Human mineralocorticoid receptor expression renders cells responsive for nongenotropic aldosterone actions

The steroid hormone aldosterone is important for salt and water homeostasis as well as for pathological tissue modifications in the cardiovascular system and the kidney. The mechanisms of action include a classical genomic pathway, but physiological relevant nongenotropic effects have also been described. Unlike for estrogens or progesterone, the mechanisms for these nongenotropic effects are not well understood, although pharmacological studies suggest a role for the mineralocorticoid receptor (MR). Here we investigated whether the MR contributes to nongenotropic effects. After transfection with human MR, aldosterone induced a rapid and dose-dependent phosphorylation of ERK1/2 and c-Jun NH2-terminal kinase (JNK) 1/2 kinases in Chinese hamster ovary or human embryonic kidney cells, which was reduced by the MR-antagonist spironolactone and involved cSrc kinase as well as the epidermal growth factor receptor. In primary human aortic endothelial cells, similar results were obtained for ERK1/2 and JNK1/2. Inhibition of MAPK kinase (MEK) kinase but not of protein kinase C prevented the rapid action of aldosterone and also reduced aldosterone-induced transactivation, most probably due to impaired nuclear-cytoplasmic shuttling of MR. Cytosolic Ca2+ was increased by aldosterone in mock- and in human MR-transfected cells to the same extend due to Ca2+ influx, whereas dexamethasone had virtually no effect. Spironolactone did not prevent the Ca2+ response. We conclude that some nongenotropic effects of aldosterone are MR dependent and others are MR independent (e.g. Ca2+), indicating a higher degree of complexity of rapid aldosterone signaling. According to this model, we have to distinguish three aldosterone signaling pathways: 1) genomic via MR, 2) nongenotropic via MR, and 3) nongenotropic MR independent.     

Brain mineralocorticoid receptors in cognition and cardiovascular homeostasis

Mineralocorticoid receptors (MR) mediate diverse functions supporting osmotic and hemodynamic homeostasis, response to injury and inflammation, and neuronal changes required for learning and memory. Inappropriate MR activation in kidneys, heart, vessels, and brain hemodynamic control centers results in cardiovascular and renal pathology and hypertension. MR binds aldosterone, cortisol and corticosterone with similar affinity, while the glucocorticoid receptor (GR) has less affinity for cortisol and corticosterone. As glucocorticoids are more abundant than aldosterone, aldosterone activates MR in cells co-expressing enzymes with 11β-hydroxydehydrogenase activity to inactivate them. MR and GR co-expressed in the same cell interact at the molecular and functional level and these functions may be complementary or opposing depending on the cell type. Thus the balance between MR and GR expression and activation is crucial for normal function. Where 11β-hydroxydehydrogenase 2 (11β-HSD2) that inactivates cortisol and corticosterone in aldosterone target cells of the kidney and nucleus tractus solitarius (NTS) is not expressed, as in most neurons, MR are activated at basal glucocorticoid concentrations, GR at stress concentrations. An exception may be pre-autonomic neurons of the PVN which express MR and 11β-HSD1 in the absence of hexose-6-phosphate dehydrogenase required to generate the requisite cofactor for reductase activity, thus it acts as a dehydrogenase. MR antagonists, valuable adjuncts to the treatment of cardiovascular disease, also inhibit MR in the brain that are crucial for memory formation and exacerbate detrimental effects of excessive GR activation on cognition and mood. 11β-HSD1 inhibitors combat metabolic and cognitive diseases related to glucocorticoid excess, but may exacerbate MR action where 11β-HSD1 acts as a dehydrogenase, while non-selective 11β-HSD1&2 inhibitors cause injurious disruption of MR hemodynamic control. MR functions in the brain are multifaceted and optimal MR:GR activity is crucial. Therefore selectively targeting down-stream effectors of MR specific actions may be a better therapeutic goal.     

Effect of nitrosative stress on Schizosaccharomyces pombe: inactivation of glutathione reductase by peroxynitrite

Oxidative stress has been shown to alter cellular redox status in various cell types. Changes in expressions of several antioxidative and antistress-responsive genes along with activation or inactivation of various proteins were also reported during oxidative insult as well as during nitrosative stress. In the present study, we show the effect of nitrosative stress on cellular redox status of fission yeast Schizosaccharomyces pombe. This is the first report of S-nitrosoglutathione (GSNO) reductase activity in S. pombe and its inactivation by GSNO. We also show the inactivation of glutathione reductase (GR) and glutathione peroxidase in the presence of various reactive nitrogen species in vivo. In addition, we first observe the inactivation of GR by peroxynitrite in vivo using S. pombe cells and also similar observations under in vitro conditions. An immunoreactive band against monoclonal anti-3-nitrotyrosine antibody confirms the modification of GR under in vitro conditions. We also show the effect of nitrosative stress on Deltapap1 cells of S. pombe, which are more sensitive to nitrosative stress, indicating the involvement of Pap1 in the protection against nitrosative stress. Finally, exposure of S. pombe cells to reactive nitrogen species reveals an important role of cellular thiol pool in protection against nitrosative stress.     

EF domains are sufficient for nongenomic mineralocorticoid receptor actions

The mineralocorticoid receptor (MR) is important for salt homeostasis and reno-cardiovascular pathophysiology. Signaling mechanisms include, besides classical genomic pathways, nongenomic pathways with putative pathophysiological relevance involving the mitogen-activated protein kinases ERK1/2. We determined the MR domains required for nongenomic signaling and their potential to elicit pathophysiological effects in cultured cells under defined conditions. The expression of full-length human MR or truncated MR consisting of the domains CDEF (MR CDEF), DEF (MR DEF), or EF (MR EF) renders cells responsive for the MR ligand aldosterone with respect to nongenomic ERK1/2 phosphorylation, whereas only full-length MR and MR CDEF conferred genomic responsiveness. ERK1/2 phosphorylation depends on the EGF receptor and cSRC kinase. MR EF expression is sufficient to evoke the aldosterone-induced increase of collagen III levels, similar to full-length MR expression. Our data suggest that nongenomic MR signaling is mediated by the EF domains and present the first proof of principle showing that nongenomic signaling can be sufficient for some pathophysiological effects. The minimum amino acid motif required for nongenomic MR signaling and its importance in various effects have yet to be determined.     

Corticosteroids, receptors, and the organ-specific functions of 11 beta-hydroxysteroid dehydrogenase.

Reversible oxidation of the biologically active corticosteroids to the inactive 11-dehydrocorticosteroids is catalyzed by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). The properties of the enzyme based on clinical observations of individuals with defective 11 beta HSD expression, and laboratory studies of the properties and behavior of the enzyme, are consistent with separate 11 beta-dehydrogenase and 11-oxoreductase species. However, recombinant enzyme expressed in mammalian cells retain both activities, leading to the conclusion that 11 beta HSD is a unique, reversible enzyme. 11 beta HSD is present in most tissues, but its specific functions in most tissues are unknown. How the enzyme may mediate corticosteroid-receptor interaction is illustrated by studies using kidney, testis, and brain. In kidney, 11 beta HSD prevents glucocorticoids from competing inappropriately with aldosterone for mineralocorticoid receptor (MR). Lack of enzyme in humans due to natural causes or inhibition by pharmacological agents results in maximum activation of MR by glucocorticoids, leading to the clinical symptoms of apparent mineralocorticoid excess. Leydig cells of the testes synthesize testosterone, a process that is suppressed by events initiated by the binding of corticosteroid to glucocorticoid receptors (GR). Depletion of active steroid mediated by 11 beta HSD may initiate testosterone production at puberty and affect testosterone production during adult life, as for example during periods of stress. The heterogeneous distribution of MR and GR in the brain reflects the specific regional effects of glucocorticoids and mineralocorticoids on neural function. Colocalization of 11 beta HSD and corticosteroid receptors in brain may be important in controlling the specificity of corticosteroid interaction with GR and MR. The patterns of 11 beta HSD-steroid-receptor interaction illustrated with these three tissues may provide models applicable to other tissues in which corticosteroid receptors and 11 beta HSD coexist.     

Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C

Reactive oxygen species (ROS) formation plays a major role in diabetes-induced endothelial dysfunction, though the molecular mechanism(s) involved and the contribution of nitric oxide (NO) are still unclear. This study using bovine retinal endothelial cells was aimed at assessing (i) the role of oxygen-dependent vs. NO-dependent oxidative stress in the endothelial cell permeability alterations induced by the diabetic milieu and (ii) whether protein kinase C (PKC) activation ultimately mediates these changes. Superoxide, lipid peroxide, and PKC activity were higher under high glucose (HG) vs. normal glucose throughout the 30 d period. Nitrite/nitrate and endothelial NO synthase levels increased at 1 d and decreased thereafter. Changes in monolayer permeability to 125I-BSA induced by 1 or 30 d incubation in HG or exposure to advanced glycosylation endproduct were reduced by treatment with antioxidants or PKC inhibitors, whereas NO blockade prevented only the effect of 1 d HG. HG-induced changes were mimicked by a PKC activator, a superoxide generating system, an NO and superoxide donor, or peroxynitrite (attenuated by PKC inhibition), but not a NO donor. The short-term effect of HG depends on a combined oxidative and nitrosative stress with peroxynitrite formation, whereas the long-term effect is related to ROS generation; in both cases, PKC ultimately mediates permeability changes.