P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells

Abstract

ATP-induced Ca²⁺ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca²⁺ response was entirely dependent on Ca²⁺ influx, could not be mimicked by UTP, α,β-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP₃). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X₄ subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca²⁺ increase was due to mobilisation of intracellular Ca²⁺ stores and another to capacitative Ca²⁺ entry. UTP caused an increase in intracellular Ca²⁺, and InsP₃ formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y₂ subtype.

Introduction

PC12 cells, derived from a spontaneous rat phaeochromocytoma (Greene and Tischler, 1976), are widely used to study e.g. neuronal differentiation, effects of growth factors and regulation of neurotransmitter release (Greene et al., 1991). We have compared ATP-induced Ca²⁺ transients in undifferentiated and in nerve growth factor (NGF)-differentiated cells (Greene et al., 1991) to determine if the response to ATP is modified with the phenotype of PC12 cells.

Extracellular ATP and related nucleotides are known to influence most cells via activation of cell membrane receptors (see Ralevic and Burnstock, 1998). These receptors belong to two different families: G protein coupled P2Y receptors and transmitter gated ion channels, P2X receptors (Ralevic and Burnstock, 1998). Both P2Y receptors and P2X receptors are known to increase intracellular Ca²⁺ levels. P2Y receptors act by stimulating G_q/11 proteins, by mobilising Ca²⁺ from intracellular depots, and by permitting influx via specific receptor-operated channels (Dubyak and el-Moatassim, 1993, Clementi and Meldolesi, 1996). P2X receptors increase intracellular Ca²⁺ levels by allowing Ca²⁺ ions to pass through the opened channel along the ionic gradient (see Ralevic and Burnstock, 1998). The pharmacological characterisation of the receptor(s) responsible for mediating increases in intracellular Ca²⁺ in tissues and cells has been complicated by the fact that there are few selective antagonists and that multiple receptor subtypes may co-exist in tissues and cells (Ralevic and Burnstock, 1998).

ATP has been shown to increase cytosolic Ca²⁺ (Fasolato et al., 1990) in PC12 cells via different mechanisms: opening of a receptor-operated channel (Fasolato et al., 1990, Michel et al., 1996), activation of phospholipase C (Murrin and Boarder, 1992) and opening of voltage sensitive Ca²⁺ channels secondarily to an ATP mediated depolarisation (Fasolato et al., 1990). These data have been replicated many times, but there are often marked quantitative differences between the results of different studies. Not only do the conditions under which PC12 cells are cultured differ between laboratories, but these cells are notorious for their ability to change their phenotype, both when cultured with differentiating factors and spontaneously (Greene et al., 1991). We have therefore examined the effects of NGF-induced differentiation in a clone of PC12 cells that was isolated based on neomycin resistance (clone 1 in the original article) (Zacchetti et al., 1991, Clementi et al., 1992b). This clone shows a rather stable genotype and has proven useful in studies of Ca²⁺ movements (Fasolato et al., 1991, Zacchetti et al., 1991) as well as gene and marker expression (Clementi et al., 1992a). Since a major aim was to determine if responses to ATP could be due to activation of more than one type of receptor in a PC12 cell we have performed the studies mostly on single cells. Our results indicate that in differentiated PC12 cells both P2X and P2Y receptors contribute to the Ca²⁺ response, whereas in undifferentiated PC12 cells ATP-induced effects are only mediated via P2X receptors.