Two-substrate kinetic analysis: a novel approach linking ion and acid-base transport at the gills of freshwater trout,Oncorhynchus mykiss

Summary The novel application of a two-substrate model (Florini and Vestling 1957) from enzymology to transport kinetics at the gills of freshwater trout indicated that Na⁺/acidic equivalent and Cl^-/basic equivalent flux rates are normally limited by the availability of the internal acidic and basic counterions, as well as by external Na⁺ and Cl^- levels. Adult rainbow trout fitted with dorsal aortic and bladder catheters were chronically infused (10–16 h) with isosmotic HCl to induce a persistent metabolic acidosis. Acid-base neutral infusions of isosmotic NaCl and non-infused controls were also performed. Results were compared to previous data on metabolic alkalosis in trout induced by either isosmotic NaHCO₃ infusion or recovery from environmental hyperoxia (Goss and Wood 1990a, b). Metabolic acidosis resulted in a marked stimulation of Na⁺ influx, no change in Cl^- influx, positive Na⁺ balance, negative Cl^- balance, and net H⁺ excretion at the gills. Metabolic alkalosis caused a marked inhibition of Na⁺ influx and stimulation of Cl^- influx, negative Na⁺ balance, positive Cl^- balance, and net H⁺ uptake (=base excretion). Mean gill intracellular pH qualitatively followed extracellular pH. Classical one-substrate Michaelis-Menten analysis of kinetic data indicated that changes in Na⁺ and Cl^- transport during acid-base disturbance are achieved by large increases and decreases in J_max, and by increases in K_m. However, one-substrate analysis considers only external substrate concentration and cannot account for transport limitations by the internal substrate. The kinetic data were fitted successfully to a two-substrate model, using extracellular acid-base data as a measure of internal HCO ₃ ^- and H⁺ availability. This analysis indicated that true J_max values for Na⁺/acidic equivalent and Cl^-/basic equivalent transport are 4–5 times higher than apparent J_max values by one-substrate analysis. Flux rates are limited by the availability of the internal counterions; transport K_m values for HCO ₃ ^- and H⁺ are far above their normal internal concentrations. Therefore, small changes in acid-base status will have large effects on transport rates, and on apparent J_max values, without alterations in the number of transport sites. This system provides an automatic, negative feedback control for clearance or retention of acidic/basic equivalents when acid-base status is changing.Abbreviations Amm total ammonia in water - DMO 55-dimethyl-24-oxyzolidine-dione - J_in unidirectional inward ion movement across the gill - J_out unidirectional outward ion movement across the gill - J_net net transfer of ions (sum of J_in and J_out) across the gill - J_max maximal transport rate for ion - K_m inverse of affinity of transporter for ion - PIO₂ partial pressure of oxygen in inspired water - P_aCO₂ partial pressure of carbon dixide in arterial blood - TAlk titratable alkalinity of the water - PEG polyethylene glycol - NEN New England Nuclear