Lactate efflux from sarcolemmal vesicles isolated from rainbow trout Oncorhynchus mykiss white muscle is via simple diffusion
Department of Biology, The University of Western Ontario, London,
Ontario, Canada N6A 5B7
* Present address: Department of Biology, University of New Brunswick (Saint
John), PO Box 5050, Tucker Park Road, Saint John, New Brunswick, Canada, E2L
4M5
Author for correspondence (e-mail:
milligan{at}uwo.ca)
Accepted 8 November 2002
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Summary |
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Key words: sarcolemmal vesicle, lactate transport, white muscle, lactate, lactic acid, rainbow trout, Oncorhynchus mykiss
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Introduction |
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The mechanism for retaining lactate within the muscle is unknown. A
substantial lactate concentration gradient exists between the white muscle
intra- and extracellular compartments post-exercise. For example, immediately
post-exercise, white muscle lactate can peak at levels in excess of 50 mmol
kg-1, where extracellular levels are rarely greater than 5-8 mmol
l-1 kg-1 (Wang et
al., 1997). Unlike the immediate rise within the muscle, blood
lactate rises slowly post-exercise, peaking 2-4 h after the cessation of
exercise at maximal levels of about 16 mmol kg-1, which is still
less than levels in white muscle. In addition, extracellular pH is
approximately 0.8 units higher than intramuscular pH, establishing an
outwardly directed H+ diffusion gradient, which should promote
lactic acid efflux. Cumulatively, the pH and lactate concentration gradients
create a large diffusion potential that should drive lactate out of the
muscle. Yet despite this, only 15-20% of the lactate produced appears in the
blood (Turner et al., 1983
;
Milligan and Wood, 1986
).
Our understanding of lactate transport mechanisms in fish muscle has grown
considerably in the past 5 years. In early studies, Turner and Wood
(1983), using an isolated
perfused trout trunk, reported that lactate efflux was enhanced, relative to
controls, by the anion transport inhibitor
4-acetoamido-4'-isothiocyanstilbene-2,2'-disulphonic acid (SITS).
Their interpretation of this observation was that lactate diffuses out of the
muscle and subsequently taken back up, and this re-uptake was inhibited by
SITS. More recently, Wang et al.
(1997
), using an isolated
perfused tail-trunk preparation of rainbow trout, reported that both lactate
influx and efflux are carrier-mediated. They estimated that about 33% of
lactate influx was via a lactate-/H+ symporter
(or monocarboxylate transporter; MCT), which was inhibited by
-cyano-4-hydroxycinnamate (CIN), about 45% of the influx was
via a lactate-/Cl-HCO3-
antiporter, and the remainder via passive diffusion. Efflux is
thought to be have both a free diffusional component, of both lactic acid and
the lactate- anion, and a carrier-mediated component, probably a
monocarboxylate transporter. Interestingly, lactate efflux appeared to be
concurrent with lactate influx. Simultaneous uptake and release of lactate has
been observed in mammalian heart muscle
(Chatham et al., 2001
).
The mechanism of lactate uptake was examined using sarcolemmal vesicles
isolated from rainbow trout white muscle
(Laberee and Milligan, 1999).
The findings were similar to those of Wang et al.
(1997
) in that lactate uptake
was saturable, mediated in part by a low-affinity, high capacity carrier that
is partially inhibited by pyruvate, which is consistent with a monocarboxylate
transporter. However, lactate uptake by white muscle sarcolemmal vesicles was
not inhibited by CIN; rather both CIN and the anion-transport inhibitor,
4-acetoamido-4'-isothiocyanstilbene-2-2'-disulphonic acid (SITS)
appeared to stimulate lactate uptake. The model emerging from these studies is
that lactate uptake is carrier-mediated and its subsequent efflux is inhibited
by SITS and CIN, suggesting that efflux is also carrier mediated. Thus, it
would appear that in trout muscle lactate uptake and efflux occur via
separate pathways.
In mammalian muscle, lactate uptake and efflux occur via two
separate pathways, uptake mediated by MCT1 and efflux via MCT4
(Bröer et al., 1998;
Dimmer et al., 2000
;
Fox et al., 2000
). Both MCTs
are found in mammalian fast-twitch, glycolytic fibers, which are both
producers and consumers of lactate, as is fish white muscle
(Bonen, 2001
). MCT 1 has a high
affinity for lactate (Km approx. 3.5 mmol l-1),
but low specificity (Km for pyruvate approx. 1 mmol
l-1) (Bröer et al.,
1998
) and its abundance is positively correlated with muscle
oxidative capacity (Pilegaard et al.,
1999
; Bonen et al.,
2000
; Juel, 2001
).
The MCT4 isoform has a relatively low affinity for lactate
(Km 28-35 mmol l-1;
Dimmer et al., 2000
;
Fox et al., 2000
) but a high
substrate specificity (Km for pyruvate approx. 150 mmol
l-1; Fox et al.,
2000
) and its abundance is positively correlated with glycolytic
capacity (Bonen, 2001
).
While fish white muscle clearly takes up lactate from the extracellular
space, via a carrier with MCT1-like properties
(Wang et al., 1997;
Laberee and Milligan, 1999
),
the mechanism of efflux is unknown. It is unlikely that the mechanism for
lactate efflux in fish muscle is similar to that in mammalian muscle because
of the different metabolic fates of lactate in the two groups. In mammals,
lactate is exported from the glycolytic fibers to the blood via the
MCT4 and subsequently taken up by oxidative tissues, via the MCT1
(Bonen, 2001
;
Juel, 2001
). In contrast, in
trout muscle, lactate is retained within the muscle and used as a substrate
for in situ glycogenesis. Thus, our primary objective was to use the
giant sarcolemmal vesicle preparation to characterise lactate efflux from
trout white muscle. A second objective was to combine what is known about
lactate transport in mammalian muscle with our findings to develop a model to
explain the mechanism of lactate retention within trout white muscle.
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Materials and methods |
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Giant sarcolemmal vesicle preparation
Sarcolemmal vesicles were prepared following the protocol of Laberee and
Milligan (1999). All chemical
solutions used in the experiments were made up in buffer containing 140 mmol
l-1 KCl and 5 mmol l-1
3-[N-morpholino]propanesulphonic acid (Mops) (Sigma, Oakville, ON,
Canada), adjusted to pH 7.4 (KCl/Mops buffer).
Vesicle yield and vesicular integrity
The protein content of each vesicle preparation was assayed using the
Bradford method (Bradford,
1976) using 10 µl of the vesicle solution, with bovine serum
albumin (Fraction V, Boehringer Mannheim, Germany) as a standard. Lactate
efflux is expressed per unit of vesicle protein.
Membrane integrity was routinely examined using the Trypan Blue exclusion method. Following vesicle isolation, a 0.4% solution of Trypan Blue was added to resuspended vesicles in a 1:25 ratio and the vesicles counted using a haemocytometer under a compound microscope. Vesicles were considered disrupted or incompetent if the intravesicular space was coloured with Trypan Blue. Preparations with <90% intact vesicles were discarded.
Vesicle orientation, number and size
The orientation of the sarcolemmal vesicle membranes (i.e. the percentage
of vesicles in the right-side out orientation) was determined using the
3H-ouabain binding method (Nørgaard, 1983). In brief, the
extracellular domain of the Na+/K+-ATPase was labelled
with vanadate-facilitated 3H-ouabain in both intact and
deoxycholate-ruptured vesicles. Following isolation, vesicles were resuspended
in sucrose solution containing 250 mmol l-1 sucrose, 1 mmol
l-1 MgCl2, 1 mmol l-1 vanadate (Sigma,
Oakville, ON, Canada) and 5 mmol l-1 Mops, pH 7.4, in 1.7 ml
microfuge tubes. Deoxycholate (Fisher Scientific; Toronto, Canada) was added
to one set of tubes to a final concentration of 0.8 mg ml-1, to
permeate the membrane (permeated vesicles) and expose any intracellular
ouabain binding sites (i.e. those vesicles that were inside-out). The other
set had an equivalent volume of sucrose solution added as a control (intact
vesicles). Tubes were shaken on an orbital shaker at 0.45 g
for 30 min at 20°C. The vesicle solutions were centrifuged at 15 000
g for 30 s and the supernatant removed. Vesicles were then
resuspended in sucrose solution containing 0.08 µCi 3H-ouabain
(ICN Radiochemicals; Montreal, Canada) and incubated for 30 min on the orbital
shaker at 0.45 g. The permeated vesicles permit access of
3H-ouabain to the inside of the membrane. Any excess binding of
3H-ouabain in permeated vesicles compared to intact vesicles
indicates that the vesicular membrane is oriented inside-out. Both samples
were transferred to 2.0 ml filter tubes containing 0.45 µm
cellulose-nitrate filters. The incubation tubes were rinsed four times with a
total of 100 µl of sucrose solution and the wash was added to the filter
tube in order to retrieve the entire sample. The filter tubes were centrifuged
at 15 000 g for 30 s to draw the solution through the filter
while trapping the 3H-ouabain-labelled vesicles on the filters.
Each filter was washed four times with 100 µl of sucrose solution and
centrifuged after each wash to draw the fluid through the filter. The filters
and the filtrate (consisting of both the original incubation media plus the
washes) were collected into scintillation vials and 5 ml of ReadySafe
(Beckman; Missassauga, Canada) scintillation fluid were added to all vials.
The vials were counted on a Packard 1900TR Liquid Scintillation Counter with
automatic quench correction. The counts on the filters of the ruptured
versus intact vesicles were compared and used to determine the
proportion of vesicles in the right-side-out orientation.
Sarcolemmal vesicle diameters were measured by phase contrast microscopy and an average vesicle volume was calculated, assuming the vesicles to be a sphere. The number of vesicles that correlate with a known amount of protein (i.e. x number of vesicles present in y µg of protein) was calculated by counting vesicle samples in known volumes with a known protein content.
Vesicle loading with 14C-lactate
Lactate uptake by sarcolemmal vesicles at 25 mmol l-1 external
[lactate] was first measured following the protocol of Laberee and Milligan
(1999) to establish that the
vesicles do take up lactate and to estimate the required loading times for
efflux experiments. Once lactate uptake was established, sarcolemmal vesicles
were incubated in a reaction mixture containing a known concentration of
lactate in KCl/Mops solution (see above) plus 18.5 kBq 14C-lactate
(ICN Radiochemicals; universally labelled, specific
activity=1.11x1012 d.p.m. mol-1) in 1.7 ml
microfuge tubes. Tubes were shaken at 0.45 g on an orbital
shaker at room temperature for 30 min. The samples were then pelleted by
centrifugation at 15 000 g for 30 s. The supernatant from each
sample was collected into 20 ml scintillation vials. The pellets were washed
twice with 500 µl of cold 25 mmol l-1 lactate in KCl/Mops (pH
7.4) to remove 14C-lactate loosely bound to or trapped between the
vesicles. Within 5-10 s of washing, the samples were centrifuged at 15 000
g for 30 s to pellet the vesicles. In all experiments one
sample was snipped off immediately into a scintillation vial to estimate total
lactate loaded after 30 min of incubation. All other samples were resuspended
in 1 ml of efflux medium (see below) and sampled at times specific to the
experimental conditions.
Any facilitated efflux from the vesicles was arrested by the addition of 1
ml of ice-cold 2.5 mmol l-1 HgCl2 (STOP solution) at
designated times, followed by centrifugation at 15 000 g for
30 s to pellet the vesicles. HgCl2 alters the sulphydryl groups of
proteins and has been shown to block both the influx and efflux of lactate in
a variety of cell types (Grimditch et al.,
1985; Roth and Brooks,
1990
; Laberee and Milligan,
1999
). To assess the effectiveness of the HgCl2 STOP
solution in inhibiting lactate efflux from this preparation, preloaded
vesicles were immediately resuspended in ice-cold STOP solution and efflux
monitored after 1 min. The supernatants and vesicle pellets were collected
into scintillation vials and 10 ml scintillation fluid was added to all vials.
Each sample was shaken and left overnight in the dark and counted on a Packard
1900TR Liquid Scintillation Counter with automatic quench correction
Time course and concentration dependence of lactate efflux
These experiments were performed under zero-trans conditions, i.e.
all substrate and label were initially internal to the vesicles. The relative
time course of lactate efflux at 25 mmol l-1 internal concentration
was monitored from 10 to 600 s, sampling from the same sample at various
times. At the given time, a 20 µl sample of the vesicle suspension was
taken, added to 1 ml ice-cold STOP solution and immediately centrifuged at 15
000 g for 30 s. Both the supernatant and the vesicular pellets
were separately collected into scintillation vials and counted as
described.
The concentration of cold lactate pre-loaded into the vesicles was varied from 5 to 250 mmol l-1. At the higher lactate concentrations, the vesicles were placed in efflux medium containing the appropriate concentration of sucrose to prevent osmotic shock.
To verify that vesicles were in fact loading with lactate, vesicles were osmotically shocked to burst them and the amount of lactate lost was measured. Vesicles were prepared and preloaded as previously described. Following washing, the pellet was resuspended in 1 ml of hypo-osmotic, lactate-free, 50 mmol l-1 KCl/Mops (pH 7.4). After 1 min, 1 ml of ice-cold STOP solution was added to the sample, which was then centrifuged at 15 000 g for 30 s. Both the supernatants and the vesicular pellets were collected into scintillation vials. In a parallel series of experiments, vesicular integrity was assessed after 1 min of hypo-osmotic exposure to ensure vesicles were, in fact, ruptured. Using the Trypan Blue exclusion method previously described, less than 10% of the vesicles remained intact after 1 min of hypo-osmotic exposure.
pH and Na+ dependence
When the intravesicular pH (pHi) is greater than extravesicular
pH (pHe), a gradient is established that should diminish the
potential for lactate efflux (Wang et al.,
1997). Vesicles were prepared, preloaded with 25 mmol
l-1 lactate and washed as described. The vesicular pellet was
resuspended in 1 ml of lactate-free KCl/Mops at either pH 7.4
(pHi=pHe) or pH 5.1
(pHi>pHe).
When the pH of the intravesicular compartment is less than that of the
extravesicular space, a pH gradient is established such that more lactate is
in the non-ionic, potentially diffusible form inside the vesicles. This
condition sets up the potential for lactate to diffuse out of the vesicles
down the pH gradient, and thus should enhance lactate efflux. Vesicles were
prepared in KCl/Mops (pH 7.4) and pre-loaded with 25 mmol l-1
lactate in KCl/Mops (pH 5.1) to yield an average intravesicular pH of
5.8±0.7 (N=6). Intravesicular pH was measured on vesicular
pellets lyzed using the freezethaw method
(Zeidler and Kim, 1977). The
lyzed pellets were injected into a Radiometer pH microelectrode maintained at
20°C and linked to a Radiometer PHM 73 blood gas monitor. The
lactate-loaded vesicular pellet was washed and then resuspended into 1 ml of
lactate-free KCl/Mops, pH 7.4, such that pHi<pHe, and
sampled as per the standard protocol.
To assess potential extracellular Na+-dependent lactate efflux, vesicles were prepared, preloaded and washed as described. The vesicles were resuspended into 1 ml of lactate-free 75 mmol l-1 NaCl/Mops + 75 mmol l-1 KCl/Mops, pH 7.4 and sampled as described.
Inhibitor series
All inhibitor series were run over a transport period of 1 min at an
internal lactate concentration of 25 mmol l-1. Controls were always
run concurrently with inhibitors and contained an equivalent amount of DMSO.
Vesicles were prepared, preloaded and washed as described. The vesicles were
then resuspended in 1 ml of either 5 mmol l-1
4-acetoamido-4'-isothiocyanstilbene-2,2'-disulphonic acid (SITS)
or 5 mmol l-1 -cyano-4-hydroxycinnamate (CIN) in 0.5% DMSO
(w/v) neutralised with 5 mmol l-1 NaHCO3 in KCl/Mops, pH
7.4, and sampled as described.
Statistical analysis
The values presented are means ± 1 standard error of the mean
(S.E.M.). A one-way analysis of variance (ANOVA) was used for comparison
between treatments, followed by a Tukey-HSD multiple comparison analysis.
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Results and Discussion |
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As determined by Trypan Blue exclusion, 93.9±1.71% (N=68)
of the vesicles in a preparation excluded the dye, and thus were considered to
be intact. Vesicular integrity was monitored throughout the course of the
experiments and any preparation in which less than 90% of the vesicles were
intact was discarded. These estimates of vesicle viability are consistent with
those reported by Laberee and Milligan
(1999) for vesicles isolated
from trout muscle.
To estimate whether vesicles were oriented with the correct side of the
membrane facing outwards (i.e. the in vivo extracellular surface
facing outwards), the Na+-K+ pumps were labelled with
3H-ouabain. The vanadate-facilitated ouabain binding site of the
Na+-K+ pump is located on the extracellular surface of
the membrane (Hansen, 1979),
so if all vesicles are in the correct orientation, 3H-ouabain
binding should be the same in intact and deoxycholate-treated vesicles.
Deoxycholate has been shown to disrupt membrane integrity in a variety of
different cell types, including giant sarcolemmal vesicles isolated from
mammalian muscle (Pilegaard et al.,
1993
). In the present study, examination of deoxycholate-treated
vesicles incubated with Trypan Blue by phase-contrast microscopy indicated
that most vesicles incorporated the dye, in comparison to controls, indicating
that vesicular integrity was ruptured. The 3H-ouabain binding to
intact vesicles was 169.9±48.7 d.p.m.-1 µg
protein-1 (N=10), which was not different from that seen
in deoxycholate-treated vesicles (173.3±38.5 d.p.m.-1 µg
protein-1, N=10), indicating that the vesicles were in the
correct orientation. Thus, the terms `efflux' and `influx' as applied to the
vesicles in this study retain their in vivo meaning.
Since the primary objective of this work was to examine lactate efflux, it
was first necessary to demonstrate that the vesicles could be lactate loaded.
Vesicles took up lactate at a rate of 26.4±6.3 nmol min-1
mg-1 protein (N=8) at an external lactate concentration of
10 mmol l-1, which is comparable to that previously reported by
Laberee and Milligan
(1999).
Lactate efflux from sarcolemmal vesicles
Lactate efflux from sarcolemmal vesicles preloaded with 25 mmol
l-1 lactate proceeded linearly for up to 70 s
(Fig. 1), therefore all
subsequent measurements of efflux were carried out for a preiod of 1 min.
Lactate efflux increased with increasing intravesicular lactate concentrations
from 10-250 mmol l-1 linearly, at rates approximately 500-fold
slower than influx rates. For example, at a lactate concentration of 25 mmol
l-1, mean efflux was 0.049±0.006 nmol mg-1
protein min-1 (N=6;
Fig. 2) compared to influx
rates of 15-20 nmol mg-1 protein min-1
(Laberee and Milligan, 1999).
To determine whether these very slow efflux rates were due to poor loading of
the vesicles, despite measuring apparently good lactate uptake rates, vesicles
loaded at 25 mmol l-1 lactate were burst by osmotic shock and
lactate loss measured. Over a 1 min period, mean lactate loss was
29.3±0.2 nmol mg-1 protein (N=8), which was approx.
500-fold greater than efflux rates from intact vesicles. This indicates that
the slow efflux rates are real, and not an artefact of poor lactate loading.
Rather, it appears that the trout white muscle sarcolemmal membrane is
relatively impermeant to lactate.
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Lactate efflux was unaffected by HgCl2 (STOP solution;
Fig. 3A). Mercuric chloride is
known to modify protein sulfhydryl groups and inhibit lactate uptake in trout
sarcolemmal vesicles (Laberee and
Milligan, 1999) as well as lactate efflux from vesicles isolated
from rat muscle (Roth and Brooks,
1990
; Pilegaard et al.,
1993
). Lactate efflux was unaffected by 5 mmol l-1 CIN,
which is a competitive inhibitor of the H+-lactate cotransporter in
skeletal muscle (Poole and Halestrap,
1993
; Roth and Brooks,
1993
), but was stimulated by 5 mmol l-1 SITS, which
inhibits anion transport (Fig.
3A). These results are inconsistent with what has been reported
previously for trout muscle. Both these compounds stimulated lactate influx in
trout sarcolemmal vesicles (Laberee and
Milligan, 1999
), which was interpreted as reflecting an inhibition
of efflux. Further, in an isolated perfused trunk preparation, 5 mmol
l-1 CIN caused a 75% reduction in lactate efflux from trout muscle
and SITS was without effect (Wang et al.,
1997
). These different effects of inhibitors in the perfused trunk
and sarcolemmal vesicle preparations may reflect differences in the scale of
the preparation. In the present study, efflux rates are very low, relative to
those measured in the perfused trunk, so that small effects caused by SITS or
CIN are more likely to be seen. Furthermore, the perfused trunk model is a
multiple membrane system and both SITS and CIN are non-specific inhibitors
that may act at multiple sites (e.g. sarcolemmal membrane, mitochondrial
membrane, blood vessel endothelium)
(Halestrap, and Denton, 1974
).
The sarcolemmal vesicle, however, is a single membrane and the effect observed
is specific to the sarcolemmal membrane. Since SITS is a non-specific anion
transport inhibitor, it may have secondarily affected lactate movement by its
actions on Cl-/HCO3- exchange and alteration
of the transmembrane pH gradient which, because of the smaller scale of the
vesicle preparation compared to the perfused trunk, was a measurable
phenomenon. Lactate efflux is sensitive to alterations in the transmembrane pH
gradient, such that when intravesicular pH (pHi) was acidic
relative to extravesicular pH (pHe), lactate efflux increased
(Fig. 3B) as diffusive loss of
lactic acid increased. Even though lactate efflux was stimulated when
pHi<pHe, a situation seen in vivo, the
absolute rate of loss was still quite low, in the pmol mg-1 protein
min-1 range, compared to uptake rates in the nmol mg-1
protein min-1. Taken together, these data suggest that the trout
muscle sarcolemmal membrane is relatively impermeant to lactic acid, and that
the putative monocarboxylate transporter identified by Wang et al.
(1997
) and Laberee and
Milligan (1999
) is probably
responsible for lactate uptake only, and efflux is via passive
diffusion and is minimal.
|
This model for lactate movement across the trout muscle membrane, in which
the MCT-like transporter is responsible for facilitating lactate uptake only,
is different from that described for mammalian fast-twitch muscle (see below),
but consistent with what is known about muscle lactate metabolism in trout
in vivo. During exhaustive exercise lactate is produced via
glycogenolysis; consequently glycogen levels drop and lactate levels increase.
Only a small fraction (approx. 10-15%;
Turner and Wood, 1983;
Turner et al., 1983
) of the
total lactate produced leaves the muscle, probably as a consequence of the
resistance of the white muscle membrane to lactate loss. Since lactate is the
primary substrate for glycogenesis, facilitated transport of the lactate out
of the muscle would only serve to delay restoration of muscle glycogen stores.
Exogenous lactate is taken up by MCT-like transporter and used by the trout
white muscle, primarily as an oxidative substrate as opposed to glycogenic
substrate (J. Kam and C. L. Milligan, unpublished observation).
In mammalian fast-twitch muscle fibers, monocarboxylate transporters are
involved in both lactate influx and efflux across the muscle membrane
(Juel, 2001;
Bonen, 2001
), with diffusion
playing a minimal role. The different role for monocarboxylate transporters in
mammals versus fish is not surprising, given the different metabolic
fates of lactate. In mammals, the primary fate of muscle lactate is oxidation,
via other tissues (including other muscle tissue;
Brooks et al., 1999
). Muscle
lactate is also cleared fairly quickly (within 60-90 min in mammals compared
to 4-8 h in trout) and glucose is the main glycogenic substrate. Mammalian
fast-twitch fibers contain both the MCT1 (a high-affinity, low-capacity
transporter) and MCT4 (a low-affinity, high-capacity transporter) isoforms,
responsible for lactate uptake and efflux, respectively
(Bröer et al., 1998
;
Bonen et al., 2000
;
Dimmer et al., 2000
;
Fox et al., 2000
). It is
hypothesised that these two transporters provide the muscle with an efficient
method for transporting lactate when lactate levels are low (i.e. under
resting or low activity conditions, when lactate is used oxidatively) or high
(during intense exercise, to facilitate net efflux)
(Bonen et al., 2000
). Clearly,
in trout muscle, the phenomenon of lactate-based in situ glycogenesis
introduces a potential for redundancy if membrane transport systems
facilitated lactate efflux. The use of lactate as a glycogenic substrate
negates the need to release lactate for clearance purposes.
In conclusion, this study, in conjunction with the that of Laberee and
Milligan (1999), suggests that
in trout white muscle, a MCT-like transporter is responsible for facilitated
lactate uptake only, with efflux occurring via passive diffusion. The
500-1000-fold lower rate of efflux than influx indicates the trout muscle is
relatively impermeant to lactate and explains the phenomenon of lactate
retention observed in vivo.
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Acknowledgments |
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