[5][6][7][8][9] and are major cells of matrix protein metabolism [10][11][12] in the liver. In the injured rat liver and during cultivation, quiescent rat HSCs transform to myofibroblastlike cells. 13 This so-called ''activation'' is associated with loss of vitamin A droplets [14][15][16] and increased proliferation 17 and increased production 18 of extracellular matrix proteins. 19 The de novo expression of ␣-smooth muscle actin (␣-SMA) increases cell contractility, 20-22 which may contribute to portal hypertension in vivo. 21 Contractility of activated HSCs/myofibroblasts is regulated by endothelins (ETs) 1, 2, and 3, 6,7,20,[23][24][25] nucleotides, 26 substance P, 6 vasopressin, 27 nitric oxide, cyclic adenosine monophosphate, 28 prostaglandin I 2 , and prostaglandin E 2 . 29 In rat liver, ET-1 is secreted by sinusoidal endothelial cells and HSCs. 24,30 ET-1 is overexpressed in activated HSCs in vivo and in culture, 24,31,32 and plasma levels of ET-1 are enhanced in liver cirrhosis in humans. 33,34 HSCs are not only a site of production, but also major target cells of endothelin action in the liver. They possess both the ET A and ET B receptors in greater abundance than any other liver-resident cells. 24 The ET A receptor binds ET-1 with high and ET-3 with low affinity. 35,36 Stimulation of ET A receptors increases the cytosolic free Ca 2ϩ -concentration ([Ca 2ϩ ] i ) 7 and mediates vasoconstrictive effects of ET-1 in rat liver in vivo. 5 The ET B receptor binds ET-1 and ET-3 with similar affinity 37 and outnumbers the ET A receptors in rat liver. 24 ET B elicits proliferative activity in rat HSCs 38 and might also be involved in the contraction of HSCs. 24 The presence of both the effector synthesis and the receptor on HSCs points to a potential autocrine/paracrine regulation of HSCs. Most studies investigating effects of endothelins on [Ca 2ϩ ] i focused on activated HSCs in culture because measurements with the widely used calciumsensitive dyes Fura-2 and Indo-1 are difficult in quiescent HSCs because of the vitamin A-dependent autofluorescence. The use of the new calcium-sensitive dye Oregon Green 488 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA-1) 39 avoids these problems and allows reliable investigation of [Ca 2ϩ ] i changes in quiescent HSCs. This study describes the effects of ET-1 on [Ca 2ϩ ] i during the dynamic process of activation. MATERIALS AND METHODS MaterialsThe materials used were purchased as follows: Dulbecco' s modified Eagle medium, RPMI medium, fetal calf serum, and penicillin/ streptomycin from Biochrom (Berlin, Germany); nycodenz from Nycomed (Oslo, Norway); Pronase from Merck (Darmstadt, Germany); DNase I, collagenases, uridin 5Ј-triphosphate (UTP) and anti-␣-SMA antibody (for Immunocytochemistry) from Boehringer

Compatible organic osmolytes, such as betaine and taurine are involved in the regulation of Kupffer cell (KC) function, but nothing is known about osmolytes in liver endothelial cells. This was investigated here by studying the effect of aniso-osmotic exposure of rat liver sinusoidal endothelial cells (SEC) on osmolyte transport and the messenger RNA (mRNA) levels for the transport systems for betaine (BGT1), taurine (TAUT), and myo-inositol (SMIT). Compared with normo-osmotic exposure (305 mosmol/L), hyperosmotic exposure (405 mosmol/L) of SEC led to an increase in the mRNA levels for these transport systems and simultaneously to a stimulation of betaine, taurine, and myo-inositol uptake, which led to an increase of cell volume. Conversely, hypo-osmotic exposure decreased osmolyte uptake. When hyperosmotically pre-exposed SEC were loaded with betaine, taurine, or myoinositol, hypo-osmotic stress stimulated the efflux of these osmolytes from the cells. Studies on osmolyte tissue levels revealed that taurine was an important compatible organic osmolyte under normo-osmotic conditions and predominantly released following hypo-osmotic stress. Conversely, following hyperosmotic exposure, the increase in cellular betaine and myo-inositol exceeded that of taurine. In lipopolysaccharide (LPS)-treated SEC, hyperosmotic exposure markedly raised the mRNA levels for cyclo-oxygenase-2 (COX-2), but not for inducible nitric oxide synthase (iNOS). The increase of COX-2 mRNA levels was counteracted by betaine and taurine and, to a lesser extent, by myo-inositol. The findings indicate that SEC use taurine, betaine, and myo-inositol as compatible organic osmolytes.

No abstract

Activation of hepatic stellate cells (HSCs) results in multiple alterations of cell function, but nothing is known about organic osmolytes in these cells. Organic osmolyte transport and transporter messenger RNA (mRNA) expression was studied in quiescent rat HSCs and after their transformation into ␣ 1 -smooth muscle actin-positive myofibroblastlike cells. Quiescent stellate cells expressed in an osmosensitive manner the mRNA levels of the transporters for taurine (TAUT) and myoinositol (SMIT), whereas that for betaine was not detectable. However, these cells showed osmosensitive uptake not only of taurine and myoinositol but also of betaine. Osmosensitive betaine uptake was mediated by amino acid transport system A. After transformation into myofibroblasts, taurine and myoinositol uptake increased 5.5-fold and 4.5-fold, respectively, together with the respective transporter mRNA levels. Betaine uptake increased twofold because of osmosensitive induction of BGT1 expression. In both quiescent and activated HSCs, hypoosmotic cell swelling induced a rapid and 4,4Ј-diisothiocyanatostilbene-2,2Ј-disulphonic acid-sensitive osmolyte efflux. In quiescent HSCs, hyperosmotic exposure increased the messenger RNA (mRNA) level of cyclooxygenase-2, which was counteracted by taurine but not by betaine or myoinositol. The study identifies taurine, myoinositol, and betaine as osmolytes in HSCs. Transformation of HSCs is accompanied by enhanced osmolyte transport activity and induction of the BGT1 transporter, which may be another activation marker of HSCs. (HEPATOLOGY 1999;29: 173-180.)

The expression of glutamine synthetase (GS) was studied in cultured quiescent hepatic stellate cells (HSC) and during their transformation into myofibroblast-like cells. GS mRNA was detectable in quiescent HSC (1-day culture); however, the enzyme protein was not expressed, as assessed by Western blot analysis, immunocytochemistry and the absence of detectable enzyme activity. Similar findings were obtained after 2 days of culture; in addition, the mRNA levels had dropped by about 70%, but they increased again thereafter during the process of HSC transformation in culture, as indicated by the expression of alpha-smooth-muscle actin. In parallel with the accumulation of alpha-smooth-muscle actin, GS was expressed, as shown by Western blot analysis and immunocytochemistry, and enzyme activity increased from undetectable levels in quiescent cells to 0.13+/-0.01 micromol/h per mg of cell protein within 7-14 days. This value compares with GS activity in liver parenchymal cells of 0.57+/-0.03 micromol/h per mg of cell protein. The findings suggest that activation of HSC results in the de novo expression of GS protein and activity, and this may serve as another marker of HSC transformation.