Adaptations to diving hypoxia in the heart, kidneys and splanchnic organs of harbor seals (Phoca vitulina)
1 Department of Marine Biology, Texas A&M University at Galveston, 5007
Avenue U, Galveston, TX 77551, USA
2 Department of Pathology, University of Texas Medical Branch, 301
University Boulevard, Galveston, TX 77555-0555, USA
3 Department of Internal Medicine, University of Texas Southwestern Medical
Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8573, USA
* Author for correspondence at present address: Division of Nephrology, University of Alabama, Birmingham, AL 35294, USA (e-mail: alfuson{at}uab.edu)
Accepted 8 August 2003
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Summary |
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Key words: diving, hypoxia, harbor seal, Phoca vitulina, metabolism
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Introduction |
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A marine mammal's response to `exercise' during diving is counterintuitive
in the context of our normal understanding of the mammalian exercise response.
When terrestrial mammals exercise, they increase ventilation and cardiac
output, and peripheral vasodilation increases skeletal muscle perfusion and
allows heat dissipation through the skin
(Rowell, 1986;
Wagner, 1991
). By contrast,
marine mammals undergo apnea (breath-holding), bradycardia (reduction in heart
rate) and peripheral vasoconstriction, which are collectively known as the
dive response. As cardiac output decreases, reflex peripheral vasoconstriction
maintains central arterial blood pressure by reducing flow to all organs and
tissues except the brain. Although the degree of bradycardia and peripheral
vasoconstriction may vary with the dive duration or level of exertion, all
organs and tissues, including the heart, kidneys, and splanchnic organs,
experience a reduction in convective oxygen delivery resulting from both
hypoxic hypoxia (decrease in O2 supply without a decrease in blood
flow) and ischemic hypoxia (the condition in which blood flow is reduced or
stopped) (Butler and Jones,
1997
; Davis and Kanatous,
1999
; Kanatous et al.,
2001
). By the end of aerobic dives, the arterial oxygen partial
pressure (PaO2) in Weddell seals is as low as
3.2x102 Pa (Qvist et al.,
1986
; Davis and Kanatous,
1999
), which is equivalent to the degree of hypoxia experienced by
human climbers on the top of Mt Everest (approximately 8850 m). At this
altitude, the maximum oxygen consumption of climbers is reduced to 25% of that
at sea level (West et al.,
1983
). Nevertheless, pinnipeds maintain aerobic metabolism during
most free-ranging dives (Kooyman et al.,
1983
; Davis et al.,
1991
; Hochachka,
1992
; Butler and Jones,
1997
).
Previous research on adaptations that enable tissues to maintain normal
function during diving have focused mostly on skeletal muscle. Kanatous et al.
(1999) found that pinniped
skeletal muscle has an increased mitochondrial volume density
[VV(mt)] that is most pronounced in the muscles used for
swimming. The increased VV(mt) is thought to facilitate
aerobic metabolism under hypoxic diving conditions by decreasing the diffusion
distance between mitochondria and intracellular oxygen stores in the form of
oxy-myoglobin. The increased VV(mt) results in an
increased citrate synthase (CS) activity and maximum aerobic capacity,
although this may not be important to marine mammals that make long dives,
because they use cost-efficient modes of locomotion to conserve oxygen stores
and prolong dive duration (Williams et
al., 2000
; Davis et al.,
2001
). It was also found that the ß-hydroxyacyl-CoA
dehydrogenase (HOAD) activity in the skeletal muscle of pinnipeds was
significantly greater than in the skeletal muscle of terrestrial mammals
(Kanatous et al., 1999
). HOAD
activity is indicative of aerobic, fat-based metabolic potential
(Pette and Dölken, 1975
;
Simi et al., 1991
). Since the
skeletal muscles of pinnipeds show an increased VV(mt)
that facilitates aerobic metabolism under hypoxic conditions, the question
arises of whether other organs and tissues show similar adaptations.
The goal of the present study was to determine whether harbor seals have an elevated VV(mt) and enzymatic capacity for aerobic metabolism in their heart, kidneys and splanchnic organs. Our results show that harbor seal organs have an enhanced VV(mt) when scaled to tissue-specific resting metabolic rate (RMR) that decreases the diffusion distance of oxygen between mitochondria during hypoxia. When scaled to tissue-specific RMR, CS and HOAD activities in these organs are also elevated, indicating a reliance on aerobic, lipid-based metabolism. These adaptations enable harbor seals to maintain aerobic metabolism and physiological homeostasis under hypoxic conditions associated with voluntary dives. LDH activity was also measured in the pyruvate to lactate (anaerobic) direction, and our results point to a heightened anaerobic ability in harbor seal organs. However, the possibility of adaptation for the oxidation of lactate to pyruvate cannot be eliminated.
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Materials and methods |
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Samples taken for electron microscopy were fixed in 2% glutaraldehyde and remained in the fixative for approximately 30 days before being minced and stored in 0.1 mol l-1 cacodylate buffer at pH 7.4 prior to embedding. Since samples were taken after death and were immersion fixed, some tissue autolysis may have occurred. However, serious autolysis was not apparent in the electron micrographs of any of the tissues. Samples for enzymatic analysis were immediately frozen in liquid nitrogen until they were returned to Texas A&M University, after which they were stored at -70°C.
Mitochondrial volume density
Fixed samples were rinsed in 0.1 mol l-1 cacodylate buffer and
postfixed for one hour in a 1% solution of osmium tetroxide. They were then
rinsed with distilled water, stained en bloc with 2% uranyl acetate
for 30 min at 60°C, dehydrated with increasing concentrations of ethanol
(50-100%) and then passed through propylene oxide and increasing
concentrations of epoxy (50-100%). They were finally embedded in epoxy and
allowed to polymerize overnight at 60°C. Semi-thick sections (1 µm)
were cut with a Leica Ultratome (Reichert Division of Leica Co., Vienna,
Austria) and stained with toluidine blue. Ultrathin (50-70 nm) sections from
four randomly selected blocks per sample were cut, placed on a copper grid
(150 Mesh) and contrasted with lead citrate and/or uranyl acetate. Micrographs
were taken with a Phillips 201 transmission electron microscope (FEI Company,
Eindhoven, The Netherlands). Final image magnification was approximately 18
150x. The number of micrographs taken for each block ranged from 10 to
20, yielding a total of 40-80 micrographs per sample. We calculated
VV(mt) from digitized micrographs using a standard
point-counting technique (Hoppeler et al.,
1981; Mathieu et al.,
1981
). Electron micrographs from the cardiac muscle were used only
if the sections were transverse or oblique in orientation.
Mitochondrial distribution
The intracellular distributions of mitochondria in the liver, kidney and
stomach were semi-quantitatively characterized in 40 micrographs per
species. Micrographs in which the mitochondria were more uniformly distributed
were classified as `homogeneous', and those with tightly packed mitochondria
with areas of cytoplasm devoid of mitochondria were classified as `clustered'
(Jones, 1984
). The
classification was conducted independently by two of the authors. The results
were compared, and those micrographs with differing classifications were
discarded from the analysis. The percentages of micrographs with homogeneous
and clustered mitochondria were calculated for the tissues of each
species.
Enzyme assays
Frozen tissue samples were thawed, blotted, weighed and immediately
homogenized in a volume of buffer (1 mmol l-1 EDTA, 2 mmol
l-1 MgCl2 and 50 mmol l-1 imidazole, pH 7.0
at 37°C) according to their mass and type (300x dilution for heart,
30x for liver, 10x for kidney and 5x for stomach or
intestine) in a ground glass homogenizer
(Reed et al., 1994). The
homogenates were centrifuged at 2900 g for 50 min at 4°C.
Enzyme analyses were performed at 37°C on a PowerWaveX 340
microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The assay
conditions for CS (EC 4.1.3.7) were: 0.5 mmol l-1 oxaloacetate, 0.4
mmol l-1 acetyl CoA, 5,5'-dithiobis(2-nitrobenzoic acid) and
50 mmol l-1 imidazole, pH 7.5; DA412,
412=13.6, where DA indicates absorbance wavelength and
is the extinction coefficient. Assay conditions for HOAD (EC 1.1.1.35) were:
0.1 mmol l-1 acetoacetyl CoA, 0.15 mmol l-1 NADH, 1 mmol
l-1 EDTA and 50 mmol l-1 imidazole, pH 7.0;
DA340,
340=6.22. Assay conditions for LDH (EC
1.1.1.27) were: 1 mmol l-1 pyruvate, 0.15 mmol l-1 NADH
and 50 mmol l-1 imidazole, pH 7.0; DA340,
340=6.22. Enzyme activities in micromoles of substrate
converted per minute per g wet mass (IU g-1 wet mass tissue) were
calculated from the rate of change in absorbance at the maximum linear slope
(Reed et al., 1994
). CS
activity in the small intestine was below the limit of detection for our
system, so the samples were combined to yield only one measurement for each
species.
Statistical analysis
Results are expressed as means ± S.E.M.
VV(mt) was determined for six seals, three rats (exclusive
of kidney cortex) and three dogs. Enzyme activities were determined for 10
seals, three rats (exclusive of kidney cortex) and three dogs. Inter-organ and
inter-species comparisons of mean values of VV(mt) and
enzyme activities were analyzed using an analysis of variance (ANOVA; Tukey
HSD; P<0.05). Rat kidney was not included in either analysis due
to a sample size of only one. CS activity in the intestine was not analyzed
statistically because only a single value could be obtained for each species.
In addition to the above analyses, values for VV(mt) and
enzyme activities were scaled to each tissue's calculated specific RMR to
adjust for differences in body mass between the seals and the control species.
Based on the work of Wang et al.
(2001), the scaling exponent
for the RMR of individual organs and tissues is more variable than for
whole-body RMR. Therefore, instead of scaling VV(mt) and
enzyme activities with the whole-body RMR, estimated as
70Mb-0.25
(Schmidt-Nielsen and Duke,
1984
), where Mb is the body mass of the animal
(in kg), we used the estimated specific RMR for each organ or tissue
(Wang et al., 2001
). The
estimated tissue-specific RMRs (kJ kg-1 day-1) were as
follows: liver RMR=2861Mb-0.27, heart
RMR=3725Mb-0.12, kidney
RMR=2887Mb-0.08, stomach and intestine
RMR=125Mb-0.17. Statistical comparisons of
scaled VV(mt) and enzyme activities among species were
made using an ANOVA (Tukey HSD; P<0.05). Statistical comparisons
among species for CS/HOAD and LDH/CS ratios were also made using an ANOVA
(Tukey HSD, P<0.05). CS/HOAD was used as an index of potential
fatty acid oxidation versus the overall aerobic metabolism of the
animal, with a ratio less than one indicating that fatty acids can provide
most of the acetyl CoA for the Krebs cycle
(Pette and Dölken, 1975
;
Simi et al., 1991
). LDH/CS was
used as an index of relative anaerobic versus aerobic metabolic
capacities (Hochachka et al.,
1982
). All statistical analyses were performed with SYSTAT version
10.
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Results |
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Harbor seal inter-organ comparisons
The mean VV(mt) of the seal liver (26.4) was
significantly greater than in the heart and kidney (18.6 and 21.4,
respectively; Table 1). Stomach
VV(mt) (24.5) was also significantly greater than that in
the heart (18.6). VV(mt) in the liver, heart, kidney and
stomach were significantly greater than in the intestine (8.9). The mean CS
activity in the heart (73.8 IU g-1 wet mass tissue) was
significantly greater than in the liver (13.5 IU g-1 wet mass
tissue), kidney (15.4 IU g-1 wet mass tissue) and stomach (11.4 IU
g-1 wet mass tissue) (ANOVA, P<0.05), but there were no
significant differences among liver, kidney and stomach
(Table 2). The mean HOAD
activity in the kidney (2.4x102 IU g-1 wet mass
tissue) was significantly greater than in the heart (1.0x102
IU g-1 wet mass tissue), liver (11.2 IU g-1 wet mass
tissue), stomach (4.4 IU g-1 wet mass tissue) and intestine (3.4 IU
g-1 wet mass tissue), but there were no significant differences
between the liver and stomach. The mean LDH activity in the liver
(1.1x103 IU g-1 wet mass tissue) was significantly
greater than in the heart (6.9x102 IU g-1 wet mass
tissue), kidney (1.9x102 IU g-1 wet mass tissue),
stomach (1.6x102 IU g-1 wet mass tissue) and
intestine (2.5x102 IU g-1 wet mass tissue), and
the LDH activity in the heart was significantly greater than in the kidney,
stomach, and intestine (statistical analysis not shown). There were no
significant differences in LDH activity among the kidney, stomach and
intestine. The CS/HOAD ratio ranged from 6.0x10-2 in the
kidney to 2.6 in the stomach (Table
3). Except for the stomach, the CS/HOAD ratios were close to or
less than one, indicating that the ß-oxidation of fatty acids could
provide most of the acetyl-CoA for the citric acid cycle. The LDH/CS ratio
ranged from 9.9 in the heart to 1.58x103 in the intestine.
Only one intestinal CS value was recorded for each species.
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Mitochondrial volume density among species
Mean VV(mt) in harbor seal liver was significantly
greater than in the dog and rat (26.4%, 17.3% and 13.2%, respectively;
Table 1; Fig. 7). The
VV(mt) in harbor seal kidney was significantly greater
than in the dog (21.4% and 16.6%, respectively). Stomach
VV(mt) in the harbor seal was significantly greater than
in the rat (24.5% and 13.3%, respectively). There were no significant
differences in VV(mt) among species in either the heart or
the small intestine.
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The VV(mt)/RMR in the harbor seal heart, liver, stomach and intestine were significantly greater than in the rat. The VV(mt)/RMR in the harbor seal liver, kidney, stomach and intestine were greater than in the dog (Table 4; Fig. 8). The VV(mt)/RMR in the dog heart, liver and stomach were also significantly greater than in the rat.
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Mitochondrial distribution
Mitochondrial distribution in the liver of the harbor seal was very
homogeneous (87.8% of the micrographs; Fig.
2), whereas only 8.7% and 4.8%, respectively, of dog and rat liver
micrographs had homogeneously distributed mitochondria. For harbor seal and
dog kidney, 49% and 50%, respectively, of the micrographs were classified as
homogeneous (Fig. 3). Again,
rat kidney was not included in the analysis due to a sample size of only one.
For harbor seal stomach, 91.3% of the micrographs were classified as
homogeneous, whereas only 76% and 69%, respectively, of dog and rat stomach
micrographs were classified as homogeneous
(Fig. 5).
Enzyme activities
The mean CS activity in the rat heart was significantly greater than in the
harbor seal or dog (124.7 IU g-1 wet mass tissue, 73.8 IU
g-1 wet mass tissue and 71. 8 IU g-1 wet mass tissue,
respectively; Table 2). The CS
activities of harbor seal and rat liver (13.5 IU g-1 wet mass
tissue and 12.3 IU g-1 wet mass tissue, respectively) were not
significantly different, but both were significantly greater than in the dog
(8.2 IU g-1 wet mass tissue). CS activity in the dog kidney was
significantly greater than in the harbor seal (21.3 IU g-1 wet mass
tissue and 15.4 IU g-1 wet mass tissue, respectively). Other organs
exhibited no significant differences in CS activity among the three species.
The CS/RMR activity of the harbor seal liver was significantly greater than
that of the dog and rat (1.3x10-2, 5.2x10-3
and 3.5x10-3, respectively;
Table 5; Fig. 9). No differences among
species existed in the CS/RMR activity of the heart or stomach. An analysis
was not conducted for the intestine because only one CS activity measurement
was made for each species.
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Mean HOAD activity in harbor seal heart was significantly greater than in the heart of dogs and rats (1.0x102 IU g-1 wet mass tissue, 38.3 IU g-1 wet mass tissue and 27.5 IU g-1 wet mass tissue, respectively; Table 2). Harbor seal liver also had a significantly greater HOAD activity when compared with that of the dog and rat (11.2 IU g-1 wet mass tissue, 2.0 IU g-1 wet mass tissue and 7.1 IU g-1 wet mass tissue, respectively). The HOAD activity in the liver of rats was significantly greater than in dogs. The HOAD activity in the small intestine of the rat was significantly greater than in the harbor seal and the dog (6.0 IU g-1 wet mass tissue, 3.4 IU g-1 wet mass tissue and 2.4 IU g-1 wet mass tissue, respectively). HOAD activities in the kidney and stomach of the three species were not statistically different. Analysis of the HOAD/RMR showed that: (1) harbor seal heart was significantly greater than dog and rat heart (4.2x10-2, 1.3x10-2 and 6.8x10-3, respectively); (2) harbor seal liver was significantly greater than dog or rat liver (1.1x10-2, 1.3x10-3 and 2.0x10-3, respectively); and (3) the harbor seal intestine was significantly greater than the dog intestine (5.1x10-2 and 2.8x10-2, respectively; Table 5; Fig. 10). There were no significant differences in the HOAD/RMR activities in the kidney or stomach among the three species.
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Mean LDH activity in rat heart was significantly greater than in both the harbor seal and dog (1.3x103 IU g-1 wet mass tissue, 6.9x102 IU g-1 wet mass tissue and 5.4x102 IU g-1 wet mass tissue, respectively), but seal heart LDH activity was significantly greater than in the dog (Table 2). The liver of harbor seals had a significantly higher LDH activity when compared with that in dogs and rats (1.1x103 IU g-1 wet mass tissue, 3.1x102 IU g-1 wet mass tissue and 8.1x102 IU g-1 wet mass tissue, respectively), and LDH in the rat liver was also significantly greater than in the dog. Dog kidney LDH activity was significantly greater than that in seal kidney (2.1x102 IU g-1 wet mass tissue and 1.9x102 IU g-1 wet mass tissue, respectively). The LDH activity of the small intestine of both the harbor seal and the rat were significantly greater than in the dog (2.5x102 IU g-1 wet mass tissue, 2.7x102 IU g-1 wet mass tissue and 1.3x102 IU g-1 wet mass tissue, respectively). The stomach was statistically indistinguishable among species. The LDH/RMR ratio showed that: (1) harbor seal heart was significantly greater than dog heart (2.9x10-1 and 1.9x10-1, respectively); (2) harbor seal liver was significantly greater than dog and rat liver (1.1, 2.0x10-1 and 2.3x10-1, respectively); (3) harbor seal kidney was significantly greater than dog kidney (8.8x10-2 and 8.6x10-2, respectively); (4) harbor seal stomach was significantly greater than rat stomach (2.4 and 1.2, respectively); and (5) harbor seal intestine was significantly greater than dog or rat intestine (3.8, 1.6 and 1.9, respectively; Table 5, Fig. 11).
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The CS/HOAD ratio ranged between 0.04 in the rat intestine and 5.2 in the dog stomach (Table 3). The most extreme difference in this ratio among species occurred in the heart. At the low end of the spectrum, the harbor seal heart had a ratio of 0.7, whereas at the high end the rat heart had a ratio of 4.5, a 6-fold difference. The CS/HOAD ratio of the harbor seal heart was significantly less than that of the dog or rat, and that of the dog was significantly less than that of the rat. The CS/HOAD ratio in the liver was significantly different between the dog and harbor seal, with ratios of 4.1 and 1.2, respectively. The CS/HOAD ratio in the harbor seal liver was significantly less than that in the dog or rat, and that in the rat was less than that in the dog. The CS/HOAD ratio of the harbor seal kidney was significantly less than that in the dog (6.0x10-3 and 0.1, respectively). Ratios for the stomach were significantly different between the dog and harbor seal (5.2 and 2.6, respectively). CS/HOAD ratios for intestine were similar among species (5.0x10-2, 6.0x10-2 and 4.0x10-2 for harbor seal, dog and rat, respectively) but were not analyzed statistically due to the small sample size.
The LDH/CS ratio ranged between 5.3 in the rat kidney and 1583.1 in the harbor seal intestine. The LDH/CS ratios of the harbor seal liver and kidney (80.5 and 12.2, respectively) were significantly greater than those in the dog (38.3 and 9.8, respectively) (Table 3). The LDH/CS ratio in the rat liver was also significantly greater than that in the dog (66.3 and 38.3, respectively). There were no differences among species in the LDH/CS ratio of the heart or stomach. The LDH/CS ratio of the intestine was not included in the statistical analysis.
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Discussion |
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Harbor seal inter-organ comparisons
The function of the heart, liver, kidneys and digestive organs of mammals
relies on the delivery of oxygen from the circulation. The estimated
mass-specific RMR of harbor seal organs (taken from Weddell seal estimates;
Davis and Kanatous, 1999) is,
in descending order, heart (59.2 ml O2 min-1
kg-1), kidneys (38.4 ml O2 min-1
kg-1), liver (27.7 ml O2 min-1
kg-1) and gastrointestinal tract (10.1 ml O2
min-1 kg-1). When blood flow is reduced, these organs
increase their extraction of oxygen from the blood
(Fisher, 1963
;
Jacobsen et al., 1969
;
Granger and Shepherd, 1973
;
Lutz et al., 1975
;
Nelson et al., 1988
;
Fink, 2001
) or, in the case of
the kidneys, decrease their metabolic rate because glomerular filtration rate
(GFR) is reduced (Brezis et al.,
1984
). If convective oxygen transport is insufficient to maintain
aerobic metabolism, the tissue will become anoxic, and cellular damage or
death can result (Brezis et al.,
1984
). Although the seal heart had the highest RMR among the
organs, it did not have the highest VV(mt). However, CS
activity in the seal heart was 4.8-6.5x greater than that in the liver,
kidney and stomach, indicating a higher density of citric acid cycle enzymes
in heart mitochondria. This, combined with an elevated HOAD activity and a
CS/HOAD ratio of 0.7, shows the high aerobic capacity of the heart and its
ability to oxidize fatty acids.
Among the organs of the harbor seal, the liver had the highest VV(mt) followed by the stomach and kidneys. The HOAD activity of the seal kidney was significantly greater than that of all other organs, reinforcing the reliance on lipid metabolism. The intestine had the lowest VV(mt), which may reflect the overall low RMR of the gastrointestinal tract.
The LDH activity of the liver was the highest among the seal's organs,
reflecting its capacity to switch on anaerobic ATP production if necessary.
However, the high LDH activity may also indicate an enhanced ability to
convert lactate into pyruvate as the initial step in gluconeogenesis. Previous
studies (Davis et al., 1983)
have shown that most of the lactate produced when harbor seals are forcibly
submerged or exercising is not oxidized but recycled, most likely back into
glucose in the liver. We believe that seals rely primarily on aerobic
metabolism during diving. In rare instances, there is a survival advantage for
the seals to produce ATP anaerobically, resulting in a large lactate load in
tissues and blood. The conversion of this lactate back to pyruvate requires
LDH, but the process also requires the presence of adequate oxygen and a high
percentage of heart (H)-type LDH subunits in the tissue
(Castellini et al., 1981
). We
would therefore argue that an elevated LDH has a greater significance for the
rapid conversion of pyruvate to lactate (and the production of ATP) than the
reverse. Nevertheless, an elevated LDH will facilitate the removal of lactate
after it is produced.
Interspecies comparison of the heart
We found that the unscaled VV(mt) of harbor seal heart
was not significantly different from that of the dog and rat, but the
VV(mt)/RMR was greater (1.4x) than that in the rat.
These results indicate a small increase in the VV(mt) in
the seal heart relative to its metabolic requirements. We hypothesize that
this increase in VV(mt)/RMR aids in the maintenance of
aerobic metabolism during diving by decreasing the diffusion distance between
mitochondria and intracellular oxygen. We base this hypothesis on the rate of
diffusion within a muscle fiber described by Fick's equation:
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The unscaled CS activity in the harbor seal heart was significantly less than in the rat heart. However, when CS activity was scaled to RMR, there were no significant differences among the three species, indicating that the high RMR of the rat accounts for the high CS activity. As a result, overall aerobic capacity of harbor seal heart muscle is not elevated compared with that of the rat and dog when scaled to cardiac muscle RMR. However, there is an increase in VV(mt) that may enhance the diffusion of intracellular oxygen into mitochondria under hypoxic conditions.
Based on a mean postabsorptive respiratory quotient (ratio of
CO2 production/O2 consumption) of 0.74 in seals,
previous studies (Kooyman et al.,
1981; Davis et al.,
1991
) showed that seals rely heavily on lipid as a fuel for energy
metabolism, especially during exercise. Even without scaling for RMR, HOAD
activity in the harbor seal heart was significantly greater (2.6x and
3.6x) than in the dog and rat, respectively. When scaled to RMR, the
HOAD activity in the seal heart was 3.2x and 6.2x greater than
that in the dog and rat, respectively, indicating that seal heart relies
heavily on lipid as a source of energy. This was further supported by a
CS/HOAD ratio of 0.7 in the seal, while the ratio in the dog and rat were 1.9
and 4.5, respectively. This dependence on lipid as an energy source in seals
results from a diet rich in fatty acids and protein but containing little
carbohydrate (Roberts et al.,
1943
; Balazquez et al.,
1971
; Kettelhut et al.,
1980
; Davis et al.,
1991
). Studies of terrestrial mammals have shown that high-fat,
low carbohydrate diets increase the rate of lipid oxidation
(Roberts et al., 1996
;
Lee et al., 2001
) and that
this is accompanied by an increase in the concentration of enzymes required
for fatty acid oxidation (Gollnick and
Saltin, 1988
; Roberts et al.,
1996
). A greater reliance on fatty acid oxidation also spares
carbohydrate for red blood cells and the central nervous system, which are
obligate glucose metabolizers.
Castellini et al. (1981)
found that LDH activity in the hearts of marine and terrestrial mammals was
not significantly different. By contrast, we observed a small, but
significant, increase in LDH activity (1.3x) of the seal heart over that
of the dog. This difference was further enhanced (1.5x) when LDH
activity was scaled to RMR. Castellini et al.
(1981
) found that the mean LDH
activity for the marine mammals was not significantly different from that of
terrestrial mammals, although some marine mammals had elevated LDH activities
relative to others. This may account for the difference in the results between
Castellini et al. (1981
) and
our study, given that our LDH values for harbor seals were not greatly
elevated compared with those of the dog and not at all compared with those of
the rat. Since the heart is critical for survival, enhanced LDH activity may
have survival advantage, even if the heart normally remains aerobic during
dives. Ohtsuka and Gilbert
(1995
) studied the effects of
high-altitude hypoxemia on cardiac enzyme activities in pregnant and
non-pregnant sheep. The results showed that LDH activity increased by 24% and
27%, respectively, in the left ventricle of non-pregnant and pregnant adult
sheep. Similar results for animals exposed to high altitudes (hypoxia) have
been reported by Vergnes
(1971
), Penney
(1974
) and Barrie and Harris
(1976
). Seal cardiac muscle
shows adaptations for both aerobic and anaerobic metabolism under conditions
of hypoxia. Oxygen will be used until a critical
PaO2 is reached during a dive. At that point,
anaerobic metabolism will become increasingly important as a source of ATP. In
Weddell seals, this critical PaO2 is less than
2.9x102 Pa, and it appears that the seals rarely exceed this
threshold during aerobic dives (Qvist et
al., 1986
; Davis and Kanatous,
1999
). As a result, convective oxygen transport to the heart is
normally sufficient to maintain aerobic metabolism during a dive.
Nevertheless, enhanced anaerobic glycolytic enzyme activity is present if
needed to protect the heart against hypoxia.
Interspecies comparison of the liver
Seal liver VV(mt) was significantly greater (2x
and 1.5x) than rat and dog liver, respectively. When scaled for RMR,
seal liver VV(mt) was even greater (6.6x and
2.3x) than in rat and dog liver, respectively. As with the seal heart,
we hypothesize that this increase in VV(mt) decreases the
intracellular distance for oxygen diffusion and effectively increases
diffusive conductance to maintain aerobic metabolism and organ function during
periods of hypoxia while diving (Costa et
al., 1988). Based on the hepatic clearance of indocyanine green
(ICG) from the blood during voluntary dives, Davis et al.
(1983
) showed that hepatic
function was maintained in subadult Weddell seals during voluntary dives, even
though hepatic arterial and portal blood flow were reduced as a result of the
dive response. The liver appears to compensate for a reduction in blood flow
during a dive by increasing the extraction coefficient of ICG
(Fisher, 1963
;
Jacobsen et al., 1969
),
thereby maintaining a pre-dive level of ICG clearance. Plevris et al.
(1999
) observed that reduced
ICG clearance in laboratory animals is due mainly to impaired microcirculation
in the liver and compromised hepatocyte function. Since ICG clearance in the
seal is maintained during aerobic dives, there is no impairment of hepatic
microcirculation or function. This conclusion is further supported by data
that show little variation in the blood glucose concentration and blood urea
nitrogen (BUN) during consecutive, aerobic dives
(Castellini et al., 1988
; R. W.
Davis, unpublished results), which would not be possible if liver function
were disrupted.
Costa et al. (1988) showed
that the livers of rats exposed to chronic, hypobaric hypoxia had a more
homogeneous distribution of mitochondria than rats raised under normoxic
conditions. In our analysis of hepatic mitochondrial distribution, 88% of
micrographs from the seal were classified as homogeneous, whereas only 9% of
dog and 5% of rat liver micrographs were classified as homogeneous. Along with
an elevated VV(mt), we hypothesize that the homogeneous
distribution of hepatic mitochondria decreases the intracellular diffusion
distance for oxygen and helps maintain aerobic metabolism under hypoxic
conditions.
CS activity in the harbor seal liver was significantly greater than that in
the dog. Since CS is an enzyme found in the matrix of the mitochondria, it
follows that an increase in VV(mt) would result in an
increase in CS activity. HOAD activity in the seal liver was 5.6x and
1.6x greater than in the dog and rat, respectively, and 8.5x and
5.5x greater, respectively, when scaled to RMR. The increased HOAD
activity in the harbor seal probably results from the high-fat,
low-carbohydrate diet discussed previously. These results are similar to those
for skeletal muscle for harbor seals, Steller's sea lions (Eumetopias
jubatus) and northern fur seals (Callorhinus ursinus)
(Kanatous et al., 1999).
Kennedy et al. (2001
) found
that HOAD activity in the liver of rats decreases after exposure to chronic
hypoxia at high altitude, but HOAD activity and glycogen sparing have not been
studied widely in the liver. Although the CS/HOAD ratio in the liver is not as
low as in heart, kidneys and intestine, it indicates that lipid is an
important source of fuel for energy metabolism in the liver.
Castellini et al. (1981)
found that LDH activity (in the direction of pyruvate to lactate) in marine
mammal liver was higher than in terrestrial mammals. Our data are in
agreement, with harbor seals having a statistically greater LDH activity than
either the dog or rat regardless of scaling for RMR. However, our mean LDH
activity was double that reported by Castellini et al.
(1981
) for marine mammals, with
values of 1084.5±66.5 IU g-1 wet mass tissue and
538±188 IU g-1 wet mass tissue, respectively. However, as
noted by Castellini et al.
(1981
), some of the terrestrial
mammals had high LDH activities and some marine mammals had low LDH
activities. The LDH/CS ratio of the harbor seal liver is greater than that of
the dog, indicating a relatively high anaerobic capacity. Therefore, our
results show a heightened ability for anaerobic metabolism in the liver of the
harbor seal. Although we did not measure LDH activity in the lactate to
pyruvate direction, this aerobic process may be important in the liver for
recycling lactate back into glucose through gluconeogenesis. As with the
heart, the seal liver shows adaptations for both aerobic and anaerobic
metabolism under conditions of hypoxia. Although Davis and Kanatous
(1999
) showed that the liver
in Weddell seals receives sufficient oxygen to prevent anaerobic ATP
production during dives within the ADL, this source of energy may be important
during longer dives.
Interspecies comparison of the kidney
Studies in which seals were forcibly submerged led researchers to believe
that there was a pronounced decrease in blood flow to the kidneys during
diving resulting from an extreme dive response
(Blix et al., 1976). Bradley
and Bing (1942
) and Murdaugh et
al. (1961
) came to the same
conclusion when seals that were forcibly submerged experienced either a
decrease or complete cessation in GFR. However, a study by Davis et al.
(1983
) of Weddell seals making
voluntary dives came to a different conclusion. By injecting inulin into the
blood of the seals, they were able to measure the seal's GFR during and after
dives. They found that inulin clearance did not change from pre-dive, resting
levels and only decreased when the seals dived for longer than their ADL. They
concluded that the kidneys functioned normally during dives shorter than the
ADL due to sustained renal blood flow and glomerular filtration.
Mammalian kidneys, regardless of species, require an abundance of
mitochondria to provide ATP for active transport of electrolytes and
metabolites across the renal tubules. Since mitochondria are the source of ATP
production, they are present in the kidneys where the sodium pump enzymes
reside (Abrahams et al., 1991).
The harbor seal kidney had a VV(mt) that was significantly
greater (29%) than that of the dog. We hypothesize that this elevation in
VV(mt) is an adaptation to sustain aerobic metabolism and
renal function during the hypoxia experienced during diving. When blood flow
to the kidneys of a terrestrial mammal decreases, it concomitantly reduces its
metabolic rate since the kidney's workload is directly proportional to the
amount of plasma that must be filtered
(Brezis et al., 1984
). However,
at very low renal blood flow, the metabolic rate of the kidneys is reduced to
basal levels because there is little filtration and absorption
(Lassen, 1964
). The kidneys
can suffer damage if there is a further decrease in blood flow
(Brezis et al., 1984
), although
the seal kidney appears to recover from severe anoxia better than the dog
kidney (Halasz et al., 1974
).
The elevated VV(mt) in the harbor seal kidney may aid in
decreasing the intracellular diffusion distance of oxygen and thereby keeping
renal metabolism aerobic and functioning normally.
Mitochondrial distribution in the harbor seal and dog kidneys was nearly identical. Unlike the liver, it appears that the harbor seal kidney needs no redistribution of mitochondria to facilitate the intracellular diffusion of oxygen. The increased volume density of the mitochondria may be enough to increase the effective oxygen diffusive conductance, or the intrinsic grouping of mitochondria in the mammalian kidney may be equally divided between a homogeneous and clustered distribution.
The CS activity in the dog kidney was significantly greater (1.4x)
than in the seal, even though the VV(mt) in the seal was
greater (1.3x) than in the dog. This result indicates a greater
concentration (packing) of CS in the dog mitochondria. Kanatous et al.
(1999) obtained similar
results for an increase in CS activity in the mitochondria of pinniped
skeletal muscle. However, when scaled for RMR, there were no statistical
differences in the CS activity of harbor seal and dog kidneys. The enzymatic
design of the mammalian kidney for oxidative metabolism may depend solely on
body mass. HOAD activity in both the seal and dog kidneys was at least twice
as great as any other organ examined (the rat showed a similar trend) but was
not significantly different between the two species regardless of scaling. As
a result, the CS/HOAD ratio for the harbor seal, dog and rat kidneys was very
low (
0.1), indicating that the mammalian kidney has an elevated enzymatic
potential for aerobic lipid metabolism.
When LDH activity in the harbor seal kidney was scaled to RMR, it was
significantly greater than in the dog. The LDH/CS ratio was also significantly
greater than in the dog, indicating a higher anaerobic capacity in the harbor
seal kidney. The enhanced LDH activity may confer a survival advantage, even
though the kidneys remain aerobic during most voluntary dives. Again, when a
dive exceeding an animal's ADL is required, there is additional LDH activity
available for the glycolytic production of ATP. This ability was observed by
Halasz et al. (1974), which
explains the ability of the seal kidney to recover from severe bouts of
hypoxia that would be rare in the wild.
Interspecies comparison of the gastrointestinal tract
The VV(mt) in the stomach of the harbor seal was
significantly greater (1.8x) than in the rat and, when scaled for RMR,
it was greater (3.9x and 1.5x, respectively) than in both the rat
and the dog. When scaled for RMR, the VV(mt) of the harbor
seal small intestine was also significantly greater (2.6x and 2x,
respectively) than that of the rat and dog. We hypothesize that the increase
in VV(mt) in the stomach and small intestine of the harbor
seal is an adaptation for maintaining aerobic metabolism and gastrointestinal
function during hypoxia. This is supported by the observations of Davis et al.
(1983), who found that the
plasma of Weddell seals making foraging dives became very lipemic and opaque
from the presence of chylomicrons. The lipemic plasma was an indication that
the digestion and intestinal absorption of fat [Weddell seals usually feed on
Antarctic silverfish (Pleuragramma antarcticum), which have a very
high lipid content] was taking place during a bout of consecutive foraging
dives.
Previous research has shown that the gastrointestinal organs are capable of
compensating for alterations in blood flow by adjusting the amount of oxygen
extracted from the blood (Granger and
Shepherd, 1973). The oxygenation of the tissue is regulated by the
balance between blood flow and oxygen extraction
(Johnson, 1960
;
Garg, 1979
;
Granger and Norris, 1980
).
Kvietys and Granger (1982
)
also found that at normal intestinal blood flows, the uptake of oxygen appears
to be blood flow independent, whereas at very low perfusion, oxygen uptake
becomes flow dependent. An increased VV(mt) would support
the efficient extraction (by increasing the diffusive conductance) of oxygen
needed to support aerobic metabolism and normal function during a dive.
Analysis of the distribution of mitochondria in the stomach revealed that approximately 91.3% of harbor seal micrographs and 76% and 69% of dog and rat micrographs, respectively, were classified as homogenously distributed. As in the liver, the more homogeneous distribution of mitochondria in the mucosal surface of the stomach may aid in decreasing the effective diffusion distance of oxygen in the stomach lining.
The CS activities in the stomachs of the three species were not significantly different and, although intestinal CS activity was not included in the analyses, the single values obtained for each species were very similar. Mean HOAD activity in the stomach of the harbor seal was not significantly different from that of rat or dog. However, the HOAD/RMR of the seal intestine was significantly greater than that of the dog. The CS/HOAD ratio for the harbor seal intestine (and the rat and dog) was very low (<0.1), indicating a high enzymatic potential for aerobic lipid metabolism. The CS/HOAD ratio of the harbor seal stomach (5.2) was higher than that of the intestine.
The LDH activity in the stomach of the harbor seal was not significantly different from that of the dog or rat. However, when scaled for RMR, LDH activity in the seal stomach was significantly greater than in the rat. When scaled for RMR, LDH activity in the small intestine of the harbor seal was significantly greater than that in the dog and rat. The higher LDH activity in the harbor seal digestive organs indicates a heightened ability to undergo anaerobic metabolism during a dive if necessary. However, given that most dives are within an animal's ADL, and the LDH/CS ratio is not significantly different from that of the dog or rat, we believe that the animal may only rely on this anaerobic production of ATP when undertaking a dive beyond its ADL.
Conclusions
The elevated VV(mt)/RMR, especially in the liver of the
harbor seal, indicates adaptations to sustain aerobic metabolism during
hypoxia by enhancing the diffusion of oxygen to mitochondria at low partial
pressures. The elevated CS/RMR of the harbor seal liver is indicative of its
high aerobic capacity and poise for aerobic metabolism. The high HOAD activity
and low CS/HOAD ratio, along with a respiratory quotient that is normally less
than 0.74, indicate that lipids are the primary substrate for aerobic
metabolism. A heightened LDH activity indicates an adaptation for the
anaerobic production of ATP on dives that exceed animal's ADL. Hence, the
heart, liver, kidneys and gastrointestinal organs of harbor seals exhibit dual
adaptations that promote an aerobic, lipid-based metabolism under hypoxic
conditions but can provide ATP anaerobically if required.
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