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J. exp. Biol. 114, 329-353 (1985) 329
Printed in Great Britain © The Company of Biologists Limited 1985
AN ANALYSIS OF BRANCHIAL AMMONIA EXCRETION INTHE FRESHWATER RAINBOW TROUT:
EFFECTS OF ENVIRONMENTAL pH CHANGE AND SODIUMUPTAKE BLOCKADE
BY P. A. WRIGHT* AND C. M. WOOD
Department of Biology, McMaster University, Hamilton, Ontario,Canada, L8S 4K1
Accepted 9 July 1984
SUMMARY
Short-term treatments (3 h) designed to change the relative NH3
(AP N H 3 ) and NH4+ (ANH4
+) gradients and sodium transport (JNa)across the gills were employed to analyse the normal mechanism(s) ofbranchial ammonia excretion (J^eTm) in trout acclimated to fresh water ofpH = 8-0. Control Jne?m occurred in the absence of, or against, an apparentAPNH gradient, while ANH4
+ was positive. Severe acid exposure(pH = 4-06) raised APNHj and ANH4
+, abolished J^3, and reduced } ^ m
by 28%, while moderate acidity (pH = 6-64), which also elevated APNH3Ihad no significant influence on J^a and JneTm- Severe alkaline exposure(pH = 9-54) raised ANH4
+, reduced APNH] to a very negative value, anddecreased J,>Ja and ]^™m by equimolar amounts, representing 55 % and80% of control levels respectively. Moderate alkalinity (pH = 8-69) hadsimilar effects on APNH and ANH4
+, but reduced Jina and JneT™ by only~ 2 5 % . The sodium transport inhibitor amiloride (10~4moll - 1 in theexternal water, pH = 8-0) had very similar effects to pH = 4-06 on both jf 8
and Jne?m> but did not alter A P N H 3 or ANH4+. The results discount the
quantitative importance of NH4+ diffusion and favour a flexible com-
bination of NH3 diffusion and Na + /NH 4+ exchange as the major mechan-
isms of J^T"1. with the latter dominating under the particular controlconditions of the present study.
INTRODUCTION
Of the three major respiratory gases of aquatic animals, ammonia is probably theleast understood. Metabolic processes are generally thought to produce ammonia inthe NH3 form, but with a pK' — 9-5, the great majority exists in the NH4
+ form atphysiological pH. Early studies by Smith (1929) established that the gills were theprincipal site of ammonia excretion. While some ammonia may arise de novo in thegill tissue (Goldstein & Forster, 1961), it is now clear that the major part of
• Present address: Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T2A9.
Key words: Trout, ammonia, gills, amiloride, pH.
330 P . A. W R I G H T AND C M . W O O D
branchial ammonia excretion represents clearance from the blood (Pequin &Serfaty, 1963; Goldstein, Forster & Fanelli, 1964; Payan & Matty, 1975; Payan &Pic, 1977; Cameron & Heisler, 1983). However there is some question as to theactual mechanism(s) by which ammonia moves across the branchial epithelium (seeKormanik & Cameron, 1981a for review). It is well documented that ammonia canpassively diffuse across the gills as NH3 (de Vooys, 1968; Kerstetter, Kirschner &Rafuse, 1970; Hillaby & Randall, 1979; Kormanik & Cameron, 19816; Cameron &Kormanik, 1982; Cameron & Heisler, 1983; Holeton, Neumann & Heisler, 1983).There is also considerable evidence for a carrier-mediated Na+/NH4+ exchange(Maetz & Garcia-Romeu, 1964; Payan & Matty, 1975; Payan, Matty & Maetz,1975; Kerstetter & Keeler, 1976; Payan, 1978; Pressley, Graves & Krall, 1981) aswell as some support for simple NH4"1" diffusion (Goldstein, Claiborne & Evans,1982). It remains unclear whether one of these processes predominates, or whethera variable combination of two or more of these processes commonly occurs (e.g.Maetz, 1972, 1973; Maetz, Payan & De Renzis, 1976; Evans, 1977, 1982; Clai-borne, Evans & Goldstein, 1982; Wood, Wheatley & Hobe, 1984).
Until recently, analysis of ammonia movements was limited by the lack of reliablephysical constants (pK', solubility) for ammonia in water and fish plasma at typicalfish temperatures (cf. Kormanik & Cameron, 1981a). These parameters have nowbeen measured for the rainbow trout (Cameron & Heisler, 1983). A furtherlimitation in the past has been the absence of studies relating internal ammonialevels under resting conditions, as sampled by chronic cannulation, to externallevels, ammonia excretion and unidirectional sodium fluxes at the gills. The presentstudy employs this approach and the physical constants of Cameron & Heisler(1983) to reinvestigate the mechanism(s) of branchial ammonia efflux in therainbow trout. Our aim has been to assess the mechanism(s) under normallaboratory conditions, rather than how they might change during long-term expo-sure to abnormal conditions. Thus we have examined the short-term effects, andtheir reversibility, of various treatments (environmental pH changes, amiloride)calculated to alter the relative NH3 and NH4
+ gradients and sodium transportacross the gills.
MATERIALS AND METHODS
Experimental animals
Experiments were performed on 77 rainbow trout (Salmo gairdneri; meanweight, 430g; range 180-786g), obtained from Spring Valley Trout Farm, Peters-burg, Ontario. The trout were initially held in flowing, dechlorinated Hamiltontapwater at seasonal temperatures and fed regularly with Silver Cup 3/16" TroutChow. One week prior to experimentation, the fish were transferred in batches of10-15 to a 350-1 closed-circuit, temperature controlled (15±2°C) fibreglass tankcontaining the same tap water ([Na+]=0-6, [CP] — 0-8, [Ca2+] = l-6,[Mg2 +]=0-3, [K+] ^O-OSmequivT1; titration alkalinity- 2-0mequivl"1; totalhardness = 140mgl~' as CaCO3; pH = 8-0). The water was changed regularly tokeep ammonia levels generally below 200/u.mol 1 . During this acclimation period,
Branchial ammonia excretion in trout 331
the fish were not fed in order to eliminate the influence of feeding history onammonia excretion (Ffomm, 1963).
Trout were fitted with chronic indwelling dorsal aortic cannulae (methods ofSmith & Bell, 1964 or Soivio, Westman & Nyholm, 1972) and urinary bladdercatheters (Wood & Randall, 1973) while under anaesthesia in a 1:10000 dilution ofMS-222 (Sigma). The fish were allowed to recover at least 48h in individual fluxboxes (see diagram and description in McDonald, 19836). For measurements ofbranchial ammonia and sodium exchange, the flux boxes could be operated asclosed, low-volume (3-61) recirculating systems at 15 ± 1 °C. As urine was collectedexternally, changes in water composition in the closed system were assumed torepresent branchial fluxes, for skin and intestinal fluxes are considered negligible infresh water (Fromm, 1968; Morii, Nishikata & Tamura, 1978; Cameron, 1978).
Experimental protocols
In each series, a 3-h control period (I) was followed by a 3-h experimental periodand then a second control period (II; i.e. recovery) of equal length. At the start ofeach 3-h flux period, the box was flushed with fresh acclimation water and thevolume reduced to a minimum (3-61 depending on the fish and box size) so as tomaximize the sensitivity with which changes in water ammonia and sodiumconcentration could be measured. This caused no apparent disturbance to the fish.At each time, new 22Na (New England Nuclear; 1 /nCiP1) was added to the waterfor measurement of unidirectional sodium fluxes at the gills. Water samples weredrawn at the start of each 3-h period and every subsequent hour. Water pH wascontinually monitored and adjusted as necessary with lmoll"1 HC1 or lmoll"1
KOH. Water samples were immediately frozen and later analysed for total ammonia(TAmm). total sodium and Na radioactivity. Arterial blood samples (300JU.I) weredrawn anaerobically from the dorsal aortic cannula and replaced by an equal volumeof heparinized Cortland saline [Wolf, 1963; sodium heparin (Sigma); 100i.u.ml" ' ] , Blood samples were taken at 0-5 h and 2-5 h of each 3-h period, and analysedfor pH (pHa), total plasma CO2 content (CT) and total plasma ammonia content(TAmm).
Six series of experiments were performed.
(i) A control in which fish were subjected to unaltered acclimation water(pH = 8'07 ± 0-02, N = 8) in the experimental period to check for anyeffects of the protocol itself,
(ii) Exposure to severely acidic water (pH = 406 ± 0-03, N = 10) obtained bytitration with lmoll"1 HC1; the water was vigorously aerated for severalhours before use to avoid any complicating effects of elevated Pco •
(iii) Exposure to moderately acidic water (pH = 6'64 ± 0-04, N= 17) preparedin the same manner as the severely acidic,
(iv) Exposure to severely alkaline water (pH = 9-54 ± 0-02, N = 9) obtained bytitration with lmo lT 1 KOH.
(v) Exposure to moderately alkaline water (pH = 8-69 ± 0-04, N = 17) preparedin the same manner as the severely alkaline,
(vi) Exposure to the drug amiloride (C6H8C1N7O; Merck, Sharp & Dome
332 P . A. W R I G H T AND C M . W O O D
Laboratories), a specific sodium transport blocker (Benos, 1982) atl O ^ m o i r 1 in water of unchanged pH (8-12 ± 0-05; N = 9).
A further seven trout were fitted with both dorsal aortic and ventral aorticcannulae, the latter by the method of Holeton & Randall (1967a). These fish wereemployed to assess the relative concentrations of ammonia in pre- and post-gillblood plasma and were sampled only under control conditions.
Analytical techniques
Water pH was monitored with a Canlab polymer body, sealed reference, com-bination pH electrode (H5503-20) coupled to a Radiometer pH 71 MK2 acid-baseanalyser or Fisher 119 digital pH meter. Total water ammonia was measured by amicro-modification of the salicylate-hypochlorite assay (Verdouw, van Echted &Dekkers, 1978). Total water sodium was determined by flame photometry (EelMark II), and 22Na radioactivity by counting 5 ml of water in 10 ml ACS fluor(Amersham) on a Beckman LS 250 liquid scintillation counter. Arterial pHa andplasma CT were measured immediately upon collection using Radiometer micro-electrodes and methodology outlined by Wood & Jackson (1980). Plasma wasobtained by centrifugation at 9000 £• for 2min and frozen for later analysis of TA m m
by micro-modification of a commercial diagnostic kit (L-glutamic dehydro-genase/NAD method; Sigma, 1982). Different ammonia assays were used forplasma and water because the simpler salicylate-hypochlorite method occasionallygave spurious values for plasma. The two assays were cross-validated on the samewater- and saline-based ammonia standards.
Calculations
Free (NH3) and ionized (NH4"1") ammonia concentrations in plasma and waterwere calculated from total ammonia concentrations (TAmm) and pH by theHenderson-Hasselbalch equation, using values of pK' appropriate for trout plasmaand fresh water at experimental temperature from Cameron & Heisler (1983):
TAmm4 l + antilog(pH-pK') K>
Therefore:
NH 3 = T A m m - N H 4+ . (2)
The partial pressure of free ammonia (PNH m /^Torr: 1 Torr32133-322387 Pa)was then calculated using the appropriate solubility coefficient ((XNH3) fromCameron & Heisler (1983):
NH 3
p ( 3 )
An analogous series of equations based on the Henderson-Hasselbalch relation-ship was used to calculate plasma HCC>3~ and Paco2 from pHa and CT, usingappropriate values of pKi' and aCO2 (Severinghaus, 1965; Albers, 1970).
Branchial ammonia excretion in trout 333
Since ammonia excreted by the gills is cleared from the blood (see Introduction),it is the mean blood plasma level [(arterial TAmm+venous TAmm)-^2] which bestdefines blood to water gradients. However, the venous cannulation procedure ismore difficult, more interventive and undoubtedly more stressful than the arterial.Therefore, we chose to predict the mean level from the arterial, rather than directlymeasuring it in all cases. Twelve simultaneous arterial and venous measurementsunder control conditions in the seven fish implanted with both cannulae yielded amean venous TAmm (241 ± 31 jamoll"1) which was 1 -66-fold mean arterial TAmm
(145 ± 28yu,molF1). As this was very similar to the ratio (1-81) reported byCameron & Heisler (1983) for Salmo gairdneri and there is essentially no differencebetween pHa and pHv, mean blood plasma TAmrn was routinely calculated as l-33xarterial TAmm for use in equations (1) and (2).
Diffusion gradients for free and ionized ammonia across the gill were estimatedas:
H3 = mean plasma PNH3 ~ water PNH3 (4)
and
ANH4+ = mean plasma NH4
+ - water NH4+, (5)
recognizing that the latter does not take into account the small (unmeasured)electrical gradient across the gill (McWilliams & Potts, 1978). In these calculations,for each 3-h flux period, the initial (0-5 h) and final (2-5 h) plasma measurementswere related to the means of the Oh and 1 h, and 2h and 3h water measurements,respectively.
Net branchial flux rates of total ammonia (J^eTm) were calculated as:
t X W
where i and f refer to initial and final concentrations in water in ju,mol ml ', V is thevolume of the system in ml (corrected for sampling deficits), t is the elapsed time inh, and W is the fish weight in kg. Several experiments were run with known TAmm
but no fish present to check for ammonia loss from the water to the atmosphere inalkaline exposures (pH = 9-54); this proved to be negligible.
Net branchial sodium flux rates ( J^) were calculated by an equation analogousto (6). Unidirectional influx rate (Jjna) was calculated as outlined by Maetz (1956):
( R i - R f ) x v
S A X t x W '
where Rj and Rf are initial and final radioactivities of water in c.p.m. ml"1, SA is themean specific activity of water (c.p.m. /u,equiv~') over the flux period in question,and the other symbols are as in equation (6). In practice, reliable changes in R couldonly be detected over 3 h, so a single value for average Jina was determined for each
334 P. A. WRIGHT AND C M . WOOD
3-h period. Internal SA never exceeded 10% of external SA, so correction forradioisotopic backflux in equation (7) was unnecessary (cf. Maetz, 1956). Uni-directional efflux rates (J^u
at) were calculated by the conservation equation:
J Na jNa rNa /o\
o u t " Jnet ~ J in • (,°J
For all fluxes, losses by the animal have a negative sign, gains a positive sign.Data have been expressed as mean ± 1 S.E.M. (A/) where N equals the number of
animals sampled. Changes in selected parameters within flux periods are illustratedin the figures, while averaged values over each 3-h treatment period are summarizedin the Tables. Student's two tailed i-test (P<0t05) has been used for comparisonswithin groups, with each animal serving as its own control.
RESULTS
Control series and resting values
Jnc"nm> measured on an hourly basis, declined during each 3-h flux period, andthen recovered after the flush (Fig. ID), a pattern also seen in the control periodsand the experimental periods of all the other experimental series (Figs2D—6D). Asimilar pattern was seen by Kormanik & Cameron (19816) in their work on bluecrabs. In the present study, the fish were preadapted to the experimental chambersfor at least 48 h, so it is unlikely to be a stress effect. Rather, this decline in JneT™appeared to be correlated with the rise in external TAmm in the closed system overtime. Plasma TA m m, while variable, did not increase significantly over the sameperiod (Fig. 1A). Consequently both A P N H 3 and ANH4
+ progressively fell(Fig. 1B,C); again the same patterns were seen in the control periods of all otherprotocols (Figs 2-6). There was no significant variation in plasma acid-base status(i.e. pHa, HCO3~, Pacoz; data not shown) within the 3-h periods.
Averaged data for each 3-h period are summarized in Table 1. All measuredparameters were unchanged in the experimental period relative to control I.However, in control II, both plasma and water PNH , NH4
+, and thus TA m m, weresignificantly lower relative to control I. This lowering of average ammonia levels inthe system was due both to a small reduction in Jn\?m in control II, at least relativeto the experimental period, and to slightly lower starting ammonia levels (cf.Fig. 1A). This probably resulted from the longer flush performed prior to controlII, which was performed to duplicate the experimental protocols where thoroughflushes were employed to ensure washout of the experimental water.
Fig. 1. Changes over time in selected parameters related to branchial ammonia excretion inrainbow trout subjected to a control protocol identical to that employed in the various experimentalseries. During the 3-h experimental period, the flux box was simply flushed with fresh acclimationwater at control pH. Means ± 1 S.E.M. (A) Total ammonia levels (TAmn,) in plasma ( • ) and water(O), A' = 5. (B) The calculated partial pressure gradient for NH3 (APNHj", inside minus outside)across the gills, ;Y = 5. (C) The calculated concentration gradient for NH4
+ (ANH4+; inside
minus outside) across the gills, A' = 5. (D) The net rate of ammonia excretion at the gills (J*,.""")during each hour, A''= 8. (E) The mean unidirectional (Jin", Jom) a n d n e t ( J M I cross-hatched)flux rates of sodium across the gills during each 3-h period, A' = 8.
Branchial ammonia excretion in trout 335
400
o 200
L +100
- 5 0 0
- 5 0 0
-1000
Control I Experimental ] Control II
T
Plasma
ANH4
Jin"
4 6Time (h)
Fig. 1
Tab
le
1. M
ean
va
lues
o
f p
ara
met
ers
rela
ted
to b
ranch
ial
am
monia
ex
cret
ion
duri
ng
each
3-h
per
iod
in
rain
bow
tr
ou
tsu
bje
cted
to
the
contr
ol p
roto
col
Con
trol I
Exp
erim
enta
lC
ontro
l II
Wat
er p
HPl
asm
a pH
aPl
asm
a Pa
c<j 2
(Tor
r)Pl
asm
a H
CO
3" (
meq
uivP
1 )Pl
asm
a P
NH
j (/x
Torr
)W
ater
PN
Hj
(/xTo
rr)
AP N
Hj
(^T
orr)
Plas
ma
NH
4+ (/
lequ
ivP1 )
Wat
er N
H,+ (
pieq
uiv
T')
AN
H4+ (
Ate
quiv
P1 )
X±
IS.E
.M. *
P<
00
5 re
lativ
e to
con
trol
I. t
^<
00
5 re
lativ
e to
exp
erim
enta
l.
8-10
±7-
79 ±
2-94
+6-4
1 ±
42
±99
±
-57
+14
5 +
113
±+3
2 ±
-387
±
+579
±
00
40
02
0-27
0-42
11 17 12 38 17 34 57 121
-625
±
124
-46
±33
8-11
+7-
80 ±
2-75
±6-
2633 77
-44
108 86
+ 22
-394
+573
-513 + 60
+ ± + + + + + ± + + +
00
40
02
0-20
0-26
5 10 8 18 12 17 61 137
145 54
809
± 0
04
7-81
±
0-0
22-
54 d
5-76
d27
d
65
d-3
8 d
89
d78
d
+ 11
d
-35
8 d
+ 55
0 d
-54
9 d
+ 1
dt 0
-24
b 0
-44
b 5
*b
7*
b 1
0b
20
*b
8*
b 2
5b
51
tb
88
bll7
b 5
7
Tab
le
2.
Mea
n
valu
es
of
pa
ram
eter
s re
late
d
to bra
nch
ial
am
monia
ex
cret
ion
duri
ng
each
3-h
per
iod
in
rain
bo
w
tro
ut
sub
ject
ed
to s
ever
e aci
d e
xposu
re d
uri
ng t
he
exper
imen
tal
per
iod
Con
trol I
Seve
re a
cid
Con
trol
II
> > z D nW
ater
pH
Plas
ma
pHa
Plas
ma
PaC
Oz
(Tor
r)Pl
asm
a H
CO
3~ (
meq
uivl
"1 )Pl
asm
a P
NH
] (u
Tor
r)W
ater
PN
Hj
(^T
orr)
AP N
H,
(^T
orr)
Plas
ma
NH
/ (f
iequ
ivT
1 )W
ater
NH
4+ (j
uequ
ivT
1 )A
NH
4+ (f
Aeq
uivT
1 )
7-96
±7-
78 ±
2-64
±5-
70 ±
73
±91
±
-18
±22
0 ±
123
++9
7 ±
-379
±
+ 43
3 ±
-363
+
+70
+
00
60
02
01
80-
2621 17 15 51 22 41 53 68 73 53
4-06
±7-
72 ±
2-99
±5-
34±
70
±0
±+7
0 ±
281
±84
±
+ 19
7 +
-273
±
-55
±-3
96
±-4
51
+
00
30
03
0-21
0-24
14 0» 14 62 19*
44*
38»
13*
93 90*
7-85
+
7-73
±
31
0±
5-81
±52
±
48
±
+4
±19
4 ±
95
±+
99
±-3
54
±+
26
5 ±
-31
0 ±
-45
±
0-0
5t
00
20-
200-
53H
t 8-t
40
|22 32
f53
t44
*f10
7 82t
X±
IS.E
.M. *
P<
00
5 re
lativ
e to
con
trol
I. t
^"<
005
rela
tive
to s
ever
e ac
id.
Branchial ammonia excretion in trout 337
Resting levels of plasma TA m m, PNH . NH4+, acid-base status, ANH4
+ acrossthe gills and J ^ m were very similar to those found by Cameron & Heisler (1983),who employed comparable methodology, though APNHj across the gills was verydifferent. This difference was probably due to the very different control water pHlevels employed in the two studies (see Discussion). The present fish started eachcontrol period with APNH close to zero, which became significantly negative by 3 has water PNH:I increased (Figs 1B-6B). Thus our fish excreted ammonia in theapparent absence of or even against a PNH3 gradient, but in the direction of theNH4
+ gradient (Tables 1-6).J,^a and jjjft were rather variable both within and between different experimental
groups (400-700/i,equivkg"1 h~' in control I; Tables 1-6), but a J eat close to zero
balance was usual (Figs 1E-6E). These unidirectional fluxes were approximatelytwice as large as those measured by Wood et al. (1984) in trout in water of the sameionic composition and temperature. J^eT
m m the present study was also twice ashigh, perhaps because Wood et al. (1984) allowed external TAmm to rise to a level3-5 times greater. Elevated external TAmm may well interfere with bothNa + /NH 4
+ exchange and diffusive ammonia efflux.
Severe acid exposure
The aim here was to apply a condition [water pH = 4-06 ± 003 (10)] whichwould enhance A P N H 3 while simultaneously blocking J, a (cf. McDonald, 19836).If branchial Na + /NH 4
+ exchange is unimportant and NH3 diffusion predominates,the predicted result would be an increase in JneTm- Since at this pH essentially allammonia is protonated, the water becomes an immense sink for NH3.
J eTm> rather than increasing, declined significantly by 28% relative to control Iand then recovered in control II (Fig. 2B, Table 2). This occurred despite a reversalof APNH3 to a positive value as water PNH3 became zero (Fig. 2B, Table 2).ANH4
+ also increased (Fig. 2C) because the reduction in Jne?m both raised plasma
TAmm (and thus NH4+) and lowered water TAmm (Fig. 2A, Table2). This rise in
internal ammonia shows that the reduction in Jne?m could not be attributed to areduction in endogenous ammonia production. As anticipated, J^a was abolished sothat J e
a became highly negative and equal to j£Jft (Fig. 2E, Table2), an effectwhich was only partially reversed in control II. A slight but significant bloodacidosis occurred during severe acid exposure attributable to both respiratory(PaCoz elevation) and metabolic (HCC>3~ reduction) components, neither of whichwas significant by itself (Table 2). The magnitude of this acidosis after 3 h atpH = 4-06 was approximately equal to that which would be predicted from anearlier study under comparable conditions (McDonald & Wood, 1981).
These results indicate a significant contribution of Na + /NH 4+ exchange to
resting JneT"1- However 72% of control J^eTm continued in the complete absence of
Ji^a, suggesting that diffusive processes, probably enhanced by the elevatedgradients, were important in sustaining Jne?m under conditions where Na + /NH 4
+
exchange was blocked.
338 P. A. WRIGHT AND C M . WOOD
Moderate acid exposure
The goal of this treatment was again to elevate APNH3 by reducing water PNH3
close to zero, but without inducing a blockage of J^a. A water pH of 6-64 ± 0-04(17) was calculated to represent the minimum acidity level to ensure the formerobjective.
Jn™m increased only slightly ( + 7%) and non-significantly relative to control Iduring moderate acid exposure (Fig. 3D, Table 3). While this JneT"1 was signifi-cantly higher ( + 23 %) than in control II, the same trend was also seen in the controlseries (Table 1). As planned, APNH was reversed to a positive value (Fig.3B,Table 3), though not as high as in the severe acid series (Table 2) because internalammonia levels fell significantly (Table 3), an effect which was not corrected duringcontrol II. It is not clear whether this was the result of the marginally elevatedJneT"1) ° r reflected a true decrease in the endogenous ammonia production, thoughthe severe acid results would argue against this. In any event, one importantconsequence was a significant reduction in ANH4"1" during the experimental period(Fig. 3C, Table3). The goal of preserving sodium exchanges at control levels waslargely achieved, with only a non-significant 23% decrease in Jjn
a and continuedpositive J^e
a occurring at pH = 664 (Fig. 3E, Table 3). There were no furtherchanges in these parameters in control II. As with severe acid exposure, a slightblood acidosis of compound respiratory and metabolic origin again occurred (Table3).
Under these conditions, a significant elevation of APNH3 n a d only a very small
stimulating effect on J T™) a n d ANH4"1" again changed in the opposite directionfrom Jn\Tm- Jne?m was therefore only slightly altered by a treatment whereas Jj^a
was largely unaffected.
Severe alkaline exposure
Exposure to alkaline water was selected as a treatment to separate the relativeimportance of NH3 and NH4
+ diffusion across the gills, for elevated water pHshould increase external PNH ar>d decrease external NH4
+. An alkaline pH[9-54 ± 0-04 (9)] close to the pK' was initially tested to ensure pronounced changesin APNH ar>d ANH4"1". As alkaline effects on sodium transport in trout have notbeen studied previously, it was not known what changes, if any, might occur in Jin.
Jnentim declined dramatically by 80 % during severe alkaline exposure and then
recovered to a level significantly above control I (+22%) during control II(Fig. 4D, Table 4). Plasma TA m m increased 2-5-fold during the experimentalperiod and then dropped during recovery (Fig. 4A), showing that the reduction inJneT"" w a s not due to a blockage of endogenous ammonia production. At the sametime, ANH4
+ increased over 4-fold to a highly positive value, while APNH3 fellfrom its initial level close to zero to a very negative value (Fig. 4B,C, Table 4).These alterations in gradients, which were the intention of the experiment, were
Fig. 2. The influence of severe acid exposure (pH = 406) during the experimental period onselected parameters related to branchial ammonia excretion in rainbow trout. A' = 7 in (A) - (C), 10in (D) and 7 in (E). Other details as in legend of Fig. 1.
Branchial ammonia excretion in trout 339
400
o 200B
0+100
0 0
-100
7 +200
'5
1 +100
-500
+500
-500
-1000
Control I
- B
Severe acid Control II
TA m m
Plasma
Water
APN
ANH4
4 6
Time (h)
Fig. 2
340 P . A. W R I G H T AND C. M. W O O D
reversed during control II. High water pH also markedly affected ]^a, which fell by55 % during the experimental period, and returned to normal in control II (Fig. 4E,Table 4). J e
at mirrored these changes. A pronounced blood alkalosis (+0-3 pH
units) of wholly respiratory origin occurred during severe alkaline exposure andpartially persisted during recovery (Table 4). PaCo2 was approximately halvedwhile [HCO3~] remained unchanged because water at pH = 9-54 essentially acts asa vacuum for CO2.
Of all the experimental treatments, this caused the largest drop in J^eTm- 1° this
situation, where ANH4+ increased over 4-fold and where APNH3 w a s rendered
highly negative and therefore the net outward diffusion of NH3 clearly prevented,the decreases ( 270/u.equivkg~' h"1) in JneT"1 and J^8 were equal in size(Table 4). These data were therefore consistent with the results of the two acidseries.
Moderate alkaline exposure
A much more moderate alkaline pH was tested in the hope that a very negativeAPNH, could still be attained without affecting J^3. A pH of 8-69 ± 0-04 (17) wasselected as the minimum level of alkalinity to ensure the former objective.
As intended, moderate alkaline exposure changed APNH3 to a very negative value(Fig. 5B, Table 5) almost equal to and less variable than' that during severealkaline exposure (Fig. 4B, Table 4). However in this case, the drop in JneTm> whilesignificant, was only 23% (Fig* 5D, Table 5). Even at this much more moderatealkaline pH, J- a declined significantly by 28% (Fig. 5E, Table 5). The absolutesize of this decrease was adequate to explain the observed fall in J^J1"1. As withsevere alkaline exposure, plasma TAmm and ANH4+ both rose during the experi-mental period as ammonia accumulated internally (Fig. 5A,C), and a respiratoryalkalosis again developed (Table 5). Most changes were reversed during control II.
Thus in water of pH = 8*69, 77 % of control I J T™ persisted in the face of a verynegative A P ^ H • This small reduction in J^T"1 w a s accompanied by a comparablefall in J ^ .
Amiloride exposure
The results of the previous four experiments suggested that a large decrease inJneTm would be the predicted result of a treatment which blocked Jina withoutaltering APNHj or ANH4
+. The drug amiloride, a specific sodium transportinhibitor (Benos, 1982), was selected for this purpose.
As intended, amiloride at 10~4moll~1 in the external water (pH = 8-12 ± 0-05)had no effect on APN H j or ANH4
+ (Fig. 6B,C, Table. 6), but essentially oblit-erated Jina, with only 6 % of the control I influx persisting (Fig. 6E, Table 6). J e
at
therefore became very negative. These effects were largely reversed during controlII. However, contrary to prediction, the fall in Jne?
m, while significant, was only
Fig. 3. The influence of moderate acid exposure (pH = 6-64) during the experimental period onselected parameters related to branchial ammonia excretion in rainbow trout. N— 15 in (A)-(C) ,17 in (D) and 9 in (E). Other details as in legend of Fig. 1.
Branchial ammonia excretion in trout 341
400
i 200E
o+ 100
i »-100
'- +1003cr4
0
-
'5? 500
+ 500
> 03<T4
-500
-1000
A
-
E
-
C
-
Control I
D
-
I
-
-
T
••• T
T
y////////////
1
Moderate acid
T
1
TT
1 1
Control II
TAmm
Plasma j
• ^ V " - Water
APNH,
i
ANH4 +
i f
T
T
•L
Jou,
4 6
Time (h)
Fig. 3
8 10
Tab
le 3
. M
ean
valu
es
of p
aram
eter
s re
late
d to
bra
nchi
al
amm
onia
ex
cret
ion
duri
ng e
ach
3-h
peri
od
in r
ainb
ow
trou
tsu
bjec
ted
to m
oder
ate
acid
exp
osur
e du
ring
the
exp
erim
enta
l pe
riod
Con
trol
IM
oder
ate
acid
Con
trol
II
Wat
er p
HPl
asm
a pH
a
Plas
ma
Pac
o 2 (
Tor
r)Pl
asm
a H
CO
3~
(meq
uiv
P1)
Pla
sma
PN
H3
(n-T
orr)
Wat
er P
NH
j (/
i-T
orr)
AP
NH
] (^
To
rr)
Pla
sma
NH
4+
(/xe
quiv
l"')
Wat
er N
H4
+
(/u-
equi
vp')
AN
H4
+
(Meq
uivl
"1 )^
'1
7-85
±7-
80 ±
2'6
5±
61
2±
56
±62
+
-6
±19
8 ±
121
±+
77
±-3
75
±+
370
±
-23
8 ±
00
80
01
01
20-
326 10 14 33 18 32 38 66 86
6-64
±7-
73 ±
2-97
+5.
42 ±
32
±3
±+
29
±12
9 +
102
±+
27
±-4
03
±+
285
±-1
88
±
00
40
02
0-24
0-32
5« 0* 5* 23*
14#
31*
30 143 84
7-6
H7-
75 d
2-64
d5-
28 d
35
d32
d
+3
d13
7 db
00
6f
b 0-
02t
0-19
b 0-
38b
3»b
6«
tb
9|
b 23
»78
±
12
*t+
59
±
24-3
27
±
28
f+
297
±
43-2
61
±
69+
132
±
55
+97
±
88+
36
±
40
X+
1 S
.P
<0
05
rela
tive
to c
ontro
l I.
f^
<0
05
rela
tive
to m
oder
ate
acid
.
Tab
le 4
. M
ean
valu
es
of p
aram
eter
s re
late
d to
bra
nchi
al
amm
onia
ex
cret
ion
duri
ng
each
3-h
per
iod
in r
ainb
ow
trou
tsu
bjec
ted
to s
ever
e al
kali
ne e
xpos
ure
duri
ng t
he e
xper
imen
tal
peri
od
Con
trol
IS
ever
e al
kali
neC
ontr
ol I
I
7-99
±7-
89 +
21
2±
6-76
±83
+
95
±-1
2
±21
5
±97
±
+ 1
18
±-4
20
±
+46
5
±-3
81
±
+84
+
00
8t
00
2*t
0-47
f0-
6913
»t22
f26 2
1t
20
t34
|45
»t46
f82 66
> •z D oW
ater
pH
Pla
sma
pHa
Pla
sma
Pa C
Oz
(Tor
r)P
lasm
a H
CO
3~
(meq
uivl
"1)
Pla
sma
PN
Hj
(tiT
orr)
Wat
er P
NH
j (/
iTor
r)A
PN
Hj
(/iT
orr)
_
Pla
sma
NH
4+
(/xe
quiv
l ')
Wat
er N
H4
+
(^eq
uiv
T1)
AN
H4+
(/
xequ
ivP
1)
]^T
m(p
imol
k
g"'
h"'
)
7-97
±7-
78 +
2-95
±6-
60 ±
59
+81
+
-22
+18
6 ±
103
±83
±
-34
5 ±
+49
6 ±
-53
0 ±
-34
±
00
60'
020-
500-
8310 20 17 22 18 22 91 56 66 33
9-54
±8
08
±1-
61 ±
6-91
±28
5 ±
424
+-1
39
+40
8 ±
16
±39
2 +
-68
++
226
±-7
45
±-5
19
+
00
40-
070
12
0-86
65»
89*
83 51* 3» 49*
13*
60#
193
194*
X +
1 S
.E.M
. •P
<0
05
rela
tive
to c
ontr
ol 1
.1 f
/><
00
5 re
lati
ve t
o se
vere
alk
alin
e.
Branchial ammonia excretion in trout 343
23% relative to control I (Fig. 6D, Table 6). This inhibition disappeared duringcontrol II. Blood acid-base status was not significantly affected by amiloride (Table6). The effects of amiloride on J,^a and JneT"1 were therefore very similar to those ofsevere acid exposure (cf. Fig. 2, Table 2). This occurred despite the fact thatelevated APNHj (and ANH4
+) gradients, interpreted as sustaining Jne?m when
Na+/NH4+ exchange was blocked at pH = 4-06, were not seen with amiloride.
DISCUSSION
Ammonia gradients
There are numerous sources of uncertainty in the estimation of ammoniagradients across the gills. As Cameron & Heisler (1983) point out, relatively smallerrors in physical constants and pH may have large effects on calculated APNH3-Employing Cameron & Heisler's physical constants, we have obtained internalPN H
a n d NH4+ levels similar to their values, which at least suggests consistency
between the two studies, and that our procedure of predicting mean plasma TAmmlevels from arterial TA m m was not a major source of error. The pH values are a moreserious concern. While blood plasma pH is a routine and precise measurement(±0-01 units), the value obtained represents an equilibrium condition in theelectrode which may or may not be the same as that in gill blood plasma dependingon the extent of carbonic anhydrase catalysis. One may even question whetherblood plasma pH is the correct internal value to use. The relevant gradient could infact be from the cytoplasm of branchial epithelial cells to the water, rather thanfrom blood to water, especially if ammonia is transported rather than diffuses acrossthe baso-lateral boundary (cf. Claiborne et al. 1982). The intracellular pH of thegill epithelium is unknown, but pHi in other trout tissues is 0#4—0-5 pH units belowpHa (Hobe, Wood & Wheatly, 19846; C. L. Milligan & C. M. Wood, unpublisheddata). Furthermore, while branchial ammonia production appears to be of littleimportance (see Introduction), it must be noted that the enzymes involved inammoniagenesis have been located in the gills (Goldstein & Forster, 1961; Maka-rawicz & Zydowo, 1962). It remains conceivable that under appropriate conditions,ammonia production within the epithelial cells could be turned on.
The measurement of water pH (±0-02 units) is somewhat less precise than that ofplasma because of the lower ionic strength. However, a much more serious concernis that like other workers (e.g. Maetz, 1972, 1973; Cameron & Heisler, 1983), wehave employed equilibrium pH measurements in the external bath where waterPCo2 is kept relatively constant. Lloyd & Herbert (1960) theorized that Pco2
w a s
higher and pH lower in lamellar water, assuming that the CO2 hydration reactionwas rapid. However in the absence of external carbonic anhydrase, theCO2/HCO3~ system is undoubtedly not at equilibrium in lamellar water. Holeton& Randall (19676) reported that mixed expired water pH, at equilibrium, was~0-2pH units below inspired in rainbow trout examined in very soft water. Ourwater is more alkaline and better buffered, so a smaller difference is anticipated.However actual lamellar water pH has yet to be measured and is difficult to predictaccurately, for it will depend on the extent of this disequilibrium, the size of theunstirred layer and the magnitude of the CO2 flux relative to the ventilatory flow, as
344 P. A. WRIGHT AND C. M. WOOD
800
= 400
Control I
(2 - l o o
-200
+400
-500
+500
t 0
-500
-1000
Severe alkaline
/
I——-1 T i
4 6Time (h)
Fig. 4
8 10
Branchial ammonia excretion in trout 345
well as the fluxes of other acidifying (H+) or alkalinizing species (e.g. NH3,HC03~, 0H~) . In view of these difficulties, we have much more confidence inrelative changes in ammonia gradients caused by experimental treatments than intheir absolute values.
Experimental responsesThe immediate and complete blockage of J, a during severe acid exposure has
been observed and analysed repeatedly (see Wood & McDonald, 1982; McDonald,1983a for reviews). However, the simultaneous reduction in J^eT
n\ in the face ofgreatly elevated A P ^ H during the initial exposure period has received littleattention, though it has been noted in three other recent studies (Ultsch, Ott &Heisler, 1981; McDonald, Walker & Wilkes, 1983; Hobe et al. 1984a,6). Instead,most authors have concentrated on the fact that JnJt"" increases well above thecontrol level during longer term acid exposure despite minimal recovery of Jjna (e.g.Ultsch et al. 1981; McDonald & Wood, 1981; Booth, Jansz & Holeton, 1982;McDonald, 1983a,b; McDonald et al. 1983). Plasma TAmm also rises during longerterm exposure (McDonald, 19836; Hobe et al. 1984a) so elevated ammoniagenesisis probably involved. Moderate acid exposure, which also elevated APNH3, did notsignificantly alter J^a and only minimally elevated J T™- These observations are allconsistent with the interpretation that a large Na + /NH 4
+ exchange under thecontrol conditions of the present laboratory experiments is blocked by severe acidexposure; the continuation of ammonia efflux is then dependent upon increasedNH3 diffusion due to the elevated APNH3-
Trout branchial function appears very sensitive to alkaline conditions, for raisingthe water pH to only 8-69 depressed both J^J11" and J^3 by —25%, and 9-54reduced these parameters by 80% and 55 % respectively, the latter representing anequimolar reduction. We are aware of no previous reports of alkaline effects onammonia excretion, and only one on sodium exchange. In contrast to the presentdata, Maetz & De Renzis (1978) found a 10-fold stimulation of J^a in Tilapiamossambica raised from water pH — 7-8 to 9-7, though their tests were conductedin 10 % sea water. The inhibitory effect of low pH on J^a has been variouslyexplained by H+ competition for the carrier, H + titration of negative charges onchannels leading to the carrier, or a direct effect on the carrier itself (cf. McDonald,1983a). Only the latter would seem a reasonable mechanism for high pH inhibitionof J^a, though the effect was reversible in the present study. The persistence ofover 75 % of control Jne?m in the face of a very negative APNHj (at pH = 8-69) andthe large equimolar reduction in both J- a and Jne?m (at pH = 9-54) were againconsistent with a dominant role for Na+/NH4+ exchange under the present controlconditions.
The observed 94% reduction in J ^ caused by amiloride agrees with previousstudies showing a 70-92% reduction in various trout preparations (Kirschner,Greenwald & Kerstetter, 1973; Kerstetter & Keeler, 1976; Payan, 1978; Perry,
Fig. 4. The influence of severe alkaline exposure (pH = 954) during the experimental period onselected parameters related to branchial ammonia excretion in rainbow trout. N = 5 in (A) —(C), 9in (D) and 7 in (E). Other details as in legend of Fig. 1.
346 P. A. WRIGHT AND C. M. WOOD
400
i 200e
o
o
-100
-200
+200
3 +100
-500
+500
SP 0
-500
-1000
Control I
_ C
Moderate alkaline
A k
Control II
ANH4
T
4 6Time (h)
Fig. 5
Branchial ammonia excretion in trout 347
Haswell, Randall & Farell, 1981; Perry & Randall, 1981). Amiloride also reducedJneT"1 by 23%, a figure consistent with 30% reductions reported in perfusedand/or irrigated trout gill preparations (Kirschner et al. 1973; Payan, 1978). Amuch larger inhibition of J^e?
m would be expected if Na + /NH 4+ were the
dominant process, for no elevation of diffusion gradients occurred to sustainexcretion as during severe acid exposure. Recently Perry & Randall (1981) haveshown that 10~4 mol 1 ~' amiloride also inhibits J,? by 55 % in intact trout, an effectthey attributed to a predicted acidosis within the branchial epithelial cells. As thiscould alter the effective driving gradients for NH3 and NH4"1" movements at boththe baso-lateral and apical surfaces (see above), the interpretation of thisexperiment is unclear.
The mechanism(s) of ammonia excretion
The results of all five experimental series were consistent with the conclusion thatNa+/NH4+ exchange plays a significant role in ]n™
m in trout under the presentcontrol conditions, and all but the amiloride experiment suggest it may be thedominant mechanism. NH3 diffusion also occurs, and this becomes more importantin treatments where APNHj is elevated and/or J^3 is inhibited. While NH4
+
diffusion cannot be ruled out, it is probably of minor quantitative importance; forJne?m always changed in the opposite direction from ANH4
+. It must be notedthat we did not take the transepithelial potential (TEP) into account. However, thiswas probably unimportant to our conclusion, for McWilliams & Potts (1978)showed TEP in the brown trout, Salmo trutta, to be stable down to pH = 6-0, andthen to become progressively more positive at lower pH values. Our calculatedelevation in ANH4
+ at pH = 4-06 was therefore probably an underestimate. Theeffect of alkaline pH on TEP has not been studied in trout, but is reported toelevate the TEP in goldfish Carassius auratus (Eddy, 1975). Our calculatedelevation in ANH4
+ at pH = 8-69 and 9-54 would again be an underestimate if thiswere also true in trout. In any event, when one compares the relative gradientsavailable to drive the passive effluxes of Na+ and NH4
+ across the gills, only a verysmall NH4
+ diffusion is theoretically likely. This argument has been developed indetail by Kormanik & Cameron (1981a).
We have attempted to relate the influx of Na+ (Jin3) to NH4+ excretion (J^!"4)
in all series by assuming that at pH = 4-06, net ammonia excretion (Jn\Tm) w a s
entirely by NH3 diffusion (i.e. J^i"4 = 0) s m c e Jil!a w a s obliterated and NH4+
diffusion appears insignificant. The diffusivity of the gills to NH3
(DN H j = Jne?m/APNH3) under these conditions was 4/umolkg~1 h~' /LtTorr"1,which is about 65% of the value estimated by Cameron & Heisler (1983) for thesame species. This D N H 5 value was then applied to the other series and the NH3
contribution (J^"3) to J T™ determined from the prevailing APNHj. J^."4 wasthen calculated as the difference between Jne?m ami j j ^ 3 , signs considered. Theanalysis therefore relies on the absolute APNHj values (see above), assumes that
Fig. 5. The influence of moderate alkaline exposure (pH = 8-69) during the experimental periodon selected parameters related to branchial ammonia excretion in rainbow trout. N= 13 in(A)-(D) and 7 in (E). Other details are in legend of Fig. 1.
348 P. A. WRIGHT AND C. M. WOOD
400Control I
-k
0-100
H 0
+ 100
+ 200
I +10°4
+500
-1000
Amiloride Control II
TA
Plasma
A P N H ,
ANH4
4 6
Time (h)
Fig. 6
Branchial ammonia excretion in trout 349
NH3 excretion is diffusion limited (i.e. any ventilatory or cardiovascular effects ofthe various treatments were unimportant), and assumes that D N H j is the sameunder all treatments and in both directions of NH3 flux: all of which are possiblesources of error.
Fig. 7 illustrates the relationship between J ."* and Jina based on this analysis.The two parameters appear to be more or less linearly related and increase with pHfrom 4 1 to 8-7. If the analysis is valid, Na + /NH 4
+ exchange clearly dominates overdiffusive processes in the control pH range. At pH = 9-5, where jJJJ."4 appearsgreatly to exceed J,^a, any overestimation in the very negative (and highly variable)A P N H 3 measurement will greatly exaggerate the calculated J^c"\ Under theseconditions, there may well have been a significant branchial H + efflux or HCO3~uptake, lowering the pH of lamellar water and decreasing the A P N H 3 below themeasured value.
These conclusions about the dominance of Na + /NH 4+ exchange relative to NH3
diffusion under our control conditions agree completely with the work of Maetz(1972, 1973) on goldfish where control pH (~7-9) was similar to the present. Maetzlater questioned the absolute values of the ammonia gradients calculated in hisexperiments, but not the basic conclusions (Maetz et al. 1976). In contrast,Cameron & Heisler's (1983) investigation of rainbow trout, while not ruling outNa + /NH 4
+ exchange, concluded that NH3 diffusion was sufficient to explain allresting JneT
m, assuming that NH3 and CO2 have similar permeabilities in gill tissue.This difference is probably related to the lower control pH = 7-0 used in theirstudy. Thus comparable internal and external TA m m in the two studies gave rise to areasonable APNHj ( + 55 juTorr) to drive resting Jne?m by passive NH3 diffusion atpH = 7 0 (Cameron & Heisler, 1983), but negligible or negative APNH atpH = 8-0 (present study) due to the approximately 10-fold higher water PNH • Inany event, absolute values of APNHj should be viewed with caution and relativechanges may well be more informative (see above). Indeed, Cameron & Heisler(1983) postulated a high rate of Na + /NH 4
+ exchange to explain the response to atreatment (pH = 8-0, elevated external TAmm) which changed APNHj to a verynegative value. Hence there appears to be no conflict in observation between ourstudy and that of Cameron & Heisler (1983), but subtle differences in interpret-ation.
We favour a flexible model in which Jn™m is achieved by a variable combinationof Na + /NH 4
+ exchange and NH3 diffusion. The exact proportions occurringthrough each pathway will depend upon the relative APNHj, the extent of J-^a, andquite possibly the acid-base status of the animal. Ammonia excretion viaNa + /NH 4
+ exchange represents acidic equivalent excretion, while NH3 efflux isneutral in net acid-base terms, though inequalities between NH3 production andexcretion rates will affect internal acid-base status. Branchial N a + / H + exchangemay also occur (e.g. Kerstetter e£ al. 1970; Kirschner et al. 1973), and the relativeAH+ and ANH4
+ gradients will determine which mechanism is an energetically
Fig. 6. The influence of exposure to 10 4moll ' amiloride in water at control pH during theexperimental period on selected parameters related to branchial ammonia excretion in rainbowtrout. N = 8 in (A)- (C) , 9 in (D) and 5 in (E). Other details as in legend of Fig. 1.
Tab
le
5. M
ean
valu
es
of p
aram
eter
s re
late
d to
bra
nchi
al a
mm
onia
ex
cret
ion
duri
ng
each
3-h
per
iod
in r
ainb
ow
trou
tsu
bjec
ted
to m
oder
ate
alka
line
exp
osur
e du
ring
the
exp
erim
enta
l pe
riod
Con
trol
IM
oder
ate
alka
line
Con
trol
II
Wat
er p
HP
lasm
a pH
a
Pla
sma
Pa C
O2
(Tor
r)P
lasm
a H
CO
3~
(meq
uiv
P1)
Pla
sma
PN
Hj
(/^T
orr)
Wat
er P
NH
, 0-
iTor
r)A
PN
Hj
(//.T
orr)
Pla
sma
NH
4+
(meq
uivP
1)
Wat
er N
H4
+
(/ue
quiv
P1 )
AN
H4
+
(/xe
quiv
p1)
Jolft
(^e
qu
ivk
g"1 h
~')
J^/i
eq
uiv
kg
"1!!
"1)
7-98
±
00
67-
79 ±
0
02
31
3d
6-91
d39
d
58
d-1
9 d
144
d90
d
+ 54
d
-34
2 d
+ 68
8 d
-65
8 dt
0-2
2b
0-4
4b
6b
9b
12b
21
b 8
b 17
b 3
4b
147
b 12
1
8-69
±7-
87 ±
2 61
±7
09
±60
±
160
+-1
00
±18
7 ±
53
±+
134
+
-26
2 ±
00
40
02
01
40-
299* 16
*18
*26
* 5* 24*
28*
+49
2 ±
125
*-6
98
±-2
06
±17
110
5
8-11
±7-
83 ±
2-67
±6-
44 ±
35
±52
±
-17
+12
0 ±
60
±+
60
±-3
80
±+
600
±
-45
8 ±
+ 1
42
±
0 0
6t
0-02
01
60
-47
|6j
- 7f "t 22t
6*
20f
37f
105 91 78
t
X±
1
S.E.
M.
*P
<0
05
rela
tive
to c
ontr
ol I
. tP
<0
05
rela
tive
to m
oder
ate
alka
line
.
Tab
le
6. M
ean
valu
es
of p
aram
eter
s re
late
d to
bra
nchi
al
amm
onia
ex
cret
ion
duri
ng
each
3-h
per
iod
in r
ainb
ow
trou
tsu
bjec
ted
to 1
0~4 m
oU~
' am
ilor
ide
in w
ater
at
cont
rol p
H d
urin
g th
e ex
peri
men
tal
peri
od
Con
trol
IA
mil
orid
eC
ontr
ol I
I
2 o X H > Z D nW
ater
pH
Plas
ma
pH
a
Plas
ma
Pa C
O2
(Tor
r)Pl
asm
a H
CO
3"
(meq
uivl
"1)
Plas
ma
PN
Hj
(/iT
orr)
Wat
er P
NH
] ( M
Tor
r)A
PN
H]
(fiT
orr)
Pla
sma
NH
4+
(/ue
quiv
P1 )
Wat
er N
H4
+
(/A
equi
vl"1 )
AN
H4
+
(/ue
quiv
P1)
^'
1
jS
^c
qu
iv
kg
h)
J™*
(^-e
quiv
kg
'h
')^
'1
7-97
d7-
84 d
2-88
d'
7-05
d51
d
61
d-1
0 d
128
d75
d
+53
d
-30
6 db
01
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24
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± 1
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47
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91
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d
39
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127
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d
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d
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d
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±
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+ 3
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00
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Branchial ammonia excretion in trout 351
-800 r-
-600
• | -400crA
-200
pH = i
• pH = 9-5ControlpH = 8 1
pH = 6-6
Amiloride_ pH = 8 1
pH = 4 1
+200 +400 +600
•I*" (/icquivkg"1 h"1)+800
Fig. 7. The relationship between estimated ] ^ " 4 and measured J^" in rainbow trout in eachexperimental series. See text for details of the J™* calculation.
more favourable means of acidic equivalent excretion in any given situation.N a + / H + exchange plus NH3 diffusion is equivalent to Na+/NH4+ exchange interms of both mass and acid-base balance. Na+/Na+ exchange diffusion alsoundoubtedly occurs in the gills (cf. Maetz et al. 1976; Wood et al. 1984). Thefactors controlling the relative rates of these various processes is a challenging fieldfor future investigation.
We thank Dr W. D. Dorian of Merck Frosst Canada Inc. for the gift of amiloride,Drs J. N. Cameron and N. Heisler for generously providing us their data prior topublication, Dr Cameron for his helpful comments on an earlier version of thismanuscript, and Drs D. J. Randall, R. G. Boutilier and D. G. McDonald for usefuldiscussions. Supported by an NSERC Operating Grant and an NSERC StrategicGrant in Environmental Toxicology to CMW.
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