An investigation of the role of carbonic anhydrase in aquatic and aerial gas transfer in the African lungfish Protopterus dolloi
1 Department of Biology, University of Ottawa, 150 Louis Pasteur, Ottawa, ON
K1N 6N5, Canada
2 Veterans Affairs Puget Sound Health Care System, 1660 South Columbian Way
Seattle, WA 98108, USA
3 Department of Natural Sciences, National Institute of Education, Nanyang
Technological University, Republic of Singapore
4 Department of Biological Sciences, National University of Singapore,
Republic of Singapore
* Author for correspondence (e-mail: sfperry{at}science.uottawa.ca)
Accepted 30 June 2005
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Summary |
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The findings of this study are significant because they (i) demonstrate that, unlike in other species of African lungfish that have been examined, the gill/skin is not the major route of CO2 excretion in P. dolloi, and (ii) suggest that CO2 excretion in Protopterus may be less reliant on carbonic anhydrase than in most other fish species.
Key words: carbon dioxide excretion, oxygen uptake, gill, lung, acetazolamide, benzolamide, breathing, lungfish, Protopterus dolloi, carbonic anhydrase
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Introduction |
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Although the lung is the primary site of O2 uptake in
Protopterus, the gill (and/or the skin) is believed to be the major
route for CO2 excretion
(Lenfant and Johansen, 1968;
Lahiri et al., 1970
;
McMahon, 1970
;
Babiker, 1979
). Thus, even
though African lungfish are bimodal breathers, the gill appears to be
unimportant in O2 uptake and the lung relatively unimportant in
CO2 excretion. The reduced reliance on the lung for CO2
excretion under resting conditions is also a feature of Australian
(Lenfant et al., 1966
) and
South American (Johansen and Lenfant,
1967
) lungfish. In comparison to most fish, the gills of lungfish
have small surface areas and large blood-to-water diffusion distances, and are
ventilated by low volumes of water
(Burggren and Johansen, 1986
).
Because of the much higher (
30-fold) capacitance of the water for
CO2 relative to O2, the low ventilation volumes may be
adequate to sustain normal rates of CO2 excretion but inadequate
for similar rates of O2 uptake
(Graham, 1997
). It was
recently demonstrated that the overall contribution of the lung of
Lepidosiren to CO2 excretion can increase markedly with
increasing metabolic rate (Amin-Naves et
al., 2004
), indicating that the lungfish lung can effectively
excrete CO2 under the appropriate conditions.
CO2 transfer across the teleost gill has been demonstrated to
behave as a diffusion limited system
(Bindon et al., 1994;
Greco et al., 1995
;
Julio et al., 2000
) owing to
chemical equilibrium limitations (Julio et
al., 2000
; Desforges et al.,
2002
). Essentially, the chemical equilibrium limitations reflect
the relatively slow rate of conversion of blood HCO3- to
CO2 during the brief period of gill transit. Indeed, recent models
for branchial gas transfer in teleosts
(Perry and Gilmour, 2002
)
suggest that CO2 transfer is more apt to be affected by diffusion
limitations than is O2 transfer. Thus, the fact that the lungfish
gill is likely to display pronounced diffusion limitations implies that there
might be other factors (in addition to low ventilation volumes) to explain why
the reduced lungfish gill does not transfer O2 while excreting
CO2 effectively.
In the present study, two possible mechanisms for the preferential
excretion of CO2 by the gill of African lungfish were investigated.
First, we hypothesised that, as in dogfish, extracellular branchial
membrane-associated carbonic anhydrase (CA) aids gill CO2 excretion
by allowing the catalysed dehydration of HCO3- within
the plasma (Gilmour et al.,
2001; Gilmour and Perry,
2004
). Assuming a high enough buffer capacity in the plasma
(Desforges et al., 2001
;
Gilmour et al., 2004
), this
would allow CO2 excretion to occur without a requirement for red
blood cell (RBC) Cl-/HCO3- exchange, which is
the rate-limiting step in CO2 excretion
(Perry, 1986
) and the origin
of chemical equilibrium limitations
(Gilmour et al., 2004
). To
test this hypothesis, aerial and aquatic gas transfers were measured before
and after inhibition of extracellular CA using a relatively impermeant CA
inhibitor, benzolamide (BZ). A second possible explanation for the
preferential role of the gill in CO2 excretion is that the high
PCO2 that is characteristic of lungfish blood
(Lenfant and Johansen, 1968
)
obviates the need for catalysed dehydration of plasma
HCO3-, because blood-to-water
PCO2 partial pressure gradients are large
enough to sustain CO2 excretion by diffusion alone. While
potentially effective at the gill, a similar scenario might be less likely at
the lung owing to a much smaller blood-to-gas diffusion gradient, reflecting
the loss of CO2 at the gill and the mixing of inspired and expired
gas within the lung. To test this hypothesis, aerial and aquatic gas transfers
were measured before and after inhibition of total CA activity using a
permeant CA inhibitor, acetazolamide (AZ). In a final series of experiments,
fish were injected with exogenous CA to test the hypothesis that
CO2 excretion in Protopterus is constrained by inadequate
catalysis of HCO3- dehydration. All experiments were
performed on Protopterus dolloi, a species that, to our knowledge,
has not been used previously in studies investigating the mechanisms of gas
transfer in African lungfish.
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Materials and methods |
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Fish were shipped by air cargo in partially filled bags of oxygenated water contained within insulated containers to Ottawa. Upon their arrival, fish were placed individually into covered plastic containers containing 23 l of dechloraminated city of Ottawa tapwater warmed to 25°C. To maintain this water temperature, the air temperature was held at 25°C. The tank water was changed on alternate days or sooner if there was obvious fouling of the medium. The fish were kept in a sealed room under an artificial photoperiod of 10 h:14 hlight:dark. Fish were fed on alternate days with frozen blood worms or pieces of rainbow trout flesh. Fish were allowed to acclimate to these conditions for at least 1 month prior to beginning experiments.
Surgical procedures
Fish were anaesthetized in a solution of MS-222
(ethyl-p-aminobenzoate; 1.0 g l-1) adjusted to neutral pH
with NaHCO3 (2 g l-1). After cessation of breathing
movements, the fish were transferred to an operating table where they were
draped with paper towels soaked with anaesthetic solution. In this way, they
were kept moist and deeply anaesthetized for the duration of the surgery. To
allow periodic blood sampling, a cannula (Clay-Adams PE 50 polyethylene
tubing) was inserted into the dorsal aorta (DA) according to standard surgical
procedures (Axelsson and Fritsche,
1994). Briefly, a lateral incision (
2 cm in length) was made
at the level of the vent approximately 3 mm below the lateral line. The DA was
exposed and the cannula was inserted via a small incision and
advanced at least 3 cm in the anterior direction. The incision was sutured
using a running stitch and the cannula was then secured to the body wall with
silk ligatures, filled with heparinized (100 units ml-1) saline
(140 mmol l-1 NaCl), and heat-sealed. Fish were retuned to their
containers where they were allowed to recover from surgery for
24 h.
Experimental protocol
Fish were placed into customized respirometry chambers approximately 2 h
prior to beginning an experiment. The chambers were filled with water (1.5 l)
except for an adjustable (60 ml maximum volume) air space at one end of the
respirometer. Once aware of the presence of the air space, the fish would
typically position themselves just underneath it and begin breathing air at
regular intervals. Initially, both compartments were provided with continually
flowing media (water or air). However, during measurements of rates of
O2 consumption
(O2) or
CO2 excretion
(
CO2), the water
was recirculated using a peristaltic pump and the air chamber was sealed.
O2 and CO2 electrodes inserted into the air chamber were
used to measure aerial PO2 and
PCO2. To measure aquatic
PCO2, water was pumped via a
peristaltic pump into the sample compartment of a CO2 electrode
(Cameron Instruments, Port Aransas, TX, USA) housed within a thermostatted
(25°C) cuvette (Radiometer, Copenhagen, Denmark) and returned to the
respirometer. To measure aquatic PO2, an
O2 electrode was inserted into the recirculating water loop.
Series 1. Partitioning of gas transfer between air (lung) and water (gill/skin)
After establishing that the fish was positioned correctly under the air
chamber and breathing air at regular intervals, the flows of freshwater and
air were stopped, the water and air chambers were sealed, and the water was
re-circulated to provide mixing. Changes in aerial and aquatic
PCO2 and PO2 as
well as air breathing frequency were monitored for approximately 30 min or
until stable rates of CO2 accumulation and O2 depletion
were achieved. Preliminary experiments were performed to determine the extent
of gas transfer across the airwater interface. To determine the
potential for aerial CO2 to be transferred to the water and thus be
misinterpreted as CO2 excretion into the water, mixtures of
CO2 and air were added incrementally to the air chamber. After
sealing the air chamber, the water PCO2 was
monitored without a fish in the respirometer. To determine the potential for
aquatic CO2 to be transferred to the air space and thus be
misinterpreted as aerial CO2 excretion, fish were euthanized by
lethal injection while in the respirometer after aerial and aquatic
PCO2 had been allowed to rise naturally to high
levels. The air-chamber was then flushed with air to lower
PCO2 and after re-sealing the chamber, the
PCO2 of the air was monitored while the water
PCO2 remained at a high level.
Initial experiments revealed substantial
O2 and
CO2 in the
absence of a fish in the respirometer, or during trials using recently
euthanized fish. This background gas transfer presumably reflected cutaneous
metabolism or metabolism arising from microorganisms within the water or
attached to the fish. Thus, all experiments were terminated by euthanizing the
fish (followed by intra-arterial injection of saturated KCl to stop the heart)
in situ and performing a final respirometry trial for a further
3060 min. For each fish, the values for
O2 and
CO2 were
corrected for background metabolism by subtracting the rates determined on
such trials.
Series 2. Effects of CA inhibition
The standard experimental protocol consisted of monitoring water/air
PO2 and PCO2 in
addition to air breathing frequency for three consecutive 30 min intervals.
The first interval consisted of a control period, after which a blood sample
(0.6 ml) was withdrawn from the DA cannula and
PO2, PCO2 and pH
immediately analyzed. The remaining blood was centrifuged (1 min x 10
000 g) and the pellet was combined with 0.6 ml of distilled
water prior to being flash-frozen in liquid N2. Red blood cell
lysates were stored at 80°C for subsequent analysis of CA
activity.
After the control period, the air chamber was flushed with air and
re-sealed; the water compartment was occasionally flushed and re-sealed
depending upon the extent of CO2 accumulation during the control
run. Once baseline rates of O2 and CO2 transfer had been
re-established, fish were injected via the DA cannula with BZ (1.2 mg
kg-1 using an injection volume of 1.0 ml kg-1), a CA
inhibitor that is relatively slow to cross RBC membranes
(Travis et al., 1964).
Respirometry was performed for 30 min and blood samples were withdrawn at 10
and 30 min and treated as described above. After flushing and re-sealing, a
final 30 min respirometry period ensued following injection of AZ (30 mg
kg-1 using an injection volume of 1.0 ml kg-1); blood
samples were withdrawn at 10 and 30 min. Fish were then euthanized to
determine background
O2 and
CO2. In a
separate group of fish (N=5), gas transfer and blood gases were
monitored for 2 and 24 h, respectively, after AZ injection.
Series 3. Effects of exogenous CA
After a 30 min control period of respirometry and blood sampling, fish were
injected via the DA cannula with bovine CA (25 mg kg-1
using a 0.5 ml injection volume) dissolved in 140 mmol l-1 NaCl. A
second 30 min period of respirometry was conducted, after which a final blood
sample was withdrawn. The blood samples in this series were treated as above
except that a RBC lysate was not prepared; instead, the plasma was collected,
frozen in liquid N2 and stored at 80°C for subsequent
determination of plasma CA activities.
Series 4. Effects of hypercapnia on blood acidbase status
Aquatic hypercapnia was established by replacing the air supplying a
water/gas equilibration column with mixtures of CO2 and air
provided by a gas mixing pump (Wösthoff, Bochum, Germany). The desired
water PCO2 of 40 mmHg was pre-set by adjusting
the rate of water and/or gas flow through the column. To rapidly achieve
aquatic hypercapnia within the respirometer, the bulk of water was removed and
replaced with water derived from the water/gas equilibrium column. After 30
min, aquatic normocapnia was re-established and 30 min of aerial hypercapnia
was imposed by gassing the air chamber with a mixture of 5% CO2 and
95% air to yield a final nominal inspired PCO2
of 37.5 mmHg. Owing to the very high PCO2
gradients between air and water, which resulted in transfer of CO2
across the airwater interface, no attempts were made to measure
CO2 during
aquatic or aerial hypercapnia.
Blood samples (0.6 ml) were withdrawn from the DA cannula after 30 min of normocapnia, aquatic hypercapnia and aerial hypercapnia. These samples were analysed for pH and total CO2 (CCO2).
Analytical procedures
For experiments conducted in Singapore
In series 13 experiments, arterial blood
PO2 (PaO2) was
measured using an Ocean Optics (Dundedin, FL, USA) fibre-optic O2
sensing system. A fibre-optic O2 sensor (FOXY AL300; Dundedin, FL,
USA) was inserted into a blood-filled syringe. Arterial blood
PCO2 (PaCO2) was
measured by injecting blood into the sample compartment (maintained at
25°C) of a PCO2 (Cameron Instruments)
electrode that was connected to a three-channel blood gas analyser (Cameron
Instruments). Blood pH (pHa) was determined using a blood gas analyser
(Medica, Bedford, MA, USA) that was thermostatted to 37°C. The measured pH
was then adjusted for the temperature differential (12°C) between the
analyser and fish using a correction factor of 0.018 pH units
°C-1 (Reeves,
1977).
For experiments conducted in Ottawa
In series 13 experiments, PaO2,
PaCO2 and pHa were monitored by injecting blood
into the sample compartments (maintained at 25°C) of
PO2, PCO2
(Cameron Instruments) and pH (Metrohm combination 6.0204.100; Herizau,
Switzerland) electrodes that were connected in series; the electrodes were
linked to a three-channel blood gas analyser (Cameron Instruments). In series
4 experiments, plasma pH was measured using a Cameron pH electrode contained
within a temperature controlled chamber and connected to a Radiometer blood
gas analyser. Plasma CCO2 was determined in
duplicate on 50 µl samples using a Capnicon V total CO2 analyzer
(Cameron Instruments). Plasma PCO2 and
[HCO3-] were calculated using the
HendersonHasselbalch equation and physiochemical constants for trout
plasma (Boutilier et al.,
1984).
In both Singapore and Ottawa, the PCO2 and PO2 within the air compartment were monitored continually by inserting and sealing CO2 (Cameron Instruments) and fibre-optic O2 (Ocean Optics AL300) electrodes into the air space of the respirometer. The PO2 of the water (PWO2) was monitored continually by inserting a fibre-optic O2 electrode (Ocean Optics AL300) into the tubing through which water was recirculating. The PWO2 was monitored by moving water via a peristaltic pump through the sample compartment of a CO2 electrode (Cameron Instruments) housed within a thermostatted (25°C) cuvette (Radiometer) before returning it to the respirometer. The PCO2 electrodes were connected to a second three-channel blood gas analyzer (Cameron Instruments) that was customized to accept two CO2 inputs. Output from the blood gas analyzers was converted to digital data and stored by interfacing with a data acquisition system (Biopac Systems Inc., Goleta, CA, USA) using AcknowledgeTM data acquisition software (sampling rate set at 30 Hz) and a PentiumTM PC. Output from the fibre-optic O2 electrodes was displayed using Ocean Optics software and the data were compiled as text files for later importation into spreadsheet software. The O2 electrode for water measurements was calibrated by immersing the electrode in a zero solution (2 g l-1 sodium sulphite) or air-saturated water, until stable readings were recorded. The O2 electrode for air measurements was calibrated in situ by flowing humidified N2 gas (zero) or air continuously through the air chamber. The CO2 electrodes for water and air measurements were calibrated in a similar manner using mixtures of 0.5 and 1.0% CO2 in air that were provided by a gas mixing flowmeter (GF-3/MP, Cameron Instruments). The pH electrode was calibrated using precision buffers; all electrodes were calibrated prior to each experiment.
Aerial gas transfer was determined from the slopes of the relationships
between inspired gas tensions and time, over the period that the air chamber
was sealed; the solubility coefficients of O2 and CO2 in
air at 25°C were obtained from Boutilier et al.
(1984). Rates of aquatic gas
transfer were determined using the slopes of the relationships between water
gas tensions and time over the interval that the respirometer was
re-circulating. The solubility coefficient for O2 in water at
25°C was obtained from Boutilier et al.
(1984
). The capacitance
coefficient of CO2 in water at 25°C was determined
experimentally to be 0.041 µmol l-1 mmHg-1. This was
accomplished by measuring the changes in water total CO2
concentration (Cameron Instruments Capnicon V total CO2 analyzer)
over the range of PCO2 values encountered in
the respirometry trials. Air breathing frequency was assessed visually
throughout each experiment.
The CA activity of plasma or RBC lysates was measured using the
electrometric pH method of Henry
(1981
). In brief, samples
containing CA activity were added to 6 ml of reaction buffer (in mmol
l-1, 225 mannitol, 75 sucrose, 10 Tris base, adjusted to pH 7.40
with 30% phosphoric acid) held in a temperature-controlled (4°C) vessel,
and the reaction was initiated by the addition of 200 µl of
CO2-saturated water. The rate of change of pH was measured over a
fall of approximately 0.15 pH units. To obtain the true catalyzed rate, the
uncatalysed rate (addition of CO2-saturated water in the absence of
any CA source) was subtracted from the observed rate. A pH electrode
(Radiometer GK2401C or Metrohm 6.0204.100) and PHM 84 pH meter (Radiometer)
linked to a data acquisition system (Biopac Systems Inc.) were used to measure
pH.
In general, 50 µl samples of plasma or diluted RBC lysate were used to
assay CA activity. CA activity in RBC lysates was also titrated with
increasing volumes of 5 µmol l-1 acetazolamide (AZ). The
resulting data were plotted according to the presentation of Easson and
Stedman (1937) to yield an
inhibition constant (Ki) for lungfish RBC CA against
acetazolamide.
To determine levels of CA inhibitors in RBCs, lysates were thawed and then
heated to 100°C for 10 min to denature endogenous CA. After heating, the
samples were spun at 1000 g for 15 min and the supernatant was
assayed for inhibitor content using the micro-method of Maren
(1960), in which a fixed
amount of human RBC CA is used to test the inhibitory activity of an unknown
sample. Known concentrations of the appropriate inhibitor were used to
generate a standard curve of catalysis time vs inhibitor
concentration and this was used to back-calculate the RBC inhibitor
concentration in vivo, taking into account appropriate dilution
factors.
Statistical analysis
The data are reported as means ± 1 standard error of the mean
(S.E.M.). Treatments were compared using one-way repeated measures
analysis of variance (RM ANOVA) or paired Student's t-tests (when two
means were compared). If significant differences (P<0.05) were
found for multiple comparisons, a post-hoc multiple comparisons test
(Bonferroni t-test) was applied.
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Results |
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Casual observation suggested that air-breathing frequency tended to be higher in fish held in respirometers compared to their holding aquaria. Furthermore, it was apparent that in some fish, air-breathing frequency varied greatly during a single experiment. Several approaches were used to determine whether the partitioning of CO2 transfer between water and air was influenced by breathing frequency. First, regression analyses revealed that the relative proportion of CO2 excreted via the gills/skin was unrelated to pulmonary breathing frequency between 6 and 48 breaths min-1 (r2=0.03; P>0.05; N=19). Second, we were able to exploit several instances where fish dramatically changed their pulmonary breathing frequency. Fig. 3 illustrates two such cases where air-breathing frequency was suddenly decreased or increased. A sudden decline in air breathing frequency (Fig. 3A) was accompanied by a marked reduction in aerial CO2 excretion, yet the rate of CO2 accumulation in the water was unchanged. Conversely, a sudden increase in air breathing frequency (Fig. 3B) was accompanied by an obvious increase in aerial CO2 excretion without any change in aquatic CO2 accumulation.
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Series 3. Effects of exogenous CA
Injection of bovine CA caused a marked increase in plasma CA activity at 30
min from 39±36 to 9642±2179 µmol CO2
ml-1 min-1 (N=5). The mean CA activity in the
plasma of pre-injected fish reflected an unusually high value in a single
sample (184 µmol CO2 ml-1 min-1); in three
of five samples, CA activity was undetectable. The rise in plasma CA activity
was associated with a slight, but statistically significant, decrease in
PaCO2 and an increase in breathing frequency
(Table 3). Although
O2 was
unaffected, CA treatment was associated with a significant increase in
CO2 that largely
resulted from increased CO2 excretion by gills/skin
(Fig. 5). The apparent rise in
CO2 at the lung
was not statistically significant (P=0.641).
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Series 4. Effects of hypercapnia on blood acidbase status
Aerial hypercapnia (37.5 mmHg) caused a marked respiratory acidosis within
30 min. PaCO2 increased from 12.1±0.5 to
20.9±1.7 mmHg (N=6) and pHa decreased from 7.57±0.02 to
7.41±0.03. Aquatic hypercapnia (40 mmHg) did not affect arterial blood
acidbase status.
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Discussion |
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Although the principal aim of the present study was to investigate the
potential roles of CA in aerial and aquatic CO2 transfer, it
quickly became apparent that gas transfer partitioning in P. dolloi
was markedly different than in other lungfish because the lung appeared to be
the major site of both O2 and CO2 transfers. In this
respect, P. dolloi resembled Lepidosiren acclimated to
elevated ambient temperatures (Amin-Naves
et al., 2004). Owing to the pronounced differences between this
and previous studies on African lungfish, a series of experiments was
performed to validate the basic respirometry protocol.
Potentials for error during respirometry
Several comprehensive reviews have been published that discuss some common
problems associated with performing respirometry on aquatic animals
(Steffensen, 1989;
Kaufmann and Forstner, 1989
;
Cech, 1990
). Arguably, the
most serious problem is the potential for modification of water
PO2 and PCO2 by
bacteria or other extraneous sources. The potential for bacterial metabolism
to contribute to the measured changes in gas partial pressures is greatest in
recirculating respirometers at high water temperatures
(Kaufmann and Forstner, 1989
).
Because antibiotics are not recommended to eliminate bacterial metabolism
during respirometry trials (Kaufmann and
Forstner, 1989
), the protocol employed in the present study was to
subtract background metabolism during blank runs conducted using dead fish.
Thus, we were unable to distinguish microbial metabolism in the water, per
se, from microbial metabolism occurring on the surface of the fish.
Clearly, however, bacterial metabolism was a major source of O2
depletion and CO2 accumulation in the water during closed system
respirometry. Indeed in many cases, there were no differences in the rates of
water gas changes after euthanasia. Because all blank runs were performed with
a fish in the respirometer, it is conceivable that cutaneous metabolism, in
addition to microbial activity, may have contributed to the background
metabolism. Regardless of its source, the true rates of aquatic
O2 and
CO2 could only
be determined after taking into account these significant background
components. It is unclear whether previous respirometry studies on P.
aethiopicus utilised corrections for background metabolism.
Closed system respirometry on bimodal breathers is particularly challenging
because of the potential for gases to diffuse across the airwater
interface of the respirometer (Graham,
1997). For example, the entry of exhaled CO2 from the
air chamber into the water, if occurring, could mistakenly be attributed to
CO2 excretion across the gills/skin. Because of the high
capacitance of water for CO2, the potential for contamination of
the water with exhaled CO2 is likely to be significantly greater
than that for O2 entry from water into the air. In the present
study, we were unable to detect any transfer of gas across the airwater
interface. Regardless, to minimize diffusion gradients between the air and
water, the chambers were flushed at regular intervals.
Partitioning of gas transfer in P. dolloi
The results obtained in this study clearly implicate the lung as the
primary route of both O2 and CO2 transfer in P.
dolloi, but also demonstrate that the gill/skin is more suited to
CO2 excretion than to O2 uptake. The former finding is
in marked contrast to conclusions derived from previous studies on African
lungfish (Lenfant and Johansen,
1968; McMahon,
1969
; Lahiri et al.,
1970
). Because the data demonstrating the lung as the principal
site of CO2 excretion were unexpected and in opposition to accepted
models, we considered it important to corroborate the respirometry data using
independent methods. This was accomplished by assessing the impact of aerial
vs aquatic hypercapnia on blood acidbase status. In support of
the respirometry data, only aerial hypercapnia elicited an arterial blood
respiratory acidosis. This result plainly indicated that pulmonary
CO2 excretion was being inhibited and that the gill was ineffective
at completely clearing the accumulating CO2. However, the fact that
PaCO2 remained lower than inspired
PCO2 may have reflected an increased
involvement of branchial CO2 excretion. Alternatively, the arterial
blood may not have yet reached equilibrium with inspired
PCO2. The most likely explanation for the
absence of an effect of aquatic hypercapnia on blood acidbase status is
that inhibition of the small component of CO2 excretion occurring
at the gills/skin or any CO2 entry from the water was simply
compensated for by increased pulmonary CO2 excretion.
As reported in other studies (e.g. Johansen and Lenfant, 1968), pulmonary
breathing frequency was highly variable within, and between, individuals.
Moreover, while not quantified, it appeared that the average breathing
frequency (20 min-1) during respirometry trials was higher
than that in fish being held in aquaria. Given previous reports that pulmonary
hyperventilation in P. aethiopicus is associated with a lowering of
PaCO2
(Lahiri et al., 1970
) or
increased pulmonary
CO2
(McMahon, 1970
), we considered
the possibility that CO2 excretion into the water was artificially
low in the present experiments because of pulmonary hyperventilation. This is
unlikely, however, based on the absence of any correlation in this study
between breathing frequency and aquatic CO2 excretion over a wide
range (648 breaths min-1). Moreover, there were no obvious
changes in the rates of CO2 accumulation in the water during
periods of pulmonary apnoea.
Although CO2 transfer into the water comprised only a minor
component of overall CO2 excretion, the relative proportion of
aquatic CO2 transfer (24% of overall CO2 excretion) was
greater than for aquatic O2 transfer (9% of overall O2
uptake). These data confirm previous conclusions that the gill/skin of
lungfish is more suited to CO2 excretion than O2 uptake
(see reviews by Burggren and Johansen,
1986; Graham,
1997
). The possibility that extracellular catalysis of plasma
HCO3- dehydration underlies this phenomenon is discussed
below.
It would be presumptuous to assume that previous conclusions concerning gas
transfer partitioning in lungfish are incorrect. Discrepancies between the
present, and other studies, may simply reflect species differences. Why P.
dolloi should differ from other species of Protopterus, however,
is unclear. Alternatively, by analogy to Lepidosiren
(Amin-Naves et al., 2004), it
is possible that the fish used in the present study had increased metabolic
rates (compared to fish used in previous studies) and hence were more reliant
on the lung as a route for CO2 excretion. Interestingly, we (S. F.
Perry, R. Euvermann, S. F. Chew, Y. K. Ip and K. M. Gilmour; unpublished data)
have recently completed a series of experiments on P. annectens and
found that, as in previous reports, the gill was the predominant site of
CO2 excretion whereas the lung was the major site of O2
uptake.
Carbonic anhydrase and CO2 excretion
Initially, the present study was conceived to test the idea that the
reputed preferential excretion of CO2 by the lungfish gill
(compared to the lung) might reflect the participation of extracellular
branchial CA. As is the case in dogfish
(Wood et al., 1994;
Henry et al., 1997
;
Gilmour et al., 2001
;
Gilmour and Perry, 2004
;
Perry and Gilmour, 2002
), it
was hypothesized that a membrane-associated plasma-facing CA isoform could
participate in the dehydration of plasma HCO3-, thereby
aiding CO2 excretion. Such a scheme might be particularly
beneficial to Protopterus owing to its low rate constant for RBC
Cl-/HCO3- exchange
(Jensen et al., 2003
), the
step normally considered to be rate limiting in CO2 excretion
(Perry, 1986
;
Tufts and Perry, 1998
). In
light of the results of the initial respirometry experiments, which showed the
gill to be a minor route for CO2 elimination, clearly the original
hypothesis was no longer appropriate. Thus, the hypothesis was modified
post hoc to test instead the idea that excretion of CO2 at
either site of gas exchange (gill/skin or lung) is aided by extracellular
CA.
It is likely that all air-breathing animals possess membrane-associated
endothelial pulmonary CA that is oriented toward the vascular lumen and thus
able to catalyze extracellular reactions
(Stabenau and Heming, 2003).
However, its role in CO2 excretion is constrained by the low
buffering capacity of plasma that limits the supply of substrate
(H+) for the HCO3- dehydration reaction
(Bidani and Heming, 1991
). It
is generally accepted that less than 10% of overall excreted CO2 is
derived from the extracellular catalysis of HCO3-
(Bidani, 1991
;
Cardenas et al., 1998
;
Henry and Swenson, 2000
).
Based on the present results obtained using the functionally impermeant CA
inhibitor, BZ, pulmonary CA (if present in lungfish), as in other
air-breathers, is clearly not playing a significant role in aerial
CO2 excretion. Although the use of BZ as an impermeant CA inhibitor
has been challenged (Supuran and
Scozzafava, 2004
), results of this and previous studies
(Gilmour et al., 2001
) clearly
demonstrate that low doses of BZ do not sufficiently penetrate RBCs of
lungfish, trout or dogfish within 30 min to significantly influence
HCO3- dehydration. Currently, studies are underway to
determine if a membrane-associated CA isoform does actually exist in the lung
of Protopterus.
CO2 excretion into the water also was unaffected by BZ
treatment, and thus the apparent preferential transfer of CO2 by
the gill/skin in comparison to O2 presumably does not reflect
extracellular catalysis of HCO3-. To date, the only fish
for which extracellular CA has been shown to significantly contribute to
CO2 excretion is Squalus acanthias
(Gilmour et al., 2001;
Gilmour and Perry, 2004
), a
species known to possess high plasma buffering capacity
(Lenfant and Johansen, 1966
;
Gilmour et al., 2002
).
Results obtained using the permeant CA inhibitor, AZ, demonstrate that
CO2 excretion in P. dolloi can occur normally in the
absence of RBC CA activity. The absence of any effect of AZ on CO2
excretion or blood acidbase status could not be attributed to
insufficient inhibition of CA, based on independent measures of RBC CA
activity, and AZ concentrations in treated fish compared with the
Ki for lungfish RBC CA against AZ; this
Ki value for lungfish was also comparable to the
corresponding value for rainbow trout
(Henry et al., 1993), where a
similar dose of AZ elicits a profound respiratory acidosis (e.g.
Hoffert and Fromm, 1973
).
Moreover, the AZ solution that was used in Singapore was brought back to
Canada and injected into two rainbow trout Oncorhynchus mykiss;
arterial PCO2 increased over 30 min by
approximately 2.72 and 2.22 mmHg in the two trout (S. F. Perry and K. M.
Gilmour, unpublished observations). To our knowledge, P. dolloi is
the only fish species examined to date that is unaffected by AZ treatment
despite total inhibition of RBC CA activity. We must emphasise, however, that
this result does not necessarily exclude a role for RBC CA in CO2
excretion under normal conditions. The results simply show that RBC CA
activity is not a prerequisite for normal rates of CO2 excretion.
Examples of air breathing species for which AZ was found to inhibit
CO2 excretion or raise arterial PCO2
include Xiphister atropurpureus
(Daxboeck and Hemimg, 1982
),
Trichogaster trichopterus
(Burggren and Haswell, 1979
),
Blennius pholis (Pelster et al.,
1988
) and Lepisosteus oculatus
(Smatresk and Cameron, 1982
).
The likeliest explanation for the absence of any effect of CA inhibition on
CO2 excretion or blood acidbase status in P. dolloi
is that CO2 excretion, at both the gills/skin and lung, is
occurring in the absence of appreciable catalyzed HCO3-
dehydration during gill/skin or lung transit. As discussed by Brauner and Val
(1996
) to explain
CO2 excretion in Arapaima gigas, the high
PCO2 values that are characteristic of lungfish blood,
coupled with low rates of metabolism, may negate the requirement for catalysed
HCO3- dehydration during the passage of blood through
the respiratory organs. Nevertheless, it is puzzling that, given the presence
of RBC CA activity and the likelihood of RBC
Cl-/HCO3- exchange (based on its presence in
the related P. aethiopicus;
Jensen et al., 2003
), there
would appear to be no obligate requirement for the catalysed dehydration of
plasma HCO3- within the gills or lungs. One possible
explanation is that the RBC CA in lungfish may be a slow-turnover isoform and
thus unable to keep pace with the rate of substrate delivery by the
Cl-/HCO3- exchanger
(Tufts et al., 2003
). In
support of this idea, we (L. Kenney, S. F. Perry, S. F. Chew, Y. K. Ip and K.
M. Gilmour, unpublished data) have recently cloned and sequenced two CA
isoforms from Protopterus blood, both of which are most similar to CA
XIII, an enzyme with only moderate catalytic activity
(Lehtonen et al., 2004
).
Because injection of bovine CA increased gill/skin CO2 excretion
(without increasing O2 uptake), it would indeed appear that the
extent of catalysed HCO3- dehydration is a constraint on
aquatic CO2 transfer in P. dolloi.
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