Action of diphenylamine carboxylate derivatives, a family of non-steroidal anti-inflammatory drugs, on [Ca2+]i and Ca2+-activated channels in neurons

doi:10.1016/0304-3940(95)11518-2

Neuroscience Letters

Volume 190, Issue 2, 5 May 1995, Pages 121-124

https://doi.org/10.1016/0304-3940(95)11518-2 Get rights and content

Abstract

Ca²⁺-activated channels, including Ca²⁺-activated non-selective (CAN) channels and Ca²⁺-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 I_CAN 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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Cited by (27)

Effect of non-steroidal anti-inflammatory drugs and new fenamate analogues on TRPC4 and TRPC5 channels
2012, Biochemical Pharmacology
Citation Excerpt :
Fenamates are effective anti-inflammatory agents through COX-1 and COX-2 inhibition, and also inhibit a variety of ion channel activities in many cell types. Early studies have shown that fenamates inhibited Ca2+-activated chloride channels [36–38], Ca2+-activated potassium channels [39], and the Ca2+-activated non-selective cationic channel [40]. Recently, it has been demonstrated that FFA activated TRPC6 [28], TRPA1 [29], and an OAG-sensitive cationic current in A7r5 cells [26], but inhibited TRPM2, TRPM3, TRPM4 and TRPM5 channels [22,24,41–44].
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.
The anti-inflammatory agent flufenamic acid depresses store-operated channels by altering mitochondrial calcium homeostasis
2009, Neuropharmacology
Citation Excerpt :
Previous works showed that FFA or related compounds release stored Ca2+ in many cell types like the mandibular cell line ST885, parotid secretory cells, mandibular secretory cells, parathyroid cells, unfertilized oocytes, cultured insulinoma (RINm5F), cardiac myocytes (Poronnik et al., 1992) and smooth muscle cells (Cruickshank et al., 2003). In neurons, fenamates influence the homeostasis of Ca2+ by releasing Ca2+ from Tg-sensitive compartments in molluscan neurons (Lee et al., 1996; Shaw et al., 1995) and from Tg-insensitive Ca2+ compartments in hippocampal (Partridge and Valenzuela, 1999) and cortical neurons (present study). We provide experimental evidence for a FFA-induced alteration of the mitochondrial Ca2+ homeostasis in neurons of the central nervous system.
Fenamates like flufenamic acid (FFA) are anti-inflammatory drugs known to alter ion fluxes through the plasma membrane. They are for instance potent blockers of cation and anion channels, and FFA is now commonly used to block currents through TRP channels and receptor-operated channels. However, FFA exerts complex and multifaceted actions on ion transport systems and, in most instances, a molecular understanding of these FFA-dependent modulations is lacking. In addition, FFA is also to known to perturb the homeostasis of Ca²⁺. In the present report, we investigated whether the FFA-induced alterations of the Ca²⁺ homeostasis could play a role in the FFA-dependent modulation of transmembrane ion fluxes. Experiments performed with the Ca²⁺ indicator Fluo-4 on cultured cortical neurons and HEK-293 cells showed that FFA increased the cytosolic concentration of Ca²⁺ even in cells kept in a Ca²⁺-free medium or when the endoplasmic reticulum was depleted with thapsigargin. The FFA-dependent Ca²⁺ responses were, however, strongly reduced by bongkrekic acid, a specific ligand of the mitochondrial ADP/ATP carrier which, in addition, inhibits the permeability transition pore. Like FCCP, FFA released Ca²⁺ from isolated brain mitochondria and indirectly modulates store-operated Ca²⁺ channels. We suggest that some of the effects of FFA on plasma membrane ion channels could be explained, at least partially, by its ability to modulate the mitochondrial Ca²⁺ homeostasis.
Diphenylamine and derivatives in the environment: A review
2003, Chemosphere
Diphenylamine (DPA) is a compound from the third European Union (EU) list of priority pollutants. It was assigned by the EU to Germany to assess and control its environmental risks. DPA and derivatives are most commonly used as stabilizers in nitrocellulose-containing explosives and propellants, in the perfumery, and as antioxidants in the rubber and elastomer industry. DPA is also widely used to prevent post-harvest deterioration of apple and pear crops. DPA is a parent compound of many derivatives, which are used for the production of dyes, pharmaceuticals, photography chemicals and further small-scale applications. Diphenylamines are still produced worldwide by the chemical industries. First reports showed that DPA was found in soil and groundwater. Some ecotoxicological studies demonstrated the potential hazard of various diphenylamines to the aquatic environment and to bacteria and animals. Studies on the biodegradability of DPA and its derivatives are very sparse. Therefore, further investigation is required to determine the complete dimension of the potential environmental hazard and to introduce possible (bio)remediation techniques for sites that are contaminated with this class of compounds. This is the first detailed review on DPA and some derivatives summarizing their environmental relevance as it is published in the literature so far and this review will recommend conducting further research in the future.
Diclofenac inhibition of sodium currents in rat dorsal root ganglion neurons
2003, Brain Research
Citation Excerpt :
Acetylsalicylic acid, antipyrin, flufenamic acid and indomethacin changed the amplitude of TTX-S sodium current to 104±4%, 105±4%, 38±2% and 82±5% of control, respectively, and that of TTX-R sodium current to 102±2%, 99±1%, 96±5% and 93±0.0% of control, respectively. Besides COX inhibition many NSAIDs modulate activities of various ion channels [11,24,28,31,38,42]. In this report, we have shown that diclofenac suppresses both the fast TTX-S and the slow TTX-R sodium currents in neonatal rat DRG neurons.
The effects of diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), on the fast tetrodotoxin-sensitive (TTX-S) and the slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons were investigated using the whole-cell patch-clamp method. Diclofenac suppressed both sodium currents in a dose-dependent manner. The apparent dissociation constants for the diclofenac suppression of TTX-S and TTX-R sodium currents were estimated to be 14 and 97 μM, respectively, at a holding potential of −80 mV. Diclofenac had no effect on the kinetic parameters of the activation process in either type of sodium current. However, diclofenac produced shifts of the steady-state inactivation curves in the hyperpolarizing direction in both types of sodium currents in a dose-dependent manner. At sufficiently negative holding potentials, the inhibitory effects of diclofenac on both types of sodium currents were minimal. The results suggested that diclofenac might bind to sodium channels with a greater affinity when they are in the inactivated state than when they are in the resting state. Effects of other NSAIDs (acetylsalicylic acid, antipyrin, indomethacin and flufenamic acid) on sodium currents were tested. Among these, only flufenamic acid suppressed the sodium currents to a considerable extent. Thus, the chemical structure of each NSAID, not the inhibition of cyclooxygenase, seems to be an important determinant in the sodium current inhibition. The suppression of sodium currents in sensory neurons by diclofenac and flufenamic acid would contribute to their analgesic activity in addition to their inhibition of cyclooxygenase.
Block of hippocampal CAN channels by flufenamate
2000, Brain Research
Citation Excerpt :
Taken together, these observations support our conclusion that the effects of FFA that we report here were specific effects of FFA on CAN channels in postsynaptic CA1 neurons and not the result of effects on action potentials, Cl− channels, or presynaptic Ca2+-dependent transmitter release. FFA causes a rapid, maintained increase in [Ca2+]i. Similar increases in [Ca2+]i in the presence of FFA have been reported in a mandibular cell line [15], jejunal circular smooth muscle cells [10], and molluscan neurons [19,7]. Unlike the observation in molluscan neurons [7], however, pretreatment with thapsigargin did not eliminate the [Ca2+]i response in CA1 neurons (see Fig. 3B) and there was no further increase in [Ca2+]i when thapsigargin was applied after the FFA-induced [Ca2+]i plateau (data not shown).
Ca²⁺-activated non-selective cation (CAN) channels are activated by cytoplasmic Ca²⁺ and I_CAN underlies many slow depolarizing processes in neurons including a putative role in excitotoxicity. CAN channels in many non-neuronal cells are blocked by non-steroidal antiinflammatory drugs that are derivatives of diphenylamine-2-carboxylate (DPC). The DPC derivative flufenamate (FFA) has a complex effect on certain neurons, whereby it blocks CAN channels and increases [Ca²⁺]_i. We report here that FFA, but not the parent compound, DPC, blocks CAN channels in hippocampal CA1 neurons. As was the case in other neurons, the effects of FFA are complex and include a maintained rise in [Ca²⁺]_i. Furthermore, the CAN channel blocking ability of FFA persists even when the channels have been potentiated by a Ca²⁺-dependent process. The use of a CAN channel-blocking drug is important for delineating CAN channel-dependent processes and may provide a basis for therapy for CAN channel-dependent events in ischemia.
Fenamates protect neurons against ischemic and excitotoxic injury in chick embryo retina
1998, Neuroscience Letters
Three fenamates (flufenamate, meclofenamate and mefenamate) were examined for their protective effect on neurons under ischemic (glucose/oxygen deprivation) or excitotoxic conditions, using the isolated retina of chick embryo as a model. Retinal damage was evaluated by histology and lactate dehydrogenase assay. Whole-cell recording was used to examine the direct effect of the fenamates on glutamate receptor-mediated currents. The fenamates protected the retina against the ischemic or excitotoxic insult. Part of the neuroprotection by the fenamates derived from inhibition of N-methyl-d-aspartate receptor-mediated currents. However, kainate receptor-mediated currents were not blocked by the fenamates, which nonetheless reduced kainate receptor-mediated retinal damage. Our results raise the possibility that fenamates may serve as lead structures in the development of novel therapeutic agents against brain ischemia.

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^☆: This work was supported in part by NSF grant BNS 9024740 to L.D. Partridge. The fluorescence ratio imaging microscope is a shared instrument in the Microscopy and Image Processing Facility of the University of New Mexico Cancer Research and Treatment Center.

¹: The authors would like to acknowledge the able laboratory assistance of Ken McCann and Michael Sandquist.

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Action of diphenylamine carboxylate derivatives, a family of non-steroidal anti-inflammatory drugs, on [Ca2+]i and Ca2+-activated channels in neurons☆

Abstract

Life Sci.

Eur. J. Pharmacol.

FEBS Lett.

J. Biol. Chem.

Biochem. Pharmacol.

Trends Neurosci.

Brain Res. Rev.

Brain Res.

Biochim. Biophys. Acta

FEBS Lett.

Voltage-activated and calcium-activated currents studied in solitary rod inner segments from the salamander retina

J. Physiol.

Calcium buffering and slow recovery kinetics of calcium-dependent outward current in molluscan neurones

J. Physiol.

Cerebral hypoxia - some new approaches and unanswered questions

J. Neurosci.

Inhibition of anion permeability by amphophilic compounds in human red cell: evidence for an interaction of niflumic acid with the band 3 protein

J. Membr. Biol.

Large conductance Ca2+-activated K+ channels are involved in both spike shaping and firing regulation in Helix neurones

J. Physiol.

Action of diphenylamine carboxylate derivatives, a family of non-steroidal anti-inflammatory drugs, on [Ca²⁺]_i and Ca²⁺-activated channels in neurons☆

Large conductance Ca²⁺-activated K⁺ channels are involved in both spike shaping and firing regulation in Helix neurones