Perturbation of Voltage-Sensitive Ca Channel Function by Volatile Organic Solvents

The mechanisms underlying the acute neurophysiological and behavioral effects of volatile organic compounds (VOCs) remain to be elucidated. However, the function of neuronal ion channels is perturbed by VOCs. The present study examined effects of toluene (TOL), trichloroethylene (TCE), and perchloroethylene (PERC) on whole-cell calcium current (ICa) in nerve growth factor-differentiated pheochromocytoma (PC12) cells. All three VOCs affected ICa in a reversible, concentration-dependent manner. At 10-mV test potentials, VOCs inhibited ICa, whereas at test potentials of 20 and 10 mV, they potentiated it. The order of potency for inhibition (IC50) was PERC (270 M) TOL (720 M) TCE (1525 M). VOCs also changed ICa inactivation kinetics from a singleto double-exponential function. Voltage-ramp experiments suggested that VOCs shifted ICa activation in a hyperpolarizing direction; this was confirmed by calculating the half-maximal voltage of activation (V1/2, act) in the absence and presence of VOCs using the Boltzman equation. V1/2, act was shifted from approximately 2 mV in control to 11, 12, and 16 mV by TOL, TCE, and PERC, respectively. Similarly, VOCs shifted the half-maximal voltage of steady-state inactivation (V1/2, inact) from approximately 16 mV in control to 32, 35, and 20 mV in the presence of TOL, TCE, and PERC, respectively. Inhibition of ICa by TOL was confirmed in primary cultures of cortical neurons, where 827 M TOL inhibited current by 61%. These data demonstrate that VOCs perturb voltage-sensitive Ca channel function in neurons, an effect that could contribute to the acute neurotoxicity of these compounds. Volatile organic compounds (VOCs), such as toluene (TOL), 1,1,1-trichloroethylene (TCE), and 1,1,2,2-tetrachloroethylene (perchloroethylene; PERC), are widely used as paintthinners, industrial degreasing agents, and dry-cleaning agents. Acute exposure to sufficient concentrations of these compounds results in behavioral and neurological deficits, characterized by biphasic changes in locomotor activity (Bushnell et al., 1985), psychomotor impairment (Moser and Balster, 1986), incoordination, sedation (Tegeris and Balster, 1994), and alterations in cognitive ability (Echeverria et al., 1991; Bushnell, 1997). The mechanism(s) underlying the acute effects of these and other VOCs is not well understood and generally has received little attention. VOCs share many effects with CNS depressant compounds, such as ethanol, barbiturates, benzodiazepines, and volatile anesthetics (for review, see Evans and Balster, 1991). Previously, the neuroactivity of volatile anesthetics and related compounds was thought to be attributed to their ability to perturb the fluidity of the plasma membrane, because their potency correlates strongly with their solubility in the organic phase (Meyer, 1899). However, increasing evidence suggests that ethanol (Crews et al., 1996), volatile anesthetics (Yamakura et al., 2001; Dilger, 2002), and VOCs exert acute effects on neuronal function via interactions with a variety of voltageand ligand-gated ion channels. Specifically, TOL has been shown to potentiate currents mediated by GABAA receptors (Beckstead et al., 2000) and to inhibit currents mediated by NMDA (Cruz et al., 1998, 2000) and Preliminary results were presented at the 32nd Annual Meeting of the Society for Neuroscience; 2002 November 2–7; and at the 42nd Annual Meeting of the Society of Toxicology; 2003 March 9–13; and have been published in abstract form [Program 437.28, in 2002 Abstract Viewer/Itinerary Planner, Society for Neuroscience, Washington, DC, 2002. CD-ROM, and in Toxicol Sci (2003) 72 (Suppl 1):266]. The information in this document has been funded wholly by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.090027. ABBREVIATIONS: VOC, volatile organic compound; TOL, toluene; TCE, 1,1,1-trichloroethylene; PERC, perchloroethylene, 1,1,2,2-tetrachloroethylene; CNS, central nervous system; VSCC, voltage-sensitive calcium channel; VSSC, voltage-sensitive sodium channel; NMDA, N-methylD-aspartate; NGF, nerve growth factor; GVIA, -conotoxin GVIA; DMEM, Dulbecco’s modified Eagle’s medium; ICa, whole-cell Ca 2 current; TTX, tetrodotoxin; ANOVA, analysis of variance; I/V, current-voltage. 0022-3565/05/3153-1109–1118 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 315, No. 3 U.S. Government work not protected by U.S. copyright 90027/3060590 JPET 315:1109–1118, 2005 Printed in U.S.A. 1109 at A PE T Jornals on July 1, 2017 jpet.asjournals.org D ow nladed from nicotinic acetylcholine receptors (Bale et al., 2002, 2005). Recently, effects of TOL were demonstrated on voltage-sensitive Ca channels (VSCCs) in pheochromocytoma (PC12) cells (Tillar et al., 2002). Voltage-sensitive calcium channels regulate important neuronal functions, including neurotransmitter release. Alcohol and volatile anesthetics have been demonstrated to disrupt function of VSCCs (Study, 1994; Kamatchi et al., 1999; Kameyama et al., 1999; McMahon et al., 2000), and this action has been suggested to contribute to effects of these compounds on the nervous system. The present studies were designed to test further the hypothesis that VOCs interact with VSCCs. PC12 cells are a clonal cell line that expresses both Nand L-type VSCCs after differentiation with nerve growth factor (NGF). This cell line has been used as a model system to study effects of neurotoxicants (for review, see Shafer and Atchison, 1991) and ethanol (McMahon et al., 2000) on Ca channel function. Toluene reduced KCl-induced Ca responses measured by fura-PE3 as well as reduced whole-cell Ca currents in PC12 cells (Tillar et al., 2002). Thus, this cell line was chosen as an appropriate model to characterize the effects of TOL on VSCCs as well as to examine the effects of TCE and PERC. Specifically, the abilities of TCE and PERC to inhibit VSCC function were examined, and the interactions of TOL, TCE, and PERC with VSCC function in NGF-differentiated PC12 cells were characterized. This included examining the reversibility of VOC effects, concentration-response relationships, and VOC effects on steady-state activation and inactivation. Finally, because PC12 cells are a clonal cell line, the sensitivity of neuronal VSCCs to VOCs was confirmed by examining TOL inhibition of calcium current (ICa) in primary cultures of cortical cells. Materials and Methods