Antagonist effect of flufenamic acid on TRPM2 cation channels activated by hydrogen peroxide

The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca(2+)-permeable cation channel that is activated by hydrogen peroxide (H(2)O(2)) as a consequence of oxidative stress although the channel activation by H(2)O(2) appears to represent a cell-specific process in cells with endogenous expression of TRPM2. Flufenamic acid (FA) is a non-steroidal anti-inflammatory compound. Whether H(2)O(2) activates or FA inhibits TRPM2 channels in Chinese hamster ovary (CHO) cell is currently unknown. Due to lack of known antogonists of this channel, we demonstrate in CHO cells that FA inhibits TRPM2 activated by extracellular H(2)O(2). CHO cells were transfected with cDNA coding for TRPM2. Cells were studied with the conventional whole-cell patch clamp technique. The intracellular solution used EDTA (10 mM) as chelator for Ca(2+) and heavy metal ions. H(2)O(2) (10 mM) and FA (0.1 mM) were applied extracellularly. Non-selective cation currents were consistently induced by H(2)O(2). The time cause of H(2)O(2) effects was characterized by a delay of 2-5 min and a slow current induction to reach a plateau. The H(2)O(2)- induced inward current was effectively inhibited by 0.1 mM FA applied extracellularly. In conclusion, we have demonstrated that FA is an effective antogonist of TRPM2 channels and H(2)O(2)activated currents in CHO cells. FA in CHO cells may be considered, at best, a starting point for the development of TRPM2 channel blockers.     

Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H₂O₂), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H₂O₂ for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H₂O₂ and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H₂O₂ in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia. nonselective cation channel; transient receptor potential channel; H₂O₂; activated microglia     

Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes

TRPM2 (transient receptor potential melastatin 2) is a Ca2+-permeable cation channel gated by ADPR (ADP-ribose) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC method to determine ADPR contents in HClO4 cell extracts. The reversed-phase ion-pair HPLC method with an Mg2+-containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were approx. 5 muM in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP (N-formylmethionyl-leucylphenylalanine). We furthermore determined intracellular concentrations of cADPR (cyclic ADPR) with a cyclase assay involving enzymatic conversion of cADPR into NAD+ and fluorimetric determination of NAD+. Intracellular cADPR concentrations were approx. 0.2 microM and not altered by fMLP. In patch-clamp experiments, ADPR (0.1-100 microM) was dialysed into granulocytes to analyse its effects on whole-cell currents characteristic for TRPM2, in the presence of a low (<10 nM) or a high (1 microM) intracellular Ca2+ concentration. TRPM2 currents were significantly larger at high than at low [Ca2+] (e.g. -225+/-27.1 versus -7+/-2.0 pA/pF at 5 muM ADPR), but no currents at all were observed in the absence of ADPR (ADPR concentration < or =0.3 microM). cADPR (0.1, 0.3 and 10 microM) was without effect even in the presence of subthreshold ADPR (0.1 microM). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca2+. Thus ADPR and Ca2+ in concert behave as a messenger system for agonist-induced influx of Ca2+ through TRPM2 in granulocytes.     

Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels

TRPM2 is a member of the transient receptor potential melastatin-related (TRPM) family of cation channels, which possesses both ion channel and ADP-ribose hydrolase functions. TRPM2 has been shown to gate in response to oxidative and nitrosative stresses, but the mechanism through which TRPM2 gating is induced by these types of stimuli is not clear. Here we show through structure-guided mutagenesis that TRPM2 gating by ADP-ribose and both oxidative and nitrosative stresses requires an intact ADP-ribose binding cleft in the C-terminal nudix domain. We also show that oxidative/nitrosative stress-induced gating can be inhibited by pharmacological reagents predicted to inhibit NAD hydrolysis to ADP-ribose and by suppression of ADP-ribose accumulation by cytosolic or mitochondrial overexpression of an enzyme that specifically hydrolyzes ADP-ribose. Overall, our data are most consistent with a model of oxidative and nitrosative stress-induced TRPM2 activation in which mitochondria are induced to produce free ADP-ribose and release it to the cytosol, where its subsequent accumulation induces TRPM2 gating via interaction within a binding cleft in the C-terminal NUDT9-H domain of TRPM2.     

TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35 degrees C) apparently via direct heat-evoked channel gating. beta-NAD(+)- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25 degrees C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca(2+) and insulin release, which is K(ATP) channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca(2+) entry into pancreatic beta-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion.     

Sites of the NUDT9-H domain critical for ADP-ribose activation of the cation channel TRPM2

TRPM2 is a cation channel unique within the transient receptor potential family because of its gating by ADP-ribose (ADPR). ADPR gating is enabled by a cytosolic C-terminal Nudix box sequence motif embedded into a region homologous to the NUDT9 ADPR pyrophosphatase. A recently discovered splice variant of TRPM2 (TRPM2-DeltaC) lacks 34 amino acid residues in the NUDT9 domain and is insensitive to ADPR. To analyze in detail which parts of the deleted sequence (DeltaC-stretch) are critical for ADPR gating, we tested mutants that lacked 19, 25, and 29 amino acid residues in the N-terminal part or had amino acid residues substituted in the remaining C-terminal part of the DeltaC-stretch. All of these mutants displayed typical ADPR-induced currents. However, the deletion or substitution of the amino acid residue Asn-1326 immediately downstream of the DeltaC-stretch abrogated ADPR gating. We furthermore analyzed the mutation I1405E/L1406F in the Nudix box of TRPM2, because a considerably decreased AD-PRase activity of the TRPM2 NUDT9-H protein in comparison to the NUDT9 pyrophosphatase has been attributed to the reverse exchange EF --> IL. The I1405E/L1406F variant of TRPM2 failed to respond to ADPR even at concentrations up to 10 mM. We concluded that the DeltaC-stretch contains no individual amino acid residues essential for ADPR gating but may act as a spacer segment stabilizing a conformation necessary for the essential residue Asn-1326 to interact with other channel regions. Enhancing the enzymatic activity of the Nudix box abolishes the ADPR gating of TRPM2, pointing to the requirement of prolonged binding rather than degradation.     

The Poly(ADP-ribose) polymerase PARP-1 is required for oxidative stress-induced TRPM2 activation in lymphocytes

TRPM2 cation channels are widely expressed in the immune system and are thought to play a role in immune cell responses to oxidative stress. Patch clamp analyses suggest that TRPM2 channel activation can occur through a direct action of oxidants on TRPM2 channels or indirectly through the actions of a related group of adenine nucleotide 2nd messengers. However, the contribution of each gating mechanism to oxidative stress-induced TRPM2 activation in lymphocytes remains undefined. To better understand the molecular events leading to TRPM2 activation in lymphocytes, we analyzed oxidative stress-induced turnover of intracellular NAD, the metabolic precursor of adenine nucleotide 2nd messengers implicated in TRPM2 gating, and oxidative stress-induced TRPM2-mediated currents and Ca2+ transients in DT40 B cells. TRPM2-dependent Ca2+ entry did not influence the extent or time course of oxidative stress-induced turnover of NAD. Furthermore, expression of oxidative stress-activated poly(ADP-ribose) polymerases (PARPs) was required for oxidative stress-induced NAD turnover, TRPM2 currents, and TRPM2-dependent Ca2+ transients; no oxidant-induced activation of TRPM2 channels could be detected in PARP-deficient cells. Together, our results suggest that during conditions of oxidative stress in lymphocytes, TRPM2 acts as a downstream effector of the PARP/poly(ADP-ribose) glycohydrolase pathway through PARP-dependent formation of ADP-ribose.     

Free radical activation of N-hydroxy-2-acetylaminofluorene by methemoglobin and hydrogen peroxide

We have shown that N-hydroxy-2-acetylaminofluorene, a metabolite of 2-acetylaminofluorene, is converted via a nitroxide free radical into N-acetylaminofluorene and 2-nitrosofluorene by H2O2 in the presence of methemoglobin. Utilizing optical methods, we have demonstrated that the rate of 2-nitrosofluorene production parallels that of N-hydroxy-2-acetylaminofluorene oxidation. This evidence is consistent with a model whereby two molecules of N-hydroxy-2-acetylaminofluorene yield two nitroxide free radicals which then dismutate to form one molecule of N-acetoxy-2-acetylaminofluorene and one molecule of 2-nitrosofluorene. The Km of N-hydroxy-2-acetylaminofluorene for this reaction is 114 microM with a Vmax of 50 microM/min.     

Determination of intracellular concentrations of the TRPM2 agonist ADP-ribose by reversed-phase HPLC

Since the NAD metabolite ADP-ribose (ADPR) has recently gained attention as a putative messenger, a method was established for the quantification of intracellular ADPR by reversed-phase HPLC. Cellular nucleotides were extracted with trichloroacetic acid, and crude cell extracts purified by solid phase extraction using a strong anion exchange matrix. After optimization of the extraction procedure, cellular ADPR levels were determined using two different reversed-phase columns (C18 versus C12), operated in ion pair mode. Intracellular ADPR concentrations in human Jurkat T-lymphocytes and murine BW5147 thymocytes were determined to be 44+/-11 microM and 73+/-11 microM, respectively.     

Modulation of K+ channels by hydrogen peroxide.

External application of hydrogen peroxide (H2O2) was found to inhibit the time-dependent fast inactivation process of three cloned voltage-gated K+ channels expressed in Xenopus oocytes: KShIIIC, KShIIID and HukII. As expected from kinetic models where some channels are still opening while a significant fraction of channels is already inactivated there was a large increase in current magnitude concomitant to inactivation block. The channels otherwise functioned normally. The effects of H2O2 were specific (other cloned voltage-gated K+ channels were not affected), and reversible, the currents returned to normal upon removal of the H2O2. H2O2 is produced during normal metabolism; it could act as a modulator of excitability through effects on K+ channels if effective local concentrations are reached in neuronal regions close to the channel. KShIIIC and KShIIID currents are very similar to an O2-sensitive K+ current present in type I cells of the carotid body which is believed to underlie the modulation of excitability of these cells by changes in arterial O2 pressure. H2O2 has been proposed as an intermediary between O2 and cellular response in the carotid body; our results provide support for this model.     

Phospholipase A2 activation by hydrogen peroxide during in vitro capacitation of buffalo spermatozoa

Progressively motile, washed buffalo spermatozoa (50 x 10(6) cells in 0.5 ml) were in vitro capacitated in HEPES containing Bovine Gamete Medium 3 (BGM3) in presence of heparin (10 microg/ml), and different concentrations of hydrogen peroxide (10 to 100 microM). Spermatozoa (60%) were capacitated in presence of heparin compared to 56% in presence of 25 microM H2O2 (optimally found suitable for capacitation). The extent of capacitation was measured in terms of acrosome reaction (AR) induced by lysophosphatidyl choline (100 microg/ml). The acrosome reacted cells were counted after triple staining. Catalase (100 microg/ml) significantly reduced the sperm capacitation to 16-18% when added with H2O2, or alone in the capacitation medium. Phospholipase A2 activity of spermatozoa increased linearly up to 50 microM H2O2 concentration included in the assay system. Moreover, significant increase in phospholipase A2 activity was observed after capacitation by both, the heparin and 25 microM H2O2. The activity was always higher in acrosome reacted cells.     

Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice

Non-selective cation (NSC) channels activated by intracellular Ca2+ ([Ca2+]i) play an important role in Ca2+ signaling and membrane excitability in many cell types. TRPM4 and TRPM5, two Ca2+-activated cation channels of the TRP superfamily, are potential molecular correlates of NSC channels. We compared the functional properties of mouse TRPM4 and TRPM5 heterologously expressed in HEK 293 cells. Dialyzing cells with different Ca2+ concentrations revealed a difference in Ca2+ sensitivity between TRPM4 and TRPM5, with EC50 values of 20.2+/-4.0 microM and 0.70+/-0.1 microM, respectively. Similarly, TRPM5 activated at lower Ca2+ concentration than TRPM4 when [Ca2+]i was raised by UV uncaging of the Ca2+-cage DMNP-EDTA. Current amplitudes of TRPM4 and TRPM5 were not correlated to the rate of changes in [Ca2+]i. The Ca2+ sensitivity of both channels was strongly reduced in inside-out patches, resulting in approximately 10-30 times higher EC50 values than under whole-cell conditions. Currents through TRPM4 and TRPM5 deactivated at negative and activated at positive potentials with similar kinetics. Both channels were equally sensitive to block by intracellular spermine. TRPM4 displayed a 10-fold higher affinity for block by flufenamic acid. Importantly, ATP4- blocked TRPM4 with high affinity (IC50 of 0.8+/-0.1 microM), whereas TRPM5 is insensitive to ATP4- at concentrations up to 1 mM.     

Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose

LTRPC2 is a cation channel recently reported to be activated by adenosine diphosphate-ribose (ADP-ribose) and NAD. Since ADP-ribose can be formed from NAD and NAD is elevated during oxidative stress, we studied whole cell currents and increases in the intercellular free calcium concentration ([Ca(2+)](i)) in long transient receptor potential channel 2 (LTRPC2)-transfected HEK 293 cells after stimulation with hydrogen peroxide (H(2)O(2)). Cation currents carried by monovalent cations and Ca(2+) were induced by H(2)O(2) (5 mm in the bath solution) as well as by intracellular ADP-ribose (0.3 mm in the pipette solution) but not by NAD (1 mm). H(2)O(2)-induced currents developed slowly after a characteristic delay of 3-6 min and receded after wash-out of H(2)O(2). [Ca(2+)](i) was rapidly increased by H(2)O(2) in LTRPC2-transfected cells as well as in control cells; however, in LTRPC2-transfected cells, H(2)O(2) evoked a second delayed rise in [Ca(2+)](i). A splice variant of LTRPC2 with a deletion in the C terminus (amino acids 1292-1325) was identified in neutrophil granulocytes. This variant was stimulated by H(2)O(2) as the wild type. However, it did not respond to ADP-ribose. We conclude that activation of LTRPC2 by H(2)O(2) is independent of ADP-ribose and that LTRPC2 may mediate the influx of Na(+) and Ca(2+) during oxidative stress, such as the respiratory burst in granulocytes.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H(2)O(2)) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca(2+)/Mg(2+)-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca(2+)/Mg(2+)-supplemented medium. In Ca(2+)/Mg(2+)-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 microm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H(2)O(2) (84.40+/-0.50 ng/egg) when compared to control eggs (80.46+/-1.34 ng/egg). The higher concentration of calcium ionophore (1.6 microm) induced apoptosis and pronounced generation of intracellular H(2)O(2) (92.43+/-0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H(2)O(2) level (81.20+/-1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H(2)O(2) in rat eggs.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H₂O₂) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca²⁺/Mg²⁺-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca²⁺/Mg²⁺-supplemented medium. In Ca²⁺/Mg²⁺-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 µm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H₂O₂ (84.40±0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 µm) induced apoptosis and pronounced generation of intracellular H₂O₂ (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H₂O₂ level (81.20±1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H₂O₂ in rat eggs.     

T-butyl hydrogen peroxide increases the activities of the Maxi-K channels of rat brain

In this study, we investigated the effects of tertiary-butyl hydrogen peroxide (tBHP) on the large-conductance Ca2+-activated K+ (Maxi-K) channel of rat brain using lipid bilayer. When tBHP was applied to the cytosolic side, the open probability (Po) of both fast- and slow-gating Maxi-K channels increased within 1 min in dose-dependent manner. tBHP effects did not reverse immediately, suggesting tBHP induces some chemical modification on the channel protein. From kinetic analysis of single channel data, the increase in the Po appears to be mainly due to shortening of closed dwell time in both types of the Maxi-K channels. 50 microM diamide, a sulfhydryl-specific oxidant, irreversibly decreased the Po. However, further addition of 7.3 mM tBHP still increased the Po, suggesting that tBHP does not share the target for oxidation with diamide.     

Chemical physiology of oxidative stress-activated TRPM2 and TRPC5 channels

The ability to sense and adapt to a wide variety of environmental changes is crucial for the survival of all cells. Transient receptor potential (TRP) channels play pivotal roles in these sensing and adaptation reactions. In vertebrates, there are about 30 TRP channels; these are divided into six subfamilies by homology of the protein sequences. We have previously revealed that a group of TRP channels senses oxidative stress and induces cellular signaling and gene expression. TRPM2, a member of the TRPM subfamily, is activated by reactive oxygen species (ROS) via second-messenger production. Recently, we demonstrated that Ca²⁺ influx through TRPM2 activated by ROS induces chemokine production in monocytes, which aggravates inflammatory neutrophil infiltration. Additionally, we also revealed that nitric oxide, chemical compounds containing reactive disulfide, and inflammatory mediators directly activate the TRPC, TRPV, and TRPA subfamilies via oxidative modification of cysteine residues. In this review, we describe how these TRP channels sense oxidative stress and induce adaptation reactions, and we discuss the biological importance of oxidative stress-activated TRP channels.     

Urothelial cells produce hydrogen peroxide through the activation of Duox1

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H(2)O(2) is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H(2)O(2) production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H(2)O(2) production similar to that observed in plants.