Measuring gill paracellular permeability with polyethylene glycol-4000 in freely swimming trout: proof of principle.

The influence of swimming activity on gill paracellular permeability has not been measured previously in fishes. We critically assessed the use of tritium-labeled polyethylene glycol ([(3)H]PEG-4000) for this purpose, a substance that is also a classic marker for extracellular fluid volume, glomerular filtration rate and drinking rate. Tests (8 h) on resting freshwater trout showed that when measuring [(3)H]PEG-4000 clearance from the plasma in the efflux direction, correction for a large excretion via glomerular filtration was essential, necessitating urinary catheterization. When measuring [(3)H]PEG-4000 clearance from the water in the influx direction, correction for a significant uptake by drinking was essential, necessitating terminal gut removal, whereas glomerular filtration losses were minimal. After correction for these alternate routes of loss and uptake, [(3)H]PEG-4000 clearance rates by efflux from the plasma and by influx from the water were identical, showing that gill paracellular permeability is not rectified, and can be measured in either direction. The influx technique with terminal gut removal was used to assess gill paracellular permeability in trout without urinary catheters freely swimming at 1.2 body lengths s(-1) for 8 h. Branchial [(3)H]PEG-4000 clearance rate (by influx from the water) increased significantly by ~80% in accord with a similar measured increase in O2 consumption rate. Thus in trout, gill paracellular permeability does increase during exercise, in accord with the traditional concept of the osmorespiratory compromise.

Ionoregulatory Aspects of the Osmorespiratory Compromise during Acute Environmental Hypoxia in 12 Tropical and Temperate Teleosts.

In the traditional osmorespiratory compromise, as seen in the hypoxia-intolerant freshwater rainbow trout (Oncorhynchus mykiss), the branchial modifications that occur to improve O2 uptake during hypoxia result in unfavorable increases in the fluxes of ions and water. However, at least one hypoxia-tolerant freshwater species, the Amazonian oscar (Astronotus ocellatus), shows exactly the opposite: decreased branchial flux rates of ions, water, and nitrogenous wastes during acute hypoxia. In order to find out whether the two strategies were widespread, we used a standard 2-h normoxia, 2-h hypoxia (20%-30% saturation), 2-h normoxic recovery protocol to survey 10 other phylogenetically diverse tropical and temperate species. Unidirectional influx and efflux rates of Na(+) and net flux rates of K(+), ammonia, and urea-N were measured. The flux reduction strategy was seen only in one additional species, the Amazonian tambaqui (Colossoma macropomum), which is similarly hypoxia tolerant and lives in the same ion-poor waters as the oscar. However, five other species exhibited evidence of the increased flux rates typical of the traditional osmorespiratory compromise in the trout: the rosaceu tetra (Hyphessobrycon bentosi rosaceus), the moenkhausia tetra (Moenkhausia diktyota), the bluegill sunfish (Lepomis macrochirus), the zebra fish (Danio rerio), and the goldfish (Carassius auratus). Four other species exhibited no marked flux changes during hypoxia: the cardinal tetra (Paracheirodon axelrodi), the hemigrammus tetra (Hemigrammus rhodostomus), the pumpkinseed sunfish (Lepomis gibbosus), and the Atlantic killifish (Fundulus heteroclitus). Overall, a diversity of strategies exist; we speculate that these may be linked to differences in habitat and/or lifestyle.

Gill paracellular permeability and the osmorespiratory compromise during exercise in the hypoxia-tolerant Amazonian oscar (Astronotus ocellatus).

In the traditional osmorespiratory compromise, fish increase their effective gill permeability to O2 during exercise or hypoxia, and in consequence suffer unfavorable ionic and osmotic fluxes. However oscars, which live in the frequently hypoxic ion-poor waters of the Amazon, actually decrease ionic fluxes across the gills during acute hypoxia without changing gill paracellular permeability, and exhibit rapid paving over of the mitochondrial-rich cells (MRCs). But what happens during prolonged exercise? Gill paracellular permeability, ionic fluxes, and gill morphology were examined in juvenile oscars at rest and during aerobic swimming. Initial validation tests with urinary catheterized fish quantified drinking, glomerular filtration, and urinary flow rates, and confirmed that measurements of gill paracellular permeability as [(3)H]PEG-4000 clearances were the same in efflux and influx directions, but far lower than previously measured in comparably sized trout. Although the oscars achieved a very similar proportional increase (90%) in oxygen consumption (MO2) to trout during steady-state swimming at 1.2 body lengths sec(-1), there was no increase in gill paracellular permeability, in contrast to trout. However, oscars did exhibit increased unidirectional Na(+) efflux and net K(+) rates during exercise, but no change in drinking rate. There were no changes in MRC numbers or exposure, or other alterations in gill morphology during exercise. A substantial interlamellar cell mass (ILCM) that covered the lamellae to a depth of 30% was unchanged by 4 h of swimming activity. We conclude that a low branchial paracellular permeability which can be dissociated from changes in O2 flux, as well as the presence of the ILCM, may be adaptive in limiting ionoregulatory costs for a species endemic to ion-poor, frequently hypoxic waters.

Mechanisms of Na+ uptake, ammonia excretion, and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi, Hemigrammus rhodostomus, and Moenkhausia diktyota).

Mechanisms of Na(+) uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J in), efflux (J out), and net flux rates (J net) of Na(+) and Cl(-), and the net excretion rate of ammonia (J Amm). The fish were collected directly from the Rio Negro, and studied in their native "blackwater" which is acidic (pH 4.5), ion-poor (Na(+), Cl(-) ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l(-1)). J in (Na) , J in (Cl) , and J Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J in (Na) was unaffected by amiloride (10(-4) M, NHE and Na(+) channel blocker), but both J in (Na) and J in (Cl) were virtually eliminated (85-99 % blockade) by AgNO3 (10(-7) M). A time course study on cardinal tetras demonstrated that J in (Na) blockade by AgNO3 was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10(-4) M) caused a 50 % reduction in J in (Na) .. Additionally, J in (Na) was unaffected by phenamil (10(-5) M, Na(+) channel blocker), bumetanide (10(-4) M, NKCC blocker), hydrochlorothiazide (5 × 10(-3) M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J in (Na) (by acetazolamide and AgNO3 respectively), had any inhibitory effect on J Amm. Therefore, Na(+) uptake in Rio Negro tetras depends on an internal supply of H(+) from CA, but does not fit any of the currently accepted H(+)-dependent models (NHE, Na(+) channel/V-type H(+)-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current "Na(+)/NH4 (+) exchange metabolon" paradigm. Na(+), K(+)-ATPase and V-type H(+)-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH4Cl, 10(-3) M) significantly increased J in (Na) , and NH4 (+) was equally or more effective than K(+) in activating branchial Na(+),(K(+)) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na(+) uptake, but that Na(+) uptake (by an as yet unknown H(+)-dependent apical mechanism) depends on ammonia excretion, driven by active NH4 (+) entry via basolateral Na(+),(K(+))-ATPase.

Impact of environmental oxygen, exercise, salinity, and metabolic rate on the uptake and tissue-specific distribution of 17α-ethynylestradiol in the euryhaline teleost Fundulus heteroclitus.

17α-ethynylestradiol (EE2) is a synthetic estrogen that is an endocrine disruptive toxicant in aquatic environments. The aim of this study was to determine whether metabolic rate influenced EE2 uptake in male killifish (Fundulus heteroclitus), based on the hypothesis that the mechanism of EE2 uptake at the gills is similar to that of oxygen. F. heteroclitus were exposed to 100 ng/L radiolabeled [³H]EE2 for 2 h while swimming at 0, 15, and 40 cm/s. A positive linear correlation between the rates of oxygen consumption (MO2) and EE2 accumulation was seen (r² = 0.99, p<0.01), with more EE2 taken up at higher swimming speeds, suggesting that oxygen uptake predicts EE2 uptake. EE2 tended to accumulate in the liver (where lipophilic toxicants are metabolized), the gall bladder (where metabolized toxicants enter bile), and the gut (where bile is received). In a subsequent experiment killifish were exposed to both hypoxic and hyperoxic conditions (PO2=70-80 Torr, and PO2=400-500 Torr respectively). Despite significant decreases in MO2 during hypoxia, EE2 uptake rates increased only slightly with hypoxia, but in individual fish there was still a significant correlation between MO2 and EE2 uptake. This correlation was lost during hyperoxia, and EE2 uptake rates did not change significantly in hyperoxia. Marked influences of salinity on EE2 uptake rate occurred regardless of the oxygen condition, with higher uptake rates in 50% seawater than in freshwater or 100% seawater. Tissue distribution of EE2 in these exposures may have been influenced by changes in tissue blood flow patterns and oxygen supply. These data will be useful in eventually constructing a predictive model to manage the optimal timing for discharge of EE2 from sewage treatment plants into receiving waters.