Dataset Information

Agonist regulation of cellular Gs alpha-subunit levels in neuroblastoma x glioma hybrid NG108-15 cells transfected to express different levels of the human beta 2 adrenoceptor.

ABSTRACT: Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at least three receptors which activate adenylate cyclase via the intermediacy of the stimulatory G-protein, Gs. Sustained exposure of the cells to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of Gs (Gs alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, Mullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. By contrast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effect on cellular amounts of Gs alpha. To examine whether this is a feature specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transfected with a construct containing a human beta 2-adrenoceptor cDNA under the control of the beta-actin promoter. Two clones of these cells were examined in detail, beta N22, which expressed some 4000 fmol/mg of membrane protein, and clone beta N17, which expressed approx. 300 fmol/mg of membrane protein of the receptor. Exposure of beta N22 cells to the beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of Gs alpha, without effect on membrane levels of Gi2 alpha, Gi3 alpha, G(o) alpha or Gq alpha/G11 alpha. Dose-response curves to isoprenaline in beta N22 cells indicated that half-maximal down-regulation of Gs alpha was produced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to isoprenaline did not significantly modify levels of any of the G-protein alpha subunits, including Gs alpha. In beta N22 cells the IP prostanoid receptor was expressed at similar levels to those in wild-type NG108-15 cells, and treatment with iloprost resulted in a similar down-regulation of cellular Gs alpha levels. Iloprost was also effective in causing down-regulation of Gs alpha levels in clone beta N17. Concurrent addition of both isoprenaline and iloprost to clone beta N22 resulted in less than additive down-regulation of Gs alpha. These results demonstrate that the phenomenon of agonist-induced specific G-protein down-regulation is determined by the levels of expression of the receptor.

SUBMITTER: Adie EJ

PROVIDER: S-EPMC1138224 | biostudies-other | 1994 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Stable cell lines, derived from NG108-15 cells and transfected to express both the beta 2-adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [3H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the beta 2-adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5'-[beta, gamma-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced beta 2-adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration-effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones over-expressing adenylate cyclase type II. A maximally effective concentration of the beta-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5'-N-ethylcarbox-amidoadenosine (acting at an endogenous A2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.

Project description:Mitochondrial dysfunction is a common mediator of disease and organ injury. Although recent studies show that inducing mitochondrial biogenesis (MB) stimulates cell repair and regeneration, only a limited number of chemicals are known to induce MB. To examine the impact of the β-adrenoceptor (β-AR) signaling pathway on MB, primary renal proximal tubule cells (RPTC) and adult feline cardiomyocytes were exposed for 24 h to multiple β-AR agonists: isoproterenol (nonselective β-AR agonist), (±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL 37344) (selective β(3)-AR agonist), and formoterol (selective β(2)-AR agonist). The Seahorse Biosciences (North Billerica, MA) extracellular flux analyzer was used to quantify carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled oxygen consumption rate (OCR), a marker of maximal electron transport chain activity. Isoproterenol and BRL 37244 did not alter mitochondrial respiration at any of the concentrations examined. Formoterol exposure resulted in increases in both FCCP-uncoupled OCR and mitochondrial DNA (mtDNA) copy number. The effect of formoterol on OCR in RPTC was inhibited by the β-AR antagonist propranolol and the β(2)-AR inverse agonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol hydrochloride (ICI-118,551). Mice exposed to formoterol for 24 or 72 h exhibited increases in kidney and heart mtDNA copy number, peroxisome proliferator-activated receptor γ coactivator 1α, and multiple genes involved in the mitochondrial electron transport chain (F0 subunit 6 of transmembrane F-type ATP synthase, NADH dehydrogenase subunit 1, NADH dehydrogenase subunit 6, and NADH dehydrogenase [ubiquinone] 1β subcomplex subunit 8). Cheminformatic modeling, virtual chemical library screening, and experimental validation identified nisoxetine from the Sigma Library of Pharmacologically Active Compounds and two compounds from the ChemBridge DIVERSet that increased mitochondrial respiratory capacity. These data provide compelling evidence for the use and development of β(2)-AR ligands for therapeutic MB.

Dataset Information

Agonist regulation of cellular Gs alpha-subunit levels in neuroblastoma x glioma hybrid NG108-15 cells transfected to express different levels of the human beta 2 adrenoceptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets