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Receptor-mediated increases in cytosolic Ca2+ in the human erythroleukaemia cell line involve pertussis toxin-sensitive and -insensitive pathways.

ABSTRACT: The pluripotent human erythroleukaemia cell line, HEL, possesses erythrocytic, megakaryocytic and macrophage-like properties. With respect to signal transduction, HEL cells have been used as a model system for platelets, but little attention has been paid to their phagocytic properties. We studied the effects of various receptor agonists on the intracellular free Ca2+ concentration ([Ca2+]i) in HEL cells. Thrombin, platelet-activating factor (PAF), ATP, UTP, prostaglandins E1 and E2 (PGE1 and PGE2), the PGE2 analogue sulprostone and the stable PGI2 analogues iloprost and cicaprost increased [Ca2+]i. ADP was less effective than ATP, and UDP was unable to increase [Ca2+]i. The increases in [Ca2+]i induced by thrombin, PAF, ATP, UTP, iloprost and cicaprost were pertussis toxin-insensitive, whereas the increases induced by PGE2 and sulprostone were completely inhibited by the toxin. The increase in [Ca2+]i induced by PGE1 was partially inhibited by pertussis toxin. PGE2 did not desensitize the increase in [Ca2+]i induced by iloprost, and vice versa. PGE1 desensitized the response to PGE2 and iloprost but not vice versa. Adrenaline potentiated the iloprost- but not the PGE2-induced rise in [Ca2+]i. The phorbol ester phorbol 12-myristate 13-acetate completely blocked the rise in [Ca2+]i induced by ATP and PGE1, whereas the increases induced by thrombin and PAF were only partially inhibited. Agonists increased [Ca2+]i through release from internal stores and sustained Ca2+ influx. Thrombin stimulated Mn2+ influx, which was blocked by Ni2+. Diltiazem, isradipine, gramicidin and 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) did not affect agonist-induced rises in [Ca2+]i. HEL cells contained substantial amounts of beta-glucuronidase which, however, could not be released, and they did not aggregate or generate superoxide. Our data suggest that: (1) HEL cells possess nucleotide receptors with properties similar to those of phagocytes; (2) they possess receptors for PGE2 and PGI2, and PGE1 is an agonist at both receptors; (3) agonist-induced increases in [Ca2+]i are mediated through pertussis toxin-sensitive as well as -insensitive signal transduction pathways; and (4) agonists increase [Ca2+]i by mobilization from internal stores and influx from the extracellular space through cation channels with properties similar to those of phagocytes and platelets.

SUBMITTER: Schwaner I

PROVIDER: S-EPMC1130683 | biostudies-other | 1992 Jan

REPOSITORIES: biostudies-other

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Project description:In the present study, we determined the agonist specificity and the signalling mechanisms of a putative sphingosine 1-phosphate (S1P) receptor, AGR16. In CHO cells transiently transfected with an AGR16 expression vector, but not in cells transfected with an empty vector, the addition of a low concentration of S1P (1 nM) caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) by mobilization of Ca2+ from both intra- and extra-cellular pools. To determine the spectrum of agonists for AGR16, we employed K562 cells, which in the naive state do not respond at all to either S1P or structurally related lipids with an increase in [Ca2+]i. In K562 cells stably expressing AGR16, S1P and sphingosylphosphorylcholine (SPC) dose-dependently increased [Ca2+]i with half-maximal values of 3 nM and 100 nM respectively. In CHO cells stably expressing AGR16 (CHO-AGR16), but not in parental CHO cells, we observed specific binding of [32P]S1P, which was displaced by unlabelled S1P and SPC. In CHO-AGR16 cells, but not in parental CHO cells, S1P stimulated the production of inositol phosphates and Ca2+ mobilization which was only 30% inhibited by pertussis toxin (PTX), different from the case of the recently identified S1P receptor EDG1. Also in CHO-AGR16 cells, but not in CHO cells, S1P at higher concentrations activated mitogen-activated protein kinase (MAPK) in a PTX-sensitive and Ras-dependent manner. S1P also induced the activation of two stress-activated MAPKs, c-Jun N-terminal kinase and p38, in a manner that was totally insensitive to PTX. In CHO-AGR16 cells, S1P induced stress-fibre formation, with an increase in myosin light chain phosphorylation, in a PTX-insensitive and Rho-dependent manner. S1P also induced an increase in the cellular cAMP content in CHO-AGR16 cells, which contrasts sharply with the case of EDG1. These results establish that the S1P receptor AGR16 is coupled via both PTX-sensitive and -insensitive G-proteins to multiple effector pathways.

Project description:Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca(2+)](cyt) in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca(2+) induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca(2+) release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca(2+) transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca(2+). Ca(2+) oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca(2+) influx and Ca(2+) reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca(2+) influx, intracellular Ca(2+) release, and reuptake underlie the Ca(2+) transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.

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Receptor-mediated increases in cytosolic Ca2+ in the human erythroleukaemia cell line involve pertussis toxin-sensitive and -insensitive pathways.

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