Abstract. A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed ~, /3, and 3" rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of or, 8, and 3' subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that c~,/3, and 3' subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.XLORIDE-sensitive electrogenic transepithelial sodium transport constitutes the rate limiting step for sodium reabsorption in the epithelium of the distal nephron, the distal colon, the ducts of several exocrine glands (for instance the salivary glands and the sweat glands) and the epithelia of the lung (10). In kidney (25) and colon (29), electrogenic sodium transport is upregulated by aldosterone, a mineralocorticoid hormone, allowing the maintenance of sodium balance, blood volume, and blood pressure (30). In the distal nephron of rats submitted to a 1 wk salt restriction, the electrogenic sodium transport is highly efficient at reabsorbing sodium from the urine of collecting ducts, decreasing luminal sodium concentration to levels as low as 1 raM, while the plasma concentration is maintained at 140 raM. To establish such a gradient, the epithelia of the distal nephron expresses an electrogenic sodium transport mediated by an amiloride-sensitive sodium channel located in the apical membrane facing the external compartment (urine), and a ouabain-sensitive sodium pump restricted to the basolateral membrane facing the extracellular compartment (blood). Current physiological evidence indicates that the amiloride-sensitive epithelial sodium channel is mainly expressed in tissues that fall into t...

The sites of synthesis and action of prostaglandins (PGs) along the renal tubule are examined. We focused our attention on experiments performed on well-defined nephron segments, using direct quantitative measurements of prostaglandin synthesis by radio- or enzyme-immunoassay. On the other hand, we selected, among the described effects of PGs, those obtained on precisely defined tubular segments. Among PGs, PGE2 synthesis is largely predominant all along the tubule. Its main sites of synthesis are the medullary collecting tubule and, to a lesser extent, the cortical collecting tubule and the thin limb of Henle's loop. Synthesis of PGE2 is amplified approximately tenfold in the presence of an excess exogenous substrate, arachidonic acid, compared with values measured without addition of substrate. Other eicosanoids have roughly the same distribution along the tubule as PGE2. Their rate of synthesis is, however, much less than that of PGE2, approximately 20-fold lower for PGF2 alpha and 6-keto-PGF1 alpha, and 100-fold lower for thromboxane B2 (TxB2). This contrasts with glomerular PG synthesis, where the difference between the production of PGE2 and other eicosanoids is much less marked. Most studies agree that antidiuretic hormone (ADH) and kinins augment PGE2 synthesis, whereas corticosteroids decrease it, at least in the collecting tubule. Direct effects of PGE2 have been described mainly in the medullary thick ascending limb and collecting tubule. They generally consist of a decrease in transepithelial potential difference and reabsorptive rates of water and solutes, in particular sodium and chloride. However, whatever the solute or tubular segment concerned, some studies failed to find such effects. The bulk of evidence suggests that ADH and PGs interact in kidney tubular cells. It is generally accepted that PGs antagonize the hydrosmotic effects of ADH in the collecting tubule. The mechanisms underlying these complex interactions are still under discussion: they probably involve several types of receptors and pathways for ADH action, which intervene in the modulation of both PG synthesis and cyclic nucleotides, and several types of PG receptors, either stimulatory or inhibitory to adenylate cyclase.

The presence of mineralocorticoid receptors (MRs) and their physicochemical characteristics were investigated in the heart and blood vessels of rabbits. Immunohistochemical methods using the monoclonal anti-idiotypic antibody H10E, which interacts with the steroid binding domain of MRs, revealed the presence of immunoreactive material in the heart and large blood vessels. In the heart, a positive staining was observed in myocytes and endothelial cells of atria and ventricles. In vessels, MRs were detected in the aorta and pulmonary artery. They were localized in endothelial and vascular smooth muscle cells. No staining was present in the small vascular bed, arterioles, and capillaries. In all these studies, the mineralocorticoid specificity of the staining was assessed by in situ competition experiments with aldosterone and RU486, a glucocorticoid antagonist. The presence of MRs in the heart and vessels was further demonstrated by specific aldosterone binding to one class of high affinity binding sites in the cytosol of the adrenalectomized rabbit heart (Kd, 0.25 nM; maximum MR concentration, 15-20 fmol/mg protein), whose mineralocorticoid specificity has been clearly established by competition studies. Sedimentation gradient analyses revealed that the cardiovascular MR is an 8.5S hetero-oligomer that includes the heat shock protein 90. The physicochemical characteristics of the cardiovascular MRs are virtually identical to those of the renal MRs. Altogether, our results clearly demonstrate the presence of MRs in the cardiovascular system. This supports the possibility of direct aldosterone actions in the heart and blood vessels.

Distal colon and renal cortical collecting ducts are major effectors of aldosterone-dependent Na homeostasis. Na is absorbed by entry through an apical amiloride-sensitive Na channel and extruded by Na-K-ATPase at the basolateral membrane. Using a ribonuclease protection assay, we studied, in vivo, aldosterone regulation of alpha-, beta-, gamma-subunits of the rat epithelial Na channel (rENaC) and alpha 1- and beta 1-subunits of Na-K-ATPase. In the kidney, Na-K-ATPase mRNAs were also assayed over discrete tubular segments by in situ hybridization. In rat colon, all three rENaC mRNAs were decreased by adrenalectomy, with a major effect on beta- and gamma-subunits, and were restored with 7 days, but not 2 days, of aldosterone treatment; in the kidney, however, only alpha-transcripts varied. Na-K-ATPase alpha 1- and beta 1-subunit mRNAs in both organs were not (in the case of the beta 1-subunit) or were mildly (in the case of the alpha 1-subunit) affected after adrenalectomy. Our conclusions are as follows: 1) Transcripts of rENaC and Na-K-ATPase subunits are not coordinately regulated by aldosterone in vivo; i.e., modulation involves mainly the Na channel, not Na-K-ATPase; the effect is not of comparable magnitude on each subunit mRNA and differs between tissues. 2) The delay of the aldosterone effect on transcripts is much longer than that required to restore normal Na transport in adrenalectomized rats, indicating that rENaC and Na-K-ATPase subunit transcript levels may depend on unidentified early aldosterone-induced proteins.

We have examined whether arginine vasopressin (AVP) can induce a long-term modulation of transepithelial ion transport in addition to its well known shortterm effect. In the RCCD 1 rat cortical collecting duct cell line, an increase in both short-circuit current and 22 Na transport was observed after several hours of 10