P2u Receptor–Mediated Release of Endothelium-Derived
Relaxing Factor/Nitric Oxide and Endothelium-Derived
Hyperpolarizing Factor From Cerebrovascular
Endothelium in Rats
Junping You, MD; T. David Johnson, PhD; Sean P. Marrelli, PhD;
Jean-Vivien Mombouli, PhD; Robert M. Bryan, Jr, PhD
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Background and Purpose—Stimulation of P2u purinoceptors by UTP on endothelium dilates the rat middle cerebral artery
(MCA) through the release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) and an unknown relaxing
factor. The purpose of this study was to determine whether this unknown relaxing factor is endothelium-derived
hyperpolarizing factor (EDHF).
Methods—Rat MCAs were isolated, cannulated, pressurized, and luminally perfused. UTP was added to the luminal
perfusate to elicit dilations.
Results—Resting outside diameter of the MCAs in one study was 20967 mm (n510). The MCAs showed concentrationdependent dilations with UTP administration. Inhibition of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME)
(1 mmol/L to 1 mmol/L) did not diminish the maximum response to UTP but did shift the concentration-response curve to
the right. Scavenging NO with hemoglobin (1 or 10 mmol/L) or inhibition of guanylate cyclase with ODQ (1 or 10 mmol/L)
had effects on the UTP-mediated dilations similar to those of L-NAME. In the presence of L-NAME, dilations induced by
10 mmol/L UTP were accompanied by 1362 mV (P,0.009) hyperpolarization of the vascular smooth muscle membrane
potential (22862 to 24161 mV). Iberiotoxin (100 nmol/L), blocker of the large-conductance calcium-activated K channels,
sometimes blocked the dilation, but its effects were variable. Charybdotoxin (100 nmol/L), also a blocker of the
large-conductance calcium-activated K channels, abolished the L-NAME–insensitive component of the dilation to UTP.
Conclusions—Stimulation of P2u purinoceptors on the endothelium of the rat MCA released EDHF, in addition to
EDRF/NO, and dilated the rat MCA by opening an atypical calcium-activated K channel. (Stroke. 1999;30:1125-1133.)
Key Words: cerebrovascular circulation n endothelium-derived relaxing factor n endothelium, vascular
n muscle, smooth n potassium channels n rats
T
he naturally occurring purine and pyrimidine phosphates—ATP, ADP, and UTP— dilate cerebral vessels
by stimulating purinoceptors located on the endothelium.1– 8
In the rat middle cerebral artery (MCA), ADP likely produces
dilation through stimulation of the P2y (or P2Y1) purinoceptors with the subsequent synthesis and release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO).7 (The
term EDRF/NO is used [in place of the commonly used term
NO] to accurately reflect the fact that the molecular structure
of this relaxing factor is not known. The relaxing factor
responsible for stimulating guanylate cyclase in many circumstances may be an NO-containing compound. This is
especially true in the cerebral circulation, where there is good
evidence against the gas, NO, and evidence for a NOcontaining compound.9,10) On the other hand, ATP and UTP
See Editorial Comment, page 1132
dilate the rat MCA by stimulating P2u purinoceptors (likely
P2Y2 subtype) with the subsequent release of EDRF/NO and
possibly another endothelium-derived relaxing factor.7 NGNitro-L-arginine methyl ester (L-NAME), an NO synthase
inhibitor, did not diminish the maximum dilation but did
increase the concentration of UTP or ATP necessary to
produce one half of the maximum dilation by '10-fold.
Either EDRF/NO was not completely blocked by L-NAME
(10 mmol/L) or a relaxing factor other than EDRF/NO was
also involved with the dilation.
In this study we tested the following hypothesis: The
unknown relaxing factor involved with UTP-mediated dilations in rat MCAs is endothelium-derived hyperpolarizing
Received November 3, 1998; final revision received January 12, 1999; accepted February 3, 1999.
From the Departments of Anesthesiology (J.Y., T.D.J., S.P.M., R.M.B.) and Medicine (J-V.M.) and the Graduate Program in Cardiovascular Sciences
of the DeBakey Heart Center (S.P.M., R.M.B.), Baylor College of Medicine, Houston, Tex, and the Department of Internal Medicine, University of Lund
(J.Y.), Lund, Sweden.
Reprint requests to Robert M. Bryan, Jr, Department of Anesthesiology, Room 434D, Baylor College of Medicine, One Baylor Plaza, Houston, TX
77030. E-mail [email protected]
© 1999 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
1125
1126
EDRF/NO and EDHF From Cerebrovascular Endothelium
factor (EDHF). EDHF, which is distinct from either
EDRF/NO or prostacyclin, dilates vessels by hyperpolarizing
the vascular smooth muscle through K channel activation.11–13 Given that EDHF has not been positively identified
and that it may be more than a single compound,11–14 it would
be difficult to positively determine the exact agent responsible for the unknown component of the dilation. Consequently, we asked whether this component of the UTPmediated dilation had classic characteristics of EDHF.11–13
(1) Is it distinct from EDRF/NO? This issue is critical in light
of a recent publication indicating that endothelial EDRF/NO
may be difficult to block.15 (2) Is it distinct from prostacyclin
or another cyclooxygenase metabolite? We have previously
determined that the dilation did not involve a cyclooxygenase
metabolite,7 and therefore we will not further address this
issue in the present studies. (3) Does it hyperpolarize the
vascular smooth muscle? (4) Are K channels involved in the
dilation?
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Materials and Methods
The Animal Protocol Review committee at Baylor College of
Medicine approved the experimental protocol. One hundred twenty
male Long-Evans rats (weight, 250 to 350 g) were anesthetized with
3% isoflurane and decapitated. The brain was immediately removed
and placed in cold (4°C) physiological salt solution (PSS). With the
aid of a dissecting microscope, both MCAs were carefully harvested
beginning at the circle of Willis and extending 6 to 8 mm distally. A
section of the MCA (1 to 2 mm in length) was mounted in an
arteriograph (Living Systems) as previously described.16,17 Micropipettes were inserted into both ends of each MCA and secured in
place with nylon ties. Each MCA was bathed in PSS (37°C) that was
equilibrated with a gas consisting of 20% O2/5% CO2 with a balance
of N2.16,17 The pH of the bath was '7.40, PCO2 '35 mm Hg, and PO2
'130 mm Hg.16
Luminal pressure of the MCAs was maintained at 85 mm Hg by
raising reservoirs to the appropriate height above the MCAs.16
Luminal perfusion was adjusted to 100 mL/min by setting the 2
reservoirs at different heights. Pressure transducers on either side of
the MCA provided a measurement of perfusion pressure across the
MCA and pipettes. The vessels were magnified with an inverted
microscope equipped with a video camera and monitor. Outside
diameters of the MCAs were measured directly from the video
screen. Agonists and other drugs were added either to the extraluminal bath (smooth muscle side) or to the PSS perfusing the lumen
(endothelial side).
After mounting and pressurization, the MCAs developed spontaneous tone by constricting to '75% of the initial diameter over the
course of 1 hour. Experimental protocols were not initiated until the
MCA diameter was stable over a 15-minute period.
Adding UTP to the luminal perfusate selectively stimulated
endothelial purinoceptors.7 The change in MCA diameter was
measured after exposure to UTP concentrations from 1029 to 1024
mol/L. Only 1 concentration-response curve was conducted for each
MCA to avoid the risk of tachyphylaxis.
In 4 MCAs, membrane potential (Em) was measured in individual
vascular smooth muscle cells with the use of glass microelectrodes
filled with 3 mol/L KCl (impedances from 55 to 75 MV). The Em
measurements were made in pressurized perfused MCAs mounted in
the arteriograph so that diameters could be simultaneously recorded.18 The potential difference between the glass microelectrode
and a reference electrode, placed in the bath of the arteriograph, was
measured with a Dagan 8700 Cell Explorer with the output being
displayed on a Tektronix 5223 digitizing oscilloscope. Micropipettes
were made by pulling capillary tubing to a rapid taper (tip '0.1 mm
diameter) with the use of a model P-87 Brown-Flaming micropipette
puller (Sutter). Primary criteria for a successful impalement included
a sharp drop in voltage from baseline on entry of the microelectrode
tip into the cell and no change in microelectrode resistance after
exiting the cell. Em values from 5 different smooth muscle cells were
averaged to obtain a single Em for a given condition in a single MCA.
The number of observations (n) was the number of MCAs studied,
not the number of impalements.
Drugs and Reagents
UTP, L-NAME, BaCl2, 4-aminopyridine, apamin, and KCl were
purchased from Sigma Chemical Co. Glibenclamide, tetraethylammonium chloride (TEA), iberiotoxin, and charybdotoxin were purchased from Research Biochemicals Inc.
1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was purchased from Tocris Cookson, Inc. Oxyhemoglobin was purchased
from Calzyme Laboratories. ODQ was dissolved in ethanol; glibenclamide was dissolved in dimethyl sulfoxide; apamin was dissolved
in 0.05 mol/L acetic acid solution. All other reagents and drugs were
dissolved in distilled water.
L-NAME, oxyhemoglobin, and ODQ were added to both the
abluminal bath and the luminal perfusate; UTP was added only to the
luminal perfusate; all other drugs were added to the abluminal bath
only. Vehicles for the blockers were given to the control MCAs. The
composition of the PSS used to bathe the MCAs was previously
described.16
Statistical Analysis
All data are presented as mean6SEM. For concentration-response
curves to UTP, the results are presented as percentage of the
maximum diameter of the MCAs and calculated by the following
equation: % Maximum Diameter5[(DUTP–Dbase/(Dmax–Dbase)]3100,
where Dmax is the maximum diameter of the MCA at 85 mm Hg, Dbase
is the baseline diameter of the MCA before addition of UTP, and
DUTP is the diameter of the MCA after the luminal administration of
UTP. Dmax is the diameter of the MCA immediately after pressurization to 85 mm Hg and before development of spontaneous tone.
Preliminary results demonstrated that Dmax, as calculated above, was
identical to the diameter of the MCA in calcium-free buffer at
85 mm Hg.
For comparison of the concentration-response curves, repeatedmeasures ANOVA was used with a post hoc Student-Newman-Keuls
test for comparison of individual data points. The acceptable level of
significance was defined as P,0.05.
Results
Dilations to the luminal administration of UTP in control
MCAs and after L-NAME (1 mmol/L to 1 mmol/L) are
shown in Figure 1. Note that the dilations at 1027 and 1026
mol/L UTP (P,0.05 for each concentration compared with
control) were either attenuated or abolished by L-NAME.
However, the dilations to UTP in the presence of L-NAME
were not altered at 1025 or 1024 mol/L UTP. The dilations in
the presence of L-NAME were essentially identical regardless of the concentration (1000-fold increase in L-NAME
concentration; Figure 1).
Figure 2 shows the effects of oxyhemoglobin, a scavenger of EDRF/NO, alone or in combination with L-NAME
(10 mmol/L) on UTP-mediated dilation. Oxyhemoglobin
(10 mmol/L) altered the dilation to UTP in a manner
similar to that of L-NAME (Figure 2a), that is, the
dilations at 1027 mol/L UTP (P5NS) and 1026 mol/L UTP
(P,0.05) were attenuated by oxyhemoglobin. With the use
of repeated-measures ANOVA, the control and oxyhemoglobin groups reached statistical significance (P,0.04),
and there was a significant interaction between groups and
UTP concentration (P50.007). Dilations to UTP in the
presence of L-NAME and oxyhemoglobin were not signif-
You et al
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Figure 1. Diameter change of rat MCAs in response to luminal
applications of UTP in controls and after inhibition of NO synthase with L-NAME (1 mmol/L to 1 mmol/L). Diameters for each
group before administration of UTP: control, 20967 mm (n510);
1 mmol/L L-NAME, 17366 mm (n55); 10 mmol/L L-NAME,
16865 mm (n517); 100 mmol/L L-NAME, 17966 mm (n55);
1 mmol/L L-NAME, 197610 mm (n53). With the use of 2-way
repeated-measures ANOVA, there was a significant group effect
(P,0.0001) and a significant interaction between group and
concentration (P,0.001). *P,0.05 compared with all other
groups at the same concentration (Student-Newman-Keuls
method).
icantly different from the dilations in the presence of
L-NAME alone (Figure 2b).
Figure 3 shows the effects of ODQ, a guanylate cyclase
inhibitor, alone or in combination with L-NAME
(10 mmol/L) on UTP-mediated dilation. ODQ, at concentrations of either 1 or 10 mmol/L, altered the dilation to UTP in
a manner similar to that of L-NAME (P50.0002 compared
Figure 2. Effects of oxyhemoglobin (Hb), a scavenger of NO,
alone (a) or in combination with 10 mmol/L L-NAME (b) on UTPmediated dilation. Diameters for each group before administration of UTP: control, 21666 mm (n56); 10 mmol/L Hb,
20568 mm (n55); L-NAME, 16467 mm (n55);
L-NAME11 mmol/L Hb, 173611 mm (n55); L-NAME110 mmol/L
Hb, 164612 mm (n55). The control and Hb groups (a) were significantly different (P,0.04), and there was a significant interaction between group and concentration of UTP (P,0.0001, 2-way
repeated-measures ANOVA). *P,0.05 compared with dilation in
the control group with 1026 mol/L UTP (Student-Newman-Keuls
method).
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1127
Figure 3. Effects of ODQ, a guanylate cyclase inhibitor, alone
(a) or in combination with 10 mmol/L L-NAME (b) on UTPmediated dilation. ODQ, at concentrations of either 1 or
10 mmol/L, altered the dilation to UTP. With the use of 2-way
repeated-measures ANOVA, there was a significant group effect
(P,0.0001) and significant interaction between group and concentration of UTP (P50.0002). Diameters for each group before
administration of UTP: vehicle control, 16866 mm (n510);
1 mmol/L ODQ, 171613 mm (n56); 10 mmol/L ODQ, 15863 mm
(n54); L-NAME, 16563 mm (n55); L-NAME11 mmol/L ODQ,
15765 mm (n56). *P,0.05 compared with dilation at corresponding UTP concentration in vehicle control group; 1P,0.05
compared with dilation at the corresponding UTP concentration
in the other 2 groups.
with control; Figure 3a). In the presence of L-NAME, ODQ
did not further suppress the dilations to UTP (Figure 3b).
Figure 4 shows mean MCA diameters (n53, top) and Em
(n54, bottom) before and after dilations to 1025 mol/L UTP
Figure 4. Mean diameter of MCAs (n53, top) and Em (n54, bottom) in MCAs before and after dilations to 1025 mol/L UTP in
vessels treated with 10 mmol/L L-NAME. Note that in 1 MCA the
diameter was not simultaneously measured with the Em because
of technical difficulties. *P,0.01 compared with the corresponding measure in MCAs treated with L-NAME alone.
1128
EDRF/NO and EDHF From Cerebrovascular Endothelium
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Figure 5. Effects of 30 mmol/L KCl (abluminal bath) on the dilations produced by the luminal administration of UTP in L-NAME
(10 mmol/L)–treated MCAs. KCl negates the effects of K channels. Diameters for each group before administration of UTP:
L-NAME control, 16865 mm (n517); L-NAME130 mmol/L KCl,
154610 mm (n57). *P,0.05 compared with dilation at the corresponding UTP concentration in L-NAME control MCAs
(repeated-measures ANOVA followed by Student-NewmanKeuls method).
in L-NAME–treated (10 mmol/L) vessels. Diameter and Em
were measured simultaneously from the same MCAs with 1
exception, in which case the diameter measurement was not
obtained. The MCAs significantly dilated 10761 mm after
the administration of UTP (P50.007). The Em of the vascular
smooth muscle significantly hyperpolarized by 1362 mV
(n54; P50.009).
When 30 mmol/L KCl was added to the abluminal bath to
negate any effects of K channels,19 the L-NAME–insensitive
component of the UTP-mediated dilation was significantly
attenuated (Figure 5). The addition of 10 mmol/L glibenclamide or 75 mmol/L BaCl2, blockers of ATP-sensitive and
inward-rectifier K channels, respectively, had no effect on the
L-NAME–insensitive component of the UTP-mediated dilation (Figure 6). 4-Aminopyridine (3 mmol/L), a blocker of
the delayed-rectifier K channels, slightly but significantly
altered the dilations to luminal UTP (Figure 6; group effect,
P50.017; interaction between group and UTP concentration,
P50.047). TEA (either 3 or 10 mmol/L) attenuated the
L-NAME–insensitive component of the UTP-mediated (1025
mol/L) dilation; however, 1 mmol/L TEA had no significant
effect on the response (Figure 7a). TEA is considered
selective for the calcium-activated K (KCa) channels at 1 and
3 mmol/L but is considered a nonspecific inhibitor at
10 mmol/L.20 Apamin (either 1 or 3 mmol/L), a selective
blocker of the small-conductance KCa channels, attenuated the
L-NAME–insensitive component of the UTP-mediated dilation (Figure 7b).
Figure 8 shows that charybdotoxin, a blocker of the
large-conductance KCa channels, completely abolished the
dilation to UTP in MCAs treated with L-NAME. Iberiotoxin,
a blocker of the large-conductance KCa channels, at concentrations of 50 and 100 nmol/L attenuated the dilations to UTP
in MCAs treated with L-NAME (Figure 9a). However, the
effects of iberiotoxin at either concentration were quite
Figure 6. Effects of addition of 10 mmol/L glibenclamide (Glib),
75 mmol/L BaCl2, or 3 mmol/L 4-aminopyridine (4-AP), blockers
of ATP-sensitive, inward-rectifier, and delayed-rectifier K channels, respectively, on the L-NAME–insensitive component of
UTP-mediated dilation in rat MCAs. Diameters for each group
before administration of UTP: L-NAME control, 16865 mm
(n517); L-NAME110 mmol/L Glib, 166614 mm (n55);
L-NAME175 mmol/L BaCl2, 172610 mm (n55);
L-NAME13 mmol/L 4-AP, 15665 mm (n56). *P,0.05 compared
with dilation at the corresponding UTP concentration in L-NAME
control MCAs (repeated-measures ANOVA followed by StudentNewman-Keuls method).
variable. Figure 9b and 9c shows mean responses for the
L-NAME control and individual responses for the MCAs
treated with 50 and 100 nmol/L iberiotoxin, respectively. In
some vessels, iberiotoxin (either 50 or 100 nmol/L) substantially blocked the dilation to UTP, while in other vessels there
appeared to be no block at all.
Discussion
The purpose of the present investigation was to determine the
unknown relaxing factor associated with the stimulation of
purinoceptors with UTP on cerebrovascular endothelium.
This study demonstrated that the L-NAME–insensitive component of the UTP-mediated dilation can be attributed to
EDHF. This conclusion is based on the following: (1) The
unknown relaxing factor was not EDRF/NO. (2) The unknown relaxing factor was not a cyclooxygenase metabolite
such as prostacyclin.7 (3) The dilation produced by the
unknown relaxing factor was accompanied by hyperpolarization of the vascular smooth muscle. (4) The dilation and
hyperpolarization were accompanied by K channel activation.
L-NAME–Insensitive Component of
UTP-Mediated Dilation Is Not Attributed
to EDRF/NO
Stimulation of purinoceptors on cerebrovascular endothelium
by ATP and UTP dilates the rat MCA through a mechanism
involving EDRF/NO, since L-NAME abolished or attenuated
the dilation at lower agonist concentrations.7 However, at
higher concentrations of UTP or ATP (.1026 mol/L),
L-NAME (10 mmol/L) had no effect on the dilation.7 Either
another relaxing factor was involved at the higher concentrations of agonists, or we had not adequately blocked NO
synthase in the original study.7 This issue was critical in light
You et al
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Figure 7. Effects of TEA (a) or apamin (b) on the L-NAME–insensitive component of UTP-mediated dilation in rat MCAs.
Diameters for each group before administration of UTP: (a)
L-NAME control for TEA, 16464 mm (n522);
L-NAME11 mmol/L TEA, 18269 mm (n55); L-NAME13 mmol/L
TEA, 16868 mm (n56); L-NAME110 mmol/L TEA, 14765 mm
(n57); (b) L-NAME control for apamin, 17064 mm (n525);
L-NAME11 mmol/L apamin, 16667 mm (n511);
L-NAME13 mmol/L apamin, 15762 mm (n56). There was a significant group effect for the TEA and apamin studies (P,0.0001
and P50.0007, respectively) and a significant interaction
between group and concentration of UTP for the TEA and
apamin studies (P,0.0001 and P50.012, respectively). *P,0.05
compared with dilation at corresponding UTP concentration in
the corresponding control group (L-NAME alone) (repeatedmeasures ANOVA followed by Student-Newman-Keuls method).
of a recent publication indicating that endothelial EDRF/NO
may be difficult to block.15
The present study conclusively demonstrates that
(1) EDRF/NO was blocked, for all practical purposes, and
(2) a relaxing factor other than EDRF/NO was involved in the
UTP-mediated dilation. This conclusion is based on 3 findings. First, dilations to 1025 and 1024 mol/L UTP were
maintained in the presence of 1 mmol/L, 10 mmol/L,
100 mmol/L, or 1 mmol/L L-NAME. If NO synthase was not
sufficiently inhibited by 10 mmol/L L-NAME,7 then it would
be expected that a 10- or 100-fold concentration increase of
L-NAME (1024 or 1023 mol/L, respectively) would abolish or
at least attenuate the UTP-mediated dilations.15 Since this did
not occur (Figure 1), we conclude that NO synthase was
essentially blocked. Second, oxyhemoglobin, a scavenger of
EDRF/NO, attenuated the dilation to UTP in a manner similar
to that of L-NAME (Figure 2a). Furthermore, oxyhemoglobin, in combination with L-NAME, did not further attenuate
the dilation to UTP more than L-NAME alone (Figure 2b). If
NO synthesis was not completely blocked by L-NAME, then
oxyhemoglobin should have abolished or further attenuated
the dilation to UTP. Third, ODQ, an inhibitor of guanylate
cyclase,21 attenuated the dilations to UTP in a manner similar
to that of L-NAME (Figure 3a). EDRF/NO dilates cerebral
vessels by stimulation of soluble guanylate cyclase.21,22 In
combination with L-NAME, ODQ had no further effects on
inhibiting the dilation to UTP than did L-NAME alone
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1129
Figure 8. Effects of charybdotoxin (CHTX) on the L-NAME–insensitive component of UTP-mediated dilation in rat MCAs.
Diameters for each group before administration of UTP:
L-NAME control, 16764 mm (n512); L-NAME1100 nmol/L
CHTX, 186614 mm (n56). *P,0.05 compared with dilation at
the corresponding UTP concentration in L-NAME control MCAs
(repeated-measures ANOVA followed by Student-NewmanKeuls method).
(Figure 3b). Thus, it appears clear that NO synthase was
adequately blocked and that a relaxing factor, other than
EDRF/NO, must be involved in the UTP-mediated dilations
in rat MCAs.
L-NAME–Insensitive Component of the
UTP-Mediated Dilation Is Attributed to EDHF
Ever since Furchgott and Zawadzki23 reported the existence
of endothelium-derived relaxing factor, later identified as
EDRF/NO,24,25 the role of endothelium in the regulation of
vascular tone has been an important and fruitful area of
Figure 9. Effects of iberiotoxin (IBTX) on the L-NAME–insensitive component of UTP-mediated dilation in rat MCAs. Mean
responses are provided in a; mean responses for the L-NAME
control and the individual responses with 50 and 100 nmol/L
IBTX are provided in b and c, respectively. Diameters for each
group before administration of UTP: L-NAME control,
218614 mm (n514); L-NAME150 nmol/L IBTX, 21867 mm
(n57); L-NAME1100 nmol/L IBTX, 230613 mm (n57). *P,0.05
compared with dilation at the corresponding UTP concentration
in L-NAME control MCAs (repeated-measures ANOVA followed
by the Student-Newman-Keuls method).
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EDRF/NO and EDHF From Cerebrovascular Endothelium
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investigation. As the field progressed, it soon became apparent that EDRF/NO could not explain all endotheliumdependent relaxations. Even when one considered the family
of vasodilatory metabolites of the cyclooxygenase pathway,
at least 1 other relaxing factor derived from the endothelium
had to be postulated.26 This relaxing factor became known as
endothelium-derived hyperpolarizing factor (EDHF).11,12,14,26
EDHF is defined as a relaxant, released from the endothelium, that is distinct from both EDRF/NO and prostaglandins
and that dilates vessels by hyperpolarizing the vascular
smooth muscle.12 The hyperpolarization is a result of activation of K channels (for comprehensive reviews, see References 11–14 and 26).
We believe that the L-NAME–resistant component of the
UTP-mediated dilation in the rat MCA is mediated by EDHF.
Our conclusion is based on the following: First, this component is independent of EDRF/NO (see above) or cyclooxygenase metabolites.7 Second, this component of the UTPmediated dilation is accompanied by hyperpolarization of the
vascular smooth muscle (Figure 4). Third, the dilation was
dependent on the activation of K channels (Figures 5 through
9 ). The fact that 30 mmol/L KCl (Figure 5) or 10 mmol/L
TEA (Figure 7a) blocked or attenuated the L-NAME–insensitive component of the UTP-mediated dilation indicates that
K channels were involved.19,20,27 KCl negates any involvement of K channels by making the Nernst potential for K1
and the Em of the smooth muscle equal. Thus, opening of K
channels would produce no net movement of K1 and no
change in the Em. TEA at a concentration of 10 mmol/L is a
general blocker of K channels.20,27 The L-NAME–insensitive
component of the UTP-mediated dilation fits the criteria for
EDHF, defined in the preceding paragraph. We therefore
conclude that the unknown relaxing factor, elicited by stimulating P2u receptors on the endothelium with UTP, is EDHF.
There have been only a few reports of EDHF associated
with agonist-mediated dilations in cerebral arteries. EDHF
may be released from endothelium in rabbit MCA, guinea-pig
basilar artery, and human pial artery after stimulation with
acetylcholine or substance P.28 –30 We have now added to this
list by demonstrating the release of EDHF from cerebrovascular endothelium of the rat MCA. There have been other
published reports in cerebral arteries that might involve
EDHF4,31–34; however, involvement of a cyclooxygenase
metabolite was not ruled out. For our discussion, the term
EDHF is reserved for a substance that differs from a
cyclooxygenase metabolite (and EDRF/NO).12
EDHF may not be a single agent but rather a diverse class
of agents, all of which open K channels.12,14 The exact agent
might vary with species and/or vessel. Candidates for EDHF
include the following: (1) epoxyeicosatrienoic acids, a cytochrome P450 metabolite of arachidonic acid; (2) anandamide,
an endogenous agonist of the cannabinoid receptors; (3) hydrogen peroxide; (4) hydroxyl radicals; (5) superoxide anions; or (6) CO.12,14,35–38 Although many, if not most, investigators believe that EDHF and EDRF/NO are different
entities, EDHF may not be distinct from EDRF/NO in some
vessels. Cohen et al15 reported that an inadequate block of NO
synthase could lead to a false conclusion regarding the
existence of an EDHF distinct from EDRF/NO. For the rat
MCA, we are able to rule out an inadequate block of NO
synthase (see above). The identity of EDHF in the rat MCA
is presently not known and awaits further studies for its
identification.
Identification of the type of K channel involved with the
EDHF-mediated dilation by UTP in the rat MCA is not
straightforward. We are able to immediately rule out the
ATP-sensitive and inward-rectifier K channels since neither
10 mmol/L glibenclamide nor 75 mmol/L BaCl2 had any
effect on the L-NAME–insensitive component of the dilation
(Figure 6).39 – 43 The delayed-rectifier K channels do not
appear to be a major factor, since 4-aminopyridine, a selective blocker of this channel, had only a slight effect on the
UTP-mediated dilations (Figure 6). However, the use of
4-aminopyridine as a general inhibitor of all delayed rectifiers
has been questioned.28,44,45 It is therefore possible that
delayed-rectifier K channels, not sensitive to 4-aminopyridine, could be involved. On the other hand, the fact that TEA
(3 mmol/L), apamin (1 or 3 mmol/L), or iberiotoxin (50 or
100 nmol/L) attenuated the dilation while charybdotoxin (100
nmol/L) completely abolished the L-NAME–insensitive component of the UTP-mediated dilation points to involvement of
the KCa channels. KCa channels, although a distinct class of K
channels, belong to the delayed rectifier K channel superfamily.46 Apamin is a blocker of the small-conductance KCa
channels,41 and charybdotoxin and iberiotoxin are blockers of
the large-conductance KCa channels.41
Our data further support the idea that the largeconductance KCa channel is “atypical” or “nonclassic.” First,
1 mmol/L TEA did not alter the dilation to UTP in L-NAME–
treated MCAs. TEA at this concentration should partially or
fully block the KCa channels20,27,40,41 and therefore should
block or attenuate the dilation. Second, while charybdotoxin
completely blocked the dilation (Figure 8), the response with
iberiotoxin was quite variable (Figure 9). Iberiotoxin is a very
specific and potent inhibitor of KCa channels.41 On the other
hand, charybdotoxin affects certain delayed-rectifier K channels in addition to blocking KCa channels.28,44,45 Given that
our results are not entirely consistent with a “typical” or
“classic” KCa, we conclude that the KCa channel must be
atypical or nonclassic. Atypical K channel involvement has
also been reported for EDHF-mediated dilations by others.
While individual K channel blockers, including apamin,
charybdotoxin, and iberiotoxin, were ineffective in inhibiting
the dilation (or relaxation) by EDHF, the combination of
apamin and charybdotoxin, but not apamin and iberiotoxin,
completely abolished the dilation produced by
EDHF.28,44,45,47
In attempting to explain the type of K channel involved
with the L-NAME–insensitive component of the UTPmediated dilation, we offer 2 possible explanations. First, the
UTP-mediated dilation in the rat MCA might involve both a
small-conductance (apamin-sensitive) and a largeconductance (charybdotoxin-sensitive) KCa channel. The
large-conductance KCa channel would be nonclassic, as discussed above. A second possibility would be the involvement
of a single nonclassic K channel sensitive to charybdotoxin,
apamin, and TEA (3 and 10 mmol/L but not 1 mmol/L) and
somewhat sensitive to iberiotoxin. A nonclassic K channel
You et al
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having similarities to both delayed-rectifier and KCa channels
has been postulated to be involved with the EDHF-mediated
dilations.44 Our results do not exclude this channel type.
The natural agonists for the endothelial P2u purinoceptors in
cerebral vessels in vivo are ATP and UTP in the plasma.48 –51
These agonists appear to stimulate only the P2u receptor
subtype in rat MCAs without affecting the P2y (P2Y1) subtype, which is also present on the endothelium.7 Since UTP
may be more selective at the P2u site than ATP,52 UTP was the
agonist of choice in the present study. UTP in plasma is
derived from endothelium and platelets.49,51,53 ATP in plasma
is derived from endothelium, platelets, and erythrocytes.51,54 –56 It appears that UTP and ATP can reach concentrations of '5 and 50 mmol/L, respectively, in plasma.49 –51
However, there is reason to believe that after platelet aggregation, the concentrations of these nucleotides at the endothelial wall (receptor site) could approach or even reach
concentrations in the millimolar range.48 Thus, these agonists
may reach concentrations necessary to stimulate the synthesis
and release of not only EDRF/NO (1028 to 1026 mol/L for
either agonist) but also EDHF (above 1026 mol/L for either
agonist) (Figures 1 through 3).7
Although stimulation of endothelial purinoceptors is
known to dilate peripheral and cerebral vessels by the release
of EDRF/NO and/or prostacyclin,1,50 our laboratory is the
first to conclusively demonstrate that EDHF is associated
with purinoceptors57 (see also Reference 58). Thus, stimulation of purinoceptors by UTP on endothelium of the rat MCA
dilates the vessel by the release of EDRF/NO at concentrations up to 1026 mol/L UTP and EDHF (likely in combination
with EDRF/NO) at higher concentrations. One or more K
channels are associated with the EDHF-mediated dilation.
Although stimulation of some receptors, including P2y purinoceptors, on cerebrovascular endothelium leads to dilation
exclusively by the release of EDRF/NO or prostacyclin,
stimulation of P2u purinoceptors by UTP also involves EDHF
in the rat MCA. The extent to which EDHF is associated with
other endothelial receptor systems in the cerebral circulation
is not presently known. EDHF may be an important but
understudied relaxing factor released from the endothelium of
cerebral arteries.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Acknowledgments
This study was supported by National Institute of Neurological
Disorders and Stroke grants (PO1 NS-27616 and RO1 NS-37250)
and by a National Institutes of Health training grant (T32HL07816).
26.
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Editorial Comment
Endothelium plays a major role in regulation of vascular
tone.1 A key endothelium-derived relaxing factor in the
cerebral circulation is NO (or a closely related, NOcontaining compound).1 For example, relaxation of the carotid artery, large cerebral arteries, and cerebral microvessels
in response to acetylcholine (the classic endotheliumdependent agonist) is mediated predominantly by NO.1,2
Relaxation of human cerebral arteries in response to endothelium-dependent agonists is also mediated in large part by
NO.3,4
In addition to NO, endothelium may produce other
relaxing factors, including prostacyclin and one or more
endothelium-derived hyperpolarizing factors (EDHFs).5
Many studies define EDHF as an endothelium-derived
relaxing factor that produces hyperpolarization of vascular
muscle but which is not NO or a product of cyclooxygenase. In contrast to NO, which produces relaxation of
cerebral vascular muscle by activation of soluble guanylate
cyclase,6,7 EDHF produces hyperpolarization and relaxation of vascular muscle by activation of potassium channels. In comparison with NO, much less is known regard-
ing the functional importance of EDHF in the cerebral
circulation.
The preceding study provides evidence that uridine
triphosphate (UTP) produces relaxation of the middle cerebral artery through endothelium-dependent mechanisms. Vasorelaxation in response to lower concentrations of UTP was
mediated by NO and activation of soluble guanylate cyclase.
In contrast, relaxation of this artery in response to higher
concentrations of UTP appears to be mediated by an EDHF.
At least two aspects of the present study are noteworthy. First,
the authors tried very hard to eliminate any contribution of
NO and prostacyclin and thus provide stronger evidence that
the factor being studied was an EDHF. This latter point is
important because recent evidence suggests that it can be
difficult to completely inhibit production of NO by endothelium in some blood vessels.8 Second, the authors demonstrated that relaxation of the middle cerebral artery in response to UTP was associated with hyperpolarization of
vascular muscle and attenuated by KCl (which prevents
membrane hyperpolarization) or inhibitors of potassium
channels. Activation of potassium channels in vascular mus-
You et al
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cle is generally believed to mediate relaxation in response to
EDHF.
An important unanswered question in this study, and
studies of EDHF in general, relates to the identity of the
factor(s). Candidate factors have included epoxyeicosatrienoic acids (EETs) and anandamide (products of metabolism
of arachidonic acid), and potassium ion.2,5,9 Recent studies
suggest that EETs and anandamide do not account for
EDHF-mediated responses in cerebral arteries.7,10,11 Although
small-to-moderate increases in the concentration of extracellular potassium produce membrane hyperpolarization and
relaxation of cerebral arteries, this response is mediated by
activation of a barium-sensitive potassium channel (an
inward-rectifier potassium channel).2 This latter characteristic
not consistent with the pharmacological profile obtained for
EDHF in the present study.
Thus, although the identity of EDHF(s) in the cerebral
circulation remains unclear, findings such as those in the
present study illustrate that EDHF may contribute to regulation of cerebral vascular tone under some conditions. It is
noteworthy that recent evidence suggests EDHF may become
functionally more important in disease states that are associated with impairment of the NO/cGMP signaling pathway.2
Frank M. Faraci, PhD, Guest Editor
Department of Internal Medicine
Cardiovascular Center
University of Iowa College of Medicine
Iowa City, Iowa
May 1999
1133
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P2u Receptor −Mediated Release of Endothelium-Derived Relaxing Factor/Nitric Oxide
and Endothelium-Derived Hyperpolarizing Factor From Cerebrovascular Endothelium in
Rats
Junping You, T. David Johnson, Sean P. Marrelli, Jean-Vivien Mombouli and Robert M. Bryan,
Jr
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Stroke. 1999;30:1125-1133
doi: 10.1161/01.STR.30.5.1125
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