Takekoshi Kazuhiro, Ishii Kiyoaki, Shibuya Shunsuke, Kawakami Yasushi, Isobe Kazumasa, Nakai Toshiaki
Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan. K-takemd @md.tsukuba.ac.jp
Hypertension. 2002 Jan;39(1):142-8. doi: 10.1161/hy1201.096816.
We previously showed that CGP 42112 (an angiotensin type 2 [AT(2)] agonist) markedly reduces catecholamine biosynthesis by decreasing cGMP production mediated by AT(2), a subtype of Ang II receptor that is dominantly expressed in cultured porcine chromaffin cells. To elucidate the relationship of the 2 types of Ang II receptors, angiotensin type 1 (AT(1)) and AT(2), in the synthesis of catecholamine in adrenal medullary cells, we have examined the effect of Ang II plus CV-11974 (an AT(1) antagonist that selectively simulates AT(2) stimulation) and the effect of Ang II plus PD 123319 (an AT(2) antagonist that selectively simulates AT(1) stimulation) on catecholamine synthesis. We found that Ang II reduced cGMP production via AT(2), in a similar manner to that found with CGP 42112. Stimulation of AT(1) significantly upregulated protein kinase C activity. Tyrosine hydroxylase (TH) is a rate-limiting enzyme involved in the biosynthesis of catecholamine, and this catecholamine synthesis depends both on TH enzyme activity and on the levels of TH protein after TH gene transcription. We found that AT(2) stimulation significantly inhibited TH enzyme activity, whereas AT(1) stimulation significantly upregulated TH enzyme activity. The stimulatory effect of AT(1) was completely inhibited by Ro-32-0432 (a protein kinase C inhibitor) and PD 98059 (a MAP kinase kinase-1 [MEK-1] inhibitor). Pretreatment of cells with either 8-Br-cGMP (a membrane-permeable cGMP analog) or Zaprinast (a phosphodiesterase inhibitor) abolished the inhibitory effect of AT(2) on TH enzyme activity, indicating that the stimulatory effect of AT(2) may be mediated through a reduction in cGMP concentration. Similar to the effect on TH enzyme activity, AT(2) stimulation significantly reduced TH mRNA and protein levels and net catecholamine content below basal levels, whereas AT(1) stimulation increased them. We confirmed these findings by gel mobility shift assay. Our results show that stimulation of AT(2) reduces catecholamine biosynthesis via a decrease in cGMP levels. In contrast, stimulation of AT(1) stimulates catecholamine biosynthesis through activation of PKC. Thus, we conclude that AT(1) and AT(2) have counter-regulatory roles in the synthesis of catecholamine in adrenal medullary chromaffin cells.
我们之前的研究表明，CGP 42112（一种血管紧张素2型[AT(2)]激动剂）通过降低由AT(2)介导的cGMP生成，显著减少儿茶酚胺的生物合成。AT(2)是血管紧张素II受体的一种亚型，在培养的猪嗜铬细胞中占主导表达。为了阐明血管紧张素II的两种受体，即血管紧张素1型（AT(1)）和AT(2)，在肾上腺髓质细胞儿茶酚胺合成中的关系，我们研究了血管紧张素II加CV-11974（一种选择性模拟AT(2)刺激的AT(1)拮抗剂）以及血管紧张素II加PD 123319（一种选择性模拟AT(1)刺激的AT(2)拮抗剂）对儿茶酚胺合成的影响。我们发现，血管紧张素II通过AT(2)降低cGMP生成，其方式与CGP 42112相似。刺激AT(1)显著上调蛋白激酶C活性。酪氨酸羟化酶（TH）是儿茶酚胺生物合成中的一种限速酶，这种儿茶酚胺合成既取决于TH酶活性，也取决于TH基因转录后TH蛋白的水平。我们发现，刺激AT(2)显著抑制TH酶活性，而刺激AT(1)显著上调TH酶活性。AT(1)的刺激作用被Ro-32-0432（一种蛋白激酶C抑制剂）和PD 98059（一种丝裂原活化蛋白激酶激酶-1[MEK-1]抑制剂）完全抑制。用8-Br-cGMP（一种可透过细胞膜的cGMP类似物）或扎普司特（一种磷酸二酯酶抑制剂）预处理细胞，消除了AT(2)对TH酶活性的抑制作用，表明AT(2)的刺激作用可能是通过降低cGMP浓度介导的。与对TH酶活性的影响类似，刺激AT(2)显著降低TH mRNA和蛋白水平以及净儿茶酚胺含量至基础水平以下，而刺激AT(1)则使其增加。我们通过凝胶迁移率变动分析证实了这些发现。我们的结果表明，刺激AT(2)通过降低cGMP水平减少儿茶酚胺生物合成。相反，刺激AT(1)通过激活蛋白激酶C刺激儿茶酚胺生物合成。因此，我们得出结论，AT(1)和AT(2)在肾上腺髓质嗜铬细胞儿茶酚胺合成中具有反向调节作用。
