The amiloride-sensitive epithelial sodium channel, ENaC, is a heteromultimeric protein made up of three homologous subunits (alpha, beta and gamma) (1,2). In vitro, assembly and expression of functional active sodium channels in the Xenopus oocyte is strictly dependent on alpha-ENaC--the beta and gamma subunits by themselves are unable to induce an amiloride-sensitive sodium current in this heterologous expression system (2). In vivo, ENaC constitutes the limiting step for sodium absorption in epithelial cells that line the distal renal tubule, distal colon and the duct of several exocrine glands. The adult lung expresses alpha, beta and gamma ENaC (3,4), and an amiloride-sensitive electrogenic sodium reabsorption has been documented in upper and lower airways (3-7), but it is not established whether this sodium transport is mediated by ENaC in vivo. We inactivated the mouse alpha-ENaC gene by gene targeting. Amiloride-sensitive electrogenic Na+ transport was abolished in airway epithelia from alpha-ENaC(-/-) mice. Alpha-ENaC(-/-) neonates developed respiratory distress and died within 40 h of birth from failure to clear their lungs of liquid. This study shows that ENaC plays a critical role in the adaptation of the newborn lung to air breathing.

The role of the glucocorticoid receptor {GR1 in glucocorticoid physiology and during development was investigated by generation of GR-deficient mice by gene targeting. GR -/-mice die within a few hours after birth because of respiratory failure. The lungs at birth are severely atelectatic, and development is impaired from day 15.5 p.c. Newborn livers have a reduced capacity to activate genes for key gluconeogenic enzymes. Feedback regulation via the hypothalamic-pituitary-adrenal axis is severely impaired resulting in elevated levels of plasma adrenocorticotrophic hormone (15-fold) and plasma corticosterone (2.5-fold). Accordingly, adrenal glands are enlarged because of hypertrophy of the cortex, resulting in increased expression of key cortical steroid biosynthetic enzymes, such as side-chain cleavage enzyme, steroid 11B-hydroxylase, and aldosterone synthase. Adrenal glands lack a central medulla and synthesize no adrenaline. They contain no adrenergic chromaffin cells and only scattered noradrenergic chromaffin cells even when analyzed from the earliest stages of medulla development. These results suggest that the adrenal medulla may be formed from two different cell populations: adrenergic-specific cells that require glucocorticoids for proliferation and/or survival, and a smaller noradrenergic population that differentiates normally in the absence of glucocorticoid signaling.

Salt taste in mammals can trigger two divergent behavioural responses. In general, concentrated saline solutions elicit robust behavioural aversion, while low concentrations of NaCl are typically attractive, particularly after sodium depletion1-5. Notably, the attractive salt pathway is selectively responsive to sodium and inhibited by amiloride, while the aversive one functions as a non-selective detector for a wide range of salts1-3, 6-9. Since amiloride is a potent inhibitor of the epithelial sodium channel (ENaC), ENaC has been proposed to function as a component of the salt taste receptor system1, 3, 6-14. Here, we examine the basis of sodium sensing in the mammalian taste system. Previously, we showed that four of the five basic taste qualities, sweet, sour, bitter and umami are mediated by separate taste receptor cells (TRC) each tuned to a single taste modality, and wired to elicit stereotypical behavioural responses5, 15-18. We now demonstrate that sodium sensing is also mediated by a dedicated population of TRCs. These taste cells express the epithelial sodium channel ENaC19, 20, and mediate behavioural attraction to NaCl. We genetically engineered mice lacking ENaCα in TRCs, and produced animals exhibiting a complete loss of salt attraction and sodium taste responses. Together, these studies substantiate independent cellular substrates for all five basic taste qualities, and validate the essential role of ENaC for sodium taste in mice.

Glut-2 is a low-affinity transporter present in the plasma membrane of pancreatic beta-cells, hepatocytes and intestine and kidney absorptive epithelial cells of mice. In beta-cells, Glut-2 has been proposed to be active in the control of glucose-stimulated insulin secretion (GSIS; ref. 2), and its expression is strongly reduced in glucose-unresponsive islets from different animal models of diabetes. However, recent investigations have yielded conflicting data on the possible role of Glut-2 in GSIS. Whereas some reports have supported a specific role for Glut-2 (refs 5,6), others have suggested that GSIS could proceed normally even in the presence of low or almost undetectable levels of this transporter. Here we show that homozygous, but not heterozygous, mice deficient in Glut-2 are hyperglycaemic and relatively hypo-insulinaemic and have elevated plasma levels of glucagon, free fatty acids and beta-hydroxybutyrate. In vivo, their glucose tolerance is abnormal. In vitro, beta-cells display loss of control of insulin gene expression by glucose and impaired GSIS with a loss of first phase but preserved second phase of secretion, while the secretory response to non-glucidic nutrients or to D-glyceraldehyde is normal. This is accompanied by alterations in the postnatal development of pancreatic islets, evidenced by an inversion of the alpha- to beta-cell ratio. Glut-2 is thus required to maintain normal glucose homeostasis and normal function and development of the endocrine pancreas. Its absence leads to symptoms characteristic of non-insulin-dependent diabetes mellitus.

Serine proteases are proteolytic enzymes that are involved in the regulation of various physiological processes. We generated mice lacking the membrane-anchored channel-activating serine protease (CAP) 1 (also termed protease serine S1 family member 8 [Prss8] and prostasin) in skin, and these mice died within 60 h after birth. They presented a lower body weight and exhibited severe malformation of the stratum corneum (SC). This aberrant skin development was accompanied by an impaired skin barrier function, as evidenced by dehydration and skin permeability assay and transepidermal water loss measurements leading to rapid, fatal dehydration. Analysis of differentiation markers revealed no major alterations in CAP1/Prss8-deficient skin even though the epidermal deficiency of CAP1/Prss8 expression disturbs SC lipid composition, corneocyte morphogenesis, and the processing of profilaggrin. The examination of tight junction proteins revealed an absence of occludin, which did not prevent the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface. This study shows that CAP1/Prss8 expression in the epidermis is crucial for the epidermal permeability barrier and is, thereby, indispensable for postnatal survival.

The epithelial sodium channel (ENaC) expressed in aldosterone-responsive epithelial cells of the kidney and colon plays a critical role in the control of sodium balance, blood volume, and blood pressure. In lung, ENaC has a distinct role in controlling the ionic composition of the air-liquid interface and thus the rate of mucociliary transport. Loss-of-function mutations in ENaC cause a severe salt-wasting syndrome in human pseudohypoaldosteronism type 1 (PHA-1). Gain-of-function mutations in ENaC beta and gamma subunits cause pseudoaldosteronism (Liddle's syndrome), a severe form of salt-sensitive hypertension. This review discusses genetically defined forms of a salt sensitivity and salt resistance in human monogenic diseases and in animal models mimicking PHA-1 or Liddle's syndrome. The complex interaction between genetic factors (ENaC mutations) and the risk factor (salt intake) can now be studied experimentally. The role of single-nucleotide polymorphisms (SNPs) in determining salt sensitivity or salt resistance in general populations is one of the main challenges of the post-genomic era.

The cAMP response element bnding protein (CREB) has been implicated as a key regulator in the transcriptional control of many genes. Transcription of many genes is affected by changes in cAMP levels in response to a variety of external signals and is mediated via a cAMP response element (CRE). This DNA sequence is recognized by a diverse family of DNA binding proteins (1-5), ofwhich the CRE-binding protein (CREB) has been best characterized (6-12). Activation of the protein kinase A (PKA) pathway leads to phosphorylation of CREB at Ser 33, which is required for CREB to initiate transcription of target genes (6, 13). Since the cloning of CREB, a large number of CRE-binding proteins have been identified. They all contain a leucine-zipper DNA binding motif and for some members the potential for heterodimerization has been demonstrated in vitro (14). Transcription factor CREB heterodimerizes with activating transcription factor 1 (ATF1) (15) and CRE modulator protein (CREM) (16) Fig. 1A). The construct (20 jg) was linearized with Not I and used to electroporate 1 x 107 D3 ES cells derived from 129Sv/J mice, which were cultured on mitomycin-treated embryonic fibroblast feeder layers (19). DNA from clones surviving G418 selection (200 .g/ml) were individually analyzed on Southern blots and hybridized with probes located either 5' or 3' ofthe genomic sequence contained in the construct. Depending on the probe used blots were prepared by digestion with Pvu II (3' probe) or Nco I (5' probe).Generation of CREB -/-Mice. ES cells from two clones were used for injection into blastocysts derived from C57BL/6J mice. Blastocysts were transferred to pseudopregnant NMRI/Han females and chimeric offspring were detected by the presence of agouti hairs (genotype Aw) on a nonagouti (a) background. Chimeric males were mated to females to produce ES-cell-derived offspring that were then analyzed on Southern blots containing DNA isolated from mouse tails (20

contributed equally to this work. Conflict of interest:The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: parathyroid hormone (PTH); 1,25-dihydroxycholecalciferol (1,25-(OH)2D3); transient receptor potential (TRP); TRP cation channel subfamily V, member 5 (TRPV5); TRP cation channel subfamily V, member 6 (TRPV6); last loop of proximal tubules (LPT); hypoxanthine-guanine phosphoribosyl transferase (HPRT); bone volume (BV); total bone marrow volume including trabeculae (TV); trabecular bone volume fraction (BV/TV); trabecular thickness (Tb.Th); trabecular number (Tb.N); connectivity density (CD); structure model index (SMI); cortical volume (Ct.V); endocortical volume (Ec.V); total diaphyseal volume (Dp.V), cortical thickness (Ct.Th); cortical bone volume fraction (Ct.V/Dp.V); moment of inertia (MOI); Tartrate-resistant acid phosphatase (TRAP); number of osteoclasts per bone surface area (N.Oc/BS); surface area of osteoclasts per bone surface area (Oc.S/BS); arginine vasopressin (AVP); Na + -Ca 2+ exchanger (NCX1); vitamin D receptor (VDR). IntroductionCa 2+ is the most abundant cation in the human body and serves a number of important physiological functions, including fertilization, synaptic transmission, muscle contraction, blood clotting, and bone mineralization. The extracellular Ca 2+ concentration is controlled by the kidney, intestine, and bone through the action of the calciotropic hormones, including parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol (1,25-(OH) 2 D 3 ). In humans, the daily dietary Ca 2+ intake is less than 1,000 mg, of which only 30% is absorbed in the intestinal tract. This percentage is significantly enhanced during growth, pregnancy, and lactation by increased levels of circulating 1,25-(OH) 2 D 3 . Although there is continuous turnover of bone mass, there is no net gain or loss of Ca 2+ from bone in a young and healthy individual. This indicates that healthy adults excrete a maximum of 300 mg Ca 2+ in the urine to balance the intestinal Ca 2+ uptake and that the remaining 98% of the Ca 2+ filtered in the glomeruli is reabsorbed along the nephron. The molecular mechanism responsible for Ca 2+ absorption in the small intestine and the kidney was elusive for a long time.Renal Ca 2+ wasting, hyperabsorption, and reduced bone thickness in mice lacking TRPV5 Ca 2+ ions play a fundamental role in many cellular processes, and the extracellular concentration of Ca 2+ is kept under strict control to allow the proper physiological functions to take place. The kidney, small intestine, and bone determine the Ca 2+ flux to the extracellular Ca 2+ pool in a concerted fashion. Transient receptor potential (TRP) cation channel subfamily V, members 5 and 6 (TRPV5 and TRPV6) have recently been postulated to be the molecular gatekeepers facilitating Ca 2+ influx in these tissues and are members of the TRP family, which mediates diverse biological effects ranging from pain perception to male aggression. Genetic ablation of TRPV5 in the mouse allowed u...