herbimycin and Hypokalemia

We have previously noted that in neonatal myocytes grown in culture, reductions in extracellular K+ concentration produced a hypertrophic response as assessed by induction of early response genes, atrial natriuretic peptide and skeletal actin and repression of the alpha 3 isoform of the sodium pump in a dose dependent manner. Similarly, decreases in media K+ concentration caused increases in cytosolic calcium concentration in a dose dependent manner, which correlated with repression of alpha 3 expression. In the current study we demonstrate that decreases in media K+ concentration caused increases in cytosolic calcium concentration in isolated adult rat cardiac myocytes. These increases are potentiated by the addition of the cardiotonic steroid, ouabain and blocked by the addition of the Src kinase inhibitor Herbimycin A. In parallel studies performed in vivo, when rats subjected to dietary K+ restriction were subsequently subjected to partial (5/6th) nephrectomy for 4 weeks, cardiac growth was greater than in rats fed a control diet. These data suggest that hypokalemia may produce phenotypic alterations consistent with cardiac hypertrophy as well as potentiate the cardiovascular effects of cardiotonic steroids.

Topics: Animals; Benzoquinones; Calcium; Cardiac Glycosides; Cells, Cultured; Food, Formulated; Hypokalemia; Lactams, Macrocyclic; Male; Muscle Cells; Myocardium; Nephrectomy; Ouabain; Rats; Rats, Sprague-Dawley; Rifabutin; src-Family Kinases

We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KD) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased NP(o), a product of channel number (N) and open probability (P(o)), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent that inhibits the cytochrome P-450-dependent production of 20-hydroxyeicosatetraenoic acid, further increased NP(o) of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased NP(o) of the 70-pS K channel in the mTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the mTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the low-conductance K channels in the mTAL and that PTK-induced tyrosine phosphorylation inhibits, whereas PTP-induced tyrosine dephosphorylation stimulates, the apical 70-pS K channel in the mTAL.

Topics: Amides; Animals; Arsenicals; Benzoquinones; CSK Tyrosine-Protein Kinase; Enzyme Inhibitors; Female; Hydroxyeicosatetraenoic Acids; Hyperkalemia; Hypokalemia; Lactams, Macrocyclic; Loop of Henle; Male; Phosphorylation; Potassium; Potassium Channels; Potassium Channels, Inwardly Rectifying; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin; Sodium; Specific Pathogen-Free Organisms; src-Family Kinases; Sulfones