Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C.

Ascorbate (AH, the reduced form of vitamin C) is an important radical scavenger and antioxidant in human plasma; the resulting ascorbyl radical can disproportionate to AH and dehydroascorbic acid (DHA). Here we address potential maintenance mechanism(s) for extracellular AH by examining the ability of cells to convert extracellularly presented DHA to AH. DHA was rapidly transported into human liver (HepG2), endothelial and whole blood cells in vitro by plasma membrane glucose transporters and reduced intracellularly. Liver cells displayed the highest capacity to release the intracellularly accumulated AH. The proteins responsible for DHA uptake and AH release could be distinguished by inhibitor studies. Thus, unlike DHA uptake, AH efflux was largely insensitive to cytochalasin B and thiol-reactive agents but was inhibited by phloretin, 4,4'-di-isothiocyanostilbene-2,2'-disulphonate and isoascorbate. Efflux of AH from cells was temperature-sensitive and saturable with a low affinity (millimolar, intracellular) for AH. In addition to isolated liver cells, perfusion of intact rat and guinea-pig liver with DHA resulted in AH in the circulating perfusate. Our results show that hepatocytes take up and reduce DHA and subsequently release part of the AH formed, probably via a membrane transporter. By converting extracellular DHA to extracellular AH, the liver might contribute to the maintenance of plasma AH, a process that could be important under conditions of oxidative stress.

On the mechanism of action of econazole, the capacitative calcium inflow blocker.

The ability of bovine serum albumin to reverse the inhibitory action of econazole and the unsaturated fatty acid oleate on store-dependent Ca2+ inflow was examined in Ehrlich ascites tumour cells. We report that inhibition of Ca2+ inflow by both compounds is reversed immediately upon addition of bovine serum albumin. It is concluded that the inhibitory action of econazole resembles that of unsaturated fatty acids. The mechanism appears to be one pertaining to nonspecific events at the plasma membrane, possibly involving alterations in plasma membrane fluidity/structure.

Permeable analogues of cGMP promote hepatic calcium inflow induced by the synergistic action of glucagon and vasopressin but inhibit that induced by vasopressin alone.

Treatment of perfused rat liver with the nitric oxide-generating reagent molsidomine led to substantial increases in cGMP without itself affecting basal Ca2+ fluxes. Under these conditions the ability of glucagon plus vasopressin to induce Ca2+ influx was greatly enhanced. The permeable analogue of cGMP (8-bromo-cGMP) enhanced glucagon plus vasopressin-induced Ca2+ influx to a similar extent as that with molsidomine. This suggests that the effect of the latter is attributable to the generation of cGMP which itself enhances the ability of the two hormones to induce synergistic Ca2+ influx. While 8-bromo-cGMP (or molsidomine) did not influence Ca2+ fluxes induced by glucagon, these agents strongly inhibited Ca2+ influx induced by vasopressin alone. These data show that while 8-bromo-cGMP has no effect on basal Ca2+ fluxes, it is able to modify the Ca2+ influx induced by glucagon and vasopressin action in hepatic tissue.

Rapid Ca2+ influx induced by the action of dibutylhydroquinone and glucagon in the perfused rat liver.

Glucagon induces a slight Ca2+ efflux when administered to the perfused rat liver. However, the hormone promotes rapid and significant Ca2+ influx after the prior administration of 2, 5-di(t-butyl)-1,4-hydroquinone (BHQ), an agent that promotes Ca2+ release from the endoplasmic reticulum (ER). The concentrations of glucagon that promote Ca2+ influx are similar to those that promote glycogenolysis and gluconeogenesis in isolated hepatocytes. The permeable analogue of cAMP, but not that of cGMP, is able to duplicate the Ca2+-mobilizing effects of glucagon. The influx of Ca2+ into liver is blocked by Ni2+. Administration of sodium azide, an inhibitor of mitochondrial electron transport, also blocks the BHQ plus glucagon-induced Ca2+ influx and this is reversed when azide administration is terminated. The actions of azide are evident within 60 s after administration or withdrawal, and also occur when either oligomycin or fructose is co-administered; this provides evidence for an effect of azide independent of cellular ATP depletion. Measurement of total calcium in mitochondria that were isolated rapidly from perfused livers after the combined administration of glucagon and BHQ confirmed that large quantities of extracellular Ca2+ had entered these organelles. These experiments provide evidence that in the perfused rat liver the artificial emptying of the ER Ca2+ pool allows glucagon to promote rapid and sustained Ca2+ influx that seems to terminate in mitochondria.

Unsaturated fatty acids mobilize intracellular calcium independent of IP3 generation and VIA insertion at the plasma membrane.

Addition of oleic and arachidonic acids to Ehrlich ascites tumor cells mobilizes Ca2+ from the same intracellular pool as that mobilized by thapsigargin. Such mobilization occurs in the presence of the phospholipase C inhibitor U73122 as well as in cells treated with pertussis toxin. Co-addition of fatty acids and thapsigargin leads to initial rates of Ca2+ mobilization much greater than that induced by either compound alone. The responses induced by the fatty acids are observed also with other lipophiles like sphingosine, bromo-palmitate and the Ca2+ influx inhibitor econazole; all responses are rapidly reversed by addition of bovine serum albumin. Many of the above effects of fatty acids are observed also in Jurkat T lymphocytes and Friend erythroleukemia cells. The experiments provide evidence of lipid-induced plasma membrane perturbations that influence intracellular Ca2+ mobilization independent of the generation of currently known second messengers.

Fasciola hepatica infection in sheep: changes in liver metabolism.

Several aspects of liver function during infection with Fasciola hepatica were examined in sheep four weeks after infection and compared with the changes observed in infected rats. Previously reported respiratory abnormalities in mitochondria isolated from the left lobe of the liver of infected sheep were characterised further. Evidence is presented that the respiratory lesion is located in the mitochondrial electron transport chain and that the aberrant respiratory behaviour is not associated with an increase in nonesterified fatty acids and the depletion of mitochondrial phospholipids, as is the case in the rat. Microsomal membranes, which have also been shown to be depleted of phospholipids in the fluke-infected rat liver, showed no such changes in the sheep. However, in common with the rat, a substantial loss of cytochrome P450 was recorded in microsomes prepared from the left lobe, and the glycogen content of the left lobe was found to be less than 50 per cent of control values. No change was observed in glucose 6-phosphatase activity. All these changes were localised effects, confined to areas of fluke infiltration.

What is the concentration of calcium ions in the endoplasmic reticulum?

Consideration of the data from a number of sources indicates that the concentration of Ca2+ in the endoplasmic reticulum is very high and perhaps in the mM range. A number of implications flow from this-an important one being that the magnitude of Ca2+ gradients across the endoplasmic and plasma membranes are very similar.

Regulation of cellular calcium through signaling cross-talk involves an intricate interplay between the actions of receptors, G-proteins, and second messengers.

Various external stimuli regulate cellular Ca2+ fluxes. Information from these stimuli is transmitted to the cell interior by signal transducing systems that involve interactions between receptors, G-proteins, and enzymes that generate second messengers. The possible mechanisms by which "cross-talk" within and between these second messenger-generating systems provides an intricate molecular avenue for fine modulation of intracellular Ca2+ are reviewed.

Importance of T-cell-dependent inflammatory reactions in the decline of microsomal cytochrome P450 concentration in the livers of rats infected with Fasciola hepatica.

The concentration of cytochrome P450, measured spectrophotometrically in microsomal preparations from the livers of rats infected with 30 metacercariae of Fasciola hepatica, declined by approximately 50% at 3 weeks post-infection. Treatment of infected rats with the anti-inflammatory agent dexamethasone (2 mg/kg at 48 h intervals for 8 days prior to assay) abolished the decline in P450 content. Assay of P450 in infected congenitally athymic (nude) rats showed normal levels. These results demonstrate that the T-cell-dependent inflammatory response in the liver of the host is a necessary factor in the development of the decline in hepatic P450, which is known to compromise the metabolism of certain drugs in infected hosts.

Do nitric oxide and cGMP play a role in calcium cycling?

The biological molecule NO and its cyclic nucleotide effector molecule cGMP, are involved in a variety of biological systems. This article reviews evidence supporting a role for these molecules in signal transduction. Over the last 10 years, it has become evident that these molecules are important in Ca2+ regulation, particularly in excitable cells. In these cells, cGMP-dependent mechanisms appear to both directly and indirectly regulate Ca2+ transport. Until recently, reports of the actions of cGMP in non-excitable cells have been contradictory, presenting a confusing plethora of effects. In these cells, the cGMP-Ca2+ regulation pathway appears to be concentration-dependent, possibly representing a negative feedback mechanism. Ca2+ entry appears to be activated when low concentrations of cGMP are present, and inhibited at higher concentrations. The role of cGMP in Ca2+ regulation in non-excitable cells has been largely overlooked and further investigation of this issue may provide clues as to the nature of various unknown components that induce Ca2+ entry into these cells.

Nickel: an agent for investigating the relation between hormone-induced Ca2+ influx and bile flow in the perfused rat liver.

Influx of Ca2+ induced by the synergistic action of glucagon plus vasopressin in the perfused rat liver was progressively inhibited by infusing increasing concentrations of Ni2+ to the perfusion medium. The onset of Ca2+ influx following vasopressin administration was delayed and inhibition occurred of both the initial rate of Ca2+ influx as well as the total amount of Ca2+ taken up by the liver. Inhibition of the Ca2+ influx rate was almost maximal at approximately 500 microM Ni2+; half-maximal inhibition occurred at less than 250 microM. Added Ni2+ also delayed the onset of the early transient bile flow peak. In addition, the duration of the transient peak in bile flow was prolonged by approximately 2 min by all concentrations of Ni2+ between 25-500 microM, the greatest amount of bile being released in the presence of 250 microM Ni2+. Concentrations of Ni2+ at 100 microM and above also inhibit the decrease in bile flow to below baseline levels. The data identify a multiple role for Ca2+ mobilisation in bile flow.

A study of hepatic mitochondrial respiration and microsomal cytochrome P450 content in mice infected with the liver fluke Fasciola hepatica.

Previous studies of the effects of infection of Wistar rats with the common liver fluke, Fasciola hepatica, on liver bioenergetic and drug metabolism have demonstrated a loss of respiratory control in isolated mitochondria and reduced microsomal cytochrome P450 content, respectively, from 2 weeks post-infection throughout the acute phase of the infection. In the present study male Balb/c mice infected with F. hepatica showed a loss of respiratory control in isolated liver mitochondria only at 4 weeks post-infection. A similar time course was demonstrated for a reduction in hepatic microsomal cytochrome P450 content. Preparations from infected CBA mice showed similar changes to Balb/c mice but mitochondrial respiration in preparations from infected Swiss outbred mice was normal. A host difference between strains of mice and between mice and rats is therefore evident in the timing and extent of liver mitochondrial dysfunction and in the timing of the decrease in the cytochrome P450 content of hepatic microsomes. This difference between hosts may be related to the reported differences in cellular inflammatory responses to the migrating juvenile flukes in the livers of rats and mice.

Hormone-induced bile flow and hepatobiliary calcium fluxes are attenuated in the perfused liver of rats made cholestatic with ethynylestradiol in vivo and with phalloidin in vitro.

The actions of vasopressin and glucagon, administered alone or together, were assessed on bile flow in perfused livers from rats made cholestatic by the injection of ethynylestradiol and from those allowed to recover from such treatment. Concomitant measurements were made of biliary calcium output as well as changes in the perfusate Ca2+ concentration, glucose output, and oxygen uptake. Experiments were also conducted where cholestasis was induced in vitro in the perfused liver by the infusion of phalloidin. In each case cholestasis was demonstrated to have occurred by a reduction in bile flow by approximately 50%. The data show that the transient increase in bile flow and bile calcium seen in control rat liver soon after the administration of vasopressin, particularly when coadministered with glucagon, is largely absent in cholestasis induced by ethynylestradiol and attenuated in cholestasis induced by phalloidin. At the same time the pattern of perfusate Ca2+ fluxes in ethynylestradiol-induced cholestasis shifts to one reflecting net efflux of the ion from the liver. The responses to glucagon administration alone contrast with those of vasopressin in that in the perfused liver of ethynylestradiol-treated rats, glucagon induces a pronounced and sustained increase in bile flow. In cholestasis induced by both ethynylestradiol and phalloidin, glucagon fails to induce an initial transient decrease in bile flow. The effects of glucagon, including enhancement of vasopressin-stimulated bile flow in control and in ethynylestradiol-treated rats, can be mimicked by dibutyryl cyclic adenosine monophosphate (cAMP). Changes in glucose output and oxygen uptake induced by both hormones are only slightly attenuated. The data show that the modulation of bile flow that occurs rapidly after the administration of vasopressin and glucagon to control perfused rat liver is altered in conditions of cholestasis induced by either ethynylestradiol or phalloidin.

Does nitric oxide play a role in liver function?

Nitric oxide (NO) is becoming increasingly recognised as a signalling molecule in many organs, although its role in the liver remains to be fully elucidated. There is no doubt that liver cells can produce NO in response to a variety of stimuli including Corynebacterium parvum-infection, lipopolysaccharide (LPS) and a variety of cytokines. Within the liver, NO modulates some fundamental intracellular functions such as protein synthesis, mitochondrial electron transport and components of the citric acid cycle. Intercellular roles for NO in the liver may include drug metabolism and blood storage. Also, NO acts to protect the liver from immunological damage in models of hepatic inflammation. Understanding the role of NO in the liver may provide insight into the functioning of this organ in health and disease.

Aberrant mitochondrial respiration in the livers of rats infected with Fasciola hepatica: the role of elevated non-esterified fatty acids and altered phospholipid composition.

The non-esterified fatty acid (NEFA) content and phospholipid composition of mitochondria isolated from the livers of Wistar rats infected with Fasciola hepatica were examined in relation to the aberrant mitochondrial respiration previously reported [Rule, Behm, and Bygrave (1989) Biochem. J. 260, 517-523]. At 2 weeks post-infection, elevated NEFA levels were associated with uncoupling of mitochondrial respiration that was reversible in vitro by the addition of BSA. State IV respiration rates showed a strong correlation with NEFA content. At 3 weeks post-infection, NEFA content had increased further and uncoupled mitochondria no longer showed any response to BSA. 31P-NMR analyses of cholate extracts of mitochondria from infected livers at 3 weeks post-infection revealed a marked loss of several major phospholipid species with a concomitant increase in catabolic products, particularly glycerophosphocholine and glycerophosphoethanolamine. Similar changes were observed in microsomal extracts. The NEFA content and phospholipid composition of mitochondria isolated from infected, athymic nude rats were not significantly different from uninfected, athymic rats. These findings suggest that uncoupling of liver mitochondria during infection with F. hepatica is the result of phospholipase activation mediated by the immune system of the host.

The synergistic action (cross-talk) of glucagon and vasopressin induces early bile flow and plasma-membrane calcium fluxes in the perfused rat liver.

A study was made of the initial responses of perfusate Ca2+ fluxes and bile flow to Ca(2+)-mobilizing agonists, following refinements to the methods for analysing these parameters in the perfused rat liver. Net Ca2+ efflux induced by vasopressin commences at 15 s, reaches a maximal rate at 35 s and declines to zero by 55 s, when Ca2+ influx commences. Vasopressin-induced increases in bile flow commence by 20 s, attain a maximal rate by 35 s and begin to decline at 50 s, to reach basal values by 90 s. Concomitant administration of glucagon modifies each of these actions of vasopressin in the following ways: it decreases by 5 s the time of onset of net Ca2+ efflux, and the time and magnitude of such efflux, and the time of onset of bile flow is decreased to 15 s, and the flow reaches maximal rates by 30 s. When the alpha 1-adrenergic agonist phenylephrine is used in place of vasopressin, Ca2+ efflux commences at 17-18 s and is greater in magnitude; little bile flow is induced by this agonist. Glucagon modifies the action of phenylephrine in the following ways: the onset of Ca2+ efflux is brought forward by 2-3 s, it is of lower magnitude and Ca2+ influx begins by 45 s; bile flow commences by 15-20 s, and reaches a maximum at 30 s, where the rate is much greater than in the absence of glucagon; this rate gradually declines to be near basal by 80 s. The onset of agonist-induced oxygen uptake was also brought forward by the co-administration of glucagon. Comparison of agonist-induced plasma-membrane Ca2+ fluxes and bile flow (with or without glucagon administration) suggests that correlations can be made between net Ca2+ fluxes and the transient increases seen in bile flow.

Characterization of the oligomycin-sensitivity properties of the F1F0-ATPase in mitochondria from rats infected with the liver fluke Fasciola hepatica.

The F1F0-ATPase activity of liver mitochondria isolated from rats infected with Fasciola hepatica at 3 and 4 weeks post-infection showed a marked loss of sensitivity to oligomycin and to N,N'-dicyclohexylcarbodiimide. A loss of sensitivity to diethylstilbestrol was also demonstrated at 4 weeks post-infection. Recovery was apparent in most cases by 6 weeks post-infection. No significant difference in latent ATPase activity was observed between mitochondria from control and infected livers at any stage of the infection. The mitochondria from infected livers were therefore considered to have a full complement of the F1 moiety of the F1F0-ATPase complex. Purification of the mitochondrial ATPase from 4-week infected livers resulted in a very low yield of an oligomycin-insensitive complex. This was due to a failure to enrich specific activity during purification. The evidence presented indicates that infection with Fasciola hepatica gives rise to alterations in the function of the host liver mitochondrial ATPase, namely loss of inhibitor sensitivity and apparent structural alterations of the ATPase complex.

Crosstalk between calcium- and cyclic AMP-mediated signalling systems and the short-term modulation of bile flow in normal and cholestatic rat liver.

The flow of bile is subject to short-term modulation by glucagon and calcium-mobilizing hormones. Of potential relevance is the crosstalk between the second messenger-mediated signal transducing systems of these agonists. This latter point has revealed an area of investigation that should enable further insights to be made into a physiological network that interrelates bile flow, hepatocellular calcium movements and hormone action. This information in turn may provide insights into the etiology and treatment of human and animal diseases in which cholestasis is an underlying feature.

Evidence that stimulation of plasma-membrane Ca2+ inflow is an early action of glucagon and dibutyryl cyclic AMP in rat hepatocytes.

The ability of glucagon (1 nM) and of dibutyryl cyclic AMP (50 microM) to increase cytosolic free Ca2+ concentration ([Ca2+]i) in Fura-loaded rat hepatocytes was examined in a system wherein Ca2+ inflow was induced by the re-admission of excess Ca2+ to a nominally Ca(2+)-free medium. An increase in [Ca2+]i did not occur in the absence of either agonist, but did so after co-addition of either agonist with Ca2+. Increasing the time between addition of dibutyryl cyclic AMP (or of glucagon) and Ca2+ led to increases in [Ca2+]i; half-maximal and maximal increases were observed at 0 s (i.e. at co-addition) and 5-7 s respectively. Dibutyryl cyclic AMP and Ca2+ each exhibited a concentration-dependence when their respective concentrations were changed for a fixed time interval between additions. Half-maximal and maximal effects were obtained with 30 microM and 50 microM dibutyryl cyclic AMP and with 0.5 mM and approx. 1 mM Ca2+ respectively. The data demonstrate an early action of glucagon and dibutyryl cyclic AMP on [Ca2+]i. It is argued that the agonist-induced rise in [Ca2+]i results from an increase in plasma-membrane Ca2+ inflow, an effect that appears to occur much earlier than that on mobilization of internal stores of Ca2+.