RESUMEN
Cannabinoid CB2 receptor has emerged as a very promising target over the last decades. We have synthesized and evaluated a new fluorescent probe designated NMP6 based on 6-methoxyisatin scaffold, which exhibited selectivity and K(i) value at hCB2 of 387 nM. We have demonstrated its ability to be an effective probe for visualization of CB2 receptor binding using confocal microscopy and a flow cytometry probe for the analysis of CB2 protein expression. Furthermore, NMP6 was easily obtained in two chemical steps from commercially available building blocks.
Asunto(s)
Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Hidrazonas/síntesis química , Hidrazonas/metabolismo , Isatina/análogos & derivados , Receptor Cannabinoide CB2/análisis , Animales , Linfocitos B , Células CHO , Cricetinae , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Hidrazinas/química , Hidrazinas/metabolismo , Hidrazonas/química , Isatina/síntesis química , Isatina/química , Isatina/metabolismo , Ligandos , Pulmón , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Estructura Molecular , Oxadiazoles/química , Oxadiazoles/metabolismo , Unión Proteica , Piranos/farmacología , Pirimidinas/farmacología , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/metabolismo , Relación Estructura-ActividadRESUMEN
The cellular events that serve to regulate lung mucosal Th2 responses and limit allergic inflammatory reactions are unclear. Using the DO11.10 TCR transgenic mouse, we developed a model of T cell-mediated pulmonary inflammation and demonstrated that high levels of PGI(2) are produced in the airways following OVA inhalation. Selective inhibition of cyclooxygenase-2 in vivo specifically reduced PGI(2) synthesis and resulted in a marked increase in Th2-mediated, but not Th1-mediated, lung inflammation. The elevated Th2-mediated inflammatory response elicited by the cyclooxygenase-2 inhibitor was associated with enhanced airway hyperreactivity and was coincident with a marked increase in the levels of IL-4, IL-5, and IL-13 in the airways, but a reduction in IL-10 production. In keeping with these observations, we found that the mRNA for the PGI(2) receptor was expressed by Th2, but not Th1, cells, and transcripts for the PGI(2) receptor were induced by IL-4 and OVA peptide stimulation. Interestingly, treatment with PGI(2) or its stable analog, carbaprostacyclin, augmented IL-10 production by Th2 cells. Collectively, our findings reveal a key role for PGI(2) in differentially limiting Th2 responses, possibly by promoting production of the immunosuppressive cytokine IL-10 at the site of allergic lung inflammation. These results indicate an important role for prostanoids generated during inflammation in regulating mucosal T cell responses and highlight a potential risk in the use of cyclooxygenase-2-specific inhibitors by allergic asthmatics.