Roles of Mitogen-Activated Protein Kinases and Protein Kinase C in α1A-Adrenoceptor–Mediated Stimulation of the Sarcolemmal Na+-H+ Exchanger
Abstract—Activation of the sarcolemmal Na+-H+ exchanger (NHE) has been implicated as a mechanism of inotropic, arrhythmogenic, antiacidotic, and hypertrophic effects of α1-adrenoceptor (AR) stimulation. Although such regulation of sarcolemmal NHE activity has been shown to be selectively mediated through the α1A-AR subtype, distal signaling mechanisms remain poorly defined. We investigated the roles of various kinase pathways in α1A-AR–mediated stimulation of sarcolemmal NHE activity in adult rat ventricular myocytes. As an index of NHE activity, trans-sarcolemmal acid efflux rate (JH) was determined through microepifluorescence in single cells, during recovery from intracellular acidosis in bicarbonate-free conditions. Extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase (MAPK), and p90rsk activities were indexed on the basis of analysis of their phosphorylation status. In control cells, there was no change in JH in response to vehicle. Phenylephrine and A61603, an α1A-AR subtype–selective agonist, increased JH, as well as cellular ERK and p90rsk activities. Neither agonist affected p38 activity, which was increased with sorbitol. The MAPK kinase inhibitor PD98059 abolished phenylephrine- and A61603-induced increases in JH and cellular ERK and p90rsk activities. In contrast, the PKC inhibitor GF109203X abolished phenylephrine- and A61603-induced increases in JH but failed to prevent the increases in ERK and p90rsk activities. Our findings suggest that α1A-AR–mediated stimulation of sarcolemmal NHE activity in rat ventricular myocytes requires activation of the ERK (but not the p38) pathway of the MAPK cascade and that the ERK-mediated effect may occur via p90rsk. Activation of PKC is also required for α1A-AR–mediated NHE stimulation, but such regulation occurs through an ERK-independent pathway.
The sarcolemmal Na+-H+ exchanger (NHE) consists of the ubiquitous NHE-1 isoform of the multigene NHE family1 and is an important H+ extrusion mechanism that contributes to the integrated control of intracellular pH (pHi) in cardiac myocytes.2 Although sarcolemmal NHE activity is regulated primarily by pHi and is markedly increased in response to acidosis,2 it is also subject to modulation by several stimuli that act via Gq protein–coupled receptors (GqPCRs), such as α1-adrenergic agonists,3 endothelin,4 thrombin,5 and angiotensin II.6 These stimuli increase sarcolemmal NHE activity by enhancing the affinity of the exchanger for intracellular H+, which is the primary mechanism underlying receptor-mediated regulation of NHE-1.7
Of the various GqPCR signaling pathways that regulate sarcolemmal NHE activity, those that are activated by α1-adrenoceptors (α1-ARs) warrant attention because they are likely to mediate important physiological and pathophysiological responses. In this regard, increased sarcolemmal NHE activity and consequent increases in pHi, intracellular Na+, or both have been suggested to be causally involved in the positive inotropic,8 arrhythmogenic,9 and hypertrophic10 11 consequences of myocardial α1-AR stimulation. Furthermore, α1-AR–mediated stimulation of sarcolemmal NHE activity may contribute to the antiacidotic effect during ischemia of ischemic or pharmacological preconditioning.12 In an effort to delineate the molecular mechanisms that underlie α1-adrenergic stimulation of sarcolemmal NHE activity, we recently demonstrated that such regulation of the exchanger is mediated selectively through the α1A-AR subtype.13 Nevertheless, pertinent signaling pathways distal to the GqPCR remain controversial (eg, see Wallert and Fröhlich3 versus Pucéat et al14 on the role of protein kinase C [PKC]) and incompletely characterized.
The results of recent studies in noncardiac cells suggest that intracellular signals transduced via the extracellular signal-regulated kinase (ERK)15 16 17 and p3818 pathways of the mitogen-activated protein kinase (MAPK) cascade may be important contributors to GqPCR-mediated regulation of NHE-1 activity. Furthermore, GqPCR (including α1-AR) stimulation has been shown to activate both ERK and p38 in isolated rat hearts19 and cultured neonatal rat ventricular myocytes,20 21 through mechanisms that may involve PKC.21 22 However, the potential roles and interactions of ERK, p38, and PKC in α1A-AR–mediated regulation of sarcolemmal NHE activity have not been investigated.
The present study was undertaken to determine the involvement of ERK, p38, and PKC pathways in α1A-AR–mediated stimulation of sarcolemmal NHE activity in freshly isolated adult rat ventricular myocytes. To achieve this, we used established techniques for the determination of NHE and various kinase activities, in conjunction with 2 agonists of distinct α1-AR subtype selectivity and specific kinase inhibitors. Our data suggest that α1A-AR–mediated stimulation of sarcolemmal NHE activity in adult rat ventricular myocytes requires activation of the ERK (but not the p38) pathway of the MAPK cascade. Activation of PKC is also required for this response, but PKC and ERK appear to be independent regulators of NHE activity in response to α1A-AR stimulation.
Materials and Methods
This investigation was performed in accordance with the Home Office “Guidance on the Operation of the Animals (Scientific Procedures) Act 1986,” published by HMSO (London).
Isolation of Ventricular Myocytes
Ventricular myocytes were isolated from the hearts of adult male Wistar rats (weight 200 to 250 g) through enzymatic digestion for the study of drug effects on sarcolemmal NHE5 6 13 23 or cellular kinase6 activity.
Determination of Sarcolemmal NHE Activity
Sarcolemmal NHE activity was determined in single myocytes loaded with the pH-sensitive fluoroprobe cSNARF-1, through the use of a microepifluorescence technique.5 6 13 23 Cells were maintained in bicarbonate-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. To quantify drug-induced changes in NHE activity, JH values were determined at pHi intervals of 0.05 during recovery from intracellular acidosis.
Determination of Cellular MAPK and p90rsk Activities
MAPK activities were determined through the detection of dual phosphorylation of ERK1/2 and p38 on the Thr and Tyr residues of their regulatory Thr-Xaa-Tyr motifs, by Western analysis with dual phosphospecific antibodies (New England Biolabs).6 The activity of p90rsk was determined through the detection of Ser381 phosphorylation with a phosphospecific antibody (New England Biolabs). To confirm equal protein loading, we used nonphosphospecific antibodies for ERK2 (Santa Cruz Biotechnology), p38 (Santa Cruz Biotechnology), and p90rsk (Transduction Laboratories). Specific protein bands were detected with enhanced chemiluminescence and autoradiography, and phosphorylation status was quantified with laser densitometry.
For the determination of drug effects on NHE activity, myocytes (10 per group, obtained from 7 to 9 separate hearts in each protocol) were subjected to intracellular acidosis through transient (3 minutes) exposure to 20 mmol/L NH4Cl (first acid pulse), which was repeated ≈15 minutes later (second acid pulse).5 6 13 In control cells, both acid pulses occurred in the absence of any drug. When the effects of phenylephrine (Sigma), a non–subtype-selective α1-AR agonist, or A61603 (gift from Abbott Laboratories), an α1A-AR subtype–selective agonist, were studied, this was present during the second pulse. When the effects of either agonist in the presence of the MAPK kinase (MEK) inhibitor PD98059 (Calbiochem-Novabiochem) or the PKC inhibitor GF109203X (Calbiochem-Novabiochem) were studied, the inhibitor was present from 10 minutes before the second acid pulse. Drug vehicles were included in superfusion solutions, as appropriate. For determination of the effects on kinase activity, myocytes in suspension were exposed to the same drugs with the use of identical concentrations and exposure times (4 experiments with each protocol, with cells from 4 separate hearts).
Values are given as mean±SEM. Experiments in each microepifluorescence protocol were randomized, with contemporary controls. A paired t test was used to assess changes in JH between the first and second acid pulses. For an intergroup comparison of the change in JH at pHi 6.90 (ΔJH6.9) or of protein kinase phosphorylation, data were subjected to ANOVA; further analysis was made with Dunnett’s test to compare each treatment group with the control group. P<0.05 was considered significant.
Role of ERK1/2 in α1A-AR–Mediated Stimulation of Sarcolemmal NHE Activity
Figure 1⇓ shows the JH-versus-pHi relationships obtained after 2 consecutive acid pulses in the 6 groups of this protocol, which addressed the role of ERK1/2 in α1A-AR–mediated stimulation of sarcolemmal NHE activity. During the second acid pulse, control cells (Figure 1A⇓) continued to receive agonist-free superfusate, whereas the other groups were exposed to phenylephrine (Figure 1B⇓) or A61603 (Figure 1C⇓) in the absence or presence of PD98059. The concentrations of phenylephrine and A61603 were selected on the basis of our dose-response studies13 and were those that produced near-maximal stimulation of sarcolemmal NHE activity. In control cells, the JH-versus-pHi curves obtained after both acid pulses were superimposed (Figure 1A⇓, left), indicating that temporal changes in NHE activity do not occur in the absence of drug exposure. In these cells, PD98059 alone had no effect on the JH-versus-pHi curve (Figure 1A⇓, right). Consistent with our previous data that α1A-AR stimulation increases sarcolemmal NHE activity,13 phenylephrine and A61603 both produced rightward shifts of the JH-versus-pHi curve such that over the range of pHi 6.80 to 7.20, JH was significantly greater in the presence of either agonist (Figures 1B⇓ and 1C⇓, left). However, in the presence of PD98059, neither phenylephrine nor A61603 produced a significant shift in the JH-versus-pHi curve (Figures 1B⇓ and 1C⇓, right). Figure 3A⇓ shows ΔJH6.9 values in the 6 study groups and allows a comparison of the effects of the different stimuli on sarcolemmal NHE activity. As illustrated, in the absence of PD98059, ΔJH6.9 was significantly greater in cells that received phenylephrine or A61603. In contrast, in the presence of PD98059, there was no significant difference in ΔJH6.9 between control cells and those exposed to either α1-AR agonist. Because PD98059 inhibits Raf-mediated activation of MEK1/2,24 which in turn activates ERK1/2,25 these data suggest that activation of the ERK pathway is a necessary step in α1A-AR–mediated stimulation of sarcolemmal NHE activity.
Role of PKC in α1A-AR–Mediated Stimulation of Sarcolemmal NHE Activity
Figure 2⇓ shows the JH-versus-pHi relationships obtained in this protocol, which was analogous to that described earlier except that it tested the role of the PKC pathway. In control cells, the JH-versus-pHi curves obtained after both acid pulses were again superimposed (Figure 2A⇓, left). GF109203X had no effect on the JH-versus-pHi curve in control cells (Figure 2A⇓, right) but abolished the rightward shifts of the curve induced by phenylephrine (Figure 2B⇓) or A61603 (Figure 2C⇓). As illustrated in Figure 3B⇓, in the absence of GF109203X, ΔJH6.9 was again significantly greater in cells that received phenylephrine or A61603, reflecting α1A-AR–mediated stimulation of sarcolemmal NHE activity. In contrast, in the presence of GF109203X, there was no significant change in ΔJH6.9 in response to either α1-AR agonist. Because GF109203X is a selective inhibitor of PKC,26 these data suggest that PKC is a critical component of the distal signaling pathways of the α1A-AR that mediate the stimulation of sarcolemmal NHE activity.
Regulation of Sarcolemmal NHE Activity via PKC and ERK1/2: Contiguous or Independent Pathways?
These data suggest that in α1A-AR–mediated stimulation of sarcolemmal NHE activity, both PKC and ERK1/2 are critical components of the signaling pathways distal to the GqPCR. This situation could arise if (1) PKC and ERK1/2 are proximal and distal components of a contiguous signaling pathway or (2) PKC and ERK1/2 mediate independent signaling pathways, but activation of both is necessary to achieve the full response. To address this issue, we determined the effects on ERK activity of α1-AR stimulation in the absence or presence of GF109203X. Figure 4⇓ shows that in parallel with their effects on sarcolemmal NHE activity, phenylephrine and A61603 produced significant increases in ERK activity. GF109203X failed to prevent significant increases in ERK activity in response to phenylephrine and A61603, whereas PD98059 abolished ERK activation by each agonist. The distinct effects of the 2 kinase inhibitors on ERK activation (Figure 4⇓), despite their common ability to prevent α1A-AR–mediated stimulation of sarcolemmal NHE activity (Figure 3⇑), allow the following conclusions to be made: (1) in adult rat ventricular myocytes, α1-AR–mediated activation of ERK1/2 occurs, to a large extent, through PKC-independent mechanisms, and (2) activation of both pathways is required for α1A-AR–mediated stimulation of sarcolemmal NHE activity.
The lack of an effect of GF109203X on ERK activation might reflect the absence of PKC-mediated ERK regulation or the dissociation of such regulation from the α1-AR–mediated response. To address this issue, we determined the effects on ERK activity of direct PKC activation by phorbol 12-myristate 13-acetate (PMA). As shown in Figure 5⇓, PMA produced a significant increase in ERK activity, indicating that PKC-mediated ERK activation is functional in adult rat ventricular myocytes. Figure 5⇓ also shows that GF109203X abolished PMA-induced ERK activation, thus confirming that the concentration used was sufficient to block PKC-mediated responses.
Role of p38 in α1A-AR–Mediated Stimulation of Sarcolemmal NHE Activity
In some cardiac preparations,19 21 α1-AR stimulation has been shown to activate p38, which has been implicated in GqPCR-mediated regulation of plasma membrane NHE activity in rat vascular smooth muscle cells.18 Therefore, we tested whether p38 could also be involved in α1A-AR–mediated regulation of sarcolemmal NHE activity in adult rat ventricular myocytes. However, as illustrated in Figure 6⇓, neither phenylephrine nor A61603 produced a significant increase in p38 activity. In contrast, osmotic stress, induced by exposure to 0.5 mol/L sorbitol and used as a positive control, produced a significant increase in p38 activity (Figure 6⇓). The common inability of the α1-AR agonists to increase p38 activity at concentrations that were sufficient to increase sarcolemmal NHE activity precludes a role for the p38 pathway in α1A-AR–mediated regulation of the exchanger.
Downstream Effectors of ERK1/2
The 90-kDa ribosomal S6 kinase (p90rsk), which is activated by ERK1/2, has been shown to phosphorylate the regulatory domain of NHE-117 27 28 and may mediate serum- or endothelin-induced stimulation of NHE activity in cultured fibroblasts27 and neonatal rat ventricular myocytes.28 To determine whether p90rsk could be a downstream effector in ERK-mediated regulation of the sarcolemmal NHE in adult rat ventricular myocytes, we determined the effects of α1-AR stimulation on the activity of this kinase. As shown in Figure 7⇓, both phenylephrine and A61603 significantly increased p90rsk activity. The activation of p90rsk by α1-AR stimulation was abolished by PD98059 but unaffected by GF109203X, suggesting that such activation occurred via an ERK-dependent but PKC-independent pathway. This is consistent with an effector role for p90rsk in ERK-mediated regulation of the sarcolemmal NHE, in response to α1A-AR stimulation.
Our main novel findings, which were obtained in adult rat ventricular myocytes, are that (1) inhibition of either MEK (the upstream activator of ERK1/2) or PKC abolishes stimulation of sarcolemmal NHE activity by the α1-AR agonists phenylephrine and A61603; (2) inhibition of MEK, but not PKC, abolishes ERK activation by both agonists; (3) activity of p90rsk, a putative NHE-1 kinase, is regulated in parallel with that of ERK1/2; and (4) phenylephrine and A61603 do not activate p38.
The ability of the MEK inhibitor PD98059 to abolish phenylephrine- and A61603-induced increases in the activities of both sarcolemmal NHE and cellular ERK1/2 provides the first evidence that ERK activation is a critical step in α1A-AR–mediated stimulation of the exchanger in adult rat ventricular myocytes. This finding, considered together with our recent work in the same system on the regulation of sarcolemmal NHE activity via the angiotensin II type 1 (AT1) receptor6 and other pertinent data from noncardiac cells15 16 17 and cultured neonatal rat ventricular myocytes,28 29 suggests that the ERK pathway is a critical regulator of NHE-1 activity in response to multiple stimuli in various cell types. Furthermore, the parallel changes observed in NHE, ERK1/2, and p90rsk activities in response to α1-AR stimulation, in the absence or presence of the MEK and PKC inhibitors, are consistent with an effector role for p90rsk in ERK-mediated regulation of the sarcolemmal NHE. In this regard, recent studies have revealed that the regulatory domain of NHE-1 is a substrate for p90rsk17 27 28 and that phosphorylation of NHE-1 by p90rsk at Ser703 stimulates exchanger activity.27
Our finding that PKC inhibition by GF109203X also abolishes α1A-AR–mediated stimulation of sarcolemmal NHE activity supports earlier data from Wallert and Fröhlich,3 who studied the effects on exchanger activity of the α1-AR agonist 6-fluoronorepinephrine, and from studies with other GqPCR agonists, such as endothelin,4 thrombin,5 and angiotensin II.6 In another pertinent study,14 however, GF109203X was shown not to inhibit phenylephrine-induced stimulation of sarcolemmal NHE activity. In that study,14 phenylephrine was used at a concentration of 100 μmol/L, which is 10-fold greater than that used in our present work. Furthermore, this concentration is ≥80-fold greater than the EC50 value of phenylephrine for stimulation of sarcolemmal NHE activity13 or phosphoinositide hydrolysis30 in adult rat ventricular myocytes and for translocation of PKCε in neonatal rat ventricular myocytes.20 To determine whether the difference in agonist concentration could account for the contrasting effects of GF109203X in our study and that by Pucéat et al,14 we carried out additional experiments with a 10-fold greater concentration (100 μmol/L) of phenylephrine. In these experiments, GF109203X failed to inhibit the stimulation of sarcolemmal NHE activity by phenylephrine, which increased JH6.9 from 4.1±0.5 to 9.5±0.8 mmol · L−1 · min−1 (P<0.05) when administered alone and from 3.5±0.6 to 8.4±1.2 mmol · L−1 · min−1 (P<0.05) when administered after pretreatment with GF109203X (8 cells per group, from 3 hearts). This suggests that in the presence of a supramaximal α1-AR agonist concentration, non–PKC-mediated pathways may be sufficient to effect increased sarcolemmal NHE activity. However, with agonist concentrations that are likely to be of greater physiological relevance, PKC activation appears to be a necessary component of the pertinent signaling pathways distal to the α1A-AR.
The common ability of GF109203X and PD98059 to inhibit α1A-AR–mediated stimulation of sarcolemmal NHE activity may suggest that PKC and ERK are proximal and distal components, respectively, of a contiguous NHE-regulatory signaling pathway. Indeed, our recent work in an identical system has shown that PKC and ERK1/2 participate in such a contiguous pathway in response to AT1 receptor stimulation.6 In our present work, however, ERK activation by the α1-AR agonists was not prevented by GF109203X (Figure 4⇑). This indicates that PKC and ERK1/2 mediate largely independent signaling pathways and that the activation of both pathways is necessary to achieve α1A-AR–mediated stimulation of sarcolemmal NHE activity. With regard to the inability of GF109203X to prevent ERK activation by α1-adrenergic stimulation, it is notable that α1A-AR–mediated ERK activation has recently been reported to be PKC independent in PC12 cells stably transfected with this receptor subtype.31 Furthermore, at a concentration of 1 μmol/L, GF109203X has been shown to produce only marginal inhibition of endothelin-induced ERK activation in neonatal rat ventricular myocytes.21 This observation is similar to our present findings in adult rat ventricular myocytes, in which the same concentration of GF109203X reduced the magnitude but did not prevent the occurrence of significant ERK activation by α1-adrenergic stimulation (Figure 4⇑). We did not test higher concentrations of GF109203X because 1 μmol/L was sufficient to abolish PMA-induced ERK activation (which confirms that it was sufficient to inhibit PKC-mediated responses) and due to concern for potential nonspecific effects.26
Significant ERK activation was achieved through the exposure of adult rat ventricular myocytes to PMA (Figure 5⇑), which illustrates that PKC can function as a proximal activator of the ERK pathway in this cell type. However, our observation that GF109203X prevented ERK activation by PMA (Figure 5⇑) but not that by phenylephrine or A61603 (Figure 4⇑) indicates that the PKC-mediated mechanism is not the major mechanism of ERK activation in response to α1-adrenergic stimulation. This contrasts with our recent findings regarding ERK activation via the AT1 receptor6 and suggests the existence of receptor-specific differences in the role of PKC in GqPCR-mediated ERK activation.
In contrast to recent reports in neonatal rat ventricular myocytes21 and intact adult rat hearts,19 we found no activation of p38 in response to either α1-AR agonist. This points toward a difference between neonatal and adult myocyte preparations in GqPCR-mediated regulation of p38 activity, although it is unclear whether this reflects a maturational difference or arises from the maintenance of neonatal cells in culture. It should also be noted that in the earlier studies, neonatal myocytes21 or isolated hearts19 were exposed to 100 μmol/L phenylephrine, which produced peak p38 activation after 10 minutes19 21 In contrast, in our study, myocytes were exposed to 10 μmol/L phenylephrine for 3 minutes (which was sufficient to stimulate the sarcolemmal NHE) before the assessment of p38 activity. These differences in agonist concentration and duration of exposure may contribute to the distinct findings. Regardless of these issues, the inability of phenylephrine and A61603 to alter p38 activity in the present study precludes a role for the p38 pathway in NHE-regulatory signaling mechanisms distal to the α1A-AR in adult rat ventricular myocytes.
Our results have shown that in isolated adult rat ventricular myocytes, α1A-AR–mediated stimulation of sarcolemmal NHE activity requires activation of the ERK (but not the p38) pathway of the MAPK cascade. Activation of PKC is also required for this response, but PKC and ERK are independent regulators of NHE activity in response to α1A-AR stimulation. Stimulation of NHE activity by the ERK pathway is likely to occur via activation of p90rsk, which phosphorylates the exchanger at Ser703 and may alter its interaction with accessory proteins that regulate exchanger activity.27 Although the mechanism through which PKC contributes to α1A-AR–mediated stimulation of NHE activity is unknown, PKC does not directly phosphorylate the regulatory domain of the exchanger,32 and altered phosphorylation of accessory proteins may play an important role. In view of the potential physiological and pathophysiological significance of α1-adrenergic stimulation of sarcolemmal NHE activity, further work is required to fully characterize the relevant signaling pathways.
This work was supported in part by grants from the Dunhill Medical Trust and the Special Trustees of Guy’s Hospital. Dr Avkiran holds a Senior Lectureship Award (BS/93002) from the British Heart Foundation.
- Received September 14, 1999.
- Accepted November 10, 1999.
- © 2000 American Heart Association, Inc.
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