1 Division of Pulmonary and
Critical Care Medicine, To determine the
effects of hypoxia on energy state and intracellular pH
(pHi) in resting pulmonary and
systemic arterial smooth muscles, we used
31P nuclear magnetic resonance
spectroscopy and colorimetric and enzymatic assays to measure
pHi; intracellular concentrations of ATP, phosphocreatine, creatine, and
Pi; and phosphorylation potential
in superfused tissue segments from porcine proximal intrapulmonary and
superficial femoral arteries. Under baseline conditions
(PO2 467 ± 12.1 mmHg), energy
state and total creatine (phosphocreatine + creatine) concentration
were lower and pHi was higher in
pulmonary arteries. During hypoxia
(PO2 23 ± 2.4 mmHg), energy state
deteriorated more in femoral arteries than in pulmonary arteries.
pHi fell in both tissues but was
always more alkaline in pulmonary arteries. Reoxygenation reversed the changes induced by hypoxia. These results suggest that production and/or elimination of ATP and
H+ was different in resting
pulmonary and systemic arterial smooth muscles under baseline and
hypoxic conditions. Because energy state and
pHi affect a wide variety of
cellular processes, including signal transduction, contractile protein
interaction, and activities of ion pumps and channels, further
investigation is indicated to determine whether these differences have
functional significance.
phosphorus-31 nuclear magnetic resonance; phosphorylation
potential; adenosine 5'-triphosphate; phosphocreatine; creatine; vascular smooth muscle; intracellular water volume
CELLULAR ENERGY STATE is determined by the balance
between production and utilization of high-energy phosphagens such as
ATP, the immediate source of metabolic energy, and phosphocreatine (PCr), which provides ATP via the creatine kinase, or Lohman, reaction
(10). When the ratio of energy supply to energy demand decreases, as
during severe hypoxia, homeostatic mechanisms attempt to match ATP
production to ATP utilization. As a result, ATP concentration ([ATP]) can remain relatively constant and is therefore not
a very sensitive index of the energy supply-demand condition (4, 13).
More sensitive indexes include the ratios of PCr ([PCr]) or
Pi
([Pi]) concentration
to [ATP] or creatine (Cr; [Cr]) concentration ([PCr]/[ATP],
[Pi]/[ATP],
and [PCr]/[Cr]), phosphorylation
potential ( The hypothesis that changes in energy state signal pulmonary vasomotor
responses to hypoxia has been considered by many investigators, but the
role of energy state in these responses remains unclear (30). In
isolated pig lungs, we found that [ATP] and adenylate charge did not change during vasoconstrictor responses to moderate hypoxia or vasodilator responses to severe hypoxia (5). These findings
did not support the possibility that pulmonary vasomotor responses were
triggered by changes in energy state; however, a role for energy state
was not ruled out because ATP and adenylate charge may not be the
relevant energy state signals. Furthermore, measurements in the whole
lung may not reflect energy state in the cells responsible for hypoxic responses.
The cell that triggers pulmonary vascular responses to hypoxia may be
the pulmonary arterial myocyte itself. In support of this possibility,
hypoxia was found to decrease potassium-channel conductance, increase
membrane potential and intracellular calcium concentration, and cause
contraction in isolated pulmonary arterial myocytes (29, 35, 44, 48).
The signal responsible for these effects is unknown. Moreover, these
hypoxic effects appear to differ from those in systemic arterial tissue
where hypoxia typically causes smooth muscle relaxation, possibly due
to deterioration of smooth muscle energy state and secondary inhibition
of intracellular signal transduction (11, 18, 21, 28, 33, 38).
In addition to its effects on energy state, hypoxia can increase
production of lactic acid (34) and thereby promote a decrease in
intracellular pH (pHi). In
vascular smooth muscle, changes in
pHi can alter membrane potential,
calcium homeostasis, and myosin light chain kinase activity (1, 9, 24,
31). Furthermore, interventions designed to alter
pHi have been shown to change vasomotor tone in both systemic and pulmonary arteries (2, 24, 32) and
to modify pulmonary pressor responses to hypoxia in isolated lungs (17,
37).
Despite the potential importance of smooth muscle energy state and
pHi as determinants of vasomotor
tone, it remains unclear how hypoxia affects these variables in
pulmonary arterial smooth muscle or whether these effects differ from
those in systemic arterial smooth muscle. Thus, in the present study,
we used 31P nuclear magnetic
resonance (NMR) and enzymatic and colorimetric assays to measure energy
state and pHi in resting porcine
intrapulmonary and femoral arterial smooth muscles. Our results
demonstrate that energy state and
pHi differed markedly in these
tissues under baseline conditions and that hypoxia decreased energy
state more in femoral than in pulmonary arterial smooth muscle.
Tissue preparation. Pigs weighing
28-35 kg were anesthetized with ketamine (20 mg/kg im) followed by
pentobarbital sodium (12.5 mg/kg iv) and killed by exsanguination from
the carotid or femoral arteries. Proximal intrapulmonary (5- to
7-mm-ID) and superficial femoral (1- to 2-mm-ID) arteries were
isolated, placed in oxygenated Ringer solution at 25°C, cleaned of
adherent connective tissue, and cut into 4 × 4-mm segments. These
vessels were chosen because they provided adequate amounts of tissue
for 31P NMR spectroscopy and
appeared to have similar wall thicknesses. In addition, proximal
intrapulmonary arteries exhibited hypoxic vasoconstriction in vivo
(20), and myocytes from proximal pulmonary arteries developed
depolarization and reduced potassium-channel conductance during hypoxia
in vitro (35, 48). We studied resting unloaded tissue because we were
interested in the possibility that changes in energy state and
pHi caused changes in vasomotor tone rather than the reverse.
Approximately 25-35 segments of proximal intrapulmonary artery
(total wet weight 0.94 ± 0.05 g; total dry weight 0.17 ± 0.007 g; n = 13) or superficial femoral
artery (total wet weight 0.53 ± 0.04 g; total dry weight 0.11 ± 0.009 g; n = 11) were loosely arranged
in the lower 3 cm of a glass NMR sample tube (10-mm OD) and superfused
continuously through a multiport polyethylene catheter with
phosphate-free Krebs bicarbonate solution, which contained (in mM)
118.3 NaCl, 4.7 KCl, 1.2 MgSO4,
2.5 CaCl2, 25.0 NaHCO3, and 10.0 glucose. The
superfusate was recirculated with a roller pump from a heated reservoir
through insulated polyvinyl chloride tubing and bubble trap to the NMR
sample tube at 20 ml/min. At this flow rate, the temperature in the
sample tube was 37°C when the temperature in the reservoir was
50°C. The superfusate was gassed with 93%
O2-7%
CO2 at the reservoir, and the
fluid surface in the sample tube was continuously flushed with the same
gas mixture. As described in 31P
NMR spectroscopy, a
CO2 concentration of 7% was
necessary to achieve PCO2 of
35-45 mmHg in the NMR sample tube. Superfusate PO2,
PCO2, and pH in the sample tube were
measured with a blood gas analyzer (Radiometer model BMS3 Mk2,
Copenhagen, Denmark).
31P NMR spectroscopy.
The sample tube containing the superfused arterial tissue was placed in
the bore of an 11.8-T vertical superconducting magnet containing a
31P probe tuned to 202 MHz
connected to a Bruker MSL-500 NMR spectrometer. Field homogeneity was
optimized by shimming the free induction decay (FID) of protons at 500 MHz. Proton spectral line width was 15-35 Hz in pulmonary artery
experiments and 5-20 Hz in femoral artery experiments.
31P NMR spectra were obtained with
a 12-µs pulse width (60° flip angle) and the FID was acquired
over 100 ms. For each spectrum, 904 scans were collected with a 1-s
interpulse interval over 15 min. The FID was subjected to Fourier
transformation after applying 20-Hz line broadening to reduce noise in
the spectra. No additional signal enhancement was required. Saturation
factors determined in four pulmonary and three femoral artery
experiments with an 8-s pulse interval were as follows: 1.5 ± 0.06 for PCr, 1.18 ± 0.07 for
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
= [ATP]/[ADP][Pi],
where [ADP] is ADP concentration), and adenylate charge
([ATP + 2ADP]/[ATP + ADP + AMP]). These ratios
may also act as signals for homeostatic metabolic reactions and other
adaptive responses (4, 13, 34).
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-ATP, and 1.47 ± 0.01 for
Pi in the pulmonary arteries and
1.57 ± 0.03 for PCr, 1.08 ± 0.01 for
-ATP, and 1.9 ± 0.05 for Pi in the femoral arteries.
-ATP, and Pi peaks from
spectra collected over 30 min. The baseline used to integrate the
spectral peaks was determined by Fourier transformation of the summed
FID from the total 5-h experiment, which produced a single spectrum
with very low baseline noise. [PCr]/[ATP] and
[Pi]/[ATP]
were calculated from these integrals after multiplication by
appropriate saturation factors. Because tissue density in the coil
varied from day to day, relative rather than absolute changes in
intracellular [PCr], [ATP], and
[Pi] were determined.
Specifically, in each experiment, intracellular [PCr],
[ATP], and
[Pi] are expressed as
a percentage of values measured during the baseline period (60-120
min of superfusion). Spectral resonance positions are expressed in
parts per million (ppm) relative to that of PCr.
pHi was determined from the
chemical shift difference
(
) between the PCr
and Pi peaks according to the equation pHi = pK + log[(
B)/(
A
)], where
pK,
A, and
B are constants equal to 6.70, 5.64, and 3.18, respectively (42).
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RESULTS |
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In the 31P NMR experiments, control pulmonary and femoral arterial segments were superfused throughout the experiment with a physiological salt solution gassed with 93% O2-7% CO2. Under these conditions, PO2 was 467 ± 12.1 mmHg, PCO2 was 39.4 ± 3.4 mmHg, and pH was 7.41 ± 0.014 at the tissue in the sample tube. In arteries exposed to hypoxia, the perfusate was gassed with 0% O2-7% CO2 during the exposure period (third and fourth hours of the experiment), resulting in a sample tube PO2 of 23 ± 2.4 mmHg. Perfusate PCO2 and pH were unchanged. Differences between perfusate gas tensions measured in the sample tube and those predicted to exist in the reservoir were probably due to incomplete equilibration in the reservoir, diffusion of gas across the walls of perfusate tubing, or the difference in temperature between the reservoir and sample tube (50 vs. 37°C).
To optimize acquisition times for
31P NMR data, coefficients of
variation for areas under the -ATP, PCr, and
Pi peaks were determined in
control pulmonary and femoral arteries with data collected in 15-, 30-, or 60-min blocks (904, 1,808, and 3,616 scans, respectively) over the
entire period of perfusion. As shown in Table
1, this index of variance, which includes
both random and time-dependent physiological variations, was smaller in
femoral than in pulmonary arterial tissue and decreased as acquisition time increased. On the basis of these results, we selected an acquisition time of 30 min as a reasonable compromise between adequacy
of signal-to-noise ratio and ability to follow physiological changes
occurring over time. Thus two 15-min scans were summed to produce
spectra every 30 min.
|
Examples of spectra obtained in this manner are shown in Fig.
1, which demonstrates that 30-min
acquisitions allowed acceptable signal-to-noise ratios to be achieved
in both pulmonary and femoral arteries. The resonance positions of the
-,
-, and
-ATP peaks relative to that of PCr were the same in
pulmonary and femoral arteries (
2.43 ± 0.01,
7.54 ± 0.01, and
16.09 ± 0.01 ppm, respectively), as were
those of the phosphomonoester and phosphodiester peaks (6.78 ± 0.02 and 2.97 ± 0.01 ppm, respectively). Only the position of the
Pi peak differed between tissues
(4.97 ± 0.007 and 5.02 ± 0.006 ppm in femoral and pulmonary
arteries, respectively), indicating a difference in
pHi as discussed below.
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The time courses of energy state variables and pHi in control experiments are shown in Figs. 2 and 3. Comparison of control pulmonary and femoral arteries by analysis of variance indicated marked differences in [PCr]/[ATP] (0.410 ± 0.016 in pulmonary vs. 0.892 ± 0.015 in femoral arteries; P < 0.0001) and pHi (7.246 ± 0.005 in pulmonary vs. 7.201 ± 0.002 in femoral arteries; P < 0.01); however, [Pi]/[ATP] was not different between the tissues (0.467 ± 0.018 in pulmonary vs. 0.434 ± 0.019 in femoral arteries; P > 0.6). In control femoral arteries, none of the variables changed during the experiment. In control pulmonary arteries, [ATP], [Pi], [PCr], and [PCr]/[ATP] did not change, whereas [Pi]/[ATP] decreased and pHi increased slightly (P < 0.02 and 0.04, respectively); however, for no variable was the effect of time significantly different between control pulmonary and femoral arteries.
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|
The effects of hypoxia were determined by comparing arteries exposed to hypoxia to control arteries (Figs. 2 and 3). Results obtained during the baseline period in experimental arteries confirmed the differences described above for control vessels, namely, lower [PCr]/[ATP] and higher pHi in pulmonary arteries. Hypoxia did not significantly alter [ATP], [Pi], [PCr], [PCr]/[ATP], or [Pi]/[ATP] in pulmonary arteries; however, pHi was markedly decreased (P < 0.001), achieving a value of 7.177 ± 0.026 by the end of exposure. This change was completely reversed on reoxygenation during the recovery period. In femoral arteries, hypoxia increased [Pi] (P < 0.03) and decreased [PCr] (P < 0.0001) to 132 and 79.7%, respectively, of values measured in control vessels but did not alter [ATP]. As a result, [Pi]/[ATP] increased (P < 0.03) and [PCr]/[ATP] decreased (P < 0.0001). For example, during the last 30 min of the exposure period, [Pi]/[ATP] was 0.617 ± 0.046 and 0.433 ± 0.053 and [PCr]/[ATP] was 0.728 ± 0.059 and 0.887 ± 0.086 in hypoxic and control femoral arteries, respectively. As in pulmonary arteries, hypoxia markedly decreased pHi (P < 0.0001), which reached a value of 7.140 ± 0.010 by the end of exposure. All changes induced by hypoxia were reversed on reoxygenation. Analysis of variance indicated that the decrease in [PCr]/[ATP] induced by hypoxia was significantly greater in femoral arteries than in pulmonary arteries (P = 0.036). This comparison did not achieve significance for the other variables.
[ATP] and [Cr] measured by enzymatic and colorimetric techniques during the last 30 min of the baseline and exposure periods are shown in Table 2. Expressed as total tissue concentration (in µmol/g wet weight), ATP was lower in pulmonary than in femoral arterial smooth muscle; however, expressed as intracellular concentration (in mM), ATP was the same. This occurred because intracellular water volume was lower in pulmonary arterial tissue (0.140 ± 0.047 vs. 0.267 ± 0.051 ml/g wet weight; P = 0.04). Hypoxia did not affect [ATP] in either tissue. Cr, whether expressed as total tissue or intracellular concentration, was lower in pulmonary arteries and was increased equally by hypoxia.
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To estimate the size of the total Cr pool in pulmonary and femoral arterial smooth muscles, we added the mean values of [PCr]/[ATP] determined by 31P NMR to the mean values of [Cr]/[ATP] determined spectrophotometrically during the last 30 min of the baseline and exposure periods. As shown in Fig. 4, baseline [Cr]/[ATP] and [PCr]/[ATP] values in pulmonary arterial smooth muscle were ~60 and 40%, respectively, of values measured in femoral arterial smooth muscle ([Cr]/[ATP] = 0.705 ± 0.097 vs. 1.21 ± 0.173, P < 0.01; [PCr]/[ATP] = 0.362 ± 0.047 vs. 0.906 ± 0.045, P < 0.0001). As a result, baseline [PCr + Cr]/[ATP] in pulmonary arterial smooth muscle was about one-half of that in femoral arterial smooth muscle. Hypoxia decreased [PCr]/[ATP] in femoral but not in pulmonary arterial smooth muscle and had no effect on [Cr]/[ATP] in either tissue. Consequently, [PCr + Cr]/[ATP] changed little, if at all.
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The effects of NaCN administered to control pulmonary and femoral arteries after the recovery period are shown in Table 3. At concentrations of 1 and 10 mM, cyanide significantly decreased [PCr]/[ATP] and pHi in both arteries. At 10 mM, cyanide decreased [ATP] in pulmonary and femoral arteries to 74 and 88%, respectively, of control values measured at the end of the recovery period; however, this change achieved significance only in the femoral arteries.
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Vascular wall thickness averaged 671 ± 49 µm (range 463-911 µm) in pulmonary arterial segments and 487 ± 27 µm (range 339-607 µm) in femoral arterial segments. These values were significantly different (P < 0.01).
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DISCUSSION |
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We used 31P NMR spectroscopy
because it allowed nondestructive continuous determination of free
intracellular [ATP], [PCr], [Pi], and
H+ concentration in the same
tissue sample. The major disadvantage of this technique is its low
sensitivity. Consequently, large amounts of tissue, strong magnetic
fields, and long spectral acquisition times are usually required to
achieve adequate measurements. In previous studies of vascular tissue
(19, 23, 42), relatively low magnetic field strengths and small amounts
of tissue dictated acquisition times 1 h. In the present study, we
used an 11.8-T magnet to examine 0.5-1 g of tissue and, as shown
in Fig. 1 and Table 1, were able to obtain well-resolved spectra with
acquisition times as short as 15 min in the femoral artery and 30 min
in the pulmonary artery. Spectral peak positions were similar to those reported previously for vascular tissue (19, 23, 42). Line widths for the PCr and
-ATP peaks were small, and the
Pi peak was clearly resolved from
the phosphomono- and diester peaks. Clear demarcation of the
Pi and PCr signals allowed
pHi to be monitored continuously
from the difference in the positions of the PCr and
Pi peaks as described above.
Because ATP, PCr, and Pi were not
present in the extracellular fluid and smooth muscle cells contain
virtually all of the intracellular fluid volume in vascular tissue
(42), we assume that our measurements reflect changes occurring in
arterial myocytes. These changes could occur by mechanisms intrinsic to
myocytes or as a result of influences on myocyte energy state and pH
deriving from other cell types.
Baseline conditions. In femoral arteries under baseline conditions, mean [ATP] (0.613 ± 0.084 µmol/g) and [PCr]/[ATP] (0.892 ± 0.015) were within the range of values previously reported for resting systemic vascular smooth muscle (6, 18, 23, 25, 28, 33). Mean [Pi]/[ATP] (0.434 ± 0.019) was comparable to previous values obtained by 31P NMR (23, 42) but lower than values obtained by enzymatic and chromatographic techniques (19, 21, 25), probably because the tissue extraction required by these techniques caused release of tissue-bound Pi and overestimation of free intracellular [Pi] (19, 45).
Another index of energy state is , defined as
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(1) |
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(2) |
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(3) |
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(4) |
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Intracellular [ATP] in pulmonary arteries under baseline
conditions was the same as that in femoral arteries (Table 2) but greater than the value (1 mM) measured in rabbit aorta contracted with norepinephrine or KCl (11). Expressed as total tissue
concentration, pulmonary arterial ATP was less than the values
calculated for rat extrapulmonary arteries (1-2 µmol/g wet
weight) but similar to the values obtained in systemic vascular smooth
muscle (0.3-1.2 µmol/g wet weight) (6, 25, 39). These
discrepancies may be related to differences in species, experimental
conditions, measurement techniques, or vascular intracellular water volume.
We are unaware of previous measurements of
[Pi]/[ATP]
or [PCr]/[ATP] in pulmonary vessels;
however, mean
[Pi]/[ATP]
in control pulmonary arteries (0.467 ± 0.018) was the same as that
in control femoral arteries (0.434 ± 0.019). On the other hand,
[PCr]/[ATP] was considerably lower
(0.410 ± 0.016 vs. 0.892 ± 0.015;
P < 0.0001). Equation 4 indicates that, at the same
[Pi]/[ATP],
pulmonary [PCr]/[ATP] would be less than
femoral [PCr]/[ATP] if the pulmonary
K'ck were higher or
the pulmonary [Cr] or were lower.
K'ck is a function
of intracellular
[Mg2+] and
H+ concentration (16). A
femoral-pulmonary difference in intracellular [Mg2+] seems unlikely
because the resonance position of the -ATP peak, which depends on
intracellular [Mg2+]
(19, 42), was the same in the two vessels. As discussed below,
pHi was higher in pulmonary
arteries (Fig. 3), but a higher pHi should decrease, not increase,
K'ck (16). [Cr] and [Cr]/[ATP] were lower in
pulmonary arteries, and, as a result, the total intracellular Cr pool
([PCr + Cr]/[ATP]) was about one-half of that
in femoral arteries (Table 2, Fig. 4). This difference must have
contributed to the lower pulmonary
[PCr]/[ATP], but it was not the whole
explanation. As shown in Fig. 5,
in pulmonary arteries was 2.8 × 104
M
1 under baseline
conditions or ~40% of its value in femoral arteries. This difference
is likely to be significant because the mean values of
[PCr]/[ATP], [Cr], and
pHi used to calculate
were
significantly different. We conclude that
[PCr]/[ATP] was less in pulmonary arteries not
only because the Cr pool size was smaller but also because the energy
state was lower.
The difference in baseline energy state between pulmonary and femoral arterial smooth muscles indicates that production or consumption of ATP was different under baseline conditions; however, it is unlikely that the difference in energy state was due to a difference in ATP consumption by actin-myosin interaction because the tissues were studied under resting, unloaded conditions. In addition, several considerations suggest that the energy state difference was not caused by decreased ATP production due to limitation of oxidative phosphorylation by oxygen diffusion. First, oxygen consumption in unstimulated unloaded vascular smooth muscle was very low (34). Second, because the tissues were exposed to a high flow of perfusate containing oxygen at high partial pressures, the gradient for oxygen diffusion was very high. Third, although vascular wall thickness was higher in pulmonary arteries (671 ± 49 vs. 487 ± 27 µm), the concentration of cells in the vascular wall was lower (intracellular water volume 0.140 ± 0.047 vs. 0.267 ± 0.051 ml/g); therefore, a larger oxygen diffusion distance was offset by a smaller oxygen consumption requirement. Fourth, phosphagen concentrations and pHi in control pulmonary and femoral arteries were extraordinarily stable throughout the 5-h observation period (Figs. 2 and 3). Finally, if oxygen diffusion were limiting ATP production under baseline conditions, energy state should rapidly deteriorate under hypoxic conditions. As discussed below, this did not occur in pulmonary arterial smooth muscle. Thus the pulmonary-femoral difference in baseline energy state must have some other explanation.
As shown in Fig. 3, pHi averaged 7.201 ± 0.002 in control femoral arteries and 7.246 ± 0.005 in control pulmonary arteries (P < 0.01). Our femoral arterial measurements are consistent with pHi values (7.01-7.26) previously measured with fluorescent dyes, NMR, or microelectrodes in systemic arterial tissue under similar conditions (1, 9, 23, 32). Only a few measurements of pHi have been made in pulmonary arterial tissue, and these yielded a similar range of values. For example, with fluorescence techniques, mean values of 7.28 ± 0.03 (36) and 6.9 ± 0.06 (14) were obtained in cultured pulmonary arterial smooth muscle from guinea pigs and ferrets, respectively, whereas an average of 7.09 was obtained in isolated segments of intrapulmonary arteries from dogs (24). To our knowledge, the only previous direct comparison of pHi in systemic and pulmonary arterial tissues was that of Vadula et al. (43), whose preliminary communication reported values of 7.08 ± 0.12 and 7.27 ± 0.22 in primary cultures of smooth muscle cells from cat cerebral and pulmonary arteries > 800 µm in diameter, respectively. These data are consistent with our own observations.
The pHi difference suggests lower
H+ production, higher
H+ extrusion, or greater
intracellular buffering capacity in pulmonary arterial smooth muscle.
Vascular smooth muscle is known to produce lactic acid via glycolysis
at high rates, even under aerobic conditions (34), and to extrude
acid via
Na+-H+
exchange,
Cl-HCO
3
exchange, and
Na+-HCO
3
cotransport (1, 14, 40, 47). Furthermore, intracellular
buffering capacity in systemic vascular smooth muscle is appreciable
(3). Which of these processes explains the baseline
pHi difference is unknown;
however, a lower rate of aerobic glycolysis in pulmonary arterial
smooth muscle could explain both the higher
pHi and lower
found in this
tissue. A lower
could also signal a higher rate of oxidative
phosphorylation (4, 13), which could secondarily increase the
H+ concentration gradient across
the inner mitochondrial membrane and raise cytoplasmic pH (15).
Effects of hypoxia. In femoral
arterial smooth muscle, hypoxia did not alter [ATP] (Fig.
2, Table 2), but it decreased [PCr] and
[PCr]/[ATP] and increased
[Pi] and
[Pi]/[ATP]
(Figs. 2-4). Moreover, decreased from 7.3 × 104 to 2.7 × 104
M
1 (Fig. 5). These results
indicate that hypoxia decreased the femoral arterial energy state.
Data on the energy state effects of hypoxia in resting systemic
vascular smooth muscle are limited. Most previous studies (18, 21, 28,
38) were performed in stimulated or spontaneously contracting systemic
vessels and yielded results similar to our own. For example, in rabbit
aorta contracted with norepinephrine, Katayama et al. (21) and Scott
and Coburn (38) found that short exposures to anoxic gas
mixtures did not change [ATP] but increased
[Pi] and decreased
[PCr] and .
The most likely explanation for deterioration of femoral arterial energy state during hypoxia is hypoxic inhibition of mitochondrial electron transport and oxidative phosphorylation. Previous observations (28) that oxygen consumption fell in vascular smooth muscle exposed to similar conditions support this possibility. The stability of [ATP] (Fig. 2, Table 2) implies that ATP production remained matched to ATP utilization despite inhibition of oxidative phosphorylation. This could occur because ATP utilization decreased or, as previously demonstrated in hypoxic vascular smooth muscle (28, 34), ATP production via glycolysis increased (the Pasteur effect). The decrease in pHi observed in femoral arteries during hypoxia (Fig. 3) is consistent with the latter possibility because lactic acid is also produced via glycolysis.
It is possible that factors other than enhanced lactic acid production contributed to the fall of femoral arterial pHi during hypoxia. Decreased mitochondrial electron transport and proton pumping could lead directly to cytoplasmic acidification (15). Na+-H+ exchange, an important component of pHi regulation in vascular smooth muscle, requires a finite Na+ concentration gradient across the cell membrane, and this, in turn, depends on activity of the Na+-K+ pump, which requires energy for operation (1, 36, 47). Thus deterioration of energy state could limit Na+-K+-ATPase activity, reduce the transmembrane sodium gradient, and decrease acid extrusion via Na+-H+ exchange. Evidence that Na+-K+-ATPase preferentially utilizes ATP derived from glycolysis rather than from oxidative phosphorylation may argue against this possibility (7).
Hypoxia did not alter [ATP], [PCr],
[Pi],
[PCr]/[ATP], or
[Pi]/[ATP]
in pulmonary arterial smooth muscle (Table 2, Figs. 2 and 3). These
results differ from those of Shigemori et al. (39), who found a
progressive decrease in [ATP] in rat pulmonary arteries
exposed to 0% oxygen for 10 min; however, because their vessels were
stretched and precontracted with phenylephrine, energy utilization may
have been greater and energy state more susceptible to hypoxia than
those in unstretched, unstimulated tissue. As shown in Fig. 5,
pulmonary arterial was 1.6 × 104
M
1 at the end of the
hypoxic exposure compared with a baseline value of 2.8 × 104
M
1. Of the variables used
to calculate
(Eq. 4),
only [Cr] was altered by hypoxia (Table 2). Although
significant, this increase was small, and its effect on
was offset
by a simultaneous increase in
K'ck due to the
decrease in pHi (16), also caused
by hypoxia (Fig. 3). Thus, in contrast to femoral arterial smooth
muscle, we could not demonstrate that hypoxia altered the energy state
in pulmonary arterial smooth muscle.
Because baseline energy state was lower in pulmonary than in femoral arteries, it could be argued that the effects of hypoxia were not significant in pulmonary arteries because the energy state in this tissue could not be further reduced. This explanation cannot be correct because cyanide given to pulmonary arteries under baseline conditions decreased the energy state more severely than hypoxia. For example, [PCr]/[ATP] fell from 0.447 ± 0.031 to 0.057 ± 0.017 after exposure to a cyanide concentration of 10 mM (Table 3) but did not change during hypoxia (Fig. 3).
Analysis of variance indicated that the decrease in [PCr]/[ATP] induced by hypoxia was signifiantly greater (P = 0.036) in femoral than in pulmonary arteries (Fig. 3), suggesting that hypoxia caused a greater deterioration of energy state in femoral arteries. The explanation for this difference is unknown. Possibly, pulmonary arterial smooth muscle was able to maintain a higher ATP production. Less impairment of oxygen diffusion could allow greater mitochondrial oxygen uptake by pulmonary arteries under hypoxic conditions; however, wall thickness (and therefore oxygen diffusion distance) was greater in pulmonary arteries. Perhaps pulmonary arteries had a greater capacity to increase glycolytic ATP production under hypoxic conditions; however, hypoxia decreased pulmonary and femoral pHi by similar amounts (Fig. 3), suggesting similar increases in lactic acid production. Moreover, inhibition of oxidative phosphorylation by cyanide appeared to decrease energy state more severely in pulmonary than in femoral arteries (Table 3). This difference suggests that glycolytic capacity may have been smaller, not greater, in pulmonary arteries. Alternatively, pulmonary arterial smooth muscle may have had a greater ability to downregulate ATP utilization in the face of hypoxia (8) or a cytochrome oxidase of greater oxygen affinity. Further investigation will be required to determine which of these (or other) explanations is correct.
Summary. In this study of resting vascular smooth muscle, we found that 1) under baseline conditions, energy state and Cr pool size were lower and pHi was higher in pulmonary than in femoral arteries; 2) during hypoxia, energy state deteriorated more in femoral than in pulmonary arteries; and 3) during hypoxia, pHi fell in both vessels but was always more alkaline in pulmonary arteries. The mechanisms responsible for these differences are unknown.
Because energy state influences many cellular processes, including
interaction of contractile proteins,
Na+-K+
pump activity, and signal transduction, a difference in energy state
could be associated with a difference in contractile function. Higher
and [PCr] values are thought to characterize tissues routinely subjected to high-energy demand, such as cardiac and skeletal
muscles. Because pulmonary and femoral arteries normally change caliber
in the face of markedly different transmural pressures, the baseline
pulmonary-femoral differences in
and
[PCr]/[ATP] are consistent with this concept;
however, [PCr] in vascular smooth muscle is very low
compared with that in skeletal or cardiac muscle, where levels of
20-30 µmol/g are typical (6, 10, 13). This profound
quantitative difference, the low-energy requirements for contraction in
vascular smooth muscle (34), and the fact that our studies were
conducted in resting smooth muscle suggest that the pulmonary-femoral
difference in energy state was related to differences other than the
potential load on the contractile machinery.
pHi can also affect a wide variety of cellular processes. For example, increased pHi has been found to increase resting tone and intracellular calcium concentration in vascular smooth muscle, possibly due to the release of calcium from intracellular stores (2, 24, 32). In addition, higher pHi has been associated with membrane depolarization (1) and increases in myosin light chain kinase activity (31), myosin phosphorylation (9), and calcium sensitivity of contraction (32).
These considerations suggest that the differences in energy state and pHi we observed between pulmonary and femoral arterial smooth muscles could have functional consequences. For example, they could play a role in vasomotor responses to hypoxia, which are typically constrictor in pulmonary arteries and dilator in systemic arteries. Further investigation will be required to determine whether this is true.
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ACKNOWLEDGEMENTS |
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We express appreciation to Marilyn Banta and Greg Booth for technical assistance and to Paula Foltz for assistance in preparing the manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-51912.
R. M. Leach was supported by a Travelling Fellowship from the Medical Research Council of the United Kingdom.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. T. Sylvester, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.
Received 13 February 1998; accepted in final form 31 August 1998.
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