Modulation of hormone-induced calcium oscillations by intracellular pH in rat hepatocytes

1997 ◽  
Vol 272 (5) ◽  
pp. G954-G961 ◽  
Author(s):  
J. Y. Chatton ◽  
H. Liu ◽  
J. W. Stucki
Keyword(s):  
Intracellular Ph ◽  
Rat Hepatocytes ◽  
Calcium Oscillations ◽  
Adrenergic Stimulation ◽  
Isolated Rat Hepatocytes ◽  
Adrenergic Response ◽  
Agonist Concentration ◽  
Continuous Presence ◽  
Cellular Acidification

Single isolated rat hepatocytes were used to investigate the influence of intracellular pH (pHi) on hormone-induced cytosolic Ca2+ oscillations, using videofluorescence microscopy. Although pHi did not vary after alpha-adrenergic stimulation, manipulations of pHi induced pronounced alterations in the frequency of oscillations. Increasing the resting pHi with ammonium chloride (5-20 mM), trimethylammonium (2-10 mM), or triethylammonium (1.2-8 mM) reduced the frequency of oscillations. A change in pHi of > 0.25 was sufficient to reversibly inhibit oscillations. This effect could be overcome by increasing the agonist concentration or by adding 8-bromoadenosine 3',5'-cyclic monophosphate, an agent known to potentiate the alpha-adrenergic response. Cellular acidification, obtained by the ammonium prepulse method as well as by application of acetate or the ionophore nigericin, in the continuous presence of agonist was accompanied by a modest frequency increase of the oscillations, leading in some cases to an overstimulated state. This study indicates that pHi, within a range of values expected to occur in vivo (0.1-0.2 pH units), exerts a chronotropic effect on phenylephrine-induced Ca2+ oscillations. In contrast, oscillations induced by ADP or vasopressin were pHi invariant.

scholarly journals Comparative metabolism of the antiviral dimer 3'-azido-3'-deoxythymidine-P-2',3'-dideoxyinosine and the monomers zidovudine and didanosine by rat, monkey, and human hepatocytes.

1997 ◽  
Vol 41 (11) ◽  
pp. 2502-2510 ◽  
Author(s):  
X R Pan-Zhou ◽  
E Cretton-Scott ◽  
X J Zhou ◽  
M Y Xie ◽  
R Rahmani ◽  
...  
Keyword(s):  
High Performance ◽  
Rat Hepatocytes ◽  
Human Hepatocytes ◽  
Metabolic Fate ◽  
Extracellular Medium ◽  
Isolated Rat Hepatocytes ◽  
Cell Extracts ◽  
Comparative Metabolism ◽  
Isolated Rat

AZT-P-ddI is an antiviral heterodimer composed of one molecule of 3'-azido-3'-deoxythymidine (AZT) and one molecule of 2',3'-dideoxyinosine (ddI) linked through their 5' positions by a phosphate bond. The metabolic fate of the dimer was studied with isolated rat, monkey, and human hepatocytes and was compared with that of its component monomers AZT and ddI. Upon incubation of double-labeled [14C]AZT-P-[3H]ddI in freshly isolated rat hepatocytes in suspension at a final concentration of 10 microM, the dimer was taken up intact by cells and then rapidly cleaved to AZT, AZT monophosphate, ddI, and ddI monophosphate. AZT and ddI so formed were then subject to their respective catabolisms. High-performance liquid chromatography analyses of the extracellular medium and cell extracts revealed the presence of unchanged dimer, AZT, 3'-azido-3'-deoxy-5'-beta-D-glucopyranosylthymidine (GAZT), 3'-amino-3'-deoxythymidine (AMT), ddI, and a previously unrecognized derivative of the dideoxyribose moiety of ddI, designated ddI-M. Trace extracellular but substantial intracellular levels of the glucuronide derivative of AMT (3'-amino-3'-deoxy-5'-beta-D-glucopyranosylthymidine [GAMT]) were also detected. Moreover, the extent of the formation of AMT, GAZT, and ddI-M from the dimer was markedly lower than that with AZT and ddI alone by the hepatocytes. With hepatocytes in primary culture obtained from rat, monkey, and human, large interspecies variations in the metabolism of AZT-P-ddI were observed. While GAZT and ddI-M, metabolites of AZT and ddI, respectively, as well as AZT 5'-monophosphate (MP) and ddI-MP were detected in the extracellular media of all species, AMT and GAMT were produced only by rat and monkey hepatocytes. No such metabolites were formed by human hepatocytes. The metabolic fate of the dimer by human hepatocytes was consistent with in vivo data recently obtained from human immunodeficiency virus-infected patients.

Hepatic and extrahepatic factors critical for liver injury during lipopolysaccharide exposure

2001 ◽  
Vol 281 (6) ◽  
pp. G1423-G1431 ◽  
Author(s):  
Frederic Moulin ◽  
Bryan L. Copple ◽  
Patricia E. Ganey ◽  
Robert A. Roth
Keyword(s):  
Liver Injury ◽  
Polymorphonuclear Leukocytes ◽  
Rat Hepatocytes ◽  
Hepatocellular Injury ◽  
In Vitro Perfusion ◽  
Isolated Rat Hepatocytes ◽  
Hepatocellular Damage ◽  
Alt Activity

Bacterial endotoxin [lipopolysaccharide (LPS)] causes liver injury in vivo that is dependent on platelets, neutrophils [polymorphonuclear leukocytes (PMNs)], and several inflammatory mediators, including thrombin. We tested the hypothesis that thrombin contributes to LPS-induced hepatocellular injury through direct interactions with platelets and/or PMNs in vitro. Perfusion of isolated livers from LPS-treated rats with buffer containing thrombin resulted in a significant increase in alanine aminotransferase (ALT) activity in the perfusion medium, indicating hepatocellular damage. This effect was completely abolished by prior depletion of PMNs from the LPS-treated donor rats but not by depletion of platelets, suggesting interaction between thrombin and PMNs in the pathogenesis. Thrombin did not, however, enhance degranulation of rat PMNs in vitro, and it was not directly toxic to isolated rat hepatocytes in the presence of PMNs even after LPS exposure, suggesting that hepatocellular killing by the PMN-thrombin combination requires the intervention of an additional factor(s) within the liver. In livers from naive donors perfused with buffer containing PMNs and LPS, no injury occurred in the absence of thrombin. Addition of thrombin (10 nM) to the medium caused pronounced ALT release. These results indicate that thrombin and PMNs are sufficient extrahepatic requirements for LPS-induced hepatocellular damage in intact liver.

Biosynthesis of Methylguanidine in Isolated Rat Hepatocytes and in vivo

Nephron ◽  
1985 ◽  
Vol 40 (4) ◽  
pp. 470-475 ◽  
Author(s):  
Sohji Nagase ◽  
Kazumasa Aoyagi ◽  
Mitsuharu Narita ◽  
Shizuo Tojo
Keyword(s):  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

scholarly journals Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin

1981 ◽  
Vol 194 (1) ◽  
pp. 155-165 ◽  
Author(s):  
C J Kirk ◽  
R H Michell ◽  
D A Hems
Keyword(s):  
Glycogen Phosphorylase ◽  
Equilibrium Distribution ◽  
Rat Hepatocytes ◽  
Subcellular Fractionation ◽  
Isolated Rat Hepatocytes ◽  
Phosphatidylinositol Metabolism ◽  
Maximal Activation ◽  
Vasopressin Receptors ◽  
Stimulation Of

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.

Abstract 1333: Modulation Of Cardiac Beta-adrenergic Responsiveness By The Neuronal Nitric Oxide Synthase/Sarcolemmal Calcium Pump Complex

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Tamer M Mohamed ◽  
Delvac Oceandy ◽  
Nasser Alatwi ◽  
Florence Baudoin ◽  
Elizabeth J Cartwright ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Neonatal Rat ◽  
No Production ◽  
Adrenergic Stimulation ◽  
Rat Cardiomyocytes ◽  
Adrenergic Response ◽  
Oxide Synthase

The pivotal role of neuronal nitric oxide synthase (nNOS) in regulating cardiac function has only recently been unveiled. Notably, others have shown that responsiveness to β-adrenergic stimulation is dependent on nNOS activity. In a cellular model, we showed that the Ca 2+ /calmodulin-dependent nNOS activity is reduced by overexpression of isoform 4b of the plasma membrane Ca 2+ /Calmodulin-dependent Ca 2+ -pump (PMCA4b), which binds to nNOS. We demonstrated that PMCA4b overexpression in the heart reduced β-adrenergic responsiveness in vivo via an nNOS dependent mechanism (Oceandy et al, Circulation 2007). Here we investigated the cellular mechanisms of the regulation of the β-adrenergic response by PMCA4b. We used an adenoviral system to overexpress PMCA4b (PMCA4b cells) or LacZ (control, C) in neonatal rat cardiomyocytes. PMCA4b cells showed an 18±5% and 24±5% reduction in nitric oxide (DAF-FM fluorescence) and cGMP levels, respectively (n=6, p<0.05 each) compared to C demonstrating the regulation of NO production by the PMCA4b in this system. Since nNOS has been shown to regulate phospholamban (PLB) phosphorylation, we examined phosphorylation of PLB at Ser16. PMCA4b cells showed a significant increase in Ser16-PLB at baseline (66±17%, p<0.05) compared to C. As a result of increased baseline Ser16-PLB in PMCA4b cells, β-adrenergic stimulation of PMCA4b cells using 2μM isoproter-enol (IP) showed reduced relative induction in Ser16-PLB (23±10% vs. 78±19% in C; n=5, p<0.05). Further analysis in adult cardiomyocytes isolated from our PMCA4b transgenic mice (PMCA4b TG) demonstrated that PMCA4b TG showed 3-fold higher Ser16-PLB phosphorylation at baseline compared to wild type (WT) myocytes and the relative response following β-adrenergic stimulation was significantly reduced (1.2±0.2 fold induction after IP treatment in PMCA4b TG, vs. 3.1±0.7 in WT, n=5, p<0.05). Thus, PMCA4b regulates NO production from nNOS, which in turn modulates cGMP levels and PLB phosphorylation. These findings provide mechanistic insight into the regulation of the β-adrenergic response in the heart by PMCA4b and place this Ca 2+ -pump upstream of the recently described pathway linking nNOS and Ser16-PLB phosphorylation and downstream of the β-adrenergic receptor(s).

scholarly journals Purine catabolism in isolated rat hepatocytes. Influence of coformycin

1980 ◽  
Vol 188 (3) ◽  
pp. 913-920 ◽  
Author(s):  
Georges Van Den Berghe ◽  
Françoise Bontemps ◽  
Henri-Géry Hers
Keyword(s):  
High Speed ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Rat Hepatocytes ◽  
Amp Deaminase ◽  
Purine Nucleotides ◽  
Isolated Rat Hepatocytes ◽  
Adenine Nucleotide Pool ◽  
Isolated Rat

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [14C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [14C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.

scholarly journals Differential effects of tryptophan on glucose synthesis in rats and guinea pigs

1978 ◽  
Vol 176 (3) ◽  
pp. 817-825 ◽  
Author(s):  
S A Smith ◽  
K R F Elliott ◽  
C I Pogson
Keyword(s):  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glucose Production ◽  
Rat Hepatocytes ◽  
Liver Cells ◽  
Isolated Rat Hepatocytes ◽  
Rat Liver Cells ◽  
Glucose Output ◽  
Isolated Rat

1. Tryptophan inhibition of gluconeogenesis in isolated rat liver cells is characterized by a 20 min lag period before linear rates of glucose output are attained. 2. Half-maximal inhibition of gluconeogenesis in isolated rat hepatocytes is produced by approx. 0.1 mM-tryptophan. 3. Tryptophan inhibits gluconeogenesis from all substrates giving rise to oxaloacetate, but stimulates glycerol-fuelled glucose production. 4. Gluconeogenesis in guinea-pig hepatocytes is insensitive to tryptophan. 5. Changes in metabolite concentrations in rat liver cells are consistent with a locus of inhibition at the step catalysed by phosphoenolpyruvate carboxykinase. 6. Inhibition of gluconeogenesis persists in cells from rats pretreated with tryptophan in vivo. 7. Tryptophan has no effect on urea production from alanine, but decreases [1-14C]palmitate oxidation to 14CO2 and is associated with an increased [hydroxybutyrate]/[acetoacetate] ratio. 8. These results are discussed with reference to the control of gluconeogenesis in various species.

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