Integrative Physiology |
From the Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio.
Correspondence to Dr Richard Paul, Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, 231 Bethesda St, Cincinnati, OH 45267-0576. E-mail Richard.Paul{at}uc.edu
| Abstract |
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Key Words: coronary arteries hypoxia pH Ca2+ metabolism
| Introduction |
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The first class involves some form of limitation on ATP synthesis. Relaxation is attributable to the inability of cellular ATP production under hypoxia to support the actin-myosin ATPase activity and hence contractile activity. This could be simply because of an inherently low glycolytic capacity for ATP synthesis under hypoxic conditions, or the capacity could be compromised; for example, acidic pH associated with hypoxia could inhibit glycolysis. It is also possible that regulatory sites for oxidative or anaerobic metabolism, such as phosphofructokinase, may be oxygen sensing modulatory elements.4 We have reported that an energy limitation mechanism is likely to underlie hypoxic relaxation observed in guinea pig taenia coli5 6 ; however, the available evidence does not support an energy limitation mechanism for the oxygen sensitivity of vascular smooth muscle. Pittman and Duling7 reported that contractility was depressed only when the smooth muscle cells in the core of a hypoxic artery became anoxic, suggesting that vascular sensitivity to PO2 could involve an energy limitation. However, because PO2 for arterioles would be unlikely to reach these low levels, a direct role for oxygen in situ could not be supported. There is also evidence that vascular reactivity of both large and small arteries is depressed for PO2s in the 20 to 100 torr range,2 8 which is higher than the range generally associated with inhibition of mitochondrial oxidative phosphorylation. Evidence for an oxygen sensing mechanism in vessels independent of mitochondrial metabolism was also provided by Coburn,9 who reported that even after inhibition of respiration by cyanide, contractility in rabbit aorta could still be depressed by hypoxia. More recent studies4 10 indicate that hypoxic relaxation is not simply a function of energy stores, and oxidative metabolism-contraction coupling is regulated by energy delivery to a reaction- or reactions-controlling muscle force.
An alternative class of hypotheses involves oxygen sensing mechanisms that would modulate the cell signaling pathways involved in excitation-contraction coupling, including mechanisms in which [Ca2+]i is the regulated parameter, for example by oxygen-dependent or ATP-dependent changes in Ca2+ permeability.11 12 A hypothesis of this class with considerable experimental support is that lowered [Ca2+]i concomitant with hyperpolarization is attributable to activation of K+ channels.13 14 Alternatively, evidence for signaling changes at constant [Ca2+]i is accumulating.3 5 Mechanisms involving altered Ca2+ sensitivity of the contractile apparatus, such as those potentially associated with pHi changes with hypoxia, would be in this category. These categories are not necessarily mutually exclusive.
Although vessel sensitivity to oxygen is well documented, the mechanisms are not known with certainty. To further characterize the mechanisms of oxygen sensing in the coronary artery, we investigated the effects of hypoxia on energetics and [Ca2+]i, the central second messenger for control of vascular smooth muscle contractility. In addition, we studied the roles of K+ channels and pHi, major factors known to modulate contractility. In these coronary arteries, both Ca2+-dependent and -independent mechanisms underlie hypoxic vasodilatation.
| Materials and Methods |
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Isometric force measurements were previously described.16 17 Hypoxia was operationally defined as aeration with 95% N2/5% CO2, which decreases the bath PO2 to <1%, as we have reported.18
Intracellular Ca2+ measurements were made with the fluorescent probe fura 2-AM in an apparatus that permitted simultaneous measurements of force and [Ca2+]i, as previously described.19 20 In some experiments, the rings were mounted isometrically on a wire loop that was fitted into a spectrofluorometer cuvette21 ; no differences were noted in the [Ca2+]i values, and the data were averaged.
[Ca2+]i Calibration
The fluorescent intensity at 340-nm excitation was
divided by fluorescent intensity 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 29 mmol/L KCl. This
protocol was chosen as a general routine over absolute calibration of
the fura 2 fluorescence because of the major uncertainties of
absolute calibration.21 Using standard calibration
procedures,22 we previously reported values of 50.4±17.2
nmol/L and 441±163 nmol/L for
[Ca2+]i under baseline
and KCl stimulation, respectively.20 21 In this limited
range, the 340/380 ratio is nearly linearly dependent on
[Ca2+]i, as reported by
others.23
Intracellular pH measurements were made with the fluorescence pH indicator dye BCECF, as previously described,21 and calibrated using the high K+nigericin technique.24 25 26
High-energy phosphagens, metabolites, and lactate production measurements have been previously reported in detail.27 Briefly, artery rings were frozen in liquid N2, pulverized, and mixed with frozen extraction solution (1 vol methanol and 1 vol 2 mmol/L EDTA) at liquid N2 temperature. An LKB analytical isotachophoresis apparatus was used to measure negatively charged metabolites in the thawed solution. Lactate production was measured using standard procedures previously reported in detail.15
Solutions
The PSS contained (in mmol/L) NaCl 122, KCl 4.73,
NaHCO3 15, MgCl2 1.19, EDTA
0.02, KH2PO4 1.19, glucose
11.10, and CaCl2 2.50. When aerated with 95%
O2/5% CO2, the pH of the
PSS was 7.3 to 7.4 at 37°C.
Analysis of Data
The values given are mean±SEM. N values for sample size
represent the number of hearts from which arteries were taken.
Students t test for paired data and standard ANOVA with
Bonferroni discrimination for multiple groups were used; a significance
level of P<0.05 was chosen for rejection of the null
hypothesis.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
| Results |
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50% of the
initial value, as previously reported.17 The half-time of
this decline of 4.8±0.6 minutes was considerably greater than that of
the change in PO2. The response to
hypoxia was reversible (Figure 2A
20% of the maximum KCl
contracture.15 16 28 Addition of 29 mmol/L KCl under
hypoxia elicited a markedly diminished contraction, with
maximum force similar to that obtained in the steady state after
imposition of hypoxia on a control KCl contracture, as in
Figure 2A
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Interestingly, increasing the level of stimulation can override the
inhibitory effects of hypoxia. The dependence of
the inhibitory effects of hypoxia on isometric
force on the level of stimulus is shown in Figure 3
. Cumulative concentration-response
curves for KCl (Figure 3A
) and receptor-mediated stimulation
using U46619, a thromboxane A2 analog
(Figure 3B
), were generated under aerobic and hypoxic
conditions. Both stimuli elicit similar levels of maximum isometric
force; however, hypoxia clearly has a more pronounced
inhibitory effect on the U46619 response. The extent of the
inhibition by hypoxia was strongly dependent on the
concentration of the stimuli used. For example, at 20 mmol/L KCl,
which elicits
80% of the maximum force, hypoxia reduces
this response to only 20%. However, increasing KCl to 80 mmol/L
increases isometric force under hypoxia to
75% of that
under oxygenated conditions. If metabolism were
limited by hypoxia, this would be expected to set an absolute
limit to force production independent of stimulus intensity.
Thus, the effects of stimulus intensity on the inhibition of isometric
force by hypoxia argue against an energy limitation hypothesis
based on a limited anaerobic metabolic
capacity.
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In addition to energy limitation or mechanisms involving the mitochondrial respiratory electron transport chain29 as the sensing element, a number of potential sites for oxygen sensing have been proposed. They include the Na+ pump,1 eicosanoid pathways,30 31 alterations in plasma membrane electrical properties,32 33 Ca2+ permeability,11 12 hydrogen peroxide formation,34 potentially oxygen free radicals,35 36 37 cGMP,38 and more recently, ATP-dependent K+ channels13 and the inositol phosphate pathway.29 To delimit the potential mechanisms of oxygen sensing in porcine coronary artery, we also screened a number of these potential pathways using similar pharmacological approaches. We have previously reported17 the effects of inhibitors, including ouabain, aminotriazole, superoxide dismutase, catalase, indomethacin, and propranolol, on the response to hypoxia. Although some minor modulatory effects were seen, as might be anticipated, on the whole these agonists had little effect on hypoxic relaxation.
To add to this database, we further tested the effects of chelerythrine
(3 µmol/L), an inhibitor of the C-kinase pathway,
and LY83583 (3 µmol/L), an inhibitor of the G-kinase
pathway. Table 1
shows that neither
inhibitor had significant effects on the force generated by
29 mmol/L KCl or the relaxation to subsequent hypoxia.
Thus, within the limitations of a pharmacological approach, the oxygen
sensing pathway in porcine coronary artery does not seem to
conform to any of the proposed hypotheses.
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Effects of Hypoxia on [Ca2+]i
Central to most theories of hypoxic vasodilatation are mechanisms
for decreasing [Ca2+]i.
We studied the effects of hypoxia on
[Ca2+]i using the
ratiometric fluorescent dye fura 2. Figure 4
shows representative
recordings of the effects of hypoxia on
[Ca2+]i at high (Figures 4A
and 4C
) and low (Figures 4B
and 4D
) stimulation
intensity. At 40 mmol/L KCl, the 340/380 ratio rapidly increases
and then is characterized by a small, slow decline. A decline in
[Ca2+]i despite
maintained force is characteristic of smooth muscle.39 40
After imposition of hypoxia,
[Ca2+]i slightly
increases (during which time isometric force is decreasing) and then
resumes a slow decline. After 30 minutes of hypoxia,
[Ca2+]i is
indistinguishable from the level just before the hypoxia
intervention. Reoxygenation elicits a small rapid
decrease in [Ca2+]i
followed by a return to the original small, slow decrease. The rise of
[Ca2+]i is less steep
with 20 mmol/L KCl than with 40 mmol/L KCl. The effects of
hypoxia with 20 mmol/L KCl are considerably different than
those with 40 mmol/L KCl. Imposition of hypoxia elicits a
rapid and steady decline of
25%, which is reversed on
reoxygenation. Average values for these experiments are
graphically summarized in Figure 5
.
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Ishida and Honda41 reported that
[Ca2+]i was not affected
by hypoxia when stimulated with KCl, whereas hypoxia
decreased [Ca2+]i for
receptor-mediated stimulation in guinea pig aorta. Thus, we measured
the effects of hypoxia on
[Ca2+]i for U46619
contractures. Typical experiments are shown in Figure 4
, and the
average values are graphically summarized in Figure 5
. The
general results are similar to those with KCl stimulation. At 1
µmol/L U46619, hypoxia increased
[Ca2+]i by 23% but force
decreased by 50%. At 0.1 µmol/L U46619, hypoxia
decreased both force (
80%) and
[Ca2+]i (
50%).
Role of K+ Channels in the Ca2+-Dependent
Hypoxic Vasodilatation
Activation of K+ channels is one of the more
extensively studied hypotheses for hypoxic vasodilatation. This
hypothesis involves K+ channel activation,
hyperpolarization, and consequent closing of
voltage-dependent Ca2+ channels.13
Because [Ca2+]i was
decreased by hypoxia at low levels of stimulation, the
potential for K+ channel involvement was
suggested. We used a pharmacological approach to identify whether
K+ channels were involved in the low-stimulus
hypoxic vasodilatation. Arteries were contracted with U46619 (0.1
µmol/L) and treated with K+ channel
inhibitors before exposure to hypoxia. As with any
pharmacological approach, the specificity of K+
channel inhibitors is open to interpretation. We made the
following operational assignments in our investigation based on the
literature42 and our recent studies on
K+ channels in porcine coronary
artery43 : KCa channel
inhibition, tetraethylammonium (TEA) (1
mmol/L), apamin (1 µmol/L), and charybdotoxin (0.1
µmol/L); KV channel inhibition,
4-aminopyridine (4-AP) (1 mmol/L);
KATP channel inhibition, glibenclamide (10
µmol/L); KCa and KV, TEA
(10 mmol/L), KIR and
KATP, BaCl2 (10
µmol/L); non-specific, tetrabutylammonium (TBA) (5
mmol/L). The effects of these K+ channel
inhibitors on isometric force in response to U46619 and
subsequent hypoxia are summarized in Table 2
. Inhibition of
KATP or KCa channels was
associated with only small effects. This is somewhat surprising because
the literature13 suggests that KATP
channels could be an important mediator of hypoxic vasodilatation.
However, our results are consistent with a recent study from
our laboratory indicating that KV channels are
the predominant K+ channels in porcine
coronary artery.43 TBA was associated with a
significant reduction (60%) of the response to hypoxia. More
specific inhibition of KV channels with 4-AP was
associated with a small (<20%) but significant increase in force and
a moderate (15%) reduction in the relaxation to hypoxia.
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Mechanisms of Ca2+-Independent Hypoxic
Vasodilatation
The data in Figure 5
demonstrate that at high levels of
stimulation, hypoxia decreases force with a little change or
increase in [Ca2+]i. To
further demonstrate a Ca2+-independent
relaxation, we attempted to maximize
[Ca2+]i by exposure of
the arteries to KCl (40 mmol/L), added ionomycin (1
µmol/L), a Ca2+ ionophore, and finally added
U46619 (1 µmol/L) to promote sarcoplasmic reticulum
Ca2+ release. Data from these experiments,
graphically summarized in Figure 6
, show
that isometric force under normoxic conditions increased to 150% of a
KCl contracture by the cumulative treatment with ionomycin and U46619.
Even under these conditions to fix
[Ca2+]i at maximal
levels, hypoxia was still able to elicit a substantial
relaxation. In light of this Ca2+-independent
relaxation to hypoxia, we investigated
pHi and energy metabolism, 2
alternative mechanisms proposed for hypoxic
vasodilatation.5 6
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Effects of Hypoxia on pHi
Hypoxia is known to increase both lactate content (see
below) and lactate production in porcine coronary
artery.44 Moreover, we have reported that acidic
pHi is associated with an inhibition of force in
coronary arteries.21 Thus, it is plausible that
the inhibition of force by hypoxia could be attributed to an
acidification of pHi. We measured the effects of
hypoxia on pHi using the ratiometric
fluorescent dye BCECF, shown in Figure 7
. As previously reported,21
KCl addition had only minor effects on pHi
despite the known increase in lactate
production.45 Similarly, imposition of
hypoxia did not acidify, as might be anticipated, because of
the significant increase in lactate production (see below). The
trend with hypoxia is alkalinization, although the differences
were not statistically significant. Because alkalinization was shown to
increase force in previous studies,21 the effects of
hypoxia on pHi are unlikely to play any
role in the observed Ca2+-independent
relaxation.
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Effects of Hypoxia on Phosphagen Content and
Metabolite Profile
To gain more insight into the mechanisms of hypoxic
vasorelaxation, we measured the phosphagen content and metabolite
profile in freeze-clamped coronary arteries using
isotachophoresis.27 The results are graphically summarized
in Figure 8
. As previously reported for
carotid artery, stimulation of maximum isometric force with KCl was
associated with little change in high-energy phosphagens or other
metabolites. Hypoxia, as might be expected, was associated with
a 3-fold increase in lactate content. The only other measured change
was a decrease of
50% in phosphocreatine (PCr) content.
Significantly, there was no change in ATP or
Pi.
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As shown in Figure 9A
, addition of U46619
(1 µmol/L) reverses the hypoxic relaxation of a KCl (29
mmol/L) contracture. This large increase in force under hypoxic
conditions was not associated with a proportional increase in
[Ca2+]i (Figure 9B
). The order of stimulation was not important, because adding
KCl reversed the effects of hypoxia on a U46619 contracture
(88.7±5.5% of normoxic force, n=4, not significant). These data would
again support the hypothesis that metabolic ATP
production is not limiting, for presumably there is additional
ATP mobilized to support the higher level of isometric force. In
general, ATP utilization is proportional to the level of isometric
force.46
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It is possible that the increase in force with additional stimulation
under hypoxia could be accomplished by increasing the force per
ATP utilization (economy) rather than increasing ATP utilization at a
constant economy. Thus, from an energetics viewpoint, it is of
particular interest to know what, if any, changes in
metabolism were associated with the increase in force
observed when U46619 was added to the KCl contracture under hypoxic
conditions (Figure 10
).
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Under these hypoxic conditions (
1% O2),
anaerobic glycolysis is virtually the only source for
production of ATP. The rate of lactate production
(Jlac) was equal to that measured in the bath,
and no significant differences in tissue lactate content were found.
Under hypoxia with KCl stimulation (Figure 10
),
Jlac was
4 times the rate of oxygen
consumption.47 This would suggest that the capacity for
anaerobic glycolysis is adequate to stoichiometrically
replace the ATP attributable to oxidative metabolism. After
the addition of U46619 under hypoxia, force increased and
remained elevated for the entire 60-minute measurement period at levels
similar to the original force in the presence of KCl under aerobic
conditions. Jlac did not increase with U46619
treatment, but in fact was somewhat lower, although not significantly
(P=0.08), than that observed in KCl alone under
hypoxia. This was surprising, because an increase in glycolytic
ATP production would have been predicted to match the increase
in force elicited by U46619. The ATP and PCr content measured in
freeze-clamped arteries was reduced by the end of the measurement
(P<0.05). However, the decrease of ATP was inconsequential
when compared with the ATP produced over 60 minutes by aerobic
glycolysis (
60x Jlac). Thus, it would seem
that hypoxia not only decreases the Ca2+
sensitivity (ie, less force at similar
[Ca2+]i concentrations)
but also can alter the relation between force production and
ATP utilization (ie, less ATP utilization per unit force maintained).
| Discussion |
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A most striking observation in these studies is that the relaxation or inhibition of force associated with hypoxia could be ascribed to at least two different mechanisms. At low stimulus levels, the inhibition of force was accompanied by a decrease in [Ca2+]i. At high stimulus levels, hypoxia decreased force in the presence of constant or increasing [Ca2+]i. In our conditions, hypoxia (bubbling with N2) was such that bath PO2 was <1%, and the tissue PO2 was likely below that for mitochondrial oxidative phosphorylation throughout. In physiological terms, this is severe hypoxia, but it does enable one to set defined conditions for the tissue as a whole, which is useful to separate Ca2+-dependent and -independent mechanisms. The dependence of these mechanisms on the PO2 is currently under investigation and will be important to understanding tissue oxygen sensing, which can be observed at considerably higher PO2.52
The decrease in [Ca2+]i
at low stimulus levels was not affected by inhibitors of
KATP or KCa channels. There
was a moderate inhibition (
15%) of the hypoxic relaxation
associated with inhibition of KV channels. These
results are consistent with a recent study from our laboratory
indicating that KV channels are the predominant
K+ channels in porcine coronary
artery.43 Interpretation of these data, however, is not
straightforward, because the specificity of KV
channel inhibitors is not absolute. In addition, 4-AP and
TEA (but not TBA) also increased the force (<20%) before exposure to
hypoxia, which could also account for some of the reduction in
the relaxation to hypoxia.
A direct effect of hypoxia on Ca2+ channels11 12 or Ca2+ uptake or extrusion systems, such as enhanced Na+-Ca2+ exchange,1 cannot be ruled out. These arteries are able to relax to hypoxia after inhibition of the sarcoplasmic reticulum Ca2+ pump with cyclopiazonic acid (G.T. and R.J.P., unpublished observations, 1999). Thus, a major role for the sarcoplasmic reticulum in hypoxic relaxation is unlikely in these arteries.
The Ca2+-independent relaxation to hypoxia was observed for both receptor-mediated stimulation and depolarization, as long as the intensity was high. A Ca2+-independent pathway has been suggested for several smooth muscles.3 The Ca2+-independent effects of hypoxia are by definition a decrease in Ca2+ sensitivity; ie, a lower force maintained under hypoxic conditions was not associated with a change in [Ca2+]i.
A change in pHi would be the most obvious choice
as an effector of Ca2+ sensitivity. It is known
that pHi can modify, although not dramatically,
the Ca2+ sensitivity in
permeabilized preparations.53 Our studies
on the effects of pHi on intact coronary
artery21 in fact show a larger dependence of force on
pHi than would be predicted from studies on
permeabilized smooth muscle. One might predict an
inhibition of force caused by a reduction of pHi
under hypoxic conditions, based on the increases in lactate
production previously reported44 and specifically
shown here (Figures 8
and 10
). This would be
consistent with the inhibition of force seen in response to the
acidification elicited by removal of
NH4Cl.21 However, our studies
indicate that pHi is not affected by
hypoxia. This finding, although initially surprising, is
consistent with other studies on smooth muscle showing that
hypoxia and cyanide do not affect pHi
measured with nuclear magnetic resonance methodology54 or
weak acid distribution.55 The increased acid load may be
partially offset by the decrease in PCr, which would exert an
alkalinizing effect. Although it is an attractive hypothesis, our
results indicate that the hypoxic relaxation and the change in
Ca2+ sensitivity observed here for porcine
coronary artery cannot be attributed to alterations in
pHi.
To further circumscribe the mechanisms of the inhibition of force by
hypoxia, we measured its effects on the high-energy phosphagen
and metabolite profile. Hypoxia was characterized by a
significant decrease in PCr to
60% of control; ATP, however, was
not altered. These values are similar to those reported56
for porcine femoral artery, measured during 6 hours of hypoxia
using nuclear magnetic resonance techniques. The only other significant
change in metabolite content seemed to be an increase in lactate
content, as expected. Mechanisms that are dependent on decreasing ATP
content as the signal for vasorelaxation, such as those involving
ATP-dependent K+ channels, would seem to be ruled
out by these measurements. Alternatively, measurement of bulk ATP may
not reflect changes in the localized compartments involved, such as a
purported subsarcolemmal region that may be distinct from the cytosol.
Similar comments would apply to Pi, which has
been associated with the effects of hypoxia on striated
muscle57 but did not change with hypoxia in our
experiments (Figure 8
).
In a related set of experiments, we observed that although
hypoxia inhibited force, increasing stimulation could override
this inhibition. As shown in Figure 9
, addition of U46619 to
KCl-stimulated arteries under hypoxia (or vice versa) restored
force to near initial levels. No major changes in high-energy
phosphagens from those of hypoxia alone were measured (Figure 10
). Thus, changes in ATP levels do not seem to be a major
signaling pathway.
To further investigate the effects of hypoxia on energetics, we
measured the rate of ATP production by arterial
lactate production in parallel experiments of similar design.
Because changes in high-energy phosphagen were negligible, the ATP
production is equivalent to utilization. Interestingly, whereas
U46619 increased force under hypoxia, there was no parallel
increase in Jlac (Figure 10
). Thus,
hypoxia seems to affect the coupling between force
production and ATP utilization. This may simply mean that ATP
was diverted to the myosin ATPase from other processes. It is also
possible that the crossbridge cycle might be modified under hypoxic
conditions. Because PCr was decreased by hypoxia, the
calculated free ADP levels would increase, assuming that the creatine
kinase reaction was in equilibrium.58 Thus, it is
plausible that changes in [ADP] may underlie hypoxic relaxation. One
might anticipate that increasing ADP could affect the crossbridge
detachment rate, which may also underlie the change in energetics.
Changes in [ADP] are known to alter the crossbridge cycle rate in
smooth muscle, but the effective concentration ranges seem to be too
high59 or too low60 based on the calculated
[ADP] in vascular smooth muscle.
It is also plausible that changes in myosin regulatory light chain phosphorylation (MLC-Pi) may be the regulated step for hypoxic relaxation. Coburn et al4 reported that hypoxia decreased MLC-Pi in receptor-mediated contractures but was less affected in KCl contractures in rabbit aorta. Because ATP was not unaffected, presumably myosin light chain kinase activity would not be the site for the Ca2+-independent oxygen sensing. There is evidence that myosin phosphatase may be a site for MLC-Pi regulation,61 which may involve a G protein and C kinase. Within the scope of limited pharmacological data, our negative results with chelerythrine lessen the likelihood for the involvement of C kinase in the mechanism of hypoxic relaxation. However, oxygen sensing at the level of MLC-Pi remains to be tested.
In conclusion, hypoxic vasorelaxation in porcine coronary arteries involves both Ca2+-dependent and -independent mechanisms. The hypoxic vasodilatation cannot be attributed to changes in pHi, Pi, or ATP. Under hypoxia, Ca2+ sensitivity is decreased but the force per ATP seems to be increased. Large coronary arteries can play a significant role in the regulation of coronary circulation.62 The relaxation induced by hypoxia in coronary arteries likely plays a protective role in coronary ischemia. Our results rule out many of the current theories for hypoxic relaxation, but the sites for this important oxygen sensing mechanism remain to be elucidated.
| Acknowledgments |
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Received August 31, 1999; accepted February 29, 2000.
| References |
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