NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice

The mechanism of apical Na(+)-dependent H(+) extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH(i)) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na(+) stimulated similar rates of pH(i) recovery in cells at the base of distal colonic crypts of wild-type or Na(+)/H(+) exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 microM HOE694 (NHE2 inhibitor) blocked 68-75% of the pH(i) recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 microM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 microM), inhibited pH(i) recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na(+) leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na(+)-dependent pH(i) recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH(i) recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na(+)-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.     

Basolateral membrane Na(+)-independent Cl-/HCO3- exchange in the inner stripe of the rabbit outer medullary collecting tubule

The inner stripe of the outer medullary collecting tubule is a major distal nephron segment in urinary acidification. To examine the mechanism of basolateral membrane H+/OH-/HCO3- transport in this segment, cell pH was measured microfluorometrically in the inner stripe of the rabbit outer medullary collecting tubule perfused in vitro using the pH-sensitive fluorescent dye, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Decreasing peritubular pH from 7.4 to 6.8 (changing [HCO3-] from 25 to 5 mM) caused a cell acidification of 0.25 +/- 0.02 pH units, while a similar luminal change resulted in a smaller cell acidification of only 0.04 +/- 0.01 pH units. Total replacement of peritubular Cl- with gluconate caused cell pH to increase by 0.18 +/- 0.04 pH units, an effect inhibited by 100 microM peritubular DIDS and independent of Na+. Direct coupling between Cl- and base was suggested by the continued presence of peritubular Cl- removal-induced cell alkalinization under the condition of a cell voltage clamp (K(+)-valinomycin). In addition, 90% of basolateral membrane H+/OH-/HCO3- permeability was inhibited by complete removal of luminal and peritubular Cl-. Peritubular Cl(-)-induced cell pH changes were inhibited two-thirds by removal of exogenous CO2/HCO3- from the system. The apparent Km for peritubular Cl- determined in the presence of 25 mM luminal and peritubular [HCO3-] was 113.5 +/- 14.8 mM. These results demonstrate that the basolateral membrane of the inner stripe of the outer medullary collecting tubule possesses a stilbene-sensitive Cl-/HCO3- exchanger which mediates 90% of basolateral membrane H+/OH-/HCO3- permeability and may be regulated by physiologic Cl- concentrations.     

Effect of Cytosolic pH on Epithelial Na+ Channel in Normal and Cystic Fibrosis Sweat Ducts

The activities of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel and the amiloride-sensitive epithelial Na(+) channel (ENaC) are acutely coordinated in the sweat duct. However, the mechanisms responsible for cross-talk between these ion channels are unknown. Previous studies indicated that luminal pH of sweat ducts varies over 3 pH units and that the cytoplasmic pH affects both CFTR and ENaC. Therefore, using basolaterally alpha-toxin-permeabilized apical membrane preparations of sweat ducts as an experimental system, we tested the hypothesis that the cytosolic pH may mediate the cross-talk between CFTR and ENaC. We showed that while luminal pH had no effect, cytosolic pH acutely affected ENaC activity. That is, acidic pH inhibited, while basic pH activated, ENaC. pH regulation of ENaC appears to be independent of CFTR or endogenous kinase activities because basic pH independently stimulated ENaC (1) in normal ducts even when CFTR was deactivated, (2) in CF ducts that lack CFTR in the plasma membranes and (3) after blocking endogenous kinase activity with staurosporine. Considering the evidence of Na(+)/H(+) exchange (NHE) activity as shown by the expression of mRNA and function of NHE in the basolateral membrane of the sweat duct, we postulate that changes in cytosolic Na(+) ([Na(+)]( i )) may alter cytosolic pH (pH( i )) as salt loads into the cell during electrolyte absorption. These changes may play a role in coordinating the activities of ENaC and CFTR during transepithelial salt transport.     

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca(2+) overload and acidosis

We investigated the question of whether inhibition of the Na(+)/H(+) exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca(2+) accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na(+)-HCO(3)(-) symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca(2+) (fura 2), Na(+) [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 micromol/l HOE 642 or 1 micromol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na(i) and Ca(i), respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 +/- 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH(i)), Na(i), and Ca(i), but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 +/- 1.4%, EIPA: 68.2 +/- 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca(i) overload.     

Kinetic dissection of two distinct proton binding sites in Na+/H+ exchangers by measurement of reverse mode reaction

We examined the effect of intracellular acidification on the reverse mode of Na+/H+ exchange by measuring 22Na+ efflux from 22Na+-loaded PS120 cells expressing the Na+/H+ exchanger (NHE) isoforms NHE1, NHE2, and NHE3. The 5-(N-ethyl-N-isopropyl)amiloride (EIPA)- or amiloride-sensitive fraction of 22Na+ efflux was dramatically accelerated by cytosolic acidification as opposed to thermodynamic prediction, supporting the concept that these NHE isoforms are activated by protonation of an internal binding site(s) distinct from the H+ transport site. Intracellular pH (pHi) dependence of 22 Na+ efflux roughly exhibited a bell-shaped profile; mild acidification from pHi 7.5 to 7 dramatically accelerated 22Na+ efflux, whereas acidification from pHi 6.6 gradually decreased it. Alkalinization above pHi 7.5 completely suppressed EIPA-sensitive 22Na+ efflux. Cell ATP depletion and mutation of NHE1 at Arg440 (R440D) caused a large acidic shift of the pHi profile for 22Na+ efflux, whereas mutation at Gly455 (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na+/H+ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.     

Muscarinic activation of Na+-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acinus

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage (V(m)) and intracellular concentrations of Ca(2+), Na(+) ([Na(+)](c)), Cl(-), and H(+) (pH(i)) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 muM, 20 s), RSMGA showed strong delayed hyperpolarization (V(h,ACh); -95 +/- 1.8 mV) that was abolished by ouabain. In the HCO(3)(-)-free condition, the V(h,ACh) was also blocked by bumetanide, a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC). In the presence of HCO(3)(-) (24 meq, bubbled with 5% CO(2)), however, the V(h,ACh) was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a Na(+)/H(+) exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na(+)](c) was totally blocked by bumetanide in the absence of HCO(3)(-), but only by one-half in the presence of HCO(3)(-). ACh induced a prominent acidification of pH(i) in the presence of HCO(3)(-), and the acidification was further increased by EIPA treatment. Without HCO(3)(-), an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH(i) during the application of NH(4)(+) (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO(3)(-). In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na(+)](c). The intracellular Na(+)-induced activation of electrogenic Na(+)/K(+)-ATPase causes V(h,ACh). The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO(3)(-).     

Apical Na+/H+ exchange near the base of mouse colonic crypts

Colonic crypts can absorb fluid, but the identity of the absorptive transporters remains speculative. Near the crypt base, the epithelial cells responsible for vectorial transport are relatively undifferentiated and often presumed to mediate only Cl- secretion. We have applied confocal microscopy in combination with an extracellular fluid marker [Lucifer yellow (LY)] or a pH-sensitive dye (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) to study mouse colonic crypt epithelial cells directly adjacent to the crypt base within an intact mucosal sheet. Measurements of intracellular pH report activation of colonocyte Na+/H+ exchange in response to luminal or serosal Na+. Studies with LY demonstrate the presence of a paracellular fluid flux, but luminal Na+ does not activate Na+/H+ exchange in the nonepithelial cells of the lamina propria, and studies with LY suggest that the fluid bathing colonocyte basolateral membranes is rapidly refreshed by serosal perfusates. The apical Na+/H+ exchange in crypt colonocytes is inhibited equivalently by luminal 20 microM ethylisopropylamiloride and 20 microM HOE-694 but is not inhibited by luminal 20 microM S-1611. Immunostaining reveals the presence of epitopes from NHE1 and NHE2, but not NHE3, in epithelial cells near the base of colonic crypts. Comparison of apical Na+/H+ exchange activity in the presence of Cl- with that in the absence of Cl- (substitution by gluconate or nitrate) revealed no evidence of the Cl--dependent Na+/H+ exchange that had been previously reported as the sole apical Na+/H+ exchange activity in the colonic crypt. Results suggest the presence of an apical Na+/H+ exchanger near the base of crypts with functional attributes similar to those of the cloned NHE2 isoform.     

Expression of Na+/HCO3- cotransporter and its role in pH regulation in mouse parotid acinar cells

Ion transporters such as Na(+)/H(+) exchanger (NHE), Cl(-)/HCO(3)(-) exchanger (AE), and Na(+)/HCO(3)(-) cotransporter (NBC) are known to contribute to the intracellular pH (pH(i)) regulation during agonist-induced stimulation. This study examined the mechanisms for the pH(i) regulation in the mouse parotid and sublingual acinar cells using the fluorescent pH-sensitive probe, BCECF. The pH(i) recovery from agonist-induced acidification in the sublingual acinar cells was completely blocked by EIPA, a NHE inhibitor. However, the parotid acinar cells required DIDS, a NBC1 inhibitor, in addition to EIPA in order to block the pH(i) recovery. Moreover, RT-PCR analysis detected the expression of pancreatic NBC1 (pNBC1) only in the parotid acinar cells. These results provide strong evidence that the mechanisms for the pH(i) regulation are different in the two types of acinar cells, and pNBC1 contributes to pH(i) regulation in the parotid acinar cells, whereas NHE is likely to be the exclusive pH(i) regulator in the sublingual acinar cells.     

Increased luminal pH in the epididymis of infertile c-ros knockout mice and the expression of sodium-hydrogen exchangers and vacuolar proton pump H+-ATPase

Transgenic mice targeted for the c-ros gene, which are fertile when heterozygous (HET), but infertile when homozygous (knockout, KO) and associated with failure in pubertal differentiation of the epididymal initial segment, provide a model for studying the role of the epididymal luminal environment in sperm development. Luminal fluid from the cauda epididymidis was measured by both ion-selective microelectrodes and pH strips to be 0.3 pH units higher in the KO than HET. Of the genes responsible for luminal acidification, expression of mRNA of vacuolar H(+)-ATPase was found in all epididymal regions, but with no difference between KO and HET. Immunohistochemistry showed its presence in epithelial apical cells and clear cells. The Na(+)-hydrogen exchanger NHE2 was expressed at mRNA and protein levels in the caput but only marginally detectable if at all in the distal epididymis. This was compensated for by NHE3 which was expressed strongest in the cauda region, in agreement with immunohistochemical staining. Quantification of Western blot data revealed slight, but significant, decreases of NHE2 in the caput and of NHE3 in the cauda in the KO mice. The increase in luminal fluid pH in the KO mice could also be contributed to by other epithelial regulating factors including the Na(+)-dependent glutamate transporter EAAC1 formerly reported to be down regulated in the KO.     

Differential effects of superoxide on luminal and basolateral Na+/H+ exchange in the thick ascending limb

Superoxide (O2-) increases Na+ reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of O2- on other THAL transporters, such as Na(+)/H+ exchangers, are unknown. We hypothesized that O2- stimulates Na(+)/H+ exchange in the THAL. We assessed total Na(+)/H+ exchange activity by measuring recovery of intracellular pH (pH(i)) after acid loading in isolated perfused THALs before and after adding xanthine oxidase (XO) and hypoxanthine (HX). We found that XO and HX decreased total pH(i) recovery rate from 0.26 +/- 0.05 to 0.21 +/- 0.04 pH units/min (P < 0.05), and this net inhibition decreased steady-state pH(i) from 7.52 to 7.37. Because THALs have different Na(+)/H+ exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral Na(+)/H+ exchange by adding dimethylamiloride to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal Na(+)/H+ exchange from 3.5 +/- 0.8 to 6.7 +/- 1.4 pmol.min(-1).mm(-1) (P < 0.01) but decreased basolateral Na(+)/H+ exchange from 10.8 +/- 1.8 to 6.8 +/- 1.1 pmol.min(-1).mm(-1) (P < 0.007). To ascertain whether these effects were caused by O2- or H2O2, we examined the ability of tempol, a superoxide dismutase mimetic, to block these effects. In the presence of tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral Na(+)/H+ exchange. We conclude that O2- inhibits basolateral and stimulates luminal Na(+)/H+ exchangers, perhaps because different isoforms are expressed on each membrane. Inhibition of basolateral Na(+)/H+ exchange may enhance stimulation of luminal Na(+)/H+ exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of O2- on Na(+)/H+ exchange may increase net HCO3- reabsorption by the THAL.     

Na+-H+ exchange at the apical membrane of Necturus gallbladder. Extracellular and intracellular pH studies

The mechanism of luminal solution acidification was studied in Necturus gallbladder by measurement of mucosal solution and intracellular pH with glass electrodes. When the gallbladder was bathed by a Na-Ringer's solution it acidified the luminal side by a Na+-dependent, amiloride- inhibitable process. In the presence of ouabain, acidification was reduced but could be stimulated to a rate greater than that under control conditions by the imposition of an inwardly directed Na+ gradient. These results suggest that luminal acidification results from Na+-H+ exchange at the apical membrane and not by diffusion of metabolic CO2. Li+ can substitute for Na+ but K+, Rb+, Cs+, and tetramethylammonium (TMA+) cannot. The maximal rate of exchange was about five times greater for Na+ than for Li+. Intracellular pH (pHi) was measured with recessed-tip glass microelectrodes; with the tissue bathed in Na-Ringer's solution (pH 7.75), pHi was 7.51 +/- 0.04. After inhibition of Na+-H+ exchange by mucosal perfusion with amiloride (1 mM) or by complete Na+ replacement with TMA+, phi fell reversibly by 0.15 and 0.22 pH units, respectively. These results support the conclusion that Na+-H+ exchange at the apical membrane is the mechanism of luminal acidification and is involved in the maintenance of steady state pHi.     

Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na(+)-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl- /HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na(+)-independent vacuolar-type H+ pump, and (c) BLM-located, Na(+)- dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene- 2,2'-disulfonic acid sensitive mechanism, possibly a Na(+)-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na(+)-dependent HCO3- transporter. An Na(+)-independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.     

Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes

Na+/H+ exchanger (NHE) isoforms play important roles in intracellular pH regulation and in fluid absorption. The isoform NHE3 has been localized to apical surfaces of epithelia and in some tissues may facilitate the absorption of NaCl. To determine whether the apical isoform NHE3 is present in cholangiocytes and to examine whether it has a functional role in cholangiocyte fluid secretion and absorption, immunocytochemical studies were performed in rat liver with NHE3 antibodies and functional studies were obtained in isolated bile duct units from wild-type and NHE3-/- mice after stimulation with forskolin, using videomicroscopic techniques. Our results indicate that NHE3 protein is present on the apical membranes of rat cholangiocytes and on the canalicular membrane of hepatocytes. Western blots also detect NHE3 protein in rat cholangiocytes and isolated canalicular membranes. After stimulation with forskolin, duct units from NHE3-/- mice fail to absorb the secreted fluid from the cholangiocyte lumen compared with control animals. Similar findings were observed in isolated bile duct units from wild-type mice and rats in the presence of the Na+/H+ exchanger inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. In contrast, we could not demonstrate absorption of fluid from the canalicular lumen of mouse or rat hepatocyte couplets after stimulation of secretion with forskolin. These findings indicate that NHE3 is located on the apical membrane of rat cholangiocytes and that this NHE isoform can function to absorb fluid from the lumens of isolated rat and mouse cholangiocyte preparations.     

Zinc and 17beta-estradiol induce modifications in Na+/H+ exchanger and pyruvate kinase activity through protein kinase C in isolated mantle/gonad cells of Mytilus galloprovincialis

We investigated the transduction pathway mediated by Zn and 17beta-estradiol in isolated mantle/gonad cells of the mussel Mytilus galloprovincialis. Both the essential metal Zn, and the estrogen 17beta-estradiol, caused an increase in intracellular pH (pHi) of isolated mantle/gonad cells of the mussel M. galloprovincialis, thus indicating the activation of the Na+/H+ exchanger (NHE). The observed effect was inhibited by EIPA (20 nM), a specific NHE inhibitor, thus verifying NHE activation. Protein kinase C (PKC) also seemed to play an activating role in zinc and 17beta-estradiol effects on NHE and PK activity. In addition, the glycolytic enzyme pyruvate kinase (PK) was increased after zinc, while it was decreased after 17beta-estradiol treatment. It is noteworthy that, both the latter effects were reversed in the presence of EIPA, indicating the involvement of NHE in the signaling mechanism. cAMP seems to participate in the signaling mechanism induced by Zn but not to that induced by 17beta-estradiol. The potential implication of the heavy metal and 17beta-estradiol on the reproductive activity of the marine animals is discussed.     

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 Ca(2+)-activated Cl(-) secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca(2+)-activated Cl(-) secretion was accompanied by secretion of HCO(3)(-), possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO(2)-25 mM HCO(3)(-)). During carbachol (CCh) stimulation, pH(i) 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 pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO(2)-HCO(3)(-)-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO(3)(-) efflux by ion substitution or exposure to the Cl(-) channel inhibitor niflumic acid (100 microM) 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 pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO(3)(-) during Ca(2+)-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl(-) channel, with HCO(3)(-) secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na(+)-dependent pH(i) regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na(+)-free media.     

Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process

In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH-sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium-coupled H+/OH-/HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule.     

Increased tolerance to oxygen and glucose deprivation in astrocytes from Na(+)/H(+) exchanger isoform 1 null mice

The ubiquitously expressed Na(+)/H(+) exchanger isoform 1 (NHE1) functions as a major intracellular pH (pH(i)) regulatory mechanism in many cell types, and in some tissues its activity may contribute to ischemic injury. In the present study, cortical astrocyte cultures from wild-type (NHE1(+/+)) and NHE1-deficient (NHE1(-/-)) mice were used to investigate the role of NHE1 in pH(i) recovery and ischemic injury in astrocytes. In the absence of HCO(3)(-), the mean resting pH(i) levels were 6.86 +/- 0.03 in NHE1(+/+) astrocytes and 6.53 +/- 0.04 in NHE1(-/-) astrocytes. Removal of extracellular Na(+) or blocking of NHE1 activity by the potent NHE1 inhibitor HOE-642 significantly reduced the resting level of pH(i) in NHE1(+/+) astrocytes. NHE1(+/+) astrocytes exhibited a rapid pH(i) recovery (0.33 +/- 0.08 pH unit/min) after NH(4)Cl prepulse acid load. The pH(i) recovery in NHE1(+/+) astrocytes was reversibly inhibited by HOE-642 or removal of extracellular Na(+). In NHE1(-/-) astrocytes, the pH(i) recovery after acidification was impaired and not affected by either Na(+)-free conditions or HOE-642. Furthermore, 2 h of oxygen and glucose deprivation (OGD) led to an approximately 80% increase in pH(i) recovery rate in NHE1(+/+) astrocytes. OGD induced a 5-fold rise in intracellular [Na(+)] and 26% swelling in NHE1(+/+) astrocytes. HOE-642 or genetic ablation of NHE1 significantly reduced the Na(+) rise and swelling after OGD. These results suggest that NHE1 is the major pH(i) regulatory mechanism in cortical astrocytes and that ablation of NHE1 in astrocytes attenuates ischemia-induced disruption of ionic regulation and swelling.