The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.     

Amiloride blocks cell-free protein synthesis at levels attained inside cultured rat hepatocytes

Amiloride, a passive Na⁺ influx inhibitor, lowers initial rates and plateau levels of [³⁵S]met uptake into proteins in cell-free rabbit reticulocyte lysates (ID₅₀∽0.4 mM). Isolated hepatocytes take up amiloride through a saturable (K_m∽0.02 mM; V_max∽1.43 nmol/ 10⁶ cells/min) Na⁺-dependent process. Similar temperature dependent uptake occurs in cultured hepatocyte monolayers. In chemically defined media, under growth reinitiation conditions, amiloride lowers overall rates of cellular protein and albumin synthesis (ID₅₀∽0.4 and ∽0.028 mM, respectively). Amiloride concentrations (0.02 mM) that half-maximally inhibit reinitiation of hepatocyte DNA synthesis reach, within 30 min, cellular levels (∽0.14 mM) that block reticulocyte lysate protein synthesis by 25%. These findings complicate interpretations, from studies in many eukaryotic systems, of cause and effect between mitogen-activated membrane Na⁺ influxes and the reinitiation of DNA synthesis.     

Characteristics of an amiloride-sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells

We have studied the induction of an amiloride-sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na⁺ influxes of approximately 25–30 nmol/mg protein min; < 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na⁺ influx to 15–20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na⁺ influx, depending on the cell type, of 20–400% within 2 min. This increment in Na⁺ influx represented an increase in amiloride-sensitive Na⁺ transport with an apparent K′, of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride-sensitive Na⁺ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 μg/ml of cycloheximide blocked the increase in Na⁺ transport, indicating that de novo protein synthesis mediated this serum deprivation–induced increase in Na⁺ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na⁺ influx did not saturate with Na⁺ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na⁺ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.     

A Na+-DEPENDENT SYSTEM A AND ASC-INDEPENDENT AMINO ACID TRANSPORT SYSTEM STIMULATED BY GLUCAGON IN RAT HEPATOCYTES

The effects of glucagon on amino acid transport in rat hepatocytes are not fully understood. We examined the effect of this hormone on alanine, serine and cysteine preferring system (system ASC)-mediated amino acid transport in rat hepatocyte monolayers using 2-aminoisobutyric acid (AIB) and L -cysteine. Glucagon induced a time and protein synthesis-dependent stimulation of Na⁺-dependent alanine preferring system (system A)-independent AIB transport. The glucagon-induced increase in transport activity was not modified by substrate starvation and not related to changes in the intracellular pool of amino acids. Glucagon did not modify system ASC activity measured by L -cysteine. Therefore the transport activity of AIB independent of system A stimulated by glucagon cannot be attributed to system ASC. This suggests a Na⁺-dependent transport system in rat hepatocytes not identified until now.     

Demonstration of a late amiloride-sensitive event as a necessary step in initiation of DNA synthesis by thrombin

Amiloride, a Na⁺ influx inhibitor, has been shown to inhibit initiation of DNA synthesis by thrombin in mouse embryo fibroblast-like cells. Long exoosures (24 hr) to high concentrations of amiloride inhibited incorporation of thymidine into the DNA of both thrombin-stimulated and nonstimulated cells, suggesting that this inhibition might not be specific for thrombin-initiated DNA synthesis. Fluorescence microscopy and spectrofluorimetry showed that amiloride was internalized with an apparent mitochondrial association and that the internalized amiloride was readily released from the cells after removing amiloride from the medium. Based on this reversibility, cells were exposed to amiloride for short periods of time during thrombin treatment to determine the temporal relationship between any amiloride-sensitive event(s) and initiation of DNA synthesis. The presence of amiloride (100 μM) during a 12-hr exposure to thrombin did not block thrombin-initiated DNA synthesis or cell division but did delay the onset of DNA synthesis and the peak of thymidine incorporation into DNA by approximately 3 hr, suggesting that early initiation events might proceed in the presence of amiloride. ⁸⁶Rb⁺ transport studies demonstrated that in this system ouabain-sensitive K⁺ uptake via the Na, K-ATPase was stimulated by thrombin during both an early and a late period. This stimulation was amiloride-sensitive under the same conditions used for growth experiments, suggesting that amiloride was inhibiting thrombin-stimulated Na⁺ transport in this system. Additional experiments showed that exposing cells to amiloride only during the first 8 hr after thrombin addition did not inhibit initiation. The presence of amiloride from 8–12 hr after thrombin addition maximally inhibited thrombin-stimulated DNA synthesis. Together these results demonstrate that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8–12 hr after thrombin addition.     

Induction of DNA synthesis in adult rat hepatocytes cultured in a serum-free medium

Isolated hepatocytes from adult rats were cultured for 3 days in a serum-free synthetic medium. Supplementation with fibrinogen digests, glucagon and insulin remarkably increased DNA synthesis in hepatocytes. DNA synthesis began to increase at 35 h and reached a maximum at 41 to 54 h after plating. At this time, cells were morphologically identifiable as hepatocytes. Glucagon could be replaced by dibutyryl cyclic AMP or isobutyl-methyl-xanthine. Addition of amiloride (a Na⁺ influx inhibitor) during the initial 22 h completely inhibited DNA synthesis. These results suggest that influx of Na⁺ during early prereplicative period and increase in cellular cyclic AMP levels during late prereplicative period are necessary for the induction of DNA synthesis in hepatocytes.     

Membrane potential and sodium flux in neuroblastoma X glioma hybrid cells: Effects of amiloride and serum

The Na⁺ uptake into neuroblastoma x glioma hybrid cells was measured in Hepes-buffered EMEM containing 10% calf serum and 5 mM ouabain in the presence and absence of amiloride (1.0 mM). Amiloride was found to markedly inhibit net Na⁺ influx (by approximately 50%). Examination of the effect of amiloride on net Na⁺ influx in the absence of calf serum revealed that a significant amiloride-sensitive Na⁺ influx remains even under serum-deprived conditions, although the degree of amiloride inhibition (35%) is substantially lower than that found in the presence of serum. The amiloride-insensitive portion of Na⁺ influx was found to be independent of serum effects. Estimation of resting membrane potential was made by measurement of the steady state distribution of the lipophilic cation, TPP⁺, in the presence and absence of amiloride. A large, immediate increase in TPP⁺ uptake, indicative of a membrane hyperpolarization, was seen upon addition of amiloride. Determination of the effect of amiloride on resting membrane potential of serum-deprived cells showed that cells are hyperpolarized to a greater extent in the presence than in the absense of amiloride, and that serum exerts a depolarizing effect on the cells. Thus, serum-stimulation of Na⁺ influx results in a depolarization of resting membrane potential, while amiloride inhibition of Na⁺ influx causes a hyperpolarization. These data strongly suggest that NG108-15 cells possess an electrogenic Na⁺ influx pathway that is sensitive to amiloride inhibition and enhanced by serum.     

Amiloride, protein synthesis, and activation of quiescent cells

Amiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0 to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.     

Inhibition by amiloride of sodium transport into rabbit kidney medulla microsomes

Sodium transport into rabbit kidney medulla microsomes was 50% inhibited by amiloride. This Na⁺ uptake was shown to represent transport when the uptake process was reversed by the ionophore nigericin. The transport was complete within 60 min and proportional to the microsomal protein concentration. The effect of amiloride on transport was specific since the similar compound sulfaguanidine failed to affect microsomal Na⁺ transport. Amiloride-sensitive Na⁺ transport into microsomes was inhibited 70% by decreasing the pH (from 7.0 to 5.9), but was unaffected by the presence of a pH gradient. The kinetics of Na⁺ transport could be explained by a simple model, assuming that amiloride lowered the rate of Na⁺ entrance into the vesicles but had no effect on the rate of efflux. The failure of amiloride to effect efflux from the vesicles was also demonstrated directly.     

Amiloride is a cholinergic antagonist in the rabbit pancreas

The effect of amiloride on fluid and protein secretion in the isolated rabbit pancreas and on amylase secretion in rabbit pancreatic acini has been studied. Amiloride (1 mM) has no effect on the pancreatic fluid secretion either in a normal incubation medium (143 mM Na⁺), or in a medium containing only 25 mM Na⁺. The carbachol-induced enzyme secretion is inhibited by amiloride in both systems, whereas the enzyme secretion induced by the C-terminal octapeptide of cholecystokinin (PzO) is not affected. Amiloride also inhibits the carbachol-induced ⁴⁵Ca efflux from rabbit pancreatic acini, but again not that induced by PzO. The amiloride concentrations for half-maximal inhibition of carbachol-induced amylase secretion and ⁴⁵Ca efflux are 40 and 80 μM, respectively. Amiloride also competitively inhibits the specific binding of [³H]quinuclidinyl benzylate ([³H]QNB) to rabbit pancreatic acini, suggesting that the amiloride effect is due to competition on the level of the muscarinic acetylcholine receptor.     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.     

Osmoregulation of amino acid transport activity in cultured fibroblasts

The effect of exposure of chick embryo cells to increasing concentrations of Na⁺ in the culture medium on the subsequent amino acid transport as determined at physiological osmolarity was investigated in detail. It was found that the hyperosmolar treatment stimulated amino acid transport in a dose-dependent manner up to 200 mM Na⁺. Changes were measurable as early as 1 h after altering Na⁺ and reached a maximum after 4 h, remaining constant thereafter. The maintenance of this effect required continuous exposure of the cell to high Na⁺ in the culture medium. Hyperosmolarity-mediated increases in amino acid transport activity by system A have been detected with l-proline and l-alanine. Transport activities of systems ASC and L did not change appreciably after exposure of the cells to high Na⁺. Inhibition of protein synthesis by cycloheximide or RNA synthesis by actinomycin D (actD) prevented these uptake changes. Kinetic analysis indicated that the stimulation of the activity of transport system A by high Na⁺ treatment occurred through a mechanism affecting V_max rather than K_m.     

Participation of protein phosphatases in regulation of sodium transport across the erythrocyte membrane of the lamprey Lampetra fluviatilis

Erythrocytes of lamprey Lampetra fluviatilis were incubated in standard isotonic medium at 20°C with ²²Na to determine the unidirectional Na⁺ influx. Cell incubation in the presence of various protein phosphatase inhibitors (NaF, cantharidin, calyculin A) led to a considerable increase of Na⁺ transport into erythrocytes. The stimulation of Na⁺ influx into erythrocytes rose with increase of concentration of calyculin A within the range of 10–100 nM. The calyculin A concentration producing a 50% activation of Na⁺ transport amounted to 41.5 nM. Under optimal experimental conditions, the Na⁺ influx increased from control level of 5–8 to 20–40 mmol/l cells/h under effect of protein phosphatase blockers. The Na⁺ transport induced by these inhibitors was completely suppressed on addition of amiloride to the incubation medium. The treatment of lamprey erythrocytes with protein phosphatase inhibitors was accompanied by a small (～12%), but statistically significant decrease of intracellular Na⁺ content. A small decrease of intracellular K⁺ content in erythrocyte was observed only under the effect of NaF. The obtained data allow making the conclusion that protein phosphatases of the PP1 and PP2A types play a significant role in regulation of Na⁺ transport across the lamprey erythrocyte membrane in both directions.     

Receptor Binding Profiles of Amiloride Analogues Provide no Evidence for a Link Between Receptors and the Na+/H+ Exchange,But Indicate a Common Structure on Receptor Proteins

Abstract

Amiloride and its analogues affect radioligand binding to the adenosine-A₁ receptor. In this paper, the specificity of this effect is investigated by generating receptor binding profiles for amiloride and two of its analogues. A limited structure-activity relationships study is performed to probe the relationship between inhibition of receptor binding by amiloride analogues and the effects of these compounds on Na⁺ transport, in particular Na⁺/H⁺ exchange. The receptor binding profiles of amiloride, benzamil and 5′-(N,N-hexamethylene)amiloride (HMA) indicate that the compounds affect a variety of receptors and that none of the compounds is highly selective for any of these. The SAR study indicates that it is very unlikely that a direct coupling between receptors and Na⁺/H⁺ exchange or another amiloride-sensitive ion transport system is responsible for the inhibition of receptor binding. A correlation between the signal transduction systems coupled to the receptors involved and the potency of the amiloride analogues is also absent. The varying nature of the receptors, affected by amiloride or its analogues, suggests a wide-spread presence of an amiloride binding site on receptors and other membrane proteins.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions II. Relationship between counter-flows of fluorescein and sodium ion across cell layer

With the aid of a direct microfluorimetric method a dependence of organic onion (fluorescein) transport into proximal tubules of surviving frog kidney on Na⁺-flow in the opposite direction was studied. It was shown that the complete removal of Na⁺ from the tubules lumen resulted in inhibition of fluorescein transport of about 30%. After a specific inhibitor of sodium channels, amiloride (10^-3M) having been introduced into lumen of the tubules, the fluorescein transport was inhibited to the same extent. Amiloride affects only when Na⁺ is present in the tubular lumen. S present in the tubular lumen. Strophantin K (5 · 10^?5 M), a specific inhibitor of (Na⁺, K⁺)-ATPase, reduced fluorescein transport about twice. Substances increasing the 3′,5′-AMP level in cells (theophylline, NaF) and exogenous 3′,5′-AMP inhibited fluorescein transport while substance that decreased the 3′,5′-AMP level intracellularly (carbachol) stimulated it. For realization of these effects Na⁺ should be present in proximal tubules lumen.Thus, the various effects on the Na⁺ flow from lumen of the tubules to medium at the level of both the basal and apical membranes alter the rate of organic acid active transport from medium to lumen as a result of changes in the maximum rate of transport ($\text{V}$) with unchanged $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. It is suggested that the system of Na⁺ extrusion from proximal tubules produces peritubular membrane-side (near the membrane) gradient of Na⁺ concentration which may be higher than the summary Na⁺ gradient between the medium and the cytoplasm. The magnitude of this gradient affects the maximal rate value of Na⁺-dependent organic acid transport. So, there is a double dependence of the active transport system on Na⁺, and the stages where Na⁺ is needed are: (1) the formation of a carrier-substrate-Na⁺ complex and (2) the production of substantial membrane-side Na⁺ gradient at the expense of Na⁺ extrusion from the tubules.     

Alanine and leucine transport in unfertilized pig oocytes and early blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. In mouse oocytes and cleavage-stage conceptus Na⁺-dependent L-alanine and L-leucine transport are nearly undetectable. Sodium-dependent transport via system B^O,+ in the mouse conceptus increases greatly between the 8-cell and blastocyst stages. By contrast, data presented here for the pig show that L-alanine and L-leucine transport is mainly Na⁺-dependent in the oocyte; this Na⁺-dependent component of transport becomes undetectable by the blastocyst stage. The Na⁺-dependent component of transport in oocytes is inhibited by BCH (2-aminoendo-bicyclo[2.2.1] hexane-2-carboxylic acid) and L-lysine and thus could be a form of system B^O,+. In both oocytes and blastocysts Na⁺-independent L-leucine transport is inhibited by BCH, which is consistent with the presence of system L. The dramatic decrease in Na⁺-dependent amino acid transport activity could occur in pig conceptuses in association with the onset of RNA synthesis during the 4-cell stage. Regardless of the precise time during development at which it occurs, however, this dramatic, developmentally regulated decrease in Na⁺-dependent alanine and leucine transport activity contrasts sharply with the large increase in Na⁺-dependent system B^O,+ activity that occurs during preimplantation development of murine conceptuses. Elucidation of the molecular mechanisms by which these changes occur should contribute to an understanding of regulation of gene expression during early development. © 1993 Wiley-Liss, Inc.     

Transport of lithium and rectification by frog skin

The isolated frog skin, bathed with Li⁺-Ringer (Na⁺-free) on the outside and Na⁺-Ringer on the inside, can maintain a normal potential difference (PD) and short-circuit current (s.c.c.) for more than 6. h. The s.c.c. correspondended to the Li⁺ influx. The Na⁺ efflux was 4% of the s.c.c. 10⁻⁵ M ouabain depressed Li⁺ influx and s.c.c. 10¹⁰⁻⁵ M amiloride abolished the Li⁺ s.c.c., while 0.1 unit/ml oxytocin stimulated it. When the inside of the skin was bathed with Li⁺-Ringer, PD and s.c.c. fell to zero within 2 h. The oxygen consumption of skin slices bathed in Li⁺-Ringer was 29% lower than controls bathed in Na⁺-Ringer.When the isolated frog skin is bathed in Na₂SO₄-Ringer it shows electrical rectification which has been correlated with the active transport of Na⁺. In skins transporting Li⁺, rectification characteristics are similar to those of skins transporting Na⁺. When the inner face of the skin is bathed with Li⁺-Ringer, rectification, PD and s.c.c. decline in a parallel fashion.It is concluded that: (1) Li⁺ can be transported when Na⁺ is present at the inner face. (2) Amiloride, ouabain and oxytocin affect Li⁺ and Na⁺ transport in a similar manner. (3) Li⁺ transport, like Na⁺ transport, is associated with rectification. (4) Active transport of Na⁺ and Li⁺ seems to depend on two different but associated proceses; one taking place at the external barrier (where rectification occurs) as shown by the effect of amiloride; and the other of an inner site related to energy requirements and affected by ouabain and Li⁺. (5) The cation being transported is not necessarily activating the (Na⁺-K⁺-ATPase.     

Na+ transport by human placental brush border membranes: Are there several mechanisms?

Na⁺ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na⁺ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na⁺ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na⁺ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na⁺ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na⁺ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride-sensitive Na⁺ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na⁺ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na⁺. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na⁺ uptake, whereas benzamil, the Na⁺ channel blocker, had no effect, it was concluded that the amiloride sensitive Na⁺ uptake was predominantly or exclusively due to a Na⁺-H⁺ exchanger activity. K⁺ in trans-position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis-position had no effect. The amiloride resistant Na⁺ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 μM or 1 mM dibutyryl cAMP, 0.1 or 1 μM phorbol myristate acetate, 10⁻⁷ M insulin, 10⁻¹⁰ M angiotensin II, or 10⁻⁸ M human parathyroid hormone (PTH) did not influence Na⁺ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na⁺-H⁺ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of co-substrates such as phosphate and aminoacids, the Na⁺-H⁺ exchange is probably the unique mechanism of Na⁺ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane. © 1996 Wiley-Liss, Inc.     

Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues

Summary Dicyclohexylcarbodiimide (DCCD) and the 5-ethylisopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na⁺/H⁺ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na^–/H⁺ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na⁺, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na⁺-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na⁺/H⁺ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na⁺ and Li⁺ protected. [¹⁴C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na⁺ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [¹⁴C]-DCCD that was identified as the -subunit of the Na⁺, K⁺-ATPase. [¹⁴C]-Br-EIPA was mainly incorporated into a brushborder membrane polypeptide with apparent molecular weight of 65,000. Na⁺ decreased the labeling of this protein. Similar to the Na⁺/H⁺ exchanger this Na⁺-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na⁺/H⁺ exchange.