Morphine, a powerful analgesic, and norepinephrine, the principal neurotransmitter of sympathetic nerves, exert major inhibitory effects on both peripheral and brain neurons by activating distinct cell-surface G protein-coupled receptors-the mu-opioid receptor (MOR) and alpha2A-adrenergic receptor (alpha2A-AR), respectively. These receptors, either singly or as a heterodimer, activate common signal transduction pathways mediated through the inhibitory G proteins (G(i) and G(o)). Using fluorescence resonance energy transfer microscopy, we show that in the heterodimer, the MOR and alpha2A-AR communicate with each other through a cross-conformational switch that permits direct inhibition of one receptor by the other with subsecond kinetics. We discovered that morphine binding to the MOR triggers a conformational change in the norepinephrine-occupied alpha2A-AR that inhibits its signaling to G(i) and the downstream MAP kinase cascade. These data highlight a new mechanism in signal transduction whereby a G protein-coupled receptor heterodimer mediates conformational changes that propagate from one receptor to the other and cause the second receptor's rapid inactivation.
Previously, we demonstrated that the vacuolar-type H(+)-ATPase (V-ATPase) a2-subunit functions as an endosomal pH sensor that interacts with the ADP-ribosylation factor (Arf) guanine nucleotide exchange factor, ARNO. In the present study, we showed that ARNO directly interacts not only with the a2-subunit but with all a-isoforms (a1-a4) of the V-ATPase, indicating a widespread regulatory interaction between V-ATPase and Arf GTPases. We then extended our search for other ARNO effectors that may modulate V-ATPase-dependent vesicular trafficking events and actin cytoskeleton remodeling. Pull-down experiments using cytosol of mouse proximal tubule cells (MTCs) showed that ARNO interacts with aldolase, but not with other enzymes of the glycolytic pathway. Direct interaction of aldolase with the pleckstrin homology domain of ARNO was revealed by pull-down assays using recombinant proteins, and surface plasmon resonance revealed their high avidity interaction with a dissociation constant: K(D) = 2.84 × 10(-10) M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed that the expression of aldolase is inversely correlated with gelsolin levels in HeLa cells. In summary, we have shown that aldolase forms a complex with ARNO/Arf6 and the V-ATPase and that it may contribute to remodeling of the actin cytoskeleton and/or the trafficking and redistribution of V-ATPase-dependent acidic compartments via a combination of protein-protein interaction and gene expression mechanisms.
The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology. Bone morphogenetic proteins (BMPs) represent a large subfamily of the transforming growth factor  (TGF-) superfamily of ligands that play roles in numerous physiologic and pathologic processes including cell proliferation, differentiation, apoptosis, and specification of developmental fate during embryogenesis and in adult tissues. 1 In the kidney, BMPs play an important role in nephrogenesis. During normal development, BMPs are expressed in the metanephric mesenchyme and the ureteric bud and play a key role in the epithelialization of the metanephric mesenchyme and reciprocal induction of collecting duct differentiation. Loss of BMP expression has profound effects on kidney development. [2][3][4][5][6][7][8][9][10][11] The role of BMPs in the adult kidney is less well understood. The expression of multiple BMP ligands, including BMP4, BMP6, and BMP7, and BMP receptors persists in the adult kidney, 12 supporting the notion that the adult kidney can respond to BMP stimulation. 13 Response to injury and repair frequently recapitulates development,
Neutrophils infiltration in liver is one of the typical histological characteristics of nonalcoholic steatohepatitis (NASH) in both animal models and human subjects. This study was aimed to investigate the role of neutrophils in the process of NASH and its underling mechanisms. C57BL/6J mice were fed with either standard chow (SC) or methionine/choline-deficient (MCD) diet for 1, 2, 4, 8 weeks, respectively. C57BL/6J APOE(-/-) mice were fed with SC or high-fat high-cholesterol (HFHC) diet. Anti-Ly6G antibody was employed to deplete neutrophils and sivelestat was used to inhibit neutrophil elastase (NE). MCD-diet-receiving mice with neutrophil depletion had much lower serum ALT activity, liver inflammation, and mRNA levels of proinflammatory genes in the early stage of NASH (1 and 2 weeks) when compared to non-neutrophil-depleted mice. NE inhibitor sivelestat could recapitulate the effects of neutrophil depletion in APOE(-/-) mice fed with HFHC diet. As the disease progressed (4 and 8 weeks), neutrophil depletion did not lower serum ALT levels and liver lesions due to activation of Kupffer cells. Finally, we found neutrophils also affected anti-inflammation cytokine interleukin-1 receptor antagonist mRNA expression. Neutrophils play a crucial role in the early stage of NASH via NE.
The aim of the present study was to investigate Toll-like receptor-4 (TLR4) signalling at different stages of non-alcoholic fatty liver disease (NAFLD) induced by a high-fat, high-fructose (HFHFr) diet in mice. Both TLR4 wild-type (WT) and mutant (TLR4(mut) ) mice were fed either standard chow (SC) or the HFHFr diet for different periods of time from 4 to 16 weeks. Pathological characteristics and function of the liver were assessed. Simple steatosis, steatohepatitis and hepatic fibrosis occurred sequentially in Week 4, 8 and 16 in WT mice fed with the HFHFr. Expression of TLR4, myeloid differentiation factor 88 (MyD88), interferon regulatory factor (IRF) 3 and IRF7 started to increase at Week 4, peaked at Week 8 and then declined to basal levels at Week 16. This pattern was consistent with changes in inflammation in the liver revealed by haematoxylin and eosin staining. However, lipid accumulation, inflammation and fibrosis in livers of TLR4(mut) mice fed the HFHFr diet were significantly alleviated. In addition, the expression of activin A in WT mice fed the HFHFr diet increased at Week 16. The data suggest that TLR4 signalling mediates non-alcoholic steatohepatitis before fibrosis and that activin A is subsequently involved in NAFLD.
Zhuang Z, Marshansky V, Breton S, Brown D. Is caveolin involved in normal proximal tubule function? Presence in model PT systems but absence in situ. Am J Physiol Renal Physiol 300: F199-F206, 2011. First published October 27, 2010 doi:10.1152/ajprenal.00513.2010.-Kidney proximal tubule (PT) cells are specialized for the uptake and transport of ions, solutes, peptides, and proteins. These functions are often regulated by hormones that signal at the cell surface and are internalized by clathrin-mediated endocytosis. However, the caveolin/caveolae pathway has also been implicated in normal PT function, often based on data from isolated PTs or PT cells in culture. Although we reported previously that caveolae and caveolin 1 are not detectable in PTs in vivo, reports of caveolin expression and function in PT cells appear periodically in the literature. Therefore, we reexamined caveolin expression in PTs in vivo, in isolated "purified" PTs following collagenase digestion, and in cultured PT cells. Caveolin 1 and 2 protein, mRNA, or immunofluorescence was undetectable in PTs in vivo, but PT cell cultures expressed caveolin 1 and/or 2. Furthermore, caveolin 1 and 2 mRNAs were detected in isolated PTs along with the endothelial markers CD31 and ICAM1. In contrast, no caveolin or endothelial marker mRNAs were detectable in samples isolated from snap-frozen kidneys by laser cut microdissection, which eliminates contamination by other cell types. We conclude 1) caveolin 1 and 2 are not normally expressed by PT cells in situ, 2) caveolin expression is "activated" in cultured PT cells, 3) contamination with non-PT, caveolin-expressing cells is a potential source of caveolin 1 and 2 that must be taken into account when isolated PTs are used in studies to correlate expression of these proteins with PT function. kidney; cell culture; isolated tubules; endocytosis RENAL PROXIMAL TUBULE (PT) cells are highly specialized for the bulk reabsorption of many ions, solutes, and smaller proteins, including receptor ligands and hormones, which penetrate the glomerular filtration barrier and appear in the tubule lumen. Furthermore, PT cells respond to circulating hormones and have distinct transport properties associated with their apical and basolateral membranes. As such, they display a very distinct polarized expression of many channels, transporters, enzymes, and receptors that all contribute toward their physiological function (4 -6, 8, 14, 41, 50). As part of their vectorial transporting and reabsorptive activity, many membrane proteins undergo extensive constitutive and regulated trafficking and recycling (8). In the PT, the endocytotic arm of the trafficking pathway is intimately related to a variety of specialized functions including the reabsorption of filtered molecules from the luminal fluid, as well as downregulation of membrane transporters such as NaPi1a in response to activation of PTH receptors (5), and downregulation of angiotensin II receptors (56). Plasma membrane hormone receptors themselves also undergo endocytosis ...
Background:The functional role of the V-ATPase as a pH-sensing receptor remains unknown. Results: N-terminal peptides from the a-subunit isoforms of the V-ATPase modulate the enzymatic GEF activity of cytohesin-2. Conclusion: V-ATPase is a novel cytohesin-signaling receptor. Significance: These data reveal an evolutionarily conserved signaling process between V-ATPase, cytohesin-2, and Arfs, which is crucial for understanding the cell biological functions of these proteins.
The non-alcoholic fatty liver disease (NAFLD) fibrosis score (NFS) has emerged as a useful predictor of long-term outcome in NAFLD patients. We evaluated the predictive performance of the NFS for overall mortality in a Chinese population with NAFLD. All NAFLD patients diagnosed ultrasonographically at Xixi Hospital of Hangzhou between 1996 and 2011 were retrospectively recruited to the study. Outcome was determined by interview and causes of death were confirmed by medical records. The area under the receiver operating characteristic curve (AUCROC ) was used to determine the predictive accuracy of the NFS, BARD (body mass index, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio, diabetes) score, FIB-4 index and the AST/platelet ratio index (APRI) for mortality. Data from a total of 180 eligible patients (median age 39 years; 96 men) were analysed, with 12 deaths over a median follow-up period of 6.6 years (range 0.5-14.8 years). Using Cox model analysis, the NFS as a continuous variable was identified as the only predictor for all-cause mortality (hazard ratio 2.743, 95% confidence interval (CI) 1.670-4.504). The NFS yielded the highest AUCROC of 0.828 (95% CI 0.728-0.928, P < 0.05), followed by the FIB-4 index, APRI and BARD score (AUCROC 0.806 (P < 0.05), 0.732 (P < 0.05) and 0.632, respectively). The data indicated that the NFS is a useful predictor of 6.6-year all-cause mortality for Chinese patients with NAFLD.
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