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. ? ? ?
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