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The increased production of reactive oxygen species (ROS) has been postulated to play a key role in the progression of nonalcoholic fatty liver disease (NAFLD). However, the source of ROS and mechanisms underlying the development of NAFLD have yet to be established. We observed a significant up-regulation of a minor isoform of NADPH oxidase, NOX1, in the liver of nonalcoholic steatohepatitis (NASH) patients as well as of mice fed a high-fat and high-cholesterol (HFC) diet for 8 weeks. In mice deficient in Nox1 (Nox1KO), increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 demonstrated in HFC diet-fed wild-type mice (WT) were significantly attenuated. Concomitantly, increased protein nitrotyrosine adducts, a marker of peroxynitrite-induced injury detected in hepatic sinusoids of WT, were significantly suppressed in Nox1KO. The expression of NOX1 mRNA was much higher in the fractions of enriched liver sinusoidal endothelial cells (LSECs) than in those of hepatocytes. In primary cultured LSECs, palmitic acid (PA) up-regulated the mRNA level of NOX1, but not of NOX2 or NOX4. The production of nitric oxide by LSECs was significantly attenuated by PA-treatment in WT but not in Nox1KO. When the in vitro relaxation of TWNT1, a cell line that originated from hepatic stellate cells, was assessed by the gel contraction assay, the relaxation of stellate cells induced by LSECs was attenuated by PA treatment. In contrast, the relaxation effect of LSECs was preserved in cells isolated from Nox1KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impaired hepatic microcirculation through the reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the progression of NAFLD.

Recognition of sweet, bitter and umami tastes requires the nonvesicular release from taste bud cells of ATP, which acts as a neurotransmitter to activate afferent neural gustatory pathways (1). However, how ATP is released to fulfil this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel (2,3), is indispensable for taste-stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas their recognition of sour and salty tastes remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells by taste stimuli. Thus, CALHM1 is a voltage-gated ATP-release channel required for sweet, bitter and umami taste perception., Tastes are sensed by dedicated receptor cells in taste buds, which are composed of three anatomically distinct types of cells: types I, II and III. Only type III cells, which [...]

In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates the amiloride-sensitive [Na.sup.+] transport. Hypotonic action on [Na.sup.+] transport consists of two phases, a nongenomic early phase and a genomic delayed phase. Although it has been reported that, during the genomic phase, hypotonic stress stimulates transcription of [Na.sup.+] transport-related genes, such as serum- and glucocorticoid-inducible kinase 1 (SGK1) and subunits of the epithelial [Na.sup.+] channel (ENaC), increasing [Na.sup.+] transport, the mechanism remains unknown. We focused the present study on the role of intracellular [Ca.sup.2+] in hypotonicity-induced SGK1 and ENaC subunit transcription. Since hypotonic stress raises intracellular [Ca.sup.2+] concentration in A6 cells, we hypothesized that [Ca.sup.2+]-dependent signals participate in the genomic action. Using real-time quantitative RT-PCR and Western blot techniques and measuring short-circuit currents, we observed that 1) BAPTA-AM and W7 blunted the hypotonicity-induced expression of SGK1 mRNA and protein, 2) ionomycin dose dependently stimulated expression of SGK1 mRNA and protein under an isotonic condition and the time course of the stimulatory effect of ionomycin on SGK1 mRNA was remarkably similar to that of hypotonic action on SGK1 mRNA, 3) hypotonic stress stimulated transcription of three ENaC subunits in an intracellular [Ca.sup.2+]-dependent manner, and 4) BAPTA-AM retarded the delayed phase of hypotonic stress-induced [Na.sup.+] transport but had no effect on the early phase. These observations indicate for the first time that intracellular [Ca.sup.2+] plays a role as the second messenger in hypotonic stress-induced [Na.sup.+] transport by stimulating transcription of SGK1 and ENaC subunits. sodium transport; calmodulin; W7

We previously reported that hypotonic stress stimulated transepithelial [Na.sup.+] transport via a pathway dependent on protein tyrosine kinase (PTK; Niisato N, Van Driessche W, Liu M, Marunaka Y. J Membr Biol 175: 63-77, 2000). However, it is still unknown what type of PTK mediates this stimulation. In the present study, we investigated the role of receptor tyrosine kinase (RTK) in the hypotonic stimulation of [Na.sup.+] transport. In renal epithelial A6 cells, we observed inhibitory effects of AG1478 [an inhibitor of the EGF receptor (EGFR)] and AG1296 [an inhibitor of the PDGF receptor (PDGFR)] on both the hypotonic stress-induced stimulation of [Na.sup.+] transport and the hypotonic stress-induced ligand-independent activation of EGFR. We further studied whether hypotonic stress activates members of the MAP kinase family, ERK1/2, p38 MAPK, and JNK/SAPK, via an RTK-dependent pathway. The present study indicates that hypotonic stress induced phosphorylation of ERK1/2 and JNK/SAPK, but not p38 MAPK, that the hypotonic stress-induced phosphorylation of ERK 1/2 and JNK/SAPK was diminished by coapplication of AG 1478 and AG1296, and that only JNK/SAPK was involved in the hypotonic stimulation of [Na.sup.+] transport. A further study using cyclohexamide (a protein synthesis inhibitor) suggests that both RTK and JNK/SAPK contributed to the protein synthesis-independent early phase in hypotonic stress-induced [Na.sup.+] transport, but not to the protein synthesis-dependent late phase. The present study also suggests involvement of phosphatidylinositol 3-kinase (PI3-kinase) in RTK-JNK/SAPK cascade-mediated [Na.sup.+] transport. These observations indicate that 1) hypotonic stress activates JNK/SAPK via RTKs in a ligand-independent pathway, 2) the RTK-JNK/SAPK cascade acts as a mediator of hypotonic stress for stimulation of [Na.sup.+] transport, and 3) PI3-kinase is involved in the RTK-JNK/SAPK cascade for the hypotonic stress-induced stimulation of [Na.sup.+] transport. ENaC; epidermal growth factor doi:10.1152/ajprenal.00011.2007