Astrid Hagelüken

Activation of GTP formation and high-affinity GTP hydrolysis by mastoparan in various cell membranes. G-protein activation via nucleoside diphosphate kinase, a possible general mechanism of mastoparan action.

The wasp venom, mastoparan (MP), is a direct activator of reconstituted pertussis toxin-sensitive G-proteins and of purified nucleoside diphosphate kinase (NDPK) [E.C. 2.6.4.6.]. In HL-60 membranes, MP activates high-affinity GTPase [E.C. 3.6.1.-] and NDPK-catalyzed GTP formation, but not photolabeling of G-protein alpha-subunits with GTP azidoanilide; this suggests that the venom activates G-proteins in this system indirectly via stimulation of NDPK. Moreover, the MP analogue, mastoparan 7 (MP 7), is a much more effective activator of reconstituted G-proteins than MP, whereas with regard to NDPK and GTPase in HL-60 membranes, the two peptides are similarly effective. In our present study, we investigated NDPK- and G-protein activation by MP in membranes of the human neuroblastoma cell line, SH-SY5Y, the human erythroleukemia cell line, HEL, the rat basophilic leukemia cell line, RBL 2H3, and the hamster ductus deferens smooth muscle cell line, DDT1MF-2. All these membranes exhibited high NDPK activities that were increased by MP. Compared to basal GTP formation rates, basal rates of high-affinity GTP hydrolysis in cell membranes were low. MP activated high-affinity GTP hydrolysis in cell membranes but did not enhance incorporation of GTP azidoanilide into G-protein alpha-subunits. As with HL-60 membranes, MP and MP 7 were similarly effective activators of NDPK and GTPase in SH-SY5Y membranes. Pertussis toxin inhibited MP-stimulated GTP hydrolyses in SH-SY5Y- and HEL membranes, whereas NDPK activations by MP were pertussis toxin-insensitive. Our data suggest that indirect G-protein activation via NDPK is not restricted to HL-60 membranes but is a more general mechanism of MP action in cell membranes. Pertussis toxin-catalyzed ADP-ribosylation of alpha-subunits may inhibit the transfer of GTP from NDPK to G-proteins. NDPK may play a much more important role in transmembrane signal transduction than was previously appreciated and, moreover, the GTPase of G-protein alpha-subunits may serve as GDP-synthase for NDPK.

Histamine receptor-dependent and/or -independent activation of guanine nucleotide-binding proteins by histamine and 2-substituted histamine derivatives in human leukemia (HL-60) and human erythroleukemia (HEL) cells.

In dibutyryl cAMP-differentiated human leukemia (HL-60) cells, the potent histamine H1-receptor agonist, 2-(3-chlorophenyl)histamine, activates pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins) of the Gi-subfamily by a mechanism which is independent of known histamine receptor subtypes (Seifert et al. Mol Pharmacol 45: 578-586, 1994). In order to learn more about this G-protein activation, we studied the effects of histamine and various 2-substituted histamine derivatives in various cell types and on purified G-proteins. In HL-60 cells, histamine and 2-methylhistamine increased cytosolic Ca2+ concentration ([Ca2+]i) in a clemastine-sensitive manner. Phenyl- and thienyl-substituted histamines increased [Ca2+]i as well, but their effects were not inhibited by histamine receptor antagonists. 2-Substituted histamines activated high-affinity GTPase in HL-60 cell membranes in a PTX-sensitive manner, with the lipophilicity of substances increasing their effectiveness. Although HEL cells do not possess histamine receptors mediating rises in [Ca2+]i, 2-(3-bromophenyl)histamine increased [Ca2+]i in a PTX-sensitive manner. It also increased GTP hydrolysis by Gi-proteins in HEL cell membranes. All these stimulatory effects of 2-substituted histamine derivatives were seen at concentrations higher than those required for activation of H1-receptors. In various other cell types and membrane systems, 2-substituted histamine derivatives showed no or only weak stimulatory effects on G-proteins. 2-Substituted histamine derivatives activated GTP hydrolysis by purified bovine brain Gi/Go-proteins and by pure Gi2 (the major PTX-sensitive G-protein in HL-60 and HEL cells). Our data suggest the following: (1) histamine and 2-methylhistamine act as H1-receptor agonists in HL-60 cells; (2) incorporation of bulky and lipophilic groups results in loss of H1-agonistic activity of 2-substituted histamine derivatives in HL-60 cells but causes a receptor-independent G-protein-stimulatory activity; (3) the effects of 2-substituted histamine derivatives on G-proteins are cell-type specific.

Direct and indirect receptor‐independent G‐protein activation by cationic‐amphiphilic substances. Studies with mast cells, HL‐60 human leukemic cells and purified G‐proteins

Abstract Studies from several laboratories have revealed that structurally diverse substances including the wasp venom, mastoparan (MP), activate purified regulatory heterotrimeric guanine nucleotidc‐binding proteins (G‐proteins) in a receptor‐independent manner, presumably by mimicking the effects of heptahelical receptors. Mast cells and differentiated HL‐60 human leukemic cells are useful model systems for the analysis of receptor‐independent G‐protein activation. We compared the effects of 2‐phenylhistamines which are cationic‐amphiphilic, too, and of MP on G‐protein activation in dibutyryl cAMP‐differentiated HL‐60 cells and in the rat basophilic leukemia cell line, RBL 2H3. In HL‐60 cells, 2‐phenylhistamines show stimulatory effects which resemble those of formyl peptide receptor agonists but which cannot be attributed to agonism at classical receptors. 2‐phenylhistamines do not, however, activate RBL 2H3 cells and various other myeloid cell types, pointing to cell type‐specificity of receptor‐independent G‐protein activation. In HL‐60 cells, MP shows effects on G‐protein activation which differ substantially from those of formyl peptides. In RBL 2H3 membranes, MP shows similar effects on G‐prolein activation as in HL‐60 membranes. We develop a model according to which receptor‐independent G‐protein activation can be subdivided into direct and indirect receptor‐independent G‐protein activation. In case of the former mechanism, substances like 2‐phenylhislamines interact with G‐protein α‐subunits and in case of the latter mechanism, substances like MP interact with nucleoside diphosphate kinase which catalyzes the formation of GTP. This newly formed GTP is then transferred to, and cleaved by, G‐protein a‐subunits. NDPK is a novel target for the design of drugs which interfere with G‐protein‐mcdiated signal transduction at a post‐receptor level and may modulate the function of various cell types including mast cells.

2-Substituted histamines with G-protein-stimulatory activity

Summary The cationic-amphiphilic 2-substituted histamines, 2-(3-chlorophenyl)histamine (2-[2-(3-chlorophenyl)-1H-imidazol-4-yl]ethanamine) and 2-(2-cyclohexylethyl)histamine, activate pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins) of the Gi-subfamily by a receptor-independent mechanism. We studied structure-activity relationships of 2-substituted histamine derivatives for this G-protein activation using six known and 12 newly synthesized compounds. Elongation of the alkyl chain between imidazole and the ring system enhanced the potency and efficiency of substances in activating high-affinity GTP hydrolysis, ie the enzymatic activity of G-protein α-subunits, in membranes of HL-60 leukemic cells. Cyclopentyl-, cyclohexyl and norbornyl-substituted histamines were more effective and potent than phenyl-substituted histamines in mediating G-protein activation in HL-60 membranes and in activating reconstituted bovine brain Gi/Go-proteins. Our data show that the chain length and the type of ring system are important determinants for receptor-independent G-protein activation by 2-substituted histamines. With respect to histamine Hi-receptors, most of the substances studied displayed weak antagonistic activity.