1,721,084 research outputs found
Assay of CB1 Receptor Binding.
No full textType-1 cannabinoid receptor (CB1), one of the main targets of endocannabinoids, plays a key role in several pathophysiological conditions that affect both central nervous system and peripheral tissues. Today, its biochemical identification and pharmacological characterization, as well as the screening of thousands of novel ligands that might be useful for developing CB1-based therapies, are the subject of intense research. Among available techniques that allow the analysis of CB1 binding activity, radioligand-based assays represent one of the best, fast, and reliable methods. Here, we describe radioligand binding methods standardized in our laboratory to assess CB1 binding in both tissues and cultured cells. We also report a high-throughput radioligand binding assay that allows to evaluate efficacy and potency of different compounds, which might represent the basis for the development of new drugs that target CB1 receptor-dependent human diseases
Inactivation of multiple targets by nitric oxide in CD95-triggered apoptosis
No full textNitric oxide (NO) plays an important anti-apoptotic role by inactivating both upstream and downstream apoptotic molecules. We now report that exogenously supplied NO protected Jurkat T cells from anti-CD95-stimulated apoptosis. We have recently shown that nitrosation of the activator protein-1 (AP-1) transcriptional factor is crucial for NO-mediated inhibition of cell death triggered by etoposide or ceramide. Since the inhibition of apoptosis by NO has been reported to involve AP-1, we evaluated its involvement in in CD95-mediated cell death. Cross-linking of CD95 enhanced AP-1 DNA binding activity and AP-1-dependent CD95L transactivation, which were both significantly reduced by different NO-donors compounds. However, AP-1 induction does not seem to significantly contribute to anti-CD95-triggered apoptosis, as cell death could not be prevented by using the recombinant Fas-Fc fusion protein which inhibits the CD95/CD95L interaction. We observed that caspase 3-like activity was negatively modulated by several NO-donors in vitro and that titratable thiol groups of purified caspases 3, 7, and 9 decreased in the presence of NO-releasing compounds. In conclusion, we demonstrated that NO-mediated inhibition of other targets, possibly caspases, but not AP-1, is a crucial event responsible for protection against anti-CD95-stimulated apoptosis. Even though NO affects multiple molecular mechanisms, the relevant target for exerting the cellular effects, may vary among different models
Nitric oxide can inhibit apoptosis or switch it into necrosis
No full textNitric oxide (NO) and its related molecules are important messengers that play central roles in pathophysiology. Redox modulation of thiol groups on protein cysteine residues by S-nitrosylation can modulate protein function. NO has emerged as a potent regulator of apoptosis in many cell types, either preventing cell death or driving an apoptotic response into a necrotic one. NO protects neuroblastoma cells from retinoid- and cisplatin-induced apoptosis, without significantly increasing necrotic cell damage. Nitrosylation of thiol groups of several critical factors may be important for cell survival. Indeed, S-nitrosylation of the active-site cysteine residue of apoptotic molecules, such as caspases and tissue transglutaminase, results in the inhibition of their catalytic activities and has important implications for the regulation of apoptosis by NO. On the other hand, NO is able to shift the anti-CD95- and ceramide-triggered apoptotic response of Jurkat T cells into necrotic cell death. In these apoptotic models, NO is therefore unable to solely inhibit cell death, indicating that it may act below the point of no return elicited by CD95-ligation and ceramide stimulation
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