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Cardiovascular Research 2002 53(2):470-480; doi:10.1016/S0008-6363(01)00464-3
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Copyright © 2001, European Society of Cardiology

Regulation of sarcolemmal Na+/H+ exchange by hydrogen peroxide in adult rat ventricular myocytes

Andrew K Snabaitis, David J Hearse and Metin Avkiran*

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

metin.avkiran{at}kcl.ac.uk

* Corresponding author. Tel.: +44-20-7928-9292, ext. 3375; fax: +44-20-7928-0658

Received 20 July 2001; accepted 14 September 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: To characterise the effects of exogenous H2O2 on sarcolemmal Na+/H+ exchanger (NHE) activity and determine the roles of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC) in observed effects. Methods: Sarcolemmal H+ efflux rate (JH) was determined by microepifluorescence at a pHi of 6.70 in adult rat ventricular myocytes, after two consecutive acid pulses in HCO3–-free medium; before the second pulse, cells (n=7–10/group) were exposed to H2O2 or vehicle and the change in JH ({Delta}JH) was used to quantify the change in NHE activity. ERK and p38 MAPK activities were determined by immunoblotting with phosphospecific antibodies. Results: Relative to control, {Delta}JH was increased by a 10-min exposure to 100, but not 1 or 10 µM H2O2 (1000 µM was not tolerated); 3 or 6 min exposure to 100 µM H2O2 was without effect. ERK and p38 MAPK activities were both increased by 100 µM H2O2 (peak at 6 min); the ERK kinase inhibitor PD98059 (10 µM), but not the p38 MAPK inhibitor SB203580 (1 µM), inhibited the H2O2-induced increase in {Delta}JH. H2O2-induced ERK activation was inhibited not only by PD98059 (10 µM), but also by the non-selective tyrosine kinase inhibitor genistein (3–100 µM), the EGF receptor kinase inhibitor AG1478 (3–300 nM) and the Src family kinase inhibitor PP2 (0.1–10 µM). The PKC inhibitors GF109203X (0.3–10 µM) and chelerythrine (1–30 µM) were without effect on ERK activation, although the former abolished the H2O2-induced increase in {Delta}JH. Conclusions: Our data demonstrate that, in adult rat ventricular myocytes, (i) hydrogen peroxide stimulates sarcolemmal NHE activity, (ii) this response requires activation of ERK and PKC, but not p38 MAPK, (iii) ERK activation occurs through tyrosine kinase-mediated, but PKC-independent, mechanisms

KEYWORDS Free radicals; Myocytes; Na/H-exchanger; Signal transduction; Protein kinases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The sarcolemmal Na+/H+ exchanger (NHE) is encoded by the NHE-1 gene [1] and contributes to the integrated control of intracellular pH (pHi) in cardiac myocytes [2]. Basal activity of the sarcolemmal NHE is low under physiological conditions [2] but increased exchanger activity and resultant increases in intracellular Na+ and/or pHi may mediate the inotropic responses of myocardium to neurohormonal stimuli [3–6] and myocardial stretch [7]. With respect to cardiac pathophysiology, sarcolemmal NHE activity may play a permissive role in the hypertrophic response of cardiac myocytes to neurohormonal [8] and mechanical [9] stimuli in vitro and in the adverse ventricular remodelling that occurs following myocardial infarction in vivo [10–12]. Perhaps the strongest experimental evidence for a pathophysiological role for the sarcolemmal NHE, however, is that which implicates the exchanger in the development of myocardial injury and dysfunction during ischaemia and reperfusion (see reviews by Avkiran [13] and Karmazyn et al. [14]). Importantly, recent studies suggest that NHE inhibition by the NHE-1-selective inhibitor cariporide may provide cardioprotective benefit in certain clinical settings also, such as in high-risk patients who are subjected to elective myocardial ischaemia and reperfusion during coronary artery bypass graft surgery [15].

There is strong evidence that oxidative stress also contributes to the development of myocardial injury and dysfunction during ischaemia and reperfusion [16,17]. Intriguingly, data from Karmazyn's laboratory suggest that the adverse myocardial effects of oxidative stress (induced by exposure to H2O2), when applied either alone [18] or in combination with ischaemia and reperfusion [19], are attenuated by NHE inhibition. Although sarcolemmal NHE activity was not determined in either study, these findings raise the possibility that an increase in such activity may mediate some of the detrimental consequences of exposure to H2O2, or that NHE inhibitors (or the consequences of NHE inhibition) may enhance myocardial tolerance to such oxidative stress. Recent data from neonatal rat ventricular myocytes are consistent with the former possibility [20]. However, the effects of oxidative stress induced by H2O2 on sarcolemmal NHE activity in adult myocardium and the signalling mechanism(s) underlying any such effects are unknown. Notably, qualitative and quantitative changes are known to occur during post-natal development in the myocardial expression of NHE-regulatory signalling molecules, such as ERK [21] and protein kinase C (PKC) [22–24]. Furthermore, the basal expression and activity of NHE differs significantly between neonatal and adult rat ventricular myocytes [25]. Thus, the regulation of NHE activity by H2O2 may differ between adult and neonatal myocardium.

The present study used adult rat ventricular myocytes to characterise the time- and dose-dependent effects of H2O2 on the activities of the sarcolemmal NHE and the putative NHE-regulatory signalling pathways mediated by ERK and p38 MAPK. Subsequently, any causal link between the observed changes was probed through the use of specific inhibitors of ERK kinase (MEK) and p38 MAPK. Finally, we explored the proximal signalling mechanisms that are responsible for H2O2-induced ERK activation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
The present investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by HMSO, London.

2.1 Isolation of ventricular myocytes
Ventricular myocytes were isolated from the hearts of adult male Wistar rats (200–250 g) using a collagenase-based enzymatic digestion technique that provides a yield of >80% rod-shaped myocytes, as described previously [26].

2.2 Determination of sarcolemmal NHE activity
Sarcolemmal NHE activity was determined in quiescent single myocytes loaded with the pH-sensitive fluoroprobe cSNARF-1, using a microepifluorescence technique [26–29]. Cells were maintained in HCO3–-free medium (34°C) throughout each experiment, thus enabling the rate of acid efflux (JH) to be used as the indicator of sarcolemmal NHE activity. JH was determined at a pHi of 6.70 during recovery from intracellular acidosis before and after exposure to H2O2, and the change in JH ({Delta}JH) used to quantify H2O2-induced changes in NHE activity.

2.3 Determination of cellular MAPK, p90 ribosomal S6 kinase (p90rsk) and Src tyrosine kinase activities
Protein samples (40 µg) from whole cell lysates were separated by SDS–PAGE on 9% polyacrylamide gels. MAPK activities were determined through the detection of dual phosphorylation of ERK1/2 and p38 MAPK by immunoblot analysis using phosphospecific antibodies (New England BioLabs, MA), as described previously [27,28]. Activities of p90rsk and Src tyrosine kinase were determined by the detection of Ser381 or Tyr416 phosphorylation, respectively, using phosphospecific antibodies (New England BioLabs) [27,28]. To confirm equal protein loading, non-phosphospecific antibodies for ERK2, p38, p60Src (all from Santa Cruz Biotechnology, CA) and p90rsk (Transduction Laboratories, KY) were used. Specific protein bands were detected by enhanced chemiluminescence and autoradiography, and phosphorylation status was quantified by using a LKB 2222 Bromma Ultroscan XL laser densitometer.

2.4 Determination of cellular MAPK-activated protein kinase-2 (MAPKAPK-2) activity
The inhibitory effect of SB203580 on the cellular activity of p38 MAPK was determined by immunoprecipitating MAPKAPK-2 (which is phosphorylated and activated by p38 MAPK) from cell lysates and subjecting it to an in vitro kinase assay (Upstate Biotechnology, NY). In this assay, activity of MAPKAPK-2 is determined by incubation of the immune complex with the specific MAPKAPK-2 substrate peptide KKLNRTLSVA in a reaction mixture containing [{gamma}-32P]:ATP and quantifying the resulting incorporation of 32P into the peptide by scintillation counting [30].

2.5 Experimental protocols
For determination of H2O2-induced changes in NHE activity, myocytes were subjected to intracellular acidosis by transient exposure to 20 mM NH4Cl (first acid pulse), which was repeated 17–27 min later (second acid pulse). Dose-dependent effects of H2O2 were assessed by exposing cells to vehicle or 1, 10, 100 or 1000 µM H2O2 (Sigma–Aldrich, Poole, UK) from 10 min before the second acid pulse (see Fig. 1A). Time-dependent effects were assessed by exposing cells to vehicle or 100 µM H2O2 from 3, 6 or 10 min before the second acid pulse (see Fig. 1B). When studying the effects of the MEK inhibitor PD98059 (10 µM), the p38 MAPK inhibitor SB203580 (1 µM) or the PKC inhibitor GF109203X (1 µM) (all from Calbiochem–Novabiochem, Nottingham, UK), the kinase inhibitor was present from 10 min before the start of exposure to 100 µM H2O2 (see Fig. 1C). The concentrations of PD98059 and GF109203X were selected on the basis of our earlier work on receptor-mediated NHE regulation in the same preparation [27,28] and the concentration of SB203580 was selected since it fully inhibits p38 MAPK activity while not affecting other kinase pathways in neonatal myocytes [31].


Figure 1
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  		Fig. 1 Experimental protocols used to determine the effects of H2O2 on sarcolemmal Na+/H+ exchanger activity. Protocols are shown for the determination of (A) dose-dependent, (B) time-dependent and (C) protein kinase-mediated effects of H2O2 on sarcolemmal Na+/H+ exchanger activity in adult rat ventricular myocytes. Solid bars represent the periods of exposure to 20 mM NH4Cl, whose washout produced intracellular acidosis; JH was determined during recovery from such acidosis (shaded areas).

 
For the determination of H2O2 effects on kinase activity (n=4 experiments in each protocol, with each experiment using cells from a separate heart), myocytes in suspension were exposed to H2O2, using identical concentrations and exposures to those described above; in time–response studies, duration of H2O2 exposure was additionally extended to 20 and 30 min. When studying the effects of PD98059 (10 µM) or SB203580 (1 µM) on H2O2-induced kinase activation, this was present from 10 min before the start of exposure to 100 µM H2O2 (10 min), as above. When studying the effects of PKC inhibitors (GF109203X and chelerythrine), the non-selective tyrosine kinase inhibitor genistein, the Src family kinase inhibitor PP2 and the epidermal growth factor (EGF) receptor kinase inhibitor AG1478 (all from Calbiochem-Novabiochem) on H2O2-induced kinase activation, various concentrations of each inhibitor were present from 30 min before the start of exposure to 100 µM H2O2 (10 min).

Stock solutions of all kinase inhibitors were dissolved in DMSO, which was also included in superfusion solutions (at its final concentration of 0.05%) in control and H2O2-only experiments, for the appropriate periods.

2.6 Statistical analysis
Data are mean±S.E.M. For the microepifluorescence experiments, cells from one heart were used on each day and these were allocated in a randomised manner to the experimental groups of the protocol under study; eight to 10 separate hearts were required to complete each protocol (see figure legends), within which each experimental group comprised seven to 10 myocytes. The biochemical experiments were repeated four times, each time using myocytes from a separate heart. For inter-group comparison of {Delta}JH or protein kinase phosphorylation, data were subjected to ANOVA; further analysis was by Dunnett's test, to compare each treatment group with the control group. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Effects of H2O2 on sarcolemmal NHE activity
3.1.1 Dose–response studies
In control cells that were exposed to vehicle, there was little change in JH during the second acid pulse relative to the first, as reflected by a {Delta}JH of around zero (Fig. 2A). Relative to the control group, a 10-min exposure to 100 µM H2O2 produced a significant increase in {Delta}JH; in contrast, {Delta}JH was not changed significantly by a similar exposure to 1 or 10 µM H2O2 (Fig. 2A). Following a 10-min exposure to 1000 µM H2O2, cells became hyper-contracted and detached from the cover slip during the second acid pulse; thus, {Delta}JH could not be determined in this group. These data indicate that H2O2 stimulates sarcolemmal NHE activity in a dose-dependent manner, such that >10 µM H2O2 is required to achieve a significant effect.


Figure 2
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  		Fig. 2 Dose- and time-dependent effects of H2O2 on sarcolemmal Na+/H+ exchanger activity. The change in JH ({Delta}JH) during the second acid pulse relative to the first is shown in (A) cells exposed to vehicle or 1, 10 or 100 µM H2O2 from 10 min before the second acid pulse, and (B) cells exposed to vehicle or 100 µM H2O2 from 3, 6 or 10 min before the second acid pulse. *P<0.05 versus control (seven to 10 cells per group, obtained from 10 hearts).

 
3.1.2 Time–response studies
These experiments were performed to delineate the temporal profile of H2O2-induced stimulation of NHE activity. Consistent with the above results, a 10-min exposure to 100 µM H2O2 again produced a significant increase in {Delta}JH (Fig. 2B). However, when exposure time was limited to 3 or 6 min, there was no significant change in {Delta}JH in response to 100 µM H2O2 (Fig. 2B). These data indicate that H2O2-induced stimulation of sarcolemmal NHE activity requires an exposure time of >6 min.

3.2 Effect of H2O2 on cellular ERK and p38 MAPK activity
We next determined the dose- and time-dependent effects of H2O2 on ERK and p38 MAPK activity, to test whether either pathway is activated in a manner that is consistent with a potential NHE-regulatory role. Exposure of cells to 1–1000 µM H2O2 for 10 min produced dose-dependent activation of both ERK1/2 and p38 MAPK, with significant increases in activity achieved with 100 µM H2O2 (Fig. 3A). These data also showed that in adult rat ventricular myocytes, ERK2 (cf. ERK1) was the pre-dominant isozyme detected by the ERK1/2 phosphospecific antibody with readily detectable basal phosphorylation in the untreated control cells. Therefore, subsequent quantitation of the effects of H2O2 on ERK activity was based on the change in the phosphorylation status of ERK2. The temporal profiles indicated that peak ERK and p38 MAPK activation occurred after 6 min of exposure to 100 µM H2O2, and that such activation was sustained for approximately 10 min (Fig. 3B). The concordance between the dose- and time-dependence characteristics of H2O2-induced activation of ERK and p38 MAPK and H2O2-induced stimulation of sarcolemmal NHE activity suggests that either kinase may play a role in the latter response.


Figure 3
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  		Fig. 3 Dose- and time-dependent effects of H2O2 on ERK and p38-MAPK activity. Cells were exposed to (A) vehicle or 1, 10, 100 and 1000 µM H2O2 for 10 min, and (B) vehicle or 100 µM H2O2 for 3, 6, 10, 20 or 30 min. Autoradiograms show representative Western blots with phosphospecific (top panel) and non-phosphospecific (bottom panel) for ERK and p38-MAPK antibodies. *P<0.05 versus control (four experiments, with cells from four hearts).

 
3.3 Role of ERK and p38 MAPK in H2O2-induced stimulation of sarcolemmal NHE activity
This protocol was designed to test whether activation of the ERK and/or the p38 MAPK pathway was necessary for H2O2-induced stimulation of sarcolemmal NHE activity. In the absence of any pre-treatment, a 10-min exposure to 100 µM H2O2 again produced a significant increase in {Delta}JH (Fig. 4A). This effect was abolished by pre-treatment of cells with the MEK inhibitor PD98059 but was unaffected by pre-treatment with the p38 MAPK inhibitor SB203580 (Fig. 4A). The lack of effect of SB203580 was not due to inadequate inhibition of p38 MAPK activity, since H2O2-induced activation of MAPKAPK-2 was abolished by the SB203580 pre-treatment protocol (Fig. 4B). These data indicate that H2O2-induced stimulation of sarcolemmal NHE activity requires activation of the ERK but not the p38 MAPK pathway.


Figure 4
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  		Fig. 4 Effects of MAPK inhibition on H2O2-induced increases in sarcolemmal Na+/H+ exchanger and cellular MAPKAPK-2 activities. In (A), cells were subjected to two consecutive acid pulses. The change in JH ({Delta}JH) during the second acid pulse relative to the first is shown in cells exposed to vehicle or 100 µM H2O2 from 10 min before the second acid pulse, in the absence or presence of pre-treatment with the MEK inhibitor PD98059 (10 µM) or the p38 MAPK inhibitor SB203580 (1 µM). *P<0.05 versus control (seven to 10 cells per group, obtained from 10 hearts). In (B), cells were exposed to vehicle or 100 µM H2O2 for 10 min in the absence or presence of pre-treatment with the p38 MAPK inhibitor SB203580 (1 µM). *P<0.05 versus control (four experiments, with cells from four hearts).

 
To explore whether the inhibitory effect of PD98059 on NHE stimulation occurred through the predicted mechanism of MEK inhibition, we next determined its effect on ERK activity. As shown in Fig. 5, a 10-min exposure to 100 µM H2O2 activated not only ERK (thus confirming our earlier observation) but also its downstream substrate p90rsk, which is a putative NHE-1 kinase [32,33]. PD98059 abolished H2O2-induced increases in ERK and p90rsk activity, whilst having no effect on basal activity (Fig. 5). These data support an effector role for ERK and/or p90rsk in H2O2-induced stimulation of sarcolemmal NHE activity.


Figure 5
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  		Fig. 5 Effects of MEK inhibition on H2O2-induced increases in ERK and p90rsk activity. Cells were exposed to vehicle or 100 µM H2O2 for 10 min in the absence or presence of pre-treatment with the MEK inhibitor PD98059 (10 µM). Autoradiograms show representative Western blots with phosphospecific (top panel) and non-phosphospecific (bottom panel) antibodies for (A) ERK and (B) p90rsk. *P<0.05 versus control (four experiments, with cells from four hearts).

 
3.4 Role of PKC in H2O2-induced stimulation of sarcolemmal NHE activity
Our previous work has shown that, in addition to ERK/p90rsk activation, PKC activation is also necessary for the stimulation of sarcolemmal NHE activity through {alpha}1A-adrenergic [28] and angiotensin II AT1 [27] receptors. Here, we tested whether this holds true also for H2O2-induced stimulation of sarcolemmal NHE activity. As shown in Fig. 6, pre-treatment with the PKC inhibitor GF109203X (used at a concentration of 1 µM, which we have shown previously to inhibit phorbol ester-induced effects in an identical preparation [28]) completely abolished the H2O2-induced increase in {Delta}JH. This suggests that, in addition to ERK/p90rsk activation, PKC activation is also necessary for the stimulation of sarcolemmal NHE activity by H2O2.


Figure 6
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  		Fig. 6 Effects of PKC inhibition on H2O2-induced increases in sarcolemmal Na+/H+ exchanger activity. The change in JH ({Delta}JH) during the second acid pulse relative to the first is shown in cells exposed to vehicle or 100 µM H2O2 from 10 min before the second acid pulse in the absence or presence of pre-treatment with the PKC inhibitor GF109203X (1 µM). *P<0.05 versus control (seven to 10 cells per group, obtained from eight hearts).

 
3.5 Proximal regulators of ERK activity: role of PKC
In cultured neonatal rat ventricular myocytes, H2O2-induced ERK activation has been suggested to occur through a PKC-mediated mechanism [20,34]. However, when we examined the effects of GF109203X (Fig. 7A) and the structurally distinct PKC inhibitor chelerythrine (Fig. 7B), we found no inhibition of the H2O2-induced increase in ERK activity over a broad concentration range. Thus, it appears that H2O2-induced ERK activation in freshly isolated adult rat ventricular myocytes occurs through PKC-independent mechanisms.


Figure 7
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  		Fig. 7 Effects of PKC inhibition on H2O2-induced increases in ERK activity. Cells were exposed to vehicle or 100 µM H2O2 for 10 min in the absence or presence of pre-treatment with the PKC inhibitor (A) GF109203X (0.3–10 µM) or (B) chelerythrine (1–30 µM). Autoradiograms show representative Western blots with phosphospecific (top panel) and non-phosphospecific (bottom panel) antibodies for ERK. *P<0.05 versus control (four experiments, with cells from four hearts).

 
3.6 Proximal regulators of ERK activity: role of tyrosine kinases
In the light of the above, we examined whether H2O2-induced ERK activation might be mediated through tyrosine kinase pathways. To address this, we initially determined the effects of the non-selective tyrosine kinase inhibitor genistein, which produced a dose-dependent inhibition of the H2O2-induced increase in ERK activity (Fig. 8A). Daidzein, a structural analog of genistein without tyrosine kinase inhibitory activity, had no effect on the H2O2-induced response. Thus, relative to control, H2O2 increased ERK phosphorylation by 4.1±0.5-fold in the absence of daidzein and by 4.0±0.3-, 3.3±0.4-, 3.1±0.8- and 4.8±1.2-fold in the presence of 3,10, 30 and 100 µM daidzein, respectively (four experiments, with cells from four hearts). These findings indicate that H2O2-induced ERK activation in the adult myocyte occurs through tyrosine kinase-mediated mechanisms.


Figure 8
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  		Fig. 8 Effects of tyrosine kinase inhibition on H2O2-induced increases in ERK activity. Cells were exposed to vehicle or 100 µM H2O2 for 10 min in the absence or presence of pre-treatment with (A) the non-selective tyrosine kinase inhibitor genistein (3–100 µM), (B) the Src-family kinase inhibitor PP2 (0.1–10 µM) or (C) the EGF receptor kinase inhibitor AG1478 (3–300 nM). Autoradiograms show representative Western blots with phosphospecific (top panel) and non-phosphospecific (bottom panel) antibodies for ERK. *P<0.05 versus control (four experiments, with cells from four hearts).

 
To explore the role of non-receptor tyrosine kinases of the Src family, which have been shown to play an important role in H2O2 signalling in neonatal myocytes [35], we next determined the effects of the Src-selective inhibitor PP2. This agent, like genistein, produced a dose-dependent inhibition of the H2O2-induced increase in ERK activity (Fig. 8B), indicating a key role for Src family tyrosine kinases in this response. Indeed, Src tyrosine kinase was found to be activated rapidly upon exposure to H2O2 (Fig. 9), which supports a proximal role for Src-family tyrosine kinases in the H2O2-induced increase in ERK activity. In other cell types, c-Src (a member of the Src family) can phosphorylate the EGF receptor and thereby facilitate its full activation [36], and activation of this receptor tyrosine kinase has been implicated in ERK activation by H2O2 [37]. We therefore also investigated the effects of the EGF receptor kinase inhibitor AG1478 in our adult myocyte system. The data revealed that AG1478 inhibited H2O2-induced ERK activation in a dose-dependent manner (Fig. 8C), suggesting that the signalling mechanisms underlying this response are likely to involve activation of the EGF receptor.


Figure 9
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  		Fig. 9 Time-dependent effects of H2O2 on Src tyrosine kinase activity. Cells were exposed to vehicle or 100 µM H2O2 for 1, 3, 6, 10, 20 or 30 min. Autoradiograms show representative Western blots with phosphospecific (top panel) and non-phosphospecific (bottom panel) antibodies for Src tyrosine kinase. *P<0.05 versus control (four experiments, with cells from four hearts).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The present study has shown that exposure to H2O2 stimulates sarcolemmal NHE activity in freshly isolated adult rat ventricular myocytes. Our data extend earlier findings of accelerated recovery from intracellular acidosis in response to H2O2 in cultured neonatal rat ventricular myocytes [20], and provide additional evidence that H2O2-induced stimulation of NHE activity requires activation of not only the ERK (but not the p38) pathway of the MAPK cascade, but also PKC. Intriguingly, unlike in neonatal myocytes [20], in our adult myocyte preparation PKC and ERK activation are not proximal and distal components, respectively, of a contiguous signalling pathway. Instead, H2O2-induced ERK activation in the adult myocyte appears to occur through the activation of tyrosine kinases of both receptor and non-receptor families.

Fig. 10 is based on the data obtained in the present study with rationally selected concentrations of specific kinase inhibitors (shown in italics) and the information that is available in the literature, and illustrates the signalling processes that are likely to mediate H2O2-induced stimulation of sarcolemmal NHE activity in the adult myocyte. According to this model, H2O2-induced stimulation of sarcolemmal NHE activity requires the parallel activation of PKC and ERK, in an analogous manner to {alpha}1A-adrenoceptor-mediated stimulation of the exchanger [28]. In response to H2O2, activation of both PKC and ERK may be facilitated through the activation of non-receptor tyrosine kinases, including members of the Src family. Exposure to H2O2 has been shown to activate a variety of PKC isozymes, including PKC{delta} and PKC{varepsilon} (the predominant PKC isozymes in adult rat myocardium [24]), in transfected COS-7 cells, through tyrosine phosphorylation by unidentified kinases [38]. ERK is also activated by H2O2, as shown by previous work in neonatal myocytes [20,34,35] and the present study in adult myocytes. Although it has been suggested that this activation may occur through a PKC-mediated pathway in cultured neonatal myocytes [20,34], contradictory data have also been reported [35]. Our present data with GF109203X and chelerythrine oppose a PKC-mediated mechanism for H2O2-induced ERK activation in freshly isolated adult myocytes. Instead, our data suggest that ERK activation in these cells occurs through tyrosine kinase-mediated mechanisms that involve both Src family enzymes (which are inhibited selectively by PP2) and the EGF receptor (which is inhibited selectively by AG1478). EGF receptor activation has been suggested previously to mediate ERK activation in response to H2O2 in vascular smooth muscle cells [37]; furthermore, recent evidence suggests that c-Src can facilitate EGF receptor activation by direct phosphorylation of the receptor [36]. Such complementary interaction between Src family kinases and the EGF receptor would be consistent with our observations that genistein (a non-selective tyrosine kinase inhibitor), PP2 and AG1478 each inhibited H2O2-induced ERK activation in adult ventricular myocytes. In this context, it is noteworthy that, in adult guinea pig hearts, perfusion with H2O2 activates Src, as well as ERK and p90rsk [39]. Furthermore, in neonatal myocytes, overexpression of C-terminal Src kinase (a negative regulator of Src family tyrosine kinases) by transfection has been shown to inhibit H2O2-induced ERK activation [35]. On the basis of the above, it is likely that H2O2 activates PKC through tyrosine phosphorylation (by an as yet unidentified kinase) and ERK through Src-dependent EGF receptor activation and subsequent stimulation of the classical Ras-Raf-MEK pathway.


Figure 10
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  		Fig. 10 Potential integration of signalling pathways in H2O2-induced stimulation of sarcolemmal Na+/H+ exchanger activity in adult rat ventricular myocytes. The kinase inhibitors used in the present study are shown in italics, with their target enzymes indicated (see Section 4 for details).

 
A potential limitation of the present study is the reliance on pharmacological kinase inhibitors to dissect the intracellular signalling pathways that mediate H2O2-induced stimulation of sarcolemmal NHE activity. In previous studies in a variety of cell types, these inhibitors have been used widely as pharmacological tools and have contributed significantly to the delineation of the physiological roles of pertinent pathways. Furthermore, extensive characterisation of inhibitors such as PD98059 [40], SB203580 [41], GF109203X [42], chelerythrine [43], AG1478 [44], and PP2 [45] indicates that, over the carefully selected concentration ranges used in the present study, they inhibit their target kinases but not a variety of other common kinases. Indeed, recent reports of non-specific effects of some of these kinase inhibitors [31,46] have observed such effects at concentrations that are considerably higher than those which produced significant effects in the present study.

An issue that is pertinent to the scheme proposed in Fig. 10 is how parallel activation of ERK and PKC may result in the stimulation of sarcolemmal NHE activity. Activation of the ERK pathway can lead to the phosphorylation of serine residues in the regulatory C-terminal domain of the NHE-1 protein, either directly by ERK itself [47] or indirectly through p90rsk [32], in a manner that may alter the interaction of this domain with accessory protein(s) that regulate exchanger activity. PKC, in contrast, does not directly phosphorylate the regulatory domain of NHE-1 [48] and, as we have suggested previously [28], may promote the stimulation of NHE activity by producing a concomitant change in the phosphorylation status of pertinent accessory protein(s).

There are likely to be cell type-dependent differences in the manner in which p90rsk activity is regulated in response to H2O2. The ability of the MEK inhibitor PD98059 to inhibit H2O2-induced p90rsk activation in the present study suggests that such activation in adult rat ventricular myocytes occurs predominantly through ERK. In contrast, Abe et al. [49] have shown that, in cultured fibroblasts and Jurkat cells, H2O2-induced p90rsk activation occurs through an ERK-independent pathway that is mediated via Fyn and Ras.

In conclusion, the present study has shown that H2O2 stimulates sarcolemmal NHE activity of adult rat ventricular myocytes, through the concomitant (but independent) activation of PKC- and ERK-mediated signalling pathways. In these cells, H2O2-induced stimulation of ERK occurs through Src family kinase- and EGF receptor kinase-mediated mechanisms. H2O2-induced stimulation of sarcolemmal NHE activity is likely to have functional consequences, particularly in the setting of myocardial ischaemia and reperfusion.

Time for primary review 25 days.


   Acknowledgements
 
This work was supported by a grant from the Dunhill Medical Trust. MA holds a Basic Science Award (BS/93002) from the British Heart Foundation.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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