Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an ... more Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an activation of the epidermal growth factor receptor (EGFR) and subsequent cell proliferation (1). Free fatty acids (FFAs) are known to induce lipoapoptosis in liver cells in a c-Jun-NH2terminal kinase (JNK)-dependent, but death receptor-independent way (2). As nonalcoholic steatohepatitis (NASH) is associated with hyperinsulinemia and increased FFAblood levels, the interplay between insulin and FFA was studied with regard to hepatocyte proliferation and apoptosis in isolated rat and mouse hepatocytes. Saturated long chain FFAs induced apoptosis and JNK activation in primary rat hepatocytes, but did not activate the CD95 (Fas, APO-1) system, whereas insulin triggered EGFR activation and hepatocyte proliferation. Coadministration of insulin and FFAs, however, abolished hepatocyte proliferation and triggered CD95-dependent apoptosis due to a JNK-dependent association of the activated EGFR wi...
We studied the downregulation of hepatobiliary transport systems and the effect of pharmacologica... more We studied the downregulation of hepatobiliary transport systems and the effect of pharmacological heme oxygenase-1 (HO-1) preinduction by Hemoglobin-Glutamer 200 (HbG200) in cold ischemia-reperfused rat liver (I/R). Cold I/R reduced bile flow in the reperfusion period from 3.10±0.10 ml/3 h to 0.54±0.20 ml/3 h…
The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate... more The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single-cell fluorescence. Regardless of the duration of HSC culture, ET-1 caused a BQ-123-sensitive but IRL-1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETA but not ETB receptors. HSCs in early culture ("quiescent HSCs") were mildly responsive to ET-1: the ET-1 concentration required to obtain a [Ca2+]i transient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET-1 concentrations above 40 nmol/L. With culture time, -smooth muscle actin (-SMA) expression increased, as did the ET-1 sensitivity of cells, resulting in a shift of the RC50 value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET-1 sensitivity was higher in -SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]i response to extracellular uridin 5'-triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+ stores, which could be mobilized by ET-1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET-1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to -adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET-1-mobilizable Ca2+ store is contained in and is smaller than the Ca2+ pool, which is mobilized by phenylephrine or UTP.
The calcium-activated potassium channel KCa3.1 controls different cellular processes such as prol... more The calcium-activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tet...
Hydrophobic bile salts activate NADPH oxidase through a ceramide- and PKCzeta-dependent pathway a... more Hydrophobic bile salts activate NADPH oxidase through a ceramide- and PKCzeta-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis. The mechanisms underlying bile salt-induced ceramide formation have remained unclear to date and thus were studied in rat hepatocytes. Proapoptotic bile salts, such as taurolithocholylsulfate (TLCS), lowered the apparent pHves within seconds from 6.0 to 5.6 in an FITC-dextran-accessible endosomal compartment that also contains acidic sphingomyelinase. Simultaneously, a rapid decrease in N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) fluorescence was observed, suggestive of an increase in cytosolic [Cl-], which is known to activate vacuolar-type H+-ATPase. No vesicular acidification or increase in cytosolic [Cl-] was found in response to the non-apoptotic bile salt taurocholate or the anti-apoptotic bile salt tauroursodesoxycholate. Inhibition of TLCS-induced endosomal acidification by bafilomycin or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid largely abolished the TLCS-induced ceramide-formation and downstream ceramide-dependent processes, such as p47phox-serine phosphorylation, NADPH oxidase activation, CD95 activation and apoptosis. These responses were also abolished after knockdown of acidic sphingomyelinase in rat hepatocytes. In conclusion, hydrophobic, proapoptotic bile salts stimulate ceramide formation through chloride-dependent acidification of endosomes, with subsequent activation of acidic sphingomyelinase. Our data suggest that changes in ion homeostasis underlie the stimulation of ceramide formation in response to hydrophobic bile acids as an important upstream event of bile salt-induced apoptosis.
Proliferation and matrix synthesis of activated pancreatic stellate cells (PSCs) participate in t... more Proliferation and matrix synthesis of activated pancreatic stellate cells (PSCs) participate in the development of chronic pancreatitis. Besides other substances, endothelin-1 (ET-1) may influence the activation process of PSCs. Until now, ET-1 has not been studied in this particular cell type. To characterize PSCs in rat pancreas with respect to expression of ET(A)-receptors, production of ET-1, and physiological effects induced by ET-1 during PSC activation. Immunocytochemical and ELISA techniques and cDNA microarray analysis were used. Physiologic effects were characterized by single cell measurements of free cytosolic Ca2+-concentration and of PSC contractility on collagen lattices. Activation of PSCs in vitro, as assessed by alpha-smooth muscle actin expression, was accompanied by the de novo expression of ET(A)-receptors and synthesis of ET-1 mRNA and protein. Cytosolic Ca2+-concentration was increased upon ET-1 stimulation in activated but not in quiescent PSCs. Contractility of activated PSCs was significantly reduced by the selective ET(A)-receptor antagonist BQ123 but not by the ET(B)-receptor antagonist IRL-1038. The results suggest that ET-1 may act as a paracrine and autocrine factor for activated PSCs and may mediate contractions of activated, but not quiescent, PSCs.
Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an ... more Insulin is known to induce hepatocyte swelling, which triggers via integrins and c-Src kinase an activation of the epidermal growth factor receptor (EGFR) and subsequent cell proliferation (1). Free fatty acids (FFAs) are known to induce lipoapoptosis in liver cells in a c-Jun-NH2terminal kinase (JNK)-dependent, but death receptor-independent way (2). As nonalcoholic steatohepatitis (NASH) is associated with hyperinsulinemia and increased FFAblood levels, the interplay between insulin and FFA was studied with regard to hepatocyte proliferation and apoptosis in isolated rat and mouse hepatocytes. Saturated long chain FFAs induced apoptosis and JNK activation in primary rat hepatocytes, but did not activate the CD95 (Fas, APO-1) system, whereas insulin triggered EGFR activation and hepatocyte proliferation. Coadministration of insulin and FFAs, however, abolished hepatocyte proliferation and triggered CD95-dependent apoptosis due to a JNK-dependent association of the activated EGFR wi...
We studied the downregulation of hepatobiliary transport systems and the effect of pharmacologica... more We studied the downregulation of hepatobiliary transport systems and the effect of pharmacological heme oxygenase-1 (HO-1) preinduction by Hemoglobin-Glutamer 200 (HbG200) in cold ischemia-reperfused rat liver (I/R). Cold I/R reduced bile flow in the reperfusion period from 3.10±0.10 ml/3 h to 0.54±0.20 ml/3 h…
The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate... more The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single-cell fluorescence. Regardless of the duration of HSC culture, ET-1 caused a BQ-123-sensitive but IRL-1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETA but not ETB receptors. HSCs in early culture ("quiescent HSCs") were mildly responsive to ET-1: the ET-1 concentration required to obtain a [Ca2+]i transient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET-1 concentrations above 40 nmol/L. With culture time, -smooth muscle actin (-SMA) expression increased, as did the ET-1 sensitivity of cells, resulting in a shift of the RC50 value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET-1 sensitivity was higher in -SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]i response to extracellular uridin 5'-triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+ stores, which could be mobilized by ET-1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET-1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to -adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET-1-mobilizable Ca2+ store is contained in and is smaller than the Ca2+ pool, which is mobilized by phenylephrine or UTP.
The calcium-activated potassium channel KCa3.1 controls different cellular processes such as prol... more The calcium-activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tet...
Hydrophobic bile salts activate NADPH oxidase through a ceramide- and PKCzeta-dependent pathway a... more Hydrophobic bile salts activate NADPH oxidase through a ceramide- and PKCzeta-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis. The mechanisms underlying bile salt-induced ceramide formation have remained unclear to date and thus were studied in rat hepatocytes. Proapoptotic bile salts, such as taurolithocholylsulfate (TLCS), lowered the apparent pHves within seconds from 6.0 to 5.6 in an FITC-dextran-accessible endosomal compartment that also contains acidic sphingomyelinase. Simultaneously, a rapid decrease in N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) fluorescence was observed, suggestive of an increase in cytosolic [Cl-], which is known to activate vacuolar-type H+-ATPase. No vesicular acidification or increase in cytosolic [Cl-] was found in response to the non-apoptotic bile salt taurocholate or the anti-apoptotic bile salt tauroursodesoxycholate. Inhibition of TLCS-induced endosomal acidification by bafilomycin or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid largely abolished the TLCS-induced ceramide-formation and downstream ceramide-dependent processes, such as p47phox-serine phosphorylation, NADPH oxidase activation, CD95 activation and apoptosis. These responses were also abolished after knockdown of acidic sphingomyelinase in rat hepatocytes. In conclusion, hydrophobic, proapoptotic bile salts stimulate ceramide formation through chloride-dependent acidification of endosomes, with subsequent activation of acidic sphingomyelinase. Our data suggest that changes in ion homeostasis underlie the stimulation of ceramide formation in response to hydrophobic bile acids as an important upstream event of bile salt-induced apoptosis.
Proliferation and matrix synthesis of activated pancreatic stellate cells (PSCs) participate in t... more Proliferation and matrix synthesis of activated pancreatic stellate cells (PSCs) participate in the development of chronic pancreatitis. Besides other substances, endothelin-1 (ET-1) may influence the activation process of PSCs. Until now, ET-1 has not been studied in this particular cell type. To characterize PSCs in rat pancreas with respect to expression of ET(A)-receptors, production of ET-1, and physiological effects induced by ET-1 during PSC activation. Immunocytochemical and ELISA techniques and cDNA microarray analysis were used. Physiologic effects were characterized by single cell measurements of free cytosolic Ca2+-concentration and of PSC contractility on collagen lattices. Activation of PSCs in vitro, as assessed by alpha-smooth muscle actin expression, was accompanied by the de novo expression of ET(A)-receptors and synthesis of ET-1 mRNA and protein. Cytosolic Ca2+-concentration was increased upon ET-1 stimulation in activated but not in quiescent PSCs. Contractility of activated PSCs was significantly reduced by the selective ET(A)-receptor antagonist BQ123 but not by the ET(B)-receptor antagonist IRL-1038. The results suggest that ET-1 may act as a paracrine and autocrine factor for activated PSCs and may mediate contractions of activated, but not quiescent, PSCs.
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Papers by Roland Reinehr