Life and times of a cellular bleb

Blebs are spherical cellular protrusions that occur in many physiological situations. Two distinct phases make up the life of a bleb, each of which have their own biology and physics: expansion, which lasts ∼30 s, and retraction, which lasts ∼2 min. We investigate these phases using optical microscopy and simple theoretical concepts, seeking information on blebbing itself, and on cytomechanics in general. We show that bleb nucleation depends on pressure, membrane-cortex adhesion energy, and membrane tension, and test this experimentally. Bleb growth occurs through a combination of bulk flow of lipids and delamination from the cell cortex via the formation and propagation of tears. In extreme cases, this can give rise to a traveling wave around the cell periphery, known as “circus movement.” When growth stalls, an actin cortex reforms under the bleb membrane, and retraction starts, driven by myosin-II. Using flicker spectroscopy, we find that retracting blebs are fivefold more rigid than expanding blebs, an increase entirely explained by the properties of the newly formed cortical actin mesh. Finally, using artificially nucleated blebs as pressure sensors, we show that cells rounded up in mitosis possess a substantial intracellular pressure.     

Apical phosphatidylserine externalization in auditory hair cells

In hair cells of the inner ear, phosphatidylserine (PS), detected with fluorescent annexin V labeling, was rapidly exposed on the external leaflet of apical plasma membranes upon dissection of the organ of Corti. PS externalization was unchanged by caspase inhibition, suggesting that externalization did not portend apoptosis or necrosis. Consistent with that conclusion, mitochondrial membrane potential and hair-cell nuclear structure remained normal during externalization. PS externalization was triggered by forskolin, which raises cAMP, and blocked by inhibitors of adenylyl cyclase. Blocking Na(+) influx by inhibiting the mechanoelectrical transduction channels and P2X ATP channels also inhibited external PS externalization. Diminished PS externalization was also seen in cells exposed to LY 294002, which blocks membrane recycling in hair cells by inhibiting phosphatidylinositol 3-kinase. These results indicate that PS exposure on the external leaflet, presumably requiring vesicular transport, results from elevation of intracellular cAMP, which can be triggered by Na(+) entry into hair cells.     

Calcium dependence of bleb formation and cell death in hepatocytes

Calcium dependence of bleb formation and cell death was evaluated in rat hepatocytes following ATP depletion by metabolic inhibition with KCN and iodoacetate ('chemical hypoxia'). Cytosolic free Ca2+ was measured in single cells by ratio imaging of Fura-2 fluorescence using multiparameter digitized video microscopy. Cells formed surface blebs within 10 to 20 minutes after chemical hypoxia and most cells lost viability within an hour. An increase of cytosolic free Ca2+ was not required for bleb formation to occur. One to a few minutes prior to the onset of cell death, free Ca2+ increased rapidly in high Ca2+ buffer (1.2 mM) but not in low Ca2+ buffer (less than 1 microM). In either buffer, the rate of cell killing was the same. As the onset of cell death was approached in both high and low Ca2+ buffers, Fura-2 began to leak from the cells at an accelerating rate indicating rapidly increasing plasma membrane permeability. In high Ca2+ buffer, cytosolic free Ca2+ increased in parallel with dye leakage. No regional changes in cytosolic free Ca2+ were observed during this metastable period of increased membrane permeability. In many experiments, actual rupture of cell surface blebs could be observed which led to micron-size discontinuities of the cell surface and cell death. We conclude that a metastable period characterized by increasing plasma membrane permeability marked the onset of cell death in cultured hepatocytes which culminated in rupture of a cell surface bleb. An increase of cytosolic free Ca2+ was not required for the metastable state to develop or cell death to occur.     

Extracellular ATP causes ROCK I-dependent bleb formation in P2X7-transfected HEK293 cells

The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1-2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.     

Differences in cortical actin structure and dynamics document that different types of blebs are formed by distinct mechanisms

Bleb formation has been studied by specifically targeting major factors controlling this process, such as microtubule disassembly, local actin depolymerization, and increased pressure. At least two different types of blebs (types 1 and 2) formed by different mechanisms and possibly a third type (type 3) can be documented at the front of living polarized cells expressing green fluorescent protein-actin and/or in fixed and stained cells. Type 1 blebs (membrane/cortex dissociation blebs) formed by dissociation of the plasma membrane from cortical actin develop cytoplasmic actin layers associated with restriction rings. They can be induced by the microtubule-disassembling agent colchicine. Type 2 blebs (cortical actin disassembly blebs) form after disassembly of the cortical actin layer in the presence of latrunculin A. Restriction rings without a cytoplasmic actin layer occur in a transition zone between the intact cortical actin layer of the cell body and the compromised actin layer of the bleb. Evidence for a third type of bleb (type 3), showing an intact cortical actin layer but no cytoplasmic actin layer and no recognizable relationship between the actin cytoskeleton and the restriction ring, has been obtained by passive cell deformation in micropipettes, which increases pressure. Repolymerization of the cortical actin layer does not necessarily result in bleb retraction. Once formed, restriction rings do not narrow, suggesting that they result from isometric contraction. A simplified classification scheme has been developed to relate the type of bleb to specific signals or cell functions. Its application shows that spontaneously blebbing cells form almost exclusively type 1 blebs.     

Toxic injury from mercuric chloride in rat hepatocytes

The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2. In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min. Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers. Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy. In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium. Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+. Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM). After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential. The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+. HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.     

Membrane tether formation from blebbing cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.     

Role of luminal ATP in regulating electrogenic Na(+) absorption in guinea pig distal colon

Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na(+) absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive I(sc) and (22)Na(+) absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive I(sc) had an IC(50) value of 20-30 microM, with the maximum inhibition being approximately 50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+ and Ca2+ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+ did not affect the percent inhibition of amiloride-sensitive I(sc) induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive I(sc), this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na(+) absorption. This effect was probably mediated by a P2Y(2) receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+ concentration may not be necessary for this process.     

Regulation of plasma membrane blebbing by the cytoskeleton

When neuroblastoma cells are exposed to lysophosphatidic acid (LPA), they undergo a vigorous, but transient blebbing phase. The effect is sensitive to inhibition by staurosporine, KT 5926 (an inhibitor of myosin light chain kinase), and cytochalasin B, suggesting that LPA activates the phosphorylation of myosin light chain and increases the contractile activity of the actomyosin network. Cell contractions increase the intracellular pressure driving bleb formation. Calyculin, an inhibitor of protein phosphatase2A, also causes blebbing which continues as long as the drug is present, presumably by keeping myosin light chain in the phosphorylated state. Blebbing of neuroblastoma cells is regulated by the status of all three cytoskeletal systems: disassembly of microtubules by nocodazole and of intermediate filaments by acrylamide increased the number of blebbing cells. Cytochalasin B, on the other hand, prevents bleb retraction and, after prolonged incubation, bleb formation. These results are discussed in terms of a model viewing the cytoskeleton as an integrated network transmitting force throughout the cell. Bleb retraction was studied by transfecting neuroblastoma cells with a vector containing the gene for gamma-cytoplasmic actin fused to the green fluorescent protein EGFP (EGFP-actin). EGFP-actin was not detected on the membranes of extending blebs, but started accumulating along the cytoplasmic surface of blebs as soon as the extension phase came to an end and retraction set in. These results confirm earlier suggestions that actin polymerization is required for bleb retraction and for the first time directly relate the two events.     

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphthoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

The formation of plasma membrane blebs in hepatocytes exposed to agents that increase cytosolic Ca2+ is mediated by the activation of a non-lysosomal proteolytic system

Exposure of isolated hepatocytes to extracellular ATP, cystamine or ionophore A23187 was associated with an increase in cytosolic Ca2+ concentration, a stimulation of intracellular proteolysis, and the appearance of plasma membrane blebs which preceded the loss of cell viability. Both bleb formation and cell killing were prevented when inhibitors of Ca2+-activated neutral proteases, such as antipain or leupeptin, were included in the incubation medium, whereas inhibitors of lysosomal proteases had no effect. Thus, the activation of a Ca2+-dependent, non-lysosomal proteolytic system appears to be responsible for the plasma membrane blebbing and, ultimately, the cytotoxicity associated with treatment of hepatocytes with agents that disrupt intracellular Ca2+ homeostasis.     

Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability

Using low-light digitized video microscopy, the onset, progression, and reversibility of anoxic injury were assessed in single hepatocytes isolated from fasted rats. Cell-surface bleb formation occurred in three stages over 1-3 h after anoxia. Stage I was characterized by formation of numerous small blebs. In stage II, small blebs enlarged by coalescence and fusion to form a few large terminal blebs. Near the end of stage II, cells began to swell rapidly, ending with the apparent breakdown of one of the terminal blebs. Breakdown of the bleb membrane initiated stage III of injury and was coincident with a rapid increase of nonspecific permeability to organic cationic and anionic molecules. On reoxygenation, stages I and II were fully reversible, and plasma membrane blebs were resorbed completely within 6 min of reoxygenation without loss of viability. Stage III, however, was not reversible, and no morphological changes occurred on reoxygenation. The results indicate that onset of cell death owing to anoxia is a rapid event initiated by a sudden increase of nonspecific plasma membrane permeability caused by rupture of a terminal bleb. Anoxic injury is reversible until this event occurs.     

Subcapsular sinus macrophage fragmentation and CD169+ bleb acquisition by closely associated IL-17-committed innate-like lymphocytes

Subcapsular sinus macrophages (SSMs) in lymph nodes are rapidly exposed to antigens arriving in afferent lymph and have a role in their capture and display to B cells. In tissue sections SSMs exhibit long cellular processes and express high amounts of CD169. Here, we show that many of the cells present in lymph node cell suspensions that stain for CD169 are not macrophages but lymphocytes that have acquired SSM-derived membrane blebs. The CD169 bleb(+) lymphocytes are enriched for IL-17 committed IL-7Rα(hi)CCR6(+) T cells and NK cells. In addition, the CD169 staining detected on small numbers of CD11c(hi) dendritic cells is frequently associated with membrane blebs. Counter intuitively the CD169 bleb(+) lymphocytes are mostly CD4 and CD8 negative whereas many SSMs express CD4. In situ, many IL-7Rα(hi) cells are present at the subcapsular sinus and interfollicular regions and migrate in close association with CD169(+) macrophages. These findings suggest SSMs undergo fragmentation during tissue preparation and release blebs that are acquired by closely associated cells. They also suggest an intimate crosstalk between SSMs and IL-17 committed innate-like lymphocytes that may help provide early protection of the lymph node against lymph-borne invaders.     

Immunocytochemical Identification and Localization of Active and Inactive alpha-Amylase and Pullulanase in Cells of Clostridium thermosulfurogenes EM1

Clostridium thermosulfurogenes EM1 formed blebs, i.e., protrusions still in contact with the cytoplasmic membrane, that originated from the cytoplasmic membrane during growth in batch culture and continuous culture. They could be observed squeezed between the cell wall and cytoplasmic membrane in cells with seemingly intact wall layers (surface layer and peptidoglycan layer) as well as in cells with wall layers in different states of degradation caused by phosphate limitation or high dilution rates. Blebs were found to turn into membrane vesicles by constriction in cases when the cell wall was heavily degraded. Bleb and vesicle formation was also observed in the absence of substrates that induce alpha-amylase and pullulanase synthesis. No correlations existed between bleb formation and the presence of active enzyme. Similar blebs could also be observed in a number of other gram-positive bacteria not producing these enzymes, but they were not observed in gram-negative bacteria. For immunoelectron-microscopic localization of alpha-amylase and pullulanase in C. thermosulfurogenes EM1, two different antisera were applied. One was raised against the enzymes isolated from the culture fluid; the other was produced against a peptide synthesized, as a defined epitope, in analogy to the N-terminal amino acid sequence (21 amino acids) of the native extracellular alpha-amylase. By using these antisera, alpha-amylase and pullulanase were localized at the cell periphery in samples taken from continuous culture or batch culture. In samples prepared for electron microscopy by freeze substitution followed by ultrathin sectioning, blebs could be seen, and the immunolabel pinpointing alpha-amylase enzyme particles was seen not only randomly distributed in the cell periphery, but also lining the surface of the cytoplasmic membrane and the blebs. Cells exhibiting high or virtually no enzyme activity were labeled similarly with both antisera. This finding strongly suggests that alpha-amylase and pullulanase may occur in both active and inactive forms, depending on growth conditions.     

Interaction and fusion dynamics between cellular blebs

Membrane blebbing, as a mechanism for cells to regulate their internal pressure and membrane tension, is believed to play important roles in processes such as cell migration, spreading and apoptosis. However, the fundamental question of how different blebs interact with each other during their life cycles remains largely unclear. Here, we report a combined theoretical and experimental investigation to examine how the growth and retraction of a cellular bleb are influenced by neighboring blebs as well as the fusion dynamics between them. Specifically, a boundary integral model was developed to describe the shape evolution of cell membrane during the blebbing/retracting process. We showed that a drop in the intracellular pressure will be induced by the formation of a bleb whose retraction then restores the pressure level. Consequently, the volume that a second bleb can reach was predicted to heavily depend on its initial weakened size and the time lag with respect to the first bleb, all in quantitative agreement with our experimental observations. In addition, it was found that as the strength of membrane-cortex adhesion increases, the possible coalescence of two neighboring blebs changes from smooth fusion to abrupt coalescence and eventually to no fusion at all. Phase diagrams summarizing the dependence of such transition on key physical factors, such as the intracellular pressure and bleb separation, were also obtained.     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pH(i)) and Na(+) activity ([Na(+)](i)) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pH(o)). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pH(o) to 2.0 or 10.3. At constant pH(o), buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO(3)(-)/CO(2) inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO(3)(-)/CO(2) buffer, suggesting a regulatory role for pH(i). In polarized TRCs step changes in apical pH(o) from 10.3 to 2.0 induced a linear decrease in pH(i) that remained within the physiological range (slope = 0.035; r(2) = 0.98). At constant pH(o), perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO(3)(-)/CO(2) decreased resting TRC pH(i), and MK-507 or MK-417 attenuated the decrease in pH(i) in TRCs perfused with HCO(3)(-)/CO(2) buffer. In parallel experiments, TRC [Na(+)](i) decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na(+), and (d) acid loading the cells with NH(4)Cl or sodium acetate at constant pH(o). Diethylpyrocarbonate and Zn(2+), modification reagents for histidine residues in proteins, attenuated the CO(2)-induced inhibition of NaCl CT responses and the pH(i)-induced inhibition of apical Na(+) influx in TRCs. We conclude that TRC pH(i) regulates Na(+)-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.