Porcine spermatozoa contain more than one membrane progesterone receptor.

Progesterone has been shown to be a physiologically relevant inducer of the sperm acrosome reaction. A novel protein intrinsic to microsomal membranes, membrane progesterone receptor (mPR, now termed progesterone membrane receptor component 1, PGMRC1) that binds progesterone with high affinity has been cloned from porcine liver previously, and corresponding antibodies mitigate the progesterone induced acrosome reaction. In this study we aimed at the localization of mPR in porcine spermatozoa. Immunostaining suggested the exclusive occurrence of mPR in a hardly accessible place, possibly the inner acrosomal membrane, with digitonin dramatically increasing the number of positively stained cells. Consistent with the structure prediction for mPR, its short N-terminus (NT) but not the large C-terminal part becomes accessible from outside after digitonin treatment as evidenced by the staining pattern of antibodies directed against different regions of the protein. However, digitonin treatment solubilizes a progesterone binding activity of approximately 140 kDa molecular weight, that is different from mPR, which remains in the cell membrane as demonstrated by Western blotting. Ligand binding studies confirm the dissimilarity of mPR and the digitonin-soluble progesterone binding protein. Chemical modification studies also indicate that the digitonin-soluble progesterone binding protein has a binding site that differs from that of mPR. It is concluded that more than one progesterone receptor is present in porcine spermatozoa.

Mineralocorticoid receptor antagonists do not block rapid ERK activation by aldosterone.

Aldosterone can elicit rapid nongenomic effects both in vivo and in vitro, often mediated by signal transduction cascades. However, it is not understood how these rapid effects are initiated. In this study we show that aldosterone leads to rapid activation of mitogen activated protein kinases ERK1/2 in the cortical collecting duct cell line M-1. Inhibitors of transcription and translation could not block this activation, which suggests an extranuclear (nongenomic) mechanism. Although it is known that M-1 cells do not contain a transcriptionally functional MR, it is not known whether a closely related protein still could mediate the effects, or an unrelated nonclassic receptor. To test this hypothesis, the effects of four classical mineralocorticoid receptor antagonists were studied. None of the compounds could block the response to aldosterone. Altogether, the data suggest that rapid aldosterone effects in M-1 cells are initiated by a receptor different from the classical mineralocorticoid receptor.

Early aldosterone up-regulated genes: new pathways for renal disease?

BACKGROUND: The use of aldosterone antagonists has important beneficial effects on the progression of renal and cardiac disease reflected in a decrease of cardiovascular mortality and renal failure. Nevertheless, the incidence of heart and end-stage renal failure continues to increase. This leads to the conclusion that mechanisms independent of the classical aldosterone/mineralocorticoid receptor system may contribute to the pathogenesis of cardiac and renal disease. METHODS: The mRNA expression profile of human renal epithelial cells in response to aldosterone was characterized using cDNA arrays covering approximately 1430 genes. Differentially expressed genes were further evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and estimating the gene products by Western blotting. RESULTS: Aldosterone treatment of cells resulted in significant up-regulation of several genes within 1 hour, with sgk, p21/waf1, gadd45, and gadd153 being the most significant ones. Long-term treatment (>4 hours) with aldosterone induced the mRNA expression of pparalpha and puralpha. The mineralocorticoid receptor inhibitor spironolactone decreased the mRNA levels of sgk, p21/waf1, and gadd45, whereas the glucocorticoid receptor inhibitor RU 486 reduced the mRNA level of sgk and p21/waf1. Gadd153 was not affected by any of the inhibitors, probably indicating regulation by nonclassic mechanisms. CONCLUSION: Among the early genes investigated in this study, one transcript has been identified that is not suppressed by antagonists of either glucocorticoid or mineralocorticoid receptor. Further studies should be able to identify other genes regulated in a similar manner that could explain the inefficacy of spironolactone in some cases of aldosterone-mediated kidney disease.

Nongenomic steroid action: controversies, questions, and answers.

Steroids may exert their action in living cells by several ways: 1). the well-known genomic pathway, involving hormone binding to cytosolic (classic) receptors and subsequent modulation of gene expression followed by protein synthesis. 2). Alternatively, pathways are operating that do not act on the genome, therefore indicating nongenomic action. Although it is comparatively easy to confirm the nongenomic nature of a particular phenomenon observed, e.g., by using inhibitors of transcription or translation, considerable controversy exists about the identity of receptors that mediate these responses. Many different approaches have been employed to answer this question, including pharmacology, knock-out animals, and numerous biochemical studies. Evidence is presented for and against both the participation of classic receptors, or proteins closely related to them, as well as for the involvement of yet poorly understood, novel membrane steroid receptors. In addition, clinical implications for a wide array of nongenomic steroid actions are outlined.

Nongenomic effects of aldosterone: cellular aspects and clinical implications.

Nongenomic action of aldosterone has been observed in many cell types which often are different from the classic target tissues for mineralocorticoid action, such as vascular cells. As judged from their time scale and insensitivity toward inhibitors of protein synthesis, effects are not mediated by the classic mineralocorticoid receptor pathway. Here we summarize studies on rapid in vitro aldosterone effects, e.g. ion fluxes, and second messengers involved therein. Furthermore, several clinical studies on in vivo aldosterone action have shown rapid effects on cardiovascular parameters, among them baroreflex and vascular resistance. Taken together with the beneficial effect of aldosterone antagonism in heart failure patients that was demonstrated in the Randomized Aldactone Evaluation Study (RALES), aldosterone may be an equally important factor of the renin-angiotensin-aldosterone system in cardiovascular pathogenesis.