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.

The P2U Purinoceptor Obligatorily Engages the Heterotrimeric G Protein G16 to Mobilize Intracellular Ca2+ in Human Erythroleukemia Cells

“…This suggests that both nucleotides act through a common nucleotide receptor, most likely of the P 2U purinoceptor subtype. Consistently, the presence of receptors in HEL cells for UTP and ATP has been shown in earlier studies (28). As shown above, UTP did not depend on extracellular Ca 2ϩ to evoke its effects, suggesting that the receptor for UTP is not ionotropic (such as P 2 receptors of the X subtype).…”

“…2B). PGE 2 , which acts via G i -coupled receptors (28), was completely ineffective after PTX treatment, demonstrating that PTX treatment was exhaustive (not shown). The similar relative inhibitions of agonist-induced Ca 2ϩ changes that were observed after PTX treatment in parental and G ␣16 -suppressed cells make it unlikely that the expression pattern of G ␣ -and G ␤␥ -sensitive PLC-␤ isoforms differed considerably between G ␣16 -suppressed cells and parental HEL cells.…”

“…The sensitivity to PTX can help to identify the subclass of G proteins and, consequently, the possible subtypes of PLC-␤ which may be involved in intracellular Ca 2ϩ mobilization. Schwaner et al (28) reported that in HEL cells, the transient increase of [Ca 2ϩ ] i induced by thrombin is rather insensitive to PTX, whereas the effects of PGE 1 and PGE 2 are partially or completely inhibited by the toxin, respectively. In our experiments in parental HEL and G ␣16 -suppressed 3D4 cells, PTX inhibited thrombin-induced changes in [Ca 2ϩ ] i by 23 and 14%, respectively (Fig.…”

“…A variety of ligands are known to induce a transient increase in [Ca 2ϩ ] i in HEL cells in a G protein-dependent fashion (28). ] i over resting levels after adding different agonists at maximum effective concentrations.…”

Cloning and expression of a cDNA for the human prostacyclin receptor

“…3b, iloprost gen- hydrolysis. In previous studies, PGI, or its analogues evoked an increase in the cytosolic Ca2+ concentration in human cell lines, MEG-01 cells [8], and HEL cells, a pluripotent human erythroleukaemia cell line [19]. The present study revealed that the human PGI, receptor as well as the mouse receptor can couple to multiple signalling pathways, adenylate cyclase and phospholipase C. sequence of the human PGI, receptor is 73% identical to that of the mouse receptor.…”