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.

Bile acid synthesis in isolated rat hepatocytes

1978 ◽  
Vol 56 (8) ◽  
pp. 780-783 ◽  
Author(s):  
I. M. Yousef ◽  
J. Ho ◽  
K. N. Jeejeebhoy
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Rat Hepatocytes ◽  
Acid Synthesis ◽  
Isolated Hepatocytes ◽  
Bile Acid Synthesis ◽  
Isolated Rat Hepatocytes ◽  
Adult Rat ◽  
Vitro Model

Normal adult rat hepatocytes were incubated for 48 h and the concentration of total and individual bile acids in homogenized samples of the culture was measured at intervals during the incubation, using radiogas chromatography and isotope derivative assay. The net increase in bile acids over the value observed at the start of the culture was taken as synthesis. The results showed that bile acid synthesis was linear up to 24 h of incubation, at a rate of 20 nmol/g hepatocytes per hour, and that 85% of the newly synthesized bile acid was cholic acid. The bile acid synthesized was mainly conjugated with taurine. These results suggest that isolated hepatocytes cultured in the way described could be a useful in vitro model for the study of bile acid synthesis.

Nature of taurodehydrocholic acid uptake in rat hepatocytes

1988 ◽  
Vol 254 (2) ◽  
pp. G269-G274 ◽  
Author(s):  
W. G. Hardison ◽  
P. J. Lowe ◽  
E. Gosink
Keyword(s):  
Membrane Potential ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Acid Uptake ◽  
Isolated Rat Hepatocytes ◽  
Acid Analogue ◽  
Sodium Gradient ◽  
Sodium Dependent ◽  
Independent Process ◽  
Transport Site

We studied uptake into isolated rat hepatocytes of the bile acid analogue taurodehydrocholate (TDHC) over a concentration range of 2.5-4,000 microM. Uptake was mainly by a saturable sodium-dependent process with a Km of approximately 50 microM and a Vmax of 0.036 nmol.s-1.mg protein-1. A lesser sodium-independent process was evident but was linear in the range studied. Both processes were inhibited by incubation at 37 degrees C under nitrogen in the presence of 3 mM sodium cyanide or by incubation at 0 degrees C. A single transport site was suggested by the Eadie-Hofstee plot of TDHC uptake from 2.5 to 750 microM. TDHC was a weak competitive inhibitor of taurocholic acid (TCA) uptake (Ki = 236 microM) but was not itself taken up by the TCA transport site. TCA exhibited moderately potent mixed inhibition of TDHC uptake. Uptake of both compounds was strongly inhibited by bromosulfophthalein (BSP) and Rose Bengal, whereas 0.5 mM alanine uptake was not affected. BSP exhibited a complex pattern of inhibition of TDHC uptake: mixed partial inhibition. Degree of inhibition of both TDHC and TCA uptake did not increase as BSP concentrations were increased from 50 to 100 microM. BSP did not exert its inhibitory effects by alteration of membrane potential or sodium gradients; 50 microM BSP changed membrane potential less than 10% and sodium gradient not at all. The data indicate that despite close structural analogy between TDHC and TCA, the two compounds are taken up by different sodium-dependent mechanisms. Nonetheless, the similar qualitative and quantitative effects of BSP on their uptakes suggests the mechanisms are related.

scholarly journals Vasopressin-stimulated Ca2+ influx in rat hepatocytes is inhibited in high-K+ medium

1989 ◽  
Vol 260 (3) ◽  
pp. 821-827 ◽  
Author(s):  
A L Savage ◽  
M Biffen ◽  
B R Martin
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Rat Hepatocytes ◽  
Initial Increase ◽  
Phosphorylase Activity ◽  
Plasma Membrane Potential ◽  
Control Value ◽  
High K ◽  
Increased Activity ◽  
Ca2 Channels

We examined the effects of K+ substitution for Na+ on the response of hepatocytes to vasopressin, and on the hepatocyte plasma-membrane potential. (1) High K+ (114 mM) had no effect on the initial increase in phosphorylase a activity in response to vasopressin, but abolished the ability of the hormone to maintain increased activity beyond 10 min. With increasing concentrations a decrease in the vasopressin response was first observed at 30-50 mM-K+. (2) High K+ (114 mM) had no effect on basal 45Ca2+ influx, but abolished the ability of vasopressin to stimulate influx. This effect was also first observed at a concentration of 30-50 mM-K+. (3) Increasing K+ had little effect on the plasma-membrane potential until a concentration of 40 mM was reached. With further increases in concentration the plasma membrane was progressively depolarized. (4) Replacement of Na+ with N-methyl-D-glucamine+ depolarized the plasma membrane to a much smaller extent than did replacement with K+, and was also much less effective in inhibiting the vasopressin response. (5) The plasma-membrane potential was restored to near the control value by resuspending cells in normal-K+ medium after exposure to high-K+ medium. The effects of vasopressin on phosphorylase activity were also restored. (6) We conclude that the Ca2+ channels responsible for vasopressin-stimulated Ca2+ influx are closed by depolarization of the plasma membrane.

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.

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.

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.

scholarly journals Effects of glucagon and Ca2+ on the metabolism of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in isolated rat hepatocytes and plasma membranes

1987 ◽  
Vol 241 (3) ◽  
pp. 835-845 ◽  
Author(s):  
D E Whipps ◽  
A E Armston ◽  
H J Pryor ◽  
A P Halestrap
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Mitochondrial Fraction ◽  
Ruthenium Red ◽  
Isolated Rat Hepatocytes ◽  
Guanine Nucleotide Regulatory Protein ◽  
Phosphatidylinositol 4

Rat hepatocytes whose phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) had been labelled for 60 min with 32P were treated with glucagon for 10 min or phenylephrine for 2 min. Glucagon caused a 20% increase in PIP but no change in PIP2 whereas phenylephrine caused a similar increase in PIP but a 15% decrease in PIP2. Addition of both hormones together for 10 min produced a 40% increase in PIP. A crude liver mitochondrial fraction incubated with [32P]Pi and ADP incorporated label into PIP, PIP2 and phosphatidic acid. The PIP2 was shown to be in contaminating plasma membranes and PIP in both lysosomal and plasma-membrane contamination. A minor but definitely mitochondrial phospholipid, more polar than PIP2, was shown to be labelled with 32P both in vitro and in hepatocytes. The rate of 32P incorporation into PIP was faster in mitochondrial/plasma-membrane preparations from rats treated with glucagon or if 3 microM-Ca2+ and Ruthenium Red were present in the incubation buffer. Loss of 32P from membranes labelled in vitro was shown to be accompanied by formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate, and was faster in preparations from glucagon-treated rats or in the presence of 3 microM-Ca2+. It is concluded that glucagon stimulates both PIP2 phosphodiesterase and phosphatidylinositol kinase activities, as does the presence of 3 microM-Ca2+. The resulting formation of IP3 may be responsible for the observed release of intracellular Ca2+ stores. The roles of a guanine nucleotide regulatory protein and phosphorylation in mediating these effects are discussed.

scholarly journals Glucocorticoids Regulate Plasma Membrane Potential During Rat Thymocyte Apoptosis in Vivo and in Vitro

Endocrinology ◽  
2001 ◽  
Vol 142 (1) ◽  
pp. 421-429 ◽  
Author(s):  
Cynthia L. Mann ◽  
John A. Cidlowski
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Glucocorticoid Receptors ◽  
De Novo ◽  
Strong Association ◽  
Glucocorticoid Treatment ◽  
Plasma Membrane Potential ◽  
Induced Apoptosis

Abstract Glucocorticoids induce a series of profound biochemical changes in thymocytes that initiate apoptosis; however, the pathways beyond receptor transactivation that lead to this form of cell death are not fully understood. In this study, we report a novel site of action for glucocorticoids at the site of the plasma membrane. Specifically, we find that glucocorticoids induce the loss of plasma membrane potential both in vivo and in vitro. The glucocorticoid-induced loss of plasma membrane potential in cultured primary isolated rat thymocytes was both dose and time dependent. Other steroid hormones, including progesterone, estrogen, and testosterone, fail to alter the depolarization state of the thymocyte plasma membrane. Interestingly, other nonsteroid stimuli that also activate apoptosis in thymocytes also lead to cellular depolarization. In contrast, HeLa cells, which contain functional glucocorticoid receptors but do not die in response to hormone, do not alter their plasma membrane potential in response to glucocorticoids, indicating a strong association between depolarization and apoptosis. Furthermore, the ability of glucocorticoids to depolarize the plasma membrane of thymocytes required the interaction of glucocorticoids with their cognate receptor, because RU486 failed to depolarize thymocytes and antagonized the effect of glucocorticoids. Finally, experiments using inhibitors of transcription and translation indicated that the loss of plasma membrane potential in thymocytes following glucocorticoid treatment required de novo gene expression. The results of these studies establish that the loss of plasma membrane potential is an early important feature of glucocorticoid-induced apoptosis of thymocytes.

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