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PubMed2005

IL-4 inhibits calcium transients in bovine trachealis cells by a ryanodine receptor-dependent mechanism.

IL-4 and IL-13 have important roles in the pathogenesis of asthma. A novel finding was that brief exposure of airway smooth muscle cells to IL-4 inhibited carbachol-stimulated calcium transients. We hypothesized that IL-4 inhibits transients by decreasing calcium store content and tested this by measuring the effects of IL-4 on transients induced by a nonspecific ionophore. Bovine trachealis cells were loaded with fura 2-AM, and cytosolic calcium concentrations ([Ca2+]i) were measured in single cells by digital microscopy. Stimulation (S1) with carbachol (10 microM) caused rapid, transient increases in [Ca2+]i to 1299 +/- 355 nM (n=5). After recovery of calcium stores, stimulation (S2) of the same cells with ionomycin (10 microM), in the absence of extracellular calcium, also increased [Ca2+]i to give S2/S1 ratio of 1.03 +/- 0.29. However, after 20 min of IL-4 (50 ng/ml), but not IL-13, ionomycin transients were decreased to 0.50 +/- 0.16 (S2/S1, P=0.02, n=6). IL-4 did not inhibit transients with ryanodine receptor calcium release channels (RyR) blocked by ryanodine (200 microM) (S2/S1=1.01+/-0.11) but still did in the presence of 8-bromo cyclic ADP-ribose, an antagonist of cyclic ADP-ribose (cADPR) signaling at RyR (S2/S1=0.48+/-0.13). Together, findings suggest that IL-4 decreases intracellular calcium stores by mechanisms dependent on RyR, but not on cADPR signaling.

Animals↗
PubMed1995

Inhibition by volatile anesthetics of endogenous glutamate release from synaptosomes by a presynaptic mechanism.

BACKGROUND: Synaptic transmission is more sensitive than axonal conduction to the effects of general anesthetics. Previous studies of the synaptic effects of general anesthetics have focused on postsynaptic sites of action. We now provide direct biochemical evidence for a presynaptic effect of volatile anesthetics on neurotransmitter release. METHODS: Rat cerebrocortical synaptosomes (isolated presynaptic nerve terminals) were used to determine the effects of general anesthetics on the release of endogenous L-glutamate, the major fast excitatory neurotransmitter. Basal and evoked (by 4-aminopyridine, veratridine, increased KCl, or ionomycin) glutamate release were measured by continuous enzyme-coupled fluorometry. RESULTS: Clinical concentrations of volatile halogenated anesthetics, but not of pentobarbital, inhibited 4-aminopyridine-evoked Ca(2+)-dependent glutamate release. Halothane also inhibited veratridine-evoked glutamate release but not basal, KCl-evoked, or ionomycin-evoked glutamate release. Halothane inhibited both the 4-aminopyridine-evoked and the KCl-evoked increase in free intrasynaptosomal [Ca2+]. CONCLUSIONS: Inhibition of glutamate release from presynaptic nerve terminals is a potential mechanism of volatile anesthetic action. Comparison of the sensitivity of glutamate release evoked by secretogogues that act at various steps in the neurotransmitter release process suggests that halothane does not affect Ca(2+)-secretion coupling or vesicle exocytosis but inhibits glutamate release at a step proximal to Ca2+ influx, perhaps by blocking presynaptic Na+ channels. Synaptosomal glutamate release evoked by 4-aminopyridine should provide a useful system for further characterization of the presynaptic effects of anesthetics.

4-Aminopyridine↗
PubMed1999

Dibucaine and tetracaine inhibit the activation of mitogen-activated protein kinase mediated by L-type calcium channels in PC12 cells.

BACKGROUND: An elevation of the intracellular calcium level, which is mediated by N-methyl-D-aspartate receptors and L-type Ca2+ channels both, activates the mitogen-activated protein (MAP) kinase signaling pathway involved in synaptic modification. It has recently been suggested that MAP kinase plays a role in coupling the synaptic excitation to gene expression in the nucleus of postsynaptic neurons. Because the effects of local anesthetics on cellular signal transduction in neuronal cells are not well-known, the authors investigated whether they affect the MAP kinase signaling pathway using PC12 cells. METHODS: The cells were stimulated with either 50 mM KCl or 1 microM ionomycin, and activated MAP kinase was thus immunoprecipitated. The immunocomplexes were then subjected to an Elk1 phosphorylation assay. Both the phosphorylation of MAP kinase and the induction of c-Fos were detected by immunoblotting. RESULTS: Pretreatment of the cells with 1 mM (ethylenedioxy)-diethyl-enedinitrilotetraacetic acid or 5 micron nifedipine blocked the MAP kinase activation induced by 50 mM KCl, whereas pretreatment with 2 microM omega-conotoxin GIVA did not. The expression of c-Fos induced by potassium chloride was also suppressed by dibucaine, tetracaine (concentrations that inhibited 50% of the activity of positive control [IC50s] were 16.2+/-0.2 and 73.2+/-0.7 microM, respectively), and PD 98059, a mitogen-activated/extracellular receptor-regulated kinase inhibitor. Higher concentrations of dibucaine and tetracaine were needed to suppress the activation of MAP kinase induced by ionomycin (the IC50 values of dibucaine and tetracaine were 62.5+/-2.2 and 330.5+/-32.8 microM, respectively) compared with potassium chloride (the IC50 values of dibucaine and tetracaine were 17.7+/-1.0 and 70.2+/-1.2 microM, respectively). Although probable targets of these local anesthetics might be L-type Ca2+ channels or components between Ca2+ and Ras in MAP kinase pathway, the possibility that they directly affect MAP kinase still remains. CONCLUSIONS: Dibucaine and tetracaine at clinical concentrations were found to inhibit the activation of MAP kinase and the expression of c-Fos mediated by L-type Ca2+ channels in PC12 cells. The suppression of MAP kinase pathway may thus be a potential target site for the actions of dibucaine and tetracaine, including the modification of the synaptic functions.

Anesthetics, Local↗
PubMed1994

Effects of hypoxia on interleukin-2 mRNA expression by T lymphocytes.

OBJECTIVE: To investigate the effects of hypoxia on T-lymphocyte expression of IL-2 messenger RNA (mRNA), after cell activation with phorbol ester and ionomycin. DESIGN: Prospective, controlled, cellular trial. SETTING: University research laboratory. SUBJECTS: EL4.6.1 cells, a murine T-cell lymphoma line. INTERVENTIONS: Tissue culture media was deoxygenated and flushed continuously with 100% helium to maintain a PO2 of 30 to 40 torr (< 40 torr [< 5.3 kPa]), or flushed with 10% oxygen/90% helium to maintain a PO2 of 45 to 55 torr (> 45 torr [> 6.0 kPa]). The pH was maintained between 7.3 and 7.6. The media was inoculated with EL4 cells. Aliquots of cells were obtained at intervals and divided into two groups: an immediate group, stimulated immediately, and an overnight group that was returned to normal incubator conditions of 5% CO2/humidified room air for 18 hrs before stimulation. MEASUREMENTS AND MAIN RESULTS: Gas tension, pH, cell count, and viability were determined for each aliquot. Cells were stimulated with phorbol myristate acetate and ionomycin for 4 hrs, at which time levels of interleukin-2 (IL-2) messenger RNA (mRNA) and gamma actin mRNA were measured by solution hybridization and enzyme immunoassay. The results were expressed as IL-2 mRNA/gamma actin mRNA ratio, normalised to baseline room air values. Cell viability and housekeeping functions (gamma actin mRNA expression) were unaffected by hypoxia. Cells exposed to a PO2 of < 40 torr (< 5.3 kPa) demonstrated a dramatic reduction in IL-2 mRNA expression with increasing duration of hypoxia. These effects persisted after an 18-hr recovery period. There was no effect on IL-2 mRNA expression when cells were exposed to a PO2 of > 45 torr (> 6.0 kPa). CONCLUSIONS: The regulation of IL-2 transcription in the T lymphocyte appears to be exquisitely sensitive to changes in oxygen tension. Exposure to a PO2 of < 40 torr (< 5.3 kPa) causes prolonged impairment of IL-2 mRNA expression. IL-2 is an important growth factor for T and NK cells, and plays a pivotal role in the regulation of the host's immune response. The long-lasting effects of brief hypoxic exposure may, in part, explain the critically ill patient's predisposition to infectious complications.

Animals↗
PubMed1990

Platelet volume responses to cytoplasmic acidification in essential hypertension.

Using a Coulter-based cell sizing method, we have previously demonstrated that, in response to cytoplasmic acidification by 140 mmol/l sodium propionate, both the mean initial rate of amiloride-sensitive platelet volume swelling and the net volume change achieved at steady-state are greater in essential hypertensives than in normotensives. In the present study, we extend this observation by showing that, in response to graded propionate exposure (56-140 mmol/l), steady-state amiloride-sensitive volume responsiveness (as percentage increase over baseline) increases linearly, and the mean slope of the line relating amiloride-sensitive volume change and propionate concentration is increased in hypertensives (0.40 +/- 0.02 versus 0.32 +/- 0.02% per mmol/l propionate, P less than 0.003). In 56 mmol/l propionate, average amiloride-sensitive platelet swelling is significantly less in hypertensives than in normotensives (7.6 +/- 0.8 versus 11.1 +/- 0.9%, P less than 0.05), but in 140 mmol/l propionate, swelling is significantly increased in hypertensives (40.8 +/- 1.7 versus 36.2 +/- 1.5%, P less than 0.05). Since platelet intracellular calcium concentration is elevated in some hypertensives and Ca2+ is known to stimulate Na(+)-H+ antiport, the transport system that is the primary determinant of amiloride-sensitive cell swelling during propionate incubation, we studied the effects of the Ca2+ ionophore, ionomycin, on volume regulation. In both normotensives and hypertensives, ionomycin (2 x 10(-10 to 2 x 10(-7) mol/l) causes dose-related increases in amiloride-sensitive platelet swelling during graded propionate exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2002

Expression and regulation of calpain in rat pancreatic acinar cells.

INTRODUCTION: Calpains, cytosolic Ca(2+)-dependent cysteine proteases, are expressed in a variety of mammalian cells and have been found to participate in stimulus-secretion coupling in platelets and alveolar cells. AIMS: In pancreatic acinar cells, expression of calpains and their role in the secretory process have not yet been elucidated. Both subjects, therefore, were examined in the current study. METHODOLOGY: mu-calpain and m-calpain were detected immunochemically. Calpain activation was measured by fluorescence spectrophotometry and single-cell fluorometry using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Amylase secretion and cell damage, characterized by lactate dehydrogenase release, were measured by colorimetric assays. RESULTS: Immunochemistry revealed cytoplasmic localization of both calpain isoforms. Immediately after increasing the cytosolic Ca(2+) concentration with ionomycin, a marked dose-dependent protease activation and cellular damage were observed. Inhibition of ionomycin-mediated enzyme activation through preincubation of cells with Ca(2+)-free medium, BAPTA-AM, or Z-Leu-Leu-Tyr-CHN(2) significantly reduced cell injury. Cholecystokinin (100 pM) also induced proteolytic activity, preceding cholecystokinin-stimulated amylase secretion. Protease activity and amylase release were significantly inhibited by Z-Leu-Leu-Tyr-CHN(2 ) retreatment. CONCLUSION: Calpains are expressed in pancreatic acinar cells and may participate in stimulus-secretion coupling. In addition, our study indicates that pathologic calpain activation may contribute to Ca(2+)-mediated acinar cell damage.

Amylases↗
PubMed1991

Discodermolide--a new, marine-derived immunosuppressive compound. I. In vitro studies.

The in vitro immunosuppressive properties of a novel, marine-derived compound, discodermolide, are reported here. Discodermolide suppressed the proliferative responses of splenocytes in the murine two-way mixed lymphocyte reaction (MLR) and concanavalin A stimulated cultures, with IC50 values of 0.24 microM and 0.19 microM, respectively. There was no evidence of cytotoxicity for murine splenocytes at concentrations of discodermolide as high as 1.26 microM. Similarly, discodermolide suppressed the proliferative responses of human peripheral blood leukocytes (PBL) in the two-way MLR, and Con A and phytohemagglutinin mitogenesis. The IC50 values were 5.65 microM, 28.02 microM, and 30.12 microM for the MLR, Con A, and PHA mitogenic responses, respectively. There was no evidence of cytotoxicity toward human PBL at discodermolide concentrations as high as 80.64 microM. Discodermolide was equally effective, compared with cyclosporine, in suppressing the PMA-ionomycin induced proliferation of purified, murine T cells, with IC50 values of 9.0 nM and 14.0 nM for discodermolide and CsA, respectively. The production of IL-2 by PMA-ionomycin stimulated T cells was not inhibited by discodermolide; however, the percentage of IL-2 receptor-bearing cells as measured by immunofluorescence with 7D4 antibody, specific for the 55-kDa chain (p55) comprising the murine IL-2 receptor, was reduced. The expression of a similar chain comprising the human IL-2 receptor (Tac antigen, p55) by PHA or Con-A-stimulated PBL was similarly suppressed by discodermolide. The precise mechanism of action of discodermolide remains to be elucidated.

Alkanes↗
PubMed1996

Calcium signaling restitution prevents T-cell proliferative suppression by prostaglandin E2.

Previous studies from our laboratory have implicated a role for Ca2+ in prostaglandin E2 (PGE2)-induced suppression in T-cell proliferation during sepsis. The present study further elucidated the mechanism of PGE2-induced suppression in T-cell proliferation. We assessed whether prevention of the suppression in Ca2+ mobilization in PGE2-treated T-cells would restore proliferation. Rat splenic T-cell Ca2+ mobilization and proliferation were measured after stimulation of cells with concanavalin A (Con A) employing Fura-2 spectroscopy and cellular [3H]thymidine uptake techniques, respectively. PGE2 and other agents that directly up-regulate the PGE2-mediated cell signaling events (e.g., cholera toxin and forskolin), substantially suppressed both Con A-induced proliferation (p < .01) and Ca2+ mobilization in T-cells (p < .01). When stimulated with Con A plus ionomycin, [Ca2+]i in PGE2 treated T-cells (395 +/- 21, nM) was not significantly different (p > .05) from that observed in Con A-stimulated T-cells without the PGE2 exposure (351 +/- 8.6). The stimulation of PGE2-treated T-cells with ionomycin and Con A also significantly (p < .025), if not completely, prevented the PGE2-induced suppression in T-cell proliferation. These results suggest that the cross-talk between the TCR- and PGE2-mediated signaling in T-cells negatively modulates the TCR signals at the Ca2+ mobilization step and/or earlier to it.

Animals↗
PubMed1986

The Wiskott-Aldrich syndrome: studies of platelets, basophils and polymorphonuclear leucocytes.

Platelets, basophils and neutrophils from a patient with the Wiskott-Aldrich syndrome (WAS) were exposed to stimuli that activate specific membrane receptor or directly initiate biochemical events (e.g. the Ca2+ ionophore A23187 and ionomycin or arachidonic acid). Platelets from this patient did not aggregate in response to ADP, collagen, thrombin or adrenaline, which activate specific membrane receptors. Platelet aggregation, however, was normal in response to compound A23187, ionomycin or exogenous arachidonic acid. Histamine release from basophils of the WAS patient was normal in response to anti-IgE, a formylated peptide (f-met peptide), and to A23187. Similarly, the release of the lysosomal enzymes, beta-glucuronidase and lysozyme, from neutrophils of the WAS patient in response to serum treated zymosan (Zx), f-met peptide, and A23187 was not significantly different from that of his parents and 13 normal donors. These results suggest that the primary defect in WAS is selectively present in platelets and is located in a biochemical step between receptor activation and Ca2+ influx and/or initiation of arachidonate metabolism.

Arachidonic Acid↗
PubMed1991

The control of class II expression on T cells is independent of the regulation of Tac and the induction of proliferation.

In order to define the association between class II expression and other markers of T cell activation we tested the ability of various modes of stimulation to induce the expression of class II, Tac, and to stimulate proliferation. Stimulation of T cells with phytohaemagglutinin (PHA) in the presence of accessory cells strongly induced proliferation, Tac and the class II antigen DR. When purified T cells without accessory cells were stimulated with the phorbol ester, PdB, and the calcium ionophore, ionomycin, strong proliferation and Tac expression were induced, but only low levels of surface class II were observed. In contrast, stimulation of the same cells with PHA resulted in weak proliferation, strong Tac, but again low class II levels. The addition of PdB to the PHA increased the proliferative response, but did not affect Tac expression, which remained high, or class II expression, which remained low. Subsequent culture in conditioned medium of purified T cells which had been activated with either PdB and ionomycin or with PHA resulted in increased surface class II levels in both cases. Additional experiments suggested that neither IL-2, IL-4, nor interferon-gamma (IFN-gamma) alone was responsible. These results demonstrate that class II expression can be separated from the induction of proliferation and the upregulation of Tac and that the mode of T cell stimulation influences the resulting activation pathway. Furthermore, they suggest that the control of class II expression on T cells is more tightly regulated than it is on other cells.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2006

Vimentin-Ser82 as a memory phosphorylation site in astrocytes.

In astrocytes, the PGF(2alpha) or ionomycin treatment induces the phosphorylation at Ser38 and Ser82 of vimentin, a type III intermediate filament, by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We found here that vimentin phospho-Ser82 was dephosphorylated much slower than phospho-Ser38. Vimentin phospho-Ser38 was dephosphorylated quickly by purified PP1 catalytic subunit (PP1c) in vitro, whereas phospho-Ser82 was insensitive to PP1c. Because PP1c directly bound to vimentin through a VxF motif (Val83-Asp84-Phe85), the PP1c active site appeared to be unable to approach phospho-Ser82, leading to the prolongation of the phosphorylation at Ser-82. In astrocytes, PP1calpha was in vivo associated with vimentin filaments. The repetitive treatment by ionomycin at a short interval resulted in the sustained elevation of Ser82 phosphorylation, leading to the marked disassembly of vimentin filaments. Taken together, these results suggest that vimentin is a novel member of binding partner of PP1c in astrocytes, and vimentin-Ser82 may act as a memory phosphorylation site.

Amino Acid Motifs↗
PubMed1989

Enhancement of human B-cell proliferation by a monoclonal antibody to CD43.

Monoclonal antibodies (MoAb) to human leucocyte sialoglycoprotein, CD43, have been shown to deliver mitogenic signals to human T cells or to enhance T-cell proliferation induced by concanavalin A, anti-CD3 antibodies or phorbol ester. In this paper, we studied the effects of anti-CD43 MoAb B1B6 on the activation of human B cells. Anti-CD43 MoAb B1B6 was not mitogenic by itself for human B cells. However, when added together with TPA, both resting and in vivo activated tonsillar B cells, containing 5-10% and about 35% CD43+ respectively, responded with three- to fivefold higher proliferation compared to that obtained with TPA alone. A peak in the proliferative response was reached on day 3. Optimal proliferation was obtained when the antibody was present from the start of culturing. Addition of MoAb B1B6 together with a calcium ionophore, ionomycin, did not induce B-cell proliferation. Neither did mAb B1B6 sustain the growth of B cells that were already in the cell cycle, i.e. precultured with phorbol ester (PDB) and ionomycin for 3 days. The results are similar to those obtained with antibodies to CD22 and CD23 and show that early progression signals are delivered to resting B cells through CD43 in the presence of primary activators of protein kinase C.

Adjuvants, Immunologic↗
PubMed1989

Translocation of protein kinase C to subcellular fractions of human neutrophils.

The subcellular localization of protein kinase C in unstimulated human neutrophils and neutrophils stimulated by phorbol-myristate-acetate (PMA), 1-oleoyl-2-acetyl-rac-glycerol (OAG), and ionomycin was investigated in subcellular fractions obtained by nitrogen cavitation and Percoll density gradient centrifugation. Protein kinase C was found to be localized mainly in the cytosol in unstimulated cells, whereas significant translocation to fractions containing the plasma membrane was observed after stimulation by PMA, OAG, and ionomycin. At the same time, phospholipid-insensitive protein kinase activity appeared in the cytosol and the plasma membrane fractions. To determine whether binding of protein kinase C occurred to the plasma membrane or to intracellular membranes that had translocated to the plasma membrane, we investigated the ability of isolated azurophil, specific and secretory granules, and plasma membrane vesicles to bind protein kinase C in response to addition of PMA and OAG. Only fractions containing plasma membranes and secretory granules were able to bind protein kinase C. The observation explains the selective activation of plasma membrane structures by protein kinase C.

Binding Sites↗
PubMed1989

Induction of high-affinity interleukin 2 receptors on human T lymphocytes. The role of calcium and protein kinase C.

The relationship between free cytoplasmic calcium, activation of protein kinase C (PKC) and expression of high-affinity interleukin 2 receptors (HA-IL-2R) on human T lymphocytes was studied. Induction of HA-IL-2R by phytohaemagglutinin (PHA) was associated with an increase in free cytoplasmic calcium and a transient increase in membrane-associated PKC. However, whereas addition of EGTA inhibited induction of receptors by PHA, addition of the PKC-inhibitor H7 did not. 12-o-tetradecanoyl-phorbol-13-acetate (PMA) and 1-oleoyl-2-acetyl-rac-glycerol (OAG) were both found to activate and translocate PKC. However, only PMA induced expression of HA-IL-2R. Not surprisingly, the effect of PMA was independent of extracellular calcium, but was inhibited by H7. Furthermore, a correlation between the number of HA-IL-2R and free cytoplasmic calcium upon stimulation with ionomycin was observed. Associated with the rise in intracellular calcium, the ionophore caused a slight increase in membrane-associated PKC. Also, addition of H7 inhibited expression of HA-IL-2R. Finally, OAG and ionomycin acted synergistically on expression of HA-IL-2R. In conclusion, induction of HA-IL-2R requires at least two different signals and neither activation of PKC nor an increase in free cytoplasmic calcium is sufficient. However, these two signals may act synergistically. There is evidence for both a PKC- and calcium-independent pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Phagocytosis following translocation of the neutrophil b-cytochrome from the specific granule to the plasma membrane is associated with an increased leakage of reactive oxygen species.

The effect of neutrophil b-cytochrome translocation on the respiratory burst activation generated during phagocytosis of yeast particles was investigated. Secretion of neutrophil specific granules was induced by the calcium ionophore ionomycin prior to phagocytosis. The secretory process is associated with a translocation from the specific granules to the plasma membrane of the respiratory burst b-cytochrome. Respiratory burst activity was measured as release of hydrogen peroxide in the absence of azide (extracellular leakage) and in the presence of azide (total production). The subcellular localization of the b-cytochrome was found to affect the extracellular release of hydrogen peroxide in that a plasma membrane localization was associated with a significantly increased release during phagocytosis. It should be pointed out, however, that most of the hydrogen peroxide, both in control and in ionomycin-treated cells, is produced intracellularly, probably in the phagosomes.

Adult↗
PubMed1987

Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes.

The exocytotic release of L-glutamate from guinea-pig cerebral cortical synaptosomes can be extensively inhibited by preincubation with botulinum neurotoxin type A at 37 degrees C for 1-2 h. The toxin has no effect on synaptosomal respiratory control, respiratory capacity, ATP synthesis, plasma-membrane 86Rb+ permeability or plasma-membrane potential, does not inhibit the entry of 45Ca2+ into the synaptosome upon depolarization and does not alter the ability of intrasynaptosomal mitochondria to sequester Ca2+. The blockade of Ca2+-dependent glutamate release may be totally reversed by the Ca2+/2 H+-exchange ionophore ionomycin, but not by increasing extracellular Ca2+ concentration. It is suggested (a) that exocytosis is triggered by the penetration of Ca2+ into an intracellular hydrophobic milieu; (b) that this stage is blocked by the toxin and (c) that ionomycin is able to bypass this block and deliver Ca2+ to the exocytotic apparatus.

Animals↗
PubMed1989

Intracellular Ca2+ potentiates Na+/H+ exchange and cell differentiation induced by phorbol ester in U937 cells.

The human cell line U937 differentiates to monocyte macrophage-like cells in response to tumour-promoting phorbol esters. This effect is attributed to activation of protein kinase C. We show here that U937 cell differentiation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) is associated with cytoplasmic alkalinization. Ethyl-isopropyl-amiloride (EIPA), a potent inhibitor of Na+/H+ exchange, blocked both cytoplasmic alkalinization and cell differentiation. Cell acidification by addition of 2-4 mM sodium propionate also blocked TPA-induced U937 cell differentiation. These results suggest that a sustained cell alkalinization mediated by activation of Na+/H+ exchange is essential for TPA-induced differentiation in U937 cells. The increase of cytoplasmic free calcium concentration ([Ca2+]i) by addition of the calcium ionophore ionomycin enhanced TPA-induced alkalinization by increasing the apparent affinity of the Na+/H+ antiporter for intracellular H+. Treatment with ionomycin also potentiated differentiation of U937 cells induced by TPA. This synergism suggests that [Ca2+]i either potentiates the activation of protein kinase C or triggers additional transducing mechanisms. The key events of this interaction occur during the first 30 min of treatment, even though cell differentiation manifests much later.

Amiloride↗
PubMed1994

Contribution of phospholipases A2 and D to arachidonic acid liberation and prostaglandin D2 formation with increase in intracellular Ca2+ concentration in rat peritoneal mast cells.

The contribution of phospholipases A2 (PLA2) and D (PLD) activation to arachidonic acid liberation and prostaglandin D2 (PGD2) formation was studied in stimulated rat peritoneal mast cells. Stimulation of the cells with ionomycin induced time-dependent and Ca(2+)-concentration-dependent increase in arachidonic acid liberation and PGD2 formation, and the Ca(2+)-dependent increase was especially remarkable at extracellular Ca2+ concentration higher than 200 microM. Staurosporine did not induce any effect on the arachidonic acid liberation, indicating that protein kinase C is not involved in the liberation. Addition of ethanol to the cells decreased the ionomycin-stimulated arachidonic acid liberation to 40% of the control, while it decreased the PGD2 formation almost completely, with the increase in phosphatidylethanol formation. Propranolol, a phosphatidate phosphohydrolase inhibitor, caused similar effects. p-Bromophenacyl bromide, a PLA2 inhibitor, inhibited partially the arachidonic acid liberation. The inhibition of the liberation by combination of p-bromophenacyl bromide and ethanol was additive and reached approximately 90%. Under the conditions used p-bromophenacyl bromide did not influence significantly the PLD activity assessed by the phosphatidylethanol formation. Histamine release was decreased by ethanol treatment to 35% of the control. These results suggest that more than half of the total arachidonic acid liberation is mediated by the sequential pathway of PLD/phosphatidate phosphohydrolase/diacylglycerol lipase and more than half of histamine release is also dependent on PLD activation, while the PGD2 formation is fully mediated by the pathway. PLA2 also contributes to arachidonic acid liberation but to a lower extent.

Acetophenones↗