Dynamics of Ca -Dependent Cl Channel Modulation by Niflumic Acid in Rabbit Coronary Arterial Myocytes

Calcium-activated chloride channels (ClCa) are crucial regulators of vascular tone by promoting a depolarizing influence on the resting membrane potential of vascular smooth muscle cells. Niflumic acid (NFA), a potent blocker of ClCa in vascular myocytes, was shown recently to cause inhibition and paradoxical stimulation of sustained calcium-activated chloride currents [ICl(Ca)] in rabbit pulmonary artery myocytes. The aims of the present study were to investigate whether NFA produced a similar dual effect in coronary artery smooth muscle cells and to determine the concentration-dependence and dynamics of such a phenomenon. Sustained ICl(Ca) evoked by intracellular Ca clamped at 500 nM were dose-dependently inhibited by NFA (IC50 159 M) and transiently augmented in a concentration-independent manner (10 M to 1 mM) 2-fold after NFA removal. However, the time to peak and duration of NFAenhanced ICl(Ca) increased in a concentration-dependent fashion. Moreover, the rate of recovery was reduced by membrane depolarization, suggesting the involvement of a voltage-dependent step in the interaction of NFA, leading to stimulation of ICl(Ca). Computer simulations derived from a kinetic model involving low (Ki 1.25 mM) and high (Ki 30 M) affinity sites could reproduce the properties of the NFA-modulated ICl(Ca) fairly well. Calcium-activated chloride currents [ICl(Ca)] are expressed in various cell types, including some types of vascular smooth muscle cells, and are considered a key player in the regulation of cell-membrane potential. These currents are evoked by an elevation of intracellular Ca concentration ([Ca ]i) and have distinctive biophysical properties that include a discriminating selectivity pore that conforms to type I Eisenman sequence, voltage-dependent kinetics of activation and deactivation (Greenwood et al., 2001; Ledoux et al., 2003), a calcium sensitivity within the submicromolar range (EC50 365 nM with full activation 600 nM [Ca ]i) (Pacaud et al., 1992; Ledoux et al., 2003), and a small single-channel conductance (1–3 pS) with multiple subconductance states (Klöckner, 1993; van Renterghem and Lazdunski, 1993; Hirakawa et al., 1999; Piper and Large, 2003). However the molecular identity of the channel underlying ICl(Ca) in smooth muscle is still a matter of debate (Britton et al., 2002). Therefore, investigation of the physiological relevance of ICl(Ca) relies on the effectiveness of pharmacological tools. A number of chemically disparate compounds block ICl(Ca) in smooth muscle cells ranging from stilbene derivatives (4,4-diisothiocyanato-stilbene-2,2-disulfonic acid) (Hogg et al., 1994b), anthracene-9-carboxylic acid (A-9-C) (Hogg et al., 1994b), and fenamates (flufenamic acid, niflumic acid) (Hogg et al., 1994a; Greenwood and Large, 1995). Niflumic acid (NFA) is a nonsteroidal anti-inflammatory drug that is recognized as the most potent inhibitor of ICl(Ca) in smooth muscle cells owing to an IC50 within the low micromolar range (Hogg et al., 1994a; Greenwood and Large, 1995). Therefore, NFA has been widely used to assess the physiological role of these currents in the regulation of vascular tone (Criddle et al., 1996, 1997; Yuan, 1997; Lamb and Barna, 1998; Remillard et al., 2000; Dai and Zhang, 2001). However, NFA is not an ideal probe for investigating the This work was supported by a Doctoral Studentship Award (to J.L.) from the Canadian Institute of Health Research (CIHR), a Wellcome Trust Research Career Development Fellowship (number 53793) (to I.A.G.), and grants from the CIHR (MOP-10863), the Montréal Heart Institute Fund, the Western Affiliate of the American Heart Association (0355060Y), and the Center of Biomedical Research Excellence (COBRE; NCRR 5 P20 RR15581), University of Nevada School of Medicine, Reno, Nevada (to N.L.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.004168. ABBREVIATIONS: ICl(Ca), calcium-activated chloride current; NFA, niflumic acid; A-9-C, anthracene-9-carboxylic acid; ClCa, calcium-activated chloride channel; ANOVA, analysis of variance; STIC, spontaneous transient inward current; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N tetraacetic acid; HP, holding potential. 0026-895X/05/6701-163–173$20.00 MOLECULAR PHARMACOLOGY Vol. 67, No. 1 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 4168/1187047 Mol Pharmacol 67:163–173, 2005 Printed in U.S.A. 163 at A PE T Jornals on July 6, 2017 m oharm .aspeurnals.org D ow nladed from role of ICl(Ca), because this agent, along with other members of the fenamate family, modulates a number of other ion channels. Niflumic acid and other fenamates stimulate largeconductance Ca -dependent K channels (Hogg et al., 1994a; Ottolia and Toro, 1994; Greenwood and Large, 1995). Flufenamic acid and tolfenamic acids relaxed guinea pig tracheal contractions through a direct inhibition of voltagegated Ca channels (Li et al., 1998). Niflumic acid stimulated Ca release from internal stores in rat pulmonary artery smooth muscle cells (Cruickshank et al., 2003) and was shown, along with 5-nitro-2-(3-phenylpropylamino)-benzoic acid and 4,4-diisothiocyanato-stilbene-2,2-disulfonic acid, to inhibit endothelin-1–induced contractions of rat pulmonary arterial myocytes by a mechanism independent of block of ClCa channels (Kato et al., 1999). Moreover, the IC50 for NFA block of spontaneously occurring ICl(Ca) is significantly lower than that required to block ICl(Ca) evoked by caffeine or an agonist such as norepinephrine (Large and Wang, 1996). This phenomenon is extended further when the channel is stimulated by a sustained level of [Ca ] provided by the pipette solution in which a number of paradoxical effects are observed. In rabbit pulmonary myocytes, NFA only blocked sustained ICl(Ca) at positive potentials and augmented the tonic current at the holding potential of 50 mV (Piper et al., 2002). Upon washout of NFA, there was a marked enhancement of current amplitude greater than control values that was associated with a marked increase in the activation kinetics. Similar effects were observed for the chemically related agent dichloro-diphenyl 2-carboxylic acid (Piper et al., 2002), whereas A-9-C, which inhibits ICl(Ca) in a strongly voltage-dependent manner, produced a marked 3-fold increase in the deactivating tail current at 80 mV (Piper and Greenwood, 2003). These data suggest that in pulmonary artery myocytes, fenamateand A-9-C–induced inhibition of ICl(Ca) masks a parallel stimulation that is revealed either by washout of the drug (NFA) or by voltagedependent dissociation from the channel (A-9-C). It is presently unknown whether the NFA-induced stimulation of ICl(Ca) in pulmonary artery myocytes (Piper et al., 2002) is unique to the vascular smooth muscle preparation. In addition, Piper et al. (2002) have used a slow superfusion system to monitor the effects of NFA on ICl(Ca), and thus, the true time course of stimulation of this current after washout of NFA is unclear. Finally, information on the concentration dependence of the enhancing effect of NFA on ICl(Ca) is lacking, because Piper et al. (2002) have only examined the effect of a single concentration of NFA (100 M). The aim of the present study was to determine whether a similar anomalous effect of NFA was observed on sustained ICl(Ca) in rabbit coronary artery myocytes and to investigate the dynamics and concentration dependence of this stimulation using a computer-assisted fast-flow superfusion system. We report here that the relative increase of ICl(Ca) is independent of drug concentration, whereas the time to reach the maximal stimulation and the duration of the enhanced state increased in a concentration-dependent manner. The rate of recovery from stimulation was also voltage-dependent, with negative potentials accelerating recuperation from enhanced ICl(Ca). We propose a model whereby NFA stimulates ICl(Ca) by binding to highand low-affinity sites, which are occluded by the interaction of NFA with the inhibitory site. Preliminary results have been presented previously (Ledoux and Leblanc, 2002). Methods and Materials Isolation of Coronary Myocytes. Cells were freshly dissociated from coronary arteries isolated from New Zealand white rabbits (2–3 kg) that had been killed by anesthetic overdose in accordance with Canadian and U.S. regulations. All animal-handling protocols received the approval of local ethics committees. Arterial myocytes were isolated from the left descending and circumflex coronary arteries. After dissection and removal of connective tissue, the coronary arteries were cut into small strips and placed in a physiological salt solution containing no added Ca and 100 M EGTA at 22°C for 30 min. The coronary arteries were then incubated in a physiological salt solution containing 10 M Ca (no EGTA) and 1 mg/ml collagenase type 2 (Worthington Biochemicals, Lakewood, NJ) and 0.05 mg/ml protease type I (Sigma-Aldrich, St. Louis, MO) for 20 to 25 min at 35°C. In all cases, cells were released by gentle agitation with a wide-bore Pasteur pipette. Cells were stored at 4°C and were