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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 COsub2/sub). 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 COsub2/sub, hagfish can mount a rapid elevation of plasma HCOsub3/subsup-/supconcentration (hypercarbia). Once PCOsub2/subis restored, hagfish quickly excrete their HCOsub3/subsup-/supload, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCOsub3/subsup-/supdehydration into COsub2/subat the hagfish gills. We aimed to characterize the role of branchial CA in COsub2/sub/HCOsub3/subsup-/supclearance from the plasma at the gills of E. stoutii, under control and high PCOsub2/sub(hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCOsub3/subsup-/supexcretion by measuring in situ [sup14/supC]-HCOsub3/subsup-/supfluxes. 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. [sup14/supC]-HCOsub3/subsup-/supefflux 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 HCOsub3/subsup-/supflux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCOsub3/subsup-/supfluxes were much higher and perfusion with acetazolamide caused a reduction of HCOsub3/subsup-/supflux 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 PCOsub2/subconditions. In summary, our data suggests that there are additional pathways for HCOsub3/subsup-/supexcretion at the gills of hagfish that are independent of plasma-accessible CA. |
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