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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该项目的最初目标是利用原位对比成像技术评估野生型小鼠肺、脑、心脏和肾脏的血流。在敲除小鼠中，对诱导血管收缩和血管松弛的激动剂的反应可能发生改变。对这些激动剂的血压测量也将进行。全身肾上腺素能性血管收缩可由α激动剂苯肾上腺素或α诱导1,2；-1激动剂去甲肾上腺素。对苯肾上腺素（0.5-8 ug/kg）和去甲肾上腺素（100-600 ng/kg）进行剂量反应。这些药物的最大剂量预计会使平均动脉血压（MAP）比基础水平（基础MAP 90-120 mmHg）高出约40mmHg，因此对血流的影响应该是明显的。肾素-血管紧张素-醛固酮系统依赖性血压升高可以通过血管紧张素II和血管加压素的施用来评估。通过长期服用L-NAME （400 ug/g IP，持续7天），一种一氧化氮合酶抑制剂，可以测量一氧化氮依赖性血压升高。评价肾血流时，多巴胺（2 ug/kg）应收缩肾的传出小动脉，放松肾的传入小动脉。在分离的32P标记平滑肌中，CHASM被鉴定为蛋白激酶PKG的早期靶点。该蛋白通过从头序列数据被鉴定为人类基因组中的理论蛋白。因此，它的功能是未知的。我们随后用一种抗体对重组蛋白进行了验证。组织学染色显示CHASM仅在肌纤维和支配肌肉的神经内分泌细胞中表达。PKG是小鼠平滑肌松弛的主要介质，蛋白激酶的缺失导致该组织中所有激素介导的信号通路完全丧失。结果，这些动物出现高血压，并表现出与肠道协调运动丧失相关的胃肠道疾病。PKG一旦被激活，导致平滑肌放松的分子机制尚不清楚。我们的假设表明PKG通过磷酸化平滑肌中表达的一种或多种特定蛋白来调节其肌肉松弛作用。有趣的是，PKG磷酸化蛋白的补充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主要表达于强直性平滑肌，如股动脉或主动脉。因此，我们认为，在CHASM无效的小鼠中，血管系统对已知激活PKG的药物激动剂的正常反应性会发生变化。正常情况下不表达CHASM的肌肉的反应性预计会正常。由于该蛋白在回肠神经内分泌细胞中高度表达，我们也可以预测CHASM缺失小鼠肠道运动的改变。因此，实时成像研究将为评估CHASM在PKG调节平滑肌收缩中的生理作用提供一种非常简便的方法。我们想要检查的参数是激动剂/拮抗剂诱导的对通过循环和每个主要器官（卵子）的正常血流的影响。肾脏和心血管)。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。野生型小鼠的血管松弛将通过内皮依赖性血管松弛剂缓动素（0.1-10 ug/kg）、一氧化氮供体、硝普钠（3 ug/kg）和乙酰胆碱（1 mg/kg）进行评估。确定在平滑肌中表达的环GMP依赖性蛋白激酶（PKG）的新靶点CHASM的生理意义

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