Hypoxic vasodilation in porcine coronary artery
is preferentially inhibited by organ culture
George D.
Thorne,
Shunichi
Shimizu, and
Richard J.
Paul
Department of Molecular and Cellular Physiology, University of
Cincinnati, College of Medicine, Cincinnati, Ohio 45267 - 0576
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ABSTRACT |
Hypoxia (95% N2-5%
CO2) elicits an endothelium-independent relaxation
(45-80%) in freshly dissected porcine coronary arteries. Paired
artery rings cultured at 37°C in sterile DMEM (pH ~7.4) for 24 h contracted normally to KCl or 1 µM U-46619. However, relaxation in
response to hypoxia was sharply attenuated compared with control (fresh
arteries or those stored at 4°C for 24 h). Hypoxic
vasorelaxation in organ cultured vessels was reduced at both high and
low stimulation, indicating that both Ca2+-independent and
Ca2+-dependent components are altered. In contrast,
relaxation to G-kinase (sodium nitroprusside) or A-kinase (forskolin
and isoproterenol) activation was not significantly affected by organ
culture. Additionally, there was no difference in relaxation after
washout of the stimulus, indicating that the inhibition is specific to
acute hypoxia-induced relaxation. Simultaneous force and intracellular
calcium concentration ([Ca2+]i) measurements
indicate the reduction in [Ca2+]i concomitant
with hypoxia at low stimulus levels in these tissue is abolished by
culture. Our results indicate that organ culture at 37°C specifically
attenuates hypoxic relaxation in vascular smooth muscle by altering
dynamics of [Ca2+]i handling and decreasing a
Ca2+-independent component of relaxation. Thus organ
culture can be a novel tool for investigating the mechanisms of
hypoxia-induced vasodilation.
hypoxia
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INTRODUCTION |
CHANGES IN OXYGEN
TENSION (PO2) profoundly affect the
normal physiology of an organism. Systemic vascular smooth muscle (VSM) relaxes in response to acute hypoxia or decrease in
PO2 (6). Vasodilation to hypoxia
in systemic VSM is likely a protective response to increase blood flow
to areas of low PO2. A variety of theories have
been proposed to explain the mechanism of hypoxia-induced vasodilation,
such as release of vasoactive substances (25, 29),
activation of ATP-sensitive K+
(K+ATP) channels (5, 18), and
energy limitation due to a limited anaerobic metabolic capacity
(4, 10, 16). The last is unlikely in light of evidence
that suggests relaxation in response to hypoxia occurs without
compromising energy metabolism (26). It has also been
shown that hypoxic vasorelaxation is insensitive to various ion channel
and metabolic antagonists (3, 26). Recently we showed that
mechanisms involving reduction in ATP, decrease in pH, increased
Pi, or activation of K+ATP or
calcium-activated potassium (KCa) channels cannot
explain hypoxic relaxation in pig coronary artery
(26). Moreover, both
Ca2+-dependent and Ca2+-independent mechanisms
were required to account for hypoxia-induced vasodilation.
The role of intracellular calcium concentration
([Ca2+]i) handling in hypoxic sensitivity has
been studied extensively. Hypoxia-induced pulmonary vasoconstriction
has been linked to increases in [Ca2+]i
attributed to release from intracellular stores (17) and inhibition of sarcolemmal K+ currents (15,
22), both of which activate Ca2+ channels.
Ca2+ modulation in hypoxic vasodilation of systemic VSM is
less clear. Although several studies have shown an inhibition of L-type
Ca2+ channels by hypoxia (8, 13, 24), our
studies (26) and other (1) indicate that
hypoxia-induced relaxation can occur independently of the decreases in
[Ca2+]i that are normally associated with
smooth muscle vasodilation. Still others report dilation of arterial
smooth muscle via KCa channel activation by release of
Ca2+ from intracellular stores (21). Although
Ca2+ modulation by hypoxia has been studied extensively, no
consistent theory has emerged.
Hypoxia is known to cause an increase in coronary blood flow in vivo
(7). Although many studies have demonstrated this effect
of hypoxia on small coronary resistance vessels, hypoxia-induced vasodilation of larger coronary arteries has been shown both in vivo
and in vitro (6, 6a, 7) and may be of physiological
significance (30). This relaxation response to hypoxia can
be demonstrated in isolated porcine coronary arterial rings (Fig.
1). Recently, we observed that organ
culture severely inhibits coronary relaxation to hypoxia, whereas
contractility appears normal (27). In this study, we
characterize the effects of organ culture in detail to probe further
the mechanisms of O2 sensing. We demonstrate that
1) the inhibition of relaxation is specific for hypoxia, 2) loss in ability to respond to hypoxia is linked to
changes in [Ca2+]i handling properties after
organ culture, and 3) Ca2+-independent
components of vasorelaxation are also impaired. Thus organ culture may
provide a novel way to investigate the mechanisms of hypoxic
vasorelaxation.

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Fig. 1.
Typical recording of a freshly isolated porcine coronary arterial
ring in response to KCl stimulation (40 mM) and hypoxia (95%
N2-5% CO2). The right axis bar shows
%O2 as determined by the output of an O2
electrode in the bath and calibrated as 100% = 760 mmHg.
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METHODS |
Coronary Artery Culture Conditions
Adult pig hearts were obtained from a slaughterhouse immediately
after death. Left descending coronary arteries were dissected, and two
rings per artery were cleaned of adhering connective tissue. One ring
was cultured in sterile DMEM + 1% antibiotic solution at 37°C.
A paired ring (control) was stored in the same solution at 4°C. In a
subset of these experiments, arteries were mounted isometrically on
wires set to a diameter slightly greater than the resting diameter.
After 24 h, rings were prepared for organ bath studies. All organ
culture preparations were performed under sterile conditions in a
culture hood. Arterial rings were prepared immediately after dissection
for organ bath studies, and these results are designated as the fresh
artery response. In a separate group of experiments, several rings from
the same artery were cultured/stored under the conditions described
above for 6, 12, 18, 24, and 30 h and then prepared for organ bath
studies. In another group of experiments, paired rings were cultured
for 24 h with 10 µM cycloheximide, a protein synthesis inhibitor
in the media.
Organ Bath Studies
All tissue used for these experiments were mechanically
de-endothelialized by gentle rubbing of the luminal surface with a cotton-tipped applicator. Rings were mounted onto two wires, one of
which was fixed and the other was connected to a force transducer. One
cultured ring and its paired control were placed into a bath containing
physiological salt solution (PSS) of the following composition (in
mmol/l): 118.3 NaCl, 15.0 NaHCO3, 11.1 dextrose, 4.7 KCl,
1.2 MgSO4, 1.2 KH2PO4, 0.026 EDTA,
and 2.5 CaCl2. In experiments using cycloheximide, the 10 µM concentration was maintained in the baths of those rings cultured
with the inhibitor. pH was 7.4 when the bath was aerated with 95%
O2-5% CO2 at 37°C. Tissues were allowed to
equilibrate for 1 h. Tension was adjusted to 40 mN, which sets the
tissue length in the range for optimal force generation. At least two
contraction/relaxation cycles to 80 mM KCl were performed until maximum
reproducible muscle forces were observed. The absence of the
endothelium was confirmed by lack of a response to substance P
(10
8 M). A test contraction was elicited using 0.1 µM
U-46619 or 40 mM KCl. After steady force was generated, hypoxia was
obtained by bubbling 95% N2-5% CO2 through
the baths for
20 min. The final oxygen tension of the bath solution,
measured polarographically, was
1-2%. We define these
conditions as hypoxia. Upon steady contracture (40 mM KCl), rings were
also challenged cumulatively with increasing concentrations
(10
9-10
5 M) of one of three vasodilators
[sodium nitroprusside (SNP), forskolin, or isoproterenol].
The hypoxic response was characterized in terms of the maximum hypoxic
relaxation, expressed as a percentage of the initial isometric force,
and the time course, quantified by time to half-maximal relaxation
(t1/2). All organ bath measurements were
recorded using a digital data acquisition system (Biopac). Force was
normalized to cross-sectional area [F/A = (change in force × circumference)/(2 × wet weight)].
Intracellular Calcium Imaging
A Photon Technology International spectrofluorimeter adapted for
force acquisition permitted simultaneous force and
[Ca2+]i measurements as previously described
(2, 20). Briefly, rings were mounted onto a force
transducer and submersed in a cuvette containing a solution for loading
the fluorescent probe fura 2-AM (MOPS-buffered PSS, BSA, 0.2% Pluronic
acid, and 5 µM fura 2-AM) for 3 h at room temperature. After a
steady baseline tension was attained, rings were rinsed and constantly
perfused (20 ml/min) with 95% O2-5%
CO2-bubbled PSS at 37°C. Tissues were stimulated by
adding agonists to the perfusate PSS. Hypoxia was obtained by perfusing
with the same solution but aerated with 95% N2-5%
CO2.
[Ca2+]i calibration.
The fluorescent emission at 510 nm for intensity at 340 nm excitation
was divided by that measured at 380 nm, and this ratio was used as an
index of [Ca2+]i. For statistical analysis,
the ratio was assigned values of 0% for resting muscle and 100% for
tissue stimulated with 40 mM KCl. This protocol was chosen as a general
routine over absolute calibration of the fura 2 fluorescence in lieu of
the major uncertainties in terms of absolute calibration
(2). Using standard calibration procedures
(9), we previously reported values of 50.4 ± 17.2 nM
and 441 ± 163 nM for [Ca2+]i under
baseline and KCl simulation, respectively (2, 20). In this
limited range, the 340/380 ratio is nearly linearly dependent on
[Ca2+]i as reported by others
(19).
Statistical Analysis
Data were analyzed using the t-test for paired
two-sample means or two-way repeated ANOVA with one-factor balance
design. Statistical significance was accepted for P < 0.05. Values are expressed as the mean ± SE. Values of
n represent the number of hearts from which arteries were isolated.
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RESULTS |
Hypoxia-induced Vasodilation in Fresh, Cold, Stored, and Cultured
Arteries
Fresh coronary artery relaxes upon exposure to hypoxia
(PO2 < 10 mmHg; Fig. 1). The magnitude of
relaxation depended on the type of stimulus used for the
precontraction. Fresh arterial rings relaxed up to 45 and 80% of
maximum force when stimulated with KCl or the thromboxane
A2 analog U-46619, respectively. Similar responses were
seen in arteries stored at 4°C for 24 h (Fig.
2, top; Fig.
3, B and D). The
paired arteries that were stored at 4°C were subsequently used as the
control for cultured arteries. Figure 2 illustrates a typical response
of control (stored at 4°C for 24 h) and organ-cultured (37°C
for 24 h) arterial rings to 40 mM KCl or 1 µM U-46619 followed
by hypoxia. Organ culture results in a marked attenuation in magnitude
of relaxation to hypoxia. Data from these types of experiments for
U-46619 and KCl are summarized in Fig. 3. Similar experiments were
performed on control and cultured arterial rings maintained
isometrically on wires so that they were under tension for 24 h.
As with the untethered conditions, hypoxic relaxation was reduced in
the organ cultured arteries compared with their paired controls
(n = 4; data not shown).

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Fig. 2.
Effect of differential organ culture on porcine coronary artery.
Cultured rings (bottom) have an attenuated hypoxia-induced
relaxation relative to control (top, n = 24). Tracings show relaxation to hypoxia during 40 mM KCl
(left) and 1 µM U-46619 (right) activation.
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Fig. 3.
A
summary of the effects of organ culture on maximum isometric force
generation and hypoxia-induced vasodilation for 40 mM KCl
(top) and 1 µM U-46619 (bottom) contractions.
B and D: maximum relaxation in response to acute
hypoxia (15-20 min) is attenuated by 40-60% after organ
culture at 37°C. A and C: the ability to
generate isometric force is not significantly altered by this
treatment. Results represent the means ± SE for 12 experiments.
*P < 0.05 for differences between organ cultured and
control.
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The reduction in hypoxic relaxation after organ culture occurred
without any significant change in maximum force developed in response
to either KCl or U-46619 (Fig. 3, A and C).
Figure 4 shows the average time course of
isometric force after bubbling with N2 from experiments
such as that shown in Fig. 1. Organ culture can be seen to result in a
significant decrease in the rate of hypoxia-induced vasodilation and
increased t1/2 compared with control.

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Fig. 4.
Average (n = 12) time course of isometric force
after bubbling with N2 from experiments such as that shown
in Fig. 1. The ordinate represents the amount of force remaining after
imposition of hypoxia. Organ culture can be seen to result in a
significant decrease in the rate of hypoxia-induced vasodilation
and to increase the half-maximal time (t1/2)
compared with control. Time course for KCl (A) and U-46619
(B) activation are both slowed by about 50%
(n = 12). C: average
t1/2 for hypoxic relaxation in control and
cultured arteries for 40 mM KCl and 1 µM U-46619 activation. Bar
graph shows the means ± SE for 8 (KCl) and 6 (U-46619)
experiments. *Differences were significant at P < 0.05.
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Figure 5 shows the effects of the
duration of time under control or culture conditions on the hypoxic
relaxation. Isometric force generating capability with depolarization
or receptor-mediated activation in either group was not affected until
30 h of storage (Fig. 5, A and C). In
contrast, reduced relaxation to hypoxia occurred as early as 6 h
with KCl stimulation and 12 h with U-46619 stimulation in cultured
rings. Control rings exhibited a small attenuation in relaxation to
hypoxia at 30 h compared with shorter durations of storage when
stimulated with 40 mM KCl (Fig. 5B). There was no loss of
relaxation to hypoxia with U-46619 stimulation in control rings (Fig.
5D).

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Fig. 5.
Effects of time in storage on control and cultured coronary
arterial rings. The ability to generate isometric force was not
significantly altered in response to 40 mM KCl (A) and 1 µM U-46619 (C) stimulation. Only after 30 h did
control or cultured rings exhibit a decline in force. Relaxation
response to hypoxia was significantly different between control and
culture groups as early as 12 h (B and D).
Bar graph shows the means ± SE for 8 (KCl) and 6 (U-46619)
experiments. *Differences were significant at P < 0.05.
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We investigated whether organ culture impaired relaxation in general by
studying the response to various smooth muscle vasodilators. Figure
6 shows the average force remaining after
increasing concentrations of SNP, isoproterenol, and forskolin.
Sensitivity to increasing concentrations of the nitric oxide donor SNP
was not significantly different between experimental groups (Fig.
6C). Similarly, organ culture did not significantly
affect the sensitivity of coronary rings to receptor-mediated
(isoproterenol) or direct (forskolin) activation of adenylate cyclase
(Fig. 6, A and C). Thus the ability of porcine
coronary arterial rings to relax to G- or A-kinase mediated pathways
was not affected by organ culture at 37°C for 24 h. Analysis of
the decrease in isometric force upon washout of agonist (Fig.
7) indicates that organ culture also does
not alter this mechanism of relaxation as well.

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Fig. 6.
Concentration-response curves for sodium nitroprusside
(A), forskolin (B), and isoproterenol
(C). The ordinate represents the average force remaining
after treatment with increasing concentrations of each drug. Results
indicate no change in G- or A-kinase-mediated smooth muscle relaxation
pathways after organ culture at 37°C. There was no significant
difference in sensitivity to these vasodilators at any concentration
used (n = 8).
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Fig. 7.
A: typical recording of a control and cultured
arterial ring stimulated with 40 mM KCl and rinsed with fresh
physiological salt solution under normoxic conditions. B:
summary of t1/2 to relaxation for 8 experiments.
The bar graph shows the means ± SE.
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We recently showed that with high stimulus levels hypoxic
vasorelaxation occurred without a decrease in
[Ca2+]i (26). The response to
hypoxia under high and low stimulus conditions thus gives an index of
the relaxation's dependence on [Ca2+]i.
Figure 8 illustrates that in all cases
(low and high activation, U-46619 and KCl), organ-cultured arteries had
a significantly reduced relaxation in response to hypoxia. The largest
difference was seen with 20 mM KCl stimulation (lane 4), a
low-level depolarization, where cultured arteries relaxed 60% less
than control. Interestingly, the smallest difference between control
and cultured arteries was observed during low-level receptor-mediated
stimulation, 0.1 mM U-46619 (lane 3). These data suggest
that both Ca2+-dependent and -independent components of
hypoxia-induced relaxation are affected by organ culture.

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Fig. 8.
Hypoxia-induced relaxation during high (1 µM U-46619
and 40 mM KCl) and low (0.1 µM U-46619 and 20 mM KCl) activation.
Organ culture at 37°C for 24 h significantly reduces relaxation
response to hypoxia regardless of stimulation type or level. Bar graph
shows means ± SE; n = 8-12. *Differences
were significant at P < 0.05.
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Effects of Protein Synthesis Inhibition on Relaxation in Response
to Hypoxia
To investigate whether an enhancement or inhibition of protein
synthesis underlies the loss of relaxation response to hypoxia, we
incubated both control and cultured coronary rings with 10 µM
cycloheximide, a protein synthesis inhibitor. Figure
9 shows that after 24 h of
incubation with cycloheximide, control rings exhibit a significant
reduction in relaxation in response to hypoxia compared with control
rings not incubated with the inhibitor (Fig. 9B). This was
true for hypoxia during both KCl and U-46619 stimulation. Organ
cultured rings incubated with cycloheximide showed no change in their
inhibited relaxation response to hypoxia. Similar to organ culture,
relaxation to SNP and forskolin were unaffected by cyclohexamide. In
all cases, isometric force generation was not significantly affected by
incubation of these rings with cycloheximide (Fig. 9A).

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Fig. 9.
The effects of 10 µM cycloheximide (chx) incubation on force
(A) and hypoxic relaxation (B) for control and
cultured porcine coronary arteries. Inhibition of protein synthesis
causes an attenuated relaxation to hypoxia in control arterial rings
but no further changes in cultured rings. Bar graph shows means ± SE; n = 8. Differences were significant at
P < 0.05 (*control vs. cultured; §control + chx
vs. control chx).
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Intracellular Calcium Measurements
Organ culture has been reported to alter
[Ca2+]i handling through modification of
Ca2+ channel expression and intracellular Ca2+
store function (11, 17, 17a). We thus measured
[Ca2+]i and force generation during hypoxic
challenge to determine if Ca2+ handling was affected by
organ culture. Figure 10 shows typical [Ca2+]i response to hypoxia with low (20 mM)
and high (40 mM) KCl stimulation in fresh coronary artery. A
significant decrease in [Ca2+]i during
hypoxia with 20 mM KCl was observed, but little change with 40 mM KCl,
as previously reported (26). The decrease in [Ca2+]i concomitant with hypoxia during low
stimulation was also observed in control arteries (Fig.
11B). In contrast, in organ
cultured arteries [Ca2+]i does not decrease
in response to hypoxia (Fig. 11A). Data from these types of
experiments are summarized in Table 1.
These data suggest an impaired [Ca2+]i
handling capacity during hypoxia after organ culture.

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Fig. 10.
Calcium tracings during hypoxia on 40 (A) and
20 (B) mM KCl contractures for a fresh coronary ring.
Although hypoxia decreases force in either case (not shown in this
figure), intracellular calcium concentration
([Ca2+]i) is decreased only at 20 mM
(B).
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Fig. 11.
A: simultaneous force and
Ca2+ measurements showing a typical tracing of a coronary
artery after organ culture that has lost the ability to reduce
[Ca2+]i during hypoxia under low stimulus
levels (20 mM KCl). B: consistent with freshly isolated
vessels, there is a concomitant decrease in
[Ca2+]i during hypoxia with low activation in
control vessels.
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Table 1.
Decrease in [Ca2+]i during
hypoxia-induced vasorelaxation under low-level activation in porcine
coronary artery
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DISCUSSION |
In this investigation we show that organ culture at 37°C for
24 h significantly reduces the relaxation response to hypoxia in
porcine coronary artery. The ability to generate control levels of
isometric force after organ culture suggests that significant smooth
muscle cell alteration does not occur. Moreover, the ability to respond
to both depolarization (KCl) and receptor-mediated (U-46619)
contractile stimuli suggests that the machinery necessary for these two
different excitation pathways is not altered by organ culture. These
results are quite different from those of cultured smooth muscle cells
where both contractility and activation pathways can be significantly
altered (8, 13, 24).
The attenuation of the hypoxic relaxation was dependent on the duration
of organ culture (Fig. 5), with a reduction compared with control seen
as early as 6 h. In control rings, a significant reduction in the
hypoxic relaxation did not occur until 30 h. The importance of
this observation is less clear, as there was also a significant drop in
force generation at 30 h as well, possibly simply reflecting
tissue degradation. Although control rings showed some minor loss in
relaxation in response to hypoxia at various time points compared with
earlier ones, they always relaxed significantly more than cultured
rings. These results suggest that the specific attenuation of hypoxic
vasorelaxation depends both on time and storage condition. The most
consistent results in terms of a maximal loss of relaxation response to
hypoxia without a concomitant loss of force production were obtained
with a duration of 24 h, which is when all other experiments were performed.
Importantly, relaxation in response to activation of cGMP and
cAMP-mediated vasodilatory pathways was not affected by organ culture.
Sensitivity to these pathways activated directly (SNP, forskolin) or
through receptor stimulation (isoproterenol) was not significantly
different (Fig. 6). This evidence implies that organ culture does not
impair the innate ability of coronary arteries to respond to the major
smooth muscle pathways of relaxation. Therefore, the loss in hypoxic
relaxation in these tissues cannot be attributed to a generalized loss
in inherent relaxation capability. Moreover, this also suggests that
O2 sensing does not involve the effector pathways
downstream from A- or G-kinase activation. This is consistent with
previous studies based on pharmacological approaches (3,
26). The idea that organ culture specifically reduces
hypoxia-induced vasodilation is strengthened further by the fact that
cultured vessels will relax when the stimulus is removed (Fig. 7).
We previously identified both Ca2+-independent and
-dependent components of the mechanism(s) underlying hypoxic relaxation
in coronary arteries (24, 26). The
Ca2+-dependent component is visible at lower K+
stimulus levels such as 20 mM KCl (Figs. 8 and 9) or at low-level receptor-mediated stimulation with 0.1 µM U-46619 (Fig. 8). In response to hypoxia, both freshly dissected coronary artery and those
stored at 4°C (control) decrease [Ca2+]i
when activated at low levels (Fig. 10). However, after 24 h in
organ culture, both the decrease in [Ca2+]i
(Table 1) and the decrease in force (Fig. 8) in response to hypoxia are
lost. A reduction in the capacity to lower
[Ca2+]i could directly translate into a
reduced ability to relax in response to hypoxia in cultured arteries.
The fact that relaxation in response to washout of agonist is not
affected by organ culture (Fig. 7) suggests that this impairment of
Ca2+ handling appears to be specific to hypoxia-induced
vasodilation. Our results indicate that both
Ca2+-independent and -dependent mechanisms are markedly
reduced after organ culture (Fig. 8).
At high stimulus intensities, 40 mM KCl or 1 µM U-46619, little
change in [Ca2+]i occurs during acute hypoxic
challenge in fresh or control arteries despite a significant decrease
in force (Fig. 8, lanes 1 and 2). In cultured
arteries under these high-intensity stimulus conditions, the relaxation
response to hypoxia is severely inhibited (Fig. 8). Our results thus
indicate that both Ca2+-independent and -dependent
mechanisms of the relaxation response to hypoxia are markedly reduced
after organ culture (Fig. 8).
Organ culture elicits a very specific reduction in the relaxation
response to hypoxia, which may help delimit the underlying O2-sensing mechanisms. As the mechanisms regulating smooth
muscle contraction are complex, mechanisms of hypoxic relaxation would be expected to be dependent on the nature of the stimulus. It is of
interest to note that with KCl stimulation, the hypoxia relaxation in
control arteries is attenuated at high stimulus levels (40 mM) relative
to low (20 mM). This suggests that K+ channels, some
classes of which are known to be sensitive to oxygen tension (15,
22), may be involved, because their effects would be expected to
decrease with increasing KCl depolarization. The loss of the
hypoxia-induced decrease in [Ca2+]i could
reflect a loss of K+ channels or Ca2+ channels,
which have also been shown to be oxygen sensitive (8a). This would be consistent with the reduction of the hypoxic relaxation after cycloheximide treatment in control arteries (Fig. 9).
With U-46619 stimulation, one might anticipate that both
Ca2+-dependent mechanisms, as do K+ channel
activation and Ca2+-independent mechanisms, could play a
role in hypoxic relaxation. This would be expected particularly
at low intensity (0.1 µM U-46619), for which decreases in
[Ca2+]i with hypoxia have been observed. But
the magnitude of hypoxic relaxation at 0.1 and 1 µM was similar (Fig.
8), thus the effects are not additive in any simple fashion. This is
not surprising, because relaxation is complex, particularly with
U-46619, when Ca2+ sensitization of the contractile
elements is likely (14).
There is also a change in the Ca2+-independent component of
relaxation in response to hypoxia. Again, this could be due to an decrease or increase in some factor(s) not involved in Ca2+
handling, but the evidence here suggests the loss of function by
degradation. Many genes and proteins change during 24 h of organ
culture of VSM (28). Presumably an alteration in
contractile proteins, heat shock proteins, or redox proteins could make
the tissue less responsive to hypoxia, directly or indirectly. With the
recent development of molecular biology techniques such as subtraction
cDNA libraries and proteomics for identification of differential mRNA
or protein changes, the specific loss of hypoxic relaxation may provide
a new approach to elucidating the mechanism(s) of hypoxic vasorelaxation.
In this investigation summary, our results show that hypoxic
vasorelaxation is lost after 24 h in organ culture at 37°C,
while contractility and other relaxation pathways are unaffected. Thus organ culture may provide a tool for identifying new targets for understanding the elusive mechanisms underlying this important physiological response.
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FOOTNOTES |
Address for reprint requests and other correspondence: R. J. Paul, Dept. of Molecular and Cellular Physiology, Univ. of
Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH
45267-0576.
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. Section 1734 solely to indicate this fact.
Received 30 June 2000; accepted in final form 12 January 2001.
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