RESUMO
Cyclic ADP-ribose (cADP-ribose) is a putative second messenger or modulator. However, the role of cADP-ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6-deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimulated cyclase activity. GTP-gamma-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108-15 neuroblastoma x glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pre-treatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGluRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal functions are mediated by cADP-ribose.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Glioma/metabolismo , Neuroblastoma/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Retina/metabolismo , Gânglio Cervical Superior/metabolismo , ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/efeitos dos fármacos , Adenosina Difosfato Ribose/biossíntese , Animais , Membrana Celular/enzimologia , Células Cultivadas , Toxina da Cólera/farmacologia , Ativação Enzimática/efeitos dos fármacos , Glioma/química , Ácido Glutâmico/farmacologia , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Camundongos , Camundongos Knockout , Neuroblastoma/química , Neurônios/química , Neurônios/citologia , Ratos , Ratos Wistar , Retina/química , Retina/citologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Gânglio Cervical Superior/química , Gânglio Cervical Superior/citologiaRESUMO
A procedure for isolation of cyclic AMP (cAMP) by thin-layer chromatography on silica gel is described. One-dimensional ascending chromatograms were developed using [H(2)O/C(2)H(5)OH/NH(4)HCO(3) (30%:70%:0.2M)] as the mobile phase. This procedure separated [32P]cAMP from other radioactive metabolites of [32P]ATP in up to 19 samples on one sheet (20 x 10 cm) over 40-60 min at room temperature (21 degrees C). This simple and rapid isolation method provides a novel and convenient technique for the assay of adenylyl cyclase.
