(Stroke. 1999;30:2727.)
© 1999 American Heart Association, Inc.
Original Contributions |
Contribution of 20-HETE to Vasodilator Actions of Nitric Oxide in the Cerebral Microcirculation
From the Departments of Physiology (M.A.-G., R.J.R.) and Anesthesiology (A.G.H., H.S.) and the Cardiovascular Research Center (D.R.H.), Medical College of Wisconsin, Milwaukee.
Correspondence to Richard J. Roman, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226-0509. E-mail rroman{at}mcw.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Background and Purpose—The present study examined the contributions of a rise in cGMP versus a fall in 20-HETE levels to the vasodilator response to nitric oxide (NO) in the cerebral circulation of the rat.
Methods—Intact rat middle cerebral and basilar arteries were bathed in physiological saline solution containing indomethacin (5 µmol/L) and baicalein (0.5 µmol/L) and pressurized at 90 mm Hg. Relaxations to sodium nitroprusside (SNP) were studied before and after addition of [1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one] (ODQ, a guanylyl cyclase blocker), 8R,9S,11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT5823, a protein kinase G blocker), and 20-hydroxyeicosatetraenoic acid (20-HETE). Cerebral blood flow was measured by using a laser Doppler flow probe over a thin cranial window in anesthetized rats, and the effects of intracerebroventricular infusion of 1-hexamine,6-(2-hydroxy-1-methyl-2-nitrosohydrazino)N-methyl (MAHMA nonoate) and dibromododecenyl methylsulfimide (DDMS) were determined.
Results—SNP-induced dilation of serotonin-preconstricted (0.2 µmol/L) middle cerebral arteries (10-7 to 10-3 mol/L) was attenuated in arteries treated with ODQ (10 µmol/L) or KT5823 (1 µmol/L) by 52% and 27%, respectively. Preventing the NO-induced fall in intracellular 20-HETE, by adding 20-HETE (100 nmol/L) to the bath, reduced the dilation to SNP by 62%. Simultaneous administration of ODQ and 20-HETE markedly attenuated the SNP-induced dilation by 90%. In basilar arteries, ODQ (10 µmol/L) alone completely blocked the response to SNP. Infusion of MAHMA nonoate (10 nmol/min ICV) in anesthetized rats increased cerebral blood flow by 52% before and 8% after blockade of the endogenous production of 20-HETE with DDMS (50 pmol/min).
Conclusions—These results suggest that NO dilates cerebral arteries through both cGMP-dependent and cGMP-independent pathways and that inhibition of 20-HETE formation contributes to the cerebral vasodilator response to NO both in vitro and in vivo.
Key Words: cerebral vessels • cyclic GMP • cytochrome P-450 • hydroxyeicosatetraenoic acids • nitric oxide • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Recent studies indicate that nitric oxide (NO) plays a central role in the regulation of cerebral blood flow (CBF).1 2 3 4 5 6 7 8 9 In this regard, the finding that inhibitors of NO synthase lower CBF indicates that the tonic release of NO plays an important role in the regulation of basal cerebral vascular tone.3 4 NO also mediates the cerebral vascular responses to a wide variety of stimuli. For example, endothelium-derived NO contributes to the vasodilator responses to acetylcholine, substance P, bradykinin, and
2-adrenergic receptor
agonists.5 6 7 There is also evidence that NO contributes
to the rise in CBF produced by hypercapnia (5%
CO2)3 4 6 9 and inhalational
anesthetics.10 11 Moreover, several studies have indicated
that inhibitors of NO synthase attenuate the changes in CBF
in response to electrical stimulation,12
seizures,6 and nociceptive stimulation.13
These results suggest that NO couples CBF to changes in neuronal
activity. The vasodilator actions of NO in the cerebral circulation are generally thought to be due to the stimulation of guanylyl cyclase (GC), as has been reported for the aorta and large conduit arteries.2 14 NO and cGMP activate Ca2+-activated K+ channels in vascular smooth muscle cells from a variety of vascular beds.15 16 17 18 Activation of these channels hyperpolarizes vascular smooth muscle and limits Ca2+ influx through voltage-gated channels.2 In the cerebral circulation, several pharmacological and patch-clamp studies have been performed to directly examine the mechanism by which NO alters cerebral vascular tone. Recent studies reporting that inhibitors of GC attenuate the dilator response to NO in pial arterioles in vivo19 20 21 support a primary role for cGMP in mediating the vasodilator response to NO. On the other hand, the results of other studies indicating that NO can still dilate cerebral arteries and large conduit arteries treated with inhibitors of GC in vitro suggest that other mechanisms may also be involved.2 18 22 23 24 25
We have recently reported that NO donors inhibit enzymes of the cytochrome P-450 4A (CYP 4A) family that catalyze the formation of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) in renal arterioles.26 27 Cerebral arteries also express mRNA and protein for the CYP 4A2 enzyme and produce 20-HETE when incubated with arachidonic acid.28 20-HETE is a potent vasoconstrictor of cerebral arteries that inhibits Ca2+-activated K+ channels.28 29 Given our recent findings that inhibition of the formation of 20-HETE contributes to the vasodilator response to NO in renal arterioles,26 27 the purpose of the present study was (1) to evaluate the relative contribution of cGMP-dependent and cGMP-independent pathways to the vasodilator actions of NO in cerebral arteries and (2) to examine whether inhibition of the formation of the vasoconstrictor 20-HETE mediates the cGMP-independent actions of NO in cerebral arteries both in vitro and in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Adult male Sprague-Dawley rats (10 to 12 weeks old) were purchased from Harlan Sprague-Dawley Laboratories (Indianapolis, Ind). The rats were housed in the animal care facility at the Medical College of Wisconsin, which is approved by the American Association for the Accreditation of Laboratory Animal Care. The animals had free access to food and water. All protocols involving animals received approval by the Animal Care Committee of the Medical College of Wisconsin.
Isolated MCA and BA Studies
Rats were anesthetized with sodium pentobarbital (50
mg/kg body wt IP). The brain was removed, and small branches of middle
cerebral artery (MCA, inner diameter <65 µm) or basilar artery
(BA, inner diameter >140 µm) were microdissected and mounted on
glass micropipettes in a perfusion chamber containing
physiological saline solution equilibrated with a
95% O2/5% CO2 gas mixture
and maintained at 37°C. The inflow pipette was connected to a
pressurized reservoir to allow for control of intraluminal perfusion
pressure, which was monitored by use of a transducer (Cobe). The
outflow cannula was clamped off, and intraluminal pressure was
maintained at 90 mm Hg during the experiment. The composition of
the perfusate and the bath was (mmol/L) NaCl 119, KCl 4.7,
MgSO4 1.17, CaCl2 1.6,
NaHCO3 12,
NaH2PO4 1.18, EDTA 0.03,
and glucose 10, pH 7.4. Vascular diameters were measured by using a
video system composed of a stereo microscope (Carl Zeiss, Inc), a CCTV
television camera (model KP-130AU, Hitachi), a videocassette
recorder (model AG-7300, Panasonic), a television monitor (model
CVM-1271, Sony), and a video measuring system (model VIA-100, Boeckeler
Instrument Co). Indomethacin (5 µmol/L) and
baicalein (0.5 µmol/L) were added to the bath to block the
endogenous metabolism of
arachidonic acid via the
cyclooxygenase and lipoxygenase
pathways, as we have previously described.26
Measurement of NO Concentration
The concentration of NO generated in the bath solution by
various doses of sodium nitroprusside (SNP) was measured with a
2-mm-diameter gas-permeant membrane NO sensor (Iso-NOP) and a NO meter
(World Precision Instruments). The meter was calibrated by chemically
reducing known amounts of NO2 to NO in the
presence of KI and H2SO4.
SNP (10-5 to 10-3 mol/L)
was added to the bath containing physiological salt
solution saturated with 95% O2/5%
CO2 at 37°C, and the NO concentration was
recorded 3 minutes after addition of the NO donor.
Relative Contribution of cGMP-Dependent and -Independent Pathways
to Vasodilator Actions of NO in Cerebral Arteries
To determine whether the cGMP pathway mediates the dilator
actions of NO, the response to SNP (10-7 to
10-3 mol/L) was examined in MCAs and BAs
preconstricted with serotonin (0.2 µmol/L) before
and after blocking GC with
[1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one] (ODQ,
10 µmol/L) or cGMP-dependent protein kinase (PKG) with
8R,9S,11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one
(KT5823, 1 µmol/L). Vascular diameter was measured 3 minutes
after the addition of each dose of SNP to the bath. The effectiveness
of blockade of PKG with KT5823 was assessed by comparing the
vasodilator response to 8-bromo-cGMP (10-4
mol/L) before and after addition of KT5823 to the bath.
To examine whether inhibition of the formation of the vasoconstrictor 20-HETE contributes to the cGMP-independent actions of NO in cerebral arteries, the response to SNP (10-7 to 10-3 mol/L) was examined in MCAs preconstricted with serotonin (0.2 µmol/L) before and after fixing 20-HETE levels at 100 nmol/L by adding exogenous 20-HETE to the bath. Vascular diameter was measured 3 minutes after the addition of each dose of SNP to the bath.
To examine whether the effects of ODQ were specific to NO donors, we studied the effects of ODQ on the vascular response to the NO-independent dilator adenosine. Cumulative concentration-response curves to adenosine (10-7 to 10-3 mol/L) were obtained in MCAs preconstricted with serotonin (0.2 µmol/L) under control conditions and after blockade of GC with ODQ (10 µmol/L).
To determine whether different mechanisms mediate the NO-induced vasodilation in large versus small cerebral vessels, we compared the vascular responses of BAs and MCAs to the stable cell-permeable cGMP analogue 8-bromo-cGMP. Cumulative dose-response curves to 8-bromo-cGMP (10-8 to 10-4 mol/L) were generated in these arteries after 30 minutes of incubation with indomethacin (5 µmol/L) and baicalein (0.5 µmol/L) to block the endogenous metabolism of arachidonic acid via the cyclooxygenase and lipoxygenase pathways. Vascular diameter was measured 2 minutes after the addition of each dose of 8-bromo-cGMP to the bath.
To examine whether the effects of dibromododecenyl methylsulfimide (DDMS) were specific to NO donors, we studied the effects of DDMS on the vascular response to the NO-independent vasodilator adenosine. Cumulative concentration-response curves to adenosine (10-7 to 10-3 mol/L) were obtained in MCAs preconstricted with serotonin (0.2 µmol/L) under control conditions and after blockade of 20-HETE synthesis with DDMS (25 µmol/L).
Western Blot Analysis of Soluble GC and CYP 4A
Proteins
To determine whether differences in the expression of soluble GC
(sGC) or CYP 4A proteins in large versus small cerebral vessels may
explain the differences in the NO-induced responses in MCAs and BAs, we
compared the levels of GC and CYP 4A protein in these arteries. MCAs
and BAs were microdissected from the brain of 5 Sprague-Dawley rats.
The vessels were homogenized in a 10 mmol/L potassium
phosphate buffer at pH 7.7 containing 250 mmol/L sucrose, 1
mmol/L EDTA, 2 µmol/L leupeptin, 1 µmol/L pepstatin, 2
µg/mL aprotinin, and 0.1 µmol/L phenylmethylsulfonyl
fluoride. Microsomes prepared from liver of Sprague-Dawley rats
were used as a positive control. Protein concentrations were measured
by the Bradford method (Bio-Rad) with bovine serum albumin used
as a standard. Sample protein (2 µg) was subjected to 7.5%
SDS–polyacrylamide gel electrophoresis on 8x10-cm gels at 100
V for 1 hour. The separated proteins were transferred
electrophoretically to polyvinylidene fluoride membranes at 100
V for 1 hour. After transfer, nonspecific binding was blocked by
incubating the membranes overnight at 4°C in TBS-T buffer (6
mmol/L Tris-HCl, 4 mmol/L Tris-base, 150 mmol/L NaCl, and
0.08% Tween 20, pH 7.5) containing 10% nonfat dry milk. The next day,
the membranes were washed 4 times with TBS-T and subsequently incubated
for 2 hours with a polyclonal antibody raised against the
1 and ß1 subunits of
sGC (Cayman Chemical) at a 1:5000 dilution in TBS-T buffer containing
2% milk or a polyclonal antibody raised against rat CYP 4A1 (Gentest
Corp), which cross-reacts with CYP 4A2 and 4A3, at a 1:5000 dilution.
After incubation with the primary antibody, the membranes were washed 6
times with TBS-T buffer and incubated with either goat anti-rabbit IgG
conjugated with alkaline phosphatase for the sGC (Santa Cruz
Biotechnology) at a 1:5000 dilution or anti-goat IgG conjugated with
alkaline phosphatase (Bio-Rad) at a 1:5000 dilution in 2% milk for 1
hour for the CYP 4A. After 6 more washes, the immunoblots
were developed by using Vistra ECF substrate (Amersham), and the
conversion of the substrate to a fluorescent product by the
alkaline phosphatase–coupled second antibody was captured by use of a
FluorImager scanner (Molecular Dynamics).
Measurement of CBF In Vivo
To determine whether inhibition of the formation of the
vasoconstrictor 20-HETE contributes to the NO-induced vasodilation in
vivo, we measured the effects of an
intracerebroventricular infusion of the
short-acting NO donor
1-hexamine,6-(2-hydroxy-1-methyl-2-nitrosohydrazino)N-methyl
(MAHMA nonoate) on CBF in anesthetized rats before and after
blockade of 20-HETE formation with DDMS. Experiments were performed in
7 male rats anesthetized with ketamine (70 mg/kg IM)
and a low dose of pentobarbital (10 mg/kg IP) and in 3 rats
anesthetized with chloralose (60 mg/kg IP) and urethane (400
mg/kg IP) followed by a constant intravenous infusion of
chloralose alone (50 mg/kg per hour). The rats were tracheostomized,
paralyzed with gallamine triethiodide (80 mg/kg IP), and artificially
ventilated with a small animal ventilator (model SAR-830, CWE) with
30% O2 in N2. Cannulas
were placed in the femoral artery for measurement of
arterial pressure and in the jugular vein for
intravenous infusions. Expired
PO2 and
PCO2 were measured with a gas
analyzer, and the ventilation rate was controlled to maintain
constant PCO2. CBF was measured
at 2 to 4 different sites by use of a laser Doppler flowmeter
(Oxford Optronix, Inc), as we have previously described in
detail.9 Briefly, the head of the rat was placed in a
stereotaxic apparatus, and the bone covering
the right parietal cranium was thinned by using a low-speed air drill
until a thin translucent cranial window remained. A laser Doppler
flow (LDF) probe was positioned over the thin cranial window, and a
30-gauge cannula was inserted into the left lateral ventricle for
intracerebroventricular infusions.
After surgery, a 1-hour equilibration period was allowed. The LDF
response to intracerebroventricular
infusion of the short-acting NO donor MAHMA nonoate (10 nmol/min) was
measured during a control period and after 30 minutes of blocking
20-HETE production with a constant
intracerebroventricular infusion of the
selective CYP 4A inhibitor DDMS (50 pmol/min). Results are
expressed as a percent change from the baseline value measured at each
site and then averaged to a single value per rat.
Drugs and Chemicals
All chemicals were of analytical grade.
Indomethacin, sodium nitroprusside,
serotonin, adenosine, 8-bromo-cGMP, leupeptin,
pepstatin, aprotinin, and phenylmethylsulfonyl fluoride were
purchased from Sigma Chemical Co. MAHMA nonoate was purchased from
Cayman Chemical. ODQ was purchased from Alexis Co. Baicalein and KT5823
were obtained from Biomol. DDMS and 20-HETE were synthesized by Dr J.R.
Falck (Southwestern Medical Center, Dallas, Tex).
Statistics
Values are expressed as mean±SEM. Statistical differences in
mean values within and between groups were examined by ANOVA for
repeated measures followed by the Duncan multiple range test. A value
of P<0.05 was considered significant.
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Results |
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Role of cGMP in Mediating the Dilator Response to NO in MCAs
A schematic figure summarizing the effects of various agonists and antagonists used in our studies is presented in Figure 1
. The long-acting NO donor SNP
was used in the isolated-vessel studies because it produces a steady
release of NO that lasts for 30 minutes. The short-acting NO donor
MAHMA nonoate was used in the CBF studies because it has a half-life of
only 2 to 3 minutes, and its vasodilator effects are readily reversed.
ODQ was used to inhibit sGC activity. KT5823 was used to block PKG. The
formation of 20-HETE in cerebral arteries in vivo was blocked with
DDMS, a selective competitive inhibitor of 20-HETE
synthesis.30 The NO-induced fall in 20-HETE formation was
prevented by adding exogenous 20-HETE (100 nmol/L) to the bath.
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The contribution of cGMP to the vasodilator response to NO was assessed
by comparing the concentration-dependent response to SNP under control
conditions and after blocking GC with ODQ. The results of these studies
are summarized in Figure 2A. The
baseline inner diameter of these arteries measured at 90 mm Hg
averaged 63±3 µm (n=6 vessels, 5 rats). Serotonin
(0.2 µmol/L) reduced vessel diameter by 52±3%, and SNP
(10-7 to 10-3 mol/L)
dose-dependently increased vessel diameter to a maximum of 89±6% of
the serotonin-constricted value. The concentration of NO
generated averaged 25±2, 40±5, and 139±29 nmol/L at 3 minutes after
addition of 10-5,10-4,
and 10-3 mol/L SNP to the bath. Blockade of GC
with ODQ (10 µmol/L) reduced the basal diameter of
serotonin-preconstricted arteries by 15±6%, and it
attenuated the vasodilator response to all doses of SNP by 
50%.
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To determine whether ODQ had any nonspecific inhibitory effects, the effects of ODQ on the response to the NO-independent dilator adenosine were examined in MCAs. The inner diameter of these MCAs measured at 90 mm Hg averaged 77±5 µm (n=3 vessels, 3 rats), and it fell to 43±3% of control after adding serotonin to the bath. Under control conditions, adenosine (10-7 to 10-3 mol/L) dose-dependently increased vessel diameter to a maximum of 92±2% of the serotonin-constricted value. Blockade of GC with ODQ (10 µmol/L) did not impair the vasodilator response to adenosine. After ODQ, adenosine (10-7 to 10-3 mol/L) dose-dependently increased vessel diameter to a maximum of 92±4% of the serotonin-constricted value.
The effects of blocking PKG with KT5823 on the vasodilator response to
SNP in MCAs are summarized in Figure 2B
. The baseline inner
diameters of these arteries averaged 64±2 µm (n=5 vessels, 4
rats), and it fell to 51±2% of control after adding
serotonin to the bath. Under control conditions, SNP
(10-7 to 10-3 mol/L)
dose-dependently increased vessel diameter to a maximum of 103±2% of
the serotonin-constricted value. Blockade of PKG activity
with KT5823 (1 µmol/L) reduced the response to SNP by 27±9%.
The effectiveness of the blockade of PKG activity was assessed by
comparing the vasodilator response to 8-bromo-cGMP before and after
adding KT5823 to the bath. In serotonin-preconstricted
cerebral arteries, addition of 10-4 mol/L
8-bromo-cGMP increased vascular diameter to 88±3% of the
serotonin-constricted value (Figure 2B, inset), and
KT5823 (1 µmol/L) completely blocked the vasodilator response to
8-bromo-cGMP in these vessels.
Role of 20-HETE in Mediating the Dilator Response to NO in
MCAs
The contribution of a fall in 20-HETE levels to the dilator
response to NO in cerebral arterioles was evaluated by comparing the
response to SNP under control conditions and after preventing the fall
in endogenous 20-HETE levels produced by the NO donor. This
was achieved by adding a high concentration of exogenous 20-HETE (100
nmol/L) to the bath. The results of these experiments are summarized in
Figure 3. The control inner diameter of
these vessels averaged 63±3 µm (n=6 vessels, 5 rats), and it
fell to 51±2% of control after adding serotonin to the
bath. Under these conditions, SNP (10-7 to
10-3 mol/L) dose-dependently dilated these
arteries to a maximum of 92±2% of the
serotonin-constricted value. 20-HETE had no significant
effect on the baseline diameter of these preconstricted vessels.
However, it did reduce the vasodilator response to SNP by 63%. In
the presence of 100 nmol/L 20-HETE, SNP dose-dependently dilated these
arteries to a maximum of 34±5% of the
serotonin-constricted value. Simultaneous
blockade of both pathways by adding ODQ and 20-HETE to the bath
completely abolished the vasodilator response to SNP (Figure 3).
The control inner diameter of these vessels averaged 63±4 µm
(n=6 vessels, 5 rats), and it fell to 50±3% of control after adding
serotonin to the bath. Under these conditions, SNP
(10-7 to 10-3 mol/L)
dose-dependently dilated these arteries to a maximum of 97±5% of the
serotonin-constricted value. However, in the presence of
20-HETE (100 nmol/L) and ODQ (10 µmol/L), vessel diameter
increased only by 8±3% from the serotonin-constricted
value in response to the highest dose of SNP
(10-3 mol/L).
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Role of cGMP in Mediating the Dilator Response to NO in
BAs
The relative contribution of the cGMP pathway to the vasodilator
responses to NO in larger cerebral arteries was also assessed. The
results of these experiments are summarized in Figure 4. The control inner diameter of the BAs
studied averaged 147±12 µm (n=4 vessels, 4 rats), and it fell
to 46±4% after adding serotonin (0.2 µmol/L) to
the bath. SNP (10-7 to
10-3 mol/L) dose-dependently dilated these
arteries to a maximum of 64±4% of the
serotonin-preconstricted diameter. Blocking GC with ODQ
completely eliminated the vasodilator response to SNP in BAs. Vessel
diameter increased only to a maximum of 2±0.2% in response to the
highest dose of the NO donor.
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cGMP Sensitivity in MCAs and BAs
To determine the mechanisms underlying the difference in the
importance of the cGMP pathway to the vasodilator response to NO in BAs
versus MCAs, we compared cumulative dose-response curves to
8-bromo-cGMP (10-8 to
10-4 mol/L) in MCAs and BAs in the presence of
indomethacin (5 µmol/L) and baicalein (0.5
µmol/L). The results of these studies are presented in Figure 5. The dilator response to cGMP after
preconstriction with serotonin was similar in BAs versus
MCAs except at the lowest concentration of agonist studied. The control
inner diameter of BAs averaged 154±10 µm (n=5 vessels, 5 rats).
8-Bromo-cGMP (10-8 to
10-4 mol/L) dose-dependently dilated BAs to a
maximum of 66±8% and MCAs to a maximum of 70±4%.
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Immunodetection of CYP 4A and sGC Proteins in MCAs and BAs
We also examined whether differences in the expression of CYP 4A
and sGC could explain the difference in the relative contribution of
the cGMP pathway to the NO-mediated vasodilation in BAs versus MCAs.
Typical examples of immunoblots for CYP 4A and sGC are
presented in Figure 6.
Only one CYP 4A immunoreactive band (52 kDa) was detected in both
MCAs and BAs, and it migrated like the CYP 4A2 and 4A3 isoforms
expressed in the positive control (rat liver). There was no significant
difference in the levels of CYP 4A protein in MCAs and BAs. We also
performed immunoblot experiments for sGC using a polyclonal
antibody. Liver, MCAs, and BAs exhibited one sGC immunoreactive band
(77 kDa), and there was no significant difference in pixel density
between bands, suggesting that they express similar levels of sGC
protein.
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Contribution of 20-HETE to the CBF Response to NO Donors in the
Intact Rat
To determine whether inhibition of 20-HETE production
contributes to the dilator actions of NO in the cerebral circulation of
rats in vivo, we examined the effects of an
intracerebroventricular infusion of the
CYP 4A inhibitor DDMS (50 pmol/min) on the CBF response to
an intracerebroventricular infusion of
the short-acting NO donor MAHMA nonoate (10 nmol/min) in
anesthetized rats. The results of these studies are
presented in Figure 7. In
preliminary experiments, we found that 10 nmol/min was the largest dose
of NO donor that could be infused
intracerebroventricularly that did not
lower systemic blood pressure. The responses to NO donor and DDMS were
similar in ketamine pentobarbital–anesthetized rats
compared with chloralose-treated rats; thus, the results from these 2
groups were combined. In the present experiments, mean
arterial pressure averaged 106±5 mm Hg during
intracerebroventricular infusion of
MAHMA nonoate and 104±4 mm Hg during
intracerebroventricular infusion of the
NO donor and DDMS (n=10 rats). Cerebral LDF signal increased by 52±6%
during intracerebroventricular infusion
of the NO donor. After blockade of 20-HETE formation with DDMS (50
pmol/min), the NO donor increased the cerebral LDF signal by only
8±2%. In time-control experiments (n=4 rats),
intracerebroventricular infusion of
vehicle alone had no effect on the second response to
intracerebroventricular infusion of the
NO donor (data not shown).
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To determine whether DDMS had nonspecific inhibitory effects on cerebral vascular responses, the effects of DDMS on the response to the NO-independent vasodilator adenosine were examined in isolated arteries. The inner diameter of MCAs averaged 77±7 µm (n=4 vessels, 3 rats), and it fell to 42±5% of control after adding serotonin to the bath. Under these conditions, adenosine (10-7 to 10-3 mol/L) dose-dependently increased vessel diameter to a maximum of 91±7% of the serotonin-constricted value. Blockade of 20-HETE formation with DDMS (25 µmol/L) did not impair the vasodilator response to adenosine. After DDMS, adenosine (10-7 to 10-3 mol/L) dose-dependently increased vessel diameter to a maximum of 99±9% of the serotonin-preconstricted diameter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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Although NO plays an important role in the control of cerebrovascular tone, its mechanism of action is still not completely understood. It is generally thought that the effects of NO in the cerebral circulation are due to stimulation of GC, followed by a rise in cGMP levels and activation of K+ channels.2 14 19 20 21 31 However, the results of several recent studies showing that inhibitors of GC cannot completely prevent NO-induced vasodilation in arteries from different vascular beds, including cerebral vessels, suggest that a "cGMP-independent" mechanism may also be involved.2 18 22 23 24 25 32 Therefore, the present study examined the relative contributions of the cGMP-dependent and -independent pathway to the NO-induced vasodilation of rat cerebral arteries. We found that in small lenticulostriate branches of MCAs, blockade of GC with ODQ attenuated the vasodilator response to NO donors by only
50%. In contrast, ODQ had no effect on the
vasodilator response to adenosine, suggesting that the effects
of ODQ were selective for the NO donor. Similarly, blockade of PKG with
KT5823 decreased the response to NO by only 25%. The inability of
KT5823 to block the response to SNP was not due to a failure to inhibit
PKG activity. Indeed, KT5823 completely blocked the vasodilator
response to 8-bromo-cGMP in these vessels. Together, the results
suggest that in rat MCAs studied in vitro, a large component, between
50% and 75%, of the response to NO is cGMP independent.
We have recently reported that NO inhibits the CYP 4A enzyme
responsible for the formation of 20-HETE in the kidney and that a fall
in 20-HETE levels mediates the cGMP-independent actions of NO in the
renal microcirculation.26 27 These findings led us to
consider the hypothesis that a similar mechanism may contribute to the
vasodilator response to NO in the cerebral microcirculation. Our
results indicate that preventing the NO-induced fall in 20-HETE levels
markedly impairs the vasodilator response to low doses of SNP and
attenuates the response to higher doses of SNP by 65% in MCAs.
Simultaneous addition of 20-HETE (100 nmol/L) and ODQ to
the bath completely blocks the vasodilator response to SNP in these
arteries. Moreover, the residual dilator response to the NO donor seen
after fixing 20-HETE levels was blocked by ODQ, indicating that it is
mediated by cGMP. These findings suggest that the vasodilator response
to NO in MCAs of the rat can be completely explained on the basis of a
rise in cGMP and a fall in 20-HETE levels and that the direct effect of
NO on K+ channels does not contribute to this
response.
Our findings in MCAs are not consistent with the results of recent studies demonstrating that GC inhibition blocks 80% to 90% of the vasodilator response to NO donors in rat,19 mouse,20 or rabbit21 pial arterioles and rat BAs33 in vivo. We considered the possibility that different mechanisms may mediate the NO-induced vasodilation in different types of cerebral vessels. Indeed, our findings in BAs are entirely consistent with the results of the previous studies showing that blockade of GC completely impaired the dilator response to NO donors in pial and BAs in vivo and large conduit arteries in vitro.2 14 21 31 33 At the present time, we cannot explain why the relative contribution of the cGMP pathway to the NO-mediated vasodilation differs in BAs versus MCAs. One possible explanation could be that the expression of GC and CYP 4A enzymes might differ in BAs and MCAs. However, our immunoblot results do not support this idea. We found that the expression of sGC and CYP 4A protein is similar in rat MCAs and BAs. It is also possible that the response of K+ channels and other proteins to cGMP and/or 20-HETE may differ in BAs versus MCAs. However, in the present study, both BAs and MCAs exhibited a similar sensitivity to exogenous administration of 8-bromo-cGMP. It is also possible that NO donors may activate different types of K+ channels in BAs versus MCAs. In this regard, Sobey and Faraci33 reported that vasodilatation caused by NO in rat BAs is mediated by activation of voltage-dependent K+ channels through a cGMP-dependent pathway. In contrast, studies by Onoue and Katusic17 indicate that in canine MCAs, most of the NO-induced vasodilation is cGMP independent and is mediated by activation of large-conductance Ca2+-activated K+ channels.
The conclusion that the NO-induced vasodilation of cerebral vessels is cGMP-dependent is based on the evidence that ODQ is a selective inhibitor of sGC. Recently, Feelisch et al25 reported that ODQ inhibits not only sGC but also NO synthase and markedly attenuates the release of NO from SNP and glycerol trinitrate by tissue. These side effects of ODQ may explain why this inhibitor appears to be more effective at blocking the vasodilator response to SNP and NO-dependent vasodilators in vivo compared with in vitro preparations. Studies showing that ODQ completely blocks NO-induced vasodilation were performed by using SNP and cranial window preparations in vivo,19 20 21 33 whereas the studies showing incomplete blockade of NO responses by ODQ were performed by using isolated vessel preparations.18 22 25 The surrounding parenchymal tissue in experimental in vivo preparations catalytically releases large quantities of NO from SNP and other donors, whereas the spontaneous hydrolysis of these donors in aqueous solutions used in vitro is limited. Thus, the nonspecific effects of ODQ would be more pronounced in studies using cranial window preparations. Regardless of the mechanisms involved, the present in vitro studies demonstrate that a fall in 20-HETE levels contributes in a major way to the vasodilator response to NO in the MCA of the rat but not in the larger BA.
Additional studies were performed to determine to what extent, if any,
changes in 20-HETE levels that occur in some but not all cerebral
arteries contribute to the CBF response to NO in the intact rat. We
measured CBF through a thin cranial window by use of laser Doppler
flowmetry to obtain an idea of the integrated blood flow
effects of NO on cerebral vessels of various sizes. We found that an
intracerebroventricular infusion of the
NO donor increased CBF by 50%. After blockade of 20-HETE formation
with DDMS, the NO donor increased CBF by only 8%. These results
indicate that blockade of the formation of 20-HETE attenuated the
cerebral vasodilator response to NO by 80%. Because CYP 4A activity
can be affected by anesthetics, we performed our experiments with 2
different anesthetics, ie, ketamine and a low dose of
pentobarbital or chloralose and urethane. We found that DDMS attenuated
the cerebral vasodilator response to the NO donor by about the same
degree regardless of the anesthetic used. The reason the blockade of
20-HETE formation had such a large effect on the cerebral vasodilator
response to NO in vivo remains to be determined, but it may indicate
that an intracerebroventricular
infusion of the NO donor could preferentially affect the small
resistance vessels in the cerebral cortex.
In summary, the present results indicate that in the rat BA, nearly all of the vasodilator response to NO can be blocked by ODQ; thus, it is cGMP dependent. In rat MCA, however, about half of the dilator response to NO is cGMP independent, and this is largely mediated by a NO-induced fall in 20-HETE production. Moreover, the results of our in vivo studies indicate that inhibition of the formation of 20-HETE does contribute to the cerebral vasodilator response to NO in the intact rat.
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Acknowledgments |
|---|
This study was supported by National Institutes of Health grants HL-29587, HL-36279, and GM-56398–01. Dr Alonso-Galicia was supported by a research fellowship award from the National Kidney Foundation. The authors wish to thank Dr John R. Falck (Southwestern Medical Center, Dallas, Tex) for supplying DDMS and 20-HETE.
Received May 18, 1999; revision received August 10, 1999; accepted September 8, 1999.
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Editorial Comment
Associate Editor for Basic Science, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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The mechanisms of regulation of cerebral vascular tone are complex. Multiple vasodilator and vasoconstrictor agents are involved. There is a strong possibility that these agents may interact with each other. A fuller understanding of the mechanisms of regulation of the cerebral circulation requires a more complete understanding of the mechanisms by which vasoactive agents exert their action and also a more detailed understanding of the mechanisms by which they interact with each other.
The article above by Alonso-Galicia and colleagues addresses the mechanisms by which NO dilates cerebral arteries. The authors showed that two mechanisms are involved. First, NO activates guanylyl cyclase to generate cyclic GMP. The second mechanism involves the inhibition by NO of the synthesis of 20-HETE, a vasoconstrictor agent. The authors showed that both of these mechanisms are involved in vitro and in vivo. Another important finding was that the relative contributions of the two mechanisms were different in different cerebral arteries. For example, in the basilar artery the vasodilator action of NO was due mostly to guanylyl cyclase activation, while in the middle cerebral arteries both mechanisms contributed. It is likely that differences between vessels as well as species differences may be uncovered by future investigation. Studies such as this emphasize that the great complexity of the mechanisms of regulation of vascular tone is a fertile field of investigation.
Received May 18, 1999; revision received August 10, 1999; accepted September 8, 1999.
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