Decrease in dietary K intake stimulates the generation of superoxide anions in the kidney and inhibits K secretory channels in the CCD.
ABSTRACT We previously demonstrated that K depletion inhibited ROMK-like small-conductance K channels (SK) in the cortical collecting duct (CCD) and that the effect was mediated by superoxide anions that stimulated Src family protein tyrosine kinase (PTK) and mitogen-activated protein kinase (MAPK) (51). However, because animals on a K-deficient diet had a severe hypokalemia, superoxide-dependent signaling may not regulate ROMK channels under physiological conditions with a normal plasma K concentration. In the present study, we used the patch-clamp technique and Western blot to examine the effect of a moderate K restriction on ROMK-like SK channels and the role of PTK and MAPK in regulating apical K channels in the CCD of animals on a low-K diet (LK; 0.1% K). Rats and mice fed a LK diet for 7 days had a normal plasma K concentration. However, a LK intake increased the expression of angiotensin II type 1 receptor in the kidney. Moreover, patch-clamp experiments demonstrated that LK intake decreased the probability finding SK channels and channel activity defined by NP(o) (a product of channel number and open probability) in the CCD of both rat and mouse kidneys. Also, LK intake significantly stimulated the production of superoxide anions in the renal cortex and outer medulla in both rats and mice and increased superoxide level in the rat CCD. Moreover, LK intake augments the phosphorylation of p38 and ERK MAPK, the expression of c-Src and tyrosine phosphorylation of ROMK channels. However, treatment of animals with tempol abolished the effect of LK intake on MAPK and c-Src and increased ROMK channel activity in comparing with those of nontreated rats on a LK diet. Inhibiting p38 and ERK with SB202190 and PD98059 significantly stimulated SK in the CCD in rats on a LK diet. In addition, inhibition of PTK with herbimycin A activated SK channels in the CCD from rats on a LK diet. We conclude that LK intake stimulates the generation of superoxide anion and related products and that MAPK and Src family PTK play a physiological role in inhibiting apical K channels in the principal cells in response to LK intake.
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ABSTRACT: In a variety of cell types, insulin stimulation elicits the rapid production of H(2)O(2), which causes the oxidative inhibition of protein-tyrosine phosphatases and enhances the tyrosine phosphorylation of proteins in the early insulin action cascade (Mahadev, K., Zilbering, A., Zhu, L., and Goldstein, B. J. (2001) J. Biol. Chem. 276, 21938-21942). In the present work, we explored the potential role of insulin-induced H(2)O(2) generation on downstream insulin signaling using diphenyleneiodonium (DPI), an inhibitor of cellular NADPH oxidase that blocks insulin-stimulated cellular H(2)O(2) production. DPI completely inhibited the activation of phosphatidylinositol (PI) 3'-kinase activity by insulin and reduced the insulin-induced activation of the serine kinase Akt by up to 49%; these activities were restored when H(2)O(2) was added back to cells that had been pretreated with DPI. Interestingly, the H(2)O(2)-induced activation of Akt was entirely mediated by upstream stimulation of PI 3'-kinase activity, since treatment of 3T3-L1 adipocytes with the PI 3'-kinase inhibitors wortmannin or LY294002 completely blocked the subsequent activation of Akt by exogenous H(2)O(2). Preventing oxidant generation with DPI also blocked insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane, providing further evidence for an oxidant signal in the regulation of the distal insulin-signaling cascade. Finally, in contrast to the cellular mechanism of H(2)O(2) generation by other growth factors, such as platelet-derived growth factor, we also found that insulin-stimulated cellular production of H(2)O(2) may occur through a unique pathway, independent of cellular PI 3'-kinase activity. Overall, these data provide insight into the physiological role of insulin-dependent H(2)O(2) generation, which is not only involved in the regulation of tyrosine phosphorylation events in the early insulin signaling cascade but also has important effects on the regulation of downstream insulin signaling, involving the activation of PI 3'-kinase, Akt, and ultimately cellular glucose transport in response to insulin.Journal of Biological Chemistry 01/2002; 276(52):48662-9. · 4.65 Impact Factor
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ABSTRACT: Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha) are known to induce production of reactive oxygen species (ROS), which have been suggested to act as second messengers. Here we demonstrate that ROS production by bovine chondrocytes upon cytokine stimulation induces c-jun expression. Since c-jun expression is regulated by its own gene product via phosphorylation by c-Jun NH2-terminal kinases (JNKs), we investigated if cytokines and ROS could modulate JNK activity in chondrocyte monolayer cultures. Treatment of bovine chondrocytes with both IL-1 and TNFalpha leads to rapid induction of JNK activity, stimulating JNK activity 7- and 20-fold, respectively. Importantly, the observation that antioxidant treatment antagonizes IL-1 and TNFalpha activation of JNK provides strong evidence that ROS can act as mediators of JNK activity. Moreover, potent activation of JNK is also observed by direct addition of the ROS hydrogen peroxide (H2O2) to the chondrocyte cultures. Nitric oxide (NO), a multifunctional ROS, also appears to simulate JNK, albeit to a lesser extent. These findings identify JNK as another molecular target for the actions of NO and H2O2. In addition, the inhibitory effect of diphenyleneiodonium on JNK activation implicates the involvement of flavonoid-containing enzymes in the ROS-mediated signaling process. Overstimulation of JNK activity by excessive production of ROS may, therefore, underlie pathological conditions such as arthritis and cancer.Journal of Biological Chemistry 07/1996; 271(26):15703-7. · 4.65 Impact Factor
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ABSTRACT: Extracellular K must be kept within a narrow concentration range for the normal function of neurons, skeletal muscle, and cardiac myocytes. Maintenance of normal plasma K is achieved by a dual mechanism that includes extrarenal factors such as insulin and beta-adrenergic agonists, which stimulate the movement of K from extracellular to intracellular fluid and modulate renal K excretion. Dietary K intake is an important factor for the regulation of K secretion: An increase in K intake stimulates secretion, whereas a decrease inhibits K secretion and enhances absorption. This effect of changes in dietary K intake on tubule K transport is mediated by aldosterone-dependent and -independent mechanisms. Recently, it has been demonstrated that the protein tyrosine kinase (PTK)-dependent signal transduction pathway is an important aldosterone-independent regulatory mechanism that mediates the effect of altered K intake on K secretion. A low-K intake stimulates PTK activity, which leads to increase in phosphorylation of cloned inwardly rectifying renal K (ROMK) channels, whereas a high-K intake has the opposite effect. Stimulation of tyrosine phosphorylation also suppresses K secretion in principal cell by facilitating the internalization of apical K channels in the collecting duct.Annual Review of Physiology 02/2004; 66:547-69. · 19.55 Impact Factor