胰岛素通过快速激活一种组成型一氧化氮合酶，影响来自阴茎海绵体的培养人平滑肌细胞中的一氧化氮环核苷酸途径。

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase.

作者信息

Anfossi Giovanni, Massucco Paola, Mattiello Luigi, Balbo Alessandra, Russo Isabella, Doronzo Gabriella, Rolle Luigi, Ghigo Dario, Fontana Dario, Bosia Amalia, Trovati Mariella

机构信息

Diabetes Unit, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, 10043 Orbassano, Turin, Italy.

出版信息

Eur J Endocrinol. 2002 Nov;147(5):689-700. doi: 10.1530/eje.0.1470689.

DOI:10.1530/eje.0.1470689

PMID:12444902

Abstract

AIMS

We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP).

METHODS

cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as l-[3H]-citrulline production from l-[3H]-arginine and cyclic nucleotides by radioimmunoassay.

RESULTS

cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 micro mol/l calcium ionophore ionomycin (from 0.1094+/-0.0229 to 0.2685+/-0.0560 pmol/min per mg cell protein, P=0.011) and to 2 nmol/l insulin (from 0.1214+/-0.0149 to 0.2045+/-0.0290 pmol/min per mg cell protein, P=0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71+/-0.10 to 6.80+/-0.40 pmol/10(6) cells at 30 min, P=0.0001; cAMP from 1.26+/-0.06 to 3.02+/-0.30 pmol/10(6) cells at 60 min, P=0.0001). NOS inhibitor N(G)-monomethyl-l-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin.

CONCLUSION

Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.

摘要

目的

我们在培养的人海绵体平滑肌细胞中评估了钙依赖性组成型一氧化氮合酶（cNOS）的表达和活性，以及胰岛素诱导一氧化氮（NO）生成和增加细胞内环核苷酸鸟苷 3',5'-环磷酸（cGMP）和腺苷 3',5'-环磷酸（cAMP）的能力。

方法

通过 RT-PCR 扩增检测 cNOS mRNA，通过免疫荧光检测 cNOS 蛋白，以 l-[3H]-精氨酸生成 l-[3H]-瓜氨酸来检测 cNOS 活性，通过放射免疫测定法检测环核苷酸。

结果

在培养的细胞中发现了 cNOS mRNA 和 cNOS 蛋白；暴露于 1 μmol/l 钙离子载体离子霉素 5 分钟（从 0.1094±0.0229 增加到 0.268±0.0560 pmol/分钟每毫克细胞蛋白，P = 0.011）和 2 nmol/l 胰岛素（从 0.1214±0.0149 增加到 0.2045±0.0290 pmol/分钟每毫克细胞蛋白，P = 0.041）可增加 cNOS 活性。胰岛素以剂量和时间依赖性方式增加 cGMP 和 cAMP（即使用 2 nmol/l 胰岛素时，30 分钟时 cGMP 从 2.71±0.10 增加到 6.80±0.40 pmol/10⁶ 个细胞，P = 0.0001；60 分钟时 cAMP 从 1.26±0.06 增加到 3.02±0.30 pmol/10⁶ 个细胞，P = 0.000）。一氧化氮合酶抑制剂 N(G)-单甲基-l-精氨酸以及磷脂酰肌醇 3-激酶（PI 3-激酶）抑制剂渥曼青霉素和 LY 294002 可减弱胰岛素这些作用。在存在磷酸二酯酶抑制、NO 供体激活鸟苷酸环化酶以及伊洛前列素或福斯可林激活腺苷酸环化酶的情况下，胰岛素对环核苷酸的作用仍然存在。