guanosine-triphosphate and ethylisopropylamiloride

At low concentrations (50 nM), okadaic acid (OA), an inhibitor of phosphatases 1 and 2A, inhibits platelet-derived growth factor-induced cell proliferation in late G1 (A. Simm et al., Exp. Cell Res., 210: 160-165, 1994). This inhibition is caused by the interference of OA in the induction and activation of the cell division protein kinases cdk1 and cdk2. OA alone has no effect on cell number, but induces a pronounced increase in cell size. The OA-induced hypertrophy can be divided into two phases. The first phase is characterized by a swelling of the cells. This increase in cellular volume is not accompanied by a change in the level of cellular macromolecules, i.e., protein and RNA. Inhibitor studies indicated a possible role of the Na+/H+ antiporter and Cl- channels in this process. In the second phase, an increase in the cellular protein and RNA content was observed along with a minor change in cell volume. To delineate a possible signaling pathway, the involvement of numerous protein kinases was analyzed. Low concentrations of OA lead to pronounced and sustained activation of the p70S6 kinase. There was little or no effect on various other kinases that can be activated by extracellular signals, e.g., mitogen-activated kinase, ribosomal S6 kinase, or other S6 peptide kinases. Likewise, at these concentrations, OA did not activate the genes for fos, myc, or ornithine decarboxylase. At very low concentrations (ED50, 0.5 nM), rapamycin, a specific inhibitor of the activation of p70S6 kinase, reversed the activation of the p70S6 kinase and the enhancement of RNA synthesis and partially the increase in cell volume and protein synthesis. The OA-induced hypertrophy of AKR-2B fibroblasts may serve as a model system for investigations aimed at the identification of signaling pathways leading to hypertrophy of differentiated nonproliferating cells.

Topics: Adenine Nucleotides; Amiloride; Animals; Becaplermin; CDC2 Protein Kinase; CDC2-CDC28 Kinases; Cell Line; Cell Size; Chloride Channels; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Guanosine Triphosphate; Mice; Mice, Inbred AKR; Nitrobenzoates; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Platelet-Derived Growth Factor; Polyenes; Protein Biosynthesis; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins c-sis; Ribosomal Protein S6 Kinases; RNA, Ribosomal; Signal Transduction; Sirolimus; Sodium-Hydrogen Exchangers

We have previously shown that the beta-adrenergic receptor (beta-AR) stimulates activity of the ubiquitous Na-H exchanger (NHE-1) independently of changes in cAMP accumulation and independently of a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). To further investigate the potential role of a GTP-binding protein in coupling the beta-AR to NHE-1, we have used a recently available nonhydrolyzable GTP analog, "caged" guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), to study time-dependent effects of GTP on NHE-1 in intact cells. By monitoring intracellular pH (pHi) in cells loaded with the fluorescent pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, we determined NHE-1 activity in primary cultures of canine enteric endocrine cells, which express an endogenous beta-AR, and in mouse L cells stably transfected with either the wild type hamster beta 2-AR or a mutant construct of the hamster beta 2-AR containing a deletion in amino acid residues 222-229. This D(222-229)beta 2-AR is functionally uncoupled from Gs and adenylylcyclase. In all three cell types, NaF and GTP gamma S induced an increase in activity of the exchanger, determined by assessing the rate of pHi recovery from an acute intracellular acid load (dpHi/dt). This increase in pHi recovery was dependent on extracellular Na+ and sensitive to the amiloride analog ethylisopropylamiloride. GTP gamma S, but not NaF, also increased beta-adrenergic stimulation of resting NHE-1 activity. The alkalinization in response to isoproterenol was reversed by propranolol in the absence, but not the presence, of GTP gamma S and was completely blocked by GDP beta S. The ability of guanine nucleotides to regulate beta-adrenergic activation of NHE-1 in cells expressing the mutant D(222-229)beta 2-AR suggests that functional coupling of the beta-AR to NHE-1 may be mediated by a GTP-binding protein other than Gs.

Topics: Amiloride; Animals; Carrier Proteins; Cells, Cultured; Cricetinae; Dogs; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Hydrogen-Ion Concentration; Ileum; Intestinal Mucosa; Isoproterenol; Kinetics; L Cells; Meglumine; Mice; Propranolol; Receptors, Adrenergic, beta; Sodium; Sodium Fluoride; Sodium-Hydrogen Exchangers; Thionucleotides; Transfection