Nerve growth factor inhibits HCO3- absorption in renal thick ascending limb through inhibition of basolateral membrane Na+/H+ exchange

Nerve growth factor (NGF) inhibits transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi recovery after lumen or bath Na+ addition. NGF (0.7 nM in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3- absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525-12529). To determine whether a similar mechanism could explain inhibition of HCO3- absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mM Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3- absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3- absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3- absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3- absorption.     

Neurotrophin-3 inhibits HCO3- absorption via a cAMP-dependent pathway in renal thick ascending limb

Neurotrophins are expressed in the adult kidney, but theirsignificance is unclear. We showed previously that nerve growth factor(NGF) inhibits HCO absorption in the rat medullarythick ascending limb (MTAL) via an extracellular signal-regulatedkinase (ERK)-dependent pathway. Here we examined whether otherneurotrophic factors affect MTAL HCO absorption.Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor had no effect. In contrast, neurotrophin-3 (NT-3,0.7 nM) inhibited HCO absorption by 40%(half-maximal inhibition at ~0.4 nM). Inhibition by NT-3 was additiveto inhibition by NGF. Inhibitors of ERK activation that blockinhibition by NGF had no effect on inhibition by NT-3. In contrast,8-bromo-cAMP or forskolin pretreatment blocked inhibition by NT-3 butnot NGF. Inhibition by NT-3 was also blocked by the specific proteinkinase A (PKA) inhibitor myristoylated PKI(14-22) amide and by vasopressin, which inhibits HCO absorption via cAMP. Inhibitors of phosphatidylinositol 3-kinase orprotein kinase C did not affect NT-3-induced inhibition, but inhibitionby NT-3 was eliminated by genistein, consistent with involvement of areceptor tyrosine kinase. These results demonstrate that NT-3 inhibitsHCO absorption via a cAMP- and PKA-dependentpathway. NT-3 and NGF regulate MTAL ion transport through differentsignal transduction mechanisms. These studies establish a direct rolefor NT-3 in regulation of renal tubule transport and identify the MTALas an important target for neurotrophins, which may be involved in thecontrol of renal acid excretion.

     

Polarization of Na(+)/H(+) and Cl(-)/HCO (3)(-) exchangers in migrating renal epithelial cells

Cell migration is crucial for processes such as immune defense, wound healing, or the formation of tumor metastases. Typically, migrating cells are polarized within the plane of movement with lamellipodium and cell body representing the front and rear of the cell, respectively. Here, we address the question of whether this polarization also extends to the distribution of ion transporters such as Na(+)/H(+) exchanger (NHE) and anion exchanger in the plasma membrane of migrating cells. Both transporters are required for locomotion of renal epithelial (Madin-Darby canine kidney, MDCK-F) cells and human melanoma cells since their blockade reduces the rate of migration in a dose-dependent manner. Inhibition of migration of MDCK-F cells by NHE blockers is accompanied by a decrease of pH(i). However, when cells are acidified with weak organic acids, migration of MDCK-F cells is normal despite an even more pronounced decrease of pH(i). Under these conditions, NHE activity is increased so that cells are swelling due to the accumulation of organic anions and Na(+). When exclusively applied to the lamellipodium, blockers of NHE or anion exchange inhibit migration of MDCK-F cells as effectively as when applied to the entire cell surface. When they are directed to the cell body, migration is not affected. These data are confirmed immunocytochemically in that the anion exchanger AE2 is concentrated at the front of MDCK-F cells. Our findings show that NHE and anion exchanger are distributed in a polarized way in migrating cells. They are consistent with important contributions of both transporters to protrusion of the lamellipodium via solute uptake and consequent volume increase at the front of migrating cells.     

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.     

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.     

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.     

Basic fibroblast growth factor stimulates surface expression and activity of Na(+)/H(+) exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase

Na(+)/H(+) exchanger NHE3 is a plasma membrane (PM) protein, which contributes to Na(+) absorption in the intestine. Growth factors stimulate NHE3 via phosphatidylinositol 3-kinase (PI3-K), but mechanism of this process is not clear. To examine the hypothesis that growth factors stimulate NHE3 by modulating NHE3 recycling, and that PI3-K participates in this mechanism, we used PS120 fibroblasts expressing a fusion protein of NHE3 and green fluorescent protein. At steady state, approximately 25% of cellular NHE3 content was expressed at PM. Inhibition of PI3-K decreased PM expression of NHE3, which correlated with retention of the exchanger in recycling endosomal compartment. In contrast, basic fibroblast growth factor (bFGF) increased PM expression of NHE3, which was associated with a 2-fold increase in rate constant for exit of the exchanger from the recycling compartment. Qualitatively similar effects of bFGF were observed in cells pretreated with PI3-K inhibitors, but their magnitude was only approximately 50% of that in intact cells. These data suggest that: (i) bFGF stimulates NHE3 by increasing PM expression of the exchanger; (ii) PI3-K mediates PM expression of NHE3 in both basal and bFGF-stimulated conditions, and (iii) not all of the effects of bFGF on NHE3 expression are mediated by PI3-K, suggesting additional regulatory mechanisms.     

A chloride-activated Na(+)/HCO(3)(-)-coupled transport activity in corneal endothelial membranes

Investigations of corneal endothelium were made to resolve the apparent contradiction of the presence of sodium/bicarbonate cotransporter (NBC) in fresh and cultured cells and NBC's reported absence in isolated plasma membrane vesicles. Gradient-driven ion fluxes into the vesicles were measured. Short-term incubations (0-30 s) showed the presence of a bicarbonate-dependent inward sodium flux (BDSF), which was active when the insides of the vesicles were preloaded with chloride ions. The BDSF was absent if chloride was present only externally to the vesicles. Chloride at concentrations between 30 and 40 mM inside the vesicle had its maximum effect on BDSF. Other anions (acetate, thiocyanate, or gluconate) inside the vesicles did not mimic the chloride effect. Associated with the net inward sodium flux was a net inward bicarbonate flux. Hill plots of sodium influx with respect to external bicarbonate concentrations indicated that the stoichiometry of the net transfer was 1.7 +/- 0.2 (mean +/- standard error, n = 5) bicarbonate ions for each sodium ion transported. There was no net chloride flux found across the membrane vesicles. The finding of a novel chloride-activated NBC activity fully resolves the apparent contradiction between whole-cell and membrane vesicle preparations.     

Na(+)/H(+) exchange in mosquito Malpighian tubules

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na(+)/H(+) exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 microM 5-(N-ethyl-n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na(+)/H(+) exchange. Steady-state intracellular pH was measured microfluorometrically by using 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na(+) or in the presence of 100 microM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na(+)/H(+) exchanger by using the NH(4)Cl pulse technique resulted in a rate of recovery from the NH(4)Cl-induced acute acid load of 8.7 +/- 1.0 x 10(-3) pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na(+) or in the presence of 100 microM EIPA were 0.7 +/- 0.6 and -0.3 +/- 0.3 x 10(-3) pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na(+)/H(+) exchanger.     

CO2-stimulated NaCl absorption in the mouse renal cortical thick ascending limb of Henle. Evidence for synchronous Na +/H+ and Cl-/HCO3- exchange in apical plasma membranes

These experiments evaluated salt transport processes in isolated cortical thick limbs of Henle (cTALH) obtained from mouse kidney. When the external solutions consisted of Krebs-Ringer bicarbonate (KRB), pH 7.4, and a 95% O2-5% CO2 gas phase, the spontaneous transepithelial voltage (Ve, mV, lumen-to-bath) was approximately mV; the net rate of Cl- absorption (JnetCl) was approximately 3,600 pmols s-1 cm-2; the net rate of osmotic solute absorption Jnetosm was twice JnetCl; and the net rate of total CO2 transport (JnetCO2) was indistinguishable from zero. Thus, net Cl- absorption was accompanied by the net absorption of a monovalent cation, presumably Na+, and net HCO3- absorption was negligible. This salt transport process was stimulated by (CO2 + HCO3- ): omission of CO2 from the gas phase and HCO3- from external solutions reduced JnetCl, Jnetosm, and Ve by 50%. Furthermore, 10(-4) M luminal furosemide abolished JnetCl and Ve entirely. The lipophilic carbonic anhydrase inhibitor ethoxzolamide (10(-4) M, either luminal or peritubular) inhibited (CO2 + HCO3-)-stimulated JnetCl, Jnetosm, and Ve by approximately 50%; however, when the combination (CO2 + HCO3-) was absent, ethoxzolamide had no detectable effect on salt transport. Ve was reduced or abolished entirely by omission of either Na+ or Cl- from external solutions, by peritubular K+ removal, by 10(-3) M peritubular ouabain, and by 10(-4) M luminal SITS. However, Ve was unaffected by 10(-3) M peritubular SITS, or by the hydrophilic carbonic anhydrase inhibitor acetazolamide (2.2 x 10(-4) M, lumen plus bath). We interpret these data to indicate that (CO2 + HCO3-)-stimulated NaCl absorption in the cTALH involved two synchronous apical membrane antiport processes: one exchanging luminal Na+ for cellular H+; and the other exchanging luminal Cl- for cellular HCO3- or OH-, operating in parallel with a (CO2+ HCO3-)-independent apical membrane NaCl cotransport mechanism.     

Na(+)/Ca(2+) exchange regulates Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion in mice

Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca(2+), which regulates ion transport, including HCO(3)(-) secretion. However, the underlying Ca(2+) handling mechanisms are poorly understood. The aim of the present study was to determine whether Na(+)/Ca(2+) exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and HCO(3)(-) secretion by controlling Ca(2+) homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current (I(sc)), and HCO(3)(-) secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot, and cytosolic Ca(2+) in duodenocytes was measured by fura 2. Carbachol (100 muM) increased I(sc) in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced HCO(3)(-) secretion peaked at 10 min. 2-Aminoethoxydiphenylborate (2-APB, 100 muM) or LiCl (30 mM) significantly reduced the initial peak in I(sc) by 51 or 47%, respectively, and abolished the plateau phase of I(sc) without affecting HCO(3)(-) secretion induced by carbachol. Ryanodine (100 muM), caffeine (10 mM), and nifedipine (10 muM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10-30 muM) significantly inhibited carbachol-induced increases in duodenal mucosal I(sc) and HCO(3)(-) secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae, and functional NCX in duodenocytes was demonstrated in Ca(2+) imaging experiments where Na(+) depletion elicited Ca(2+) entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion that results from stimulation of muscarinic receptors.     

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.     

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.     

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.     

Effects of omega-3 polyunsaturated fatty acids on cardiac sarcolemmal Na(+)/H(+) exchange

Myocardial ischemia-reperfusion activates the Na(+)/H(+) exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The omega-3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the Na(+)/H(+) exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) Na(+)/H(+) exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10-100 microM eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). H(+)-dependent (22)Na(+) uptake was inhibited by 30-50% after treatment with > or =50 microM EPA or > or =25 microM DHA. This was a specific effect of these PUFAs, because 50 microM linoleic acid or linolenic acid had no significant effect on Na(+)/H(+) exchange. The SL vesicles did not exhibit an increase in passive Na(+) efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL Na(+)/H(+) exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion.     

Extracellular HCO(3)(-) dependence of electrogenic Na/HCO(3) cotransporters cloned from salamander and rat kidney

We studied the extracellular [HCOabstract (3) (-)] dependence of two renal clones of the electrogenic Na/HCO(3) cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (DeltaV(m)) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (DeltaI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract (3 )(-)/CO(2) (0.33-99 mM HCOabstract (3)(-), pH(o) 7.5) elicited an immediate, DIDS (4, 4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na(+)-dependent hyperpolarization (or outward current). In DeltaV(m) experiments, the apparent K(m ) for HCOabstract (3)(-) of akNBC (10. 6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCOabstract (3)(-) of rkNBC was less (6.5 mM). Because it has been reported that SOabstract (3)(=)/HSO abstract (3)(-) stimulates Na/HCO(3 ) cotransport in renal membrane vesicles (a result that supports the existence of a COabstract (3)(=) binding site with which SOabstract (3)(=) interacts), we examined the effect of SOabstract (3)(=)/HSO abstract (3)(-) on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract (4)(=) nor 33 mM SOabstract (3) (=)/HSOabstract (3)(-) substantially affects the apparent K(m) for HCO abstract (3)(-). We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract (3 )(-)/0.5% CO(2). We found that SO abstract (3)(=)/HSOabstract (3 )(-) did not significantly affect the DIDS-sensitive component of the pH(i) recovery from the initial CO(2 )-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO(2)](o), pH(o), and [COabstract (3)(=)](o) at a fixed [HCOabstract (3)(-)](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract (3)(=)](o ). However, a pH titration curve nicely fitted the data expressed as current versus pH(o). Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract (3 )(=), HSOabstract (3)(-), or COabstract (3)(=).