okadaic-acid and ethylisopropylamiloride

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

Topics: Amiloride; Animals; Arachidonic Acids; Brefeldin A; Cytochrome P-450 Enzyme System; Endocytosis; Enzyme Activation; Enzyme Inhibitors; Kidney Cortex; Kinetics; Male; Models, Biological; Nocodazole; Okadaic Acid; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphatidylinositol 3-Kinases; Protein Kinase C; Rats; Rats, Wistar; Sodium; Sodium-Potassium-Exchanging ATPase; Staurosporine; Tetradecanoylphorbol Acetate

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