NITRIC OXIDE SYNTHASE IN THE JUXTAGLOMERULAR APPARATUS

描述（由申请方提供）：血管内皮依赖性舒张因子/一氧化氮（EDRF/NO）功能障碍伴随盐敏感性高血压、糖尿病（DM）和心血管疾病。不对称二甲基精氨酸（ADMA）通过与NOS底物L-精氨酸竞争经由系统y+的膜转运或通过竞争性抑制NOS来损害EDRF/NO。因此，ADMA可能是导致心血管疾病的EDRF/NO反应缺陷的基础。然而，ADMA的正常调节尚未被广泛研究。ADMA由二甲基精氨酸二甲氨基水解酶（DDAH-1和-2）代谢。我们发现DDAH-2在肾脏中大量表达，尤其是在致密斑细胞和远端肾单位中。DDAH-1在血管内皮和近端小管中大量表达。我们的研究表明，血管紧张素II上调DDAH-2（从而可能减少致密斑ADMA），但下调DDAH-1（从而可能增加内皮ADMA）。饮食限制盐和早期胰岛素减少DM都损害EDRF/NO。这种缺陷可以通过过量精氨酸克服，这表明NO可能被ADMA抑制。事实上，我们已经发现，盐限制降低血浆精氨酸，但增加血浆ADMA和削弱EDRF/NO反应的乙酰胆碱的离体肠系膜阻力血管。相反，我们发现DM增加肾DDAH-2表达，导致肾ADMA水平降低。致密斑NO的增加可阻断血管收缩性肾小管球蛋白反馈（TGF）反应，从而选择性地舒张肾传入小动脉，导致肾小球高滤过。我们建议测试的假设，精氨酸的交付，运输和代谢的NOS在肾脏和微血管阻力血管的差异调节DDAH，从而导致网站和细胞特异性生成的ADMA和NO在生理适应盐的摄入量和病理生理反应的变化，胰岛素缺乏型糖尿病。我们已经开发了一种靶向DDAH-1和-2的基因沉默策略，以测试这些异构体在阻力血管中的EDRF/NO和肾脏中的TGF的生理研究中的特定作用。前两个目的测试的假设，饮食中的盐限制限制NO生成血管内皮细胞，因为血浆精氨酸浓度的降低和DDAH-1活性在内皮细胞中的减少。这可能会降低细胞内精氨酸：ADMA浓度，从而抑制EDRF/NO。我们建议检查血管紧张素和盐皮质激素受体的具体作用。第三个目的是检验以下假设：在早期胰岛素缺乏性DM期间，内皮中DDAH-1的下调是血管病变之前的缺陷性EDRF/NO的基础，而致密斑中DDAH-2的上调增强了局部NO，其阻断了肾小管肾小球反馈（TGF）反应，导致肾病之前的hvperfiltration。

DESCRIPTION (provided by applicant): Dysfunction of the endothelium dependent relaxation factor/nitric oxide (EDRF/NO) in blood vessels accompanies salt sensitive hypertension, diabetes mellitus (DM) and cardiovascular disease. Asymmetric dimethylarginine (ADMA) impairs EDRF/NO either by competing with the NOS substrate, L-arginine for membrane transport via system y+, or by competitive inhibition of NOS. Therefore, ADMA could underlie defective EDRF/NO responses that contribute to cardiovascular disease. However, the normal regulation of ADMA has not been extensively studied. ADMA is metabolized by dimethylarginine dimethylaminohydrolases (DDAH-1 and -2). We have found that DDAH-2 is heavily expressed in the kidney, especially in the macula densa cells and distal nephron. DDAH-1 is heavily expressed in the vascular endothelium and proximal tubules. Our studies show that Angiotensin II upregulate DDAH-2 (and thereby may reduce macula densa ADMA) but downregulates DDAH-1 (and thereby may increase endothelial ADMA). Dietary salt restriction and early insulinopenic DM both impairs EDRF/NO. This defect can be overcome by excess arginine, suggesting that NO may be inhibited by ADMA. Indeed, we have found that salt restriction decreases plasma arginine, yet increases plasma ADMA and impairs the EDRF/NO responses to acetylcholine of isolated mesenteric resistance vessels. In contrast, we have found that DM increases renal DDAH-2 expression, leading to reduced renal ADMA levels. A consequent increase in macula densa NO could block the vasoconstrictive tubuloglomrular feedback (TGF) response and thereby vasodilate the renal afferent arteriole selectively, leading to glomerular hyperfiltration. We propose to test the hypothesis that arginine delivery, transport and metabolism by NOS in the kidneys and microvascular resistance vessels is differentially regulated by DDAH thereby leading to site- and cell-specific generation of ADMA and NO during physiologic adaptations to changes in salt intake and pathophysiologic responses to insulinopenic DM. We have developed a gene silencing strategy targeting DDAH-1 and -2 in the rat to test the specific roles of these isoforms in physiologic studies of EDRF/NO in resistance vessels and TGF in the kidney. The first two aims test the hypothesis that dietary salt restriction limits NO generation in vascular endothelium because of a reduction in plasma arginine concentration and a decrease in DDAH-1 activity in endothelial cells. This could reduce the intracellular arginine: ADMA concentration, thereby inhibiting EDRF/NO. We propose to examine the specific roles of angiotensin and mineralocorticosteroid receptors. The third aim tests the hypothesis that during early insulinopenic DM downregulation of DDAH-1 in endothelium underlies the defective EDRF/NO that precedes vasculopathy whereas upregulation of DDAH-2 in the macula densa enhances local NO that blocks the tubuloglomerular feedback (TGF) response causing hvperfiltration that precedes nephropathv.