Mibefradil is a Ca 2ϩ channel antagonist that inhibits both Ttype and high-voltage-activated Ca 2ϩ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride , that exerts a selective inhibitory effect on T-type channels. The acute IC 50 of NNC 55-0396 to block recombinant ␣ 1 G T-type channels in human embryonic kidney 293 cells was ϳ7 M, whereas 100 M NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca 2ϩ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca 2ϩ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca 2ϩ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca 2ϩ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca 2ϩ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of Ltype Ca 2ϩ channels, thus rendering it more selective to T-type Ca 2ϩ channels.Voltage-gated Ca 2ϩ channels are transmembrane proteins involved in the regulation of cellular excitability and intracellular Ca 2ϩ signaling. Calcium channels have been divided into various categories based on functional and pharmacological criteria. High-voltage-activated (HVA) channels, which have been further subdivided into L-, N-, P/Q-, and R-types, require strong depolarizations for activation, whereas lowvoltage-activated or T-type channels activate over a much more negative voltage range and exhibit unique inactivation and deactivation kinetics (Armstrong and Matteson, 1985;Catterall, 1998;Perez-Reyes, 1998). The main structural component of the voltage-gated calcium channel is the ␣ 1

Hemoproteins undergo degradation during hypoxic/ischemic conditions, but the pro-oxidant free heme that is released cannot be recycled and must be degraded. The extracellular heme associates with its high-affinity binding protein, hemopexin (HPX). Hemopexin is shown here to be expressed by cortical neurons and it is present in mouse cerebellum, cortex, hippocampus, and striatum. Using the transient ischemia model (90-min middle cerebral artery occlusion followed by 96-h survival), we provide evidence that HPX is protective in the brain, as neurologic deficits and infarct volumes were significantly greater in HPX−/− than in wild-type mice. Addressing the potential protective HPX cellular pathway, we observed that exogenous free heme decreased cell survival in primary mouse cortical neuron cultures, whereas the heme bound to HPX was not toxic. Heme–HPX complexes induce HO1 and, consequently, protect primary neurons against the toxicity of both heme and prooxidant tert-butyl hydroperoxide; such protection was decreased in HO1−/− neuronal cultures. Taken together, these data show that HPX protects against heme-induced toxicity and oxidative stress and that HO1 is required. We propose that the heme–HPX system protects against stroke-related damage by maintaining a tight balance between free and bound heme. Thus, regulating extracellular free heme levels, such as with HPX, could be neuroprotective.

Epidemiological and experimental reports have linked mild-to-moderate wine and/or grape consumption to a lowered incidence of cardiovascular, cerebrovascular, and peripheral vascular risk. This study revealed that resveratrol, an enriched bioactive polyphenol in red wine, selectively induces heme oxygenase 1 (HO1) in a dose-and time-dependent manner in cultured mouse cortical neuronal cells and provides neuroprotection from free-radical or excitotoxicity damage. This protection was lost when cells were treated with a protein synthesis or heme oxygenase inhibitor, suggesting that HO1 induction is at least partially required for resveratrol's prophylactic properties. Furthermore, resveratrol pretreatment dose-dependently protected mice subjected to an optimized ischemicreperfusion stroke model. Mice in which HO1 was selectively deleted lost most, if not all, of the beneficial effects. Together, the data suggest a potential intracellular pathway by which resveratrol can provide cell/organ resistance against neuropathological conditions.