Purpose:We investigated the effects of BTX-A on visceral afferent nerve transmission by measuring bladder tissue NGF levels in patients with neurogenic detrusor overactivity before and after intravesical treatment with BTX-A. We also compared the bladder tissue NGF content with clinical and urodynamic data. Materials and Methods: A total of 23 patients underwent clinical evaluation and urodynamics with detection of the UDC threshold, maximum pressure and maximum cystometric capacity before, and at the 1 and 3-month followups. Endoscopic bladder wall biopsies were also obtained at the same time points. NGF levels were measured in tissue homogenate by enzyme-linked immunosorbent assay (Promega, Madison, Wisconsin). Results: At 1 and 3 months mean catheterization and incontinent episodes were significantly decreased (p Ͻ0.05 and Ͻ0.001, respectively). On urodynamics we detected a significant increase in the UDC threshold and maximum cystometric capacity, and a significant decrease in UDC maximum pressure at the 1 and 3-month followups compared to baseline (each p Ͻ0.001).At the same time points we detected a significant decrease in NGF bladder tissue content (each p Ͻ0.02). Conclusions: BTX-A intravesical treatment induces a state of NGF deprivation in bladder tissue that persists at least up to 3 months. As caused by BTX-A, the decrease in acetylcholine release at the presynaptic level may induce a decrease in detrusor contractility and in NGF production by the detrusor muscle. Alternatively BTX-A can decrease the bladder level of neurotransmitters that normally modulate NGF production and release.
Activation of brain mitochondrial phospholipase(s) A 2 (PLA 2 ) might contribute to cell damage and be involved in neurodegeneration. Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown. Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca 2؉ -dependent PLA 2 having biochemical properties characteristic to secreted PLA 2 (sPLA 2 ) and immunoreacting with the antibody raised against recombinant type IIA sPLA 2 (sPLA 2 -IIA). Under identical conditions, no release of fumarase in the extramitochondrial medium was observed. The release of sPLA 2 from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC 6 (3) as a probe. The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA 2 release. The increase of sPLA 2 specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts. The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA 2 has mainly nuclear localization. In addition to sPLA 2 , mitochondria contain another phospholipase A 2 that is Ca 2؉ -independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane. We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA 2 -IIA that might contribute to cell damage.
Phospholipases A(2) (PLA(2)) participate in neuronal death signalling pathways because of their ability to release lipid mediators, although the contribution of each isoform and mechanism of neurotoxicity are still elusive. Using a novel fluorogenic method to assess changes in a PLA(2) activity by flow cytometry, here we show that the group IIA secretory phospholipase A(2) isoform (GIIA) was specifically activated in cortical neurons following stimulation of N-methyl-d-aspartate glutamate receptor subtype (NMDAR). For activation, GIIA required Ca(2+) and reactive oxygen/nitrogen species, and inhibition of its activity fully prevented NMDAR-mediated neuronal apoptotic death. Superoxide, nitric oxide or peroxynitrite donors stimulated GIIA activity, which mediated neuronal death. Intriguingly, we also found that GIIA activity induced mitochondrial superoxide production after NMDAR stimulation. These results reveal a novel role for GIIA in excitotoxicity both as target and producer of superoxide in a positive-loop of activation that may contribute to the propagation of neurodegeneration.
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