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Clinical and Experimental Pharmacology and Physiology
(2004)
31
,
563–570
BRIEF REVIEW
FACTORS, FICTION AND ENDOTHELIUM-DERIVED
HYPERPOLARIZING FACTOR
Shaun L Sandow
Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra,
Australian Capital Territory, Australia and Department of Pharmacy and Pharmacology, University of Bath,
Claverton Down, Bath, UK
SUMMARY
1. The principal mediators of vascular tone are neural,
endothelial and physical stimuli that result in the initiation of
dilator and constrictor responses to facilitate the control of
blood pressure. Two primary vasodilatory stimuli produced by
the endothelium are nitric oxide (NO) and prostaglandins. An
additional endothelium-dependent vasodilatory mechanism is
characterized as the hyperpolarization-mediated relaxation
that remains after the inhibition of the synthesis of NO and
prostaglandins. This mechanism is due to the action of a
so-called endothelium-derived hyperpolarizing factor (EDHF)
and is dependent on either the release of diffusible factor(s)
and/or to a direct contact-mediated mechanism.
2. Most evidence supports the concept that ‘EDHF’ activity
is dependent on contact-mediated mechanisms. This involves
the transfer of an endothelium-derived electrical current, as an
endothelium-derived hyperpolarization (EDH), through direct
heterocellular coupling of endothelial cells and smooth muscle
cells via myoendothelial gap junctions (MEGJ). However, there
is a lack of consensus with regard to the nature and mechanism
of action of EDHF/EDH (EDH(F)), which has been shown to
vary within and between vascular beds, as well as among
species, strains, sex and during development, ageing and
disease.
3. In addition to actual heterogeneity in EDH(F), further
heterogeneity has resulted from the less-than-optimal design,
analysis and interpretation of data in some key papers in the
EDHF literature; with such views being perpetuated in the
subsequent literature.
4. The focus of the present brief review is to examine what
factors are proposed as EDH(F) and highlight the correlative
structural and functional studies from our laboratory that
demonstrate an integral role for MEGJ in the conduction of
EDH, which account for the heterogeneity in EDH(F), while
incorporating the reported diffusible mechanisms in the
regulation of this activity. Furthermore, in addition to the
reported heterogeneity in the nature and mechanism of action
of EDH(F), the contribution of experimental design and
technique to this heterogeneity will be examined.
Key words: artery, connexin, endothelium, experimental
design, gap junction, smooth muscle, vascular tone, vaso-
constrictor, vasodilator.
WHAT IS EDH(F)?
The aim of the present brief review is to provide a critical overview
of the endothelium-derived hyperpolarizing factor/endothelium-
derived hyperpolarization (EDH(F)) field, with a focus on the role
of gap junctions in the EDH(F) phenomenon. More extensive
reviews on endothelium-derived hyperpolarizing factor (EDHF)
are provided by McGuire
et al.
,
1
Campbell and Gauthier,
2
Ding and
Triggle
3
and Griffith.
4
Briefly, the arterial endothelium produces three vasodilatory
factors: nitric oxide (NO), prostaglandins and EDH(F). Classically,
EDH(F) is the hyperpolarization and associated relaxation
remaining after the inhibition of the synthesis of NO synthase
(and, thus, NO) and prostaglandins
.
The two primary mechanisms
that can account for EDH(F) activity rely on either diffusible- and/
or contact-mediated mechanisms. Those that are dependent on the
release of a diffusible substance, for which there is yet to be
unequivocal evidence, are due to EDHF. Those that are dependent
on the direct contact of endothelial cells (EC) and smooth muscle
cells (SMC) via myoendothelial gap junctions (MEGJ) are due to
the transfer of an electrical current, as an endothelium-derived
hyperpolarization (EDH).
4–9
In both cases, the net result is the
hyperpolarization of the adjacent smooth muscle with subsequent
vessel dilation. For clarity, the term EDH(F) will be used here to
refer to both a diffusible- or contact-mediated mechanism.
Regardless of whether a diffusible- or contact-mediated mech-
anism is involved in EDH(F) activity, it is accepted that its action
is dependent on the release of intracellular calcium and the
activation of a specific pattern of potassium channels. The activ-
ation of receptors and/or application of physical stimuli, such as
shear stress, results in a rise in intracellular EC calcium.
1,4,10
Correspondence: Dr Shaun L Sandow, Division of Neuroscience, John
Curtin School of Medical Research, Australian National University,
Canberra, ACT 0200, Australia. Email: Shaun.Sandow@anu.edu.au
Presented at the Australian Physiological and Pharmacological Society
Symposium Potassium Channels and Endothelium-Derived Hyperpolariz-
ing Factor: Physiological and Clinical Roles, September 2003. Published
with the permission of the APPS and initially published on the APPS
website (http://www.apps.org.au).
Received 9 January 2004; revision 30 April 2004; accepted 7 June 2004.
564
SL Sandow
Subsequently, this results in the activation of small (S) and inter-
mediate (I) conductance calcium-activated potassium channels
(K
Ca
) located on EC and, in some cases, the activation of EC or
SMC large (B) K
Ca
.
1
This channel activation results in the gener-
ation of an EDH or the release of an EDHF, which is subsequently
transmitted to the adjacent SMC layer either via MEGJ or
diffusion.
1,2
Indeed, it is agreed that EDH(F) activity is blocked
by the application of K
Ca
antagonists, such as apamin (SK
Ca
antagonist) and charybdotoxin (non-selective IK
Ca
and BK
Ca
antagonist, with additional effects at voltage-dependent potassium
channels
3
) in combination
1,2
or apamin and 1-[(2-chloro-
phenyl)diphenylmethyl]-1H-pyrazole (TRAM)-34 (IK
Ca
antago-
nist) in combination
4,11,12
in the case of SK
Ca
- and IK
Ca
-dependent
responses, or by iberiotoxin in the case of BK
Ca
-dependent
responses.
2
The nature and mechanism of EDH(F) apparently varies within
and between vascular beds and among species, strains, sex and
during development, ageing and disease,
1–3
as well as with variable
experimental conditions and between laboratories.
4
A proposal for
unifying the role of EDH(F) and heterocellular coupling has
recently been put forward by Griffith.
4
This scheme incorporates
many of the proposed EDH(F) and questions others, for which
there is debatable evidence.
DIFFUSIBLE FACTORS
Contact-mediated mechanisms represent the simplest explanation
of EDH(F) activity, as a purely electrical event. However, the
release of diffusible factor/s from the endothelium, at a concen-
tration sufficient to change that of the internal elastic lamina and
the local environment surrounding the innermost layer of SMC, has
also been proposed to account for EDH(F) activity. This substance
then putatively effects the activation of SMC receptors and ion
channels to initiate smooth muscle hyperpolarization and
relaxation.
1–4
Diffusible factors proposed as EDHF include K
+
ions,
epoxyeicosatrienoic acids (EET), H
2
O
2
,
1,2
and C-type natriuretic
peptide (CNP).
13
N
G
-Nitro-
L
-arginine methyl ester (
L
-NAME)-
insensitive NO has also been suggested to account for EDHF
activity.
14,15
In addition,
S
-nitrosothiols have been suggested to
contribute to EDHF activity,
16
although the evidence for the
endothelial dependence of this response requires further
investigation.
Potassium ions
Several studies have supported the proposal that K
+
ions are an
EDHF in some vessels.
1,3,4,17
Indeed, since the original proposition
that K
+
ions were an EDHF, this hypothesis has received much
attention. Basically, this scheme involves the activation of EC K
Ca
and the subsequent EC efflux of K
+
from these channels. The
resultant potassium ‘cloud’
17
then reportedly diffuses across the
internal elastic lamina to act as an EDHF by evoking smooth
muscle hyperpolarization and relaxation via the activation of
smooth muscle Na
+
/K
+
-ATPase and inwardly rectifying potassium
channels,
17
key channels for the modulation of ionic mechanisms
that are reportedly sensitive to the application of ouabain and
barium, respectively. Antagonism of the EDHF response by these
blockers is used as defining evidence for K
+
as an EDHF. In its
current form, this mechanism is referred to as the ‘potassium cloud
hypothesis’.
17
A complication to this hypothesis is the efflux of K
+
from SMC
that arises as a result of depolarization, which would contribute to
the basal level of K
+
surrounding vascular cells and thus suppress
the K
+
/EDHF effect. At a simplistic level, the term ‘potassium
cloud’ is misleading, in that it implies the presence of a global
cloud of potassium surrounding the vascular cells when, in fact, any
physiologically relevant change in the K
+
concentration will be
transient and localized. Indeed, a more plausible scenario is that the
K
+
flux acts at restricted localized sites (microdomains), as has
been described in SMC and other cell types.
18
Interestingly, the most recent version of the ‘potassium cloud
hypothesis’ includes a role for MEGJ in the action of K
+
as
EDHF.
17
However, once a role for MEGJ is included in this
mechanism, a role for K
+
as a diffusible EDHF may be redundant,
because the EDHF phenomenon can be explained simply through
the action of EDH. As alluded to above, a potential scenario where
the diffusion of K
+
may play a role in the EDHF activity could arise
if there is a close spatial relationship between MEGJ and K
Ca
distribution (as well as, perhaps, with sites of calcium extrusion),
in the form of microdomains, where highly localized changes in K
+
concentrations could play a role in the coordination and modulation
of heterocellular EDH(F) signalling (CJ Garland and SL Sandow,
unpubl. obs., 2004). Although evidence for similar functional
microdomains in SMC and other cell types is well documented,
18
it is interesting to speculate that this scenario may be the case in
EC of resistance vessels, such as the mesenteric bed of the rat,
where functional studies have suggested this to occur.
19
Further
anatomical support for the existence of microdomains in EC is not
currently available in resistance vessels and, thus, a role for K
+
in
this scenario is speculative.
Epoxyeicosatrienoic acids
There is evidence of a role for EET in EDH(F) activity in some
vascular beds.
1,2
Epoxyeicosatrienoic acids are cytochrome P450
expoxygenase metabolites of phospholipase-dependent arachi-
donic acid production, which putatively activate smooth muscle
BK
Ca20
to result in hyperpolarization and arterial relaxation in
cerebral, coronary and renal arteries of several species.
1,2
Indeed,
although there is evidence that EET play an integral role in EDH(F)
activity in some vascular beds, EET are not a universal EDH(F), in
that in many vascular beds, EDH(F) activity is not sensitive to the
application of iberiotoxin, a BK
Ca
antagonist.
4
Furthermore, it is
not clear whether EET activity is related to the participation of EET
in the facilitation of autocrine pathways that generate hyper-
polarization via mechanisms that are indistinct from alternative
agonist-induced pathways that result in an analogous activation of
an EDH(F)-type response.
4
Hydrogen peroxide
In human and mouse mesenteric and porcine coronary arteries,
H
2
O
2
has been proposed to act as an EDHF.
21–24
However, a
primary problem with these studies is that the appropriate time and
concentration controls for catalase, as an H
2
O
2
antagonist, were not
undertaken and, indeed, the proposal that H
2
O
2
is an EDHF in these
vascular beds is not consistent with several other studies under-
Factors, fiction and EDH(F)
565
taken in the same vascular beds (see below). Beny and von der
Weid,
25
for example, have shown that EDHF and H
2
O
2
are distinct
factors in porcine coronary arteries, where Pomposiello
et al
.
26
have demonstrated that catalase, an enzyme inhibitor of H
2
O
2
activity, has no effect in porcine coronary vessels, although, at
300 U/mL, catalase did abolish the endothelium-independent
relaxation to exogenously generated H
2
O
2
after 45 min incubation.
Catalase has been shown to have no effect on EDHF in the bovine
ciliary, rat saphenous and mesenteric and human radial and sub-
cutaneous arteries.
9,27–30
In this light, several studies have shown
that H
2
O
2
can cause a vasoconstriction
31,32
that can be attenuated
by a 20 min incubation in 100 U/mL catalase.
33
Furthermore, in a
membrane potential-independent manner, reactive oxygen species,
such as H
2
O
2
, have been reported to variably activate SMC K
Ca
,
ATP-sensitive potassium channels and Na
+
/K
+
-ATPase and to
modulate the sensitivity of the contractile apparatus to
calcium,
4,10
thus playing additional roles unrelated to EDHF, but
complicating any speculative role for H
2
O
2
in EDHF activity.
Indeed, in contrast with the original proposition that H
2
O
2
was an
EDHF in mouse mesenteric vessels, Ellis
et al
.
34
provide evidence
that H
2
O
2
is not an EDHF in these vessels. Indeed, Ellis
et al
.
34
found that an inhibitory effect of catalase does not provide
definitive evidence that H
2
O
2
is critical to a given vascular
response.
10
In any event, the physiological relevance of H
2
O
2
as an EDHF
is simply questioned based on the observation that the concen-
tration of H
2
O
2
produced in response to endothelial stimulation
(10–60 nmol/L;
35
see also Griffith
4
) is substantially less than the
3–100
mol/L of H
2
O
2
required to elicit a 30–90% relaxation in
human mesenteric vessels
22
or the 0.1 and 1 mmol/L of H
2
O
2
required to elicit a 60 and 100% relaxation, respectively, in porcine
coronary arteries.
25
In addition, concentration-dependent effects of
H
2
O
2
are critical to the question of whether physiological or
pathophysiological effects are observed, because H
2
O
2
can mediate
vascular cell proliferation, apoptosis, hyperplasia, cell adhesion
and migration, as well as having effects on arterial tone.
10
Indeed,
predominant evidence supports the proposition that H
2
O
2 is not
involved in the hyperpolarization-dependent EDHF response and
that it is not an EDHF.10,36
C-Type natriuretic peptide
C-Type natriuretic peptide (CNP) has been proposed to act as an
EDHF13 and, indeed, the data presented by Chauhan et al.13 are
consistent with the activation of the CNP receptor C subtype
playing a role in the EDH(F) phenomenon. However, in the same
mesenteric vessels as examined in Sprague-Dawley rats by
Chauhan et al.13 but in the mature Wistar rat, Sandow et al.37
demonstrate that heterocellular coupling of EC and SMC accounts
for EDH activity in this bed. Although the difference between the
two studies could be related to strain variation, such a fundamental
difference is unlikely and the specific reason for the discrepancy is
unknown. Interestingly, in this light, the use of the non-selective
gap junction antagonist glycyrrhetinic acid (GA) and its derivatives
have implicated a primary role for gap junctional coupling in
EDH(F) activity in this vascular bed.38–41 Indeed, Chauhan et al.13
implicate a role for MEGJ in the proposal that CNP is EDH(F) via
the use of -GA, although at present this role is currently unknown
but is being investigated (A Ahluwalia, pers. comm.). In any case,
a role for MEGJ in the activity of CNP as EDH(F) is based on the
assumption that GA is a specific antagonist for MEGJ and because
no control studies for the effects of GA were undertaken in the
study of Chauhan et al.,13 this claim is open to question. Indeed,
GA and its derivatives have been shown to block homocellular and
MEGJ in this vessel,39,41 as well as having direct effects on the EC
hyperpolarization to acetylcholine (ACh) via effects on phospho-
lipase activity and EC SKCa, IKCa and Na+/K+-ATPase, irrespective
of their putative effects at gap junctions.6,42 A limitation of future
studies examining a potential role for CNP as an EDH(F) is the lack
of availability of selective antagonists for the CNP receptor C
subtype that is reported to mediate this response. Furthermore,
specific limitations of the study of Chauhan et al.13 include the lack
of a demonstration that the CNP-mediated relaxation can occur
independently of the endothelium (which would thus demonstrate
CNP action at the smooth muscle) and a lack of explanation of the
observations that CNP evokes approximately 60–70% relaxation,
whereas EDHF evokes approximately 100% relaxation. In addi-
tion, there is also a lack of explanation as to why the (non-specific)
blockade of gap junctions with GA suppresses CNP activity or
what effects barium alone has on the CNP- and EDHF-mediated
relaxations, or the inclusion of appropriate control data to deter-
mine whether there was a basal release of CNP in these mesenteric
vessels. Thus, a definitive role for CNP in EDH(F) activity remains
to be elucidated.
L-NAME-insensitive NO
Endogenous or basal NO activity, which is insensitive to the
application of NO synthase antagonists used in the routine study of
EDH(F), has been suggested to account for EDH(F) activity.14,15
Current evidence suggests that, in some vascular beds under
specific experimental conditions, this L-NAME-insensitive NO
may account for a minor degree of EDH(F) activity and one not
consistently observed in studies of the same vascular bed. For
example, in the study of Chauhan et al.15 purporting to show that
L-NAME-insensitive NO accounts for a significant portion of
EDH(F) activity, 63% of hyperpolarization and 70% of
relaxation to ACh remain after the addition of the NO scavenger
oxyhaemoglobin (in the presence of L-NAME and indomethacin).
Furthermore, in the caudal and saphenous arteries of the rat and
mesenteric artery of the mouse, the NO scavengers hydroxo-
cobalamin and carboxy–2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-
tetramethyl-1H-imidazol-1-yloxy-3-oxide (PTIO) have no effect
on EDH(F),9,24,43 thus demonstrating a lack of an L-NAME-
insensitive NO component in these vascular beds. Therefore, the
contribution of endogenous NO to EDH(F) activity appears
variable and, in many cases, non-existent. Further studies are
required to determine the physiological relevance of this
phenomenon.
CONTACT-MEDIATED MECHANISMS
Evidence supporting the critical role of MEGJ in EDH(F) activity
comes primarily from structural and functional studies from our
laboratory in Canberra and Tudor Griffith’s4,36,44,45 laboratory in
Cardiff. These studies, which illustrate the simplest explanation of
EDH(F) activity, use electron microscopic identification of MEGJ,
electrophysiological recordings from dye-identified EC and SMC
566 SL Sandow
and myography with pharmacological interventions, as well as
immunohistochemical methods for identifying the connexins
and ion channels involved in the EDH(F) phenomenon. These
studies are consistent with the hypothesis that EDH(F) is an
electrical phenomenon involving the gap junctional transfer of an
EDH from the EC to the innermost layer of intimal SMC in
the arterial wall for the subsequent generation of an arterial
relaxation.
Studies from our laboratory, which are the focus of this section
of the present review, have examined the role of MEGJ in EDH
activity. We have found that the distribution and activity of MEGJ
is correlated with the presence of EDH within and between vas-
cular beds, during development and in disease. In the proximal and
distal mesenteric arteries of the rat, for example, gap junctions play
a critical role in EDH activity39,41 where MEGJ are prevalent.46 In
this vascular bed, in collaborative studies with Marianne Tare in
Helena Parkington’s laboratory in Melbourne, we showed that the
presence of EDH is correlated with the presence of MEGJ, whereas
in the femoral artery a lack of MEGJ is correlated with the absence
of EDH.37 A similar situation is present in the lateral saphenous
artery of the juvenile rat, where MEGJ are prevalent and EDH-
mediated relaxation present.9 This is in contrast with the saphenous
artery of the adult, where MEGJ were rare and EDH absent.9 The
relationship between EDH and MEGJ is somewhat more
complicated in disease states, such as in hypertension. In a com-
parative study of the caudal artery of the spontaneously hyper-
tensive and normotensive Wistar-Kyoto rats, EDH activity was
maintained in spite of an increase in the number of SMC layers
in vessels from the hypertensive rat. This maintenance was found
to be correlated with a concomitant increase in the incidence of
MEGJ in the caudal artery of the hypertensive rat.43
The above studies demonstrate that there is a direct relationship
between the degree of EDH and the incidence of MEGJ. Indeed,
EDH increases with an increase in the number of MEGJ per EC,
whereas, conversely, it generally decreases with an increase in the
number of SMC layers and vessel diameter (Fig. 1). Interestingly,
although EDH is the predominant vasodilator in smaller vessels, it
is present in some larger vessels (Fig. 1), such as the rabbit iliac,
rat caudal and superior mesenteric arteries.41,43,45 In the rabbit iliac
artery, cAMP has been proposed to enhance the spread of EDH via
modulating gap junctional coupling within the multiple SMC
layers, as well as at MEGJ.47 Although conclusive biophysical
evidence for this mechanism being relevant in larger vessels is
lacking,48 this mechanism may be of some importance for EDH
activity.
These studies demonstrate that there is a consistent positive
correlation between MEGJ and EDH activity within and between
vascular beds and during development and disease. Although this
correlation is not definitive evidence that contact-mediated mech-
anisms account for EDH(F) activity, to date these data provide the
most conclusive and plausible explanation for this activity.
Fig. 1 Summary data demonstrating the relationship between acetyl-
choline (ACh)-induced endothelium-derived hyperpolarizing factor/
endothelium-derived hyperpolarization (EDH(F)) activity and arterial
morphology as the number of myoendothelial gap junctions (MEGJ) per
endothelial cell (EC), per number of medial smooth muscle cell (SMC)
layers and per vessel diameter. Individual data points are presented as the
meanSEM, with data being derived from earlier studies.9,37,43,46 Data were
fitted with a one-phase exponential curve using Graphpad Prism (Graphpad
Software, San Diego, CA, USA). PE, phenylephrine. (), tertiary mesen-
teric (juvenile); (), primary mesenteric (juvenile); (), primary/secondary
mesenteric (juvenile); (), caudal (adult); (), saphenous (adult); (),
femoral (adult).
Factors, fiction and EDH(F) 567
ROLE OF DIFFUSIBLE FACTORS IN
CONTACT-MEDIATED MECHANISMS
Direct electrical coupling is the most plausible mechanism to fully
account for EDH activity. Indeed, there is increasing evidence that
the diffusible factors that act as credible EDHF may, in fact, be
associated with the modulation of gap junction activity and,
specifically, of MEGJ4 for the transfer of EDH as the most plausible
mechanism of their activity. These mechanisms are outlined
below.
Potassium ions
The original hypothesis regarding the mechanism of action of K+
as EDHF has been modified to include a role for MEGJ.17 However,
although K+ is involved in mediating EDH(F) activity, once a role
for such MEGJ is included, no direct role for K+ as a diffusible
EDHF is necessary for the transfer of an EDH. Indeed, in a series
of experiments that repeated those in the original proposal that K+
was EDHF, the data in the original study could not be repeated.49
In addition, several studies have questioned the nature of K+ as
EDHF because barium and ouabain, which are used to define the
role of K+ as an EDHF, do not universally block EDHF-mediated
responses.1,3,4,17,50 Indeed, the efficacy of ouabain as a selective
Na+/K+-ATPase antagonist has been questioned,4,51,52 whereby it
has inhibitory effects on cell coupling via modulating gap junction
function.53 Indeed, ouabain may directly attenuate the transfer of
EDH by its action at gap junctions.4,51,52 This action includes direct
effects on gap junctional coupling, such as reducing connexin (Cx)
expression through reduced Cx trafficking to the cell membrane, as
well as modulating gap junction conductance.52 The implication of
these observations is that the attenuation of an EDH(F) response
by ouabain, as with high concentrations of potassium, does not
necessarily provide evidence of the EDH(F) nature of the
response.4,6,52 The demonstration that ouabain has direct effects on
gap junctions and, thus, on EDH(F) are essentially control studies
for the earlier work that relied on the use of ouabain to show that
K+ was EDHF. Thus, based on these ‘control’ data,4,51,52 K+ ions are
not an EDH(F) but, rather, may simply be involved in the modu-
lation of the signal transduction pathways associated with gap
junction function54,55 and, thus, with EDH activity.4 Further investi-
gation is required to elucidate any potentially specific effects of
ouabain on vasomotor responses and those at gap junctions. Indeed,
this point is critical for the accurate interpretation of future EDH(F)
data.
Epoxyeicosatrienoic acids
In studies of cultured EC, EET have been shown to modulate
homocellular gap junctions,56 thus providing a potential mech-
anism for a modulatory role for EET in EDH action.4 Griffith4
suggests that EET activity may be related to a complex interaction
of calcium and potassium homeostasis, cAMP and arachidonic acid
activity and electrotonic signalling (see Fig. 3 in Griffith4 and also
Dhein50). Indeed, EET have been suggested to modulate EC KCa
activity,57 thus providing a further mechanism for their potential
role in modulating EDH, independent of acting directly as an
EDHF. Further studies of the role of EET in EDH activity in intact
vessels are required to clarify these proposals.
Hydrogen peroxide
There is some evidence that H2O2 can affect gap junction activity
and calcium homeostasis, two factors that are integral for EDH
activity. Depending on the experimental conditions, studies have
shown that H2O2 can both increase58 and decrease59 gap junctional
coupling and effect changes in intracellular calcium homeostasis,
both in cultured cells and in intact arteries.59–61 Although no
specific evidence is currently available to support this proposal,
these observations provide potential support for a mechanism to
link the putative role of H2O2 as an EDH(F) with the MEGJ
dependence of the EDH phenomenon.
C-Type natriuretic peptide
The putative action of CNP as an EDH(F)13 may be via acting as
yet another factor that facilitates electrical coupling through gap
junctions, although any putative mechanism for this is unknown.
Indeed, any putative action for CNP as EDH(F) cannot be associ-
ated directly with the gap junctional transfer of CNP from EC to
SMC because gap junctions are limited to passing substances of
1 kDa and CNP has a molecular weight of approximately
2.2 kDa (A Ahluwalia, pers. comm.). Interestingly, in the study of
Chauhan et al.13 proposing that CNP is an EDH(F), the response is
sensitive to the combination of barium and ouabain, an observation
that this is not a universal characteristic of EDH(F) in this, the rat
mesenteric vascular bed.49 Indeed, because ouabain is recognized
as a non-specific gap junction antagonist, this result may, in fact,
reflect an MEGJ dependence of EDH(F) in the mesenteric bed of
the rat, as demonstrated by Sandow et al.37
Myoendothelial gap junctions, EDH and gap junction
inhibitors
The demonstration of the dependence of EDH activity on gap
junctions relies, in part, on the specific pharmacological inhibition
of gap junctions. Unfortunately, there are a number of limitations
regarding this methodology. The primary one of these relates to the
dependence on the use of gap junction inhibitors that have not been
characterized adequately in terms of their specificity and mech-
anism of action. Currently, there is no unequivocal evidence that
the available gap junction inhibitors are specific,62 let alone selec-
tive, for gap junctions, be they heterocellular or homocellular.
Indeed, unfortunately, to date few studies have examined this
problem in detail and few have performed the defining experiment
of examining the effect of these agents on cell input resistance,
whereby an increase in input resistance would provide key data on
the gap junction antagonist effects of these agents. Of the studies
that have undertaken such technically demanding experiments, the
data are not consistent and are incomplete, although this may
reflect, in part, the heterogeneity in the Cx composition of vascular
gap junctions.63
Much of the current evidence for the gap junction and, specific-
ally, MEGJ dependence of EDH relies on the use of the licorice
derivatives (the GA and carbenoxolone; see above for an outline of
non-specific actions), the Cx-mimetic peptides (Gap26,43 Gap2740
and Gap27,37,43 which, based on putative selectivity, are the current
gap junction inhibitors of choice4,9,44) and, decreasingly, with the
long-chain alcohols, such as heptanol.64,65 However, there is little
568 SL Sandow
equivocal evidence that these agents are gap junction specific and
that they do not induce other non-gap junctional effects. Although
there is well-documented (and often ignored) evidence for the non-
specific effects of the licorice derivatives (see above) and hepta-
nol,64,65 the Cx-mimetic peptides have not yet been equivocally
tested for specificity and nor is their mechanism of action known.
In this regard, a primary issue with the use of the Cx-mimetic
peptides relates to the apparent requirement to use very high
concentrations and long incubation times to attenuate gap junction
activity.4 Interestingly, others report significant effects with lesser
concentrations of the peptide/s and reduced incubation times.62,66,67
Clearly, there is a pressing need for these issues to be addressed.
WHY IS THERE SUCH A DISPARITY OF VIEWS
AS TO THE NATURE AND MECHANISM OF
ACTION OF EDH(F)?
The conventional reason given for the disparity of views as to the
nature and mechanism of action of EDH(F) is that there is hetero-
geneity within and between arteries, species, sex, strain and disease
states.1–4,10,17 However, a further cause of the heterogeneity relates
to the less-than-optimal design, analysis and interpretation of data
present in some key papers in the EDH(F) literature. Although
some earlier studies can be seen as flawed with hindsight, this is
not necessarily the case, because they may, in fact, represent
significant contributions to the EDHF literature through their role
in advancing the evolution of the field. Unfortunately, this is not
always the case and the perpetuation of now potentially misleading
data is problematic. In any case, it is recognized that there is
variation in the nature and mechanism of EDH(F) between labor-
atories,4 thus questioning the relevance of the data and conclusions
of some studies.
The problems of experimental technique, with regard to the
design, analysis and interpretation of data that contribute to the
reported heterogeneity in the nature and mechanism of action of
EDH(F) in the literature include the following:
1. The use of selected agonists, antagonists and/or modulators
of the investigators’ choice and interest, but not those that may
indicate an alternative nature or mechanism of EDH(F).1,3,4,17 That
is, for example, an investigator may be interested in EET or gap
junctions to account for EDH(F) activity, but may limit the
investigation to the use of antagonists of the mechanism of their
interest, rather than of alternative pathways. This results in a
potential for a bias in favour of a particular putative EDH(F).1,3,4,17
2. The lack of control data for the effects of agonists, antago-
nists and other modulators. For instance, in the studies of Matoba
et al.21–23 examining the role of H2O2 as an EDH(F), justification
should be provided as to the incubation time with catalase (2 h or
longer), as well as the high (and variable) concentration of catalase
that was used.
3. The clear need for greater transparency with regard to
variability in cell, vessel and species specific responses, as a result
of a specific receptor and channel population and associated signal
transduction pathways.63
4. Making inappropriate comparative analyses between
studies, including a lack of consideration of strain,68,69 age,9,70–72
sex,73–76 the use of intact versus isolated tissue and tension versus
pressurized myography,63 as well as variation in the classification
of arterial branching patterns.39,41,46 Indeed, such characteristics are
often not stated in the methods section of papers and, thus, result
in an inability to make comparative analyses between studies.
5. Lack of clarity and relevance as to the experimental pro-
tocol. For instance, under conditions of little or no vascular tone,
the use of buffers (such as HEPES)77 that have non-physiological
effects, the use of preconstrictor agents that adversely effect
channel activity4 (such as the effect of U46619 on SKCa78 and the
effect of the GA and related compounds on a variety of cell
processes), as outlined above.
6. Extrapolation of data to other vascular beds. For example,
Chauhan et al.13,15 examined EDH(F) in mesenteric vessels of the
mature male Sprague-Dawley rat, but extrapolate the data to be
applicable to the vasculature as a whole. Although several studies
have made such claims,1,3,4 this contention merely confuses the
field because there is no evidence to justify this point of view.
CONCLUSIONS
The nature and mechanism of action of EDH(F) can apparently
differ along and between vascular beds, between species, strains,
sex and during development, ageing and disease. This hetero-
geneity can be explained through the action of heterocellular
coupling. Indeed, contact-mediated mechanisms represent the
simplest explanation of EDH(F) activity and involve the transfer of
an endothelium-derived electrical signal to the smooth muscle via
MEGJ, as EDH. Of the putative diffusion-mediated mechanisms,
K+ ions have received much attention in the literature and although
they may not be an EDHF, they are involved in the signal trans-
duction pathways associated with the generation of the EDH and
they may be involved in the modulation of gap junction activity. In
a similar manner, there is good evidence for a role of EET in
EDH(F) activity in some vascular beds, although this role may be
confined to a modulatory role of homo- and heterocellular
coupling, as well as modulating the KCa component of the EDH
mechanism. The role of CNP as an EDH(F) is yet to be clarified,
but may also be related to the modulation of EDH activity. Pre-
dominant evidence supports the proposition that H2O2 is not an
EDH(F), although, again, its activity may be related to the
modulation of gap junction function and, thus, of EDH. NG-Nitro-
L-arginine methyl ester-insensitive NO may account for a degree of
EDH(F) activity in some vascular beds, but the extent of this is
limited to only a minor part of such activity. Although the nature
and mechanism of action of EDH(F) is due, in part, to actual
heterogeneity, it is also unfortunately due to a lack of consistent and
sound scientific methodology.
ACKNOWLEDGEMENTS
The author’s work was supported by the National Heart Foundation
and National Health and Medical Research Council of Australia
(NHMRC) and the Wellcome Trust. SLS was supported by a
NHMRC Peter Doherty Fellowship. The author thanks Dr Alister
McNeish (Department of Pharmacy and Pharmacology, University
of Bath, Bath, UK) for critical comments on the manuscript.
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