In Vitro Studies of freshwater Teleost Ionoregulation in the Common Killifish (Fundulus heteroclitus), Nile Tilapia (Oreochromis niloticus) and Rainbow Trout (Onocorhynchus mykiss)

Abstract

Largely through the use ofin vitro preparations there is now a generally accepted theory for ion transport for the seawater (SW) gill. However, to date there is no generally accepted freshwater (FW) model for the mechanisms of NaCl transport in the teleost gill. By using an Ussing chamber approach with the opercular epithelia of Fundulus heteroclitus and Oreochromis niloticus, and the urinary bladder of Oncorhynchus mykiss, all acclimated to FW, we hoped to establish one as a possible model for the study of FW ion regulation. FW Fundulus opercular epithelia displayed a serosal negative transepithelial potential (Vₜ) of-43.9 mV, transepithelial conductance (Gₜ) of 1.94 mS·cm⁻², and active transport of Cl⁻ from the mucosal FW against a strong electrochemical gradient. Na⁺ movement was dominated by passive diffusion. The opercular epithelia of Fundulus adapted to 10% SW exhibited properties similar to SW Fundulus by actively extruding Cl⁻ while Na⁺ moved passively into the mucosal 10% SW. With FW bathing the mucosal surface, FW Oreochromis opercular epithelia displayed a serosal positive Vₜ of +8.0 mV, Gₜ of 1.78 mS·cm⁻², and active reabsorption of Na⁺, Cl⁻ and Ca²⁺ against large electrical and/or chemical gradients. The FW Oreochromis opercular epithelia is the only FW in vitro preparation to date that exhibits active absorption (albeit at small absolute rates) of both Na⁺ and Cl⁻. FW 0. mykiss urinary bladders mounted in vitro under symmetrical saline conditions exhibited a transepithelial conductance (Gₜ) of-9.15 mS·cm⁻² and electroneutral active absorption of Na⁺ and Cl⁻ from the mucosal urine side. The transport of Na⁺ and Cl⁻ was a partially coupled process whereby removal of Na⁺ from the mucosal saline decreased Cl⁻ absorption by a 56% and removal of Cl⁻ inhibited Na⁺ absorption by 69%. However, active net absorption of both ions persisted when the counter-ion was replaced with a non-permeant ion. Under more realistic conditions with artificial urine bathing the mucosal surface, Vₜ increased to a serosal positive ~+7.6 mV and Gₜ decreased to ~1.47 mS·cm⁻² Unidirectional influx rates of both Na⁺ and Cl⁻ were much lower, but active absorption of both ions still occurred. Replacement of Na⁺ in the mucosal artificial urine caused no change in unidirectional influx of Cl⁻ and vice versa. The mucosal addition of DIDS, amiloride or bumetanide (10⁻⁴M) all had no affect on absorption rates of Na⁺ and/or Cl⁻, under either artificial urine or symmetrical saline conditions. When the mucosal surface was bathed in artificial urine, removal of mucosal Cl⁻· significantly reduced the maximum transport rate (Jₘₐₓ of Na⁺ (6.1 ~2.1 μmol·cm⁻²·h⁻¹) but had no effect on affinity for Na⁺ (Kₘ~27 mM). Similarly, removal of mucosal Na⁺ significantly reduced the Jₘₐₓ for Cl⁻ uptake (11.4 ~2.4 μmol·cm⁻²·h⁻¹) but had no effect on Cl⁻ Kₘ(~37 mM). The anterior portion of the urinary bladder transported Na⁺ and Cl⁻ at a faster rate than the posterior portion under symmetrical saline conditions, but there was no difference in measured Na⁺/K⁺ -ATPase activities between the two portions. In conclusion, opercular epithelia of Fundulus and Oreochromis demonstrated transport properties believed to be typical of FW teleosts; either preparation may eventually prove to be a good working model for studying FW transport mechanisms. The urinary bladder of Oncorhynchus mounted in vitro did not behave characteristically as the teleost gill is thought to function, but this model may be useful in characterizing various other mechanisms of ionic transport.