© 2000 American Heart Association, Inc.
Vascular Biology |
Thiol Oxidation Activates a Novel Redox-Regulated Coronary Vasodilator Mechanism Involving Inhibition of Ca2+ Influx
Presented in part at the Experimental Biology ’99 Meeting, Washington, DC, April 17–21, 1999, and published in abstract form (FASEB J. 1999;13:A198) and at the 72nd Scientific Sessions of the American Heart Association, Atlanta, Ga, November 7–10, 1999, and published in abstract form (Circulation. 1999;100[suppl I]:I-418).
From the Department of Physiology, New York Medical College, Valhalla, NY.
Correspondence to Michael S. Wolin, PhD, Department of Physiology, New York Medical College, Valhalla, NY 10595. E-mail mike_wolin{at}nymc.edu
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Abstract |
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Abstract—This study examines the mechanism of relaxation of isolated endothelium-removed bovine coronary arteries (BCAs) to the thiol oxidant diamide. BCAs precontracted with KCl or the thromboxane A2 receptor agonist U46619 showed a concentration-dependent reversible relaxation on exposure to 10 µmol/L to 1 mmol/L diamide. This relaxation was enhanced by an inhibitor of glutathione reductase, and it was not altered by severe hypoxia, the presence of inhibitors of soluble guanylate cyclase, K+ channels, tyrosine kinases, or probes that modulate levels of superoxide. The relaxation was almost eliminated when BCAs were precontracted with a phorbol ester that causes a contraction that is largely independent of extracellular Ca2+. The initial transient contraction elicited by 5-hydroxytryptamine in Ca2+-free solution was not altered by the presence of 1 mmol/L diamide; however, a subsequent tonic contraction on addition of CaCl2 was inhibited by diamide. Diamide also inhibited contractions caused by the addition of CaCl2 to Ca2+-free Krebs’ buffer containing Bay K8644 (an L-type Ca2+ channel opener) or KCl. Relaxation to diamide was attenuated by L-type Ca2+ channel blockers (nifedipine and diltiazem). Thus, thiol oxidation elicited by diamide appears to activate a novel redox-regulated vasodilator mechanism that seems to inhibit extracellular Ca2+ influx.
Key Words: calcium • diamide • redox signaling • thiol redox • vasodilation
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Introduction |
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Intracellular thiols are known to be a major cellular defense mechanism for oxidant stress, and changes in cellular redox status have been proposed to play a role in vascular tone responses. There is evidence that alterations in the thiol redox of key sites on regulatory proteins is a potential mechanism of controlling the function of these systems in a signaling-like manner.1 The thiol oxidant diamide is known to promote the reversible oxidation of glutathione (GSH) and adjacent protein thiols (RSH) to their disulfide forms [GSSG and R(S-S)] and to promote the S-thiolation (eg, RSSG) of reactive protein thiols.2 Diamide was initially observed by Weir et al3 to cause vasodilation in the precontracted pulmonary circulation. This observation evolved into studies showing that oxidants opened plasma membrane K+ channels in pulmonary arterial smooth muscle through a mechanism potentially mediated through thiol oxidation.4 These studies also contributed to the development of a hypothesis for the involvement of thiol redox regulated K+ channels in the process in pulmonary hypoxic vasoconstriction.4 5 There is also substantial evidence that soluble guanylate cyclase (sGC) is an additional vasodilator mechanism that could be controlled by interactions between oxidants and thiol redox.6 7 8 The oxidation of GSH is also likely to increase the levels of intracellular peroxides and reactive hydroxyl radical–like species derived from peroxides in a manner that could enhance the magnitude of the signaling actions of these species. Multiple additional cellular control systems, such as tyrosine phosphorylation–initiated activation of the G protein p21ras, phospholipases A2, C, and D, protein kinase C, and mitogen-activated protein kinases,9 10 11 key enzymes in energy metabolism,12 and systems that control the storage and release of Ca2+13 14 could be regulated by processes linked to oxidants and thiol redox. Thus, thiol redox could control multiple systems that are involved in the regulation of vascular force.
In the present study, we examined the mechanism through which diamide causes relaxation of isolated endothelium-removed bovine coronary arteries (BCAs). Endothelium was removed to avoid the complicating effects of redox changes on endothelial mediator release. The present study began with investigating the role of stimulation of increases in sGC activity, K+ channel opening, and reactive O2 species in the mechanism of relaxation of BCAs elicited by diamide because of the existing evidence1 that these processes could potentially control vascular contractile function through a regulatory mechanism normally modulated by changes in thiol redox. However, additional regulatory mechanisms potentially influenced by thiol redox had to be explored when the initial studies did not detect a role for these mechanisms in the relaxant actions of diamide. The subsequent detection of evidence of a role of inhibiting Ca2+ influx in the relaxation to diamide resulted in a characterization of the properties of the processes that were involved.
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Methods |
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Materials
Many of the reagents used in the present study were obtained from sources previously described.15 16 17 18 19 Diamide, dithiothreitol (DTT), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), tetraethylammonium chloride (TEA), charybdotoxin (CTX), glibenclamide, 4-aminopyridine, genistein, phorbol 12,13-dibutyrate (PDBu), Bay K8644, EGTA, nifedipine, and diltiazem were from Sigma Chemical Co. LY83583 was from Cayman Chemicals.
Measurement of Changes in Force in BCAs
Isolated endothelium-removed left circumflex
coronary arterial rings were prepared from
slaughterhouse-derived bovine hearts and studied for changes in
isometric force, as previously described.16 17 18 The rings
were incubated in individually thermostated (37°C) 10-mL baths (Metro
Scientific) for 2 hours at an optimal passive tension of 5 g in
Krebs’ bicarbonate buffer (pH 7.4) containing the following (in
mmol/L): NaCl 118, KCl 4.7, CaCl2 1.5,
NaHCO3 25, MgSO4 1.1,
KH2PO4 1.2, and glucose
5.6, gassed with 21% O2/5%
CO2 (balance N2). After a
2-hour equilibration and a brief depolarization with 123 mmol/L
KCl, the BCAs were then allowed to reequilibrate for 30 minutes before
the experiments were conducted. After a 30-minute equilibration period,
the experiments described in Results were conducted. The doses of
contractile agents were selected to generate 
70% of maximal force
(typically 14 g of force). In studies examining the actions of
mechanistic inhibitors on the response to diamide, probes
were usually added together with contractile agents, and they were
incubated with BCAs for at least 15 minutes before exposure to
increasing cumulative concentrations of diamide or other relaxing
agents. BCNU,
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ),
LY83583, glibenclamide, genistein, and nifedipine were
dissolved in dimethyl sulfoxide, and the 0.05% to 0.1% concentrations
used did not alter the vascular responses. None of the probes or
treatments, except those mentioned in Results, had a statistically
significant effect on force generation.
Statistical Analysis
Results are expressed as mean±SE, with n equal to the number of
animals used. Comparisons between groups were made by ANOVA and Student
t test with a Bonferroni correction for multiple
comparisons. A value of P<0.05 was used to determine
statistical significance.
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Results |
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Diamide Causes a Reversible Relaxation of BCAs
Figure 1A
contains a typical
experiment using BCAs initially precontracted with the
thromboxane A2 receptor agonist
U46619 (0.1 µmol/L) before exposure to the thiol oxidant
diamide. As shown in Figure 1A (top), a dose of diamide
(0.3 mmol/L) that oxidizes thiols in
cardiovascular preparations20 21 22 caused
BCAs to relax (74±8% relaxation, n=4) in a manner that was reversed
(106±3% recovery) by the thiol reductant DTT (1 mmol/L). DTT did
not reverse the relaxation (90±4%, n=6) elicited by stimulation of
adenylate cyclase by 1 µmol/L forskolin (-2±4%
recovery, Figure 1A, middle). Washing out of the diamide with
37°C Krebs’ buffer containing U46619 also caused a rapid reversal
(107±2% recovery, n=8) of the relaxation (64±6% relaxation) to
diamide (See Figure 1A, bottom).
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An Inhibitor of GSH Reductase Enhances BCA Relaxation
to Diamide
Inhibition of GSH reductase23 by a 30-minute
pretreatment with 0.1 mmol/L BCNU followed by washout was used to
examine whether preventing the reduction of oxidized GSH enhanced the
response to the thiol oxidant diamide. The conditions used for the BCNU
treatment were optimized in preliminary experiments to minimize its
previously reported inhibitory effects on force generation
and relaxation to NO.22 Although BCNU pretreatment did not
significantly depress the contraction caused by the subsequent addition
of 0.1 µmol/L U46619, the dose of U46619 was increased to
0.3 µmol/L in BCNU-pretreated vessels, which showed a slow
development of force. Under these conditions, as shown in Figure 1B
, BCNU pretreatment enhanced relaxation to increasing
cumulative concentrations of diamide. Under similar conditions, BCNU
pretreatment did not significantly alter the relaxation responses to
increasing cumulative concentrations of the NO donor
S-nitroso-N-acetyl-penicillamine (SNAP; see panel
C of Figure I, which can be accessed online at
http://atvb.ahajournals.org) or forskolin, a stimulator of cAMP
production (see panel D of online Figure I).
Effects of Inhibitors of sGC on BCA Relaxation to
Diamide
Pretreatment with inhibitors of the activation of sGC,
including 10 µmol/L ODQ, methylene blue, and LY83583, was used
to examine their potential effects on BCA relaxation to diamide. BCAs
were initially precontracted with either 0.1 µmol/L U46619 or
30 mmol/L KCl in the absence or presence of the
inhibitors of sGC activation, and then the relaxant effects
of diamide (10 µmol/L to 1 mmol/L) were examined. As shown
in Figure 2A, none of these
inhibitors altered relaxation to diamide in BCAs
precontracted with either U46619 or KCl. The data in Figure 2A
also indicate that relaxation responses to each dose of diamide were
markedly reduced (P <0.05) in BCAs precontracted with
30 mmol/L KCl compared with BCAs precontracted with 0.1
µmol/L U46619.
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Effects of K+ Channel Blockers on BCA Relaxation
to Diamide
K+ channel blockers for channels including
the Ca2+-dependent K+
channels (10 mmol/L TEA and 50 nmol/L CTX), ATP-dependent
K+ channels (10 µmol/L glibenclamide), and
voltage-gated K+ channels (1 mmol/L
4-aminopyridine)24 were used to examine
their potential effects on BCA relaxation to diamide. BCAs were
initially precontracted with 0.1 µmol/L U46619 in the absence or
presence of the K+ channel blockers, and then the
relaxant effects of diamide (10 µmol/L to 1 mmol/L) were
examined. As shown in Figure 2B, pretreatment with these
blockers did not alter the BCA relaxation to diamide in
U46619-precontracted BCAs. The combined presence of CTX and
glibenclamide also did not alter the relaxation to diamide (n=4, not
shown).
Effects of Probes for O2 Metabolism on BCA
Relaxation to Diamide
To examine whether O2 tension affects
diamide-elicited relaxation, experiments were performed in either
normoxic or severely hypoxic conditions. BCAs were initially
precontracted with either 0.1 µmol/L U46619 or 30 mmol/L
KCl in the absence or presence of a severely hypoxic atmosphere (95%
N2/5% CO2,
PO2 8 to 10 mm Hg). Hypoxic conditions did
not affect the relaxation to diamide in BCAs precontracted with either
U46619 or KCl (see Figure 3A). The
effects of altering BCA levels of superoxide on the response to diamide
were examined to determine whether the levels of this species
influenced relaxation to this agent. Superoxide levels were increased
by inhibition of CuZn superoxide dismutase either by using a 30-minute
pretreatment with 10 mmol/L diethyldithiocarbamic acid (DETCA),
followed by washout before contraction,15 or by
contracting BCAs with either 0.1 µmol/L U46619 or 30 mmol/L
KCl in the presence of 1 mmol/L NaCN.25 Superoxide
levels were lowered by contracting BCAs with either 0.1 µmol/L
U46619 or 30 mmol/L KCl in the presence of an intracellular
scavenger of superoxide that promotes
H2O2 formation (10
mmol/L 4,5-dihydroxy-1,3-benzenedisulfonic acid
[Tiron]).16 As shown by the data in Figure 3B, these methods of altering BCA levels of superoxide did not alter the
relaxation elicited by subsequent exposure of the BCAs to increasing
cumulative concentrations of diamide (10 µmol/L to 1
mmol/L) in BCAs precontracted with either U46619 or KCl. None of the
probes examined had a significant effect on contraction to U46619,
whereas N2 and DETCA decreased (P
<0.05) contraction to KCl by 26% and 35%, respectively.
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Effects of Inhibition of Tyrosine Kinases on BCA Relaxation to
Diamide
The effects of inhibiting tyrosine kinase activity in BCAs with
10 µmol/L genistein on the relaxation to diamide was examined to
determine whether any of the thiol redox–sensitive processes that
influence signaling through tyrosine phosphorylation
participate in this response. BCAs were initially precontracted with
either 0.1 µmol/L U46619 or 30 mmol/L KCl in the absence or
presence of genistein, and then the relaxant effects of diamide
(10 µmol/L to 1 mmol/L) were examined. This agent did not
significantly alter the response to diamide in BCAs precontracted with
either U46619 or KCl (panel C of Figure II, which can be accessed
online at http://atvb.ahajournals.org).
Effects of Different Types of Contractile Stimuli on BCA Relaxation
to Diamide
To examine whether the sensitivity of BCA relaxation to diamide is
dependent on the signaling mechanisms involved in the stimuli used for
contraction, vessels were precontracted to similar levels of force with
either 0.1 µmol/L U46619, 1 µmol/L Bay K8644 (L-type
Ca2+ channel opener), or 10 µmol/L PDBu
(protein kinase C activator), and then the concentration
dependence of the diamide-elicited relaxation was compared. Bay K8644
was applied in the presence of 14.7 mmol/L KCl (a concentration of
KCl that did not affect vascular tone), because Bay K8644 requires a
subthreshold depolarization to cause contraction.26 As
shown in Figure 4A, diamide-elicited
relaxation was almost eliminated when vessels were precontracted with
PDBu. Although there was a right shift in the concentration-response
curve when vessels were precontracted with Bay K8644 compared with
U46619, 1 mmol/L diamide caused almost full relaxation of BCAs
precontracted with either agent. To elucidate whether PDBu-elicited
contraction is modulated by the presence of extracellular
Ca2+, vessels were contracted in
Ca2+ -free solution containing 1 mmol/L
EGTA, and then the maximal stable contraction was compared with that in
normal Krebs’ solution. PDBu (10 µmol/L) caused substantial
contraction even in Ca2+-free solution, and this
contraction was only 29% (n=15) less than that in
Ca2+-containing solution, indicating that
PDBu-elicited contraction is largely independent of extracellular
Ca2+.
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To assess whether other relaxants cause vascular relaxation when
vessels are precontracted with PDBu, the NO donor SNAP or the cAMP
stimulant forskolin were applied to BCAs precontracted with U46619, Bay
K8644, or PDBu. Although each contractile agent showed different
sensitivity to vasorelaxant agents, SNAP (Figure 4B) and
forskolin (see panel C of Figure III, which can be accessed online at
http://atvb.ahajournals.org) caused substantial relaxation of
PDBu-precontracted BCAs.
Effects of Diamide on Contraction to Different Types of Stimuli
Elicited in Ca2+-Free Solution Followed by Addition of
CaCl2
In this series of experiments, vessels were first contracted with
30 mmol/L KCl in normal Krebs’ solution, followed by washout, and
subsequent contractile responses were expressed as a percentage of the
initial response to 30 mmol/L KCl. BCAs were then placed in
Ca2+-free Krebs’ solution containing 0.1
mmol/L EGTA, and then the contractile effects of 10 µmol/L
5-hydroxytryptamine (5-HT), 30 mmol/L KCl, or
1 µmol/L Bay K8644 (14.7 mmol/L KCl) were examined in the
absence or presence of 1 mmol/L diamide, followed by addition of
1.5 mmol/L CaCl2 to detect contractile
responses originating from the initial release of intracellular
Ca2+ and the subsequent influx of extracellular
Ca2+, respectively. Diamide was added 10 minutes
before the application of contractile agents, and the initial response
was observed for 5 to 10 minutes before the addition of
CaCl2.
5-Hydroxytryptamine
Figure 5A (top) shows a typical BCA
response to 10 µmol/L 5-HT in Ca2+-free
solution and the subsequent contraction elicited by the addition of
1.5 mmol/L CaCl2 either in the absence or
presence of 1 mmol/L diamide. In the absence of diamide, 5-HT
caused rapid transient contraction, followed by sustained tonic
contraction on addition of CaCl2. In the presence
of 1 mmol/L diamide, the initial transient contraction was not
significantly affected, but subsequent contraction elicited by
CaCl2 was markedly inhibited. These responses are
summarized in Figure 5B. In some separate experiments in which
5-HT was washed out after the initial transient contraction, no
apparent contraction was observed after adding
CaCl2, indicating that capacitative
Ca2+ entry27 had a minimum role, if
any, in the contraction elicited by 5-HT under these experimental
conditions (n=4, not shown).
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Potassium Chloride
Figure 5A (middle) shows a typical BCA response to 30
mmol/L KCl in Ca2+-free solution and the
subsequent contraction elicited by the addition of 1.5 mmol/L
CaCl2 in the absence or presence of 1 mmol/L
diamide. No significant contraction was observed in
Ca2+-free solution in the absence or presence of
1 mmol/L diamide. Subsequent addition of 1.5 mmol/L
CaCl2 caused a sustained contraction in the
absence of diamide; however, this sustained contraction was markedly
inhibited by the presence of 1 mmol/L diamide. These responses are
summarized in Figure 5B. In some separate experiments in which
KCl was washed out after 5 minutes, no apparent contraction was
observed after adding CaCl2, indicating that
capacitative Ca2+ entry had a minimum role, if
any, in the contraction elicited by KCl under these experimental
conditions (n=4, not shown).
Bay K8644
Figure 5A (bottom) shows a typical BCA response to an
L-type Ca2+ channel opener, 1 µmol/L Bay
K8644, in Ca2+-free solution and the subsequent
contraction elicited by the addition of 1.5 mmol/L
CaCl2 in the absence or presence of 1 mmol/L
diamide. Similar to the responses to 30 mmol/L KCl, Bay K8644 did
not cause any significant contraction in
Ca2+-free solution, and the subsequent addition
of 1.5 mmol/L CaCl2 caused a sustained
contraction in the absence of diamide; this sustained contraction was
markedly inhibited by the presence of 1 mmol/L diamide. These
responses are summarized in Figure 5B.
Effects of L-Type Ca2+ Channel Blockers
on BCA Relaxation to Diamide
The effects of L-type Ca2+ channel blockers
nifedipine and diltiazem on the response to diamide were
examined to determine whether these channels contributed to its
relaxant actions. In initial experiments, these agents were observed to
cause a similar degree of inhibition of contraction to 0.1
µmol/L U46619 or 30 mmol/L KCl at each dose examined. The
inhibition of contraction of 53.5±2.0% by 1 µmol/L
nifedipine (n=4) and 72.0±4.2% by 10 µmol/L
diltiazem (n=4) could be reversed by increasing the concentration of
U46619 from 0.1 to 0.3 to 1.0 µmol/L. In contrast, it was
difficult to obtain similar levels of force in the absence or presence
of nifedipine or diltiazem through increasing the
concentration of KCl. As shown in Figure 6A, 1 µmol/L
nifedipine and 10 µmol/L diltiazem inhibited the
relaxation to increasing cumulative concentrations of diamide. Under
similar conditions, the presence of nifedipine or diltiazem
had minimal effects on relaxation responses to increasing cumulative
concentrations of SNAP (Figure 6B) or forskolin (see panel C of
Figure IV, which can be accessed online at
http://atvb.ahajournals.org).
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Discussion |
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In the present study, the thiol oxidant diamide was observed to cause a reversible concentration-dependent relaxation of endothelium-removed BCAs, which appeared to be mediated through alterations in a thiol redox process, inasmuch as the response was enhanced by an inhibitor of GSH reductase, and it was reversed by the thiol reductant DTT or by removal of the diamide. Previous studies on a variety of cardiovascular tissue and cellular preparations (eg, see References 20 ) have demonstrated that the concentrations of diamide observed to cause coronary arterial relaxation in the present study consistently cause severe degrees of reversible GSH oxidation and S-thiolation of proteins. The actions of probes used in the present study suggest that the relaxing mechanism activated by diamide does not seem to involve the stimulation of sGC or tyrosine kinases, the opening of K+ channels, or a process involving O2 metabolism or the modulation of reactive O2 species. Under the conditions of the present study, diamide appears to function primarily through inhibiting a mechanism of Ca2+ influx, and evidence of its promotion of relaxation through influencing the signaling mechanisms associated with the 5-HT receptor–mediated release of Ca2+ from intracellular organelles or contractile mechanisms regulated through the stimulation of protein kinase C could not be detected.
Thiol redox has multiple ways of potentially interacting with systems that regulate the activity of sGC. Because the metabolism of H2O2 by GSH peroxidase appears to function as a modulator of the stimulation of sGC by peroxide metabolism by catalase in BCAs,17 diamide could cause relaxation by increasing the intracellular levels of H2O2 and its metabolism by catalase. However, this mechanism does not appear to mediate the relaxation to diamide, inasmuch as it has been shown in the present study that inhibitors of sGC stimulation do not alter relaxation to this agent. In a previous study, it has also been observed that the inactivation of catalase does not alter the relaxation of endothelium-removed bovine coronary arteries to diamide.17 In addition, the catalytic activity of sGC appears to be inactivated by disulfide formation or S-thiolation through reactions with disulfides, including the oxidized form of GSH,6 and this process may function as an endogenous mechanism of inhibiting the vascular form of sGC.7 Thus, the stimulation of sGC does not appear to be involved in the relaxation to diamide.
There is substantial evidence that many of the different forms of K+ channels are potentially regulated by thiol redox processes.4 28 In the present study, it was observed that agents that inhibit most of the known forms of K+ channels did not alter the response to diamide. Because hypoxia did not alter relaxation to diamide, it appears that processes associated with the PO2-dependent regulation of K+ channels4 5 did not contribute to preventing the detection of a role for K+ channels in the actions of this thiol oxidant. Thus, a relaxing mechanism activated by hyperpolarization as a result of the opening of plasma membrane K+ channels does not seem to participate in the mechanism of relaxation to diamide.
The absence of effects of other probes used in the present study that altered O2 metabolism on relaxation to diamide were consistent with the absence of a role for several additional mechanisms. Because modulating superoxide levels, NaCN, and severe hypoxia did not alter the response to diamide, it appears that relaxation to this agent does not involve alterations in a signaling mechanism dependent on the formation of reactive O2 species (eg, H2O2) or other processes controlled by PO2, such as energy metabolism. Because superoxide-derived oxidants and thiol redox signaling appear to activate multiple genistein-inhibitable tyrosine kinase–dependent signaling mechanisms, including processes dependent on activation of p21ras, mitogen-activated protein kinases, and phospholipases,9 10 11 these processes may also not be involved in relaxation to diamide.
Various approaches were investigated to probe the role of systems that control the levels of intracellular Ca2+ in BCAs because systems such as sarcoplasmic reticulum Ca2+ uptake is thought to be regulated by oxidant mechanisms through changes in thiol redox.13 14 The absence of a relaxation to diamide in BCAs contracted with PDBu, an activator of protein kinase C, suggested that a key aspect of the mechanism of action of diamide could involve either a process that is inhibited by protein kinase C or a system that controls the levels of intracellular Ca2+. This latter hypothesis is based on the observation that contraction to PDBu is largely independent of the presence of extracellular Ca2+ under the conditions examined in the present study. It is important to note that PDBu does not prevent BCA relaxation in a nonspecific manner, because SNAP and forskolin caused substantial relaxation in BCAs contracted with PDBu. Because the initial contraction to 5-HT in the absence of extracellular Ca2+ was not altered by diamide, this thiol oxidant does not appear to be influencing the processes involved in the release of sarcoplasmic reticulum Ca2+ by the 5-HT receptor–regulated mechanism. The absence of an inhibition of force generation by the protein kinase C activator PDBu or the initial contraction to 5-HT in the absence of Ca2+ is also consistent with diamide not directly altering the function of contractile proteins involved in the generation of force under the conditions of these experiments. However, further increases in thiol oxidation could influence these thiol-dependent signaling systems. Observation of a marked inhibition of contraction caused by the readdition of Ca2+ in the presence of 5-HT or agents that activate voltage-dependent Ca2+ channels implicates a role for inhibition of sarcolemmal Ca2+ influx in the mechanism of relaxation to diamide.
The L-type Ca2+ channel is one of the best identified Ca2+ channels in smooth muscle, and this channel may be the primary channel that mediates Ca2+ influx associated with receptor and voltage-dependent contraction in smooth muscle.27 Because L-type Ca2+ channel blockers selectively attenuated the response to diamide, these channels appear to contribute to its mechanism of relaxation. The observation that L-type Ca2+ channel blockers had a similar potency in relaxing BCAs contracted with KCl and U46619, compared with the markedly reduced potency of diamide on arteries contracted with KCl, suggests that diamide may also be simultaneously activating an additional mechanism that could potentially function through enhancing the contractile actions of KCl. Interestingly, studies on the cloned smooth muscle L-type Ca2+ channel have identified a key role for a thiol redox process in controlling its activity, in which oxidation closes the channel.29 Although diamide could be functioning in a manner similar to that of L-type Ca2+ channel blockers, the observation that relaxation to diamide is enhanced in the presence of inhibition of GSH reductase is consistent with a mechanism controlled by intracellular thiol redox. Thus, diamide appears to function through activating a thiol oxidation mechanism that inhibits L-type Ca2+ channels.
The present study has identified a potentially novel vasodilator mechanism in BCAs that appears to be mediated through closure of a plasma membrane channel that seems to be the L-type Ca2+ channel by a thiol oxidation mechanism. Because removal of the diamide caused a rapid reversal of this relaxation, enzymes that normally control cellular thiol redox processes, including GSH and thioredoxin reductases,12 are likely to participate in reversal of the alterations caused by diamide. Although it is possible that some of the other thiol redox–related signaling mechanisms examined in the present study are altered by diamide, it appears that closure of a plasma membrane Ca2+ channel is the dominant relaxing mechanism activated in BCAs under the conditions examined. The coronary vasodilator mechanism investigated in the present study may be of importance in situations associated with thiol oxidation caused by oxidant and/or nitrosative stress, such as cardiac ischemia/reperfusion and inflammation.
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Acknowledgments |
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This work was supported by US Public Health Service Grants HL-31069 and HL-43023. T. Iesaki was partly supported by a Research Fellowship from the Uehara Memorial Foundation.
Received June 29, 2000; accepted July 21, 2000.
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