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1Departments of Medical Pharmacology and Physiology and 2Department of Internal Medicine, 3The Center for Diabetes & Cardiovascular Health, University of Missouri, Columbia, Missouri 65212; and 4Division of Molecular Medicine, 5Department of Anesthesiology, 6Department of Molecular & Medical Pharmacology, and 7Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, California 90095
Submitted 22 October 2002 ; accepted in final form 30 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Ca2+-dependent K+ channel; sarcoplasmic reticulum; Ca2+ release; dyslipidemia; voltage clamp; porcine; vascular smooth muscle
The contribution of K+ channel activity in the regulation of vascular smooth muscle tone has been clearly demonstrated (12, 20, 46). The proposed mechanism for K+ channel-dependent relaxation involves activation of K+ channels leading to hyperpolarization of the membrane potential (Vm), which then inactivates the voltage-gated Ca2+ channel (VGCC), ultimately leading to a decrease in Ca2+ influx and reduction in cytosolic Ca2+ concentration ([Ca2+]i). The role of the ATP-sensitive K+ channel (KATP) channel in regulation of vascular tone is minor owing to lack of change in the metabolic demands of smooth muscle under normal healthy conditions (28, 59); therefore, the voltage-sensitive K+ channel (Kv) and Ca2+-sensitive K+ channel (KCa) play the dominant roles in the regulation of vascular tone (12, 46). Because a prominent function of the K+ channel is to provide a means for smooth muscle relaxation, any alteration in the activity or expression of K+ channels would have a direct impact on vascular tone.
Alterations in the expression and activity of K+ channels have been shown to occur in several disease states (15, 37, 38, 40, 42). An increase in KCa channel expression has been interpreted in the particular case of hypertension as a compensatory response to the elevated [Ca2+]i (40). Additionally, there is a large body of evidence demonstrating that [Ca2+]i is elevated in vascular smooth muscle cells obtained from hypercholesterolemic and diabetic models, both animal and human (9, 30, 47, 67, 72). Although many investigators have shown diabetes-induced increases in vasoreactivity of several different vascular tissue beds (1-3, 53), the effect of diabetes on K+ channel activity or expression, Ca2+ regulation, and vascular smooth muscle reactivity has not been thoroughly studied. A better understanding of this relationship could help explain the enhanced vascular reactivity to agonists and elucidate potential therapeutic targets for the treatment of altered vascular reactivity.
Endurance exercise training, which is generally thought to provide cardioprotection, has several well-described effects on coronary smooth muscle (CSM) that can be summarized as an enhanced ability to relax and an attenuated response to vasoconstrictors (9, 16, 54). Although the exact mechanism for this protection is unclear, altered Ca2+ regulation is a potential candidate because of its critical role in the contraction-relaxation cycle of CSM, and, most importantly, Ca2+ homeostasis is affected by chronic endurance exercise (10, 62, 63). The close functional relationship of Ca2+/current and the activity of K+ channels make alterations in K+ channel activity another potential candidate for the observed effect of exercise-induced cardioprotection. Indeed, Bowles et al. (11) have shown that CSM cells obtained from exercise-trained miniature swine exhibit an increase in the contribution of KCa channels to tone, suggesting that K+ channel activity is another target for the signal(s) that results from endurance exercise training that ultimately affects coronary blood flow.
The purpose of this study was to determine whether the effects of hyperlipidemia (H) and diabetes (D) independently or in combination [diabetic dyslipidemia (DD)] result in alterations in the coupling of Ca2+ regulation and K+ channel activity and whether the changes in diabetic dyslipidemia can be prevented by exercise training. We chose the porcine model of diabetes because it possesses many qualities similar to humans, including cardiac and coronary anatomy and diet-induced atherosclerosis (17, 29, 49, 52, 69), and is also a well-established model of endurance exercise training (62). We found that hyperlipidemia, diabetes, and diabetic dyslipidemia resulted in greater coupling of sarcoplasmic reticulum (SR) Ca2+ release to whole cell potassium current (IK) activation but no increase in K+ channel protein expression as a compensatory mechanism in an attempt to prevent increased vasoconstriction. All of these events were prevented by endurance exercise training.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Diabetes was induced in the D, DD, and DDX animals by injecting alloxan
(125 mg/kg, Sigma Chemical, St. Louis, MO) into the superior vena cava via a
surgically implanted vascular access port
(48). Alloxan specifically
destroys the insulin-producing 
-cells of the pancreas
(69). Animals were maintained
at a fasting blood glucose concentration between 300 and 400 mg/dl
(6). Additionally, to mimic the
dyslipidemia that is common in the human diabetic population, DD and DDX
animals were fed a high-fat feed (see above). Blood urea nitrogen, creatinine,
and liver enzymes all remained within normal limits in all animals as
previously reported (17).
Treadmill exercise protocol. DDX animals underwent an acclimatization period to a motorized treadmill (Good Horse-keeping, Ash Grove, MO) over a 2-wk period during which the grade and speed were incrementally increased so that, by the end of the 2 wk, a workload was reached that elicited an exercise heart rate between 65 and 75% of maximal heart rate. The grade was adjusted during the remaining 14-wk exercise training regimen to maintain this target heart rate. Total running time at the target heart rate was 30 min 4 days/wk (8).
In vivo assessment of coronary reactivity. The right femoral
artery was accessed by arterial cutdown or with an 18-gauge needle through
which a 0.035-in. J-guidewire was introduced. After an introducer and an 8-Fr
sheath were inserted over the guidewire, an 8-Fr Amplatz L (sizes 0.75-2.0)
guiding catheter was advanced into the aortic arch. The guidewire was removed,
and a manifold apparatus was attached that allowed direct blood pressure
measurements to be obtained as well as injection of contrast media or
experimental solutions. The ostium of the left main artery was engaged with
the guiding catheter, and a 3.2-Fr intravascular ultrasound catheter (35 MHz,
Ultracross, Boston Scientific/SciMed, Maple Grove, MN) was advanced through
the guiding catheter and positioned in a proximal segment of left circumflex
artery. A bolus of prostaglandin F2
(PGF2, 8 µg/kg) was injected into the left
main artery via the manifold. The contraction to
PGF2 was recorded on videotape for off-line
analysis. Peak response to PGF2 across all groups
was obtained by assessment of the luminal area during diastole and systole.
Distensibility index, which is a measure of vessel stiffness, was calculated
by using the equation distensibility index = [(dA/A)/dP]
x 1,000, where dA is the difference between the smallest and
largest luminal areas, dP is the pulse pressure, and A is the
diastolic luminal area. Finally, an automated pullback was performed in the
circumflex artery at a rate of 0.5 mm/s to visualize atheroma. Images were
captured on videotape for off-line quantitative segmental analysis. At 1-mm
intervals (segments) of the pullback through typically 60-100 mm of circumflex
and left anterior descending arteries, the presence of atheroma was defined as
any fibrous or soft plaque less echogenic than the adventitia
(34). This characteristic was
easily resolved as distinct from the nonlayered appearance of all arteries
from control pigs (26).
Luminal area was defined by fine scintillations from red blood cells and
larger scintillations and/or turbulence from injection of saline. Percent
atheroma was defined as number of segments having atheroma divided by total
number of segments x 100. This analysis was performed for each pig; the
total number of segments studied for each group is indicated in
Fig. 2.
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In vitro assessment of coronary reactivity. The right coronary
artery (RCA) was removed from all animals immediately after exsanguination
under isoflurane anesthesia and placed in ice-cold sterile storage media. Four
3-mm serial segments were obtained and denuded of endothelium by rubbing a
thin dowel along the luminal surface. Denudation was confirmed by the lack of
a response to the endothelium-dependent vasodilator bradykinin after
preconstricting with 30 x 10-6 M
PGF2. Ring segments were hung on force
transducers (Grass-Telefactor, West Warwick, RI) and placed in a modified
Krebs-Henseleit bicarbonate-based buffer that contained (in mM) 2
CaCl2, 118 NaCl, 1 MgCl2, 5 KCl, 24.8 NaHCO3,
and 10 glucose. The pH was adjusted to 7.4, and the buffer was continuously
gassed with a mixture of 5% CO2-21% O2-75%
N2. All data were collected at a sampling frequency of 0.2 Hz with
AxoBASIC software (Axon Instruments, Union City, CA) and stored on a personal
computer for off-line analysis.
The rings were stretched to a length that elicited maximal force and were
allowed to equilibrate for 1 h before the experimental protocol began. A
predetermined amount of stock KCl solution was added to the tissue bath to
reach a final depolarizing concentration of 60 mM KCl. After 5 min, the
solution was washed out and replaced with the Krebs-Henseleit.
PGF2 (30 x 10-6 M
final concentration) or endothelin-1 (ET-1, 1 x
10-8 M final concentration) was added to evaluate vessel
contractility. Force development to both constrictors was calculated by
determining steady-state force and subtracting the initial baseline force. The
developed force was then normalized to the response to the 60 mM KCl
application.
Isolation of smooth muscle cells from RCA. Coronary smooth muscle cells were dispersed from segments of freshly dissected RCA as previously described (64). Briefly, small (0.5-1.0 cm) segments of the proximal RCA were cut longitudinally to allow exposure of the luminal side of the RCA. The segment was pinned to the bottom of a Sylgard-coated specimen jar into which a solution containing elastase (5 U/ml, Worthington Biochemical, Lakewood, NJ), collagenase (CLS II, Worthingon Biochemical), 2 mg/ml bovine serum albumin (fraction V, Sigma Chemical), 1 mg/ml soybean trypsin inhibitor (type-I-S, Sigma Chemical), and 0.4 mg/ml DNase I (type IV, Sigma Chemical) was added. The jar was placed in a shaking water bath (37°C, 100/min) for an additional 30-60 min. The cell suspension was centrifuged at 150 g for 4 min, and the pellet was resuspended in a low-Ca2+-containing storage media.
Whole cell Ca2+ measurement. A second cell suspension was obtained from the section of the RCA. These CSM cells were loaded with the acetoxymethyl ester form of the fluorescent Ca2+ indicator fura 2 (fura 2-AM, 2.5 µM). Fura 2-AM-loaded cells were placed in a constant-flow perfusion chamber for the determination of epifluorescence under resting and stimulated conditions. Light was excited with a 300-W xenon arc lamp and directed through alternating 340- and 380-nm band-pass filters with a liquid light guide. The resultant fluorescence from the selected smooth muscle cell was reflected to a photomultiplier tube (Products for Research, Danvers, MA) onto which a photon counter (Hamamatsu, Bridgewater, NJ) was attached. An optical processor (OP400) received the data, which were stored on a personal computer for off-line analysis. Ratiometric data obtained at 340 and 380 nm were used to estimate myoplasmic Ca2+. Background fluorescence was subtracted for each cell before the experimental protocol was performed (64).
Perforated patch electrophysiology. The cell suspension (30 µl)
was pipetted onto a microscope coverslip secured in a custom-made patch-clamp
perfusion chamber. The perforated patch-clamp technique was used to preserve
the intrinsic cellular constituents
(11). Glass capillary tubes
(Fisher Scientific, Atlanta, GA) were pulled to 4- to 10-M
tips, fire
polished, and dipped in an intracellular solution that did not contain
amphotericin B (Sigma Chemical). The pipette was then back-filled with an
amphotericin B-containing solution (240 µg/ml) with the following
additional constituents (in mM): 75 K2SO4, 45 KCl, 10
NaCl, 8 MgSO4, and 10 HEPES, pH 7.10. All experiments were
performed with the use of a Dagan 8900 patch-clamp amplifier, which was
interfaced with a Labmaster analog-to-digital converter and personal computer
equipped with AxoBASIC 1.0 software (Axon Instruments, Foster City, CA) for
data acquisition. Data were filtered through an eight-pole low-pass filter
after digitization at 495-µs intervals. A gigaohm seal was formed, and the
protocol was begun when series resistance had reached a value of 25 M
or less. A Ca2+-containing solution (2CaNa) was
superfused through the chamber by using gravity-assisted flow. The
constituents of 2CaNa were (in mM) 2 CaCl2, 138 NaCl, 0.1
MgCl2, 1.0 KCl, 1.0 HEPES, and 10 glucose, pH 7.4. A
current-voltage step protocol was performed by setting the holding potential
(HP) to -80 and applying serial test pulses in increments of 10 mV (267 ms)
from -80 to +60 mV. Peak steady-state currents at each step potential were
determined off-line and normalized to cell surface area by use of cell
capacitance. A current density-voltage relationship was determined for all
groups.
STOC measurement. Spontaneous transient outward currents (STOCs) occur when KCa channels are activated by quantal releases of Ca2+ from the SR, which increases the subsarcolemmal Ca2+ concentration (7, 51). STOCs have been identified in many types of vascular smooth muscle cells from different animal species (7, 24, 33, 44), including porcine coronary artery (63, 64). The outward current resulting from activation of KCa channels is a useful bioassay for transient increases in Ca2+ (64), quantal releases of Ca2+ from the SR and/or agonist-sensitive SR Ca2+ stores (63, 65). In the presence of constant superfusion of 2CaNa solution, we used an electrophysiological protocol designed to optimize the measurement of STOC events, which is shown in Fig. 1. During the "loading" portion of the protocol (Fig. 1A), the holding potential was maintained at -40 mV for 3 min to facilitate Ca2+ influx via VGCC and loading of the SR Ca2+ store. To prevent channel inactivation, Vm was hyperpolarized transiently (45 ms) to -80 mV before depolarizing to +30 mV (263 ms) (64). The depolarizing step to +30 mV was chosen because it has been shown that the sensitivity of the KCa channels to Ca2+ is relatively high, approximating Ca2+ sensitivity of fura 2 (64, 65). An 11-min "unloading" protocol followed in which the HP was maintained at -80 mV to facilitate vectoral Ca2+ release from the SR toward the sarcolemma (63, 64) (Fig. 1B). Vectoral Ca2+ release would either be extruded from the cell via Na+/Ca2+ exchanger or plasmalemmal Ca2+-ATPase, or activate KCa channels, thus stimulating STOC events. A STOC event was defined as a transient excursion of current with an amplitude greater than 10% of the steady-state outward current at measured at +30 mV with a duration of greater than 10 ms (see Fig. 1C).
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Caffeine-sensitive SR Ca2+. After the unloading protocol, cells were superfused with 2CaNa solution containing caffeine (5 mM), which causes the release of the SR Ca2+ stores (64, 65) and activates the KCa channel. During the caffeine application, the HP was maintained at -80 mV and the Vm was stepped to +30 mV for 380 ms every second to determine peak caffeine-induced KCa current. Peak caffeine-induced KCa current was normalized to cell capacitance.
Western blotting. A small portion of the RCA was homogenized in
(in mM) 20 HEPES-KOH, 1 EDTA, and 250 sucrose at pH 7.4 supplemented with 0.1
mmol/l phenylmethylsulfonyl fluoride, 1 µmol/l pepstatin A, 1 µg/ml
aprotinin, 1 µg/ml leupeptin, and 10 mmol/l CHAPS and centrifuged at 1,000
g. The supernatant was fractionated by standard SDS-PAGE and
subjected to Western blotting for identification and quantification of
KCa-. Briefly, the proteins (30 µg) were separated by 6%
SDS-polyacrylamide gels under reducing conditions and electrophoretically
transferred to nitrocellulose paper. The resulting blots were blocked in TBS
containing 5% nonfat dry milk for1hat room temperature. The blots were
incubated with 1:250 affinity-purified KCa883-896 rabbit polyclonal
antibody or 1:50,000 monoclonal antibody anti- smooth muscle actin in
1% nonfat milk TBS for 12 h at 4°C. Blots were washed three times in TBS
for 30 min and then incubated in horseradish peroxidase-conjugated secondary
antibody (1:4,000) (Amersham Biosciences, Piscataway, NJ) for 1 h. After
washing was completed, the blots were incubated in substrate for enhanced
chemiluminescence for 1 min and autoradiographed on Kodak BioMax film. The
bands were quantified by use of the Image-Pro Plus (MediaCybernetics, Des
Moines, IA) program. The specificity of the labeling was tested by
preincubating the antibody with the corresponding peptide.
For Kv1.4 protein Western blotting, coronary artery segments
(5-30 mg) were minced on ice and extracted for overnight in the cold room with
immunoprecipitation buffer
(57) containing 1% Triton
X-100, 60 mM n-octyl -D-glucopyranoside, 150 mM
NaCl, 20 mM Tris, pH 8.0, 2 mM EDTA, 50 mM NaF, 30 mM sodium pyrophosphate,
100 µM sodium orthovanadate, 1 mM PMSF, and 2 µg/ml leupeptin. Extracts
were centrifuged, and the supernatants were transferred to the fresh tubes and
incubated for 1 h (37°C) with 3 M urea and sample buffer
(43) containing 0.31 M Tris,
pH 6.8, 2.5% (wt/vol) SDS, 50% (vol/vol) glycerol, 0.125% (wt/vol) bromophenol
blue, and 1.2% (vol/vol) -mercaptoethanol. The volumes of extracts
containing 15 µg of total tissue protein were loaded and run on triple-wide
3-20% gradient polyacrylamide gels. The proteins were transferred at 400 mA
for 2.5 h in transfer buffer containing 15% methanol to Immobilon-P membrane.
After transfer, blots were blocked for overnight with 4% milk in Tween
20-Tris-buffered saline (T-TBS) in the cold room on a rocker. Blots were
washed three times with T-TBS, incubated for 3.5 h with rabbit polyclonal
anti-Kv1.4 antibody (Alomone Labs, Jerusalem, Israel), washed three
times with T-TBS, incubated with goat anti-rabbit antibody conjugated with
alkaline phosphatase (Zymed, San Francisco, CA), washed twice with T-TBS and
once with TBS, and stained with BCIP/NBT (Promega, Madison, WI). Dry blots
were scanned and analyzed by use of NIH Image software.
Immunostaining. A small segment of the RCA was fixed (4%
paraformaldehyde, 2% picric acid in 0.1 M phosphate-buffered saline, pH 7.4)
for 2 h. Transverse cryostat sections (10 µm) were processed for
immunostaining. The sections were incubated for 12 h at 4°C with
anti-KCa- subunit 883-896 affinity-purified
antibody (1:150). After washing was completed, the tissue sections were
incubated for 1 h at room temperature with Cy5-donkey anti-rabbit IgG (Jackson
ImmunoResearch Laboratory, West Grove, PA). In the control section, the
KCa- antibody was inactivated by addition of excess amount
of corresponding antigenic peptide (100 µg/ml). Images were acquired by use
of a confocal microscope (Olympus, Melville, NY). The intensity of the
immunofluorescent staining was measured in each section with NIH Scion
Image.
Citrate synthase assay. Citrate synthase assay was performed on skeletal muscle homogenates of the right biceps muscle to determine the efficacy of the treadmill training protocol by using the methods of Srere (61). Citrate synthase is a mitochondrial enzyme that responds to a program of endurance exercise training by increasing in activity.
Statistical analysis. One-way ANOVA was performed to determine treatment effects for all variables. Statistical significance was established if the P value was <0.05. When treatment effects were statistically different, the least significant difference test was used to perform multiple comparisons.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Percent atheroma, in vivo and in vitro responses to agonists.
Figure 2 represents the
protocol used to assess the precise measurement of luminal diameters during a
cardiac cycle as well as visualization of atheroma and summary data of percent
atheroma. Figure 2A
contains a representative intravascular ultrasound image of the lumen of the
circumflex artery. The dotted white line marks the luminal border that was
used to calculate lumen areas for in vivo assessment of the distensibility
index as well as responses to the vasoconstrictor
PGF2. Figure
2B represents a schematic of the segment of the coronary
artery from which these functional data were recorded. The dashed lines
represent the segment from which the image in
Fig. 2A was obtained.
Figure 2C shows
summary data of percent atheroma across groups. There was a significantly
higher percent atheroma in vessels from H, DD, and DDX compared with the C and
D groups (P < 0.05).
Figure 3 represents summary
data for the constriction response to the intracoronary application of
PGF2 and distensibility index. The data for the D
group were not included because of lack of sufficient data because there were
unexpected equipment malfunctions and one incident of premature animal death.
As shown in Fig. 3A,
there was trend toward greater percent constriction to
PGF2 in the H and DD groups compared with the C
group, but this difference did not reach statistical significance. Similarly,
there was a trend of a normalization toward the control level of percent
constriction in the DDX. Importantly, the relative magnitude of changes in
constriction in vivo was greater than those noted in vitro in response to
PGF2.
Figure 3B shows that
the baseline distensibility was lower in the H and DD groups compared with the
C and DDX groups although the differences did not reach statistical
significance largely because of the low number of experimental animals. In
other words, these measures indicate that both hyperlipidemia and diabetic
dyslipidemia increased the stiffness of the coronary artery. Together, albeit
not statistically significant partially because of the variability in the
responses, the in vivo measurements are consistent with in vitro results
showing that coronary artery function is altered as a result of hyperlipidemia
and diabetic dyslipidemia.
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Figure 4 represents summary
data of the developed force in response to PGF2
and ET-1 in the tissue bath for all experimental groups. There was a
significant increase in the PGF2-induced
contraction in the D, DD, and DDX groups compared with the C group (P
< 0.05) as seen in Fig.
2A. In response to the agonist ET-1 (see
Fig. 2B), there was
also a significant increase in the steady-state contraction in the H, D, and
DD groups compared with the C group, and this increase was prevented by
exercise training (DDX) (P < 0.05). Overall, the 
25% increase
in PGF2-induced contraction in DD was more modest
than the 60% increase in ET-1-induced contraction.
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Exercise effect on K+ current density in
hyperlipidemic and/or diabetic animals.
Figure 5 represents the
IK density-vs.-voltage relationship of all groups at step
potentials from -80 to 70 mV in steps of 10 mV. The IK
densities of the H, D, and DD groups were greater from the untreated animals
(DD H > D > C) at all membrane potentials between -10 and 40 mV.
Interestingly, the high-fat diet and diabetes (DD) effects were not additive
because DD animals had IK densities similar to those
induced by a high-fat diet alone (H). Nevertheless, the increase in
IK induced by a high-fat diet in diabetic animals (DD vs.
D) was completely reversed by exercise (DDX) even to levels lower than those
found in controls. These results indicate that endurance exercise training may
prevent the increase in IK that occurs not only with
diabetic dyslipidemia, but also with hyperlipidemia and diabetes.
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Caffeine-induced outward current. To investigate whether
KCa channels play a role in the increase of IK
in pathological conditions, we examined their activation on
Ca2+ release from caffeine-sensitive stores.
Figure 6A represents
an example of peak outward current recorded during caffeine application after
the membrane potential was stepped from a holding potential of -80 to +30 mV.
The current was then normalized to cell capacitance to obtain the
caffeine-induced outward current density for all groups, which is shown in
Fig. 6B. It is
noteworthy that the changes in KCa density followed a similar trend
as for IK, being H, D, and DD significantly (P
< 0.05) larger compared with the control group. Also, the increase in
KCa by hyperlipidemia or diabetes was not additive and reached
similar levels as in animals with both pathologies (DD). Moreover, exercise
training reversed the effect of diabetic dyslipidemia on KCa
current density to control levels. The increase in KCa current
density in H, D, and DD animals could be due to several mechanisms, including
1) an increased Ca2+ release from
caffeine-sensitive pools, 2) an increased efficiency of coupling
between Ca2+ release and KCa activation, or
3) an increase in KCa- subunit protein. We tested
these possibilities by examining in the different pathologies the amount of
Ca2+ released by caffeine, the STOC frequency as a
measure of KCa coupling with spontaneous quantal releases of
Ca2+, and the amount of total KCa-
subunit protein by immunochemical analysis.
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To measure possible changes in the caffeine-sensitive pools of Ca2+, we measured whether the fura 2 ratio changed in H, D, or DD with respect to untreated animals. As shown in Fig. 6B, there was no treatment effect on the caffeine-sensitive Ca2+ pool; thus changes in KCa current density on caffeine application in H, D, DD, and DDX animals seem not to be due to changes in the amount of Ca2+ released from caffeine-sensitive stores.
STOC events. Numbers of STOC events for each experimental group are shown in Figs. 7 and 8. Figure 7 represents the STOCs during the SR Ca2+ loading portion of the protocol (63, 64). In contrast to an increase of KCa current, the number of STOCs induced by hyperlipidemia and/or diabetes had a tendency to decrease, which was significant in the diabetic group compared with control animals. Nevertheless, a high-lipid diet in diabetic animals caused an increase in STOC frequency (DD vs. D) that was clearly reversed by exercise training (DD vs. DDX). A similar pattern was observed during the unloading portion of the protocol shown in Fig. 8. It seems, therefore, that the increase in KCa current by caffeine observed in H, D, and DD animals cannot be explained by an increase in coupling efficiency between spontaneous Ca2+ releases from SR. Instead, there is increased coupling efficiency primarily on "massive" caffeine-induced Ca2+ releases from the SR and activation of vicinal KCa channels.
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Western blotting for KCa channel.
Western blot analysis in Fig. 9
shows that total KCa- subunit protein was not significantly
changed by hyperlipidemia (H), diabetes (D), or diabetic dyslipidemia (DD).
Figure 9A shows
representative gels showing signals for KCa channel and their
corresponding blockade by preincubation of the antibody with the antigenic
peptide in control, DD, and DDX animals.
Figure 9B shows that
signals were not significantly different as assessed by densitometry. Similar
results were obtained in intact RCA segments immunostained with the same
specific antibody against the subunit of the KCa channel
(see Fig. 10). Immunolabeling
of tissue sections was also blocked by preincubation with the antigenic
peptide. Figure 10B
represents the mean intensity of immunofluorescent staining obtained from all
groups. There was no significant difference in intensity between groups. These
results suggest that the amount of KCa channel -subunit
protein that is expressed in RCA tissue is unaffected by any of the
experimental treatments. Alternatively, the sensitivity of this method may not
be sufficient to detect changes in expression of KCa channel
-subunit in the plasma membrane of smooth muscle cells that are
effectively detected with electrophysiological methods. In fact, from the
images in Fig. 10, it is not
possible to separate the KCa channel in the plasma membrane from
that in the intracellular organelles. This limitation is also true for
measurements of total membrane proteins using Western blot analysis. Increases
in KCa current density could also result from altered open
probability of the channel brought about by changes in its metabolic state
(e.g., phosphorylation) or changes in expression of splice variants and/or
-subunits.
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Although our patch-clamp studies largely assessed KCa current that dominates whole cell K+ current in these CSM cells (63, 64), we determined whether voltage-dependent K+ channel protein might be increased. Figure 11A shows a Western blot of Kv1.4 protein. Each lane under the group letter designations represents a sample from each animal in the group. Molecular weight markers separate the groups. Kv1.4 runs at about 50 kDa. Note that there was no difference in Kv1.4 protein between groups when densitometric analysis of the bands was performed (see Fig. 11B). There was not sufficient tissue to probe successfully for Kv1.3 and Kv1.5 isoforms. These data are consistent with a lack of effect of the experimental treatments on the density of the Kv1.4 channel.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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In 1995, 4% of the world population was diagnosed with diabetes, and the number of people with diabetes in industrialized countries is projected to increase by 42% by the year 2025 (32). Diabetes is a major independent cardiovascular risk factor (31) leading to atherosclerosis in more than 80% of patients (4, 5, 35, 60). Diabetes results in altered vascular reactivity, but some studies have shown an increase (2, 3, 27, 53, 71), and others have shown a decrease (53, 66, 23) in the response of vascular tissue when stimulated to contract by agonists. Furthermore, several studies have documented alterations in ionic currents in response to chronic diabetes both indirectly (3, 73) and directly (68, 70); however, the effect of diabetes on smooth muscle cell membrane currents has not been fully described, nor has the mechanism(s) for these effects been clearly elucidated. The reasons for some of the disparate results are not completely clear; however, the conflicting data could be a result of the use of the different species and different vessels studied or the duration of the diabetic state. These results provide strong evidence, however, for diabetes-induced alterations in Ca2+ handling not only at the level of the isolated arteries but also at the level of vascular smooth muscle cells. These findings are significant because under normal circumstances Ca2+ regulation is tightly coupled to KCa channel activity and together they are critical in the development of arterial tone. A potential negative consequence of the diabetes-induced increase in arterial reactivity is a decrease in coronary blood flow and myocardial perfusion that could then contribute to the increase in morbidity and mortality (5, 60). Because the porcine model of diabetes mimics many human qualities, including cardiac and coronary anatomy and response to consuming an atherogenic diet (29, 49, 52), these findings may have implications regarding human morbidity and mortality in diabetes. Furthermore, the similar body weights between groups show that it is possible to overcome the difficulty of maintaining positive energy balance in experimental models of diabetes (8), again providing more similarity to the course of diabetes in humans (13, 14). Finally, the significantly lower resting heart rate (exercise-induced bradycardia) and increased skeletal muscle oxidative enzyme activity in exercise-trained pigs indicate that appropriate cardiac and skeletal muscle adaptations occurred in these grossly diabetic and dyslipidemic animals.
Altered coronary artery contractility and atheroma. The in vivo
and in vitro measurements of coronary artery contractility confirmed that
animals in the H, D, and DD groups exhibited enhanced contractility in
response to agonists. The in vivo measurements indicated that hyperlipidemia
and diabetic dyslipidemia reduced the distensibility index compared with the
control conditions as well as increased the percent constriction to
PGF2. The lack of statistically significant
effects in the response to the intracoronary application of
PGF2 could be a result of the documented
vasodilatory effects of isoflurane anesthesia
(56) masking the acute effects
of PGF2 application. Generalized vasodilation to
isoflurane was evident by the lower blood pressures (90/60 mmHg, data not
shown). Endurance exercise prevented the enhanced contractility to ET-1 but
not to PGF2 under in vitro conditions, and there
was a tendency for the increase in contractility to
PGF2 measured in vivo to be prevented by
exercise. The early atheroma observed in the H, DD, and DDX animals suggests
that hyperglycemia alone does not accelerate the development of atheroma,
which has been shown by Gerrity et al.
(21) in a porcine model of
diabetic dyslipidemia. Furthermore, although exercise did not reduce the
percent atheroma in the present study, this finding is entirely consistent
with the findings in a study of atherosclerosis and exercise in monkeys
(74). We speculate that with a
more extended duration of diabetic dyslipidemia and exercise training, which
would produce more flow-limiting atheroma, there would be a significant
reduction in the percent atheroma with exercise training.
Whole cell K+ current. Our data suggest that chronic hyperlipidemia and diabetic dyslipidemia result in an increase in whole cell IK density, which was prevented by endurance exercise training. The mechanism for this increase is not clear; however, it has been shown by others that K+ channel function is modified by conditions associated with cardiovascular disease (15, 39, 40, 42, 15). Liu et al. (38) have suggested that the increase in KCa current and KCa protein expression in a hypertensive animal model may be a compensatory mechanism whereby K+ efflux is increased to hyperpolarization the membrane and consequently reduces Ca2+ influx. The predictable effect of membrane hyperpolarization and attenuated Ca2+ influx is enhanced vasodilatation (39, 40). Similarly, Mathew and Lerman (42) have reported an increase in the functional dependence on KATP channels in hypercholesterolemia during in vivo coronary reactivity studies, although they performed no direct measurement of K+ current as in the present study. We found no evidence for elevated KATP in our single cell voltage-clamp studies (not shown). The discrepancy with the in vivo data could be the different levels of metabolic demand. Elevated KATP channel activity could also be a mechanism in normalizing the enhanced CSM cell membrane depolarization that occurs in pathophysiological conditions. It is therefore plausible that in our model the significant increase in IK density in hyperglycemia, hyperlipidemia, and diabetic dyslipidemia is also an adaptive response necessary to counteract the changes in Ca2+ regulation that favor an enhanced basal Ca2+ concentration (75). Enhanced basal Ca2+ concentration would result in increased basal tone or increased reactivity in intact coronary vessels.
Enhanced basal tone or reactivity has been reported by many investigators in many animal models of hypercholesterolemia and diabetes (19, 36, 47, 58, 67, 70, 72, 73, 78). Factors that lead to increased basal tone and elevated vessel reactivity include an increased basal cytosolic Ca2+, increased circulating concentration of agonist, increased number of agonist receptors, a reduction in Ca2+ removal mechanisms, or a combination of these factors. C. A. Witczak and M. Sturek (unpublished observations) have studied fura 2-loaded CSM cells dispersed from the same experimental model to determine basal Ca2+. They reported a significantly greater basal Ca2+ in diabetic animals fed an atherogenic diet (diabetic dyslipidemia) compared with control animals. Furthermore, basal cytosolic Ca2+ levels in diabetic dyslipidemic exercise-trained animals were less than control values, which is consistent with an exercise-induced augmentation of Ca2+ efflux (75).
There is little information on the effect of diabetes and diabetic dyslipidemia on Ca2+ efflux mechanisms in CSM from conduit arteries. We have reported that the activity of the Na+/Ca2+ exchanger was blunted in CSM from diabetic dyslipidemic swine and that this reduction in Na+/Ca2+ exchange activity was also prevented by endurance exercise training (45). The net result of reduced Na+/Ca2+ exchange activity could also lead to the elevated basal Ca2+ levels seen in the CSM cells in this animal model. Taken together, the elevation in IK density in the CSM cells from the diseased animals appears to be a compensatory mechanism in response to elevated basal Ca2+ levels.
Immunohistochemistry and Western blotting for
KCa and Kv
channel. Two different techniques were used to determine the amount
of KCa channel -subunit protein that was expressed in the
vessels. Analysis of the Western blots showed that there was no difference in
the density of the bands located at the 125-kDa position. Furthermore, by
using immunocytochemistry techniques, tissue was stained with a fluorescently
labeled antibody directed against the KCa channel. The mean pixel
intensity for all groups indicated that there was no difference between the
experimental treatments.
To date, the distribution of Kv channels in porcine vascular
smooth muscle is unknown, largely owing to the Kv channel
dependence on vessel location but also because of the difficulty in
identifying all the different K+ channel subtypes. Immunoblotting
for the K+ channel subtype Kv1.4 was performed as an
initial probe to begin to determine which K+ channel subtype was
responsible for the change in whole cell K+ current in this present
animal model. Several investigators have confirmed that the mRNA for the
-subunit for Kv1.4 are indeed expressed in vascular smooth
muscle cells (77,
79). The data show that the
Kv1.4 member of the K+ channel family was not affected
by the experimental treatments. Taken together, these data suggests that
diabetes, diabetic dyslipidemia, and endurance exercise training did not
affect the expression of KCa or the Kv1.4 channel
proteins in the coronary arteries. It is possible, however, that functional
gating or second messenger, i.e., Ca2+, modulation of
the channels was altered. These alternative explanations represent an area for
further research.
Caffeine-induced outward current. The application of a
caffeine-containing superfusate (5 mM) was performed to activate the
KCa channel and also to indirectly assess the caffeine-sensitive
Ca2+ stores. Previous investigators have shown that such
an application to CSM cells releases the majority of
Ca2+ from the SR
(62,
63). This release of
Ca2+ from the caffeine-sensitive stores causes rapid
activation of the KCa channels and a significant transient increase
in current. A larger increase in the current density on activation by caffeine
in one group compared with another could occur for several reasons. A greater
caffeine-sensitive current could be the result of greater caffeine-sensitive
stores, a greater number of KCa channels, or an alteration in the
coupling of the SR Ca2+ release channels to the
KCa channels such that the same amount of
Ca2+ released from the SR activated more KCa
channels. It is unlikely that the size of the caffeine-sensitive
Ca2+ stores was greater in the diabetic dyslipidemic
group because there were no differences in the peak fura 2 ratios on caffeine
application between groups. Because the immunohistochemistry and
immunoblotting showed no statistical differences between groups, it is
unlikely that there were a greater number of KCa channel
subunits in the group that exhibited the greatest caffeine-induced outward
current. A more likely explanation is that the function of the channels was
altered, despite a lack of change in protein expression.
Another plausible explanation for the increase in the caffeine-activated outward current in CSM cells from animals with diabetic dyslipidemia is that the coupling of the ryanodine receptor to the KCa channel was altered in a way to cause greater activation of the KCa channel. Such an alteration could stem from a change in the distribution of ryanodine receptor or a difference in the regulation of the activation of the KCa channel. Indeed, Löhn et al. (41) have provided evidence for a regulatory function of the ryanodine receptor isoform that is expressed predominantly in vascular smooth muscle, ryanodine receptor 3. They have shown that mice deficient of this isoform of the ryanodine receptor have a remarkable change in the voltage dependence of the activation of KCa channel, which was measured in STOCs.
STOC events. It has been clearly demonstrated that quantal releases of Ca2+ from the SR in the form of Ca2+ sparks plays a important role in activating the KCa channel, initiating STOCs (18, 24, 33), and altering Vm (12, 38) or vessel lumen diameter (50, 40). Hyperpolarization by activation of the KCa channel decreases Ca2+ influx via VGCC and leads to vessel relaxation (40, 50). Several groups have shown that, in pathological conditions, a compensatory increase in KCa channel activity or protein expression occurs, in an attempt to augment membrane hyperpolarization (38, 55). STOCs were not quantified in these previous studies, however. Importantly, Stehno-Bittel et al. (63) have proposed that STOC frequency is reduced in CSM obtained from exercise-trained miniature swine because of an enhanced ability to extrude Ca2+ without activating the KCa channel. Taken together, these studies suggest that STOC events can be altered under certain conditions, and there may be a functional role related to alterations in STOC frequency.
The functional role of STOCs in conduit-sized arteries of the pig is still not entirely known; however, it is clear that there is low basal activity of STOCs. The fact that there is basal activity of STOCs suggests that there may be a functional role of STOCs in CSM. Our whole cell patch-clamp experiments consisted of two distinct voltage-step protocols to better elucidate the effect of hyperlipidemia and hyperglycemia on STOC events. The first protocol was an SR Ca2+ loading protocol in which holding potential was set to -40 mV and depolarizing step pulses were applied at 0.2 Hz. The purpose of this protocol was to facilitate Ca2+influx to gradually load the SR with Ca2+ by the slight activation of VGCC and the reverse mode action of the Na+/Ca2+ exchanger. Our goal for the first portion of the protocol was to have similar SR Ca2+ stores in all groups, to determine how STOC frequency was affected by our experimental conditions.
The STOC frequency in the D group was significantly lower than in C, H, and DD, suggesting that hyperglycemia is partially responsible for the diminution of STOC frequency in these cells. The STOC events in the DD group were not different from in C or H, but there were significantly fewer STOC events in the DDX group compared with the DD group. Finally, when animals are diabetic, dyslipidemic, and exercise-trained, the number of STOC events was normalized to levels seen in the C group. Changes in STOC frequency can be the result of the amount of KCa channel protein expressed in the cells, the activation characteristics of the KCa channel, the amount of Ca2+ in the SR, the juxtaposition of the ryanodine receptor to the KCa channel (microdomain sublocalization), and the gating characteristics of the ryanodine receptor. There was a reduction in the STOC frequency in the D group despite an increase in KCa current, an increase in the caffeine-sensitive IK, and similar caffeine-induced [Ca2+]i. One explanation for these results is that the coupling of the ryanodine receptor to the KCa channel was impaired as a result of chronic diabetes such that, under steady-state activation of the KCa channel (SR Ca loading and unloading protocols), fewer KCa channels were activated. This reduction in steady-state activation of the KCa channel could be due to fewer quantal Ca2+ release events (sparks) or the same number of sparks but a larger physical distance between the two proteins.
The result with the DD group was somewhat puzzling because we had expected even fewer STOCs in the DD group than in the D group. Instead, we observed an increase in STOC frequency in DD compared with D animals. It is possible that the combination of diabetes and dyslipidemia resulted in more of an increase in the KCa current density that senses spontaneous quantal release of Ca2+ and therefore a relative increase in STOC activation. An additional explanation involves an improved coupling of the ryanodine receptor with the KCa channel in the DD group. In this group, caffeine-sensitive IK was also elevated above C and DDX, with no between-group differences in the caffeine-induced myoplasmic Ca2+ concentration. A relocalization of the ryanodine receptor with the KCa channel or a relative increase in the amount of vectoral Ca2+ release toward the subsarcolemmal space might have occurred in response to diabetic dyslipidemia. Regardless of the mechanism (changes in KCa protein, ryanodine receptor and KCa channel coupling, or spark frequency), the diabetic dyslipidemia-induced increase in STOC events with respect to those induced by diabetes alone was returned to control conditions after endurance exercise training. The ryanodine receptor channel number, spark frequency, or colocalization studies were not determined in the present study.
In conclusion, our data indicate that 20 wk of chronic hyperlipidemia, diabetes, and diabetic dyslipidemia significantly increased whole cell IK in CSM cells in swine. The immunoblotting results suggest that KCa and Kv protein was not changed; instead, functional activation of KCa channels was increased. Moderate endurance exercise training appeared to prevent the changes in whole cell IK and either normalized the altered coupling of the KCa channel and ryanodine receptor or rescued the pattern of bolus Ca2+ release.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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10 ms) excursion of current with an amplitude >10% of the
steady-state outward current measured at a step potential of +30 mV.
C: representative current tracing obtained during the loading
protocol. Arrows indicate occurrences of STOCs.
significantly greater than H,
P < 0.05. B: summary data from in vitro studies with ET-1
indicate enhanced contractility in H, D, and DD whereas exercise training
prevented this increase in contractility (P < 0.05).
*Significantly greater than C, #significantly greater than DDX.
