Pathway In Rat Hepatocytes Research Articles

Lysosomal cysteine protease, cathepsin B, is targeted to lysosomes by the mannose 6-phosphate-independent pathway in rat hepatocytes: site-specific phosphorylation in oligosaccharides of the proregion.

Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [(32)P]phosphate, (32)P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of (32)P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated (35)S- or (32)P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular (35)S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the (32)P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH(4)Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized (35)S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.

Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment

BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF- induced PLC gamma tyrosine phosphorylation and inositol 1,4,5- trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism. (Gastroenterology 1997 Jun;112(6):2073-88)

Characterization of sphingosine 1-phosphate-induced actions and its signaling pathways in rat hepatocytes.

Sphingosine 1-phosphate (S-1-P) and lysophosphatidic acid (LPA) stimulated glycogen phosphorylase, a rate-limiting enzyme responsible for glycogenolysis, in association with Ca2+ mobilization and phospholipase C (PLC) activation in rat hepatocytes. S-1-P, but not LPA, also inhibited adenosine 3',5'-cyclic monophosphate accumulation reflecting adenylyl cyclase inhibition. S-1-P-induced PLC activation, Ca2+ mobilization, and phosphorylase activation were markedly enhanced by primary culture of the cells for 24 h, whereas the inhibitory adenosine 3',5'-cyclic monophosphate response was unchanged by increasing culture time. Activation of the PLC-Ca2+ system during primary culture was specific to the lysosphingolipid; PLC and Ca2+ responses to LPA and NaF were unchanged or slightly attenuated by increasing culture time. Pertussis toxin treatment almost completely suppressed the S-1-P-induced inhibition of adenylyl cyclase but hardly influenced the lipid-induced activation of PLC and its cascade reactions. We conclude that S-1-P, through an LPA receptor-independent mechanism, stimulates two signaling pathways, i.e., activation of the PLC-Ca2+ system and inhibition of adenylyl cyclase, through distinct S-1-P receptor-transducer systems, resulting in the modulation of glycogenolysis in rat hepatocytes.

Regulation of gamma-glutamylcysteine synthetase by protein phosphorylation.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS; EC 6.3.2.2), the rate-limiting enzyme in GSH synthesis, can be acutely inhibited approximately 20-40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg(2+)-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its K(m). Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity.

Evidence of differential effects produced by ethanol on specific phospholipid biosynthetic pathways in rat hepatocytes.

1. The aim of the present study was to investigate the effects of ethanol in vitro on the phospholipid biosynthetic pathways in hepatocytes isolated from the rat. We have used [methyl-14C]-choline, [1-3H]-ethanolamine and L-[3-3H]-serine as exogenous precursors of the corresponding phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). 2. Incubation of hepatocytes in the presence of ethanol significantly alters the incorporation of radiolabel from [14C]-choline and [3H]-ethanolamine into the metabolic intermediates and the final products of the CDP-choline and CDP-ethanolamine pathways. Radioactivity in the metabolic intermediates of both pathways was significantly decreased and the amount of label in PE was reduced whilst that of PC was not modified. 3. In the presence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH) activity, ethanol produces a reduction in the label of choline phosphate, ethanolamine phosphate and a significant decrease in the amount of PC and PE radiolabel. 4. On the other hand, ethanol increases the incorporation of serine into phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, although this effect is observed only in the absence of 4-methylpyrazole, indicating that this alteration is produced by some metabolite generated as a consequence of hepatic alcohol metabolism. 5. Ethanol also interferes with the methylation of phosphatidylethanolamine produced via the CDP-ethanolamine pathway but it does not alter phosphatidylethanolamine methylation when this phospholipid is produced by mitochondrial phosphatidylserine decarboxylation, suggesting the existence of different intramembrane pools of phosphatidylethanolamine, which may exhibit different sensitivity to alcohol. 6. Our results indicate that ethanol exerts two different effects on phospholipid metabolism in hepatocytes: a stimulatory effect on the incorporation of exogenous substrates into different phospholipids probably related to an alteration in the availability of lipogenic substrates as a consequence of ethanol metabolism, and another inhibitory effect produced by ethanol per se, which can be observed only when ethanol metabolism is inhibited by the presence of a specific inhibitor of alcohol dehydrogenase activity.

Cholestasis-induced alterations of the trans- and paracellular pathways in rat hepatocytes

Bile secretion depends on the vectorial transport of solutes from blood to bile and involves three different pathways: transcellular pathways mediated by transport proteins distributed asymmetrically in the basolateral and canalicular plasma membrane and by transcytotic vesicles, and a paracellular pathway allowing selective diffusion through tight junctions. All three pathways are impaired differentially by extrahepatic (bile duct ligation) or intrahepatic (ethinyloestradiol) cholestasis. Ethinyloestradiol treatment leads to tight junctional defects that are less severe than those induced by bile duct ligation. Junctional impairment is reflected functionally in increased permeability for horseradish peroxidase and structurally by decreased strand numbers and increased junctional length, but not by alterations at the level of the individual strands. The parallelism of physiological and morphological perturbations indicates a structure-function relationship in hepatocellular tight junctions. In addition, impaired functional integrity of tight junctions following bile duct ligation is reflected in a partial loss of hepatocellular surface polarity owing to redistribution of some, but not all, domain-specific plasma membrane antigens, which might mimic the behaviour of transport systems. After ethinyloestradiol treatment no alterations of surface polarity were observed. Thus, immunohistochemistry supports the view that ethinyloestradiol results in less severe impairment of the tight junctions than bile duct ligation. Finally, bile duct ligation, but not ethinyloestradiol, affects the transcytotic vesicular pathway; severe impairment of this is reflected in the absence of a late horseradish peroxidase peak in bile and also in the accumulation of pericanalicular vesicles that are immunopositive for canalicular membrane proteins and accessible for bulk phase endocytic markers.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoflavonoids, genistein, psi-tectorigenin, and orobol, increase cytoplasmic free calcium in isolated rat hepatocytes

Isoflavonoid compounds, genistein, psi-tectorigenin and orobol have been implicated as inhibitors of tyrosine-specific protein kinase and phosphatidylinositol turnover. These compounds have been frequently used as a pharmacological tool to assess signal transduction pathways in various cell systems. In the course of analyzing signaling pathways in rat hepatocytes, we obtained an unexpected finding that these compounds transiently increase cytoplasmic free calcium. Since the Ca 2+ mobilizing effect was observed in 1 μM calcium containing buffer, the source of the Ca 2+ may be intracellular stores. Thus, when interpreting data obtained using these compounds, caution is needed.

Characterization of H+ efflux pathways in rat hepatocytes.

A pH-stat method was used to characterize H+ efflux pathways in hepatocytes in order to determine if Na+/H+ and Ca++/H+ exchange are involved in H+ efflux from hepatocytes under basal conditions and if cyclic AMP analogs affect Na+/H+ exchange. Total H+ efflux of freshly prepared hepatocytes ranged from 10 to 15 nmoles per min per mg protein. A part of total H+ efflux (35 to 50%) was dependent on extracellular Na+. This Na+-dependent H+ efflux was (i) inhibited by amiloride with a half-maximal effect at 0.3 mM, (ii) inhibited by ouabain, (iii) dependent on extracellular pH and (iv) characterized by a Km of 15 +/- 3 mM Na+ and a Vmax of 9 +/- 0.07 nmoles per min per mg protein. Amiloride, ouabain and replacement of Na+ by choline also decreased intracellular pH determined from equilibrium distribution of dimethyloxazolidinedione. Li+ could partially substitute for Na+ in Na+-dependent H+ efflux and in maintaining intracellular pH. Efflux of CO2 and lactic acid from hepatocytes represented 80% of Na+-independent H+ efflux. Efflux of H+ in the presence and absence of Na+ was not significantly altered by extracellular Ca++ (less than 10 microM and 1.0 mM). Thus, Ca++/H+ exchange is unlikely to contribute significantly to total H+ efflux from hepatocytes. Cyclic AMP analogs, dibutyryl cyclic AMP and 8-bromo cyclic AMP, inhibited amiloride-sensitive Na+-dependent H+ efflux, and dibutyryl cyclic AMP decreased intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments.

The endocytic compartments of the asialoglycoprotein (ASGP) pathway in rat hepatocytes were studied using a combined morphological and biochemical approach in the isolated perfused liver. Use of electron microscopic tracers and a temperature-shift protocol to synchronize ligand entry confirmed the route of ASGP internalization observed in our previous in vivo studies (1) and established conditions under which we could label the contents of successive compartments in the pathway for subcellular fractionation studies. Three endosomal compartments were demonstrated in which ASGPs appear after they enter the cell via coated pits and vesicles but before they reach their site of degradation in lysosomes. These three compartments could be distinguished by their location within the hepatocyte, by their morphological appearance in situ, and by their density in sucrose gradients. The distributions of ASGP receptors, both accessible and latent (revealed by detergent permeabilization), were also examined and compared with that of ligand during subcellular fractionation. Most accessible ASGP receptors co-distributed with conventional plasma membrane markers. However, hepatocytes contain a substantial intracellular pool of latent ASGP binding sites that exceeds the number of cell surface receptors and whose presence is not dependent on ASGP exposure. The distribution of these latent ASGP receptors on sucrose gradients (detected either immunologically or by binding assays) was coincident with that of ligand sequestered within the early endosome compartments. In addition, both early endosomes and the membrane vesicles containing latent ASGP receptors had high cholesterol content, because both shifted markedly in density upon exposure to digitonin.

Effect of chloroquine on low-density lipoprotein catabolic pathway in rat hepatocytes.

Injection of 125I-low-density lipoprotein (LDL) into estradiol-treated rats substantially increases the volume of the multivesicular body compartment in hepatocytes. When these animals were additionally given chloroquine 60 and 120 min before injection of 125I-LDL, the amount of 125I retained by the liver increased threefold and the amount of label in multivesicular bodies increased fivefold, as determined by quantitative analysis of autoradiograms. Lamellar inclusions that appeared after chloroquine also contained a substantial fraction of 125I, whereas the fraction of 125I in secondary lysosomes was reduced. From these data it is concluded that the multivesicular bodies comprise an intermediate endocytic compartment, the conversion of which to secondary lysosomes is impeded by chloroquine.