Cortisol-sensitive urea transport across the gill basolateral membrane of the gulf toadfish (Opsanus beta)

Abstract

Gulf toadfish (Opsanus beta) use a unique pulsatile urea excretion mechanism that allows urea to be voided in large pulses via the periodic insertion or activation of a branchial urea transporter. The precise cellular and subcellular location of the facilitated diffusion mechanism(s) remains unclear. An in vitro basolateral membrane vesicle (BLMV) preparation was used to test the hypothesis that urea movement across the gill basolateral membrane occurs through a cortisol-sensitive carrier-mediated mechanism. Toadfish BLMVs demonstrated two components of urea uptake: a linear element at high external urea concentrations, and a phloretin-sensitive saturable constituent (K(m) = 0.24 mmol/l; V(max) = 6.95 micromol x mg protein(-1) x h(-1)) at low urea concentrations (<1 mmol/l). BLMV urea transport in toadfish was unaffected by in vitro treatment with ouabain, N-ethylmaleimide, or the absence of sodium, conditions that are known to inhibit sodium-coupled and proton-coupled urea transport in vertebrates. Transport kinetics were temperature sensitive with a Q(10) > 2, further suggestive of carrier-mediated processes. Our data provide evidence that a basolateral urea facilitated transporter accelerates the movement of urea between the plasma and gills to enable the pulsatile excretion of urea. Furthermore, in vivo infusion of cortisol caused a significant 4.3-fold reduction in BLMV urea transport capacity in lab-crowded fish, suggesting that cortisol inhibits the recruitment of urea transporters to the basolateral membrane, which may ultimately affect the size of the urea pulse event in gulf toadfish.

The Effect of Stress on Gill Basolateral Membrane Binding Kinetics of 5‐HT2 Receptor Ligands: Potential Implications for Urea Excretion Mechanisms

“…Isolating the basolateral membrane of the gill through differential centrifugation allowed for the removal of any non‐membrane bound 5‐HT receptors that may have been internalized either prior to membrane insertion or post‐activation (Perry and Flik, ; Bury et al, ; Gray and Roth, ; Rodela et al, ). Using this technique it was established that there was no difference in [ 3 H]‐5‐HT binding to gill basolateral membranes of uncrowded and crowded toadfish, representing no net change in the gill's entire 5‐HT receptor population.…”

“…Gill basolateral membranes (BLMs) were isolated using differential centrifugation using a method specifically adapted for toadfish by Rodela et al, using methods originally for salmonids by Perry and Flik () and Bury et al (). Toadfish were removed from their respective tanks (see above), wrapped in a paper towel and a blood sample (0.2 mL) was taken immediately from the caudal vein using a 23 gauge needle on a 1 mL disposable, heparinized syringe (50 IU heparin mL saline −1 ).…”

Interactions between cortisol and Rhesus glycoprotein expression in ureogenic toadfish, Opsanus beta

“…Although elevated cortisol levels have been linked to increased proteolytic activity and ammonia excretion in many teleosts (see Mommsen et al, 1999), cortisol does not seem to elicit the same extent of proteolysis and endogenous nitrogen production in toadfish. Toadfish reduce nitrogen excretion when exposed to elevated cortisol Wood et al, 2003;Rodela et al, 2009a). At extremely high concentrations of circulating cortisol (~1500ngml -1 ), the proteolytic effect swamps the ammonia-trapping mechanisms in the toadfish and high rates of ammonia excretion resume (see .…”

“…The cortisol concentration was chosen based on previous studies Rodela et al, 2009a). A third group (mean mass0.065±0.003kg, N8) was infused with ammonia [(NH 4 ) 2 HPO 4 ; Sigma-Aldrich] in isosmotic saline.…”

“…The concentration of urea in water samples was determined using the diacetyl monoxime method described by Rahmatullah and Boyde (Rahmatullah and Boyde, 1980), adapted for use with a microplate reader. The excretion (mmolNkg -1 ) of ammonia or urea was calculated as reported in previous studies (Wright and Wood, 1985;Rodela et al, 2009a). These values were used to calculate the percent ureotelism.…”