Inhibition of depolarisation-evoked [3H]naradrenaline release from SH-SY5Y human neuroblastoma cells by muscarinic (M1) receptors is not mediated by changes in [Ca2+]i

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 ([Ca2+](i)) and (b) activation of protein kinase C (PKC). This study used specific inhibitors of PKC and conditions which deplete Ca-i(2+) stores to examine the role of protein kinase C and changes in [Ca2+](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 Ca2+-free Hepes buffered saline in the presence of thapsigargin, conditions which prevented muscarine induced increases in [Ca2+](i), failed to prevent inhibition of K+-evoked NA release by muscarine. The effect of muscarine on K+-evoked uptake of Ca-e(2+) was examined in SH-SY5Y cells loaded with Fura-2. Muscarine inhibited Ca-e(2+)-uptake by decreasing the rate at which Ca2+ entered SH-SY5Y cells via voltage sensitive Ca2+-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 Ca2+ from internal stores. (C) 2001 Elsevier Science B.V. All rights reserved.