Mineralocorticoid hormones exert dramatic effects on pluripotent human stem cell progeny.

The authors studied mineralocorticoid receptor (MCR)-mediated effects of steroids on CD34(+) progenitor cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed the presence of mRNA for both the MCR and the alpha subunit of the epithelial sodium channel, a member of the amiloride-sensitive sodium channel (ASSC) superfamily, in human CD41(+) megacaryoblastic cells derived from cultured bone marrow CD34(+) isolates, as well as in the human erythromegakaryoblastic leukemia (HEL) cell line. Immunofluorescence also revealed the presence of both the MCR and ASSC in circulating CD34(+) and medullar CD41(+) megacaryoblastic cells, the former as a nucleocytoplasmic protein and the latter as a membrane-bound protein, as expected from earlier studies using MCR-specific targets. In a selective medium, the formation of erythrocyte burst-forming units, and of the granulocyte-macrophage colony-forming units, by circulating CD34(+) cells was influenced by the agonists deoxycorticosterone and aldosterone, as well as by the antagonists RU 26752 and ZK 91587, targeted for the MCR. The multiplication of the leukemic HEL progeny, derived from CD41(+) cells, was similarly altered by these steroids targeted for the MCR. In contrast, in the optimal growth medium, the multiplication, and colony formation by bone marrow CD34(+) progenitor cells were not altered by either aldosterone or ZK 91587. These and other studies reveal that the receptor-mediated action of mineralocorticoids may influence the functional maturation of the hematopoietic progenitor lineage, contrary to the classical notion where the mineralotropic effect would be a unique feature of the epithelial cell.

The blockade of mineralocorticoid hormone signaling provokes dramatic teratogenesis in cultured rat embryos.

Although the administration of adrenocortical hormones to pregnant rats provokes only limited effect on the growth and development of the fetus, the direct influence of these steroids on cultured embryos has never been studied. The disruption of cell signaling by ZK 91587, which specifically occupies the mineralocorticoid receptor, resulted within 2 days in significant and pronounced adverse effects on the total length, the somite number, the embryo curvature, the communication between vitelline and umbilical blood vessels in the allantoid, and the vascularization of the vitelline sac, in 244-hour Wistar rat embryos in culture. The average score of 16 organs declined in a dose-dependent manner, following exposure to ZK 91587, and this was totally reversed by 10 microM aldosterone which, by itself, did not at all influence the embryonic development. The organogenesis was inhibited in the order: hind limb > fore limb > optic stalk > brain > olfactory pit > otic vesicle. ZK 91587 was completely ineffective in embryos that had attained the age of 260 hours. Similar, but less dramatic, results were obtained with the mineralocorticoid antagonist RU 26752, and with the antiglucocorticoid RU 38486. Sprague-Dawley rat embryos responded in a manner similar to the Wistar conceptuses. Thus, steroid receptor-mediated cell signaling is of critical importance to the growth and development of cultured rat embryos, which form a new model system to unravel adrenocortical hormone action.

Epithelial sodium channel and the mineralocorticoid receptor in cultured rat Müller glial cells.

Müller glial cells are the major non-neuronal cells of the retina. They are involved in retinal function and exert a profound influence on the function of retinal neurons. We present an in vitro study of the localization of the mineralocorticoid receptor (MCR) and of the epithelial sodium channel (ENaC) in rat Müller glial cells isolated from rat retina, using respectively, a polyclonal antiserum raised against the rat purified MCR, and a rabbit polyclonal antibody against the 14-amino acid (aa) peptide QGLGKGDKREEQGL, which corresponds to the N-terminal region (44-58aa) of the alpha-subunit of the ENaC. In an immunocytochemical study using anti-MCR and anti-ENaC antibodies, the MCR was detected as a protein present in the cytoplasm and in the nucleus, whereas ENaC was detected as a membrane-bound protein. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers, 5'-CTGCCTTTATGGATGATGGT-3' (sense), 5'-GTTCAGCTCGAAGAAGA-3' (antisense) for ENaC and 5'-AGGCTACCACAGTCTCCCTG-3' (sense) and 5'-GCAGTGTAAAATCTCCAGTC-3' (antisense) for MCR, showed expression of the ENaC and MCR genes in Müller cells. The presence of ENaC and MCR was detected as the predicted bands of 520 bp and 843 bp, respectively. In both cases, 100% identity was observed between the sequences of rat Müller cell (RMC) PCR products and rat kidney. Interestingly, the basal levels of ENaC were increased in vitro by the MCR-specific hormone, aldosterone. Thus, our results strongly suggest that the Müller glial cells may play a role in the regulation of extracellular Na+ concentration, which could be regulated by steroid-mediated sodium uptake.

Mineralocorticoid receptor-mediated signaling regulates the ion gated sodium channel in vascular endothelial cells and requires an intact cytoskeleton.

The PCR analysis followed by sequence alignment showed that both the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes were expressed in the human vascular endothelial cell line (ECV). The growth and multiplication of the ECV in culture were influenced by both aldosterone and the MCR-specific antagonist ZK 91587. Following double labelled immunofluorescence recorded by confocal microscopy, both the MCR and the ENaC were found to colocalize with the tubulin filaments in ECV cells in situ; no association was observed with cellular actin. ZK 91587 not only eliminated the basal expression, but it also impaired the transactivation of the ENaC gene by aldosterone. The disruption of actin and tubulin by cytochalasin D and colchicine, respectively, resulted in the total elimination of ENaC induction by aldosterone. These studies suggest that (i) the transcriptional regulation of the ENaC gene by the MCR-mediated signalling is not restricted to epithelial cells and requires cytoskeleton integrity in ECV cells in situ, (ii) tubulin may form a new and novel mediator in cell regulation, and (iii) the vascular tone may actually be regulated via transactivation of the ion gated sodium channel in the endothelial cell of the blood vessels under direct, receptor-mediated action of aldosterone.

Mineralocorticoid hormone signaling regulates the 'epithelial sodium channel' in fibroblasts from human cornea.

We investigated the regulation of sodium absorption by steroid hormones in embryologically diverse cells from the human eye. A cell extract from human corneal fibroblasts was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as 82- to 85-kD and 102-kD bands, respectively, by the Western blot technique. In fluorescent, confocal and electron microscopy, the MCR was revealed as a nucleocytoplasmic protein, whereas the ENaC was almost exclusively membrane bound; both appeared aligned along actin filaments of corneal keratocytes, and both were widely colocalized in various cell types of human cornea in situ. Following reverse transcription and amplification of total RNA isolated from corneal fibroblasts, the ENaC and MCR genes in the PCR product were evident as predicted bands of 520 and 843 bp, respectively, whose sequence exhibited 100% identity with those from known human sources. The multiplication of corneal fibroblasts was influenced by both the MCR-specific antagonist RU 26752 and the natural hormone aldosterone, and these steroids also stimulated protein phosphorylation. In quantitative PCR, both the basal and aldosterone-induced levels of ENaC were diminished by the MCR-specific antagonist ZK 91587. Consequently, the ocular sodium channel appears to be regulated by steroid signalling in cells of diverse embryological origins, contrary to the existing notions where (a) this process would be limited exclusively to the epithelial cells and (b) ocular sodium transport would be regulated via the Na(+)-K(+)-ATPase in the basolateral membrane.

The epithelial sodium channel (ENaC) in rodent retina, ontogeny and molecular identity.

PURPOSE: To assess the appearance of the epithelial sodium channels (ENaC) during retinal development and establish its molecular identity. METHODS: Photoreceptors were isolated by horizontal sectioning of rat retina with a vibratome series 1000. Intact retina and isolated photoreceptors were analyzed for the developmental appearance of ENaC using the Polymerase Chain Reaction (PCR) technique. Immunofluorescence was conducted with the aid of an antibody raised against the 14 amino acids peptide QGLGKGDKREEQGL, corresponding to the N-terminal region (44-58 aa) of alpha ENaC. RESULTS: ENaC message was present in rat retina photoreceptors, isolated just one day after birth. The bipolar and ganglion cell layers, separated from whole retina by vibratome, also contained the ENaC message. The partial sequence of the photoreceptor ENaC (496 base) exhibited one hundred percent homology with the channel from rat known sources. Immunochemistry revealed that only the outer nuclear layer was positive for the ENaC in one-day-old rat; the inner segment became immunopositive at the age of 9 days. CONCLUSION: The ENaC is present in the retina and visible soon after birth. These observations suggest that ENaC may prove to have physiological importance in the retina but until now its function is unknown.

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines.

We studied the expression of the mineralocorticoid receptor (MCR), and of the amiloride-sensitive sodium channel (ASSC) regulated by the MCR, in human leukemic cell lines. Cell extracts from TF1 (proerythroblastic), HEL (human erythroblastic leukemia) and U937 (myeloblastic) cell line were positive for the ASSC, as a 82 kDa band in Western blots developed with the aid of a polyclonal antibody raised against the peptide QGLGKGDKREEQGL, corresponding to the region 44-58 of the alpha subunit of the epithelial sodium channel (ENaC) cloned from rat colon, linked to KLH. The polyclonal antibody against the MCR revealed a single band of about 102 kDa in extracts from HEL and TF1 cells. The immunofluorescent labelling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor but the ASSC was exclusively membrane-bound and these results were confirmed by confocal microscopy. The expression of the MCR in the HEL cells was evident as a predicted band of 843 bp (234 amino acids) in electrophoresis of the PCR product obtained after total RNA had been reverse transcribed and then amplified using the primers 5'-AGGCTACCACAGTCTCCCTG-3' and 5'-GCAGTGTAAAATCTCCAGTC-3' (sense and antisense, respectively). The ENaC was similarly evident with the aid of the primers 5'-CTGCCmATG GATGATGGT-3' (sense) and 5'-GTTCAGCTCGAAGAAGA-3' (antisense) as a predicted band of 520 bp. In both cases, 100% identity was observed between the sequences of the PCR products compared to those from known human sources. The multiplication of the HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was selectively reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population. These permit new and novel possibilities to understand the pathobiology of human leukemia and to delineate sodium-water homeostasis in nonepithelial cells.

Paradoxical effects of mineralocorticoids on the ion gated sodium channel in embryologically diverse cells.

PCR analysis and Western blotting revealed the expression of the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes at the level of RNA, DNA, and protein in several leukemic cell lines, fibroblasts from human cornea, and epithelial cells from ocular tissues. Following immunofluorescence, the MCR appeared to be primarily nuclear whereas the ENaC was almost exclusively membrane-bound. Paradoxically, the MCR-specific antagonist ZK 91587 actually stimulated the multiplication of human erythroblastic leukemia cells, contrary to the inhibitory effect of the antagonist RU 26752 on the multiplication of corneal fibroblasts; both effects were opposed by aldosterone. In quantitative PCR, both basal and aldosterone-induced levels of ENaC were diminished by ZK 91587 in the corneal fibroblast, in contrast to the stimulation observed in the retinal pigmentary epithelium. Thus, contrary to the existing notions, (a) antimineralocorticoids can act both as agonists and antagonists, and (b) the receptor-mediated action of mineralocorticoids on the sodium channel is not restricted to the epithelial cell.

Immunochemical analysis of the sodium channel in rodent and human eye.

The presence of the amiloride-sensitive sodium channel (ASSC) in ocular tissues was studied with the aid of a polyclonal antiserum raised against the 14 amino acid peptide QGLGKGDKREEQGL. This sequence corresponds to the region 44-58 of the alpha subunit of the channel, termed ENaC, cloned from rat colon. The antibody titers, measured by the ELISA technique, rose to 1∶2560 4 weeks after immunization, and this bleed was used in all subsequent experiments. Immunoblotting with the polyclonal anti-alphaENaC serum, revealed a major band of 82-86 kDa in extracts prepared from whole bovine or rat retina; a minor component of 92 kDa in the extract from bovine ciliary body may represent a glycosylated species. Immunohistochemistry, using the alphaENaC-specific antiserum, revealed strong fluorescence in specific areas of the rat and human eye. Pronounced labelling was observed in the epithelial cell layer of the retina, the lens, as well as both the pigmented and the nonpigmented epithelium of the ciliary body and the iris. All of the cell layers (epithelium, endothelium and fibroblasts) in the cornea, the blood vessels in the iris, and iris epithelium, were also strongly immunopositive. The somatic body of the photoreceptor cells (cones and rods) in the inner and outer segments could be traced to forming a synapse in both the internal and external portions of the internal nuclear layer. The bipolar cells and ganglia in the neuronal compartment also exhibited occasional immunofluorescence. The method of fixation and the source of the tissue were important parameters for the immunochemical localization of the ENaC. The resolution was very poor when rat eye was fixed in Bouin's solution but this method was satisfactory for human tissues. For rat eye, optimum resolution was obtained with AMeX fixation. This widespread distribution of the ENaC generally colocalizes with the previously observed immunopositivity for the mineralocorticoid receptor such that steroid hormone-mediated ion regulation would appear to add a new parameter to the functional expression of ocular tissues.

Mineralocorticoid hormone receptor and the epithelial sodium channel in a human leukemic cell line.

A Cell extract from the HEL (human erythroblastic leukemia) cell line was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as glycosylated 82-84 kDa bands, and a single 102 kDa band, respectively, in Western blots using polyclonal antibodies raised against these proteins. The immunofluorescent labeling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor whereas the ENaC was exclusively membrane-bound. These results were confirmed by confocal microscopy. The expression of the MCR in HEL cells was evident as a predicted band of 843 bp (234 amino acids) after total RNA from HEL cells had been reverse transcribed and then amplified by PCR; the ENaC was similarly evident as a predicted band of 520 bp. In both cases, near 100% identity was observed between the deduced amino acid sequences of the PCR products and those from known human sources. The multiplication of HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population.