|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online May 21, 2007
Journal of Experimental Biology 210, 1944-1959 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02776
Mechanisms of acid–base regulation in the African lungfish Protopterus annectens
1 Department of Biology and Centre for Advanced Research in Environmental
Genomics, University of Ottawa, Ottawa, ON, Canada
2 Natural Sciences and Science Education, National Institute of Education,
Nanyang Technological University, Republic of Singapore
3 Department of Biological Sciences, National University of Singapore,
Republic of Singapore
* Author for correspondence (e-mail: kgilmour{at}uottawa.ca)
Accepted 13 March 2007
 |
Summary |
|---|
 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Key words: acid–base balance, ventilation, lung, gill, kidney, acidosis, alkalosis, African lungfish, Protopterus annectens
|
Introduction |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
;
Swenson, 2000;
McNamara and Worthley, 2001).
In water-breathing fish, on the other hand, respiratory compensation is of
negligible importance and acid–base balance is restored metabolically
(for reviews, see Goss et al.,
1998; Claiborne et al.,
2002; Hirose et al.,
2003; Perry et al.,
2003b; Evans et al.,
2005; Perry and Gilmour,
2006). A systemic acidosis is corrected by the increased output of
acidic equivalents (thereby raising the plasma HCO
–3 concentration), whereas the response to
alkalosis consists of decreased net efflux of acid (equivalent to increased
base efflux). In fish, the gill accounts for 90% or more of such direct
transfer of acid–base equivalents
(Claiborne et al., 2002).
Adjustment of renal acid secretion plays an important complementary role to
the gills (Wood et al., 1999;
Georgalis et al., 2006a) (for
reviews, see Perry and Fryer,
1997; Perry et al.,
2003b; Perry and Gilmour,
2006). Tetrapods also employ metabolic mechanisms to compensate
for acid–base disturbances, with the kidney being the main regulatory
site (Heisler, 1986;
Swenson, 2000;
McNamara and Worthley, 2001).
Aquatic amphibians, however, parallel fish in utilizing extra-renal transfer
of acid–base equivalents, in their case, via the skin
(Stiffler and Bachoura, 1991;
Stiffler, 1991;
Talbot and Stiffler, 1992).
Regardless of the tissue site, work on a variety of organisms suggests that
similar cellular and molecular mechanisms of acid–base equivalent
transfer are employed (for reviews, see
Claiborne et al., 2002;
Kirschner, 2004;
Wall, 2005;
Perry and Gilmour, 2006).
With this background in mind, the present study focused on characterizing
strategies for acid–base regulation in the African lungfish
Protopterus annectens. Mechanisms of acid–base compensation in
lungfish have received little attention to date
(Sanchez et al., 2005), but
several factors argue compellingly for their investigation. African lungfish
(Protopterus sp.) are obligate air breathers that possess true lungs
and cannot survive without access to air, but gills are also present,
permitting bimodal breathing (reviewed by
Burggren and Johansen, 1986).
The lung is the primary site of O2 uptake in all four
Protopterus species, but the majority of CO2 excretion
occurs across the gill (and/or the skin)
(Lenfant and Johansen, 1968;
Lahiri et al., 1970;
McMahon, 1970), except in
P. dolloi, where the lung appears to be a major route of both
O2 and CO2 transfer
(Perry et al., 2005). Because
most O2 uptake occurs from the air, branchial ventilation in
lungfish need not be constrained by convection requirements for O2
uptake. Consequently, the gill is ventilated by low volumes of water
(Jesse et al., 1967) and this
factor, coupled with the low surface area and high blood-to-water diffusion
distances of the lungfish gill (Sturla et
al., 2001), results in arterial PCO2 values
that are unusually high for fish (
20–30 mmHg)
(Lenfant and Johansen, 1968;
Perry et al., 2005). Thus, the
respiratory status of lungfish is more suitable for using ventilation to
regulate acid–base disturbances than is the case for water-breathing
fish. At the same time, however, the gills, although reduced in surface area
relative to those of water-breathing fish, are covered by a heterogeneous
epithelium containing several cell types, including pavement cells,
mitochondria-rich (MR) cells, and sensory cells
(Sturla et al., 2001). MR
cells are thought to be responsible for the excretion of acid–base
equivalents by the gills of freshwater fish (for a review, see
Perry and Gilmour, 2006), and
the presence of MR cells therefore implies the potential for the lungfish gill
to function in metabolic acid–base compensation.
Two hypotheses were tested in the present study. First, the hypothesis that ventilatory adjustments are used by P. annectens to regulate acid–base disturbances was tested. It was predicted that lungfish would increase pulmonary and/or branchial ventilation in response to metabolic acidosis, but hypoventilate under conditions of metabolic alkalosis. Second, the transfer of acid–base equivalents across the gill (and/or skin) was hypothesized to play a significant role in recovery from acid–base disturbances. Under these conditions, net acid excretion to the water would be predicted to increase during metabolic acidosis and decrease during metabolic alkalosis. Finally, the molecular mechanisms underlying the transfer of acid–base equivalents at the gill and kidney were investigated by cloning fragments of lungfish V-type H+-ATPase, Na+/HCO –3 cotransporter (NBC) and carbonic anhydrase (CA), and examining the mRNA expression of these genes in the gill and kidney prior to and following acid–base disturbances.
|
Materials and methods |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
To allow periodic blood sampling as well as acid or base infusion, a
cannula (Clay-Adams PE50 polyethylene tubing; VWR, Montreal, QC, Canada) was
inserted into the dorsal aorta of all fish, as described by Perry et al.
(Perry et al., 2005). Surgery
was carried out on lungfish that were first anaesthetized by immersion in a
solution of MS-222 (ethyl-p-aminobenzoate; 0.67 g
l–1) adjusted to neutral pH with NaHCO3 (1.3 g
l–1); anaesthesia was maintained during surgery by wrapping
the fish in paper towels soaked with the anaesthetic solution. A subset of
lungfish (N=7) was fitted with an external urinary catheter according
to the procedure of Curtis and Wood
(Curtis and Wood, 1991). The
use of an external urinary catheter allows the collection of urine as it is
naturally discharged from the urogenital papilla. Following surgery, lungfish
were returned to their containers for a 24 h recovery period. Cannulae were
flushed daily with Cortland saline (Wolf,
1963).
Experimental protocol
Series 1. Respiratory versus metabolic compensation of acid–base disturbances
Fish (N=19) that previously had been fitted with a dorsal aortic
cannula were placed into customized respirometry chambers approximately 2 h
before an experiment was initiated. The cylindrical chambers were filled with
water (1 l) apart from an adjustable air space (60 ml maximum volume;
typically set to 30 ml) at one end of the respirometer; exact water and
air volumes were noted for each fish. Lungfish quickly became aware of the
presence of the air space, and normally would begin breathing air at regular
intervals, adopting a position in the respirometer where their head was just
below the airspace. The occurrence of each air breath was captured by a
custom-built device that detected interruptions in two infrared light beams
aimed across the surface of the water. Both compartments were provided with
continually flowing media (water or humidified air, at 25°C) except during
measurements of the rates of O2 consumption
(
O2),
CO2 excretion
(CO2), and net
acid excretion into the water. During these measurements, the water was
re-circulated using a peristaltic pump, and the air chamber was sealed.
Fibre-optic O2 electrodes (Ocean Optics AL300, Dunedin, FL, USA)
sealed into the air chamber and into the tubing through which water was
re-circulating were used to measure aerial and aquatic
PO2, respectively. Aerial PCO2 was
measured using a CO2 electrode (Analytical Sensors E201, Sugarland,
TW, USA) inserted into the air chamber, while measurement of aquatic
PCO2 involved using a small peristaltic pump to move water
past a CO2 electrode (Analytical Sensors E201) housed within a
thermostatted (25°C) cuvette (Radiometer) before returning it to the
respirometer. These respirometers were used in a previous study
(Perry et al., 2005) during
which the extent of gas transfer across the air–water interface was
quantified and found to be negligible.
Once fish were breathing air at regular intervals and had been doing so for
at least 1 h, baseline (`pre') measurements of
O2,
CO2, ventilation
frequency, net acid excretion and blood acid–base status were carried
out over the course of 1 h. The water and air chambers were sealed after
stopping the flow of media, and the re-circulating pump was started to provide
mixing. Changes in aerial PO2 and PCO2
were monitored for two 30-min periods, between which the air within the
chamber was refreshed. Changes in aquatic PO2 and
PCO2 were monitored for approximately 30 min or until
stable rates of CO2 accumulation and O2 depletion were
achieved. In addition to the automatic detection of air breaths,
water-breathing frequency was determined by visually counting water breaths at
periodic intervals. To measure net acid excretion, 20 ml water samples were
withdrawn at the beginning and end of the 1–h flux period. A 0.5 ml
blood sample was withdrawn at the end of the measurement period. The blood
sample was centrifuged and plasma was removed for immediate analysis of pH and
total CO2 content (CCO2). The remaining red blood cells
were re-suspended in heparinized (50 i.u. ml–1 ammonium
heparin; Sigma, Oakville, ON, Canada) Cortland saline and re-injected into the
fish. At the end of this measurement period, the respirometer was returned to
a flow-through condition and acid or base infusion was initiated.
Lungfish were infused with acid (0.9 mol l–1
NH4Cl; N=11) or base (0.9 mol l–1
NaHCO3; N=8) at a rate of 3 mmol kg–1
h–1 for 1 h. A syringe pump (SAGE instruments 355) was used
to infuse acid or base via the dorsal aortic cannula. Once acid or
base infusion had been initiated, the water and air chambers were sealed and
the series of measurements described above was repeated, culminating with the
withdrawal of a blood sample immediately following the infusion period. Blood
samples were also withdrawn at 3, 6 and 18 h post-infusion, and were preceded
in each case by the same series of measurements of
O2,
CO2, ventilation
frequency and net acid excretion. After withdrawal of the 18 h blood sample,
lungfish were anaesthetized in situ by intra-arterial injection of
400 mg kg–1 of sodium pentobarbital (Somnotol), and then
sacrificed by an intra-arterial injection of saturated KCl. A final
measurement period for
O2,
CO2 and net acid
excretion was carried out over 45 min to estimate background levels of gas
transfer and net acid excretion. These background values were assumed to
reflect cutaneous metabolism (which they may underestimate) and/or metabolism
arising from microorganisms in the water or on the surface of the fish
(Perry et al., 2005). For each
fish, the values for
O2,
CO2 and net acid
excretion were corrected for background metabolism by subtracting the rates
determined in this final respirometry/flux period.
Series 2. Partitioning of net acid excretion between gills/skin and kidney in base-infused fish
Lungfish (N=7) were placed into the customized respirometry
chambers and experiments were not initiated until at least 1 h of breathing
air at regular intervals had occurred. Fish used in these experiments were
fitted with both a dorsal aortic cannula (for base infusion; blood samples
were not collected in these experiments) and an external urinary catheter.
Urine was collected continuously throughout the experimental period by
allowing the urinary catheter to drain, by gravity, into a vial located
outside the respirometer and held approximately 5 cm below water level. The
urinary catheter was checked for leaks by raising the catheter 5 cm above
water level. Under these conditions, a fall of the urine level in the catheter
indicated a leak, and the experiment was terminated. First, a flux period was
carried out to determine baseline (`pre') levels of net acid excretion into
water and urine, as well as urine flow rate (UFR), pH and CCO2.
Water flow to the respirometer was halted, the chamber was sealed and the
re-circulating pump was started to provide mixing. Water samples (20 ml) were
collected at the beginning and at 3 h of the 4 h flux period. To ensure that
the dead space volume within the urinary catheter was cleared, urine collected
during the initial 60 min of the flux period was discarded. At the end of
the flux period, water flow to the respirometer was re-established and base
infusion was initiated. Base (0.9 mol l–1 NaHCO3)
was infused into the dorsal aortic cannula, using a syringe pump, at a rate of
3 mmol kg–1 h–1 for 1 h. Flux periods were
carried out over the initial 4 h following base infusion, and from 17–21
h post base infusion.
Series 3. The impact of acid or base infusion on gene expression
Following recovery from surgery, lungfish fitted with a dorsal aortic
cannula were infused intra-arterially with saline (Cortland saline, 0.5 ml
h–1; N=3), acid (0.9 mol l–1
NH4Cl; N=8) or base (0.9 mol l–1
NaHCO3; N=7) at a rate of 3 mmol kg–1
h–1 for 1 h using a syringe pump. Lungfish were sacrificed 4
h (N=8) or 8 h (N=7) after the acid or base infusion period,
and gill and kidney tissue samples were collected, flash frozen in liquid
N2 and stored at –80°C for later analysis of gene
expression. Saline-infused fish were sampled 8 h after the infusion period to
provide control data.
Analytical procedures
For respirometry, PCO2 electrodes were connected to a
blood gas analyzer (Cameron Instruments BGM200, Port Aransas, TX, USA) that
was customized to accept two CO2 inputs. Output from the blood gas
analyzer and the infrared air-breath detector was converted to digital data
and stored by interfacing with a data acquisition system (Biopac Systems Inc.,
Harvard Apparatus Canada, Saint-Laurent, QC, Canada) using AcknowledgeTM
data acquisition software (sampling rate set at 30 Hz) and a PC. Output from
the fibre-optic O2 electrodes was collected using Ocean Optics
software running on the same PC. These data were compiled as text files for
later importation into spreadsheet software for storage and analysis. To
calibrate the fibre-optic O2 electrode used for water
PO2 measurements, the electrode was immersed in zero
solution (2 g l–1 sodium sulphite) or air-saturated water
until stable readings were recorded. The fibre-optic O2 electrode
used for air PO2 measurements was calibrated in
situ in the respirometer by flowing humidified N2 gas (zero)
or air continuously through the air chamber. Both CO2 electrodes
were calibrated in situ using mixtures of 0.5% and 1.0%
CO2 in air that were provided by a gas mixing flowmeter (Cameron
Instruments GF-3/MP). Humidified gas mixtures flowing through the air chamber
of the respirometer were used to calibrate the air PCO2
electrode, whereas water equilibrated with the gas mixtures and pumped through
the cuvette was used to calibrate the CO2 electrode used for water
PCO2 measurements.
Rates of aerial gas transfer were determined from the slopes of the
relationships between inspired gas tensions and time, over the period that the
air chamber was sealed, taking into account air chamber volume, fish mass, and
the solubility coefficients of O2 and CO2 in air at
25°C (Boutilier et al.,
1984). Similarly, aquatic gas transfer was calculated using the
slopes of the relationships between water gas tensions and time over the
interval that the water in the respirometer was re-circulated, taking into
account water chamber volume, and fish mass. For O2, the solubility
coefficient in fresh water at 25°C was obtained from Boutilier et al.
(Boutilier et al., 1984). For
CO2, the capacitance coefficient in dechloraminated city of Ottawa
tap water at 25°C was determined experimentally to be 0.041 µmol
l–1 mmHg–1 by measuring the total
CO2 concentrations (Cameron Instruments Capni-Con 5) of water
samples equilibrated to the range of PCO2 values
encountered in respirometry trials.
Blood samples were centrifuged (10 000 g for 1 min)
immediately following withdrawal to yield plasma. Plasma total CO2
concentration was determined in duplicate on 50 µl samples (Cameron
Instruments Capni-Con 5). Plasma pH was measured using a pH electrode and
calomel reference (Analytical Sensors E301 glass pH electrode) that were
housed in a temperature-controlled, low-volume pH chamber (Cameron
Instruments) and connected to a PHM 72 acid–base analyzer (Radiometer).
The arterial blood PCO2 (PaCO2) and
bicarbonate concentration ([HCO –3]) were then
calculated from the Henderson–Hasselbalch equation using appropriate
values for 
CO2 and pK'
(Boutilier et al., 1984).
Net acid excretion (JnetH+) was determined
from measurements of titratable net acid flux (JnetTA) and
the change in ammonia concentration in the water samples collected at the
beginning and end of a flux period. Water JnetTA and
ammonia concentration were assessed within 24 h of sample collection and the
remainder of the water sample was frozen for later analysis of ion
concentrations. JnetTA was determined by titrating (using
a Gilmont precision microburet) 5 ml water samples from the beginning and end
of each flux period to pH 4.00 with 0.02 mol l–1 HCl. Samples
were continuously aerated prior to and during titration to ensure mixing and
removal of CO2. A micro-modification of the
salicylate–hypochlorite colorimetric assay of Verdouw et al.
(Verdouw et al., 1978) was
used to measure total ammonia levels in water samples.
JnetH+ was then calculated as the sum of
JnetTA and the ammonia flux
(JnetNH3), signs considered, as described by
McDonald and Wood (McDonald and Wood,
1981).
Net renal acid excretion was also determined as the sum of titratable net
acid flux and ammonia efflux (Wood and
Caldwell, 1978). Urinary JnetTA was measured
by lowering the pH of a 200 µl aliquot of urine below 5.0 through the
addition of a known volume of 0.02 mol l–1 HCl. The sample
was then aerated for 20 min to remove CO2. While continuing to
aerate, the pH of the urine sample was titrated back to the pH of blood
representative of the particular sampling period through the addition of 0.02
mol l–1 NaOH using a precision microburet (Gilmont). The
titratable component of net renal acid excretion is given by the difference in
the quantities of acid and base added to the urine. Urine flow rates were
determined gravimetrically. As with water samples, urine
JnetTA and ammonia concentration were assessed within 24 h
of sample collection together with urine pH and CCO2. Urine ammonia
concentration, pH and total CO2 concentration were assessed using
the procedures described above for water or blood. For the calculation of
urine [HCO –3], constants derived for freshwater
were employed (Boutilier et al.,
1984).
Molecular cloning and analysis of lungfish carbonic anhydrase, Na+/HCO3– cotransporters and H+ V-ATPase
Cloning procedures
Tissues for the cloning of lungfish CA, NBC and H+ V-ATPase were
collected from fish that were terminally anaesthetized by immersion in a
solution of MS-222 (see above). Total RNA was isolated from a given tissue
using Trizol (Invitrogen, Burlington, ON, Canada) according to the
manufacturer's instructions. RNA quantity and quality were verified by
spectrophotometry (Eppendorf BioPhotometer) and/or by inspection of an RNA
gel. First strand cDNA was synthesized from RNA using Superscript reverse
transcriptase (Invitrogen) and an oligo(dT) primer (Sigma). Gene-specific
primers (Table 1) were then
used to amplify cDNA fragments by PCR. PCR was performed using 1 µl of cDNA
template in 25 µl reaction mixture containing 3.5 mmol l–1
MgCl2, 200 µmol l–1 of each dNTP, 250 nmol
l–1 each of forward and reverse primers, and 1 i.u. Taq
polymerase (New England Biolabs, Toronto, ON, Canada) in PCR buffer supplied
with the enzyme. All PCR reactions involved an initial denaturation at
94°C for 3 min followed by 39 cycles of 94°C for 30 s, annealing
temperature for 30 s, 72°C for 60 s, and ending with a final extension for
10 min at 72°C. PCR products obtained in this manner were gel-purified,
cloned into pCR2.0-TOPO vector (TOPO TA cloning kit, Invitrogen) and
sequenced. The resulting sequences were compared against those in the GenBank
database using BLASTX to identify the gene, and also to design primers (where
needed) for 3'- and 5'-rapid amplification of cDNA ends (RACE) to
extend the amplified sequence lengths. For 3'-RACE, cDNA was synthesized
using a 3'-RACE adapter primer (Invitrogen) and Superscript II reverse
transcriptase (Invitrogen). Semi-nested PCR was performed on the cDNA using
abridged universal amplification primers (AUAP; Invitrogen) and gene-specific
3'-RACE primers (Table
1). For 5'-RACE, cDNA was synthesized using a gene-specific
primer (Table 1) and
Superscript II reverse transcriptase (Invitrogen), and then purified using a
PCR purification kit (Sigma). The newly purified cDNA was tailed with dCTP
using a recombinant terminal transferase TdT (Invitrogen) with final reaction
conditions; 10 mmol l–1 Tris–HCl (pH 8.4), 25 mmol
l–1 KCl, 1.5 mmol l–1 MgCl2, 200
µmol l–1 dCTP, 1 µl cDNA, and 1 µl rTdT. The tailed
cDNA was then used for two rounds of PCR using a gene-specific forward primer
(Table 1) and a 5'
abridged anchor primer (5AP; Invitrogen) for the first round, and a
semi-nested gene-specific forward primer
(Table 1) and AUAP (Invitrogen)
for the second round. PCR products were cloned into the pCR2.0-TOPO vector
using TOPO TA cloning kits (Invitrogen). All RACE product sequences were
confirmed by overlap with the initial fragment of cDNA. After repeated
bi-directional sequencing of both RACE products, a consensus sequence was
created by multiple sequence alignment using DNAMAN (v4.0, Lynnon Biosoft,
Vaudreuil, QC, Canada).
|
Using red blood cell cDNA as template, a 315 base pair (bp) internal segment of a CA coding region (denoted lfCAb) was PCR amplified at an annealing temperature of 46°C using the primer pair CA (Table 1). These degenerate primers were designed based on alignments of vertebrate cytoplasmic CA isoforms exhibiting a high degree of amino acid conservation. The sequence was then extended by three rounds of 3'-RACE using the gene-specific primers CA3R1, CA3R2 and CA3R3.
A 665 bp internal segment of the coding region of NBC was PCR amplified at
an annealing temperature of 57°C using cDNA derived from lungfish gill
tissue and the primer pair NBC. These primers corresponded to the nucleotides
at positions 1492–1512 and 2183–2203 of the tiger salamander
(Ambystoma tigrinum) kidney NBC (GenBank accession no. AF001958)
(Romero et al., 1997). The
resultant clone, lfNBC, was deemed of sufficient length for the purposes of
the present study and therefore no attempt was made to extend the sequence
length using RACE.
Using lungfish gill tissue, an internal segment of the coding region of the
B subunit of V-type H+-ATPase (denoted lfH+ V-ATPase)
was PCR amplified using the primer pair HV1 at an annealing temperature of
50°C. These primers, which were used successfully by Perry et al.
(Perry et al., 2000) to clone
the rainbow trout V-type H+-ATPase B subunit, correspond to the
nucleotides at positions 441–461 and 1395–1415 of the bovine
kidney V-type H+-ATPase B subunit sequence (GenBank accession no.
M88691) (Nelson et al., 1992).
A second round of PCR was required to obtain an 810 bp fragment. This PCR
round used the primer pair HV2 at an annealing temperature of 49°C. The
new forward primer corresponded to the nucleotides at positions 606–626
of the bovine kidney H+ V-ATPase. The sequence length was then
further extended by two rounds of 3'-RACE using gene-specific primers
HV3R1 and HV3R2, and by 5'-RACE. For 5'-RACE, the gene-specific
primer HV5R1 was used for cDNA synthesis. Two rounds of 5'-RACE were
then carried out using gene-specific primers HV5R2 and HV5R3.
|
) with
penalties for gap opening and gap extension set to 30 and 0.75, respectively,
for pairwise alignments, and 15 and 0.3, respectively, for multiple
alignments. Note that full-length sequences from other vertebrates were
truncated appropriately so as to compare only regions overlapping with the
lungfish sequences. The PHYLIP package was then used to carry out
neighbour-joining phylogenetic analysis
(Saitou and Nei, 1987) on a
matrix of mean character distances, with a bootstrapping resampling option to
assess the support for nodes (100 pseudoreplicates). In general, default
parameters were used except that the outgroup was specified (as
Drosophila) and the input order of species was randomized where
possible. The accession numbers for sequences used in alignments or
phylogenetic analysis are presented in the appropriate figure legends.
|
) with lungfish
18S mRNA expression as a normalizing factor.
|
|
Results |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Rapid correction of the acid–base disturbances was accomplished, at
least in part, through adjustment of pulmonary and branchial ventilation
frequencies (Figs 2 and
3). Acid-infused lungfish
(Fig. 2) approximately doubled
both air (1.8-fold) and water (1.7-fold) breathing frequencies, thereby
increasing total CO2 excretion
(CO2) 2.4-fold.
This increase in total
CO2 was
accomplished through a doubling of CO2 transfer to water together
with a 4.8-fold increase in aerial CO2 excretion, illustrating the
fact that P. annectens is capable of effective CO2
excretion via the lungs, even though under control conditions aquatic
CO2 transfer accounted for 78.2±4.6% (N=18) of
total CO2. By
contrast with the enhancement of CO2 excretion, O2
uptake (O2) was
unaffected by acid infusion, causing the respiratory exchange ratio (RER) to
increase, although not significantly (Table
2). Air-breathing frequency and consequently aerial CO2
transfer were unaffected by the infusion of a base load
(Fig. 3), whereas
water-breathing frequency in base-infused lungfish was halved
(Fig. 3), accounting for the
2.6-fold decrease in total
CO2 and driving
the RER value down significantly (Table
2).
|
Adjustment of the net excretion of acidic equivalents into the water (JnetH+) was also important in correcting acid–base disturbances, particularly following the infusion of a base load (Fig. 4). Acid infusion resulted in a significant 5.7-fold increase in net ammonia excretion (JnetNH3). However, owing to more variable titratable acid net flux (JnetTA) data in which only seven of 11 fish increased JnetTA in response to acid infusion, there was no significant increase in the net excretion of acidic equivalents. By contrast, both JnetH+ and JnetTA values were strongly negative following base infusion, indicating that net excretion of base to the water occurred; net ammonia excretion was unaffected by base infusion.
|
|
|
|
Because the responses of lungfish to acid–base disturbances included
changes in the net excretion of acid–base equivalents via the
gills and kidneys, the expression of mRNA of three genes thought to be
involved in both renal and branchial mechanisms of acid–base excretion
was assessed. Using homology cloning strategies, a 683 base pair cDNA was
assembled from lungfish gill tissue. This cDNA included a deduced protein
fragment of 226 amino acid residues. A BLAST search of the GenBank protein
database determined that this lungfish gene shared high sequence identity with
several vertebrate NBCs, and in particular, NBC1. Protein alignment revealed
that the lungfish NBC fragment (dubbed lfNBC) corresponded to residues
498–723 of the human NBC (GenBank acc. no. AAG47773), and was
73–77% identical to amphibian and mammalian NBC1s, and 65–67%
identical to NBCs from other fish (Fig.
7A). A second cDNA, of 921 base pairs, was assembled from lungfish
gill tissue and included a deduced protein fragment of 307 amino acid residues
that was found to share high sequence identity with the B subunit,
particularly the type 2 B subunit, of the V-type H+-ATPase from
several vertebrates. Using protein alignment, the lungfish H+
V-ATPase (lfVATPase) corresponded to residues 195–511 of the human B2
subunit (GenBank acc. no. CAA44721) and was 
95% identical and 98%
similar to B subunits from a range of vertebrates including fish, amphibians,
birds and mammals (Fig. 7B).
Finally, degenerate primers were used to obtain a 663 base pair cDNA from
lungfish blood (Fig. 8). This
cDNA encoded a deduced protein fragment of 221 amino acid residues. The
collective evidence of a BLAST search of the GenBank protein database, protein
alignment and phylogenetic analysis suggests that this lungfish gene is a
cytoplasmic carbonic anhydrase (CA) that, based on its tissue of origin, was
labelled lfCAb. Phylogenetic analysis grouped lfCAb with fish cytoplasmic
(blood-specific and general cytoplasmic) isoforms as well as mammalian CA I,
II, III and XIII. Similarly, lfCAb was found to be 61–65% identical and
77% similar to blood CA isoforms from zebrafish, trout, carp and gar (GenBank
protein acc. no. NP_571185, AAP73748, AAZ83748 and AAM94169, respectively) as
well as 60% identical and 78% similar to mouse and rat CA XIII (NP_078771 and
XP_574890, respectively). Examination of the mRNA expression for these three
lungfish genes, lfNBC, lfVATPase and lfCAb, in gill and kidney tissue 4 h and
8 h following a 1 h infusion of acid or base did not reveal any statistically
significant changes (Fig.
9).
|
|
|
|
Discussion |
|---|
|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
).
By contrast, metabolic compensation of pH imbalances in P. annectens
relies predominantly on the exchange of acid–base equivalents across the
gills (and/or skin, although with a possible role for the intestine) rather
than on urinary excretion of acid–base equivalents, a strategy that is
distinctly fish-like (for a review, see
Perry and Gilmour, 2006).
Because CO2 excretion and net H+ excretion were not
monitored continuously following acid or base infusion, it was not possible to
accurately quantify the relative contributions of respiratory and metabolic
mechanisms to acid–base compensation. However, estimates can be obtained
by examination of the 1 h periods of greatest excretion (`peak' values),
providing at least a snapshot of the relative contributions of the different
mechanisms. Lungfish received an acid or base load of 3 mmol
kg–1. Following acid infusion, CO2 excretion into
air and water peaked at 0.62 mmol kg–1 above the pre-infusion
value, while net H+ excretion peaked at 0.18 mmol
kg–1 in excess of the pre-infusion value. Thus, peak
CO2 and H+ excretion (for a 1 h period) amounted to a
loss of 27% of the total acid load, with respiratory compensation accounting
for 77% of this value, and metabolic compensation, 23%. Respiratory and
metabolic contributions were more balanced following a base load, accounting
for 46% and 54%, respectively, of the 0.91 mmol kg–1 of the
base load that was lost during the 1 h period of greatest excretion (30% of
the total base load).
The compensatory strategies of P. annectens reflect the unique
opportunities available to bimodally breathing animals that inhabit an aquatic
environment. Yet despite the opportunity afforded by such animals to
understand acid–base strategies, few studies have investigated, and none
appear to have attempted to quantify, the relative involvement of respiratory
versus metabolic compensatory mechanisms in bimodal breathers
(reviewed by Truchot, 1987).
The involvement of air breathing in the recovery from acid–base
disturbances elicited by exhaustive exercise was assessed for two fish species
that are facultative air-breathers, bowfin Amia calva and spotted gar
Lepisosteus oculatus (Burleson et
al., 1998; Gonzalez et al.,
2001). Both studies concluded that air breathing delayed
compensation of the post-exercise acid–base disturbance because
branchial ventilation was not increased to the extent that occurs in unimodal
water breathers, thereby hindering branchial exchange of acid–base
equivalents and CO2 elimination, processes critical for the
restoration of acid–base balance
(Burleson et al., 1998;
Gonzalez et al., 2001).
Neither study, however, assessed metabolic compensation post-exercise. The
blood acid–base responses of lungfish in the present study were very
comparable to those of channel catfish, a unimodal water-breather, subjected
to virtually the same infusion protocol
(Cameron and Kormanik, 1982),
implying that air-breathing in lungfish did not appear to delay recovery.
Indeed, CO2 loss via the lungs of P. annectens
should have aided recovery from an infused acid load, a difference from bowfin
and gar that may reflect the obligate nature of pulmonary gas transfer in
lungfish versus the facultative role it plays in bowfin and gar.
The best-studied bimodal breathers are amphibians, among which responses to
acid–base disturbances reflect the degree of terrestrialization of the
species (reviewed by Toews and Boutilier,
1986; Truchot,
1987; Toews and Stiffler,
1989). Amphibians that are predominantly aquatic rely on the
cutaneous exchange of acid–base equivalents to adjust extracellular pH
(reviewed by Stiffler, 1989).
By contrast, semi-terrestrial amphibians with well-developed lung ventilation,
such as adult anurans, exhibit respiratory compensation supported to a
variable extent by the exchange of acid–base equivalents via
the kidney/bladder or skin (e.g. Boutilier
et al., 1979; Tufts and Toews,
1985; Boutilier and Heisler,
1988; Stiffler,
1991).
The use of ventilatory adjustments to compensate for acid–base
disturbances by semi-terrestrial but not predominantly aquatic amphibians
parallels the situation in fish – water-breathing fish do not appear to
modulate ventilation to correct pH imbalances (for a review, see
Perry and Gilmour, 2006),
whereas P. annectens clearly utilized respiratory compensation (Figs
2 and
3). The implication of this
finding is that P. annectens, like tetrapod vertebrates, possesses
CO2 and/or pH-sensitive chemoreceptors that monitor the status of
the body fluids. In tetrapods, central and peripheral
CO2/pH-sensitive chemoreceptors initiate ventilatory adjustments
for acid–base regulation (reviewed by
Gonzalez et al., 1994;
Milsom, 1995;
Milsom, 2002;
Taylor et al., 1999). By
contrast, the balance of evidence suggests that strictly water-breathing fish
lack internally oriented CO2/pH chemoreceptors – central
chemoreceptors appear to be absent, and ventilatory chemoreflexes are
dominated by branchial chemoreceptors that respond primarily to changes in
water CO2 (reviewed by Gilmour
and Perry, 2007). Although the locations of CO2/pH
chemoreceptors in P. annectens remain to be determined, work on the
South American lungfish Lepidosiren paradoxa suggests the existence
of both central and peripheral CO2 and/or pH chemoreceptors
(Sanchez et al., 2001;
Amin-Naves et al., 2007). In
Lepidosiren, pulmonary ventilation increased as cerebrospinal fluid
(CSF) pH was lowered by perfusing the fourth cerebral ventricle with mock CSF,
providing evidence of central CO2/pH chemosensitivity
(Sanchez et al., 2001).
Similar manipulation of CSF pH moderated but did not eliminate pulmonary
ventilation responses to (combined) aerial and aquatic hypercapnia, a result
that supports the existence of peripheral CO2/pH chemoreceptors in
Lepidosiren but without providing information on their orientation
(water/air versus blood)
(Amin-Naves et al., 2007).
Interestingly, the data for P. annectens indicate that branchial as
well as pulmonary ventilation is sensitive to internal acid–base status,
a possibility that was not examined in the studies on Lepidosiren.
Similarities (e.g. in acid–infused lungfish;
Fig. 2) and differences (e.g.
in base-infused lungfish; Fig.
3) between pulmonary and branchial ventilatory responses to
acid–base disturbances suggest that both shared and independent control
systems are present.
Although the modulation of ventilation to correct acid–base
disturbances, and the underlying implication that internal CO2/pH
sensors must be present evokes a tetrapod scenario of acid–base
regulation for P. annectens, the primary reliance on branchial
(and/or cutaneous) rather than renal routes for the exchange of
acid–base equivalents is typical of fish and predominantly aquatic
amphibians. In P. annectens, partitioning of the peak excretion of a
base load into branchial/cutaneous versus renal contributions
revealed that 62% was eliminated into the water, and 38% into urine. Urine is
similarly a minor route of net acid efflux during acid–base disturbances
in fish (Kobayashi and Wood,
1980; Wood and Jackson,
1980; Cameron and Kormanik,
1982; Wood, 1991;
Curtis and Wood, 1992) and
aquatic amphibians (Stiffler and Bachoura,
1991; Stiffler,
1991; Talbot and Stiffler,
1992), with extra-renal excretion of acid–base equivalents
playing a dominant role. In fish, up to 90% or more of acid–base
movements occur across the branchial epithelium
(Claiborne et al., 2002;
Evans et al., 2005), whereas
cutaneous acid–base excretion appears to predominate in aquatic
amphibians (Stiffler, 1989).
These widely accepted views are based primarily on morphological
considerations rather than on explicit attempts to partition extra-renal net
acid efflux into branchial versus cutaneous components. For example,
the role of the gill in correcting pH imbalances in fish is strongly supported
by the abundance of ion-transporting MR cells in the branchial epithelium
coupled with remodelling of this epithelium in response to acid–base
disturbances (reviewed by Goss et al.,
1992; Goss et al.,
1994; Goss et al.,
1995; Laurent and Perry,
1995; Perry and Gilmour,
2006). MR cells are found in both the branchial epithelium and
skin of P. annectens (Sturla et
al., 2001), indicating that either surface could contribute to the
movement of acid–base equivalents, but the gills may be more likely to
play a larger role. Whereas the skin is covered by a protective coating of
scales, the gills are actively ventilated and involved in CO2
excretion, as evidenced by the relationship between ventilation frequency and
CO2 (Figs
2 and
3), and the hydration of
CO2 provides H+ and HCO –3
for exchange with the environment (see below). However, whether the branchial
epithelium of P. annectens is a more important contributor to
metabolic acid–base regulation than the skin requires empirical
confirmation.
The molecular mechanisms involved in the movement of acid–base
equivalents across the gills of freshwater fish remain a subject of debate.
Recent models (reviewed by Perry and
Gilmour, 2006) suggest that acid secretion reflects the combined
actions of apical membrane H+ efflux, probably via a
V-type H+-ATPase in freshwater fish, and basolateral membrane HCO
–3 efflux by means of Cl–/HCO
–3 exchange and/or NBC. Mechanisms for base
excretion are probably located in a different cell type, and involve apical
membrane HCO –3 efflux via
Cl–/HCO –3 exchange coupled to
basolateral H+ efflux by a V-type H+-ATPase. In both
cases, the hydration of molecular CO2 catalyzed by a high activity
cytosolic CA provides the necessary protons and HCO
–3 ions. Several components of these mechanisms,
specifically NBC, V-type H+-ATPase, and cytosolic CA, were cloned
from lungfish tissues (Figs 7
and 8), but no evidence of
changes in mRNA expression was detected in the gills following acid or base
infusion (Fig. 9). This result
was somewhat surprisingly given the considerable support for transcriptional
regulation of branchial proteins during acid–base disturbances in other
fish (Galvez et al., 2002;
Hirata et al., 2003;
Perry et al., 2003a;
Perry et al., 2003b;
Georgalis et al., 2006b). It
is possible that the acid–base challenges were not sufficient to
activate transcriptional regulation, that isolation of discrete cell types
(acid versus base excreting) is necessary to detect such changes, or
that post-translational mechanisms are involved, as recently reported for
dogfish (Tresguerres et al.,
2005; Tresguerres et al.,
2006). Alternatively, the sampling times (4 and 8 h post infusion)
and/or animal numbers may not have been appropriate or sufficient to detect
changes that did occur; unfortunately, these were constrained by animal
availability.
The kidney of P. annectens, although quantitatively less important
than the gill/skin, nevertheless played a significant role in eliminating an
infused base load, a finding that is true for other freshwater fish
(Cameron and Kormanik, 1982;
Curtis and Wood, 1992;
Wood et al., 1999). Moreover,
the freshwater teleost kidney exhibits a flexibility of response that is
comparable to that of the mammalian kidney
(Wood et al., 1999), a
statement that arguably can also be applied to the lungfish. Urine flow rate
in P. annectens in the present study, at 6 ml
kg–1 h–1, was within the range of values
reported previously for Protopterus sp., 2–6 ml
kg–1 h–1
(Sawyer, 1965;
Sawyer, 1970;
Babiker and Rankin, 1979),
although somewhat higher than values for rainbow trout (Oncorhynchus
mykiss) or channel catfish (Ictalurus punctatus), 2–4
ml kg–1 h–1
(Cameron and Kormanik, 1982;
Curtis and Wood, 1992;
Wood et al., 1999). This
result is surprising in view of the reduced gill surface area of lungfish, and
may indicate a degree of stress-induced diuresis
(Babiker and Rankin, 1979). A
high control value may also have masked any tendency for metabolic alkalosis
to increase urine flow rate, as observed previously in trout and catfish
(Cameron and Kormanik, 1982;
Curtis and Wood, 1992;
Wood et al., 1999). As in
other freshwater fish as well as mammals
(Cameron and Kormanik, 1982;
Curtis and Wood, 1992;
Wood et al., 1999), the renal
response of P. annectens to a metabolic alkalosis was characterized
by greatly elevated urinary HCO –3 loss
(Fig. 6), presumably reflecting
increased HCO –3 filtration that was not matched
by a corresponding increase in tubular HCO –3
reabsorption.
The mechanisms involved in renal HCO3–
reabsorption appear to be similar in freshwater fish and mammals
(Perry et al., 2003b;
Perry and Gilmour, 2006;
Georgalis et al., 2006a). In
mammals, filtered HCO –3 ions combine with protons
secreted via apically localized Na+/H+
exchangers (NHE3) and/or V-type H+-ATPase. Molecular CO2
formation is catalyzed by membrane-associated CA IV; CO2 diffuses
into the tubule cell and is hydrated in the presence of cytosolic CA. While
protons are recycled into the tubule lumen, HCO –3
ions move across the basolateral membrane via NBC1, resulting in a
net transfer of HCO –3 ions
