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

Abstract

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

Activity-Dependent Autocrine-Paracrine Activation of Neuronal P2Y Receptors

“…In hepatoma cells (Junankar et al, 2002), for example, released ATP is degraded to ADP to stimulate P2Y 1 receptors. In epithelial (Torres et al, 2002) and osteoblastic (You et al, 2002) cells, in contrast, released ATP itself activates P2Y 11 and P2Y 2 receptors, respectively.…”

“…Therefore, neuronal ATP release is depolarization and Ca 2ϩ dependent (von Kugelgen et al, 1994). In non-neural tissues, such as liver, kidney, bones, and blood vessels, released nucleotides subserve autocrine-paracrine functions by activating certain P2Y receptors (Gerasimovskaya et al, 2002;Junankar et al, 2002;Schwiebert et al, 2002;Torres et al, 2002;You et al, 2002). In neurons, action potential-and Ca 2ϩ -dependent release of ATP contributes to synaptic transmission via P2X receptors (Robertson et al, 2001); however, much less is known about the activation of neuronal P2Y receptors by released nucleotides.…”

Metabotropic P2Y Purinoceptor-Mediated Presynaptic and Postsynaptic Enhancement of Cerebellar GABAergic Transmission

“…It is also likely that the P2Y 1 purinoceptor subtype mediates not only the ADPinduced short-term enhancement of GABAergic transmission at cerebellar interneuron3 PC inhibitory synapses but also purinoceptor-mediated LTP of postsynaptic GABA A receptor currents in PCs via the ADP-induced [Ca 2ϩ ] i elevation. P2Y 1 R has been classified as a member of G-protein-coupled metabotropic receptors linking to IP 3 -sensitive Ca 2ϩ stores in a variety of cells (Marrelli, 2001;Junankar et al, 2002;Gallagher and Salter, 2003;Hu et al, 2003;Ju et al, 2003). Thus, using combined techniques of electrophysiology and Ca 2ϩ imaging, we examined whether purinoceptor activation can increase Ca 2ϩ influx or stimulate internal Ca 2ϩ stores.…”

P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

“…For instance, some groups suggest that purinergic receptor activation is mandatory for VSOR Cl Ϫ activation (23,38), nevertheless, others authors propose that purinergic receptors only modulate VSOR Cl Ϫ channel activation (47). Similarly, in intestinal epithelial and HTC cells, VSOR Cl Ϫ activation has been shown to be independent of ATP release and purinergic receptor activation (37,48).…”