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The Na+- and Cl+-dependent GABA-betaine transporter (BGT1) has received attention mostly as a protector against osmolarity changes in the kidney and as a potential controller of the neurotransmitter GABA in the brain. Nevertheless, the cellular distribution of BGT1, and its physiological importance, is not fully understood. Here we have quantified mRNA levels using TaqMan real-time PCR, produced a number of BGT1 antibodies, and used these to study BGT1 distribution in mice. BGT1 (protein and mRNA) is predominantly expressed in the liver (sinusoidal hepatocyte plasma membranes) and not in the endothelium. BGT1 is also present in the renal medulla, where it localizes to the basolateral membranes of collecting ducts (particularly at the papilla tip) and the thick ascending limbs of Henle. There is some BGT1 in the leptomeninges, but brain parenchyma, brain blood vessels, ependymal cells, the renal cortex, and the intestine are virtually BGT1 deficient in 1- to 3-mo-old mice. Labeling specificity was assured by processing tissue from BGT1-deficient littermates in parallel as negative controls. Addition of 2.5% sodium chloride to the drinking water for 48 h induced a two- to threefold upregulation of BGT1, tonicity-responsive enhancer binding protein, and sodiummyo- inositol cotransporter 1 (slc5a3) in the renal medulla, but not in the brain and barely in the liver. BGT1-deficient and wild-type mice appeared to tolerate the salt treatment equally well, possibly because betaine is one of several osmolytes. In conclusion, this study suggests that BGT1 plays its main role in the liver, thereby complementing other betaine-transporting carrier proteins (e.g., slc6a20) that are predominantly expressed in the small intestine or kidney rather than the liver. [ABSTRACT FROM AUTHOR]

It has been proposed that key enzymes of ureagenesis and the alanine aminotransferase activity predominate in periportal hepatocytes. However, ureagenesis from alanine, when measured in the perfused liver, did not show periportal predominance and even the release of the direct products of alanine transformation, lactate and pyruvate, was higher in perivenous cells. An alternative way of analyzing the functional distributions of alanine aminotransferase and the urea cycle along the hepatic acini would be to measure alanine and urea production from precursors such as lactate or pyruvate plus ammonia. In the present work these aspects were investigated in the bivascularly perfused rat liver. The results of the present study confirm that gluconeogenesis and the associated oxygen uptake tend to predominate in the periportal region. Alanine synthesis from lactate and pyruvate plus ammonia, however, predominated in the perivenous region. Furthermore, no predominance of ureagenesis in the periportal region was found, except for conditions of high ammonia concentrations plus oxidizing conditions induced by pyruvate. These observations corroborate the view that data on enzyme activity or expression alone cannot be extrapolated unconditionally to the living cell. The current view of the hepatic ammonia-detoxifying system proposes that the small perivenous fraction of glutamine synthesizing perivenous cells removes a minor fraction of ammonia that escapes from ureagenesis in periportal cells. However, since urea synthesis occurs at high rates in all hepatocytes with the possible exclusion of those cells not possessing carbamoyl-phosphate synthase, it is probable that ureagenesis is equally important as an ammonia-detoxifying mechanism in the perivenous region. [ABSTRACT FROM AUTHOR]

Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase (Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups. [ABSTRACT FROM AUTHOR]

Progressive liver fibrosis (with the resultant cirrhosis) is the primary cause of chronic liver failure. Hepatic stellate cells (HSCs) are critically important mediators of liver fibrosis. In the healthy liver, HSCs are quiescent lipid-storing cells limited to the perisinusoidal endothelium. However, in the injured liver, HSCs undergo myofibroblastic transdifferentiation (activation), which is a critical step in the development of organ fibrosis. HSCs express P2Y receptors linking extracellular ATP to inositol (1,4,5)- trisphosphate-mediated cytosolic Ca2+ signals. Here, we report that HSCs express only the type I inositol (1,4,5)-trisphosphate receptor and that the receptor shifts into the nucleus and cell extensions upon activation. These cell extensions, furthermore, express sufficient machinery to enable local application of ATP to evoke highly localized Ca2+ signals that induce localized contractions. These autonomous units of subcellular signaling and response reveal a new level of subcellular organization, which, in turn, establishes a novel paradigm for the local control of fibrogenesis in the liver. [ABSTRACT FROM AUTHOR]

The contractile force generated by hepatic stellate cells in response to endothelin-1 contributes to sinusoidal blood flow regulation and hepatic fibrosis. This study's aim was to directly test the widely held view that changes in cytosolic Ca2+ concentration ([Ca2+]i) mediate stellate cell force generation. Contractile force generation by primary cultures of rat hepatic stellate cells grown in three-dimensional collagen gels was directly and quantitatively measured using a force transducer. Stellate cell [Ca2+]i, myosin activation, and migration were quantified using standard techniques. [Ca2+]i was modulated using ionomycin, BAPTA, KCl, and removal of extracellular Ca2+ Removal of extracellular Ca2+ did not alter endothelin-1-stimulated force development or [Ca2+]i. lonomycin, a Ca2+ ionophore, triggered an increase in [Ca2+]i that was three times greater than that stimulated by endothelin-1, but only induced 16% of the force and 38% of the myosin regulatory light chain (MLC) phosphorylation induced by endothelin-1. Physiological increases in [Ca2+]i induced by hyperkalemia had no effect on contractile force. Loading BAPTA, a Ca2+ chelator, in stellate cells completely blocked endothelin-1-induced increases in [Ca2+]i but had no effect on endothelin-1-stimulated force generation or MLC phosphorylation. In contrast, Y-27632, a selective rho-associated kinase inhibitor, inhibited endothelin-1-stimulated force generation by at least 70% and MLC phosphorylation by at least 80%. Taken together, these observations indicate that changes in [Ca2+]i are neither necessary nor sufficient for contractile force generation by rat stellate cells. Our results challenge the current model of contractile regulation in hepatic stellate cells and have important implications for our understanding of hepatic pathophysiology. [ABSTRACT FROM AUTHOR]

Betaine-homocysteine S-methyltransferase (BHMT) is the only enzyme known to catabolize betaine. In addition to being a substrate for BHMT, betaine also functions as an osmoprotectant that accumulates in the kidney medulla under conditions of high extracellular osmolarity. The mechanisms that regulate the partitioning of betaine between its use as a methyl donor and its accumulation as an osmoprotectant are not completely understood. The aim of this study was to determine whether BHMT expression is regulated by salt intake. This report shows that guinea pigs express BHMT in the liver, kidney, and pancreas and that the steady-state levels of BHMT mRNA in kidney and liver decrease 68% and 93% in guinea pigs consuming tap water containing high levels of salt compared with animals provided untreated tap water. The animals consuming the salt water also had ∼50% less BHMT activity in the liver and kidney, and steady-state protein levels decreased ∼30% in both organs. Pancreatic BHMT activity and protein levels were unaffected by the high salt treatment. The complex mechanisms involved in the downregulation of hepatic and renal BHMT expression in guinea pigs drinking salt water remain to be clarified, but the physiological significance of this downregulation may be to expedite the transport and accumulation of betaine into the kidney medulla under conditions of high extracellular osmolarity. [ABSTRACT FROM AUTHOR]

Fas-Fas ligand (FasL)-dependent pathways exert a suppressive effect on inflammatory responses in immune-privileged organs. FasL expression in hepatic Kupffer cells (KC) has been implicated in hepatic immunoregulation. In this study, modulation of FasL expression of KC by endogenous gut-derived bacterial LPS and the role of reactive oxygen species (ROS) as potential mediators of FasL expression in KC were investigated. LPS stimulation of KC resulted in upstream ROS generation and, subsequently, increased FasL expression and consequent Jurkat cell (Fas-positive) apoptosis. The NADPH oxidase and xanthine oxidase enzymatic pathways appear to be major sources of this upstream ROS generation. Increased FasL expression was blocked by antioxidants and by enzymatic blocking of ROS generation. Exogenous administration of H2O2 stimulated KC FasL expression and subsequent Jurkat cell apoptosis. Intracellular endogenous ROS generation may therefore represent an important signal transduction pathway for FasL expression in KC. [ABSTRACT FROM AUTHOR]

TonEBP is a transcription factor that, when activated by hypertonicity, increases transcription of genes, including those involved m organic osmolyte accumulation. Surprisingly, it is expressed in virtually all tissues, including many never normally exposed to hypertonicity. We measured TonEBP mRNA (real-time PCR) and protein (Western blot analysis) in tissues of control plasma osmolality 294 ± 1 mosmol/kgH[sub 2]O) and hyposmotic (dDAVP infusion plus water loading for 3 days, 241 ± 2 mosmol/kgH[sub 2]O) rats to test whether the ubiquitous expression of TonEBP mRNA is osmotically regulated around the normal plasma osmolality. TonEBP protein is reduced by hyposmolality in thymus and liver, but not in brain and is not detected in heart and skeletal muscle. TonEBP mRNA decreases in brain and liver but is unchanged in other tissues. There are no general changes in mRNA of TonEBP-mediated genes: aldose reductase (AR) does not change in any tissue, betaine transporter (BGT1) decreases only in liver, taurine transporter (TauT) only in brain and thymus, and inositol transporter (SMIT) only in skeletal muscle and liver. Heat shock protein (Hsp)70-1 and Hsp70-2 mRNA increase greatly in most tissues, which cannot be attributed to decreased TonEBP activity. The conclusions are ss follows: 1) TonEBP protein or mRNA expression is reduced by hyposmolality in thymus, liver, and brain. 2) TonEBP protein and mRNA expression are differentially regulated in some tissues. 3) Although AR, SMIT, BGT1, and TauT are regulated by TonEBP in renal medullary cells, other sources of regulation may predominate in other tissues. 4) TonEBP abundance and activity are regulated by factors other than tonicity in some tissues. [ABSTRACT FROM AUTHOR]

Discusses the mechanisms regulating amino acid and glucose transporters in vascular endothelial cells. Mechanisms controlling membrane transport activity and expression in vascular smooth muscle cells; Comparison between amino acid and glucose transport systems in terms of specificity, ionic dependence and kinetic properties.

Presents a study which investigated whether hemorrhagic shock and resuscitation alters the vascular responsiveness of the portohepatic circulation to endothelins. Materials and methods; Results; Discussion.

Focuses on a study which discussed the responses to hypertonicity of cultured endothelial cells from swine pulmonary arteries. Materials and methods; Effects of hypertonicity on cell protein synthesis, growth, and survival; Cell volume, cation and ninhydrin-positive substances content; Induction of transport activities.

Deals with a study which examined the abundance and nuclear distribution of tonicity-responsive enhancer binding protein (TonEBP). Materials and methods; Stimulation of TonEBP; Effect of cellular accumulation of betaine and inositol on TonBEP.