scholarly journals Evidence that lanthanum ions stimulate calcium inflow to isolated hepatocytes

1981 ◽  
Vol 200 (1) ◽  
pp. 109-114 ◽  
Author(s):  
J C Parker ◽  
G J Barritt
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Initial Rate ◽  
Compartmental Analysis ◽  
Isolated Hepatocytes ◽  
Lanthanum Ions ◽  
Isolated Liver Cells ◽  
Isolated Liver ◽  
Predominant Effect ◽  
Stimulation Of

LaCl3 stimulated the initial rate of 45Ca2+ exchange measured under steady-state conditions in isolated liver cells. Cu2+ greater than La3+ = Fe3+ greater than Fe2+ = Zn2+ greater Ni2+ greater than Mn2+ also stimulated 45Ca2+ exchange. Compartmental analysis of 45Ca2+-exchange curves obtained in the presence or absence of La3+, and in the presence or absence of adrenaline, showed that the predominant effect of La3+ is to stimulate the inflow of Ca2+ to the cell from the medium. No evidence for an inhibition of Ca2+ outflow from the cell was obtained. In the presence of La3+, adrenaline caused no further stimulation of Ca2+ inflow to the cell. In the absence of adrenaline, La3+ increased the uptake of Ca2+ (measured by atomic-absorption spectroscopy) by isolated hepatocytes incubated at 1 degree C. The proposal that La3+ stimulates Ca2+ inflow to the liver cell by inducing a conformational change in the Ca2+-inflow transporter of the plasma membrane is briefly discussed.

scholarly journals The role of insulin in the modulation of glucagon-dependent control of phenylalanine hydroxylation in isolated liver cells

1987 ◽  
Vol 242 (3) ◽  
pp. 655-660 ◽  
Author(s):  
M J Fisher ◽  
A J Dickson ◽  
C I Pogson
Keyword(s):  
Cyclic Amp ◽  
Insulin Action ◽  
Phenylalanine Hydroxylase ◽  
Liver Cells ◽  
Phosphorylation State ◽  
Isolated Liver Cells ◽  
The Impact ◽  
Isolated Liver ◽  
Stimulation Of

The stimulation of phenylalanine hydroxylation in isolated liver cells by sub-maximally effective concentrations of glucagon (less than 0.1 microM) is antagonized by insulin (0.1 nM-0.1 microM). This phenomenon is a consequence of a decrease in the glucagon-stimulated phosphorylation of phenylalanine hydroxylase from liver cells incubated in the presence of insulin. The impact of insulin on the phosphorylation state and activity of the hydroxylase is mimicked by incubation of liver cells in the presence of orthovanadate (10 microM). A series of cyclic AMP and cyclic GMP analogues enhanced phenylalanine hydroxylation: in each case insulin diminished the stimulation of flux. These results are discussed in the light of the characteristics of insulin action on other metabolic processes.

scholarly journals Polyribosomes in isolated liver cells. Preparative procedures, effects of incubation and correlation with protein synthesis

1980 ◽  
Vol 186 (1) ◽  
pp. 35-45 ◽  
Author(s):  
A J Dickson ◽  
C I Pogson
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Liver Protein ◽  
Isolated Cells ◽  
Isolated Liver Cells ◽  
Rat Liver Cells ◽  
Isolated Liver

Methods have been derived which permit the isolation of undergraded polyribosomes from isolated rat liver cells. Under the conditions used the polyribosome profile of hepatocytes immediately after isolation was essentially identical with that from intact liver. However, during incubation of cells in complex physiological media there was a progressive dissociation of polyribosomes. The addition of a variety of factors that produce reaggregation of polyribosomes in rat liver in vivo did not prevent dissociation during cell incubations. Although large polyribosomes were lost most rapidly, the albumin-synthesizing capacity of isolated cells was not selectively lost when compared with total protein synthesis. The significance of these results for the use of isolated hepatocytes in the study of liver protein synthesis is discussed.

scholarly journals Effect of treatment in vivo of rats with bacterial endotoxin on fructose 2,6-bisphosphate metabolism and L-pyruvate kinase activity and flux in isolated liver cells

1992 ◽  
Vol 284 (3) ◽  
pp. 761-766 ◽  
Author(s):  
E D Ceppi ◽  
R G Knowles ◽  
K M Carpenter ◽  
M A Titheradge
Keyword(s):  
Pyruvate Kinase ◽  
Intact Cell ◽  
Total Activity ◽  
Liver Cells ◽  
Bacterial Endotoxin ◽  
Phosphorylation State ◽  
Isolated Liver Cells ◽  
Effect Of Treatment ◽  
Isolated Liver ◽  
Stimulation Of

The effect of treatment of rats with bacterial endotoxin on fructose 2,6-bisphosphate (Fru-2,6-P2) metabolism was investigated in isolated liver cells prepared from 18 h-starved animals. The results obtained support the hypothesis that a stimulation of 6-phosphofructo-1-kinase (PFK-1) activity and an inhibition of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) may be one mechanism underlying the inhibition of gluconeogenesis from lactate and pyruvate by endotoxin. We suggest that the stimulation of PFK-1 and inhibition of Fru-1,6-P2ase activity is the result of a 2-3-fold increase in Fru-2,6-P2. The latter is not due to changes in the total activity or phosphorylation state of the bifunctional 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase, but appears to be the result of a decrease in the cytosolic concentration of phosphoenolpyruvate (PEP), an inhibitor of PFK-2 activity. The effect of endotoxin is resistant to the presence of glucagon, which has comparable effects in cells prepared from both control and endotoxin-treated animals. The mechanism by which endotoxin treatment of the rat decreases PEP and gluconeogenesis remains to be established. However, it does not involve alterations in either the total activity or the phosphorylation state of pyruvate kinase, nor does it involve increased flux through this enzyme in the intact cell, which is in fact decreased in this model of septic shock. It is suggested that the decreased flux may result from a lower rate of formation of PEP, suggesting that the prime lesion in sepsis is an inhibition of one or more of the steps leading to PEP formation.

scholarly journals Constitutive endocytosis and recycling of NKCC2 in rat thick ascending limbs

2010 ◽  
Vol 299 (5) ◽  
pp. F1193-F1202 ◽  
Author(s):  
Gustavo R. Ares ◽  
Pablo A. Ortiz
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Acute Treatment ◽  
Apical Membrane ◽  
Surface Expression ◽  
Membrane Cholesterol ◽  
Thick Ascending Limb ◽  
Apical Surface ◽  
Total Pool ◽  
Stimulation Of

The Na-K-2Cl cotransporter (NKCC2) mediates NaCl absorption by the thick ascending limb of Henle's loop (THAL). Exocytosis and endocytosis regulates surface expression of most transporters. However, little is known about the mechanism of NKCC2 trafficking in the absence of stimulating hormones and whether this mechanism contributes to regulation of steady-state surface expression of apical NKCC2 in the THAL. We tested whether NKCC2 undergoes constitutive endocytosis that regulates steady-state surface NKCC2 and NaCl reabsorption in THALs. We measured steady-state surface NKCC2 levels and the rate of NKCC2 endocytosis by surface biotinylation and Western blot and confocal microscopy of isolated perfused rat THALs. We observed constitutive NKCC2 endocytosis over 30 min that averaged 21.5 ± 2.7% of the surface pool. We then tested whether methyl-β-cyclodextrin (MβCD), a compound that inhibits endocytosis by chelating membrane cholesterol, blocked NKCC2 endocytic retrieval. We found that 30-min treatment with MβCD (5 mM) blocked NKCC2 endocytosis by 81% ( P < 0.01). Blockade of endocytosis by MβCD induced accumulation of NKCC2 at the apical membrane as demonstrated by a 60 ± 16% ( P < 0.05) increase in steady-state surface expression and enhanced apical surface NKCC2 immunostaining in isolated, perfused THALs. Acute treatment with MβCD did not change the total pool of NKCC2. MβCD did not affect NKCC2 trafficking when it was complexed with cholesterol before treatment. Inhibition endocytosis with MβCD enhanced NKCC2-dependent NaCl entry by 57 ± 16% ( P < 0.05). Finally, we observed that a fraction of retrieved NKCC2 recycles back to the plasma membrane (36 ± 7%) over 30 min. We concluded that constitutive NKCC2 trafficking maintains steady-state surface NKCC2 and regulates NaCl reabsorption in THALs. These are the first data showing an increase in apical membrane NKCC2 in THALs by altering the rates of constitutive NKCC2 trafficking, rather than by stimulation of hormone-dependent signaling.

scholarly journals Characterization of the binding of diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) to rat liver cell membranes

1996 ◽  
Vol 314 (2) ◽  
pp. 687-693 ◽  
Author(s):  
Mandy EDGECOMBE ◽  
Alexander G. McLENNAN ◽  
Michael J. FISHER
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Liver Cell ◽  
Plasma Membranes ◽  
Rank Order ◽  
Liver Cells ◽  
Diadenosine Polyphosphates ◽  
Cell Plasma ◽  
Isolated Liver Cells ◽  
Isolated Liver

Diadenosine polyphosphates present in the extracellular environment can, through interaction with appropriate purinoceptors, influence a range of cellular activities. Here we have investigated the nature of the ligand:receptor interactions involved in diadenosine 5′,5″-P1,P4-tetraphosphate (Ap4A)-mediated stimulation of glycogen breakdown in isolated rat liver cells. [2-3H]Ap4A showed specific binding to both intact isolated liver cells and plasma membrane fractions prepared from isolated liver cells. HPLC analysis confirmed that binding was mediated by intact Ap4A and not by potential breakdown products (e.g. ATP, adenosine etc). Binding of [2-3H]Ap4A, to isolated liver cell plasma membrane preparations, was successfully displaced by a range of both naturally occurring and synthetic diadenosine polyphosphates with the rank order potency Ap4A ⩾Ap5A > Ap6A > Ap3A > Ap2A. [2-3H]Ap4A binding was not displaced by P1 effectors but was successfully displaced by a range of P2 effectors with the rank order potency 2-methylthio-ATP > ATP > ADP ⩾adenosine 5′-[αβ-methylene]triphosphate > adenosine 5′-[βγ-methylene]triphosphate. These findings are consistent with the interaction of Ap4A with a P2y-like subclass of purinoceptor and are discussed in relation to (1) the known purinoceptor populations in liver cell plasma membranes and (2) observations concerning the binding of diadenosine polyphosphates to purinoceptors in other tissues.

Kinetics of high-affinity Ca2+ sequestration in permeabilized rat pancreatic acini

1988 ◽  
Vol 254 (5) ◽  
pp. C621-C627 ◽  
Author(s):  
T. W. Hurley
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Initial Rate ◽  
Ruthenium Red ◽  
Ionophore A23187 ◽  
Pancreatic Acini ◽  
High Affinity ◽  
Energy Dependent ◽  
Kinetics Of

Energy-dependent subcellular Ca2+ sequestration was studied in the presence of ruthenium red using rat pancreatic acini, which had been permeabilized by exposure to medium nominally free of Ca2+. The initial rate of Ca2+ uptake (approximately 2,800 pmol.min-1.mg acinar protein-1) quickly slowed, and a mean steady-state Ca2+ content of approximately 3,000 pmol/mg was reached after 5-10 min of incubation at 37 degrees C. Ca2+ uptake was stimulated by submicromolar Ca2+ concentrations (K0.5 = 156 nM); required Mg2+-ATP (K0.5 = 0.78 mM) was greatest at a pH of 7.0 and was abolished by the Ca2+ ionophore A23187. Other nucleotide phosphates as well as p-nitrophenylphosphate were relatively poor substrates, supporting Ca2+ uptake at initial rates that were 6-14% of those measured in the presence of ATP. These results show that pancreatic acini permeabilized without detergents possess a nonmitochondrial Ca2+ transporting system not located in the plasma membrane but with the properties expected of a major regulator of acinar cytosolic Ca2+ concentration.

Acid extrusion in S3 segment of rabbit proximal tubule. I. Effect of bilateral CO2/HCO3-

1995 ◽  
Vol 268 (2) ◽  
pp. F179-F192 ◽  
Author(s):  
L. K. Chen ◽  
W. F. Boron
Keyword(s):  
Steady State ◽  
Proximal Tubule ◽  
Initial Rate ◽  
Iodoacetic Acid ◽  
Buffering Power ◽  
Acid Load ◽  
S3 Segment ◽  
Series Of Experiments ◽  
Acid Extrusion ◽  
Stimulation Of

Monitoring the absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein, we studied intracellular pH (pHi) regulation in the isolated perfused S3 segment of rabbit proximal tubule. To explain a previous observation, that steady-state pHi is higher in the presence than in the absence of CO2/HCO3- (N. L. Nakhoul, L. K. Chen, and W. F. Boron. J. Gen. Physiol. 102: 1171-1205, 1993), we examined the effect of bilateral (i.e., luminal and basolateral) CO2/HCO3- on the acid extrusion processes responsible for recovery of pHi from acid loads. To compute fluxes from rates of pHi change, we determined the pHi dependence of intrinsic intracellular buffering power, which was approximately 50 mM/pH at pHi 6.5 and fell linearly to approximately 20 mM at pHi 7.4. In one series of experiments, we monitored the rate of pHi recovery from an acid load imposed by an NH4+/NH3 prepulse. Over a broad range of pHi values, total net acid extrusion was approximately four times higher in bilateral presence of CO2/HCO3- than in its absence. In a second group of experiments, which were designed to determine the effect of CO2/HCO3- on luminal Na+/H+ exchange, we monitored the rate of pHi recovery elicited by adding Na+ back to only the lumen, after first removing Na+ bilaterally. Initial rate of luminal Na(+)-dependent net acid extrusion in presence of CO2/HCO3- was approximately 229 microM/s (pHi 6.92), approximately 1.8 times higher than the flux of approximately 127 microM/s (P < 0.005) obtained in absence of CO2/HCO3- (pHi 6.66). CO2/HCO3- alkali-shifted the flux vs. pHi relationship by 0.3-0.4 pH units. In a final series of experiments, we examined the effect of CO2/HCO3- on the Na(+)-independent alkalinization that follows the rapid, initial acidification elicited by bilateral Na+ removal. In the presence of CO2/HCO3-, lag time for initiation of the Na(+)-independent alkalinization was only approximately 36 vs. approximately 211 s (P < 0.002) in absence of CO2/HCO3-. Also, Na(+)-independent net acid extrusion rate was approximately two to three times higher in presence than in absence of CO2/HCO3- at comparable pHi. This Na(+)-independent acid extrusion was insensitive to N-ethylmaleimide (2 mM), but was inhibited approximately 94% by efforts to deplete intracellular ATP (i.e., removal of glucose and amino acids, plus addition of 2 mM cyanide and 10 mM iodoacetic acid). Stimulation of luminal Na+/H+ exchange and Na(+)-independent acid extrusion appears to be the major, if not the entire, explanation for the higher steady-state pHi caused by bilateral addition of CO2/HCO3-.

scholarly journals Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition

1986 ◽  
Vol 238 (3) ◽  
pp. 793-800 ◽  
Author(s):  
B P Hughes ◽  
S E Milton ◽  
G J Barritt
Keyword(s):  
Plasma Membrane ◽  
Glycogen Phosphorylase ◽  
Subcellular Fractionation ◽  
Isolated Hepatocytes ◽  
Ionophore A23187 ◽  
Phosphorylase Activity ◽  
45Ca Uptake ◽  
Maximal Stimulation ◽  
Glycogen Phosphorylase Activity ◽  
Stimulation Of

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [(Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.

Abstract 5385: Transport of Free Fatty Acids in Cardiac Myocytes is Mediated by a Plasma Membrane Pump as Revealed by Monitoring Cytoplasmic Unbound Free Fatty Acids

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Andrew N Carley ◽  
J P Kampf ◽  
Alan M Kleinfeld
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Steady State ◽  
Free Fatty Acids ◽  
Cardiac Myocytes ◽  
Time Course ◽  
Plasma Membranes ◽  
Initial Rate ◽  
Ffa Metabolism ◽  
Ffa Transport

The transport of FFA across the plasma membrane represents one of the earliest points at which FFA metabolism can be controlled by cardiac myocytes. Using novel methods to measure the intracellular unbound concentration of FFA ([FFA i ]), the first direct measurements of FFA transport across cardiac plasma membranes have been performed in freshly isolated cardiac myoctyes. Measurements of the unbound concentrations of FFA (FFA u ) in the aqueous phase were performed using the fluorescent ratio probe ADIFAB. Cardiac myocytes were microinjected with ADIFAB, and the transport of oleate and palmitate was determined by monitoring [FFA i ] using fluorescence ratio microscopy. FFA influx was initiated by rapidly increasing the extracellular concentration of FFA u ([FFA o ]) using FFA-BSA complexes, which clamped [FFA o ] at fixed values. The time course of influx was monitored from the change in [FFA i ], which rose exponentially to a steady state level (k influx ~ 0.01 s −1 ). Once steady state was achieved, efflux was initiated by changing the extracellular media back to zero [FFA o ]. Efflux was monitored by the decrease in [FFA i ] which, like influx, revealed exponential behavior (k efflux ~ 0.02 s −1 ). At steady state [FFA i ] was greater than [FFA o ] by a factor of ~3.5, indicating that during influx FFA are pumped up a concentration gradient. Both the initial rate of transport and the gradient ([FFA i ] > [FFA o ]) revealed saturation with increasing [FFA o ]. The initial rate of influx saturated at [FFA o ] > 200 nM, while the [FFA i ] > [FFA o ] gradient was relatively constant (~ 3.5) but began to decrease and approached 1 at [FFA o ] > 200 nM. The efflux rate constant decreased for [FFA o ] > zero, suggesting that efflux may be regulated by a mechanism that senses the level of circulating FFA u . Our results indicate that the mechanism of FFA transport across cardiac myocytes is regulated by the plasma membrane and allows for the efficient storage and release of FFA from cardiac myocytes. We suggest that this mechanism involves an as yet unknown membrane protein pump which enables the cells to accumulate surprisingly high concentrations of FFA. The ability to measure [FFA i ] and the demonstration of efflux are significant steps in understanding cardiac FFA metabolism. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).

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