ATP Receptor-Mediated Increase of Ca Ionophore-Stimulated Arachidonic Acid Release from PC12 Pheochromocytoma Cells

Abstract

ABSTRACT-PhospholipaseA2 has recently been proposed as the effector enzyme involved in the receptor-mediated release of arachidonic acid (AA). Released AA and its metabolites have been demonstrated to play an important role in the regulation of cell functions.[3H]AA release from prelabeled PC12 cells was stimulated by a Ca ionophore such as ionomycin or A23187. Although ATP and its effective analogs, adenosine 5'-O-(3-thiotrisphosphate) (ATPrS), 2-methylthio ATP and 3'-O-(4-benzoyl)benzoyl ATP, did not stimulate [3H]AA release on their own, they did enhance Ca ionophore-stimulated[3H]AA release. The effect of ATP analogs was dose-dependent. ADP, UTP, GTP, ITP, a f3-methylene ATP, i37-methylene ATP and 8-bromo ATP showed no effect or very limited effect. The effect of ATPrS was antagonized by suramin, a putative P2Y receptor antagonist. The effective ATP analogs also increased [Ca 2+], (cytosolic free Ca2+ concentration) via Ca 2+ influx. However, the addition of 50 mM KCl or 10 PM bradykinin, which are well-known to increase [Ca 2+]; by different pathways, did not stimulate [3H]AA release, either with or without the Ca ionophore. The addition of phorbol 12-myristate 13-acetate, an activator of protein kinase C, showed no effect on [3H]AA release, either with or without the Ca ionophore. These data suggest that 1) ATP increased Ca ionophore-stimulated AA release via a P2Y-like ATP receptor, and that 2) the elevation of [Ca 2+]; by ATP does not quantitatively explain the ATP-stimulated AA release in PC12 cells.Keywords: ATP, Arachidonic acid, Intracellular calcium, PC12 cell Purine nucleotides and nucleosides have widespread and potent extracellular actions on a variety of excitable and non-excitable cells. Burnstock (1) proposed two types of receptors: the P1 receptor for adenosine, and the P2 receptor for ATP. Different subtypes of the P2 receptor have been defined classically as follows (1-3): P2x and P2Y receptor subclasses have been differentiated on the basis of relative potencies of the ATP analogs and selective antagonism. Thus, for the P2X receptor: aj3-methylene ATP = (3r-methylene ATP > ATP; P2Y receptor: 2-methylthio ATP >_ ATP > a~-methylene ATP = Prmethylene ATP. P2x receptors are preferentially located in vascular smooth muscle cells and mediate vasoconstriction. P2Y receptors are located on a variety of cell types and regulate cell functions via the activation of phospholipase C. Suramin and reactive blue 2 have been claimed to be specific antagonists to P2Y receptors, at least over a limited concentration range (4, 5). Since the receptors for ATP on platelets, mast cells, lymphocytes and fibroblasts do not seem to fit this subclassification, they have been termed P2T and P2z receptors, respectively (1-3). The P2T receptors found in platelets are unique in being activated by ADP rather than ATP. The actual agonist form for P2z receptors is thought to be the tetrabasic acid ATP4-(6), because Mg2+ addition increased the ED50 (dose required to achieve 50010 of maximal effect) value for ATP. The rank o...

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

“…Previous studies have suggested that PC12 cells also contain P2Y (Sela et al 1991;Nikodijevic et al 1994;Murayama et al 1995) and P2U (Raha et al 1993;Majid et al 1993;Nikodijevic et al 1994;Barry and Cheek 1994) purinoceptors and it is possible that activation of these G-protein coupled receptors could have stimulated 45Ca 2+ influx through mobilisation of intracellular calcium and refilling of intracellular stores, particularly through capacitative mechanisms involving calcium-influx across the cell membrane. The potential involvement of such a mechanism in the 45Caa+ influx measurements made in the present study is unlikely, however, for a number of reasons.…”

“…Ideally for such studies stable cell lines expressing one or other of the various recombinant receptor types would be of choice. However, in their absence PC12 cells were utilised as an alternative since there is extensive evidence that they contain a number of P2 purinoceptor subtypes, and in particular an ATP-gated channel receptor, obtained using a wide range of techniques such as electrophysiology (Inoue et al 1989;Nakazawa et al 1990) fura-2 calcium measurements (Fasolato et al 1990;Nakazawa and Inoue 1992;Raha et al 1993;Kim and Rabin 1994;Barry and Cheek 1994;Gollasch and Hailer 1995), neurotransmitter release (Inoue et al 1989;Sela et al 1991;Majid et al 1992), arachadonic acid release (Murayama et al 1995), inositol phosphate accumulation (Murrin and Boarder 1992;Majid et al 1993;Raha et al 1993;Barry and Cheek 1994) and 45Ca2+ influx (Seta et al 1991;Kim and Rabin 1994). Indeed the P2X2 purinoceptor gene was cloned from this cell line by Brake et al (1994) and our own studies have identified mRNA for the P2Xa purinoceptor in the cells used in this study (C.B.…”

Purinergic signalling and cancer

“…Reduction of ACh-activated current by ATP has also been observed [709]. ATP, acting through P2Y receptors, leads to the release of arachidonic acid from PC12 cells [710]. Diadenosine tetraphosphate, probably acting via a P2Y receptor, increased [Ca 2+ ] i in PC12 cells [711].…”

Direct dopamine D2‐receptor‐mediated modulation of arachidonic acid release in transfected CHO cells without the concomitant administration of a Ca2+‐mobilizing agent

“…The method of detecting release of incorporated [ 3 H]‐arachidonic acid as an index of living whole cell membrane AA metabolism is well established. Thus, HPLC and TLC analyses have revealed that high amounts (≈ 90–99%) of released radioactivity in the presence of BSA is attributable to free AA (Kanterman et al , 1991; Piomelli et al , 1991; Muruyama et al , 1995). Baseline control levels of d.p.m.…”