2-SUBSTRATE KINETIC-ANALYSIS - A NOVEL-APPROACH LINKING ION AND ACID-BASE TRANSPORT AT THE GILLS OF FRESH-WATER TROUT, ONCORHYNCHUS-MYKISS

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 HCO3- 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 HCO3- 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 feed-back control for clearance or retention of acidic/basic equivalents when acid-base status is changing.