Slusher B S, Zacco A E, Maslanski J A, Norris T E, McLane M W, Moore W C, Rogers N E, Ignarro L J
Department of Pharmacology, ZENECA Pharmaceuticals Group, Wilmington, Delaware 19897.
Mol Pharmacol. 1994 Jul;46(1):115-21.
Rat neurotensin (NT) receptor (NTR) cDNA was subcloned into the pRC-CMV expression vector and transfected into 293 cells, and cellular clones that stably expressed the NTR were isolated and characterized. [3H]NT binding to membranes prepared from the NTR cDNA-transfected cells displayed specificity and saturability, with an apparent Kd of 1.25 nM and a Bmax of 43.4 pmol/mg of protein (approximately 3.5 x 10(6) binding sites/cell). NT stimulated an increase in [3H]inositol phosphate levels in the NTR-expressing cells up to 2500% of basal levels. The response was time and dose dependent, with an EC50 of 10.4 nM. NT also stimulated cAMP formation in these cells, with an EC50 of 27.0 nM. In addition, NT evoked an increase in the level of intracellular calcium. Approximately 60% of the calcium rise was attributable to the release of intracellular stores and 40% was attributable to calcium influx. Although NTR occupancy has been shown to stimulate cGMP formation in several brain preparations and cell lines, NT was unable to mediate cGMP synthesis in the NTR-expressing 293 cells. We found that 293 cells have guanylate cyclase activity but have undetectable levels of nitric oxide synthase (NOS) activity. Because it was possible that the production of nitric oxide is required as the mediator of NT-induced cGMP synthesis, we subcloned NOS cDNA into the pCEP4 expression vector and transiently expressed it in the NTR cells. We report that NT increased cGMP levels up to 375% of basal levels when NOS cDNA was coexpressed and that the increase was completely inhibited by the NOS inhibitor N omega-nitro-L-arginine. NT-induced cGMP accumulation was time and dose dependent, with an EC50 of 1.7 nM. To our knowledge, this is the first report of NT mediating cGMP formation with a cloned receptor and the first evidence that NT-induced cGMP accumulation requires the production of nitric oxide.
将大鼠神经降压素（NT）受体（NTR）cDNA亚克隆到pRC-CMV表达载体中，并转染到293细胞中，然后分离并鉴定稳定表达NTR的细胞克隆。用[3H]NT与从转染了NTR cDNA的细胞制备的膜进行结合实验，结果显示具有特异性和饱和性，其表观解离常数（Kd）为1.25 nM，最大结合容量（Bmax）为43.4 pmol/mg蛋白质（约3.5×10(6)个结合位点/细胞）。NT刺激表达NTR的细胞中[3H]肌醇磷酸水平增加，最高可达基础水平的2500%。该反应具有时间和剂量依赖性，半数有效浓度（EC50）为10.4 nM。NT还刺激这些细胞中cAMP的形成，EC50为27.0 nM。此外，NT引起细胞内钙水平升高。钙升高的约60%归因于细胞内储存钙的释放，40%归因于钙内流。虽然在几种脑标本和细胞系中已表明NTR占据可刺激cGMP形成，但NT在表达NTR的293细胞中无法介导cGMP合成。我们发现293细胞具有鸟苷酸环化酶活性，但一氧化氮合酶（NOS）活性水平检测不到。因为一氧化氮的产生可能是NT诱导cGMP合成的介导物，所以我们将NOS cDNA亚克隆到pCEP4表达载体中，并在NTR细胞中瞬时表达。我们报告说，当共表达NOS cDNA时，NT使cGMP水平增加至基础水平的375%，并且该增加被NOS抑制剂Nω-硝基-L-精氨酸完全抑制。NT诱导的cGMP积累具有时间和剂量依赖性，EC50为1.7 nM。据我们所知，这是关于NT通过克隆受体介导cGMP形成的首次报道，也是NT诱导的cGMP积累需要一氧化氮产生的首个证据。
