Redox status changes exert critical impacts on necrotic/apoptotic and normal cellular processes. We report here a widely expressed Ca2+-permeable cation channel, LTRPC2, activated by micromolar levels of H2O2 and agents that produce reactive oxygen/nitrogen species. This sensitivity of LTRPC2 to redox state modifiers was attributable to an agonistic binding of nicotinamide adenine dinucleotide (beta-NAD+) to the MutT motif. Arachidonic acid and Ca2+ were important positive regulators for LTRPC2. Heterologous LTRPC2 expression conferred susceptibility to death on HEK cells. Antisense oligonucleotide experiments revealed physiological involvement of "native" LTRPC2 in H2O2- and TNFalpha-induced Ca2+ influx and cell death. Thus, LTRPC2 represents an important intrinsic mechanism that mediates Ca2+ and Na+ overload in response to disturbance of redox state in cell death.

Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca 2 þ -responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP 3 ) is responsible for Ca 2 þ increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP 3 is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca 2 þ oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca 2 þ channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAGinduced Ca 2 þ signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.

Canonical transient receptor potential (TRPC) channels control influxes of Ca 2؉ and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca 2؉ influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptorinduced Ca 2؉ response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca 2؉ -dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.Ca 2ϩ signaling ͉ pyrazole compounds ͉ TRPC channels ͉ TRPC3

An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS−) regulates the metabolism and signaling actions of various electrophiles. HS− reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS− biosynthesis. Cardiac HS−, in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS− on myocardial infarction–associated heart failure. Thus, this study reveals HS−-induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling.

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, Gprotein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as protonsensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0 -6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1-and GPR4-expressing cells but not for prostaglandin E 2 -and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pHdependent action, although to a lesser extent. Psychosinesensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against proteinsensing receptors, including TDAG8, GPR4, and OGR1. TDAG81 was initially cloned as an orphan GPCR, which is up-regulated during the programmed cell death of T lymphocytes (1-3). This gene product has recently been reported (4) to be a receptor for psychosine, a lysosphingolipid, which induces the formation of multinuclear cells. OGR1, which shares 41% identical amino acids with TDAG8, was initially reported (5) to be a receptor for sphingosylphosphorylcholine (SPC). GPR4 also shares homology with TDAG8 and was identified as a receptor for lysolipids, including lysophosphatidylcholine (LPC) and SPC (6). It has recently been reported (7), however, that OGR1 and GPR4 sense extracellular protons through histidine residues of receptors and are coupled to G-proteins to stimulate intracellular signaling pathways. Thus, OGR1 stimulation causes inositol phosphate production, and the subsequent mobilization of intracellular calcium and GPR4 stimulation induces cAMP accumulation, probably reflecting the activation of adenylyl cyclase in response to an extracellular pH change (7). These results raise the possibility that TDAG8 may also respond to extracellular pH change and stimulate intracellular signaling pathways.If TDAG8 is prov...