RESUMO
The role of cyclic ADP-ribose (cADPR) and its synthetic enzyme, CD38, as a downstream signal of muscarinic acetylcholine receptors (mAChRs) was examined in neuroblastoma cells expressing M1 mAChRs (NGM1). NGM1 cells were further transformed with both wild-type and mutant (C119K/C201E) human CD38. The dual transformed cells exhibited higher cADPR formation than ADPR production and elevated intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in response to ACh. These phenotypes were analyzed in detail in a representative CD38 clone. The intracellular cADPR concentration by ACh application was significantly increased by CD38 overexpression. Digital image analysis by a confocal microscopy revealed that topographical distribution of the sites of Ca(2+) release was unchanged between control and overexpressed cells. These results indicate that cADPR is an intracellular messenger of Ca(2+) signalling, suggesting that CD38 can contribute to mAChR-cADPR signalling.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , ADP-Ribosil Ciclase/metabolismo , Acetilcolina/metabolismo , Sinalização do Cálcio/fisiologia , ADP-Ribose Cíclica/metabolismo , Receptores Muscarínicos/metabolismo , ADP-Ribosil Ciclase 1/genética , Acetilcolina/farmacologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Ratos , Receptores Muscarínicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
ADP-ribosyl cyclase activity in the crude membrane fraction of neuroblastomaxglioma NGPM1-27 hybrid cells was measured by monitoring [(3)H] cyclic ADP-ribose (cADPR) formation from [(3)H] NAD(+). Bradykinin (BK) at 100nM increased ADP-ribosyl cyclase activity by about 2.5-fold. Application of 300nM BK to living NGPM1-27 cells decreased NAD(+) to 78% of the prestimulation level at 30s. In contrast, intracellular cADPR concentrations were increased by 2-3-fold during the period from 30 to 120s after the same treatment. Our results suggest that cADPR is one of the second messengers downstream of B(2) BK receptors.