Inhibition of depolarisation-evoked [ 3H]noradrenaline release from SH-SY5Y human neuroblastoma cells by muscarinic (M1) receptors is not mediated by changes in [Ca 2+] i

Abstract

The aim of this study was to obtain further understanding of the mechanism by which activation of muscarinic M 1 receptors inhibits K +-evoked noradrenaline (NA) release in the human neuroblastoma SH-SY5Y. Previous studies have found that muscarinic M 1 and M 3 receptors couple to the activation of phospholipase C in SH-SY5Y cells leading to an increase in (a) intracellular calcium ([Ca 2+] i) and (b) activation of protein kinase C (PKC). This study used specific inhibitors of PKC and conditions which deplete Ca 2+ i stores to examine the role of protein kinase C and changes in [Ca 2+] i in mediating the inhibition of K +-evoked NA release by muscarine. Our data show that pretreatment of SH-SY5Y cell layers with bisindolylmaleimide I (BIM-I) (i) failed to reverse inhibition of K +-evoked NA release by muscarine but (ii) did overcome the attenuation of muscarine inhibition following pretreatment with TPA. Furthermore pretreating cell layers with Ca 2+-free Hepes buffered saline in the presence of thapsigargin, conditions which prevented muscarine induced increases in [Ca 2+] i, failed to prevent inhibition of K +-evoked NA release by muscarine. The effect of muscarine on K +-evoked uptake of Ca 2+ e was examined in SH-SY5Y cells loaded with Fura-2. Muscarine inhibited Ca 2+ e-uptake by decreasing the rate at which Ca 2+ entered SH-SY5Y cells via voltage sensitive Ca 2+-channels. Thus this study shows that muscarine inhibits depolarisation-evoked NA release by a mechanism which is not dependent on activation of PKC or release of Ca 2+ from internal stores.