Peripheral contribution of NGF and ASIC1a to colonic hypersensitivity in a rat model of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with idiopathic colonic hypersensitivity (CHS). However, recent studies suggest that low-grade inflammation could underlie CHS in IBS. The pro-inflammatory mediator nerve growth factor (NGF) plays a key role in the sensitization of peripheral pain pathways and several studies have reported its contribution to visceral pain development. NGF modulates the expression of Acid-Sensing Ion Channels (ASICs), which are proton sensors involved in sensory neurons sensitization. This study examined the peripheral contribution of NGF and ASICs to IBS-like CHS induced by butyrate enemas in the rat colon. METHODS: Colorectal distension and immunohistochemical staining of sensory neurons were used to evaluate NGF and ASICs contribution to the development of butyrate-induced CHS. KEY RESULTS: Systemic injection of anti-NGF antibodies or the ASICs inhibitor amiloride prevented the development of butyrate-induced CHS. A significant increase in NGF and ASIC1a protein expression levels was observed in sensory neurons of rats displaying butyrate-induced CHS. This increase was specific of small- and medium-diameter L1 + S1 sensory neurons, where ASIC1a was co-expressed with NGF or trkA in CGRP-immunoreactive somas. ASIC1a was also overexpressed in retrogradely labeled colon sensory neurons. Interestingly, anti-NGF antibody administration prevented ASIC1a overexpression in sensory neurons of butyrate-treated rats. CONCLUSIONS & INFERENCES: Our data suggest that peripheral NGF and ASIC1a concomitantly contribute to the development of butyrate-induced CHS NGF-ASIC1a interplay may have a pivotal role in the sensitization of colonic sensory neurons and as such, could be considered as a potential new therapeutic target for IBS treatment.

Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors.

Nonsteroid anti-inflammatory drugs (NSAIDs) are major drugs against inflammation and pain. They are well known inhibitors of cyclooxygenases (COXs). However, many studies indicate that they may also act on other targets. Acidosis is observed in inflammatory conditions such as chronic joint inflammation, in tumors and after ischemia, and greatly contributes to pain and hyperalgesia. Administration of NSAIDs reduces low-pH-induced pain. The acid sensitivity of nociceptors is associated with activation of H(+)-gated ion channels. Several of these, cloned recently, correspond to the acid-sensing ion channels (ASICs) and others to the vanilloid receptor family. This paper shows (1) that ASIC mRNAs are present in many small sensory neurons along with substance P and isolectin B4 and that, in case of inflammation, ASIC1a appears in some larger Abeta fibers, (2) that NSAIDs prevent the large increase of ASIC expression in sensory neurons induced by inflammation, and (3) that NSAIDs such as aspirin, diclofenac, and flurbiprofen directly inhibit ASIC currents on sensory neurons and when cloned ASICs are heterologously expressed. These results suggest that the combined capacity to block COXs and inhibit both inflammation-induced expression and activity of ASICs present in nociceptors is an important factor in the action of NSAIDs against pain.

Dehydroepiandrosterone sulfate and beta-cell function: enhanced glucose-induced insulin secretion and altered gene expression in rodent pancreatic beta-cells.

Administration of dehydroepiandrosterone (DHEA), or its sulfated form (DHEAS), controls hyperglycemia in diabetic rodents without directly altering insulin sensitivity. We show that DHEAS enhanced glucose-stimulated insulin secretion when administered in vivo to rats or in vitro to beta-cell lines, without changing cellular insulin content. Insulin secretion increased from 3 days of steroid exposure in vitro, suggesting that DHEAS did not directly activate the secretory processes. DHEAS selectively increased the beta-cell mRNA expression of acyl CoA synthetase-2 and peroxisomal acyl CoA oxidase in a time-dependent manner. Although DHEAS is a peroxisomal proliferator, it did not alter the mRNA expression of peroxisomal proliferator-activated receptor (PPAR) alpha or beta, or enhance the activity of transfected PPAR alpha, beta, or gamma in vitro. Thus, DHEAS directly affected the beta-cell to enhance glucose-stimulated insulin secretion and increased the mRNA expression of specific beta-cell mitochondrial and peroxisomal lipid metabolic enzymes. This effect of DHEAS on insulin secretion may contribute to the amelioration of hyperglycemia seen in various rodent models of diabetes.

Characterization of rat liver malonyl-CoA decarboxylase and the study of its role in regulating fatty acid metabolism.

In the liver, malonyl-CoA is central to many cellular processes, including both fatty acid biosynthesis and oxidation. Malonyl-CoA decarboxylase (MCD) is involved in the control of cellular malonyl-CoA levels, and functions to decarboxylate malonyl-CoA to acetyl-CoA. MCD may play an essential role in regulating energy utilization in the liver by regulating malonyl-CoA levels in response to various nutritional or pathological states. The purpose of the present study was to investigate the role of liver MCD in the regulation of fatty acid oxidation in situations where lipid metabolism is altered. A single MCD enzyme of molecular mass 50.7 kDa was purified from rat liver using a sequential column chromatography procedure and the cDNA was subsequently cloned and sequenced. The liver MCD cDNA was identical to rat pancreatic beta-cell MCD cDNA, and contained two potential translational start sites, producing proteins of 50.7 kDa and 54.7 kDa. Western blot analysis using polyclonal antibodies generated against rat liver MCD showed that the 50.7 kDa isoform of MCD is most abundant in heart and liver, and of relatively low abundance in skeletal muscle (despite elevated MCD transcript levels in skeletal muscle). Tissue distribution experiments demonstrated that the pancreas is the only rat tissue so far identified that contains both the 50.7 kDa and 54. 7 kDa isoforms of MCD. In addition, transfection of the full-length rat liver MCD cDNA into COS cells produced two isoforms of MCD. This indicated either that both initiating methionines are functionally active, generating two proteins, or that the 54.7 kDa isoform is the only MCD protein translated and removal of the putative mitochondrial targeting pre-sequence generates a protein of approx. 50.7 kDa in size. To address this, we transiently transfected a mutated MCD expression plasmid (second ATG to GCG) into COS-7 cells and performed Western blot analysis using our anti-MCD antibody. Western blot analysis revealed that two isoforms of MCD were still present, demonstrating that the second ATG may not be responsible for translation of the 50.7 kDa isoform of MCD. These data also suggest that the smaller isoform of MCD may originate from intracellular processing. To ascertain the functional role of the 50. 7 kDa isoform of rat liver MCD, we measured liver MCD activity and expression in rats subjected to conditions which are known to alter fatty acid metabolism. The activity of MCD was significantly elevated under conditions in which hepatic fatty acid oxidation is known to increase, such as streptozotocin-induced diabetes or following a 48 h fast. A 2-fold increase in expression was observed in the streptozotocin-diabetic rats compared with control rats. In addition, MCD activity was shown to be enhanced by alkaline phosphatase treatment, suggesting phosphorylation-related control of the enzyme. Taken together, our data demonstrate that rat liver expresses a 50.7 kDa form of MCD which does not originate from the second methionine of the cDNA sequence. This MCD is regulated by at least two mechanisms (only one of which is phosphorylation), and its activity and expression are increased under conditions where fatty acid oxidation increases.

Cloning and expression of rat pancreatic beta-cell malonyl-CoA decarboxylase.

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

Mutations causing neurodegeneration in Caenorhabditis elegans drastically alter the pH sensitivity and inactivation of the mammalian H+-gated Na+ channel MDEG1.

The mammalian degenerin MDEG1 belongs to the nematode degenerin/epithelial Na+ channel superfamily. It is constitutively activated by the same mutations that cause gain-of-function of the Caenorhabditis elegans degenerins and neurodegeneration. ASIC and DRASIC, which were recently cloned, are structural homologues of MDEG1 and behave as H+-gated cation channels. MDEG1 is also a H+-activated Na+ channel, but it differs from ASIC in its lower pH sensitivity and slower kinetics. In addition to the generation of a constitutive current, mutations in MDEG1 also alter the properties of the H+-gated current. Replacement of Gly-430 in MDEG1 by bulkier amino acids, such as Val, Phe, or Thr, drastically increases the H+ sensitivity of the channel (half-maximal pH (pHm) approximately 4.4 for MDEG1, pHm approximately 6.7 for the different mutants). Furthermore, these replacements completely suppress the inactivation observed with the wild-type channel and increase the sensitivity of the H+-gated channel to blockade by amiloride by a factor of 10 without modification of its conductance and ionic selectivity. These results as well as those obtained with other mutants clearly indicate that the region surrounding Gly-430, situated just before the second transmembrane segment, is essential for pH sensitivity and gating.

The amiloride-sensitive Na+ channel: from primary structure to function.

Three homologous subunits of the amiloride-sensitive Na+ channel, entitled alpha, beta, and gamma, have been cloned either from distal colon of a steroid-treated rat or from human lung. The alpha, beta, and gamma subunits have similarities with degenerins, a family of proteins found in the mechanosensory neurons of the nematode Caenorhabditis elegans. All these proteins are characterized by the presence of a large extracellular domain, located between two transmembrane alpha-helices, and by short NH2 and COOH terminal cytoplasmic segments. They constitute the first members of a new gene super-family of ionic channels. The epithelial Na+ channel is specifically expressed at the apical membrane of Na(+)-reabsorbing epithelial cells. Its activity is controlled by several distinct hormones, especially by corticosteroids. These hormones act either transcriptionally (such as aldosterone in distal colon, or glucocorticoids in lung) and/or post-transcriptionally (such as aldosterone in kidney). Recent works have provided new insights in the function of that important osmoregulatory system.

Molecular biology of the amiloride-sensitive epithelial Na+ channel.

The amiloride-sensitive epithelial Na+ channel is formed by the assembly of three homologous subunits, alpha, beta and gamma. The channel is characterized by its sensitivity to amiloride and to some amiloride derivatives, such as phenamil and benzamil, by its small unitary conductance (approximately 5 pS), by its high selectivity for lithium and sodium, and by its slow kinetics. The alpha-, beta-, and gamma-proteins share significant identity with degenerins, a family of proteins found in the mechanosensory neurons and interneurons of the nematode Caenorhabditis elegans. They are also homologous to FaNaCh, a protein from Helix aspersa nervous tissues, which corresponds to a neuronal ionotropic receptor for the Phe-Met-Arg-Phe-NH2 peptide. All these proteins contain a large extracellular loop, located between two transmembrane alpha-helices. The NH2 and COOH terminal segments are cytoplasmic and contain potential regulatory segments that are able to modulate the activity of the channel. Accordingly, in Liddle syndrome, in which patients develop a form of genetic hypertension, mutations within the cytoplasmic COOH terminal of the beta- and gamma-chains of the epithelial Na+ channel lead to a hyperactivity of the channel. Epithelial Na+ channel activity is tightly controlled by several distinct hormonal systems, including corticosteroids and vasopressin. In kidney and colon, aldosterone is the major sodium-retaining hormone, acting by stimulation of Na+ reabsorption through the epithelium. In the distal colon from steroid-treated animals, a large increase in beta- and gamma-subunit transcription is observed, whereas the alpha-subunit remains constitutively transcribed. In kidney, RNA levels of the three subunits are not altered by aldosterone, suggesting that other mechanisms control Na+ channel activity in that tissue. In lung, the glucocorticoids are positive regulators of the channel activity, especially around birth, and act via an increased transcription of the three subunits.

The mammalian degenerin MDEG, an amiloride-sensitive cation channel activated by mutations causing neurodegeneration in Caenorhabditis elegans.

Mutations of the degenerins (deg-1, mec-4, mec-10) are the major known causes of hereditary neurodegeneration in the nematode Caenorhabditis elegans. We cloned a neuronal degenerin (MDEG) from human and rat brain. MDEG is an amiloride-sensitive cation channel permeable for Na+, K+, and Li+. This channel is activated by the same mutations which cause neurodegeneration in C. elegans. Like the hyperactive C. elegans degenerin mutants, constitutively active mutants of MDEG cause cell death, suggesting that gain of function of this novel neuronal ion channel might be involved in human forms of neurodegeneration.

[The amiloride sensitive sodium channel]. / Le canal Na+ sensible à l'amiloride.

The amiloride-sensitive epithelial Na+ channel is formed by the assembly of three homologous subunits alpha, beta and gamma. The channel is characterized by its sensitivity to amiloride and to some amiloride derivatives, such as phenamil and benzamil, by its small unitary conductance (approximately 5pS), by its high selectivity for lithium and sodium, and by its slow kinetics. The alpha, beta, and gamma proteins share significant identity with degenerins, a family of proteins found in the mechanosensory neurons and interneurons of the nematode Caenorhabditis elegans. They are also homologous to FaNaCh, a protein from Helix aspersa nervous tissues, which corresponds to a neuronal ionotropic receptor for the Phe-Met-Arg-Phe-amide peptide. All these proteins contain a large extracellular loop, located between two transmembrane alpha-helices. The NH2 and COOH terminal segments are cytoplasmic, and contain potential regulatory segments that are able to modulate the activity of the channel. In Liddle syndrome, in which patients develop a form of genetic hypertension, mutations within the cytoplasmic COOH terminal of the beta and gamma chains of the epithelial Na+ channel lead to a hyper-activity of the channel. Epithelial Na+ channel activity is tightly controlled by several distinct hormonal systems, including corticosteroids and vasopressin. In kidney and colon, aldosterone is the major sodium-retaining hormone, acting, by stimulation of Na+ reabsorption through the epithelium. In the distal colon from steroid-treated animals, a large increase of the beta and gamma subunits transcription is observed, whereas the alpha subunit remains constitutively transcribed. In kidney, RNA levels of the three subunits are not significantly altered by aldosterone, suggesting that other mechanisms control Na+ channel activity in that tissue. In lung, the glucocorticoids are the positive regulators of the channel activity, especially around birth, and act via an increased transcription of the three subunits.

Molecular cloning and functional expression of a novel amiloride-sensitive Na+ channel.

We have isolated a cDNA for a novel human amiloride-sensitive Na+ channel isoform (called delta) which is expressed mainly in brain, pancreas, testis, and ovary. When expressed in Xenopus oocytes, it generates an amiloride-sensitive Na+ channel with biophysical and pharmacological properties distinct from those of the epithelial Na+ channel, a multimeric assembly of alpha, beta, and gamma subunits. The Na+ current produced by the new delta isoform is increased by two orders of magnitude after coexpression of the beta and gamma subunit of the epithelial Na+ channel showing that delta can associate with other subunits and is part of a novel multisubunit ion channel.

Cloning, chromosomal localization, and physical linkage of the beta and gamma subunits (SCNN1B and SCNN1G) of the human epithelial amiloride-sensitive sodium channel.

Three subunits of the amiloride-sensitive Na+ channel, named alpha, beta, and gamma, have previously been cloned in rat colon. The human lung alpha chain (SCNN1A) has also been cloned and its gene localized on chromosome 12p13. We now report the molecular cloning of the human lung beta (SCNN1B) and gamma (SCNN1G) chains. In situ hybridization and pulsed-field electrophoresis experiments demonstrate that both genes are located within a common 400-kb fragment on chromosome 16p12-p13. Screening of the cDNA library reveals two forms of the beta subunit that differ by the presence or absence of a 464-bp fragment in the 3' region. A frameshift in the short form modifies the COOH terminal sequence of the corresponding protein. Since several similar frameshifts mutations have recently been reported in patients affected by a rare form of hypertension, the existence of COOH truncated forms of the beta chain might be of physiological importance.

Localization and regulation by steroids of the alpha, beta and gamma subunits of the amiloride-sensitive Na+ channel in colon, lung and kidney.

Polyclonal antibodies have been raised against the alpha, beta and gamma subunits of the amiloride-sensitive Na+ channel. The three subunits were detected by immunohistochemistry at the apical membrane of epithelial cells from the distal colon, the lung and the distal segments of the kidney tubules. No significant labelling was detected in lung alveoli, suggesting that it is not a major site of expression of the Na+ channel. Effects of a low Na+ diet or of dexamethasone treatment were measured at the mRNA level and at the protein level by immunohistochemistry. In the colon, steroids controlled Na+ channel activity via the stimulation of the transcription of beta and gamma subunits. The alpha mRNA was constitutively expressed. However, while neither alpha, beta nor gamma proteins were detected in the colon of control animals, they were all detected in the colon of steroid-treated animals. In the lung, Na+ channel expression was regulated by glucocorticoids the circulating level of which was sufficiently high to induce a maximal expression of the three subunits, even in control animals. Adrenalectomy drastically reduced expression of the three subunits. A surprising finding was the apparent absence of steroid effects on alpha, beta and gamma subunit expression in the kidney. Neither the expression of the mRNAs nor the expression of the proteins were significantly altered by aldosterone or by dexamethasone. These results could be due to mixed gluco- and mineralocorticoid regulations in different segments of the kidney tubule, but their interpretation also requires regulations that are apparently not found in the lung or colon.

Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone.

Long term regulation of the amiloride-sensitive Na+ channel activity by steroid hormones occurs via de novo protein synthesis. The messenger level of RCNaCh1, previously shown by expression cloning to be a component of this channel, was measured in colons from rats fed with a low sodium diet. After 1 week of this diet, the channel activity was increased in an all-or-none fashion, whereas the level of RCNaCh1 messenger remained constant. A cDNA coding for another subunit of the Na+ channel was obtained by polymerase chain reaction. The 650-amino acid protein, entitled RCNaCh2, is 58% homologous to RCNaCh1 and displays a similar structure. It had no intrinsic activity when expressed alone in Xenopus oocytes, but its co-expression with RCNaCh1 increased the channel activity 18 +/- 5-fold. The increase in messenger level for RCNaCh2 during the time course of the diet is likely to explain the positive regulation of the rat colon Na+ channel by steroids. Immunocytochemical localization of the RCNaCh1 subunit revealed an apical labeling in colon from sodium-depleted rats. No labeling was observed in colon from control animals. These results suggest that oligomerization is needed for the proper expression of RCNaCh1 at the cell surface.

Regulation of expression of the lung amiloride-sensitive Na+ channel by steroid hormones.

Molecular cloning of the amiloride-sensitive Na+ channel has permitted analysis of the mechanisms of its stimulation by steroids. In rat lung cells in primary culture, where its mRNA has been detected, the activity of an amiloride-sensitive channel, highly selective for Na+, is controlled by corticosteroids. Dexamethasone (0.1 microM) or aldosterone (1 microM) induced, after a minimum 10 h treatment, a large increase of the amiloride-induced hyperpolarization and of the amiloride-sensitive current. A parallel increase in the amount of the mRNA was observed. The corresponding gene is thus a target for steroid action. Using synthetic specific agonists and antagonists for mineralo- and glucocorticoid receptors, it has been shown that the steroid action on Na+ channel expression is mediated via glucocorticoid receptors. Triiodothyronine, known to modulate steroid action in several tissues, had no effect on both the amiloride-sensitive Na+ current and the level of the mRNA for the Na+ channel protein, but potentiates the stimulatory effect of dexamethasone. The increase in Na+ channel activity observed in the lung around birth can thus be explained by a direct increase in transcription of the Na+ channel gene.

Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel.

A key protein component of the amiloride-sensitive sodium channel has been cloned from rat colon and human lung. It may represent the first member of a new family of ionic channels expressed from nematode to human. The biochemical properties of the rat protein, a 699 amino acids long polypeptide, have been analyzed. Four polyclonal antibodies raised against distinct parts of the channel immunoprecipitated a glycosylated protein of 96 kDa after cRNA expression in oocytes as well as after in vitro translation. When expressed alone into oocytes, the protein was not stable; most of it remains stacked into the endoplasmic reticulum. This results in a very low yield of complete maturation of the protein at the cell surface after expression from the pure cRNA. To determine the membrane topology of the protein, in vitro translation by a rabbit reticulocyte lysate was performed followed by insertion into canine pancreatic microsomes and protease digestion. Analysis revealed a model with only two transmembrane alpha helices and a large extracellular domain of about 500 amino acids. The NH2 and COOH termini are cytoplasmic. Protease digestion results suggest the possible presence of a structural element that could have a function similar to that of the H5 segment in K+ channels. The model indicates that there is no cytoplasmic site for protein kinase A phosphorylation. The well known regulation of the channel activity by hormones that activate this kinase such as vasopressin might thus be situated on another channel component.

The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning.

Water balance in the lung is controlled via active Na+ and Cl- transport. Electrophysiological measurements on lung epithelial cells demonstrated the presence of a Na+ channel that is inhibited by amiloride (K0.5 = 90 nM) and some of its derivatives such as phenamil (K0.5 = 19 nM) and benzamil (K0.5 = 14 nM) but not by ethylisopropylamiloride. An amiloride-sensitive Na+ channel of 4 pS was recorded from outside-out patches excised from the apical membrane. This channel is highly selective for Na+ (PNa+/PK+ > or = to 10). Isolation of a human lung cDNA led to the primary structure of the lung Na+ channel. The corresponding protein is 669 residues long and has two large hydrophobic domains. An amiloride-sensitive Na(+)-selective current apparently identical to the one observed in lung epithelial cells was recorded after expression of the cloned channel in oocytes. The level of the mRNA for the Na+ channel was highly increased from fetal to newborn and adult stages. This observation indicates that the increased Na+ reabsorption that occurs at birth as a necessary event to pass to an air-breathing environment is probably associated with control of transcription of this Na+ channel. The human gene for the lung Na+ channel was mapped on chromosome 12p13.

Molecular cloning and functional expression of different molecular forms of rat amiloride-binding proteins.

The colon and lung amiloride-binding proteins were cloned from rat tissues. Two sizes of transcripts were identified. The 2.7-kb transcript codes for an 85-kDa protein, whereas the 1.2-kb transcript codes for a 25-kDa polypeptide. The 2.7-kb transcript was detected in the proximal and distal colon and in duodenum, liver, placenta and thymus. The 1.2-kb transcript was the only form present in lung and spleen, and it was also detected in placenta and colon. The short form corresponds to the 3' terminus of the longer one. It is formed by alternative transcription under the control of an internal promoter. Cells stably transfected with cDNAs encoding these two proteins were used for binding studies using [3H]phenamil, a potent blocker of the epithelial Na+ channel, derived from amiloride. Both the long and short forms of the protein bind amiloride and some of its derivatives, but they have distinct pharmacologies. The order of potency of the different amiloride derivatives to inhibit [3H]phenamil binding was phenamil (K0.5 = 10 nM) > benzamil (K0.5 = 43 nM) > amiloride (K0.5 = 1.4 microM) approximately ethylisopropylamiloride (K0.5 = 1.6 microM) for the long form, whereas it was phenamil (K0.5 = 68 nM) > amiloride (K0.5 = 3.2 microM) approximately ethylisopropylamiloride (K0.5 = 4 microM) approximately benzamil (K0.5 = 6.3 microM) for the short form. Although the binding proteins described here are distinct from the pore-forming protein of the epithelial Na+ channel, the pharmacological profile of the long form of the ABP is identical to that described previously in pig and human kidney, and similar to that expected for an epithelial Na+ channel. The pharmacological profile of the short form resembles that previously described for an amiloride-binding protein in pneumocytes. Results presented in this paper suggest that previously purified preparations showing Na+ channel activity contain different forms of the amiloride-binding protein, possibly associated with other proteins. The similarity between amiloride-binding proteins and a protein identified in seminal vesicles suggests that amiloride-binding proteins are the first members of a new family of epithelia-specific proteins.