GABA-and glycine-induced depolarization is thought to provide important developmental signals, but the role of the underlying chloride gradient has not been examined from the onset of development. We therefore overexpressed globally the potassium-chloride cotransporter 2 (KCC2) in newly fertilized zebrafish embryos to reverse the chloride gradient. This rendered glycine hyperpolarizing in all neurons, tested at the time that motor behaviors (but not native KCC2) first appear. KCC2 overexpression resulted in fewer mature spontaneously active spinal neurons, more immature silent neurons, and disrupted motor activity. We observed fewer motoneurons and interneurons, a reduction in the elaboration of axonal tracts, and smaller brains and spinal cords. However, we observed no increased apoptosis and a normal complement of sensory neurons, glia, and progenitors. These results suggest that chloride-mediated excitation plays a crucial role in promoting neurogenesis from the earliest stages of embryonic development.

It is well known that K restriction suppresses renal K excretion (2). This is achieved, at least in part, by decreasing the apical K conductance in the cortical collecting duct (CCD) 1 and by stimulating K absorption in the outer medullary collecting duct (3, 4). However, the mechanism by which low K intake suppresses the apical K channels is not completely understood. We previously demonstrated that low K intake increased the expression of Src family protein tyrosine kinase (PTK) such as c-Src and c-Yes (1) and that inhibition of PTK increased the apical ROMK-like small conductance (SK) channels (1). This suggests that PTK is involved in mediating the effect of low K intake on the apical K channels and that increases in PTK activity and expression are important for suppression of renal K secretion during K depletion.Low K intake has been reported to increase the production of O 2. anion in rabbit carotid arteries (5). Moreover, it has beenshown that H 2 O 2 stimulates the phosphorylation of c-Jun in endothelial cells, an indication of activation of transcription factor (6). Therefore, it is possible that increases in O 2 . or related products induced by low K intake may be an upstream signal responsible for mediating the effect of low K intake on PTK expression and K secretion in the kidney. This hypothesis was tested in the present study by examining whether O 2 . and related products such as H 2 O 2 can mimic the effect of low K intake and stimulate the expression of PTK in the CCD. We also examined whether decreases in O 2 . and related products with tempol could attenuate the effect of low K intake on c-Src expression, ROMK channel activity, and renal K excretion. EXPERIMENTAL PROCEDURESAnimals-Sprague-Dawley rats (6 -8 weeks, either sex) were purchased from Taconic Farms (Germantown, NY). Rats were housed in metabolic cages for 7 days to study urinary K excretion. After 3 days of training in the cage, rats were divided into three groups: 1) control group in which animals were kept on a normal K (1.1%) diet and had a daily intraperitoneal injection of saline for 1 week; 2) the low K group in which rats were maintained on a K-deficient (KD) diet and received a daily intraperitoneal injection of saline for 7 days; and 3) the tempol-treated group in which rats were also fed with KD diet and had a daily intraperitoneal injection of tempol (15 mg/kg) for 1 week. Data regarding the 24-h food intake, body weight, and urine output were recorded. Urinary Na and K concentrations were measured by a flame photometer, and daily Na and K excretion were calculated as mEq/24 h. Animals were anesthetized with pentobarbital (60 mg/kg), and blood samples were drawn from the heart to measure the plasma K and Na concentrations. Rats were then killed, and the abdomens were opened to remove the kidneys.Tissue Preparation-The renal cortex and the outer medulla were separated under a dissecting microscope and suspended in radioimmune precipitation assay buffer solution (1:8 ratio, w/v) containing 1ϫ phosphate-buffered saline, 1% No...

We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P-450 (CYP) epoxygenasedependent pathway (34). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney (11) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)-positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11,12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 M AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport.11,12-epoxyeicosatrienoic acid; Na absorption; cortical collecting duct THE KIDNEY PLAYS AN IMPORTANT role in maintaining the balance between Na intake and Na excretion and thus in the regulation of extracellular volume (31). Although only 10% filtered Na is reabsorbed in the distal nephron, the Na absorption in the distal nephron plays a key role in determining the final renal Na excretion and is also tightly regulated by hormones and dietary Na intake (31). Defective regulation of Na transport in the distal nephron has been shown to cause diseases such as Liddle syndrome and pseudohypoaldosteronism type II (2, 13, 29).The cortical collecting duct (CCD) is responsible for the hormone-regulated Na absorption which takes place by a two-step process: Na enters the cells across the apical membrane via epithelial Na channels (ENaC) (1, 7, 23) and is extruded from the cell across the basolateral membrane by Na-K-ATPase (12, 22). It is well established that the ratelimiting step for Na absorption in the CCD is the apical Na conductance (15), which is regulated by aldosterone and Na intake: a high aldosterone level or low Na intake augments (24), whereas an increased Na intake de...

It was demonstrated previously that low dietary potassium (K) intake stimulates Src family protein tyrosine kinase (PTK) expression via a superoxide-dependent signaling. This study explored the role of mitogen-activated protein kinase (MAPK) in mediating the effect of superoxide anions on PTK expression and ROMK (Kir 1.1) channel activity. Western blot analysis demonstrated that low K intake significantly increased the phosphorylation of P38 MAPK (P38) and extracellular signalregulated kinase (ERK) but had no effect on phosphorylation of c-JUN N-terminus kinase in renal cortex and outer medulla.