PubMed · 4334846
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A C Guyton, T G Coleman, H J Granger. 1972. Circulation: overall regulation.. https://doi.org/10.1146/annurev.ph.34.030172.000305
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Acute nitric oxide (NO) inhibits angiotensin II (ANG II)-stimulated aldosterone synthesis in zona glomerulosa (ZG) cells. In this study, we investigated the effects of chronic administration of NO on the ANG II receptor type 1 (AT1) expression and aldosterone synthesis. ZG cells were treated daily with DETA NONOate (10(-4) M), an NO donor, for 0, 12, 24, 48, 72, and 96 h. Chinese hamster ovary (CHO) cells, stably transfected with the AT1B receptor, were used as a positive control. Western blot analysis indicated that AT1 receptor expression was decreased as a function of time of NO administration in both CHO and ZG cells. ANG II binding to its receptors was determined by radioligand binding. NO treatment of ZG cells for 96 h resulted in a decrease in ANG II binding compared with control. The receptor density was decreased to 1,864 +/- 129 fmol/mg protein from 3,157 +/- 220 fmol/mg protein (P < 0.005), but the affinity was not changed (1.95 +/- 0.22 vs. 1.88 +/- 0.21 nM). Confocal Raman microspectroscopy and immunocytochemistry both confirmed that the expression of AT1 receptors in ZG cells decreased with chronic NO administration. In addition, chronic NO administration also decreased the expression of cholesterol side-chain cleavage enzyme in ZG cells and inhibited ANG II- and 25-hydroxycholesterol-stimulated aldosterone synthesis in ZG cells. This study demonstrates that chronic administration of NO inhibits aldosterone synthesis in ZG cells by downregulation of the expression of both AT1 receptors and cholesterol side-chain cleavage enzyme.
We have investigated the functional consequences of three naturally occurring amino acid substitutions of the human mineralocorticoid receptor (hMR). These mutations are located in the DNA-binding domain and the ligand-binding domain (LBD) and are associated with autosomal dominant or sporadic type I pseudohypoaldosteronism. All mutant receptors bound specifically to glucocorticoid-responsive elements but presented modified transcriptional properties. The DNA-binding domain mutant G633R, which possesses a normal affinity for a glucocorticoid-responsive element, displayed altered interaction with, and a reduced dissociation rate from, DNA. Its intracellular localization in the absence of hormone was predominantly nuclear in comparison with predominant cytoplasmic location of hMR. Hormone-dependent nuclear cluster formation was comparable to wild-type hMR. These results and the three-dimensional modeling of the interaction of R633 with DNA suggest that altered interaction dynamics with DNA as well as modified intracellular localization may be responsible for submaximal transcriptional potency of hMR. Two LBD mutations, Q776R and L979P, were also investigated. Our data confirm the fundamental role of amino acid Q776 for anchoring the C3 ketone group of steroids in the ligand-binding pocket. Analysis of LBD conformation of mutant P979 demonstrates the relevance of hydrophobic interactions in the extreme C-terminal tail of the hMR for the correct ligand-binding competent state of the receptor. Our data underline the importance of studying naturally occurring mutants to identify crucial residues involved in hMR function.
Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both alpha- and beta- but not gamma-ENaC. Western blots showed that both alpha- and beta- but not gamma-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against alpha-ENaC but not by antisense oligonucleotide against beta-ENaC, indicating that only the alpha subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 microm) reduced the frequency of observance of 21-pS alpha-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from alpha- and beta-ENaC. After expression of exogenous gamma-ENaC, aldosterone again reduced the frequency of observance of the 21-pS alpha-ENaC channel but induced the appearance of a 5-pS channel, presumably a alphabetagamma-ENaC channel. In the absence of aldosterone, the alpha subunit forms an alpha-cryptic channel that is activated by stretch, and in the presence of aldosterone, beta and alpha subunits together form an active channel that is modulated by aldosterone.