Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura-2 responded to exogenous platelet-activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF-induced [CA2+]i transients consisted of a dose-dependent phasic peak response followed by a sustained tonic phase of increased [Ca2+]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+]i: a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor-operated channels. Lyso-PAF had no effect on basal [Ca2+]i and the PAF-receptor antagonist L652,731 selectively inhibited responses to PAF. PAF-stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium-mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF-induced [Ca2+]i transient, suggesting a regulatory role for PKC in PAF-activation of mesangial cells. An increase in [Ca2+]i, as a result of receptor-linked activation of phospholipase C, may mediate PAF-induced hemodynamic and inflammatory events in renal glomeruli.     

Serotonin-induced cytosolic free calcium transients in cultured vascular smooth muscle cells

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.     

Nature of thrombin-induced sustained increase in cytosolic calcium concentration in cultured endothelial cells

It has recently been appreciated that thrombin induces the retraction of endothelial cells resulting in an alteration of the integrity of the monolayers. We studied thrombin-induced changes in cytosolic calcium concentration (Ca2+i) using microfluorometry of fura-2-loaded single cells, cell topography (scanning electron microscopy), and cytoskeleton (rhodamine phalloidin) in endothelial cells. Thrombin caused an initial and sustained phase of an increase in Ca2+i. Pretreatment with pertussis toxin abolished both phases of Ca2+i response. Sustained phase of thrombin effect required extracellular calcium. Pretreatment of endothelial cells with indomethacin protracted the sustained phase, whereas a lipoxygenase inhibitor, nordihydroguaiaretic acid curtailed it. Thrombin caused a marked retraction of confluent endothelial cells coincident with the sustained phase of Ca2+i response. This was paralleled by the formation of gaps in F-actin distribution at the periphery of the cells. Pretreatment of endothelial cells with nordihydroguaiaretic acid blunted the thrombin-induced cell retraction. Microinjection of various putative messengers into the endothelial cells showed that initial Ca2+ mobilization is not sufficient to account for sustained elevation of Ca2+i. The sustained response required microinjection of phospholipase A2 or co-injection of phospholipase A2 with phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, phosphatidylinositol 1,4,5-trisphosphate, or CaCl2, further implying that thrombin receptor(s) can be coupled to both phospholipases C and A2. Sustained elevation of Ca2+i was a necessary prerequisite for the thrombin-induced changes in endothelial cell topography.     

Regulation of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin

The regulating mechanisms of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin were investigated. The formation of PAF-acether was maximal at 5 min after stimulation and gradually decreased for up to 30 min. Thrombin induced a rapid 3-4-fold increase in the activity which was maximal by 1 min after stimulation and returned progressively to basal level within 10 min. The thrombin-induced enhancement in acetyltransferase activity was due to an increase of the Vmax of the acetylation reaction without a significant effect on the apparent Km of the enzyme for acetyl-CoA. Human endothelial cells also exhibited a basal PAF-acether acetylhydrolase activity which was not altered upon thrombin stimulation. The pretreatment with 2 mM phenylmethylsulfonyl fluoride (PMSF), a serine proteinase inhibitor reported to block the acetylhydrolase, induced about 2-times more PAF-acether production in response to 2.5 U/ml thrombin stimulation. However, this enhancement of PAF-acether formation seems to be not only due to the inhibition of the acetylhydrolase, but also to the influences on the activities of the acetyltransferase and other enzymes such as phospholipase A2. These results suggest a key role for acetyltransferase and acetylhydrolase in the regulation of PAF-acether formation and catabolism in thrombin-stimulated human endothelial cells.     

Stimulation of eosinophil adherence to human vascular endothelial cells in vitro by platelet-activating factor

111In-Labeled eosinophils from mildly eosinophilic subjects have been examined for their capacity to adhere to cultured human umbilical vein endothelial cells. In assay buffer alone, 32.0% +/- 2.6 eosinophils adhered spontaneously to endothelial cells. Platelet-activating factor (PAF) (1-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) at concentrations as low as 10(-9) M increased this adherence to a level of 46.7% +/- 2.0. The effects of PAF were confirmed to be on eosinophils by parallel adherence assays done on serum-coated plastic plates where comparably enhanced adhesion of the eosinophils was seen. Lyso-PAF, the biologically inactive precursor/metabolite of PAF, had no stimulatory properties. FMLP caused an increase in eosinophil adherence, comparable to that of PAF, but only at high concentrations (10(-6) to 10(-7) M). Further examination of eosinophil subpopulations separated on metrizamide gradients indicated that "hypodense" eosinophils had a significantly higher ability to adhere spontaneously to endothelial cells than "normal" dense eosinophils, (35.5% +/- 4.2 vs 23.8% +/- 2.5, respectively) and could be stimulated with PAF to higher levels, although the magnitude of stimulation was similar for both populations. A mouse mAb TS1/18 to the common beta-subunit of the Mac-1 cell surface glycoprotein complex (CDw18) reduced by up to 94.6% the PAF-induced increase in adherence, but had no effect on the spontaneous adhesion. Eosinophils were also shown by cytofluorography to be capable of binding the TS1/18 antibody on their cell surface, and in some experiments to exhibit an increased expression of the Mac-1 complex on stimulation with PAF. These studies indicate that eosinophils are capable of binding to endothelial cells in culture, that PAF is a potent stimulator of eosinophil adherence, and that the Mac-1 complex has a critical role in this adhesion process.     

Mediation by platelet-activating factor of 12-hydroxyeicosatetraenoic acid-induced cytosolic free calcium concentration elevation in neutrophils

12(R)-hydroxyeicosatetraenoic acid (HETE) shows biphasic increase in cytosolic free calcium concentration ([Ca2+]i) in rabbit and human neutrophils; the initial transient phase and the continuous falling phase. 12(S)-HETE was less potent in both species. BN50739, a platelet-activating factor (PAF) receptor antagonist, inhibited both phases of 12(R)-HETE-induced [Ca2+]i rise but did not affect leukotriene B4 (LTB4)-induced [Ca2+]i rise. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a PAF synthesis inhibitor, and manoalide, a phospholipase A2 inhibitor, reduced 12(R)-HETE-induced [Ca2+]i rise. These blockers inhibited the continuous phase of [Ca2+]i rise induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) with little effect on the initial phase. It had no significant effect on LTB4-induced [Ca2+]i rise. SC-41930, a LTB4-receptor antagonist, did not block 12-HETE-induced [Ca2+]i rise. In 12(R)-HETE-, FMLP- and LTB4-stimulated cells, accumulations of cell-associated PAF and released PAF were detected but not in unstimulated cells. BN50739 did not affect the accumulation of cell-associated PAF and release of PAF in 12(R)-HETE-stimulated cells. These results suggest that 12(R)-HETE-induced and partially, FMLP-induced, but not LTB4-induced [Ca2+]i rise are mediated by PAF, which is produced and released by stimulation of the cells by 12(R)-HETE and FMLP, respectively.     

Correlation between thrombin-induced prostacyclin production and inositol trisphosphate and cytosolic free calcium levels in cultured human endothelial cells

Cultured human umbilical vein endothelial cells (HUVEC) stimulated with thrombin are known to synthesize prostacyclin at least in part from arachidonate released by phospholipase A2, an enzyme directly activated by calcium. In this study, thrombin stimulation of Quin 2-loaded HUVEC caused rapid and dose-dependent rises in inositol trisphosphate (IP3) and cytosolic free calcium (Ca2+i) levels which preceded a similarly dose-dependent rise in prostacyclin production measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) by radioimmunoassay (ED50 = 0.6-0.7 units/ml for all three effects). Thrombin induced these effects in the absence of extracellular calcium (EGTA) or in the presence of either 8-bromo-cAMP or the calmodulin inhibitor W7. Thrombin inactivated with either diisopropyl fluorophosphate or D-Phe-Pro-Arg-chloromethyl ketone was inactive. In contrast, Quin 2-loaded cultured bovine aortic endothelial cells failed to respond to thrombin, although stimulation with trypsin elevated IP3 and Ca2+i levels and increased 6-keto-PGF1 alpha production. Restimulation of HUVEC with thrombin or histamine 5 min after an initial stimulation with thrombin (2 units/ml for 5 min) failed to induce a second rise in either IP3 or Ca2+i levels or further production of 6-keto-PGF1 alpha, whereas restimulation with ionomycin in the presence or absence of extracellular calcium elevated Ca2+i levels and induced further 6-keto-PGF1 alpha production. However, if the initial stimulation with thrombin was terminated by addition of D-Phe-Pro-Arg-chloromethyl ketone within 10-60 s, restimulation with a second dose of thrombin induced second rises in both IP3 and Ca2+i levels and additional 6-keto-PGF1 alpha production that were greatest when the initial thrombin stimulus was briefest. These results are consistent with the conclusion that IP3 acts as a second messenger by which thrombin elevates Ca2+i levels and initiates prostacyclin synthesis in HUVEC and that in vivo endothelial cells may be stimulated multiple times to synthesize prostacyclin if each period of stimulation is brief.     

Effect of superoxide and lipid peroxide on cytosolic free calcium concentration in cultured pig aortic endothelial cells

The effect of reactive oxygen on cytosolic free calcium concentration [( Ca++]i) in pig aortic endothelial cells (ECs) was studied. Linoleate hydroperoxide (LHO) and superoxide radicals generated from xanthine with xanthine oxidase (X-XO) were used as sources of reactive oxygen. [Ca++]i in ECs was measured with quin 2 and the value for quiescent ECs was 112 +/- 11 nM. Both LHO and X-XO increased [Ca++]i in a dose-dependent manner without accompanying the significant cellular damage. Nifedipine suppressed the increase in [Ca++]i provoked by LHO and X-XO. Thus, the biological effects of reactive oxygen might be mediated, at least in part, by the activation of voltage-dependent calcium channels in ECs.     

Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells.

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+ channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni(2+)-, La(3+)-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 mumol l-1 and 2.5 mumol l-1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mumol l-1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mumol l-1 and 95.29 mumol l-1, respectively. The depolarization was significantly inhibited at 50 mumol l-1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.     

Receptor binding activity and cytosolic free calcium response by synthetic endothelin analogs in cultured rat vascular smooth muscle cells

Using a variety of synthetic analogs of porcine endothelin (pET), we have studied the effects of these analogs on receptor binding activity and cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMC). Removal of C-terminal Trp21 residue, truncated derivatives pET(1-15) and (16-21), substitution of disulfide bond, Cys(3-11) or Cys(1-15), by Cys (Acm), all resulted in a complete loss of receptor binding activity and [Ca2+]i response, while N-terminal elongation of Lys-Arg residues, but not oxidation of Met7 residue, decreased receptor binding activity and [Ca2+]i response. [Cys1-15,Cys3-11]pET was far more potent than [Cys1-11,Cys3-15]pET in receptor binding and [Ca2+]i response. These data indicate that the C-terminal Trp21 as well as the proper double cyclic structure formed by the intramolecular disulfide bonds of the pET molecule are essential for receptor binding and subsequent [Ca2+]i increase in rat VSMC.     

State-dependent calcium mobilization by urotensin-II in cultured human endothelial cells

Human endothelial cells express urotensin-II (U-II) as well as its receptor GPR14. Using microfluorimetric techniques, the effect of human U-II on cytosolic Ca2+ concentrations [Ca2+]i in cultured human aortic endothelial cells (HAECs) loaded with Fura-2 was evaluated in static or flow conditions. Under the static state, U-II (100 nM) abolished spontaneous Ca2+ oscillations, which occurred in a population of cultured HAEC. Similarly, U-II reduced thrombin-, but not ATP-induced calcium responses, suggesting that the peptide does not alter the Gq/11/IP3 pathway; rather, it modifies the coupling between protease-activated receptors and Gq/11/IP3. Under the flow condition, U-II (1, 10 and 100 nM) produced a dose-dependent increase in [Ca2+]i, which was subjected to desensitization. The result demonstrates a state-dependent effect of U-II in cultured HAEC, which may explain the variable responses to U-II under different experimental conditions.     

Biphasic increase in intracellular calcium induced by platelet-activating factor in macrophages

In single mouse macrophages stimulated by platelet-activating factor (PAF), the intracellular calcium concentration (Cai) monitored with fura-2 at room temperature presents a biphasic increase, including a transient and a more sustained component. After pulse administration of PAF, the first phase lasts for a few seconds and reaches a peak value of 0.5-1 microM Ca2+ at high PAF concentration. The amplitude of this peak is independent of extracellular Ca2+ concentration, suggesting that the initial Ca2+ transient is due to the release of Ca2+ from intracellular stores. The second phase of the response lasts for several minutes; its maximum amplitude is reached 1-2 min after the brief initial PAF stimulation. This phase, suppressed in zero external Ca2+ and increased in 10 mM Ca2+, is probably due to influx of Ca2+ through the plasma membrane. This secondary Ca2+ increase is blocked by 10-50 microM lanthanum. At low PAF concentration, the initial Ca2+ transient is not followed by a second phase, showing that the initial rises of Ca2+ and of its activator (presumably inositol trisphosphate) are not sufficient to trigger the second phase of Ca2+ increase.     

Stimulation of cytosolic free calcium and inositol phosphates by prostaglandins in cultured rat mesangial cells

We studied the effects of four products of arachidonate cyclo-oxygenation on a phospholipase C-dependent signal transduction system in cultured rat glomerular mesangial cells. PGF2 alpha, PGE2 and the thromboxane A2/endoperoxide analogue U-46619 rapidly increased cytosolic free Ca2+, measured in monolayers loaded with the fluorescent intracellular probe fura-2. Peak responses were dose-dependent and unaffected by chelation of extracellular Ca2+, indicating release from internal stores. The thromboxane A2-receptor antagonist SQ 27,427 selectively inhibited responses to U-46619. The PGI2 analogue Iloprost had no effect on cytosolic Ca2+. PGF2 alpha, PGE2 and U-46619 also stimulated accumulation of total inositol phosphates during 15 min incubations. We conclude that phospholipase C activation mediates the effects of certain eicosanoids on the glomerular mesangium.     

Synthesis and release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin 1 alpha

Human endothelial cells synthesize large amounts of platelet-activating factor (PAF) after 30-min treatment with recombinant tumor necrosis factor (TNF). Synthesis of PAF peaks at 4-6 h, whereas in endothelial cells treated with interleukin 1 alpha (IL-1) it peaks at 8-12 h. More than twice as much PAF is synthesized in response to optimal concentrations of TNF than in response to IL-1. However, PAF synthesis is stimulated by lower molar concentrations of IL-1 than TNF. About 30% of PAF produced in response to either TNF or IL-1 is released into the medium, whereas approximately 70% remains cell-associated. Experiments with labeled precursors show that PAF is synthesized de novo in response to TNF. This activity of TNF is inhibited by treating endothelial cells with the inhibitors of protein or RNA synthesis cycloheximide or actinomycin D. This finding may be explained by the observation that TNF induces in endothelial cells an acetyltransferase required for PAF synthesis. The induction of this enzymatic activity precedes the peak of PAF synthesis in TNF-treated cells. After prolonged incubation with either TNF or IL-1, endothelial cells no longer respond to the same monokine, but are still capable of producing PAF when treated with the other monokine. The finding that these monokines do not show reciprocal tachyphylaxis in endothelial cells may be explained by their binding to different receptors. In cells treated simultaneously with different concentrations of TNF and IL-1, PAF synthesis is stimulated in an additive rather than synergistic way. This suggests that PAF is synthesized by the same pathway in response to TNF or IL-1.     

Characterization of platelet-activating factor-induced elevation of cytosolic free calcium concentration in eosinophils

In order to evaluate the role of calcium in the activation processes in eosinophils induced by platelet-activating factor (PAF), we investigated the changes in free cytoplasmatic Ca2+ concentration using fura-2. PAF causes a rapid and transitory rise of the intracellular free calcium ion concentration [( Ca2+]i) in purified guinea pig eosinophils of approx. 1000 nM above a basal level of 120.7 +/- 36.5 nM (n = 10). The effect was dose-related with a maximum rise at 1000 nM PAF and an EC50 of 17.4 nM and specifically inhibited by the PAF antagonist WEB 2086 with an IC50 of 95.5 nM. WEB 2086 did not affect either the leukotriene B4- or the fMet-Leu-Phe-induced elevation of [Ca2+]i. The response to PAF was dependent on external Ca2+ as it was significantly inhibited by EGTA (85.6 +/- 5.4%) and Ni2+ (95.8 +/- 2.1%) but not by the dihydropyridine antagonist nimodipine. We conclude that Ca2+ entry via receptor-operated Ca2+ channels may be involved in PAF-induced degranulation of eosinophils.     

Copper elicits an increase in cytosolic free calcium in cultured tobacco cells

At concentrations greater than 0.1 mM, CuSO(4) provoked a rapid and sustained increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), in tobacco suspension culture cells expressing apoaequorin, a Ca(2+)-sensitive photoprotein. The increase was suppressed by treatment with LaCl(3), indicating that the increase is due to an influx of Ca(2+) from the apoplast through plasma membrane Ca(2+) channels. Although stimulation of H(2)O(2) production upon the CuSO(4) treatment (0.1 mM) was observed, treatment with catalase did not inhibit the increase in [Ca(2+)](cyt), and treatment with H(2)O(2) dose-dependently suppressed or delayed the increase. These results suggested that active oxygen species generated through copper-mediated reactions, or copper-mediated oxidative damages to plasma membrane, are not responsible for the increase. Treatment with sulfhydryl reagents, which alkylate or oxidize thiol groups, or acidification of the culture medium suppressed the increase in [Ca(2+)](cyt). These results demonstrated that copper causes an influx of Ca(2+) through plasma membrane Ca(2+) channels, and that plasma membrane thiol groups play an important role in activating the Ca(2+) channels.