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)(-).     

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.     

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.     

High sodium intake increases HCO(3)- absorption in medullary thick ascending limb through adaptations in basolateral and apical Na+/H+ exchangers

A high sodium intake increases the capacity of the medullary thick ascending limb (MTAL) to absorb HCO(3)(-). Here, we examined the role of the apical NHE3 and basolateral NHE1 Na(+)/H(+) exchangers in this adaptation. MTALs from rats drinking H(2)O or 0.28 M NaCl for 5-7 days were perfused in vitro. High sodium intake increased HCO(3)(-) absorption rate by 60%. The increased HCO(3)(-) absorptive capacity was mediated by an increase in apical NHE3 activity. Inhibiting basolateral NHE1 with bath amiloride eliminated 60% of the adaptive increase in HCO(3)(-) absorption. Thus the majority of the increase in NHE3 activity was dependent on NHE1. A high sodium intake increased basolateral Na(+)/H(+) exchange activity by 89% in association with an increase in NHE1 expression. High sodium intake increased apical Na(+)/H(+) exchange activity by 30% under conditions in which basolateral Na(+)/H(+) exchange was inhibited but did not change NHE3 abundance. These results suggest that high sodium intake increases HCO(3)(-) absorptive capacity in the MTAL through 1) an adaptive increase in basolateral NHE1 activity that results secondarily in an increase in apical NHE3 activity; and 2) an adaptive increase in NHE3 activity, independent of NHE1 activity. These studies support a role for NHE1 in the long-term regulation of renal tubule function and suggest that the regulatory interaction whereby NHE1 enhances the activity of NHE3 in the MTAL plays a role in the chronic regulation of HCO(3)(-) absorption. The adaptive increases in Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL may play a role in enabling the kidneys to regulate acid-base balance during changes in sodium and volume balance.     

Expression of the Na+-HCO3- cotransporter and its role in pHi regulation in guinea pig salivary glands

Patterns of salivary HCO(3)(-) secretion vary and depend on species and gland types. However, the identities of the transporters involved in HCO(3)(-) transport and the underlying mechanism of intracellular pH (pH(i)) regulation in salivary glands still remain unclear. In this study, we examined the expression of the Na(+)-HCO(3)(-) cotransporter (NBC) and its role in pH(i) regulation in guinea pig salivary glands, which can serve as an experimental model to study HCO(3)(-) transport in human salivary glands. RT-PCR, immunohistochemistry, and pH(i) measurements from BCECF-AM-loaded cells were performed. The amiloride-sensitive Na(+)/H(+) exchanger (NHE) played a putative role in pH(i) regulation in salivary acinar cells and also appeared to be involved in regulation in salivary ducts. In addition to NHE, NBC also played a role in pH(i) regulation in both acini and ducts. In the parotid gland, NBC1 was functionally expressed in the basolateral membrane (BLM) of acinar cells and the luminal membrane (LM) of ducts. In the submandibular gland, NBC1 was expressed only in the BLM of ducts. NBC1 expressed in these two types of salivary glands takes up HCO(3)(-) and is involved in pH(i) regulation. Although NBC3 immunoreactivity was also detected in submandibular gland acinar cells and in the ducts of both glands, it is unlikely that NBC3 plays any role in pH(i) regulation. We conclude that NBC1 is functionally expressed and plays a role in pH(i) regulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands.     

The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na(+)-dependent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO(3)(-) transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO(3)(-)-transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.     

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter

Regulation of intra- and extracellular ion activities (e.g. H(+), Cl(-), Na(+)) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na(+)/HCO(3)(-) cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO(3)(-) transporters are known: a neutral Na(+)/ HCO(3)(-) cotransporter, a K(+)/ HCO(3)(-) cotransporter, and a Na(+)-dependent Cl(-)-HCO(3)(-) exchanger. We report the cloning and characterization of a Na(+)-coupled Cl(-)-HCO(3)(-) exchanger and a physiologically unique bicarbonate transporter superfamily member. This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughout Drosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl(-), Na(+), H(+), and HCO(3)(-) but does not require HCO(3)(-). Transport is blocked by the stilbene 4,4'-diisothiocyanodihydrostilbene- 2, 2'-disulfonates and may not be strictly electroneutral. Our functional data suggest this Na(+) driven anion exchanger (NDAE1) is responsible for the Na(+)-dependent Cl(-)-HCO(3)(-) exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Activation of Na+/H+ exchanger-directed protein kinases in the ischemic and ischemic-reperfused rat myocardium

The activity of the Na(+)/H(+) exchanger has been implicated as an important contributing factor in damage to the myocardium that occurs during ischemia and reperfusion. We examined regulation of the protein in ischemic and reperfused isolated hearts and isolated ventricular myocytes. In isolated myocytes, extracellular signal-regulated kinases were important in regulating activity of the exchanger after recovery from ischemia. Ischemia followed by reperfusion caused a strong inhibitory effect on NHE1 activity that abated with continued reperfusion. Four major protein kinases of size 90, 55, 44, and 40 kDa phosphorylated the Na(+)/H(+) exchanger. The Na(+)/H(+) exchanger-directed kinases demonstrated dramatic increases in activity of 2-10-fold that was induced by 3 different models of ischemia and reperfusion in intact hearts and isolated myocytes. p90(rsk) was identified as the 90-kDa protein kinase activated by ischemia and reperfusion while ERK1/2 was identified as accounting for some of the 44-kDa protein kinase phosphorylating the Na(+)/H(+) exchanger. The results demonstrate that MAPK-dependent pathways including p90(rsk) and ERK1/2 and are important in regulating the Na(+)/H(+) exchanger and show their dramatic increase in activity toward the Na(+)/H(+) exchanger during ischemia and reperfusion of the myocardium. They also show that ischemia followed by reperfusion have important inhibitory effects on Na(+)/H(+) exchanger activity.     

cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO3- cotransporter subtype pNBC1

Basolateral Na(+)-HCO(3)(-) cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na(+)-HCO(3)(-) cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na(+)-HCO(3)(-) transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na(+)-HCO(3)(-) cotransport rates were 5.07 +/- 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na(+)-HCO(3)(-) cotransport activation was secondary to secretion-associated changes in HCO(3)(-) or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na(+)-HCO(3)(-) transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na(+)-HCO(3)(-) transport rates in CFTR -/- compared with CFTR +/+ crypts but similar Na(+)-HCO(3)(-) cotransport activation by forskolin. These studies establish the existence of an intracellular HCO(3)(-) concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.     

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).     

Nerve growth factor inhibits Na+/H+ exchange and formula absorption through parallel phosphatidylinositol 3-kinase-mTOR and ERK pathways in thick ascending limb

In the medullary thick ascending limb, inhibiting the basolateral NHE1 Na(+)/H(+) exchanger with nerve growth factor (NGF) induces actin cytoskeleton remodeling that secondarily inhibits apical NHE3 and transepithelial HCO(3)(-) absorption. The inhibition by NGF is mediated 50% through activation of extracellular signal-regulated kinase (ERK). Here we examined the signaling pathway responsible for the remainder of the NGF-induced inhibition. Inhibition of HCO(3)(-) absorption was reduced 45% by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 and 50% by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), a downstream effector of PI3K. The combination of a PI3K inhibitor plus rapamycin did not cause a further reduction in the inhibition by NGF. In contrast, the combination of a PI3K inhibitor plus the MEK/ERK inhibitor U0126 completely eliminated inhibition by NGF. Rapamycin decreased NGF-induced inhibition of basolateral NHE1 by 45%. NGF induced a 2-fold increase in phosphorylation of Akt, a PI3K target linked to mTOR activation, and a 2.2-fold increase in the activity of p70 S6 kinase, a downstream effector of mTOR. p70 S6 kinase activation was blocked by wortmannin and rapamycin, consistent with PI3K, mTOR, and p70 S6 kinase in a linear pathway. Rapamycin-sensitive inhibition of NHE1 by NGF was associated with an increased level of phosphorylated mTOR in the basolateral membrane domain. These findings indicate that NGF inhibits HCO(3)(-) absorption in the medullary thick ascending limb through the parallel activation of PI3K-mTOR and ERK signaling pathways, which converge to inhibit NHE1. The results identify a role for mTOR in the regulation of Na(+)/H(+) exchange activity and implicate NHE1 as a possible downstream effector contributing to mTOR's effects on cell growth, proliferation, survival, and tumorigenesis.     

Regulation of in vivo cardiac contractility by phospholemman: role of Na+/Ca2+ exchange

Phospholemman (PLM), when phosphorylated at serine 68, relieves its inhibition on Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger 1 (NCX1) in cardiac myocytes. Under stress when catecholamine levels are high, enhanced Na(+)-K(+)-ATPase activity by phosphorylated PLM attenuates intracellular Na(+) concentration ([Na(+)](i)) overload. To evaluate the effects of PLM on NCX1 on in vivo cardiac contractility, we injected recombinant adeno-associated virus (serotype 9) expressing either the phosphomimetic PLM S68E mutant or green fluorescent protein (GFP) directly into left ventricles (LVs) of PLM-knockout (KO) mice. Five weeks after virus injection, ～40% of isolated LV myocytes exhibited GFP fluorescence. Expression of S68E mutant was confirmed with PLM antibody. There were no differences in protein levels of α(1)- and α(2)-subunits of Na(+)-K(+)-ATPase, NCX1, and sarco(endo)plasmic reticulum Ca(2+)-ATPase between KO-GFP and KO-S68E LV homogenates. Compared with KO-GFP myocytes, Na(+)/Ca(2+) exchange current was suppressed, but resting [Na(+)](i), Na(+)-K(+)-ATPase current, and action potential amplitudes were similar in KO-S68E myocytes. Resting membrane potential was slightly lower and action potential duration at 90% repolarization (APD(90)) was shortened in KO-S68E myocytes. Isoproterenol (Iso; 1 μM) increased APD(90) in both groups of myocytes. After Iso, [Na(+)](i) increased monotonically in paced (2 Hz) KO-GFP but reached a plateau in KO-S68E myocytes. Both systolic and diastolic [Ca(2+)](i) were higher in Iso-stimulated KO-S68E myocytes paced at 2 Hz. Echocardiography demonstrated similar resting heart rate, ejection fraction, and LV mass between KO-GFP and KO-S68E mice. In vivo closed-chest catheterization demonstrated enhanced contractility in KO-S68E compared with KO-GFP hearts stimulated with Iso. We conclude that under catecholamine stress when [Na(+)](i) is high, PLM minimizes [Na(+)](i) overload by relieving its inhibition of Na(+)-K(+)-ATPase and preserves inotropy by simultaneously inhibiting Na(+)/Ca(2+) exchanger.     

The basolateral NHE1 Na+/H+ exchanger regulates transepithelial HCO3- absorption through actin cytoskeleton remodeling in renal thick ascending limb

In the renal medullary thick ascending limb (MTAL), inhibiting the basolateral NHE1 Na(+)/H(+) exchanger with amiloride or nerve growth factor (NGF) results secondarily in inhibition of the apical NHE3 Na(+)/H(+) exchanger, thereby decreasing transepithelial HCO3- absorption. MTALs from rats were studied by in vitro microperfusion to identify the mechanism underlying cross-talk between the two exchangers. The basolateral addition of 10 microM amiloride or 0.7 nM NGF decreased HCO3- absorption by 27-32%. Jasplakinolide, which stabilizes F-actin, or latrunculin B, which disrupts F-actin, decreased basal HCO3- absorption by 30% and prevented the inhibition by amiloride or NGF. Jasplakinolide had no effect on HCO3- absorption in tubules bathed with amiloride or a Na(+)-free bath to inhibit NHE1. Jasplakinolide and latrunculin B did not prevent inhibition of HCO3- absorption by vasopressin or stimulation by hyposmolality, factors that regulate HCO3- absorption through primary effects on apical Na(+)/H(+) exchange. Treatment of MTALs with amiloride or NGF for 15 min decreased polymerized actin with no change in total cell actin, as assessed both by fluorescence microscopy and by actin Triton X-100 solubility. Jasplakinolide prevented amiloride-induced actin remodeling. Vasopressin, which inhibits HCO3- absorption by an amount similar to that observed with amiloride and NGF but does not act via NHE1, did not affect cellular F-actin content. These results indicate that basolateral NHE1 regulates apical NHE3 and HCO3- absorption in the MTAL by controlling the organization of the actin cytoskeleton.     

14-3-3 Binding to Na+/H+ exchanger isoform-1 is associated with serum-dependent activation of Na+/H+ exchange

Na(+)/H(+) exchanger isoform-1 (NHE1), the ubiquitous form of the Na(+)/H(+) exchanger, has increased activity in hypertensive patients and in animal models of hypertension. Furthermore, NHE1 is activated in cells stimulated with growth factors. We showed previously that activation of the exchanger is dependent on phosphorylation of serine 703 (Ser(P)(703)) by p90 ribosomal S6 kinase (RSK). Because the NHE1 sequence at Ser(P)(703) (RIGSDP) is similar to a consensus sequence (RSXSXP) specific for 14-3-3 ligands, we evaluated whether serum stimulated 14-3-3 binding to NHE1. Five different GST-NHE1 fusion proteins spanning amino acids 515-815 were phosphorylated by RSK and used as ligands in a far Western analysis; only those containing Ser(P)(703) exhibited high affinity 14-3-3 binding. In PS127A cells (NHE1-overexpressing Chinese hamster fibroblasts) stimulated with 20% serum, NHE1 co-precipitation with GST-14-3-3 fusion protein increased at 5 min (5.2 +/- 0.4-fold versus control; p < 0.01) and persisted at 40 min (3.9 +/- 0.3-fold; p < 0.01). We confirmed that binding occurs at the RIGSDP motif using PS120 (NHE1 null) cells transfected with S703A-NHE1 or P705A-NHE1 (based on data indicating that 14-3-3 binding requires phosphoserine and +2 proline). Serum failed to stimulate association of 14-3-3 with these mutants. A GST-NHE1 fusion protein was phosphorylated by RSK and used as a ligand to assess the effect of 14-3-3 on protein phosphatase 1-mediated dephosphorylation of Ser(P)(703). GST-14-3-3 limited dephosphorylation (66% of initial state at 60 min) compared with GST alone (27% of initial state; p < 0.01). The protective effect of GST-14-3-3 was lost in the GST-NHE1 P705A mutant. Finally, the base-line rate of pH recovery in acid-loaded cells was equal in unstimulated cells expressing wild-type or P705A-NHE1. However, activation of NHE1 by serum was dramatically inhibited in cells expressing P705A-NHE1 compared with wild-type (0.13 +/- 0.02 versus 0.48 +/- 0.06 mmol of H(+)/min/liter, p < 0.01). These data suggest that 14-3-3 binding to NHE1 participates in serum-stimulated exchanger activation, a new function for 14-3-3.     

Glucose-induced cytosolic pH changes in beta-cells and insulin secretion are not causally related: studies in islets lacking the Na+/H+ exchangeR NHE1

The contribution of Na(+)/H(+) exchange (achieved by NHE proteins) to the regulation of beta-cell cytosolic pH(c), and the role of pH(c) changes in glucose-induced insulin secretion are disputed and were examined here. Using real-time PCR, we identified plasmalemmal NHE1 and intracellular NHE7 as the two most abundant NHE isoforms in mouse islets. We, therefore, compared insulin secretion, cytosolic free Ca(2+) ([Ca(2+)](c)) and pH(c) in islets from normal mice and mice bearing an inactivating mutation of NHE1 (Slc9A1-swe/swe). The experiments were performed in HCO(-)(3)/CO(2) or HEPES/NaOH buffers. PCR and functional approaches showed that NHE1 mutant islets do not express compensatory pH-regulating mechanisms. NHE1 played a greater role than HCO(-)(3)-dependent mechanisms in the correction of an acidification imposed by a pulse of NH(4)Cl. In contrast, basal pH(c) (in low glucose) and the alkalinization produced by high glucose were independent of NHE1. Dimethylamiloride, a classic blocker of Na(+)/H(+) exchange, did not affect pH(c) but increased insulin secretion in NHE1 mutant islets, indicating unspecific effects. In control islets, glucose similarly increased [Ca(2+)](c) and insulin secretion in HCO(-)(3) and HEPES buffer, although pH(c) changed in opposite directions. The amplification of insulin secretion that glucose produces when [Ca(2+)](c) is clamped at an elevated level by KCl was also unrelated to pH(c) and pH(c) changes. All effects of glucose on [Ca(2+)](c) and insulin secretion proved independent of NHE1. In conclusion, NHE1 protects beta-cells against strong acidification, but has no role in stimulus-secretion coupling. The changes in pH(c) produced by glucose involve HCO(-)(3)-dependent mechanisms. Variations in beta-cell pH(c) are not causally related to changes in insulin secretion.     

Gene expression of Na+/H+ exchanger in zebrafish H+ -ATPase-rich cells during acclimation to low-Na+ and acidic environments

In mammalian nephrons, most of the Na(+) and HCO(3)(-) is reabsorbed by proximal tubular cells in which the Na(+)/H(+) exchanger 3 (NHE3) is the major player. The roles of NHEs in Na(+) uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish (Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1-8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H(+)-ATPase but not with Na(+)-K(+)-ATPase, indicating that H(+)-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na(+) FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H(+)-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H(+)-ATPase are probably involved in Na(+) uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.     

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia

The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO(3)(-) transporter family (Na(+)/HCO(3)(-) cotransporter NBC1 and Cl(-)/HCO(3)(-) exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO(2). Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na(+)/H(+) exchangers (NHE1A, NHE1B) during the first days (by 50-60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na(+)-K(+)-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO(2) exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na(+)-K(+)-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.