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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。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，大脑，心脏和肾脏的血液流动。敲除小鼠对诱导血管收缩和血管结构化的激动剂的反应可能会改变。还将对这些相同的激动剂进行血压测量。 α-激动剂苯肾上腺素或α1,2; beta-1激动剂去甲肾上腺素很好地诱导了全身性肾上腺素血管收缩。将对苯肾上腺素（0.5-8 ug/kg）和去甲肾上腺素（100-600 ng/kg）的剂量反应进行。这些药物的最大剂量预计将升高平均动脉血压（MAP）高于基础水平（基底MAP 90-120 mmHg），因此应对血流的影响显而易见。肾素 - 血管紧张素 - 醛固酮系统依赖性血压的升高可以通过给予血管紧张素II和加压素评估。一氧化氮依赖性升高可以通过长期施用L-名称（400 ug/g IP 7天），一种一氧化氮合酶抑制剂来测量。为了评估肾脏血流，多巴胺（2 ug/kg）应收缩传出动脉，并放松肾脏的传入小动脉。 使用质谱法标记了蛋白激酶PKG的蛋白激酶PKG的早期靶标。该蛋白通过从头序列数据鉴定为人类基因组中的理论蛋白。因此，其功能未知。随后，我们用对重组蛋白的抗体验证了蛋白质。组织学染色表明，裂缝仅在肌肉纤维和神经内分泌细胞中支配肌肉。 PKG是小鼠中平滑肌松弛和蛋白激酶缺失的主要介体，导致该组织中所有激素介导的信号传导完全丧失。结果，这些动物是高血压，表现出与肠道衰减相关的胃肠道疾病。 PKG曾经激活的分子机制使平滑肌放松是未知的。我们的假设表明，PKG通过在平滑肌中表达的一种或多种特异性蛋白质的磷酸化来介导其肌肉放松作用。有趣的是，PKG磷酸化的蛋白质的称赞随平滑肌亚型而变化。我们认为，表达的这种差异会影响这些肌肉的特定收缩特性。裂缝在很大程度上以股动脉或主动脉等强调平滑肌表示。因此，我们认为，在裂口无效的小鼠中，脉管系统的正常反应性会改变，以响应已知激活PKG的药理学激动剂。通常不会表达裂缝的肌肉的反应能力是正常的。我们还将预测裂口无效小鼠肠运动的改变，因为该蛋白在回肠的神经内分泌细胞中高度表达。因此，实时成像研究将提供一种非常优雅的手段，以评估鸿沟在PKG调节平滑肌收缩中的生理作用。我们要检查的参数是激动剂/拮抗剂对正常血液流过循环和每个主要器官（卵肾脏和心血管）的影响。我们还想监测激动剂对肠道运动的影响。我们还想检查所有血管和肠道结构，以确保鸿沟不会引起任何发育异常。 野生型小鼠的血管瘤化将通过给药依赖于内皮的血管肌发育蛋白（0.1-10 ug/kg），一氧化氮供体，硝丙胺钠（3 ug/kg）和乙酰胆碱（1 mg/kg）来评估。 确定Chasm的生理意义，Chasm是在平滑MUSCL中表达的环环GMP蛋白激酶（PKG）的新靶标