Nitric Oxide and gastric motility in female diabetics

描述(申请人提供)：胃轻瘫是一种主要影响年轻女性的致残疾病。这种疾病的生物学基础及其相关的性别偏见仍然知之甚少。最近的研究表明神经元型一氧化氮合酶(NNOS)在肌间神经元中的失调起作用，我们推测氮能控制胃运动的性别差异可能解释了观察到的女性对糖尿病胃轻瘫的易感性。我们的初步数据表明，在链脲佐菌素(STZ)诱导的糖尿病后，女性选择性地表现出明显的氮能松弛和固体胃排空延迟的损害。最重要的是，我们还表明，健康和糖尿病大鼠的氮能松弛变化与nNOS1的二聚化状态有很好的相关性，但与总nNOS(1、2和3)的表达无关。NNOS的二聚化对该酶的活性是必不可少的，但以前还没有在胃肠道组织中进行过研究。在这项提议中，我们重点研究了四氢生物蝶呤(BH4)的作用，四氢生物蝶呤是维持nNOS同源二聚体结构所必需的nNOS活性的重要辅助因子。组织中BH4的水平反过来又取决于GTP环水解酶1(GTPCH1)的活性，GTPCH1是BH4从头合成的限速酶。在初步实验中，我们已经证明了GTPCH1在糖尿病女性胃组织中的表达下调，同时BH4含量显著减少，这表明生物蝶呤的合成减少导致了氮能松弛的减少。为此，GTPCH1抑制剂2，4-二氨基-6-羟基嘧啶(DAHP)在体外可减少正常雌性大鼠胃组织中nNOS1的二聚化、NO释放和氮能松弛。相反，在体外向女性胃组织中加入外源性BH4可以逆转高血糖引起的氮能松弛下降。此外，体内BH4治疗可减轻糖尿病大鼠胃组织中nNOS活性的降低。因此，我们假设胃BH4生物合成受损是女性糖尿病胃轻瘫患者nNOS活性降低和氮能松弛的原因。为了验证这一假说，我们提出了以下具体目标：具体目标1：确定糖尿病是否导致雌性大鼠胃组织中BH4生物合成途径、nNOS二聚化、nNOS活性和氮能松弛的损害。特异性目的2：探讨补充BH4能否恢复雌性糖尿病大鼠胃排空延迟、胃nNOS二聚化、nNOS活性、NO合成和氮能松弛。这些研究数据将为了解正常和糖尿病雌性大鼠胃组织中nNOS功能的调节机制提供重要信息，从而加深我们对胃轻瘫病理生理学的理解。此外，它可能导致发现新的治疗靶点来管理这一具有挑战性的临床综合征。糖尿病是一种慢性进行性疾病，影响着1600万美国人。糖尿病会引起多种并发症，影响视网膜、肾脏、血管、胃肠道和神经系统。糖尿病并发症的发生机制尚不清楚。胃动力障碍或胃病是糖尿病临床上的严重并发症之一，可导致恶心、呕吐和体重减轻等致残症状，并经常导致胃排空延迟或加速。报告表明，80%的患者是患有这一问题的妇女。尽管确切的发病机制尚不清楚，但在实验模型中有相当令人信服的证据表明，糖尿病导致产生神经递质一氧化氮(NO)的特定神经元发生故障。这种情况是由于负责产生NO的酶(神经元型一氧化氮合酶：nNOS)的表达缺失造成的，胰岛素替代治疗可以逆转这种情况。导致糖尿病胃nNOS表达缺失和胃排空改变(延迟或加速)的分子事件(由于失去对胃肌肉收缩和松弛的神经元控制而引起的机械性事件)仍不清楚。这些研究数据将为研究糖尿病雌性大鼠NO介导的胃排空和胃收缩的调节机制提供重要信息。此外，它可能导致发现新的治疗靶点来管理这一具有挑战性的临床综合征。

DESCRIPTION (provided by applicant): Gastroparesis is a disabilitating disease affecting predominantly young women. The biological basis of this disorder and its associated gender bias remains poorly understood. Recent studies have implicated a role for dysregulation of neuronal nitric oxide synthase (nNOS) in myenteric neurons and we surmised that gender differences in nitrergic control of gastric motility may possibly account for observed vulnerability of females to diabetic gastroparesis. Our preliminary data suggests that a significant impairment of nitrergic relaxation and delayed gastric emptying for solids was demonstrated selectively in females after diabetes induction with streptozotocin (STZ). Most importantly, we have also shown that changes in nitrergic relaxation in both healthy and diabetic rats correlates well with the state of dimerization of nNOS1 but not with the expression of total nNOS (1, 2 and 3). Dimerization of nNOS is essential for activity of this enzyme but has not been previously studied in gastrointestinal tissue. In this proposal, we have focused on the role of tetrahydrobiopterin (BH4), an essential cofactor for nNOS activity that is required to maintain the homodimeric structure of nNOS. BH4 levels in tissue in turn depend on the activity of GTP cyclohydrolase1 (GTPCH1), the rate limiting enzyme in the de novo synthesis of BH4. In preliminary experiments we have demonstrated that GTPCH1 mRNA and protein expression are down regulated in the diabetic female gastric tissues along with significantly reduced BH4 content, suggesting reduced synthesis of biopterins leading to decreased nitrergic relaxation. In support of this, the GTPCH1 inhibitor, 2, 4 diamino-6-hydroxypyrimidine (DAHP), reduced nNOS1 dimerization, NO release and nitrergic relaxation of gastric tissue in normal female rats in vitro. Conversely, the exogenous addition of BH4 to female gastric tissues in vitro reverses a hyperglycemia-induced decrease in nitrergic relaxation. In addition, in vivo BH4 treatment attenuated reduced nNOS activity in diabetic gastric tissue. We therefore hypothesize that impaired biosynthesis of gastric BH4 accounts for the decrease in nNOS activity and nitrergic relaxation in female diabetic gastroparesis. To test this hypothesis we propose the following specific aims: Specific Aim 1: To determine whether diabetes results in impairment of the BH4 biosynthetic pathway, nNOS dimerization, nNOS activity and nitrergic relaxation in female rat gastric tissues. Specific Aim 2: To investigate whether BH4 supplementation restores delayed gastric emptying, impaired gastric nNOS dimerization, nNOS activity, NO synthesis and nitrergic relaxation in female diabetic rats. The data from these studies will provide important information as to the mechanisms of regulation of nNOS function in normal and diabetic female rat gastric tissues and thereby enhance our understanding of the pathophysiology of gastroparesis. Further, it may lead to the discovery of novel therapeutic targets for the management of this challenging clinical syndrome.Diabetes mellitus is a chronic progressive disease that affects 16 million Americans. Diabetes causes several complications that affect retina, kidney, vascular, gastrointestinal, and nervous system. The mechanism through which diabetic complications develop is unclear. Gastric dysmotility or gastropathy is one of the vigorous complications of diabetic mellitus in clinics and can cause disabling symptoms including nausea, vomiting and weight loss and often leads to delayed or accelerated gastric emptying. Reports indicate that 80% of patients are women suffering with this problem. Although the exact pathogenesis remains unknown, there is fairly convincing evidence in experimental models that diabetes results in the malfunctioning of specific neurons that produce the neurotransmitter nitric oxide (NO). This condition is due to loss of expression of the enzyme responsible for NO production (neuronal nitric oxide synthase: nNOS) and can be reversed with insulin replacement. The molecular events leading to loss of nNOS expression and altered (delay or accelerate) gastric emptying (mechanical due to loss of neuronal control of stomach muscle contractions and relaxations) in diabetic stomachs remain unknown. The data from these studies will provide important information as to the mechanisms of regulation of NO mediated gastric emptying and stomach contractions in diabetic female rat. Further, it may lead to the discovery of novel therapeutic targets for the management of this challenging clinical syndrome.