Expression and function of the guanylin ligand family

说明(申请人提供)：鸟苷和尿鸟苷是主要在哺乳动物肠道表达的多肽，由于它们与细菌耐热肠毒素(ST)的同源性以及它们与ST受体鸟苷环化酶C(GC-C)的结合能力而被鉴定。ST是分泌性腹泻的世界性病因，而鸟苷和尿鸟苷被认为在不引起腹泻的情况下调节肠道分泌。因此，更好地了解这些多肽的作用机制和受体将可能有助于了解毒素性腹泻的病理生理学。为了进一步探索鸟苷和尿鸟苷的生理作用，我们建立了鸟苷和尿鸟苷基因靶向的小鼠。我们在初步实验中发现，尿鸟苷在盐的动态平衡中具有重要的调节作用。因此，这一应用的首要假设是，尿鸟苷和鸟苷是肠内形成局部旁分泌调节系统的肠道激素系统，也是作为肠-肾轴的一部分的激素系统，以调节钠尿。我们将解决以下具体目标。I)确定鸟苷和尿鸟苷作为内分泌激素调节基因靶标小鼠的盐和水代谢的作用。我们将检验以下假设：1)尿鸟苷，而不是鸟苷素或GC-C缺乏的小鼠，将表现出尿钠和钾排出障碍。2)尿鸟苷基因缺陷小鼠表现为盐敏感型高血压。3)尿鸟苷的这些作用是通过一种新的非GC-C受体介导的。Ii)确定鸟苷和尿鸟苷表达的调控机制。我们将检验以下假设：1)鸟苷和尿鸟苷的基础表达水平受蛋白激酶C(PKC)亚型的调节。2)高张力通过转录调控的PKC依赖机制上调鸟苷和尿鸟苷。3.干扰素-γ通过转录调控机制下调鸟苷和尿鸟苷的表达。Iii)确定肠和肾脏中由于尿鸟苷丢失而导致的细胞分泌缺陷。我们将检验以下假设：1)尿鸟苷丢失会导致肠道氯的分泌受损。2)在肾脏中，尿鸟苷的丢失将导致近端肾小管细胞水和溶质转运失调，导致细胞内空泡形成。3)尿鸟苷缺失将导致新的尿鸟苷受体基因(S)代偿性上调，并对近端小肠和肾小管上皮细胞转运蛋白和水通道的表达产生代偿性逆调节。这些研究将进一步描述这些多肽和ST在肠道中的作用和作用，以及它们在肠-肾轴中作为肠道激素的作用。此外，这些研究将为将这些多肽作为液体超负荷状态的治疗剂进行翻译研究奠定基础。

DESCRIPTION (provided by applicant): Guanylin and uroguanylin are peptides that are expressed primarily in the mammalian intestine and were identified because of their homology to the bacterial heat stable enterotoxin (ST) and their ability to bind to the ST receptor, guanylyl cyclase C (GC-C). ST is a worldwide cause of secretory diarrhea whereas guanylin and uroguanylin are thought to modulate intestinal secretion without causing diarrhea. Therefore, a better understanding of the mechanisms and receptors through which these peptides act will likely shed light on the pathophysiology of toxigenic diarrhea. To further explore the physiologic role of guanylin and uroguanylin we created guanylin and uroguanylin gene-targeted mice. We found in preliminary experiments that uroguanylin has an important regulatory role in salt homeostasis. Therefore, the overarching hypothesis of this application is that uroguanylin and guanylin are enteric hormones forming both a local paracrine regulatory system in the intestine and a hormonal system as part of an enteric-renal axis to regulate natriuresis. We will address the following specific aims. I) Define the role of guanylin and uroguanylin as endocrine hormones regulating salt and water metabolism in gene targeted mice. We will test the hypotheses that: 1) Uroguanylin but not guanylin or GC-C deficient mice, will demonstrate impaired urinary sodium and potassium excretion. 2) Uroguanylin deficient mice will demonstrate salt sensitive hypertension. 3) These actions of uroguanylin are mediated via a novel, non-GC-C receptor. II) Determine the mechanisms regulating guanylin and uroguanylin expression. We will test the hypotheses that: 1) Basal levels of guanylin and uroguanylin expression are regulated by protein kinase C (PKC) isoforms. 2) Hypertonicity upregulates guanylin and uroguanylin by a transcriptionally-regulated, PKC-dependent mechanism. 3. Interferon-gamma (IFN-gamma) downregulates guanylin and uroguanylin via a transcriptionally-regulated mechanism. III) Define the cellular secretory defects resulting from uroguanylin loss in intestine and kidney. We will test the hypotheses that: 1) Loss of uroguanylin will result in impaired intestinal secretion of chloride. 2) In the kidney, loss of uroguanylin will result in dysregulated water and solute transport in proximal tubular cells leading to intracellular vacuole formation. 3) Loss of uroguanylin will result in compensatory upregulation of novel uroguanylin receptor gene(s) and compensatory counter-regulation of transporters and water channels expressed in enterocytes in the proximal small intestine and proximal tubule cells in the kidney. These studies will further delineate the role and action of these peptides and ST in the intestine as well as their roles as intestinal hormones in an enteric-renal axis. Furthermore, these studies will lay the groundwork for translational research involving these peptides as therapeutic agents for fluid overload states.