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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Extracellular ATP is known to cause uptake of Ca2+ by rat liver cells. The specific pathway permitting influx of Ca2+ has not yet been identified. In the present investigations, we studied the properties of ATP-evoked 45Ca2+ uptake in rat hepatoma cell monolayers and then used patch-clamp electrophysiology to identify the channel that may account for this uptake. The results suggest that ATP-stimulated 45Ca2+ uptake occurs as a result of P2-purinergic receptor interaction because uptake was inhibited by Reactive Blue (100 microM), a blocker of this type of receptor. Furthermore, the ability of other adenine nucleotides to stimulate 45Ca2+ uptake was related to the selectivity sequence for binding to the P2-purinergic receptor. ATP-stimulated 45Ca2+ uptake occurs primarily through a conductance pore since it was inhibited by 70% upon dissipation of the membrane potential using the K+ ionophore valinomycin. The calcium channel blockers nifedipine and verapamil failed to inhibit 45Ca2+ uptake, but gadolinium (GdCl3) was an effective blocker. In cell-attached patch-clamp experiments, a single type of channel was activated with ATP (100 microM) addition to the bath in 18 of 32 trials. The current-voltage relationship of the ATP-activated channel is identical to that of the stretch-activated channel previously characterized in this laboratory as a calcium-permeable cation-nonselective channel [Am. J. Physiol. 258 (Cell Physiol. 27): C421-C428, 1990]. There are several lines of evidence which suggest that this cation-nonselective channel may account for ATP-stimulated 45Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calcium-permeable channels in rat hepatoma cells are activated by extracellular nucleotides. 166 1

Acetyl-CoA carboxylase (ACC) can be regulated in vitro via phosphorylation by a 5'-AMP-activated protein kinase. A potential intracellular role for this kinase has been studied in the Fao hepatoma cell by manipulating the intracellular adenine nucleotide pool with ATP-depleting agents. Three different ATP depletors, antimycin A, dinitrophenol, and sodium azide, all promote the rapid loss of ACC activity characterized by a marked reduction in enzyme Vmax, abolition of citrate-independent activity, an increase in the Ka for citrate and a reduction in the mass of a complex between the two major ACC isozymes. These effects persist through enzyme purification on monomeric avidin-Sepharose and are accompanied by an increase in 32P-content, both consistent with depletor-induced covalent enzyme modification. The effects of ATP depletors in intact cells are mimicked in vitro on phosphorylation of ACC by the 5'-AMP-activated protein kinase and are reversible on dephosphorylation. These data indicate that ACC activity is sensitive to the intracellular adenylate charge, but that changes in the state of enzyme phosphorylation, rather than direct allosteric regulation by adenine nucleotides, underly this mode of enzyme control. This kinase-mediated modulation provides a mechanism for altering the rate of fatty acid synthesis and, secondarily, fatty acid oxidation, depending on the rate of ATP generation from carbohydrate-derived precursors in several tissues in vivo.
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PMID:Regulation of intracellular acetyl-CoA carboxylase by ATP depletors mimics the action of the 5'-AMP-activated protein kinase. 168 96

Biochemical and morphological studies compared the ATP requirements for and the internalization routes of alpha 2-macroglobulin and insulin in H35 hepatoma cells. Cellular ATP concentrations were decreased more than 94% by 1 mM 2,4-dinitrophenol or 10 mM sodium azide, potassium cyanide, or oligomycin. ATP depletion decreased total cell-associated alpha 2-macroglobulin 70-90% by inhibiting binding 67-77% and receptor-mediated internalization 90-96%. Under the same conditions, insulin binding was decreased less than 10%, and endocytosis and intracellular accumulation were not affected. Quantitative electron microscopic analysis of the distribution of occupied receptors on the surface of control and treated cells was performed using colloidal gold-labeled alpha 2-macroglobulin or insulin. alpha 2-Macroglobulin concentrated in and was internalized almost exclusively by coated pits. Insulin was rarely associated with coated pits, but was found in and internalized by noncoated invaginations. ATP depletion did not affect receptor mobility or ligand-induced aggregation of either receptor. There was an increase in the amount of alpha 2-macroglobulin found in coated pit-like structures. The coat underlying pits in ATP-depleted cells was poorly defined and may account for the inability of coated pits to form and/or internalize. These results showed that receptor-mediated internalization via coated pits was ATP dependent, whereas internalization via pinocytotic invaginations was energy independent, which explained the difference in the ATP dependency of uptake for the two ligands. These observations suggested that autophosphorylation of the insulin receptor may not be involved in either the aggregation or internalization of the insulin-receptor complex, since ATP depletion did not affect either process. This study provided evidence that specialized mechanisms exist for the internalization of insulin which may be related to some of its intracellular effects.
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PMID:Differences in adenosine triphosphate dependency of receptor-mediated endocytosis of alpha 2-macroglobulin and insulin correlate with separate routes of ligand-receptor complex internalization. 168 53

1. Insulin receptors were partially purified from rat liver by chromatography on wheat-germ-lectin-Sepharose. Incubation with [gamma-32P]ATP in the presence of insulin resulted in increased phosphorylation of the beta-subunit on both tyrosine and serine residues. Two-dimensional mapping of tryptic peptides showed that, in agreement with previous studies using preparations of receptors from other sources, the tyrosine residues involved were the three tyrosines in the kinase domain (corresponding to tyrosines 1158, 1162 and 1163 of the human receptor) plus two tyrosines close to the C-terminus (corresponding to tyrosines 1328 and 1334). 2. The effects of insulin on the phosphorylation of receptors within intact rat liver cells were determined by incubating cells in the presence of [32P]Pi for 50 min and then with or without insulin for a further 10 min. The labelled receptors were then rapidly isolated by sequential use of wheat-germ-lectin-Sepharose chromatography and immuno-isolation using a monoclonal antibody to the C-terminal end of the beta-subunit. 3. Insulin was found to increase overall phosphorylation of the receptor nearly 3-fold. Two-dimensional mapping was then carried out in combination with phosphoamino acid analysis. This revealed that the pattern of phosphorylation of the receptors in cells incubated in the absence and presence of insulin exhibited a number of marked differences from that observed in previous studies on intact cells, which had been restricted to cells expressing very high levels of insulin receptors such as certain hepatoma-derived cells or cells transfected with insulin receptor cDNA. The differences in the effects of insulin included a larger increase in the proportion of receptors being phosphorylated on the three tyrosine residues of the kinase domain, no apparent phosphorylation of the two tyrosine residues close to the C-terminus and no increase in either threonine or overall serine phosphorylation. 4. The receptors appeared to be phosphorylated on a number of different serine residues in cells incubated in the absence of insulin. Evidence for both increases and decreases in the phosphorylation of specific serine residues on addition of insulin was obtained. 5. It is concluded that care should be taken when extrapolating findings on the phosphorylation of the insulin receptor within cultured cells to more physiological situations.
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PMID:Analysis of insulin receptor phosphorylation sites in intact rat liver cells by two-dimensional phosphopeptide mapping. Predominance of the tris-phosphorylated form of the kinase domain after stimulation by insulin. 170 33

We previously observed that Ser378 in the heparin-binding domain of vitronectin becomes phosphorylated by a protein kinase in plasma upon addition of ATP and divalent cations. We now report that purified plasma vitronectin contains approximately 2.5 mol of phosphate per mol of protein and that vitronectin becomes phosphorylated during biosynthesis in human hepatoma (HepG2) cells. In vitro, rabbit muscle cAMP-dependent protein kinase specifically phosphorylates Ser378 in single-chain (75 kDa) vitronectin but does not phosphorylate the two-chain (65/10 kDa) form cleaved at Arg379. Heparin affects neither the time course nor the extent of phosphorylation of Ser378 at neutral pH. The extent of phosphorylation of Ser378 achieved with cAMP-dependent protein kinase (greater than or equal to 0.3 mol phosphate per mol vitronectin) is greater than that obtainable in plasma and should enable comparisons to be made of the activities of the native and phosphorylated forms.
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PMID:Cyclic AMP-dependent protein kinase phosphorylates serine378 in vitronectin. 171 1

Hypolipidemic drugs and phthalic ester plasticizers induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. Recent evidence from this laboratory suggests that many agents in this class of chemicals are uncouplers of mitochondrial oxidative phosphorylation both in vitro and in vivo. Uncoupling of oxidative phosphorylation decreases ATP required for ion pumps and could thereby indirectly increase intracellular free calcium. The goal of these experiments, therefore, was to compare the effect of the potent uncoupler and non-genotoxic carcinogen Wy-14,643 with the weaker agent 2-ethylhexanol on intracellular free calcium in cultured Kupffer cells. Kupffer cells, the resident hepatic macrophages, are activated by calcium and release a variety of mitogenic growth factors that may modulate cell proliferation. In this study, the cytosolic free calcium concentration in Fura-2-loaded cultured Kupffer cells was increased significantly from 78 +/- 11 to 838 +/- 112 nM following incubation with Wy-14,643 (1.25 mM). The increase in intracellular calcium due to Wy-14,643 was both time- and dose-dependent. At equimolar concentrations, ethylhexanol had no effect on intracellular calcium (65 +/- 20 nM). However, at higher concentrations (3 mM), ethylhexanol also increased intracellular calcium. These data suggest that elevation of intracellular calcium in Kupffer cells may be involved in the mechanism of action of this interesting class of non-genotoxic carcinogens.
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PMID:Wy-14,643 but not 2-ethylhexanol increases intracellular free calcium in cultured Kupffer cells. 175 31

Plasma membrane fractions from normal, regenerating liver and the AS-30D ascites hepatocarcinoma exhibited a high degree of enrichment when a set of plasma membrane enzyme markers were studied in comparison to the ones associated to the mitochondrial and cytosolic compartments. While the (Ca2+, Mg2+)-ATPase observed for the plasma membrane fraction isolated from normal liver showed an activity of 1.2 mumoles/mg/min, the regenerating liver and the AS-30D plasma membrane fractions presented a much lower ATPase activity (0.3 and 0.22 mumoles/mg/min respectively). Despite the differences in ATPase activity observed between models, the plasma membrane fraction from the AS-30D hepatocarcinoma presented a calcium transport activity similar to the value observed for the normal system (5.9 and 5.5 nmoles Ca2+/mg/10 min, respectively). Interestingly, the ATP in equilibrium with Pi exchange experiments carried out with the different plasma membrane fractions revealed that the (Ca2+, Mg2+)-ATPase contained in the plasma membrane from the AS-30D cells shows an exchange activity of 26 nmoles ATP in equilibrium with Pi/mg/min, similar to the one observed fo the enzyme from normal liver (30 nmoles ATP in equilibrium with Pi/mg/min). Our results suggest that the plasma membrane from the transformed model presents a more efficient mechanism to regulate the movement of calcium through the calcium pump, with an optimum expenditure of energy.
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PMID:Altered coupling states between calcium transport and (Ca2+, Mg2+)-ATPase in the AS-30D ascites hepatocarcinoma plasma membrane. 182 60

A decrease in the rate of ATP hydrolysis was observed after preincubation of intact mitochondria from hepatoma 22a with an uncoupler. This effect is due both to a decrease in the rate of ATP transport and to an inactivation of the F0F1-ATPase. The former effect is shown to result from an uncoupler-induced ADP efflux. In de-energized mitochondria from hepatoma (but not from mice liver), the concentration of adenine nucleotides in the matrix equilibrates with the medium concentration via a carboxyatractyloside (CATR)-insensitive transport system. CATR-insensitive accumulation of medium ADP and stoichiometric exchange of added ATP are observed in energized hepatoma mitochondria. The dependence of the uncoupler-induced inactivation of ATPase activity on delta mu H+, pH, and ATP is consistent with the effect being caused by the natural protein inhibitor (IF1) of F0F1. ATP- and pH-dependent inactivation of the enzyme is also observed after disruption of mitochondria with the detergent Lubrol-WX. Almost all F0F1 in hepatoma mitochondria have IF1 bound in a noninhibitory manner. In the presence of uncoupler, this complex converts, via a reversible pH-dependent and an irreversible ATP-dependent process, to an inhibitory complex. The pH-dependent step can be blocked by Zn2+ and Cd2+ ions which probably bind to negatively charged residues on IF1, thereby preventing their protonation and conversion of the protein to an inhibitory conformation.
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PMID:Regulation of ATP hydrolysis in hepatoma 22a mitochondria. 183 36

Viability, glycolytic capacity and energy metabolism under anaerobic conditions were studied in the hepatoma cell lines HTC, FU5 and HepG2 and in rat and human hepatocytes using glucose and fructose as glycolytic precursors. During 6 hr of anaerobic incubation without additional substrate, viability decreased rapidly in FU5 and HTC cells, whereas viability of HepG2 cells was not significantly affected. In all tumor cells, 10 mmol/L glucose prevented hypoxic cell injury almost completely. Lactate formation from glucose was about five times higher than in hepatocytes under these circumstances. ATP content of the tumor cells remained almost constant under anaerobic conditions in the presence of glucose. Ten millimoles per liter of fructose diminished glycolysis in the hepatoma cells compared with glucose, ranging from 87% reduction in HTC cells to 43% reduction in HepG2 cells. Accordingly, ATP content decreased rapidly in the FU5 and slowly in the HepG2 cells. Viability was strongly diminished in the HTC and FU5 cells in the presence of fructose, whereas in the HepG2 cells no effect of fructose on viability was detectable. In contrast to the hepatoma cells, rat and human hepatocytes exhibited higher rates of anaerobic glycolysis in the presence of fructose and thus were able to maintain their viability under these conditions. These differences in glycolytic capacity, energy metabolism and hypoxia tolerance of hepatoma cells compared with hepatocytes may be used for the treatment of liver cancer by isolated liver perfusion and ex situ revision of the organ.
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PMID:Differences in glycolytic capacity and hypoxia tolerance between hepatoma cells and hepatocytes. 184 50

A comparative study revealed that Ehrlich ascites carcinoma (EAC) cells use glutamine plus inosine for regeneration of adenylates via the purine nucleotide cycle, whereas AS 30D hepatoma cells use adenosine instead. This observation can be correlated with the very low production of aspartate from glutamine in hepatoma cells. Although glucose is an important energy fuel for EAC, it cannot maintain a high enough level of adenylates unless glutamine is also present. Kinetic analysis of hydrolysis of ATP and ADP in the presence of rotenone suggests that deamination of AMP does not maintain a high enough ATP/ADP ratio and probably does not act as energy buffer after inhibition of cell respiration. It seems that, compared with normal cells, malignant cells have the ability for a very rapid regeneration of adenylates. It is proposed that instability of the adenine nucleotide pool, owing to frequent aerobic-anaerobic transitions, represents an essential feature of neoplasia, with profound impact on the whole metabolism of tumour cells.
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PMID:Mechanism and control of degradation and resynthesis of adenylates in tumour cells. 199 Oct 26


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