(Stroke. 1999;30:1125-1133.)
© 1999 American Heart Association, Inc.
Original Contributions |
P2u Receptor–Mediated Release of Endothelium-Derived Relaxing Factor/Nitric Oxide and Endothelium-Derived Hyperpolarizing Factor From Cerebrovascular Endothelium in Rats
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.
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Abstract |
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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 209±7 µm (n=10). The MCAs showed concentration-dependent dilations with UTP administration. Inhibition of NO synthase with NG-nitro-L-arginine methyl ester (L-NAME) (1 µmol/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 µmol/L) or inhibition of guanylate cyclase with ODQ (1 or 10 µmol/L) had effects on the UTP-mediated dilations similar to those of L-NAME. In the presence of L-NAME, dilations induced by 10 µmol/L UTP were accompanied by 13±2 mV (P<0.009) hyperpolarization of the vascular smooth muscle membrane potential (-28±2 to -41±1 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.
Key Words: cerebrovascular circulation • endothelium-derived relaxing factor • endothelium, vascular • muscle, smooth • potassium channels • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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The naturally occurring purine and pyrimidine phosphates—ATP, ADP, and UTP—dilate cerebral vessels by stimulating purinoceptors located on the endothelium.1 2 3 4 5 6 7 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 NO-containing compound.9 10 ) On the other hand, ATP and UTP 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 NG-Nitro-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 µmol/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 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 12 13 Given that EDHF has not been positively identified and that it may be more than a single compound,11 12 13 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 UTP-mediated dilation had classic characteristics of EDHF.11 12 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 |
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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 µL/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 10-9 to 10-4 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
M
). 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 µm 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 mean±SEM. 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
Diameter=[(DUTP–Dbase/(Dmax–Dbase)]x100,
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, repeated-measures 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.
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Results |
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Dilations to the luminal administration of UTP in control MCAs and after L-NAME (1 µmol/L to 1 mmol/L) are shown in Figure 1
. Note that the dilations at
10-7 and 10-6 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
10-5 or 10-4 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).
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Figure 2 shows the effects of
oxyhemoglobin, a scavenger of EDRF/NO, alone or in combination with
L-NAME (10 µmol/L) on UTP-mediated dilation. Oxyhemoglobin
(10 µmol/L) altered the dilation to UTP in a manner similar to
that of L-NAME (Figure 2a), that is, the dilations at
10-7 mol/L UTP (P=NS) and
10-6 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 (P=0.007). Dilations to UTP in
the presence of L-NAME and oxyhemoglobin were not significantly
different from the dilations in the presence of L-NAME alone (Figure 2b).
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Figure 3 shows the effects of ODQ, a
guanylate cyclase inhibitor, alone or in
combination with L-NAME (10 µmol/L) on UTP-mediated dilation.
ODQ, at concentrations of either 1 or 10 µmol/L, altered the
dilation to UTP in a manner similar to that of L-NAME
(P=0.0002 compared with control; Figure 3a). In the
presence of L-NAME, ODQ did not further suppress the dilations to UTP
(Figure 3b).
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Figure 4 shows mean MCA diameters (n=3,
top) and Em (n=4, bottom) before and after
dilations to 10-5 mol/L UTP in L-NAME–treated
(10 µmol/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 107±1 µm after the administration of
UTP (P=0.007). The Em of the vascular
smooth muscle significantly hyperpolarized by 13±2 mV (n=4;
P=0.009).
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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 µmol/L
glibenclamide or 75 µmol/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,
P=0.017; interaction between group and UTP concentration,
P=0.047). TEA (either 3 or 10 mmol/L) attenuated the
L-NAME–insensitive component of the UTP-mediated
(10-5 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
µmol/L), a selective blocker of the small-conductance
KCa channels, attenuated the L-NAME–insensitive
component of the UTP-mediated dilation (Figure 7b).
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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 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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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 (>10-6
mol/L), L-NAME (10 µmol/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 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
10-5 and 10-4 mol/L UTP
were maintained in the presence of 1 µmol/L, 10 µmol/L,
100 µmol/L, or 1 mmol/L L-NAME. If NO synthase was not
sufficiently inhibited by 10 µmol/L L-NAME,7 then
it would be expected that a 10- or 100-fold concentration increase of
L-NAME (10-4 or 10-3
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
(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 investigation. As the field
progressed, it soon became apparent that EDRF/NO could not explain all
endothelium-dependent 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 1111 –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 UTP-mediated 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 K+ and the Em
of the smooth muscle equal. Thus, opening of K channels would produce
no net movement of K+ 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 29 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 32 33 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 36 37 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 µmol/L glibenclamide nor 75 µmol/L
BaCl2 had any effect on the L-NAME–insensitive
component of the dilation (Figure 6).39 40 41 42 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 µmol/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 large-conductance
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 UTP-mediated 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 large-conductance (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 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 49 50 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 55 56 It appears that UTP and ATP can
reach concentrations of 5 and 50 µmol/L, respectively, in
plasma.49 50 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 (10-8 to
10-6 mol/L for either agonist) but also EDHF
(above 10-6 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 5858 ). 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 10-6 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.
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Acknowledgments |
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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).
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Footnotes |
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Reprint requests to Robert M. Bryan, Jr, Department of Anesthesiology, Room 434D, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
Received November 3, 1998; revision received January 12, 1999; accepted February 3, 1999.
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Editorial Comment
Department of Internal Medicine, Cardiovascular Center, University of Iowa College of Medicine, Iowa City, Iowa
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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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, NO-containing compound).1 For example, relaxation of the carotid artery, large cerebral arteries, and cerebral microvessels in response to acetylcholine (the classic endothelium-dependent 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 regarding 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 muscle 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
Received November 3, 1998; revision received January 12, 1999; accepted February 3, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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1. Faraci FM, Heistad DD. Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol Rev.. 1998;78:53–97.
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