MAB3140 Sigma-AldrichAnti-Na+/H+ Exchanger-1 Antibody, CT, clone 4E9

Cl(-)/HCO(-) 3 exchangers are expressed abundantly in cardiac muscle, suggesting that HCO(-) 3 extrusion serves an important function in heart. Mice lacking Anion Exchanger Isoform 3 (AE3), a major cardiac Cl(-)/HCO(-) 3 exchanger, appear healthy, but loss of AE3 causes decompensation in a hypertrophic cardiomyopathy (HCM) model. Using intra-ventricular pressure analysis, in vivo pacing, and molecular studies we identified physiological and biochemical changes caused by loss of AE3 that may contribute to decompensation in HCM. AE3-null mice had normal cardiac contractility under basal conditions and after β-adrenergic stimulation, but pacing of hearts revealed that frequency-dependent inotropy was blunted, suggesting that AE3-mediated HCO(-) 3 extrusion is required for a robust force-frequency response (FFR) during acute biomechanical stress in vivo. Modest changes in expression of proteins that affect Ca(2+)-handling were observed, but Ca(2+)-transient analysis of AE3-null myocytes showed normal twitch-amplitude and Ca(2+)-clearance. Phosphorylation and expression of several proteins implicated in HCM and FFR, including phospholamban (PLN), myosin binding protein C, and troponin I were not altered in hearts of paced AE3-null mice; however, phosphorylation of Akt, which plays a central role in mechanosensory signaling, was significantly higher in paced AE3-null hearts than in wild-type controls and phosphorylation of AMPK, which is affected by Akt and is involved in energy metabolism and some cases of HCM, was reduced. These data show loss of AE3 leads to impaired rate-dependent inotropy, appears to affect mechanical stress-responsive signaling, and reduces activation of AMPK, which may contribute to decompensation in heart failure.

The effect of an alteration from regular endurance to interval (10-20-30) training on the health profile, muscular adaptations, maximum oxygen uptake (Vo(2max)), and performance of runners was examined. Eighteen moderately trained individuals (6 females and 12 males; Vo(2max): 52.2 ± 1.5 ml·kg(-1)·min(-1)) (means ± SE) were divided into a high-intensity training (10-20-30; 3 women and 7 men) and a control (CON; 3 women and 5 men) group. For a 7-wk intervention period the 10-20-30 replaced all training sessions with 10-20-30 training consisting of low-, moderate-, and high-speed running (less than 30%, less than 60%, and greater than 90% of maximal intensity) for 30, 20, and 10 s, respectively, in three or four 5-min intervals interspersed by 2 min of recovery, reducing training volume by 54% (14.0 ± 0.9 vs. 30.4 ± 2.3 km/wk) while CON continued the normal training. After the intervention period Vo(2max) in 10-20-30 was 4% higher, and performance in a 1,500-m and a 5-km run improved (P less than 0.05) by 21 and 48 s, respectively. In 10-20-30, systolic blood pressure was reduced (P less than 0.05) by 5 ± 2 mmHg, and total and low-density lipoprotein (LDL) cholesterol was lowered (P less than 0.05) by 0.5 ± 0.2 and 0.4 ± 0.1 mmol/l, respectively. No alterations were observed in CON. Muscle membrane proteins and enzyme activity did not change in either of the groups. The present study shows that interval training with short 10-s near-maximal bouts can improve performance and Vo(2max) despite a ∼50% reduction in training volume. In addition, the 10-20-30 training regime lowers resting systolic blood pressure and blood cholesterol, suggesting a beneficial effect on the health profile of already trained individuals.

It was investigated if athletes subjected to 4 wk of living in normobaric hypoxia (3,000 m; 16 h/day) while training at 800-1,300 m ["live high-train low" (LHTL)] increase muscular and systemic capacity for maintaining pH and K(+) homeostasis as well as intense exercise performance. The design was double-blind and placebo controlled. Mean power during 30-s all-out cycling was similar before and immediately after LHTL (650 ± 31 vs. 628 ± 32 W; n = 10) and placebo exposure (658 ± 22 vs. 660 ± 23 W; n = 6). Supporting the performance data, arterial plasma pH, lactate, and K(+) during submaximal and maximal exercise were also unaffected by the intervention in both groups. In addition, muscle buffer capacity (in mmol H(+)·kg dry wt(-1)·pH(-1)) was similar before and after in both the LHTL (140 ± 12 vs. 140 ± 16) and placebo group (145 ± 2 vs. 140 ± 3). The expression of sarcolemmal H(+) transporters (Na(+)/H(+) exchanger 1, monocarboxylate transporters 1 and 4), as well as expression of Na(+)-K(+) pump subunits-α(1), -α(2), and -β(1) was also similar before and after the intervention. In conclusion, muscular and systemic capacity for maintaining pH and K(+) balance during exercise is similar before and after 4 wk of placebo-controlled normobaric LHTL. In accordance, 30-s all-out sprint ability was similar before and after LHTL.

We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na(+)/H(+) exchanger, hNHE1 (Pedersen, S. F., King, S. A., Nygaard, E. B., Rigor, R. R., and Cala, P. M. (2007) J. Biol. Chem. 282, 19716-19727). Here, we present a structural model of the transmembrane part of hNHE1 that further supports this conclusion. The hNHE1 model was based on the crystal structure of the Escherichia coli Na(+)/H(+) antiporter, NhaA, and previous cysteine scanning accessibility studies of hNHE1 and was validated by EPR spectroscopy of spin labels in TM IV and TM XI, as well as by functional analysis of hNHE1 mutants. Removal of all endogenous cysteines in hNHE1, introduction of the mutations A173C (TM IV) and/or I461C (TM XI), and expression of the constructs in mammalian cells resulted in functional hNHE1 proteins. The distance between these spin labels was ∼15 A, confirming that TM IV and TM XI are in close proximity. This distance was decreased both at pH 5.1 and in the presence of the NHE1 inhibitor cariporide. A similar TM IV·TM XI distance and a similar change upon a pH shift were found for the cariporide-insensitive Pleuronectes americanus (pa) NHE1; however, in paNHE1, cariporide had no effect on TM IV·TM XI distance. The central role of the TM IV·TM XI arrangement was confirmed by the partial loss of function upon mutation of Arg(425), which the model predicts stabilizes this arrangement. The data are consistent with a role for TM IV and TM XI rearrangements coincident with ion translocation and inhibitor binding by hNHE1.

Myocardial stretch induces a biphasic force response: a first abrupt increase followed by a slow force response (SFR), believed to be the in vitro manifestation of the Anrep effect. The SFR is due to an increase in Ca²⁺ transient of unclear mechanism. We proposed that Na⁺/H⁺ exchanger (NHE-1) activation is a key factor in determining the contractile response, but recent reports challenged our findings. We aimed to specifically test the role of the NHE-1 in the SFR. To this purpose small hairpin interference RNA capable of mediating specific NHE-1 knockdown was incorporated into a lentiviral vector (l-shNHE1) and injected into the left ventricular wall of Wistar rats. Injection of a lentiviral vector expressing a nonsilencing sequence (scramble) served as control. Myocardial NHE-1 protein expression and function (the latter evaluated by the recovery of pH(i) after an acidic load and the SFR) were evaluated. Animals transduced with l-shNHE1 showed reduced NHE-1 expression (45 ± 8% of controls; P less than 0.05), and the presence of the lentivirus in the left ventricular myocardium, far from the site of injection, was evidenced by confocal microscopy. These findings correlated with depressed basal pH(i) recovery after acidosis [(max)dpH(i)/dt 0.055 ± 0.008 (scramble) vs. 0.009 ± 0.004 (l-shNHE1) pH units/min, P less than 0.05], leftward shift of the relationship between J(H⁺) (H⁺ efflux corrected by the intrinsic buffer capacity), and abolishment of SFR (124 ± 2 vs. 101 ± 2% of rapid phase; P less than 0.05) despite preserved ERK1/2 phosphorylation [247 ± 12 (stretch) and 263 ± 23 (stretch l-shNHE1) % of control; P less than 0.05 vs. nonstretched control], well-known NHE-1 activators. Our results provide strong evidence to propose NHE-1 activation as key factor in determining the SFR to stretch.

The Na(+)/H(+)Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na(+)/H(+) exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na(+)/H(+) exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na(+) transport capacity without affecting transport affinity (K(m)=44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32)P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na(+) transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.

The forkhead transcription factor FoxM1 controls expression of a large number of genes that are specifically expressed during the G(2) phase of the cell cycle. Throughout most of the cell cycle, FoxM1 activity is restrained by an autoinhibitory mechanism, involving a repressor domain present in the N-terminal part of the protein. Activation of FoxM1 in G(2) is achieved by Cyclin A/Cyclin-dependent kinase (Cdk)-mediated phosphorylation, which alleviates autoinhibition by the N-terminal repressor domain. Here, we show that FoxM1 interacts with B55α, a regulatory subunit of protein phosphatase 2A (PP2A). B55α binds the catalytic subunit of PP2A, and this promotes dephosphorylation and inactivation of FoxM1. Indeed, we find that overexpression of B55α results in decreased FoxM1 activity. Inversely, depletion of B55α results in premature activation of FoxM1. The activation of FoxM1 that is observed upon depletion of B55α is fully dependent on Cyclin A/Cdk-mediated phosphorylation of FoxM1. Taken together, these data demonstrate that B55α acts to antagonize Cyclin A/Cdk-dependent activation of FoxM1, to ensure that FoxM1 activity is restricted to the G(2) phase of the cell cycle.

Invadopodia are invasive protrusions with proteolytic activity uniquely found in tumor cells. Cortactin phosphorylation is a key step during invadopodia maturation, regulating Nck1 binding and cofilin activity. The precise mechanism of cortactin-dependent cofilin regulation and the roles of this pathway in invadopodia maturation and cell invasion are not fully understood. We provide evidence that cortactin-cofilin binding is regulated by local pH changes at invadopodia that are mediated by the sodium-hydrogen exchanger NHE1. Furthermore, cortactin tyrosine phosphorylation mediates the recruitment of NHE1 to the invadopodium compartment, where it locally increases the pH to cause the release of cofilin from cortactin. We show that this mechanism involving cortactin phosphorylation, local pH increase, and cofilin activation regulates the dynamic cycles of invadopodium protrusion and retraction and is essential for cell invasion in 3D. Together, these findings identify a novel pH-dependent regulation of cell invasion.

It is investigated if exercise-induced mRNA changes cause similar protein expression changes of Na(+)-K(+) pump isoforms (α(1), α(2), β(1), β(2)), FXYD1, and Na(+)/K(+) exchanger (NHE1) in rat skeletal muscle. Expression was evaluated (n = 8 per group) in soleus and extensor digutorum longus after 1 day, 3 days, and 3 wk (5 sessions/wk) of either sprint (4 × 3-min sprint + 1-min rest) or endurance (20 min) running. Two hours after exercise on day 1, no change in protein expression was apparent in either training group or muscle, whereas sprint exercise increased the mRNA of soleus α(2) (4.9 ± 0.8-fold; P less than 0.05), β(2) (13.2 ± 4.4-fold; P less than 0.001), and NHE1 (12.0 ± 3.1-fold; P less than 0.01). Two hours after sprint exercise, protein expression normalized to control samples was higher on day 3 than day 1 for soleus α(1) (41 ± 18% increase vs. 15 ± 8% reduction; P less than 0.05), α(2) (64 ± 35% increase vs. 37 ± 12% reduction; P less than 0.05), β(1) (17 ± 21% increase vs. 14 ± 29% reduction; P less than 0.05), and FXYD1 (35 ± 16% increase vs. 13 ± 10% reduction; P less than 0.05). In contrast, on day 3, soleus α(1) (0.1 ± 0.1-fold; P less than 0.001), α(2) (0.2 ± 0.1-fold; P less than 0.001), β(1) (0.4 ± 0.1-fold; P less than 0.05), and β(2)-mRNA (2.9 ± 1.7-fold; P less than 0.001) expression was lower than after exercise on day 1. After 3 wk of training, no change in protein expression relative to control existed. In conclusion, increased expression of Na(+)-K(+) pump subunits, FXYD1 and NHE1 after 3 days exercise training does not appear to be an effect of increased constitutive mRNA levels. Importantly, sprint exercise can reduce mRNA expression concomitant with increased protein expression.

Angiotensin (ANG) II via AT1 receptors (AT1Rs) maintains sodium homeostasis by regulating renal sodium transporters including Na(+)/H(+) exchanger 3 (NHE3) in a biphasic manner. Low-ANG II concentration stimulates whereas high concentrations inhibit NHE3 activity. Oxidative stress has been shown to upregulate AT1R function that could modulate the ANG II-mediated NHE3 regulation. This study was designed to identify the signaling pathways responsible for ANG II-mediated biphasic regulation of proximal tubular NHE3 and the effect of oxidative stress on this phenomenon. Male Sprague-Dawley rats were chronically treated with a pro-oxidant L-buthionine sulfoximine (BSO) with and without an antioxidant tempol in tap water for 3 wk. BSO-treated rats exhibited oxidative stress and high blood pressure. At low concentration (1 pM) ANG II increased NHE3 activity in proximal tubules from all animals. However, in BSO-treated rats, the stimulation was more robust and was normalized by tempol treatment. ANG II (1 pM)-mediated NHE3 activation was abolished by AT1R blocker, intracellular Ca(2+) chelator, and inhibitors of phospholipase C (PLC) and Ca(2+)-dependent calmodulin (CaM) but it was insensitive to Giα and protein kinase C inhibitors or AT2R antagonist. A high concentration of ANG II (1 μM) inhibited NHE3 activity in control and tempol-treated rats. However, in BSO-treated rats, ANG II (1 μM) continued to induce NHE3 stimulation. Tempol restored the inhibitory effect of ANG II (1 μM) in BSO-treated rats. The inhibitory effect of ANG II (1 μM) involved AT1R-dependent, cGMP-dependent protein kinase (PKG) activation and was independent of AT2 receptor and nitric oxide signaling. We conclude that ANG II stimulates NHE3 via AT1R-PLC-CaM pathway and inhibits NHE3 by AT1R-PKG activation. Oxidative stress impaired ANG II-mediated NHE3 biphasic response in that stimulation was observed at both high- and low-ANG II concentration.