© 1999 American Heart Association, Inc.
Integrative Physiology |
cAMP-Independent Dilation of Coronary Arterioles to Adenosine
Role of Nitric Oxide, G Proteins, and KATP Channels
From the Department of Medical Physiology, Cardiovascular Research Institute, Texas A&M University System Health Science Center, College Station, Tex.
Correspondence to Lih Kuo, PhD, Department of Medical Physiology, Cardiovascular Research Institute, Texas A&M University System Health Science Center, College Station, TX 77843-1114. E-mail lkuo{at}tamu.edu
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Abstract |
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Abstract—Adenosine is known to play an important role in the regulation of coronary blood flow during metabolic stress. However, there is sparse information on the mechanism of adenosine-induced dilation at the microcirculatory levels. In the present study, we examined the role of endothelial nitric oxide (NO), G proteins, cyclic nucleotides, and potassium channels in coronary arteriolar dilation to adenosine. Pig subepicardial coronary arterioles (50 to 100 µm in diameter) were isolated, cannulated, and pressurized to 60 cm H2O without flow for in vitro study. The arterioles developed basal tone and dilated dose dependently to adenosine. Disruption of endothelium, blocking of endothelial ATP-sensitive potassium (KATP) channels by glibenclamide, and inhibition of NO synthase by NG-nitro-L-arginine methyl ester and of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one produced identical attenuation of vasodilation to adenosine. Combined administration of these inhibitors did not further attenuate the vasodilatory response. Production of NO from coronary arterioles was significantly increased by adenosine. Pertussis toxin, but not cholera toxin, significantly inhibited vasodilation to adenosine, and this inhibitory effect was only evident in vessels with an intact endothelium. Tetraethylammonium, glibenclamide, and a high concentration of extraluminal KCl abolished vasodilation of denuded vessels to adenosine; however, inhibition of calcium-activated potassium channels by iberiotoxin had no effect on this dilation. Rp-8-Br-cAMPS, a cAMP antagonist, inhibited vasodilation to cAMP analog 8-Br-cAMP but failed to block adenosine-induced dilation. Furthermore, vasodilations to 8-Br-cAMP and sodium nitroprusside were not inhibited by glibenclamide, indicating that cAMP- and cGMP-induced dilations are not mediated by the activation of KATP channels. These results suggest that adenosine activates both endothelial and smooth muscle pathways to exert its vasodilatory function. On one hand, adenosine opens endothelial KATP channels through activation of pertussis toxin–sensitive G proteins. This signaling leads to the production and release of NO, which subsequently activates smooth muscle soluble guanylyl cyclase for vasodilation. On the other hand, adenosine activates smooth muscle KATP channels and leads to vasodilation through hyperpolarization. It appears that the latter vasodilatory process is independent of G proteins and of cAMP/cGMP pathways.
Key Words: adenosine • microcirculation • nitric oxide • K+ channel
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nucleoside adenosine is a potent vasodilator that has been suggested to play a major role in the control of coronary blood flow during metabolic stress, such as hypoxia,1 ischemia,2 and increase in myocardial oxygen consumption.3 Recently, adenosine receptors have been characterized in coronary vascular smooth muscle4 and endothelial cells5 ; however, the cellular signal pathway responsible for the vasodilation has not been systematically examined. The mechanisms implicated in adenosine-induced dilation of coronary arteries are controversial. For example, evidence suggests that endothelial production of nitric oxide (NO),6 7 8 stimulation of receptor-coupled G proteins,9 and elevation of intracellular cAMP levels10 11 12 are involved in the dilation of isolated coronary arteries to adenosine. In an intact heart preparation, the increased coronary flow by adenosine was partly inhibited by the ATP-sensitive potassium (KATP) channel blocker glibenclamide1 and by inhibition of adenylyl cyclase activity,13 suggesting the putative role of KATP channels and cAMP in this process. Conversely, evidence against the involvement of endothelium,14 15 NO,16 17 KATP channels,18 and cAMP19 20 in vasodilation to adenosine was also reported. Despite these discrepancies, it is important to note that the findings derived from both isolated conduit vessel and intact heart studies are generally confounded by various undefined factors. For example, because of the lack of vascular tone in vitro, the study of vasodilation in conduit vessels is performed with various pharmacological constrictors that potentially influence the adenosine response by activating and superimposing different contractile mechanisms. Most importantly, large-vessel studies are not representative of the resistance microvessels, where the blood flow is actually controlled,21 and thus it is difficult to extrapolate the results from conduit vessel to microvessel because of the functional differences. In addition, the limitation of in vivo studies is also apparently caused by the inevitable influences from neurohumoral and local control mechanisms. Therefore, the cellular mechanism and signal transduction pathway for the vasomotor response to adenosine in coronary resistance vessels are still unclear.
Recently, Kuo and Chancellor22 reported that dilation of isolated coronary arterioles to adenosine is attenuated by endothelial removal, NO synthase inhibitor, and glibenclamide, suggesting the involvement of endothelial NO release and KATP channel activation. However, the release of NO from these microvessels has not been directly assessed, and the cellular pathways for NO and KATP channel activation leading to vasodilation have not been elucidated. Whether cyclic nucleotides (cAMP/cGMP) are involved in this signal transduction process is also unclear. It has been shown that inhibition of G protein signal transduction by pertussis toxin (PTX) prevents the opening of KATP channels in pancreatic cells23 and cardiac myocytes,24 suggesting the involvement of PTX-sensitive G proteins in KATP channel activation. However, it is not known whether this transduction process also occurs in the vascular tissue, especially in response to adenosine. Therefore, in the present study, we focused on the cellular mechanism for adenosine-induced dilation. The role of endothelial NO, G proteins, cyclic nucleotides, and potassium channels in vasodilation to adenosine was examined in the isolated and pressurized coronary resistance vessels.
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Materials and Methods |
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General Preparation
Pigs (8 to 12 weeks old of either sex) were sedated with an intramuscular injection of tiletamine and zolazepam (1:1, 4.4 mg/kg) and xylazine (2.2 mg/kg) and then anesthetized and heparinized with an intravenous administration of pentobarbital sodium (20 mg/kg) and heparin (1000 U/kg), respectively, via the marginal ear vein. Pigs were intubated and ventilated with room air. After a left thoracotomy was performed, the heart was electrically fibrillated, excised, and immediately placed in cold (5°C) saline solution. The procedures followed were in accordance with guidelines set by the Laboratory Animal Care Committee at Texas A&M University.
Isolation and Cannulation of Microvessels
The techniques for identification and isolation of porcine
coronary microvessels were described previously.25
In brief, a mixture of India ink and gelatin in
physiological salt solution (PSS) containing
(in mmol/L) NaCl 145.0, KCl 4.7, CaCl2 2.0,
MgSO4 1.17,
NaH2PO4 1.2, glucose 5.0,
pyruvate 2.0, EDTA 0.02, and MOPS 3.0 was perfused into the left
anterior descending artery (0.3 mL) and the circumflex artery (0.4 mL)
to enable visualization of coronary microvessels. Subepicardial
arteriolar branches (50 to 100 µm in internal diameter and 0.6
to 1.0 mm in length without branches) from the left anterior
descending or circumflex arteries were selected and carefully dissected
from the surrounding cardiac tissue under cold (5°C) PSS containing
BSA (1%; Amersham) at pH 7.4. Each isolated arteriole was then
transferred for cannulation to a methacrylic acid (Lucite) vessel
chamber containing PSS-albumin equilibrated with room air at
ambient temperature. One end of the microvessel was cannulated with a
glass micropipette (40 µm in tip diameter) filled with filtered
PSS-albumin, and the outside of the microvessel was securely
tied to the pipette with 11-0 ophthalmic suture (Alcon). The
ink-gelatin solution inside the vessel was flushed out at a low
perfusion pressure (<20 cm H2O). Then, the other
end of the vessel was cannulated with a second micropipette and tied
with suture. We have previously shown that the ink-gelatin solution has
no detectable detrimental effect on either endothelial
or vascular smooth muscle function.26 In our previous
studies,22 25 26 we found that the isolated arterioles
tend to maintain viability for a longer period of time when a small
amount of albumin (1%) is present. The albumin
used in our studies has no detectable effect on common vasomotor
function, eg, endothelium-dependent and –independent
agonist-induced dilations as well as myogenic and flow-induced
responsiveness.
Instrumentation
The cannulated vessel was transferred to the stage of an
inverted microscope (model IM35, Zeiss) coupled to a charge-coupled
device camera (KP-161, Hitachi) and video micrometer
(Microcirculation Research Institute, Texas A&M University Health
Science Center). Internal diameters of the vessel were measured
throughout the experiment using video microscopic techniques
incorporated into the MacLab (ADInstruments Inc) data acquisition
system.26 The micropipettes were connected to independent
reservoir systems, and intraluminal pressures were measured through
sidearms of the 2 reservoir lines. The isolated vessels were
pressurized without flow by setting both reservoirs at the same
hydrostatic level. Leaks were detected by differences between reservoir
pressure and intraluminal pressure. Preparations with leaks were
excluded from further study.
Experimental Protocols for Mechanistic Study of
Adenosine-Induced Dilation
Cannulated arterioles were bathed in PSS-albumin at
36°C to 37°C to allow development of basal tone. After vessels
developed a stable basal tone (
40 to 60 minutes), the dose-diameter
relationship for adenosine (0.1 nmol/L to 10 µmol/L) was
established. The role of endothelium in the
adenosine-induced coronary arteriolar dilation was
evaluated by comparing the response before and after
endothelial removal. A nonionic detergent, CHAPS
(0.4%), was perfused into the vessel for 1 to 2 minutes to remove
endothelial cells.27 To ensure that the
vascular smooth muscle function was not compromised by CHAPS treatment,
dose-dependent dilation of the vessel in response to sodium
nitroprusside (1 nmol/L to 10 µmol/L) was examined before and
after denudation. Only vessels that exhibited normal basal tone, showed
no vasodilation to endothelium-dependent vasodilator
bradykinin (1 nmol/L),27 and showed unaltered vasodilation
to sodium nitroprusside after endothelial removal were
accepted for data analysis.
The following studies were performed to elucidate the possible mechanisms involved in the coronary arteriolar dilation to adenosine. First, the involvement of NO and prostaglandins in adenosine-induced dilation was examined by extraluminal incubation (30 minutes) of isolated arterioles with the specific inhibitors NG-nitro-L-arginine methyl ester (L-NAME; 10 µmol/L) and indomethacin (10 µmol/L), respectively. Second, the possible involvement of endothelial KATP channels in adenosine-induced vasodilation was examined by intraluminal incubation of vessels with glibenclamide (5 µmol/L) for 10 minutes. This technique has been shown to specifically inhibit endothelial KATP channel function in our previous study.22 The role of vascular smooth muscle potassium channels was evaluated in denuded vessels treated with a nonspecific potassium channel inhibitor, tetraethylammonium (TEA, 10 and 20 mmol/L, extraluminal).28 The involvement of KATP and calcium-activated potassium (KCa) channels in vasodilation to adenosine was examined by extraluminal incubation of intact or denuded vessels with the specific inhibitors glibenclamide (5 µmol/L, 30 minutes)29 and iberiotoxin (0.1 µmol/L, 60 minutes),28 respectively. Third, the role of G proteins in adenosine-induced dilation was examined by extraluminal incubation (60 minutes) of the vessels with PTX (100 ng/mL)30 31 or cholera toxin (CTX, 2 µg/mL).9 Fourth, the dilation of denuded vessel to adenosine was performed in the presence of a depolarizing PSS solution containing 40 mmol/L KCl. The isotonic depolarizing solution was prepared by substituting 35 mmol/L NaCl with an equimolar amount of KCl. Finally, to examine the contribution of cAMP and cGMP, the vessels were treated with the cAMP-competitive antagonist Rp-8-Br-cAMPS (Biolog)32 and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 0.1 µmol/L; Research Biochemicals International),33 respectively. The vasodilation to adenosine was examined before and after extraluminal exposure of the vessels to each inhibitor. The efficacy of cAMP and cGMP inhibition was verified by the absence of vasodilation to 8-Br-cAMP (Research Biochemicals International) and sodium nitroprusside, respectively. It should be noted that glibenclamide has been shown to bind strongly, >99%, to albumin34 35 ; we performed studies with this KATP channel blocker in the PSS without albumin.
Measurement of Adenosine-Induced NO Release from
Coronary Microvessels
The porcine coronary arterioles (10 segments, 1 to
2 mm in length, 70 to 100 µm in situ diameter) were
isolated and placed in a microcentrifuge tube containing 100
µL PSS at 37°C. After a 30-minute initial incubation,
adenosine (10 µL, final concentration 1 µmol/L) was
added to the vessel bath. The bathing solution was then collected for
NO measurement after a 25-minute incubation with adenosine. The
production of NO was evaluated by measuring nitrite using a
chemiluminescence NO analyzer (Siever Instruments). Basically,
the collected sample was injected into a reflux chamber containing
glacial acetic acid and 1% potassium iodide at room temperature. Under
these conditions, nitrite is quantitatively converted to NO. The NO gas
was then purged into the chemiluminescence NO analyzer and
quantified by reference to NaNO2 standards.
Another series of experiments was performed in the presence of NO
synthase inhibitor. The vessels were treated with L-NAME
(10 µmol/L) for 30 minutes during the initial incubation period.
Then, adenosine (1 µmol/L final concentration) was added
to the vessel bath and NO production was assayed after 25
minutes of incubation. In addition, a separate experiment was run as a
control by adding vehicle solution (PSS) instead of adenosine
and L-NAME. The background level of nitrite in the solution was
measured from the tube containing PSS only, and this value was
subsequently subtracted from the sampled solution to obtain NO
production. The protein levels in each vessel tube were
quantified by bicinchoninic acid protein assay (Pierce) and were used
as a basis to normalize the NO production. The above NO
measurements were performed in 4 independent sets of experiments, and
the values were averaged.
Chemicals
Drugs were obtained from Sigma, except as specifically stated.
Adenosine, bradykinin, iberiotoxin,
indomethacin, L-NAME, CTX, PTX, sodium nitroprusside,
8-Br-cAMP, Rp-8-Br-cAMPS, and TEA were dissolved in PSS. Pinacidil was
dissolved in ethanol. ODQ and glibenclamide were dissolved in DMSO as
stock solutions (10 mmol/L). Subsequent concentrations of pinacidil,
ODQ, and glibenclamide were diluted in PSS. The final concentrations of
ethanol and DMSO in the vessel bath were 0.1 and 0.03%, respectively.
Vehicle control studies indicated that these final concentrations of
ethanol and DMSO had no effect on the arteriolar function.
Data Analysis
At the end of each experiment, the vessel was relaxed with
sodium nitroprusside 100 µmol/L to obtain its maximal diameter
at 60 cm H2O intraluminal pressure. We have
previously shown that this concentration of sodium nitroprusside
produced maximal relaxation of isolated vessels.36
Therefore, all diameter changes in response to agonists were normalized
to the vasodilation in response to 100 µmol/L sodium
nitroprusside and expressed as a percentage of maximal dilation. All
data are presented as mean±SEM. Statistical comparisons of
vasomotor responses and NO production under various treatments
were performed with 1- or 2-way ANOVA when appropriate and tested with
the Fisher protected least significant difference multiple-range test.
Differences in resting diameter before and after pharmacological
interventions and vasodilations to bradykinin and 8-Br-cAMP before and
after intervention were compared by paired Student t tests.
Significance was accepted at P<0.05.
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Results |
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Role of Endothelium-Derived Factors in Vasodilation to Adenosine
In this study, all vessels developed a similar level of basal tone (68±1% of maximal diameter) at 36°C to 37°C bath temperature with 60 cm H2O intraluminal pressure. The average resting and maximal diameters of the vessel were 84±3 and 125±3 µm, respectively. Indomethacin (10 µmol/L) did not alter vasodilation elicited by adenosine, but L-NAME (10 µmol/L) significantly inhibited the vasodilatory response (Figure 1
). The inhibitory
effect produced by L-NAME was identical to that produced by soluble
guanylyl cyclase blocker ODQ (0.1 µmol/L, Figure 1).
Incubation of these vessels with L-NAME or ODQ did not significantly
affect resting diameter.
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Measurement of Adenosine-Induced NO Release From
Coronary Arterioles
In the absence of adenosine, the NO production
from coronary arterioles was 1.4±0.7 nmol/g protein. Adding
adenosine (1 µmol/L) to the vessels produced an 8-fold
increase in NO production (Figure 2). The increased NO production
by adenosine (1 µmol/L) was not seen in the vessels
treated with L-NAME (10 µmol/L) (Figure 2). There was no
statistical difference in the NO production between control and
L-NAME-treated vessels.
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Role of Endothelial KATP Channels in
Adenosine-Induced Vasodilation
Intraluminal incubation of vessels with glibenclamide (5
µmol/L, 10 minutes) did not alter resting diameter, but attenuated
arteriolar dilation to adenosine (Figure 3). It is worth noting that the extent of
attenuation by intraluminal glibenclamide was similar to that by L-NAME
or ODQ (Figure 1
). Furthermore, the addition of ODQ (0.1
µmol/L, 30 minutes) to these intraluminal glibenclamide-treated
vessels did not further attenuate vasodilation to adenosine
(Figure 3). These results suggest that intraluminal
glibenclamide and ODQ inhibit the same vasodilatory pathway.
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Role of G Proteins in Adenosine-Induced Vasodilation
Inhibition of arteriolar Gi and
Go proteins by extraluminal PTX (100
ng/mL, 60 minutes) did not alter resting vascular diameter or
vasodilation to nitroprusside (Table) but significantly attenuated
adenosine-induced vasodilation (Figure 4A). Removal of
endothelium attenuated vasodilation to
adenosine, and this attenuation was identical to that produced
by PTX (Figure 4A). In these denuded vessels, PTX did not have
an additional inhibitory effect on
adenosine-induced vasodilation (Figure 4A). To evaluate
the role of Gs proteins in vasodilation, the
intact vessels were treated with extraluminal CTX (2 µg/mL) for 60
minutes, and its effect on adenosine-induced dilation was
reexamined. Initially, CTX produced a slight vasodilation (17±5%) of
coronary arterioles, but the vessels regained tone after a
15-minute exposure to CTX. In contrast to PTX, coronary
arteriolar dilation to adenosine was not affected by CTX
(Figure 4B).
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Role of Vascular Smooth Muscle K+ Channels and Membrane
Hyperpolarization in Arteriolar Dilation to
Adenosine
The role of smooth muscle potassium channels and membrane
hyperpolarization in vasodilation to
adenosine is elucidated in Figures 5 and 6. In
this series of studies, the endothelium was initially
removed to eliminate its contribution to vasodilation. Disruption of
endothelium attenuated adenosine-induced
vasodilation, which is consistent with the data shown in Figure 4A. Extraluminal incubation of denuded vessels with a
nonspecific potassium channel blocker TEA (10
mmol/L)28 attenuated vasodilation to adenosine
(Figure 5). A higher concentration of TEA (20 mmol/L)
almost completely blocked the vasodilation (10% of maximal dilation,
Figure 5). It is noted that 10 mmol/L TEA did not alter
resting vessel tone, whereas 20 mmol/L TEA increased vessel tone
from 68% to 61% of its maximal diameter without affecting dilation to
sodium nitroprusside (Table). To probe the role of
KATP channels in adenosine-induced
dilation, the denuded vessels were treated with extraluminal
glibenclamide for 30 minutes. Glibenclamide (5 µmol/L) did not
affect the resting diameter but completely blocked dilation of denuded
vessels to adenosine (Figure 6). It should be noted that
the dilation of these glibenclamide-treated vessels to sodium
nitroprusside was not affected (data not shown). In the presence of a
high concentration of extraluminal KCl (40 mmol/L),
adenosine-induced dilation was significantly attenuated (Figure 6). This concentration of KCl had a tendency to increase
vascular tone but not in a significant manner and did not alter sodium
nitroprusside-induced dilation (Table). In contrast to TEA and
glibenclamide, inhibition of KCa channels by
extraluminal iberiotoxin (0.1 µmol/L) did not alter the
vasodilatory response to adenosine (n=4, data not shown).
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Role of cGMP in KATP Channel-Mediated
Vasodilation
Because the cGMP pathway is involved in the
adenosine-induced dilation (Figure 1), it is likely that
the opening of smooth muscle KATP channels is
mediated by the activation of this pathway as shown in porcine pial
arteries.37 To examine this possibility, the NO donor
sodium nitroprusside (10 nmol/L to 10 µmol/L) was used to
activate guanylyl cyclase for cGMP production, and the
vasodilation produced by nitroprusside was examined after extraluminal
administration of the KATP channel
antagonist glibenclamide. Coronary arteriolar
dilation to sodium nitroprusside was not altered by glibenclamide
(5 µmol/L, Table), but it was inhibited by the soluble
guanylyl cyclase inhibitor ODQ (0.1 µmol/L,
Table). These results indicate that activation of guanylyl
cyclase does not lead to KATP channel-mediated
coronary arteriolar dilation.
Role of cAMP in Adenosine-Induced Vasodilation
The contribution of cAMP in coronary arteriolar dilation
to adenosine was examined before and after extraluminal
incubation of vessels with cAMP antagonist Rp-8-Br-cAMPS
(10 µmol/L, 40 minutes). As shown in Figure 7A, Rp-8-Br-cAMPS abolished
coronary arteriolar dilation in response to the direct increase
in cAMP by a cell-permeable cAMP analog, 8-Br-cAMP (2 mmol/L);
however, this cAMP antagonist failed to block
adenosine-induced vasodilation (Figure 7B). To eliminate
the possible confounding effects from endothelium,
another series of experiments was performed in denuded vessels.
Similarly, Rp-8-Br-cAMPS abolished dilation of denuded vessels to
8-Br-cAMP but failed to inhibit vasodilation to adenosine (n=3,
data not shown). It is worth noting that resting diameter and sodium
nitroprusside-induced dilation (Table) in these vessels were not
altered by Rp-8-Br-cAMPS.
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Role of cAMP in KATP Channel-Mediated
Vasodilation
To examine whether KATP channels were
involved in the cAMP-mediated dilation of coronary arterioles,
the effect of glibenclamide (5 µmol/L) on vasodilation to
8-Br-cAMP was investigated. 8-Br-cAMP (2 mmol/L) produced a
significant vasodilation by increasing diameter from a control level of
89±3 µm to 105±2 µm, corresponding to 48% of maximal
dilation. However, this dilation was not affected by glibenclamide
(n=4, data not shown), indicating that cAMP-induced coronary
arteriolar dilation is not mediated by the opening of
KATP channels.
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Discussion |
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The role of the endothelium in dilation of coronary arteries to adenosine is controversial. For example, previous studies have shown that adenosine-induced dilation is independent,14 15 partially dependent,7 22 38 or exclusively dependent39 on the presence of an intact endothelium. Our previous22 and the present studies using isolated and pressurized coronary arterioles demonstrated that the vasodilation to adenosine is partially dependent on the endothelium, given that the vasodilatory response was not abolished by endothelial removal (Figures 4A
, 5, and 6). However, in some in vitro studies,
the investigators did not observe the endothelial
component of adenosine-induced dilation in large
coronary arteries.14 15 This discrepancy may be
derived from the difference in vascular preparations, ie, pressurized
microvessel segment versus stretched vascular ring. In addition, the
use of preconstrictors for large vessel preparations may have masked or
interfered with the endothelial function. It is
important to note that the development of basal tone in our
coronary arterioles precluded the use of preconstrictors and
thus eliminated these potential confounding effects.
The activation of endothelial adenosine
receptors may stimulate the production and release of
endothelium-derived vasodilators. Administration of
indomethacin to the coronary arterioles, with a
concentration (10 µmol/L) sufficient to block
prostaglandin synthesis in our previous
study,26 did not alter adenosine responsiveness
(Figure 1). This result indicates that adenosine-induced
vasodilation is not mediated by the release of
prostaglandins. Because adenosine-induced
coronary arteriolar dilation was attenuated by the NO synthase
inhibitor
NG-monomethyl-L-arginine
(L-NMMA) in our previous study22 and by L-NAME in
the present study (Figure 1) and was inhibited to the same
extent by endothelial removal (Figures 4A, 5, and 6), it is believed that
endothelial production/release of NO is
partially responsible for the observed adenosine response. This
contention is directly supported by the result of NO measurement, which
shows that NO production was increased after incubation of the
coronary arterioles with adenosine (Figure 2).
However, another possible explanation for the inhibitory
effect by L-NAME and denudation is that basal production of NO
plays a permissive or amplifying role in the adenosine-induced
response. If NO indeed plays a permissive/amplifying role, we should
see a restoration or enhancement of vasodilation in the denuded vessels
in response to adenosine in the presence of a threshold
concentration of NO donor sodium nitroprusside. In fact, we found that
sodium nitroprusside (10 nmol/L) produced a slight dilation (5%;
Table) of the denuded vessels but did not restore or enhance the
adenosine response (data not shown). Collectively, our data
indicate that NO is actively participating in the vasodilation rather
than acting as a permissive or amplifying agent for the relaxing effect
of adenosine.
Interestingly, our data showed that the endothelium was
more sensitive than the vascular smooth muscle to adenosine for
vasodilation, ie, dilation threshold 1 nmol/L and 0.1 µmol/L for
endothelium and smooth muscle, respectively (Figures 4A, 5, and 6). This may suggest a dose-dependent
activation of the NO-dependent versus NO-independent pathways for
adenosine-induced vasodilation. For instance, the vasodilation
at lower doses (
10–8 mol/L) of
adenosine is exclusively mediated by the NO (eg, abolished by
L-NAME and denudation). In contrast, the higher doses
(
10–7 mol/L) of adenosine
activate both NO-dependent and NO-independent pathways (Figures 1 and 3), but the latter pathway becomes more obvious and
masks the NO-dependent component. Therefore, the contribution of
NO/endothelium to the observed vasodilation is
diminished with increasing adenosine concentration. At the
highest dose of adenosine (10–5 mol/L),
the dilation occurs independently of NO or
endothelium (Figures 1 and 4).
This might explain the seemingly conflicting results on NO dependency
reported in vivo. For instance, flow responses to an
intracoronary infusion (10 µg/min)40 or a bolus
injection (3 µg total)6 of low doses of
adenosine were attenuated by NO inhibitors. On the
other hand, higher doses of adenosine (0.5
mg/min41 or 8 mg/min42 ) that produced maximal
coronary dilation were not affected by NO inhibition.
Therefore, these in vivo and our in vitro data support the notion that
the contribution of NO/endothelium to
adenosine-induced dilation is inversely related to the
adenosine concentration. The dose-dependent activation of
adenosine vasodilatory pathways in endothelial
and smooth muscle cells is schematically illustrated in Figure 8 and discussed below.
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Em; ie,
hyperpolarization), leading to an influx of calcium
for NO production by NO synthase (NOS). The
endothelium-released NO diffuses to the underlying
smooth muscle and stimulates guanylyl cyclase to produce cGMP for
relaxation. At the higher concentrations of adenosine
(
As discussed above, the released NO is an important mediator for
vasodilation in response to the lower concentrations of
adenosine (10–7 mol/L). In general, NO
released from the endothelium subsequently
activates soluble guanylyl cyclase in underlying vascular
smooth muscle cells and thus produces vasodilation.43
However, a direct activation of vascular smooth muscle
KATP and KCa channels by NO
was also reported.44 45 Therefore, signal transduction
mediating dilation of coronary arteries to adenosine
downstream from NO remains unclear. In the present study,
inhibition of soluble guanylyl cyclase by ODQ attenuated the vasomotor
response to adenosine. In addition, ODQ attenuated
adenosine-induced dilation similarly to that by L-NAME (Figure 1) and by denudation (Figures 4A, 5, and 6), and combined administration of these 2
inhibitors did not further enhance the inhibition (data not
shown), suggesting the involvement of the guanylyl cyclase/cGMP pathway
in this vasodilatory process. On the other hand, glibenclamide and
iberiotoxin did not affect vasodilation to NO donor sodium
nitroprusside (Table), indicating that adenosine-induced
dilation associated with NO is not mediated by the opening of
KATP and KCa channels in
our preparation. In addition, ODQ attenuated adenosine-induced
dilation in a manner identical to that by L-NAME (Figure 1) and
denudation (Figures 4A, 5, and 6), and combined
administration of these 2 inhibitors did not further
enhance the inhibition (data not shown). It appears that activation of
the NO/guanylyl cyclase pathway is responsible for the
endothelium-dependent component of vasodilation to
adenosine (Figure 8).
It has been shown that the membrane
hyperpolarization leading to the influx of calcium
into endothelial cells is necessary for the NO
production from constitutive NO synthase during agonist
stimulation.46 Interestingly, adenosine has been
shown to produce a sustained hyperpolarization of
cultured coronary endothelial
cells.47 Recently, Kuo and Chancellor22
demonstrated that prevention of endothelial
hyperpolarization by a high luminal KCl solution
attenuated dilation of isolated porcine coronary arterioles to
adenosine. This inhibitory effect is
consistent with that elicited by L-NMMA, indicating that
hyperpolarization of the
endothelium by adenosine might be responsible
for the release of NO (Figure 8). In our previous study, we
speculated that the membrane hyperpolarization
might be a result of opening of endothelial
KATP channels, given that specific inhibition of
endothelial KATP channel and
prevention of endothelial
hyperpolarization by intraluminal administration of
glibenclamide and KCl, respectively, produce identical inhibition of
coronary arteriolar dilation to
adenosine.22 In the present study, inhibition
of endothelial KATP channels by
luminal glibenclamide attenuated adenosine-induced vasodilation
in a manner identical to that produced by L-NAME (Figure 1), ODQ
(Figure 1), and endothelial removal (Figures 4A, 5, and 6). Furthermore, subsequent
administration of ODQ to vessels pretreated with luminal glibenclamide
did not further enhance the inhibitory effect (Figure 3), suggesting that these inhibitors act on the same
pathway for vasodilation. Because luminal glibenclamide did not affect
vasodilation to NO donor nitroprusside,22 the inhibition
of adenosine-induced dilation by luminal glibenclamide is
believed to be upstream of the NO-cGMP pathway. Altogether, these
results suggest that the opening of endothelial
KATP channels leads to membrane
hyperpolarization for calcium influx and thus
produces vasodilation through subsequent activation of NO synthase for
NO production (Figure 8).
The opening of endothelial KATP
channels by adenosine may involve activation of G proteins as
suggested by patch-clamp studies in cardiac myocytes.24 It
has been shown that adenosine receptors can be coupled to both
Gs and Gi proteins in
various types of tissues.48 49 In the present study,
we found that Gs proteins do not play a role in
coronary arteriolar dilation to adenosine, because CTX
did not alter the vasodilatory response. In contrast, PTX attenuated
adenosine-induced dilation to the same extent as L-NAME, ODQ,
intraluminal glibenclamide, or denudation. It appears that the
component of PTX-sensitive dilation resides in the
endothelium, because the adenosine-induced
response was not inhibited by PTX in denuded vessels (Figure 4A). Furthermore, PTX did not further enhance the
inhibitory effect produced by L-NAME, ODQ, or luminal
glibenclamide (n=3, data not shown). Collectively, our present
results support the contention that the activation of
endothelial PTX-sensitive G proteins is coupled to the
opening of endothelial KATP
channels for membrane hyperpolarization and
subsequently leads to vasodilation through the release of NO (Figure 8).
At the higher concentrations of adenosine (>0.1
µmol/L), the contribution of endothelium-released NO
to vasodilation is diminished, and the direct action of
adenosine on smooth muscle predominates. Because potassium
channels have been shown to play an important role in smooth muscle
relaxation,28 in the present study we examined the
possible involvement of these channels in adenosine-induced
dilation of denuded coronary arterioles. The role of potassium
channels in the vasodilation to adenosine was evident because
the adenosine response was inhibited by a nonspecific potassium
channel blocker, TEA. Although the concentrations of TEA (10 and
20 mmol/L) are likely to inhibit both KATP
and KCa channels,28 the role of
KCa channels is not supported, because the
dilation was not altered by iberiotoxin. In contrast, inhibition of
KATP channels by glibenclamide abolished the
adenosine response, suggesting that opening of
KATP channels in vascular smooth muscle is
responsible for the endothelium-independent response to
adenosine (Figure 6). It is likely that vascular
hyperpolarization after the opening of smooth
muscle KATP channels results in vasodilation,
because administration of a depolarizing agent, KCl (40 mmol/L),
significantly attenuated adenosine-induced dilation (Figure 6). A higher concentration of KCl (60 mmol/L) nearly
abolished the vasodilatory response (7% dilation, data not shown).
However, the latter data are difficult to interpret, because the
resting vessel diameter was significantly decreased, from 63% to 44%
of its maximal diameter, by 60 mmol/L KCl. Nevertheless, these
results suggest that the activation of smooth muscle
KATP channels leading to vascular
hyperpolarization is an essential pathway for
vasodilation to adenosine (Figure 8). It is important to
note that adenosine-induced dilation of large porcine
coronary arteries was unaffected by
glibenclamide,18 indicating that heterogeneous
vasodilatory mechanisms might exist in the coronary
circulation. On the other hand, it should be noted that this disparate
result might be related to the vessel size, different vascular
preparations, and the use of preconstrictors in these large
vessels.18
It is generally believed or assumed that stimulation of
adenosine receptors activates adenylyl cyclase in
vascular smooth muscle to produce cAMP, which may in turn relax
coronary arteries by activating cAMP-dependent protein kinase
A.10 11 12 However, there is no direct evidence to support
this contention in the coronary microcirculation. In fact,
there are some doubts as to the role of cAMP pathway in
adenosine-induced dilation of coronary arteries,
because adenosine and adenosine receptor agonists seem
not to increase intracellular levels of cAMP in coronary
arteries in vitro.19 20 In the present study, we found
that the dilation elicited by adenosine was not affected by
cAMP antagonist Rp-8-Br-cAMPS, suggesting a minimal role of
this pathway, if any, in vasodilation. The concentration of cAMP
antagonist used in the present study was effective and
selective, given that vasodilation to cAMP analog 8-Br-cAMP (Figure 7A) was abolished, and the cGMP-mediated dilation in response to
sodium nitroprusside (Table) remained intact. The present
study also ruled out the possibility of activation of
KATP channels by the cAMP–protein kinase A
pathway,50 because vasodilation to cAMP agonist was not
altered by glibenclamide. Collectively, the ability of glibenclamide
but not of Rp-8-Br-cAMPS to inhibit vasodilation indicates that the
activation of KATP channels by adenosine
is not mediated by the cAMP pathway. Furthermore, our data do not
support the idea that activation of KATP channels
by adenosine is through the cGMP pathway, because vasodilation
in response to sodium nitroprusside was not affected by glibenclamide
(Table). Unlike the endothelium, the
KATP channel activation in smooth muscle appears
to be independent of Gi/Go
proteins, because PTX (100 ng/mL, 60-minute incubation) had no effect
on adenosine-induced dilation in the denuded vessels. In our
previous study, we have shown that the same PTX treatment produced an
effective inhibition of acidosis-induced dilation mediated by the
opening of smooth muscle KATP
channels.31 Therefore, the failure of inhibiting
adenosine-induced dilation of denuded vessels by PTX in the
present study is unlikely the result of the ineffectiveness of this
drug. Nevertheless, these findings highlight the involvement of
different signaling pathways in the activation of smooth muscle
KATP channel by adenosine (PTX
insensitive) and by acidosis (PTX sensitive).
In conclusion, porcine coronary arteriolar dilation to adenosine could be explained by at least 2 mechanisms. First, adenosine, at lower concentrations (eg, nmol/L range), selectively opens endothelial KATP channels through activation of PTX-sensitive G proteins. This signaling leads to the production and release of NO, which subsequently activates smooth muscle soluble guanylyl cyclase for vasodilation. Second, at higher concentrations of adenosine (eg, µmol/L range), the opening of smooth muscle KATP channels associated with membrane hyperpolarization takes over the role of NO for vasodilation. It appears that the latter vasodilatory pathway is independent of G proteins and of cAMP/cGMP pathways. Currently, the detailed mechanism for the inverse relationship between adenosine concentration and the involvement of endothelium in vasodilation is unclear. It is possible that the G proteins in endothelium might serve as an amplifier for the cellular signaling linked to KATP channels and thus might contribute to the high sensitivity of endothelium to adenosine. Therefore, a low concentration of adenosine is sufficient to activate the endothelial KATP channel pathway for vasodilation. In contrast, a higher concentration of adenosine is required to activate smooth muscle KATP channels, because the PTX-sensitive G protein is not involved in this vasodilatory pathway. This might explain the differential sensitivity of the adenosine response, in terms of endothelium versus smooth muscle pathway for vasodilation.
|
Acknowledgments |
|---|
This study was supported by National Heart, Lung, and Blood Institute (Bethesda, Md) grants HL-48179 and K02 HL-03693 (Research Career Award) to L.K. We thank Dr Cuihua Zhang and Chiung-I Chang for their technical assistance.
Received March 26, 1999; accepted July 9, 1999.
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References |
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 K. Murphy, V. Gerzanich, H. Zhou, S. Ivanova, Y. Dong, G. Hoffman, G. A. West, H. R. Winn, and J. M. Simard Adenosine-A2a Receptor Down-Regulates Cerebral Smooth Muscle L-Type Ca2+ Channel Activity via Protein Tyrosine Phosphatase, Not cAMP-Dependent Protein Kinase Mol. Pharmacol., September 1, 2003; 64(3): 640 - 649. [Abstract] [Full Text] [PDF]
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D. Merkus, D. B. Haitsma, T.-Y. Fung, Y. J. Assen, P. D. Verdouw, and D. J. Duncker Coronary blood flow regulation in exercising swine involves parallel rather than redundant vasodilator pathways Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H424 - H433. [Abstract] [Full Text] [PDF]
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H.-Y. Sohn, F. Krotz, T. Gloe, M. Keller, K. Theisen, V. Klauss, and U. Pohl Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine Cardiovasc Res, June 1, 2003; 58(3): 638 - 646. [Abstract] [Full Text] [PDF]
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J. J. Bosnjak, K. Terata, H. Miura, A. Sato, A. C. Nicolosi, M. McDonald, S. A. Manthei, T. Saito, O. A. Hatoum, and D. D. Gutterman Mechanism of thrombin-induced vasodilation in human coronary arterioles Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1080 - H1086. [Abstract] [Full Text] [PDF]
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C. Zhang, T. W. Hein, W. Wang, and L. Kuo Divergent Roles of Angiotensin II AT1 and AT2 Receptors in Modulating Coronary Microvascular Function Circ. Res., February 21, 2003; 92(3): 322 - 329. [Abstract] [Full Text] [PDF]
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R. Rubio and G. Ceballos Sole activation of three luminal adenosine receptor subtypes in different parts of coronary vasculature Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H204 - H214. [Abstract] [Full Text] [PDF]
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 L. M. Riou, M. Ruiz, J. M. Rieger, T. L. Macdonald, D. D. Watson, J. Linden, G. A. Beller, and D. K. Glover Influence of propranolol, enalaprilat, verapamil, and caffeine on adenosine A2A-receptor-mediated coronary vasodilation J. Am. Coll. Cardiol., November 6, 2002; 40(9): 1687 - 1694. [Abstract] [Full Text] [PDF]
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A. Tawakol, M. A. Forgione, M. Stuehlinger, N. M. Alpert, J. P. Cooke, J. Loscalzo, A. J. Fischman, M. A. Creager, and H. Gewirtz Homocysteine impairs coronary microvascular dilator function in humans J. Am. Coll. Cardiol., September 18, 2002; 40(6): 1051 - 1058. [Abstract] [Full Text] [PDF]
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A. Aaker and M. H. Laughlin Differential adenosine sensitivity of diaphragm and skeletal muscle arterioles J Appl Physiol, September 1, 2002; 93(3): 848 - 856. [Abstract] [Full Text] [PDF]
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A. Yaghi, S. Mehta, and D. G. McCormack Delayed rectifier potassium channels contribute to the depressed pulmonary artery contractility in pneumonia J Appl Physiol, September 1, 2002; 93(3): 957 - 965. [Abstract] [Full Text] [PDF]
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G.F Baxter Role of adenosine in delayed preconditioning of myocardium Cardiovasc Res, August 15, 2002; 55(3): 483 - 494. [Abstract] [Full Text] [PDF]
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P. O. Bonetti, S. H. Wilson, M. Rodriguez-Porcel, D. R. Holmes Jr, L. O. Lerman, and A. Lerman Simvastatin preserves myocardial perfusion and coronary microvascular permeability in experimental hypercholesterolemia independent of lipid lowering J. Am. Coll. Cardiol., August 7, 2002; 40(3): 546 - 554. [Abstract] [Full Text] [PDF]
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 R. Weiss, M. Mevissen, D. S. Hauser, G. Scholtysik, C. J. Portier, B. Walter, U. E. Studer, and H. Danuser Inhibition of Human and Pig Ureter Motility in Vitro and in Vivo by the K+ Channel Openers PKF 217-744b and Nicorandil J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 651 - 658. [Abstract] [Full Text] [PDF]
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K. Sato, T. Komaru, H. Shioiri, S. Takeda, K. Takahashi, H. Kanatsuka, and K. Shirato Vasodilator signals from the ischemic myocardium are transduced to the coronary vascular wall by pertussis toxin-sensitive g proteins: A new experimental method for the analysis of the interaction between the myocardium and coronary vessels J. Am. Coll. Cardiol., June 5, 2002; 39(11): 1859 - 1865. [Abstract] [Full Text] [PDF]
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H. M. O. Farouque, S. G. Worthley, I. T. Meredith, R. A. P. Skyrme-Jones, and M. J. Zhang Effect of ATP-Sensitive Potassium Channel Inhibition on Resting Coronary Vascular Responses in Humans Circ. Res., February 8, 2002; 90(2): 231 - 236. [Abstract] [Full Text] [PDF]
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C. L. Heaps and D. K. Bowles Gender-specific K+-channel contribution to adenosine-induced relaxation in coronary arterioles J Appl Physiol, February 1, 2002; 92(2): 550 - 558. [Abstract] [Full Text] [PDF]
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D. Wothe, A. Hohimer, M. Morton, K. Thornburg, G. Giraud, and L. Davis Increased coronary blood flow signals growth of coronary resistance vessels in near-term ovine fetuses Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R295 - R302. [Abstract] [Full Text] [PDF]
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O. Sorop, J. A. E. Spaan, and E. VanBavel Pulsation-induced dilation of subendocardial and subepicardial arterioles: effect on vasodilator sensitivity Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H311 - H319. [Abstract] [Full Text] [PDF]
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R. Nakamura, K. Egashira, K. Arimura, Y. Machida, T. Ide, H. Tsutsui, H. Shimokawa, and A. Takeshita Increased inactivation of nitric oxide is involved in impaired coronary flow reserve in heart failure Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2619 - H2625. [Abstract] [Full Text] [PDF]
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R. J. Rivers, T. W. Hein, C. Zhang, and L. Kuo Activation of Barium-Sensitive Inward Rectifier Potassium Channels Mediates Remote Dilation of Coronary Arterioles Circulation, October 9, 2001; 104(15): 1749 - 1753. [Abstract] [Full Text] [PDF]
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B. M. Stavrou, D. J. Sheridan, and N. A. Flores Contribution of Nitric Oxide and Prostanoids to the Cardiac Electrophysiological and Coronary Vasomotor Effects of Diadenosine Polyphosphates J. Pharmacol. Exp. Ther., August 1, 2001; 298(2): 531 - 538. [Abstract] [Full Text] [PDF]
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S. Dube and J. M. Canty Jr. Shear stress-induced vasodilation in porcine coronary conduit arteries is independent of nitric oxide release Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2581 - H2590. [Abstract] [Full Text] [PDF]
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R. D. Lasley, M. S. Jahania, and R. M. Mentzer Jr. Beneficial effects of adenosine A2a agonist CGS-21680 in infarcted and stunned porcine myocardium Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1660 - H1666. [Abstract] [Full Text] [PDF]
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C. Zhang, T. W. Hein, and L. Kuo Transmural difference in coronary arteriolar dilation to adenosine: effect of luminal pressure and KATP channels Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2612 - H2619. [Abstract] [Full Text] [PDF]
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R. M Mohan and D. J Paterson Activation of sulphonylurea-sensitive channels and the NO-cGMP pathway decreases the heart rate response to sympathetic nerve stimulation Cardiovasc Res, July 1, 2000; 47(1): 81 - 89. [Abstract] [Full Text] [PDF]
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C. L. Heaps, M. Sturek, J. A. Rapps, M. H. Laughlin, and J. L. Parker Exercise training restores adenosine-induced relaxation in coronary arteries distal to chronic occlusion Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1984 - H1992. [Abstract] [Full Text] [PDF]
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H. M. O. Farouque, S. G. Worthley, I. T. Meredith, R. A. P. Skyrme-Jones, and M. J. Zhang Effect of ATP-Sensitive Potassium Channel Inhibition on Resting Coronary Vascular Responses in Humans Circ. Res., February 8, 2002; 90(2): 231 - 236. [Abstract] [Full Text] [PDF]
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P. Vequaud and E. Thorin Endothelial G Protein {beta}-Subunits Trigger Nitric Oxide- but not Endothelium-Derived Hyperpolarizing Factor-Dependent Dilation in Rabbit Resistance Arteries Circ. Res., October 12, 2001; 89(8): 716 - 722. [Abstract] [Full Text] [PDF]
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