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First published online November 17, 2006
Journal of Experimental Biology 209, 4701-4716 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02564
A critical analysis of carbonic anhydrase function, respiratory gas exchange, and the acid-base control of secretion in the rectal gland of Squalus acanthias
1 Department of Pharmacology and Physiology, University of Rochester School
of Medicine and Dentistry, Rochester, NY 14642, USA
2 Bamfield Marine Sciences Centre, 100 Pachena Drive, Bamfield, British
Columbia, VOR 1BO, Canada
3 Canadian Nuclear Safety Commission, PO Box 1046, Station B, 280 Slater
Street, Ottawa, Ontario, K1P 5S9, Canada
4 Department of Biology, McMaster University, 1280 Main St. West, Hamilton,
Ontario, L8S 4K1, Canada
* Author for correspondence (e-mail: woodcm{at}mcmaster.ca)
Accepted 27 September 2006
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and
increased in linear relation to increases in Cl- secretion rate. CA
inhibition (10-4 mol l-1 acetazolamide) had no effect on
Cl- secretion rate or pHi in the perfused gland, in contrast to
in vivo, but caused a transitory 30% inhibition of
(relative to stable
) and
elevation in secretion PCO2 effects, which
peaked at 2 h and attenuated by 3.5-4 h. Secretion was inhibited by acidosis
and stimulated by alkalosis; the relationship between relative Cl-
secretion rate and pHe was almost identical to that seen in vivo.
Experimental manipulations of perfusate pH,
PCO2 and HCO3-
concentration, together with measurements of pHi, demonstrated that these
responses were most strongly correlated with changes in pHe, and were not
related to changes in PCO2, extracellular
HCO3-, or intracellular HCO3-
levels, though changes in pHi may also have played a role. The acid-base
status of the secreted fluid varied with that of the perfusate, secretion pH
remaining about 0.3-0.5 units lower, and changing in concert with pHe rather
than pHi; secretion HCO3- concentrations remained low,
even in the face of greatly elevated perfusate HCO3-
concentrations. We conclude that pH effects on rectal gland secretion rate are
adaptive, that CA functions to catalyze the hydration of CO2,
thereby maintaining a gradient for diffusive efflux of CO2 from the
working cells, and that differences in response to CA inhibition likely
reflect the higher perfusion-to-secretion ratio in vitro than in
vivo.
Key words: chloride secretion, O2 consumption, CO2 excretion, gas exchange ratio, pHi, pHe, acidosis, alkalosis, shark, acetazolamide
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;
Burger, 1962), who demonstrated
that the rectal gland of the intact dogfish shark (Squalus acanthias)
secreted an almost pure NaCl solution at a concentration of approximately 500
mmol l-1, almost twice the NaCl level in blood plasma, but close to
isosmotic with seawater and the urea-rich plasma. Subsequently, the gland has
been used at many levels as a powerful in vitro model for
understanding the mechanism and control of NaCl secretion. These analyses have
included the use of in situ preparations in pithed, artificially
ventilated dogfish, isolated-perfused whole gland preparations, perfused
rectal gland tubules, cultured tubular epithelial cells, and membrane vesicles
of the tubules (reviewed by Epstein et al.,
1983; Shuttleworth,
1988; Riordan et al.,
1994; Silva et al.,
1990; Silva et al.,
1996; Silva et al.,
1997; Olson, 1999;
Hazon et al., 2003). The
isolated-perfused rectal gland preparation, first developed by Hayslett et al.
(Hayslett et al., 1974) and
subsequently improved by many others, has been a particularly useful approach,
in which secretion can be directly stimulated using forskolin
(Moran and Valentich, 1991) to
activate the adenyl cyclase pathway (e.g.
Forrest, Jr et al., 1997;
Walsh et al., 2006).
The following model has emerged. The oral intake of seawater or salty food
during feeding (Mackenzie et al.,
2002) is assumed to cause volume expansion in vivo
(Solomon et al., 1984a;
Solomon et al., 1984b;
Solomon et al., 1985). The
latter is known to mobilize C-type natriuretic peptide (CNP) from the heart
(Silva et al., 1987), which
stimulates increased rectal gland blood flow and secretion, by both direct and
indirect mechanisms. The direct action seems to involve activation of both
protein kinase C and guanyl cyclase signalling pathways
(Silva et al., 1999). However,
at least in Squalus acanthias, there is also a very potent indirect
action, by which CNP causes the release of vasoactive intestinal polypeptide
(VIP) from nerve endings in the rectal gland, and VIP in turn activates the
adenyl cyclase pathway (Stoff et al.,
1979; Stoff et al.,
1988; Silva et al.,
1987). A cascade of events (reviewed by
Silva et al., 1997) is
initiated by the various intracellular signals, in which apical CFTR-like
channels are activated for Cl- extrusion, together with basolateral
Na+,K+,2Cl- co-transporters (NKCC),
Na+,K+-ATPase and K+ channels. The
Na+ electrochemical gradient powers the NKCC-mediated entry of both
Cl- and K+. These ions exit via their
respective apical and basolateral channels, while Na+ is secreted
in equimolar amounts to Cl- through a paracellular pathway, driven
by the negative potential created by apical Cl- extrusion. The net
NaCl transport into the tubule entrains an isosmotic flux of water.
The rectal gland contains high carbonic anhydrase (CA) activity
(Maren, 1967;
Lacy, 1983) but its function
has never been satisfactorily integrated into this model. Several in
vitro studies with isolated-perfused glands have reported a complete lack
of effect of CA inhibition on secretory performance
(Siegel et al., 1975;
Silva et al., 1977;
Swenson and Maren, 1984).
However, using an in situ preparation in a pithed, artificially
ventilated whole dogfish, Swenson and Maren
(Swenson and Maren, 1984)
reported that CA inhibition reduced rectal gland secretion. They speculated
that the normal role of CA is to facilitate the diffusive excretion of
CO2 from the metabolically active gland cells, in accord with the
pioneering work of Gros in muscle tissue
(Gros et al., 1976;
Gros, 1991), but their results
may have been confounded by the loss of neural control and depressed
circulation in this preparation, as well as systemic effects of CA
inhibition.
Recently we developed a method for assessing rectal gland performance in
intact, unanaesthetized dogfish subjected to volume loading
(Wood et al., 2006).
Therefore, our first objective was to use this approach to further investigate
the role of CA in the gland by examining the effect of CA blockade with
acetazolamide in the intact animal on secretory performance, secretion
acid-base status, and intracellular pH in the rectal gland. We followed this
up with comparable studies on the effects of CA inhibition using the
isolated-perfused rectal gland preparation to separate gland-specific effects
from systemic effects of the CA blockade.
One advantage of the isolated-perfused preparation is that it allows the
direct collection of not only the secreted fluid itself, but also the outflow
through the rectal gland vein. Respiratory gas exchange by the secreting cells
can therefore be directly measured, though to our knowledge, this has only
been done for O2 uptake (e.g.
Silva et al., 1980), and not
for CO2 excretion. Our second objective was therefore to measure
both O2 and CO2 exchange of the perfused gland, as well
as the acid-base status of the secreted fluid, at rest, after activation with
forskolin, under various acid-base manipulations, and during blockade of CA
function with acetazolamide, to assess whether CA is involved in facilitating
the diffusive excretion of CO2, as suggested by Swenson and Maren
(Swenson and Maren, 1984).
Several early studies reported that acidic perfusates inhibited secretion
in the isolated-perfused gland in vitro
(Siegel et al., 1975;
Silva et al., 1992), and
Swenson and Maren demonstrated that severe metabolic or respiratory acidosis
inhibited secretion of their in situ gland preparation
(Swenson and Maren, 1984).
Recently, we have shown that the NaCl secretion rate of the rectal gland in
intact unanaesthetized dogfish is very responsive to blood acid-base status
in vivo, being stimulated by metabolic alkalosis, and inhibited by
both respiratory acidosis and metabolic acidosis in volume-loaded animals
(Wood et al., 2006). The
nature of the acid-base stimulus (i.e. extracellular or intracellular pH,
HCO3- level or PCO2) was
not determined in any of these investigations. Our final objective was
therefore to use the forskolin-stimulated isolated-perfused preparation
together with independent manipulations of perfusate
PCO2, HCO3- level and pH
to analyze the precise nature of the extracellular and/or intracellular
acid-base stimuli controlling secretion rate in the rectal gland.
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In vivo preparations
In order to economize on radioisotopes, the smallest animals (0.3-1.0 kg)
were selected for intracellular pH measurements, while larger dogfish (1.0-5.0
kg) proved more suitable for cannulation of the secretory duct of the rectal
gland. Dogfish were anaesthetized with MS-222 (0.1-0.2 g l-1;
Syndel Labs., Vancouver, BC, Canada), weighed, and irrigated on an operating
table. Dogfish intended for intracellular pH measurements (N=20) were
fitted with only an indwelling mesenteric artery catheter (PE50 polyethylene
tubing; Clay-Adams, Parsipanny, NJ, USA) by the method of Graham et al.
(Graham et al., 1990), whereas
those intended for secretion collections (N=13) were also fitted with
a rectal gland duct catheter (PE50 or PE90 depending on animal size) by the
method described (Wood et al.,
2006). The arterial catheter was filled with dogfish saline
[recipe as in Pärt et al. (Pärt
et al., 1998), but with the omission of PVP-40 and albumin]
containing 50 i.u. ml-1 sodium heparin (Sigma-Aldrich, St Louis,
MO, USA), and the rectal gland catheter with 500 mmol l-1 NaCl to
duplicate normal secretion composition. The animal was then revived and placed
in an individual PlexiglasTM chamber served with aeration and a constant
flow (>0.5 l min-1) of fresh seawater. Dogfish were allowed to
recover for at least 36 h before experiments commenced.
In vivo protocols
Secretion measurements
Rectal gland secretion flow proved to be negligible in these unfed resting
dogfish. Therefore the animals were infused via the arterial catheter
with 500 mmol l-1 NaCl at a rate of 15 ml kg-1
h-1 using a Minipuls peristaltic pump (Gilson, Middleton, WI, USA),
France). This created a brisk, stable secretory flow from the gland, reaching
a stable plateau from 1.5 to 6 h (cf. Wood
et al., 2006), against which the effects of carbonic anhydrase
inhibition could be assessed. The 1.5-3.0 h period served as the pre-treatment
control, and the experiment was terminated at 6 h. Control animals
(N=7) simply received the infusion alone for 6 h, whereas the
experimental animals (N=6) received 20 mg kg-1 of
acetazolamide (as the HCl salt, Wyeth-Lederle, Madison, NJ, USA) in 0.5 ml
kg-1 dogfish saline via the arterial catheter at 3 h, and
the experiment continued with 500 mmol l-1 NaCl infusion up to 6 h.
This acetazolomide dose was calculated to produce an in vivo
circulating concentration of about 10-4 mol l-1,
representing the level generally recognized as the maximum safe dose needed to
achieve significant CA inhibition while avoiding non-specific effects
(Maren, 1977). This
concentration has been commonly used to produce effective CA inhibition in
previous shark studies (Gilmour et al.,
1997; Perry et al.,
1999; Wilson et al.,
2000; Gilmour and Perry,
2004). Rectal gland secretion flow was collected over successive
0.5 h periods, quantified gravimetrically, analysed immediately for acid-base
status (pH and total CO2) and frozen at -20°C for later ionic
analysis. Blood samples (0.6 ml) were drawn from the arterial catheter at 1 h
intervals, and analysed immediately for arterial blood gases and pH, while
plasma was separated (2 min at 9000 g) and frozen (-20°C)
for later ion measurements. The red cell pellet was resuspended in dogfish
saline, combined with blood recovered from the electrodes, and the original
volume re-infused, so the impact of blood sampling on hematocrit was
minimal.
Intracellular pH and fluid volume measurements
Intracellular pH was measured in the rectal gland as well as in white
muscle (as a point of reference), together with extracellular acid-base status
and fluid volume distribution in these tissues, in three treatments. These
were unstimulated control (N=8), stimulated control (N=6),
and stimulated plus acetazolamide-treated (N=6). In the first
treatment, no infusion was given. The stimulated control (infusion with 500
mmol l-1 NaCl for 6 h) and stimulated acetazolamide treatments
(infusion with 500 mmol l-1 NaCl for 6 h with administration of 20
mg kg-1 of acetazolamide at 3 h) were identical to the comparable
treatments in the secretion measurement experiments.
The DMO (5,5-dimethyloxazolidine-2,4-dione) method
(Waddell and Butler, 1959) was
employed, using mannitol as the extracellular fluid volume marker. The two
radiolabelled compounds - [3H]mannitol (28 µCi kg-1,
specific activity 27.4 mCi mmol-1) and [14C]DMO (7
µCi kg-1, specific activity 50.0 mCi mmol-1), both
from Dupont-NEN, Boston, MA, USA - were administered via the arterial
catheter in 1 ml kg-1 of 500 mmol l-1 NaCl approximately
12 h prior to sampling to allow adequate equilibration time
(Munger et al., 1991).
Immediately prior to sacrifice, an arterial blood sample (1.0 ml) was drawn
for blood gas and pH measurements, with a portion of plasma immediately
separated by centrifugation (2 min at 9000 g) and divided into
aliquots for radioactivity measurements. The dogfish was then killed by a
sharp cephalic blow and spinal section. The rectal gland and a section of
epaxial muscle were immediately removed, blotted, weighed and processed for
radioactivity measurements as described below. The capsule was removed from
the rectal gland prior to processing.
In vitro perfused rectal gland preparation
Isolated-perfused rectal gland preparations (N=75) were made from
dogfish in the 1.5-3.5 kg range. Each dogfish was anaesthetized with MS-222
(0.2 g l-1), injected with 5000 i.u kg-1 of sodium
heparin (Sigma-Aldrich, St Louis, MO, USA) via the caudal haemal
arch, then killed by pithing with a steel wire inserted via the
rostrum through the brain and spinal cord. The rectal gland artery, vein and
duct were cannulated in situ as described
(Shuttleworth, 1983) using
PE50 polyethylene tubing (Clay-Adams, Parsipanny, NJ, USA). The arterial and
venous catheters were filled with standard dogfish saline, with the following
composition: 257 mmol l-1 NaCl, 7 mmol l-1
Na2SO4, 6 mmol l-1 NaHCO3, 4 mmol
l-1 KCl, 3 mmol l-1 MgSO4.7H2O, 2
mmol l-1 CaCl2.2H2O, 0.1 mmol l-1
Na2HPO4, 30 mmol l-1 glucose, 20 mmol
l-1 trimethylamine oxide, 400 mmol l-1 urea. The saline
was passed through a 0.45 µm filter and equilibrated with a precision gas
mixture (0.256% CO2, 99.744% O2) so as to achieve
control acid-base conditions (see below). The rectal gland duct catheter was
filled with 500 mmol l-1 NaCl. The preparation was then dissected
free and placed on a thermostatted platform.
Perfusion with control saline was initiated using the peristaltic
pump/overflow system (Shuttleworth,
1983) to maintain an inflow pressure of 
20 mmHg. A windkessel
reduced pressure pulsatility to 3 mmHg and served as a bubble trap. The
perfusion reservoirs were fitted with gassing ports and a combination pH
electrode (GK2401C, Radiometer-Copenhagen, Copenhagen, Denmark) for monitoring
of gas equilibration. The reservoirs and the perfusion platform were jacketed
with flowing seawater and the perfusion lines were immersed so as to maintain
the experimental temperature at 11±1°C. Perfusion pressure was
monitored by a P23 dB pressure transducer (Statham, Hato Rey, Puerto Rico)
connected via a sidearm to the perfusion line immediately proximal to
the gland. This T-junction also served as an arterial sampling point. A second
T-junction just distal to the gland served as the venous sampling point. The
venous outflow and the secretion outflow from the rectal gland duct were set
to 0 mmHg relative to the gland, and drained through infrared drop-sensors
connected to custom-built microprocessor-controlled digital flowmeters, so as
to provide continuous flow records. The venous outflow (which is always less
than the arterial inflow) represents the actual perfusion flow through the
secretory parenchyma of the gland, whereas various non-secretory shunt
pathways drain diffusely (Kent and Olsen,
1982). Therefore determinations of O2 consumption
() and
CO2 production
()
rates by the perfused gland employed the Fick principle using the measured
venous outflow rate and simultaneous arterial and venous measurements of total
O2 or CO2 concentrations (see below).
In vitro protocols
Secretion measurements
After a series of pilot experiments, the following general protocol was
established. The preparation was first perfused at 20 mmHg (1 mmHg=133.3
Pa) with standard saline equilibrated with the 0.256% CO2, 99.744%
O2 gas mixture so as to establish control acid-base conditions of
PCO2 1.9 mmHg, pH 7.8,
HCO3- 5.3 mmol l-1 and a
PO2 >400 mmHg. After 0.75 h, samples of the
rectal gland secretion, venous perfusate and arterial perfusate were collected
for gas and acid-base analyses, and the secretion was frozen (-20°C) for
the later analysis of ions. These sampling procedures took 15 min, and then at
1 h, 5x10-6 mol l-1 forskolin (Sigma-Aldrich) was
added to the perfusion reservoir to stimulate secretion
(Moran and Valentich, 1991;
Forrest, Jr et al., 1997;
Walsh et al., 2006). After a
further 0.75-1 h, the sampling and analyses were repeated, and then the
perfusion pressure was lowered to 12 mmHg, a procedure chosen to decrease
the perfusion flow through the gland without altering secretion flow so as to
increase the precision of measurement of arterial-venous differences (see
Results). A third set of sampling and analyses was performed after a further
0.75-1 h; these represented the pre-treatment control measurements. The
experimental treatment was then implemented, while maintaining stimulation
with 5x10-6 mol l-1 forskolin. In later
preparations, only the pre-treatment control measurements were performed so
the first two sets of analyses were omitted, but the gland was put through the
same protocol so as to maintain consistency with earlier preparations.
N=6 was the minimum employed for each experimental treatment. The
experimental treatments involved either the addition of 10-4 mol
l-1 acetazolamide HCl (Wyeth-Lederle, Madison, NJ, USA) to the
inflowing perfusate or alterations in its
pH-PCO2-HCO3- status. The
latter were accomplished by altering either its gassing or the concentration
of NaHCO3 or both. The objective was to achieve desired
manipulations of extracellular and intracellular acid-base status (see
Results). Manipulations were guided by the Henderson-Hasselbach equation:
 |
using the solubility of carbon dioxide (
CO2)
and the apparent pK (pKapp) for dogfish at the experimental
temperature (Boutilier et al.,
1984). When NaHCO3 was experimentally elevated, NaCl
was reduced on an equimolar basis, and vice versa so as to maintain
osmolality. Alterations in PCO2 were achieved
by gassing the perfusate with the output from a 301-af gas mixing pump
(Wosthof, Bochum, Germany), in which the mixing pistons were fed from various
certified mixtures of CO2 in O2.
Experimental treatments lasted routinely 2 h, except for acetazolamide
where the experimental period was 4 h. Sampling and analysis of rectal gland
secretion, venous perfusate, and arterial perfusate were performed at 1 h
intervals, except in the acetazolamide treatment where they were performed at
0.5 h intervals. In two series (control acid-base conditions, 12 mmHg,
5x10-6 mol l-1 forskolin; 10-4 mol
l-1 acetazolamide, 12 mmHg, 5x10-6 mol
l-1 forskolin), at the end of the experiment, a portion of the
gland tissue was immediately freeze-clamped in liquid N2 and stored
at -80°C for later determination of tissue buffer capacity. In addition,
in three experiments (glands from three different series), samples of the
secreted fluid and venous effluent were immediately fixed in two volumes of
ice-cold 8% HClO3 for the assay of lactate, and the glands were
freeze-clamped and stored as above. This tissue was later homogenized in ten
volumes of ice-cold 8% HClO3 for lactate analysis.
Intracellular pH and fluid volume measurements
The DMO method (Waddell and Butler,
1959), with mannitol as the extracellular fluid volume marker, was
again employed, and measurements were made in each of the acid-base series,
and in the acetazolamide treatment. Additional series (N=5-6) were
run to measure fluid volume distribution and intracellular pH in the two
treatments at the start of the perfusion protocol prior to lowering of the
perfusion pressure: (i) non-stimulated preparations (control acid-base
conditions, 20 mmHg, no forskolin); and (ii) stimulated preparations
(control acid-base conditions, 20 mmHg, 5x10-6 mol
l-1 forskolin).
[14C]DMO (12.5 µCi l-1) and [3H]mannitol (50 µCi l-1), both from NEN-Dupont, Boston, MA, USA, were added to the perfusion reservoirs 2 h before terminal sampling - i.e. at the start of most experimental treatments or after 2 h of acetazolamide treatment. Pilot experiments demonstrated that a time period of 2 h was sufficient to achieve full equilibration. At termination, immediately after the final set of perfusion analyses, the rectal gland was blotted and weighed, and the capsule was removed prior to processing for radioactivity and water content measurements.
Analyses for intracellular pH and fluid volume calculations
The same methods were used for both in vivo and in vitro
samples. Notably, the response time of this [14C]DMO technique is
less than 15 min, even in poorly perfused trout trunk muscle
(Milligan and Wood, 1985),
whereas a minimum of 2 h in vitro and 3 h in vivo was
allowed for label redistribution in the present study on the much better
perfused rectal gland. Triplicate samples (50-150 mg each) of the tissue were
used for radioactivity measurements, and the remaining tissue was dried to a
constant mass at 70°C in order to determine its total water content.
Plasma water content was determined by refractometry using a Goldberg
refractometer (American Optical TS meter, Buffalo, NY, USA) recalibrated for
dogfish plasma. For radioactivity measurements, samples were added to 2 ml NCS
(Amersham, Piscataway, NJ, USA) digest medium and incubated at 35-40°C for
12 h in sealed glass scintillation vials. The clear digests were then
neutralized with glacial acetic acid, diluted with 10 ml OCS fluor (Amersham),
and stored in the dark overnight to reduce chemiluminescence prior to
scintillation counting for [14C]DMO and [3H]mannitol
d.p.m. on an LKB Rackbeta 1217 (LKB-Wallac, Turku, Finland). Triplicate plasma
samples from in vivo experiments, or inflow and outflow perfusate
samples from in vitro experiments (100 µl each) were similarly
processed and counted. Separation of 3H and 14C d.p.m.
was achieved using on-board dual label quench-correction programs in the
Rackbeta 1217, calibrated by the external standard ratio method. The
coordinates of the programs were experimentally generated using a range of
quenched samples from perfusate, plasma, rectal gland, and muscle tissue in
the NCS/OCS system. Recovery of d.p.m. was regularly checked by spiking with
known amounts of [3H]mannitol and [14C]DMO (internal
standardization).
Tissue intracellular pHi, [HCO3-]i,
extracellular fluid volume (ECFV) and intracellular fluid volume (ICFV) were
calculated from measurements of extracellular acid-base status, water contents
of tissue, plasma or perfusate, and [14C]DMO and
[3H]mannitol radioactivities in tissue, and plasma or perfusate,
using equations given by Wright et al.
(Wright et al., 1988). For
in vivo experiments, arterial measurements of all parameters were
used. For in vitro experiments, extracellular pH and
PCO2 were taken as the average of arterial and
venous values, and inflow and outflow [14C]DMO and
[3H]mannitol d.p.m. were similarly averaged for these calculations.
The pK of DMO at the experimental temperature was interpolated from the
measurements of Heisler et al. (Heisler et
al., 1976) at comparable ionic strength.
Buffer capacity measurements
In two series, rectal gland tissue buffer capacity was measured by an acid
titration of tissue homogenate using the protocol described
(Wood et al., 1990). The slope
of the curve relating mmol HCl added versus pH was linear over the pH
range 7.0-7.8 (the operative pHi range in vivo) and taken as the
buffer capacity of the tissue in mmol pH unit-1 kg-1.
This was then converted to mmol pH unit-1 l-1
intracellular water (slykes), taking into account the measured intracellular
fluid volume. This technique measures total physico-chemical buffer capacity
(i.e. non-HCO3- plus HCO3-
buffering). However, as the original HCO3- content of
the tissues is low, and the PCO2 is close to
zero during titration, the HCO3- component is
negligible, and the results in essence yield an estimate of the
non-HCO3- buffer capacity.
Analytical techniques
For in vivo analyses, blood samples were withdrawn via
the arterial catheter into ice-cold, gas-tight Hamilton syringes (Reno, NV,
USA). Arterial blood pH (pHa) and oxygen tension
(PaO2) were measured using Radiometer
micro-electrodes (Radiometer-Copenhagen, Copenhagen, Denmark) kept at the
experimental temperature with water jackets. Electrode outputs were displayed
on Radiometer pHM 71 and pHM 72 acid-base analysers. True plasma
CO2 was measured on plasma obtained from blood samples centrifuged
in sealed tubes. In some experiments, the measurements were made using a
Corning 965 total CO2 analyzer (Loughborough, UK) and in others the
method of Cameron (Cameron,
1971); the two yielded identical results, though the former was
more rapid. The same analytical methods (pH, total CO2) were used
for rectal gland secretions. Because of the low buffer capacity of the
secretions, it was necessary to flush the pH electrode capillary 3 times with
the sample over a 2 min period followed by a further 2 min of equilibration to
achieve stable, reproducible values. Carbon dioxide tensions
(PCO2) and bicarbonate concentrations
([HCO3-]) were calculated using the solubility of carbon
dioxide (CO2), the apparent pK (pKapp)
for dogfish at the experimental temperature, and rearrangements of the
Henderson-Hasselbalch equation (Boutilier
et al., 1984).
For in vitro analyses, venous followed by arterial perfusate
samples (typically 300 µl) were obtained anaerobically from the outflow and
inflow ports of the perfusion set-up, by gentle withdrawal into ice-cold,
gas-tight Hamilton syringes. The rectal gland secretion was diverted from the
drop counter into the needle of a Hamilton syringe; the secretion was allowed
to overflow directly through the open syringe barrel for 10 min, which was
then sealed to yield a sample with negligible air exposure. Arterial (pHa),
venous (pHv), and secretion pH, arterial (PaO2)
and venous oxygen tensions (PvO2), and arterial
(CaCO2) and venous
(CvCO2) total CO2 concentrations,
were measured using the same equipment as for the in vivo samples.
Again, because of the low buffer capacity of the perfusate and secretion
fluid, it was necessary to pre-condition the pH capillary electrode when
reading these samples, as outlined above. PaO2
and PvO2 values were converted to total
O2 concentrations (CaO2,
CvO2) using tabulated solubility coefficients
(Boutilier et al., 1984).
Na+ and Cl- in blood plasma and rectal gland
secretions were measured by atomic absorption spectrophotometry (Varian 1275
AA, Mulgrave, Victoria, Australia) and coulometric titration (Radiometer
CMT-10, Radiometer-Copenhagen, Copenhagen, Denmark), respectively. For lactate
analyses of fluid and gland samples, the deproteinized HClO3
extracts were neutralized and assayed enzymatically by the
L-lactate dehydrogenase method
(Bergmeyer, 1983) using
reagents from Sigma-Aldrich.
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0.35 units to 7.4
due to a tripling of PaCO2 to 4 mmHg, and
rectal gland secretion rate had fallen by 53%. Na+ and
Cl- concentrations in the secretion (both 520 mmol
l-1) were not altered, but its pH was significantly depressed
(6.85±0.16 at 5.5-6 h versus 7.30±0.07 in the control
fish) and its PCO2 elevated (4.38±2.01
versus 0.90±0.10 mmHg) by a comparable amount to the elevation
in PaCO2. Secretion HCO3-
concentration was only marginally increased (0.99±0.19 versus
0.77±0.18 mmol l-1 in the control treatment). These changes
in secretion PCO2 and
HCO3- concentration accompanying acetazolamide treatment
were significant relative to the pre-treatment period in the same animals, but
not relative to the control group. Plasma Na+ and Cl-
levels (both 320 mmol l-1) and
PaO2 (90 mmHg) were unaffected (data not
shown). Intracellular pHi and fluid volume distribution were measured in three experimental treatments in vivo: unstimulated (non-infused) control, stimulated control (infused), and stimulated (infused) plus acetazolamide-treated (Table 1). In these smaller dogfish, infusion alone caused a small decrease in pHa, probably associated with dilution of plasma HCO3- (i.e. slight metabolic acidosis). Acetazolamide treatment caused the expected respiratory acidosis in the extracellular fluid. Intracellular pHi in the rectal gland and white muscle tended to fall with infusion, though the difference was only significant for the latter (Table 1). However the combination of infusion plus acetazolamide caused a significant intracellular acidosis in the gland tissue. Intracellular HCO3- concentration in the rectal gland rose significantly with acetazolamide treatment of volume-loaded fish, whereas there were no differences relative to non-infused fish. Similar trends were seen with intracellular HCO3- concentrations in the white muscle. There was also an increase in total rectal gland water content and ECFV in both treatments, as well as an increase in rectal gland ICFV in the infusion + acetazolamide treatment. Muscle water and ICFV increased in the infusion + acetazolamide treatment.
|
Characteristics of the perfused gland in vitro
In the unstimulated rectal gland preparation perfused initially at an
inflow pressure of 20 mmHg, there was no detectable secretion flow (<2
µl g-1 gland min-1). After stimulation with
5x10-6 mol l-1 forskolin, perfusion flow increased
modestly (by 18%), and secretion flow was activated at a brisk rate (25-30
µl g-1 gland min-1). This was accompanied by
increases in the gradients of pH, PCO2, total
O2 and total CO2 between arterial inflow and venous
outflow, achieved almost entirely by changes in the venous outflow values
(Table 2). The activation of
secretion was therefore accompanied by 3-4-fold increases in
and
(Table 2). Notably
was
approximately twice as large as
in
both treatment conditions, but the measurements of
were
of lower precision, because of the difficulty of resolving small
arterial-venous differences against the background total CO2
concentrations in the perfusate.
|
To increase the precision of measurement of arterial-venous differences in
the stimulated gland, the perfusion pressure was decreased to approximately 12
mmHg, which reduced the perfusion flow rate by 34%
(Table 2). There was no
significant decrease in rectal gland secretion rate, but the arterial-venous
gradients in pH, PCO2, total O2, and
total CO2 all increased. Again, these were achieved largely by
changes in the venous outflow values, though there were also some small
differences in arterial inflow values.
and
did
not change significantly, but the discrepancy between them was reduced
(Table 2). Notably, while the
pH of the secretion was well below the venous outflow level, its
PCO2 remained fairly close to arterial rather
than venous values. Secretion total CO2 concentrations were only
about 20% of those in the perfusate. In all cases, HCO3-
concentrations (data not shown) remained similar to but slightly smaller than
total CO2 concentrations in perfusate and secretion samples. Pilot
experiments demonstrated that all parameters remained approximately stable for
up to 6 h of perfusion under these conditions.
Intracellular pHi (terminal measurements) and [HCO3-]i, together with fractional fluid volume distributions, were determined in separate groups of glands under the three perfusion conditions (Table 3). Total O2 and CO2 concentrations, as well as acid-base characteristics of both inflowing and outflowing perfusate (data not shown), did not differ significantly from the values for the respective treatments in Table 2. Intracellular pHi was about 0.3 units below mean extracellular pHe at rest, and did not change significantly when the glands were stimulated with 5x10-6 mol l-1 forskolin, or when inflow pressure was reduced in the continued presence of forskolin. However, all these treatments exhibited a significantly greater [HCO3-]i relative to the unstimulated control. The stability of pHi was seen despite the observed decreases in pHe (Table 3) under these conditions due to the falls in pHv (cf. Table 2). Fractional water content remained unchanged but was clearly re-distributed, with ECFV increasing, and ICFV decreasing reciprocally after stimulation with forskolin, a situation which persisted after reduction of the inflow pressure.
|
There was a very strong linear relationship (r=0.99,
N=127, P<0.0001) between secretion flow rate and
Cl- secretion rate in the perfused, forskolin-stimulated rectal
glands, over a wide secretory range (Fig.
2A). The slope of the relationship indicated a mean Cl-
concentration of 520 mmol l-1 in the secretion, which may be
compared with 270 mmol l-1 in the perfusate. While data were
initially analysed according to whether the gland was at high pressure
(20 mmHg), reduced pressure (12 mmHg), or subjected to a particular
experimental treatment, it became clear that a single linear relationship
fitted all the data. In those samples (N=32) where Na+
concentration was monitored simultaneously, it was indistinguishable from
Cl- concentration (both 520 mmol l-1). Because of
this consistency, Cl- secretion rate was used as the indicator of
secretory performance in all further analyses, as in vivo.
|
) of
the perfused glands (Fig. 2B).
The relationship was not altered or improved by considering any of the
perfusion conditions or experimental treatments separately. The intercept at
zero Cl- secretion was positive, indicating that unstimulated
glands consume O2 at a rate of about 0.11 µmol g-1
gland min-1, in accord with the mean measured value (0.11 µmol
g-1 gland min-1) of
Table 2.
The rate of carbon dioxide excretion
() by
the perfused glands was more difficult to measure, and essentially
undetectable when high HCO3- salines were used because
it was impossible to resolve small arterial-venous differences in total
CO2 concentrations. The analysis of
(versus Cl- secretion rate) therefore uses the same set of
data points as for the analysis of
, but
without those obtained with high HCO3- saline, or under
acetazolamide treatment (which could potentially inhibit CO2
excretion). The relationship between Cl- secretion rate and
was
much more variable than for
, but
still highly significant (r=0.48, N=117,
P<0.0001; Fig. 2B).
The intercept at zero Cl- secretion was 0.22 mol g-1
gland min-1, comparable to the mean measured value (0.20 mol
g-1 gland min-1) for unstimulated glands in
Table 2. However, the two
regression lines were not statistically different, and there was no
significant difference when all simultaneous measurements of
versus
were
compared (N=117; paired t-test).
Lactate concentrations measured in three perfused glands were 7.15±1.19 mmol kg-1; lactate concentrations in the secreted fluid and venous effluent were below the resolution of the assay (<0.06 mmol l-1).
Responses to acetazolamide in the perfused gland in vitro
Addition of 10-4 mol l-1 acetazolamide to the
perfusate had no significant effect on Cl- secretion rate
(Fig. 3A),
(Fig. 3B), or perfusion rate
(data not shown) in the stimulated rectal gland. However
exhibited a transient decline of about 30%, which became significant at 2 h,
2.5 h and 3 h, but recovered by 4 h (Fig.
3C). The gas exchange ratio
(
)
declined significantly from 1.41±0.06 under control conditions to
1.04±0.17 at 2 h and 1.01±0.22 at 2.5 h, and then recovered to
1.30±0.08 by 4 h. PaCO2 (controlled
experimentally) and PvCO2 did not change
significantly, but the PCO2 of the secretion
was significantly elevated by about 1.5 mmHg at most times from 1.0 to 3.5 h
of acetazolamide treatment (Fig.
4). There were no significant changes in pHa, pHv,
[HCO3-]a, or secretion pH, whereas
[HCO3-]v rose in mirror image to the transient decline
in ,
and secretion HCO3- levels increased slightly in accord
with increases in secretion PCO2 (data not
shown).
|
|
The non-HCO3- buffer capacity of the forskolin-activated gland perfused under control conditions (normal perfusate PCO2 and HCO3-, 12 mmHg) was 35.28±2.10 (6) slykes per unit intracellular fluid [16.83±0.79 (6) mmol pH unit-1 kg-1 on a whole tissue basis] and this value remained unchanged at 33.97±1.03 (6) slykes [16.87±0.32 (6) mmol pH unit-1 kg-1] after treatment with 10-4 mol l-1 acetazolamide for 4 h.
Responses of the perfused gland to acid-base manipulations in vitro
PCO2 and [HCO3-] in
the inflowing perfusate were experimentally manipulated in order to discern
the relative importance of extracellular and intracellular pH,
[HCO3-], and/or PCO2, in
controlling the rate of secretion in the stimulated rectal gland. The measured
acid-base status of the inflowing and outflowing perfusate and estimated
[HCO3-]i for all treatments are detailed in
Table 4, and the secretory
response of the rectal gland relative to pHe or pHi is illustrated in
Fig. 5. The control glands
(control PCO2, control
[HCO3-]) were simply put through the same manipulations
but without change in the acid-base status of the inflowing perfusate, and
exhibited a 14% decline in secretion rate. There were no significant changes
in fractional water content, ECFV or ICFV of the glands in any of the
treatments (data not shown), mean values staying close to the control data of
Table 3.
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|
Approximately equal depressions of both pHe (by 0.3 units) and pHi (by
about 0.15 units) were accomplished in one group by raising
PCO2 at control [HCO3-],
and in another by lowering [HCO3-] at control
PCO2. Both treatments resulted in comparable
marked inhibitions (by 50%) of rectal gland secretion, despite very
different changes in intracellular and extracellular
[HCO3-] and PCO2,
suggesting that pH rather than PCO2 or
[HCO3-] was the important factor
(Fig. 5,
Table 4). This conclusion was
reinforced by a treatment where both PCO2 and
[HCO3-] were lowered so as to maintain pHe and pHi
unaltered. Rectal gland secretion rate remained unchanged relative to the
control treatment despite the changes in intracellular and extracellular
[HCO3-] and PCO2. Further
support for this conclusion and some distinction between the importance of pHi
and pHe were obtained by a treatment where both
PCO2 and [HCO3-] were
raised. Rectal gland secretion rate again remained unchanged despite a marked
rise in [HCO3-]i and a marked fall in pHi, the latter
equal to that seen in the two treatments that had caused about a 50%
inhibition of rectal gland secretion rate
(Fig. 5,
Table 4). This suggests that
pHe, rather than pHi, may be the key controlling factor. Notably, however, pHe
was significantly elevated in this treatment. When pHe was further raised to
about 1.0 unit above control pHe, by increasing [HCO3-]
at control PCO2, a treatment that did not
significantly alter pHi or [HCO3-]i, there was a
dramatic stimulation (2.3-fold) of rectal gland secretion rate
(Fig. 5,
Table 4). A smaller increase in
pHe (0.6 units) achieved by reduction of
PCO2 at control [HCO3-]
resulted in a very similar pHi, but a reduced [HCO3-]i,
and a secretion rate which was slightly elevated but not-significantly
different from the control. Overall, these results suggest that pHe, rather
than PCO2, or intracellular or extracellular
[HCO3-], may be the dominant influence on rectal gland
secretion rate, but do not eliminate a role for pHi (see Discussion).
As expected from the relationship in
Fig. 2B, changes in rectal
gland
varied in parallel to changes in rectal gland secretion rate in the various
experimental treatments (data not shown). However, on a relative basis they
tended to be smaller - e.g. 24% versus 50% inhibitions in
the two extracellular acidosis treatments, 1.9-fold versus 2.3-fold
stimulation in the extracellular alkalosis treatment, and only the latter
change in
was
statistically significant. This reflected the fact that a portion of rectal
gland
is devoted to routine metabolism and occurs even in the absence of secretion,
as well as the greater variability in
measurements (Table 2,
Fig. 2B).
The acid-base characteristics of the secretions from the rectal glands subjected to the various acid-base manipulations are summarized in Table 5. In general, secretion pH was about 0.3-0.5 units below pHa and a more variable amount (0.1-0.5 units) below pHv. Secretion pH declined in those treatments where the extracellular fluid was acidotic, and increased in those treatments where it was alkalotic, though only some of these changes were statistically significant. Thus secretion pH correlated with changes in pHe rather than pHi: note for example, the unchanged secretion pH associated with the high PCO2, high [HCO3-] treatment (where pHi was depressed), and the greatly elevated secretion pH associated with the control PCO2, high [HCO3-] treatment where pHi was unchanged. Changes in secretion PCO2 reflected changes in the PaCO2 of inflowing perfusate, but were generally smaller than those in PaCO2. In all cases (Table 5), secretion PCO2 remained well below PvCO2 (Table 4). Secretion HCO3- concentrations reflected changes in perfusate HCO3- concentrations, but remained only a small fraction (8-42%) of the latter.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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) on
the in situ gland preparation in pithed dogfish subjected to a
similar volume-loading protocol, where the degree of inhibition by another CA
blocker (methazolamide) was about 50%. However, key points differ. In the
present study, there was also a significant fall in rectal gland pHi
(Table 1) accompanying CA
inhibition, a point to which we return later in our discussion of role of
carbonic anhydrase in rectal gland function. Swenson and Maren
(Swenson and Maren, 1984) did
not measure pHi, but reported a paradoxical 40% decrease in rectal gland
CO2 content after CA inhibition (from 18 to 11 mmol
kg-1), for which the mechanism remained unexplained. In the present
study, total gland CO2 content was not measured directly, but
[HCO3-]i in the rectal gland increased significantly
(from 2.08 to 3.15 mmol l-1 ICF;
Table 1), as might be expected
during respiratory acidosis. The gross discrepancy in absolute values and
opposite direction of the changes between the two studies suggests that the
in situ preparation (Swenson and
Maren, 1984) may have been severely perfusion-limited due to a
depressed circulation resulting in CO2 retention, such that
methazolamide treatment of the whole animal put the gland into metabolic
depression or metabolic acidosis. Swenson and Maren attempted a more specific
inhibition of gland CA alone using a low dose of benzolamide
(Swenson and Maren, 1984), but
a systemic PCO2 rise still occurred; they
reported a small fall in secretion rate and a large rise in secretion
PCO2. While the present results on the intact
animal appear more coherent, in neither study can we attribute the decreases
in secretion rate specifically to an effect on rectal gland CA. This is
because the accompanying systemic respiratory acidosis, which has been
demonstrated in previous studies on CA inhibition in Squalus
acanthias (e.g. Swenson and Maren,
1984; Swenson and Maren,
1987; Gilmour et al.,
1997; Gilmour et al.,
2001; Perry et al.,
1999; Gilmour and Perry,
2004), could have contributed a major portion of the effect.
Indeed Swenson and Maren (Swenson and
Maren, 1984) demonstrated inhibitory effects on secretion of
severe respiratory and metabolic acidoses alone (without CA inhibition), and
fully recognized this complication. Recently, we have established a
relationship between pHa and secretion rate in intact Squalus
acanthias subjected to an identical volume-loading protocol, and in which
acid-base status was experimentally manipulated without using acetazolamide
(Wood et al., 2006). Based on
this regression, the fall in pHa (to 7.455±0.034;
Fig. 1B) alone caused by
acetazolamide would have caused a 31% inhibition of secretion, whereas the
actual inhibition by acetazolamide in the intact animal was 53±6%,
right at the 95% confidence limit of the relationship. We therefore turned to
the in vitro perfused gland preparation to more clearly dissect these
influences.
Acid-base control of rectal gland secretion
The present results confirm that secretion in the isolated-perfused rectal
gland is very sensitive to acid-base status, in agreement with several early
studies on the perfused gland in vitro
(Siegel et al., 1975;
Silva et al., 1992), on the
in situ gland preparation in the pithed, artificially ventilated
dogfish (Swenson and Maren,
1984), and our recent investigation on the intact, unanaesthetized
dogfish (Wood et al., 2006).
However our data extend these studies by dissecting out the influences of
various extracellular and intracellular acid-base parameters. As noted in
Results, the results overall point to pHe as the dominant influence, but pHi
may also be involved. Thus the inhibitory action associated with low pHe
occurred to the same extent regardless of whether pHe was reduced by raising
PCO2 at control [HCO3-]e
and [HCO3-]i (respiratory acidosis) or by lowering
[HCO3-]e and [HCO3-]i at control
PCO2 (metabolic acidosis)
(Fig. 5). Furthermore, lowering
both PCO2 and [HCO3-]e
(and [HCO3-]i) so as to leave pHe unchanged had no
effect, as did lowering only intracellular pHi (and not pHe) by raising both
PCO2 and [HCO3-]e (and
[HCO3-]i). Raising pHe (and not pHi or
[HCO3-]i) by high [HCO3-]e
treatment at normal PCO2 (metabolic alkalosis)
markedly stimulated secretion, whereas simultaneous treatment with both high
[HCO3-]e (and [HCO3-]i) and high
PCO2 had no effect.
|
Fig. 6 integrates the in
vitro data of the present study with the responses of the rectal gland
in vivo from Wood et al. (Wood et
al., 2006) where only extracellular acid-base parameters were
measured. Overall, there was good agreement with the in vivo data
reinforcing the present in vitro data, such that the relationship
between pHe and relative secretion rate did not change, but became more
significant due to the greater number of treatment means (r=0.94,
P<0.0001; exponential model). Again there was no significant
relationship with [HCO3-]e. These responses of rectal
gland secretion to pHe are probably adaptive in the context of the intact
animal because feeding will cause both a metabolic alkalosis (`alkaline tide';
Wood et al., 2005) and volume
loading due to the ingestion of seawater and/or salty food
(Mackenzie et al., 2002),
necessitating activation of rectal gland secretion
(Walsh et al., 2006). The
highest pHe of about 8.6 from the in vitro experiments is probably
outside the physiological range and may explain the non-linearity of response
in this range (Fig. 6). However
significant stimulation did occur in vivo at a pHe of 8.15, which is
within the physiological range (Wood et
al., 2005). Conversely inhibition of rectal gland output by
acidosis would be useful, because metabolic acidosis normally occurs after
severe exercise, a time when volume is contracted due to a shift of
extracellular fluid into white muscle because of the high intracellular
lactate load (Holeton and Heisler,
1983; Richards et al.,
2003).
As the effects in vivo are similar to those in the
forskolin-stimulated gland completely removed from the dogfish
(Fig. 6), these data place the
site of acid-base action at the level of the gland itself, rather than at the
volume detection, neuroendocrine communication, or surface receptor activation
level. One possible site of action might be basolateral K+
channels, which are known to be activated as part of the secretory mechanism
(Valentich and Forrest, Jr,
1991; Greiger et al.,
1999), and which are known to be very sensitive to pHi in the
appropriate fashion (Kerst et al.,
2001). Other possibilities might include the apical Cl-
channels, the basolateral Na+,K+,2Cl-
co-transporter (NKCC) or the basolateral Na+,K+-ATPase;
work at the cellular level will be needed to separate these possibilities.
Gas exchange of the rectal gland and acid-base status of the secretion
The present measurements of
in
relation to secretion rate are in reasonable agreement with the only other
detailed study on this topic in the isolated-perfused rectal gland. Using very
similar methodology but a higher perfusion pressure (40 mmHg), Silva et al.
(Silva et al., 1980) reported
that the
of the
unstimulated gland was 0.30 µmol g-1 gland min-1, but
that extrapolation of the regression of
on
Cl- secretion rate back to zero Cl- secretion gave a
much lower value, 0.14 µmol g-1 gland min-1. Our
values were 0.11 µmol g-1 gland min-1 by both
techniques (Table 2,
Fig. 2B). From the slope of
their regression, Silva et al. calculated that the cost of transporting 1
µmol Cl- was about 0.030 µmol O2
(Silva et al., 1980), whereas
our value indicated slightly greater efficiency, 0.022 µmol O2
per µmol Cl- (Fig.
2B).
is a
more difficult measurement, and we are aware of no previous data in the
isolated-perfused rectal gland. Our determinations of
were
generally higher than simultaneous measurements of
. For
example, resting
was
0.20-0.22 µmol g-1 gland min-1 by direct measurement
or extrapolation of the regression relationship
(Table 2,
Fig. 2B) and the cost of
Cl- transport from the slope was 0.036 µmol O2 per
µmol Cl-. However, these differences were not statistically
significant. While lactate concentrations in the secreted fluid and venous
effluent were below detection [as also reported by Silva et al.
(Silva et al., 1980
values significantly different (P<0.05) from
the pre-treatment reference values in the same treatment group.
