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+)].
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.
Institute for Cardiovascular Research, University of Leeds, LS2 9JT, Leeds, UK.
D J
DJ
Roberts
N
N
Khan
R L
RL
McDonald
N J
NJ
Webster
C
C
Peers
P F
PF
Vaughan
D002118:Calcium / Q000378:metabolism*; D004546:Elapid Venoms / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D009116:Muscarine / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism*; D009638:Norepinephrine / Q000493:pharmacokinetics*; D010704:Phorbols / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D013268:Stimulation, Chemical; D014316:Tritium; D014407:Tumor Cells, Cultured
D004546:Elapid Venoms; D004963:Estrenes; D018721:Muscarinic Agonists; D010704:Phorbols; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D014316:Tritium; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C034916:toxin F7; D009116:Muscarine; D011493:Protein Kinase C; D011188:Potassium; D002118:Calcium; D009638:Norepinephrine; C033085:phorbol