Multiple WW domains, but not the C2 domain, are required for inhibition of the epithelial Na+ channel by human Nedd4.

The epithelial Na+ channel (ENaC) absorbs Na+ across the apical membrane of epithelia. The activity of ENaC is controlled by its interaction with Nedd4; mutations that disrupt this interaction increase Na+ absorption, causing an inherited form of hypertension (Liddle's syndrome). Nedd4 contains an N-terminal C2 domain, a C-terminal ubiquitin ligase domain, and multiple WW domains. The C2 domain is thought to be involved in the Ca2+-dependent localization of Nedd4 at the cell surface. However, we found that the C2 domain was not required for human Nedd4 (hNedd4) to inhibit ENaC in both Xenopus oocytes and Fischer rat thyroid epithelia. Rather, hNedd4 lacking the C2 domain inhibited ENaC more potently than wild-type hNedd4. Earlier work indicated that the WW domains bind to PY motifs in the C terminus of ENaC. However, it is not known which WW domains mediate this interaction. Glutathione S-transferase-fusion proteins of WW domains 2-4 each bound to alpha, beta, and gammaENaC in vitro. The interactions were abolished by mutation of two residues. WW domain 3 (but not the other WW domains) was both necessary and sufficient for the binding of hNedd4 to alphaENaC. WW domain 3 was also required for the inhibition of ENaC by hNedd4; inhibition was nearly abolished when WW domain 3 was mutated. However, the interaction between ENaC and WW domain 3 alone was not sufficient for inhibition. Moreover, inhibition was decreased by mutation of WW domain 2 or WW domain 4. Thus, WW domains 2-4 each participate in the functional interaction between hNedd4 and ENaC in intact cells.

Method for serum-free culture of late fetal and early postnatal rat brainstem neurons.

Primary culture of postnatal brainstem neurons in defined medium has not been described in the literature. Successful primary culture of brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of late fetal and early postnatal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27 (Life Technologies). Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. This paper describes the first reliable method for culturing brainstem neurons from late fetal and early postnatal stages of the rat for up to 6 days postpartum.

Serum-free culture of rat post-natal and fetal brainstem neurons.

Serum-free medium is essential for cell culture studies in which complete control of the environment is required. Primary culture of post-natal brainstem neurons in defined medium has not been described in the literature, and successful culture of primary brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of fetal and post-natal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27. Media and supplements are commercially available products from Life Technologies. Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. Fetal E14, E16, E20, and post-natal P3 and P6 cultures were examined after 4, 7, and 9 days in culture. Neuron and glial cells present in the cultures were identified using immunocytochemistry with antibodies raised against microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), respectively. Fetal E14 cultures had more bipolar neurons than multipolar neurons compared with developmentally older P6 cultures. Early fetal cultures had a higher percentage of neurons than late fetal and early post-natal cultures. Neuron survival was similar between 4 and 9 days in culture for all age groups tested. This is the first reliable, defined culture medium that supports brainstem neurons from late fetal and early post-natal stages of the rat for up to 6 days post-partum.

Human Nedd4 interacts with the human epithelial Na+ channel: WW3 but not WW1 binds to Na+-channel subunits.

The epithelial Na(+) channel (ENaC) regulates Na(+) absorption in epithelial tissues including the lung, colon and sweat gland, and in the distal nephrons of the kidney. When Na(+)-channel function is disrupted, salt and water homoeostasis is affected. The cytoplasmic regions of the Na(+)-channel subunits provide binding sites for other proteins to interact with and potentially regulate Na(+)-channel activity. Previously we showed that a proline-rich region of the alpha subunit of the Na(+) channel bound to a protein of 116 kDa from human lung cells. Here we report the identification of this protein as human Nedd4, a ubiquitin-protein ligase that binds to the Na(+)-channel subunits via its WW domains. Further, we show that WW domains 2, 3 and 4 of human Nedd4 bind to the alpha, beta and gamma Na(+)-channel subunits but not to a mutated beta subunit. In addition, when co-expressed in Xenopus oocytes, human Nedd4 down-regulates Na(+)-channel activity.

Disruption of the beta subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype.

The epithelial Na+ channel (ENaC) is composed of three homologous subunits: alpha, beta and gamma. We used gene targeting to disrupt the beta subunit gene of ENaC in mice. The betaENaC-deficient mice showed normal prenatal development but died within 2 days after birth, most likely of hyperkalemia. In the -/- mice, we found an increased urine Na+ concentration despite hyponatremia and a decreased urine K+ concentration despite hyperkalemia. Moreover, serum aldosterone levels were increased. In contrast to alphaENaC-deficient mice, which die because of defective lung liquid clearance, neonatal betaENaC deficient mice did not die of respiratory failure and showed only a small increase in wet lung weight that had little, if any, adverse physiologic consequence. The results indicate that, in vivo, the beta subunit is required for ENaC function in the renal collecting duct, but, in contrast to the alpha subunit, the beta subunit is not required for the transition from a liquid-filled to an air-filled lung. The phenotype of the betaENaC-deficient mice is similar to that of humans with pseudohypoaldosteronism type 1 and may provide a useful model to study the pathogenesis and treatment of this disorder.

Ectopic expression of Fgf-4 in chimeric mouse embryos induces the expression of early markers of limb development in the lateral ridge.

The biological consequences of constitutive fibroblast growth factor-4 (fgf-4) expression were investigated in chimeric embryos prepared between wild-type host embryos and murine ES cells transfected with a construct in which expression of the murine fgf-4 gene was directed by the phosphoglycerate kinase (PGK-1) promoter. The embryos exhibit abnormalities of the limbs and the anterior central nervous system (CNS). The limb phenotype comprised the induction of outgrowth along the lateral ridge between the definitive fore and hind limbs resembling the early phases of limb development. The CNS defects comprised a complete absence, or marked reduction in forebrain and midbrain structures and rudimentary or absent eye development. Constitutive expression of fgf-4 was also accompanied by ectopic expression of the sonic hedgehog (shh) and msx-1 genes in the lateral ridge. These findings indicate that FGF exhibits multiple activities in early development which include the ability to induce the expression of early markers of limb development in the lateral ridge.

Binding of the proline-rich region of the epithelial Na+ channel to SH3 domains and its association with specific cellular proteins.

The amiloride-sensitive epithelial Na+ channel (ENaC) is an important component of the Na(+)-reabsorption pathway in many epithelia. The identification of three subunits of ENaC (alpha, beta and gamma), as well as results from a number of functional and biochemical studies, suggests that functional Na+ channels are composed of a complex of proteins. To learn about possible interactions of the channel with other proteins, we studied the alpha-subunit of rat and human ENaC. We found that the proline-rich C-terminal domains of both rat and human alpha-ENaC, expressed as glutathione S-transferase fusion proteins, bound to SH3 domains in vitro. A 116 kDa protein from a human lung adenocarcinoma cell line (H441) was specifically bound by the human alpha-ENaC C-terminal fusion protein and by a shorter 18-amino acid proline-rich peptide derived from the larger fusion protein. The 116 kDa protein was not glycosylated and was not phosphorylated on tyrosine or by cyclic AMP-dependent protein kinase (PKA). A 134 kDa protein which was also bound by the human alpha-ENaC C-terminal fusion protein was a substrate for phosphorylation by PKA. These data suggest that the proline-rich C-terminal tail of alpha-ENaC may interact with other proteins that control its function, regulation or localization.

Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel.

Liddle's syndrome is an inherited form of hypertension caused by mutations that truncate the C-terminus of human epithelial Na+ channel (hENaC) subunits. Expression of truncated beta and gamma hENaC subunits increased Na+ current. However, truncation did not alter single-channel conductance or open state probability, suggesting there were more channels in the plasma membrane. Moreover, truncation of the C-terminus of the beta subunit increased apical cell-surface expression of hENaC in a renal epithelium. We identified a conserved motif in the C-terminus of all three subunits that, when mutated, reproduced the effect of Liddle's truncations. Further, both truncation of the C-terminus and mutation of the conserved C-terminal motif increased surface expression of chimeric proteins containing the C-terminus of beta hENaC. Thus, by deleting a conserved motif, Liddle's mutations increase the number of Na+ channels in the apical membrane, which increases renal Na+ absorption and creates a predisposition to hypertension.

rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct.

The terminal nephron segment, the inner medullary collecting duct (IMCD), absorbs Na+ by an electrogenic process that involves the entry through an apical (luminal) membrane Na+ channel. To understand the nature of this Na+ channel, we employed the patch clamp technique on the apical membrane of primary cultures of rat IMCD cells grown on permeable supports. We found that all ion channels detected in the cell-attached configuration were highly selective for Na+ (Li+) over K+. The open/closed transitions showed slow kinetics, had a slope conductance of 6-11 pS, and were sensitive to amiloride and benzamil. Nonselective cation channels with a higher conductance (25-30 pS), known to be present in IMCD cells, were not detected in the cell-attached configuration, but were readily detected in excised patches. The highly selective channels had properties similar to the recently described rat epithelial Na+ channel complex, rENaC. We therefore asked whether rENaC mRNA was present in the IMCD. We detected mRNA for all three rENaC subunits in rat renal papilla and also in primary cultures of the IMCD. Either glucocorticoid hormone or mineralocorticoid hormone increased the amount of alpha-rENaC subunit mRNA but had no effect on the mRNA level of the beta-rENaC or gamma-rENaC subunits. From these data, taken in the context of other studies on the characteristics of Na+ selective channels and the distribution of rENaC mRNA, we conclude that steroid stimulated Na+ absorption by the IMCD is mediated primarily by Na+ channels having properties of the rENaC subunit complex.

Cloning and expression of the beta- and gamma-subunits of the human epithelial sodium channel.

Amiloride-sensitive Na+ channels are an important component of the Na+ reabsorption pathway in a number of epithelia. Here we report the cloning and characterization of cDNAs encoding two subunits of the human kidney epithelial Na+ channel (beta- and gamma-hENaC). Their predicted amino acid sequences were highly homologous (83-85% identical) to the corresponding subunits reported from rat colon (beta- and gamma-rENaC). Both beta- and gamma-hENaC mapped to human chromosome 16. Northern blot analysis showed high expression of beta- and gamma-hENaC in kidney and lung and differential expression of the three subunits in other tissues. Coexpression of beta- and gamma-hENaC with alpha-hENaC in Xenopus oocytes produced Na+ channels with high selectivity for Na+ and high sensitivity to amiloride. In addition, human subunits were able to substitute for the corresponding rat subunits in forming functional Na+ channels, suggesting conservation of function and suggesting that differences in sequence do not disrupt interactions between subunits. These results suggest that human alpha-, beta-, and gamma-ENaC together form Na+ channels with properties that are similar to those observed in epithelia, and will allow further investigation into the role that these channels may play in human disease.

Membrane topology of the amiloride-sensitive epithelial sodium channel.

The amiloride-sensitive epithelial sodium channel (ENaC) is involved in fluid and electrolyte absorption across a number of epithelia, and cloning of several ENaC subunits has begun to facilitate investigation of the structure, function, and regulation of this channel. Analysis of the amino acid sequence has revealed two potential membrane-spanning domains, but little else is known about the structure of ENaC. To investigate the membrane topology of one subunit, alpha rENaC, we used in vitro transcription, translation, and translocation into microsomal membranes. This generated a glycosylated protein of 93 kDa. Sequence analysis also revealed eight potential sites for N-glycosylation, six of which were found to be glycosylated (Asn190, Asn259, Asn320, Asn339, Asn424, and Asn538), indicating that they are extracellular. The C terminus was localized as intracellular based on antibody recognition and protease sensitivity of a tagged epitope at the C terminus. The N terminus was also found to be intracellular, based on its protease sensitivity. Similar results were obtained by expression in Xenopus oocytes. Together, these results support a model of alpha rENaC consisting of an intracellular N terminus and C terminus, a large N-glycosylated extracellular domain, and two membrane-spanning domains that each pass once through the plasma membrane. Because of their sequence similarity, it is likely that this structure is shared by other ENaC subunits and possibly the degenerins of Caenorhabditis elegans as well.

Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel.

Amiloride-sensitive Na+ channels control, in part, fluid and electrolyte transport across epithelia in many organs. In the lung, they control the quantity and composition of the respiratory tract fluid and play a key role in the transition from a fluid-filled lung at the time of birth. Their function may also be altered in a number of diseases. The recent identification of an epithelial Na+ channel from rat colon allowed us to use a probe from that sequence to clone an amiloride-sensitive Na+ channel from human kidney, alpha hENaC. The cDNA had an open reading frame of 2,007 nucleotides and encoded a protein predicted to contain 669 amino acids. The amino acid sequence of alpha hENaC was 83% identical to that of the rat. The gene was mapped to chromosome 12 by polymerase chain reaction (PCR) of somatic cell hybrids. Transcripts of alpha hENaC were detected in human kidney, lung, liver, and pancreas. No message was detected in first- and second-trimester human fetal lung, indicating that alpha hENaC expression is developmentally regulated. In vitro transcription and translation of alpha hENaC produced a 74-kDa protein and translation in the presence of microsomal membranes produced a glycosylated 87-kDa protein. Expression of alpha hENaC in Xenopus oocytes produced currents that were amiloride sensitive and Na+ selective, properties consistent with the function of epithelial Na+ channels in native tissues.

Developmentally regulated expression of fibroblast growth factor receptor genes and splice variants by murine embryonic stem and embryonal carcinoma cells.

The expression of the four fibroblast growth factor receptor (FGF-R) genes was examined in murine embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and their differentiated derivatives. FGF-R1 and FGF-R4 were found to be expressed constitutively in all samples examined. The expression of FGF-R2 and FGF-R3 was, however found to increase significantly upon differentiation of both ES and EC cells. Examination of splice variants of the third immunoglobulin domain (IgIII) of the extracellular region of the FGF-R2 revealed that whilst IgIIIc transcripts were expressed upon ES cell differentiation, IgIIIb transcripts (which confer specificity for the ligand FGF-7) were expressed in both ES cells and their differentiated progeny. FGF-R3 transcripts were also expressed in ES cells, but variant FGF-R3 transcripts containing the IgIIIb region were expressed upon differentiation. The findings suggest that the repertoire of FGF-R expression in embryonic cell types is developmentally regulated at the level of both gene expression, and alternative splicing and different members of the FGF-R family can exhibit distinct patterns of both gene and splice variant expression.

Transferrin-gene expression in the rat mammary gland. Independence of maternal iron status.

Transferrin mRNA concentrations were measured in total RNA isolated from liver and mammary tissue of lactating rats at different stages of lactation. The mammary transferrin mRNA concentration varied in a biphasic manner, increasing up to parturition and then decreasing to undetectable levels at days 5 and 10 of lactation before increasing again markedly in late lactation. The values obtained at day 20 of lactation were double those found in livers of lactating rats. The concentrations of total RNA and mRNA for alpha-casein and alpha-lactalbumin did not change between days 5 and 20 of lactation. Transferrin concentrations were measured in milk from rats fed on an iron-free, a control and an iron-supplemented diet. Although there was a 5-fold difference in the transferrin concentration of samples taken between day 5 and day 20 of lactation, the dietary treatments did not result in significant changes. Maternal serum transferrin concentrations were, however, elevated, and pup haemoglobin concentrations were suppressed for the rats receiving the iron-free diet, indicating an alteration of the iron status of these rats.

Determination of hepatic cholesterol 7alpha-hydroxylase activity in man.

Methods were developed to determine the activity of the microsomal enzyme cholesterol 7alpha-hydroxylase in human liver. The enzyme assay could be performed with as little as 20 mg of fresh liver tissue, thus making the procedure applicable to specimens obtained by percutaneous liver biopsy. Optimal assay conditions were determined and the identity and radioactive purity of the reaction product, cholest-5-ene-3beta,7alpha-diol (7alpha-hydroxycholesterol) were established. Specific enzyme activity was measured in a number of patients with disorders of lipid metabolism.

Isolation and identification of cerebrosides from the marine sponge Chondrilla nucula.

A cerebroside mixture has been isolated from the marine sponge Chondrilla nucula. Acid-catalyzed methanolysis of this cerebroside mixture afforded methyl glucosides, three long-chain bases, and a mixture of alpha-hydroxy fatty acid methyl esters. The bases were identified as saturated C(17), C(18), and C(19) trihydroxy bases (1,3,4-trihydroxy-2-aminoalkanes) by gas-liquid chromatographic-mass spectrometric analysis of their corresponding trimethylsilyl derivatives. The methyl ester fraction consisted of a mixture of homologous C(16) to C(26) saturated straight-chain alpha-hydroxy esters plus a trace of saturated C(25) iso alpha-hydroxy ester.