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We report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na+ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH3BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current of in vitro frog skin and 22Na+ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH3BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na+ channel. [ABSTRACT FROM AUTHOR]

Hypoxia and epithelial stretch that are commonly observed in patients with acute lung injury have been shown to promote the release of serotonin (5-hydroxytryptamine, 5-HT) in vitro. However, whether 5-HT contributes to the decrease of alveolar epithelial fluid transport, which is a hallmark of lung injury, is unknown. Thus, we investigated the effect of 5-HT on ion and fluid transport across the alveolar epithelium. 5-HT caused a dose-dependent inhibition of the amiloride-sensitive current across primary rat and human alveolar epithelial type II cell monolayers, but did not affect Na(+)/K(+) ATPase function. Furthermore, we found that the 5-HT induced inhibition of ion transport across the lung epithelium was receptor independent, as it was not prevented by the blockade of 5-HT2R (5-HT receptor 2), 5-HT3R (5-HT receptor 3), or by pretreatment with an intracellular calcium-chelating agent, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester). In addition, the stimulation of 5-HT1R (5-HT receptor 1), 5-HT2R (5-HT receptor 2), 5-HT4R (5-HT receptor 4), and 5-HT7R (5-HT receptor 7) failed to reproduce the 5-HT effect on amiloride-sensitive sodium transport. We ascertained that 5-HT directly inhibited the function of rat alphabetagamma epithelial sodium channel (ENaC), as determined by heterologous expression of rat ENaC in Xenopus oocytes that do not express endogenous ENaC nor 5-HT receptors (5-HTR). Exposure of mice to hypoxia for 1 hour induced a 30% increase of 5-HT secretion into the distal airways of mice. Finally, the intratracheal instillation of 5-HT inhibited the amiloride-sensitive fraction of alveolar fluid clearance in mice. Together, these results indicate that 5-HT inhibits the amiloride-sensitive fraction of the alveolar epithelial fluid transport via a direct interaction with ENaC, and thus can be an endogenous inhibitor of this ion channel.

Essential polyunsatured fatty acids have been shown to modulate enzymes, channels and transporters, to interact with lipid bilayers and to affect metabolic pathways. We have previously shown that eicosapentanoic acid (EPA, C20:5, n-3) activates epithelial sodium channels (ENaCs) in a cAMP-dependent manner involving stimulation of cAMP-dependent protein kinase (PKA). In the present study, we explored further the mechanism of EPA stimulation of ENaC in A6 cells. Fluorescence resonance energy transfer experiments confirmed activation of PKA by EPA. Consistent with our previous studies, EPA had no further stimulatory effect on amiloride-sensitive transepithelial current (INa) in the presence of CPT-cAMP. Thus, we investigated the effect of EPA on cellular pathways which produce cAMP. EPA did not stimulate adenylate cyclase activity or total cellular cAMP accumulation. However, membrane-bound phosphodiesterase activity was inhibited by EPA from 2.46 pmol/mg of protein/min to 1.3 pmol/mg of protein/min. To investigate the potential role of an A-kinase-anchoring protein (AKAP), we used HT31, an inhibitor of the binding between PKA and AKAPs as well as cerulenin, an inhibitor of myristoylation and palmitoylation. Both agents prevented the stimulatory effect of EPA and CPT-cAMP on INa and drastically decreased the amount of PKA in the apical membrane. Colocalization experiments in A6 cells cotransfected with fluorescently labeled ENaC beta subunit and PKA regulatory subunit confirmed the close proximity of the two proteins and the membrane anchorage of PKA. Last, in A6 cells transfected with a dead mutant of Sgk, an enzyme which up-regulates ENaCs, EPA did not stimulate Na+ current. Our results suggest that stimulation of ENaCs by EPA occurs via SGK in membrane-bound compartments containing an AKAP, activated PKA, and a phosphodiesterase.

During inhalational anesthesia, halogenated gases are in direct contact with the alveolar epithelium, in which they may affect transepithelial ion and fluid transport. The effects of halogenated gases in vivo on epithelial Na+ and K+ channels, which participate in alveolar liquid clearance, remain unclear. In the present study, the effects of halothane (1, 2, and 4% atm) on ion-channel function in cultured human alveolar cells were investigated using the patch-clamp technique. After exposure to 4% halothane, amiloride-sensitive whole-cell inward currents increased by 84+/-22%, whereas tetraethylammonium-sensitive outward currents decreased by 63+/-7%. These effects, which occurred within 30 s, remained for 30-min periods of exposure to the gas, were concentration-dependent, and were reversible upon washout. Pretreatment with amiloride prevented 90+/-7% of the increase in inward currents without change in outward currents, consistent with an activation of amiloride-sensitive epithelial sodium channels. Tetraethylammonium obliterated 90+/-9% of the effect of halothane on outward currents, without change in inward currents, indicating inhibition of Ca2+-activated K+ channels. These channels were identified in excised patches to be small-conductance Ca2+-activated K+ channels. These effects of halothane were not modified after the inhibition of cytosolic phospholipase A2 by aristolochic acid. Exposure of the cells to either trypsin or to low Na+ completely prevented the increase in amiloride-sensitive currents induced by halothane, suggesting a release of Na+ channels self-inhibition. Thus, halothane modifies differentially and independently Na+ and K+ permeabilities in human alveolar cells.

Confluent monolayers of epithelial cells grown on nonporous support form fluid-filled hemicysts called domes, which reflect active ion transport across the epithelium. Clara-like H441 lung adenocarcinoma cells grown on glass supports and exposed to 50 nM dexamethasone developed domes in a time-dependent fashion. Uplifting of small groups of cells occurred within 6-12 h, well formed domes appeared between 24 and 48 h, and after 7 days, individual domes started to merge. Cells inside of domes compared with those outside domes, or with monolayers not exposed to dexamethasone, differed by higher surfactant production, an increased cytokeratin expression, and the localization of claudin-4 proteins to the plasma membrane. In patch clamp studies, amiloride-blockable sodium currents were detected exclusively in cells inside domes, whereas in cells outside of domes, sodium crossed the membrane through La3+-sensitive nonspecific cation channels. Cells grown on permeable support without dexamethasone expressed amiloride-sensitive currents only after tight electrical coupling was achieved (transepithelial electrical resistance (R(t)) > 1 kilohm). In real-time quantitative PCR experiments, the addition of dexamethasone increased the content of claudin-4, occludin, and Na+ channel gamma-subunit (gamma-ENaC) mRNAs by 1.34-, 1.32-, and 1.80-fold, respectively, after 1 h and was followed by an increase at 6 h in the content of mRNA of alpha- and beta-ENaC and of alpha1- and beta1-Na,K-ATPase. In the absence of dexamethasone, neither change in gene expression nor cell uplifting was observed. Our data suggest that during epithelial differentiation, coordinated expression of tight junction proteins precedes the development of vectorial transport of sodium, which in turn leads to the fluid accumulation in basolateral spaces that is responsible for dome formation.

The epithelial sodium channel is found in apical membranes of a variety of native epithelial tissues, where it regulates sodium and fluid balance. In vivo, a number of hormones and other endogenous factors, including polyunsaturated fatty acids (PUFAs), regulate these channels. We tested the effects of essential n-3 and n-6 PUFAs on amiloride-sensitive sodium transport in A6 epithelial cells. Eicosapentaenoic acid [EPA; C20:5(n-3)] transiently stimulated amiloride-sensitive open-circuit current (I(Na)) from 4.0 +/- 0.3 to 7.7 +/- 0.3 microA/cm2 within 30 min (P < 0.001). No activation was seen in the presence of 10 microM amiloride. In cell-attached but not in cell-excised patches, EPA acutely increased the open probability of sodium channels from 0.45 +/- 0.08 to 0.63 +/- 0.10 (P = 0.02, paired t-test). n-6 PUFAs, including linoleic acid (C18:2), eicosatetraynoic acid (C20:4), and docosapentanoic acid (C22:5) had no effect, whereas n-3 docosahexanoic acid (C22:6) activated amiloride-sensitive I(Na) in a manner similar to EPA. Activation of I(Na) by EPA was prevented by H-89, a PKA inhibitor. Similarly, PKA activity was stimulated by EPA. Nonspecific stimulation of phosphodiesterase activity by CoCl2 completely prevented the effect of EPA on sodium transport. We conclude that n-3 PUFAs activate epithelial sodium channels downstream of cAMP in a cAMP-dependent pathway also involving PKA.

The activity of epithelial Na(+) selective channels is modulated by various factors, with growing evidence that membrane lipids also participate in the regulation. In the present study, Triton X-100 extracts of whole cells and of apical membrane-enriched preparations from cultured A6 renal epithelial cells were floated on continuous-sucrose-density gradients. Na(+) channel protein, probed by immunostaining of Western blots, was detected in the high-density fractions of the gradients (between 18 and 30% sucrose), which contain the detergent-soluble material but also in the lighter, detergent-resistant 16% sucrose fraction. Single amiloride-sensitive Na(+) channel activity, recorded after incorporation of reconstituted proteoliposomes into lipid bilayers, was exclusively localized in the 16% sucrose fraction. In accordance with other studies, high- and low-density fractions of sucrose gradients likely represent membrane domains with different lipid contents. However, exposure of the cells to cholesterol-depleting or sphingomyelin-depleting agents did not affect transepithelial Na(+) current, single-Na(+) channel activity, or the expression of Na(+) channel protein. This is the first reconstitution study of native epithelial Na(+) channels, which suggests that functional channels are compartmentalized in discrete domains within the plane of the apical cell membrane.

We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6-fold decrease in apical sodium channel activity by decreasing channel open probability (P(o)). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone. Sodium channel activity in excised "inside-out" patches usually decreased within 1-2 min; in the presence of S-adenosyl-l-methionine (AdoMet), activity persisted for 5-8 min. Sodium channel mean time open (t(open)) before and after patch excision was higher in the presence of AdoMet than in untreated excised patches but less than t(open) in cell-attached patches. Sodium channel activity in excised patches exposed to both AdoMet and GTP usually remained stable for more than 10 min, and P(o) and the number of active channels per patch were close to values in cell-attached patches from untreated cells. These findings suggest that a methylation reaction contributes to the activity of epithelial sodium channels in A6 cells and is directed to some regulatory element closely connected with the channel, whose activity also depends on the presence of intracellular GTP.

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPgammaS revealed a class of binding sites with an apparent Kd value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Galphai-3, Galpha0, Galphas, and Galphaq were not increased after aldosterone. The patterns of small Mr G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [alpha-32P]GTP, in control membranes, showed either no labeling or a faint band of Mr 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of Mr 59.5 kDa.

In order to detect possible differences in the energy metabolism between normal and neoplastic lymphoid cells, we studied purified normal human lymphocytes (FL) and transformed lymphoblastoid cell lines derived from umbilical cord blood (CL) and compared them to cell lines derived from American Burkitt's lymphoma (BL). The total adenosine triphosphate production rate by these cells was estimated by measuring O2 consumption and lactic acid production rates. O2 consumption (nmol/min/mg protein) was 4.9 +/- 0.3 (S.D.) in CL, 4.4 +/- 0.3 in FL, and 4.9 +/- 0.3 in BL. Lactic acid production (nmol/min/mg protein) was 30.9 +/- 3.0 in CL, 29.9 +/- 3.0 in FL, and 23.4 +/- 4.0 in BL. Using these values of O2 consumption and lactic acid production, the average adenosine triphosphate production rates (nmol/min/mg protein) were calculated to be 60 in CL, 56 in FL, and 53 in BL. We conclude that the BL do not have more aerobic glycolysis than do normal lymphoid cells, suggesting that the lactic acidosis seen in American Burkitt's lymphoma is not due to a preferential glycolytic metabolism of the tumor. More likely, the lactic acidosis is simply due to the large total mass of these neoplastic cells and not due to a modification of their energy metabolism.

High resistance epithelia actively transport sodium from the luminal side to the blood. Aldosterone and vasopressin stimulate this sodium transport system; the diuretic drug amiloride inhibits it in a reversible fashion. The first step in the transepithelial transport of Na+ is the facilitated diffusion of Na+ across the apical membrane via Na+-specific, amiloride-sensitive channels. We report here the first direct measurements of single, amiloride-sensitive Na+ channel activity. The channel was isolated after incorporation of purified apical membrane vesicles from A6 cells into planar lipid bilayers. The channel had the following characteristics: single-channel conductance ranged from 4 to 80 pS at 200 mM NaCl; it was perfectly cation-selective; amiloride reduced the open-state conductance in a dose-dependent fashion when present in the cis compartment, and induced flickering when present in the trans chamber; channel conductance and gating were voltage-independent; and the Na+/K+ selectivity ratio of the channel was 2:1.

The amiloride-binding protein from cultured toad kidney cells (A6) was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), functionally reconstituted into liposomes, and partially purified. The specific binding of [3H]methylbromoamiloride ([3H]CH3BrA) was measured in intact A6 epithelia, A6 cell homogenate (H), apical plasma membrane vesicle (V1), and CHAPS-solubilized V1 and on material obtained after affinity chromatography of CHAPS-solubilized plasma membrane vesicles on agarose-immobilized wheat germ agglutinin (WGA). Specific [3H]CH3BrA binding to H, V1, and WGA material reached equilibrium after 10 min. Scatchard analysis of [3H]CH3BrA binding to V1 and WGA material revealed a homogeneous class of binding sites with KD's of 130 and 128 nM, respectively. These KD values were similar to the apparent inhibitory dissociation constant determined from amiloride inhibition of 22Na+ influx in both intact A6 epithelia and V1. The total number of specific binding sites was 4 pmol/mg of V1 protein, which represented a 10-fold enrichment compared to H, and 66.6 pmol/mg of WGA material (a 148-fold enrichment). From association/displacement kinetic studies of specific [3H]CH3BrA binding to V1, the rate constants of association (ka) and dissociation (kd) were calculated to be 3.6 X 10(5) M-1 s-1 and 49.5 X 10(-3) s-1, respectively. These values yield an equilibrium dissociation constant of 138 nM. In solubilized V1 protein, binding activity was enriched approximately 20-fold over H and was markedly dependent upon the relative concentrations of detergent and phospholipid. CHAPS solubilization of V1 resulted in an average 44% recovery of protein with 90% retention of the total number of specific [3H]CH3BrA binding sites. After WGA chromatography 2.7% of the applied protein and 46% of the specific binding sites were recovered.(ABSTRACT TRUNCATED AT 250 WORDS)