Rat Hepatocyte Plasma Membrane Research Articles

Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes κ1 for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients κ1 probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/l) and DMSO (2250 mOsm/l) the filtration coefficient L(p) in the presence of a penetrating cryoprotectant (L(pDMSO) = (4.45 ± 0.04) x 10(-14) m3/Ns) is 3 orders lower compared to the case with electrolyte (L(pNaCl) = (2.25 ± 0.25) x 10(-11) m3/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.

The effect of Amaranth oil on monolayers of artificial lipids and hepatocyte plasma membranes with adrenalin-induced stress

In this paper the oil from seeds of Amaranthus cruentus L. (AmO) was shown to be an efficient modulator of the physical chemical properties of artificial lipid and rat hepatocyte plasma membranes. AmO improved the membrane stability, their stress resistance and the adsorption of neurotensin to plasma membranes with the distinct biphasic interactions being observed even after adrenalin stress exposure. The analysis of pro-/antioxidant balance in rat blood revealed a mild prooxidant activity after AmO intake, which was accompanied by accumulation of oxidative destruction products in plasma membranes. This prooxidant action of AmO was corroborated in vitro in an adrenalin autooxidation model. On the other hand, the observed improved resistance to adrenalin stress in AmO supplemented rats was associated with an antioxidant response in blood and plasma membrane studies. The AmO effects can be attributed to the modulation of the metabolic pathways involved into oxygen and free radical homeostasis.

Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.

Protective role of unconjugated bilirubin on complement-mediated hepatocytolysis

Hyperbilirubinemia and complement-mediated immune attack on hepatocyte membrane are common features of certain hepatic diseases. To assess whether unconjugated bilirubin (UB) counteracts complement-mediated hepatocytolysis, we first generated a rabbit polyclonal antibody (Ab) against rat hepatocyte plasma membrane (RHPM). An assay performed with isolated rat hepatocytes in the presence of the polyclonal Ab and rat serum as complement donor demonstrated that UB inhibits cell lysis, as lactate dehydrogenase release into the medium was inhibited by the pigment in a dose-dependent manner. Immunofluorescence microscopy studies showed that UB significantly attenuates the binding of C3 to the hepatocyte–Ab complex. Further enzyme immunoassay studies showed that UB interferes the binding of C1q to purified anti-RHPM IgG, also in a dose-dependent manner. A dot-blot assay showed that [ 14C]-UB binds to C1q and human serum albumin (HSA) to a similar extent. A differential spectrum analysis of UB in the presence of C1q further confirmed that the pigment interacts with this protein. In conclusion, we demonstrated an inhibitory action of UB on complement-mediated Ab-induced hepatocytolysis, this action being evidenced at pathophysiological pigment concentrations (171 μM and higher). A direct binding of the pigment to C1q is likely involved.

Replacement of Cyclosporin A from its Binding to Hepatocyte Plasma Membrane by Silymarin Flavonoids

Interactions between cyclosporin A and three silymarin flavonoids (silybin, silydianin, and silychristin) were studied on the rat hepatocyte plasma membrane using the replacement of [3H]cyclosporin A by silymarin flavonoids. It was found that flavonoids significantly replaced CsA from its binding to the rat hepatocyte plasma membrane. It seems that these two types of compounds are competitors at the same binding site of the plasma membrane receptor.

Synthetic heteroprostanoids of A- and E-types as novel non-comprehensive inhibitors of adenylyl cyclase in rat hepatocytes

Treatment of rat hepatocyte plasma membranes with five novel synthetic heteroprostanoids of A- and E-types significantly decreased basal activity of adenylyl cyclase. Inhibition of forskolin-stimulated activity of the enzyme was seen as well. The maximal effective concentration for all substances tested was found at approximately 5 × 10 −6–1 × 10 −5 M. The values of half maximal concentration (IC 50) for all prostanoids were at the region of 0.7–1.1 μM. Prostanoids belonging to cyclopentenone group A (U-39, U-26) were less active than analogs of 11-deoxy-PGE 1 (TA-227, TA-280, and TA-239). The strongest inhibitory effect of adenylyl cyclase activity (more than 3 times) was determined in the presence of prostanoid TA-227 possessing hydrophobic 15-phenyl ring and isoxazol group in ω-chain. The investigation of AC activity in the presence of different concentrations of prostanoids and varying concentrations of Mg·ATP has demonstrated that a non-comprehensive mechanism with particular effect takes place in case of AC inhibition by the heteroprostanoids.

Isolation and characterization of lipid microdomains from apical and basolateral plasma membranes of rat hepatocytes

Canalicular bile is formed by the osmotic filtration of water in response to osmotic gradients generated by active transport at the apical and basolateral plasma membrane domains of hepatocytes. We recently demonstrated that mixed plasma membrane fractions isolated from rat hepatocyte couplets contain lipid microdomains ("rafts") enriched in cholesterol and sphingolipids and AQP8 and 9. We isolated lipid microdomains from hepatocyte apical and basolateral plasma membrane domains using Triton X-100 as detergent, and characterized their lipid and protein composition. A Triton-insoluble band ("raft fraction") at the 5%/30% sucrose interface in both apical and basolateral fractions was enriched for alkaline phosphatase (apical) and Na/K ATPase (basolateral) and was negative for amino peptidase-N. This detergent-insoluble band was also positive for caveolin-1 (a "raft" associated protein) and negative for clathrin (a "raft" negative protein). Lipid analysis showed that, the Triton-insoluble fraction was highly enriched in cholesterol and sphingolipids. Immunofluorescence staining on hepatocyte couplets for both caveolin-1 and cholera toxin B showed a punctate distribution on both the apical and basolateral plasma membranes, consistent with localized membrane microdomains. Dot blot analysis showed that the "raft" associated ganglioside GM1 was enriched in the detergent-insoluble fraction both domains. Furthermore, exposure of isolated hepatocytes to glucagon, a choleretic agonist, significantly increased the expression of AQP8 associated with the apical microdomain fractions but had no effect on AQP9 expression in the basolateral microdomain fractions. In conclusion, "rafts" represent target microdomains for exocytic insertion and retrieval of "flux proteins", including AQPs, involved in canalicular bile secretion.

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors.

This study describes a novel mechanism of regulation of the high-affinity Na(+)-dependent adenosine transporter (CNT2) via the activation of A(1) adenosine receptors (A(1)R). This regulation is mediated by the activation of ATP-sensitive K(+) (K(ATP)) channels. The high-affinity Na(+)-dependent adenosine transporter CNT2 and A(1)R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (-)-N(6)-(2-phenylisopropyl)adenosine is fully inhibited by K(ATP) channel blockers and mimicked by a K(ATP) channel opener. A(1)R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A(1)R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A(1)R, CNT2, and K(ATP) channels. These results also suggest that the activation of K(ATP) channels under metabolic stress can be mediated by the activation of A(1)R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for K(ATP) channels.

Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains

This unit describes a method for isolation of plasma membrane sheets from rat liver. It also includes protocols for preparation of plasma membrane domains isolated from plasma membrane sheets and indirect immunofluorescence localization of marker proteins associated with plasma membrane sheets. The unit has been updated with assays for the marker enzymes alkaline phosphodiesterase I, 5' nucleotidase, and K+-stimulated.

Molecular Mechanisms for the Hepatic Uptake of Magnetic Resonance Imaging Contrast Agents

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Kmof 92 ± 15 μM and a Vmaxof 143 ± 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection intoXenopus laevisoocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a ≥2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms.

Arginine-nitric oxide pathway in plasma membrane of rat hepatocytes during early and late sepsis.

To study the transported L-arginine in rat hepatocytes during different stages of sepsis. A prospective, controlled study. Thirty-six Sprague-Dawley male rats (250 to 300 g) were anesthetized and studied. Early sepsis was produced 9 hrs after cecal ligation and puncture (CLP) and late sepsis developed 18 hrs after CLP. The control group underwent sham operation. Plasma membrane of rat hepatocytes was prepared by differential centrifugation. The [3H] L-arginine uptake of plasma membrane vesicles during sepsis was measured and inhibition studies employing omega-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine were performed. L-arginine transport was saturable, increased linearly with plasma membrane protein concentration, and increased with uptake time up to 5 mins. [3H] L-arginine uptake increased by 77% to 121% (p < .05) during early sepsis, with no significant changes during late sepsis. Comparing inhibitors of nitric oxide synthase, L-NAME was effective in inhibiting L-arginine transport while aminoguanidine was not. L-arginine transport was enhanced in rat hepatocytes during the early stage of sepsis. The increased uptake of L-arginine could contribute to the increase production of nitric oxide by hepatocyte during sepsis.

Characterization of a sodium-dependent transport system for butyrobetaine into rat liver plasma membrane vesicles.

Butyrobetaine transport into the liver was studied using isolated rat hepatocyte plasma membrane vesicles. In the presence of a sodium chloride gradient, an overshoot could be observed, indicating active sodium-dependent transport. A similar overshoot was recorded in the presence of lithium, but not of potassium, cesium, or choline chloride. Investigation of several sodium salts revealed that an overshoot could only be observed in the presence of chloride, but not of nitrate, thiocyanate, sulfate, or gluconate. An osmolarity plot in the presence of sodium chloride revealed a slope different from zero and a positive intercept, indicating active transport and nonspecific binding, respectively. In agreement with the osmolarity plot, the kinetic characterization of butyrobetaine transport revealed a binding and a saturable component. The saturable component could be described by Michaelis-Menten kinetics, with a Km of 4.88 +/- 0.70 mmol/L and a Vmax of 4.16 +/- 0.73 picomoles per milligram of protein per second. Butyrobetaine transport could be inhibited significantly (30%) by 250 micromol/L propionylcarnitine, but not by D- or L-carnitine, other acylcarnitines (acetylcarnitine, isovalerylcarnitine, palmitoylcarnitine), trimethyllysine, or quinine. Butyrobetaine transport activity was also expressed in Xenopus laevis oocytes by injecting mRNA isolated from rat liver or kidney. After 5 days of cultivation, the endogenous butyrobetaine transport activity was increased by 82% in oocytes injected with liver mRNA and by 99% in oocytes injected with kidney mRNA. The studies show that butyrobetaine is transported actively across the basolateral plasma membrane of hepatocytes and that this transport is driven by sodium and chloride gradients. This transport is quite specific for butyrobetaine and is not rate-limiting for carnitine biosynthesis.

Identification of B10, an alkaline phosphodiesterase of the apical plasma membrane of hepatocytes and biliary cells, in rat serum: increased levels following bile duct ligation and during the development of cholangiocarcinoma.

Alkaline phosphodiesterase (APDE) is associated with the cellular plasma membrane of many organs. Several isoforms are also detected in normal human serum and their respective amounts vary in liver diseases but their significance is unknown. The aims of this study were: 1) to identify a serum form of B10, an APDE exclusively localized at the apical pole of the plasma membrane of rat hepatocytes and biliary cells; 2) to gain insight into its origin; and 3) to investigate its behavior, in two liver diseases in which an abnormal membrane expression of B10 has been reported, namely cholestasis and cholangiocarcinoma. A soluble form of B10 was immunoprecipitated from normal rat serum, which amounted to 13% of total serum APDE activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size of the serum enzyme was 125 kd, which is slightly lower than that found in the plasma membrane (130 kd). In bile, a 120-kd and a 130-kd form was found. A sixfold and fivefold increase of B10 APDE activity was observed in the serum of bile duct-ligated rats and in the Long-Evans Cinnamon (LEC) rats which spontaneously develop cholangiocarcinoma. The molecular size of the form present in serum was unchanged. A threefold increase was also observed in LEC rats which had not yet developed a cholangiocarcinoma. In conclusion, we identified a soluble form of B10 in normal rat serum. The increase in serum B10 in the experimental and pathological conditions investigated does not seem to result from passage of the biliary form to the serum but seems to be caused by increased cleavage of the membrane form. Its rise early during the onset of cholangiocarcinoma suggests that B10 in the serum might be a marker of carcinogenesis and/or be involved in the development of cholangiocarcinoma.

The influence of dietary lipids on the composition and membrane fluidity of rat hepatocyte plasma membrane.

Weanling male Wistar rats were fed for five weeks on standard rat chow (23 g fat/kg diet) or one of four synthetic diets with butterfat, coconut oil, corn oil, or fish oil as the main lipid source (100 g fat/kg diet). In all diets, 10% of the fat was provided as corn oil to prevent essential fatty acid deficiency. Significant differences were observed in the saturated, monounsaturated, and polyunsaturated fatty acid composition, and in the ratio of cholesterol to phospholipid, in the hepatocyte membranes. The fluidity of hepatocyte plasma membranes was assessed using the fluorescence recovery after photobleaching technique and steady-state fluorescence anisotropy of diphenylhexatriene. No significant differences were found in the fluidity of plasma membranes between animals on the different fat diets, despite diet-induced changes in their fatty acid composition. However, the proportion of lipid free to diffuse in the plasma membrane varied with diet, being significantly greater (P < 0.05) in animals fed chow (63.7%), coconut oil (61.5%), and butterfat (57.6%) diets than in those fed the corn oil (47.3%) diet. Animals fed fish oil showed an intermediate (50.0%) proportion of lipid free to diffuse. The data support the hypothesis that dietary lipids can change both the chemical composition and lateral organization (lipid domain structure) of rat hepatocyte plasma membranes.

Antigenic targets in tienilic acid hepatitis. Both cytochrome P450 2C11 and 2C11-tienilic acid adducts are transported to the plasma membrane of rat hepatocytes and recognized by human sera.

Patients with tienilic acid hepatitis exhibit autoantibodies that recognize unalkylated cytochrome P450 2C9 in humans but recognize 2C11 in rats. Our aim was to determine whether the immune reaction is also directed against neoantigens. Rats were treated with tienilic acid and hepatocytes were isolated. Immunoprecipitation, immunoblotting, and flow cytometry experiments were performed with an anti-tienilic acid or an anti-cytochrome P450 2C11 antibody. Cytochrome P450 2C11 was the main microsomal or plasma membrane protein that was alkylated by tienilic acid. Inhibitors of vesicular transport decreased flow cytometric recognition of both unalkylated and tienilic acid-alkylated cytochrome P450 2C11 on the plasma membrane of cultured hepatocytes. Tienilic acid hepatitis sera that were preadsorbed on microsomes from untreated rats (to remove autoantibodies), poorly recognized untreated hepatocytes in flow cytometry experiments, but better recognized tienilic acid-treated hepatocytes. This recognition was decreased by adsorption with tienilic acid or by preexposure to the anti-tienilic acid or the anti-cytochrome P450 2C11 antibody. We conclude that cytochrome P450 2C11 is alkylated by tienilic acid and follows a vesicular route to the plasma membrane. Tienilic acid hepatitis sera contain antibodies against this tienilic acid adduct, in addition to the previously described anticytochrome P450 autoantibodies.

The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat liver organic anion-transporting polypeptide.

The peptidomimetic thrombin inhibitor CRC 220, 4-methoxy-2,3,6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinop henylalanyl- piperidide, is taken up into isolated rat hepatocytes through active, carrier-mediated transport. This uptake is inhibited by bile acids. Functional expression in Xenopus laevis oocytes was performed to identify the transport system responsible for the hepatocellular CRC 220 uptake. Injection of poly(A)+RNA in X. laevis oocytes resulted in a two- to three-times higher uptake of CRC 220, compared with uninjected or water-injected control oocytes. Taurocholate (200 mumol/L) inhibited this uptake completely. No uptake of the peptidomimetic thrombin inhibitor was observed, when X. laevis oocytes were injected with complementary RNA (cRNA) encoding either the cloned rat liver Na(+)-dependent taurocholate transporter Ntcp, the renal oligopeptide carrier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na(+)-independent organic anion transporting polypeptide oatp, a specific and saturable CRC 220 uptake was observed (Michaelis-Menten constant 29.5 mumol/L). Cis-inhibition with known oatp-substrates, e.g., 20 mumol/L Bromsulphalein (BSP), 2007 mumol/L taurocholate and 2007 mumol/L cholate, occurred in oatp-expressing X. laevis oocytes, whereas substrates of the two peptide carriers as well as dipeptide- and single-amino acid constituents of the thrombin inhibitor itself lacked any significant inhibitory effects. These data show that the modified dipeptide CRC 220 is a highly selective substrate of the organic anion transporting polypeptide oatp in the basolateral plasma membrane of rat hepatocytes.

The peptide-based thrombin inhibitor CRC 220 is a new substrate of the basolateral rat liver organic anion-transporting polypeptide

The peptidomimetic thrombin inhibitor CRC 220, 4-methoxy-2,3,6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinop henylalanyl- piperidide, is taken up into isolated rat hepatocytes through active, carrier-mediated transport. This uptake is inhibited by bile acids. Functional expression in Xenopus laevis oocytes was performed to identify the transport system responsible for the hepatocellular CRC 220 uptake. Injection of poly(A)+RNA in X. laevis oocytes resulted in a two- to three-times higher uptake of CRC 220, compared with uninjected or water-injected control oocytes. Taurocholate (200 mumol/L) inhibited this uptake completely. No uptake of the peptidomimetic thrombin inhibitor was observed, when X. laevis oocytes were injected with complementary RNA (cRNA) encoding either the cloned rat liver Na+- dependent taurocholate transporter Ntcp, the renal oligopeptide carrier rhaPT or the intestinal oligopeptide transporter PepT1. However, after injection of cRNA of the cloned rat liver Na+-independent organic anion transporting polypeptide oatp, a specific and saturable CRC 220 uptake was observed (Michaelis-Menten constant 29.5 mumol/L). Cis-inhibition with known oatp-substrates, e.g., 20 mumol/L Bromsulphalein (BSP), 2007 mumol/L taurocholate and 2007 mumol/L cholate, occurred in oatp-expressing X. laevis oocytes, whereas substrates of the two peptide carriers as well as dipeptide- and single-amino acid constituents of the thrombin inhibitor itself lacked any significant inhibitory effects. These data show that the modified dipeptide CRC 220 is a highly selective substrate of the organic anion transporting polypeptide oatp in the basolateral plasma membrane of rat hepatocytes. (Hepatology 1996 Aug;24(2):380-4)

First-pass elimination of a peptidomimetic thrombin inhibitor is due to carrier-mediated uptake by the liver: Interaction with bile acid transport systems

CRC 220 (4-methoxy-2, 3, 6-trimethylphenylsulfonyl-L-aspartyl-D-4-amidinophenylalanyl-piperidide) is a competitive peptide-based trombin inhibitor with high affinity to human α-thrombin ( K i 2.5 nM). The amphiphilic compound exhibits virtually no systemic bioavailability despite proteolytic stability and proven enteral absorption. After intravenous application (V. jejunalis) in rats CRC 220 is almost completely excreted into bile. Simultaneous administration of bile acids considerably decreases this first-pass elimination. CRC 220 is extensively taken up in isolated rat hepatocytes by a saturable carrier-mediated transport with K m 23.7 μM and V max 775 pmol × mg −1 × min −1. A large part of this transport is energy-dependent. At temperatures above 20 °C, the uptake is accelerated exponentially. The activation energy amounts to 82 kJ/mol. A minor portion of CRC 220 uptake occurs by physical diffusion with a permeability coefficient of 7.83 × 10 −7cm/sec at 12 °C. Sodium ions energize CRC 220 uptake. Replacement of sodium by choline or lithium decreases the transport rate of 23–40%. In addition, a negative membrane potential facilitates the uptake. CRC 220 transport is only observed in hepatocytes: it is absent in BHK, FAO, HepG2, HPCT 1E3, and HPCT 1E3-TC cells. In the presence of 4-amidinophenylalanine derivatives, CRC 220 uptake is considerably decreased. Inhibition also occurs with bile acids and bromosulfophthalein, but less with bumetanide. Because CRC 220 inhibits bile acid uptake into hepatocytes and vice versa, the results suggest that the first-pass elimination of this amphiphilic thrombin inhibitor is due to an active carrier-mediated transport process in the basolateral plasma membrane of rat hepatocytes, and that this transport occurs via a bile acid transport system.

Accumulation of ATP by plasma membranes of human and rat hepatocytes induced by some growth factors and phosphatidylcholine

Using the method of ATP luminometry it is shown that crude membrane preparations from human and rat hepatocytes accumulate ATP 20–100 nmol/mg protein during a 1-min incubation under conditions of oxidative phosphorylation. Application of appropriate inhibitors shows that a possible contamination of the membrane preparations with mitochondria does not contribute to this ATP accumulation. Phosphatidylcholine, tumor necrosis factor, and cell proliferation factor markedly stimulate the accumulation of ATP by plasma membraneenriched particles isolated from rat and human liver. The hepatocyte plasma membrane is shown to be able to synthesize ATP from inorganic phosphate and ADP using the aerobic mechanism. ATP in the plasma membrane is assumed to participate in the transmembrane signal transduction from growth factors to the cell effector systems.