Ari Karjalainen

The Role of P2Y1 Purinergic Receptors and Cytosolic Ca2+ in Hypotonically Activated Osmolyte Efflux from a Rat Hepatoma Cell Line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca2+ concentration ([Ca2+] i ) to increase transiently by ∼90 nm. This rise in [Ca2+] i was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca2+] i increase. The hypotonically induced rise in [Ca2+] i was prevented by the prior discharge of the intracellular Ca2+ store of the cells by thapsigargin. Removal of extracellular Ca2+ or inhibition of Ca2+ influx by 1–10 μm Gd3+depleted the thapsigargin-sensitive Ca2+ stores and thereby diminished the rise in [Ca2+] i . The hypotonically induced rise in [Ca2+] i was prevented by adenosine 2′-phosphate-5′-phosphate (A2P5P) and pyridoxyl-5′-phosphate-6-azophenyl-2′,4′-disulfonate, inhibitors of purinergic P2Y1 receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca2+] i elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca2+] i elicited by the addition of the P2Y2 and P2Y4 receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y1, P2Y2, and P2Y6 receptors. Inhibition of the hypotonically induced rise in [Ca2+] i blocked hypotonically induced K+ (86Rb+) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl− (125I−) efflux. The interaction of extracellular ATP and/or ADP with P2Y1purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.

Osmotic swelling activates two pathways for K+ efflux in a rat hepatoma cell line.

The pathways for the efflux of K⁺ from osmotically-swollen HTC rat hepatoma cells were investigated using ⁸⁶Rb⁺ as a tracer for K⁺. Exposure of HTC cells to a hypotonic solution (< 250 mOsm kg^-1) resulted in a transient efflux of ⁸⁶Rb⁺ that reached a maximal value after ñ1 min, and inactivated within 3 min. This initial ⁸⁶Rb⁺ efflux was inhibited by charybdotoxin, clotrimazole and Ba²⁺, but not by apamin or paxilline, consistent with it being via an intermediate-conductance Ca²⁺-activated K⁺ channel. For cells exposed to an extracellular osmolality < 180 mOsm kg^-1 there was an additional ⁸⁶Rb⁺ efflux component which was slower to activate, taking 4 - 6 min to reach a maximum, and remaining active for > 20 min. The second ⁸⁶Rb⁺ efflux component was not inhibited by K⁺ channel blockers but was inhibited by the anion channel blockers, tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumate. The time-courses for its activation and inactivation, as well as its dependence on the extracellular osmolality, were very similar to those observed for the hypotonically-activated efflux of the organic osmolyte, taurine. The data are consistent with the second component of ⁸⁶Rb⁺ efflux and the efflux of taurine from osmotically-swollen cells occurring via a common pathway having a marked selectivity for taurine over ⁸⁶Rb⁺.

Passive calcium influx by plasma membrane vesicles isolated from rat liver

Passive Ca2+ influx independent of ATP addition to the incubation medium, took place in plasma membrane vesicles isolated from rat liver. The rate of Ca2+ influx was found to depend on the concentration of added Ca2+, and on the incubation temperature, and was inhibited by La3+, Hg2+ and by p-chloromercuribenzoate. Influx was not blocked by calcium channel blockers, or affected by a range of uncouplers. Addition of the Ca2+ ionophore A23187 to vesicles that had taken up the ion induced a rapid efflux of Ca2+ especially when EGTA also was added to the incubation medium. A number of divalent cations inhibited Ca2+ influx. The vesicles could be frozen and stored overnight with little loss in activity. The kinetics of Ca2+ influx could be related to that which occurs in the unstimulated perfused rat liver. The data suggest that the plasma membrane vesicle preparation may be useful for further studies on the basal liver cell Ca2+ influx system in vitro.

Exposure to depolarizing concentrations of K+ inhibits hormonally-induced calcium influx in rat liver

The exposure of perfused rat livers to depolarizing concentrations of K+ (60 mM) by partial substitution of the NaCl in the medium with KCl induces glycogenolysis, respiratory changes and vasoconstriction. These responses were found to be inhibited 70-80% by 20 microM indomethacin and by 20 microM bromophenacyl bromide. This suggests that eicosanoids, namely prostaglandins, are involved in mediating these effects, and hence that the action of K+ involves primarily an effect on eicosanoid-producing cells (Kupffer and endothelial cells) within the liver. A 5 min pre-exposure of perfused livers to depolarizing concentrations of K+ (in the presence of indomethacin) was found to inhibit (by approx. 85%) the influx of Ca2+ induced by the co-administration of 10 nM glucagon and 10 nM vasopressin. A similar result was observed in isolated hepatocytes. The inhibition was probably not due to a decrease in the concentration of Na+ in the medium since the substitution of 80 mM NaCl with 80 mM choline chloride resulted in significantly less inhibition (30-40%). These results suggest that under these conditions the influx of Ca2+ in liver occurs through a pathway that is inhibited by high K+ concentration and/or a depolarization of the plasma membrane.

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

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 for the involvement of carboxyl groups in passive calcium uptake by liver plasma membrane vesicles and in agonist-induced calcium uptake by hepatocytes

The hydrophobic reagents DCCD and EEDQ, each of which reacts with protein carboxyl groups, were found to inhibit both passive Ca2+ uptake by plasma membrane vesicles isolated from rat liver and agonist‐induced Ca2+ uptake by hepatocytes. The data raise the possibility that the Ca2+ inflow pathway(s) in liver has a specific requirement for a reactive carboxyl group or groups.

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.