© 1999 American Heart Association, Inc.
Original Contribution |
Nitric Oxide–Independent Relaxations to Acetylcholine and A23187 Involve Different Routes of Heterocellular Communication
Role of Gap Junctions and Phospholipase A2
From the Departments of Diagnostic Radiology (I.R.H., A.T.C., T.M.G.) and Medical Biochemistry (W.H.E.), Cardiovascular Sciences Research Group, University of Wales College of Medicine, Cardiff, United Kingdom.
Correspondence to Prof Tudor M. Griffith, Department of Diagnostic Radiology, Cardiovascular Sciences Research Group, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK. E-mail griffith{at}cardiff.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—NO- and prostanoid-independent relaxations are generally assumed to be mediated by an endothelium-derived hyperpolarizing factor (EDHF) that has been postulated to be an arachidonic acid metabolite. Recent evidence also suggests that direct heterocellular gap junctional communication (GJC) between endothelium and smooth muscle contributes to NO-independent relaxations. In the present study we have investigated the contribution of phospholipase A2 (PLA2)-linked metabolites and GJC to EDHF-type relaxations in rabbit mesenteric artery. In isolated rings preconstricted with 10 µmol/L phenylephrine in the presence of NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin, acetylcholine (ACh) and the Ca2+ ionophore A23187 evoked relaxations that were markedly attenuated by the Ca2+-dependent PLA2 inhibitors 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (3 µmol/L) and arachidonyl trifluoromethyl ketone (3 µmol/L), but were potentiated by the sulfhydryl agent thimerosal (300 nmol/L). In intact rings, relaxations to ACh were attenuated synergistically by L-NAME and Gap 27 peptide, an inhibitor of GJC, whereas ACh-evoked relaxations of "sandwich" preparations were unaffected by the peptide but were abolished by L-NAME. In both ring and sandwich preparations A23187-induced relaxations were attenuated by inhibition of PLA2 but were insensitive to L-NAME and Gap 27 peptide. We conclude that EDHF-type relaxations of rabbit mesenteric artery to ACh and A23187 depend on a common pathway that involves activation of PLA2. In the case of ACh, relaxation requires transfer of a factor or factors from the endothelium to smooth muscle via gap junctions, whereas A23187 permits release directly into the extracellular space.
Key Words: EDHF • phospholipase A2 • gap junction • acetylcholine • A23187
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The endothelium plays a central role in the control of vascular tone through the release of vasoactive autacoids in response to agonist stimulation and shear stress.1 Mediators include NO, prostanoids, and in many vessel types a distinct endothelium-derived hyperpolarizing factor (EDHF).2 3 The chemical identity of EDHF remains controversial, although it seems probable that it mediates smooth muscle hyperpolarization by activating K+ channels (see References 2 and 32 3 for review). Recent reports suggest that epoxyeicosatrienoic acids (EETs), which are cytochrome P-450 monooxygenase metabolites of arachidonic acid, and the endocannabinoid anandamide, which is also derived from arachidonic acid, possess the characteristics of EDHF in certain artery types.3 4 5 6 It has been suggested that the source of arachidonic acid for EDHF production is the Ca2+-dependent cytosolic form of phospholipase A2 (PLA2), since specific inhibitors of this enzyme attenuate NO-independent relaxations to acetylcholine (ACh), histamine, and bradykinin in the perfused rat heart and mesenteric bed.7 8
EDHF has been detected both in cascade bioassay9 10 and in sandwich preparations,11 suggesting that this factor can diffuse freely in the extracellular space. However, recent evidence suggests that NO-independent relaxations in rabbit mesenteric artery and aorta could involve the preferential direct transfer of a mediator through gap junctions, rather than via/by an extracellular route.12 13 Intercellular continuity is facilitated by gap junctions that are assembled when 2 connexon hemichannels supplied by neighboring cells interact and dock. Each connexon consists of 6 connexin protein subunits arranged around a central pore,14 and the characteristic pentalaminar appearance of sections of gap junction plaques has previously been demonstrated in rabbit conduit arteries.15 Connexin 43 is the most prevalent subtype in endothelium-denuded rabbit superior mesenteric artery, and connexins 37, 40, and 43 have been identified in endothelial cells.16 17 Previous studies have shown that the inhibitory Gap 27 peptide (amino acid sequence SRPTEKTIFII), which possesses conserved sequence homology with a region of the second extracellular loop of these connexins, rapidly and reversibly attenuates NO-independent relaxations to the agonists ACh and adenosine triphosphate and also to cyclopiazonic acid.12 Direct heterocellular communication may therefore contribute to EDHF-mediated smooth muscle relaxations evoked by receptor-dependent and -independent activation of the endothelial cell. Although the exact mode of action of Gap 27 peptide remains to be elucidated at the molecular level, possibilities include (1) inhibition or reversal of connexon docking before or after penetration of the peptide into the intercellular gap and (2) induction of a conformational change that results in channel closure.16 18
In the present study we have used isolated ring and sandwich preparations to evaluate the contributions of gap junctional communication (GJC) and PLA2 to NO-independent relaxations evoked by ACh and the calcium ionophore A23187 in rabbit superior mesenteric artery. Arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of the cytosolic form of PLA219 ; [E-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-1] (HELSS), a specific inhibitor of the secretory form of PLA220 ; 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082), an inhibitor of both forms of PLA221 ; 1-(6-{[17ß-3-methoxyestra-1,3,5(10)-triene-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), a specific inhibitor of phospholipase C (PLC)22 ; and thimerosal, an organic mercury sulfhydryl reagent that is known to inhibit the acyl-coenzyme A:lysolecithin acyltransferase23 and to modulate mobilization of intracellular Ca2+ from stores,24 were used to characterize the pathways involved in the mediation of these relaxations. Gap 27 peptide was used to elucidate the predominant diffusion pathway of the putative mediator once formed. Previous studies have shown that this peptide does not directly affect ambient force development in the rabbit mesenteric artery.16
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Isolated Ring Preparations
Experiments were performed with superior mesenteric arteries from male New Zealand White rabbits (2.5 kg) that had been sacrificed by injection of sodium pentobarbitone (120 mg/kg IV). The tissues were transferred to cold Holman's solution of the following composition (in mmol/L): NaCl 120, KCl 5, NaH2PO4 1.3, NaHCO3 25, CaC12 2.5, glucose 11, and sucrose 10. Rings (2 to 3 mm wide) were cut and suspended by thread from FT102 force transducers connected to a MacLab/4e (ADInstruments) in 3-mL organ baths containing gassed (95% O2 and 5% CO2, pH 7.4) Holman's solution at 37°C to measure isometric force development. The rings were placed under a resting tension of 0.5g and allowed to equilibrate for
1 hour with frequent readjustment of
tension to allow for stress relaxation.
Experimental Protocol
The rings were precontracted with 10 µmol/L
phenylephrine, and cumulative concentration-response curves
to ACh (1 nmol/L-10 µmol/L) and the Ca2+
ionophore A23187 (0.1 nmol/L-1 µmol/L) were constructed before
and after a 45-minute incubation with
NG-nitro-L-arginine
methyl ester (L-NAME) (300 µmol/L) alone or a combination of
L-NAME and 1H-(1,2,4)-oxadiazolo-(4,3a)-quinoxaline-1-1 (ODQ, 10
µmol/L), an inhibitor of guanylate cyclase.
Experiments were also performed in the presence of
indomethacin (10 µmol/L) to determine the
involvement of prostanoid synthesis. The role of GJC was investigated
by preincubating for 20 minutes with Gap 27 peptide (300
µmol/L). To analyze the role of PLA2
and PLC in the L-NAME–sensitive and –insensitive components of the
relaxations, cumulative concentration-response curves were constructed
following a 45-minute incubation with ONO-RS-082 (1 to 10
µmol/L), AACOCF3 (3 µmol/L), HELSS (300
nmol/L), or U-73122 (500 nmol/L), respectively. In other studies,
thimerosal (300 nmol/L) was added 20 minutes before construction of
concentration-response curves to ACh and A23187 in the absence and
presence of L-NAME. These experiments were then repeated in the
presence of Gap 27 peptide (300 µmol/L).
To assess the vasoactivity of EETs in this preparation, cumulative concentration-response curves to 5,6-, 11,12-, and 14,15-EET (100 nmol/L to 3 µmol/L) were constructed in the absence and presence of L-NAME. In studies in which relaxation was observed, the experiments were repeated in the presence of Gap 27 peptide. Responses to ACh and A23187 were also investigated before and after a 30-minute incubation with 5 µmol/L of each of the 3 EET isomers.
Sandwich Experiments
In a separate series of experiments, rings of superior
mesenteric artery were cut open and small strips of
endothelium intact vessel were sutured onto strips of
endothelium-denuded artery, with their intimal surfaces
in close apposition, to provide composite preparations. These sandwich
preparations were allowed to equilibrate at a tension of 0.6 g for
1 hour before precontraction with phenylephrine (10
µmol/L), with tension monitored only in the
endothelium-denuded vessel. The transferable nature of
endothelium-dependent relaxations to ACh and A23187
were assessed in the absence and presence of L-NAME (300
µmol/L), ONO-RS-082 (3 µmol/L), and Gap 27 peptide (300
µmol/L).
Materials
ACh, phenylephrine, indomethacin,
thimerosal, A23187, and L-NAME were obtained from Sigma. U-73122 was
obtained from Calbiochem-Novabiochem; ONO-RS-082 was obtained from
Alexis Corp; and 5,6-EET, 11,12-EET, 14,15-EET, HELSS,
AACOCF3, and ODQ were obtained from Affiniti
Research Products Ltd. Gap 27 peptide (SRPTEKTIFII) was synthesized
by Severn Biotech Ltd. All drugs were dissolved in Holman's buffer
with the exception of U-73122, HELSS, AACOCF3,
ODQ, and ONO-RS-082 (DMSO); 5,6-, 11,12-, and 14,15-EET (ethanol); and
indomethacin (5% wt/vol NaHCO3
in distilled water).
Statistics
All data are given as mean±SEM, and n denotes the number of
animals studied for each data point. Concentration-response curves were
assessed by 1-way ANOVA followed by the Bonferroni multiple comparisons
test. EC50 and maximal responses were compared by
the Student's t test for paired and unpaired data as
appropriate. P<0.05 was considered as significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Effects of PLA2 and PLC Inhibition on ACh- and A23187-Induced Relaxations in Intact Rings
ACh and A23187 both evoked endothelium-dependent relaxations of mesenteric rings that were maximal at
3 µmol/L
and 300 nmol/L, respectively. Addition of L-NAME (300 µmol/L)
to the organ bath increased tone by 18.8±1.5% (n=20) and attenuated
ACh-induced relaxations, causing an increase in the
EC50 value from 0.16±0.02 to 0.76±0.11
µmol/L (P<0.001, n=25) and a reduction in the maximal
response (control, 70.9±2.8%; L-NAME, 26.4±2.7%;
P<0.001, n=25). The EC50 value for
relaxation to A23187 was similarly increased from 50.0±1 to 134.5±30
nmol/L following incubation with L-NAME (n=21), and there was a smaller
but significant reduction in maximal relaxation from 73.4±3.4% to
61.1±2% (n=21, P<0.01). These values were obtained by
pooling data from all experiments. Addition of ODQ (10 µmol/L)
in the presence of L-NAME had no further inhibitory effect
on either ACh- or A23187-induced relaxations (Figure 1
). Similarly,
indomethacin was without effect on the
EC50 values and maximal relaxations to ACh or
A23187 both in the absence and presence of L-NAME (Figure 1).
Indomethacin was included in the buffer for all
remaining studies.
|
In the absence of L-NAME, inhibition of
PLA2 with ONO-RS-082 (3 µmol/L) had no
effect on ACh-induced relaxations (Figure 2A) in terms of either the
EC50 value (control, 0.18±0.05 µmol/L;
ONO-RS-082, 0.18±0.09 µmol/L; n=5) or maximal relaxations
(control, 73.5±2.4%; ONO-RS-082, 72.7±4.2%; n=5) but increased the
EC50 values for A23187 from 50±0.1 to 340±80
nmol/L (P<0.01, n=4, Figure 2B), although there was
no significant effect on maximal relaxations (control, 70.2±3%;
ONO-RS-082, 64.5±3.3%). There was also no effect of
AACOCF3 (3 µmol/L) on ACh-induced
relaxations in terms of either the EC50 value
(control, 0.16±0.01 µmol/L; AACOCF3,
0.13±0.03 µmol/L; n=3 for both) or maximal relaxations
(control, 70.8±4.7%; AACOCF3, 72.5±2.1%; n=3
for both), but this agent significantly increased the
EC50 value for A23187 from 51±0.3 to 122±29
nmol/L (P<0.05, n=3) and caused a small but not significant
reduction in the maximal response from 67.3±2.9% to 53.3±7.9%
(n=3). In the presence of L-NAME, ONO-RS-082 almost abolished
ACh-induced relaxations, with maximal responses being reduced from
37.7±4.6% to 8.8±3% (P<0.01, n=8, Figure 2A).
AACOCF3 also virtually abolished
relaxations to ACh, with maximal responses being reduced from
26.4±3.2% to 6.6±1.4% (P<0.01, n=4, Figure 2A).
Furthermore, both ONO-RS-082 and AACOCF3
significantly attenuated the maximal L-NAME–insensitive relaxations to
A23187 from 54.2±7% to 23.4±7.2% (P<0.01, n=5, Figure 2B) and from 66.5±3.4% to 27.3±7.8% (P<0.05,
n=6, Figure 2B), respectively. HELSS (300 nmol/L) had no effect
on ACh-induced relaxations in the presence of L-NAME either in terms of
the EC50 value (control, 0.18±0.03
µmol/L; HELSS, 0.27±0.07 µmol/L; n=4) or maximal response
(control, 23.1±5.1%; HELSS, 24.4±6%; n=4). Similarly, HELSS was
without effect on the L-NAME–insensitive responses to A23187, with
EC50 values (control, 108±21 nmol/L; HELSS,
93.5±32 nmol/L; n=4) and maximal responses (control, 66.5±3.4%;
HELSS, 60.5±9.5%; n=4) being unchanged.
|
Preincubation with U-73122 (500 nmol/L), a specific PLC
inhibitor, in the presence of L-NAME attenuated relaxations
to ACh, with EC50 values being increased from
0.36±0.12 to 1.06±0.3 µmol/L and maximal relaxations reduced
from 29.6±7.6% to 10.2±3.1% (P<0.05, n=4, Figure 2C
). U-73122 had no significant effect on the
L-NAME–insensitive response to A23187 (Figure 2D).
Effects of Gap 27 Peptide on ACh- and A23187-Induced Relaxations in
Intact Rings
Preincubation of rings with Gap 27 peptide (300 µmol/L) had
no effect on phenylephrine-induced tone (not shown) but
significantly attenuated relaxations to ACh, reducing the maximal
response from 81.3±4% to 58.8±5.5% (P<0.01, n=8) and
causing a rightward shift in the concentration-response curve and an
increase in the EC50 value from 200±20 to
480±80 nmol/L (P<0.01, n=8, Figure 3A). By contrast, A23187-induced
relaxations were completely unaffected by pretreatment with Gap 27
peptide (n=4, Figure 3B), with no change in either
EC50 values (52±3 to 50±4 nmol/L) or maximal
responses (75.9±6% to 67.5±2%). In the presence of L-NAME (300
µmol/L), Gap 27 peptide significantly attenuated the residual
relaxation to ACh, causing a further increase in the
EC50 value from 281±93 to 428±52 nmol/L (n=4)
and a significant reduction in the maximal response from 54±3.2% to
33.3±4.9% (P<0.05, n=4, Figure 3A). Gap 27 peptide
was without effect on the L-NAME–insensitive component of the
relaxations to A23187 either in terms of the EC50
values (54±6 to 58±8 nmol/L, n=4) or maximal response (61.5±1.3% to
61.8±4.1%) (n=4, Figure 3B). Figure 3C illustrates that
for relaxations greater than 20%, the time taken from onset to
maximum relaxation was substantially longer for A23187 than for ACh in
the presence of L-NAME.
|
Correlations With the Initial Relaxant Response to ACh
Figure 4 illustrates that in any
given preparation there was an inverse relationship between the
magnitude of the initial relaxations to ACh and those subsequently
obtained in the presence of L-NAME. Linear regression analysis
confirmed a negative correlation between maximal responses to ACh in
the presence and absence of L-NAME (P<0.01, n=19,
r=0.60). There was a direct relationship between the
magnitude of the initial relaxations to ACh and those subsequently
obtained in the presence of Gap 27 peptide, with a positive correlation
coefficient being confirmed by linear regression analysis
(P<0.001, n=8, r=0.94).
|
Effects of Thimerosal on ACh- and A23187-Induced Relaxations in
Intact Rings
Thimerosal (300 nmol/L) did not affect
phenylephrine-induced tone (not shown). Incubation of
rings for 20 minutes with this agent, in the absence of L-NAME,
enhanced relaxations to ACh (Figure 5A),
with a significant reduction in EC50 values from
0.11±0.03 µmol/L to 21±0.3 nmol/L (P<0.05, n=5)
but no effect on maximal responses (control, 73.5±5.5%; thimerosal,
76.6±5.3%; n=5). Gap 27 peptide (300 µmol/L) had no effect on
the thimerosal-induced enhancement in terms of the
EC50 value (25±0.4 nmol/L, n=4) but
significantly reduced the maximal relaxations to 51.9±2.5% (n=4).
Similarly, A23187-induced relaxations were enhanced by thimerosal in
the absence of L-NAME (Figure 5B), with
EC50 values being reduced from 70±9 nmol/L to
33±10 nmol/L (P<0.05, n=4) and the maximal response
showing a small, but not significant, rise from 75.1±8.7% to
86.2±4.3% (n=4). In the presence of L-NAME, thimerosal significantly
reduced the EC50 values for both ACh (from
0.51±0.13 to 0.13±0.05 µmol/L, P<0.05, n=10) and
A23187 (from 0.18±0.06 to 0.083±0.03 µmol/L, n=6, Figure 5C and 5D). Furthermore, thimerosal increased the maximal
response of the residual relaxations to both ACh (from 29.8±8.1% to
42.1±6.1%, P<0.05, n=10, Figure 5C) and A23187
(from 56.9±3.5% to 70.4±3.5%, P<0.05, n=5, Figure 5D).
|
Concentration-response curves to ACh in the presence of L-NAME
(300 mmol/L) and thimerosal (300 nmol/L) showed significant
reductions in maximal responses to 18.8±4.9% (n=4) in the presence of
Gap 27 peptide (300 µmol/L) although EC50
values remained similar at 0.17±0.05 µmol/L (n=4, Figure 5C). A23187-induced relaxations observed in the presence of
L-NAME and thimerosal were not significantly affected by Gap 27 peptide
in terms of either the EC50 value (35±17 nmol/L,
n=4) or maximal response (66.9±2.1%, n=4, Figure 5).
Sandwich Preparations
Relaxations of sandwich preparations to ACh exhibited an
EC50 of 1.1±0.27 µmol/L and a maximal
relaxation of 21.8±2.7% of phenylephrine-induced tone
(n=4, Figure 6A). In marked contrast to
the intact ring studies, ACh-induced relaxations were almost abolished
in the presence of L-NAME, whereas preincubation with Gap 27 peptide
was without effect (Figure 6A). A23187 caused
concentration-dependent relaxations of the sandwich preparations, with
maximal relaxations constituting 25.4±3.0% of developed tension with
an EC50 value of 0.24±0.09 µmol/L (n=8,
Figure 6B). In contrast to intact ring studies, preincubation
with L-NAME had no effect on A23187-induced relaxations, whereas
ONO-RS-082 had no effect on the EC50 value
(0.16±0.08 µmol/L, n=4) but markedly attenuated the maximal
relaxation to 11±5.8% (P<0.05, n=4, Figure 6B). As
in the case of ACh, Gap 27 peptide was without effect on either
L-NAME–sensitive or –insensitive relaxations to A23187 in sandwich
preparations (Figure 6B).
|
Vascular Effects of EETs
The effects of 3 EET regioisomers, 5,6-, 11,12-, and 14,15-EET,
were examined in rabbit superior mesenteric arterial ring
preparations. Only 5,6-EET evoked concentration-dependent relaxations
of endothelium-intact rings that were abolished
following endothelial denudation of the vessel (Figures 7 and 8).
Addition of either L-NAME (300 µmol/L) or Gap 27 peptide
(300 µmol/L) significantly attenuated these
endothelium-dependent responses (P<0.05
[n=4], and P<0.05 [n=6], respectively), and in
combination L-NAME and Gap 27 peptide abolished 5,6-EET-induced
relaxations (n=4, Figures 7 and 8A). Preincubation of
ring preparations with either 5,6-, 11,12-, or 14,15-EET (5
µmol/L for each agent) had no effect on either ACh- or A23187-induced
relaxations or maximal relaxations (n=4 for each agent, Figure 9).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have used isolated ring and sandwich preparations to characterize the role of GJC in the NO-independent responses of the rabbit mesenteric artery to ACh and A23187. The major findings are that EDHF-type relaxations evoked by ACh are attributable to an agent that preferentially crosses between endothelium and smooth muscle via gap junctions, and that a similar agent also mediates relaxation to A23187, but transfer to smooth muscle then involves diffusion via the extracellular space. The formation and/or mode of action of this mediator(s) is intimately related to the metabolism of phospholipids by a Ca2+-dependent PLA2.
Previous studies have shown that heterocellular GJC between the
endothelium and smooth muscle contributes to
NO-independent relaxations of rabbit conduit
arteries.12 13 The present experiments confirm that
Gap 27 peptide, which contains the SRPTEK motif common to the second
extracellular loop of connexins present in the vascular wall,
attenuates L-NAME–insensitive relaxations to ACh in rabbit mesenteric
artery. In ring preparations, 60% of the initial relaxation to ACh
was mediated by NO, whereas in sandwich preparations, in which GJC
cannot contribute to responses, relaxations were exclusively NO
dependent. In marked contrast, only 15% of the relaxation evoked by
A23187 was mediated by NO in rings, and in sandwich preparations
relaxation was not susceptible to inhibition by L-NAME. Furthermore,
Gap 27 peptide was completely without effect on the responses to A23187
either in rings or in sandwich preparations. These findings indicate
that, unlike ACh, A23187 mediates relaxation predominantly through the
release of a freely diffusible factor, which is not NO, into the
extracellular space. This conclusion is supported by comparison of the
time course of the relaxations to the 2 agents. At concentrations
producing equivalent mechanical responses in the presence of L-NAME,
relaxations to A23187 were approximately twice as slow as those to ACh.
This is consistent with the idea that the EDHF released by
A23187 would have to negotiate 2 cell membranes before exerting a
relaxant effect on vascular smooth muscle.
Our findings are consistent with observations by Plane and coworkers, who found that A23187, but not ACh, evokes the release of a diffusible, L-NAME–insensitive factor from the endothelium in sandwich preparations of rabbit femoral artery.11 These authors suggested that the EDHF released from this artery type was stimulus specific. In the present study, however, NO-independent relaxations to ACh and A23187 were both abolished by the structurally unrelated Ca2+-dependent PLA2 inhibitors ONO-RS-082 and AACOCF3 but not by the Ca2+-independent PLA2 inhibitor, HELSS. Although the experimental protocols cannot differentiate between activation of PLA2 within the endothelium or smooth muscle cell, the simplest explanation of these findings is that a closely similar agent mediates EDHF-type responses to ACh and A23187. Alternatively, if different mediators are involved, the production and/or action of both are crucially dependent on activation of PLA2 by Ca2+ ions. Inhibition of the Ca2+-dependent cytosolic form of PLA2 has also previously been shown to attenuate L-NAME–insensitive relaxations to ACh, histamine, and bradykinin in the perfused rat coronary and mesenteric beds.7 8 Cohen and colleagues have suggested that residual NO synthesis in the presence of the NO synthase inhibitors L-NAME and L-NG-nitroarginine could account for endothelium-dependent hyperpolarization.25 We excluded this possibility, however, by demonstrating that the selective inhibitor of guanylate cyclase, ODQ, did not modulate the relaxations to ACh and A23187 observed in the presence of L-NAME. The ability of ONO-RS-082 and AACOCF3 to virtually abolish L-NAME–insensitive relaxations, without affecting NO-mediated relaxations, provides further evidence that L-NAME completely inhibits NO formation in the rabbit mesenteric artery at the concentration used.
Several possibilities may be advanced to explain why A23187-induced relaxations were not susceptible to inhibition by Gap 27 peptide. First, it is possible that incorporation of A23187 into the plasmalemma alters local membrane structure and phospholipid metabolism, allowing easier access of an EDHF linked to the activity of a Ca2+-dependent PLA2 to the extracellular space. Second, there is evidence that molecules of A23187 aggregate in lipid bilayers to form channels, as well as behaving as a carrier ionophore with selectivity for Ca2+ ions,26 that could potentially allow egress of an EDHF into the extracellular space and its subsequent diffusion to adjacent smooth muscle, thereby "bypassing" gap junctions. Third, A23187 has been shown to cause closure of gap junctions between endothelial cells,27 a mechanism that could promote the egress of an EDHF via the membrane. In other cell types, ACh has also been reported to promote closure of gap junctions,28 but clearly this cannot be an important factor in our experiments, as Gap 27 peptide was highly effective in reducing relaxations to ACh. Conventionally, increases in [Ca2+]i have been thought to mediate closure of gap junctions.29 However, a G-protein/tyrosine kinase-dependent mechanism linked to activation of receptors has recently been shown to promote the closing of gap junctions in cells expressing connexin 43, independently of elevations in local [Ca2+]i.30 Conversely, other evidence suggests that elevated [Ca2+]i may also open gap junctions. In cardiac myocytes, which express connexin 43 as the dominant connexin protein, channel permeability increases monotonically as a function of [Ca2+]i over the physiological range of 0.1 to 1 µmol/L, and decreases only for [Ca2+]i within the supraphysiological range.31 Further experiments are therefore necessary to elucidate the principal mechanisms gating heterocellular GJC between endothelial and vascular smooth muscle cells.
Cascade bioassay experiments have demonstrated that NO inhibits the formation and/or release of EDHF by endothelial cells.32 This inverse relationship between the activities of the 2 mediators may explain why ONO-RS-082 and AACOCF3 had no effect on ACh-evoked relaxations in ring preparations in the absence of L-NAME but reduced the potency of A23187. Under these conditions, ACh-induced responses were substantially more dependent on NO synthesis than those evoked by A23187, thus suggesting that NO masks the effects of PLA2 inhibition by suppressing EDHF activity. In the presence of L-NAME, however, EDHF activity will be enhanced, and relaxations to ACh then become susceptible to ONO-RS-082 and AACOCF3, as observed experimentally. The positive correlation between the magnitude of the ACh-induced relaxations obtained in the presence and absence of Gap 27 peptide is also consistent with a dynamic interaction between NO and EDHF, with inhibition of GJC having a smaller effect in the presence of high levels of NO release. A new observation was that in preparations exhibiting large relaxations to ACh in the absence of L-NAME, the subsequent L-NAME–insensitive response to this agonist was small. This suggests that EDHF-type relaxations are chronically diminished in preparations exhibiting high NO activity, although the mechanisms underlying this downregulation remain to be determined.
Activation of PLC following agonist stimulation results in generation of inositol trisphosphate (IP3) and release of Ca2+ from internal stores. The resulting increase in [Ca2+]i activates the Ca2+-dependent cytosolic form of PLA2 resulting in cleavage of arachidonic acid from membrane glycerophospholipids.33 The specific PLC inhibitor U-7312222 abolished L-NAME–insensitive relaxations to ACh but did not affect responses to A23187. The probable explanation for these findings is that EDHF-type relaxations to ACh require activation of PLC and IP3 formation to release Ca2+ from intracellular stores and activate PLA2, whereas the ionophore properties of A23187 increase [Ca2+]i by directly facilitating Ca2+ influx into the cell, as well as Ca2+ release from stores through second-messenger–independent mechanisms.34 Activation of PLC also generates diacylglycerol (DAG), which can be hydrolyzed by a DAG lipase to provide a second source of arachidonic acid.35 However, inhibition of NO-independent relaxations to ACh by U-73122 is unlikely to reflect loss of DAG as a substrate for arachidonic acid production, as the DAG lipase inhibitor, RHC-80267, has no effect on EDHF-type responses in the perfused rat heart and mesenteric prearteriolar bed.7 8
Further evidence for the common identity of the EDHF released by ACh and A23187 was obtained with the sulfhydryl agent thimerosal, which has previously been reported to hyperpolarize porcine coronary arterial smooth muscle in an endothelium-dependent manner36 and enhance the release of an EDHF from canine carotid artery in cascade bioassay.10 In the present study, low concentrations of thimerosal enhanced relaxations to ACh and A23187 both in the absence and presence of L-NAME and indomethacin. Importantly, the potentiation of EDHF-type relaxations to ACh in the presence of L-NAME remained susceptible to inhibition by Gap 27 peptide, whereas those to A23187 were not. The effects of thimerosal can consequently be attributed to factors other than alterations in the pathways allowing transfer of EDHF between the endothelium and smooth muscle by ACh and A23187. Two known mechanisms of action of thimerosal could contribute to an apparent increase in EDHF activity. First, thimerosal inhibits the acyl-coenzyme A:lysolecithin acyltransferase,23 thereby elevating free levels of arachidonic acid within the cell,33 an action that would be consistent with the inhibition of relaxation observed with ONO-RS-082 and AACOCF3. Second, nanomolar concentrations of thimerosal equivalent to those used in the present study, are also known to sensitize the IP3 receptor and thereby promote Ca2+ release from intracellular stores.24 This mechanism could potentially contribute to a secondary activation of the Ca2+-dependent PLA2 and enhancement of EDHF activity, which, like NO, is known to be Ca2+ dependent.37 While receptor-independent Ca2+ influx induced by cyclopiazonic acid also stimulates NO-independent relaxations, in marked contrast to A23187, these responses are dependent on GJC.12 A dominant effect of Ca2+ mobilization by thimerosal therefore seems less likely than its potential effects on arachidonate metabolism. Furthermore, A23187 does not require IP3-mediated release of Ca2+ from stores to evoke NO-independent relaxations, as demonstrated by the lack of effect of U-73122, yet the responses to A23187 were enhanced by thimerosal. It remains to be determined whether thimerosal exerts additional, as yet unknown, effects such as sensitization of vascular smooth muscle to the action of EDHF, but these would not detract from the logic of our argument.
Several groups have suggested that EDHF could be an EET metabolite of arachidonic acid, synthesized by an endothelial cytochrome P-450 epoxygenase, that evokes smooth muscle relaxation through activation of K+ channels and subsequent membrane hyperpolarization.3 4 5 38 39 Other workers, however, have reported that relaxation is observed only with micromolar concentrations of EETs, and EETs do not induce hyperpolarization and relaxation in all artery types.40 41 A number of groups have demonstrated that inhibitors of cytochrome P-450 attenuate EDHF-mediated relaxations,3 4 5 consistent with the involvement of an EET, but it has become apparent that such inhibitors can also directly inhibit K+ channel activity.42 Furthermore, highly specific cytochrome P-450 inhibitors appear to have little effect on NO-independent relaxations in rat mesenteric arteries.43 In the present study with rabbit arteries, micromolar concentrations of 11,12-EET and 14,15-EET were found to be devoid of vasorelaxant activity, and relaxation to 5,6-EET was a strictly endothelium-dependent phenomenon. Other studies have also shown significantly more potent dilator effects of 5,6-EET than 14,15-EET in rat intestinal and cat cerebral microvessels.44 45 5,6-EET may promote Ca2+ influx and stimulate NO release from endothelial cells,46 a mechanism that seems to operate in rabbit mesenteric arteries, as its effects were partially susceptible to inhibition by L-NAME. A surprising new finding, however, was that L-NAME–insensitive relaxations induced by 5,6-EET were abolished by Gap 27 peptide. In a recent study, Weintraub et al41 reported that preincubation with EETs potentiates endothelium-dependent relaxations to bradykinin in porcine coronary arteries. In the present study we repeated this experiment and failed to demonstrate potentiation with 5,6-, 11,12-, or 14,15-EET after preincubation for 30 minutes at a concentration of 5 µmol/L. Taken together, our findings therefore provide no evidence that the biologically active EDHF in rabbit arteries is an EET.
We conclude that ACh and A23187 both stimulate the production of an EDHF in rabbit superior mesenteric artery the vasodilator activity of which crucially depends on GJC in the case of ACh and on extracellular diffusion in the case of A23187. The similarity of the effects of 2 structurally dissimilar Ca2+-dependent inhibitors of PLA2 and thimerosal on relaxations obtained to ACh and A23187 point to the operation of common mechanistic pathways. The primary signaling mechanism for relaxation appears to be chemical rather than electrical in nature. The exact identity of the EDHF(s) has yet to be established, and it remains to be determined at which point distal to activation of PLA2 an active agent is mobilized and within which cell type. Since the central pore of gap junctions is aqueous and thought to allow preferential diffusion of small, water-soluble charged molecules, a role for hydrophobic arachidonic acid derivatives, such as EETs, might be challenged on a purely physiochemical basis.
|
Acknowledgments |
|---|
This work was supported by grants 9307148 and 9305117 from the Medical Research Council.
Received June 3, 1998; accepted October 10, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Griffith TM. Modulation of blood flow and tissue perfusion by endothelium-derived relaxing factor. Exp Physiol. 1994;79:873–913.[Medline] [Order article via Infotrieve]
2. Garland CJ, Plane F, Kemp BK, Cocks TM. Endothelium-dependent hyperpolarization: a role in the control of vascular tone. Trends Pharmacol Sci. 1995;16:23–30.[Medline] [Order article via Infotrieve]
3. Mombouli J-V, Vanhoutte PM. Endothelium-derived hyperpolarizing factor(s): updating the unknown. Trends Pharmacol Sci. 1997;18:252–256.[Medline] [Order article via Infotrieve]
4.
Campbell WB, Gebremedhin D, Pratt PF, Harder DR.
Identification of epoxyeicosatrienoic acids as
endothelium-derived hyperpolarizing factors. Circ
Res. 1996;78:415–423.
5.
Hecker M, Bara AT, Bauersachs J, Busse R.
Characterization of endothelium-derived hyperpolarizing
factor as a cytochrome P450-derived arachidonic acid
metabolite in mammals. J Physiol (Lond). 1994;481:407–414.
6. Randall MD, Kendall DA. Endocannabinoids: a new class of vasoactive substances. Trends Pharmacol Sci. 1998;19:55–58.[Medline] [Order article via Infotrieve]
7. Adeagbo AS, Henzel MK. Calcium-dependent phospholipase A2 mediates the production of endothelium-derived hyperpolarizing factor in perfused rat mesenteric prearteriolar bed. J Vasc Res. 1998;35:27–35.[Medline] [Order article via Infotrieve]
8.
Fulton D, McGiff JC, Quilley J. Role of phospholipase
C and phospholipase A2 in the nitric oxide-independent vasodilator
effect of bradykinin in the rat perfused heart. J Pharmacol
Exp Ther. 1996;278:518–526.
9.
Popp R, Bauersachs J, Hecker M, Fleming I, Busse R. A
transferable, beta-naphthoflavone-inducible, hyperpolarizing factor is
synthesized by native and cultured porcine coronary
endothelial cells. J Physiol (Lond). 1996;497:699–709.
10. Mombouli JV, Bissiriou I, Agboton VD, Vanhoutte PM. Bioassay of endothelium-derived hyperpolarizing factor. Biochem Biophys Res Commun. 1996;221:484–488.[Medline] [Order article via Infotrieve]
11. Plane F, Pearson T, Garland CJ. Multiple pathways underlying endothelium-dependent relaxation in the rabbit isolated femoral artery. Br J Pharmacol. 1995;115:31–38.[Medline] [Order article via Infotrieve]
12.
Chaytor AT, Evans WH, Griffith TM. Central role of
heterocellular gap junctional communication in
endothelium-dependent relaxations of rabbit arteries.
J Physiol (Lond). 1998;508:561–573.
13.
Taylor HJ, Chaytor AT, Evans WH, Griffith TM.
Inhibition of the gap junctional component of
endothelium-dependent relaxations in rabbit iliac
artery by 18-
glycyrrhetinic acid. Br J Pharmacol. 1998;125:1–4.[Medline]
[Order article via Infotrieve]
14. Yeager M, Nicholson BJ. Structure of gap junction intercellular channels. Curr Opin Struct Biol. 1996;6:183–192.[Medline] [Order article via Infotrieve]
15. Spagnoli LG, Villaschi S, Neri L, Palmieri G. Gap junctions in myo-endothelial bridges of rabbit carotid arteries. Experientia. 1982;38:124–125.[Medline] [Order article via Infotrieve]
16.
Chaytor AT, Evans WH, Griffith TM. Peptides homologous
to extracellular loop motifs of connexin 43 reversibly abolish rhythmic
contractile activity in rabbit arteries. J Physiol
(Lond). 1997;503:99–110.
17. Carter TD, Chen XY, Carlile G, Kalapothakis E, Ogden D, Evans WH. Porcine aortic endothelial gap junctions: identification and permeation by caged InsP3. J Cell Sci. 1996;109:1765–1773.[Abstract]
18.
Warner A, Clements DK, Parikh S, Evans WH, DeHaan RL.
Specific motifs in the external loops of connexin proteins can
determine gap junction formation between chick heart myocytes.
J Physiol (Lond). 1995;488:721–728.
19. Street IP, Lin H-K, Laliberte F, Ghomashchi F, Wang Z, Perrier H, Tremblay NM, Huang Z, Weech PK, Gelb MH. Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2. Biochemistry. 1993;32:5935–5940.[Medline] [Order article via Infotrieve]
20.
Hazen SL, Zupan LA, Weiss RH, Getman DP, Gross RW.
Suicide inhibition of canine myocardial cytosolic calcium-independent
phospholipase A2: mechanism-based discrimination
between calcium-dependent and -independent phospholipases
A2. J Biol Chem. 1991;266:7227–7232.
21.
Banga HS, Simons ER, Brass LF, Rittenhouse SE.
Activation of phospholipases A and C in human platelets exposed to
epinephrine: role of glycoproteins IIb/IIIa and
dual role of epinephrine. Proc Natl Acad Sci
U S A. 1986;83:9197–9201.
22. Bleasdale JE, Bundy GL, Bunting S, Fitzpatrick FA, Huff RM, Sun FF, Pike JE. Inhibition of phospholipase C dependent processes by U-73, 122. Adv Prostaglandin Thromboxane Leukot Res. 1989;19:590–593.[Medline] [Order article via Infotrieve]
23.
Forstermann U, Goppelt-Strube M, Frolich JC, Busse R.
Inhibitors of acyl-coenzyme A:lysolecithin
acyltransferase activate the production of
endothelium-derived vascular relaxing factor.
J Pharmacol Exp Ther. 1986;238:352–359.
24.
Bootman MD, Taylor CW, Berridge MJ. The thiol reagent,
thimerosal, evokes Ca2+ spikes in HeLa cells by
sensitizing the inositol 1,4,5-trisphosphate receptor. J
Biol Chem. 1992;267:25113–25119.
25.
Cohen RA, Plane F, Najibi S, Huk I, Malinski T, Garland
CJ. Nitric oxide is the mediator of both
endothelium-dependent relaxation and
hyperpolarization of the rabbit carotid artery.
Proc Natl Acad Sci U S A. 1997;94:4193–4198.
26. Balasubramanian SV, Sikdar SK, Easwaran KR. Bilayers containing calcium ionophore A23187 form channels. Biochem Biophys Res Commun. 1992;189:1038–1042.[Medline] [Order article via Infotrieve]
27.
Rabito CA, Jarrell JA, Scott JA. Gap junctions and
synchronization of polarization process during epithelial
reorganization. Am J Physiol. 1987;253:C329–C336.
28. Randriamampita C, Giaume C, Neyton J, Trautmann A. Acetylcholine-induced closure of gap junction channels in rat lacrimal glands is probably mediated by protein kinase C. Pflugers Arch. 1988;412:462–468.[Medline] [Order article via Infotrieve]
29. Xie HQ, Hu VW. Modulation of gap junctions in senescent endothelial cells. Exp Cell Res. 1994;214:172–176.[Medline] [Order article via Infotrieve]
30.
Postma FR, Hengeveld T, Alblas J, Giepmans BN, Zondag
GC, Jalink K, Moolenaar WH. Acute loss of cell-cell communication
caused by G protein coupled receptors: a critical role for c-Src.
J Cell Biol. 1998;140:1199–1209.
31. Delage B, Deleze J. Increase of the gap junction conductance of adult mammalian heart myocytes by intracellular calcium ions. In: Werner R, ed. Gap Junctions. Amsterdam, the Netherlands: IOS Press; 1998:72–75.
32.
Bauersachs J, Popp R, Hecker M, Sauer E, Fleming I,
Busse R. Nitric oxide attenuates the release of
endothelium-derived hyperpolarizing factor.
Circulation. 1996;94:3341–3347.
33. Irvine RF. How is the level of free arachidonic acid controlled in mammalian cells? Biochem J. 1982;204:3–16.[Medline] [Order article via Infotrieve]
34.
Williams JA. Regulation of pancreatic acinar cell
function by intracellular calcium. Am J Physiol. 1980;238:G269–G279.
35.
Bell RL, Kennerly DA, Stanford N, Majerus PW.
Diglyceride lipase: a pathway for arachidonate release from
human platelets. Proc Natl Acad Sci U S A. 1979;76:3238–3241.
36. Beny JL. Thimerosal hyperpolarizes arterial smooth muscles in an endothelium- dependent manner. Eur J Pharmacol. 1990;185:235–238.[Medline] [Order article via Infotrieve]
37. Fukao M, Hattori Y, Kanno M, Sakuma I, Kitabatake A. Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. Br J Pharmacol. 1997;120:1328–1334.[Medline] [Order article via Infotrieve]
38.
Li PL, Zou AP, Campbell WB. Regulation of potassium
channels in coronary arterial smooth muscle by
endothelium-derived vasodilators.
Hypertension. 1997;29:262–267.
39.
Li PL, Campbell WB. Epoxyeicosatrienoic acids
activate K+ channels in coronary
smooth muscle through a guanine nucleotide binding protein.
Circ Res. 1997;80:877–884.
40. Chataigneau T, Feletou M, Duhault J, Vanhoutte PM. Epoxyeicosatrienoic acids, potassium channel blockers and endothelium-dependent hyperpolarization in the guinea-pig carotid artery. Br J Pharmacol. 1998;123:574–580.[Medline] [Order article via Infotrieve]
41.
Weintraub NL, Fang X, Kaduce TL, VanRollins M,
Chatterjee P, Spector AA. Potentiation of
endothelium-dependent relaxation by epoxyeicosatrienoic
acids. Circ Res. 1997;81:258–267.
42.
Alvarez J, Montero M, Garcia-Sancho J. High affinity
inhibition of Ca2+-dependent K+ channels by
cytochrome P-450 inhibitors. J Biol Chem. 1992;267:11789–11793.
43. Fukao M, Hattori Y, Kanno M, Sakuma I, Kitabatake A. Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery. Br J Pharmacol. 1997;120:439–446.[Medline] [Order article via Infotrieve]
44.
Proctor KG, Falck JR, Capdevila J. Intestinal
vasodilation by epoxyeicosatrienoic acids: arachidonic
acid metabolites produced by a cytochrome P450
monooxygenase. Circ Res. 1987;60:50–59.
45.
Ellis EF, Police RJ, Yancey L, McKinney JS, Amruthesh
SC. Dilation of cerebral arterioles by cytochrome P-450 metabolites of
arachidonic acid. Am J Physiol. 1990;259:H1171–H1177.
46.
Graier WF, Simecek S, Sturek M. Cytochrome P450
mono-oxygenase-regulated signalling of
Ca2+ entry in human and bovine aortic
endothelial cells. J Physiol (Lond). 1995;482:259–274.
This article has been cited by other articles:
 |
|
 |
|
  H. C. Hercule, W.-H. Schunck, V. Gross, J. Seringer, F. P. Leung, S. M. Weldon, A. Ch. da Costa Goncalves, Y. Huang, F. C. Luft, and M. Gollasch Interaction Between P450 Eicosanoids and Nitric Oxide in the Control of Arterial Tone in Mice Arterioscler. Thromb. Vasc. Biol., January 1, 2009; 29(1): 54 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Edwards, Y. Li, and T. M. Griffith Hydrogen Peroxide Potentiates the EDHF Phenomenon by Promoting Endothelial Ca2+ Mobilization Arterioscler. Thromb. Vasc. Biol., October 1, 2008; 28(10): 1774 - 1781. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Sorensen, M. Salomonsson, T. H. Braunstein, M. S. Nielsen, and N.-H. Holstein-Rathlou Connexin mimetic peptides fail to inhibit vascular conducted calcium responses in renal arterioles Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2008; 295(3): R840 - R847. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. V. Matchkov, A. Rahman, L. M. Bakker, T. M. Griffith, H. Nilsson, and C. Aalkjaer Analysis of effects of connexin-mimetic peptides in rat mesenteric small arteries Am J Physiol Heart Circ Physiol, July 1, 2006; 291(1): H357 - H367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Sokoya, A. R. Burns, C. T. Setiawan, H. A. Coleman, H. C. Parkington, and M. Tare Evidence for the involvement of myoendothelial gap junctions in EDHF-mediated relaxation in the rat middle cerebral artery Am J Physiol Heart Circ Physiol, July 1, 2006; 291(1): H385 - H393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Siegl, M. Koeppen, S. E. Wolfle, U. Pohl, and C. de Wit Myoendothelial Coupling Is Not Prominent in Arterioles Within the Mouse Cremaster Microcirculation In Vivo Circ. Res., October 14, 2005; 97(8): 781 - 788. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Scotland, M. Madhani, S. Chauhan, S. Moncada, J. Andresen, H. Nilsson, A. J. Hobbs, and A. Ahluwalia Investigation of Vascular Responses in Endothelial Nitric Oxide Synthase/Cyclooxygenase-1 Double-Knockout Mice: Key Role for Endothelium-Derived Hyperpolarizing Factor in the Regulation of Blood Pressure in Vivo Circulation, February 15, 2005; 111(6): 796 - 803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B Vanheel and J Breyne Endothelium-dependent hyperpolarization in small gastric arteries Cardiovasc Res, August 1, 2004; 63(2): 331 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. T. Chaytor, D. H. Edwards, L. M. Bakker, and T. M. Griffith Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H2O2 in NO-independent relaxations of rabbit arteries PNAS, December 9, 2003; 100(25): 15212 - 15217. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. T. Udosen, H. Jiang, H. C. Hercule, and A. O. Oyekan Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H2054 - H2063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Matsumoto, T. Kobayashi, and K. Kamata Alterations in EDHF-type relaxation and phosphodiesterase activity in mesenteric arteries from diabetic rats Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H283 - H291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gschwend, R. H. Henning, D. de Zeeuw, and H. Buikema Coronary Myogenic Constriction Antagonizes EDHF-Mediated Dilation: Role of KCa Channels Hypertension, April 1, 2003; 41(4): 912 - 918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gschwend, H. Buikema, R. H. Henning, Y. M. Pinto, D. de Zeeuw, and W. H. van Gilst Endothelial dysfunction and infarct-size relate to impaired EDHF response in rat experimental chronic heart failure Eur J Heart Fail, March 1, 2003; 5(2): 147 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-J. Chen, H. Jiang, and J. Quilley The Nitric Oxide- and Prostaglandin-Independent Component of the Renal Vasodilator Effect of Thimerosal Is Mediated by Epoxyeicosatrienoic Acids J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1292 - 1298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fuloria, T. K. Smith, and J. L. Aschner Role of 5,6-epoxyeicosatrienoic acid in the regulation of newborn piglet pulmonary vascular tone Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L383 - L389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Griffith, A. T. Chaytor, H. J. Taylor, B. D. Giddings, and D. H. Edwards cAMP facilitates EDHF-type relaxations in conduit arteries by enhancing electrotonic conduction via gap junctions PNAS, April 30, 2002; 99(9): 6392 - 6397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. T. Chaytor, H. J. Taylor, and T. M. Griffith Gap junction-dependent and -independent EDHF-type relaxations may involve smooth muscle cAMP accumulation Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1548 - H1555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. T. Chaytor, P. E. M. Martin, D. H. Edwards, and T. M. Griffith Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2441 - H2450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Coats, F. Johnston, J. MacDonald, J. J. V. McMurray, and C. Hillier Endothelium-Derived Hyperpolarizing Factor : Identification and Mechanisms of Action in Human Subcutaneous Resistance Arteries Circulation, March 27, 2001; 103(12): 1702 - 1708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Boitano and W. H. Evans Connexin mimetic peptides reversibly inhibit Ca2+ signaling through gap junctions in airway cells Am J Physiol Lung Cell Mol Physiol, October 1, 2000; 279(4): L623 - L630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Emerson and S. S. Segal Electrical Coupling Between Endothelial Cells and Smooth Muscle Cells in Hamster Feed Arteries : Role in Vasomotor Control Circ. Res., September 15, 2000; 87(6): 474 - 479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Vanheel and J. Van de Voorde EDHF and residual NO: different factors Cardiovasc Res, June 1, 2000; 46(3): 370 - 375. [Full Text] [PDF]
|
|
|
|
|
|
F. S. Lamb and T. J. Barna Endothelium modulates anion channel-dependent aortic contractions to iodide Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1527 - H1536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Huang, D. Sun, C. J. Smith, J. A. Connetta, E. G. Shesely, A. Koller, and G. Kaley In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF Am J Physiol Heart Circ Physiol, March 1, 2000; 278(3): H762 - H768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Fleming Myoendothelial Gap Junctions : The Gap Is There, but Does EDHF Go Through It? Circ. Res., February 18, 2000; 86(3): 249 - 250. [Full Text] [PDF]
|
|
|
|
|
|
S. L. Sandow and C. E. Hill Incidence of Myoendothelial Gap Junctions in the Proximal and Distal Mesenteric Arteries of the Rat Is Suggestive of a Role in Endothelium-Derived Hyperpolarizing Factor-Mediated Responses Circ. Res., February 18, 2000; 86(3): 341 - 346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A T Chaytor, P E M Martin, W H Evans, M D Randall, and T M Griffith The endothelial component of cannabinoid-induced relaxation in rabbit mesenteric artery depends on gap junctional communication J. Physiol., October 15, 1999; 520(2): 539 - 550. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Griffith, A. T. Chaytor, H. J. Taylor, B. D. Giddings, and D. H. Edwards cAMP facilitates EDHF-type relaxations in conduit arteries by enhancing electrotonic conduction via gap junctions PNAS, April 30, 2002; 99(9): 6392 - 6397. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||