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.

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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

To determine the effect of intravenous recombinant tissue plasminogen activator (rt-PA) on vascular and neurologic outcomes, we enrolled 31 patients with acute carotid artery-territory ischemic stroke within 6 hours from symptom onset in a randomized, double-blind, placebo-controlled study. We gave either rt-PA (duteplase at the dose of 20 or 30 mega-international units [MIU]) or placebo intravenously for 60 minutes in patients randomly assigned to the three groups. A comparison between the baseline and postinfusion angiograms showed that complete or partial reperfusion occurred in 50% (5/10) of patients treated with 30 MIU rt-PA, 44% (4/9) of those treated with 20 MIU rt-PA, and 17% (2/12) in the control group. In patients with middle cerebral artery occlusions, reperfusion occurred in 71% (5/7) of the 30-MIU group, in 67% (4/6) of the 20-MIU group, and in 13% (1/8) of the control group. Patients treated with 30 MIU rt-PA showed a significantly early and better clinical improvement, as measured by the neurologic scale, than did those treated with placebo. Parenchymal hemorrhage occurred in one patient in each group, and frequency of clinically insignificant hemorrhagic infarction was comparable among the treatment groups. No major systemic complications occurred in any group. These results support the efficacy of intravenous infusion of rt-PA soon after the onset of stroke in producing rapid thrombolysis and neurologic recovery; it may be of particular value in patients with thromboembolic occlusion in the middle cerebral artery.