Action of diphenylamine carboxylate derivatives, a family of non-steroidal anti-inflammatory drugs, on [Ca2+]i and Ca(2+)-activated channels in neurons.
ABSTRACT Ca(2+)-activated channels, including Ca(2+)-activated non-selective (CAN) channels and Ca(2+)-activated Cl- channels play important roles in regulating the electrical activity of neurons. No blockers of neuronal CAN channels have been previously reported. We used 2-electrode voltage clamping to measure membrane currents and fura-2 fluorescence imaging to measure [Ca2+]i in molluscan neurons. We show that the diphenylamine carboxylate derivative flufenamate (FFA), but not mefenamate or the parent compound, cause a transient increase in ICAN and a slow outward current, and a maintained increase in [Ca2+]i. We interpret this as a FFA-dependent release of Ca2+ from intracellular stores and Ca2+ influx, [Ca2+]i-dependent activation of the CAN and slow outward currents, and slow FFA-dependent channel block.
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ABSTRACT: Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used anti-inflammatory therapeutic agents, among which the fenamate analogues play important roles in regulating intracellular Ca²⁺ transient and ion channels. However, the effect of NSAIDs on TRPC4 and TRPC5 is still unknown. To understand the structure-activity of fenamate analogues on TRPC channels, we have synthesized a series of fenamate analogues and investigated their effects on TRPC4 and TRPC5 channels. Human TRPC4 and TRPC5 cDNAs in tetracycline-regulated vectors were transfected into HEK293 T-REx cells. The whole cell current and Ca²⁺ movement were recorded by patch clamp and calcium imaging, respectively. Flufenamic acid (FFA), mefenamic acid (MFA), niflumic acid (NFA) and diclofenac sodium (DFS) showed inhibition on TRPC4 and TRPC5 channels in a concentration-dependent manner. The potency was FFA>MFA>NFA>DFS. Modification of 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with F, CH₃, OCH₃, OCH₂CH₃, COOH, and NO₂ led to the changes in their channel blocking activity. However, 2-(2'-methoxy-5'-methylphenyl)aminobenzoic acid stimulated TRPC4 and TRPC5 channels. Selective COX1-3 inhibitors (aspirin, celecoxib, acetaminophen, and indomethacin) had no effect on the channels. Longer perfusion (> 5 min) with FFA (100 μM) and MFA (100 μM) caused a potentiation of TRPC4 and TRPC5 currents after their initial blocking effects that appeared to be partially mediated by the mitochondrial Ca²⁺ release. Our results suggest that fenamate analogues are direct modulators of TRPC4 and TRPC5 channels. The substitution pattern and conformation of the 2-phenylamino ring could alter their blocking activity, which is important for understanding fenamate pharmacology and new drug development targeting the TRPC channels.Biochemical pharmacology 04/2012; 83(7):923-31. DOI:10.1016/j.bcp.2012.01.014 · 4.25 Impact Factor
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ABSTRACT: TMEM16A/Anoctamin-1 (ANO1) is a protein widely expressed in mammalian tissues and has the properties of the classical calcium-activated chloride channel (CaCC). This protein has been implicated in numerous major physiological functions. However, the lack of effective and selective blockers has hindered a detailed study of the physiological functions of this channel. In this study, we have developed a potent and selective blocker for endogenous ANO1 in Xenopus laevis oocytes (xANO1) using a drug screening method we had previously established (Oh et al., 2008). We have synthesized a number of anthranilic acid derivatives and have determined the correlation between biological activity and the nature and position of substituents in these derived compounds. A structure-activity relationship (SAR) revealed novel chemical classes of xANO1 blockers. The derivatives contain an -NO2 group on position 5 of a naphthyl group substituted anthranilic acid, and they fully blocked xANO1 chloride currents with an IC50 < 10 μM. The most potent blocker, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) had an IC50 of 0.08 μM for xANO1. Selectivity tests revealed that other chloride channels such as bestrophin-1, CLC2 and cystic fibrosis transmembrane conductance regulator (CFTR) were not appreciably blocked by 10 - 30 μM MONNA. The potent and selective blockers for ANO1 identified here should permit pharmacological dissection of ANO1/CaCC function and serve as potential candidates for drug therapy of related diseases such as hypertension, cystic fibrosis, bronchitis, asthma and hyperalgesia.Molecular pharmacology 08/2013; DOI:10.1124/mol.113.087502 · 4.12 Impact Factor
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ABSTRACT: BACKGROUND AND PURPOSE Fenamate analogues, econazole and 2-aminoethoxydiphenyl borate (2-APB) are inhibitors of transient receptor potential melastatin 2 (TRPM2) channels and are used as research tools. However, these compounds have different chemical structures and therapeutic applications. Here we have investigated the pharmacological profile of TRPM2 channels by application of newly synthesized fenamate analogues and the existing channel blockers. EXPERIMENTAL APPROACH Human TRPM2 channels in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells and the expression was induced by tetracycline. Whole cell currents were recorded by patch-clamp techniques. Ca(2+) influx or release was monitored by fluorometry. KEY RESULTS Flufenamic acid (FFA), mefenamic acid (MFA) and niflumic acid (NFA) concentration-dependently inhibited TRPM2 current with potency order FFA > MFA = NFA. Modification of the 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with -CH(3) , -F, -CF(3) , -OCH(3) , -OCH(2) CH(3) , -COOH, and -NO(2) at various positions, reduced channel blocking potency. The conservative substitution of 3-CF(3) in FFA by -CH(3) (3-MFA), however, gave the most potent fenamate analogue with an IC(50) of 76 µM, comparable to that of FFA, but unlike FFA, had no effect on Ca(2+) release. 3-MFA and FFA inhibited the channel intracellularly. Econazole and 2-APB showed non-selectivity by altering cytosolic Ca(2+) movement. Econazole also evoked a non-selective current. CONCLUSION AND IMPLICATIONS The fenamate analogue 3-MFA was more selective than other TRPM2 channel blockers. FFA, 2-APB and econazole should be used with caution as TRPM2 channel blockers, as these compounds can interfere with intracellular Ca(2+) movement.British Journal of Pharmacology 05/2012; 167(6):1232-43. DOI:10.1111/j.1476-5381.2012.02058.x · 5.07 Impact Factor