okadaic-acid and tyrphostin-25

Insulin enhances Na(+)-K(+) pump activity in various noncardiac tissues. We examined whether insulin exposure in vitro regulates Na(+)-K(+) pump function in rabbit ventricular myocytes. Pump current (I(p)) was measured using the whole-cell patch-clamp technique at test potentials (V(m)s) from -100 to +60 mV. When the Na(+) concentration in the patch pipette ([Na](pip)) was 10 mM, insulin caused a V(m)-dependent increase in I(p). The increase was approximately 70% when V(m) was at near physiological diastolic potentials. This effect persisted after elimination of extracellular voltage-dependent steps and when K(+) and K(+)-congeners were excluded from the patch pipettes. When [Na](pip) was 80 mM, causing near-maximal pump stimulation, insulin had no effect, suggesting that it did not cause an increase in membrane pump density. Effects of tyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I in pipette solutions suggested that the insulin-induced increase in I(p) involved activation of tyrosine kinase, phosphatidylinositol 3-kinase, and protein phosphatase 1, whereas protein phosphatase 2A and protein kinase C were not involved.

Topics: Androstadienes; Animals; Cells, Cultured; Electric Stimulation; Enzyme Inhibitors; Heart; Heart Ventricles; Indoles; Insulin; Male; Maleimides; Membrane Potentials; Myocardium; Okadaic Acid; Patch-Clamp Techniques; Phosphatidylinositol 3-Kinases; Rabbits; Sodium; Sodium-Potassium-Exchanging ATPase; Tyrphostins; Wortmannin

We have previously demonstrated that the sarcolemmal Na+-K+ pump current (Ip) in cardiac myocytes is stimulated by cell swelling induced by exposure to hyposmolar solutions. However, the underlying mechanism has not been examined. Because cell swelling activates stretch-sensitive ion channels and intracellular messenger pathways, we examined their role in mediating Ip stimulation during exposure of rabbit ventricular myocytes to a hyposmolar solution. Ip was measured by the whole cell patch-clamp technique. Swelling-induced pump stimulation altered the voltage dependence of Ip. Pump stimulation persisted in the absence of extracellular Na+ and under conditions designed to minimize changes in intracellular Ca2+, excluding an indirect influence on Ip mediated via fluxes through stretch-activated channels. Pump stimulation was protein kinase C independent. The tyrosine kinase inhibitor tyrphostin A25, the phosphatidylinositol 3-kinase inhibitor LY-294002, and the protein phosphatase-1 and -2A inhibitor okadaic acid abolished Ip stimulation. Our findings suggest that swelling-induced pump stimulation involves the activation of tyrosine kinase, phosphatidylinositol 3-kinase, and a serine/threonine protein phosphatase. Activation of this messenger cascade may cause activation by the dephosphorylation of pump units.

Topics: Animals; Calcium; Cell Size; Chromones; Enzyme Inhibitors; Homeostasis; Hypotonic Solutions; Male; Membrane Potentials; Morpholines; Myocardium; Okadaic Acid; Patch-Clamp Techniques; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoprotein Phosphatases; Protein Kinase C; Protein Phosphatase 1; Protein-Tyrosine Kinases; Rabbits; Sodium-Potassium-Exchanging ATPase; Tyrphostins

To increase our understanding of the role of the Src homology 2 (SH2) domain-containing protein Shb in the mitogenic signal transduction, Shb mRNA contents were determined in the fibroblast-like NIH3T3 cells and the insulin producing beta TC-1 cells under various conditions. In NIH3T3 cells, the serine/ threonine phosphatase inhibitor okadaic acid and the tyrosine kinase inhibitor genistein increased Shb mRNA contents, the protein kinase C activating phorbol ester 12-O-tetradecanoyl 13-acetate (TPA) decreased the Shb mRNA content, whereas the tyrosine kinase inhibitor tyrphostin 25 and the mitogen platelet-derived growth factor (PDGF-BB) had no effect. In beta TC-1 cells, okadaic acid and genistein increased the Shb mRNA content, whereas tyrphostin 25 and serum were without effect. Okadaic acid and genistein decreased the rates of beta TC-1 cell DNA synthesis. It is concluded that expression of the SHB gene is under a complex mode of regulation involving at least three different protein kinases. As a consequence of this, it is likely that SHB gene expression is significantly modulated by conditions of specific activation of certain pathways, whereas its expression appears little influenced by serum and a mitogen.

Topics: 3T3 Cells; Animals; Becaplermin; Enzyme Inhibitors; Ethers, Cyclic; Gene Expression Regulation; Genistein; Isoflavones; Mice; Nitriles; Okadaic Acid; Phosphorylation; Platelet-Derived Growth Factor; Protein Processing, Post-Translational; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-sis; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Tyrphostins