The cultured branchial epithelium of the rainbow trout as a model for diffusive fluxes of ammonia across the fish gill
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1
*Author for correspondence (e-mail: woodcm{at}mcmail.cis.mcmaster.ca)
Accepted September 19, 2001
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Summary |
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Key words: Oncorhynchus mykiss, rainbow trout, gill, cultured epithelium, ammonia diffusion, transepithelial resistance, transepithelial conductance, PNH3 gradient, NH4+ electrochemical gradient, pH gradient, paracellular pathway.
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Introduction |
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Even in situations in which simple diffusion appears to predominate, it has not been possible to determine whether diffusion is transcellular, paracellular or both. Differences amongst species, salinities and in the ammonia, ionic and acidbase status of the animals in different studies have undoubtedly contributed to this uncertainty. However, the complex geometry of the intact gills with respect to both blood and water flow has also been an important factor. In particular, it has not been possible to determine the exact PNH3, NH4+ and pH levels on the two sides of the branchial epithelium because of unknown boundary layer and transit time effects, considerable differences in the composition of blood and water entering and leaving the gills and the possible heterogeneity of different parts of the gill surface.
An alternative approach is to develop simple model systems in which some of these factors can be better controlled. Recently, our laboratory has developed a cultured branchial epithelial preparation (double-seeded preparation) that incorporates the two major cell types of the freshwater gill, respiratory cells (pavement cells, approximately 85 %) and mitochondria-rich cells (chloride cells, approximately 15 %) in the approximate proportions in which they occur in vivo (Fletcher et al., 2000; Kelly et al., 2000
). The freshwater rainbow trout Oncorhynchus mykiss, the species that has been studied most for ammonia excretion, is the source of the cells, and the preparation is an extension of an earlier single-seeded trout gill epithelium that contained only respiratory cells (Wood and Pärt, 1997
; Wood et al., 1998
; Gilmour et al., 1998
). These preparations are flat and thus amenable to manipulation and sampling of the medium on either side for measurements of flux, pH and electrical characteristics.
The cultured epithelia are grown with culture medium (a blood-plasma-like substance) on both surfaces (symmetrical conditions), but are tolerant of subsequent exposure to fresh water on the apical surface (asymmetrical conditions), as in vivo. This exposure causes an overall increase in epithelial resistance. Simultaneous flux measurements with the paracellular marker polyethlene glycol-4000 (PEG-4000) have shown that the phenomenon is due to a decrease in transcellular permeability since paracellular permeability actually increases at this time (Wood et al., 1998; Gilmour et al., 1998
; Fletcher et al., 2000
), although it remains far lower than if sea water is placed on the apical surface (Fletcher, 1997
). While the double-seeded preparation does not exhibit active Na+ uptake (i.e. transport from the apical to the basolateral surface), it appears faithfully to duplicate the passive electrical and diffusive properties of the intact gill for Na+ and Cl movements (Fletcher et al., 2000
). It may, therefore, be particularly suitable for analysing passive ammonia movements across the gill. In the present study, we evaluate this preparation for the general study of ammonia excretion and use it to examine the roles of pH and electrical gradients and of paracellular versus transcellular pathways and the importance of NH3 versus NH4+ diffusion.
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Materials and methods |
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All procedures for gill cell isolation were conducted in a laminar flow hood using sterile techniques. Methods for initial gill cell isolation were based on those originally developed by Pärt et al. (1993), with modifications described by Wood and Pärt (1997
), and methods for epithelial preparation and culture were based on those outlined by Fletcher et al. (2000
). Briefly, gill cells were initially obtained from excised gill filaments by two consecutive cycles of tryptic digestion (Gibco BRL Life Technologies, 0.05 % trypsin in phosphate-buffered saline, PBS, with 5.5 mmol l1 EDTA) and resuspended in culture medium (Leibovitzs L-15 supplemented with 2 mmol l1 glutamine, 5 % foetal bovine serum, 100 i.u. ml1 penicillin, 100 µg ml1 streptomycin and 200 µg ml1 gentamycin). The cells were then directly seeded onto permeable Falcon cell culture inserts (Cyclopore polyethylene terephthalate filters; Becton Dickinson, Franklin Lakes, New Jersey, USA; pore density 1.6x106 pores cm2, pore size 0.45 µm, growth surface 0.9 cm2) at a density of 2x1062.5x106 viable cells cm2. Initially, 0.8 ml and 1.0 ml of medium plus antibiotics (see above) were added to the apical (insert) and basolateral (companion wells) sides of the preparations respectively. One day after seeding, inserts were rinsed with 0.4 ml of PBS (pH 7.7) to remove non-adherent cells and mucus, and new cells freshly isolated from a second fish were then seeded at the same density onto the adherent layer of cells established in the inserts. After 24 h, non-adherent cells and mucus were again rinsed off the inserts, and 1.5 ml and 2.0 ml of antibiotic-free medium (Leibovitzs L-15 supplemented with 2 mmol l1 glutamine and 5 % foetal bovine serum) were added to the apical and basolateral sides of the preparations respectively. Media were changed every 48 h thereafter and remained antibiotic-free throughout the culture period. Epithelial preparations were incubated at 18°C in an air atmosphere. Full details of the procedures for the preparation and culture of rainbow trout epithelia can be found in Kelly et al. (2000
).
All experiments were conducted on epithelia 67 days after initial seeding, a time when transepithelial resistance was close to maximum plateau values (Fletcher et al., 2000). When fresh water was used to replace the apical medium (asymmetrical or simulated in vivo conditions), it (sterilized, chemical composition same as original holding water) was added to the apical side of the insert after several rinses to ensure removal of any residual medium.
Electrophysiological measurements
Transepithelial resistance (TER) was monitored using STX-2 chopstick electrodes connected to an EVOM epithelial voltohmeter, custom-modified by the manufacturer (World Precision Instruments, Sarasota, Florida, USA) to measure resistances up to 100 000 . To correct for background resistance, the TER of an identical vacant culture insert was determined and subtracted from the TER of the culture inserts containing cultured epithelia. The corrected value in ohms (
) was then multiplied by the effective growth area (0.9 cm2) to yield the final value for TER (in
cm2). Conductance was calculated as the inverse of TER. In a parallel series of experiments, we determined strong linear regression relationships between measured transepithelial potential (TEP; expressed with respect to the apical surface as 0 mV) and measured TER under both symmetrical and asymmetrical conditions, as reported in Fig. 6 of Fletcher et al. (2000
); these relationships were used to predict TEP for the purposes of calculation in the present experiments.
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Series 1
Selected epithelia with almost identical TERs under symmetrical culture conditions (L-15 apical/L-15 basolateral) were exposed to asymmetrical conditions (fresh water apical/L-15 basolateral) after adding varying levels of NH4Cl (BDH, Toronto, Ontario, Canada) to the basolateral medium (N=10 for each group). The added nominal concentrations of NH4Cl were 0, 250 and 1000 µmol l1, but since the L-15 medium contains 100150 µmol l1 of endogenous ammonia, final measured total ammonia concentrations (TAmm) on the basolateral side were 123±2, 400±5 and 1055±10 µmol l1 respectively. After a 3 h flux period, apical fresh water was collected for the analysis of total ammonia concentration (TAmm). TER across the epithelia was recorded prior to experimental manipulation and at 0 and 3 h after the addition of fresh water. On the basis of our observations in this experiment, a final TAmm concentration of approximately 650 µmol l1 was selected for all subsequent experiments, achieved by adding 500 µmol l1 NH4Cl.
Series 2
To determine whether basolateral pH influenced ammonia flux rates (JAmm) from the basolateral to the apical compartment of cultured epithelia under asymmetrical conditions, basolateral medium pH (pHBl) was manipulated by adding appropriate amounts of either 140 mmol l1 HCl or 140 mmol l1 NaOH, and TAmm was set to 650 µmol l1. Availability of inserts allowed for six groups (N=3 for each group) with pHBl values of 7.0, 7.4, 7.6, 7.8, 8.2 and 8.6 (±0.05 pH units). Apical water samples were collected before and after a 3 h flux period for analysis of TAmm. TER measurements were recorded immediately before and after the introduction of asymmetrical conditions and just prior to the collection of apical water at the end of the 3 h flux period.
Series 3
This most detailed series examined the effects of basolateral pH manipulations, of symmetrical versus asymmetrical conditions and of directionality of gradients (i.e. basolateral-to-apical versus apical-to-basolateral gradients) under symmetrical conditions. The pH of the basolateral medium (set to 650 µmol l1 TAmm) was manipulated as described previously (see above), and a range of values from 7.0 to 8.6 was assessed. Separate preparations were used for apical freshwater treatments (asymmetrical; N=3 at each pHBl) and apical L-15 (symmetrical; N=6 at each pHBl) treatments at each pH. Apical water and basolateral L-15 samples were taken at 0, 3 and 6 h for analysis of TAmm, apical pH (pHAp), pHBl (and adjustment of pH if necessary), together with TER measurements.
Using preparations derived from the same batch of fish, a concurrent experiment was performed to determine whether the cultured epithelia exhibited any signs of rectification. This was done under symmetrical culture conditions in which the apical medium of another set of epithelia, matched to approximately the same TER, was pH-manipulated and loaded to 650 µmol l1 TAmm (N=56 at each pHAp). Both apical and basolateral L-15 samples were collected for the analysis of TAmm, pHAp, pHBl (and adjustment of pH if necessary) and TER measurements at 0, 3 and 6 h.
Analytical techniques and calculations
In water, TAmm was determined by the salicylate/hypochlorite method (Verdouw et al., 1978). Components of the L-15 medium interfere with colour development in this assay so, in medium, TAmm was determined using the enzymatic method of Mondzac et al. (1965
) employing Sigma ammonia kit no. 171-UV according to the manufacturers instructions for blood plasma samples. The two assays were cross-validated to produce the same results using both saline- and water-based standards, although the precision of the colorimetric assay was greater than that of the enzymatic method. Both water and medium pH were measured with a Radiometer E5021 microelectrode system, thermostatically controlled to the experimental temperature (18°C).
The concentrations of NH3 and NH4+ and the partial pressure of ammonia (PNH3) in both water and medium samples were calculated from the respective pH and TAmm measurements in the two solutions using the HendersonHasselbalch equation and appropriate values of pK and ammonia solubility (NH3) in plasma and fresh water from Cameron and Heisler (1983
), as detailed in Wright and Wood (1985
). As L-15 medium is very similar in ionic composition to trout plasma (Wood and Pärt, 1997
), plasma values were employed for L-15. The difference (
PNH3) between simultaneous determinations of PNH3 in apical water or medium and basolateral medium was calculated as a measure of the NH3 diffusion gradient. The electrochemical gradient for NH4+ diffusion (e.g. basolateral-to-apical) was calculated as the driving force, i.e.
| (1) |
where concentrations are in molar units, TEP is in volts, and R, T, z and F in the Nernst component have their usual thermodynamic values. PNH3, pH and electrochemical gradients were averages from measurements made at the start and end of the associated ammonia flux measurements.
Ammonia flux rates (JAmm, nmol cm2 h1), from either apical-to-basolateral or basolateral-to-apical compartments of the cultured epithelia, were calculated in the standard fashion from the appearance of TAmm (nmol ml1) in the unmanipulated compartment (the one with a lower TAmm ), multiplied by volume (ml) and factored by time (h) and surface area (cm2). In the experiments of series 3, we checked whether disappearance from the basolateral compartment (L-15, higher concentration) corresponded to appearance in the apical compartment (fresh water, lower concentration). There were never any significant differences between the two measurements of flux, but the lower precision of the enzymatic assay, coupled with the problem of detecting small changes against high background levels in the basolateral compartment, resulted in higher variability in the disappearance measurement, so only appearance measurements are reported. We also ran tests with vacant filter inserts to check whether the ammonia diffusion resistance of the filter was significant relative to that of the cultured epithelium. The results demonstrated that the ammonia permeability of the filter alone was at least 10-fold greater than the permeability of the epithelium plus filter under symmetrical conditions and at least fivefold greater under asymmetrical conditions, indicating that this was not an important source of error.
Data have been expressed as means ± 1 S.E.M. Statistical comparisons were made by Students paired or unpaired t-test (two-tailed) or by one-factor or two-factor analysis of variance (ANOVA) followed by a StudentNewmanKeuls test to detect specific differences, as appropriate (Nemenyi et al., 1977). Regression relationships were fitted by the method of least squares, and the significance of Pearsons correlation coefficient was assessed. A fiducial limit of P
0.05 was used throughout.
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Results |
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Series 1
The 30 preparations used in this experiment were selected for initial uniform TER under symmetrical conditions (30003500 cm2), and the addition of various levels of TAmm as NH4Cl to the basolateral medium did not affect TER (Fig. 1A). However, when the apical medium was changed to fresh water, TER increased significantly, approximately threefold, and became more variable in all groups (Fig. 1A). Basolateral pH was not controlled in this series but was approximately 7.47.6. Under these asymmetrical conditions, increasing basolateral TAmm from 123 to 400 and 1055 µmol l1 resulted in significant increases in ammonia flux rates (JAmm) from basolateral to apical surfaces across cultured epithelia (Fig. 1B), although there was again no effect on TER. There was no significant relationship between JAmm and TER at the two lower TAmm levels, but a significant negative relationship at a basolateral TAmm of 1055 µmol l1 (Fig. 1C).
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Series 3
Basolateral TAmm was again set to 650 µmol l1, pHBl was experimentally manipulated and pHAp was closely monitored to keep it close to starting values (pH 7.5 for apical L-15, pH 8.0 for apical fresh water) in preparations under either symmetrical (apical L-15/basolateral L-15) or asymmetrical (apical fresh water/basolateral L-15) conditions. Flux periods of 6 h (average of two 3 h periods, with pH adjustment at 3 h) were employed to control pH levels better. In this series, there were no significant differences in TER associated with apical freshwater exposure, and no significant influence of pHBl on TER, which averaged 4443±548 cm2 (N=45).
As in series 2, basolateral-to-apical JAmm increased significantly with increasing pHBl (one-way ANOVA), and this overall effect was seen under both asymmetrical and symmetrical test conditions (Fig. 3A). Furthermore, the rates at pHBl 8.6 were significantly greater than those at all other pHBl levels below 7.8 under both conditions. (Because of the limited number of matched preparations available, pHBl 7.8 was not tested under symmetrical conditions.) When a two-way ANOVA was applied, there was a significant overall effect of asymmetrical versus symmetrical conditions, with consistently greater JAmm values when the apical medium was fresh water. Individual differences were significant at pHBl values of 7.0, 7.4 and 8.2 (Fig. 3). However, since L-15 contains an endogenous level of TAmm whereas the starting fresh water did not, and since the pHAp values were different in the two media, the pH, PNH3 and NH4+ gradients were not necessarily the same under the two conditions. Indeed, the basolateral-to-apical pH gradients were substantially lower or more negative at every pHBl value when the apical medium was fresh water (Fig. 3B), but the PNH3 gradients were very similar in the two treatments (Fig. 3C). Very clearly, the ammonia fluxes (Fig. 3A) tended to track the PNH3 gradient (Fig. 3C) in each treatment. Relationships between JAmm and the respective PNH3 and NH4+ gradients are examined in greater detail in Fig. 5 and Fig. 6 (see below).
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Under all three conditions, there was a positive electrochemical driving force for NH4+ in the direction of JAmm, principally due to the addition of NH4Cl to the driving side. In themselves, these analyses provided no evidence for diffusion of NH4+ along its electrochemical gradient as a major pathway of ammonia flux because, under all three conditions, the relationship between JAmm and electrochemical driving force was actually negative (Fig. 6), although this was significant only under asymmetrical conditions (Fig. 6A). The negative relationships reflect the fact that JAmm was greater at high pH and PNH3 gradients, resulting in greater NH4+ accumulation on the recipient side and, therefore, lower electrochemical gradients when averaged over 6 h.
Nevertheless, the range of electrochemical driving forces was clearly higher under asymmetrical conditions (Fig. 6A) (generally +40 mV to +65 mV) than under symmetrical conditions (Fig. 6B,C) (generally +5 mV to +40 mV). This difference was reflected in mean values that were more than twice as high: +54.6±2.1 mV, N=18, under asymmetrical conditions versus +22.2±0.9 mV, N=33, in basolateral-to-apical trials and +16.9±2.3 mV, N=29, in apical-to-basolateral trials under symmetrical conditions. This difference was not due to the TEP component, which actually reduced the driving force under asymmetrical conditions (mean TEP 8.9±0.1 mV, N=18) and had a marginal influence under symmetrical conditions (mean TEP +2.4±0.3 mV, N=33, in basolateral-to-apical trials and +2.0±0.2 mV, N=29, in apical-to-basolateral trials). Rather, it was due to the higher starting TAmm levels and NH4+ concentrations, and therefore lower Nernst component, when L-15 was used on the non-manipulated (recipient) side in the symmetrical trials.
In theory, if NH4+ is diffusing across the epithelium, then its flux should be a linear function of conductance (the inverse of TER), all other factors being equal. The results of series 1 suggested that JAmm decreased as TER increased in the expected fashion, at least at high basolateral concentrations of TAmm when the apical medium was fresh water (Fig. 1). The results of the present series provide an opportunity to analyse possible conductance versus flux relationships in detail under conditions of both apical fresh water and apical L-15. To remove the influence of NH3 flux, the product of the particular PNH3 gradient and the slope of the relevant regression line (i.e. JAmm per nmHg PNH3 from Fig. 5; 1 nmHg=0.133 Pa) was subtracted from each value of total JAmm to yield the apparent NH4+ flux (Fig. 7). Under asymmetrical conditions, this flux was strongly correlated with transepithelial conductance (r=0.68, N=18, P<0.05), whereas there was no relationship under symmetrical conditions (r=0.01, N=29, P>0.05). In neither case was the relationship improved by expressing JAmm per millivolt driving force. This analysis suggests that diffusive NH4+ flux is important under asymmetrical conditions but not under symmetrical conditions.
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Discussion |
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Leaving aside for the moment the curious influence of directionality (Fig. 4), several conclusions can be drawn about the passive movement of ammonia across the epithelium. The results confirm that the diffusion of NH3 occurs across the epithelium and that this responds to variations in PNH3 gradients achieved through pH manipulations (Fig. 3, Fig. 4, Fig. 5). Within any one condition, variations in NH4+ electrochemical gradients do not appear to be an important factor in variations in ammonia flux because there was either no relationship or a negative relationship between ammonia flux and net electrochemical driving force (Fig. 6). Nevertheless, the results indicate an important role for NH4+ diffusion under asymmetrical conditions. In this regard, the intercept of the regression of basolateral-to-apical ammonia flux on PNH3 gradient was positive (i.e. some ammonia flux still occurred in the absence of a PNH3 gradient) and much higher when fresh water was present on the apical surface, while the slopes (representing NH3 permeability) were the same in the two treatments (Fig. 5A,B). A significant diffusive flux of NH4+ may, therefore, have contributed to the greater net ammonia fluxes seen when fresh water was present on the apical surface (Fig. 3).
This possibility is supported by three other pieces of evidence. First, the mean basolateral-to-apical electrochemical driving force for NH4+ was more than twice as high (+55 mV versus +22 mV) when the apical medium was fresh water. Second, the apparent NH4+ flux was strongly correlated with transepithelial conductance in the expected fashion, but only under asymmetrical conditions (Fig. 7). Lastly, it is now well-documented that paracellular permeability (measured with PEG-4000) increases (generally by 50100 %) when these preparations are exposed to apical fresh water (Wood et al., 1998; Gilmour et al., 1998
; Fletcher et al., 2000
). This increase in paracellular permeability is responsible for increased diffusive fluxes of ions such as Na+, Cl and Ca2+ upon exposure of these cultured epithelia to fresh water, so it is not surprising that greater diffusion of NH4+ may also occur. However, it should be noted that this increase in paracellular permeability when the apical medium is changed from iso-osmotic L-15 medium (osmotically equivalent to approximately 30 % sea water) to fresh water is far smaller than when the apical medium is changed to full-strength sea water (Fletcher, 1997
). Thus, the permeability characteristics of the cultured epithelium are in accord with the well-documented pattern that gill permeability in vivo is much lower in freshwater fish than in seawater fish (Evans, 1984
).
The greater overall rates of JAmm when the gradients were reversed under symmetrical conditions was unexpected (Fig. 4). However, rectification of transport of many non-electrolytes across the fish gill is a well-documented phenomenon, with differences of up to 10-fold, although the phenomenon has not previously been reported for ammonia, and the mechanisms of rectification remain poorly understood (for a review, see Isaia, 1984). In the present study, there was no significant relationship between the apical-to-basolateral JAmm and the PNH3 gradient (Fig. 5C), the net driving force for NH4+ (Fig. 6C) or the transepithelial conductance (data not shown), so the mechanism is again unclear. A priori, it may seem counterintuitive for the gill to favour ammonia uptake (apical-to-basolateral flux) over ammonia excretion (basolateral-to-apical flux). However, we have recently presented evidence that rainbow trout may actually use low levels of ammonia in the environmental water to promote protein synthesis and growth (Linton et al., 1997
; Morgan et al., 2001
). Thus, a rectification mechanism favourable to ammonia uptake may be adaptive in some circumstances. Clearly, the phenomenon deserves further detailed study.
Comparison with in vivo data
Several studies of ammonia excretion in freshwater trout in vivo have estimated the diffusivity of the gill to NH3 as approximately 46 µmol kg1 h1 nmHg1 (Cameron and Heisler, 1983; Wright and Wood, 1985
). These estimates were achieved by establishing conditions in which it was thought that only NH3 diffusion was occurring and then dividing the measured JAmm (approximately 300 µmol kg1 h1) by the apparent PNH3 gradient from blood to water (approximately 65 nmHg). Assuming a gill surface area of 2500 cm2 kg1 for freshwater trout (Wood, 1974
), this yields an apparent gill NH3 permeability of approximately 1.1x102 cm s1. Contrary to popular belief, NH3 has low lipophilicity (Evans and Cameron, 1986
), so this is a high permeability value for NH3 relative to most other preparations (generally in the range 103 to 104 cm s1) (see Cameron and Heisler, 1985
; Evans and More, 1988
) and comparable with the very high value seen in the rabbit proximal kidney tubule (Garvin et al., 1987
). By way of comparison, at a basolateral-to-apical PNH3 gradient of 65 nmHg, JAmm across the cultured epithelium was approximately 25.5 nmol cm2 h1 (from Fig. 3 or Fig. 5A), equivalent to only 64 µmol kg1 h1, or approximately 20 % of the flux in vivo. However, on the basis of the slope of Fig. 5A (JAmm per nmHg PNH3 gradient), only approximately 6 % (4 µmol kg1 h1) of this would be due to NH3 diffusion, with the remainder presumably due to NH4+ diffusion. Thus, NH3 permeability across the cultured epithelium is at most 2.3x103 cm s1 and could be as low as 1.5x104 cm s1, in general accord with values in the literature for other tissues and in accord with the predicted range (Isaia, 1984
) for a substance whose oil:water partition coefficient is less than 0.1 (Evans and Cameron, 1986
). NH4+ permeability across cultured epithelia under asymmetrical conditions (in the mid-range of conductance) would be approximately 105 cm s1, in accord with other analyses suggesting that the permeability ratio of NH4+ to NH3 is somewhat higher in the tissues of ammoniotelic fish than in those of ureotelic higher vertebrates (for a review, see Wood, 1993
). It is problematic whether significant NH4+ permeability occurs under symmetrical conditions; if so, it is small and hidden in the noise of the data (e.g. Fig. 7).
While these permeability estimates are only approximations, an inescapable conclusion from the present results is that passive fluxes of ammonia alone (as both NH4+ and NH3) are insufficient to explain the rates of branchial ammonia excretion observed in vivo. This is especially striking since passive fluxes of Na+ and Cl across this same preparation nicely match branchial Na+ and Cl efflux rates recorded in intact freshwater rainbow trout (Fletcher et al., 2000). However, the conclusion is not surprising when one considers that concentrations of Na+ and Cl in trout plasma are generally approximately 2501000 times higher than those of TAmm (e.g. 150 mmol l1 versus 150600 µmol l1). Indeed, in hindsight, it is curious why so much emphasis has been placed on diffusion as the supposed dominant pathway for ammonia excretion across the gills of freshwater fish (Cameron and Heisler, 1983
; Cameron and Heisler, 1985
; Heisler, 1990
; Wilson et al., 1994
), an emphasis that has necessitated the implicit assumption of an unusually high permeability to NH3. In vivo, there is abundant, though controversial, evidence for carrier-mediated NH4+ transport across the gills of freshwater fish (Maetz and Garcia-Romeu, 1964
; Maetz, 1972
; Maetz, 1973
; Payan, 1978
; Wright and Wood, 1985
; Cameron, 1986
; McDonald and Prior, 1988
; McDonald and Milligan, 1988
; Balm et al., 1988
; Wilkie, 1997
; Salama et al., 1999
), generally associated directly or indirectly with active inward Na+ transport. This active Na+ transport does not occur in the cultured branchial epithelium (Fletcher et al., 2000
), which probably explains the discrepancy.
In summary, the present results demonstrate that both NH4+ and NH3 diffusion occur across the gill epithelium, that NH4+ diffusion, which probably proceeds via the paracellular pathway, is quantitatively the more important component, at least in apical fresh water, and that these fluxes are substantially less than rates of ammonia excretion in vivo. As such, they provide impetus to re-examine carrier-mediated ammonia flux in the intact gill and to investigate methods of promoting transepithelial active transport in the cultured branchial epithelium.
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Acknowledgments |
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