Collaborative Research: Differential Expression of Oxygen-binding Proteins in Antarctic Fishes Affects Nitric Oxide-mediated Pathways of Angiogenesis and Mitochondrial Biogenesis.

目前环绕南极洲的极地海洋是地球上最冷、最热稳定的海洋环境。由于海水中氧的溶解度与温度成反比，寒冷的南极海域是一个异常富氧的水生栖息地。在南极洲周围的鱼类动物群中，有8个科属于一个单一的波斯亚目，即Notothenioidei亚目。南极极锋以南约35%的鱼类物种和90%的鱼类生物量为类石斑鱼。因此，在一套非常不寻常的条件下，近亲诺氏体类物种的辐射迅速发生：由于绕极洋流和围绕大陆的深海海沟，在海洋上与其他动物群相对隔绝；长期严重寒冷的水温；非常高的氧气可利用量；在中第三纪至今的某个时候，南大洋鱼类物种多样性急剧下降，导致物种数量急剧减少。这些特征使南极诺氏类鱼类成为研究生理和生化适应寒冷体温的一个独特的有吸引力的群体。极少有南极鱼类的独特之处在于其氧结合蛋白的表达模式在一个无脊椎科鱼类(南极冰鱼)中表现得如此独特。所有的银鱼都缺乏循环中的氧结合蛋白--血红蛋白(Hb)，而细胞内的氧结合蛋白--肌红蛋白(Mb)在该科物种中的表达并不统一。这两种蛋白质通常被认为是将氧气充分输送到动物有氧平衡组织所必需的。为了弥补Hb的缺失，冰鱼发育出了较大的心脏，快速循环大量的血液，并拥有比红血鱼更大的管腔直径的精细血管。氧化肌肉中Mb表达的丧失与细胞内线粒体密度的显著增加有关，尽管每个细胞器都不那么密集地堆积着呼吸蛋白。在氧气运动的框架内，更大的血管密度和线粒体种群的适应意义是可以理解的，但这些特征的潜在发展机制仍不清楚。答案可能在于Hb和Mb的另一个主要功能，即降解普遍存在的生物活性化合物--一氧化氮(NO)。这项研究将检验这样一种假设，即冰鱼中血红素蛋白表达的丧失导致了一氧化氮水平的增加，而一氧化氮水平介导了血管系统的改变和氧化组织中线粒体数量的扩大。该建议的目的是量化+HB和-HB类鱼类视网膜的血管密度，描述南极类鱼类视网膜和心肌中一氧化氮合酶的亚型和催化活性，评估参与血管生成(在视网膜组织)和线粒体生物发生(在心肌)的下游因子的表达水平，并确定体内抑制一氧化氮合酶是否会导致血管生成和线粒体生物起源反应的倒退。影响范围更广，从基础生物学到年轻科学家的培训，再到加强对临床相关生物医学过程的理解。

The polar ocean presently surrounding Antarctica is the coldest, most thermally stable marine environment on earth. Because oxygen solubility in seawater is inversely proportional to temperature, the cold Antarctic seas are an exceptionally oxygen-rich aquatic habitat. Eight families of a single perciform suborder, the Notothenioidei, dominate the present fish fauna surrounding Antarctica. Notothenioids account for approximately 35% of fish species and 90% of fish biomass south of the Antarctic Polar Front. Radiation of closely related notothenioid species thus has occurred rapidly and under a very unusual set of conditions: relative oceanographic isolation from other faunas due to circumpolar currents and deep ocean trenches surrounding the continent, chronically, severely cold water temperatures, very high oxygen availability, very low levels of niche competition in a Southern Ocean depauperate of species subsequent to a dramatic crash in species diversity of fishes that occurred sometime between the mid-Tertiary and present. These features make Antarctic notothenioid fishes an uniquely attractive group for the study of physiological and biochemical adaptations to cold body temperature. Few distinctive features of Antarctic fishes are as unique as the pattern of expression of oxygen-binding proteins in one notothenioid family, the Channichthyidae (Antarctic icefishes). All channichthyid icefishes lack the circulating oxygen-binding protein, hemoglobin (Hb); the intracellular oxygen-binding protein, myoglobin (Mb) is not uniformly expressed in species of this family. Both proteins are normally considered essential for adequate delivery of oxygen to aerobically poised tissues of animals. To compensate for the absence of Hb, icefishes have developed large hearts, rapidly circulate a large blood volume and possess elaborate vasculature of larger lumenal diameter than is seen in red-blooded fishes. Loss of Mb expression in oxidative muscles correlates with dramatic elevation in density of mitochondria within the cell, although each individual organelle is less densely packed with respiratory proteins. Within the framework of oxygen movement, the adaptive significance of greater vascular density and mitochondrial populations is understandable but mechanisms underlying development of these characteristics remain unknown. The answer may lie in another major function of both Hb and Mb, degradation of the ubiquitous bioactive compound, nitric oxide (NO). The research will test the hypothesis that loss of hemoprotein expression in icefishes has resulted in an increase in levels of NO that mediate modification of vascular systems and expansion of mitochondrial populations in oxidative tissues. The objectives of the proposal are to quantify the vascular density of retinas in +Hb and -Hb notothenioid species, to characterize NOS isoforms and catalytic activity in retina and cardiac muscle of Antarctic notothenioid fishes, to evaluate level of expression of downstream factors implicated in angiogenesis (in retinal tissue) and mitochondrial biogenesis (in cardiac muscle), and to determine whether inhibition of NOS in vivo results in regression of angiogenic and mitochondrial biogenic responses in icefishes. Broader impacts range from basic biology, through training of young scientists, to enhanced understanding of clinically relevant biomedical processes.