Normalization of the calcineurin pathway underlies the regression of hypertensive hypertrophy induced by Na+/H+ exchanger-1 (NHE-1) inhibition

Na+/H+ exchanger-1 (NHE-1) inhibition induces cardiac hypertrophy regression and (or) prevention in several experimental models, although the intracellular events involved remain unclarified. We aimed to determine whether the calcineurin/NFAT pathway mediates this effect of NHE-1 inhibitors. Spontaneously hypertensive rats (SHR) with cardiac hypertrophy were treated with the NHE-1 inhibitors cariporide and BIIB723 for 30 days. Wistar rats served as normotensive controls. Their hearts were studied by echocardiography, immunoblotting, and real-time RT-PCR. Cytoplasmic Ca2+ and calcineurin Abeta expression were measured in cultured neonatal rat ventricular myocytes (NRVM) stimulated with endothelin-1 for 24 h. NHE-1 blockade induced cardiac hypertrophy regression (heart mass/body mass=3.63+/-0.07 vs. 3.06+/-0.05 and 3.02+/-0.13 for untreated vs. cariporide- and BIIB723-treated SHR, respectively; p<0.05) and decreased myocardial brain natriuretic peptide, calcineurin Abeta, and nuclear NFAT expressions. Echocardiographic evaluation demonstrated a reduction in left ventricular wall thickness without changes in cavity dimensions or a significant decrease in blood pressure. NHE-1-inhibitor treatment did not affect myocardial stiffness or endocardial shortening, but increased mid-wall shortening, suggesting that a positive inotropic effect develops after hypertrophy regression. Cariporide normalized the increased diastolic Ca2+ and calcineurin Abeta expression observed in ET-1-stimulated NRVM. In conclusion, our data suggest that inactivation of calcineurin/NFAT pathway may underlie the regression of cardiac hyper-trophy induced by NHE-1 inhibition.     

Stimulation of the plasma membrane Na+/H+ exchanger NHE1 by sustained intracellular acidosis. Evidence for a novel mechanism mediated by the ERK pathway

Activity of the Na+/H+ exchanger (NHE) isoform 1 (NHE1) is increased by intracellular acidosis through the interaction of intracellular H+ with an allosteric modifier site in the transport domain. Additional regulation is achieved via kinase-mediated modulation of the NHE1 regulatory domain. To determine if intracellular acidosis stimulates NHE1 activity solely by the allosteric mechanism, we subjected cultured neonatal rat ventricular myocytes (NRVM) with native NHE1 expression to intracellular acidosis (pHi approximately 6.6) for up to 6 min by transient exposure to NH4Cl and its washout in the presence of NHE inhibition (by zero [Na+]o or the NHE1 inhibitor cariporide) in HCO3- -free medium. After the desired duration of acidosis, NHE was reactivated (by reintroduction of [Na+]o or removal of cariporide), and the rate of recovery of pHi (dpHi/dt) was measured as the index of NHE activity. Regardless of the method used when intracellular acidosis was sustained for > or =3 min, subsequent NHE activity was significantly increased (>4-fold). Similar NHE stimulatory effects of sustained acidosis were observed in adult rat ventricular myocytes and COS-7 cells. Sustained (3 min) intracellular acidosis activated several NHE1 kinases in NRVM, in an in-gel kinase assay using as substrate a glutathione S-transferase fusion protein of the NHE1 regulatory domain. Detailed investigation of ERK and its downstream effector p90RSK, two putative NHE1 kinases, revealed time-dependent activation of both by intracellular acidosis in NRVM. Furthermore, inhibition of MEK1/2 by pretreatment of NRVM with two structurally distinct inhibitors, PD98059 (30 microM) or UO126 (3 microM), inhibited the activation of ERK and p90RSK and abolished the stimulation of NHE activity by sustained (3 min) intracellular acidosis. Our data show that not only the extent but also the duration of intracellular acidosis regulates NHE1 activity and suggest that the stimulatory effect of sustained intracellular acidosis occurs through a novel mechanism mediated by activation of the ERK pathway.     

Focal localization of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH.

Na+/H+ exchange (antiport) is a major pathway for the regulation of intracellular pH. Antiport activity is stimulated when suspended cells adhere to the substratum. In this report, immunofluorescence was used to study the subcellular localization of the ubiquitous NHE-1 isoform of the antiport. NHE-1 was not distributed homogeneously on the surface of the cells. Instead, antiports were found to accumulate along the border of lamellipodia and near the edge of finer processes. Dual immunofluorescence experiments demonstrated that vinculin, talin and F-actin are concentrated at sites of NHE-1 accumulation. A mutated construct of NHE-1 lacking residues 566-635 of the cytosolic domain also accumulated near marginal lamellae. In contrast, the focal distribution observed in adherent cells was not detectable in cells grown in suspension. Fluorescence ratio imaging was used to define the functional consequences of focal accumulation of NHE-1. In the steady state, the pH was virtually identical throughout the cytosol. Moreover, no pH gradients were found to develop when cells recovered from an acid load by activation of Na+/H+ exchange. This is probably because of the presence of high concentrations of mobile buffers in the cytosol. The focal accumulation of antiporters near the cell margins may be involved in stimulation by adherence and/or generation of local osmotic gradients.     

A comparative study on defense systems for lipid peroxidation by free radicals in spontaneously hypertensive and normotensive rat myocardium.

1. Antiperoxidation ability and lipid peroxidation in myocardium were examined in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) at 6 and 16 weeks of age. 2. Glutathione peroxidase activity was higher in SHR at 6 weeks of age, but lower at 16 weeks compared to that in WKY. alpha-Tocopherol content was lower in SHR at both 6 and 16 weeks of age than in WKY. 3. In vitro formation of free malondialdehyde was more pronounced in SHR myocardium than in WKY. 4. Coincidence of lower antiperoxidation ability and higher peroxidation of membrane phospholipid indicate myocardial cell vulnerability in SHR hypertrophied myocardium.     

The Na+/H+ exchanger isoform 1

The Na+/H+ exchanger (NHE) isoform 1 is a ubiquitously expressed integral membrane protein which regulates intracellular pH in mammalian cells. Nine isoforms of the Na+/H+ exchanger have been identified. The isoform first discovered has two domains: an N-terminal membrane domain containing approximately 500 amino acids and a C-terminal regulatory domain containing approximately 315 amino acids. The exchanger, which resides in the plasma membrane, exchanges an intracellular proton for an extracellular sodium, thereby regulating intracellular pH. It is involved in cell growth and differentiation, cell migration, and regulation of sodium fluxes. The Na+/H+ exchanger plays an important role in myocardial damage during ischemia and reperfusion and has recently been implicated as a mediator of cardiac hypertrophy. Inhibitors of the Na+/H+ exchanger, which may prove useful in the clinical treatment of these conditions, are currently being developed and clinical trials are underway.     

Na+/H+ metabolic activity in the thrombocytes of spontaneously hypertensive rats

The sodium-proton exchange was determined in platelets of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The platelets were suspended in sodium propionate; the cytoplasmic acidification activated the exchanger and intracellular pH (the increasing) and volume of the platelets (the swelling) were registered. The activity of Na+/H+ exchange was inhibited by isopropyl amiloride. The platelets' volume and the exchange rate constant of SHR were increased on 30-40% as compared with those of WKY.     

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 (32)P-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, Ser(766), Ser(770), and Ser(771), showed that Ser(770) and Ser(771) are responsible for mediating increases in NHE1 activity through sustained acidosis. Both intact Ser(770) and Ser(771) were required for sustained acidosis-mediated activation of NHE1. Our results suggest that amino acids Ser(770) and Ser(771) mediate ERK-dependent activation of the Na(+)/H(+) exchanger in vivo.     

Renal Na+-K+-ATPase in weanling and adult spontaneously hypertensive rats

The interrelationships among plasma renin activity (PRA, ng AI/ml plasma/hr), aldosterone concentration (ng%), and renal Na+-K+-ATPase activity (mumole PO4/mg protein/hr) were studied in 9 weanling normotensive spontaneously hypertensive rats (SHR), 9 adult hypertensive SHR, and 9 weanling and 9 adult normotensive Wistar-Kyoto rats (WKY). All groups were placed on a normal (0.4% sodium) diet. PRA and plasma aldosterone, measured in samples drawn from the ether-anesthetized rat, were higher in weanling SHR (15.2 +/- 2.0, 37 +/- 4.2) than in WKY. PRA measured in samples collected from a separate group of unanesthetized weanling SHR was also greater than in age-matched WKY. In adult SHR, PRA (6.1 +/- 0.9) and plasma aldosterone (20.0 +/- 2.7) were decreased. During the weanling period Na+-K+-ATPase activity in SHR was not only greater than in age-matched WKY but was also increased compared to adult normotensive and hypertensive rats (137 +/- 9 weanling SHR, 89 +/- 7 weanling WKY, 73 +/- 11 adult SHR, 84 +/- 17 adult WKY). Thus, during the weanling period the renin-angiotensin-aldosterone (R-A-A) system and renal Na+-K+-ATPase activity are activated in SHR. The elevation of Na+-K+-ATPase activity may be due to increased aldosterone levels. It was noted, however, that plasma aldosterone was similar in adult WKY and weanling SHR, while Na+-K+-ATPase activity was higher in SHR. These findings involving R-A-A and renal Na+-K+-ATPase activity prior to the elevation of blood pressure suggest that the kidneys may play a role in the initiation of hypertension in SHR.     

Involvement of AE3 isoform of Na(+)-independent Cl(-)/HCO(3)(-) exchanger in myocardial pH(i) recovery from intracellular alkalization

Myocardial pH(i) recovery from intracellular alkalization results in part from the acid load (-J(H+)) carried by Cl(-)/HCO(3)(-) anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pH(i) homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pH(i) regulation. We developed rabbit polyclonal antibodies against the extracellular "loops": one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pH(i) regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased -J(H+) in response to myocardial alkalization (from a mean control value of 1.06+/-0.26 to 0.32+/-0.13 mmol/L/min, n=7, P<0.05) without affecting the baseline pH(i) (7.22+/-0.03 vs. 7.21+/-0.04). The anti-AE3 loop 4 antibody did not modify either pH(i) recovery or baseline pH(i). Under control conditions, endothelin-1 (ET-1) increased -J(H+) in response to myocardial alkalization from 1.30+/-0.18 to 2.01+/-0.33 mmol/L /min (n=5, P<0.05). This effect of ET-1 on -J(H+) was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pH(i) recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pH(i).     

Cannabinoid receptor CB1 activates the Na+/H+ exchanger NHE-1 isoform via Gi-mediated mitogen activated protein kinase signaling transduction pathways

We previously showed that the cannabinoid receptor CB1 stably transfected in Chinese hamster ovary cells was constitutively active and could be inhibited by the inverse agonist SR 141716A. In the present study, we demonstrate that the cannabinoid agonist CP-55940 induced cytosol alkalinization of CHO-CB1 cells in a dose- and time-dependent manner via activation of the Na+/H+ exchanger NHE-1 isoform. By contrast, the inverse agonist SR 141716A induced acidification of the cell cytosol, suggesting that the Na+/H+ exchanger NHE-1 was constitutively activated by the CB1 receptor. CB1-mediated NHE1 activation was prevented by both pertussis toxin treatment and the specific MAP kinase inhibitor PD98059. NHE-1 and p42/p44 MAPK had a similar time course of activation in response to the addition of CP-55940 to CHO-CB1 cells. These results suggest that CB1 stimulates NHE-1 by G(i/o)-mediated activation of p42/p44 MAP kinase and highlight a cellular physiological process targeted by CB1.     

Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+ exchanger in sperm motility

The Na,K-ATPase generates electrochemical gradients that are used to drive the coupled transport of many ions and nutrients across the plasma membrane. The functional enzyme is comprised of an alpha and beta subunit and families of isoforms for both subunits exist. Recent studies in this laboratory have identified a biological role for the Na,K-ATPase alpha4 isoform in sperm motility. Here we further investigate the role of the Na,K-ATPase carrying the alpha4 isoform, showing again that ouabain eliminates sperm motility, and in addition, that nigericin, a H+/K+ ionophore, and monensin, a H+/Na+ ionophore, reinitiate motility. These data, along with the observation that the K+ ionophore valinomycin has no effect on the motility of ouabain-inhibited sperm, suggest that ouabain may change intracellular H+ levels in a manner that is incompatible with sperm motility. We have also localized NHE1 and NHE5, known regulators of intracellular H+ content, to the same region of the sperm as the Na,K-ATPase alpha4 isoform. These data highlight the important role of the Na,K-ATPase alpha4 isoform in regulating intracellular H(+) levels, and provide evidence suggesting the involvement of the Na+/H+ exchanger, which is critical for maintaining normal sperm motility.     

Sustained intracellular acidosis activates the myocardial Na/H exchanger independent of amino acid Ser and p90

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the myocardium and other tissues. It is an important mediator of the myocardial damage that occurs after ischemia-reperfusion injury and is implicated in heart hypertrophy. Regulation of NHE1 has been proposed as a therapeutic target for cardioprotection. We therefore examined mechanisms of control of NHE1 in the myocardium. Several different amino acids have been implicated as a being critical to NHE1 regulation in a number of tissues including Ser⁷⁰³, Ser⁷⁷⁰, and Ser⁷⁷¹. In the myocardium, NHE1 is activated in response to a variety of stimuli including activation by an ERK-dependent sustained intracellular acidosis. In this study, we determined whether Ser⁷⁰³ and p90^rsk activity are critical in activation of NHE1 by sustained intracellular acidosis. In vitro phosphorylation of NHE1 C-terminal fusion proteins determined that ERK-dependent phosphorylation of the cytoplasmic region was not dependent on Ser⁷⁰³; however, phosphorylation by p90^rsk required Ser⁷⁰³. A Ser703Ala mutation decreased basal NHE1 activity in CHO cells but not in cardiomyocytes. NHE1 with a Ser703Ala mutation was activated in response to sustained intracellular acidosis in CHO cells. In addition, sustained intracellular acidosis also activated the Ser703Ala mutant protein in isolated cardiomyocytes and phosphorylation levels were also increased by acidosis. The presence of a dominant-negative p90^rsk kinase also did not prevent activation and phosphorylation of NHE1 by sustained intracellular acidosis in isolated cardiomyocytes. We conclude that Ser⁷⁰³ and p90^rsk are not required for activation by sustained intracellular acidosis and that p90^rsk phosphorylation of Ser⁷⁰³ is independent of this type of activation.     

Na + /H + exchanger isoform 1 activity in AQP2-expressing cells can be either proliferative or anti-proliferative depending on extracellular pH

We have previously shown in renal cells that expression of the water channel Aquaporin-2 increases cell proliferation by a regulatory volume mechanism involving Na+/H+ exchanger isoform 2. Here, we investigated if Aquaporin-2 (AQP2) also modulates Na+/H+ exchanger isoform 1-dependent cell proliferation. We use two AQP2-expressing cortical collecting duct models: one constitutive (WT or AQP2-transfected RCCD1 cell line) and one inducible (control or vasopressin-induced mpkCCDc14 cell line). We found that Aquaporin-2 modifies Na+/H+ exchanger isoform 1 (NHE1) contribution to cell proliferation. In Aquaporin-2-expressing cells, Na+/H+ exchanger isoform 1 is anti-proliferative at physiological pH. In acid media, Na+/H+ exchanger isoform 1 contribution turned from anti-proliferative to proliferative only in AQP2-expressing cells. We also found that, in AQP2-expressing cells, NHE1-dependent proliferation changes parallel changes in stress fiber levels: at pH 7.4, Na+/H+ exchanger isoform 1 would favor stress fiber disassembly and, under acidosis, NHE1 would favor stress fiber assembly. Moreover, we found that Na+/H+ exchanger-dependent effects on proliferation linked to Aquaporin-2 relied on Transient Receptor Potential Subfamily V calcium channel activity. In conclusion, our data show that, in collecting duct cells, the water channel Aquaporin-2 modulates NHE1-dependent cell proliferation. In AQP2-expressing cells, at physiological pH, the Na+/H+ exchanger isoform 1 function is anti-proliferative and, at acidic pH, Na+/H+ exchanger isoform 1 function is proliferative. We propose that Na+/H+ exchanger isoform 1 modulates proliferation through an interplay with stress fiber formation.     

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent,but can couple to an H+ conductance

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT- PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time- course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH- sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.     

Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon

Bicarbonate and butyrate stimulate electroneutral Na absorption via apical membrane Na-H exchange (NHE) in rat distal colon. cAMP downregulates NHE-3 isoform and inhibits HCO3-dependent, but not butyrate-dependent, Na absorption. This study sought to determine whether 1) the apical membrane NHE-2 and NHE-3 isoforms differentially mediated HCO3- and butyrate-dependent Na absorption, and 2) cAMP had different effects on NHE-2 and NHE-3 isoforms. The effect of specific inhibitors of NHE-2 and NHE-3 isoforms (50 microM HOE 694 and 2 microM S3226, respectively) on unidirectional 22Na transepithelial fluxes performed across isolated mucosa from rat distal colon under voltage-clamp conditions was examined. HCO3 stimulation of Na absorption was inhibited by EIPA, a nonspecific inhibitor of all NHE isoforms, by S3226 and dibutyryl cAMP but not by HOE 694. In contrast, butyrate stimulation of Na absorption was not altered by dibutyryl cAMP and was not inhibited by HOE 694 in the absence of dibutyryl cAMP, but in the presence of dibutyryl cAMP was HOE694 sensitive. In contrast, S3226 inhibited butyrate-stimulated Na absorption in the absence of dibutyryl cAMP, but not in its presence. We conclude that 1) HCO3-stimulated Na absorption is mediated solely by NHE-3 isoform, whereas butyrate-stimulated Na absorption is mediated by either NHE-3 or NHE-2 isoform, and 2) dibutyryl cAMP selectively inhibits NHE-3 isoform but stimulates NHE-2 isoform. Dibutyryl cAMP does not inhibit butyrate-stimulated Na absorption as a result of its differential effects on NHE-2 and NHE-3 isoforms.     

Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism.

A variety of cell types regulate their volume in anisotonic media by stimulating Na+/H+ exchange. Like growth factors, osmotic challenge activates the Na+/H+ antiport by increasing its sensitivity to intracellular [H+]. To investigate the molecular mechanism underlying this shift in pH sensitivity, the antiporter of 32P-labeled human bladder carcinoma cells and of Chinese hamster ovary cells was immunoprecipitated using antibodies raised against the cytosolic domain of the NHE-1 isoform of the Na+/H+ exchanger. Unlike the effects of growth promoters, activation of the antiport during volume regulation was not associated with increased phosphorylation. The possible coexistence of multiple antiporter isoforms was considered. The cytosolic alkalosis normally elicited by hypertonic media was found to be absent in Na+/H+ exchange-deficient fibroblasts. Responsiveness to osmotic challenge was restored by stable transfection of these cells with the cDNA encoding NHE-1. In these transfectants, phosphorylation of the antiporter was also unaffected during osmotic activation. The unchanged phosphate content of the antiporter might be explained by dephosphorylation of one site with concomitant phosphorylation at a different site. However, this possibility appears unlikely since phosphoamino acid analysis revealed that serine was the only residue phosphorylated in immunoprecipitated antiports of both control and osmotically stimulated cells. Moreover, phosphopeptide maps of control and hypertonically activated antiports were identical. These findings reveal a novel mode of activation of Na+/H+ exchange not requiring direct phosphorylation of the antiporter. We propose the existence of dual control of Na+/H+ exchange by phosphorylation-dependent and -independent mechanisms.     

Increased Na(+)/H(+) exchanger isoform 1 activity in spontaneously hypertensive rats: lack of mutations within the coding region of NHE1

Enhanced Na(+)/H(+) exchange, measured as amiloride derivative-sensitive Na(+) and H(+) fluxes in cells with a preliminary acidified cytoplasm (Deltamu(H+)-induced Na(+)/H(+) exchange), is one of the most prominent intermediate phenotypes of altered vascular smooth muscle cell (VSMC) function in spontaneously hypertensive rats (SHR). Analysis of Na(+)/H(+) exchange in F(2) hybrids of SHR and normotensive rats seems to be the most appropriate approach in the search for the genetic determinants of abnormal activity of this carrier. However, the measurement of Deltamu(H+)-induced Na(+)/H(+) exchange is hardly appropriate for precise analysis of the carrier's activity in VSMC derived from several hundred F(2) hybrids. To overcome this problem, we compared the rate of (22)Na influx under baseline conditions and in Na(+)-loaded (ouabain-treated) VSMC. The dose-dependency of the rate of Deltamu(H+)-induced H(+) efflux as well as of (22)Na influx in control and ouabain-treated cells on ethylisopropylamiloride (EIPA) concentration were not different (K(0.5) approximately 0.3 microM), suggesting that these ion transport pathways are mediated by the same carrier. EIPA-sensitive (22)Na influx in Na(+)-loaded cells was approximately 6-fold higher than in ouabain-untreated VSMC and was increased by 50-70% in two different substrains of SHR. About the same increment of EIPA-sensitive (22)Na influx in Na(+)-loaded VSMC was observed in 5- to 6-week-old SHR (an age at which hypertension has not yet developed) as well as in stroke-prone SHR (SHRSP) with severe hypertension, indicating that the heightened activity of Na(+)/H(+) exchange is not a consequence of long-term blood pressure elevation. To examine whether or not the augmented activity of Na(+)/H(+) exchange in SHR is caused by mutation of NHE1, i.e. the only isoform of this carrier expressed in VSMC, we undertook single-stranded conformational polymorphism analysis of 23 NHE1 cDNA fragments from SHR and SHRSP and sequencing of the 456-2421 NHE1 cDNA fragment. This study did not reveal any mutation in the entire coding region of NHE1. The lack of mutation in the coding region of NHE1 indicates that the augmented activity of the ubiquitous Na(+)/H(+) exchanger in primary hypertension is caused by altered regulation of carrier turnover number or/and its plasma membrane content.     

Na(+)/H(+) exchange inhibition-induced cardioprotection in dogs: effects on neutrophils versus cardiomyocytes

Numerous studies have examined the effect of Na(+)/H(+) exchanger (NHE) inhibition on the myocardium; however, the effect of NHE-1 inhibition on neutrophil function has not been adequately examined. An in vivo canine model of myocardial ischemia-reperfusion injury in which 60 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion was used to examine the effect of NHE-1 inhibition on infarct size (IS) and neutrophil function. BIIB-513, a selective inhibitor of NHE-1, was infused before ischemia. IS was expressed as a percentage of area at risk (IS/AAR). NHE-1 inhibition significantly reduced IS/AAR and reduced neutrophil accumulation in the ischemic myocardium. NHE-1 inhibition attenuated both phorbol 12-myristate 13-acetate- and platelet-activating factor-induced neutrophil respiratory burst but not CD18 upregulation. Furthermore, NHE-1 inhibition directly protected cardiomyocytes against metabolic inhibition-induced lactate dehydrogenase release and hypercontracture. This study provides evidence that the cardioprotection induced by NHE-1 inhibition is likely due to specific protection of cardiomyocytes and attenuation of neutrophil activity.