Intracellular pH regulatory mechanism in human atrial myocardium: functional evidence for Na(+)/H(+) exchanger and Na(+)/HCO(3)(-) symporter

Intracellular pH (pH(i)) exerts considerable influence on cardiac contractility and rhythm. Over the last few years, extensive progress has been made in understanding the system that controls pH(i) in animal cardiomyocytes. In addition to the housekeeping Na(+)-H(+) exchanger (NHE), the Na(+)-HCO(3)(-) symporter (NHS) has been demonstrated in animal cardiomyocytes as another acid extruder. However, whether the NHE and NHS functions exist in human atrial cardiomyocytes remains unclear. We therefore investigated the mechanism of pH(i) recovery from intracellular acidosis (induced by NH(4)Cl prepulse) using intracellular 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein fluorescence in human atrial myocardium. In HEPES (nominally HCO(3)(-)-free) Tyrode solution, pH(i) recovery from induced intracellular acidosis could be blocked completely by 30 microM 3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE 694), a specific NHE inhibitor, or by removing extracellular Na(+). In 3% CO(2)-HCO(3)(-) Tyrode solution, HOE 694 only slowed the pH(i) recovery, while addition of HOE 694 together with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (an NHS inhibitor) or removal of extracellular Na(+) inhibited the acid extrusion entirely. Therefore, in the present study, we provided evidence that two acid extruders involved in acid extrusion in human atrial myocytes, one which is HCO(3)(-) independent and one which is HCO(3)(-) dependent, are mostly likely NHE and NHS, respectively. When we checked the percentage of contribution of these two carriers to pH(i) recovery following induced acidosis, we found that the activity of NHE increased steeply in the acid direction, while that of NHS did not change. Our present data indicate for the first time that two acid extruders, NHE and NHS, exist functionally and pH(i) dependently in human atrial cardiomyocytes.     

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.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

Gastrointestinal distribution and kinetic characterization of the sodium-hydrogen exchanger isoform 8 (NHE8).

NHE8 is a newly identified NHE isoform expressed in rat intestine. To date, the kinetic characteristics and the intestinal segmental distribution of this NHE isoform have not been studied. This current work was performed to determine the gene expression pattern of the NHE8 transporter along the gastrointestinal tract, as well as its affinity for Na(+), H(+), and sensitivity to known NHE inhibitors HOE694 and S3226. NHE8 was differentially expressed along the GI tract. Higher NHE8 expression was seen in stomach, duodenum, and ascending colon in human, while higher NHE8 expression was seen in jejunum, ileum and colon in adult mouse. Moreover, the expression level of NHE8 is much higher in the stomach and jejunum in young mice compared with adult mice. To evaluate the functional characterictics of NHE8, the pH indicator SNARF-4 was used to monitor the rate of intra-cellular pH (pH(i)) recovery after an NH(4)Cl induced acid load in NHE8 cDNA transfected PS120 cells. The NHE8 cDNA transfected cells exhibited a sodium-dependent proton exchanger activity having a Km for pH(i) of approximately pH 6.5, and a Km for sodium of approximately 23 mM. Low concentration of HOE694 (1 microM) had no effect on NHE8 activity, while high concentration (10 microM) significantly reduced NHE8 activity. In the presence of 80 microM S3226, the NHE8 activity was also inhibited significantly. In conclusion, our work suggests that NHE8 is expressed along the gastrointestinal tract and NHE8 is a functional Na(+)/H(+) exchanger with kinetic characteristics that differ from other apically expressed NHE isoforms.     

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.     

Mechanisms of Mg(2+) transport in cultured ruminal epithelial cells

Net Mg(2+) absorption from the rumen is mainly mediated by a transcellular pathway, with the greater part (62%) being electrically silent. To investigate this component of Mg(2+) transport, experiments were performed with isolated ruminal epithelial cells (REC). Using the fluorescent indicators mag-fura 2, sodium-binding benzofuran isophthalate, and 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, we measured the intracellular free Mg(2+) concentration ([Mg(2+)](i)), the intracellular Na(+) concentration ([Na(+)](i)), and the intracellular pH (pH(i)) of REC under basal conditions, after stimulation with butyrate and HCO(-)(3), and after changing the transmembrane chemical gradients for Mg(2+), H(+), and Na(+). REC had a mean resting pH(i) of 6.83 +/- 0.1, [Mg(2+)](i) was 0.56 +/- 0. 14 mM, and [Na(+)](i) was 18.95 +/- 3.9 mM. Exposure to both HCO(-)(3) and HCO(-)(3)/butyrate led to a stimulation of Mg(2+) influx that amounted to 27.7 +/- 5 and 29 +/- 10.6 microM/min, respectively, compared with 15 +/- 1 microM/min in control solution. The increase of [Mg(2+)](i) was dependent on extracellular Mg(2+) concentration ([Mg(2+)](e)). Regulation of pH(i) has been demonstrated to be Na(+) dependent and is performed, for the most part, by a Na(+)/H(+) exchanger. The recovery of pH(i) was fully blocked in nominally Na(+)-free media, even if [Mg(2+)](e) was stepwise increased from 0 to 7.5 mM. However, an increase of [Mg(2+)](i) was observed after reversing the transmembrane Na(+) gradient. This rise in [Mg(2+)](i) was pH independent, K(+) insensitive, dependent on [Mg(2+)](e), imipramine and quinidine sensitive, and accompanied by a decrease of [Na(+)](i). The results are consistent with the existence of a Na(+)/Mg(2+) exchanger in the cell membrane of REC. The coupling between butyrate, CO(2)/HCO(-)(3), and Mg(2+) transport may be mediated by another mechanism, perhaps by cotransport of Mg(2+) and HCO(-)(3).     

Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 +/- 0.14 to 0.76 +/- 0.06 mM, corresponding to a Mg2+ uptake rate of 15 microM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 +/- 19%) in the presence of CO2/HCO(-)3. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in HCO(-)3-buffered high-KCl medium (22.3 +/- 4 microM/min) rather than in HEPES-buffered KCl medium (37.5 +/- 6 microM/min). After switching from high- to low-Cl- solution, [Mg2+]i was reduced from 0.64 +/- 0.09 to 0.32 +/- 0.16 mM and the CO2/HCO(-)3-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl- and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.     

Relative contributions of Na+/H+ exchange and Na+/HCO3- cotransport to ischemic Nai+ overload in isolated rat hearts

The Na(+)/H(+) exchanger (NHE) and/or the Na(+)/HCO(3)(-) cotransporter (NBC) were blocked during ischemia in isolated rat hearts. Intracellular Na(+) concentration ([Na(+)](i)), intracellular pH (pH(i)), and energy-related phosphates were measured by using simultaneous (23)Na and (31)P NMR spectroscopy. Hearts were subjected to 30 min of global ischemia and 30 min of reperfusion. Cariporide (3 microM) or HCO(3)(-)-free HEPES buffer was used, respectively, to block NHE, NBC, or both. End-ischemic [Na(+)](i) was 320 +/- 18% of baseline in HCO(3)(-)-perfused, untreated hearts, 184 +/- 6% of baseline when NHE was blocked, 253 +/- 19% of baseline when NBC was blocked, and 154 +/- 6% of baseline when both NHE and NBC were blocked. End-ischemic pH(i) was 6.09 +/- 0.06 in HCO(3)(-)-perfused, untreated hearts, 5.85 +/- 0.02 when NHE was blocked, 5.81 +/- 0.05 when NBC was blocked, and 5.70 +/- 0.01 when both NHE and NBC were blocked. NHE blockade was cardioprotective, but NBC blockade and combined blockade were not, the latter likely due to a reduction in coronary flow, because omission of HCO(3)(-) under conditions of NHE blockade severely impaired coronary flow. Combined blockade of NHE and NBC conserved intracellular H(+) load during reperfusion and led to massive Na(+) influx when blockades were lifted. Without blockade, both NHE and NBC mediate acid-equivalent efflux in exchange for Na(+) influx during ischemia, NHE much more than NBC. Blockade of either one does not affect the other.     

Mechanisms contributing to intracellular pH homeostasis in an immortalised human chondrocyte cell line

The maintenance of chondrocyte pH is an important parameter controlling cartilage matrix turnover rates. Previous studies have shown that, to varying degrees, chondrocytes rely on Na(+)/H(+) exchange to regulate pH. HCO(3)(-)-dependent buffering and HCO(3)(-)-dependent acid-extrusion systems seem to play relatively minor roles. This situation may reflect minimal carbonic anhydrase activity in cartilage cells. In the present study, the pH regulation of the human chondrocyte cell line, C-20/A4 has been characterised. Intracellular pH (pH(i)) was measured using the H(+)-sensitive fluoroprobe BCECF. In solutions lacking HCO(3)(-)/CO(2), pH(i) was approximately 7.5, and the recovery from intracellular acidification was predominantly mediated by a Na(+)-dependent, amiloride- and HOE 694-sensitive process. A small additional component which was sensitive to chloro-7-nitrobenz-2-oxa-1,3-diazole, an inhibitor of the V-type H(+)-ATPase, was also apparent. In solutions containing HCO(3)(-)/CO(2), pH(i) was approximately 7.2. Comparison of buffering capacity in the two conditions showed that this variable was not significantly augmented in HCO(3)(-)/CO(2)-containing media. The recovery from intracellular acidification was more rapid in the presence of HCO(3)(-)/CO(2), although under these conditions it was again largely dependent on Na(+) ions and inhibited by amiloride and HOE 694. A small component was inhibited by SITS, although this effect did not reach the level of statistical significance. These findings indicate that HCO(3)(-)-dependent processes play only a minimal role in pH regulation in C-20/A4 chondrocytes. pH regulation instead relies heavily on the Na(+)/H(+) exchanger together with a H(+)-ATPase. The absence of extrinsic (HCO(3)(-)/CO(2)) buffering is likely to reflect the low levels of carbonic anhydrase in these cells. In addition to providing fundamental information about a widely-used cell line, these findings support the contention that the unusual nature of pH regulation in chondrocytes reflects the paucity of carbonic anhydrase activity in these cells.     

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.     

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.     

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.     

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)(-).     

Immunolocalization of anion exchanger AE2, Na(+)/H(+) exchangers NHE1 and NHE4, and vacuolar type H(+)-ATPase in rat pancreas.

We have studied the expression and localization of several H(+) and HCO(3)(-) transporters, whose presence in the rat pancreas is still unclear. The Cl(-)/HCO(3)(-) exchanger AE2, the Na(+)/H(+) exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H(+)-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at approximately 160 kD for AE2, at approximately 90 kD and approximately 103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl(-) uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO(3)(-) secretion. The molecular identity of the ductal Cl(-)/HCO(3)(-) exchanger remains unclear.     

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.     

Cl--dependent and Cl--independent Na+/ HCO3- acid extrusion in cultured rat cerebellar astrocytes

It is still uncertain whether the Na+-dependent Cl--HCO3- exchanger (NCBE) is expressed in mammalian astrocytes. Using fluorescent indicators to monitor the intracellular pH (pHi) and intracellular Na+ or Cl- levels, the NCBE in cultured rat cerebellar astrocytes was examined in detail. In nominally bicarbonate-free (Hepes-buffered) medium, a marked pHi recovery from internal acid load was seen which could be blocked completely by 30 microM HOE 694, a specific Na+-H+ exchanger isoform 1(NHE-1) inhibitor, at a pHi above 6.9. These conditions were therefore used to block NHE activity in CO2/HCO3-buffered media when the NCBE was being studied at pHi above 6.9. After internal acid loading in completely Cl--free bicarbonate-buffered medium (containing HOE 694), the rates of pHi recovery and transient Na+ influx were considerably slowed, and the Cl--dependent acid extrusion was both Na+- and 4,4-diisothiocyano-stilbene-disulphonic acid (DIDS)-sensitive. Moreover, a HCO3-dependent Cl- efflux during internal acid injection was seen. These results suggest that the NCBE is present in astrocytes. Following repetitive internal acid loading by addition of 5% CO2 to internal Cl- depleted cells, a similar rate of pHi recovery was consistently seen, suggesting Cl--independent pHi regulation also occurred in astrocytes. Moreover, this pHi recovery was completely blocked in the absence of sodium or on addition of DIDS, confirming that the Na+-HCO3 cotransporter (NBC) is present. Thus, the present study provides evidence that both the NCBE and NBC play important roles in acid extrusion in cultured mammalian astrocytes.     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.