Reduced Vasorelaxant Effect of Carbon Monoxide in Diabetes and the Underlying Mechanisms
Rui Wang,
Zunzhe Wang,
Lingyun Wu,
Salma Toma Hanna, and
Robert Peterson-Wakeman
From the Departments of Physiology (R.W., S.T.H., R.P.-W.) and Anatomy
and Cell Biology (L. W.), University of Saskatchewan, Saskatoon, Saskatchewan,
Canada; and the Laboratory of Cellular Morphology (Z. W.), Weifang Medical
College, Weifang, China.
Address correspondence and reprint requests to Dr. Rui Wang, Department of
Physiology, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, SK, Canada
S7N 5E5. Email:
wangrui{at}duke.usask.ca
.
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ABSTRACT
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Carbon monoxide (CO) is an endogenous gaseous factor that relaxes vascular
tissues by acting on both the cGMP pathway and calcium-activated K+
(KCa) channels. Whether the vascular effect of CO is altered in
diabetes had been unknown. It was found that the CO-induced relaxation of tail
artery tissues from streptozotocin-induced diabetic rats was significantly
decreased as compared with that of nondiabetic control rats. The blockade of
the cGMP pathway with ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one)
completely abolished the CO-induced relaxation of diabetic tissues but only
partially inhibited the CO effect in normal tissues. Single-channel
conductance of KCa channels in diabetic smooth muscle cells (SMCs)
was not different from that of normal SMCs. However, the sensitivity of
KCa channels to CO in diabetic SMCs was significantly reduced. CO
(10 µmol/l) induced an 81 ± 24% increase in the mean open
probability of single KCa channels in normal SMCs but had no effect
in diabetic SMCs. Longterm culture of normal vascular SMCs with 25 mmol/l
glucose or 25 mmol/l 3-OMG (3-O-methylglucose) but not 25 mmol/l
mannitol significantly reduced the sensitivity of KCa channels to
CO. On the Other hand, the sensitivity of KCa channels to CO was
regained in diabetic SMCs that were cultured with 5 mmol/l glucose for a
prolonged period. The decreased vasorelaxant effect of CO in diabetes
represents a novel mechanism for the vascular complications of diabetes, which
could be closely related to the glycation of KCa channels in
diabetic vascular SMCs.
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INTRODUCTION
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The vasorelaxant effects of carbon monoxide (CO) have been demonstrated
(1,2,3,4),
and heme oxygenase (HO) that cleaves the heme ring to form biliverdin and CO
has been located in many different types of vascular smooth muscles
(5,6).
The production of CO from vascular tissues has also been directly measured
(7). These studies emphasize
the importance of CO as an endogenous vasorelaxant factor under physiological
(8) or pathophysiological
(9,10)
conditions. The elevation of cellular cGMP levels and the opening of plasma
membrane K+ channels are the main mechanisms that have been
proposed to explain the vascular effects of CO. CO may increase cGMP content
via its stimulatory interaction with the heme in the regulatory subunit of
guanylyl cyclase. Increased cGMP would consequently decrease the intracellular
Ca2+ concentration ([Ca2+]i) in smooth muscle
cells (SMCs) through the inhibition of inositol triphosphate formation, the
activation of Ca2+-ATPase, and the inhibition of Ca2+
channels. The opening of K+ channels leads to membrane
hyperpolarization, which in turn inhibits the agonist-induced increase in
inositol triphosphate, reduces Ca2+ sensitivity and resting
Ca2+ level, and relaxes SMCs
(11). Our studies have
demonstrated that CO directly enhanced the activity of the big-conductance
calcium-activated K+ (KCa) channels in rat tail artery
SMCs via a cGMP-independent mechanism
(2,3).
Whether the vascular effects of CO are mediated by cGMP or specific types of
K+ channels or both depends on the vascular tissue types, the
developmental stages of the tissue, and the animal species.
Vascular complications of diabetes are largely manifested as hypertension
(12,13),
peripheral vessel occlusion, atherosclerosis, retinopathy, and nephropathy
(14). These vascular
complications are responsible for most of the morbidity and mortality of
patients with diabetes and have been reproduced in both insulinopenic
(15) and insulin-resistant
rats (16). Altered vascular
sensitivity to different vasoconstricting substances, such as bradykinin
(17), changed properties of
ion channels (18), and
abnormal calcium handling in diabetic vascular SMCs
(19) are among many putative
mechanisms for the vascular complications of diabetes. The involvement of CO
in the etiology of diabetes has been implicated as CO upregulated, whereas
nitric oxide downregulated insulin secretion from pancreatic islets
(20). The altered metabolism
of CO in cardiac tissues from streptozotocin-induced diabetic rats has also
been shown recently (21). It
was hypothesized that the vasorelaxing effects of CO may change in diabetes,
constituting a novel mechanism for the diabetic vascular complications.
In the present study, the vasorelaxant effect of CO on tail artery tissues
from streptozotocin-induced diabetic rats was examined and compared with that
from normal control rats. The relative contributions of cGMP pathway and
KCa channels to the putatively altered effect of CO on the
contractility of diabetic vascular tissues were investigated. The direct
effect of CO on single KCa channel activity was determined. The
role of glycation of KCa channels in the altered effect of CO was
further analyzed. Finally, the vascular responses to the endogenously
generated CO in diabetic rat tail artery tissues were assayed. Our results
provide evidence that corroborates the altered vascular effect of CO in
diabetes and unravels the underlying cellular mechanisms.
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RESEARCH DESIGN AND METHODS
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Animal model of diabetes. Adult male Sprague-Dawley rats weighing
150-180 g were maintained on standard rat diet and tap water and libitum with
12-h light:dark cycles in a quiet environment. After the rats were
anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg
body wt), diabetes was induced by a single injection via the lateral tail vein
or penis vein of streptozotocin (STZ) (60 mg/kg body wt) dissolved in sodium
citrate buffer (pH 4.5) (19).
Age-matched control rats were injected with an equal volume of vehicle (sodium
citrate buffer). The STZ-injected rats were used in the present study 1 month
after the induction of diabetes. Glycosuria was determined using Chemstrip
(Boehringer Mannheim). Mean blood pressure of the anesthetized rats (sodium
pentobarbital, 60 mg/kg i.p.) was determined through the right femoral artery
by a pressure transducer connected to a Biopac system (Biopac System), and
recorded by a computer. At the time of tail artery removal, 1.5-ml blood
samples were collected from the rats in the fasted state. The measurement of
blood levels of glucose and glycated hemoglobin was performed by Chemistry
Laboratory at the Royal University Hospital, University of Saskatchewan.
The laboratory animal care guidelines (National Institutes of Heath) were
followed, and animal experimental protocols were approved by the University
Committee on Animal Care and Supply of the University of Saskatchewan.
Measurement of isometric tension development of isolated rat tail artery
tissues. The method for measurement of isometric tension development of
isolated rat-tail artery tissues has been described previously
(4,17,22).
Briefly, tail arteries were isolated from rats. After cleaning connective
tissues, the arteries were cut into helical strips (
1.5 cm in length).
The vascular tissues were mounted in 10-ml organ baths filled with Krebs'
bicarbonate solution (bubbled with 95% O2 and 5% CO2),
which was composed of the following (in mmol/l): 115 NaCl, 5.4 KCl, 1.2
MgSO4, 1.2 NaH2PO4, 25 NaHCO3, 11
glucose, and 1.8 CaCl2. The pH and osmolality of Krebs' solution
were adjusted to 7.4 and 290 mOsm, respectively. The tail artery strip was
mounted with one end immobilized and another end tied to a transducer. These
strips were mechanically stretched to achieve a basal tension of
0.7 g
and were allowed to equilibrate for 1 h before the start of experiments.
Because the vascular effect of CO was not dependent on the presence of an
intact endothelium (4),
endothelium was removed from vascular strips by a rubbing procedure. The
absence of a functional endothelium was verified by the inability of
acetylcholine (1 µmol/l) to induce relaxation of tail artery tissues as
shown in our previous study
(4,17).
Concentration-response curves of the tissues to CO were obtained by
accumulative addition of CO to the organ bath.
The isometric tension development was measured with FT 03 force
displacement transducers (Grass Instruments). Data acquisition and analysis
were accomplished using a Biopac system (Biopac System), including MP 100 WS
acquisition units, TCI 100 amplifiers, Acknowledge software (3.01), universal
modules, and a Macintosh computer.
Cell preparation. Single SMCs were dispersed enzymatically following
our established procedure (23)
with modifications. Rats were anesthetized by intraperitoneal injection of
sodium pentobarbital (60 mg/kg body weight). Tail arteries were isolated and
connective tissue removed under a dissecting microscope. The arteries were cut
open longitudinally and immersed in a Ca2+- and
Mg2+-free Hanks' buffered saline solution (HBSS) (Gibco) at
4°C. The arterial strips were then processed in the following solutions at
37°C: 1) low-Ca2+ (0.2 mmol/l) HBSS containing
collagenase/dispase (1.5 mg/ml, Boehringer Mannheim), elastase (0.5 mg/ml,
type II, Sigma), trypsin inhibitor (1 mg/ml, Sigma) and bovine serum albumin
(BSA) (2 mg/ml, Sigma) for 50 min; 2) Ca2+-free HBSS in
which the tissue was rinsed twice; and 3) Ca2+-free HBSS
with collagenase 1 mg/ml (type II, Sigma) and BSA 2 mg/ml for 20 min. Next,
arterial pieces were transferred to a Ca2+-free HBSS at 4°C and
triturated for 5 min. Calcium concentration of the incubating solution was
gradually increased to 1.7 mmol/l. The dispersed cells were plated onto 35 mm
Petri dishes in Dulbecco's modified Eagle's medium (DMEM) (Gibco) containing
penicillin (100 U/ml, Sigma) and streptomycin (0.1 mg/ml, Sigma) and
maintained at 4°C for at least 4 h. These freshly isolated cells were used
in electrophysiological recording within 8-24 h of isolation.
In some experiments, the isolated cells were primarily cultured in DMEM
containing 5% fetal calf serum (FCS) (Gibco) at 37°C. Glucose,
3-O-methyl-glucose, or mannitol was added to the culture medium at
different concentrations, and the medium was changed every other day. The
cells were used for the patch-clamp study 8-35 days after in culture.
Single-channel recording. The inside-out and outside-out
configurations of the patch-clamp technique were used to record single
KCa channel currents as described previously
(2,3).
Pipettes with a resistance of 6-8 M
were used and the seal resistance
was usually >10 G
. Membrane patches with no more than 3 channels
were used for experiments. Single-channel currents were filtered at 2 KHz
(8-pole Bessel, -3 dB) and recorded with a 5 µs sampling interval in a
gap-free mode. For each concentration of CO tested, at least 60 s of channel
activity was recorded directly on a computer hard disk. The open probability
(NPo), with N representing the number of single channels in
one patch, and the unit amplitude of KCa channels were determined
from all-point histograms with a Fetchan program (Axon Instruments).
NPo of KCa channels was averaged over 2- to 5-min
recordings to describe the changes in channel activity after different
treatments. Membrane patches with unstable NPo over time were
excluded from further analysis. A current level >50% of the unit channel
current was considered to reflect a channel opening. The external surface of
membrane patches was bathed in a solution containing the following (in
mmol/l): 145 KCl, 10 HEPES, and 10 glucose. The internal surface of membrane
patches was exposed to a solution containing the following (in mmol/l): 145
KCl, 10 HEPES, 1.2 MgCl2, 10 glucose, 1 EGTA, and 0.5 µmol/l of
free Ca2+, [Ca2+]i.
[Ca2+]i of the recording solution was calculated using a
computer program (EQCAL, Biosoft), which takes account of the individual
impact of pH, EGTA, calcium, and magnesium on the final free-calcium
concentration.
Osmolalities and pH of all solutions used for electrophysiological
recordings were adjusted to 290 mOsm and 7.4, respectively. All
electrophysiological experiments were carried out at room temperature.
Total cell protein preparation and Western immunoblot assay. Total
cell proteins were prepared as described in our previous publication
(24). Briefly, rat-tail
arteries, aortae, mesenteric artery, and spleen were respectively homogenized
with a polytron homogenizer in 0.5 ml of Tris-buffered saline (20 mmol/l
Tris-HCl [pH 7.4], 0.25 mmol/l sucrose, and 1 mmol/l EDTA) containing protease
inhibitor mixture (2 µl of 1 mol/l phenanthroline, 300 mmol/l
iodoacetamide, 10 mmol/l phenylmethylsulfonyl fluoride, 1 mg/ml antipain, 1
mg/ml leupeptin, 1 mmol/l pepstatin A, and 1 mol/l benzamidine). The
homogenate was centrifuged at 6000g for 15 min at 4°C to remove
nuclei and undisrupted cells. Protein concentration was determined using a
Bio-Rad protein assay solution with BSA as standard. For Western blot, total
cell proteins were loaded and run on standard 7.5% SDS-polyacrylamide gel in
Trisglycine electrophoresis buffer (25 mmol/l Tris, 200 mmol/l glycine [pH
8.3], and 0.1% SDS). Proteins were transferred onto nitrocellulose membrane in
192 mmol/l glycine, 25 mmol/l Tris (pH 8.3), and 20% methanol at 100 V for 1.5
h in a water-cooled transfer apparatus. The membrane was blocked in a blocking
buffer, phosphate-buffered saline (PBS) containing 3% nonfat milk at room
temperature for 2 h. The membrane was then probed overnight at 4°C with
monoclonal antibodies against HO-1 (1:300) and HO-2 (1:100) (Stressgen) in the
blocking buffer. After washing five times in PBS, the membrane was
subsequently incubated with goat anti-mouse IgG or goat anti-rabbit IgG
(Bio-Rad) conjugated with horseradish peroxidase diluted 1:5000 in the
blocking buffer for 2 h at room temperature. Bound antibodies were detected
using chemiluminescent substrate kit (NEN Life Science Products).
Chemicals and data process. To prepare the CO solution, 20 ml of
stock solution in a sealed glass tube was bubbled with a stream of CO (purity
99.9999%, Canadian Liquid Air) for 20 min under the pressure of 100 kPa at
37°C. Of this CO-saturated solution, 1 µl contains 30 ng of the gas
(4). The stock solution of CO
was prepared freshly before each experiment and then diluted immediately to
the desired concentrations with bath solution. The estimated CO concentration
was based on the solubility of CO at 37°C, the extent of dilution of the
CO-saturated solution, and the assumption that the loss of the added CO from
the bath solution at the time of experiments was negligible. Because the
assumption was not strictly correct, the actual concentration of CO might be
somewhat lower than the estimated concentration. During the experiments, the
cells or vascular tissues were continuously superfused and a complete solution
change in the recording chamber was accomplished within 30 s. The effects of
CO on KCa channel currents were continuously recorded before and
after the beginning of superfusing cells with a CO-containing bath solution.
Usually, the stable effect of CO was observed within 1-3 min of CO application
and correspondingly recorded.
Phenylephrine (PHE), acetylcholine, indomethacin, STZ, 3-OMG
(3-O-methylglucose), and other chemicals were purchased from Sigma
Chemical (St. Louis, MO). Charybdotoxin (ChTX), iberiotoxin, and apamin were
from Alomone Labs (Jerusalem, Israel). ODQ
(1H-[1,2,4]oxadiazolo[4,3,-a]quinoxaline-1-one) was from Tocris (Ballwin,
MO).
The data were expressed as means ± SE. Concentration-response curves
were analyzed using a computerized curve-fitting software (Microcal Origin,
version 4.1, Microcal Software) to obtain EC50. The comparison of
EC50 under different conditions was performed by analysis of
variance followed by Student's t test in conjunction with the
Newman-Keuls test when applicable. The significant difference between
treatments was defined at a level of P < 0.05.
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RESULTS
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Severe diabetes developed in the STZ-treated diabetic rats. Compared with
normal control rats, diabetic rats lost body weight, had glycosuria, and
developed hyperglycemia with the fasting glucose concentration of plasma
elevated to 32.2 ± 2.2 mmol/l (n = 10). The mean blood
pressure and serum concentrations of K+ and Ca2+ were
not different between diabetic rats and the controls. However, serum
concentrations of Na+ and the appearance of blood cells and ketones
in urine were higher in diabetic rats than in control rats
(Table 1).
CO induced a concentration-dependent relaxation of the PHE-precontracted
endothelium-free tail artery tissues (Fig.
1). This vasorelaxant effect of CO was significantly reduced in
diabetic vascular tissues. EC50 of the vasorelaxant effect of CO
was 58 ± 24 µmol/l in normal tissues (n = 8) but 131
± 38 mmol/l in diabetic tissues (n = 8, P < 0.05)
(Fig. 1).

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FIG. 1. The CO-induced concentration-dependent relaxation of normal and diabetic
rat tail artery tissues. The vascular tissues were pre-contracted with
phenylephrine (1 µmol/l). The CO-induced vasorelaxation was significantly
reduced in diabetic vascular tissues. Each data point represents eight
experiments.
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Whether the decreased CO effect on diabetic vascular tissues was due to the
altered responsiveness of cGMP pathway or KCa channels in vascular
SMCs was further examined by incubating vascular tissues with 10 µmol/l
ODQ, a specific inhibitor for the soluble guanylyl cyclase. Without
pretreating vascular tissues with ODQ, CO (300 µmol/l) induced a 60
± 7% relaxation of normal vascular tissues (n = 8). In the
presence of ODQ, CO only induced a 38 ± 8% relaxation of normal
vascular tissues (n = 8). This represents a 63% inhibition of CO
effect by ODQ (Fig. 2). Further
prolonging the ODQ incubation time from 10 to 20 min (n = 4) or
increasing the concentration of ODQ from 10 to 30 µmol/l (n = 4)
did not induce additional inhibition of the CO effect (data not shown). In
contrast, 10-min incubation of diabetic rat tail artery tissues with 10
µmol/l ODQ completely abolished the vasorelaxant effect of CO
(Fig. 3).

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FIG. 2. The blockade of the cGMP-mediated signaling pathway reduced but not
completely abolished the CO-induced relaxation of normal rat vascular tissues.
The vascular tissues were precontracted with PHE (1 µmol/l). A:
Actual tension development trace from one normal tail artery tissue.
B: The CO-induced vasorelaxation was partially inhibited by ODQ in
normal vascular tissues. n = 8 for each group. *P
< 0.05 between the groups with or without CO treatment; P
< 0.05 between the CO-treated groups in the absence or presence of
ODQ.
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FIG. 3. The blockade of the cGMP-mediated signaling pathway completely abolished
the CO-induced vasorelaxation in the vascular tissues of 1-month diabetic
rats. The vascular tissues were precontracted with PHE (1 µmol/l).
A: Actual tension development trace from one diabetic rat tail artery
tissue. B: The CO-induced vasorelaxation was completely inhibited by
ODQ in diabetic vascular tissues. n = 8 for each group.
*P < 0.05 between the groups with or without CO
treatment. P < 0.05 between the CO-treated groups in the
absence or presence of ODQ.
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Because CO relaxes normal rat tail arteries by stimulating the cGMP
pathway, as well as high-conductance KCa channels
(1,2,3,4),
the altered functions of KCa channels in vascular SMCs would also
affect the vascular effects of CO. Therefore, the subsequent study focused on
the characteristics of KCa channels and their modulation by CO in
diabetic tail artery SMCs.
As demonstrated in our previous report, in normal rat tail artery SMCs, a
high-conductance KCa channel was also identified in diabetic rat
tail artery SMCs. The characteristics of this KCa channel were not
different among normal and diabetic rat tail artery SMCs
(Fig. 4). With symmetric KCl
(145 mmol/l) on both sides of the patch membrane, single-channel conductance
was linearly related to membrane potentials over the range of -100 to +60 mV
with no evidence of rectification. The single-channel conductances were 239
± 8 pS (n = 8) in normal rat tail artery SMCs and 230 ±
6 pS (n = 6) in diabetic SMCs (P > 0.05). The
NPo of KCa channels was decreased by ChTX (100 nmol/l) or
iberiotoxin (100 nmol/l), but not by apamin (100 nmol/l) in both normal and
diabetic tail artery SMCs (data not shown). Single-channel conductance of
KCa channels was not modified by CO in either normal rat tail
artery SMCs or diabetic SMCs. However, CO significantly increased the
NPo of KCa channels in normal tail artery SMCs in a
concentration-dependent manner (3-30 µmol/l) in both outside-out and
inside-out patches. This stimulatory effect of CO was greatly reduced in
diabetic artery SMCs (Fig. 5).
For instance, the mean NPo over 3 min of recording was increased by
CO (10 µmol/l) by 81 ± 24% in normal SMCs (n = 6,
P < 0.05). At the same concentration, CO had no effect on the mean
NPo of KCa channels in diabetic SMCs. When the
concentration of CO was increased to 30 µmol/l, the mean NPo of
single KCa channels over 3 min of recording was increased by 173
± 14% in normal SMCs (n = 5) but only by 48 ± 30% in
diabetic SMCs (n = 4, P < 0.05 vs. the effect of CO on
normal SMCs).

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FIG. 4. Characterization of high-conductance KCa channels in diabetic
tail artery SMCs. A: The actual single-channel current traces
recorded at different membrane potentials from one inside-out patch of
diabetic SMC. The dashed lines indicate the closed states. B: I-V
relationships of single KCa channels recorded from inside-out
patches of normal vascular SMCs or diabetic vascular SMCs. n = 5 for
each data point. The single-channel conductance was determined by least-square
linear regression analysis.
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FIG. 5. The concentration-dependent effect of CO on single KCa
channel currents in tail artery SMCs. A: The actual single-channel
current traces recorded at different membrane potentials from one outside-out
patch of a diabetic SMC. The dashed lines indicate the closed states. Membrane
potential, +50 mV. B: The effect of CO on the NPo of single
KCa channels recorded from outside-out patches of diabetic vascular
SMCs was significantly decreased compared with those in normal vascular SMCs.
*P < 0.05 before and after the application of CO in the
same patch. n = 4-8 for each data point.
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The decreased KCa channel sensitivity to CO might be explained
by the altered glycation status of KCa channel proteins or
hyperosmolality-induced structural and/or functional alterations in diabetic
SMCs. To test the influence of glycation on KCa channels, tail
artery SMCs were isolated and incubated in vitro under different conditions
(Fig. 6). After culturing
normal vascular SMCs for 8 days with 5 mmol/l glucose in the culture medium,
the single-channel conductance and the sensitivity of ChTX or iberiotoxin of
high-conductance KCa channels recorded in the cell-free patches
were similar to those of freshly isolated normal SMCs. CO (10 µmol/l)
increased the mean NPo over 3 min of the recording of the single
KCa channels by 95 ± 23%, taking the NPo level
before the application of CO as 100% (n = 4, P < 0.05).
After culturing SMCs isolated from diabetic rats for 8 days with 25 mmol/l
glucose, high-conductance KCa channels had similar characteristics
to those of freshly isolated diabetic SMCs. In these 8-day cultured diabetic
cells, KCa channels were also not sensitive to CO, with the 3-min
mean NPo showing no change after the application of CO (10 µmol/l)
(n = 4). In another set of experiments, normal SMCs were cultured
with 25 mmol/l glucose or 25 mmol/l 3-OMG for a period of 2-7 days. This
culture scheme yielded inconsistent results. KCa channels in some
cells were sensitive, but in others were insensitive to CO (data not shown).
Interestingly, after 8 days in culture the sensitivity of KCa
channels of normal SMCs to CO was consistently diminished. In these cells, CO
(10 µmol/l) had no effect on the 3-min mean NPo of single
KCa channels. In a similar previous study, a 10-day culture period
was applied for the study of the influence of high concentrations of glucose
or 3-OMG on the cellular functions of pericytes
(25). Also shown in
Fig. 6 is that culturing normal
SMCs for 8 days with 25 mmol/l mannitol did not alter the effect of CO on
KCa channels. A 73 ± 9% increase in the mean NPo of
KCa channels in these cells was observed in the presence of CO (10
µmol/l) (n = 4, P < 0.05).

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FIG. 6. The diminished effect of CO on high-conductance KCa channel
is due to the glycation of KCa channel proteins. Tail artery SMCs
from normal or diabetic rats were cultured under different conditions for 8
days with 5% FCS. Then, the changes in the mean NPo over 3 min of
recording of single KCa channels in outside-out patches (membrane
potential, +50 mV) before and after the application of CO were determined.
*P < 0.05 before and after the application of CO to the
same patch. n = 3-5 for each data point.
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To examine whether deglycation of KCa channels of diabetic SMCs
would regain the channel sensitivity to CO, tail artery SMCs from 1-month
diabetic rats were cultured for different periods. After culturing diabetic
SMCs for 8 days with 5 mmol/l glucose KCa channels in these cells
(Group 5G/8D, n = 5) still lacked responsiveness to CO
(Fig. 7). To avoid the
insufficient nonenzymatic deglycation, further experiments were carried out to
prolong the incubation period up to 35 days. The activity of KCa
channels in these chronic deglycated diabetic SMCs was significantly increased
by CO. As shown in Fig. 7, CO
(10 µmol/l) increased the mean NPo over 3 min of recording of the
single KCa channels in these cells (Group 5G/35D) by 62 ± 8%
(n = 5, P < 0.05). The regained sensitivity to CO was
unlikely to be related to the prolonged incubation period because CO (10
µmol/l) still had no effect on NPo of KCa channels in
diabetic SMC cultured for 35 days but with 25 mmol/l glucose (Group 25G/35D,
n = 4).

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FIG. 7. Deglycation of KCa channel proteins in diabetic SMCs regained
the sensitivity to CO. A: Representative single-channel recordings
showing the regained KCa channel sensitivity to CO after 35 days of
culture with 5 mmol/l glucose (left panel) and the insensitivity of
KCa channels to CO after 8 days of culture (right panel). The
dashed lines beside the original current traces denote the close states of
KCa channels. B: Relative changes in the mean NPo
over 3 min of recording of single KCa channels in outside-out
patches before and after the application of CO. *P <
0.05 before and after the application of CO to the same patch. n =
3-5 for each data point. Membrane potential, +50 mV. The culture condition of
each group was specified by concentration of glucose (G) in the medium and the
culture duration in days (D).
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Whether the vascular responses to the endogenous CO were altered in
diabetic vascular tissues was further studied in the next series of
experiments. Tail artery tissues were preincubated in the dark for 6 h with
hemin (20 µmol/l). Hemin acts as the inducer for the expression of the
inducible HO (HO-1) and as the substrate of HO to promote the endogenous CO
production from vascular tissues, thus decreasing the PHE-induced
vasoconstriction (4). In
agreement with our previous study
(4), 6-h incubation of tail
artery tissues from normal rats or diabetic rats without hemin added did not
alter the resting tension level or the PHE-induced concentration-dependent
vasoconstriction (data not shown). The concentration-dependent
vasoconstruction of normal tissues induced by PHE was significantly inhibited
by hemin incubation (Fig.
8A). The EC50 of PHE effects was 0.24 ±
0.03 and 1.19 ± 0.12 µmol/l without or with hemin incubation,
respectively (P < 0.05). In contrast, the vasoconstrictive effect
of PHE on diabetic tail artery tissues was not affected by 6-h hemin (20
µmol/l) incubation (Fig.
8B).

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FIG. 8. The effects of hemin (20 µmol/l) incubation for 6 h on the
phenylephrine (PHE)-induced constriction of tail artery tissues from normal or
diabetic rats. A: The contractile response of normal vascular tissues
to PHE was significantly reduced after incubating the tissues with hemin for 6
h. B: The contractile response of diabetic vascular tissues to PHE
was not changed after incubating the tissues with hemin for 6 h. n =
8 per data point.
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To determine whether the lack of relaxant effect of hemin on diabetic
vascular tissues was due to the loss of expression of HO in diabetic tissues,
Western blot assay was employed to examine the expression level of HO in
normal and diabetic rat tissues. The expression of HO-1 proteins was hardly
detected in normal rat vascular tissues but apparently visible in rat spleen.
In 1-month diabetic tissues, the expression of HO-1 proteins was significantly
induced in all vascular tissues tested. In contrast, there was no difference
in the abundance levels of HO-1 proteins between normal and diabetic spleen
tissues (Fig. 9). These results
demonstrated that the induced HO-1 protein expression was not a nonspecific
general tissue response to diabetes.
Figure 10 illustrates the
expression of constitutive HO (HO-2) protein in normal and diabetic rat
tissues. Different from the expression pattern of HO-1, HO-2 proteins were
detected in all normal rat tissues examined. More interestingly, the
expression levels of HO-2 proteins were not altered in all diabetic vascular
tissues (P > 0.05).

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FIG. 9. Increased expression of HO-1 proteins in 1-month diabetic tissues
determined by Western immunoblot assay. A: Representative immunoblots
of rat aorta, tail artery, mesenteric artery, and spleen proteins (40
µg/lane). Except for two bands for the spleen microsomes, a single band of
32 kDa was identified in all vascular tissue microsomes, which corresponds to
the known molecular mass of HO-1. ß-actin proteins were detected as the
housekeeping control. B: The relative abundance of HO-1 proteins in
normal and 1-month diabetic tissues. The immunoblot gel images were scanned
and digitized. The pixel densities of HO-1 proteins were then digitized and
analyzed using software (UN-SCAN-IT; Silk Scientific). A, artery; n =
3 for each group; *P < 0.01.
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FIG. 10. Expression levels of HO-2 proteins in normal or 1-month diabetic tissues
determined by Western blotting assay. A: Representative immunoblots
of rat aorta, tail artery, mesenteric artery, and spleen proteins (40
µg/lane). The identified HO-2 bands were 36 kDa, which corresponds to
the known molecular mass of HO-2. ß-actin proteins were detected as the
housekeeping control. B: The relative abundance of HO-2 proteins in
normal and 1-month diabetic tissues. The immunoblot gel images were scanned
and digitized. The pixel densities of HO-1 proteins were then digitized and
analyzed using software (UNSCAN-IT). A, artery; n = 3 for each group;
*P < 0.05.
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DISCUSSION
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CO actively participates in the fine regulation of vascular contractilities
under physiological conditions. Altered metabolism or vascular actions of CO
in many cases are coupled to cardiovascular diseases. For instance, the
increased endogenous production of CO was reported in chronic uremic patients
(10). Increased HO-1
expression and activity in hypoxia, ischemia-reperfusion, and subarachnoid
hemorrhage have also been reported
(1). The application of the
HO-inhibitor zinc protoporphyrin IX more significantly enhanced the blood
pressure in SHR-SP rats than in normotensive control WKY rats
(26). The mRNA levels of HO-2
in the aorta and kidney and of HO-1 in the ventricle were also significantly
higher in SHR-SP than in WKY rats
(26). Not only important for
the peripheral resistance regulation in genetically hypertensive rats, CO also
plays an important role in regulating cardiac function. A recent study showed
that the decreased endogenous CO production in SHR-SP may contribute to the
cardiac hypertrophy (27).
Furthermore, endogenous-produced CO has been shown to suppress the acute
hypertension responses of conscious rats induced by various vasoconstrictive
agents (28). Alm et al.
(29) demonstrated the
immunoreactivity of HO in the rat islets of Langerhans, including
insulin-immunoreactive ß-cells. After treatment with alloxan to induce
diabetes, the immunoreactivity of HO in rat islet cells virtually disappeared.
This study provided evidence for the pathophysiological role of the HO/CO
system in the development of insulin-dependent diabetes, for this system
appears to modulate the islet hormone secretion. In a recent study on type 1
and type 2 diabetic patients, it was found that the levels of exhaled CO were
higher when compared with those in healthy subjects. There was a positive
correlation between exhaled CO levels and the incidence of hyperglycemia in
all subjects and the duration of diabetes
(30). To date, the altered
functions of CO or the expression pattern of HO in diabetic vascular tissues
had not been reported. The STZ-induced experimental diabetes is a
well-established animal model of diabetes, characterized with hyperglycemia
and hypoinsulinemia
(17,18,19).
Using this diabetic animal model, we studied the vascular effects of CO in
diabetes and the underlying mechanisms. Our results are summarized as the
following: 1) The CO concentration-dependent relaxation of tail
artery tissues from streptozotocin-induced diabetic rats was significantly
decreased as compared with that of nondiabetic control rats; 2) the
vasorelaxant effect of CO in diabetes was solely mediated by cGMP; 3)
the sensitivity of KCa channels in diabetic vascular SMCs to CO was
significantly reduced; 4) the glycation of KCa channel
proteins may account for the reduced channel sensitivity to CO; and
5) the vasorelaxation induced by endogenous CO was also significantly
reduced in diabetes although no reduction in the expression levels of HO was
detected in diabetic vascular tissues. Taken together, our data demonstrated a
decreased vasorelaxant function of CO in diabetes. This altered vascular
effect of CO could be at least in part related to a diminished sensitivity of
KCa channels of diabetic SMCs to CO.
The mechanisms for the reduced sensitivity of KCa channels to CO
in diabetic SMCs were investigated. After chronically culturing normal tail
artery SMCs with 25 mmol/l glucose or 25 mmol/l 3-OMG, the sensitivity of
KCa channels in these SMCs to CO was consistently diminished.
Because 3-OMG is a nonmetabolizable glucose analog, these results indicate
that the glycation of KCa channels rather than the metabolism of
glucose by cultured vascular SMCs might be the mechanism for the altered
KCa channel functionality in diabetes
(25). On the other hand,
chronically culturing normal SMCs with 25 mmol/l mannitol did not alter the
effect of CO on KCa channels. The role played by the
hyperosmolality in the diminished effect of CO on KCa channels in
diabetes would thus be correspondingly minimized. If glycation of
KCa channels was responsible for the decreased sensitivity to CO,
deglycation of KCa channels should regain the channel sensitivity
to CO. Following this line of thought, we cultured diabetic SMCs with 5 mmol/l
glucose for different periods of culture time. A short culture duration (8
days) did not improve the sensitivity of KCa channels to CO in
these cells. This result could underline the notion that the nonenzymatic
deglycation of KCa channels would not be completed within the short
incubation scheme. Even in the presence of high concentrations of certain
deglycosylating enzymes, the deglycation of proteins was hardly completed in
10-20 days (31). In our
preliminary experiments, the cell viability significantly deteriorated when
the deglycation enzyme aminoguanidine (0.1 mol/l) was included in the culture
medium (R.W., Z.W., L.W., S.T.H., R.P.-W., unpublished observations).
Therefore, a prolonged cultured period beyond 8 days in the absence of
deglycation enzyme became our choice for the purpose of deglycating
KCa channels. As expected, culturing diabetic SMCs with 5 mmol/l
glucose for
5 weeks (35 days) restored the sensitivity of KCa
channels to CO. The control experiment showed that KCa channels in
diabetic SMCs cultured for the same period (35 days) but with 25 mmol/l
glucose still exhibited significantly lower sensitivity to CO
(Fig. 7). All afore-mentioned
data supported the idea that glycation of KCa channels affected
their responsiveness to CO. Future studies to isolate KCa channel
proteins from diabetic SMCs would provide direct evidence confirming the
glycation/deglycation status of these channel proteins under different
conditions.
Glycation of proteins involves a complex series of reactions, including
initial attachment of glucose to proteins by Schiffbase formation followed by
Amadori rearrangement to generate stable ketoamine. It is known that
hyperglycemia-induced protein glycation and lipid peroxidation are enhanced in
diabetic subjects (32).
Several mechanisms may account for the glycation-induced diminished
sensitivity of KCa channels to CO. Glycation of KCa
channels may produce steric hindrance for the exposure of the CO acting sites
on the KCa channel proteins
(33). Although the major
glycation sites on proteins are asparagine, glutamine, and lysine
34,35),
the glycation process will also affect the microenvironment of other amino
acid residues in proteins, including histidine. An altered accessibility to
the surface histidines has been shown in the glycated RNase A protein
(34). CO mainly interacts with
a histidine residue located on the extracellular domain of KCa
channels (3). This interaction
of CO with histidine may be impeded by the glycation of KCa
channels. Glycation of channel proteins may lead to protein thiol oxidation,
protein aggregation, and cross-linking
(36). The glycation of the gap
junction protein has been shown to alter the COOH-terminus arm, thus
decreasing the channel permeability
(36). In addition, the
glycation of KCa channels may cause channel gate inflexibility as
one of the consequences of the conformational changes, thus decreasing their
open probability to be otherwise enhanced by CO. Glycation of ion channel
proteins may facilitate the formation of advanced glycation end products. The
latter would damage the structural integrity of channel proteins by targeting
on histidine and other side chains
(35).
Due to the presence of vasculopathy and/or neuropathy, minor trauma in
diabetic patients will result in cutaneous ulceration and failure of wound
healing, eventually necessitating lower-extremity amputations. Of diabetic
patients,
51% receive lower-extremity amputation
(37). However, the nature of
vasculopathy of diabetic lower extremities remains unclear. The anatomy of the
rat tail vascular bed is suggestive of the study of distal blood circulation
in human limbs and digits
(38). The main artery
supplying cutaneous blood flow to the rat tail is the tail artery. The
function of this artery has been used as an assay to evaluate the impaired
cutaneous vascular functions in hypertension
(39) and other related
peripheral vascular diseases
(40). Therefore, the
constrictive responses of rat tail artery smooth muscles to different
vasoactive factors, including CO, may be relevant to the altered vascular
functions in diabetic distal vasculopathy. Abnormal blood flow patterns in the
diabetic foot unrelated to distal ischemia have been shown previously. In one
study on insulin-dependent diabetic patients who had previous surgeries for
diabetic foot infections, it was found that the most significant change in
peripheral circulation was the loss of the normal triphasic pattern of
arterial blood flow in lower limbs
(41). The forward blood flow
in systole was increased but the reversed flow in diastole decreased to the
diabetic foot. Consequently, transcutaneous venous oxygen tension was
increased with vein distension
(42,43).
Using the STZ-induced diabetes as a model of type 1 diabetes and using the rat
tail artery as a representative peripheral blood vessel, our study showed
significant changes in vasoconstrictive properties of peripheral vascular
tissues from diabetic rats. The altered properties of L-type calcium channels
(18), decreased responsiveness
to vasoactive bradykinin (17),
abnormal calcium handling by diabetic SMCs
(19), and decreased
vasorelaxant effect of CO as demonstrated in the present study may all
contribute to the abnormal regulation of peripheral resistance and altered
peripheral blood flow patterns in diabetes.
 |
ACKNOWLEDGMENTS
|
---|
This study was supported by research grants from Heart and Stroke
Foundation of Saskatchewan (Canada) and by the Natural Sciences and
Engineering Research Council of Canada. R.W. has been supported by a Scientist
Award of Medical Research Council of Canada. L.W. has been supported by a
postdoctoral fellowship from the Heart and Stroke Foundation of Canada.
The excellent technical assistance from Ginger Beal is greatly
appreciated.
 |
FOOTNOTES
|
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3-OMG, 3-O-methylglucose; BSA, bovine serum albumin;
[Ca2+]i, intracellular Ca2+ concentration;
ChTX, charybdotoxin; CO, carbon monoxide; DMEM, Dulbecco's modified Eagle's
medium; FCS, fetal calf serum; HBSS, Hank's buffered saline solution; HO, heme
oxygenase; Kca, calcium-activated K+; NPo, open
probability with N representing the number of single channels in one
patch; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PBS,
phosphate-buffered saline; PHE, phenylephrine; SMC, smooth muscle cell; STZ,
streptozotocin.
Received for publication February 28, 2000
and accepted in revised form October 2, 2000
 |
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