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Cardiovascular Research 2003 57(4):942-952; doi:10.1016/S0008-6363(02)00782-4
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Copyright © 2003, European Society of Cardiology

Regulatory effects of G protein-coupled receptors on cardiac sarcolemmal Na+/H+ exchanger activity: signalling and significance

Metin Avkiran* and Robert S Haworth

Centre for Cardiovascular Biology and Medicine, King's College London, The Rayne Institute, St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK

* Corresponding author. Tel.: +44-20-7928-9292x3375; fax: +44-20-7928-0658. metin.avkiran{at}kcl.ac.uk

Received 14 October 2002; accepted 13 November 2002


   Abstract
Top
 Abstract
1. Introduction
 2. GPCR-mediated stimulation of...
 3. GPCR-mediated inhibition of...
 4. Distal signalling mechanisms
 5. Functional significance
 6. Conclusion
 References
 
In cardiac myocytes, sarcolemmal Na+/H+ exchanger (NHE) activity is subject to regulation by a variety of G protein-coupled receptor (GPCR) systems. This regulation usually manifests as an increase in NHE activity (e.g. in response to the stimulation of {alpha}1-adrenergic, angiotensin AT1, endothelin and thrombin receptors), although some GPCR systems have been shown to inhibit sarcolemmal NHE activity (e.g. β1-adrenergic receptors) or to attenuate its stimulation by other ligands (e.g. adenosine A1 and angiotensin AT2 receptors). The pertinent molecular signalling mechanisms are only now beginning to be unravelled, with the extracellular signal regulated kinase/ribosomal S6 kinase pathway and the protein kinase C pathway both appearing to play critical roles in the stimulation of sarcolemmal NHE activity. GPCR-mediated regulation of sarcolemmal NHE activity is likely to play significant roles in modulating myocardial function in both physiological and pathophysiological conditions. These roles include the regulation of (1) myocardial pHi and contractility, (2) myocardial susceptibility to injury and dysfunction during ischaemia and reperfusion, and (3) myocardial hypertrophy in response to neurohormonal and mechanical stimuli. Greater understanding of the pertinent molecular signalling mechanisms distal to GPCR stimulation may reveal novel targets for therapeutic manipulation.

KEYWORDS Contractile function; G-proteins; Hypertrophy; Ischemia; Myocytes; Na/H-exchanger; Receptors; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. GPCR-mediated stimulation of...
 3. GPCR-mediated inhibition of...
 4. Distal signalling mechanisms
 5. Functional significance
 6. Conclusion
 References
 
In myocardium, as in other tissues, intracellular pH (pHi) affects numerous cellular processes and therefore has to be tightly regulated. Under physiological conditions, this regulation is achieved principally by the integrated action of four different sarcolemmal ion transporters (two ‘acid extruders’ and two ‘acid loaders’) that operate at low basal activity [1]. As pHi drops, however, the Na+/H+ exchanger (NHE) and the Na+/HCO3– cotransporter (NBC) become progressively more active and extrude acid from the cell. Conversely, as pHi increases, the Cl–/HCO3– exchanger (also known as the anion-exchanger (AE)) and the Cl–/OH– exchanger (CHE) become activated and effectively import acid. In studies designed to investigate the regulation of NHE activity, experiments are often performed in the absence of HCO3– in the medium, thereby rendering NBC inactive and making NHE the sole acid extrusion pathway.

In cardiac myocytes, the steep increase in sarcolemmal NHE activity that occurs in response to lowering pHi (i.e. increasing [H+]i) exhibits a Hill coefficient of around three [2]. This indicates that more than one H+ ion binds to the NHE protein during the transport cycle, and has led to the suggestion that the NHE protein contains a non-transporting H+-binding site, which regulates NHE activity. This is consistent with previous observations in other cells that the activity of NHE isoform 1 (NHE1), which is the molecular homologue of the cardiac sarcolemmal NHE [3], is regulated through the interaction of intracellular H+ with an allosteric modifier site (sometimes referred to as the ‘proton sensor’) within the transmembrane domain of the exchanger [4,5]. Thus, as pHi falls, the proton sensor site becomes increasingly occupied and, through an allosteric regulatory mechanism, leads to a greater increase in NHE activity than would be expected from a simple increase in the availability of transportable H+ ions.

In cardiac myocytes, the NHE1 protein appears to be localised predominantly to the intercalated disc region and transverse-tubules [6], although the functional significance of this distribution is not clear. The NHE1 protein is known to consist of a 500 amino acid transport domain that is located in the membrane and contains 12 transmembrane-spanning segments, and a 300 amino acid regulatory domain that extends into the cytoplasm [7]. The proton sensor site is within the transport domain and remains functional in the absence of the regulatory domain; nevertheless, the pHi-sensitivity of the proton sensor (and therefore the maintenance of pHi in the normal range) depends upon its interaction with the regulatory domain [8]. This interaction may be modified in response to a variety of extracellular stimuli, such as growth factors and neurohormonal mediators, which most commonly increase the H+ affinity of the proton sensor and thereby increase NHE activity at any given pHi. Such dual regulation of NHE activity, primarily by pHi and secondarily by extracellular stimuli (through altered pHi sensitivity), is illustrated schematically in Fig. 1.


Figure 1
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  		Fig. 1 Schematic illustration of the dual regulation sarcolemmal NHE activity, as reflected by the rate of H+ efflux (JH) in HCO3–-free medium, at various values of intracellular pH (pHi). Sarcolemmal NHE activity changes (1) in response to a change in pHi, with intracellular acidosis stimulating NHE activity, or (2) through a change in the pHi sensitivity of the exchanger, as commonly occurs in response to stimulation of Gq protein-coupled receptors. The shift in the JH-versus-pHi relationship illustrated in (2) is based on the effect of the {alpha}1A-adrenoceptor selective agonist A61603 on sarcolemmal NHE activity in adult rat ventricular myocytes, as reported by Snabaitis et al. [47].

 
In recent years, considerable evidence has accumulated that, in cardiac myocytes, sarcolemmal NHE activity is subject to exquisite regulation by a variety of extracellular stimuli, most of which act through G protein-coupled receptors (GPCRs). Intriguingly, although the majority of the GPCR systems that have been studied to date have been shown to stimulate sarcolemmal NHE activity, there is also evidence that some may inhibit NHE activity or its stimulation through other pathways.


   2. GPCR-mediated stimulation of sarcolemmal NHE activity
Top
 Abstract
 1. Introduction
 2. GPCR-mediated stimulation of...
3. GPCR-mediated inhibition of...
 4. Distal signalling mechanisms
 5. Functional significance
 6. Conclusion
 References
 
Available evidence suggests that GPCR systems that are linked to heterotrimeric G proteins of the Gq subtype (GqPCRs) mediate an increase in sarcolemmal NHE activity, through a change in the pHi sensitivity of the exchanger. These GqPCRs include receptors for a variety of important endogenous mediators, such as catecholamines, angiotensin II, endothelin and thrombin, which can have a significant impact on myocardial function in both physiological and pathophysiological conditions.

2.1 {alpha}1-Adrenergic receptors
The first evidence that {alpha}1-adrenergic receptors ({alpha}1-ARs) stimulate sarcolemmal NHE activity was obtained in isolated rat ventricular myocytes, in which noradrenaline was shown to produce an increase in resting pHi in a manner that was inhibited by the {alpha}1-AR antagonist prazosin or by the NHE inhibitor hexamethylamiloride [9]. Similar data were reported subsequently with {alpha}1-AR-selective agonists, such as phenylephrine [10] and 6-fluoronorepinephrine [11]. Wallert and Fröhlich [11] additionally studied the effect of {alpha}1-AR stimulation on the rate of H+ efflux (JH) via NHE, following intracellular acidification. This revealed a significant shift in the JH-versus-pHi relationship (similar to that depicted in Fig. 1), in response to {alpha}1-AR stimulation [11]. Such an effect of {alpha}1-AR stimulation has been reported also in guinea-pig ventricular myocytes [12]. These studies provide strong evidence that {alpha}1-AR stimulation leads to increased sarcolemmal NHE activity, through an altered pHi sensitivity of the exchanger. Our recent work with a range of {alpha}1-AR antagonists and agonists that have distinct selectivities for {alpha}1A-, {alpha}1B- and {alpha}1D-ARs ({alpha}1-AR subtypes which have been identified pharmacologically and by molecular cloning) has indicated that the stimulation of sarcolemmal NHE activity is mediated by the {alpha}1A-AR subtype [13]. This observation may have clinical significance, since the {alpha}1A-AR is believed to be the predominant {alpha}1-AR subtype expressed in human myocardium [14].

2.2 Angiotensin AT1 receptors
A possible signalling role for increased sarcolemmal NHE activity in mediating the myocardial actions of angiotensin II was first suggested on the basis of the negative effect of NHE inhibition on the angiotensin II-induced increase in open-state probability of the L-type Ca2+ channel, in rabbit ventricular myocytes [15]. In subsequent work, again in rabbit ventricular myocytes, angiotensin II was shown to increase resting pHi through a mechanism dependent on NHE activity, and to produce a small but significant shift in the pHi sensitivity of the exchanger [16]. Grace et al. [17] showed that, in the perfused ferret heart, angiotensin II increased H+ efflux via the sarcolemmal NHE following an intracellular acid load, and that this effect was inhibited by a selective antagonist of the angiotensin type 1 (AT1) receptor. However, in adult rat ventricular myocytes, angiotensin II produced only a small (0.02 pH unit) increase in resting pHi, an effect that was absent in hypertrophied cells [18]. More recently, we have reported that angiotensin II, when used alone, had no significant effect on sarcolemmal NHE activity in adult rat ventricular myocytes [19]. To the contrary, when given together with PD123319, a selective antagonist of the angiotensin type 2 (AT2) receptor, angiotensin II was able to stimulate sarcolemmal NHE activity in a concentration-dependent manner [19]. This effect of angiotensin II was abolished in the presence of the AT1 antagonist losartan, suggesting that activation of the AT1 receptor by angiotensin II stimulates sarcolemmal NHE activity, but that simultaneous activation of the AT2 receptor counteracts this stimulation (see below) [19]. Thus, it appears that angiotensin II is capable of stimulating sarcolemmal NHE activity through the AT1 receptor, although the magnitude of the response may be modulated by altered availability of angiotensin receptor subtypes, for example as a consequence of disease, pharmacological manipulation or species difference.

2.3 Endothelin receptors
Krämer et al. have shown that endothelin increases resting pHi in adult rat ventricular myocytes and that this effect is blocked by NHE inhibition [20]. In subsequent work, endothelin has been shown to stimulate H+ efflux via the sarcolemmal NHE, following an intracellular acid load, in both canine cardiac Purkinje fibres [21] and rat ventricular myocytes [18]. There is no direct evidence available on the identity of the receptor subtype that mediates the stimulation of sarcolemmal NHE activity by endothelin. Nevertheless, the ETA receptor is known to be the predominant endothelin receptor subtype expressed in rat myocardium [22] and is therefore likely to be responsible for endothelin-induced stimulation of sarcolemmal NHE activity, at least in this species. Consistent with this mechanism, in isolated rat hearts, pharmacological NHE inhibition and a selective antagonist of the ETA receptor have been shown to similarly attenuate the deleterious effects of endothelin during ischemia and reperfusion [23].

2.4 Thrombin receptors
In addition to its well-established role in blood coagulation and thrombus formation, thrombin induces a variety of cellular responses through receptor-mediated pathways [24]. With respect to the cardiac myocyte, both thrombin and the synthetic thrombin receptor activating peptide SFLLRN have been shown to increase sarcolemmal NHE activity [25]. The common ability of thrombin and SFLLRN to stimulate sarcolemmal NHE activity is consistent with this action being mediated by the thrombin receptor that was first cloned by Vu et al. [26], and is now known as protease-activated receptor 1 (PAR1). Indeed, the mRNA for PAR1 is expressed in adult rat ventricular myocytes [25]. Nevertheless, targeted disruption of the PAR1 gene in mice has revealed that additional thrombin receptors exist [27] and a second thrombin receptor, PAR3, has been cloned and shown to be expressed in human myocardium [28]. Furthermore, PAR2, a trypsin-activated receptor that is activated also by SFLLRN, has been shown to be present in neonatal rat ventricular myocytes [29]. Therefore, at present, there is some uncertainty regarding the identity of the receptor(s) that mediate thrombin-induced stimulation of sarcolemmal NHE activity.

2.5 Muscarinic receptors
There is very little information available on the regulation of sarcolemmal NHE activity by cholinergic stimuli. In canine Purkinje fibres, the application of carbachol, an esterase-resistant cholinergic agonist, has been shown to increase both resting pHi and the rate at which pHi recovered following an intracellular acid load [21]. These effects were inhibited by both amiloride and atropine, suggesting that carbachol stimulates sarcolemmal NHE activity via muscarinic receptors. The identity of the muscarinic receptor subtype(s) that regulate sarcolemmal NHE activity remains unknown. Based on the observation that, in many mammalian species, the predominant muscarinic receptor in the heart is the M2 receptor [14], it is likely that cholinergic stimulation of sarcolemmal NHE activity is mediated through this receptor.

2.6 Orphan receptors
Although over 300 GPCRs have been identified through genomic research, the endogenous ligands for over 100 of these receptors are not yet known [30]. It is likely that several of these ‘orphan’ receptors are expressed in the cardiac myocyte, and that some of these may regulate sarcolemmal NHE activity and thereby cardiac function. Indeed, apelin, the endogenous ligand for the recently ‘de-orphanised’ APJ receptor, has been shown to have a positive inotropic effect in the adult rat heart [31]. The positive inotropic effect of apelin could be partially blocked by pretreatment of hearts with the NHE inhibitors methylisobutylamiloride and zoniporide [31], suggesting that APJ receptors stimulate sarcolemmal NHE activity. Nevertheless, such a role for APJ receptors remains to be confirmed by direct measurements of NHE activity.


   3. GPCR-mediated inhibition of sarcolemmal NHE activity
Top
 Abstract
 1. Introduction
 2. GPCR-mediated stimulation of...
 3. GPCR-mediated inhibition of...
4. Distal signalling mechanisms
 5. Functional significance
 6. Conclusion
 References
 
In contrast to the evidence that various GqPCRs (e.g. {alpha}1A-ARs and angiotensin AT1, endothelin—most likely ETA—and thrombin—most likely PAR1—receptors) mediate an increase in sarcolemmal NHE activity (see above), GPCRs that signal through other G protein families (Gs and Gi) may attenuate NHE activity or its stimulation.

3.1 β1-Adrenergic receptors
The available evidence indicates that, in contrast to the stimulatory effect of {alpha}1-AR stimulation, β1-AR stimulation inhibits sarcolemmal NHE activity, at least in some species. Thus, in sheep Purkinje fibres, isoprenaline has been shown to produce a fall in resting pHi (i.e. relative intracellular acidosis), in HCO3–-free solution [32]. Furthermore, in guinea-pig ventricular myocytes, isoprenaline has been shown to slow pHi recovery after an intracellular acid load, again in HCO3–-free solution [12]. In dog Purkinje fibres, isoprenaline had no effect on resting pHi, but pHi recovery from an intracellular acid load was again slowed, indicating reduced NHE activity [21]. In the same study, pretreatment with atenolol was shown to abolish the negative effect of isoprenaline on pHi recovery, confirming that the effect was mediated by the β1-AR [21]. In view of these opposing effects of the principal myocardial post-synaptic adrenergic receptors (i.e. {alpha}1- and β1-ARs), the net effect of the endogenous catecholamines noradrenaline and adrenaline on sarcolemmal NHE activity is likely to be modulated by the relative density or functionality of these receptors. This in turn may result in age- and species-dependent differences in the response of sarcolemmal NHE activity to catecholamines, as well as altered responses in disease. For example, in heart failure, reduced β1-AR responsiveness [33] coupled with enhanced {alpha}1-AR signalling [34] may favour stimulation of sarcolemmal NHE activity by catecholamines, although direct evidence for this is currently lacking.

3.2 Angiotensin AT2 receptors
As noted above, in rat ventricular myocytes, the NHE-stimulatory effect of angiotensin II is revealed only when AT2 receptors are blocked, suggesting that this receptor subtype may play a counter-regulatory role [19]. Nevertheless, unlike β1-AR stimulation, AT2 receptor stimulation (by angiotensin II plus the AT1 antagonist losartan, or by the AT2-selective agonist CGP42112A) does not produce a significant inhibition of NHE activity [19]. Therefore, it appears that AT2 receptor activation may set in train intracellular processes that inhibit NHE-stimulatory signalling, rather than inhibit NHE activity directly [19].

3.3 Adenosine A1 receptors
Somewhat analogous to the counter-regulatory effects of AT2 versus AT1 receptors, we have recently shown that activation of the adenosine A1 receptor by the selective agonist GR79236 inhibits {alpha}1-AR-mediated stimulation of sarcolemmal NHE activity [35]. Once again, A1 receptor activation alone was not sufficient to significantly inhibit basal NHE activity, suggesting that attenuation of the {alpha}1-AR-mediated response was unlikely to arise from direct NHE inhibition. The counter-regulatory effect of GR79236 could be prevented by pretreatment of myocytes with either the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine or pertussis toxin, the latter indicating a Gi protein-mediated mechanism. Intriguingly, the NHE-stimulatory effect of thrombin was also inhibited by adenosine A1 receptor activation [35], suggesting a broader counter-regulatory cross-talk between the Gi protein-coupled adenosine A1 receptor and multiple GqPCRs.


   4. Distal signalling mechanisms
Top
 Abstract
 1. Introduction
 2. GPCR-mediated stimulation of...
 3. GPCR-mediated inhibition of...
 4. Distal signalling mechanisms
5. Functional significance
 6. Conclusion
 References
 
The intracellular regulatory domain of the NHE1 protein contains a number of potential phosphorylation sites [8]. Indeed, in non-myocyte cells, activation of several kinase pathways has been shown to produce an increase in NHE1 phosphorylation concomitantly with an increase in NHE activity [36,37]. Phosphorylation-mediated stimulation of NHE activity most likely arises from a conformational change that alters the interaction of the regulatory domain with the transport domain, resulting in increased H+ affinity of the proton sensor [4]. The list of putative NHE1 kinases thus far identified includes Ca2+/calmodulin-dependent kinase II (CaMKII) [38], extracellular signal-regulated kinase (ERK) [39], the 160 kDa Rho-associated kinase (p160 ROCK) [40], the 90 kDa ribosomal S6 kinase (p90RSK) [41], p38 mitogen-activated protein kinase (p38 MAPK) [42], and Nck-interacting kinase (NIK) [43]. There is emerging evidence that at least some of these NHE1 kinases play an important role in GPCR-mediated regulation of sarcolemmal NHE activity in cardiac myocytes (Fig. 2).


Figure 2
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  		Fig. 2 Schematic summary of the molecular signalling pathways that regulate sarcolemmal NHE activity, in a positive (+) or negative (–) manner, in response to GPCR stimulation. Although there is good evidence that the NHE-stimulatory effects of some GPCRs (e.g. {alpha}1-adrenergic receptors) are mediated through the activation of the ERK/p90RSK and PKC pathways, much less is known about the signalling mechanisms employed by GPCRs that either inhibit NHE activity (e.g. β1-adrenergic receptors) or inhibit its stimulation by other GPCRs (e.g. adenosine A1 receptors). See text for details. AC, adenylate cyclase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; PLC, phospholipase C; RSK, p90 ribosomal S6 kinase (p90RSK).

 
Moor and Fliegel [44] have shown that, following exposure to endothelin, extracts from adult rat hearts and cultured neonatal rat ventricular myocytes show an enhanced ability to phosphorylate in vitro a recombinant fusion protein that contains the regulatory domain of NHE1. In this study [44], two of the relevant NHE1 kinases were identified as ERK and p90RSK (which is itself regulated by ERK-mediated phosphorylation [45]) and pharmacological inhibition of the ERK pathway was shown to abolish an endothelin-induced increase in resting pHi. In our own work in adult rat ventricular myocytes, inhibiting {alpha}1A-AR-mediated activation of ERK, using either PD98059 or UO126 (which are structurally-distinct inhibitors of the ERK kinase MEK1 [46]), was found to inhibit both p90RSK activation and stimulation of sarcolemmal NHE activity [47]. Importantly, MEK1 inhibition has also been shown to inhibit the stimulation of sarcolemmal NHE activity via the angiotensin AT1 receptor [19], as well as by non-GPCR-mediated stimuli [48]. These findings, which indicate that the ERK/p90RSK pathway plays a critical role in the regulation of sarcolemmal NHE activity in cardiac myocytes, are consistent with earlier data that have shown reduced thrombin-induced stimulation of NHE1 activity in non-myocyte cells that had been transfected with a dominant-negative ERK mutant [49]. Nevertheless, further work is required to determine the relative contributions of direct phosphorylation of the NHE1 protein by ERK itself versus its downstream mediator p90RSK in GPCR-mediated stimulation of sarcolemmal NHE activity.

Protein kinase C (PKC) also appears to play a critical role in the stimulation of sarcolemmal NHE activity by some GPCR agonists, on the basis of studies with a variety of pharmacological PKC inhibitors. Thus, agents such as GF109203X (also known as bisindolylmaleimide I) [19,25,47], chelerythrine [25], H-7 [11,20], staurosporine [21] and sphingosine [11,20] have all been shown to inhibit the stimulation of sarcolemmal NHE activity, in response to diverse stimuli such as {alpha}1-AR agonists [11,47], angiotensin II [19], thrombin [25], endothelin [20] and carbachol [21]. Nevertheless, the regulatory domain of NHE1 has been shown not to be a substrate for in vitro phosphorylation by a mixture of PKC isoforms [38], and the mechanism(s) through which PKC may regulate sarcolemmal NHE activity remain unclear. In response to some stimuli, such as activation of angiotensin AT1 receptors, PKC may stimulate sarcolemmal NHE activity via the activation of the ERK pathway [19]. In contrast, PKC and ERK appear to mediate independent but necessary signalling pathways in the stimulation of NHE activity by {alpha}1-AR agonists [47].

A major concern in the interpretation of data obtained with PKC inhibitors is the suspect specificity of these agents. In this regard, recent in vitro kinase assays have revealed that widely-used PKC inhibitors, such as the bisindolylmaleimides, also inhibit both p70 S6 kinase and p90RSK with similar potency [50,51]. Any non-specific inhibition of p90RSK by PKC inhibitors would be of particular concern in investigations of the signalling mechanisms that regulate sarcolemmal NHE activity. This is because, as noted above, p90RSK is known to phosphorylate the NHE1 protein [41] and is believed to play a key role in GPCR-mediated stimulation of sarcolemmal NHE activity in cardiac myocytes [44,47]. Thus, the possibility exists that the ability of some PKC inhibitors to attenuate GPCR-mediated stimulation of sarcolemmal NHE activity may arise from non-specific inhibition of p90RSK. On the basis that the in vitro profile of a pharmacological inhibitor may not necessarily reflect its in vivo profile, there is an important need to determine whether PKC inhibitors inhibit p90RSK activity, within the concentration range that inhibits PKC activity, in intact cardiac myocytes. Further investigation is also required to determine the molecular mechanisms through which PKC may regulate sarcolemmal NHE activity and to identify the PKC isoform(s) involved.

Little is known about the distal signalling mechanisms through which some GPCRs attenuate sarcolemmal NHE activity or its stimulation via other receptors. In dog Purkinje fibres, the NHE-inhibitory effect of β1-AR stimulation could be mimicked by the adenylate cyclase activator forskolin, or the phosphodiesterase inhibitors theophylline and 3-isobutyl-1-methylxanthine (IBMX) [21]. IBMX has been shown to inhibit sarcolemmal NHE activity also in sheep Purkinje fibres [52]. These observations suggest a role for the second messenger cyclic adenosine monophosphate (cAMP) in the β1-AR-mediated response, although the molecular mechanism(s) through which cAMP may inhibit sarcolemmal NHE activity remain unclear. A regulatory role for protein kinase A (PKA), which is activated by cAMP, is well established for NHE isoform 3, but there are no consensus phosphorylation sites for PKA in the regulatory domain of NHE1 and it is unlikely that direct phosphorylation of NHE1 by PKA occurs [53]. The molecular mechanism(s) through which the counter-regulatory effects of GPCRs, such as the angiotensin AT2 receptor [19] and the adenosine A1 receptor [35], are mediated are also unknown, although they do not appear to involve the direct inhibition of sarcolemmal NHE activity [19,35] or the attenuation of ERK activation [19]. Investigation of the role of protein kinase D, whose over-expression inhibits phorbol ester-induced stimulation of plasma membrane NHE activity in other cell types [54], may yield novel information in this area.

Although direct phosphorylation is likely to be an important mechanism in GPCR-mediated regulation of sarcolemmal NHE activity, it should be noted that, in non-myocyte cells, removal of the last 180 amino acids from the regulatory domain of NHE1 abolishes its phosphorylation but only partially inhibits its stimulation by thrombin [4,55]. This suggests that mechanisms other than direct phosphorylation also contribute to the regulation of NHE1 activity. Indeed, deletion experiments have suggested that the binding of Ca2+-calmodulin to the regulatory domain increases NHE1 activity [5,56,57]. In contrast, the association of a Ca2+-binding calcineurin-homologous protein (CHP) with a separate site in the regulatory domain has been suggested to inhibit NHE1 activation [58], although more recent data suggest that CHP may instead be an essential co-factor for NHE1 activity [59]. The relative importance of these complementary mechanisms in regulating sarcolemmal NHE activity in cardiac myocytes, particularly in response to GPCR stimulation, remains unclear. It is interesting to note, however, that the inhibition of sarcolemmal NHE activity by IBMX in sheep Purkinje fibres was abolished in the presence of trifluorperazine, a non-specific Ca2+-calmodulin inhibitor [52], which suggests a role for Ca2+-dependent signalling mechanisms in cAMP-mediated regulation of the exchanger.


   5. Functional significance
Top
 Abstract
 1. Introduction
 2. GPCR-mediated stimulation of...
 3. GPCR-mediated inhibition of...
 4. Distal signalling mechanisms
 5. Functional significance
6. Conclusion
 References
 
5.1 Role in regulation of pHi and contractility
The main function of the sarcolemmal NHE is to remove acid equivalents from the cell. Under physiological conditions (i.e. in the presence of HCO3–), however, NBC activity also contributes to such acid extrusion [1]. Therefore, the net effect of GPCR stimulation on the rate of acid extrusion will depend on the responses of both NHE and NBC to the pertinent stimulus. There is only limited information available on GPCR-mediated regulation of NBC activity. Lagadic-Gossmann et al. [12] have shown that, in guinea-pig ventricular myocytes, adrenergic stimulation has opposing effects on NHE versus NBC activity; thus, {alpha}1-AR stimulation was shown to activate NHE but inhibit NBC, while β1-AR stimulation inhibited NHE but activated NBC. In the same study, the net effect of the physiological adrenergic agonists noradrenaline and adrenaline, which stimulate both {alpha}1- and β1-ARs, was to slow the rate of recovery of pHi following the induction of intracellular acidosis in HCO3–-containing medium. In contrast to the opposite effects of adrenergic stimulation on NHE versus NBC activity, recent evidence suggests that AT1 receptor stimulation by angiotensin II increases sarcolemmal NBC activity [60], as it does sarcolemmal NHE activity [19]. In the case of NBC, however, the AT1 receptor-mediated response does not appear to be subject to counter-regulation via the AT2 receptor [60]. For the variety of other GPCR agonists that have been shown to regulate NHE activity (see above), information on their effects on NBC activity and the net rate of acid extrusion in HCO3–-containing medium is not available. Furthermore, little is known about GPCR-mediated regulation of the activity of the AE and the CHE, the two sarcolemmal acid loaders [61,62]. Clearly, the sustained effect of any given stimulus on pHi will be determined by its net effect on acid equivalent flux across the sarcolemma, as determined by the sum of its actions on inward or outward flux through all four pH-regulatory transporters. Nevertheless, as proposed by Leem et al. [1], a GPCR-mediated change in the pHi sensitivity (and therefore the activity) of an individual transporter, such as the sarcolemmal NHE, can produce a displacement of pHi within a permissive range (pHi 6.95–7.25 in the guinea pig ventricular myocyte), with functional consequences.

A GPCR-mediated increase in sarcolemmal NHE activity would of course produce not only an increased rate of acid extrusion (and thereby relative intracellular alkalinization) but also an increased rate of Na+ influx into the cardiac myocyte. Each of these processes can have a significant impact on myocardial contractility, through distinct mechanisms. Altered pHi can regulate contractility at almost every stage of excitation–contraction coupling, with intracellular alkalosis producing a positive inotropic effect that is mediated largely through increased myofilament Ca2+ responsiveness (see review by Orchard and Kentish [63]). In parallel with this, any increase in the intracellular Na+ concentration ([Na+]i) that may arise from increased Na+ influx via the sarcolemmal NHE can also produce a positive inotropic effect, principally through the modulation of sarcolemmal Na+/Ca2+ exchanger activity and thereby [Ca2+]i regulation [64]. Indeed, a variety of neurohormonal stimuli that act via their cognate GPCRs, such as {alpha}1-AR agonists [10,65], angiotensin II [16,66] and endothelin [20,67] have been shown to produce positive inotropic responses that are attenuated by inhibition of the sarcolemmal NHE. Interestingly, the positive inotropic effect of {alpha}1-AR stimulation is attenuated also by stimulation of the adenosine A1 receptor [68], in a manner that mirrors the inhibitory effect of this receptor on {alpha}1-AR-mediated stimulation of sarcolemmal NHE activity [35]. These data suggest that GPCR-mediated regulation of sarcolemmal NHE activity makes an important contribution to the inotropic responses that are produced by a variety of neurohormonal stimuli; nevertheless, the relative contributions of altered pHi (and thereby myofilament Ca2+ responsiveness) versus altered [Na+]i (and thereby [Ca2+]i regulation) have not been clearly demonstrated in each case. Interestingly, the slow increase in force development that is observed in response to myocardial stretch has also been shown to be sensitive to inhibition by NHE inhibitors [69]. There is recent evidence that this slow force response occurs through autocrine/paracrine mechanisms that are mediated by the release of angiotensin II and endothelin, which produce increases in sarcolemmal NHE activity and [Na+]i via the stimulation of their cognate AT1 and ETA receptors [70].

5.2 Role in myocardial ischaemia and reperfusion
Since the late 1980s, substantial evidence has accumulated that increased activity of the sarcolemmal NHE contributes to the development of myocardial injury and dysfunction during ischaemia and reperfusion [71,72]. Indeed, selective NHE inhibitors have been shown to be cardioprotective in this setting in numerous animal studies (see reviews by Avkiran [73] and Karmazyn et al. [74]). Importantly, recent data suggest that NHE inhibition may provide cardioprotective benefit in high-risk patients who undergo global myocardial ischaemia and reperfusion during coronary artery bypass graft surgery [75], an observation which awaits confirmation by current clinical trials [76].

It is well-established that myocardial ischaemia results in activation of the sympathetic nervous system, as well as the local release of noradrenaline within the ischaemic zone [77]. This appears to be accompanied by enhanced {alpha}1-adrenergic signalling, through increased receptor density and enhanced receptor coupling to distal steps in the relevant signalling cascade [78]. It is likely therefore that {alpha}1-AR stimulation may play an important role in regulating sarcolemmal NHE activity during ischaemia. In this context, it is important to note that {alpha}1-AR stimulation appears to retain its ability to increase sarcolemmal NHE activity even in the presence of extracellular acidosis, which accompanies myocardial ischaemia [79]. Furthermore, Khandoudi et al. have shown that, in isolated rat hearts subjected to global ischaemia, {alpha}1-AR stimulation exacerbates post-ischaemic contractile dysfunction, through mechanisms that are counteracted by NHE inhibition [80]. In our own laboratory, we have shown that, in rat hearts subjected to regional ischaemia, {alpha}1-AR stimulation within the ischaemic zone exacerbates reperfusion-induced arrhythmias, and that this pro-arrhythmic effect is abolished not only by {alpha}1A-AR blockade but also by NHE inhibition [81]. These data support the possibility that {alpha}1-AR-mediated stimulation of sarcolemmal NHE activity influences the outcome of myocardial ischaemia and reperfusion. It should be noted, however, that during myocardial ischaemia in vivo, simultaneous stimulation of β1-ARs by endogenous catecholamines may offset the effects of {alpha}1-AR stimulation on sarcolemmal NHE activity.

Other GPCR-mediated stimuli that alter sarcolemmal NHE activity are also likely to be of pathophysiological importance during myocardial ischaemia and reperfusion. Intracoronary thrombosis, the commonest cause of acute myocardial ischaemia in man [82], is associated with elevated levels of thrombin in the vicinity of the evolving thrombus [83]. Thus, thrombin may increase sarcolemmal NHE activity through the stimulation of its cognate receptors on neighbouring myocytes. Indeed, thrombin receptor stimulation exacerbates the increase in [Na+]i that occurs during ischaemia [84], although the role of NHE activity in mediating this response has not been determined. Nevertheless, there is preliminary evidence that thrombin induces a potent pro-arrhythmic effect during regional ischaemia and reperfusion in the rat heart, by a mechanism that is blocked by NHE inhibition [85]. Endothelin is also released during myocardial ischaemia and reperfusion [86], and may lead to the stimulation of sarcolemmal NHE activity. Certainly, exposure of hearts to exogenous endothelin during ischaemia has been shown to depress the post-ischaemic recovery of contractile function, an effect which could be blocked by NHE inhibitors [23,87].

In view of the apparent detrimental effects of some GPCR-mediated stimuli that increase sarcolemmal NHE activity, it is tempting to speculate that other GPCR systems that suppress NHE activity may have the opposite effect and afford myocardial protection during ischaemia and reperfusion. Consistent with this, there is evidence that cardiac-specific overexpression of the adenosine A1 receptor in transgenic mice preserves myocardial integrity and function following regional ischaemia and reperfusion [88]; nevertheless, whether this effect is mediated through a reduced sarcolemmal NHE activity is not known.

5.3 Role in myocardial hypertrophy
There is substantial evidence that GqPCR signaling pathways contribute significantly to the development of cardiac hypertrophy, as well as its decompensation that can lead to heart failure [89]. In in vitro studies, many GqPCR agonists that stimulate sarcolemmal NHE activity (e.g. {alpha}1-AR agonists, endothelin, angiotensin II, thrombin; see earlier) have been shown also to induce myocyte hypertrophy [90–93]. This raises the possibility that the association between the stimulation of NHE activity and the induction of hypertrophy may be of a causal nature, which is consistent with recent data obtained with NHE inhibitors. Thus, the hypertrophic response of adult rat ventricular myocytes to adrenergic stimulation has been shown to be attenuated by NHE inhibition [94,95]. Intriguingly, NHE inhibition attenuated the hypertrophic responses to the sustained (24 h) stimulation of either {alpha}1-ARs or β1-ARs [95], despite the fact that acute β1-AR stimulation inhibits sarcolemmal NHE activity (see earlier). Furthermore, the ventricular hypertrophy that develops spontaneously in transgenic mice with cardiac-specific overexpression of the β1-AR has been shown recently to require NHE activity, on the basis that the hypertrophic phenotype was almost completely abolished by chronic treatment with the NHE inhibitor cariporide [96]. These observations raise the possibility that NHE activity may play a permissive role in the development of cardiac hypertrophy, such that in the absence of such activity (i.e. in the presence of NHE inhibitors) the phenotype is attenuated regardless of the nature of the hypertrophic stimulus. This is consistent with increasing evidence that NHE inhibitors suppress the development of hypertrophy in a diverse range of in vitro and in vivo models. Thus, in recent studies, NHE inhibitors have been shown to attenuate stretch-induced hypertrophy of cultured neonatal rat ventricular myocytes in vitro [97], post-myocardial infarction myocardial hypertrophy in rats in vivo [98–101], and myocardial hypertrophy in response to left [102] or right [103] ventricular pressure overload in rats in vivo. It appears therefore that, at least in the rodent heart, NHE activity plays a fundamental role in the induction and evolution of myocardial hypertrophy. Nevertheless, the relative importance of increased (cf. maintained) sarcolemmal NHE activity, as occurs in response to GqPCR stimulation, and the molecular mechanism(s) through which NHE activity may regulate myocardial hypertrophy remain to be elucidated.


   6. Conclusion
Top
 Abstract
 1. Introduction
 2. GPCR-mediated stimulation of...
 3. GPCR-mediated inhibition of...
 4. Distal signalling mechanisms
 5. Functional significance
 6. Conclusion
References
 
GPCR-mediated regulation of sarcolemmal NHE activity is likely to play significant roles in modulating myocardial function in both physiological and pathophysiological conditions. Greater understanding of the molecular signalling pathways that mediate the stimulatory or inhibitory effects of various GPCR systems on sarcolemmal NHE activity requires further investigation, and may ultimately lead to the development of novel approaches for therapeutic manipulation of these pathways.

Time for primary review 21 days.


   Acknowledgements
 
The authors’ work is supported by the British Heart Foundation (BS/93002; FS/02/001), the Medical Research Council, The Dunhill Medical Trust and the Charitable Foundation of Guy's and St Thomas’ Hospitals.


   References
Top
 Abstract
 1. Introduction
 2. GPCR-mediated stimulation of...
 3. GPCR-mediated inhibition of...
 4. Distal signalling mechanisms
 5. Functional significance
 6. Conclusion
 References
 

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