Butyl benzyl phthalate blocks Ca2+ signaling coupled with purinoceptor in rat PC12 cells

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Abstract

Butyl benzyl phthalate (BBP) is a plasticizer and causes public concern because of its genomic estrogenic effects via estrogen receptors. We previously found that BBP has non-genomic effects, exerting inhibitory effects on the functional activities of nicotinic acetylcholine receptors (nAChR) in bovine adrenal chromaffin cells. nAChR belongs to the superfamily of neurotransmitter-gated channels, so does P2X purinoceptor that is widely distributed in the nervous system and play a role in pain reactions. In this study, we investigated the effects of BBP on the change of [Ca2+]c (cytosolic calcium ion concentration) under the stimulation of purinoceptors in PC12 cells and found that BBP inhibited ATP-induced [Ca2+]c rise (IC50 = 8.3 μM). The inhibitory rate of BBP remained under the increase of ATP concentration; therefore, the possibility of competitive inhibition was excluded. The inhibition of BBP on P2Y was excluded because its inhibition on ATP-induced [Ca2+]c rise was not found in the absence of extracellular Ca2+. BBP might have some actions on voltage-operated Ca2+ channels (VOCCs) since BBP inhibited the Ca2+ signaling responding to high K+ stimulation (IC50 = 1.2 μM). We suggest that BBP inhibits the ATP-induced [Ca2+]c rise via its non-competitive inhibition on P2X purinoceptors and VOCCs in the plasma membrane.

Introduction

Phthalates (benzenedicarboxylic acid esters) are a class of widely used plasticizers in PVC. Phthalates are environmental pollutants because they are not chemically bound to the PVC polymer; up to 1% of all phthalates from PVC products seeps out into the environment each year (Furtmann, 1996). Phthalates are generally viewed as endocrine disruptors that interfere with hormone production, release, transport, metabolism, and binding actions (Colborn et al., 1993, Eubanks, 1997, Safe, 1995) and contribute to the development of hormone-dependent cancers (Colborn et al., 1993, Davis et al., 1993). Butyl benzyl phthalate (BBP), a plasticizer commonly found in floor covering, has been found in indoor air at approximately 35 ng/m3 (California Environmental Protection Agency, 1992). BBP also exists in adhesives, sealants, and vinyl foams, potentially subjecting humans to an exposure of 2.6 μg/kg/day (Health Canada, 1999). Based on its wide distributions, BBP is thought to be one of the most important manmade pollutants and has become a high concern in public health.

BBP has recently been shown to induce various estrogen receptor-mediated responses affecting gene expression, cancer cell proliferation, and development (Jobling et al., 1995, Sharpe et al., 1995, Wine et al., 1997). Those responses were revealed by the fact that phthalates compete with estradiol for binding to rainbow trout hepatic estrogen receptor (Jobling et al., 1995) and rat uterine estrogen receptor (Zacharewski et al., 1998). BBP estrogen receptor-mediated responses have also been shown in the recombinant yeast human estrogen receptor screening system (Harris et al., 1997). Estrogen has long-term genomic effects and various short-term, non-genomic effects (Chen and Farese, 1999, Joels, 1997). Estrogen and some other stereo compounds have an effect on membrane neurotransmitter-gated ion channels, including GABAA receptor, nicotinic acetylcholine receptors (Ke and Lukas, 1996, Liu et al., 1996), and purinoceptors (Liu et al., 2001). Using phthalates with similar characteristics as estrogen, we previously demonstrated that phthalates act as inhibitors of the nicotinic acetylcholine receptor (Liu and Lin, 2002, 2003, Liu and Wang, 2004).

Nicotinic acetylcholine receptors (nAChRs), members of the ligand-gated ion channel superfamily of neurotransmitter receptors, are widely distributed in the nervous system (Sargent, 1993). P2X purinoceptors also belong to ligand-gated cation channel receptors. Some steroids act on several types of ligand-gated ion channel receptors: for instance, DHEA suppresses the functional activity of nAChR and P2X purinoceptors (Liu et al., 1996, Liu et al., 2001), and progesterone modulates the activity of the GABA receptor, glycine receptor, and nAChRs (Wu et al., 1991, Wu et al., 1993, Ke and Lukas, 1996). Due to BBP estrogenic effects, we suspected that BBP could affect the activity of various ligand-gated ion channel superfamily receptors. In this study, we investigated the effects of BBP on P2 purinoceptors-mediated Ca2+ signaling using PC12 cells that possess a high number of P2 purinoceptors (Liu et al., 2001).

Section snippets

Chemicals

ATP, UTP, digitonin, and EGTA were all obtained from Sigma Chemical Co. NaCl, KCl, and other salts were obtained from Merck. Fura-2 AM was obtained from Molecular Probes. Butyl benzyl phthalate (BBP; 98% pure) were purchased from Tokyo Kasei Kogyo Co., Ltd. BBP was dissolved in DMSO first and then diluted (at least 100-fold) into the concentration we used in loading buffer. In all the treatments of BBP, the equipment are made of glass or quartz.

Cell culture

Rat pheochromocytoma PC12 cells were obtained from

Results

P2 purinoceptors were stimulated by addition of ATP to PC 12 cells. ATP alone was capable of inducing a transient [Ca2+]c rise, and BBP suppressed the ATP-induced [Ca2+]c rise in a dose-dependent manner with an IC50 of 8.3 μM (Fig. 1). To determine whether a competitive inhibition occurred, ATP concentrations were increased in order to block BBP binding on the agonist binding sites of the P2 purinoceptor. If BBP competed with the agonist (ATP) for binding the P2 purinoceptor, then BBP

Discussion

In this study, we found a novel characteristic of BBP—its ability to act as a channel blocker of the P2X purinoceptor and VOCCs in PC 12 cells. The evidence includes the following: (1) BBP inhibited ATP-induced [Ca2+]c rise only in the presence of extracellular Ca2+, but not in the absence of extracellular Ca2+; (2) the inhibitory effect of BBP on ATP-induced [Ca2+]c rise was maintained when the concentration of ATP was increased; (3) BBP also inhibited high K+-induced [Ca2+]c rise, and this

Acknowledgments

We wish to thank professor Steven Schaufele and Li-Ching Wang for help editing the manuscript. This study was supported by grants from the National Science Council, Taiwan, ROC (NSC-93-2320-B-031-002).

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