Department of Biological Sciences, University of Alberta, CW 405, Biological Sciences Bldg., Edmonton, AB, T6G 2E9, Canada.
Bamfield Marine Science Centre, Bamfield, BC, V0R 1B0, Canada.
J Comp Physiol B. 2022 Nov;192(6):713-725. doi: 10.1007/s00360-022-01459-0. Epub 2022 Sep 13.
Pacific hagfish (Eptatretus stoutii) are marine scavengers and feed on decaying animal carrion by burrowing their bodies inside rotten carcasses where they are exposed to several threatening environmental stressors, including hypercapnia (high partial pressures of CO). Hagfish possess a remarkable capacity to tolerate hypercapnia, and their ability to recover from acid-base disturbances is well known. To deal with the metabolic acidosis resulting from exposure to high CO, hagfish can mount a rapid elevation of plasma HCO concentration (hypercarbia). Once PCO is restored, hagfish quickly excrete their HCO load, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCO dehydration into CO at the hagfish gills. We aimed to characterize the role of branchial CA in CO/HCO clearance from the plasma at the gills of E. stoutii, under control and high PCO (hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCO excretion by measuring in situ [C]-HCO fluxes. To accomplish this, we employed a novel surgical technique of individual gill pouch arterial perfusion combined with perifusion of the gill afferent to efferent water ducts. [C]-HCO efflux was measured at the gills of fish exposed to control, hypercapnic (48 h) and recovery from hypercapnia conditions (6 h), in the presence of two well-known pharmacological inhibitors of CA, the membrane impermeant C18 (targets membrane bound, plasma accessible CA) and membrane-permeant acetazolamide, which targets all forms of CA, including extracellular and intracellular cytosolic CAs. C18 did not affect HCO flux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCO fluxes were much higher and perfusion with acetazolamide caused a reduction of HCO flux by 38%. The same pattern was observed for fish in recovery, where in all three experimental conditions, there was no significant inhibition of plasma-accessible CA. We also observed no change in CA enzyme activity (measured in vitro) in any of the experimental PCO conditions. In summary, our data suggests that there are additional pathways for HCO excretion at the gills of hagfish that are independent of plasma-accessible CA.
太平洋盲鳗（Eptatretus stoutii）是海洋清道夫，通过将身体钻入腐烂的尸体内部来吞噬腐烂的动物尸体，从而进食。在这个过程中，盲鳗暴露于多种威胁性的环境压力源中，包括高碳酸血症（高 CO 分压）。盲鳗具有很强的耐受高碳酸血症的能力，并且它们从酸碱失衡中恢复的能力是众所周知的。为了应对暴露于高 CO 引起的代谢性酸中毒，盲鳗可以迅速提高血浆 HCO 浓度（高碳酸血症）。一旦 PCO 恢复，盲鳗会迅速排出其 HCO 负荷，这一过程可能涉及到酶碳酸酐酶（CA），它在盲鳗鳃上将 HCO 脱水为 CO。我们的目的是在控制和高 PCO（高碳酸血症）暴露条件下，研究鳃部 CA 在 E. stoutii 血浆中 CO/HCO 清除中的作用。我们通过测量原位 [C]-HCO 通量来评估血浆可及性与细胞内（细胞质）CA 对鳃 HCO 排泄的相对贡献。为了实现这一目标，我们采用了一种新的手术技术，即单独的鳃袋动脉灌注与鳃入流至出流水导管的灌注相结合。我们在暴露于对照、高碳酸血症（48 小时）和高碳酸血症恢复条件（6 小时）的鱼的鳃上测量 [C]-HCO 流出，同时使用两种已知的 CA 药理学抑制剂：膜不可渗透的 C18（针对膜结合的、血浆可及的 CA）和膜可渗透的乙酰唑胺，后者靶向所有形式的 CA，包括细胞外和细胞内细胞质 CA。C18 对对照鱼的 HCO 通量没有影响，而乙酰唑胺导致 HCO 通量显著减少了 72%。在高碳酸血症鱼中，HCO 通量要高得多，并且用乙酰唑胺处理会导致 HCO 通量减少 38%。在恢复鱼中也观察到相同的模式，在所有三种实验条件下，对血浆可及性 CA 没有明显的抑制作用。我们还观察到在任何实验 PCO 条件下，CA 酶活性（在体外测量）都没有变化。总之，我们的数据表明，盲鳗鳃部存在不依赖于血浆可及性 CA 的 HCO 排泄的其他途径。
