© 1999 American Heart Association, Inc.
Original Contribution |
Alteration of Endothelium-Dependent Hyperpolarizations in Porcine Coronary Arteries With Regenerated Endothelium
From the Institut de Recherches Servier, Suresnes, France.
Correspondence to Catherine Thollon, PhD, Institut de Recherches Servier, Division pathologies cardiaques et vasculaires, 11 rue des Moulineaux, 92150 Suresnes, France.
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Abstract |
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Abstract—The present study was designed to test the ability of regenerated endothelium to evoke endothelium-dependent hyperpolarizations. Hyperpolarizations induced by serotonin and bradykinin were compared in isolated porcine coronary arteries with native or regenerated endothelium, 4 weeks after balloon endothelial denudation. The experiments were performed in the presence of inhibitors of nitric oxide synthase (N
-nitro-L-arginine) and
cyclooxygenase (indomethacin). The
transmembrane potential was measured using conventional glass
microelectrodes. Smooth muscle cells from coronary arteries
with regenerated endothelium were depolarized in
comparison with control coronary arteries from the same hearts.
Spontaneous membrane potential oscillations of small
amplitude or spikes were observed in some of these arteries but never
in arteries with native endothelium. In
coronary arteries from control pigs, both serotonin
and bradykinin induced concentration-dependent
hyperpolarizations. In the presence of ketanserin,
10 µmol/L serotonin induced a transient
hyperpolarization in control coronary
arteries. Four weeks after balloon denudation, the response to
serotonin was normal in arteries with native
endothelium, but the
hyperpolarization was significantly lower in
coronary arteries with regenerated endothelium.
In control arteries, the endothelium-dependent
hyperpolarization obtained with bradykinin (30
nmol/L) was reproducible. Four weeks after balloon denudation,
comparable hyperpolarizations were obtained in
coronary arteries with native endothelium. By
contrast, in arteries with regenerated endothelium, the
hyperpolarization to bradykinin became
voltage-dependent. In the most depolarized cells, the
hyperpolarization to bradykinin was augmented. The
changes in resting membrane potential and the alteration in
endothelium-dependent
hyperpolarizations observed in the coronary
arteries with regenerated endothelium may contribute to
the reduced response to serotonin and the unchanged
relaxation to bradykinin described previously.
Key Words: regenerated endothelium • endothelium-derived hyperpolarizing factor • serotonin • bradykinin
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Introduction |
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By releasing endothelium-derived relaxing factors (EDRFs) such as nitric oxide (NO),1 prostacyclin,2 and endothelium-derived hyperpolarizing factor (EDHF),3 4 the endothelium plays a key role in modulating the responses of the underlying vascular smooth muscle cells.5 All the major cardiovascular risk factors have been associated with endothelial dysfunction, which precedes clinically apparent vascular disease and its complications.6 Hypertension,7 diabetes mellitus,8 hypercholesterolemia,9 and atherosclerosis10 are associated with decreased responses to endothelium-dependent vasodilators. Experiments in animal models suggest that the occurrence of abnormal endothelium-dependent relaxations is an early event in the development of vascular disease.11 12 13 Because the turnover of endothelial cells is accelerated under most of these conditions,14 the functional alterations in the regenerated endothelium could play an important role in the genesis of coronary disease. Previous studies13 demonstrated that, in porcine coronary arteries with regenerated endothelium after balloon denudation, endothelium-dependent relaxations are normalized 8 days after the procedure. However, 4 weeks after denudation, the relaxations to UK 14,304 (a selective
2-adrenergic agonist), serotonin
and aggregating platelets were reduced, whereas those induced by
ADP, bradykinin, and the calcium ionophore A23187 were maintained.
Because the decrease in endothelium-dependent
relaxations seems restricted to stimuli that activate
endothelial Gi-2 proteins,15 16 a
selective Gi protein dysfunction seems likely.17 18 19 This
selective endothelium impairment and the resulting
marked dysfunction in the NO-EDRF pathway does not result from a change
in the expression of Gi proteins but rather reflects their reduced
function in regenerated endothelial
cells.18 However, because EDHF contributes to the
endothelium-dependent relaxations to bradykinin and
A23187,20 an upregulation of the release of this factor
rather than a selective Gi protein dysfunction may explain the better
preservation of endothelium-dependent relaxations to
these agonists. The present experiments were designed to test the
ability of regenerated endothelium to evoke
hyperpolarizations of the underlying smooth muscle
of the porcine coronary artery. |
Materials and Methods |
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Animals
Sixty-three Large White pigs (male or female), 8 to 12 weeks of age, were used for the electrophysiological study. The animals were purchased from a commercial swine producer (Frenelles E.A.R.L., Boisemont, France) in accordance with the French Ministry of Agriculture. All experiments were performed in accordance with governmental and institutional guidelines for the use and care of animals.
Coronary Endothelium Denudation
Thirty-four pigs, 8 weeks old (mean weight 21±0.5 kg),
were anesthetized with an intramuscular injection of a mixture
composed of tiletamine plus zolazepam (20 mg/kg) and atropine sulfate
(50 µg/kg). Additional doses of anesthetic (sodium thiopental) were
given intravenously as necessary. Animals were intubated
with an endotracheal tube and mechanically ventilated with a
respirator. Heparin (250 IU/kg) and lysin acetylsalicylate (10 mg/kg)
were administrated intravenously to prevent thrombus
formation and limit inflammation, respectively. Using aseptic surgical
techniques, a percutaneous transluminal
coronary angioplasty (PTCA) guide catheter (Cordis, model AR1
7F) was introduced via the femoral artery into the left or right
coronary ostium, under fluoroscopic guidance (Radioselectan
contrast product, x-ray imager, model BV25, Philips). A PTCA
dilatation catheter (Cordis, balloon of 2 to 3 mm in diameter and
20 to 30 mm long) was then introduced into the chosen
coronary artery through the guide catheter. The diameter of the
balloon was adapted according to the size of the coronary
artery. The endothelium was removed by inflating the
balloon 3 times for 30 seconds. The pressure of inflation was adjusted
so that the blood vessel was not overstretched (from 2 atm at the
distal side to 8 atm at the proximal side). At the end of the surgical
procedure, Terramycin (20 mg/kg) was given intramuscularly as an
antibiotic prophylaxis. After recovery from anesthesia, the
animals were housed in individual cages for 4 weeks. At the time of
killing (12 weeks of age), the body weight of these animals was 32±0.7
kg. The control animals (nonoperated) were killed at 10 weeks of age
(n=29, body weight 25±0.8 kg).
Membrane Potential Recording
After anesthesia with tiletamine plus
zolazepam (20 mg/kg, IM), the heart was removed and rapidly placed in
an ice-cold oxygenated Krebs-Ringer solution. Then the left
anterior descending, left circumflex, and right coronary
arteries were dissected-free, cleaned of adherent fat and connective
tissue, and maintained in an oxygenated Krebs-Ringer
solution at room temperature. All procedures did not last more than 15
minutes. Rings of coronary arteries (approximately 4 mm
long) were cut open along the longitudinal axis and pinned on the
bottom of an experimental chamber (2.5 mL) covered with Sylgard, the
endothelial side upward. The strips were superfused
continuously at 5 mL/min with an oxygenated modified
Krebs-Ringer solution (95% O2/5%
CO2, 36.5±0.5°C, pH 7.4) of the following
composition (mmol/L): NaCl 118, KCl 4.7, MgSO4
1.2, KH2PO4 1.2,
CaCl2 2.5, NaHCO3 25, EDTA
0.026, and glucose 11. After a resting period of 30 to 45 minutes,
membrane potential was measured with conventional glass microelectrodes
(30 to 40 M
) filled with 3 mol/L KCl. The microelectrode was
connected to an amplifier (model V180, Biologic), and the membrane
potential was monitored simultaneously on a digital storage
oscilloscope (model 2211, Tektronix,) and a pen-chart recorder
(model 3400, Gould). Criteria for considering the impalement successful
were a sudden negative shift in potential at the cell penetration and a
rapid return to the previous potential at the withdrawal of the
microelectrode. A small potential deflection was often observed before
abrupt penetration of the cell and on dislodgement of the
microelectrode, probably as a result of crossing the
endothelial cells.
Protocol
In all experiments, the response of the coronary
arteries with regenerated endothelium was compared with
that of control coronary arteries from the same heart. Because
the same vascular preparation was never used for 2 consecutive
experiments, the number of experiments performed corresponded to the
number of vascular strips used. Furthermore, when unspecified, the
number of experiments (n) corresponded to the number of animals. All
experiments were performed in the presence of
indomethacin (10 µmol/L) to inhibit
cyclooxygenase and
N-nitro-L-arginine
(L-NA, 30 µmol/L) to inhibit NO synthase (NOS).
For the experiments with serotonin, ketanserin (10 µmol/L) was added to the physiological solution to inhibit the 5-hydroxytryptamine 2 (5-HT2) receptors present on the vascular smooth muscle of the porcine coronary artery.13 The membrane potential was measured at the end of the resting period (control values), after the incubation with different drugs and during the addition of agonists (serotonin or bradykinin).
Drugs
The following drugs were used:
indomethacin, L-NA, ketanserin, 5-HT
creatinine sulfate, and bradykinin (all from Sigma Chemical
Co). Indomethacin (10 mmol/L, in ethanol),
ketanserin (10 mmol/L, in H2O), and
bradykinin (1 mmol/L, in H2O) were prepared
as stock solutions (-20°C) and diluted in Krebs-Ringer solution to
reach final concentrations. L-NA and serotonin were
dissolved daily in H2O (10 mmol/L). Further
dilutions were performed directly in Krebs-Ringer solution.
Serotonin and bradykinin were added to the Krebs-Ringer
solution to reach final concentrations of 10 µmol/L and 30
nmol/L, respectively, immediately before the application to the
vascular preparation.
Statistical Analysis
Data are expressed as mean±SEM from n experiments. For
the experiments performed on coronary arteries from control
animals, 1-way ANOVA with repeated measures was first carried out and
followed by a Newman-Keuls test to analyze the effect of
treatments. For the comparison between control coronary
arteries from operated animals versus coronary arteries from
control pigs, 2-way ANOVA with repeated measures on 1 factor followed
by a Newman-Keuls test was performed. To compare the responses between
the coronary arteries with regenerated
endothelium to those with native
endothelium from the same animals, Student t
test for paired observations was used and followed with 2-way ANOVA
with repeated measures on 2 factors. Differences were considered to be
statistically significant at a value of P<0.05.
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Results |
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Resting Membrane Potential
In the control arteries taken from animals not subjected to a surgical procedure, the resting membrane potential of the coronary smooth muscle cells averaged -56.3±1.7 mV (n=15). Twenty-eight days after endothelium denudation, the resting membrane potential of smooth muscle cells from the coronary arteries with native endothelium was not significantly different from that observed in control animals (-57.1±0.7 mV, n=34). In coronary arteries with regenerated endothelium, before any pharmacological intervention, the smooth muscle cells were significantly less polarized than in the corresponding control arteries (-49.9±1.2 mV; P
0.01,
n=34). Under control conditions, in 4 strips with regenerated
endothelium, membrane potential
oscillations (amplitude 4.9±1.0 mV), with a spontaneous
frequency of 6.5±0.4/min, were recorded (Figure 1A
). All impaled cells from these 4
vascular preparations showed similar fluctuations in resting membrane
potential. Such oscillations were never observed in either
coronary arteries from control animals or coronary
arteries with native endothelium from pigs subjected to
angioplasty 4 weeks earlier. When the vascular strips were superfused
with the incubation solution, before the addition of agonists causing
hyperpolarization, a slow depolarization on the
first 20 minutes was observed, ie, approximating 5 mV under all
experimental conditions and all vascular tissues. Bradykinin or
serotonin was given when the resting membrane potential had
stabilized (Table). After the superfusion
with the solution containing pharmacological inhibitors,
the resting membrane potential was still significantly different
between smooth muscle cells from coronary arteries with
regenerated endothelium and native
endothelium (Table). Some coronary
arteries with regenerated endothelium (n=7) that had
stable resting membrane potential under control conditions showed
fluctuations of the membrane potential during incubation with the NOS
blocker L-NA. Under these conditions, the amplitude of the
oscillations was higher (6.4±1.8 mV, n=4) (Figure 1B) and could even generate spikes (Figure 1c), with an
averaged maximal amplitude of 18.8±5.7 (n=3). The upstroke of these
action potentials attained maximal levels, ranging between -15 and
-20 mV and never showed an overshoot. The frequency of spontaneous
fluctuations in membrane potential during exposure to L-NA was
identical as under control conditions (7.6±0.8/min).
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Hyperpolarization to Serotonin
In the presence of indomethacin (10
µmol/L), L-NA (30 µmol/L), and ketanserin (1 µmol/L),
serotonin induced endothelium-dependent
hyperpolarization in coronary strips from
control animals. The response to serotonin was
concentration-dependent (Figure 2) with
maximal changes in membrane potential of -3.0±1.1 mV (n=6),
-7.8±1.6 mV (n=7), and -13.2±1.9 mV (n=6), at 0.1, 1, and 10
µmol/L, respectively. These hyperpolarizations
were transient. Thus, at 10 µmol/L serotonin, the
hyperpolarization was maximal after 25.1±1.6
seconds. The membrane potential returned to control level within 1
minute (58.6±4.1 seconds, n=7).
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Four weeks after balloon endothelial denudation, the
hyperpolarization induced by serotonin
at 10 µmol/L in the presence of indomethacin
(10 µmol/L), L-NA (30 µmol/L), and ketanserin (10
µmol/L) was not significantly different from control in
coronary arteries with native endothelium:
-10.6±1.6 mV (n=8) versus -10.8±1.2 mV in coronary strips
(n=7) from unoperated animals. In arteries with regenerated
endothelium (Figure 3),
the response to serotonin was significantly less than the
corresponding control strips (-5.6±1.7 mV, Figure 5;
P0.05, n=8). Although the amplitude of the
hyperpolarization was not influenced by the
presence or absence of L-NA in control arteries, in arteries with
regenerated endothelium, studied in the absence of
L-NA, serotonin did not cause significant
hyperpolarizations but rather evoked a small
depolarization (Figure 3). Such depolarizations were not
obtained in the presence of inhibitors of NOS, even in
presence of spikes (Figure 3A) and also were never observed in
coronary segments with native endothelium.
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Hyperpolarization to Bradykinin
In control porcine coronary arteries, bradykinin evoked
concentration-dependent hyperpolarizations (Figure 4) resistant to
indomethacin (10 µmol/L) and L-NA (30
µmol/L). The threshold concentration to obtain
endothelium-dependent
hyperpolarization was 3 nmol/L. With higher
concentrations, the amplitude of membrane potential changes increased
and the time to peak of hyperpolarization decreased
(not shown). The maximal value of hyperpolarization
was obtained at 100 nmol/L. Because at the concentration of 30 nmol/L
the amplitude of hyperpolarization was
reproducible, albeit not maximal, this concentration was used for the
evaluation of the endothelium-dependent
hyperpolarization induced by the kinin in arteries
with regenerated endothelium.
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Four weeks after balloon endothelial denudation, the
response to bradykinin (30 nmol/L) was not altered in coronary
strips with native endothelium (Figure 5
): -20.9±0.7 mV (n=16) versus
-21.5±0.8 mV hyperpolarization in arteries from
control animals (n=8). The temporal changes in membrane potential were
not modified: the more hyperpolarized potential was reached after 
1
minute, and the hyperpolarization lasted longer
than 3 minutes. In the corresponding coronary segments with
regenerated endothelium, although the average change in
membrane potential was not different (-20.1±1.6 mV, n=16), the values
of membrane potential before administration of bradykinin and that
reached in its presence were significantly lower (Figure 5B).
When examining the individual experiments, a high variability in the
response to bradykinin was noticed in preparations with regenerated
endothelium, showing identical, reduced, or increased
hyperpolarizations (Figure 5A). The
analysis of individual pairs of results showed that although
similar responses were recorded in control arteries, the changes in
membrane potential for segments with regenerated
endothelium were related to the value of membrane
potential immediately before the administration of the kinin (Figure 6). In the more depolarized cells,
bradykinin evoked a larger hyperpolarization (up to
-40 mV) (Figure 5A, bottom panel, and Figure 6).
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Discussion |
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There are 2 main findings in the present study: (1) Four weeks after balloon endothelial denudation, the smooth muscle cells from porcine coronary arteries with regenerated endothelium showed intrinsic alterations of the resting membrane potential. (2) The changes in the endothelium-dependent hyperpolarizations of these cells may contribute to the reduced response to serotonin and the unchanged relaxation to bradykinin described previously.13,17,18
Resting Membrane Potential in Arteries With Regenerated
Endothelium
The present study demonstrates that the smooth muscle
cells from coronary arteries with regenerated
endothelium are depolarized in comparison to those from
the corresponding control arteries. The depolarization of these cells
imply alterations of ionic conductance(s) implicated in the control of
the resting membrane potential. This could be achieved by an increase
in a depolarizing current (inward calcium or nonspecific currents
and/or outward chloride current), a decrease in an outward repolarizing
or hyperpolarizing potassium current, or both. Such alterations could
include changes in the number of active channels, variations in their
open probability, or differences in their regulation. Obviously,
alterations in ionic homeostasis could also change the driving force
for ions and thus affect the ionic current. Whatever the cause of this
relative depolarization, the change in resting membrane potential in
these coronary arteries is relevant, because it could modify
the myogenic tone of these vessels. This interpretation is confirmed by
the observation of spontaneous electrical activities in some arteries
with regenerated endothelium. Such membrane potential
instability was not observed in the corresponding control arteries. The
resting membrane potential of arterial smooth muscle cells
is generally stable22 except in the
microvasculature23 ; in the cerebral microcirculation,
spontaneous contractions have been associated with an unstable membrane
potential of smooth muscle cells.24 The membrane
potential of smooth muscle cells in rhythmically constricting arteries
is also significantly less negative than that of quiescent
ones.25 In the porcine coronary artery, cell
membrane potential oscillations, spikes, and associated
spontaneous contractions have been observed, under marked
depolarization with tetrabutylammonium, a potassium channel
blocker.26 The membrane potential oscillations
obtained in the present study may be partly the result of the
depolarized state of the smooth muscle cells. Indeed, action potentials
can be generated only within a certain range of cell membrane potential
(-40 to -20 mV).24 Furthermore, rhythmic spontaneous
activity has been demonstrated in human coronary arteries and
with higher frequency in vessels from older patients or those with
cardiovascular diseases27 and
atherosclerotic changes.28 Hence, the depolarization of
smooth muscle cells, observed 4 weeks after balloon denudation, may be
a key factor in the development of alterations in vasomotion of these
coronary arteries.
Previous studies have shown, in the same porcine model, a decreased response to serotonin while the relaxation to bradykinin remained normal,13 17 18 despite the reduction in the EDRF-NO pathway. Therefore, the endothelium-dependent hyperpolarizations induced by both agonists were compared in porcine coronary arteries 4 weeks after denudation and in control arteries.
Endothelium-Dependent
Hyperpolarization Induced by Serotonin
and Bradykinin
In the coronary arteries from control pigs, both
serotonin and bradykinin induced concentration-dependent
hyperpolarizations that are resistant to
indomethacin and L-NA. The
hyperpolarizations observed in the present
study during exposure to bradykinin are in agreement with previous
observations.29 30 Under the experimental conditions
imposed, bradykinin given at 30 nmol/L as a single concentration
induced an endothelium-dependent
hyperpolarization of smooth muscle cells of -20
mV. This level of hyperpolarization was slightly
higher than that observed when concentrations of bradykinin were
applied in a cumulative manner.29 The temporal aspects of
the membrane hyperpolarization were also in
agreement with previous reports.30 31 32 On the contrary,
the endothelium-dependent
hyperpolarization induced by serotonin
was transient, and it was observed at higher concentrations. A similar
response to serotonin has been reported in the same
preparation.33 In preliminary studies, it was noted that
the response to serotonin is labile, in that many factors
can influence the amplitude of the
hyperpolarization: (1) rapidity of dissection of
coronary arteries after heart excision; (2) stretch of the
blood vessels during dissection; (3) a long period before the
beginning of the experiments (the response was markedly reduced after 8
hours); and (4) previous exposure to serotonin even
after a long washout (desensitization).
Although an EDRF has been demonstrated to be NO,34 the nature of EDHF is still unknown. It could be a cytochrome P450 metabolite, derived from arachidonic acid.35 36 37 38 When considering differences between hyperpolarizations induced by bradykinin and serotonin in the present study or those provoked by bradykinin and A23187 compared with that of thrombin,30 one cannot exclude the existence of several EDHFs. These hyperpolarizations, resistant to indomethacin and L-NA, differ by their amplitude and their kinetics. The fact that in the presence of regenerated endothelium, the transient response to serotonin was reduced while the sustained hyperpolarization induced by bradykinin could be increased would be also in favor of the existence of 2 different mechanisms. It is generally accepted that EDHF increases a potassium conductance leading to the hyperpolarization of the smooth muscle cells.20 Because all potassium channels implicated in the control of resting membrane potential of smooth muscle cells are voltage-dependent, the depolarization of smooth muscle cells in coronary arteries with regenerated endothelium would be expected to influence the response to EDHF. In the vascular strips with regenerated endothelium, the response to bradykinin was correlated with the value of membrane potential before the administration of the kinin. The maximal opening of potassium channels would lead the membrane to reach potential values close to the equilibrium potential for potassium. Therefore, in preparations with regenerated endothelium, the depolarization of smooth muscle cells induced an increase in the response to bradykinin, illustrating an augmentation of the EDHF pathway in the most altered cells. This result correlates well with previous experiments showing a maintained relaxation to bradykinin in coronary strips with regenerated endothelium,17 18 whereas it was blunted under depolarizing conditions.18 Therefore, the lack of impairment of the bradykinin-evoked relaxation, despite a reduction in the NO pathway, could be the result of a greater contribution of the EDHF pathway. Furthermore, although an altered relaxation to serotonin was described in the same experimental model, the present study demonstrates a reduction in the hyperpolarization induced by serotonin. The fact that relaxations to serotonin in porcine coronary arteries are almost completely inhibited by the presence of NOS inhibitors39 is in favor of a poor participation of the EDHF pathway to the response. This interpretation is supported by the weak hyperpolarizations obtained at high concentrations of serotonin in the present study. Because this small endothelium-dependent hyperpolarization induced by serotonin is decreased 4 weeks after balloon denudation, no compensatory effect of EDHF for the reduced response to this agonist is plausible. On the contrary, depolarizations were observed that were not blocked by the presence of a high concentration of ketanserin, excluding the implication of 5-HT2 receptor on the vascular smooth muscle. These depolarizations may have curtailed the endothelium-dependent hyperpolarizations. Shimokawa et al13 have previously shown an increase in the contraction to serotonin in the same model and have suggested that the regenerated endothelium releases endothelium-derived contracting factor in response to the monoamine.
The present findings are in favor of a minor implication of EDHF in the endothelium-dependent response of porcine coronary artery to serotonin, but the participation of EDHF in the relaxation response to bradykinin is more important. After balloon endothelial denudation, the endothelium-dependent hyperpolarization induced by serotonin is markedly reduced in the presence of regenerated endothelium, whereas that generated by bradykinin is maintained or even increased in coronary arteries showing an alteration of resting membrane potential. Because in coronary arteries with regenerated endothelium the EDRF-NO pathway is markedly reduced,17 18 the present results help to explain the better preservation of endothelium-dependent relaxations to bradykinin in this model.
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Acknowledgments |
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We thank C. Thomas-Haimez for expert statistical analysis.
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Footnotes |
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Previously presented in part in abstract form (J Vasc Res. 1997;34:40. Abstract 140).
Received September 24, 1998; accepted December 2, 1998.
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