin effect was also observed on thapsigargin-induced Ba2+ and Mn2+ influx. Furthermore, capsaicin's inhibitory effect on the thapsigargin-induced Ca2+ rise overlapped with that of SK&F96365, an inhibitor of SOCE. Both capsaicin and SK&F96365 also inhibited PAF-induced cytosolic superoxide generation in HL-60 cells differentiated by all-trans-retinoic acid. Our data suggest that capsaicin exerts its anti-inflammatory effect by inhibiting SOCE elicited via PLC activation, which occurs upon PAF activation and results in the subsequent superoxide production.     

Anchorage dependence and Ca2+ requirement are independently modulated by hydrocortisone hormone in rat C6 glioma cells

We had previously shown that hydrocortisone (Hy), a glucocorticoid hormone, regulates the expression of the transformed phenotype of rat C6 glioma cells. The hormone reversibly renders C6 cells dependent on anchorage and high Ca2+ concentration for growth. We had also isolated Hy-resistant C6 variants in agarose suspension cultures. Here we report that Hy-resistant variants selected in high (1.8mM) Ca2+ medium become growth-arrested in low (30 microM) Ca2+ medium upon hormone treatment. We conclude that Hy-induced anchorage dependence and Hy-induced high Ca2+ requirement for growth of C6 glioma cells, are two independent phenotypes.     

Cholesterol depletion affects the Ca2+ influx but not the Ca2+ pump in human erythrocytes

Control and cholesterol-depleted human erythrocytes were loaded with permeant Ca2+ chelators (Benz2-AM or Quin2-AM) in order to increase their exchangeable Ca2+ pool and to measure both Ca2+ fluxes and [Ca]i (free cytoplasmic calcium concentration). The fluxes were independent of the concentration and of the nature of the intracellular chelator. The ATP content was not decreased by more than 50% under our experimental conditions. Cholesterol depletion (up to 28%) induced a decrease in both Ca2+ fluxes and [Ca]i which was proportional to the extent of the depletion. It is shown that cholesterol depletion primarily altered the properties of the system responsible for Ca2+ entry causing a diminution of the [Ca]i. This, in turn, induced a diminution of the activity of the Ca2+ pump without affecting the properties of this pump.     

Release of calcitonin gene-related peptide from rat enteric nerves is Ca2+-dependent but is not induced by K+ depolarization

The effect of extracellular calcium on the release of calcitonin gene-related peptide (CGRP) induced by electrical field stimulation from enteric nerves of isolated rat ileum was studied; the effect of high potassium, veratridine and caffeine was also examined. Release of endogenous substance P from enteric nerves was also measured for comparison. Electrical field stimulation (10 Hz, 0.3 ms for 2 min) of the ileum preparation caused a significant (P less than 0.001) increase in the release of CGRP and substance P from enteric nerves. The evoked, but not the basal, release of both CGRP and substance P was inhibited in the presence of tetrodotoxin (TTX). The release of CGRP and substance P induced by electrical stimulation was abolished in Ca2+-free medium containing CDTA and also in normal medium containing the calcium channel blocker cadmium chloride (CdCl2), with no change in the level of the basal release of both peptides. However, potassium depolarization (76 and 110 mM) failed to evoke an increase in the release of endogenous CGRP, although it did cause an increase in the release can be induced by mobilization of calcium from intracellular Ca2+ stores. Veratridine, on the other hand, did not cause an increase in CGRP release, although substance P and VIP release was induced by veratridine from the same preparations. The results of the present study have demonstrated that CGRP release from enteric nerves requires the presence of extracellular calcium but, unlike substance P and most other transmitters reported to show calcium-dependent release, potassium depolarization does not induce CGRP release from enteric nerves of rat ileum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).     

The sequestration of Ca2+ by mitochondria in rat heart cells

Rat heart ventricular cells, purified by Percoll density gradient centrifugation, were incubated in the presence of 1.3 mM CaCl2. After 20 min incubation, samples of the cells were lysed in medium containing 0.3 mM digitonin, ruthenium red and EGTA, and a mitochondrial fraction was isolated at intervals thereafter. Extrapolation of the mitochondrial 45Ca2+ contents to zero time enabled the endogenous 45Ca2+ to be estimated at the time of cell lysis. The lysis conditions yielded essentially complete release of lactate dehydrogenase from the cells, but caused negligible damage to the mitochondria as judged by their retention of glutamate dehydrogenase, and their ability to accumulate and retain Ca2+ in the absence of ruthenium red and EGTA. The data indicate that about 13% of total cell Ca2+ only may be mitochondrial in vivo.     

Spontaneous [Ca2+]i fluctuations in rat chromaffin cells do not require inositol 1,4,5-trisphosphate elevations but are generated by a caffeine- and ryanodine-sensitive intracellular Ca2+ store

A considerable fraction (65%) of single rat chromaffin cells loaded with the fluorescent [Ca2+]i indicator fura-2 exhibited spontaneous rhythmic fluctuations with an average period of approximately 100 s. Parallel patch clamp experiments as well as fura-2 experiments carried out in Ca2(+)-free and other modified media in the presence of Ca2+ and Na+ channel blockers indicated an origin from intracellular stores. Appropriate concentrations of agonists (bradykinin and histamine) for receptors (B2 and H1) that trigger generation of inositol 1,4,5-trisphosphate induced increased fluctuation frequency, recruitment of silent cells, and large [Ca2+]i changes at high doses. These effects were blocked by cell pretreatment with neomycin, a drug that inhibits inositol 1,4,5-trisphosphate generation. In contrast, spontaneous fluctuations and the effects of another drug, caffeine, which also induced increased frequency and recruitment, were unaffected by neomycin. Ryanodine caused first a prolongation and then (approximately 10 min) a block of both spontaneous fluctuations and caffeine effects, where the single transients after bradykinin and histamine were maintained. Caffeine and ryanodine are known to affect selectively the process of calcium-induced Ca2+ release; this is the first demonstration of [Ca2+]i fluctuation activity arising from Ca2(+)-induced Ca2+ release in nonmuscle cells with no strict requirement for inositol 1,4,5-trisphosphate involvement.     

ADP requirement for prevention by a cytosolic factor of Mg2+ and Ca2+ release from rat liver mitochondria

One hundred micromolar Ca²⁺ added to rat liver mitochondria induces a transient uptake of Ca²⁺ plus a rapid efflux of the mitochondrial Mg²⁺. Addition of a cytosolic molecule, cytosolic metabolic factor, to mitochondria prevents the efflux of the two divalent cations. ADP is required for this cytosolic metabolic factor action. This requirement for ADP is specific as it is shown by experiments with traps for nucleotides and inhibitors of the translocase. The implication of cytosolic metabolic factor in the mitochondrial regulation process is discussed.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

Regulation of tissue transglutaminase by prolonged increase of intracellular Ca2+, but not by initial peak of transient Ca2+ increase

Tissue transglutaminase (tTGase) is a member of calcium-dependent transamidation enzyme family, but a detailed regulation mechanism of tTGase by intracellular Ca(2+) is not clearly understood. Arachidonic acid (AA) and maitotoxin (MTX) activated tTGase in a dose- and time-dependent manner. Transfection of tTGase siRNA largely inhibited tTGase expression and tTGase activation by MTX. AA induced an initial increase of intracellular Ca(2+) followed by a prolonged increase. Removal of extracellular Ca(2+) with EGTA blocked the prolonged Ca(2+) increase in response to AA, although the initial Ca(2+) increase remained. In contrast, EGTA completely blocked the increase of intracellular Ca(2+) by MTX. The activation of tTGase by AA or MTX was significantly inhibited by EGTA. Moreover, EGTA prevented the prolonged increase of intracellular Ca(2+) and tTGase activation by lysophosphatidic acid, but had no effect on the initial Ca(2+) increase. These results suggested that tTGase is regulated by the prolonged increase of intracellular Ca(2+) originated from Ca(2+) influx, rather than by the initial peak of transient Ca(2+) increase.