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1 Department of Physiology and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 2 Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study examined the response to
nitric oxide (NO) in rat middle cerebral arteries (MCA). NO donors
increased the activity of a 205-pS K+ channel recorded from
vascular smooth muscle (VSM) cells isolated from MCA 10-fold. Blockade
of guanylyl cyclase activity with
1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10
5 M) did not alter the effect of NO on this
channel. In contrast, adding 20-hydroxyeicosatetraenoic acid (20-HETE)
to the bath (107 M) abolished the response to NO. NO
donors also increased the diameter of serotonin-preconstricted MCA to
85% of control. Blockade of K+ channels with iberiotoxin
or a high-K+ medium reduced this response by 50%. ODQ
(105 M) reduced this response by 47 ± 3%, whereas
preventing the fall of 20-HETE levels reduced the response by 59 ± 2% (n = 5). Blockade of both pathways eliminated
the response to NO donors. These results indicate that activation of
K+ channels contributes 50% to vasodilator response to NO
in rat MCA. This is mediated by a fall in 20-HETE levels rather than a
rise in cGMP levels or a direct effect of NO.
vascular smooth muscle; cytochrome P-450; potassium ion channels; cerebral circulation; 20-hydroxyeicosatetraenoic acid
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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RECENT STUDIES
HAVE INDICATED that nitric oxide (NO) plays a central role in the
tonic regulation of vascular tone (7, 10,
22) and contributes to the vasodilator responses to
acetylcholine, substance P, bradykinin, 
2-adrenergic
receptor agonists, and angiotensin II (7, 8,
10, 11, 41) in the cerebral circulation. NO also contributes to the increase in cerebral blood flow
produced by hypercapnia and inhalational anesthetics, and impairments
in NO-mediated vascular relaxation are thought to play a role in the
cerebral vasospasm following subarachnoid hemorrhage (11,
38).
Despite the importance of NO in the control of cerebral vascular tone, considerable uncertainty exists about its mechanism of action. It has been generally assumed that the vasodilator response to NO in the cerebral circulation is secondary to stimulation of guanylyl cyclase (11) and involves activation of K+ channels (20, 28, 29, 31, 34, 37) similar to what has been described in the peripheral circulation (3, 5, 6, 9, 21, 23, 26, 30, 31, 39). Activation of these channels hyperpolarizes VSM cells and limits Ca2+ influx through voltage-sensitive Ca2+ channels (11, 27). Elevations in cGMP also reduce vascular tone by stimulating the reuptake of Ca2+ in the sarcoplasmic reticulum and by diminishing the sensitivity of the contractile mechanism to Ca2+ (19, 24, 33, 42, 43, 45).
The role that activation of K+ channels plays in the vasodilator response to NO in the cerebral circulation, however, remains controversial. A few investigators have reported that blockade of Ca2+-sensitive K+ (KCa) channels with iberiotoxin (IbTX) has little effect on the vasodilator response to NO and acetylcholine in rabbit pial arterioles (40) and the basilar artery of the rat (36). However, blockade of KCa channels diminishes the response of canine middle cerebral arteries (28, 29), rat basilar arteries (20), and rat pial arterioles (31) to acetylcholine and NO donors. The reasons for the divergent results are unknown.
There is also a lack of a consensus regarding the exact role of cGMP in the vasodilator response to NO in the cerebral circulation. The results of in vivo studies indicating that an inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), blocks 80% of the vasodilator response to acetylcholine and NO in pial arteries of the mouse, rat, and rabbit (4, 12, 35) and in the basilar artery of the rat (37) support a primary role for cGMP in mediating the response to NO. However, this conclusion is based on the assumption that ODQ is a specific inhibitor of soluble guanylyl cyclase, and this view has recently been challenged. In this regard, Feelish et al. (13) found that ODQ blocks many heme-containing enzymes, including NO synthase and the P-450 enzyme that catalyzes the release of NO from sodium nitroprusside (SNP) and other nitrate-containing donors. Moreover, there are reports that the dilator response to NO in airway smooth muscle (32, 44), the aorta (6, 13), and pulmonary (17), mesenteric (9, 26), carotid (33), cerebral (25, 29), and renal arteries (1, 2, 39) cannot be blocked by inhibitors of guanylyl cyclase or protein kinase G. These observations have led to a search for cGMP-independent pathways by which NO may promote vasodilation. In this regard, Bolotina et al. (6) reported that NO has a direct effect to activate KCa channels in vascular smooth muscle (VSM) isolated from rabbit aorta. They further proposed that this mechanism may mediate the cGMP-independent actions of NO on vascular tone. Roman and co-workers (1, 39) have also demonstrated that NO binds to heme in enzymes of the P-450 4A family and inhibits the formation of a potent vasoconstrictor, 20-hydroxyeicosatetraenoic acid (20-HETE). The subsequent fall in 20-HETE levels appears to mediate the activation of K+ channels and much of the vasodilator response to NO in the renal circulation (1, 2, 39). However, the contribution of this pathway to the vasodilator response to NO in the cerebral circulation has yet to be explored.
The purpose of the present study was to evaluate the relative contribution of cGMP-dependent versus -independent pathways in mediating the vasodilator response to NO in rat middle cerebral arteries. Given the evidence that cerebral arteries express mRNA and protein for the P-450 4A enzymes that produce 20-HETE (15, 16), we further examined whether a fall in 20-HETE levels contributes to the cGMP-independent actions of NO on K+ channels and vascular tone in the cerebral circulation.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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General. Experiments were performed on 83 10- to 12-wk-old male Sprague-Dawley rats purchased from Harlan Sprague Dawley (Indianapolis, IN). The rats were housed in an animal care facility at the Medical College of Wisconsin that is approved by the American Association for the Accreditation of Laboratory Animal Care. The protocols involving animals received approval from the Animal Care Committee of the Medical College of Wisconsin.
Patch-clamp studies. VSM cells were isolated from first-order branches of middle cerebral arteries (inner diameter <100 µm) microdissected from the brain of rats. The arteries were incubated for 10 min at room temperature in a low-Ca2+ dissociation solution containing (in mM) 145 NaCl, 1 MgCl2, 4 KCl, 0.05 CaCl2, 10 glucose, and 10 HEPES (pH 7.4) as well as 1 mg/ml albumin. The vessels were incubated for 15 min at 37°C in the same solution containing 1.5 mg/ml papain (14 U/mg) and 1 mg/ml dithiothreitol (DTT). This was followed by incubation for 15 min at 37°C in a solution containing 2 mg/ml collagenase (196 U/ml), 0.5 mg/ml elastase (90 U/ml), and 1 mg/ml soybean trypsin inhibitor (10,000 U/ml). The supernatant was collected, and the cells were spun down at 500 g for 5 min. The pellet was resuspended in fresh dissociation solution and stored at 4°C. Patch-clamp experiments were completed within 4 h after the cells were isolated.
Single-channel K+ currents were recorded from VSM cells isolated from rat middle cerebral arteries using the cell-attached, patch-clamp mode at room temperature. The bath solution contained (in mM) 145 KCl, 0.37 CaCl2, 1.1 MgCl2, 10 HEPES, and 1 EGTA (pH 7.4), and the pipettes were filled with a solution containing (in mM) 145 KCl, 1.8 CaCl2, 1.1 MgCl2, and 5 HEPES (pH 7.4). In preliminary experiments, we found that NO donors had similar effects to activate K+ channels in VSM cells bathed in normal physiological salt solution (PSS) and a solution containing a high K+ (145 mM) and a low Ca2+ (107 M) concentration. Thus all of
the present studies were performed using the high-K+,
low-Ca2+ solution to null membrane potential and minimize
the possibility that changes in intracellular Ca2+
concentration could contribute to the response of the K+
channels to NO. We originally began these studies using SNP as the sole
NO donor. Subsequently, a new class of NO donors that spontaneously
release NO in aqueous solutions without generating cyanide became
available. We found that one of these compounds, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate) has effects similar
to SNP on vascular tone and K+ channels in rat middle
cerebral arteries. Thus both compounds were used as NO donors in the
present studies.
The patch-clamp pipettes were constructed from 1.5-mm borosilicate
glass capillaries pulled using a two-stage micropipette puller (model
PC-87; Sutter Instrument, San Rafael, CA) and heat-polished using a
microforge. The pipettes had a tip resistance of 8-10 M
.
Cerebral VSM cells were allowed to attach to a glass coverslip on the
bottom of a 1-ml perfusion chamber mounted on the stage of an inverted
microscope. After the tip of a pipette was positioned on a cell, a
high-resistance seal (5-20 G) was formed by applying light
suction. An Axopatch 200B amplifier (Axon Instruments, Foster City, CA)
was used to clamp pipette potential and record single-channel currents.
The amplifier output signals were filtered at 2 kHz using an eight-pole
Bessel filter (Frequency Devices, Haverhill, MA). The currents were
digitized at a rate of 10 kHz and stored on the hard disk of a computer
for off-line analysis. Data acquisition and analysis were performed
using pCLAMP software (version 7.02, Axon Instruments). Open-state
probability (NPo) for single-channel currents,
expressed as a percentage of the total recording time in which a
channel was open, was calculated using the following equation
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Effects of NO on K+ channel activity.
Single-channel K+ currents were recorded from cell-attached
patches on VSM cells isolated from the middle cerebral arteries at a
pipette potential of 
40 mV during a 2-min control period. The bath
was then exchanged with a solution containing 105 M SNP,
and K+ channel activity was recorded again after a 5-min
equilibration period.
40 mV during a
2-min control period. SNP at concentrations of 105,
104, and 103 M was then added to the bath,
and K+ channel activity was recorded again after a 3-min
equilibration period.
Effect of blockade of cGMP pathway on the response to NO.
Baseline K+ currents were recorded from cell-attached
patches on rat middle cerebral artery VSM cells during a 2-min control period at a pipette potential of 40 mV. The bath was exchanged with a
solution containing an inhibitor of soluble guanylyl cyclase, ODQ (10 µM), or vehicle (0.1% ethanol). After a 10-min equilibration period,
the effects of ODQ on K+ channel activity were determined
during a 2-min experimental period. SNP (105 M) was then
added to the bath, and after a 5-min equilibration period,
K+ currents were recorded again during a second
experimental period.
Effect of blockade of the 20-HETE pathway on the response to NO
donors.
We examined whether preventing the fall in intracellular 20-HETE levels
by adding 20-HETE (100 nM) to the bath could block the effects of NO on
K+ channel activity. We assumed that adding 20-HETE to the
bath would clamp intracellular levels during the experiment, because 20-HETE is highly lipid soluble and can easily equilibrate between the
intracellular and extracellular compartments. In these experiments, baseline K+ channel activity was recorded during a 2-min
control period at a patch potential of 40 mV. Vehicle (0.1% ethanol)
or 20-HETE (100 nM) was added to the bath, and K+ channel
activity was recorded during a 2-min experimental period. SNP
(105 M) was then added to the bath, and after a 5-min
equilibration period, K+ channel activity was recorded
again during a second experimental period.
Effect of 20-HETE and 17-octadecynoic acid on K+
currents.
We examined whether blockade of the production of 20-HETE in rat middle
cerebral artery VSM cells would activate K+ channels such
as NO and determined whether this effect could be reversed by adding nM
concentrations of 20-HETE to the bath. Baseline K+ channel
activity was recorded from cell-attached patches on rat middle cerebral
artery VSM cells during a 2-min control period at a pipette potential
of 40 mV. 17-Octadecynoic acid (17-ODYA; 1 µM) was added to the
bath, and after a 15-min equilibration period, K+ channel
activity was recorded during a 2-min experimental period. 20-HETE at a
concentration of 10 or 100 nM was then added to the bath, and after a
2-min equilibration period, K+ channel activity was
recorded again during a second 2-min experimental period.
Isolated middle cerebral artery studies. Adult male rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt), the brain was removed, and small branches of the middle cerebral artery (inner diameter <100 µm) were microdissected under a microscope. The arteries were mounted on glass micropipettes in a perfusion chamber filled with PSS containing (in mM) 119 NaCl, 4.7 KCl, 1.17 MgSO4, 1.6 CaCl2, 12 NaHCO3, 1.18 NaH2PO4, 0.03 EDTA, and 10 glucose, pH 7.4. The PSS was equilibrated with 95% O2-5% CO2 and maintained at 37°C. The vessels were secured to the pipettes with 10-0 silk suture, and the side branches were tied off. The inflow pipette was connected to a reservoir to allow for control of intraluminal pressure that was monitored with a transducer (Cobe, Lakewood, CO). After they were mounted, the vessels were stretched to their in vivo length using an eyepiece micrometer. The outflow cannula was clamped off, and intraluminal pressure was maintained at 90 mmHg during the experiment. Vascular diameters were measured with a video system composed of a stereomicroscope (Carl Zeiss), a CCTV camera (model KP-130AU, Hitachi), a videocassette recorder (model CVM-1271, Sony), and a video measurement system (VIA-100, Boeckeler Instrument, Tucson, AZ).
Preliminary experiments were performed to determine the best experimental condition in which to study the effects of NO donors on rat middle cerebral arteries in vitro. Pressure-diameter relations were first studied in rat middle cerebral arteries (n = 6). In these experiments, vascular diameter increased as the luminal pressure was increased from 0 to 30 mmHg and reached a maximum at 40 mmHg. The vessels then constricted by 15 ± 3% as pressure was increased from 40 to 100 mmHg. The minimum diameter was reached at a transmural pressure of 90 mmHg. Thereafter, vascular diameters increased when transmural pressure was raised above 100 mmHg. Because the vessels reached a minimum diameter at 90 mmHg, we chose this as the pressure at which to study the effects of NO on rat middle cerebral arteries. Other studies examined the effects of changes in transmural pressure and baseline vascular tone on the vasodilator responses to SNP. SNP (105 M) increased the diameter of six vessels on average
by 44 ± 4% at a transmural pressure of 60 mmHg, by 56 ± 3% at a pressure of 90 mmHg, and by 98 ± 5% at a transmural
pressure of 90 mmHg after the vessels were preconstricted with
serotonin (107 M). Therefore, all of the present studies
were performed in serotonin-preconstricted vessels using a transmural
pressure of 90 mmHg because the response to NO donor rat middle
cerebral arteries was greatest under these conditions.
Role of K+ channels in vasodilator response to NO.
The contribution of activation of KCa channels to the
vasodilator response to NO in rat middle cerebral arteries was assessed by comparing the response to SNP in the presence or absence of the
KCa channel blocker IbTX. Concentration-response curves to SNP (107-103 M) were constructed under
control conditions and after addition of IbTX (107 M) to
the bath. The contribution of other types of K+ channels to
the vasodilator response to NO was also assessed by comparing the
response to SNP before and after the addition of a depolarizing
concentration of KCl (80 mM) to the bath. The composition of the
high-K+ bath solution consisted of (in mM) 43.7 NaCl, 80 KCl, 1.17 MgSO4, 1.6 CaCl2, 12 NaHCO3, 1.18 NaH2PO4, 0.03 EDTA,
and 10 glucose, pH 7.4.
Effects of cGMP-dependent and -independent mechanisms on the
vasodilator response to NO donors.
The contribution of changes in cGMP versus 20-HETE levels to the
vasodilator effect of NO was evaluated by comparing the response of the
middle cerebral artery to DEA-NONOate (109 to
105 M) before and after soluble guanylyl cyclase was
inhibited with ODQ (105 M) or before and after
intracellular 20-HETE levels were fixed at 107 M by
adding 20-HETE to the bath. The effects of blocking both pathways
simultaneously were also studied by adding ODQ and 20-HETE to the bath.
These experiments were performed by using DEA-NONOate as the NO donor
to eliminate the possibility that the effect of ODQ was due to
inhibition of the catalytic release of NO from SNP by the tissue
(13).
7 M), and the
vasodilator response to acetylcholine (106 M) was
measured to verify that the endothelium was removed. The bath was
exchanged with fresh PSS, and the vessels were again preconstricted
with serotonin. The vasodilator responses to SNP (103 M)
and to inhibitors of the formation of 20-HETE, 17-ODYA (1 µM),
and N-methylsulfonyl-12,12-dibromododec-11-enamide
(DDMS; 20 µM) were compared. In other experiments, the effects of the KCa channel activator
1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619; 100 µM) were examined. Finally, we compared the vasodilator responses to DEA-NONOate (109-105 M)
under control conditions and after blockade of the formation of 20-HETE
with 17-ODYA (1 µM) or DDMS (20 µM). The concentrations of the
inhibitors used were based on the results of previous studies indicating that 1 µM 17-ODYA completely blocks the formation of 20-HETE in renal (47) and cerebral vascular tissue
(15) and that 20 µM DDMS selectively inhibits the
formation of 20-HETE in microsomes prepared from the renal cortex of
rats (1).
Measurement of NO concentration.
The steady-state concentration of NO following the addition of various
concentrations of DEA-NONOate and SNP to the bath was measured using a
2-mm-diameter gas-permeable membrane NO Sensor (Iso-NOP) and a NO Meter
(World Precision Instruments, Sarasota, FL). The meter was calibrated
by chemically reducing known amounts of potassium nitrite in the
presence of KI and H2SO4. In these experiments,
various concentrations of SNP (107-103
M) or DEA-NONOate (109-105 M) were
added to the isolated vessel chamber containing 20 ml of PSS saturated
with 95% O2-5% CO2 at 37°C. The NO
concentration in the bath was continuously recorded. Typically, we
found that the NO concentration reached a steady-state value 3 min
after addition of the NO donors, and this gradually declined with a half-life of ~20 min for DEA-NONOate and 40 min for SNP.
Effect of NO donors on cGMP levels in cerebral arterioles. Cerebral microvessels were prepared using a modification of an Evans blue sieving method, recently developed for the isolation of renal arterioles (2). In each of these experiments, six rats were anesthetized with pentobarbital sodium (50 mg/kg body wt), and the carotid arteries were cannulated in a retrograde manner. The chest was opened to stop the heart, and the cerebral circulation was flushed with 10 ml of a Tyrode solution containing (in mM) 145 NaCl, 6 KCl, 4.2 NaHCO3, 1 MgCl2, 0.05 CaCl2, 10 HEPES, and 10 glucose (pH 7.4). The cerebral circulation was filled with 10 ml of a Tyrode solution containing 6.0% albumin stained with 0.1% Evans blue. The brains of the six rats were rapidly removed and pushed through a 180-µm sieve with the barrel of a 30-ml glass syringe. Intact vascular trees were collected from the sieve and incubated for 30 min at 37°C in 10 ml of a Tyrode solution containing collagenase (196 U/ml), soybean trypsin inhibitor (10,000 U/ml), DTT (1 mg/ml), and albumin (1 mg/ml) to remove brain tissue. This digest was filtered onto a 75-µm nylon mesh and rinsed with 20 ml of fresh Tyrode solution. The filter was immersed in ice-cold Tyrode solution, and cerebral arteries filled with Evans blue were identified with a stereomicroscope and collected by microdissection.
Approximately 500 mg of cerebral microvessel tissue were isolated in each experiment. The vessels were preincubated for 30 min at 37°C in 5 ml of PSS on a shaking water bath. The microvessel preparation was then transferred to glass vials containing 1 ml of PSS and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) to prevent breakdown of cGMP generated during the incubation. Control vessels were incubated with PSS, IBMX, and vehicle. DEA-NONOate (106 M) with or
without 10 µM ODQ was added to the experimental incubations. The
samples were incubated for 30 min at 37°C with constant shaking. The
reactions were terminated by adding trichloroacetic acid to a final
concentration of 2.5%. The samples were homogenized and centrifuged at
11,000 g for 20 min at 4°C, and cGMP levels in the
supernatant were assayed using a direct cGMP enzyme-linked immunoassay
purchased from Assay Designs (Ann Arbor, MI). The range of the
standard curve was 0.1-100 pg/tube, and the intra-assay coefficient of variation of control samples averaged <5%. The pellet
was resuspended in 0.1 ml of 1 N NaOH, and the protein concentration of
each sample was determined using the Bradford dye-binding procedure
(Bio-Rad, Hercules, CA) to allow for normalization of cGMP levels per
milligram of tissue protein.
Drugs and chemicals. All chemicals were of analytic grade. Collagenase type II was purchased from Worthington Chemical (Freehold, NJ). DEA-NONOate was purchased from Calbiochem (La Jolla, CA). ODQ was purchased from Alexis (San Diego, CA). NS-1619 was purchased from RBI (Natick, MA). 17-ODYA was obtained from Biomol (Plymouth Meeting, PA). DDMS and 20-HETE were synthesized by J. R. Falck. Other chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO).
Statistics. Values are presented as means ± SE. Statistical differences in values between and within groups were examined with the use of ANOVA for repeated measures followed by Duncan's multiple range test. A value of P < 0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of NO on K+ channel activity.
K+ channel activity was recorded from cell-attached patches
on cerebral VSM cells before and after addition of the NO donor SNP
(105 M) to the bath (Fig.
1). Under control conditions, three types of K+ channels with large (Fig. 1A),
intermediate (Fig. 1B), and small (Fig. 1C)
single-channel currents were recorded at a pipette potential of 40
mV. The opening of the large-amplitude K+ channel gradually
increased and reached a stable plateau value 5 min after SNP
(105 M) was added to the bath. The time course of the
rise in K+ channel activity paralleled the time needed to
achieve a steady-state concentration of NO in the bath after SNP was
added. Typically, the increase in channel activity was sustained for 20 min, and channel activity returned to control after SNP was removed
from the bath. On average, SNP (105 M) increased the
NPo of the large-conductance K+
channel 10-fold. Mean open time increased from 0.51 ± 0.13 to 0.68 ± 0.19 ms. The number of channel openings increased from 8 ± 2 to 53 ± 9 events/2 min. Identical effects were seen
when DEA-NONOate (107 M) was used as the NO donor. At the
concentrations used in these experiments, both SNP and DEA-NONOate
produced equivalent steady-state levels of NO of ~40 nM in the bath.
The NO donors also increased the NPo of the
intermediate conductance K+ channel from 0.018 ± 0.006 to 0.058 ± 0.011 (n = 5, P < 0.05). They had no significant effect on the
NPo of the small-conductance K+
channel recorded from these cells.
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8 to
106 M) and was blocked by tetraethylammonium (1 mM) and
IbTX (107 M) (data not shown). Thus the pharmacological
and biophysical properties of the channel activated by NO are
consistent with those of the large-conductance KCa channel
(14, 27).
The effects of various concentrations of SNP
(105-103 M) on K+ channel
activity in inside-out patches excised from rat middle cerebral artery
VSM cells are presented in Fig.
2. Addition of SNP to the bath
(cytosolic face of the patch) had no significant effect on
K+ channel activity in excised patches at concentrations of
105 and 104 M. However, a higher
concentration of SNP (103 M), which raised bath NO
concentration to 200 nM, increased the NPo of
the large-conductance K+ channel 10-fold.
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Effect of blockade of cGMP versus 20-HETE pathways on the
effects of NO on K+ channel activity.
Administration of vehicle (0.1% ethanol) had no effect on the
ability of the NO donor to activate KCa channels. In these
experiments, addition of SNP (105 M) to the bath
produced a 10-fold increase in the NPo of
the KCa channel from 0.010 ± 0.002 to 0.123 ± 0.040 (Fig.
3A).
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Effect of blockade of 20-HETE production on K+ channel
activity in VSM cells isolated from middle cerebral arteries.
Representative tracings depicting the effects of 17-ODYA on
K+ channel activity recorded from cell-attached patches of
VSM cells isolated from rat middle cerebral arteries are presented in
Fig. 5. Addition of 17-ODYA (1 µM) to the bath produced a sixfold increase in the
NPo of the large-conductance KCa
channel. However, the unitary current amplitude was not significantly
altered. Addition of 20-HETE (100 nM) to the bath reversed the effects
of 17-ODYA on K+ channel activity. These results indicate
that sufficient 20-HETE is endogenously produced in rat cerebral artery
VSM cells to modulate K+ channel activity and that resting
intracellular levels of 20-HETE are normally in the range of
10-100 nM.
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Role of K+ channels in vasodilator response to NO.
The contribution of activation of K+ channels to the
vasodilator response to NO in middle cerebral artery of rats was
assessed by comparing the response to an NO donor before and after
blockade of KCa channels with IbTX (107 M) or
after the addition of a depolarizing concentration of KCl (80 mM) to
the bath (Fig. 6). Under control
conditions, SNP (107-103 M) dose
dependently dilated serotonin (107 M)-preconstricted
middle cerebral arteries by 69 ± 7% (n = 6 vessels from 6 rats). Blocking KCa channels with IbTX
(107 M) reduced the vasodilator response to SNP by about
two-thirds. Adding a depolarizing concentration of KCl to the bath
reduced the vasodilator response to NO by one-half (Fig. 6).
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Contribution of cGMP to the vasodilator response to NO in middle
cerebral arteries.
The contribution of cGMP to the vasodilator response to NO was
determined by comparing the response to DEA-NONOate under control conditions and after blockade of soluble guanylyl cyclase with ODQ (10 µM). These results are summarized in Fig.
7. The control inner diameter of this
group of vessels averaged 93 ± 6.4 µm (n = 5 vessels from 5 rats). Serotonin (107 M) reduced vascular
diameter to 46 ± 3 µm. DEA-NONOate
(109-105 M) increased the diameter of
these vessels to a maximum of 84 ± 4% of control. After blockade
of guanylyl cyclase with ODQ, the diameter of these vessels only
increased by 47 ± 3% in response to the highest
(105 M) concentration of DEA-NONOate. To rule out the
possibility that the effects of DEA-NONOate were due to a hydrolysis
product of the donor rather than NO, we examined the effects of
diethylamine (2 × 109-2 × 105 M) on vascular diameter in five additional
vessels. Diethylamine had no significant effect on the
diameter of these vessels at any concentration studied.
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Contribution of 20-HETE to the dilator response to NO in middle
cerebral arteries.
The contribution of 20-HETE to the vasodilator response to NO in
cerebral arteries was determined by comparing the
concentration-response curves to DEA-NONOate under control conditions
and after prevention of the NO-induced fall in endogenous 20-HETE
levels by the addition of 20-HETE to the bath (Fig. 7). The basal
diameter of these vessels averaged 94 ± 6 µm. It fell to
46 ± 3 µm after serotonin (107 M) was added to
bath. DEA-NONOate dose dependently increased the diameter of
serotonin-preconstricted middle cerebral arteries by 90 ± 2%.
20-HETE had no effect on the diameter of vessels after they were
preconstricted with serotonin. Vascular diameter averaged 51 ± 2 and 49 ± 1 µm before and after addition of 20-HETE to
serotonin-preconstricted vessels. Nevertheless, fixing 20-HETE levels
at 100 nM in the bath reduced the vasodilator response to DEA-NONOate
by 59 ± 2%. Simultaneous blockade of the cGMP and 20-HETE
pathways nearly abolished the vasodilator response to DEA-NONOate, and
vascular diameter only increased by 9.2 ± 2.4% in response to
the highest concentration of DEA-NONOate (105 M) (Fig.
7).
5 M)
increased the inner diameter of these arteries by 44%, from 104 ± 13 to 151 ± 20 µm. ODQ (10 µM) reduced baseline diameter to 91 ± 11 µm and attenuated the vasodilator response to NO by 50%. Similar to the results obtained in serotonin-preconstricted vessels, the cGMP-independent component of the vasodilator response to
NO was eliminated by adding 20-HETE (100 nM) to the bath. Vascular diameter only increased by 2 ± 2% in response to DEA-NONOate
(105 M) in vessels treated with 20-HETE and ODQ together.
To determine whether the failure of ODQ to block the vasodilator
response to NO was due to incomplete blockade of the guanylyl cyclase
activity, we measured cGMP levels in cerebral microvessels treated with
DEA-NONOate (105 M) and DEA-NONOate plus ODQ. The results
of these experiments are summarized in Fig.
8. DEA-NONOate (105 M)
increased cGMP levels fourfold in cerebral microvessels. ODQ (10 µM)
completely blocked the ability of DEA-NONOate to increase cGMP levels
in these vessels.
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Evidence that inhibitors of the formation of 20-HETE mimic and
block the response to NO in middle cerebral arteries.
The effects of SNP, 17-ODYA, DDMS, and NS-1619 on the diameters of
serotonin (107 M)-preconstricted pressurized rat middle
cerebral arteries are presented in Fig.
9. Control inner diameter
averaged 100 ± 14 µm (n = 6 vessels), and it
fell to 52 ± 8 µm after serotonin (107 M) was
added to the bath. Acetylcholine (1 µM) had no significant effect on
the diameter of these vessels, in which the endothelium was removed,
whereas it increased the diameter of these vessels by 40% before the
endothelium was removed. SNP (103 M) increased vascular
diameter by 65 ± 4% in the serotonin-preconstricted denuded
arteries. Blockade of 20-HETE formation with DDMS (20 µM) increased
the diameter of these vessels by 47 ± 4%. Similar effects were
seen when the formation of 20-HETE was blocked with a chemically and
mechanistically different inhibitor, 17-ODYA (1 µM). A direct
activator of KCa channels, NS-1619 (100 µM), also
produced the same degree of vasodilation and increased vascular diameter by 53 ± 2%.
|
9-105 M)
under control conditions and after blockade of 20-HETE formation with
DDMS or 17-ODYA (Fig. 10). The control
inner diameter of these vessels averaged 95 ± 10 µm
(n = 5 vessels from 5 rats). Serotonin (107 M) reduced baseline diameter to 47 ± 5 µm.
Blockade of 20-HETE production had no effect on the diameter of these
vessels. Baseline diameters averaged 49 ± 1 or 50 ± 2 µm after
blockade of 20-HETE formation with 17-ODYA or DDMS, respectively, in
serotonin-preconstricted vessels. DEA-NONOate
(109-105 M) increased the diameter of
serotonin-preconstricted vessels by 97 ± 5%. After inhibition of
20-HETE production with DDMS, the vasodilator response to DEA-NONOate
was attenuated by 61%. Similar effects were seen after 20-HETE
production was blocked using 17-ODYA.
|
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study examined the roles that a rise in cGMP versus a fall in 20-HETE levels play in activating K+ channels and in the vasodilator response to NO in rat middle cerebral arteries studied in vitro. Our results indicate that a concentration of ODQ that completely blocks the rise in cGMP levels in cerebral microvessels attenuates the vasodilator response to NO donors in serotonin-preconstricted middle cerebral arteries by 50%. Similar results were seen in vessels studied at a lower transmural pressure of 60 mmHg that were not preconstricted with serotonin. These results suggest that a large component of the vasodilator response to NO in the rat middle cerebral artery is cGMP independent. They are consistent with previous findings that blockade of guanylyl cyclase with ODQ or methylene blue has little effect on the vasodilator response to NO in canine middle cerebral arteries studied in vitro (25, 29). These results also support a large body of emerging evidence that ODQ does not fully block the vasodilator response to NO donors that directly release NO in aqueous media (2, 13, 17, 29, 39). However, the present results differ from those of previous studies indicating that ODQ blocks most of the vasodilator response to NO in pial arteries of the mouse, rat, and rabbit (4, 12, 35) and the basilar artery of the rat (37) in vivo. The reasons for these divergent results are still unknown. They could reflect species differences or differences in the type of K+ channels activated by NO in middle cerebral arteries versus basilar arteries (11, 20, 28, 29, 37). It is also possible that ODQ may be much more effective at blocking the vasodilator response to SNP than the response to other NO donors because ODQ blocks the catalytic release of NO from SNP by tissue (13). In the present study, this potential nonspecific action of ODQ was avoided by using a NO donor that spontaneously releases NO in aqueous media.
Previous studies have indicated that the cerebral arteries avidly express mRNA and protein for the P-450 4A2 enzyme that produces 20-HETE and that 20-HETE is a potent constrictor of cerebral arteries (15, 16). 20-HETE promotes Ca2+ entry by depolarizing vessels secondary to blockade of the KCa channel in VSM cells (14, 46) and has been reported to play an important role in the myogenic response of cerebral arteries to elevations in transmural pressure (15). Recent studies have indicated that NO binds heme in the P-450 4A enzymes that catalyze the formation of 20-HETE (1, 2, 39). The subsequent fall in 20-HETE levels appears to play a major role in the activation of K+ channels and in the vasodilator response to NO in the renal microcirculation (39). Therefore, we examined whether a fall in 20-HETE levels might have a similar effect to mediate the cGMP-independent effects of NO in the cerebral circulation. Fixing 20-HETE levels at 100 nM by adding 20-HETE to the bath markedly impaired the vasodilator response of pressured rat middle cerebral arteries to NO. We believe that this concentration of 20-HETE reflects the endogenous level in cerebral arterial VSM cells because it was the concentration needed to reverse the activation of K+ channels following inhibition of 20-HETE formation in the patch-clamp experiments (Fig. 5). In other studies, we found that blocking the formation of 20-HETE with 17-ODYA or DDMS had an effect similar to that of NO to dilate denuded rat middle cerebral arteries. 17-ODYA and DDMS also attenuated the vasodilator response to NO donors. Finally, simultaneous blockade of both the cGMP and 20-HETE pathways with ODQ and 20-HETE had an additive effect and abolished the vasodilator response to NO donors. Taken together, these results indicate that inhibition of the endogenous formation of 20-HETE mediates a large portion of the cGMP-independent component of the vasodilator response to NO in rat middle cerebral arteries.
We also examined what role, if any, activation of K+ channels plays in the vasodilator response to NO in rat middle cerebral arteries studied in vitro. The results of the present study indicate that exposure of these vessels to a depolarizing concentration of K+ diminishes the vasodilator response to NO by ~50%. Similar effects were observed after the KCa channels were blocked with IbTX (100 nM). These results indicate that NO activates the KCa channel in rat middle cerebral arteries and that activation of this channel contributes to the vasodilator response to NO, at least in vitro. Our findings are consistent with previous results demonstrating that activation of K+ channels plays a major role in mediating the vasodilator response to NO in several vascular beds (3, 6, 9, 17, 21, 25, 26, 30, 34, 39, 44), including cerebral arteries (20, 28, 29, 31). However, it is important to recognize that there is not unequivocal support for a role for KCa channels in mediating the response to NO in the cerebral circulation (11, 36, 40). Indeed, studies in basilar arteries (37) and bovine coronary arteries (23) have indicated that NO also activates a 4-aminopyridine-sensitive K+ channel. In the present study as well, we found that NO activated an intermediate-amplitude channel with a conductance consistent with that of a 4-aminopyridine-sensitive K+ channel. Moreover, in the basilar artery of rats (37), there is evidence that activation of the 4-aminopyridine-sensitive K+ channel may be more important than activation of the KCa channel in the vasodilator response to NO. The reason for the differences in results among studies remains to be determined. It may reflect differences in the ability of vessels from different species to produce 20-HETE versus cGMP under various experimental conditions. Alternatively, there may be differences in the types of K+ channels expressed in arteries in different parts of the cerebral circulation.
The present study also explored the mechanism by which NO activates KCa channels in VSM cells isolated from rat middle cerebral arteries. We found that blockade of guanylyl cyclase has no effect on the activation of K+ channels following administration of NO. However, preventing the NO-induced fall in intracellular 20-HETE levels completely eliminated the activation of the KCa channels produced by NO donors. This finding was surprising because there is evidence (6) that NO can directly activate KCa channels in excised membrane patches of VSM cells. We expected to find that NO would still activate K+ channels in the presence of 20-HETE via its direct effects on channels. Moreover, we further expected to find that blockade of the cGMP and 20-HETE pathways would attenuate, but not eliminate, the vasodilator response to NO in rat middle cerebral arteries. Thus the finding that simultaneous blockade of the cGMP and 20-HETE pathways abolished the vasodilator response to NO in rat middle cerebral arteries suggests that the direct effects of NO on K+ channels play little role in this response.
In other experiments, we confirmed that NO has a direct effect on KCa channels in inside-out patches excised from VSM cells isolated from rat middle cerebral arteries. However, the concentration of the NO donor needed to activate this channel in detached membrane patches was two orders of magnitude higher than that needed to activate this channel in intact VSM cells. This finding suggests that a fall in 20-HETE levels rather than a direct effect of NO mediates the cGMP-independent activation of KCa channels in rat middle cerebral arteries following administration of NO donors.
A recent in vivo study (36) in rat basilar arteries indicated that blockade of the endogenous production of NO may sensitize KCa channels to the effects of NO. Because the endothelium was absent in our patch-clamp studies, it is possible that the loss of the endogenous production of NO may have exaggerated the importance that activation of KCa channels plays in the vasodilator response to NO. To address this possibility, we performed additional experiments using intact cerebral arteries treated with IbTX or a depolarizing concentration of K+. The results of these experiments indicate that one-half of the response to NO is mediated by activation of KCa channels in intact rat middle cerebral arteries. This finding is consistent with the component of the vasodilator response to NO that was cGMP independent and that could be blocked by preventing the fall in 20-HETE levels, which in turn blocks NO-induced activation of K+ channels.
The present results also indicate that blockade of guanylyl cyclase reduces the vasodilator response to NO by 50% in the middle cerebral artery of rats even though it has no effect on NO-induced activation of KCa channels. Thus cGMP must alter the vasodilator response to NO in the middle cerebral arteries by some mechanism that is independent of K+ channels. There is evidence that cGMP and NO can lower intracellular Ca2+ concentrations and dilate vessels exposed to depolarizing concentrations of K+. In addition, several investigators have reported that cGMP activates a cGMP-dependent kinase that phosphorylates several proteins involved in determining intracellular Ca2+ concentration and contraction of VSM. These effects include inhibition of Ca2+ release, inactivation of L-type Ca2+ channels, enhancement of Ca2+ sequestration, and decreases in the Ca2+-sensitivity of the contractile mechanism (19, 33, 42, 43, 45). The results of the present study suggest that one or more of these mechanisms probably mediate the cGMP-dependent component of the vasodilator response to NO in the middle cerebral artery of the rat.
In summary, the results of the present study indicate that the vasodilator response to NO in the middle cerebral artery of rats is mediated by both cGMP-dependent and cGMP-independent signaling pathways. Activation of the KCa channel contributes ~50% to the vasodilator response to NO in the middle cerebral artery of rats. This appears to be largely due to a fall in 20-HETE rather than a rise in cGMP levels or a direct effect of NO on this channel. The remainder of the vasodilator response is cGMP dependent. The cGMP-dependent component of the response to NO is not associated with activation of K+ channels and is probably mediated by cGMP-induced changes in the reuptake of Ca2+ and/or the sensitivity of the contractile mechanism to changes in intracellular Ca2+ concentration.
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ACKNOWLEDGEMENTS |
|---|
We thank Melissa Mascarenhas for technical assistance with the cGMP measurement.
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FOOTNOTES |
|---|
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-29587 and HL-59996.
Address for reprint requests and other correspondence: R. J. Roman, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: rroman{at}post.its.mcw.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. §1734 solely to indicate this fact.
Received 14 June 1999; accepted in final form 13 January 2000.
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 A. J. Dahly-Vernon, M. Sharma, E. T. McCarthy, V. J. Savin, S. R. Ledbetter, and R. J. Roman Transforming Growth Factor-{beta}, 20-HETE Interaction, and Glomerular Injury in Dahl Salt-Sensitive Rats Hypertension, April 1, 2005; 45(4): 643 - 648. [Abstract] [Full Text] [PDF]
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 V. Randriamboavonjy, L. Kiss, J. R. Falck, R. Busse, and I. Fleming The synthesis of 20-HETE in small porcine coronary arteries antagonizes EDHF-mediated relaxation Cardiovasc Res, February 1, 2005; 65(2): 487 - 494. [Abstract] [Full Text] [PDF]
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 I. Fleming Brain in the Brawn: The Neuronal Nitric Oxide Synthase as a Regulator of Myogenic Tone Circ. Res., October 3, 2003; 93(7): 586 - 588. [Full Text] [PDF]
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X. Peng, J. R. Carhuapoma, A. Bhardwaj, N. J. Alkayed, J. R. Falck, D. R. Harder, R. J. Traystman, and R. C. Koehler Suppression of cortical functional hyperemia to vibrissal stimulation in the rat by epoxygenase inhibitors Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H2029 - H2037. [Abstract] [Full Text] [PDF]
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M. Yu, R. P. McAndrew, R. Al-Saghir, K. G. Maier, M. Medhora, R. J. Roman, and E. R. Jacobs Nitric oxide contributes to 20-HETE-induced relaxation of pulmonary arteries J Appl Physiol, October 1, 2002; 93(4): 1391 - 1399. [Abstract] [Full Text] [PDF]
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M. Alonso-Galicia, K. G. Maier, A. S. Greene, A. W. Cowley Jr., and R. J. Roman Role of 20-hydroxyeicosatetraenoic acid in the renal and vasoconstrictor actions of angiotensin II Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2002; 283(1): R60 - R68. [Abstract] [Full Text] [PDF]
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 R. J. Roman P-450 Metabolites of Arachidonic Acid in the Control of Cardiovascular Function Physiol Rev, January 1, 2002; 82(1): 131 - 185. [Abstract] [Full Text] [PDF]
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I. Fleming Cytochrome P450 and Vascular Homeostasis Circ. Res., October 26, 2001; 89(9): 753 - 762. [Abstract] [Full Text] [PDF]
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K. Niwa, C. Haensel, M. E. Ross, and C. Iadecola Cyclooxygenase-1 Participates in Selected Vasodilator Responses of the Cerebral Circulation Circ. Res., March 30, 2001; 88(6): 600 - 608. [Abstract] [Full Text] [PDF]
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 S. P. Didion, D. D. Heistad, and F. M. Faraci Mechanisms That Produce Nitric Oxide-Mediated Relaxation of Cerebral Arteries During Atherosclerosis Stroke, March 1, 2001; 32(3): 761 - 766. [Abstract] [Full Text] [PDF]
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J. C. Frisbee, R. J. Roman, U. M. Krishna, J. R. Falck, and J. H. Lombard 20-HETE modulates myogenic response of skeletal muscle resistance arteries from hypertensive Dahl-SS rats Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1066 - H1074. [Abstract] [Full Text] [PDF]
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M. Yu, C.-W. Sun, K. G. Maier, D. R. Harder, and R. J. Roman Mechanism of cGMP contribution to the vasodilator response to NO in rat middle cerebral arteries Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1724 - H1731. [Abstract] [Full Text] [PDF]
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F. Kehl, L. Cambj-Sapunar, K. G. Maier, N. Miyata, S. Kametani, H. Okamoto, A. G. Hudetz, M. L. Schulte, D. Zagorac, D. R. Harder, et al. 20-HETE contributes to the acute fall in cerebral blood flow after subarachnoid hemorrhage in the rat Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1556 - H1565. [Abstract] [Full Text] [PDF]
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Significant difference from cells treated with 17-ODYA alone
and cells treated with 17-ODYA + 10 nM 20-HETE.
