h-89 and tyrphostin-25

Exposure of cardiac myocytes to hyposmotic solution stimulates slowly-activating delayed-rectifying K(+) current (I(Ks)) via unknown mechanisms. In the present study, I(Ks) was measured in guinea-pig ventricular myocytes that were pretreated with modulators of cell signaling processes, and then exposed to hyposmotic solution. Pretreatment with compounds that (i) inhibit serine/threonine kinase activity (10-100 microM H89; 200 microM H8; 50 microM H7; 1 microM bisindolylmaleimide I; 10 microM LY294002; 50 microM PD98059), (ii) stimulate serine/threonine kinase activity (1-5 microM forskolin; 0.1 microM phorbol-12-myristate-13-acetate; 10 microM acetylcholine; 0.1 microM angiotensin II; 20 microM ATP), (iii) suppress G-protein activation (10 mM GDPbetaS), or (iv) disrupt the cytoskeleton (10 microM cytochalasin D), had little effect on the stimulation of I(Ks) by hyposmotic solution. In marked contrast, pretreatment with tyrosine kinase inhibitor tyrphostin A25 (20 microM) strongly attenuated both the hyposmotic stimulation of I(Ks) in myocytes and the hyposmotic stimulation of current in BHK cells co-expressing Ks channel subunits KCNQ1 and KCNE1. Since attenuation of hyposmotic stimulation was not observed in myocytes and cells pretreated with inactive tyrphostin A1, we conclude that TK has an important role in the response of cardiac Ks channels to hyposmotic solution.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cell Line; Chromans; Colforsin; Cricetinae; Cyclic AMP-Dependent Protein Kinases; Cytochalasin D; Cytoskeleton; GTP-Binding Proteins; Guinea Pigs; Indoles; Isoquinolines; KCNQ1 Potassium Channel; Maleimides; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Osmotic Pressure; Phosphatidylinositol 3-Kinases; Potassium Channels; Protein Kinase C; Protein-Tyrosine Kinases; Sulfonamides; Tetradecanoylphorbol Acetate; Tyrphostins

Riboflavin (RF), a water-soluble vitamin, is essential for normal cellular functions, growth, and development. Normal RF body homeostasis depends on intestinal absorption and recovery of the filtered vitamin in renal tubules. The mechanism and cellular regulation of the RF renal reabsorption process, especially in the human situation, are poorly understood. The aim of this study was therefore to address these issues, using a recently established human normal renal epithelial cell line, HK-2, as a model. Uptake of RF by HK-2 cells was found to be 1) linear with time for 5 min of incubation and occurring with minimal metabolic alterations, 2) temperature dependent, 3) Na+ independent, 4) saturable as a function of concentration [apparent Michaelis constant (K(m)) of 0.67 +/- 0.21 microM and maximal velocity (Vmax) of 10.05 +/- 0.87 pmol.mg protein-1.3 min-1], 5) inhibited by structural analogs and anion transport inhibitors, and 6) energy dependent. Protein kinase C-, protein kinase A-, and protein tyrosine kinase-mediated pathways were found to have no role in regulating RF uptake. On the other hand, a Ca2+/calmodulin-mediated pathway appeared to play a role in the regulation of RF uptake by HK-2 cells via an effect on the Vmax, as well as on the apparent K(m) of the RF uptake process. The uptake process of RF was also found to be adaptively regulated by the level of the substrate in the growth medium, with the effect being mediated through changes in the apparent K(m) and the Vmax of the uptake process. These results demonstrate that RF uptake by the human-derived renal epithelial cell line HK-2 is via a carrier-mediated system that is temperature and energy dependent and appears to be under the regulation of a Ca2+/calmodulin-mediated pathways and substrate level in the growth medium.

Topics: 1-Methyl-3-isobutylxanthine; Biological Transport; Bucladesine; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Epithelial Cells; Genistein; Humans; Isoquinolines; Kidney Tubules, Proximal; Kinetics; Nitriles; Protein Kinase C; Protein-Tyrosine Kinases; Riboflavin; Sulfonamides; Temperature; Tyrphostins