scholarly journals Selective release of plasma-membrane enzymes from rat hepatocytes by a phosphatidylinositol-specific phospholipase C

1980 ◽  
Vol 187 (1) ◽  
pp. 277-280 ◽  
Author(s):  
S D Shukla ◽  
R Coleman ◽  
J B Finean ◽  
R H Michell
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Phospholipase C ◽  
Inositol Phosphate ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Membrane Enzymes

When isolated hepatocytes are incubated with phosphatidylinositol-specific phospholipase C, three cell-surface enzymes show markedly different behaviour. Most of the alkaline phosphatase is released at very low values of phosphatidylinositol hydrolysis, whereas further phosphatidylinositol hydrolysis releases only a maximum of about one-third of the 5′-nucleotidase. Alkaline phosphodiesterase I is not released. If cells containing phosphatidyl[3H]inositol are similarly treated, then the released [3H]inositol is in the form of inositol phosphate: no evidence has been obtained for any covalent association between released [3H]inositol and alkaline phosphatase.

scholarly journals Novel evidence for an ecto-phospholipid methyltransferase in isolated rat hepatocytes

1998 ◽  
Vol 330 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Françoise BONTEMPS ◽  
Georges VAN DEN BERGHE
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Methyl Group ◽  
Methyl Donor ◽  
External Face ◽  
Subsequent Addition ◽  
Isolated Rat

Phospholipids of isolated rat hepatocytes were labelled by preincubation with either 2 μM [methyl-14C]S-adenosylmethionine (AdoMet) or 2 μM [methyl-14C]methionine. Subsequent addition of phospholipase C to the suspension removed 95% of the radioactivity from phospholipids methylated by [methyl-14C]AdoMet within a few minutes, but was without effect on phospholipids methylated by [methyl-14C]methionine radioactivity from the latter could, nevertheless, be removed by phospholipase C after permeabilization of the cells with digitonin. The results clearly show that the methyl group of exogenous AdoMet, contrary to that of methionine, is transferred on to phospholipids located on the external face of the plasma membrane. Accordingly, pretreatment of isolated hepatocytes with trypsin prevented the methylation of phospholipids from exogenous AdoMet by 60-80%, whereas it was almost without effect when exogenous methionine was the methyl donor. Our data corroborate previous work [Bontemps and Van den Berghe (1997) Biochem. J. 327, 383-389], which indicated that AdoMet methylates hepatocyte phospholipids without penetrating the cells.

Hormonal modulation of hepatic plasma membrane lactate transport in cultured rat hepatocytes

1990 ◽  
Vol 10 (6) ◽  
pp. 573-577 ◽  
Author(s):  
H. K. Metcalfe ◽  
R. D. Cohen ◽  
J. P. Monson
Keyword(s):  
Plasma Membrane ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Similar Magnitude ◽  
Potential Mechanism ◽  
Lactate Transport ◽  
Hormonal Modulation ◽  
Cultured Rat Hepatocytes

Hormonal modulation of hepatic plasma membrane lactate transport was studied in primary cultures of isolated hepatocytes from fed rats to examine the mechanism for the known enhancement of lactate transport in starvation and diabetes. Total cellular lactate entry was increased by 14% in the presence of dexamethasone; this was accounted for by an approximately 40% increase in the carrier-mediated component of entry with no effect on diffusion. A trend of similar magnitude was evident with glucagon. The effects of dexamethasone and glucagon on lactate transport constitute an additional potential mechanism for enhancement of gluconeogenesis by these hormones.

scholarly journals Sugar uptake by fluid-phase pinocytosis and diffusion in isolated rat hepatocytes

1987 ◽  
Vol 243 (3) ◽  
pp. 655-660 ◽  
Author(s):  
P B Gordon ◽  
H Høyvik ◽  
P O Seglen
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Free Sugar ◽  
Sugar Uptake ◽  
Isolated Rat Hepatocytes ◽  
Fluid Phase Endocytosis ◽  
And Diffusion ◽  
Isolated Rat

Measurements of sugar pinocytosis (fluid-phase endocytosis of radiolabelled sucrose, lactose and raffinose) in freshly isolated rat hepatocytes are disturbed by sugar diffusing into the cells through plasma-membrane blebs. Non-pinocytic entry may be even more pronounced at 0 degrees C, and is a major contributor to ‘background’ radioactivity. By electrodisruption of the plasma membrane, a distinction can be made between pinocytotically sequestered sugar and free sugar that has entered the cytosol by diffusion. Pinocytosis proceeds at a rate of 2%/h (relative to the intracellular fluid volume), whereas the rate of sucrose entry by diffusion is more than twice as high. Three pinocytotic compartments are distinguishable in isolated hepatocytes: (1) a rapidly recycling compartment, which is completely destroyed by electrodisruption, and which may represent pinocytic channels continuous with the plasma membrane; (2) a non-recycling (or very slowly recycling) electrodisruption-resistant compartment, which allows accumulation of the lysosomally hydrolysable sugar lactose, and which therefore must represent non-lysosomal vacuoles (endosomes?); (3) a lysosomal compartment (non-recycling, electrodisruption-resistant), which accumulates raffinose and sucrose, but which hydrolyses lactose. The last two compartments can be partially resolved in metrizamide/sucrose density gradients by the use of different sugar probes.

Effect of glucagon on hepatic taurocholate uptake: relationship to membrane potential

1985 ◽  
Vol 249 (4) ◽  
pp. G427-G433
Author(s):  
J. W. Edmondson ◽  
B. A. Miller ◽  
L. Lumeng
Keyword(s):  
Plasma Membrane ◽  
Bile Acid ◽  
Membrane Potential ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hill Coefficient ◽  
Isolated Rat Hepatocytes ◽  
Plasma Membrane Potential ◽  
Sodium Dependent

Since glucagon can hyperpolarize hepatic plasma membrane and stimulate biliary bile acid secretion in vitro, we studied the effect of glucagon on taurocholate uptake and its relationship to plasma membrane potential in isolated rat hepatocytes. [14C]taurocholate uptake was linear through 1 min and contained a saturable sodium-dependent and a nonsaturable sodium-independent component. Km of taurocholate uptake by the sodium-dependent system was 18.4 microM. Hill coefficient for Na+ was 2.59 and for taurocholate was 1.1, suggesting that the stoichiometry is 2 Na+:1 bile acid. Stimulation of taurocholate uptake by glucagon was limited to the sodium-dependent component, detected within 5 min of hormone exposure, and was maximum at 30 min. Glucagon, from 10(-8) to 10(-5) M, stimulated taurocholate uptake and hyperpolarized concurrently the plasma membrane potential. Because valinomycin produced a dose-related depolarization of plasma membrane potential, this agent was used to counteract the effects of glucagon. With 10(-6) M glucagon, valinomycin (10(-10) M) depolarized membrane potential from -35.50 to -28.00 mV and inhibited taurocholate uptake from 60% above the control rate to 5% below. These data strongly suggest that taurocholate uptake by isolated hepatocytes is an electrogenic process, and its stimulation by glucagon may be mediated by changes in plasma membrane potential.

scholarly journals Methyl group transfer from exogenous S-adenosylmethionine on to plasma-membrane phospholipids without cellular uptake in isolated hepatocytes

1982 ◽  
Vol 206 (3) ◽  
pp. 481-487 ◽  
Author(s):  
L Van Phi ◽  
H D Söling
Keyword(s):  
Plasma Membrane ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Methyl Groups ◽  
Membrane Phospholipids ◽  
Small Pool ◽  
Cellular Compartments ◽  
Phospholipid Species ◽  
Water Space ◽  
Methyl Group Transfer

At external concentration of 50 microM, L-methionine was rapidly taken up by hepatocytes, whereas almost no S-adenosylmethionine (SAM) was removed from the incubation medium. SAM did not enter the intracellular water space but equilibrated with a very small pool, which was most likely to be situated on the external side of the plasma membrane. Methyl groups from external L-methionine, but not from external SAM, were incorporated into total and nuclear RNA. A significant incorporation of methyl groups into phospholipids occurred not only with methionine but also with SAM. After subfractionation of hepatocytes it became evident that methyl groups from SAM were mainly incorporated into plasma-membrane phospholipids, and that phospholipid methylation in other cellular compartments resulted from contamination with plasma membrane. The pattern of methylation of the various phospholipid species with SAM as precursor was different from that obtained with L-methionine. In contrast with external L-methionine, external SAM did not enter the intracellular SAM pool. According to these results a transport system for SAM does not exist in rat hepatocytes, although methyl groups from external SAM can be incorporated into plasma-membrane phospholipids from the outside.

scholarly journals Effects of bile salts on the plasma membranes of isolated rat hepatocytes

1980 ◽  
Vol 188 (2) ◽  
pp. 321-327 ◽  
Author(s):  
D Billington ◽  
C E Evans ◽  
P P Godfrey ◽  
R Coleman
Keyword(s):  
Alkaline Phosphatase ◽  
Bile Salt ◽  
Bile Salts ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Soluble Form ◽  
Isolated Rat Hepatocytes ◽  
Low Concentrations

The conjugated trihydroxy bile salts glycocholate and taurocholate removed approx. 20–30% of the plasma-membrane enzymes 5′-nucleotidase, alkaline phosphatase and alkaline phosphodiesterase I from isolated hepatocytes before the onset of lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase. The conjugated dihydroxy bile salt glycodeoxycholate similarly removed 10–20% of the 5′-nucleotidase and alkaline phosphatase activities, but not alkaline phosphodiesterase activity; this bile salt caused lysis of hepatocytes at approx. 10-fold lower concentrations (1.5–2.0mM) than either glycocholate or taurocholate (12–16mM). At low concentrations (7 mM), glycocholate released these enzymes in a predominantly particulate form, whereas at higher concentrations (15 mM) glycocholate further released these components in a predominantly ‘soluble’ form. Inclusion of 1% (w/v) bovine serum albumin in the incubations had a small protective effect on the release of enzymes from hepatocytes by glycodeoxycholate, but not by glycocholate. These observations are discussed in relation to the possible role of bile salts in the origin of some biliary proteins.

scholarly journals Cleavage of membrane secretory component to soluble secretory component occurs on the cell surface of rat hepatocyte monolayers.

1987 ◽  
Vol 104 (6) ◽  
pp. 1725-1733 ◽  
Author(s):  
L S Musil ◽  
J U Baenziger
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Rat Hepatocytes ◽  
Integral Membrane Protein ◽  
Secretory Component ◽  
Soluble Form ◽  
Membrane Domain ◽  
Transcellular Transport ◽  
Cultured Rat Hepatocytes ◽  
Plasma Membrane Domain

Rat liver secretory component is synthesized as an integral membrane protein (mSC) and cleaved to an 80-kD soluble form (fSC) sometime during transcellular transport from the sinusoidal to the bile canalicular plasma membrane domain of hepatocytes. We have used 24-h monolayer cultures of rat hepatocytes to characterize the conversion of mSC to fSC. Cleavage of mSC in cultured hepatocytes is inhibited by the thiol protease inhibitors leupeptin, antipain, and E-64, but not by other inhibitors, including disopropylfluorophosphate, pepstatin, N-ethylmalemide, p-chloromercuribenzoic acid, and chloroquine. Leupeptin-mediated inhibition of cleavage is concentration dependent and reversible. In the presence or absence of leupeptin, only 10-20% of mSC is accessible at the cell surface. To characterize the behavior of surface as opposed to intracellular mSC, cell surface mSC was labeled with 125I by lactoperoxidase-catalyzed iodination at 4 degrees C. Cell surface 125I-mSC was converted to extracellular fSC at 4 degrees C in the absence of detectable internalization. Cleavage was inhibited by leupeptin and by anti-secretory component antiserum. Cleavage also occurred at 4 degrees C after cell disruption. In contrast, 125I-mSC that had been internalized from the cell surface was not converted to fSC at 4 degrees C in either intact or disrupted cells. Hepatocytes metabolically labeled with [35S]cys also released small quantities of fSC into the medium at 4 degrees C. The properties of fSC production indicate that cleavage occurs on the surface of cultured rat hepatocytes and not intracellularly. Other features of the cleavage reaction suggest that the mSC-cleaving protease is segregated from the majority of cell surface mSC, possibly within a specialized plasma membrane domain.

Inhibition of gluconeogenesis by extracellular ATP in isolated rat hepatocytes

1991 ◽  
Vol 261 (6) ◽  
pp. R1522-R1526 ◽  
Author(s):  
M. Asensi ◽  
A. Lopez-Rodas ◽  
J. Sastre ◽  
J. Vina ◽  
J. M. Estrela
Keyword(s):  
Plasma Membrane ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Extracellular Atp ◽  
Structural Analogue ◽  
Isolated Rat Hepatocytes ◽  
Lactate And Pyruvate ◽  
Alpha Beta ◽  
High Concentrations ◽  
Isolated Rat

The aim of this study was to determine the effect of externally added ATP on gluconeogenesis by isolated hepatocytes from starved rats. High concentrations of extracellular ATP inhibited gluconeogenesis from lactate and pyruvate but not from glycerol or fructose. This inhibition was associated with an increase in intracellular adenosine contents. ADP, AMP, or adenosine but not guanosine 5'triphosphate, inosine 5' triphosphate, or adenine also inhibited gluconeogenesis. alpha, beta-Methylene-ATP, a nonmetabolizable structural analogue of ATP, did not affect the rate of gluconeogenesis. Intracellular ATP levels were increased by externally added ATP or adenosine, but ATP-to-ADP ratios in the cytosolic and mitochondrial compartments were diminished. Malate and phosphoenolpyruvate contents were decreased by extracellular ATP or adenosine. Our results show that inhibition of gluconeogenesis by high levels of extracellular ATP may be mediated by adenosine derived from ATP catabolism at the plasma membrane.

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