The effect of protein kinase C activation on muscarinic-M3- and K(+)-evoked release of [3H]noradrenaline and increases in intracellular Ca2+ in human neuroblastoma SH-SY5Y cells.
Short-term pretreatment (9 min) with the phorbol ester 12-myristate 13-acetate (PMA) alone had no effect on the basal release of [3H]noradrenaline ([3H]NA), but enhanced K+ (100 mM)-, acetylcholine (0.1 mM)-, carbachol (1 mM)-, muscarine (1 mM)- and arecoline (1 mM)-evoked release by 2.3-, 6.4-, 3.0-, 2.0- and 2.0-fold respectively in SH-SY5Y cells. Maximum effects of PMA were observed after a 10 min preincubation at a concentration of 0.1 microM. There was a 4-fold decrease in the EC50 values (concentration required for 50% of maximal stimulation) observed for carbachol- and acetylcholine-evoked release of [3H]NA in the presence of PMA. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not alter the K(+)- or carbachol-evoked release of [3H]NA. The enhancement of release in the presence of PMA was more potently inhibited by the protein kinase C inhibitors RO 31-7549 [concentration required for 50% inhibition (IC50) = 0.18 microM/bd and RO 31-8220 (IC50 = 0.56 microM) than by either polymyxin-B or H-7. Furthermore, in the absence of PMA, both K(+)- and carbachol-evoked release was inhibited by these antagonists. Atropine, hexahydro-sila-difenidol and pirenzepine antagonized the PMA-enhanced carbachol-evoked release of [3H]NA, with Ki values of 2.75 +/- 0.25 nM, 2.6 +/- 0.64 nM and 294 +/- 17 nM respectively. These values were consistent with the coupling of an M3 muscarinic receptor to the release of [3H]NA in SH-SY5Y cells. Whereas pretreatment with PMA (5 min) enhanced M3-evoked release of [3H]NA, it decreased the muscarinic-agonist-evoked initial peak (greater than 85%) and plateau phase in intracellular Ca2+. These results suggest that noradrenaline release evoked by muscarinic agonists was triggered not only by relatively small changes in Ca2+ but also by activation of protein kinase C.
Murphy
N P
NP
Department of Cardiovascular Studies, University of Leeds, U.K.
McCormack
J G
JG
Ball
S G
SG
Vaughan
P F
PF
D002118:Calcium / Q000378:metabolism*; D002794:Choline / Q000502:physiology; D004789:Enzyme Activation; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009638:Norepinephrine / Q000378:metabolism*; D011188:Potassium / Q000502:physiology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium; D014407:Tumor Cells, Cultured
D011976:Receptors, Muscarinic; D014316:Tritium; D011493:Protein Kinase C; D002794:Choline; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium; D002118:Calcium; D009638:Norepinephrine