Amiloride Sensitivity Research Articles

Ion transport in rat tongue epithelium in vitro: A developmental study

The responsiveness of the rat gustatory system to monochloride salts changes during development. Neurophysiological recordings in the chorda tympani indicate that a) the taste responses to NaCl and KCl in early postnatal rats are small relative to NH 4Cl, b) both salts become more potent stimuli as the animal matures, and c) the developmental increase is accompanied by an increase in sensitivity of the NaCl response to the sodium transport blocker amiloride. We measured ion transport properties of in vitro tongue epithelia from Wistar rats. When the tissue is mounted in an Ussing chamber, the short-circuit current responses to NaCl and KCl are small in the neonatal rat and increase during development in postweaning and adult animals. Amiloride sensitivity of the NaCl response also increases with age. This study confirms that increased sensitivity of the rat gustatory system to NaCl with age reflects changes in the peripheral membranes. The results support the hypothesis that the increased sensitivity is due to amiloride-sensitive membrane components being added or becoming functional.

Vasopressin and protein kinase A activate G protein-sensitive epithelial Na+ channels

To determine the molecular steps involved in the vasopressin-induced renal Na+ reabsorption, the patch-clamp technique was utilized to study the role of this hormone in the regulation of apical Na+ channels in renal epithelial A6 cells. Addition of arginine vasopressin (AVP) induced and/or enhanced Na+ channel activity within 5 min of addition under cell-attached conditions. The AVP-induced channel activity was a reflection of both an increase in the average apparent channel number (0.2-1.7) and the percent open time (2-56%). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, the adenosine 3',5'-cyclic monophosphate (cAMP) analogues, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, or forskolin elicited a comparable effect to that of AVP. The induced channels had similar properties to Na+ channels previously reported, including a channel conductance of 9 pS, Na(+)-to-K+ selectivity of 3-5:1, and high amiloride sensitivity. The cAMP-dependent protein kinase A (PKA) in the presence of ATP induced and/or enhanced Na+ channel activity in excised inside-out patches with a change in average apparent channel number and percent open probability similar to those observed with either AVP or cAMP analogues in intact cells. Addition of activated pertussis toxin (100 ng/ml) completely blocked the AVP- or PKA-induced Na+ channel activity in excised inside-out patches, whereas incubation of intact cells with the toxin completely prevented the effect of both activators. The data indicate that AVP mediates its effect through a cAMP-dependent pathway involving PKA activation whose target is the G protein pathway that regulates apical epithelial Na+ channel activity.

Leu143 in the Putative Fourth Membrane Spanning Domain Is Critical for Amiloride Inhibition of an Epithelial Na+/H+ Exchanger Isoform (NHE-2)

A family of Na+/H+ exchanger isoforms (called NHE1, NHE2, and NHE3) which exhibits a wide range of amiloride sensitivity has recently been cloned and characterized. A part of the domain, which determines amiloride sensitivity in the epithelial Na+/H+ exchanger isoform, NHE2, was identified by site-directed mutagenesis and functional studies using cDNAs stably expressed in a fibroblast cell line. It has previously been reported that AR300, an amiloride resistant mutant of the ubiquitous Na+/H+ exchanger isoform, NHE1, is 30-fold more resistant to methylpropyl amiloride (MPA) compared to NHE1 and contains a single amino acid substitution of L167F in the fourth putative transmembrane helix, which corresponds to L143 in NHE2. Therefore, in the present study point mutational substitutions were introduced into the equivalent of this fourth transmembrane helix of rabbit NHE2 (including Y144F; L143F; L143F and Y144F) to mimic the corresponding amino acids in NHE1, NHE3 (another epithelial isoform) and AR300, respectively. NHE2/L143F (mimicking NHE3) increased the IC50 for amiloride by 5-fold and for ethylisopropyl amiloride (EIPA) by 20-fold. Similarly, NHE2/L143F and Y144F (mimicking AR300) increased the resistance to both amiloride and EIPA by 10-fold. On the other hand, NHE2/Y144F (mimicking NHE1) did not affect the sensitivity to amiloride or EIPA, and this mutant, like wild type NHE2, is partially resistant to EIPA. Thus, amino acid 143 of NHE2 is critical for, but is not the only amino acid responsible for, amiloride and EIPA inhibition of Na+/H+ exchange. That none of the mutations studied altered the Na+ affinity of these Na+/H+ exchangers further suggests that amiloride binding and Na+ transport sites are not identical.

Proton currents through amiloride-sensitive Na + channels in isolated hamster taste cells: Enhancement by vasopressin and cAMP

Amiloride has been suggested to inhibit responses to a variety of taste stimuli, including salty, sweet, and sour (acid). To test for the involvement of amiloride-sensitive Na+ channels in the transduction of acid stimuli, fungiform taste receptor cells were examined using patch-clamp techniques. Approximately one-half of all cells had amiloride-sensitive Na+ currents (INa) with a Ki value near 0.2 microM amiloride. After blocking voltage-gated conductances, cells having amiloride sensitivity were tested for responses to acid stimuli. Over three-fourths of cells showed an inward proton current (IH+) with an extrapolated reversal potential near approximately +150 mV, which was completely blocked by amiloride (30 microM). Treatment of isolated taste cells with arginine8-vasopressin caused equivalent increases in both INa and IH+; each effect was mimicked by 8-Br-cAMP. Taken together, these results indicate that protons permeate amiloride-sensitive Na+ channels in hamster fungiform taste cells and contribute to acid transduction.

Altered gustatory development in Na +-Restricted rats is not explained by low Na + levels in mothers' milk

Placing pregnant rats on a sodium-deficient diet (0.03% NaCl) very early in gestation and then weaning the offspring (sodium-restricted rats) to the same diet precludes development of amiloride-sensitive sodium taste transduction pathways in the offspring. However, normal amiloridesensitive sodium taste responses can be restored by permitting sodium ingestion by sodium-restricted rats. The present study tested the hypothesis that the concentration of sodium in sodium-restricted mothers' milk must be abnormally low in order to preserve altered gustatory function in the offspring. Other milk electrolyte and total protein concentrations were determined as well. Milk sodium was similar between sodium-restricted and control rat mothers at 10–13 and 16–20 days postpartum, as were levels of potassium and chloride. At 10–13 days postpartum, total protein was higher in milk from sodium-restricted mothers. Sodium-restricted mothers' milk calcium concentrations were higher versus controls at 16–20 days postpartum. These results indicate that the lack of gustatory amiloride sensitivity in sodium-restricted rats cannot be attributed to deficient dietary sodium levels during the suckling period.

Cold activation of Na influx through the Na-H exchange pathway in guinea pig red cells

Previous work showed that amiloride partially inhibits the net gain of Na in cold-stored red cells of guinea pig and that the proportion of unidirectional Na influx sensitive to amiloride increases dramatically with cooling. This study shows that at 37 degrees C amiloride-sensitive (AS) Na influx in guinea pig red blood cells is activated by cytoplasmic H+, hypertonic incubation, phorbol ester in the presence of extracellular Ca2+ and is correlated with cation-dependent H+ loss from acidified cells. Cytoplasmic acidification increases AS Na efflux into Na-free medium. These properties are consistent with the presence of a Na-H exchanger with a H+ regulatory site. Elevation of cytoplasmic free Mg2+ above 3 mM greatly increases AS Na influx: this correlates with a Na-dependent loss of Mg2+, indicating the presence of a Na-Mg exchanger. At 20 degrees C activators of Na-H exchange have little or no further stimulatory effect on the already elevated AS Na influx. AS Na influx is much larger than either Na-dependent H+ loss or AS Na efflux at 20 degrees C. The affinity of the AS Na influx for cytoplasmic H+ is greater at 20 degrees C than at 37 degrees C. Depletion of cytoplasmic Mg2+ does not abolish the high AS Na influx at 20 degrees C. Thus, elevation of AS Na influx with cooling appears to be due to increased activity of a Na-H exchanger (operating in a "slippage" mode) caused by greater sensitivity to H+ at a regulatory site.

Characterization of a distinct Na(+)-H+ exchanger in basolateral membranes of human jejunum

Kinetically distinct Na(+)-H+ exchangers exist on the apical and basolateral membranes of rabbit ileal enterocytes. The apical Na(+)-H+ exchanger appears to function in electroneutral NaCl transport, whereas the basolateral Na(+)-H+ exchanger may function in homeostatic intravesicular pH (pHi) regulation and volume regulation. This study is designed to determine the presence and characteristics of the Na(+)-H+ exchanger in basolateral membrane vesicles (BLMV) prepared from jejunal tissues of human organ donors. A well-validated Percoll-gradient technique was used to prepare BLMV. An outwardly directed H+ gradient [pHi/extravesicular pH (pHo) = 5.2/7.5] resulted in a Na+ uptake overshoot (1.45 +/- 0.21 nmol/mg protein) 2.5-fold above equilibrium values (0.59 +/- 0.13 nmol/mg protein). Na+ uptake at equilibrium represented transport into an osmotically sensitive intravesicular space as validated by an osmolality study. Na+ uptake represented an electroneutral process, as shown by studies in which negative membrane potentials were induced by K+ and the ionophore valinomycin. Na+ uptake was linear for the first 15 s of transport as depicted by y = 0.042x + 0.002, r2 = 0.98. Dixon plot analysis of amiloride sensitivity revealed an ID50 value for amiloride of 29 microM (fourfold lower than ID50 for brush-border Na(+)-H+ exchanger). Kinetic studies of amiloride-sensitive Na+ uptake revealed a maximal velocity = 1.53 +/- 0.19 nmol.mg protein-1.5 s-1 and Michaelis constant = 9.83 +/- 3.5 mM. By varying pHi a sigmoidal effect of internal H+ on Na+ uptake was noted consistent with an internal modifier site for protons. To confirm this finding, the effect of pHi on Na+ efflux and Na(+)-Na+ exchange was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosolic Ca 2+-induced modulation of ion selectivity and amiloride sensitivity of a cation channel and beta agonist action in fetal lung epithelium

The cytosolic Ca 2+ concentration ([Ca 2+] i) affects many cell functions, including the modulation of ion channel activity. In this study patch clamp experiments using primary cultures of fetal distal lung epithelium (FDLE) demonstrated that the elevation of [Ca 2+] i modulated a 25pS amiloride-blockable non selective cation (NSC) channel's ion selectivity and sensitivity to amiloride. An elevation of [Ca 2+] i from 0.1μM to 1mM both increased open probability (P o) and decreased the ratio of the premeability to Na to the permeability to K ( P Na P K ) from 1.96±0.11 (SEM, n=6) to 0.88±0.04 (n=6). Within the range of [Ca 2+] i from 0.1μM to 100μM amiloride (0.5μM) decreased P o, however amiloride (0.5μM) no longer affected P o of the NSC channel when [Ca 2+] i was increased to 1mM under physiologic membrane potentials. A β adrenergic agonist (terbutaline, 10μM) increased P o in cellattached patches from almost 0 (P o<0.01; n=9) to 0.39±0.09 (n=9) and [Ca 2+] i from 40±6nM (n=9) to more than 1μM. This suggested that amiloride-blockable NSC channel activity and ion permeability are modulated by changes in [Ca 2+] i near physiologic membrane potentials and a β adrenergic agonist increases [Ca 2+] i to more than 1μM (unlike other epithelial including adult alveolar cells) which is associated with activation the NSC channel.

Sodium-dependent short-circuit current across the yolk sac membrane during embryonic development in normal and shell-less cultured chicks.

The transepithelial electrical characteristics of the isolated yolk sac membrane of normal in ovo or shell-less cultured chick embryos were investigated. In normal chicks the potential difference (blood side positive relative to yolk side) and short-circuit current of the membrane increased during development. Ouabain (10(-4) M) on the blood side (basolateral side, serosal side) significantly decreased potential difference and short-circuit current but was without effect on the yolk side (brush border side, mucosal side). Substitution of choline for Na+ in the bathing solutions abolished the potential difference and the short-circuit current; when Na+ replaced choline this effect was reversed. Amiloride added to both sides of the yolk sac membrane had no effect on potential difference or short-circuit current. Injection of aldosterone (50 micrograms) and T3 (10 microM) into yolk did not induce amiloride sensitivity. The short-circuit current was not altered by addition of either glucose or alanine to the bath. The short-circuit current of the yolk sac membrane of shell-less cultured embryos was significantly lower than that of normal controls. Addition of Ca2+ to the serosal bathing medium did not reverse the foregoing condition, but decreased the short-circuit current. It is concluded that the yolk sac short-circuit current is Na+ dependent and increases with developmental age in the chick embryo.

Cloning, sequencing, and expression of Na(+)-H+ antiporter cDNAs from human tissues.

Two types of Na(+)-H+ antiporter with different sensitivities to amiloride analogues have been identified in mammalian plasma membranes. A human Na(+)-H+ antiporter cDNA was obtained by Sardet and co-workers (C. Sardet, L. Counillon, A. Franchi, and J. Pouysségur. Cell 56: 271-280, 1989) using mutant mouse fibroblasts lacking Na(+)-H+ antiporter transformed with human genomic DNA. However, the amiloride sensitivity of this cloned Na(+)-H+ antiporter was not precisely determined. Furthermore, the reported cDNA sequence may be a chimera of human and mouse genes. Hence we isolated a Na(+)-H+ antiporter cDNA actually expressed in human tissues and characterized its amiloride sensitivity. Our 4 kb cDNA obtained from human kidney cortex contained the identical open reading frame to that previously reported and the entire 3' terminus, which was quite different from that reported. This discrepancy was not due to differences in tissue-specific expression because cDNAs from different human tissues were identical, and single bands were observed under high stringency on Northern blots of various human tissues. Na(+)-H+ antiporter activity of mutant mouse fibroblasts deficient in Na(+)-H+ antiporter activity transfected with the cloned cDNA was very sensitive to amiloride and 5-N substituted analogues of amiloride. Thus the cloned cDNA represents the NHE-1 isoform of the Na(+)-H+ antiporter.

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles.

Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (kH+) was determined by addition of vesicles (pHi = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 x 10(-3) (gastric parietal cell stimulation-associated vesicles; SAV) and 5 x 10(-3) (duodenal brush-border membrane vesicles; dBBMV) to 11 x 10(-3) sec-1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mM, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in kH+ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na+/H+ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pHi less than pHo) 22Na+ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K(+)-diffusion potential ([K+]i much much greater than [K+]o). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K(+)-ionophore valinomycin, indicating that the native K+ permeability is rate limiting. In the presence of valinomycin, the K(+)-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K+ permeability. Valinomycin was without effect, but the K+/H+ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride sensitivity of the chorda tympani response to sodium chloride in sodium-depleted Wistar rats.

Peripheral gustatory mechanisms that may contribute to the expression of sodium (Na) appetite have been a focus of interest for many years. Because amiloride-sensitive Na transport is involved in the generation of neural signals in response to NaCl stimulation, the present study assessed whether changes in amiloride sensitivity of the neural response to NaCl accompany the induction of a Na appetite in the rat. Na deprivation was achieved by acute depletion with the diuretic furosemide. The magnitude of the whole-nerve chorda tympani response to 0.5 M NaCl was reduced in Na-depleted, compared with Na-replete, rats, which provides qualified support for previous reports that the induction of a Na appetite is associated with reduced neural responses to NaCl. However, changes in sensitivity to the specific Na channel blocker amiloride hydrochloride as a result of Na depletion were not evident. These findings suggest that the behavioral and neural changes that occur after Na depletion are not based on changes in amiloride sensitivity in the taste bud.

Amiloride sensitivity of the chorda tympani response to sodium chloride in sodium-depleted Wistar rats.

Amiloride sensitivity of the chorda tympani response to sodium chloride in sodium-depleted Wistar rats.

Lack of amiloride sensitivity in SHR and WKY glossopharyngeal taste responses to NaCl

To explore possible functional strain differences in taste receptors located on the posterior tongue, we recorded electrophysiological taste responses from the glossopharyngeal nerve of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Multifiber responses to a concentration series (0.5 M to 2.0 M) of NaCl, KCl and NH 4Cl were recorded before and after lingual application of the epithelial sodium transport blocker, amiloride. Responses to a concentration series (0.0025 M to 0.1 M) of quinine hydrochloride were also recorded. When expressed relative to the 0.5-M NH 4Cl response, responses to the monochloride salts were equivalent between SHR and WKY. Surprisingly, NaCl responses were not suppressed by the sodium transport blocker, amiloride. This is in direct contrast to the dramatic suppression observed in the chorda tympani. Also, relative responses to quinine were greater in the glossopharyngeal nerve of SHR than WKY. These results indicate that taste receptors innervated by the glossopharyngeal nerve lack amiloride sensitivity and that posterior taste receptor function to monochloride salts is equivalent between SHR and WKY.

Epithelial sodium conductance in rabbit preimplantation trophectodermal cells

We examined the development of epithelial Na + conductance in 6- and 7-day post coitus (p.c.) preimplantation rabbit embryos using the whole-cell patch-clamp technique on dissociated rabbit trophectodermal cells and by immunocytochemical localization using a polyclonal antibody directed against subunits of an apical epithelial Na + channel on the intact blastocyst. In Day 6 and 7 p.c. trophectodermal cells, we observed an outwardly rectified whole-cell Na + current. The current-voltage characteristics did not differ between the 6- and the 7-day p.c. cells. Replacement of Na + with the impermeant cation N-methyl- d-glucamine in the pipette or bath reduced outward currents and inward currents, respectively, indicating that the current was Na +-dependent. Treatment of 7-day p.c. cells with 100 μ M amiloride, benzamil, or ethylisopropyl amiloride (EIPA) blocked the whole-cell currents within 5 min. However, the current of the Day 6 p.c. embryo was not blocked by amiloride. The amiloride block at Day 7 p.c. was only partially reversible after 15 min of continuous perfusion of the bath with an amiloride-free solution. The apparent dissociation constant ( K i ) for amiloride, benzamil, and EIPA was 12, 50, and 16 μ M, respectively, when measured 5 min after drug addition. Immunolocalization studies of blastocysts with a polyclonal antibody raised against a high amiloride affinity Na + channel isolated from bovine kidney revealed no specific binding to the trophectodermal cells at Day 6 p.c. At Day 7 p.c., however, an epitope, immunoreactive to the high amiloride affinity Na + channel antibody, was predominantly localized to the microvilli on the apical membrane of the trophectodermal cells. These findings suggest that: (1) Day 6 and 7 p.c. trophectodermal cells have a Na + conductance that displays similar current-voltage characteristics; (2) the apical expression of an epitope, possibly a protein related to an epithelial sodium channel, occurs between Days 6 and 7 p.c.; (3) an amiloride-sensitive component of the sodium current develops between Days 6 and 7 p.c.; and (4) the expression of immunoreactivity and development of amiloride sensitivity are temporally related.

Amiloride sensitivity of chorda tympani response to NaCl in Fischer 344 and Wistar rats

Fischer 344 (F-344) rats fail to prefer NaCl solutions to water at any concentration and avoid NaCl solutions preferred by other strains, including Wistar rats. Behavioral and electrophysiological responses of the mammalian gustatory system to NaCl have been shown to depend on a sodium transport system that is specifically blocked by lingual application of the sodium-transport blocker amiloride. The present study examined whether strain differences exist between F-344 and Wistar rats in the amiloride sensitivity of the chorda tympani (CT) electrophysiological response to NaCl. Whole nerve CT recordings were obtained from adult F-344 and Wistar rats during chemical stimulation of the anterior tongue. Responses to NaCl solutions ranging from 0.01 to 1.0 M were examined both before and after pretreatment with amiloride hydrochloride. Integrated whole nerve responses to NaCl solutions were expressed relative to the response to 0.5 M NH4Cl. Strain differences in the response to NaCl solutions emerged, with F-344 animals showing a significantly larger amplitude of the tonic response to NaCl, relative to NH4Cl, than Wistars. F-344 rats were also more sensitive to the sodium-channel blocker amiloride. These results suggest that strain differences in amiloride sensitive signals mediated by the CT nerve may contribute to the NaCl aversion displayed by F-344 rats.

The anion in salt taste: a possible role for paracellular pathways

It is well established from psychophysical and electrophysiological measurements that both Na and Cl contribute to the taste response to NaCl. The contribution of Na to the NaCl response can be studied using amiloride, a drug that inhibits Na transport in taste and other epithelial cells. The pathways involved in response to Cl are less well understood. We undertook a series of experiments in the rat to determine whether tonic chorda tympani responss to NaCl are inhibited by specific inhibitors of anion transport. Whole nerve responses to NaCl were unchanged by bathing the tongue in SITS, DIDS, bumetanide, furosemide, 9-anthracene car☐ylic acid, or an antibody that blocks Cl conductance pathways in many epithelia. Thus, Cl co-transporters, exchangers, and channels (at least in the apical membrane of taste cells) are probably not involved in NaCl taste responses. When other anions (acetate, isethionate, methane sulfonate, gluconate, tartrate) which, are generally impermeant in other Cl-selective pathways, were substituted for Cl, the dose-response curves for the chorda tympani response were shifted toward higher concentrations than the response to NaCl, but achieved the same maximum value at sufficiently high concentrations (1.0 M Na). For all the organic Na salts, the amiloride-insensitive portion of the response was substantially less than for NaCl. Experiments with Na acetate at different pHs showed that intracellular acidification is not responsible for the differences between NaCl and organic salts of Na. One possibility which remains is that apical stimulation with these other Na salts results in a taste cell membrane potential that is hyperpolarized with respect to the membrane potential in NaCl. The hyperpolarization could arise as a consequences of lower permeability to organic anions of the tight junctions between taste cells. Such a hyperpolarization would explain the shift in dose-response curves, the altered amiloride sensitivity, and might also explain how a salt stimulus could affect taste cells specific for other, non-salt stimuli.

Regulation of renal Na(+)-H+ exchanger by cAMP-dependent protein kinase

Octyl glucoside-extracted rabbit renal brush-border membrane (BBM) proteins were sequentially fractionated using anion exchange chromatography, and the fractions were tested for Na(+)-H+ exchange activity, amiloride sensitivity, and the effect of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) after reconstitution into artificial lipid vesicles. Compared with the initial protein extract, an anionic protein fraction eluting with 0.2-0.4 M NaCl (fraction B) demonstrated increased Na(+)-H+ exchange activity. Fraction B also demonstrated sensitivity to inhibition by amiloride but was not regulated by PKA. Co-reconstitution of fraction B with a BBM protein fraction highly enriched in a 42-kDa polypeptide restored the inhibitory response to PKA. These experiments suggest that, as assayed in a solubilized and reconstituted system, the Na(+)-H+ exchanger contains a dissociable PKA regulatory component, possibly a polypeptide of 42 kDa.

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

The developmental maturation of Na(+)-H+ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na+ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside = 5.2/7.5) significantly stimulated Na+ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na+ uptake which increased with advancing age. Na+ uptake represented an electroneutral process. The amiloride sensitivity of the pH-stimulated Na+ uptake was investigated using [amiloride] 10(-2)-10(-5) M. At 10(-3) M amiloride concentration, Na+ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed a Km for amiloride-sensitive Na+ uptake of 21.8 +/- 6.4, 24.9 +/- 10.9 and 11.8 +/- 4.17 mM and Vmax of 8.76 +/- 1.21, 5.38 +/- 1.16 and 1.99 +/- 0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na(+)-H+ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.

Regulation of intracellular pH in the rabbit cortical collecting tubule.

The cortical collecting tubule (CCT) is an important nephron segment for Na+, K+, water and acid-base transport. Differential loading characteristics of the pH sensitive dye 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein (BCECF) and basolateral Cl- removal were used to identify and study intracellular pH (pHi) regulation in each of three cell types involved in this transport. Both principal cells and beta-intercalated cells were found to have a basolateral Na+/H+ exchanger based on the Na+ and amiloride sensitivity of pHi recovery from acid loads. Intercalated cells demonstrated abrupt pHi changes with basolateral Cl- removal. alpha-intercalated cells alkalinized; beta-intercalated cells acidified. In the beta-intercalated cells, luminal Cl- removal blocked changes in pHi in response to changes in luminal HCO3- or peritubular Cl-, providing direct evidence for a luminal Cl-/HCO3- exchanger. In principal cells, brief removal of either peritubular or luminal Cl- resulted in no change in pHi; however, return of peritubular Cl- after prolonged removal resulted in a rapid fall in pHi consistent with a basolateral Cl-/HCO3- exchanger, which may be relatively inactive under baseline conditions. Therefore, Cl-/HCO3- exchange is present in all three cell types but varies in location and activity.