bafilomycin-a and phenylamil

In vitro gut-sac preparations of all four sections (stomach, anterior, mid, and posterior intestine) of the gastrointestinal tract (GIT) of freshwater rainbow trout, together with radiotracer ((22)Na) techniques, were used to study unidirectional Na(+) uptake rates (UR, mucosal → blood space) and net absorptive fluid transport rates (FTR) under isosmotic conditions (mucosal = serosal osmolality). On an area-specific basis, unidirectional Na(+) UR was highest in the mid-intestine, but when total gut area was taken into account, the three intestinal sections contributed equally, with very low rates in the stomach. The theoretical capacity for Na(+) uptake across the whole GIT is sufficient to supply all of the animal's nutritive requirements for Na(+). Transport occurs by low affinity systems with apparent K m values 2-3 orders of magnitude higher than those in the gills, in accord with comparably higher Na(+) concentrations in chyme versus fresh water. Fluid transport appeared to be Na(+)-dependent, such that treatments which altered unidirectional Na(+) UR generally altered FTR in a comparable fashion. Pharmacological trials (amiloride, EIPA, phenamil, bafilomycin, furosemide, hydrochlorothiazide) conducted at a mucosal Na(+) concentration of 50 mmol L(-1) indicated that GIT Na(+) uptake occurs by a variety of apical mechanisms (NHE, Na(+) channel/H(+) ATPase, NCC, NKCC) with relative contributions varying among sections. However, at a mucosal Na(+) concentration of 10 mmol L(-1), EIPA, phenamil, bafilomycin, and hydrochlorothiazide were no longer effective in inhibiting unidirectional Na(+) UR or FTR, suggesting the contribution of unidentified mechanisms under low Na(+) conditions. A preliminary model is presented.

Topics: Amiloride; Animals; Biological Transport; Fresh Water; Furosemide; Gastrointestinal Tract; Hydrochlorothiazide; In Vitro Techniques; Kinetics; Macrolides; Models, Biological; Oncorhynchus mykiss; Osmolar Concentration; Osmoregulation; Regression Analysis; Sodium Radioisotopes

The euryhaline fish Cyprinodon variegatus variegatus is capable of tolerating ambient salinities ranging from 0.3 to 160 PSU, but is incapable of long-term survival in freshwater (<2 mmol l(-1) Na(+)). A population isolated in several freshwater (0.4-1 mmol l(-1) Na(+)) lakes in central Florida is now designated as a subspecies (Cyprinodon variegatus hubbsi). We conducted a comparative study of Na(+) transport kinetics in these two populations when acclimated to different ambient Na(+) concentrations. Results reveal that the two subspecies have qualitatively similar low affinity Na(+) uptake kinetics (K(m)=7000-38,000 μmol l(-1)) when acclimated to 2 or 7 mmol l(-1) Na(+), but C. v. hubbsi switches to a high affinity system (K(m)=100-140 μmol l(-1)) in low-Na(+) freshwater (≤1 mmol l(-1) Na(+)). Inhibitor experiments indicate that Na(+) uptake in both subspecies is EIPA-sensitive, but sensitivity decreases with increasing external Na(+). EIPA induced a 95% inhibition of Na(+) influx in C. v. hubbsi acclimated to 0.1 mmol l(-1) Na(+), suggesting that this subspecies is utilizing a Na(+)/H(+) exchanger to take up Na(+) in low-Na(+) environments despite theoretical thermodynamic constraints. Na(+) uptake in C. v. hubbsi acclimated to 0.1 mmol l(-1) Na(+) is phenamil-sensitive but not bafilomycin-sensitive, leading to uncertainty about whether this subspecies also utilizes Na(+) channels for Na(+) uptake. Experiments with both subspecies acclimated to 7 mmol l(-1) Na(+) also indicate that a Cl(-)-dependent Na(+) uptake pathway is present. This pathway is not metolazone-sensitive (NCC inhibitor) in either species but is bumetanide-sensitive in C. v. variegatus but not C. v. hubbsi. This suggests that an apical NKCC is increasingly involved with Na(+) uptake for this subspecies as external Na(+) increases. Finally, characterization of mitochondria-rich cell (MRC) size and density in fish acclimated to different ambient Na(+) concentrations revealed significant increases in the number and size of emergent MRCs with decreasing ambient Na(+). A linear relationship between the fractional area of emergent MRCs and Na(+) uptake rate was observed for both subspecies. However, C. v. variegatus have lower Na(+) uptake rates at a given MRC fractional area compared with C. v. hubbsi, indicating that the enhanced Na(+) uptake by C. v. hubbsi at low ambient Na(+) concentrations is not strictly a result of increased MRC fractional area, and other variables, such as different

Topics: Acclimatization; Amiloride; Animals; Cell Size; Chlorides; Epithelium; Fresh Water; Gills; Introduced Species; Ion Transport; Killifishes; Kinetics; Macrolides; Metolazone; Mitochondria; Models, Animal; Sodium; Species Specificity; Time Factors