Recent reports (4 -16) have demonstrated that cells respond to mechanical stimuli, including osmotic swelling, by releasing ATP. ATP is known to bind to two main classes of purinergic receptors present on the cell plasma membrane; P2Y receptors are G-protein-linked receptors, and P2X receptors are intrinsic ion channels (17). Activation of either class of receptor can result in a transient increase in [Ca 2ϩ ] i . In a number of cell types apyrase (an ATP/ADP phosphatase) and P2 receptor antagonists such as suramin and PPADS have been shown to inhibit, at least partially, swelling and mechanically stimulated cytoplasmic Ca 2ϩ signals (5, 13, 15, 16), thus providing evidence for a link between extracellular ATP release and transient increases in [Ca 2ϩ ] i . In a study of HTC rat hepatoma cells, Fitz and co-workers (4, 6) proposed that extracellular ATP, released in response to a decrease in the extracellular osmolality, could act in an autocrine manner by binding to purinergic receptors and thereby stimulating signaling pathways responsible for activating the Cl Ϫ efflux required for RVD. A recent report (18) demonstrated a rise in [Ca 2ϩ ] i in HTC cells in response to osmotic swelling and showed that depletion of thapsigargin-sensitive intracellular stores of Ca 2ϩ inhibited both swelling-activated K ϩ and Cl Ϫ currents. In contrast to the findings in other cell types (13,15,16), however, the rise in [Ca 2ϩ ] i was not inhibited by either apyrase or inhibitors of purinergic receptors, from which it was concluded that the swelling-induced release of ATP was not responsible for the rise in [Ca 2ϩ ] i (18). In this study we have investigated the relationship between a decrease in extracellular osmolality, extracellular nucleotides, [Ca 2ϩ ] i , and ion and organic osmolyte efflux in HTC cells. In experiments comparing the actions of three enzyme preparations that hydrolyze ATP, we found that the hypotonically induced rise in [Ca 2ϩ ] i was inhibited significantly only under conditions in which extracellular ADP as well as ATP was removed efficiently. The hypotonically induced rise in [Ca 2ϩ ] i in HTC cells was compared with the increase in [Ca 2ϩ ] i elicited by the addition of ATP, UTP, or ADP to cells in isotonic medium, and the effect of submicromolar concentrations of ATP on

To test whether high-density lipoproteins (HDL) could aid in the removal in vivo of potentially atherogenic oxidized lipids, we perfused rat liver in situ with buffer supplemented with isolated human HDL containing small amounts of cholesteryl linoleate hydro(pero)xides [CH18:2-O(O)H]. Perfusion resulted in the rapid removal of Ch18:2-O(O)H from HDL with a half-life (t1/2)of 11.4 min., faster than that of unoxidized cholesteryl linoleate, and dependent of the presence of the liver. In addition, the liver enhanced the reduction of Ch18:2-OOH associated with HDL remaining in the perfusate buffer. Perfusion resulted in the release of a hepatic activity that enhanced the reduction of HDL-associated CH18:2-OOH and was resistant to heat treatment. In contrast with the situation with HDL, low-density lipoprotein (LDL)-associated CH18:2-O(O)H were neither removed nor reduced by perfused rat liver within the time course studied, in support of a possible role for HDL in the detoxification of circulating lipid hydroperoxides in vivo.

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 invitro 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.

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.

A cytosolic fraction derived from rat hepatocytes was used to investigate the regulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] kinase, the enzyme which converts Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. The activity was doubled by raising the free Ca2+ concentration of the assay medium from 0.1 microM to 1.0 microM. A 5 min preincubation of the hepatocytes with 100 microM-dibutyryl cyclic AMP (db.cAMP) plus 100 nM-tetradecanoylphorbol acetate (TPA) resulted in a 40% increase in Ins(1,4,5)P3 kinase activity when subsequently assayed at 0.1 microM-Ca2+. This effect was smaller at [Ca2+] greater than 0.5 microM, and absent at 1.0 microM-Ca2+. Similar results were obtained after preincubation with 100 microM-db.cAMP plus 300 nM-vasopressin (20% increase at 0.1 microM-Ca2+; no effect at 1.0 microM-Ca2+). Preincubation with vasopressin, db.cAMP or TPA alone did not alter Ins(1,4,5)P3 kinase activity. It is proposed that these results, together with recent evidence implicating Ins(1,3,4,5)P4 in the control of Ca2+ influx, could be relevant to earlier findings that hepatic Ca2+ uptake is synergistically stimulated by cyclic AMP analogues and vasopressin.