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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.

ABSTRACT: 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.

SUBMITTER: Murphy NP

PROVIDER: S-EPMC1130836 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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Project description:Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.

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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.

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