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1 Department of Cell Biology and Physiology and 2 Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131-5218
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Nitric
oxide (NO) synthase (NOS) inhibition with
N
-nitro-L-arginine
(L-NNA) produces L-NNA hypertensive rats (LHR),
which exhibit increased sensitivity to voltage-dependent
Ca2+ channel-mediated vasoconstriction. We hypothesized
that enhanced contractile responsiveness after NOS inhibition is
mediated by depolarization of membrane potential
(Em) through attenuated K+ channel
conductance. Em measurements demonstrated that
LHR vascular smooth muscle cells (VSMCs) are depolarized in open,
nonpressurized (
44.5 ± 1.0 mV in control vs. 36.8 ± 0.8 mV in LHR) and pressurized mesenteric artery segments (41.8 ± 1.0 mV in control vs. 32.6 ± 1.4 mV in LHR). Endothelium
removal or exogenous L-NNA depolarized control VSMCs but
not LHR VSMCs. Superfused L-arginine hyperpolarized VSMCs
from both the control and LHR groups and reversed
L-NNA-induced depolarization (44.5 ± 1.0 vs.
45.8 ± 2.1 mV). A Ca2+-activated K+
channel agonist, NS-1619 (10 µM), hyperpolarized both groups of
arteries to a similar extent (from 50.8 ± 1.0 to 62.5 ± 1.2 mV in control and from 43.7 ± 1.1 to 55.6 ± 1.2 mV
in LHR), although Em was still different in the
presence of NS-1619. In addition, superfused iberiotoxin (50 nM)
depolarized both groups similarly. Increasing the extracellular
K+ concentration from 1.2 to 45 mM depolarized
Em, as predicted by the Goldman-Hodgkin-Katz
equation. These data support the hypothesis that loss of NO activation
of K+ channels contributes to VSMC depolarization in
L-NNA-induced hypertension without a change in the number
of functional large conductance Ca2+-activated
K+ channels.
NS-1619; vascular smooth muscle cells; potassium channels; nitric oxide synthase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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VASCULAR SMOOTH MUSCLE
TONE, an important determinant of peripheral vascular resistance
and blood pressure, is largely determined by vascular smooth muscle
cell (VSMC) intracellular Ca2+ concentration. Vascular tone
is actively increased by a reduction of the absolute magnitude of VSMC
membrane potential (Em). Kuriyama et al.
(21) reported a range of Em for
VSMCs of 45 to 60 mV. In VSMCs, large conductance
Ca2+-activated K+ (BKCa) channels,
voltage-operated K+ channels, and ATP-sensitive
K+ (KATP) channels are the major contributors
to potassium conductance (gK), and activation of
any of these channels leads to VSMC hyperpolarization with a consequent
closure of voltage-dependent Ca2+ channels (VDCCs),
decreased Ca2+ entry, and vasodilation. Thus K+
channel activity is an essential determinant of vascular tone and
vessel diameter.
There is increasing evidence that changes in expression and
permeability of ion channels contribute to the vascular pathology of
hypertension by causing depolarization of VSMC
Em. Indeed, VSMC depolarizations of 5 to 20 mV
have been demonstrated in several models of hypertension (12, 24,
25, 34, 38). The mechanisms responsible for VSMC depolarization
in hypertension have not been precisely defined, although there is
evidence for a role of K+, Ca2+, and
Cl
channels. For example, studies of Ca2+
channel density have shown unchanged (40), increased
(20, 26, 32), or decreased expression (13) in
various models of hypertension. Liu et al. (23)
demonstrated an increase in expression of BKCa channels in
aortas isolated from spontaneously hypertensive rats. In contrast,
Silva et al. (38) demonstrated that triethylammonium
induced less depolarization in spontaneously hypertensive rat aortas
than in normotensive Wistar-Kyoto rat aortas, suggesting a change in
K+ channel activity or expression. Lamb et al.
(22) reported that increased Cl efflux may
contribute to the depolarization. These findings suggest that a
combination of altered channel expression and ion conductances may
account for VSMC depolarization in hypertension.
Endothelial cells release several vasoactive factors, including nitric oxide (NO), which can alter smooth muscle tone by hyperpolarizing VSMC Em (1, 2, 41). Several mechanisms have been proposed to explain NO regulation of Em. NO can elicit hyperpolarization both indirectly through activation of cGMP-dependent protein kinase and directly through opening of K+ channels. Specifically, cGMP-dependent protein kinase has been shown to increase the open probability of BKCa channels (5) and KATP channels (37), whereas others have demonstrated that NO directly stimulates BKCa channel opening (2, 27). These effects of NO are thought to contribute to its vasodilator actions.
We have reported previously that inhibition of NO synthase (NOS) with
N-nitro-L-arginine
(L-NNA) for 2 wk results in elevated vascular smooth muscle
sensitivity to depolarization-induced contraction (16).
Because NO can directly activate K+ channels and
hyperpolarize arterial smooth muscle cells, and because hypertension
has been associated with inhibition of certain K+ channels,
we hypothesized that inhibition of NO production in L-NNA
hypertensive rats (LHR) decreases K+ channel activation in
VSMCs. In the presence of other depolarizing conductances, this would
lead to a consequent depolarization of Em and
increased sensitivity to contractile agents that depend on VDCC
activation. This hypothesis was tested in VSMCs from LHR and control rats.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Male Sprague-Dawley rats (200-300 g) were divided into two groups: LHR and control. The LHR group drank water containing L-NNA (0.5 g/l), whereas control rats drank water alone. Systolic blood pressure was measured using an indirect tail-cuff method (plethysmographic detection, IITC; Woodland Hills, CA). After 2 wk of L-NNA treatment, when systolic blood pressure was elevated in the LHR group (control = 145.3 ± 1.2 mmHg and LHR = 211.9 ± 2.6 mmHg), animals were anesthetized using pentobarbital sodium (50 mg/kg) and exsanguinated. The superior mesenteric artery between the junction of the abdominal aorta and the second branch artery was rapidly removed. Arteries isolated for Em recording were placed in cold physiological saline solution (PSS) containing (in mM) 130 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4 · 7H2O, 14.9 NaHCO3, 5.5 dextrose, 0.026 CaNa2EDTA, and 1.6 CaCl2 at pH 7.4. Arteries were cleaned of connective tissue and surrounding fat. Depending on the protocol, artery segments were left intact or cut open longitudinally. In some preparations, endothelial cells were removed by gently rubbing the lumen with a cotton-tipped applicator. All procedures were approved by the animal use committee at the University of New Mexico and conform to National Institutes of Health guidelines for animal use.
Membrane potential recordings. VSMC Em was recorded in either pressurized or open, nonpressurized artery segments mounted in a temperature-controlled tissue bath at 37°C containing PSS bubbled with 95% O2-5% CO2. For most recordings, segments were cut open longitudinally, pinned lumen side up on the bottom of a small recording chamber (Warner Instruments), and superfused with oxygenated PSS. VSMCs were impaled from the luminal side to measure Em responses under basal conditions and in response to treatments including L-NNA, the NO donor S-nitroso-N-acetyl penicillamine (SNAP), endothelial removal, L-arginine, the BKCa channel agonist NS-1619, the BKCa channel antagonist iberiotoxin, and to increasing extracellular concentrations of K+ ([K+]o). For pressurized recordings, artery segments (5-7 mm) were secured at both ends with 7.0 silk to glass cannulas, stretched to in situ lengths, and pressurized to approximately in vivo pressures (80-100 mmHg, control = 85 ± 4.6 mmHg and LHR = 80 ± 2.6 mmHg). Individual smooth muscle cells were impaled with microelectrodes from the adventitial side. Em measurements performed under these conditions included baseline (before addition of an experimental drug to superfusate) responses to acute NOS inhibition with L-NNA and responses to exogenous addition of SNAP.
VSMCs were impaled with 3 M KCl-filled microelectrodes with tip resistances of 60-100 M
, and Em was
measured using standard intracellular techniques. Briefly,
Em was measured using a high-input impedance
amplifier (WPI Electro 705), filtered at 100 Hz, and recorded either
alone or simultaneously with intra-arterial pressure (Gould P-3
pressure transducer) on a dual-channel chart recorder (Gould
Scientific; Cleveland, OH) with Axoscope (Axon Instruments; Union City,
CA) software. Intracellular recordings were accepted only if
1) Em measurements exhibited sharp
negative deflections upon cell penetration; 2) a stable
recording was held for at least 1 min before experimental
manipulations; and 3) an abrupt return to baseline was
observed upon withdrawal of the electrode. Em was measured continuously both before and during the addition of
vehicle or drug.
Chemicals. Em was recorded in response to superfusion with the NO donor SNAP (10 µM in ethanol), L-NNA (10 µM in PSS), L-arginine (30 mM in PSS), the BKCa channel agonist NS-1619 (100 µM in ethanol), the BKCa channel antagonist iberiotoxin (50 nM), and to increasing extracellular concentrations of [K+]o. Vehicle alone did not affect Em in any preparation. All reagents were purchased from Sigma (St. Louis, MO).
Data analysis and statistics. The VSMC Em values are reported as means ± SE. For each experimental protocol, each n is the average Em of at least three successful individual impalements in a single tissue (stable for at least 1 min), and n represents the number of animals. Data were analyzed using two-way ANOVA, followed by the Tukey's post hoc test for all pairwise comparisons. Significant difference was determined at the P < 0.05 level.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Baseline Em.
VSMCs in open segments of superior mesenteric arteries were
significantly depolarized in arteries from LHR compared with controls (Fig. 1). Control VSMC
Em was 44.5 ± 1.0 mV versus LHR
Em of 36.8 ± 1.0 mV. Thus VSMCs in
arteries from LHR are in a relatively depolarized state compared with
control VSMCs.
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Effects of L-NNA on Em.
Effects of acute L-NNA administration on
Em were analyzed by adding L-NNA
(100 µM) to the superfusion solution. L-NNA caused a
significant depolarization in control VSMCs with
Em changing from 44.5 ± 1.0 to
39.0 ± 1.9 mV. Addition of the same concentration of
L-NNA to LHR VSMCs did not change VSMC
Em (from 36.8 ± 1.1 to 36.9 ± 2.0 mV; Fig. 2A). After
L-NNA addition, Em was not different between
LHR and control VSMCs. These data suggest that a portion of the
observed depolarization in VSMCs from LHR is due to persistent NOS
blockade.
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Effects of endothelium removal on Em.
The contribution of endothelial cell-derived factors to
Em was assessed by comparing
Em in two segments of the same artery: one with
the endothelium intact and one denuded of endothelium. Removal of the
endothelium did not change Em in VSMCs from LHR (from 36.9 ± 1.5 to 36.0 ± 2.6 mV); however,
endothelium removal resulted in depolarization in control VSMCs (from
45.3 ± 1.4 to 40.8 ± 2.4 mV; Fig. 2B).
Interestingly, this response was similar to that produced by chronic
NOS inhibition. In addition, after endothelium removal,
Em was not different between groups. These data
suggest that removal of the endothelial layer has a similar effect to
treatment with L-NNA, presumably by removing an endogenous
hyperpolarizing factor such as NO.
Effects of L-NNA and L-arginine on Em. The ability to reverse the NOS inhibition-induced depolarization was assessed in two separate experiments by adding excess NOS substrate L-arginine to the bathing solution. The first experiment assessed the ability of L-arginine to reverse depolarization after acute exposure to L-NNA. After Em was measured, segments were treated with 100 µM L-NNA and then exposed to L-arginine to determine whether acute L-NNA-induced depolarization could be reversed. In three of four arteries, L-arginine (30 mM) successfully reversed the L-NNA-induced depolarization (data not shown).
A second set of related experiments evaluated the ability of L-arginine to reverse chronic NOS inhibition depolarization of VSMCs in LHR arteries. Arteries were incubated with L-arginine (30 mM) for 30 min before Em was recorded. L-Arginine hyperpolarized VSMCs from both control rats and LHR. Em of VSMCs from LHR exposed to L-arginine was not different from Em of VSMCs from control rats in PSS (control:44.5 ± 1.0 mV vs.
L-NNA plus L-arginine: 45.8 ± 2.1 mV).
However, after L-arginine, VSMCs from LHR were still
depolarized compared with control VSMCs treated with
L-arginine (45.8 ± 2.1 vs. 55.4 ± 2.5 mV,
respectively; Fig. 3A). The
remaining Em difference between the groups after
treatment with L-arginine suggests that decreased NOS
activity does not fully account for the difference observed in resting
Em and indicates that a non-NOS component may
contribute to the depolarization in LHR arteries.
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Effects of SNAP on Em.
The ability of a NO donor (10 µM SNAP) to hyperpolarize VSMCs was
evaluated by adding SNAP or its vehicle to the bath after cell
impalement. Addition of SNAP hyperpolarized VSMCs in open segments from
LHR but did not significantly change the Em of
VSMCs from control animals. In the presence of SNAP, there was no
significant difference in the Em between LHR
(44.5 ± 2.1 mV) and control cells (48.0 ± 2.5 mV; Fig.
3B). Thus the NO donor caused a greater hyperpolarization in
LHR VSMCs, suggesting that endogenous NO activation of
gK is greater in control VSMCs. Therefore, VSMCs from LHR animals, which exhibited a larger NO-dependent
hyperpolarization, may possess fewer open NO-sensitive K+ channels.
BKCa channel agonist NS-1619 hyperpolarization.
The BKCa channel agonist NS-1619 (7, 33) was
used to determine whether maximal activation of these channels
increased Em more in VSMCs from LHR, similar to
the response to exogenous NO. NS-1619 or its vehicle was added to the
bath after cell impalement, and responses to NS-1619 were measured at
their maximum. Addition of NS-1619 caused hyperpolarization in VSMCs
from both the control and LHR groups (Fig.
4A). NS-1619 hyperpolarized
cells in both groups by a similar amount (control: from 50.8 ± 1.0 to 62.5 ± 1.2 mV and LHR: from 43.7 ± 1.1 to
55.6 ± 1.2 mV), so that the Em in the
presence of the agonist was still different between LHR and control
VSMCs. The Em difference in the presence of the BKCa agonist suggests that decreased activation of
BKCa channels does not explain the difference in
Em between VSMCs from LHR and control rats.
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Iberiotoxin-induced depolarizations.
The observation that the BKCa channel agonist NS-1619
hyperpolarized both control and LHR VSMCs by a similar magnitude
suggests that activity and/or expression of these channels are not
changed during L-NNA-induced hypertension. The
BKCa channel antagonist iberiotoxin was used to verify
these results and to determine whether blocking these channels would
depolarize control VSMCs more than LHR VSMCs. Baseline
Em was measured in at least three cells in a
segment. Iberiotoxin or its vehicle was then added to the bath, and
Em was recorded in at least an additional three cells. No change in Em was observed after the
addition of vehicle alone. In addition, at the end of each experiment,
NS-1619 was added to determine the efficacy of iberiotoxin. NS-1619 did
not affect Em in the presence of iberiotoxin
(data not shown). However, iberiotoxin depolarized VSMCs from both
groups by a similar amount (control: from 41.8 ± 0.4 to
34.7 ± 0.4 mV and LHR: from 36.6 ± 1.0 to 29.7 ± 0.6 mV; Fig. 4B). Em in the
presence of the antagonist was still different between LHR compared
with control VSMC Em. The remaining
depolarization in the presence of the antagonist provides further
evidence that decreased activation of BKCa channels does
not explain the difference in Em between VSMCs
from LHR and control rats.
Response to increasing
[K+]o.
The effect of varying [K+]o on
Em was analyzed by increasing
[K+]o in the bathing medium (equimolar
replacement of NaCl with KCl). Em was recorded
at [K+]o = 1.2, 3.0, 5.9, 10, 30, and 45 mM. KH2PO4 concentration (1.2 mM) remained
fixed to keep pH constant. Em versus log
[K+]o were fit using a least-squares linear
regression for [K+]o 
3.0 mM.
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Pressurized data.
To validate the physiological relevance of NO regulation of
Em, mesenteric segments were pressurized and
Em was recorded under basal conditions and after
the addition of L-NNA and SNAP. Pressurizing the artery
segments depolarized VSMCs in arteries from both groups compared with
pinned segments (Fig.
6A). Em
for VSMCs from control animals was 41.8 ± 1.0 mV in pressurized
segments and 32.6 ± 1.4 mV in pressurized arteries from LHR.
Thus VSMCs are still depolarized in LHR arteries after pressurization,
and the mean difference in Em between groups
becomes more pronounced with luminal pressure.
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41.2 ± 1.1 to 34.3 ± 1.3 mV). Also
similar to open segments, the addition of the same concentration of
L-NNA to pressurized segments from LHR arteries did not
cause depolarization (from 32.7 ± 0.9 to 32.9 ± 1.3 mV;
Fig. 6B). In the presence of L-NNA,
Em of pressurized vessels was not different
between groups. These data strongly suggest that a portion of the
observed depolarization in VSMCs from LHR is due to persistent NOS blockade.
Addition of SNAP to pressurized vessels caused a significant
hyperpolarization in both control and LHR vessels (control: from 42.8 ± 0.5 to 48.1 ± 0.6 mV and LHR: from 33.76 ± 0.5 to 46.0 ± 0.5 mV). However, in pressurized vessels
treated with SNAP, there was still a significant difference in
Em between LHR (46.0 ± 0.5 mV) and
control cells (48.1 ± 0.6 mV; Fig. 6C). Therefore, similar to data from open segments, the larger NO-dependent
hyperpolarization of LHR pressurized arteries further supports the
conclusion that LHR VSMCs may possess fewer open NO-sensitive
K+ channels.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The major findings of the present study are as follows. First,
VSMCs of mesenteric arteries from LHR were depolarized close to the
potential for activation of VDCCs, 30 to 40 mV (9, 36). Second, NO removal, by endothelial cell removal or addition of L-NNA, only depolarized VSMCs from control rats. Third,
increased NO availability, either endogenously (by adding
L-arginine) or exogenously (by adding SNAP) hyperpolarized
cells from both groups. Fourth, the NO donor SNAP hyperpolarized LHR
VSMCs more than controls. Fifth, the BKCa channel agonist
NS-1619 hyperpolarized VSMCs from both control rats and LHR, whereas
the BKCa channel antagonist iberiotoxin depolarized VSMCs
from both LHR and control rats. However, in the presence of the
antagonist or agonist, Em was still different
between the two groups. Sixth, increasing
[K+]o depolarized Em
in an approximately Nernstian manner with a steeper slope for control
VSMCs, suggesting that gK makes a larger contribution to Em in VSMCs from control rats.
Finally, pressurizing superior mesenteric arteries caused a
depolarization of 7-10 mV and augmented the difference in
Em between control and LHR VSMCs. In addition,
responses to L-NNA or SNAP were qualitatively similar in
open and pressurized segments. These important and novel findings are,
to our knowledge, the first report of altered Em
regulation in NOS inhibition hypertension and the first to demonstrate
that in vivo NOS inhibition elicits Em depolarization.
The purpose of this study was to determine whether membrane
depolarization, resulting from decreased NO-stimulated
gK, could account for the increased contractile
sensitivity to 
2-adrenergic agonists and to the
depolarization-induced contraction reported previously for mesenteric
arteries from NOS inhibition-treated rats (16). Many
studies have reported a lack of contractile responses to
2-agonists except in the presence of depolarizing agents. Therefore, depolarization of Em could
account for the increased 2-adrenoceptor sensitivity in
NOS inhibition hypertension. On the basis of these observations, we
hypothesized that increased contractile sensitivity of vascular smooth
muscle to 2-adrenergic agonists in arteries from
NOS-inhibited rats is caused in part by VSMC depolarization. Our
measurements of Em in both pressurized and open
arteries support this hypothesis and show that VSMCs from LHR display
an Em that is ~9 mV more depolarized than that of VSMCs from control rats. In agreement with our findings, several other studies have demonstrated VSMC depolarization in the mesenteric bed in other models of hypertension (3, 8, 10). We report here that Em in mesenteric artery VSMCs from LHR
are depolarized to a level near the activation potential for VDCCs
(9, 36). This was true in both open and pressurized artery
segments, suggesting that membrane depolarization contributes to
elevated arterial contractile responses during systemic NOS inhibition.
VSMC depolarization has been observed and attributed to changes in membrane conductances including decreased gK (24), increased calcium conductance (20), or increased chloride conductance (12) in several models of hypertension. Previous studies demonstrating that VSMCs are depolarized in arteries from hypertensive animals suggest that elevated arterial pressure may directly modulate ion channel function or expression to depolarize VSMCs. In addition, it has been shown that endothelial derived factors, including NO, endothelium-derived hyperpolarizing factor, and prostaglandins, hyperpolarize smooth muscle through activation of gK. Therefore, loss of hyperpolarizing endothelial factors in hypertension could also be responsible for the depolarization. In the present study, endothelial cell removal and exposure to exogenous L-NNA were used to separate the contribution of loss of endothelial NO activation of K+ channels and VSMC loss of channel expression to the observed depolarization in LHR VSMCs. That is, if the change in Em is caused by loss of NO, then eliminating NO synthesis should cause control VSMCs to have an Em that is similar to LHR VSMC Em. If the change was intrinsic to VSMCs, then there should still be a difference in Em, even in the absence of NO synthesis in control arteries.
Acute exposure to L-NNA or removal of the endothelium depolarized only control VSMCs, suggesting that the depolarization of Em observed in LHR VSMCs is caused mostly by the loss of endothelial NO production. Indeed, addition of a NOS inhibitor directly to control vascular segments depolarized VSMCs, as has been reported by other investigators (29, 35). Similarly, depolarization of VSMCs after endothelium removal has also been observed previously (11). In this study, endothelium removal caused depolarization only in control arteries, indicating that basal release of endothelial hyperpolarizing factors contributes to VSMC Em in these vessels. This depolarization is presumably due to the loss of NO activation of gK. However, Em was not altered by removing the endothelium from LHR arteries and was still depolarized compared with control VSMCs even in the absence of endothelium. This suggests that there is an intrinsic change in VSMCs that contributes to the depolarization of Em in VSMCs from LHR.
L-Arginine reversal of L-NNA-induced depolarization further supports the hypothesis that endothelial cell production of NO contributes to resting Em. This was observed for blockade of NOS with either acute or chronic exposure to L-NNA and suggests that loss of NO activation of gK can explain in part the depolarization in LHR VSMCs. L-Arginine restored Em in LHR VSMCs close to the resting Em of VSMCs from control rats. However, even after exposure to 300 times more L-arginine than L-NNA (30), VSMCs from LHR were still depolarized compared with control VSMCs exposed to the same L-arginine concentration. This persistent difference in Em between the two groups suggests that a non-NOS-dependent component contributes to the observed depolarization.
One potential mechanism for NO-mediated hyperpolarization is activation of K+ channels. Direct application of exogenous NO to isolated arteries has been shown to cause hyperpolarization and vasodilation through activation of BKCa channels (2, 19) as well as other K+ channels (28, 29). By this mechanism, chronic inhibition of NOS could cause VSMC depolarization by removing an endogenous activator of BKCa channels. Under these conditions, replacing NO should restore Em to control levels. In support of this, the NO donor SNAP caused a significant hyperpolarization only in VSMCs from LHR, suggesting that in the control tissue, NO activation of gK is at its maximum. Therefore, VSMCs from LHR appear to have fewer NO-sensitive K+ channels open.
BKCa channels contribute significantly to resting Em in VSMCs and provide a mechanism to oppose depolarization and subsequent vasoconstriction (4, 31). Addition of the BKCa channel agonist NS-1619 (7, 33) was used to assess the ability of BKCa channels to hyperpolarize VSMCs from control and LHR. A maximal concentration of NS-1619 hyperpolarized both control and LHR VSMCs. However, even in the presence of the agonist, VSMCs from LHR were significantly depolarized compared with controls, suggesting that decreased conductance of BKCa channels (gBKCa) does not account for the difference in Em between the two groups. If a decrease in channel open probability were causing the depolarization in LHR VSMCs, NS-1619 addition should have produced a similar Em in both groups. These data suggest a change independent of gBKCa, presumably another component of gK, accounts for the difference in Em.
The previously reported increased sensitivity to KCl depolarization of arteries from LHR (16) suggests that Em is further from EK and closer to the activation potential for VDCCs in VSMCs from LHR than in VSMCs from control rats. In this study, decreasing the driving force for K+ by a few millivolts was sufficient to depolarize Em to the activation threshold for VDCCs only in LHR arteries. This could not be caused by a change in the number of VDCCs, but could be caused by either a decreased number or decreased open probability of K+ channels. In fact, a decrease in gK is consistent with all of our observations and supports the previous findings of Borges et al. (3): that K+ channel function is impaired in hypertension and this decrease in gK contributes to the elevated Em. The approximately Nernstian relationship exhibited in response to increasing [K+]o supports the importance of gK in setting Em. Moreover, the depolarization in VSMCs from LHR and steeper slope for Em versus log [K+]o in control VSMCs suggests that there is a decrease in gK during NOS inhibition hypertension. The deviations from linearity at low [K+]o are predicted by the Goldman-Hodgkin-Katz voltage equation due to an additional depolarizing conductance and may also reflect a contribution of the electrogenic pump.
Pressurization of superior mesenteric arteries was used to determine if the alterations in Em were present under more physiological conditions and not an artifact of recording in unpressurized segments. We observed that pressurization caused depolarization of Em in the superior mesenteric artery from both LHR and control rats. Previous studies have reported that increases in intraluminal pressure cause depolarization resulting in myogenic constriction (6, 15, 17, 18, 42). Similarly, myogenic responses have been previously observed in the rat superior mesenteric artery (14, 39). Although vessel diameter was not recorded in the current study, the depolarizations observed suggest that these vessels exhibit a myogenic response to increased wall stress. However, the difference in Em between LHR and control arteries was still present in pressurized vessels, suggesting that the altered conductance(s) responsible for depolarization in the LHR arteries are not affected by stretch.
In summary, the current study demonstrates that VSMCs are depolarized in mesenteric arteries from LHR. This may contribute to augmented contractile sensitivity and thus to the elevated peripheral resistance in NOS inhibition hypertension. This depolarization is due in part to loss of NO activation of gK but does not appear to result entirely from a loss of BKCa conductances. These results suggest that endothelial dysfunction resulting in decreased NO production may be the underlying factor in at least some of the vascular hypersensitivity in NO-deficient hypertension.
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ACKNOWLEDGEMENTS |
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The authors thank Pam Allgood for expert technical assistance and Dr. Bill Shuttleworth for helpful discussion of the data and assisting in the experimental design.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute HL-03852, a Scientist Development Award from the American Heart Association, and a Research Allocations Committee grant from University of New Mexico.
Address for reprint requests and other correspondence: Ian N. Bratz, Vascular Physiology Research Division, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, Albuquerque, NM 87131-5218 (E-mail: ibratz{at}salud.unm.edu).
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.
10.1152/ajpheart.00824.2001
Received 19 September 2001; accepted in final form 7 January 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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