TRPM2 Inhibitors Research Articles

TNF-α-mediated caspase-8 activation induces ROS production and TRPM2 activation in adult ventricular myocytes.

TRPM2 is a Ca(2+)-permeable cationic channel of the transient receptor potential (TRP) superfamily that is linked to apoptotic signalling. Its involvement in cardiac pathophysiology is unknown. The aim of this study was to determine whether the pro-apoptotic cytokine tumour necrosis factor-α (TNF-α) induces a TRPM2-like current in murine ventricular cardiomyocytes. Adult isolated cardiomyocytes from C57BL/6 mice were exposed to TNF-α (10 ng/mL). Western blotting showed TRPM2 expression, which was not changed after TNF-α incubation. Using patch clamp in whole-cell configuration, a non-specific cation current was recorded after exposure to TNF-α (ITNF), which reached maximal steady-state amplitude after 3 h incubation. ITNF was inhibited by the caspase-8 inhibitor z-IETD-fmk, the antioxidant N-acetylcysteine, and the TRPM2 inhibitors clotrimazole, N-(P-amylcinnamoyl) anthranilic acid and flufenamic acid (FFA). TRPM2 has previously been shown to be activated by ADP-ribose, which is produced by poly(ADP-ribose) polymerase 1 (PARP-1). TNF-α exposure resulted in increased poly-ADP-ribosylation of proteins and the PARP-1 inhibitor 3-aminobenzamide inhibited ITNF. TNF-α exposure increased the mitochondrial production of reactive oxygen species (ROS; measured with the fluorescent indicator MitoSOX Red), and this increase was blocked by the caspase-8 inhibitor z-IETD-fmk. Clotrimazole and TRPM2 inhibitory antibody decreased TNF-α-induced cardiomyocyte death. These results demonstrate that TNF-α induces a TRPM2 current in adult ventricular cardiomyocytes. TNF-α induces caspase-8 activation leading to ROS production, PARP-1 activation, and ADP-ribose production. TNF-induced TRPM2 activation may contribute to cardiomyocyte cell death.

891 Up-Regulation of Gastric TRPV1 mRNA Expression in Functional Dyspepsia Patients of Epigastric Pain Syndrome Subtype

nerve fibers in the mucosa of sigmoid colon was increased in TNBS induced colitis model on 7 days after TNBS treatment. The TRPM2 protein were increased in the inflamed sigmoid colon when examined with western blot. The VMR to CRD significantly increased in colitis models as compared with in control rats at 40, 60, and 80 mmHg. The TRPM2 inhibitor econazole attenuated the visceral hypersensitivity to the control level. CONCLUSION: TRPM2 channel expressed in intrinsic and spinal primary afferent neuron contributes to the visceral sensitivity to noxious stimuli under physiological condition and visceral hypersensitivity under pathological condition in rats.

Abstract 93: Androgen Regulation of Protection Provided by Inhibition of TRPM2 Channels against Experimental Stroke in Mice

Introduction: Stroke is a sexually dimorphic disease that affects men more severely than women. Transient receptor potential (TRP) channels have been implicated in ischemia-induced neuronal damage. We recently demonstrated that pharmacological and genetic inhibition of TRPM2, a member of the TRP channel superfamily, protects against cerebral ischemia in male mice, while having no effect in females. In addition, TRPM2 channels are activated by adenine dinucleotide phosphate ribose (ADPr) generated by poly(ADPr) polymerase (PARP). In this study, we examined the role of male sex steroids in TRPM2 engagement following experimental stroke. Methods: Focal ischemia was induced by 60 min of middle cerebral artery occlusion in male C57Bl/6 mice. To assess the efficacy of post-insult administration of TRPM2 inhibitor clotrimazole (CTZ), intact mice were treated with either CTZ (30 mg/kg) or vehicle 1 h after reperfusion. Mice were castrated (CAST) in order to remove sex steroids and either dihydrotestosterone (DHT) or vehicle was implanted. Then, animals were treated with either CTZ or vehicle 1 hr after reperfusion. The infarct sizes were evaluated in TRPM2 -/- mice to confirm TRPM2 engagement. We further compared the PARP activity in intact male and female brain to that in ischemic brain. Results: Animals treated with CTZ had significantly smaller infarcts compared to vehicle treated animals (46.2±3.1% (n=8) in vehicle vs. 32.5±3.6% (n=8; P<0.05) in CTZ). Removal of sex steroids resulted in loss of protection by CTZ (44.6±3.1% (n=9) in vehicle treated CAST vs. 35.8±3.6% (n=11) in CTZ treated CAST). Androgen replacement with DHT (CAST+DHT) resulted in recovery of CTZ protection (40.4±2.3% (n=12) in vehicle vs. 31.5±2.8% (n=11; P<0.05) in CTZ). TRPM2 -/- mice had smaller infarcts compared to wild type (28.4±7.0 (n=5) vs. 49.0±1.2 (n=5); P<0.05). We found higher PARP activity in male penumbral tissues compared to male normal tissues (P<0.05), while having no difference in females. Conclusions: These data indicate that TRPM2 inhibition provides protection from ischemic stroke in male animals in an androgen-dependent manner. Therefore, androgen status must be considered when considering inhibition of TRPM2 channels as a potential therapeutic strategy in men.

Abstract 372: Role of Redox-Sensitive Calcium Channel TRPM2 and Oxidative Stress in Vascular Smooth Muscle Cell Differentiation to an Osteogenic Phenotype: Implications in Hypertension.

Vascular smooth muscle cell (VSMC) transformation to an osteoblast-like phenotype is a major factor contributing to vascular calcification, often associated with chronic kidney disease and hypertension. Exact molecular mechanisms underlying VSMC transformation remain unclear but intracellular calcium and ROS have been implicated. Whether these factors are interlinked is unknown. Here, we tested the hypothesis that ROS and redox-sensitive calcium channels (TRPM2) induce an osteogenic phenotype transition in VSMCs from WKY and SHRSP rats. Cultured VSMCs from WKY and SHRSP rats were exposed to calcification medium (CaM) (Ca2+ 1.8 mmol/L, PO4 2.0 mmol/L) for 10 days in the presence/absence of tempol (superoxide dismutase mimetic), and N-(p-amylcinnamoyl)anthranilic acid (ACA, TRPM2 inhibitor). Osteocalcin (OC), BMP-2, BMP-7, TRPM2 and TRPM2-S expression, as well as p47 phox translocation (cytosol:membrane), were determined by immunoblotting. ROS generation was evaluated by chemioluminescence. ROS production (Ctl: 30 AU/ug protein; CaM: 60 AU/ug protein, p<0.05) and p47 translocation (Ctl: 0.7 AU; CaM: 1.1AU, p<0.05) were increased by the CaM in VSMCs from WKY. CaM-induced increase in OC (Ctl: 5 AU; CaM: 13 AU) and BMP-2 (Ctl: 0.60 AU; CaM: 0.75AU) (p<0.05), followed by a decrease in BMP-7 (Ctl: 1.12 AU; CaM: 0.7 AU) (p<0.05), expression in VSMCs from WKY; an effect that was inhibited by tempol and ACA. In SHRSP, the increase in OC (Ctl: 1.25 AU; CaM: 2 AU) and BMP-2 (Ctl: 0.6 AU; CaM: 0.85 AU, p<0.05) expression induced by the CaM was also blocked by tempol. TRPM2 expression was higher in SHRSP (1.40 AU) than in WKY (1.05 AU) VSMCs (p<0.05). However, TRPM2-S expression, an intracellular inhibitor of TRPM2, was decreased in SHRSP compared to WKY VSMCs (SHRSP: 0.9 AU; WKY: 1.5 AU; p<0.05), and it was further decreased by the CaM (0.6 AU). In conclusion, ROS, through TRPM-2 sensitive mechanisms, seems to play an important role in VSMC transformation to an osteogenic, a phenomenon that may be exacerbated in hypertension.

Direct and Indirect Effectors of the TRPM2 Cation Channel

TRPM2 is a Ca2+ permeable cation channel which plays a role in physiological and pathophysiological processes linked to oxidative stress. TRPM2 channels are co-activated by intracellular Ca2+ and ADP ribose (ADPR). In addition, in intact cells, a large number of compounds appear to modulate TRPM2 activity. Superfusion of TRPM2-expressing cells with hydrogen-peroxide (H2O2) activates TRPM2 currents, just as intracellular dialysis of cyclic ADPR (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP). Importantly, H2O2, cADPR, and NAADP enhance ADPR-induced TRPM2 whole-cell currents. Finally, in intact cells AMP acts as a TRPM2 inhibitor. Because in whole-cell recordings the entire cellular machinary involved in nucleotide- and Ca2+-homeostasis is in place, compounds might affect TRPM2 activity either directly, by binding to the TRPM2 protein, or indirectly, by altering the local concentrations of the primary ligands ADPR and Ca2+. To identify direct modulators of TRPM2 activity, we have studied the effects of H2O2, AMP, cADPR, NAADP, and nicotinic acid adenine dinucleotide (NAAD) in inside-out patches excised from Xenopus oocytes expressing human TRPM2, by directly exposing the cytosolic faces of the patches to these compounds. H2O2 (1 mM) and enzymatically purified cADPR (10 μM) failed to activate, while AMP (200 μM) failed to inhibit TRPM2 currents. NAADP acted as a partial agonist (maximal efficacy ∼50%) while NAAD was a full agonist, but both with low affinities (K0.5=104 and 35 μM). Neither of H2O2, cADPR, and NAADP enhanced activation by ADPR. Thus, in a physiological context the above compounds do not directly affect the TRPM2 channel protein. [OTKA grant F68143]