NITRIC OXIDE SYNTHASE IN THE JUXTAGLOMERULAR APPARATUS

描述(申请人提供)：血管内皮细胞依赖性松弛因子/一氧化氮(EDRF/NO)功能障碍伴随盐敏感型高血压、糖尿病(DM)和心血管疾病。不对称二甲基精氨酸通过与一氧化氮合酶底物L-精氨酸竞争膜转运或竞争性抑制一氧化氮合酶而损害内源性精氨酸受体。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的正常调控还没有得到广泛的研究。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，特别是在致密斑细胞和远端肾单位。DDAH-1在血管内皮细胞和近端小管大量表达。我们的研究表明，血管紧张素II上调DDAH-2(从而可能减少黄斑致密ADMA)，但下调DDAH-1(从而可能增加内皮ADMA)。饮食盐限制和早期胰岛素缺乏性糖尿病均损害EDRF/NO。这一缺陷可以被过量的精氨酸克服，这表明ADMA可能抑制了NO。事实上，我们已经发现，盐限制降低了血浆精氨酸，但增加了血浆ADMA，并削弱了离体肠系膜阻力血管对乙酰胆碱的EDRF/NO反应。相反，我们发现DM增加了肾脏DDAH-2的表达，导致肾脏ADMA水平降低。致密斑区NO的增加可阻断血管收缩小管球反馈反应，从而选择性地扩张肾传入小动脉，导致肾小球高滤过。我们建议检验这一假说，即精氨酸在肾脏和微血管阻力血管中通过一氧化氮合酶的传递、运输和代谢受到DDAH的不同调节，从而导致在生理适应过程中特定部位和细胞特异性地产生ADMA和NO，以适应盐摄入量的变化和对胰岛素依赖型糖尿病的病理生理反应。我们在大鼠体内建立了针对DDAH-1和DDAH-2的基因沉默策略，以测试这些异构体在抵抗血管中EDRF/NO和肾脏中转化生长因子的生理学研究中的特定作用。前两个目的是验证这样的假设，即饮食盐限制限制了血管内皮细胞中NO的生成，这是因为血浆精氨酸浓度降低和内皮细胞中DDAH-1活性降低。这可以降低细胞内精氨酸：ADMA的浓度，从而抑制EDRF/NO。我们建议研究血管紧张素和盐皮质激素受体的特殊作用。第三个目的是验证这一假设，即在早期胰岛素缺乏性糖尿病中，内皮细胞DDAH-1的下调是血管病变之前的EDRF/NO缺陷的基础，而致密黄斑中DDAH-2的上调则增强了局部NO，从而阻断了肾小管球反馈(TGF)反应，导致了肾脏病变之前的高滤过。

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.