title: Effect of non-steroidal anti-inflammatory drugs and new fenamate analogues on TRPC4 and TRPC5 channels.
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.
authors.affiliation: Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull HU6 7RX, UK.
authors.forename: Hongni
authors.initials: H
authors.lastname: Jiang
authors.forename: Bo
authors.initials: B
authors.lastname: Zeng
authors.forename: Gui-Lan
authors.initials: GL
authors.lastname: Chen
authors.forename: David
authors.initials: D
authors.lastname: Bot
authors.forename: Sarah
authors.initials: S
authors.lastname: Eastmond
authors.forename: Sandra E
authors.initials: SE
authors.lastname: Elsenussi
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Atkin
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Boa
authors.forename: Shang-Zhong
authors.initials: SZ
authors.lastname: Xu
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques; D054361:Fenamates / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000737:chemistry; D016257:Fura-2 / Q000031:analogs & derivatives / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D018408:Patch-Clamp Techniques; D051381:Rats; D013329:Structure-Activity Relationship; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors* / Q000235:genetics; D014162:Transfection
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D054361:Fenamates; D005456:Fluorescent Dyes; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; C491780:TRPC5 protein, human; C098106:fura-PE3; D002118:Calcium; D016257:Fura-2