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Cardiovascular Research 2004 62(2):287-298; doi:10.1016/j.cardiores.2004.01.019
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

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

* Tel.: +49-341-1044; fax: +49-341-1452. Email address: dhes{at}medizin.uni-leipzig.de

Received 7 October 2003; revised 15 January 2004; accepted 16 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Conclusions
 References
 
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.

KEYWORDS Gap junctions; Connexin; Pharmacology; Cardiovascular; Regulation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Conclusions
 References
 
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 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 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.


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  		Table 1 Survey on agents acutely uncoupling cardiovascular gap junctions

 
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 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 µmol/l ouabain were shown to decrease conduction velocity [29]. Similarly, De Mello [28] observed an uncoupling effect of 0.68 µmol/l ouabain in Purkinjefibres. Strophanthidin (2–20 µM) 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 µM [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 µM [32,35]. The {omega}-6 unsaturated fatty acid arachidonic acid also uncouple cells with a KD of 4 µmol/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 neonatal 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-{alpha}-glycyrrhetinic acid, 18-β-glycyrrhetinic acid and carbenoxolone have been shown to uncouple gap junction channels in various models. Thus, several authors have used 18-{alpha}-glycyrrhetinic acid as a gap junction inhibitor [39] in concentrations of about 50 µM [40] or 18-β-glycyrrhetinic acid in a concentration of 5 µM [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 µM) 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 µM 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-{alpha}-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 β-adrenoceptors. In several tissues {alpha}-adrenergic stimulation leads to uncoupling. Similarly, {alpha}-adrenergic stimulation with phenylephrine in adult rat ventricular cardiomyocytes decreases gap junctional coupling in a PKC-dependent manner [44]. Since {alpha}-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 µM) [46]. On the other hand, uncoupling effects of PKC activators have also been described (see below). Incardiac tissue several isoforms of PKC are expressed including PKC{alpha}, PKCβ, PKC{varepsilon}, PKC{xi} and PKC{gamma} (rabbit heart) [47]. However, only PKC{gamma} was found to be located close to the intercalated disks in this study. TPA treatment is assumed to result in a rapid translocation of PKC{alpha} and PKC{varepsilon} 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 PKC-activation 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 PKC{varepsilon} by fibro blast growth factor FGF-2 leads to uncoupling of cardiomyocytes (see below) while activation of PKC{alpha} 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 PKA{alpha}/PKC{varepsilon} 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 PKC{alpha} and PKC{varepsilon} have been shown to phosphorylate Cx43, however, only PKC{varepsilon} 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 PKC-dependent phosphorylation of Cx43 enhances intercellular coupling (see next section).

Taken together, the present results regarding PKC are conflicting and future detailed studies are needed which-among 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 cacid [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-2-acetylsn-glycerol (OAG) (25–100 µg/ml) has been evaluated in neonatal rat cardiomyocytes. While 25 µg/ml OAG led to partial uncoupling, more or less complete uncoupling was observed with 100 µg/ml with regard to dye transfer and junctional current [46]. Inhibition of PKC by H7 (100 µg/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 µg/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 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 µM) or the synthetic HU210 (10 µM) 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 µM [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 µM [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.


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  		Table 2 Agents reported to acutely enhance gap junctional intercellular communication in the cardiovascular system (PDE=phosphodiesterase; TPA=12-O-tetradecanoylphorbol 13-acetate; HPP-5=N-3-(4-hydroxyphenyl)propionyl-Pro-Hyp-Gly-Ala-Gly)

 
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 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-Ala-Gly-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. A derivative of AAPnat, N-3-(4-hydroxyphenyl)propionyl-Pro-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-Pro-Tyr-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-βS and to PKC inhibitors as well as to a PKC{alpha}-specific inhibitor (CGP54345), it was concluded that AAP10 acts via a G-protein which down stream activates protein kinase C{alpha} 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-D-4Hyp-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 µM) 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 {omega}-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 µM 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/PGI2-independent 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 µM 11,12-EET had a biphasic effect on GJIC in human umbilical veinendothelial cells. 11,12-EET 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 µM 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 PKA-dependent 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 µM 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.


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  		Table 3 Agents affecting expression, synthesis, assembly, docking and degradation of gap junctions (AC=adenylycyclase; ALLN (acetyl-leucyl-leucyl-norleucinal)

 
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 5'-GTCACCCATGTCTGGGCA-3' as Cx43 antisense and 5'-GTCACCCATCTTGCCAAG-3' 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, 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, TNF{alpha}, b-FGF, VEGF). Thus, in neonatal ratcardiomyocytes 24 h endothelin-1 exposure resulted in an increase of Cx43 expression (EC50: 158±41 nM) and phosphorylation (EC50: 13±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±10 nM) and phosphorylation (EC50: 93±8 nM), while–as 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].

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 stretch-induced 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 non-stretched cells, which was also blocked by VEGF antibody [108]. Since a TGF-β antibody was also effective the authors concluded that stretch might induce the increase in Cx43 expression via a TGF-β/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 junctional coupling in a PKC{varepsilon}-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 {alpha} (TNF{alpha}) should be considered. With regard to the TNF{alpha}-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 TNF{alpha} (10 U/ml). This induction was transduced via p38 MAP-kinase. Similarly, in cultured neonatal rat cardiomyocytes exposure to low concentrations of TNF{alpha} also increased Cx43 expression via p38 MAP-kinase [115]. On the other hand, in endothelial cells, 0.5 nM TNF{alpha} 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 TNF{alpha}-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 17β-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 µM) in A7r5smooth muscle cells to inhibit coupling via Cx43 channels and a second peptide, analogue to the second extracellular loop of Cx40, P177-192 (FLDTLHVCRRSPCPHP; 50 µM) 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 µM 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
Top
 Abstract
 1. Introduction
 2. Conclusions
References
 
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 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–reperfusion-induced 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, TNF{alpha}-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 EDHF-responses 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-{alpha}-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.


   Notes
 
Time for primary review 26 days


   References
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 1. Introduction
 2. Conclusions
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