Limited extracellular but complete intracellular acid-base regulation during short-term environmental hypercapnia in the armoured catfish, Liposarcus pardalis
1 Department of Zoology, University of British Columbia, 6270 University
Boulevard, Vancouver, B.C., Canada V6T 1Z4
2 Department of Zoophysiology, University of Aarhus, 8000 Aarhus C,
Denmark
3 Department of Biology, Queens University, Kingston, Ontario, Canada K7L
3N6
4 Department of Biology, University of San Diego, San Diego, CA 92110,
USA
5 Department of Zoology, University of Guelph, Guelph, ON, Canada N1G
2W1
6 National Institute for Research in the Amazon (INPA), Laboratory of
Ecophysiology and Molecular Evolution, Manaus, AM, Brazil, CEP
69083-000
* Author for correspondence (e-mail: brauner{at}zoology.ubc.ca)
Accepted 17 June 2004
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Summary |
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Key words: acid-base regulation, hypercapnia, pHe, pHi, intracellular pH regulation, catfish, Liposarcus pardalis, Amazon, water hardness, air-breathing
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Introduction |
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Complete or significant degrees of pH compensation during environmental
hypercapnia have been demonstrated in numerous species of freshwater and
marine fish (e.g. Toews et al.,
1983; Claiborne and Heisler,
1984
; Cameron,
1985
; Goss et al.,
1995
; Larsen and Jensen,
1997
; Larsen et al.,
1997
; reviewed by Heisler,
1984
; Claiborne et al.,
2002
). Water composition clearly affects the rate of acid-base
compensation where the rate and degree of compensation is more pronounced in
seawater than in freshwater, and more pronounced in hard compared to soft
freshwater (Heisler, 1984
;
Larsen and Jensen, 1997
).
Many natural systems are characterized by soft water. One example is the
Amazon, where the ionic composition of many rivers and tributaries approaches
that of distilled water (Val and
Almeida-Val, 1995). Given that extensive vegetation covers many of
the stagnant lakes and ponds in the Amazon during the dry season, it is likely
that resident fishes experience periods of hypercapnia
(Heisler et al., 1982
). The
ability of fishes endemic to these ion-poor waters to tolerate and compensate
for environmental hypercapnia has not previously been investigated. The
armoured catfish Liposarcus pardalis is common in the Amazon and can
withstand relatively severe environmental hypercapnia and large disturbances
in plasma pH (Randall et al.,
1996
). Here, we investigate the temporal regulation of extra- and
intracellular acid-base status during environmental hypercapnia of L.
pardalis in natural softwater. The first experimental series describes
extracellular acid-base status during hypercapnia
(PCO2 of 7, 14 and 42 mmHg) in softwater, while
the second series investigates intracellular acid-base parameters of heart,
liver and muscle during hypercapnia (PCO2 of 14
and 32 mmHg). The third experimental series investigates whole animal fluxes
of Na+, Cl- and ammonia during hypercapnia
(PCO2 of 14 and 32 mmHg) to elucidate the
mechanisms for acid-base regulation.
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Materials and methods |
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Experimental protocols
Series I: Effect of hypercapnia on extracellular acid-base status
Fish were anaesthetized in 0.1 g l-1 Tricaine Metanesulfonate
(MS-222) buffered with NaHCO3-. Once anaesthetized, fish
were transferred to a surgery table, and an indwelling catheter (PE-50) was
placed in the dorsal aorta according to Soivio et al.
(1975). Fish were left to
recover for 24-48 h in individual chambers, and the cannulae were flushed
several times with heparinized saline. Following recovery, the fish were
placed in 2 l chambers without access to air and exposed to a
PCO2 of 7, 14 or 42 mmHg. The low levels were
achieved using a flow meter that mixed CO2 with air into a 500 l
recirculating system, while the high level was achieved using flow-through
well-water directly from its source without prior aeration. Water
CO2 levels stabilized within 1 h. Blood samples were withdrawn from
the cannulae at 0, 2, 6 and 24 h at each level of hypercapnia. Whole blood pH
(pHe), PO2, true plasma total
CO2 content, haemoglobin concentration ([Hb]), and haematocrit
(Hct) were measured immediately, and plasma was frozen for later analysis of
Na+, Cl- and protein concentrations (see Analytical
procedures below). Cannulated fish exposed to 42 mmHg did not survive for 24
h. Thus, non-cannulated fish were exposed to 42 mmHg for 96 h, and a blood
sample was obtained from the caudal vein within 10 s of removal from the
water.
Series II: Effect of hypercapnia on intracellular acid-base status
Non-cannulated fish were placed in individual 2 l chambers overnight before
being exposed to a PCO2 of 14 or 32 mmHg
without access to air. Tissues were sampled from normocapnic control fish and
at 6, 24 or 72 h into the hypercapnic period. For tissue sampling, water flow
to the chamber was stopped and a concentrated solution of buffered MS-222 was
slowly added to the water to achieve a final concentration of 0.1 g
l-1, according to Wang et al.
(1994). Within 2-3 min, fish
lost equilibrium and could be removed from the water without struggling. This
procedure has been shown to minimize any metabolic and acid-base changes
associated with handling and sampling. Blood (0.5 ml) was drawn from the
caudal vein into a heparinized syringe for measurement of pHe, red
cell intracellular pH (pHi), PCO2,
[Hb], Hct and plasma Na+, Cl-, Ca2+
concentrations, and osmolarity. The fish were then killed, and 0.5 g samples
from heart, liver and muscle were removed and frozen immediately in liquid
nitrogen for later analysis of pHi. Sampling was complete within
2-3 min after the fish had been removed from the water.
Series III: Effect of hypercapnia on whole animal ion fluxes
Non-cannulated fish were placed in individual chambers (500 ml) overnight
and exposed to the same regime as fishes of Series II. Fish were subjected to
a PCO2 of 14 or 32 mmHg. At 0, 6 and 24 h (and
at 72 h at PCO2 of 32 mmHg), water flow was
interrupted for 1 h for measurements of whole animal unidirectional
Na+ influx (),
efflux (
), net flux
(
), net Cl-
flux (
), and total ammonia
excretion.
Analytical procedures
Blood PO2 and
PCO2 values were measured using Radiometer
(Copenhagen, Denmark) PO2 (E5046) and
PCO2 (E5036) electrodes thermostatted at
28°C in a BMS Mk2 electrode assembly with the output displayed on a
Radiometer PHM 73. Total CO2 content of true plasma was measured
according to Cameron (1971).
Extra- and intracellular pH were measured using a Radiometer microcapillary
electrode (G299A) held within the BMS Mk2 system. Red cell pHi was
measured using the freeze-thaw method of Zeidler and Kim
(1977
), and pHi of
heart, liver and white muscle was measured according to Pörtner et al.
(1990
). Water pH was measured
using a Corning (Corning, USA) combination pH electrode.
Haematocrit was measured in duplicate after centrifuging blood in
micro-haematocrit tubes at 12 000 r.p.m. for 3 min. Blood [Hb] was measured
spectrophotometrically following conversion to cyanomethaemoglobin, applying a
millimolar extinction coefficient of 11.0. Plasma Na+ and
Ca2+ levels were measured using atomic absorption flame photometry,
and plasma Cl- concentration was measured according to Zall et al.
(1956). Plasma ammonia, urea,
glucose and lactate were measured using Sigma (St Louis, USA) diagnostic kits,
and gill homogenate Na+,K+-ATPase activity was measured
according to McCormick (1993
)
and expressed relative to total homogenate protein which was measured using
Bradford reagent (Bio-Rad, Richmond, CA, USA) and bovine serum albumin as a
standard.
was measured by adding
1.5 µCi (56 kBq) 22Na to each experimental chamber and taking a
5 ml water sample at the respective time 0 (i.e. 0, 2, 6, 24 or 72 h after
initiating exposure to hypercapnia) and 2 h following addition of isotope.
Water samples were measured for radioactivity using a Beckman (Fullerton, USA)
Coulter LS 6500 (Multi-purpose scintillation counter) for calculation of
Jin as indicated below. Water Na+ (measured
spectrophotometrically), Cl- (measured according to
Zall et al., 1956
), and total
ammonia (measured using a Sigma diagnostic kit) were determined for
calculation of net fluxes based upon the difference of the respective
parameter over the 2 h incubation duration, taking into account the weight of
individual fish.
Calculations
Plasma [HCO3-] was calculated from the measured blood
pH and PCO2 (Series II). In series I, plasma
[HCO3-] was calculated from pH and total CO2
concentration of plasma, using the Henderson-Hasselbalch equation. The
CO2 solubility coefficient and pK' for plasma were taken from
Boutilier et al. (1984).
Intracellular [HCO3-] for heart, liver and muscle were
calculated from the pHi of the respective tissue, and the
CO2 solubility coefficient and pK' for plasma as described by
(Heisler, 1982
;
Heisler et al., 1982
).
was calculated from
the disappearance of isotope from the water and the average Na+
concentration of the water during the flux period using the equation from
Gonzalez and Dunson (1987
):
![]() | (1) |
where Qout0 and Qout1 are the total counts per minute (c.p.m.) in the flux chambers at the beginning and end of the flux period, respectively. Qout is the average amount of Na+ in the flux bath during the flux period, M is the mass of the fish in g, and t is the time in h.
Statistics
Differences among mean values within a given
PCO2 treatment were determined using a one-way
ANOVA, or a one-way repeated-measures ANOVA as appropriate, followed by a
Tukey's post-hoc test. Data are presented as mean ±
S.E.M., N=6, unless otherwise
indicated.
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Results |
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|
Hypercapnia did not affect arterial PO2 (pooled value=45.4±2.7mmHg), mean cell haemoglobin concentration (MCHC) (pooled value=3.9±0.1), plasma [Na+] (pooled value= 140.5±3.0 meq l-1) or [Cl-] (pooled value=114.6±1.1 meq l-1). There was, however, a statistically significant reduction in Hct (from 40.4±1.6 at time 0, to 30.9±3.4 at time 24 h) which, most likely, relates to blood sampling. There were no significant changes in Hct in Series II.
Series II: Effect of hypercapnia on intracellular acid-base status
When non-cannulated fish were exposed to hypercapnia, blood was withdrawn
from the caudal vein following terminal anaesthesia to prevent struggling and
associated acidosis. Nevertheless, pHe of normocapnic fish was
lower than pHe of cannulated fish in Series I (Figs
1 and
3). Exposure to 14 mmHg
significantly reduced pHe and red cell pHi at 6 and 24 h
with no sign of pH compensation over 24h
(Fig. 3). There were however,
no significant changes in pHi of heart, liver or white muscle
(Fig. 3). When exposed to 32
mmHg, L. pardalis also experienced a substantial, and largely
uncompensated, reduction in pHe and red cell pHi that
persisted for up to 72 h (Fig.
4). Again, however, there were no statistically significant
reductions in pHi of heart, liver or white muscle during the 72 h
of hypercapnia. Intracellular pH regulation of heart, liver and white muscle
during the extracellular acidosis is evident when pHi is plotted
against pHe for all time periods at both levels of hypercapnia
(Fig. 5). Clearly, red cell pH
decreases with lowered pHe, while pHi of liver heart and
skeletal muscle tend to increase.
|
|
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As in series I, plasma HCO3- increased during hypercapnia (Table 1). There were no significant changes in plasma [Na+], [Cl-] or [Ca2+]; however, there was a trend toward a reduction in plasma [Cl-], consistent with the changes in plasma HCO3-. There was no significant effect of hypercapnia on haematocrit (pooled value=27.9±1.5), mixed-venous PO2 (pooled value=19.4±1.6 mmHg), plasma urea (pooled value=513±56 µmol l-1 nitrogen), plasma lactate (pooled value=0.41±0.13 mmol l-1), plasma glucose (pooled value=3.0±0.2 mmol l-1) or gill Na+,K+-ATPase activity (pooled value=0.82±0.32 mmol l-1 g-1 h-1). There was a significant increase in plasma ammonia levels at both levels of hypercapnia (Table 1). Calculated intracellular [HCO3-] for heart, liver and kidney were significantly elevated at 6 h in both levels of hypercapnia. There were no significant differences after 6 h, indicating complete intracellular acid-base regulation at this time (Table 1).
|
While most of the intracellular HCO3- accumulation
during hypercapnia is due to non-bicarbonate buffering, additional
HCO3- accumulation must have occurred to compensate for
the acidosis. Assuming a non-bicarbonate buffer value of 45 slykes for white
muscle, as determined for the facultative air-breather, Synbranchus
marmoratus (Heisler,
1982), it can be estimated that intracellular
[HCO3-], in the absence of intracellular pH
compensation, would have risen to approximately 5.5 mmol l-1 at a
PaCO2 of 14 mmHg (A in
Fig. 6). When compared to the
calculated intracellular value of 6.8 mmol l-1
(Table 1), 1.3 mmol
l-1 HCO3- must have accumulated in the white
muscle within 6 h to alleviate the acidosis. At a
PaCO2 of 32 mmHg, intracellular
[HCO3-] would be expected to increase to approximately
9.2 mmol l-1 in the absence of pHi compensation (B in
Fig. 6). When subtracted from
the calculated intracellular value of 13.0 mmol l-1
(Table 1), it appears that 3.8
mmol l-1 HCO3- accumulated in white muscle to
restore pHi.
|
Series III: Effect of hypercapnia on whole animal ion fluxes
Fish were exposed to hypercapnia simultaneously with those of Series II and
thus experienced identical levels of hypercapnia. At a
PCO2 of 14 mmHg, fish exhibited about a 70%
reduction in in
conjunction with a large reduction in
. As a result
did not change at 6 and
24 h (Fig. 7). There was a
significant decrease in
,
but no significant effect of hypercapnia exposure on total ammonia excretion
(pooled value of 396±28 nmol g-1 h-1). Urea
excretion rates prior to hypercapnia were low (11.5± 1.0 nmol
g-1 h-1) representing a total of 8.0±0.9% of
total nitrogenous waste excreted. Urea excretion was not measured during
hypercapnia.
|
The effects of exposure to 32 mmHg were qualitatively similar with large
reductions in both and
, resulting in no
significant change in
over 72 h (Fig. 8). A
significant reduction in
, relative to normocapnic
control fish, was observed during exposure to hypercapnia. However, after 6 h,
was near 0.
|
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Discussion |
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Extracellular acid-base regulation
The magnitude of the respiratory acidosis tolerated by L. pardalis
is among the greatest reported in the literature. Blood pH fell from 7.98 to
6.99 within 2 h of exposure to a water PCO2 of
42 mmHg. Eel (Anguilla anguilla) is also remarkably tolerant to
hypercapnia, and blood pH fell from 7.92 to 7.16 as
PCO2 increased from 3 to 45 mmHg over a 3 h
period (McKenzie et al.,
2002), resulting in no mortality with continued exposure to
hypercapnia (McKenzie et al.,
2003
). Following 6 weeks exposure to a
PCO2 of 45 mmHg, there was considerable
HCO3- accumulation (up to 72 mmol l-1)
associated with a large fall in plasma Cl- in the eel
(McKenzie et al., 2003
);
however, restoration of pHe did not exceed 50%. White sturgeon
Acipenser transmontanus also tolerate a reduction in pHe
from 7.7 to 7.15 when exposed to severe hypercapnia where arterial
PCO2 reaches 30 mmHg
(Crocker and Cech, 1998
).
Most fish completely or partially restore pHe, through net
accumulation of plasma HCO3- in exchange for
Cl-, within 10 to 72 h of being exposed to hypercapnia. Sturgeon,
however, show a very blunted recovery of pH during hyepercapnia, where
exposure to a PCO2 of 30 mmHg resulted in no
pHe compensation at 24 h, and a mere 35% restoration of
pHe following 72 h (Crocker and
Cech, 1998). The water used in this study was relatively hard,
which in many species facilitates acid-base regulation during hypercapnia
(Heisler, 1984
;
Larsen and Jensen, 1997
). The
degree of pH compensation observed in L. pardalis during hypercapnia
was similar to sturgeon. 24 h after exposure to 7 mmHg, pHe had
only recovered by 8% and 96 h exposure to 42 mmHg merely led to a 23% recovery
of pHe. Hypercapnia was associated with a small reduction in
, indicating that
branchial Cl-/HCO3- exchange was involved in
the small rise of intra- and extracellular HCO3-. There
was also a significant reduction of
, which may be a direct
effect of the low water pH (Wood,
1989
). When L. pardalis were exposed to a
PCO2 of 10 mmHg in the presence of a 60- to
70-fold elevation in water NaCl concentration (1.0 mmol l-1 NaCl),
there was no change in the rate or magnitude of acid-base recovery within 24 h
(data not shown), indicating that the blunted extracellular response to
hypercapnia may not be solely due to limitations associated with ionic
composition of the water.
L. pardalis can tolerate a water
PCO2 of 42 mmHg for weeks in water with low
ionic levels, but it is not known whether they do compensate extracellular
acid-base status over this duration. Interestingly, the ability of L.
pardalis to tolerate extreme hypercapnia was size-dependent; in
non-cannulated fish, all fish greater than 150 g survived, while all those
weighing between 50-80 g died. Cannulation also resulted in higher mortality.
Fish reduced spontaneous activity and remained stationary for days during
exposure to severe hypercapnia. Although L. pardalis is a facultative
air-breather, they did not attempt to air-breathe even when they had access to
air (C.J.B., unpublished observation). It has been proposed that the
exceptional tolerance to environmental hypercapnia observed in the eel is
associated with the need to tolerate air exposure
(Heisler, 1982;
McKenzie et al., 2002
). This
may also be the case for L. pardalis, which can survive aerial
exposure for days (A. L. Val, unpublished data).
A complete lack of extracellular pH regulation during the respiratory
acidosis that develops upon transition to air-breathing was observed in the
facultative air-breather, Synbranchus marmoratus
(Heisler, 1982). When exposed
to aquatic hypoxia for 4 days, S. marmoratus relied exclusively upon
air-breathing which, in combination with the reduction of water flow across
the gills, led to a fivefold increase of PaCO2
and a reduction in pHe from about 8.15 to 7.5. Plasma
HCO3- did not increase to compensate for the acidosis,
and there was very limited [HCO3-] uptake from the
water. This lack of pHe compensation was concluded to result from
the lack of gill-water contact and the associated impairment of branchial
transfer of acid-base relevant ions
(Heisler, 1982
). It remains,
however, to be established whether exposure to hypercapnic water, where
gill-water interaction is maintained, would be associated with pHe
compensation in S. marmoratus. Liposarcus pardalis is also a
facultative air-breather and it is possible that its limited compensation of
pHe during hypercapnia is associated with this trait rather than
with the low ionic content of the water in which the fish lives. However, not
all facultative air-breathers that experience a respiratory acidosis upon
air-breathing exhibit a lack of pH compensation as seen in S.
marmoratus. In another Amazonian catfish, Hypostomus sp.,
air-breathing was associated with a large reduction in gill ventilation (and
presumably gill-water interaction) and blood
PCO2 increased from 3 to 20 mmHg; however,
pHe was fully compensated after 4-7 days due to a 15 mmol
l-1 rise of plasma [HCO3-], in spite of the
low ionic content of the water (Wood et
al., 1979
).
Intracellular acid-base regulation
Regulation of intracellular pH is important for enzyme function and
metabolism and pHi is tightly regulated in most animals. Regulation
of pHi is generally associated with partial to complete
pHe compensation during an acid-base disturbance in most animals
(Heisler, 1984). A reduction
in pHi from 6.95 to 6.57 decreases glycolysis by 71% in mammalian
cardiac muscle (see Somero and White,
1985
). Severe impairment of aerobic metabolism is also likely with
relatively small reductions of pHi (0.1-0.3 pH units), due to the
pH sensitivity of enzymes such as citrate synthase and pyruvate dehydrogenase
(see Hazel et al., 1978
). The
effects of intra- and extracellular acidoses can be partially alleviated by a
high tissue buffer value, achieved through an elevation in histidyl imidazole
or phosphate groups (Somero and White,
1985
); but pHi compensation requires active
regulation.
Many teleost fishes regulate red cell pH during extracellular acidosis
through release of catecholamines that stimulate red cell
Na+/H+ exchange. This acts to safeguard oxygen
transport, due to the presence of extremely pH-sensitive haemoglobins. This
response protects blood oxygen binding that would have been reduced through
the Root effect (see Nikinmaa,
1990). The armoured catfish do not exhibit a Root effect and do
not posses adrenergic stimulation of red cell Na+/H+
exchange (Val et al., 1998
).
In contrast to the other tissues studied in L. pardalis, red blood
cell pH decreased throughout the hypercapnic exposures. The ratio of the
pHi/
pHe was 0.45, which is similar to that
of other tissues in animals that experience a large extracellular acidosis
over a 24-48 h period, and similar to that observed in red blood cells of
rainbow trout in vitro in the absence of catecholamines
(Heming et al., 1986
). Turtle
hearts, in vitro, exhibit a
pHi/
pHe of approximately 0.55
(Jackson et al., 1991
) and
whole body pHi of the catfish Ictalurus punctatus is
reduced with a
pHi/
pHe of 0.60 during
hypercapnia (Cameron, 1980
),
following which changes in pHe and pHi occurred roughly
in parallel. In the skate exposed to a PCO2 of
7 mmHg,
pHi/
pHe values for brain, heart
and white muscle are approximately 0.39, 0.45 and 0.76, respectively, over a
24 h period, where recovery of pHi is tightly correlated with that
of pHe. During continued exposure to hypercapnia (days to weeks),
pHi has been reported to be completely regulated in aquatic
animals; however, this requires some degree of extracellular compensation,
usually approaching 50% (Claiborne and
Heisler, 1986
; Pörtner et
al., 1998
; McKenzie et al.,
2003
).
The only two vertebrates reported to date that regulate pHi (of
heart and white muscle) in the face of a large, uncompensated, extracellular
acidosis are S. marmoratus
(Heisler, 1982) and the
salamander Siren lacertina
(Heisler et al., 1982
). In the
salamander, an increase in water pH or bicarbonate infusion did not alter the
acid-base strategy, indicating that the animals do not attempt to regulate
pHe, and preferentially regulate pHi. In both S.
marmoratus and salamanders, pHi of the heart and muscle was
tightly regulated within 4 days, the earliest time measured. In the heart,
liver and white muscle of L. pardalis, there was a trend towards an
increase in pHi during the pronounced extracellular acidosis, which
occurred as early as 6 h, indicating that active pH regulation must be
involved. It is not known whether S. marmoratus or S.
lacertina are capable of regulating pHi this rapidly.
The rapid, preferential regulation of pHi in the face of a largely uncompensated extracllular acidosis in L. pardalis is rare among vertebrates. It is not known whether this trait is associated with the ability to air-breathe and tolerate aerial exposure, with the low ionic content of the water, or with other environmental or evolutionary selective pressures. It is conceivable that this strategy could be selected in order to minimize problems associated with Cl- homeostasis in an aquatic environment such as the Amazon, which is dilute in ions (particularly Cl- and HCO3-), acidic, and prone to daily oscillations in hypercapnia. Complete pHe compensation during environmental hypercapnia is associated with large reductions in plasma Cl- levels that would have to be replaced rapidly when the fish moves from a hypercapnic to normocapnic environment. The ubiquity of this strategy among Amazonian fishes preferentially to regulate pHi and not regulate pHe is clearly worthy of further study.
Mechanisms of intracellular pH regulation
Intracellular pH regulation is associated with a net
HCO3- uptake into the tissue, which is accomplished by
active membrane transport of strong ions
(Reeves, 1985). The
HCO3- accumulated may ultimately come from the water in
association with gill HCO3-/Cl- or
Na+/H+ exchange. The
required to account for
the intracellular HCO3- accumulation during hypercapnia
can be calculated, but is only reported for white muscle here. White muscle
was the single largest tissue measured, and because white muscle
pHi is higher than heart and liver, it is likely to represent a
large proportion of the total intracellular HCO3-
accumulated by L. pardalis during hypercapnia. Assuming that white
muscle comprises about 50% of body mass in L. pardalis (lower than
other teleosts because of the dense bone of the skull), and that intracellular
water represents about 85% of the white muscle mass, the active accumulation
of 1.3 mmol l-1 HCO3- (see Results, Section
II), requires that approximately 0.55 µmol HCO3-
g-1 must have been accumulated within the white muscle 6 h after
exposure to a water PCO2 of 14 mmHg. Assuming
that this HCO3- ultimately came from the water in
exchange for Cl-, then a
of -92 nmol
g-1 h-1 would be required to regulate white muscle
pHi, which is very similar to the measured
value of -102 nmol
g-1 h-1 (Fig.
7). Thus, based upon the fluxes measured in this study, and
consistent with studies on other species
(Heisler, 1984
;
Larsen and Jensen, 1997
),
branchial HCO3-/Cl- exchange is most likely
to account for intracellular acid-base regulation under hypercapnia in L.
pardalis.
Net Cl-/HCO3- exchange across the gills,
however, does not appear to be sufficient to account for muscle
HCO3- accumulation at a higher
PCO2. If 3.8 mmol l-1
HCO3- was actively accumulated in the white muscle at 32
mmHg(Table 1), and given the
assumptions above, 1.6 µmol HCO3- per gram of fish
would have to be accumulated in the white muscle over the 6 h exposure
duration, which would require a
of -267 nmol
g-1 h-1. This is far greater than the highest rate of
-80 nmol g-1 h-1 measured at 6 h during this exposure,
indicating that other pathways must be involved. Hypercapnia did not affect
net Na+ uptake or ammonia excretion and L. pardalis does
not appear to excrete acid via the kidney
(Randall et al., 1996
). Thus,
the remaining HCO3- taken up by the muscle (and other
tissues) may have been shuttled from the extra to intracellular space. A
reduction in plasma [HCO3-] below the blood buffer line
was observed at 2 h, indicating that this may have occurred. A reduction in
plasma [HCO3-] and an increase in plasma
[Cl-] were observed in S. marmoratus during initial
exposure to hypercapnia, illustrating the preference of pHi over
pHe regulation (Heisler,
1982
). Liposarcus pardalis is endowed with a high bone
mass, predominantly skull, and given the role of the shell and bone in
compensating for an acidosis in turtles
(Jackson, 1997
;
Jackson et al., 2000
), bone
demineralization may be playing a role in intracellular acid-base regulation
in L. pardalis. Bone does not appear to be a route for acid-base
compensation in the channel catfish, Ictalurus punctatus, exposed to
hypercapnia (Cameron, 1985
),
and there were no statistically significant changes in plasma
[Ca2+] in L. pardalis; however, Ca2+ efflux
from the fish during hypercapnia was not measured. Whether or not bone
demineralization is involved in acid-base regulation in L. pardalis
remains to be investigated. Clearly pHi of the heart, liver, white
muscle, and probably other tissues, is tightly regulated in the face of a
large, predominantly uncompensated extracellular acidosis in L.
pardalis during hypercapnia. Elucidating the precise mechanisms involved
remains an exciting avenue for further studies.
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Acknowledgments |
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![]() |
References |
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