© 1997 American Heart Association, Inc.
Articles |
Increased Myogenic Tone and Diminished Responsiveness to ATP-Sensitive K+ Channel Openers in Cerebral Arteries From Diabetic Rats
From the Department of Pharmacology (A.S.S., H.J.K., M.T.N.) and the Division of Cardiology (P.A.Z.), The University of Vermont, Burlington.
Correspondence to Dr Mark T. Nelson, Department of Pharmacology, The University of Vermont, Given Building, Room B303, Burlington, VT 05405-0068. E-mail nelson{at}northpole.med.uvm.edu
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Abstract |
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Abstract Diabetes mellitus has profound adverse effects on vascular and, in particular, endothelial function. Although pressure-induced constriction ("myogenic tone") is a major contributor to the regulation of blood flow, little is known about the effects of diabetes on this response. Diabetes has been shown to diminish the dilation of cerebral arteries to synthetic ATP-sensitive K+ (KATP) channel openers. In this study, we explored the effects of diabetes induced in rats by streptozotocin on cerebral artery (250 to 300 µm) myogenic tone and on vasodilations to the synthetic KATP channel openers pinacidil and levcromakalim. Elevation of intravascular pressure caused a graded membrane potential depolarization and constriction, which was greater in arteries from diabetic rats compared with normal rats (at 60 mm Hg, 5 mV more depolarized and 22 µm more constricted). Pressurized arteries (at 60 mm Hg) from diabetic rats were 5- to 15-fold less sensitive to pinacidil and levcromakalim than were control arteries (EC50 values for pinacidil and levcromakalim were 1.4 and 0.6 µmol/L, respectively, in diabetic animals and 0.3 and 0.04, respectively, in control animals; P<.05). Removal of the endothelium or addition of a NO synthase inhibitor, NG-nitro-L-arginine (LNNA), in control arteries decreased the sensitivity to KATP channel openers and depolarized and constricted control arteries to levels similar to those observed in arteries from diabetic animals. Sodium nitroprusside caused a membrane potential hyperpolarization and enhanced the response to pinacidil in arteries from diabetic animals. Removal of the endothelium or LNNA had little effect on the apparent KATP channel opener sensitivity, the membrane potential, and pressure-induced constrictions of arteries from diabetic animals. The results are consistent with the hypothesis that this type of diabetes leads to a decrease in tonic NO release from the endothelium, which in turn causes membrane potential depolarization and vasoconstriction, resulting in a diminished response to KATP channel openers.
Key Words: diabetes • K+ channel • endothelium • vascular smooth muscle • nitric oxide
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Introduction |
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Diabetes mellitus is a devastating disease that affects 5% of the American population. There is a 2- to 4-fold increase in the risk of coronary heart disease, cerebrovascular disease, congestive heart failure, and other cardiovascular complications due to diabetes.1,2 The underlying mechanisms causing this increased risk remain unclear. Abnormalities in lipid metabolism, coagulation cascade, and glycation products may contribute to increased vascular disease in diabetes.3
Recent evidence suggests that diabetes causes abnormal endothelial function.4–7 For example, endothelium-dependent dilations of cerebral arteries and arterioles are profoundly affected by diabetes.8–10 Endothelium dysfunction during diabetes appears to be a major link in the pathogenesis of vascular disease.11,12
Changes in vascular smooth muscle function associated with diabetes are well documented.13,14 Of particular interest, Mayhan and Faraci15 (1993) and Mayhan16 (1994) recently demonstrated that cerebral arteries from diabetic rats dilate less than control arteries to the KATP channel openers aprikalim and levcromakalim. These studies suggested that part of the functional change occurring in vascular smooth muscle during diabetes may be directly related to alterations in ion channel function.
Elevation of intravascular pressure causes a graded membrane potential depolarization and contraction (myogenic tone) of the smooth muscle cells in intact cerebral arteries.17–19 Myogenic tone is a major contributor to the regulation of arterial diameter.20 Myogenic tone of small cerebral arteries depends on Ca2+ entry through voltage-dependent Ca2+ channels,21 since Ca2+ channel blockers or membrane potential hyperpolarization through activation of K+ channels causes vasodilation.12,19,22,23 Hypoxia,24,25 calcitonin gene-related peptide,26–29, adenosine,29 and synthetic K+ channel openers (eg, pinacidil, levcromakalim, and aprikalim)29,30 appear to dilate cerebral arteries, in part, through membrane potential hyperpolarization caused by the activation of KATP channels.
The first goal of the present study was to determine the effect of diabetes on pressure-induced changes in diameter and membrane potential in rat cerebral arteries. The second goal of this study was to explore the mechanisms for the diminished sensitivity of cerebral arteries to synthetic KATP channel openers. Our results are consistent with the hypothesis that diabetes mellitus reduces the tonic release of NO from the endothelium, leading to membrane potential depolarization and vasoconstriction. This decrease of NO release and/or the subsequent membrane potential depolarization causes a decrease in the sensitivity of the arteries to KATP channel openers. These findings have implications for the regulation of arterial diameter by pressure, NO, and KATP channel openers during diabetes.
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Materials and Methods |
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Induction of Diabetes and Preparation
Female Sprague-Dawley rats (12 weeks old,
280 g) were
studied. Diabetes was induced with a single
intraperitoneal injection of STZ (60 mg/kg)
dissolved in 1 mL sterile saline as a vehicle. Control rats received a
single intraperitoneal injection of 1 mL sterile
saline solution. Induction of diabetes was confirmed by occurrence of
glucosuria on 2 consecutive days after the
intraperitoneal injection. Blood samples were
obtained when the animals were killed for study, between 4 to 8 weeks
after the development of glucosuria. Blood glucose measurements were
determined by using a glucoscan meter (One Touch II, Lifescan Inc).
Female Sprague-Dawley rats were euthanized with pentobarbital (130
mg/kg IP) and killed by thoractomy. The chest was opened, and a
blood glucose sample was obtained. The animals were decapitated, and
the brains were removed and quickly transferred into normal PSS (for
composition, see below) at 4°C. Resistance-sized proximal middle
cerebral arteries were isolated and dissected from the surrounding
connective tissue. These cerebral arteries were then cannulated and
mounted in an arteriograph. Intravascular pressure was gradually
increased in 20 mm Hg steps, with the vessel being perfused in
PSS (for composition, see below) at 37°C, after a 15-minute
equilibration period. Concentration-response curves to various
vasodilators were performed at an intraluminal pressure of 60
mm Hg, the estimated pressure experienced by the vessel in vivo
(estimated to be 50% of systolic blood pressure).
Maximal passive diameter of pressurized arteries was determined as the arterial diameter in Ca2+-free PSS. The arteries were denuded of endothelium by placing an air bubble in the lumen for 1 minute, followed by a luminal wash with distilled water for 30 seconds. Endothelial disruption was verified by the absence of a dilator response to acetylcholine after myogenic tone had developed.
Recording Methods
Arterial diameter was measured with a video
dimension analyzer (Living Systems Instrumentations). Membrane
potential was recorded in intact pressurized arteries, using
conventional intracellular glass microelectrodes filled with 3
mol/L KCl solution and tip resistances of 40 to 60 M
. Smooth
muscle cells were impaled from the cleaned adventitial side of the
pressurized artery. Membrane potentials were measured with an AXOCLAMP
2B amplifier (at 0.5- to 1-kH bandwidth, Axon Instruments Inc), and
data were recorded using an Axotape 2.0/Digidata 1200 data
aquisition system (Axon Instruments Inc) on a Gateway 386/20DX PC.
Criteria for acceptance of recordings were as follows:
(1) an abrupt change in potential on impalement of cells, (2) stable
membrane potential for at least 2 minutes before experimental
manipulations, (3) maintained impalement throughout the experimental
protocol, and (4) unchanged tip resistance before and after impalement
and tip potentials of <3 mV.
Solutions and Drugs
A PSS was used as the bathing solution and had the following
composition (mmol/L): NaCl 119, KCl 4.7, NaHCO3
24.0, KH2PO4 1.2, CaCl2 1.6,
MgSO4 1.2, EDTA 0.023, and glucose 11.0 (pH 7.4). This
solution was continuously bubbled with 95% O2/5%
CO2. The arteries were superfused at 3 to 6 mL/min (bath
volume, 6 mL). All intact vessel experiments were performed at
37°C under continuous superfusion with PSS. All chemicals and
reagents were from Sigma Chemical Co unless otherwise specified.
Pinacidil and levcromakalim were obtained from Research Biochemicals
International.
Data Analysis and Presentation
The approximation of the half effective concentration
(EC50) of drug giving a half-maximal response (eg,
vasodilation) was calculated from fitting a logistical equation
(sigmoidal nonlinear least-squares fit) to the concentration response
curves using the ORIGIN program (Microcal Software Inc). Vessel
distension ratio (DR) values were obtained from passive
(0 mmol/L Ca2+ PSS) arterial
diameter measurements and were calculated using the following equation:
DR=Dx/D0, where Dx is
the diameter at a given pressure x, and D0
represents arterial diameter at 0 pressure obtained
through extrapolation of a third-order polynomial fitted to the passive
pressure/diameter curves for each artery.31 Membrane
potential values are expressed in millivolts as mean±sample SD from n
different arteries. Diameter values are expressed in microns as
mean±SEM for n vessels. Statistical significance was tested at the
95% (P<.05) confidence level using Student's paired
t test on independent measurements. Asterisks on figures
indicate significant difference from control values.
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Results |
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Rats were euthanized at 16 to 20 weeks of age 4 to 8 weeks after STZ treatment. The average body weight of the rats at 12 weeks was
280 g. Body weight was randomly measured in the two groups at the
time of euthanasia. The diabetic rats had an average body weight of
280±25 g (n=17). The average body weight of control animals
(saline-injected) was 337±24 g (n=11). The average blood glucose
concentration was significantly higher in diabetic rats compared with
control rats: 17.6±0.6 mmol/L (317±10 mg/dL)
(n=31) and 4.6±0.2 mmol/L (83±4 mg/dL) (n=47),
respectively (P<.05). The mean proximal middle cerebral
arterial diameter at 10 mm Hg was 169±4 µm in
control rats (n=33) and 181±3 µm in diabetic rats (n=22)
(P<.05) (Fig 1A
and 1B). The mean maximal diameters at
100 mm Hg for control rats and diabetic rats were 257±5
µm (n=33) and 275±5 µm (n=22), respectively
(P<.05) (Fig 1A and 1B).
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Pressure-Induced Constrictions Are Greater in Cerebral Arteries
From Diabetic Than Control Rats
Cerebral arteries from both control and diabetic rats constricted
in response to a graded increase in pressure (Figs 1
and 2).
As pressure was increased into the physiological
range that these arteries experience in vivo, estimated at 60
mm Hg or higher (50% of systolic pressure), arteries from
diabetic rats constricted more than control arteries (compare Figs 1A and 1B, and see Fig 2). At 60 mm Hg, arteries from diabetic rats
constricted by 85±5 µm (32±2%) (n=8) compared with 63±2
µm (27±1%) (n=6) in control arteries (P<.05) (Fig 2).
At 100 mm Hg, arteries from diabetic rats constricted by
112±6 µm (39±2%) (n=8) compared with 73±2 µm
(30±1%) (n=6) in control arteries (P<.05) (Figs 1 and 2).
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60 mm Hg compared with arteries from
control animals. Removal of the endothelium from
arteries obtained from control animals [C(-ENDO)] increased the
pressure-induced constriction, effectively eliminating the difference
in tone between control and diabetic arteries, whereas removal of the
endothelium in arteries from diabetic rats
[DM(-ENDO)] had no effect. *P<.05 vs control
conditions.
Since endothelial function is altered in cerebral
arteries from diabetic animals,8–10 the effects of the
removal of the endothelium on pressure-induced
constrictions were examined. Arteries from control animals constricted
more to pressure after the removal of the endothelium
(Figs 1C and 2). Denuded arteries from control rats constricted by
84±10 µm (35±4%) (n=6) at 60 mm Hg and by 110±10
µm (42±5%) (n=6) at 100 mm Hg (Fig 1C) compared with 63 and
73 µm in arteries with intact endothelium
(P<.05). Denuded arteries from diabetic rats had no
significant increase in tone compared with nondenuded arteries (Fig 2).
Denuded arteries from diabetic rats constricted by 86±5 µm
(33±2%) (n=7) at 60 mm Hg and 110±6 µm (39±2%) (n=7)
at 100 mm Hg (Fig 2). These results suggest that
endothelial cells from control arteries tonically
release a dilating factor and that diabetes disrupts this process.
To exclude the possibility that the diabetic state had influenced the
passive properties of the arteries, the passive properties of the
arteries from control and diabetic animals, with and without intact
endothelium, were examined in nominal
Ca2+-free buffer. Calculated distension ratios were similar
at different levels of intravascular pressure under all conditions (Fig 3). Therefore, the observed difference in
pressure-induced constriction between diabetic and control animals does
not appear to be a consequence of structural alterations in vessel
stiffness.
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Acetylcholine and SNP Responses in Cerebral Arteries From Control
and Diabetic Rats
To assess the function of the endothelium, we
studied the vasodilator response to acetylcholine, an
endothelium-dependent vasodilator, in cerebral arteries
from control and diabetic rats pressurized to 60 mm Hg (Fig 4). Acetylcholine-induced dilations were
markedly reduced in cerebral arteries from diabetic rats (Fig 4A).
Acetylcholine (10 µmol/L) dilated arteries from diabetic
animals by 7±13 µm (n=6) compared with 42±5 µm (n=6) in
control tissues (P<.05). The diminished response of
cerebral arteries to acetylcholine from diabetic rats is
consistent with previous observations by
others9,32,33 and supports the hypothesis that
endothelial cells are dysfunctional in diabetes.
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Our results suggest that diabetes interrupts basal NO release from the
endothelium.34 To explore a possible direct
effect of diabetes on smooth muscle cell responsiveness to NO, the
dilator effects of the NO donor SNP were examined. SNP fully relaxed
arteries from control and diabetic animals (Fig 4B). SNP at 100
nmol/L dilated control arteries (±endothelium)
and arteries from diabetic animals to a similar extent (Fig 4B). These
results suggest that diabetes did not significantly alter smooth muscle
responsiveness to SNP.
Dilations to KATP Channel Openers Are Reduced in
Diabetes
Vasodilations of cerebral arterioles to the KATP
channel openers aprikalim and levcromakalim are reduced in
diabetes.15,16 We extended this observation by
demonstrating that dilations to other types of KATP channel
openers, such as pinacidil,29 are also reduced in diabetes
(Figs 5 and 6). Dilations to pinacidil and
levcromakalim of pressurized (to 60 mm Hg) cerebral arteries
(±endothelium) from control and diabetic animals were
measured. Cerebral arteries from diabetic rats were less sensitive to
these KATP channel openers. For example, pinacidil at
0.3 µmol/L dilated arteries from diabetic animals by only
5±4% compared with a dilation of 34±8% in the control animals
(P<.05). Similarly, levcromakalim at 0.3
µmol/L dilated arteries from diabetic animals by 20±5%
compared with an almost maximal dilation of 76±6% in arteries from
control animals (P<.05). Both pinacidil and levcromakalim
dilated arteries from control and diabetic animals to a maximum of
80±5% (n=18). The half-maximal effective concentration
(EC50) for dilation to pinacidil in arteries from diabetic
rats was 1.4±0.1 µmol/L (n=4) compared with
0.3±0.1 µmol/L (n=4) in control rats
(P<.05), indicating a 5-fold decrease in sensitivity (Fig 5A). Similarly, EC50 for levcromakalim dilation in arteries
from diabetic rats was 0.6±0.1 µmol/L (n=5) compared
with 0.04±0.01 µmol/L (n=5) in control rats
(P<.05), a 15-fold decrease in sensitivity (Fig 6A).
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Dilations to KATP Channel Openers Are Reduced by
Endothelium Removal or by Inhibitors of
NO Synthase
Loss of endothelial function appears to be an
important contributor to enhanced pressure-induced "myogenic"
vasoconstriction during diabetes (Fig 1). We thus sought to examine the
effects of endothelium removal on dilations to
pinacidil and levcromakalim (Figs 5B and 6B). Removal of the
endothelium from control arteries reduced the
sensitivity of the pressurized arteries to pinacidil and levcromakalim
to that observed in arteries from diabetic animals. For example,
pinacidil at 0.3 µmol/L dilated arteries from control
animals from which the endothelium had been removed by
17±6% compared with a dilation of 34±8% in the control arteries.
Similarly, levcromakalim at 0.3 µmol/L dilated denuded
arteries from control animals by 25±9% compared with an almost
maximal dilation of 76±6% in control arteries. Both pinacidil and
levcromakalim dilated denuded arteries from control and diabetic
animals to a maximum of 88±5% (n=22). The EC50 for
pinacidil dilation of denuded arteries from control rats shifted
rightward from 0.3±0.1 to 1.0±0.1 µmol/L (n=4), and
similarly, the EC50 for levcromakalim dilation shifted from
0.04±0.01 to 0.7±0.1 µmol/L (n=6), respectively
(P<.05) (Figs 5B and 6B). In support of the change in
KATP channel opener sensitivity with diabetes being
mediated by the endothelium, denuded arteries from
control and diabetic animals dilated to the same extent to pinacidil
(Fig 5B) and levcromakalim (Fig 6B). None of these values were
statistically different (in denuded arteries from diabetic rats:
EC50, 1.1±0.1 µmol/L, n=5, for
pinacidil and 0.4±0.1 µmol/L, n=7, for
levcromakalim).
The above data suggested that the effectiveness of KATP
channel activation could be diminished by decreasing the release of a
relaxing factor from the endothelium. Since NO released
from the endothelium can cause vasodilation, the
effects of pinacidil were assessed in the presence of LNNA (100
µmol/L), a NO synthase inhibitor (Fig 7). LNNA constricted pressurized (at
60 mm Hg) cerebral arteries from control animals by 35±7
µm (n=11) (P<.05), whereas LNNA did not have an effect on
arteries from diabetic animals (13±13 µm, n=5). LNNA
significantly reduced the sensitivity of pressurized control arteries
to pinacidil, whereas it had little effect on arteries from diabetic
animals (Fig 7). For example, pinacidil at 0.3 µmol/L
dilated arteries from control animals in the presence of 100
µmol/L LNNA by 2±1% (n=11) compared with a dilation of
34±8% (P<.05) in the control arteries. Pinacidil at
0.3 µmol/L dilated arteries from diabetic animals in the
presence of 100 µmol/L LNNA by 4±4% (n=5) compared with
a dilation of 5±4% in the control diabetic arteries. Pinacidil
dilated arteries from control and diabetic animals in the presence of
100 µmol/L LNNA to a maximum of 83±4% (n=16). The
EC50 for pinacidil dilation of arteries treated with
100 µmol/L LNNA from control rats shifted rightward from
0.3±0.06 to 1.5±0.05 µmol/L (n=8) (P<.05).
The concentration-response curves to pinacidil for LNNA-treated control
arteries, LNNA-treated diabetic arteries, and diabetic arteries were
not statistically different (Fig 7). Further, LNNA did not
significantly alter pinacidil-induced dilations of
endothelium-denuded pressurized arteries from control
or diabetic animals (EC50, 1.3±0.4 µmol/L
and 1.5±0.9 µmol/L, respectively). These results
indicate that the difference in the apparent KATP channel
opener sensitivity is due to reduced release of
endothelium-derived NO affecting the
arterial smooth muscle cells.
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In addition, in the absence of KATP channel openers, KATP channels do not appear to contribute significantly to the regulation of myogenic tone, since a KATP channel blocker, GLIB (10 µmol/L),22,29 was without effect on arteries from normal and diabetic animals. Arterial diameters (at 60 mm Hg) were similar in PSS and in PSS+10 µmol/L GLIB for normal rats (change was +2±35 µm, n=5) and in PSS and in PSS+10 µmol/L GLIB for diabetic rats (change was -4±26 µm, n=7), respectively.
LNNA Depolarizes the Membrane Potential of Smooth Muscle Cells in
Pressurized Arteries of Control, but Not Diabetic, Rats
Since membrane potential depolarization can constrict arteries and
a number of endothelium-relaxing factors, including NO,
cause membrane potential hyperpolarization, we
explored the possibility that arteries from diabetic animals were
depolarized compared with arteries from control animals. To investigate
this issue, membrane potentials of pressurized (to 60 mm Hg)
arteries from control and diabetic rats were measured in the presence
and absence of LNNA, with and without GLIB present. Pressurized
(60 mm Hg) arteries from control animals had membrane potentials
of -45.6±1.2 mV (n=12) in PSS and -45.4±1.3 mV (n=5) in PSS+10
µmol/L GLIB. Elevating intravascular pressure from 10 to
60 mm Hg caused a membrane potential depolarization from -60
to -40 mV, as previously described.17–19 Pressurized
(60 mm Hg) arteries from diabetic animals were 5 mV more
depolarized and had membrane potentials of -40.3±1.6 mV (n=15)
(P<.05) in PSS and -40.3±1.5 mV (n=6) (P<.05)
in PSS+10 µmol/L GLIB. The addition of LNNA (50
µmol/L) caused a 5-mV depolarization in arteries from control
animals (to -40.5±1.9 mV, n=6, P<.05, Fig 8A), but it did not alter the membrane
potential of arteries from diabetic animals (-39.5±1.5 mV, n=6, Fig 8B). Similarly, in the presence of 10 µmol/L GLIB, the
addition of LNNA depolarized arteries from control animals by 4 to 5 mV
to -41.2±1.2 mV (n=6), whereas it had no significant effect on
membrane potentials in arteries from diabetic animals (-40.3±1.5 mV,
n=6). These data indicate that GLIB (10 µmol/L) had no
effect on membrane potentials of arteries from normal and diabetic
rats, consistent with the observed lack of effect of GLIB on
arterial diameter and, taken together, suggest that
KATP channels do not contribute significantly to the
arterial membrane potential under these conditions.
Furthermore, GLIB did not alter the depolarizing response to LNNA in
arteries from control animals. These results are consistent
with the idea that tonic release of NO from the
endothelium of control arteries causes a membrane
potential hyperpolarization and dilation,
independent of KATP channels.
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SNP Hyperpolarizes the Membrane Potential and Restores
Pinacidil Sensitivity
Consistent with the tonic release of NO causing a membrane
potential hyperpolarization in control arteries,
SNP (100 nmol/L) caused a membrane potential
hyperpolarization of diabetic arteries from
-40.3±1.6 to -46.1±1.2 mV, or by 5.8 mV (n=7) (P<.001)
(Fig 9A). GLIB did not affect the
SNP-induced hyperpolarization of these arteries; in
the presence of GLIB, SNP hyperpolarized the arteries to -47.3±1.0 mV
(n=4). SNP (100 nmol/L) caused a membrane potential
hyperpolarization of normal arteries from
-45.7±1.2 to -48.2±0.8 mV (n=6), or by 2.5 mV (P<.001)
(n=6). As predicted from the proposed model in Fig 10, pinacidil (300
nmol/L)–induced dilations of pressurized arteries from diabetic
animals or control arteries denuded of their
endothelium were enhanced by SNP. Pinacidil (300
nmol/L) dilated pressurized (60 mm Hg) arteries from
diabetic animals by 5±4% (n=4) and by 28±6% (n=5) in the presence
of SNP (100 nmol/L). Pinacidil (300 nmol/L) dilated
control arteries, denuded control arteries, and denuded control+SNP
arteries by 34±8% (n=4), 17±6% (n=4), and 30±2% (n=4),
respectively (Fig 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have demonstrated that diabetes mellitus induced by STZ causes a membrane potential depolarization of
5 mV (at 60 mm Hg) and
vasoconstriction. These findings can be explained by a loss in the
tonic release of NO from the endothelium. The decrease
in NO release or the membrane potential depolarization leads to a
decrease in the apparent effectiveness of KATP channel
openers, such as pinacidil and levcromakalim. Our results are
summarized in the working hypothesis scheme shown in Fig 10.
We show for the first time that pressure-induced constriction
("myogenic tone") is enhanced in cerebral vessels from
(STZ-induced) diabetic animals (Figs 1 and 2) in vitro. We did not
detect changes in the passive properties of the cerebral arteries from
diabetic animals compared with normal littermates (Fig 3). However,
consistent with observations of others,5–7 the
response of arteries to the endothelium-dependent
vasodilator acetylcholine, which stimulates release of NO from the
endothelial cells, was greatly reduced in diabetic
animals (Fig 4A). Diabetes did not appear to alter sensitivity to
nitrovasodilators (Fig 4B), consistent with the observation of
Mayhan.10 These results, taken together, are
consistent with diabetes interrupting basal NO release from the
endothelium without changing the properties of the
vascular smooth muscle in this model of insulin-dependent diabetes.
The decreased sensitivity of pressurized cerebral arteries from
diabetic rats to KATP channel openers (pinacidil and
levcromakalim) that we observed is consistent with the previous
observation of Mayhan and Faraci15 (1993) and
Mayhan16 (1994) of decreased sensitivity of cerebral
arteries to the KATP channel openers aprikalim and
levcromakalim. We found that the difference in KATP channel
opener sensitivity between normal and diabetic arteries could be
abolished by removal of the endothelium (Figs 5 and 6)
or by an inhibitor of NO synthase (LNNA) (Fig 7) (also see
scheme in Fig 10). Furthermore, in support of the proposed scheme in
Fig 10, we demonstrate that SNP can increase pinacidil responses in
denuded control arteries and in arteries from diabetic animals (Fig 9B). In contrast, Mayhan and Faraci did not observe an effect of the NO
synthase inhibitor L-NMMA at 1 µmol/L. The
reason for this discrepancy in the results is unclear and may relate to
the concentration of L-NMMA used.35–37
Removal of the endothelium or inhibition of NO synthase
by LNNA caused the smooth muscle membrane potential of pressurized
arteries from normal rats to depolarize by 5 mV, which should
contribute to the observed increase in "myogenic" constriction. In
contrast, arteries from diabetic animals were 5 mV more depolarized
than control arteries, and LNNA had no effect on the membrane potential
of these arteries. These results suggest that control arteries release
NO tonically, which causes a tonic membrane potential
hyperpolarization of 5 mV, and in arteries from
diabetic animals, this tonic release of NO and, hence, membrane
potential hyperpolarization are lacking. In further
support of this mechanism, the exogenous NO donor, SNP at 100
nmol/L, hyperpolarized arteries from diabetic animals by 6 mV
(Figs 9A and 10).
Since myogenic tone is thought to be a major contributor to vascular resistance and, hence, blood pressure,20 our observations are consistent with those of Huang et al38 that mice lacking the gene for endothelial NO synthase are hypertensive. Membrane depolarization could decrease the apparent sensitivity of a pressurized artery to KATP channel openers by increasing the contribution of other ionic conductances (voltage-dependent Ca2+ channels, KCa channels, and voltage-dependent K+ channels19) to the membrane conductance. This would decrease the relative contribution of KATP channel conductance to the overall membrane conductance; thus, a greater increase in the open state probability of KATP channels would be required (hence a higher concentration of KATP channel openers) for a change in membrane potential.22,23
Another possibility for the differences in KATP channel
opener response in diabetes would be that NO increases the contribution
of KATP channels to the membrane conductance or increases
KATP channel opener affinity for the channel, even through
voltage changes. However, GLIB (10 µmol/L) did not alter
diameter and membrane potential of arteries from normal rats (Fig 8),
suggesting that KATP channels, in the absence of
KATP channel openers, do not seem to contribute
significantly to the total membrane conductance under our in vitro
conditions.
Another key question that remains to be explored is how the tonically
released NO is hyperpolarizing the tissue. It has been shown that NO
activates guanylyl cyclase, thereby increasing levels of
cGMP.35 Subsequent stimulation of PKG can activate
KCa channels in the smooth muscle cells.39
Activation of these K+ channels would cause membrane
potential hyperpolarization, which would close
voltage-dependent Ca2+ channels.21 This would
lead to a decrease in intracellular Ca2+ and consequently
to vasodilatation. In support of this, SNP has been shown to cause
membrane potential hyperpolarization of pressurized
cerebral (Fig 9A) and coronary arteries.40 Other
mechanisms may contribute to the membrane potential–dependent
component of relaxation of arteries to NO, including the direct effect
of NO on an ion channel or "cross-activation" of PKG or PKA, which
can also activate KCa channels. In addition to the
direct activation of KCa channels by either PKA or PKG,
both these kinases could enhance KCa channel activity
through increasing the amplitude or frequency of
Ca2+-release events ("Ca2+ sparks")
originating from the SR. These release events are thought to occur
through ryanodine-sensitive Ca2+ channels in the
subsarcolemmal SR. Ca2+ sparks appear to be major
regulators of KCa channel activity in myogenic cerebral
arteries.41 Other reported cGMP-mediated effects resulting
in relaxation, such as inhibition of Ca2+ channels,
enhanced Ca2+ extrusion, and, especially, enhanced uptake
of Ca2+ into the SR, which could increase
Ca2+-spark frequency and/or amplitude, may
represent additional synergistic pathways by which NO relaxes
arterial smooth muscle.
Conclusion
In summary, we conclude that insulin-dependent diabetes mellitus
induced by STZ causes a membrane potential depolarization and
vasoconstriction42 (see Fig 10). These effects may be due
to an inhibition of tonic release of NO from the
endothelium. The mechanism by which diabetes affects NO
production remains to be determined. The decrease in NO release
or the membrane potential depolarization leads to a decrease in the
apparent effectiveness of KATP channel openers such as
pinacidil and levcromakalim (Fig 10).
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the National Heart, Lung, and Blood Institute grants HL-44455, HL-51728, and HL-67647 and National Science Foundation grant DCB-9019563. This study was performed during the tenure of a fellowship of the American Heart Association, Vermont Affiliate, Inc, to Dr Knot. We would like to thank Dr Issy Laher for helpful discussions regarding the manuscript.
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Footnotes |
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1 Both authors contributed equally to this study.
Received June 20, 1997; accepted September 19, 1997.
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References |
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