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Cannabidiol (CBD) is a non-psychoactive constituent of Cannabis sativa with potential to treat neurodegenerative diseases. Its neuroprotection has been mainly associated with anti-inflammatory and antioxidant events; however, other mechanisms might be involved. We investigated the involvement of neuritogenesis, NGF receptors (trkA), NGF, and neuronal proteins in the mechanism of neuroprotection of CBD against MPP(+) toxicity in PC12 cells. CBD increased cell viability, differentiation, and the expression of axonal (GAP-43) and synaptic (synaptophysin and synapsin I) proteins. Its neuritogenic effect was not dependent or additive to NGF, but it was inhibited by K252a (trkA inhibitor). CBD did not increase the expression of NGF, but protected against its decrease induced by MPP(+), probably by an indirect mechanism. We also evaluated the neuritogenesis in SH-SY5Y cells, which do not express trkA receptors. CBD did not induce neuritogenesis in this cellular model, which supports the involvement of trkA receptors. This is the first study to report the involvement of neuronal proteins and trkA in the neuroprotection of CBD. Our findings suggest that CBD has a neurorestorative potential independent of NGF that might contribute to its neuroprotection against MPP(+), a neurotoxin relevant to Parkinson's disease.

1 We investigated the e ects of nimesulide, a recently developed non-steroidal anti-in¯ammatory drug, and of a metabolite resulting from reduction of the nitro group to an amine derivative, on succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 20 mM Ca 2+ , 1 mM cyclosporin A (CsA) or 5 mM ruthenium red. 2 Nimesulide uncoupled mitochondria through a protonophoretic mechanism and oxidized mitochondrial NAD(P)H, both e ects presenting an EC 50 of approximately 5 mM. 3 Within the same concentration range nimesulide induced mitochondrial Ca 2+ e ux in a partly ruthenium red-sensitive manner, and induced mitochondrial permeability transition (MPT) when ruthenium red was added after Ca 2+ uptake by mitochondria. Nimesulide induced MPT even in deenergized mitochondria incubated with 0.5 mM Ca 2+ . 4 Both Ca 2+ e ux and MPT were prevented to a similar extent by CsA, Mg 2+ , ADP, ATP and butylhydroxytoluene, whereas dithiothreitol and N-ethylmaleimide, which markedly prevented MPT, had only a partial or no e ect on Ca 2+ e ux, respectively. 5 The reduction of the nitro group of nimesulide to an amine derivative completely suppressed the above mitochondrial responses, indicating that the nitro group determines both the protonophoretic and NAD(P)H oxidant properties of the drug. 6 The nimesulide reduction product demonstrated a partial protective e ect against accumulation of reactive oxygen species derived from mitochondria under conditions of oxidative stress like those resulting from the presence of t-butyl hydroperoxide. 7 The main conclusion is that nimesulide, on account of its nitro group, acts as a potent protonophoretic uncoupler and NAD(P)H oxidant on isolated rat liver mitochondria, inducing Ca 2+ e ux or MPT within a concentration range which can be reached in vivo, thus presenting the potential ability to interfere with the energy and Ca 2+ homeostasis in the liver cell.

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Sulindac is a non-steroidal antiinflammatory drug (NSAID) known to inhibit cyclooxygenases (COX) 1 and 2, and at present of interest for cancer prevention. However, its therapeutic use has been limited by its toxicity to the gastrointestinal tract and liver. We address the effects of sulindac, of the pharmacologically inactive metabolite, sulindac sulfone, and of the pharmacologically active metabolite, sudindac sulfide, on isolated rat liver mitochondria and HepG2 cells. Sulindac sulfide, but not sulindac sulfone or sulindac itself, caused mitochondrial uncoupling, released preaccumulated Ca 2π from the organelle, and decreased Hep-G2 cell viability in apparent association with cell ATP depletion resulting from mitochondrial uncoupling-associated membrane potential dissipation.

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca(2+)-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys(56) relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca(2+)-induced ANT conformational change and mitochondrial swelling. ANT-Pro(61) isomerization increased ANT-Cys(56) relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca(2+) induced ANT "c" conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca(2+)-induced ANT "c" conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca(2+) induces the ANT "c" conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.

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