ATP-induced [Ca2+]i changes and depolarization in GH3 cells

Extracellular ATP is a neurotransmitter and mediates a variety of responses. In the endocrine system, there are data suggesting a physiological role for ATP in Ca2+ signalling and hormone secretion. However, the ATP receptor subtype involved has not been clearly elucidated in GH3 cells, a rat anterior pituitary cell line. BzATP- and ATP-induced [Ca2+]i responses had EC50 values of 18 and 651 μM, respectively. The maximal response to ATP was only 59±8% of that for BzATP. The BzATP-induced [Ca2+]i increase was dependent upon the extracellular Ca2+ concentration. Preincubation with oxidized ATP (oATP) nearly abolished the ATP- and BzATP-induced [Ca2+]i increases. Both BzATP and ATP induced depolarization in GH3 cells, with EC50 values of 31 μM and 1 mM, respectively. The maximal depolarization to BzATP and ATP were 152±21 and 146±16% of that elicited by 30 mM KCl. The rank order of agonist potency for [Ca2+]i and depolarization responses was BzATP > > ATP >2-MeSATP and purine derivatives such as ADP, AMP, adenosine were ineffective. Neither UTP nor α, β-methylene ATP showed any effect. In low-divalent conditions BzATP evoked non-desensitizing inward currents, which were reversed at ∼0 mV. This nonselective cationic conductance was increased by repeated applications of BzATP and the cells became very permeable to NMDG. Longer applications (30 min) of BzATP stimulated ethidium bromide influx in low divalent conditions, suggesting increased permeability to larger molecules. We also identified the existence of P2X7 mRNA on GH3 cells by using reverse transcriptase (RT)-polymerase chain reaction (PCR). These results suggest that the GH3 cells have an endogenous P2X7 receptor and purinergic stimulation may play a potential role in neuroendocrine modulation on these cells. Keywords: P2X7 receptor, GH3 cell, intracellular calcium, depolarization, nonselective cation current, membrane permeability Introduction The role of ATP in metabolism is well established, but its potential importance as an extracellular chemical messenger has been recognized only recently. Extracellular ATP mediates a variety of responses in a number of biological systems (Burnstock, 1990). It has been proposed that ATP plays important roles in both short-term physiologic events such as neurotransmission, exocrine and endocrine secretion and regulation of immune cell function, and long-term physiologic events such as cell growth, differentiation and proliferation in development (Abbracchio & Burnstock, 1998). These effects of extracellular ATP are mediated by purine-and pyrimidine-based nucleotide receptors, designated P2-receptors, expressed in many tissues throughout the body (Burnstock, 1990; Ralevic & Burnstock, 1998). P2-receptors have been classified into two main families: an ionotropic P2X receptor family consisting of ligand-gated ion channels, and a metabotropic P2Y receptor family consisting of G protein-coupled receptors (Buell et al., 1996; Burnstock, 1997; Ralevic & Burnstock, 1998). It has been proposed that many functional effects of extracellular ATP be related with the increase in [Ca2+]i (Gargett et al., 1997). Stimulation of P2-receptors can elevate the intracellular Ca2+ concentration ([Ca2+]i), either by influx from the extracellular medium via cation channels or by release of Ca2+ from the internal stores (Dubyak & El-Moatassim, 1993). Subclasses (P2X1-P2X7 and P2Y1-P2Y6, P2X11) of these two main receptor families have been identified on the basis of recent molecular biology studies and the development of new selective agonists and antagonists (Abbracchio & Burnstock, 1994; Ralevic & Burnstock, 1998). The P2X7 receptor is known as an uniquely bifunctional molecule through which ATP can lead not only to an inward ionic currents, but also to an increase in cell membrane permeability (Surprenant et al., 1996; Rassendren et al., 1997; Virginio et al., 1997). The increased permeability caused by activation of the P2X7 receptor results in large ion fluxes and leakage of small metabolites (Relevic & Burnstock, 1998). Activation of P2X7 receptor also activates phospholipase D in murine and human macrophages (El-Moatassim & Dubyak, 1993; Humphreys & Dubyak, 1996) and human leukemic lymphocytes (Gargett et al., 1996; Fernando et al., 1999), although the mechanism is not fully understood. Surprenant et al. (1996) suggested that the P2X7 receptor cloned from rat macrophage and brain is the cytolytic ‘P2Z receptor' previously described in mast cells, macrophages, fibroblasts, lymphocytes, erythrocytes and erythroleukemia cells (Relevic & Burnstock, 1998). The P2X7 receptor has been classified by the following agonist selectivity: BzATP > > ATP > 2-MeSATP > ATPγS (Dubyak & El-Moatassim, 1993; Surprenant et al., 1996). This receptor shows selectivity for the ATP4− species, but stimulation of this receptor requires a higher concentration of ATP than any other subtypes of P2X receptor (Kaiho et al., 1996). It has been reported that 2′, 3′-dialdehyde ATP (oATP) is an irreversible antagonist of the P2Z/ P2X7 receptor (Murgia et al., 1993; Wiley et al., 1994; Surprenant et al., 1996). P2X7 receptors are generally expressed in cells of hemopoietic origin, such as macrophages and thymocytes, but their function is not clear yet (Surprenant et al., 1996). There are data suggesting a possible regulatory role for ATP in endocrine tissues, since ATP has been shown to increase [Ca2−]i in hypothalamic neurons (Chen et al., 1994). Tomic et al. (1996) suggested that ATP represents a paracrine or autocrine factor in the regulation of [Ca2+]i signaling and secretion of pituitary hormone in gonadotroph cells and this action may be mediated by P2X receptor. It is acknowledged that the rat pituitary growth hormone- and prolactin-secreting GH3 cell line is a useful and well studied model system for the study of pituitary cell signalling (Charles et al., 1999). However, it has not yet been elucidated which subtypes of P2 receptor are expressed in GH3 cells and which mechanisms are involved in purinergic stimulation. In the present study, we confirmed the presence of the endogenous P2X7 receptor and characterized the receptor by combining of fluorimetric techniques, electrophysiological experiments and RT–PCR analysis.