Larry Fliegel

Structural and functional analysis of the Na+/H+ exchanger.

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.

Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

ABSTRACT Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.

Protein Kinase-mediated Regulation of the Na+/H+ Exchanger in the Rat Myocardium by Mitogen-activated Protein Kinase-dependent Pathways

We examined regulation of the Na+/H+ exchanger isoform 1 by phosphorylation in the rat myocardium. We utilized cell extracts from adult rat hearts, adult rat extracts fractionated by fast performance liquid chromatography, and extracts from cultured neonatal cardiac myocytes. The carboxyl-terminal 178 amino acids of the Na+/H+ exchanger were expressed inEscherichia coli fused with glutathioneS-transferase. The purified protein was used as a substrate for in vitro phosphorylation and in-gel kinase assays. Unfractionated extracts from neonatal myocytes or adult hearts phosphorylated the COOH-terminal domain of the antiporter. Western blot analysis revealed that mitogen-activated protein (MAP) kinase (44 and 42 kDa) and p90 rsk (90 kDa) were present in specific fractions of cardiac extracts that phosphorylated the COOH-terminal protein. In-gel kinase assays confirmed that protein kinases of approximately 44 and 90 kDa could phosphorylate this domain. MAP kinase and p90 rsk -dependent phosphorylation of the antiporter could be demonstrated by immunoprecipitation of these kinases from extracts of neonatal cardiac myocytes. PD98059, a mitogen-activated protein kinase kinase inhibitor, decreased MAP kinase and p90 rsk phosphorylation of the antiporter and abolished serum and endothelin 1-stimulated increases in steady-state pH i . These results confirm the presence of MAP kinase-dependent phosphorylation in the regulation of the Na+/H+exchanger in the rat myocardium and suggest an important role for p90 rsk phosphorylation in regulation of the protein by endothelin-mediated stimulation of the antiporter.

Ectopic expression of human and feline CD9 in a human B cell line confers beta 1 integrin-dependent motility on fibronectin and laminin substrates and enhanced tyrosine phosphorylation

Few molecules have been shown to confer cell motility. Although the motility-arresting properties of anti-CD9 monoclonal antibody (mAb) suggest the transmembrane 4 superfamily (TM4SF) member CD9 can induce a motorgenic signal, gene transfection studies have failed to confirm this hypothesis. We report here that ectopic expression of human CD9 (CD9h) and feline CD9 (CD9f) in the CD9-negative, poorly motile, human B cell line Raji dramatically enhances migration across fibronectin- and laminin-coated polycarbonate filters. Migration of Raji/CD9h and Raji/CD9f on either substrate was inhibited by the anti-CD9 mAb 50H.19 and by the anti-β1 integrin mAb AP-138. Migration of Raji/CD9h on laminin was potently inhibited by the anti-VLA-6 integrin mAb GoH3 and by the anti-VLA-4 integrin mAb 44H6, whereas migration of Raji/CD9h on fibronectin was inhibited only by mAb 44H6. Since CD9h-transfected Raji cells adhered to fibronectin as effectively as mock transfectants, expression of CD9 enhanced motility, but not adhesion. CD9-enhanced migration was inhibited by the protein tyrosine kinase inhibitor herbimycin A suggesting that tyrosine phosphorylation played a role in the generation of a motorgenic signal. Raji/CD9h transfectants adherent to fibronectin expressed 6-fold higher levels of phosphotyrosine than Raji. Raji/CD9f transfectants also phosphorylated proteins on tyrosine more effectively than Raji including a protein of 110 kDa which was phosphorylated on the motility-inducing substrates laminin and fibronectin, but not on bovine serum albumin. Our results support a role for CD9 in the amplification of a motorgenic signal in B cells involving β1 integrins and the activation of protein tyrosine kinases.

Aldosterone Increases NHE-1 Expression and Induces NHE-1-Dependent Hypertrophy in Neonatal Rat Ventricular Myocytes

Abstract—We determined the effect of 24-hour aldosterone (100 nmol/L) treatment on hypertrophic responses in rat neonatal ventricular myocytes and the possible role of Na+-H+ exchange isoform 1 (NHE-1). Aldosterone significantly increased cell size by 61% and expression of atrial natriuretic peptide by 2-fold. NHE-1 mRNA expression and protein abundance were significantly increased, and intracellular Na+ levels were elevated. Both hypertrophy and elevated Na+ levels were prevented by the NHE-1-specific inhibitor EMD87580 as well as the aldosterone antagonist spironolactone, although the increased NHE-1 levels were prevented only by spironolactone. Aldosterone transiently (within 5 minutes) stimulated p44/42 phosphorylation, which decreased thereafter for the remaining 24 hours, whereas p38 phosphorylation was reduced. Neither a p38 nor a p44/42 inhibitor had any effect on aldosterone-induced hypertrophy or NHE-1 regulation. Our results therefore demonstrate a direct hypertrophic effect of aldosterone on cultured myocytes, which is dependent on NHE-1 activity.

The Na+/H+ Exchanger: A Target for Cardiac Therapeutic Intervention

The Na(+)/H(+) exchanger (NHE) is a ubiquitous protein present in mammalian cells. In higher eukaryotes this integral membrane protein removes one intracellular H(+) for one extracellular Na(+) protecting cells from intracellular acidification. NHE is of essential importance in the myocardium. It prevents intracellular acidosis that inhibits contractility. NHE also plays a key role in damage to the mammalian myocardium that occurs during ischemia and reperfusion and is involved in hypertrophy of the myocardium. NHE is composed of a membrane bound domain of approximately 500 amino acids plus a hydrophilic regulatory cytoplasmic domain of approximately 315 amino acids. The NHE1 isoform is the only significant plasma membrane isoform present in the myocardium. The activity of NHE1 is elevated in animal models of myocardial infarcts and in left ventricular hypertrophy. During ischemia and reperfusion of the myocardium, NHE activity catalyzes increased uptake of intracellular sodium. This in turn is exchanged for extracellular calcium by the Na(+)/Ca(2+) exchanger resulting in calcium overload and damage to the myocardium. Numerous inhibitors of NHE have been developed to attempt to break this cycle of calcium overload. In animal models excellent success has been obtained in this regard. However in humans, clinical trials have resulted in only modest success and recently, significant detrimental side effects were note of one NHE inhibitor. The mechanisms by which these inhibitors affect NHE activity are presently being investigated and regions of the protein important in NHE activity and inhibitor efficacy are related but not identical. Future studies may develop superior inhibitors that may circumvent recently reported side effects. Recently, NHE inhibition has been shown to be remarkably effective in preventing hypertrophy in some animal models. Whether this proves to be a practical treatment for hypertrophy in humans has yet to be determined.

Induction of expression of the sodium-hydrogen exchanger in rat myocardium

OBJECTIVE The aim was to examine the regulation of the cardiac Na+/H+ exchanger NHE-1 isoform mRNA in response to ischaemia and acidosis in the mammalian myocardium. METHODS Male Sprague Dawley rat hearts were perfused in a non-circulated retrograde fashion according to the Langendorff method. Hearts were perfused for 3 h at flow rates of either 10 ml.min-1 (control), or 3, 1, or 0 ml.min-1 (ischaemia) followed by 5 min of reperfusion. Hearts were immediately frozen in liquid N2, and stored at -80 degrees C until ready for RNA isolation. Northern blot analysis was used to examine expression of the NHE-1 isoform of the Na+/H+ exchanger message in these isolated perfused hearts. Activity of the Na+/H+ exchanger was assessed in primary cultures of neonatal rat myocytes under either control conditions or after treatment with chronic, low external pH. RESULTS A decrease in developed tension and an increase in resting tension was observed which was dependent upon the severity of the ischaemic episode. Low flow ischaemia of 3 ml.min-1 caused increased Na+/H+ exchanger message levels, while perfusion at more reduced flow rates eliminated the increase. Treatment of primary cultures of isolated myocytes with low external pH resulted in increased ability to recover from an acute acid load. CONCLUSIONS Low flow ischaemia can increase the Na+/H+ exchanger message in the intact mammalian myocardium. More severe ischaemia prevents the increase, suggesting that severely damaged tissue may not be capable of the ischaemic response. Primary cultures of isolated myocytes can respond to chronic low external pH by increasing Na+/H+ exchanger activity.

Regulation of the Na+/H+ Exchanger (NHE1) in Breast Cancer Metastasis

The pH gradient in normal cells is tightly controlled by the activity of various pH-regulatory membrane proteins including the isoform protein of the Na(+)/H(+) exchanger (NHE1). NHE1 is constitutively active in a neoplastic microenvironment, dysregulating pH homeostasis and altering the survival, differentiation, and proliferation of cancer cells, thereby causing them to become tumorigenic. Cytoplasmic alkalinization in breast cancer cells occurs as a result of increased NHE1 activity and, while much is known about the pathophysiologic role of NHE1 in tumor progression with regard to ion flux, the regulation of its activity on a molecular level is only recently becoming evident. The membrane domain of NHE1 is sufficient for ion exchange. However, its activity is regulated through the phosphorylation of key amino acids in the cytosolic domain as well as by its interaction with other intracellular proteins and lipids. Here, we review the importance of these regulatory sites and what role they may play in the disrupted functionality of NHE1 in breast cancer metastasis.

Mitogen-activated Protein Kinase-dependent Activation of the Na+/H+ Exchanger Is Mediated through Phosphorylation of Amino Acids Ser770 and Ser771

We investigated regulation of the type 1 isoform of the Na+/H+ exchanger by phosphorylation. Four specific groups of serine and threonine residues in the regulatory carboxyl-terminal tail were mutated to alanine residues: group 1, S693A; group 2, T718A and S723A/S726A/S729A; group 3, S766A/S770A/S771A; and group 4, T779A and S785A. The proteins were expressed in Na+/H+ exchanger-deficient cells, and the activity was characterized. All of the mutants had proper expression, localization, and normal basal activity relative to wild type NHE1. Sustained intracellular acidosis was used to activate NHE1 via an ERK-dependent pathway that could be blocked with the MEK inhibitor U0126. Immunoprecipitation of 32P-labeled Na+/H+ exchanger from intact cells showed that sustained intracellular acidosis increased Na+/H+ exchanger phosphorylation in vivo. This was blocked by U0126. The Na+/H+ exchanger activity of mutants 1 and 2 was stimulated similar to wild type Na+/H+ exchanger. Mutant 4 showed a partially reduced level of activation. However, mutant 3 was not stimulated by sustained intracellular acidosis, and loss of stimulation of activity correlated to a loss of sustained acidosis-mediated phosphorylation in vivo. Mutation of the individual amino acids within mutant 3, Ser766, Ser770, and Ser771, showed that Ser770 and Ser771 are responsible for mediating increases in NHE1 activity through sustained acidosis. Both intact Ser770 and Ser771 were required for sustained acidosis-mediated activation of NHE1. Our results suggest that amino acids Ser770 and Ser771 mediate ERK-dependent activation of the Na+/H+ exchanger in vivo.

Regulation of the Na+/H+ exchanger in the healthy and diseased myocardium

Background: Na+/H+ exchangers (NHEs) are membrane proteins that regulate ion fluxes, they extrude one intracellular proton in exchange for one extracellular sodium thereby regulating intracellular pH. Mammalian NHEs have nine isoforms, NHE1–NHE9. NHE1 is present in all mammalian cell and is the only isoform present in cardiomyocytes. NHE1 contributes to damage to the myocardium with ischemia and reperfusion and to heart hypertrophy. Objective: To summarize the current state of knowledge with regard to regulation of NHE1 in the myocardium. Methods: A review of relevant literature. Results: Inhibition of NHE reduces ischemia–reperfusion damage and development of hypertrophy. Extracellular-signal-regulated kinase (ERK)-dependent phosphorylation activates NHE1 in the myocardium. Ischemia and subsequent reperfusion activates the ERK-dependent pathway and may lead to aggravation of damage. Conclusions: Elucidation of the regulatory pathway of NHE1 in the myocardium could lead to novel approaches to reduce heart hypertrophy and ischemia–reperfusion damage.