ACTIVATION AND MULTIPLE-SITE PHOSPHORYLATION OF TYROSINE-HYDROXYLASE IN PERFUSED RAT ADRENAL-GLANDS
ACTIVATION AND MULTIPLE-SITE PHOSPHORYLATION OF TYROSINE-HYDROXYLASE IN PERFUSED RAT ADRENAL-GLANDS
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DOI：
10.1111/j.1471-4159.1992.tb09276.x
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发表时间：
1992-01-01
影响因子：
4.7
通讯作者：
WAKADE, AR
中科院分区：
文献类型：
--
作者：
HAYCOCK, JW;WAKADE, AR
用胰蛋白酶消化从大鼠肾上腺中分离的酪氨酸羟化酶（TH），用P-32（i）标记，产生五种磷酸肽。 根据这些磷酸肽与大鼠嗜铬细胞瘤细胞TH中的磷酸肽的对应关系，推断出四个磷酸化位点（Ser 8，Ser 19，Ser 31和Ser 40）。 场刺激内脏神经在1或10 Hz（300脉冲）增加P-32纳入TH。 在10 Hz时，Ser 19和Ser 40的磷酸化增加，而在1 Hz时，Ser 19、Ser 31和Ser 40的磷酸化增加。 在30或300 μ M四氢生物蝶呤的体外（pH 7.2）测定中，1或10 Hz的刺激也增加了TH的催化活性。 尼古丁（3 μ M，3分钟）增加Ser 19磷酸化，血管活性肠多肽（10 μ M，3分钟）增加Ser 40磷酸化，毒蕈碱（100 μ M，3分钟）增加主要在Ser 19和Ser 31的TH磷酸化。 血管活性肠多肽，而不是尼古丁或毒蕈碱，模仿场刺激TH活性的影响。 因此，调节大鼠肾上腺髓质TH磷酸化神经冲动介导的多个第一和第二信使系统，如先前所示的儿茶酚胺分泌。 然而，不同的第二信使参与这两个过程。 血管活性肠肽作为促分泌素的作用涉及细胞内钙的动员，而其对TH磷酸化的作用是由环AMP介导的。 血管活性肠多肽的后一种作用和随后Ser 40磷酸化的增加似乎是内脏神经刺激快速激活TH的原因。
Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with P-32(i) produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased P-32 incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300-mu-M tetrahydrobiopterin. Nicotine (3-mu-M, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10-mu-M, 3 min) increased Ser40 phosphorylation, and muscarine (100-mu-M, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.