Download Pharmacology of gap junctions in the cardiovascular system

Cardiovascular Research 62 (2004) 287 – 298
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Review
Pharmacology of gap junctions in the cardiovascular system
Stefan Dhein *
Clinic for Cardiac Surgery, Heart Centre Leipzig, University of Leipzig, Struempellstr. 39, 04289 Leipzig, Germany
Received 7 October 2003; received in revised form 15 January 2004; accepted 16 January 2004
Time for primary review 26 days
Abstract
Gap junction channels provide the basis for intercellular communication and play an important physiological role in the cardiovascular
system for maintenance of the normal cardiac rhythm, regulation of vascular tone, endothelial function and myoendothelial interaction as
well as for metabolic interchange between the cells. Thus, pharmacological influence on these channels might help to elucidate their role in
physiology and pathophysiology and might reveal new therapeutic approaches. The gap junction conductance between two cells is defined
by the number of channels, the single channel conductance and the mean open and closed time. In principle, it is possible pharmacologically
to induce closing of the channels, to change preferred single channel conductance, to open channels (or to keep them open), and to regulate
the expression, synthesis, assembly and degradation of the channels thereby controlling the number of channels. This review describes the
various substances affecting these parameters and outlines the possible pharmacological use of such drugs.
D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Gap junctions; Connexin; Pharmacology; Cardiovascular; Regulation
1. Introduction
Gap junction channels provide the basis for intercellular
communication in the cardiovascular system. They form
low resistance pathways connecting cells and allowing the
transfer of current thereby enabling action potential spreading and the exchange of small molecules (molecules < 1000
Da; there is a slightly higher conductivity for cations over
anions) between neighbouring cells. Gap junction channels
are composed from proteins the so-called connexins. Each
of the neighbouring cells provides a hexameric hemichannel, a connexon consisting of six connexins, and two
connexons form the complete gap junction channel. There
exist a number of isoforms of the connexins, which all
belong to one protein family comprising two major groups
comprising 20 connexins (for details, see Refs. [1 –3]). In
the mammalian heart the following connexins have been
identified: Cx31.9, Cx37, Cx40,Cx43, Cx45, Cx46, Cx50
and Cx57. In the cardiomyocytes the most abundant isoform
isCx43, while Cx40 is mainly found in atrial tissue and in
* Tel.: +49-341-1044; fax: +49-341-1452.
E-mail address: [email protected] (S. Dhein).
the conduction system. Cx45 has been detected predominantly during early development of the heart.
The predominant function of these channels in the heart
is to allow propagation of the action potential from cell to
cell. The normal propagation of the activation wave front is
related to the subcellular distribution of the gap junction
channels, which are more or less confined to the cell poles
in the intercalated disks with only small amount of the
protein found at the lateral borders of the cell. In principle,
an action potential propagates along the fibre by activating
the sodium current, so that the propagation velocity along
the fibre axis mainly depends on the sodium channel
availability [4]. At the end of a cell the action potential is
transferred to the next cell via the gap junction channels
located in the intercalated disk. Gap junction channels at the
lateral border of the cell provide a small transverse component of action potential spreading. This transverse component mainly depends on gap junctional coupling. Thus,
propagation velocity (V) along the fibre axis is much faster
in the myocardium than transverse to it. This biophysical
property of the heart is termed anisotropy which is defined
as the ratio Vlongitudinal/Vtransverse. Another function of cardiac gap junction is the metabolic coupling of the myocardial
cells. This allows the transfer of small molecules and may
0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2004.01.019
288
S. Dhein / Cardiovascular Research 62 (2004) 287–298
enable slow calcium wave spreading, transfer of ‘‘death’’
signals [5] or of ‘‘survival’’ signals [6,7]. In contrast to
transmembrane ionic channels gap junction channels do not
exhibit only one conductance state but can switch between
various substates, which seems to depend on the phosphorylation state of the connexins, although it should be mentioned that the role of the substates in regulation of the
channel conductance is a matter of discussion since it is
related to the portion a certain substate represents [8,9].
In the vasculature the predominant connexins are Cx37,
Cx40 and Cx43 providing intercellular coupling between
endothelial cells (Cx37, Cx40, Cx43), between smooth
muscle cells (Cx43) and myoendothelial coupling. The
function of gap junctional coupling in the vasculature has
been suggested to consist in an up stream regulation of
vascular tone [10] and seems to be involved in the release of
vasoactive factors from the endothelium such as EDHF [11]
involving myoendothelial communication [12]. NO-mediated vasorelaxation does not seem to depend on gap junctional coupling [13]. Leukocytes can also couple to
endothelial cells via gap junctions in a bidirectional manner
affecting transmigration and leakiness [14].
In the following paragraphs drugs will be reviewed
which have been reported to affect cardiovascular gap
junctions. Due to space limitations not all drugs affecting
gap junctions in other systems can be mentioned.
1.1. Agents for acute closure of gap junctions
In the following paragraphs those agents will be reviewed
which have been reported to uncouple gap junctions acutely,
i.e. within 5 –10 min, in cardiovascular tissue. Table 1 gives
an overview on the acutely uncoupling agents.
Ions which can pass the channel are involved in the
regulation of gap junctional conductance. Thus, Ca2 +-induced reduction of junctional permeability has been described in cardiomyocytes [15,16]. While low changes in
calcium do not affect gap junction conductance ( gj) in adult
heart cells [17] higher changes in [Ca2 +]i reduce gj in
guinea pig and rat hearts [18]. Maurer and Weingart [18]
concluded from their experiments that reduction in gj occurs
if the intracellular calcium concentration exceeds the range
of 320 –560 nmol/l. It has been suggested that the binding
site for Ca2 + and H+ is located on the cytoplasmic loop of
Cx43 [19].
Intracellular acidification is known to decrease junctional
electrical coupling in cardiomyocytes and in Purkinje fibres
[16,20,21]. In neonatal rat heart cells Firek and Weingart
[22] found a pKH with 5.85. Using a transfection system, it
was found that Cx45 channels are more sensitive to pH than
Cx43 channels [23]. Regarding the pH sensor, the carboxy
tail length has been demonstrated as a determinant of the pH
sensitivity [24]. Further investigations [25] revealed that the
Table 1
Survey on agents acutely uncoupling cardiovascular gap junctions
Class
Ions
Lipophilic agents
and fatty acids
Glycyrrhizic acid
metabolites
Receptor ligands
Dephoshorylating
agents
Narcotics
Phorbol esters
PKC inhibitors
Metabolites
Eicosanoids
Fenamates
IP3-receptor blocker
Weak organic acids
Cannabinoids
Cardiac glycosides
a
Agents
+
+
+
++
H , Na , Ca , Mg
heptanol, octanol arachidonic acid,
oleic acid, palmitoleic acid,
decaenoic acid, myristoleic acid
18-a-glycyrrhetinic acid,
18-h-glycyrrhetinic acid,
carbenoxolone
carbachol (M-cholinoceptor),
noradrenaline (a-adrenoceptor),
FGF-2, angiotensin-II (AT1 receptor),
Atrial natriuretic factor ANF, VEGF
2,3 butandione monoxime
halothane, ethrane, isoflurane
12-O-tetradecanoyphorbol13-acetate, (TPA)
staurosporine
ATP-decrease, Diacylglycerol/
1-oleoyl-2-acetyl-sn-glycerol
11,12-epoxyeicosatrienoic acid
(late effect) thromboxane A2
meclofenamic acid, niflumic acid,
flufenamic acid
2-aminoethoxydiphenyl borate
acetic acid, propionic acid
D9-tetrahydrocannabinol
strophanthidin, ouabain
Probably depending on the isoform of PKC.
Proposed mechanism
Ref.
direct effects?
incorporation?
[16 – 22]
[17,30 – 36]
phosphorylation?,
aggregation of Cx-subunits?
[39 – 41]
cGMP/PKG, PKC, PKCq,
PKC?, cGMP/PKG, ERK
[42 – 44,51,52,
59,60,63]
unknown mechanism
[55]
incorporation?
activation of PKCa, /Ca++?
[37,38]
[45,48,49,53]
inhibition of PKCa
dephosphorylation, unknown
mechanism/disturbance of
the lipid bilayer?
ERK1/2, unknown mechanism
[54]
[46,58]
unknown mechanism
[67]
unknown mechanism
H+
ERK1/2
elevation of [Ca2 +]i
[68]
[9,50]
[66]
[27 – 29]
[64,65]
S. Dhein / Cardiovascular Research 62 (2004) 287–298
carboxy terminal serves as an independent domain which
can bind to another separate domain of the connexin protein
(e.g. a region including His-95 [26]) and close the channel,
comparable to the ball-and-chain-model for potassium channels. Elevation in [Mg2 +]i to 1 –10 mmol/l at pH = 7.4 in the
absence of calcium has also been reported to reduce
junctional conductance in pairs of adult guinea pig cardiomyocytes [16]. Na+ also is involved in the regulation of gap
junction conductance. Thus, Na+-withdrawal in adult rat
cardiomyocytes induced electrical uncoupling within 3 min
[18]. This has been interpreted as an impairment of the Na+/
Ca2 +-exchange mechanism, since calcium extrusion via this
mechanism requires the transport of sodium [18,27]. Besides this, DeMello [28] described that an increase in [Na+]i
caused uncoupling within 500 ms in Purkinje fibres. It is
uncertain whether this was a direct effect of sodium or may
be secondary to a rise in intracellular calcium via the Na+/
Ca2 +-exchange mechanism.
The intracellular concentrations of Na+and Ca2 + can be
affected pharmacologically by cardiac glycosides such as
strophanthidin, ouabain, digitoxin or digoxin acting via
inhibition of Na+/K+-ATPase leading to enhanced Na+/
Ca2 + exchange resulting in elevated intracellular Ca2 +. In
cow hearts 2 Amol/l ouabain were shown to decrease
conduction velocity [29]. Similarly, De Mello [28] observed
an uncoupling effect of 0.68 Amol/l ouabain in Purkinjefibres. Strophanthidin (2– 20 AM) also uncoupled guinea pig
ventricular cell pairs [27]. These uncoupling effects of
cardiac glycosides may contribute to the well known
arrhythmogeneity of digitalis.
Next, the lipophilic agents should be considered. Cardiac
gap junction channels can be uncoupled using micromolar
concentrations of heptanol, octanol, myristoleic acid, decaenoic acid or palmitoleic acid [30 – 32]. Most commonly,
this is explained by an incorporation of these drugs into the
lipid bilayer leading to impairment of the transcellular gap
junction channels. Heptanol has been reported to reduce
coupling by reducing open probability of the channels by a
conformational change at the connexin-membrane lipid
interface [33]. Many investigators used heptanol which
has been shown to inhibit reversibly gj with a KD of 0.16
mmol/l [17]. Oleic acid also closes gap junctions in neonatal
rat cardiomyocytes with an EC50 in the order of about 2 AM
[30,34]. Similarly, myristoleic acid and palmitoleic acid lead
to uncoupling with similar EC50 [30]. In whole heart
Langendorff preparations of rabbit hearts palmitoleic acid
exhibited a preferential impairment of transverse conduction
by the fatty acid and concomitant increase in dispersion with
an EC50 of 3.3 AM [32,35]. The N-6 unsaturated fatty acid
arachidonic acid also uncouple cells with a KD of 4 Amol/
l [36]. It should be noted, that these lipophilic drugs are not
very specific for gap junctions and can inhibit other ion
channels as well.
Within the group of lipohilic drugs, inhalative narcotics
such as halothane or isoflurane have also been shown to
interfere with intercellular coupling. Incubation of neona-
289
tal ratcardiomyocytes with 2 mM halothane resulted in a
90% reduction of initial junctional conductance within 15
s. The single channel conductance, however, was unchanged so that the authors assumed the halothane effect
being due to a decrease in the number of functional
channels [37]. In a subsequent study it was found that
halothane reduced the mean open time while increasing
the mean closed time [38].
The glycyrrhizic acid metabolites 18-a-glycyrrhetinic
acid, 18-h-glycyrrhetinic acid and carbenoxolone have
been shown to uncouple gap junction channels in various
models. Thus, several authors have used 18-a-glycyrrhetinic acid as a gap junction inhibitor [39] in concentrations
of about 50 AM [40] or 18-h-glycyrrhetinic acid in a
concentration of 5 AM [41]. The effect of these compounds
requires a longer exposure time than the above mentioned
drugs. The molecular mechanism of the glycyrrhizic acid
metabolites is still unknown and has been suggested to
involve phosphorylation or changes in the aggregation of
connexin subunits.
Next, effects transduced via membrane receptors or
intracellular signalling cascades are to be discussed. Thus,
application of the acetylcholine analogue carbachol (100
AM) in neonatal heart resulted in a reduction in electrical
coupling, which could be mimicked by 8-Br-cGMP [42]
suggesting an action via protein kinase G. However, from a
pharmacological point of view the physiological impact
remains unclear since 100 AM is a considerably high
concentration. The situation becomes even more unclear
in the vasculature where the responses to acetylcholine can
be inhibited with the gap junction blocker 18-a-glycyrrhetinic acid and the gap junction specific peptide gap27 (see
below) [39]. The question whether on the other hand
acetylcholine influences gap junctional communication in
the vasculature is not investigated so far.
Another drug probably acting via cGMP on gap junctions is atrial natriuretic factor (ANF). In cell pairs isolated
from cardiomyopathic hamsters exposure to 10 nM ANF led
to a uncoupling in a cGMP-dependent manner [43].
Regarding the effects of catecholamines adrenaline and
noradrenaline there are mainly investigations on the coupling-increasing effects of stimulation of cAMP-pathway via
h-adrenoceptors. In several tissues a-adrenergic stimulation
leads to uncoupling. Similarly, a-adrenergic stimulation
with phenylephrine in adult rat ventricular cardiomyocytes
decreases gap junctional coupling in a PKC-dependent
manner [44]. Since a-adrenoceptors couple via Gq/11 proteins to the phospholipase C/inositoltrisphosphate /diacylglycerol/PKC pathway, it might be interesting to consider
the effects of PKC activation on gap junctional communication. PKC can be directly activated with phorbol esters
such as 12-otetradecanoyphorbol-13-acetate (TPA). Activation of PKC by these agents was described in some studies
to enhance macroscopic gap junction conductance [19,45]
(see next section). In another study on neonatal rat cardiomyocytes there was no effect of TPA (0.1 AM) [46]. On the
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S. Dhein / Cardiovascular Research 62 (2004) 287–298
other hand, uncoupling effects of PKC activators have also
been described (see below). Incardiac tissue several isoforms
of PKC are expressed including PKCa, PKCh, PKCq,
PKCs and PKCg (rabbit heart) [47]. However, only PKCg
was found to be located close to the intercalated disks in this
study. TPA treatment is assumed to result in a rapid
translocation of PKCa and PKCq in cultured neonatal rat
cardiac myocytes [48]. Thus, one may argue that not all
isoforms contribute to the gap junction regulation and that
differences between various preparations or tissues may
depend on the subtypes of PKC involved. In cardiac
myocytes, increase as well as decrease in gj have been
observed in pairs of neonatal cardiomyocytes after PKCactivation by TPA [19,49]. In addition, Kwak et al. [45]
found that TPA increases electrical conductance but
decreases permeability as assessed by dye coupling in
neonatal rat cardiomyocyte gap junction channels. Thus,
permeability for small molecules and electrical conductance
do not seem to be related to each other under all conditions
(for a detailed discussion, see Ref. [50]). The above hypothesis that the divergent results regarding PKC dependent
regulation of gap junction conductance may be due to
activation of different PKC isoforms is supported by recent
findings showing that activation of PKCq by fibro blast
growth factor FGF-2 leads to uncoupling of cardiomyocytes
(see below) while activation of PKCa by antiarrhythmic
peptides induces enhanced gap junctional coupling [51,52]
(see below). Because TPA activates both isoforms (which
seem to exert opposite effects on gj) the diverging results
obtained with TPA reported in the literature might be
explained by a different PKAa/PKCq activation ratio. In
addition, it has been suggested that PKC does not directly
phosphorylate connexins but may activate other regulatory
proteins which finally phosphorylate connexins, which also
might cause divergent results. Both PKCa and PKCq have
been shown to phosphorylate Cx43, however, only PKCq
phosphorylates directly [53]. Next, the PKC inhibitor staurosporine should be considered. In cultured neonatal ratcardiomocytes, Saez et al. [54] demonstrated an uncoupling
effect of staurosporine (300 nM) which could be reversed by
TPA. Staurosporin in these experiments reduced the incorporation of 32P into Cx43 supporting the view that PKCdependent phosphorylation of Cx43 enhances intercellular
coupling (see next section).
Taken together, the present results regarding PKC are
conflicting and future detailed studies are needed whichamong other factors mentioned above-consider the isoforms
of PKC involved an duse more specific PKC inhibitors.
Some of the diverging results might also be caused by the
Ca2 + concentration in the pipet [46] or the phosphorylation
status of the connexins prior to treatment [54].
Regarding dephosphorylation agents 2,3 butandione
monoxime (BDM) was found to reduce junctional current
[55]. However, this was not correlated with a change in the
ratio between non-phosphorylated and phosphorylated
Cx43, so that the authors concluded, that the BDM effect
might be mediated via regulatory proteins associated with
Cx43.
In another approach it was shown that dephosphorylation
by endogenous protein phosphatases leads to a run down in
channel conductance which can be antagonized by a phosphatase inhibitor such as okadaic acid [56]. Dephosphorylation of Cx43 in heart has been suggested to be mediated
via PP1-like phosphatases [57].
In line with these findings a loss of intracellular ATP has
been reported to result in gap junctional uncoupling so that
the spontaneous run down of gap junction current in double
cell patch clamp experiments can be counter acted by
addition of ATP to the pipette solution [58]. This mechanism may be involved in cellular uncoupling in the course
of cardiac ischemia and thus might contribute to arrhythmogenesis in cardiac ischemia.
Diacylglycerol (DAG) as an endogenous activator of
PKC has been assumed also to be involved in gap junctional
uncoupling. Thus, the synthetic DAG analogue 1-oleoyl-2acetylsn-glycerol (OAG) (25 –100 Ag/ml) has been evaluated in neonatal rat cardiomyocytes. While 25 Ag/ml OAG led
to partial uncoupling, more or less complete uncoupling was
observed with 100 Ag/ml with regard to dye transfer and
junctional current [46]. Inhibition of PKC by H7 (100 Ag/
ml) prior to treatment did not prevent from the uncoupling
effect of OAG indicating independence from PKC. The
authors assumed that OAG might incorporate into the lipid
bilayer and disturb gap junctions as other lipophilic drugs.
Angiotensin has also been shown to influence gap
junctional coupling in cardiac cells. The acute effects seem
to be mediated via AT1 receptors coupled to Gq/11 proteins
and protein kinase C. In adult ventricular cell pairs 1 Ag/ml
angiotensin-II rapidly decreased gj [59]. This uncoupling
effect seemed to be mediated via AT-receptors coupled to
PKC since it could be inhibited by staurosporine and by
DuP 753. In subsequent experiments intracellular dialysis of
10 nmol/l angiotensin I resulted in a decrease of gj, which
was completely inhibited byintracellular dialysis of 1 nmol/
l enalaprilat, demonstrating the possible existence of an
intracellular angiotensin converting enzyme [60], which is
supported by other findings [61,62].
The angiogenetic vascular endothelial growth factor
VEGF acts on tyrosine kinase-coupled VEGF-receptors
and was shown reversibly to inhibit gap junctional intercellular communication (GJIC) investigated by dye transfer
technique in endothelial cells within 15 – 30 min after
application of 50 ng/ml VEGF with a concomitant change
in the phosphorylation of Cx43 [63]. The effect was
mediated by VEGF-receptor type 2.
Finally, it should be mentioned that recently it has been
shown that 11,12-epoxyeicosatrienoic acid also elicits an
uncoupling effect which has been demonstrated in endothelial cells. This effect was biphasic: an initial improvement of interendothelial coupling was followed by
sustained uncoupling effect which seemed to depend on
activation of ERK1/2 [64]. This opens the interesting view
S. Dhein / Cardiovascular Research 62 (2004) 287–298
of an endogenous intracellular regulation of intercellular
communication.
Another eicosanoid reported to inhibit GJIC is thromboxane A2 (TXA2). It was shown that a TXA2 mimetic
reduced dye transfer between human endothelial cells and
led to internalization of Cx43 [65]. This was associated with
capillary formation and thus might reflect a mechanism
involved in angiogenesis.
Vascular GJIC can also be blocked using the cannabinoid
receptor agonists. 9-Tetrahydrocannabinol (10 –30 AM) or
the synthetic HU210 (10 AM) which both led to Cx43
phosphorylation in an ERK1/2-dependent manner associated with reduction in electrical coupling and dye transfer
within 15 min in cultured endothelial cells [66].
A new group of drugs resulting in reversible gap junctional uncoupling comprises the fenamates with an order of
potency meclofenamic acid > niflumic acid > flufenamic acid with IC50 values of 25 –40 AM [67]. The exact molecular
mechanism of action examined inSKHep1 cells expressing
Cx43 remains unclear, but is not related to cyclooxygenase
inhibition, PKC activation, intracellular pH, calcium, or
membrane depolarization. Another reversible gap junction
uncoupling agent is 2-aminoethoxydiphenyl borate (normally used as IP3-receptor blocker), which has been evaluated
in normal rat kidney fibroblasts leading to uncoupling with
an IC50 of 5.7 AM [68]. However, the underlying mechanism of action is still unknown.
1.2. Agents for acute opening of gap junctions
While there is a broad number of agents uncoupling gap
junctions, there are only a few agents that enhance coupling.
However, this might be of particular interest since it has
been shown that enhancing intercellular coupling can act
antiarrhythmically especially in situations of reduced coupling such as ischemia-reperfusion. Table 2 gives an overview on the agents available.
Among the mechanisms involved in enhancement of gap
junctional coupling cAMP dependent protein kinase A
(PKA) is discussed in a number of studies. Intracellular
cAMP leading to activation of PKA in cardiomyocytes can
enhance coupling [69 – 71] while in others there is no effect
[45,48]. In contrast, injection of cAMP into canine Purkinje
fibers increased gap junction coupling [69]. Thus, it might
be, that PKA activation may enhance coupling inCx40 and
Cx45 coupled cells but not in Cx43 coupled cells. In support
of this hypothesis, van Rijen et al. [72] found that human
Cx40 gap junction channels are modulated by cAMP. On
the other hand, dye transfer through Cx45 gap junction
channels and electrical coupling inCx45-transfected
SKHep1 cells is not influenced by PKA activation [45].
It has been suggested that the effect of a stimulation of
the beta-adrenoceptor/adenylylcyclase/PKA pathway may
differ between normal heart and cardiomyopathic hearts
due to the alteration of the beta-adrenoceptor/adenylylcyclase/PKA-system. Accordingly, De Mello [73] showed that
291
Table 2
Agents reported to acutely enhance gap junctional intercellular communication in the cardiovascular system (PDE = phosphodiesterase; TPA = 12-Otetradecanoylphorbol 13-acetate; HPP-5 = N-3-(4-hydroxyphenyl)propionyl-Pro-Hyp-Gly-Ala-Gly)
Class
Agents
Proposed
mechanism
Ref.
cAMP-enhancing,
drugs
Antiarrhythmic
drugs
Antiarrhythmic,
peptides
cAMP, forskolin,
isoprenaline
Tedisamil
PKA
[69 – 73]
PKA
[91]
PKCa,
?PKC
[82 – 90]
PKA
[64]
PKC
inhibition of
tyrosine, kinase,
(only after
hypoxia),
unknown
mechanism
cAMP/PKA
[19]
[92]
Eicosanoids
Phorbol ester
Unsaturated
fatty acids
AAPnat, AAP10,
cAAP10RG,
HPP-5, ZP123
11,12-epoxyeicosatrienoic
acid, (early effect)
TPA
eicosapentaenoic acid
Receptor ligands
5-hydroxytryptamine
PDE inhibitor
isobutyrylmethylxanthine
[95]
[73]
in cardiomyocytes isolated from cardiomyopathic hamsters
isoprenaline, for skolin (a direct activator of adenylylcyclase), isobutyrylmethylxanthine (an inhibitor of phosphodiesterases) failed to influence gap junction conductance
while in cells from normal hearts they increased GJIC.
However, dibutyryl-cAMP in both groups of cardiomyocytes led to an increase in gap junction coupling, showing
that the beta-adrenoceptor/adenylylcyclase system seemed
to be down-regulated or uncoupled while the cAMP/PKA
system was still effective in controlling gj.
Several authors observed enhanced coupling of cardiomyocytes in response to PKC stimulation by phorbol esters
(e.g. Ref. [19]). However, others found uncoupling effects
of PKC stimulation (see above). As outlined above these
different findings may eventually be due to different isoforms of PKC involved. Alternatively, the involvement of
other regulatory proteins down stream of the PKC may be
considered which might act on connexins [53]. Another
group of agents opening gap junctions are antiarrhythmic
peptides. In 1980, Aonuma et al. [74] identified a hexapeptide in bovine atria, designated as AAP ( = antiarrhythmic
peptide, AAPnat; structure: H2N-Gly-Pro-4Hyp-Gly-AlaGly-COOH) [75] which improved synchronisation of cultured myocardial cell clusters and was shown insubsequent
studies to possess antiarrhythmic activity in various models
(for a detailed overview, see Refs. [76,77]). AAPnat was
found in concentrations of 203 pmol/g in heart, 165pmol/g
in kidney and 3.8 pmol/ml in blood [78], while in other
organs AAP tissue levels were similar to those in blood.
AAPnat was evaluated in several in-vivo models of
arrhythmia: it suppressed CaCl2-inducedarrhythmia in mice
[79], aconitine-induced arrhythmias [80], and ouabain-induced arrhythmia but not epinephrine-induced arrhythmia.
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S. Dhein / Cardiovascular Research 62 (2004) 287–298
A derivative of AAPnat, N-3-(4-hydroxyphenyl)propionylPro-Hyp-Gly-Ala-Gly ( = HPP-5) was also shown to act
antiarrhythmically [79].
Regarding the mechanism of action of AAPnat electrophysiological experiments on canine Purkinje fibers showed
that the transmembrane action potential was not altered [81],
thus indicating that transmembrane ionic currents are not
involved. Dhein and coworkers [82 –85] demonstrated that
the effect of AAPnat and related peptides consists in an
improvement of cellular coupling and an increase in gap
junctional conductance. Regarding the mechanism of action
of these antiarrhythmic peptides it was shown that the
synthetic derivative AAP10(H2N-Gly-Ala-Gly-Hyp-ProTyr-CONH2) possesses a semicyclic structure and can bind
to a membrane protein which has been assumed to represent
a membrane receptor [51,52,85 – 87]. Since the AAP10
effect was sensitive to GDP-hS and to PKC inhibitors as
well as to a PKCa-specific inhibitor (CGP54345), it was
concluded that AAP10 acts via a G-protein which down
stream activates protein kinase Ca leading (directly or
indirectly) to a phosphorylation of connexin43 resulting in
an improvement of gap junction conductance [51,52,83 –
85]. It has been suggested that the AAP10 effect is more
pronounced in cells which are partially uncoupled.
A particular disadvantage of AAP10, however, is its
instability in solutions and under in-vivo conditions due
to rapid hydrolysis and proteolysis. Therefore, more
stable compounds were developed such as cAAP10RG
( = c(CF3(OH)C-GAGHypPY)) [86,87] or the D-amino
acidAAP10-analogue ZP123 (H2N-Gly-D-Ala-Gly-D4Hyp-D-Pro-D-Tyr-Ac) [88]. Both drugs have been
shown to reduce dispersion of action potential duration
in a 256 electrode mapping in isolated rabbit hearts [89].
All AAP10, AAPnat and ZP123 activate protein kinase C
[89]. In a recent study the effect of ZP123 on pairs of
adult guinea pig cardiomyocytes was investigated showing a similar slowly increasing coupling [90] as was
described for AAP10 [52]. Moreover, Xing et al. [90]
found that ZP123 prevented from reentrant ventricular
tachycardia at plasma concentrations ranging from 1 to
69 nM in a dog model of reproducible infarct-induced
tachycardia.
The antiarrhythmic agent tedisamil, a bradycardic drug,
has been shown to increase gap junctional conductance by
58% (0.1 AM) in cell pairs of cardiomyopathic hamsters
[91]. Because of the sensitivity of the effect to inhibition
of PKA, this was interpreted as a PKA mediated action.
However, it should be noted that tedisamil has been reported
also to act on a number of other transmembrane ionic
channels, such as sodium and potassium channels. Thus,
tedisamil does not seem to be specific for gap junctions.
Among the fatty acids, the polyunsaturated N-3 fatty acid
eicosapentaenoic acid has been assumed to enhance or
preserve gap junctional coupling in endothelial cells. Thus,
hypoxia/reoxygenation reduced GJIC in human umbilical
vein endothelial cells (HUVEC) after 2 h of reoxygenation.
This could be inhibited by a 2-day long pre-treatment with 3
AM eicosapentaenoic acid [92]. Additional investigations
showed that eicosapentaenoic acid in these experiments
inhibited tyrosine phosphorylation of Cx43 induced by
hypoxia/reoxygenation. Under normoxia, however, eicosapentaenoic acid had no effect on GJIC. Finally, an interesting aspect comes from the eicosainoids. In the vasculature
hyperpolarization and vasorelaxation can be conducted
along the vessel via interendothelial gap junctions [93,94].
In some species this seems to be linked to the NO/PGI2independent pathway coupled to the cytochrom P450 isoform CYP2C and the generation of epoxyeicosatrienoic
acids such as 11,12-epoxyeicosatrienoic acid (11,12-EET)
[64]. Interestingly, 3 AM 11,12-EET had a biphasic effect
on GJIC in human umbilical veinendothelial cells. 11,12EET transiently enhanced GJIC within 1 min, followed by a
prolonged uncoupling effect (which is discussed above, see
uncoupling agents). Since the action of 11,12-EET could be
inhibited by KT5720, an inhibitor of PKA, the authors
concluded that the initial increase in coupling seems to be
PKA-mediated [64]. The enhanced coupling could be
mimicked by 10 AM for skolin and caged cAMP. Interestingly the cAMP effect was accompanied by a translocation
of Cx43 to the Triton-X-100-insoluable cell fraction [64].
This might point to an additional mechanism of PKAdependent actions, but needs further investigation. Since
EETs are potent intracellular mediators and are involved in
several signal transduction cascades, these observations
might be of general interest.
Finally, it has been shown that serotonin (5-hydroxytryptamine) in concentrations of 1 –10 AM can increase junctional conductance in vascular smooth muscle [95].
1.3. Agents affecting expression, synthesis, assembly,
docking and degradation of gap junctions
A recent aspect of gap junction regulation is the high
turnover of connexins in the membrane with half life times
of 1– 5 h [96,97] or in the case of Cx43 around 1.6 h [98 –
100] which allows the cells to adapt their communication to
the actual situation. A number of substances and mediators
has been shown to interfere with the synthesis, formation,
docking and degradation of the connexins. Table 3 gives a
survey on the agents.
Connexin expression can be pharmacologically affected by various approaches. First, it is possible to suppress
the expression of a specific connexin by use of oligonucleotides. Thus, Moore and Burt [101] used 5VGTCACCCATGTCTGGGCA-3V as Cx43 antisense and
5V-GTCACCCATCTTGCCAAG-3V as Cx40 antisense in
A7r5 cells (embryonic rat aorta smooth muscle cell line
expressing both Cx43 and Cx40) and could show, that 24
h treatment could suppress the unitary conductances
specific for the target connexin.
The next aspect of chronic regulation of connexins, is the
induction of connexin synthesis by various pathways. Thus,
S. Dhein / Cardiovascular Research 62 (2004) 287–298
293
Table 3
Agents affecting expression, synthesis, assembly, docking and degradation of gap junctions (AC = adenylycyclase; ALLN (acetyl-leucyl-leucyl-norleucinal)
Class
Agents
Proposed mechanism
Ref.
Trafficking inhibitors
brefeldin A
[118]
Metabolic inhibitors
monensin
Peptides
GAP27, GAP26; E1 and E2,
peptides, P180-195/Cx43,
P177-192/Cx40
endothelin, angiotensin,
TNFa, VEGF, bFGF
connexin transport in
the Golgi apparatus
trapping of Cx in the
trans-Golgi network
inhibition of docking
ERK1/2, p38 MAPK,
p38MAPK/others?, TGF-h,
unknown mechanism
PKA
cAMP/PKA
inhibition of proteasomal
degradation
inhibition of lysosomal
degradation
inhibition of expression
[104,105,108,
109,112 – 116]
Receptor agonists
Second messenger
AC activator
Proteasome, inhibitors
Lysosome inhibitors
Antisense, oligonucleotides
cAMP, 8-Br-cAMP
forskolin
ALLN, lactacystin,
clastolactacystin, epoxomicin
leupeptin, chloroquine,
primaquine, balifomycin A
Cx43:GTCACCCATGTCTGGGCA,
Cx40:GTCACCCATCTTGCCAAG
it has been shown that connexin expression can be induced
by the second messenger cAMP. Thus, Darrow et al. [102]
found an up regulation of Cx43 and Cx45 following a 24
h administration of dibutyryl cAMP in cardiomyocytes.
Accordingly, Salameh et al. [103] showed an up regulation
of Cx43 in neonatal cardiomyocytes in response to forskolin, a direct activator of adenylylcyclase. Thus, there is
evidence that activation of the adenylylcyclase/cAMP/
PKA pathway can enhance Cx43 expression.
Besides the cAMP/PKA pathway it has been shown that
relevant mediators involved in physiology and pathophysiology of the cardiovascular system can regulate connexin
expression (endothelin, angiotensin, TNFa, b-FGF, VEGF).
Thus, in neonatal ratcardiomyocytes 24 h endothelin-1
exposure resulted in an increase of Cx43 expression
(EC50: 158 F 41 nM) and phosphorylation (EC50: 13 F 3
nM) while Cx40 remained unaffected [104]. The increase in
Cx43 was reflected by enhanced gap junctional conductance
in double cell patch clamp experiments, so that the authors
concluded that the increase in Cx43expression may result in
a higher number of functional channels [104]. The endothelin-effect was mediated via ETA-receptors. Downstream
it was found that the enhanced Cx43 expression was
dependent on ERK1/2.
Another agonist acting at Gq/11-coupled receptors is
angiotensin-II. Twenty-four hours angiotensin-II treatment
also increased the expression of Cx43 (EC50: 57 F 10 nM)
and phosphorylation (EC50: 93 F 8 nM), whileUas with
endothelin—there was no detectable change of Cx40. Thisenhanced Cx43 expression was associated with enhanced
electrical gap junctional intercellular coupling [104] in good
accordance with previous findings by others [105]. The
angiotensin-II-induced Cx43 expression was mediated via
the AT1-receptor [104]. Regarding the signal transduction
pathway it was demonstrated that angiotensin activates both
ERK1/2and p38 signal pathway [104].
[119]
[40,121 – 125]
[102]
[103]
[127,128]
[127,128]
[101]
Wang et al. [106] showed that cyclical mechanical stretch
in neonatal ratcardiomyocytes can cause an increase in
Cx43 while Cx40 and Cx37 expression did not change.
However, the observed increase in Cx43 did not seem to be
related to de-novo synthesis and thus might be caused by an
altered transcript steady state level. Interestingly, it was
found that mechanical stretch can induce the release of
angiotensin II which according to the experiments of
Polontchouk et al. [104] can induce Cx43 expression. In
accordance with these findings an antagonization of stretchinduced augmentation in Cx43 expression by theAT1-receptor antagonist losartan has been described [107]. On the
other hand, Pimentel et al. [108] also found increased Cx43
expression levels in cultured neonatal rat cardiomyocytes
upon stretch in association with VEGF-secretion. The increased Cx43 protein level was accompanied by enhanced
conduction. This stretch-induced enhancement of conduction could be antagonised by a VEGF antibody. A further
proof of the specificity for VEGF came from the finding that
the conditioned culture medium of cells exposed to stretch
could induce the same increase in Cx43 expression in nonstretched cells, which was also blocked by VEGF antibody
[108]. Since a TGF-h antibody was also effective the
authors concluded that stretch might induce the increase in
Cx43 expression via a TGF-h/VEGF pathway [108]. These
mechanisms might be involved in atrial fibrillation in the
course of mitral valve disease and atrial dilatation as well as
in congestive heart failure.
In cardiac remodelling processes basic fibroblast growth
factor (bFGF) plays an important role. bFGF has been
shown to induce Cx43 expression in cardiac fibroblasts
within 6 h after administration [109]. Such regulation of
intercellular fibroblast communication might play a role in
cardiac scar tissue or might be of importance in arrhythmogenesis in cardiacfibrosis. Interestingly, in cardiomyocytes
bFGF exposure acutely (within 30 min) decreased gap
294
S. Dhein / Cardiovascular Research 62 (2004) 287–298
junctional coupling in a PKCq-dependent mechanism (see
above) [110]. Unfortunately, there are at present no data
available on the effects of chronic (>24 h) bFGF stimulation
of cardiomyocytes.
Besides the above mentioned factors, cytokines may also
play a role in the chronic regulationof connexin expression.
Thus, in endocardial biopsies from heart transplant recipientsexpression of Cx43 was found to be significantly
diminished during acute cellular rejection [111]. Among
the factors that could play a role in this pathophysiology
cytokines and tumor necrosis factor a (TNFa) should be
considered. With regard to the TNFa-effect on connexin
expression, there are diverging results reported in the
literature: in bacterial lipopolysaccharide (LPS)-induced
cardiac inflammation in hearts in an in-vivo rat model,
Fernandez-Cobo et al. [112] found a down regulation of
Cx43-mRNA, while in contrast, in lungs and kidney Cx43
was increased following LPS-exposure [113]. In contrast, in
cultured transfected HeLa-Cx43-cells Salameh et al. [114]
found a significant increase in Cx43expression under the
influence of 24 h TNFa (10 U/ml). This induction was
transduced via p38 MAP-kinase. Similarly, in cultured
neonatal rat cardiomyocytes exposure to low concentrations
of TNFa also increased Cx43 expression via p38 MAPkinase [115]. On the other hand, in endothelial cells, 0.5 nM
TNFa did not influence Cx43 expression but led to downregulation of Cx40 and Cx37 [116]. Thus, in different cell
types obviously different signalling pathways connected to
Cx43 regulation are activated in response to TNFa-receptor
stimulation. Since inflammation and septic shock alter
cardiovascular homeostasis and may lead to arrhythmia
regulation of connexin expression by cytokines might be
an interesting new aspect.
In the connexin43 promotor region, an estrogen – responsive element has been described. Although from the present
literature available this does not seem to be involved in the
regulation of Cx43 in the heart, Liu et al. [117] showed that
in ovariectomized female Wistarrats Cx43 was significantly
down-regulated in media and endothelium of mesenteric
arteries associated with a decreased EDHF response. Since
this could both be normalized by 17h-estradiol, estrogen
seems to be involved in the regulation of Cx43 in the
vasculature (at least in this model) in endothelial and
myoendothelial gap junctions.
Connexins are synthesised in the endoplasmic reticulum,
folded, thereafter transported to the Golgi network undergoing oligomerisation to hexameric hemichannels (connexons), and finally transported to the plasma membrane [118],
where they are inserted adjacent to, or in conjunction with
cadherins and zonula occludens protein ZO-1 (see another
review in this issue). The process of transportation between
endoplasmic reticulum, Golgi and membrane can be
inhibited pharmacologically, e.g. by the metabolic inhibitor
monensin [119]. Using connexin 43 in rat kidney cell
cultures Musil and Goodenough [120] found that connexonformation occurs after transport through the cis, medial
and trans Golgi cisternae, since connexon assembly could
be blocked by brefeldin A, a specific blocker for assembly
processes occurring before or at these compartments.
Connexons dock to each other forming the complete gap
junction channel by interaction of their extracellular loops.
From a pharmacological point of view it should be possible
to interfere with the docking process by adding peptide
sequences resembling only the extracellular loops as first
described for Cx32 [121]. In experiments using chick
cardiomyocytes the motifs QPG and SHVR in extracellular
loop 1 and SRPTEK in loop 2 were identified [122]. In
rabbit ear arteries the peptides GAP27 and GAP26 were
used as inhibitors of GJIC [123]. GAP26 has been reported
to act primarily on hemichannels [124] and may inhibit ATP
release. Kwak and Jongsma [125] used a peptide analogous
to the second extracellular loop of Cx43, P180-195 (SLSAVYTCKRDPCPHQ; 500 AM) in A7r5smooth muscle cells
to inhibit coupling via Cx43 channels and a second peptide,
analogue to the second extracellular loop of Cx40, P177192 (FLDTLHVCRRSPCPHP; 50 AM) for inhibition of
coupling via Cx40 channels. They showed that 24 h treatment of the cultured cells was sufficient to suppress the
portion of intercellular coupling which could be ascribed to
the target connexins by suppression of the unitary conductance specific for the target connexin. Another peptide often
used for inhibition of docking is also analogous to the
extracellular loop of Cx43, called the 43GAP27 peptide
(SRPTEKTIFII). In a concentration of 300 AM it was shown
to inhibit the endothelial component of cannabinoid-induced
relaxation in rabbit mesenteric artery [40].
Finally, it is possible to interfere with the degradation of
connexins. Connexins can be degraded either by the proteasomal pathway or via the lysosomal pathway [126]. Parts
of the degradation of Cx43 seem to be dependent on prior
ubiquitinylation of the protein. The lysosomal pathway can
be inhibited by protease inhibitors such as leupeptin or by
disruption of the pH gradient in the lysosome using chloroquine, primaquine or balifomycin A, leading to enhanced
Cx43 presence in the cells [127]. The proteasome can be
inhibited by ALLN(acetyl-leucyl-leucyl-norleucinal), lactacystin, clastolactacystin and epoxomicin [128]. These agents
have been widely used to study gap junction degradation,
but there is no therapeutic approach so far. Interestingly
Laing et al. [127] showed, that heat stress led to reduced
Cx43expression, which could be prevented by lactacystin,
ALLN and chloroquine. These authors concluded that heat
stress may mimic other stress such as ischemia, which also
is known to reduce Cx43 expression.
2. Conclusions
Cardiovascular gap junction channels can be regulated
either by acute opening or closure of gap junctions or by
regulating gap junction protein expression there by altering
the number of channels. A question to be addressed briefly
S. Dhein / Cardiovascular Research 62 (2004) 287–298
is what is the pharmacological benefit from uncoupling/
coupling in heart or in vasculature? Reducing GJIC in the
heart has been shown to slow conduction, alter anisotropy,
lead to transverse conduction block and enhanced dispersion
finally resulting in arrhythmia [31,32]. Accordingly, improving GJIC by use of antiarrhythmic peptides AAP10 or
ZP123 has been shown to reduce ischemia –reperfusioninduced arrhythmia [82 – 90]. However, it has also been
suggested that reduction in GJIC may limit infarct size
[129]. On the other hand, Li et al. [7] showed that the
beneficial effect of preconditioning on infarct size was
antagonized by 0.5 mM heptanol. The authors suggested
that an unknown ‘‘survival factor’’ [6,7] rather than a ‘‘death
factor’’ [5] may be passed by the gap junctions and may
play a role in the preconditioning effect.
At present, studies on pharmacological intervention
against chronic alteration of gap junction expression are
still missing. However, since in chronic heart failure, in
atrial fibrillation and in the postischemic remodeling alterations of gap junction expression have been described and
assumed to be associated with arrhythmogeneity in these
diseases due to a change in the intercellular networking, it
might be reasonable to investigate, whether pharmacological interventions such as ACE-inhibition, AT1-blockade,
TNFa-receptor blockade, ETA inhibitors or others may
inhibit the gap junctional remodeling and thus inhibit the
formation of an arrhythmogenic substrate thereby preventing arrhythmia.
Regarding the vasculature, it has been shown that EDHFresponses are attenuated if gap junctions are blocked and in
Cx40 knock-out mice hypertension develops and upstream
regulation of vasomotor tone is impaired [10] and that the
tone of cerebral arteries can be attenuated by heptanol and
18-a-glycyrrhetinic acid. Thus, it can be argued that pharmacological interventions on vascular gap junctions may
affect blood pressure. However, at present there are no
studies on this issue.
As can be seen from the studies reviewed in this paper,
pharmacological manipulation of GJIC at present appears
confusing due to the large number of drugs being reported to
activate or inhibit GJIC with sometimes conflicting results,
and due to the many different pathways involved, which
sometimes seem to be isoform-dependent. Thus, additional
pharmacological research on regulation of GJIC is required.
However, taken together, pharmacological targeting of GJIC
may open interesting new horizons for acute and chronic
interventions in cardiovascular disease.
References
[1] Bennett MVL, Zheng X, Sogin ML. The connexin family tree. In:
Kanno Y, Kataoka K, Shiba Y, Shibata Y, Shimazu T, editors.
Intercellular communication through gap junctions. Amsterdam:
Elsevier; 1995. p. 3 – 8.
[2] Bruzzone R. Learning the language of cell – cell communication
through connexin channels. Genome Biol 2001;2:4027 Reports.
295
[3] Willeckem K, Eiberger J, Degen J, et al. Structural and functional
diversity of connexin genes in the mouse and human genome. Biol
Chem 2002;383:725 – 37.
[4] Buchanan JW, Saito T, Gettes LS. The effect of antiarrhythmic
drugs, stimulation frequency and potassium induced resting membrane potential changes on conduction velocity and dV/dtmax in
guinea pig myocardium. Circ Res 1985;56:696 – 703.
[5] Kanno S, Kovacs A, Yamada KA, et al. Connexin43 as a determinant of myocardial infarct size following coronary occlusion in mice.
J Am Coll Cardiol 2003;41:681 – 6.
[6] Yasui K, Kada K, Hojo M, et al. Cell-to-cell interaction prevents cell
death in cultured neonatal rat ventricular myocytes. Cardiovasc Res
2000;48:68 – 76.
[7] Li G, Whittaker P, Yao M, et al. The gap junction uncoupler heptanol
abrogates infarct size reduction with preconditioning in mouse
hearts. Cardiovasc Pathol 2002;11:158 – 65.
[8] Christ GJ, Brink PR. Analysis of the presence and physiological
relevance of subconducting states of connxin43-derived gap junction
channels in cultured human corporalvascular smooth muscle cells.
Circ Res 1999;85:797 – 803.
[9] Dhein S. Gap junction channels in the cardiovascular system: pharmacological and physiological modulation. Trends Pharmacol Sci
1998;19:229 – 41.
[10] De Wit C, Roos F, Bolz SS, et al. Impaired conduction of vasodilation along arterioles in connexin-40 deficient mice. Circ Res 2000;
86:649 – 55.
[11] Edwards G, Feletou M, Gardener MJ, et al. Role of gap junctions in
the responses to EDHF in rat and guinea-pig small arteries. Br J
Pharmacol 1999;128:1788 – 94.
[12] Sandow SL, Tare M, Coleman HA, et al. Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor. Circ Res 2002;90:1108 – 13.
[13] Berman RS, Martin PE, Evans WH, et al. Relative contributions of
NO and gap junctional communication to endothelium-dependent
relaxations of rabbit resistance arteries vary with vessel size. Microvasc Res 2002;63:115 – 28.
[14] Zahler S, Hoffmann A, Gloe T, et al. Gap-junctional coupling between neutrophils andendothelial cells: a novel modulator of transendothelial migration. J Leukoc Biol 2003;73:118 – 26.
[15] De Mello WC. Effects of intracellular injection of calcium and
strontium on cell communication in heart. J Physiol 1975;250:
231 – 45.
[16] Noma A, Tsuboi N. Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea
pig. J Physiol (Lond) 1987;382:193 – 211.
[17] Rüdisüli A, Weingart R. Electrical properties of gap junction channels in guinea pig ventricular cell pairs revealed by exposure to
heptanol. Pflügers Arch 1989;415:12 – 21.
[18] Maurer P, Weingart R. Cell pairs isolated from guinea pig and rat
hearts: effects of [Ca+ +]i on nexal membrane resistance. Pflügers
Arch 1987;409:394 – 402.
[19] Spray DC, Burt JM. Structure-activity relations of the cardiac gap
junction channel. Am J Physiol 1990;258:C195 – 205.
[20] Reber WR, Weingart R. Ungulate cardiac Purkinje fibers: the influence of intracellular pH on the electrical cell-to-cell coupling.
J Physiol (Lond) 1982;328:87 – 104.
[21] Burt JM. Block of intercellular communication: interaction of intracellular H+ and Ca2 +. Am J Physiol 1987;253:C607 – 9.
[22] Firek L, Weingart R. Modification of gap junction conductance by
divalent cations and protons in neonatal rat heart cells. J Mol Cell
Cardiol 1995;27:1633 – 43.
[23] Hermans MMP, Kortekaas P, Jongsma HJ, et al. pH sensitivity of the
cardiac gap junction proteins, connexin 45 and 43. Pflügers Arch
1995;431:138 – 40.
[24] Liu S, Tafet S, Stoner L, et al. A structural basis for the unequal
sensitivity of the major cardiac and liver gap junctions to intracellular
acidification: the carboxy tail length. Biophys J 1993;64:1422 – 33.
296
S. Dhein / Cardiovascular Research 62 (2004) 287–298
[25] Morley GE, Taffet SM, Delmar M. Intramolecular interactions mediate pH regulation of connexin43 channels. Biophys J 1996;70:
1294 – 302.
[26] Ek JF, Delmar M, Perzova R, et al. Role of histidine 95 on pH gating
of the cardiac gapjunction protein connexin43. Circ Res 1994;74:
1058 – 64.
[27] Weingart R, Maurer P. Cell-to-cell coupling studied in isolated ventricular cell pairs. Experientia 1987;43:1091 – 4.
[28] De Mello WC. Influence of the sodium pump on intercellular communication in heart fibers: effect of intracellular injection of sodium
ion on electrical coupling. J Physiol (Lond) 1976;263:171 – 97.
[29] Weingart R. The actions of ouabain on intercellular coupling and
conduction velocity in mammalian ventricular muscle. J Physiol
(Lond) 1977;264:341 – 65.
[30] Burt JM, Massey KD, Minnich BN. Uncoupling of cardiac cells by
fatty acids: structure activity relationships. Am J Physiol 1991;260:
C439 – 48.
[31] Delmar M, Michaels DC, Johnson T, et al. Effects of increasing
intercellular resistance on transverse and longitudinal propagation
in sheep epicardial muscle. Circ Res 1987;60:780 – 5.
[32] Dhein S, Krüsemann K, Schaefer T. Effects of the gap junction uncoupler palmitoleic acid on the activation and repolarization wavefronts in isolated rabbit hearts. Br J Pharmacol 1999;128:1375 – 84.
[33] Takens-Kwak BR, Jongsma HJ, Rook MB, et al. Mechanism of
heptanol-induced uncoupling of cardiac gap junctions: a perforated
patch-clamp study. Am J Physiol 1992;262:C1531 – 8.
[34] Hirschi KK, Minnich BN, Moore LK, et al. Oleic acid differentially
affects gap junction mediated communication in heart and vascular
smooth muscle cells. Am J Physiol 1993;265:C1517 – 26.
[35] Dhein S, Hammerrath SB. Aspects of the intercellular communication in aged hearts: effects of the gap junction uncoupler palmitoleic
acid. Naunyn Schmiedebergs Arch Pharmacol 2001;364:397 – 408.
[36] Schmilinsky-Fluri G, Valiunas V, Willi M, et al. Modulation of
cardiac gap junctions: the mode of action of arachidonic acid.
J Mol Cell Cardiol 1997;29:1703 – 13.
[37] Burt JM, Spray DC. Volatile anaesthetics block intercellular communication between neonatal rat myocardial cells. Circ Res 1989;
65:829 – 37.
[38] He DS, Burt JM. Mechanism and selectivity of the effects of halothane on gap junction channel function. Circ Res 2000;86:E104 – 9.
[39] Taylor HJ, Chaytor AT, Edwards DH, et al. Gap junction-dependent
increases in smooth muscle cAMP underpin the EDHF phenomenon
in rabbit arteries. Biochem Biophys Res Comm 2001;283:583 – 9.
[40] Chaytor AT, Martin PE, Evans WH, et al. The endothelial component of cannabinoid induced-relaxation in rabbit mesenteric artery
depends on gap junctional communication. J Physiol (Lond) 1999;
520:539 – 50.
[41] Allen T, Iftinca M, Cole WC, et al. Smooth muscle membrane
potential modulates endothelium-dependent relaxation of rat basilar
artery via myo-endothelial gap junctions. J Physiol (Lond) 2002;
545:975 – 86.
[42] Takens-Kwak BR, Jongsma HJ. Cardiac gap junctions: three distinct
channel conductances and their modulation by phosphorylating
treatments. Pflügers Arch 1992;422:198 – 200.
[43] De Mello WC. Atrial natriurectic factor reduces cell coupling in the
failing heart, an effect mediated by cyclic GMP. J Cardiovasc Pharmacol 1998;32:75 – 9.
[44] De Mello WC. Influence of alpha-adrenergic-receptor activation on
junctional conductance in heart cells: interaction with beta-adrenergicagonists. J Cardiovasc Pharmacol 1997;29:273 – 7.
[45] Kwak BR, VanVeen TAB, Analbers LJS, et al. TPA increases conductance but decreases permeability in neonatal rat cardiomyocyte
gap junction channels. Exp Cell Res 1995;220:456 – 63.
[46] Bastide B, Hervé JC, Délèze J. The uncoupling effect of diacylglycerol on gap junctional communication of mammalian heart cells is
independent of protein kinase C. Exp Cell Res 1994;214:519 – 27.
[47] Rouet-Benizeb P, Mohammadi K, Pérennec J, et al. Protein kinase C
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
isoform expressionin normal and failing rabbit hearts. Circ Res
1996;79:153 – 61.
Kwak BR, Jongsma HJ. Regulation of cardiac gap junction channel
permeability and conductance by several phosphorylating conditions. Mol Cell Biochem 1996;157:93 – 9.
Münster PN, Weingart R. Effects of phorbol ester on gap junctions
of neonatal rat heart cells. Pflügers Arch Eur J Physiol 1993;423:
181 – 8.
Dhein S. Cardiac gap junctions. Basel: Karger; 1998.
Dhein S, Weng S, Schaefer T, et al. Role of protein kinase C in the
action of antiarrhythmic peptides. FASEB J 2001;15:465.1 [Suppl.].
Weng S, Lauven M, Schaefer T, et al. Pharmacological modulation
of Gap Junction coupling by an antiarrhythmic peptide via protein
kinase C activation. FASEB J 2002;16:1114 – 6.
Bowling N, Huang X, Sandusky GE, et al. Protein kinase C-a and-q
modulate connexin-43 phosphorylation in human heart. J Mol Cell
Cardiol 2001;33:789 – 98.
Saez JC, Nairn AC, Czernik AJ, et al. Phosphorylation of connexin43 and the regulation of neonatal rat cardiac myocytes gap
junctions. J Mol Cell Cardiol 1997;29:2131 – 45.
Duthe F, Dupont E, Verrecchia F, et al. Dephosphorylation agents
depress gap junctional communication between rat cardiac cells
without modifying the connxin43 phosphorylation degree. Gen
Physiol Biophys 2000;19:441 – 9.
Duthe F, Plaisance I, Sarrouilhe D, et al. Endogenous protein phosphatase 1 runs downgap junctional communication of rat ventricular
myocytes. Am J Physiol 2001;281:C1648 – 56.
Jeyaraman M, Tanguy S, Fandrich RR, et al. Ischemia-induced dephosphorylation of cardiomyocyte connexin-43 is reduced by okadaic acid and calyculin A but not fostriencin. Mol Cell Biochem
2003;242:129 – 34.
Verrecchia F, Duthe F, Duval S, et al. ATP counteracts the rundown
of gap junctional channels of rat ventricular myocytes by promoting
protein phosphorylation. J Physiol (Lond) 1999;516:447 – 59.
De Mello WC. The role of the renin angiotensin system in the
control of cell communication in the heart: effects of enalapril and
angiotensin II. J Cardiovasc Pharmacol 1992;20:643 – 51.
De Mello WC. Is an intracellular renin-angiotensin system involved
in control of cell communication in heart? J Cardiovasc Pharmacol
1994;23:640 – 6.
Dostal DE, Rothblum KN, Chermin MJ, et al. Intracellular detection
of angiotensinogen and renin: a localized renin-angiotensin system
in neonatal rat heart. Am J Physiol 1992;263:C838 – 50.
Cook JL, Zhang Z, Re RN. In vitro evidence for an intracellular site
of angiotensin action. Circ Res 2001;89:1138 – 46.
Suarez S, Ballmer-Hofer K. VEGF transiently disrupts gap junctional communication in endothelial cells. J Cell Sci 2001;114:
1229 – 35.
Popp R, Brandes RP, Ott G, et al. Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid. Circ Res 2002;90:800 – 6.
Ashton AW, Yokota R, John G, et al. Inhibition of endothelial cell
migration, intercellular communication, and vascular tube formation
by thromboxane A(2). J Biol Chem 1999;274:35562 – 70.
Brandes RP, Popp R, Ott G, et al. The extracellular regulated
kinases (ERK) 1/2 mediate cannabinoid-induced inhibition of gap
junctional communication in endothelial cells. Br J Pharmacol
2002;136:709 – 16.
Harks EGA, de Roos ADG, Peters PHJ, et al. Fenamates: a novel
class of reversible gap junction blockers. J Pharmacol Exp Ther
2001;298:1033 – 41.
Harks EGA, Gamina JP, Peters PHJ, et al. Besides affecting intracellular calcium signaling, 2-ABP reversibly blocks gap junctional
coupling in confluent monolayers, there by allowing measurement of
single-cell membrane currents in undissociated cells. FASEB J
2003;17:941 – 3.
De Mello WC. Effect of intracellular injection of cAMP on the
S. Dhein / Cardiovascular Research 62 (2004) 287–298
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
electrical coupling of mammalian cardiac cells. Biochem Biophys
Res Comm 1984;119:1001 – 7.
De Mello WC. Cyclic AMP and junctional communication viewed
through a multibiophysical approach. In: Peracchia C, editor. Biophysics of gap junction channels. Boca Raton, USA: CRC Press;
1991. p. 229 – 39.
Burt JM, Spray DC. Inotropic agents modulate gap junctional conductance between cardiac myocytes. Am J Physiol 1988;254:
H1206 – 10.
Van Rijen HV, van Veen TA, Hermans MM, et al. Human connexin40 gap junction channels are modulated by cAMP. Cardiovasc
Res 2000;45:941 – 51.
De Mello WC. Impaired regulation of cell communication by betaadrenergic receptor activation in the failing heart. Hypertension
1996;27:265 – 8.
Aonuma S, Kohama Y, Akai K, et al. Studies on heart XIX. Isolation
of an atrial peptide that improves the rhythmicity of cultured myocardial cell clusters. Chem Pharm Bull 1980;28:3332 – 9.
Aonuma S, Kohama Y, Makino T, et al. Studies on heart XXI.
Amino acid sequence of antiarrhythmic peptide (AAP) isolated from
atria. J Pharmacobio-Dyn 1982;5:40 – 8.
Dhein S, Tudyka T. The therapeutic potential of antiarrhythmic peptides. Cellular coupling as a new antiarrhythmic target. Drugs 1995;
49:851 – 5.
Dhein S. Peptides acting at gap junctions. Peptides 2002;23:1701 – 9.
Kohama Y, Kawahara Y, Okabe M, et al. Determination of immunoreactive antiarrhythmic peptide (AAP) in rats. J Pharmacobio-Dyn
1985;8:1024 – 31.
Kohama Y, Okimoto N, Mimura T, et al. A new antiarrhythmic
peptide, N-3-(4-hydroxyphenyl)propionyl-Pro-Hyp-Gly-Ala-Gly.
Chem Pharm Bull 1987;35:3928 – 30.
Aonuma S, Kohama Y, Makino T, et al. Studies on heart XXII. In:
Yakugaku Z, editor. Inhibitory effect of anatrial peptide on several
drug induced arrhythmias in vivo, vol. 103; 1983. p. 662 – 6.
Argentieri T, Cantor E, Wiggins JR. Antiarrhythmic peptide has no
direct cardiacactions. Experientia 1989;45:737 – 8.
Dhein S, Manicone N, Müller A, et al. A new synthetic antiarrhythmic peptide reduces dispersion of epicardial activation recovery interval and diminishes alterations of epicardial activation patterns
induced by regional ischemia. Naunyn Schmiedebergs Arch Pharmacol 1994;350:174 – 84.
Müller A, Gottwald M, Tudyka T, et al. Increase in gap junction
conductance by an antiarrhythmic peptide. Eur J Pharmacol 1997;
327:65 – 72.
Müller A, Schaefer T, Linke W, et al. Actions of the antiarrhythmic
peptide AAP10 oncellular coupling. Naunyn Schmiedebergs Arch
Pharmacol 1997;356:76 – 82.
Dhein S, Weng S, Polontchouk L, et al. Pharmacological modification of gap junctional coupling by antiarrhythmic peptides. Role of
PKC. Naunyn Schmiedebergs Arch Pharmacol 2001;363:R99
[Suppl.].
Grover R, Dhein S. Spatial structure determination of antiarrhythmic
peptide using nuclear magnetic resonance spectroscopy. Peptides
1998;19:1725 – 9.
Grover R, Dhein S. Structure-activity relationships of novel peptides
related to the antiarrhythmic peptide AAP10 which reduce the dispersion of epicardial action potential duration. Peptides 2001;22:
1011 – 21.
Kjolbye AL, Knudsen CB, Jepsen T, et al. Pharmacological characterization of the new stable antiarrhythmic peptide analog Ac-D-TyrD-Pro-D-Hyp-Gly-D-Ala-Gly-NH2 (ZP123): in vivo and in vitro
studies. J Pharmacol Exp Ther 2003;306:1191 – 9.
Dhein S, Larsen BD, Petersen JS. Effects of the new antiarrhythmic
peptide ZP123 one picardial activation and repolarisation. Naunyn
Schmiedebergs Arch Pharmacol 2003;367:R94 [Suppl.].
Xing D, Kjolbye AL, Nielsen MS, et al. ZP123 increases gap junctional conductance and prevents reentrant ventricular tachycardia
[91]
[92]
[93]
[94]
[95]
[96]
[97]
[98]
[99]
[100]
[101]
[102]
[103]
[104]
[105]
[106]
[107]
[108]
[109]
[110]
[111]
[112]
297
during myocardial ischemia in open chest dogs. J Cardiovasc Electrophysiol 2003;14:510 – 20.
De Mello WC, Thormählen D. Effect of tedisamil on cell communication, impulse propagation, and excitability of the failing heart.
Eur J Pharmacol 1999;372:241 – 6.
Zhang YW, Morita I, Yao XS, et al. Pretreatment with eicosapentaenoic acid prevented hypoxia/reoxygenation-induced abnormality in
endothelial gap junctional intercellular communication through
inhibiting the tyrosine kinase activity. Prostaglandins Leukot Essent
Fatty Acids 1999;61:33 – 40.
Emerson GG, Segal GG. Endothelial pathway for conduction of
hyperpolarization and vasodilatation along hamster feed artery. Circ
Res 2000;86:94 – 100.
De Wit C, Esser N, Lehr H-A, et al. Pentobarbital-sensitive EDHF
comediates ACh induced arteriolar dilatation in the hamster microcirculation. Am J Physiol 1999;276:H1527 – 34.
Moore LK, Burt JM. Gap junction function in vascular smooth
muscle: influence of serotonin. Am J Physiol 1995;269:H1481 – 9.
Fallon RF, Goodenough DA. Five hour half-life of mouse liver gap
junction protein. J Cell Biol 1981;127:343 – 55.
Laird DW. The life cycle of a connexin: gap junction formation
removal and degradation. J Bioenerg Biomembr 1996;28:311 – 7.
Laird DW, Puranam KL, Revel JP. Turnover and phosphorylation
dynamics of connexin43 gap junction protein in cultured cardiac
myocytes. Biochem J 1991;273:67 – 72.
Beardslee MA, Laing JG, Beyer EC, et al. Rapid turnover of connexin43 in the adult rat heart. Circ Res 1998;83:629 – 35.
Darrow BJ, Laing JG, Lampe PD, et al. Expression of multiple
connexins in cultured neonatal rat ventricular myocytes. Circ Res
1995;76:381 – 7.
Moore LK, Burt JM. Selective block of gap junction channel
expression with connexin specific antisense oligonucleotides. Am
J Physiol 1994;267:C1371 – 80.
Darrow BJ, Fast VG, Kléber AG, et al. Functional and structural
assessment of intercellular communication. Increased conduction
velocity and enhanced connexin expression in dibutyryl cAMP-treated cultured cardiac myocytes. Circ Res 1996;79:174 – 83.
Salameh A, Polontchouk L, Hagendorff A, et al. Protein kinase A
and C regulate synthesis of the gap junction protein connexin43.
FASEB J 2001;15:711.9 [Suppl.].
Polontchouk L, Ebelt B, Jackels M, et al. Chronic effects of endothelin-1 and angiotensin II on gap junctions and intercellular communication in cardiac cells. FASEB J 2002;16:87 – 9.
Dodge SM, Beardslee M, Darrow BJ, et al. Effects of angiotensin II on
expression of the gap junction channel protein connexin 43 in neonatal rat ventricular myocytes. J Am Coll Cardiol 1998;32:800 – 7.
Wang TL, Tseng YZ, Chang H. Regulation of connexin43 gene
expression by cyclical mechanical stretch in neonatal rat cardiomyocytes. Biochem Biophys Res Commun 2000;267:551 – 7.
Shyu KG, Chen CC, Wang BW, et al. Angiotensin II receptor antagonist blocks the expression of connexin43 induced by cyclical
mechanical stretch in cultured neonatal rat cardiac myocytes. J Mol
Cell Cardiol 2001;33:691 – 8.
Pimentel RC, Yamada KA, Kleber AG, et al. Autocrine regulation of
myocyte Cx43 expression by VEGF. Res Circ 2002;90:671 – 7.
Doble BW, Kardami E. Basic fibroblast growth factor stimulates
connexin-43 expression and intercellular communication of cardiac
fibroblasts. Mol Cell Biochem 1995;143:81 – 7.
Doble BW, Ping P, Kardami E. The e subtype of protein kinase C is
required for cardiomyocyte connexin-43 phosphorylation. Circ Res
2000;86:293 – 301.
Lerner DL, Chapman Q, Green KG, et al. Reversible down-regulation of connexin43 expression in acute cardiac allograft rejection.
J Heart Lung Transplant 2001;20:93 – 7.
Fernandez-Cobo M, Gingalewski C, Drujan D, et al. Downregulation of connexin43 gene expression in rat heart during inflammation.
The role of tumor necrosis factor. Cytokine 1999;11:216 – 24.
298
S. Dhein / Cardiovascular Research 62 (2004) 287–298
[113] Fernandez-Cobo M, Gingalewski C, Drujan D, et al. Expression of
the connexin 43 gene is increased in the kidneys and lungs of rats
injected with bacterial lipopolysaccharide. Shock 1998;10:97 – 102.
[114] Salameh A, Polontchouk L, Dhein S, et al. Chronic regulation of
the expression of the gap junction protein connexin 43 in transfected HeLa cells. Naunyn Schmiedebergs Arch Pharmacol 2003;
368:33 – 40.
[115] Salameh A, Polonchouk L, Hagendorff A, et al. On the chronic
regulation of gap junction protein connexin43 (Cx43) expression.
Br J Pharmacol 2001;133:215 [Suppl.].
[116] Van Rijen HV, Van Kempen MV, Postma S, et al. Tumor necrosis
factor alpha alters the expression of connexin43, connexin40 and
connexin37 in human umbilical vein endothelial cells. Cytokine
1998;10:258 – 64.
[117] Liu M-Y, Hattori Y, Sato A, et al. Ovariectomy attenuates hyperpolarization and relaxation mediated by endothelium-derived hyperpolarizing factor in female rat mesenteric artery: a concomitant
decrease in connexin-43 expression. J Cardiovasc Pharmacol 2002;
40:938 – 48.
[118] Lauf U, Giepmans BN, Lopez P, et al. Dynamic trafficking and
delivery of connexons to the plasma membrane and accretion to
gap junctions in living cells. Proc Natl Acad Sci U S A 2002;99:
10446 – 51.
[119] Puranam KL, Laird DW, Revel JP. Trapping an intermediate form of
connxin43 in the Golgi. Exp Cell Res 1993;206:85 – 92.
[120] Musil LS, Goodenough DA. Biochemical analysis of connexon
assembly. In: Kanno Y, Kataoka K, Shiba Y, Shibata Y, Shimazu
[121]
[122]
[123]
[124]
[125]
[126]
[127]
[128]
[129]
T, editors. Intercellular communication through gap junctions. Progress in cell research, vol. 4. Amsterdam: Elesvier; 1995. p. 327 – 30.
Dahl G, Nonner W, Werner R. Attempts to define functional
domains of gap junctions with synthetic peptides. Biophys J
1994;67:1816 – 22.
Warner A, Clements DK, Parikh S, et al. Specific motifs in the
external loops of connexin proteins can determine gap junction formation between chick heart myocytes. J Physiol 1995;488:721 – 8.
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 1997;503:99 – 110.
Braet K, Vandamme W, Martin PE, et al. Photoliberating inositol1,4,5-trisphosphate triggers ATP release that is blocked by the connexin mimetic peptide gap. Cell Calcium 2003;33:37 – 48.
Kwak BR, Jongsma HJ. Selective inhibition of gap junction channel
activity by synthetic peptides. J Physiol (Lond) 1999;516:679 – 85.
Beyer EC, Berthoud VM. Gap junction synthesis and degradation as
therapeutic targets. Current Drug Targets 2002;3:409 – 16.
Laing JG, Tadros PN, Green K, et al. Proteolysis of connexin43containing gap junctions in normal and heat-stressed cardiac myocytes. Cardiovasc Res 1998;38:711 – 8.
Lee DH, Goldberg AL. Proteasome inhibitors: valuable new tools
for cell biologists. Trends Cell Biol 1998;8:397 – 403.
Garcia-Dorado D, Inserte J, Ruiz-Meana M, et al. Gap junction
uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion. Circulation 1997;96:3579 – 86.