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

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.