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G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

ABSTRACT: Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5'-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected.

SUBMITTER: Vaziri C

PROVIDER: S-EPMC1132627 | biostudies-other | 1992 Jun

REPOSITORIES: biostudies-other

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Project description:We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated beta-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of beta-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we attempt to unify the equilibrium and collision coupling approaches to provide a consistent theoretical basis for the G-protein-mediated beta-adrenergic receptor signalling in turkey erythrocyte membranes.

Project description:Gastric cancer (GC) is the one of the most prevalent cancers and one of the leading causes of cancer-induced deaths. Previously, we found that the expression of purinergic P2Y2 receptor (P2Y2R) is increased in GC samples as compared to adjacent healthy mucosa taken from GC-diagnosed patients. In this work, we studied in detail purinergic signaling in the gastric adenocarcinoma-derived cell lines: AGS, MKN-45, and MKN-74, and compared them to a nontumoral epithelial cell line: GES-1. In GC-derived cells, we detected the expression of several purinergic receptors, and found important differences as compared to GES-1 cells. Functional studies revealed a strong contribution of P2Y2Rs in intracellular calcium increases, elicited by adenosine-triphosphate (ATP), uridine-triphosphate (UTP), and the P2Y2R agonist MRS2768. Responses were preserved in the absence of extracellular calcium and inhibited by P2Y2R antagonists. In GES-1 cells, ATP and UTP induced similar responses and the combination of P2X and P2Y receptor antagonists was able to block them. Proliferation studies showed that ATP regulates AGS and MKN-74 cells in a biphasic manner, increasing cell proliferation at 10-100 μM, but inhibiting at 300 μM ATP. On the other hand, 1-300 μM UTP, a P2Y2R agonist, increased concentration-dependent cell proliferation. The effects of UTP and ATP were prevented by both wide-range and specific purinergic antagonists. In contrast, in GES-1 cells ATP only decreased cell proliferation in a concentration-dependent manner, and UTP had no effect. Notably, the isolated application of purinergic antagonists was sufficient to change the basal proliferation of AGS cells, indicating that nucleotides released by the cells can act as paracrine/autocrine signals. Finally, in tumor-derived biopsies, we found an increase of P2Y2R and a decrease in P2X4R expression; however, we found high variability between seven different biopsies and their respective adjacent healthy gastric mucosa. Even so, we found a correlation between the expression levels of P2Y2R and P2X4R and survival rates of GC patients. Taken together, these results demonstrate the involvement of different purinergic receptors and signaling in GC, and the pattern of expression changes in tumoral cells, and this change likely directs ATP and nucleotide signaling from antiproliferative effects in healthy tissues to proliferative effects in cancer.

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G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

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