Hypoxanthine metabolism in salmon: roles in osmoregulation and the innate immune response.

大西洋鲑鱼在海水环境中时，在皮肤和鳍中积累各种代谢物。一种名为鸟嘌呤的代谢物被认为是导致皮肤“银化”的原因，这种皮肤伴随着幼鲑鱼迁移到西南大马哈鱼的相关发育变化。皮肤中浓度的升高导致鸟嘌呤结晶的形成，使皮肤具有经典的彩虹/珍珠光泽金属光泽，这是所有鲑鱼的特征。最近，我们发现在大西洋鲑鱼的皮肤和鳍中也发现了一种与结构相关的代谢物，称为次黄嘌呤。然而，这种代谢物的功能作用似乎与鸟嘌呤完全不同。虽然鸟嘌呤在皮肤细胞的水环境中基本上不溶于水(因此可以解释其沉淀为半透明晶体)，但次黄嘌呤非常容易溶解，在西南鲑鱼中积累到非常高的浓度，这表明它可能是一种有机渗透调节剂。有机渗透物质将细胞内的浓度提高到与周围环境中的氯化钠和其他离子的浓度几乎相同的浓度。这种细胞内外溶质浓度的平衡限制了细胞水向细胞外SW环境的渗透损失，从而防止了细胞脱水、收缩和不可避免的细胞死亡。次黄嘌呤的另一个潜在功能是抗微生物化合物。次黄嘌呤除了是鸟嘌呤的前体外，还可以在酶作用下转化为尿酸，但同时这个过程也会释放过氧化氢。基本上，细胞内产生的过氧化氢是次黄嘌呤的三倍。据认为，这种高活性的代谢物起到了细胞消毒剂的作用，是宿主抵御入侵皮肤或鳍的微生物或寄生虫的第一道防线。在高等脊椎动物中，过氧化氢和尿酸被认为是所谓的化学吸引分子，刺激血淋巴细胞和巨噬细胞聚集到损伤部位，并启动对任何入侵生物的免疫反应。同样的功能也被提议在鲑鱼身上发生。初步结果表明，这一假设可能确实成立，因为长期感染海虱的皮肤区域，与过氧化氢产生途径有关的一种关键酶的表达水平较高。鲑鱼是水产养殖业的重要物种，特别是在苏格兰，这种鱼养殖的关键时期是在西南转移期间和之后。这是鲑鱼生命周期的一个非常紧张的时期，如果不在最佳时间转移鱼类，西南部生活所需的生理适应可能会受到影响，导致鱼类因渗透压干扰或感染率增加而死亡。在鲑鱼生命周期的这一关键时期，次黄嘌呤的作用几乎一无所知。该项目将表征次黄嘌呤作为渗透调节剂在细胞体积调节中的作用，以及在海虱感染期间作为皮肤和鳍细胞中过氧化氢生成器的潜在功能。该项目还将确定添加到短波转移前鱼类饲料中的各种营养补充剂是否会：i)影响与次黄嘌呤一般代谢相关的酶和转运蛋白的表达，ii)影响皮肤和鳍中的次黄嘌呤水平，iii)改变急性短波转移后鱼类的渗透调节能力，并确定iv)选择性营养补充剂是否可以降低通常在短波转移后2-3个月内发生的应激性皮肤损伤/感染和死亡率。

Atlantic salmon accumulate various metabolites in the skin and fin when fish are in seawater (SW) environments. One metabolite, called guanine, is known to be responsible for the skin "silvering" that accompanies developmental changes associated with the migration of young salmon (called smolts) to SW. The elevated concentrations in the skin result in the formation of guanine crystals that give the skin its classic iridescent/pearlescent metallic lustre, characteristic of all salmonids. Recently we have identified that a structurally related metabolite called hypoxanthine, is also found at high levels within the skin and fin of Atlantic salmon. The functional role of this metabolite however, appears to be completely different from guanine. Although guanine is essentially insoluble in the aqueous environment of the skin cells (and hence explains its precipitation as translucent crystals), hypoxanthine is eminently soluble and in SW salmon, accumulates to very high concentrations suggesting that it may function as an organic osmolyte. Organic osmolytes increase the concentration inside cells to near-equivalent concentrations to that of the sodium chloride and other ions in the surrounding SW. This equilibration of solute concentrations inside and outside the cell limits the osmotic loss of cell water to the extracellular SW environment and therefore prevents cell dehydration, shrinkage and inevitable cell death. Another potential function of hypoxanthine, is that of an anti-microbial compound. In addition to being a precursor of guanine, hypoxanthine can be enzymatically converted to uric acid, but at the same time this processes releases hydrogen peroxide. Essentially three times as much hydrogen peroxide can be generated as hypoxanthine present within the cell. It is proposed that this highly reactive metabolite acts as a cellular disinfectant and the first line of the host's defense against microorganisms or parasites that invade the skin or fin. In higher vertebrates, hydrogen peroxide and uric acid are known to function as so-called chemo-attractant molecules, stimulating the recruitment of blood lymphocytes and macrophages to sites of injury and initiating an immune response against any invading organism. The same functions are proposed to take place in the salmon. Preliminary results suggest that this hypothesis may indeed be the case as skin regions chronically infected with sea lice exhibit higher levels of expression of a key enzyme involved in this hydrogen peroxide generating pathway. Salmon are an important species in the aquaculture industry, especially in Scotland, and a critical period in the farming of this fish is during and just after SW transfer. This is a very stressful period of the salmon's life cycle and if fish are not transferred at the optimal time the physiological adaptations required for life in SW can be compromised, resulting in increased fish mortalities from osmotic disturbances or increased infection rates. Virtually nothing is known about the roles of hypoxanthine during this critical period of the salmon's life cycle. This project will characterize the role of hypoxanthine as an osmolyte in cell volume regulation and its potential function as a hydrogen peroxide generator in skin and fin cells during sea lice infestation. The project will also determine whether various nutritional supplements added to the diet of pre-SW transfer smolts will i) affect the expression of enzymes and transporters associated with the general metabolism of hypoxanthine, ii) influence hypoxanthine levels in skin and fin, iii) alter the osmoregulatory capacity of fish following acute SW transfer and determine if iv) selective nutritional supplements can reduce the incidence of stress-induced skin lesions/infections and mortalities that often arise within 2-3 months of SW transfer.