Identification, localization and expression of NHE isoforms in the alveolar epithelial cells

Na+/H+ exchangers (NHEs), encoded by Solute Carrier 9A (SLC9A) genes in human, are ubiquitous integral membrane ion transporters that mediate the electroneutral exchange of H+ with Na+ or K+. NHEs, found in the kidney and intestine, play a major role in the process of fluid reabsorption together via Na+,K+-ATPase pump and Na+ channels. Nevertheless, the expression pattern of NHE in the lung and its role in alveolar fluid homeostasis has not been addressed. Therefore, we aimed to examine the expression of NHE specific isoforms in alveolar epithelial cells (AECs), and assess their role in congestive heart failure (CHF). Three NHE isoforms were identified in AEC and A549 cell line, at the level of protein and mRNA; NHE1, NHE2 and mainly NHE8, the latter was shown to be localized in the apical membrane of AEC. Treating A549 cells with angiotensin (Ang) II for 3, 5 and 24 hours displayed a significant reduction in NHE8 protein abundance. Moreover, the abundance of NHE8 protein was downregulated in A549 cells that were treated overnight with Ang II. NHE8 abundance in whole lung lysate was increased in rats with 1-week CHF compared to sham operated rats. However, lower abundance of NHE8 was observed in 4-week CHF group. In conclusion, we herein show for the first time, the expression of a novel NHE isoform in AEC, namely NHE8. Notably, Ang II decreased NHE8 protein levels. Moreover, NHE8 was distinctly affected in CHF rats, probably depending on the severity of the heart failure.

Identification, Localization and Expression of NHE Isoforms in the Alveolar Epithelial Cells

Na+/H+ exchangers (NHEs), encoded by Solute Carrier 9A (SLC9A) genes in human, are ubiquitous integral membrane ion transporters that mediate the electroneutral exchange of H+ with Na+ or K+. NHEs, found in the kidney and intestine, play a major role in the process of fluid reabsorption together via Na+,K+-ATPase pump and Na+ channels. Nevertheless, the expression pattern of NHE in the lung and its role in alveolar fluid homeostasis has not been addressed. Therefore, we aimed to examine the expression of NHE specific isoforms in alveolar epithelium cells (AECs), and assess their role in congestive heart failure.Three NHE isoforms were identified in AEC and A549 cell line, at the level of protein and mRNA; NHE1, NHE2 and mainly NHE8, the latter was shown to be localized in the apical membrane of AEC. Treating A549 cells with angiotensin (Ang) II for 1 and 3 hours displayed a significant reduction in NHE8 protein abundance and to lesser extent at 5 hours; however, there was no effect at 24 hours. Moreover, A549 treated overnight with Ang II downregulated NHE8 protein abundance.CHF rats held for 1 week had increased abundance of NHE8 compared to sham operated rats. However, lower abundance of NHE8 was observed in CHF rats held for 4 weeks.Herein we show, for the first time, the expression of a novel NHE isoform by AEC, namely NHE8. Besides being negatively affected by Ang II, NHE8 protein levels were distinctly affected in CHF rats, which may be related to CHF severity.

Ischemic factor-induced increases in cerebral microvascular endothelial cell Na/H exchange activity and abundance: evidence for involvement of ERK1/2 MAP kinase

Brain edema forms rapidly in the early hours of ischemic stroke by increased secretion of Na, Cl, and water into the brain across an intact blood-brain barrier (BBB), together with swelling of astrocytes as they take up the ions and water crossing the BBB. Our previous studies provide evidence that luminal BBB Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) participate in ischemia-induced edema formation. NKCC1 and two NHE isoforms, NHE1 and NHE2, reside predominantly at the luminal BBB membrane. NKCC and NHE activities of cerebral microvascular endothelial cells (CMEC) are rapidly stimulated by the ischemic factors hypoxia, aglycemia, and AVP, and inhibition of NKCC and NHE activities by bumetanide and HOE642, respectively, reduces brain Na uptake and edema in the rat middle cerebral artery occlusion model of stroke. The present study was conducted to further explore BBB NHE responses to ischemia. We examined whether ischemic factor-stimulated NHE activity is sustained over several hours, when the majority of edema forms during stroke. We also examined whether ischemic factors alter NHE1 and/or NHE2 protein abundance. Finally, we conducted initial studies of ERK1/2 MAP kinase involvement in BBB NHE and NKCC responses to ischemic factors. We found that hypoxia, aglycemia, and AVP increase CMEC NHE activity through 5 h and that NHE1, but not NHE2, abundance is increased by 1- to 5-h exposures to these factors. Furthermore, we found that these factors rapidly increase BBB ERK1/2 activity and that ERK1/2 inhibition reduces or abolishes ischemic factor stimulation of NKCC and NHE activities.

Differential Expression of Na+/H+-Exchanger (NHE-1, 2, and 4) Proteins and mRNA in Rodent’s Uterus under Sex Steroid Effect and at Different Phases of the Oestrous Cycle

Precise uterine fluid pH regulation may involve the Na+/H+-exchanger (NHE). We hypothesized that NHE isoforms are differentially expressed under different sex steroid treatment and at different oestrous cycle phases which may explain the uterine fluid pH changes observed under these conditions.Method. Oestrous cycle phases of intact WKY rats were identified by vaginal smear. Another group of rats was ovariectomized and treated with 0.2 μg 17β-oestradiol (E), 4 mg progesterone (P), and E followed by P (E + P). The animals were then sacrificed and the uteri were removed for mRNA and protein expression analyses by real-time PCR and western blotting, respectively. NHE isoforms distribution was detected by immunohistochemistry (IHC).Results. NHE-1 mRNA and protein were upregulated at diestrus (Ds) and following P treatment. Meanwhile, NHE-2 and NHE-4 proteins and mRNA were upregulated at proestrus (Ps) and estrus (Es) and following E treatment. NHE-1 was found predominantly at the apical membrane, while NHE-2 and NHE-4 were found at the apical and basolateral membranes of the luminal epithelia. NHE-4 is the main isoform upregulated by E.Conclusion. Differential expressions of uterine NHE isoforms 1, 2, and 4 could explain the observed changes in the uterine fluid pH under these conditions.

pH regulating transporters in neurons from various chemosensitive brainstem regions in neonatal rats

We studied the membrane transporters that mediate intracellular pH (pHi) recovery from acidification in brainstem neurons from chemosensitive regions of neonatal rats. Individual neurons within brainstem slices from the retrotrapezoid nucleus (RTN), the nucleus tractus solitarii (NTS), and the locus coeruleus (LC) were studied using a pH-sensitive fluorescent dye and fluorescence imaging microscopy. The rate of pHi recovery from an NH4Cl-induced acidification was measured, and the effects of inhibitors of various pH-regulating transporters determined. Hypercapnia (15% CO2) resulted in a maintained acidification in neurons from all three regions. Recovery in RTN neurons was nearly entirely eliminated by amiloride, an inhibitor of Na+/H+ exchange (NHE). Recovery in RTN neurons was blocked ∼50% by inhibitors of isoform 1 of NHE (NHE-1) but very little by an inhibitor of NHE-3 or by DIDS (an inhibitor of HCO3-dependent transport). In NTS neurons, amiloride blocked over 80% of the recovery, which was also blocked ∼65% by inhibitors of NHE-1 and 26% blocked by an inhibitor of NHE-3. Recovery in LC neurons, in contrast, was unaffected by amiloride or blockers of NHE isoforms but was dependent on Na+ and increased by external HCO3−. On the basis of these findings, pHi recovery from acidification appears to be largely mediated by NHE-1 in RTN neurons, by NHE-1 and NHE-3 in NTS neurons, and by a Na- and HCO3-dependent transporter in LC neurons. Thus, pHi recovery is mediated by different pH-regulating transporters in neurons from different chemosensitive regions, but recovery is suppressed by hypercapnia in all of the neurons.

HCO3− Secretion by Murine Nasal Submucosal Gland Serous Acinar Cells during Ca2+-stimulated Fluid Secretion

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca2+-activated Cl− secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca2+-activated Cl− secretion was accompanied by secretion of HCO3−, possibly a critical ASL component, by simultaneous measurements of intracellular pH (pHi) and cell volume. Resting pHi was 7.17 ± 0.01 in physiological medium (5% CO2–25 mM HCO3−). During carbachol (CCh) stimulation, pHi fell transiently by 0.08 ± 0.01 U concomitantly with a fall in Cl− content revealed by cell shrinkage, reflecting Cl− secretion. A subsequent alkalinization elevated pHi to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO2–HCO3−-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO3− efflux by ion substitution or exposure to the Cl− channel inhibitor niflumic acid (100 μM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na+/H+ exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1–4 and 6–9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pHi recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO3− during Ca2+-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl− channel, with HCO3− secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na+-dependent pHi regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na+-free media.

Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms.

Prostaglandin-mediated inhibition of Na+/H+ exchanger isoform 2 stimulates recovery of barrier function in ischemia-injured intestine

Prostaglandins stimulate repair of the ischemia-injured intestinal barrier in the porcine ileum through a mechanism involving cAMP-dependent Cl− secretion and inhibition of electroneutral Na+/H+ exchanger (NHE) activity. In the present study, we focused on the role of individual NHE isoforms in the recovery of barrier function. Ischemia-injured porcine ileal mucosa was mounted on Ussing chambers. Short-circuit current ( Isc), transepithelial electrical resistance (TER), and isotopic fluxes of 22Na were measured in response to PGE2 and selective inhibitors of epithelial NHE isoforms. Immunoassays were used to assess the expression of NHE isoforms. Forty-five minutes of intestinal ischemia resulted in a 45% reduction in TER ( P < 0.01). Near-complete restitution occurred within 60 min. Inhibition of NHE2 with HOE-694 (25 μM) added to the mucosal surface of the injured ileum stimulated significant elevations in TER, independent of changes in Isc and histological evidence of restitution. Pharmacological inhibition of NHE3 or NHE1 with mucosal S-3226 (20 μM) or serosal cariporide (25 μM), respectively, had no effect. Ischemia-injured tissues treated with mucosal S-3226 or HOE-694 exhibited equivalent reductions in mucosal-to-serosal fluxes of 22Na+ (by ∼35%) compared with nontreated ischemia-injured control tissues ( P < 0.05). Intestinal ischemia resulted in increased expression of the cytoplasmic NHE regulatory factor EBP50 in NHE2 but not in NHE3 immunoprecipitates. Selective inhibition of NHE2, and not NHE3, induces recovery of barrier function in the ischemia-injured intestine.

Stimulation of renal sulfate secretion by metabolic acidosis requires Na+/H+ exchange induction and carbonic anhydrase

The acute effect of metabolic acidosis on SO42− secretion by the marine teleost renal proximal tubule was examined. Metabolic acidosis was mimicked in primary cultures of winter flounder renal proximal tubule epithelium (fPTCs) mounted in Ussing chambers by reducing interstitial pH to 7.1 (normally 7.7). fPTCs with metabolic acidosis secreted SO42− at a net rate that was 40% higher than in paired isohydric controls (pH 7.7 on interstitium). The stimulation was completely blocked by the carbonic anhydrase inhibitor methazolamide (100 μM). Although Na+/H+ exchange (NHE) isoforms 1, 2, and 3 were identified in fPTCs by immunoblotting, administering EIPA (20 μM) to the interstitial and luminal bath solutions had no effect on net SO42− secretion by fPTCs with a normal interstitial pH of 7.7. However, EIPA (20 μM) blocked most of the stimulation caused by acidosis when applied to the lumen but not interstitium, demonstrating that induction of brush-border NHE activity is important. In the intact flounder, serum pH dropped 0.4 pH units (pH 7.7 to 7.3, at 2–3 h) when environmental pH was lowered from 7.8 to ∼4.3. Whereas serum [SO42−] was not altered by acidosis, renal tubular SO42− secretion rate was elevated 200%. Thus metabolic acidosis strongly stimulates renal sulfate excretion most likely by a direct effect on active renal proximal tubule SO42− secretion. This stimulation appears to be dependent on inducible brush-border NHE activity.