Peter Pärt

Characterization of ion and acid‐base transport in the fresh water adapted mummichog (Fundulus heteroclitus)

We examined whether ionoregulatory mechanisms of fresh water Fundulus heteroclitus in vivo are similar to those of typical freshwater species (e.g., rainbow trout, goldfish, and catfish). Under control conditions ([NaCl]ext ~1 mmol/l), the mummichog exhibits very large Na+ influx and efflux rates but virtually no Cl– influx and a small Cl– efflux component. External NaCl levels were varied to reveal a saturable, low affinity (Km = 1,723 ± 223 μmol/l), high capacity (Jmax = 2,258 ± 288 nEq/g/h) Na+ uptake system that was independent of both Na+ efflux and ammonia excretion. A measurable Cl– influx did not occur until NaCl levels surpassed 2 mmol/l and did not saturate within the freshwater range, suggesting a completely different uptake mechanism. Cl– efflux was also independent of Cl– influx. A systemic acidosis (intraperitoneal HCl injection) was induced in order to investigate the connection between ionoregulation and acid-base balance. The acidosis did not affect influx rates but induced an elevated Cl– efflux and an attenuated Na+ efflux. This resulted in an excess of net Cl– loss over Na+ loss which effected a net acid excretion by strong ion difference theory. These results concur with the measured acid-base fluxes which indicate that over 50% of the acid load was excreted within 4 h by differential efflux modulation. Therefore an ion/acid-base link does exist in the mummichog but differs in nature from that of other freshwater fish. Indeed, virtually all of these findings differ from the current model for most other teleosts, indicating that alternate models of ionoregulation in fresh water exist. J. Exp. Zool. 279:208–219, 1997.

Urea and Water Permeability in Dogfish (Squalus acanthias) Gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.

Ammonia and urea excretion in the tidepool sculpin (Oligocottus maculosus): sites of excretion, effects of reduced salinity and mechanisms of urea transport

Tidepool sculpins live in a variable environment where water temperature, salinity, gas tensions, and pH can change considerably with the daily tide cycle. Tidepool sculpins are primarily ammoniotelic, with 8–17% of nitrogen wastes excreted as urea. The majority of net ammonia (Jnetamm; 85%) and urea (Jneturea; 74%) excretion occurred across the gill, with the remainder excreted across the skin, the kidney, and/or gut. Acute (2h) exposure to 50% seawater significantly increased Jneturea (2.8-fold), but reduced Jnetamm (3.5-fold). In fish exposed to 50% seawater for 1 week, Jneturea returned to control values, but Jnetamm remained slightly depressed. Unidirectional urea influx (Jinurea) and efflux (Jouturea) were measured using14C-urea to determine if urea was excreted across the gills by simple diffusion or by a carrier-mediated mechanism. Jinurea increased in a linear manner with increasing urea water levels (0–11 mmol N l−1), while Jouturea was independent of external urea concentrations. As well, Jneturea and Jout inurea were not significantly different from one another, indicating the absence of “back transport”. Urea analogs and transport inhibitors added to the water did not have any consistent effect on unidirectional urea flux. These results demonstrate that ammonia and urea excretion rates and sites of excretion in tidepool sculpins are very similar to those found in other marine and freshwater teleosts. Urea and ammonia may play a role in osmoregulation as excretion rates and tissue levels were influenced by changes in water salinity. Finally, we found no evidence for a specific urea carrier; branchial urea excretion is likely dependent on simple diffusion.

NITROGEN EXCRETION AND THE CARDIORESPIRATORY PHYSIOLOGY OF THE GULF TOADFISH, OPSANUS BETA

Gulf toadfish, Opsanus beta, are facultatively ureotelic and can excrete the majority of their nitrogenous waste as urea. Urea excretion occurs in “pulses.” The hypothesis that pulsatile urea excretion reflects sudden, transient, generalized increases in the branchial conductance was investigated by the simultaneous monitoring of cardiorespiratory variables, oxygen uptake, and whole‐body urea, ammonia, and/or 3H2O effluxes. The direct monitoring of both expired branchial water and water exiting a respirometer demonstrated that urea pulses were derived from the gills. No significant changes in ventilation or cardiac frequency, oxygen uptake, or ammonia efflux were observed during natural urea pulses, refuting the hypothesis that pulsatile urea excretion reflects pulsatile increases in the generalized diffusive properties of the gill for solute transfer. An alternative model for pulsatile urea excretion postulates that the gill urea permeability is increased periodically by the insertion and/or activation of specific urea transporters into gill cell membranes. Pulsatile urea excretion was abolished by pretreatment with the cytoskeletal‐disrupting agent colchicine; colchicine may block trafficking of urea transporter–containing vesicles. Exocytosis of water following the fusion of vesicles with gill cell membranes could explain the significantly elevated 3H2O efflux observed during urea pulses.

The influence of some complexing agents (EDTA) and citrate) on the uptake of cadmium in perfused rainbow trout gills

Abstract The cadmium transfer through and the retention of metal in perfused gills from rainbow trout (Salmo gairdneri) has been studied in the presence of two Cd-complexing agents, ethylenediaminetetraacetic acid (EDTA) and citrate. The transfer and retention of Cd in the presence of EDTA was almost a function of the ambient free Cd2+ activity. The transfer of the free Cd ion was about 1000 times higher than of the Cd-EDTA complex. The Cd-EDTA complex was to some extent retained in perfused tissue. The transfer of Cd in the presence of citrate was markedly greater than expected on the basis of the free Cd2+ activity. The tissue retention of Cd was not affected by the presence of citrate. It is concluded that Cd uptake in fish gills in the presence of complexing agents is not simply a function of complexed versus free metal. The uptake is also profoundly dependent on the type of complexing agent present.

Oxygen transfer, gill resistance and structural changes in rainbow trout (Salmo gairdneri, Richardson) gills perfused with vasoactive agents

1. Gill resistance (Rg) and oxygen transfer (To2) were measured in perfused rainbow trout gills. After perfusion the gills were analysed morphometrically. 2. Rg increased with 40% and To2 decreased with 91% during 60 min perfusion without vasoactive substances in the perfusion Ringer. 3. In presence of 10 micro M adrenaline both Rg and To2 remained stable at their starting levels throughout the experimental period (60 min). 4. With 0.2 micro M acetylcholine and 10 micro M adrenaline in Ringer Rg increased nearby to the same extent as in perfusions without vasoactive agents, while To2 was not significantly affected. 1 micro M acetylcholine increased Rg with 76% and decreased To2 with 51%. 5. The changes in Rg and To2 could be explained by structural changes in the secondary lamellae.

Oxygen and chromium transfer in perfused gills of rainbow trout (salmo gairdneri) exposed to hexavalent chromium at two different pH levels

Abstract An in vitro study was performed on uptake and transfer of hexavalent chromium, Cr(VI), in gills of rainbow trout (Salmo gairdneri). Gills were perfused according to the isolated head perfusion technique, and externally exposed to Na2CrO4 solutions containing 51CrO42. Experiments were conducted at a concentration of 10 mg l Cr and at pH values of 8.1 and 6.5. The results show that the transfer of chromium is directly coupled with the transfer of oxygen from the external solution to the internal perfusion medium. Under similar conditions of oxygen transfer, however, chromium transfer was significantly more effective at pH 6.5 than at pH 8.1. In addition more chromium was accumulated by the gill tissue at the lower pH. Gill preparations of trout which had been pre-exposed in vivo for 4 days to 10 mg l Cr(VI) at pH 6,5, exhibited an impaired oxygen transfer. This could be well explained by the structural alterations seen after histological examination of the perfused gills.

Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture

The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10(-6) to 10(-12) M. After 24 h, TCDD showed a maximal induction at a concentration of 10(-9) M while BNF showed a maximal induction at a concentration of 10(-7) M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 µM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6beta and 16beta and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO3. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl− or the application of 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (0.1 mmol·l−1), but fell by about 0.3 units when Na+ was removed or in the presence of amiloride (0.2 mmol·l−1). Exposure to elevated ammonia (“ammonia prepulse”; 30 mmol·l−1 as NH4Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO3−buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na+-free conditions or in the presence of amiloride (0.2 mmol·l−1). Neither bafilomycin A1 (3 μmol·l−1) nor Cl removal altered the intracellular pH recovery rate. The Km for Na+ of the intracellular pH recovery mechanism was 8.3 mmol·l−1, and the rate constant at Vmax was 0.008·s−1 (equivalent to 5.60 mmol H+·l−1 cell water·min−1), which was achieved at external Na+ levels from 25 to 140 mmol·l−1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO3−-free media, both at rest and during acidosis, is regulated by a Na+/H+ antiport and not by anion-dependent mechanisms or a vacuolar H+-ATPase.

Increased availability of cadmium to perfused rainbow trout (Salmo gairdneri, Rich.) gills in the presence of the complexing agents diethyl dithiocarbamate, ethyl xanthate and isopropyl xanthate

A study was made of the cadmium transfer through and retention of metal in perfused gill tissue from rainbow trout (Salmo gairdneri) in the presence of three cadmium complexing agents; DDC (diethyl dithiocarbamate), ethyl xanthate and isopropyl xanthate. The complexes formed are non-polar. The transfer of complexed cadmium was greater than the transfer of free cadmium ion. The retention of cadmium in gill tissue was increased about ten times in the presence of each of the two xanthates. However, the retention in gill tissue was not altered by DDC. It is concluded that cadmium uptake in fish gills in the presence of complexing agents is not simply a function of complexed versus free metal. It is also heavily dependent on the type of complexing agent present.