EVALUATE BLOOD FLOW LUNG, BRAIN, HEART, KIDNEY IN WILD-TYPE MICE W/ CONTRAST

通过对比评估野生型小鼠的肺、脑、心脏、肾的血流

• 批准号: 7358284
• 负责人: TIMOTHY A HAYSTEAD
• 金额: $ 0.51万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358284
• 关键词: EVALUATE; BLOOD; FLOW; LUNG; BRAIN

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The initial goal of this project is to evaluate blood flow in lung, brain, heart, and kidney in wild-type mice using in situ contrast imaging. Responses to agonists that induce vasoconstriction and vasorelaxation may be altered in the knockout mouse. Blood pressure measurement in response to these same agonists will also be performed. Systemic adrenergic vasoconstriction is well induced by the alpha-agonist phenylephrine or the alpha1,2;beta-1 agonist norepinephrine. Dose responses to phenylephrine (0.5-8 ug/kg) and norepinephrine (100-600 ng/kg) will be performed. The maximum doses of these drugs is expected to raise mean arterial blood pressure (MAP) approximately 40mmHg above basal levels (basal MAP 90-120 mmHg), so an effect on blood flow should be apparent. Renin-angiotensin-aldosterone system dependent increases in blood pressure can be evaluated by administration of angiotensin II and vasopressin. Nitric oxide-dependent increases in blood pressure can be measured by chronic administration of L-NAME (400 ug/g IP for 7 days), a nitric oxide synthase inhibitor. For evaluation of renal blood flow, dopamine (2 ug/kg) should constrict the efferent arteriole and relax the afferent arteriole of the kidney. CHASM was identified as a very early target of the protein kinase PKG in isolated 32P labeled smooth muscle using mass spectrometry. The protein was identified by de novo sequence data as a theoretical protein in the human genome. Its function is therefore unknown. We have subsequently validated the protein with an antibody that was raised to the recombinant protein. Histological staining shows that CHASM is exclusively expressed in muscle fibers and neuro-endocrine cells innervating muscle. PKG is a major mediator of smooth muscle relaxation and deletion of the protein kinase in mouse resulted in a complete loss of all hormone mediated signaling in this tissue. As a result the animals were hypertensive and exhibited gastrointestinal disorders associated with a loss of coordinated gut motility. The molecular mechanisms by which PKG, once activated, causes smooth muscle to relax are unknown. Our hypothesis suggests that PKG mediates its muscle relaxing effects through phosphorylation of one or more specific proteins expressed in smooth muscle. Interestingly, the compliment of proteins phosphorylated by PKG varies with smooth muscle subtype. We believe this variance of expression infers specific contractile properties upon those muscles. CHASM is expressed largely in tonic smooth muscle such as femoral artery or aorta. Therefore we believe that in the CHASM null mouse there will be alterations in the normal responsiveness of vasculature in response to pharmacological agonists known to activate PKG. Responsiveness of muscles that do not normally express CHASM would be expected to be normal. We would also predict alterations in gut motility in the CHASM null mouse, since the protein is highly expressed in neuro-endocrine cells of ileum. Real time imaging studies will therefore provide a very elegant means to evaluate the physiological role of CHASM in the regulation of smooth muscle contraction by PKG. Parameters we would like to examine are agonist/antagonist induced effects on normal blood flow through out the circulation and every major organ (egg. renal and cardiovascular). We would also like to monitor the effects of agonists on gut motility. We would also like to examine the over all vascular and gut architecture to ensure that CHASM deletion does not cause any developmental abnormalities. Vasorelaxation in wild-type mice will be evaluated by administration of endothelium dependent vasorelaxants bradykinin (0.1-10 ug/kg), the nitric oxide donor, sodium nitroprusside (3 ug/kg), and acetylcholine (1 mg/kg). Determine the physiological significance of CHASM, a novel target of cyclic GMP dependant protein kinase (PKG) that is expressed in smooth muscl

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。该项目的最初目标是使用原位对比成像评估野生型小鼠肺、脑、心脏和肾脏中的血流。在基因敲除小鼠中，对诱导血管收缩和血管舒张的激动剂的反应可能会改变。还将进行对这些相同激动剂的血压测量。α-激动剂苯肾上腺素或α 1，2; β-1激动剂去甲肾上腺素可很好地诱导全身性肾上腺素能血管收缩。将进行苯肾上腺素（0.5-8 ug/kg）和去甲肾上腺素（100-600 ng/kg）的剂量反应。这些药物的最大剂量预计将使平均动脉血压（MAP）比基础水平升高约40 mmHg（基础MAP 90-120 mmHg），因此对血流的影响应该是明显的。血压的肾素-血管紧张素-醛固酮系统依赖性升高可通过给予血管紧张素II和加压素来评估。一氧化氮依赖性血压升高可通过长期给予一氧化氮合酶抑制剂L-NAME（400 ug/g IP，持续7天）进行测量。为了评价肾血流量，多巴胺（2 ug/kg）应收缩肾的输出小动脉并舒张肾的输入小动脉。 CHASM被确定为一个非常早期的目标蛋白激酶PKG在分离的32 P标记的平滑肌使用质谱。该蛋白质通过从头序列数据鉴定为人类基因组中的理论蛋白质。因此，它的功能是未知的。我们随后用针对重组蛋白产生的抗体验证了该蛋白。组织学染色显示CHASM仅在支配肌肉的肌纤维和神经内分泌细胞中表达。PKG是平滑肌松弛的主要介质，在小鼠中蛋白激酶的缺失导致该组织中所有激素介导的信号传导的完全丧失。结果，这些动物出现高血压，并表现出与协调肠道运动丧失相关的胃肠道疾病。PKG一旦被激活，引起平滑肌松弛的分子机制尚不清楚。我们的假设表明，PKG介导的肌肉松弛作用，通过磷酸化的一个或多个特定的蛋白表达在平滑肌。有趣的是，PKG磷酸化的蛋白质的补体随平滑肌亚型而变化。我们相信这种表达的差异推断了这些肌肉的特定收缩特性。CHASM主要在紧张性平滑肌如股动脉或主动脉中表达。因此，我们认为，在CHASM敲除小鼠中，血管系统对已知激活PKG的药理学激动剂的正常反应性将发生改变。通常不表达CHASM的肌肉的反应性预计是正常的。我们还将预测CHASM敲除小鼠肠道运动的改变，因为该蛋白在回肠的神经内分泌细胞中高度表达。因此，真实的时间成像研究将提供一个非常优雅的手段来评估的生理作用，CHASM的平滑肌收缩的调节PKG。我们想要检查的参数是激动剂/拮抗剂对通过循环和每个主要器官（卵）的正常血流的诱导作用。肾和心血管）。我们还想监测激动剂对肠道运动的影响。我们还想检查所有的血管和肠道结构，以确保CHASM缺失不会导致任何发育异常。 通过给予内皮依赖性血管舒张剂缓激肽（0.1-10 μ g/kg）、一氧化氮供体硝普钠（3 μ g/kg）和乙酰胆碱（1 mg/kg）来评价野生型小鼠的血管舒张。 确定CHASM的生理意义，CHASM是平滑肌表达的环GMP依赖性蛋白激酶（PKG）的新靶点，