ERK Is Regulated by Sodium-Proton Exchanger in Rat Aortic Vascular Smooth Muscle Cells

The purposes of this study were to test 1) the relationship between two widely studied mitogenic effector pathways, and 2) the hypothesis that sodium-proton exchanger type 1 (NHE-1) is a regulator of extracellular signal-regulated protein kinase (ERK) activation in rat aortic smooth muscle (RASM) cells. Angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) stimulated both ERK and NHE-1 activities, with activation of NHE-1 preceding that of ERK. The concentration-response curves for 5-HT and Ang II were superimposable for both processes. Inhibition of NHE-1 with pharmacological agents or by isotonic replacement of sodium in the perfusate with choline or tetramethylammonium greatly attenuated ERK activation by 5-HT or Ang II. Similar maneuvers significantly attenuated 5-HT- or Ang II-mediated activation of MEK and Ras but not transphosphorylation of the epidermal growth factor (EGF) receptor. EGF receptor blockade attenuated ERK activation, but not NHE-1 activation by 5-HT and Ang II, suggesting that the EGF receptor and NHE-1 work in parallel to stimulate ERK activity in RASM cells, converging distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway. Receptor-independent activation of NHE-1 by acute acid loading of RASM cells resulted in the rapid phosphorylation of ERK, which could be blocked by pharmacological inhibitors of NHE-1 or by isotonic replacement of sodium, closely linking the proton transport function of NHE-1 to ERK activation. These studies identify NHE as a new regulator of ERK activity in RASM cells. The purposes of this study were to test 1) the relationship between two widely studied mitogenic effector pathways, and 2) the hypothesis that sodium-proton exchanger type 1 (NHE-1) is a regulator of extracellular signal-regulated protein kinase (ERK) activation in rat aortic smooth muscle (RASM) cells. Angiotensin II (Ang II) and 5-hydroxytryptamine (5-HT) stimulated both ERK and NHE-1 activities, with activation of NHE-1 preceding that of ERK. The concentration-response curves for 5-HT and Ang II were superimposable for both processes. Inhibition of NHE-1 with pharmacological agents or by isotonic replacement of sodium in the perfusate with choline or tetramethylammonium greatly attenuated ERK activation by 5-HT or Ang II. Similar maneuvers significantly attenuated 5-HT- or Ang II-mediated activation of MEK and Ras but not transphosphorylation of the epidermal growth factor (EGF) receptor. EGF receptor blockade attenuated ERK activation, but not NHE-1 activation by 5-HT and Ang II, suggesting that the EGF receptor and NHE-1 work in parallel to stimulate ERK activity in RASM cells, converging distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway. Receptor-independent activation of NHE-1 by acute acid loading of RASM cells resulted in the rapid phosphorylation of ERK, which could be blocked by pharmacological inhibitors of NHE-1 or by isotonic replacement of sodium, closely linking the proton transport function of NHE-1 to ERK activation. These studies identify NHE as a new regulator of ERK activity in RASM cells. Activation of several major effectors has been linked to mitogenic stimuli. Two of those effectors are the extracellular signal-regulated protein kinase (ERK) 1The abbreviations used are: ERK, extracellular signal-regulated protein kinase; Ang II, angiotensin II; BCECF, 2′-7′-bis2-carboxymethyl-56-carboxyfluorescein; CaM, calmodulin; EGF, epidermal growth factor; ECAR, extracellular acidification rate; EIPA, 5-N-ethyl-N-isopropyl-amiloride; FLIPR, fluorescent imaging plate reader; 5-HT, 5-hydroxytryptamine; MEK, mitogen- and extracellular signal-regulated kinase kinase; MIA, 5-N-methyl-N-isobutyl-amiloride; NHE, Na+/H+ exchanger; NHE-1, type 1 NHE; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RASM, rat aortic smooth muscle cells. type of mitogen-activated protein kinases and Na+/H+ exchangers (NHE). ERK is one member of a family of kinases that participate in mitogenic signaling through complex phosphorylation cascades that convert cell surface signals into nuclear transcription programs. In the typical scenario, GTP-bound Ras, a small G protein, activates Raf kinase. In an alternative scenario, Raf is activated by protein kinase C or other signaling molecules. In either case, Raf phosphorylates and activates mitogen and extracellular signal-regulated kinases kinase (MEK), which in turn phosphorylates and activates ERK. Activated ERK translocates to the nucleus, where it activates a number of transcription factors such as Elk-1. Thus, this pathway can be depicted in a linear form as follows: Ras-GTP (or protein kinase C) → Raf-1 kinase → MEK → ERK. NHEs are expressed at the surface of all mammalian cells, serving to regulate cell volume, intracellular pH (pHi), and transepithelial transport of Na+ and acid-base equivalents (1.Putney L.K. Denker S.P. Barber D.L. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 527-552Crossref PubMed Scopus (431) Google Scholar, 2.Noel J. Pouysségur J. Am. J. Physiol. 1995; 268: C283-C296Crossref PubMed Google Scholar). The signal transduction pathways involved in activating NHEs have been more elusive. Although ERK play relatively well defined roles in mitogenesis, those of NHEs are less clear in that NHEs are possibly permissive factors rather than absolute necessities for mitogenesis (3.Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar). Although it has been known for some time that mitogens typically activate both NHE and ERK in concert (2.Noel J. Pouysségur J. Am. J. Physiol. 1995; 268: C283-C296Crossref PubMed Google Scholar, 3.Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar, 4.Kapus A. Grinstein S. Wasan S. Kandasamy R. Orlowski J. J. Biol. Chem. 1994; 269: 23544-23552Abstract Full Text PDF PubMed Google Scholar, 5.Krump E. Nikitas K. Grinstein S. J. Biol. Chem. 1997; 272: 17303-17311Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), the exact relationships between NHE and ERK have only recently been explored in any great detail. For example, microinjection of activated Ras (6.Hagag N. Lacal J.C. Graber M. Aaronson S. Viola M.V. Mol. Cell. Biol. 1987; 7: 1984-1988Crossref PubMed Scopus (94) Google Scholar) or transfection of the Ha-Ras oncogene (7.Doppler W. Jaggi R. Groner B. Gene. 1987; 54: 145-151Crossref Scopus (79) Google Scholar, 8.Maly K. Uberall F. Loferer H. Doppler W. Oberhuber H. Groner B. Grunicke H.H. J. Biol. Chem. 1989; 264: 11839-11842Abstract Full Text PDF PubMed Google Scholar, 9.Kaplan D.L. Boron W.F. J. Biol. Chem. 1994; 269: 4116-4124Abstract Full Text PDF PubMed Google Scholar) stimulates NHE activity in fibroblasts. In those studies, Ras most likely increased NHE activity by an up-regulation of NHE message and protein via ERK-regulated transcriptional processes. Recent work has shown that short-term activation of ERK leads to rapid stimulation of NHE-1 in multiple cell types (platelets, erythrocytes, fibroblasts, MDCK-11 cells, rabbit skeletal muscle, and cultured rat neonatal and adult ventricular cardiomyocytes) when activated by diverse stimuli, including angiotensin II (Ang II), cannabinoid ligands, aldosterone, and H2O2 (10.Aharonovitz O. Granot Y. J. Biol. Chem. 1996; 271: 16494-16499Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 11.Bianchini L. L'Allemain G. Pouysségur J. J. Biol. Chem. 1997; 272: 271-279Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 12.Wang H. Silva N.L. Lucchesi P.A. Haworth R. Wang K. Michalak M. Pelech S. Fliegel L. Biochemistry. 1997; 36: 9151-9158Crossref PubMed Scopus (85) Google Scholar, 13.Sabri A. Byron K.L. Samarel A.M. Bell J. Lucchesi P.A. Circ. Res. 1998; 82: 1053-1062Crossref PubMed Scopus (161) Google Scholar, 14.Bouaboula M. Bianchini L. McKenzie F.R. 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P.A. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). 5-HT and Ang II ERK through a Na+/H+ in RASM of NHE by and angiotensin ERK in RASM cells by the of a MEK the activation of NHE by 5-HT and Ang II. studies that the MEK blocked ERK activation NHE activity and Similar were when the Ras-MEK-ERK pathway Thus, it that of MEK not the of Ang II or 5-HT to activate NHE in In of sodium isotonic with significantly attenuated ERK phosphorylation by both 5-HT and Ang II in RASM NHE activity by it of extracellular Similar were when sodium with choline were when cells were with Thus, blockade of NHE activation by of extracellular Na+ or with the ERK phosphorylation by two G receptor (Ang II and that were not of in the of activation of ERK, the of activation of ERK by Ang II and 5-HT in the and of and studies that the stimulation of ERK by both most in the of to the and that significantly the stimulation of ERK the of activation of ERK between of stimulation by either and this activation the activation of NHE, which for both Ang II and Thus, the that the activation of NHE that of ERK is with a for NHE in ERK activation in RASM cells. have recently shown that the and activate NHE in RASM cells through a protein kinase C pathway that intracellular CaM, and kinase Biochemistry. 42: PubMed Scopus Google Scholar). cells for with inhibitors and 1 that have been shown to 5-HT- and Ang activation of NHE-1 as well as a that not NHE-1 activation to the of 5-HT- and Ang phosphorylation of inhibitors blocked phosphorylation of a not 5-HT- and Ang stimulation the time a to phorbol 12-myristate ERK that blocked These are in with hypothesis that NHE-1 is a regulator of in rat aortic smooth muscle cells. Receptor-independent Activation of Na+/H+ in RASM in Activation of that activation of NHE in ERK activation, to a to stimulate Thus, used the acid loading as in the to the ERK phosphorylation an acid and the pH and that intracellular acidification not significantly the level of ERK phosphorylation when or of ERK phosphorylation not when not or in the of Thus, acidification of the cell not ERK when the cells were to the acid in the of extracellular a rapid in ERK phosphorylation These that ERK phosphorylation can be when NHE-1 is activated through a pathway an acid either that NHE-1 is to activate ERK in cells or that a rapid to is for ERK phosphorylation NHE is the pathway that the pH in those to between a for proton and intracellular acid the cells in to NHE with to a rapid and the level of ERK The in that of activation of the in ERK phosphorylation more NHE activity stimulated by isotonic to the cells, suggesting that NHE activity is for ERK of NHE to MEK and Ras in the ERK Activation NHE is a of the signaling pathway through which ERK it be between known of the ERK activation pathway. that in ERK phosphorylation by 1 5-HT or Ang II the shown in and with attenuated MEK phosphorylation by either 5-HT or Ang II suggesting that NHE is of Ras activity the both 5-HT and Ang II a of Ras activity in the of In the of MIA, a activation of Ras activated Ras the of NHE of can is that NHE is at or above the level of Ras in this pathway. ERK by NHE of EGF is that activate ERK in smooth muscle cells through and phosphorylation of the EGF receptor S. H. K. F. Y. 1999; PubMed Google Scholar, M. L. S. Biol. PubMed Scopus Google Scholar, A. G. N. Y. PubMed Scopus (85) Google Scholar, L. S. E. P. S. Am. J. Physiol. Physiol. 2002; PubMed Scopus Google Scholar, K. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to NHE ERK activation in RASM cells of the EGF receptor. that isotonic of sodium with tetramethylammonium not ERK activation. of RASM cells with not in ERK These that activation of ERK either of or in parallel with NHE activation. to study the pathway by which EGF activates NHE in RASM cells of RASM cells with not the of EGF to activate NHE, of a for pathway in NHE activation. test the of in NHE activation by EGF, cells with a for to stimulation with not NHE activation by EGF but to proton that this blocked in cells with two inhibitors of and 1 both of which NHE activation. EGF, to 5-HT and Ang II, stimulates NHE activation in RASM cells via and pathway. that of kinase function with greatly in but has Ang or in These that is not for Ang II or 5-HT to activate NHE and that the is not of NHE when NHE is activated by Ang II or that both 5-HT and Ang II phosphorylation of the and that of NHE with has Ang or transphosphorylation of the that NHE is not of phosphorylation when it is through by Ang II or The relationships between NHE and ERK have been the of a number of studies the both can mitogenic and both are activated by stimuli, it has been that one be a regulator for the in some cell types ERK a clear in either the or activation of in other cell types ERK has not been to play a in the activation of The study the roles of NHE and ERK stimulated by either 5-HT or Ang II) in the activation of other in RASM cells. is new this work is that have to a for NHE in the activation of ERK in RASM cells. 1) stimulation of NHE and ERK by Ang II and 5-HT, with the activation of NHE preceding that of 2) concentration-response relationships for the stimulation of NHE and the phosphorylation of ERK by 5-HT and blockade of the activation of ERK by 5-HT and Ang II by of NHE; blockade of the activation of ERK by 5-HT and Ang II by of sodium and phosphorylation of ERK an acid a that activation of in the of activation of ERK, NHE to be of MEK and ERK and parallel to Ang and of the EGF receptor. NHE the pathway of activation of ERK at or above the level of The of the blockade of ERK phosphorylation by maneuvers that NHE that this is not an one that can for Although the of ERK by NHE-1 has not been it is not when one that inhibitors of NHE have been shown to in a rat of M. M. Circ. Res. PubMed Scopus Google Scholar). Ang II and 5-HT are both and Ang II has been shown to play major roles in including ventricular and H. PubMed Google Scholar). Thus, have for the of as well as for The relationships between ERK and NHE to be in RASM cells NHE activation is closely with activation of ERK. of NHE activity by the exchanger of extracellular sodium or by blockade with or activation of ERK by two G receptor ligands, Ang II and maneuvers have ERK, suggesting that are some in the pathways used by Ang II and 5-HT to activate ERK when with that used by the between NHE and ERK activation is by the that activation of NHE in ERK phosphorylation only when the exchanger is to an intracellular acid Thus, NHE activation is for Ang and activation of ERK and is to activate ERK of an acid In NHE activation is not for activation of ERK. The most likely is that 5-HT or Ang II the parallel activation of NHE and the to activate ERK in RASM cells. The two pathways of transphosphorylation of the and of ERK and The most likely is at or of Ras, in both the and to be is depicted in the in Although one could that NHE is not of the 5-HT- and Ang one that Ang and of the not activation of the is that NHE an in Ang and activation of ERK by or by Na+ or in activity or The the of NHE is in of work by Barber and (1.Putney L.K. Denker S.P. Barber D.L. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 527-552Crossref PubMed Scopus (431) Google Scholar, Barber D.L. Mol. Biol. Cell. 1998; PubMed Scopus Google Scholar, S.P. Orlowski J. H. Barber D.L. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, S.P. Barber D.L. J. Biol. 2002; PubMed Scopus Google Scholar) between NHE activity and the some of NHE-1 not S.P. Barber D.L. J. Biol. 2002; PubMed Scopus Google Scholar). In that the for transport by NHE-1 in the activation of ERK by Ang II and The in that the of intracellular acidification is not to activate ERK. of pH in the of sodium is suggesting a for NHE in this not to between a for proton and intracellular is likely that the of NHE in G activation of ERK be to cell types and have only been to this relationship in cells of N. J. and J. R. have not a of cells. of the work in this has the of ERK in activating NHE, that relationship to cell type and the used to stimulate in work by Y. E. Y. J. Pharmacol. 1999; PubMed Scopus Google Scholar) in which activation of NHE in cells could be blocked by a MEK growth activation of NHE could The of the relationship between NHE-1 and ERK could be by of signaling pathway or by of signal transduction PubMed Scopus Google Scholar). In any case, the work that NHE can regulate ERK in RASM cells. In have that in RASM cells NHE activation a in Ang and activation of ERK. this stimulation of ERK through an pathway could be by acid loading of RASM cells, a of activating with EGF that EGF receptor for Ang and activation of ERK but in Ang and activation of In NHE activation in EGF stimulation of ERK. studies that the and EGF pathways distal to the EGF receptor but at or above the level of Ras in the Ras-MEK-ERK pathway Thus, studies identify NHE as a new regulator of ERK activity in RASM cells. and for with cell