Dataset Information

Effect of deoxycholate on guanine-nucleotide-dependent carbachol stimulation of phosphoinositidase C in mouse brain cortical membranes.

ABSTRACT: Demonstration of guanine-nucleotide-dependent neurotransmitter stimulation of phosphoinositide breakdown in brain membranes has generally required the presence of the detergent, deoxycholate (DOC), in the assay medium. In the present study, by using mouse brain cortical membranes labelled with [3H]inositol in the presence of CMP through the reverse PtdIns synthase reaction, we have been able to show guanosine 5'-[gamma-thio]triphosphate (GTP[S])-dependent carbachol (CCh) stimulation of the formation of [3H]inositol phosphates in the absence of DOC and have studied how the detergent affects the response. The results of our study indicate that DOC affects the muscarinic receptor-G-protein-phosphoinositidase C (PIC) transduction system in several ways. First, it enhances agonist-induced PIC activity towards [3H]PtdInsP and [3H]PtdInsP2 and, secondly, it decreases the potency for GTP[S] stimulation of PIC, thus enhancing the agonist-induced leftward shift of the dose-response curve for GTP[S]. Additionally, DOC appears to increase the activity of the enzymes of the phosphoinositide cycle, PtdIns 4-kinase, Ins(1,4,5)P3 5-phosphatase and Ins(1,4)P2 1-phosphatase, thus altering the proportion of phosphoinositide substrates and inositol phosphate products. These observations advise caution in drawing conclusions about PIC substrate specificity and the potency of both guanine nucleotides and agonists from experiments performed in membranes in the presence of DOC or related bile salts.

SUBMITTER: Bas N

PROVIDER: S-EPMC1136282 | biostudies-other | 1995 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.

Dataset Information

Effect of deoxycholate on guanine-nucleotide-dependent carbachol stimulation of phosphoinositidase C in mouse brain cortical membranes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure