Marley P D, Cheung C Y, Thomson K A, Murphy R
Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia.
J Auton Nerv Syst. 1996 Sep 12;60(3):141-6. doi: 10.1016/0165-1838(96)00044-6.
The effect of pituitary adenylate cyclase-activating polypeptide (PACAP-27) on tyrosine hydroxylase activity has been studied in intact, cultured, bovine adrenal chromaffin cells. Tyrosine hydroxylase activity was determined in situ by measuring the production of 14CO2 following the hydroxylation and rapid decarboxylation of [14C]tyr offered to the cells. PACAP-27 increased tyrosine hydroxylase activity 3-fold over 10 min. With an EC50 of 10-20 nM. PACAP-38 was approximately 2-fold less potent. Removing extracellular Ca2+ reduced basal tyrosine hydroxylase activity and the activation produced by both PACAP-27 and forskolin by about 20%. In the absence of extracellular Ca2+, chelation of intracellular Ca2+ by treating cells with BAPTA-AM (50 microM) caused a consistent 40-50% reduction in basal tyrosine hydroxylase activity and in the responses to forskolin and PACAP-27. The tyrosine hydroxylase activation produced by PACAP-27 was unaffected by the protein kinase C inhibitor Ro 3l-8220 (3 microM), but was reduced by 85% by the protein kinase A inhibitor H89 (10 microM). PACAP-27 increased cellular cyclic AMP levels 3-fold at 100 nM. The results suggest that PACAP-27 activates tyrosine hydroxylase in bovine chromaffin cells through cyclic AMP formation and protein kinase A activation, and that both extracellular and intracellular Ca2+ modulate the effect of the adenylate cyclase/cyclic AMP/protein kinase A signalling pathway on tyrosine hydroxylase activity.
在完整的、培养的牛肾上腺嗜铬细胞中，研究了垂体腺苷酸环化酶激活多肽（PACAP - 27）对酪氨酸羟化酶活性的影响。通过测量向细胞提供[14C]酪氨酸羟化和快速脱羧后14CO2的产生，原位测定酪氨酸羟化酶活性。PACAP - 27在10分钟内使酪氨酸羟化酶活性增加了3倍，半数有效浓度（EC50）为10 - 20 nM。PACAP - 38的效力约低2倍。去除细胞外Ca2 +可使基础酪氨酸羟化酶活性以及PACAP - 27和福斯高林产生的激活作用降低约20%。在没有细胞外Ca2 +的情况下，用BAPTA - AM（50 microM）处理细胞螯合细胞内Ca2 +，可使基础酪氨酸羟化酶活性以及对福斯高林和PACAP - 27的反应持续降低40 - 50%。PACAP - 27产生的酪氨酸羟化酶激活作用不受蛋白激酶C抑制剂Ro 31 - 8220（3 microM）的影响，但被蛋白激酶A抑制剂H89（10 microM）降低了85%。100 nM的PACAP - 27可使细胞内环磷酸腺苷（cAMP）水平增加3倍。结果表明，PACAP - 27通过环磷酸腺苷形成和蛋白激酶A激活来激活牛嗜铬细胞中的酪氨酸羟化酶，并且细胞外和细胞内Ca2 +均调节腺苷酸环化酶/环磷酸腺苷/蛋白激酶A信号通路对酪氨酸羟化酶活性的影响。
