Glucocorticoids play important roles in development and 'fetal programming'. Fetal exposure to excess glucocorticoids reduces birth weight and causes later hypertension. To investigate these processes further we have determined the detailed category of 11 beta-hydroxysteroid dehydrogenase type2 (11 beta-HSD2, which potently inactivates glucocorticoids) and the mineralocorticoid receptor (MR) by in situ hybridisation from embryonic day 9.5 (E9.5, term = E19) until after birth in the mouse. Widespread abundant 11 beta-HSD2 mRNA expression from E9.5-E12.5 changes dramatically at approximately E13 to a limited tissue-specific pattern (kidney, hindgut, testis/bile ducts, lung and a few brain regions (later seen in cerebellum, thalamus, roof of midbrain, neuroepithelial regions in pons and near the subicular hippocampus)). Placenta (labyrinthine zone) and extra-embryonic membranes express abundant 11 beta-HSD2 mRNA until E15.5 but this ceases = E16.5. It is unclear to what extent rodent term placental 11 beta-HSD activity is due to persisting 11 beta-HSD2 protein. Convincing MR mRNA expression is seen from E13.5 and includes pituitary, heart, muscle and meninges with expression later in gut, kidney, thymus, discrete areas of lung and several brain regions (including hippocampus, rhinencephalon and hypothalamus). 11 beta-HSD2 and MR clearly co-localise = E18.5 in kidney and colon and might do so in discrete areas of lung (E14-15) and neuroepithelia near the subicular hippocampus. Probably elsewhere MR are non-selective and 11 beta-HSD2 is involved in protecting glucocorticoid receptors in fetal fetal tissues. Comparison with previous enzymology studies suggest the changing pattern of 11 beta-HSD2 mRNA is likely to be translated into enzyme activity and have significant parallels in human development.
Non-technical summary Myocardial stretch increases force in two phases. The first one is immediate and attributed to an increase in myofilament Ca 2+ responsiveness (Frank-Starling mechanism). The second phase gradually develops and is known as slow force response (SFR) or Anrep effect due to an increase in intracellular Ca 2+ transient. We previously showed that Ca 2+ entry through reverse Na + /Ca 2+ exchange underlies the SFR, as the final step of an autocrine/paracrine loop involving release of angiotensin II/endothelin, transactivation of the epidermal growth factor receptor, increased mitochondrial oxidative stress and a Na + /H + exchanger (NHE-1) activation-mediated rise in Na + . In the present study we show that mineralocorticoid receptor activation is a necessary step between endothelin and epidermal growth factor receptor activation in the stretch-triggered reactive oxygen species-mediated NHE-1 activation leading to the SFR. AbstractThe increase in myocardial reactive oxygen species after epidermal growth factor receptor transactivation is a crucial step in the autocrine/paracrine angiotensin II/endothelin receptor activation leading to the slow force response to stretch (SFR). Since experimental evidence suggests a link between angiotensin II or its AT1 receptor and the mineralocorticoid receptor (MR), and MR transactivates the epidermal growth factor receptor, we thought to determine whether MR activation participates in the SFR development in rat myocardium. We show here that MR activation is necessary to promote reactive oxygen species formation by a physiological concentration of angiotensin II (1 nmol l −1 ), since an increase in superoxide anion formation of ∼50% of basal was suppressed by blocking MR with spironolactone or eplerenone. This effect was also suppressed by blocking AT1, endothelin (type A) or epidermal growth factor receptors, by inhibiting NADPH oxydase or by targeting mitochondria, and was unaffected by glucocorticoid receptor inhibition. All interventions except AT1 receptor blockade blunted the increase in superoxide anion promoted by an equipotent dose of endothelin-1 (1 nmol l −1 ) confirming that endothelin receptors activation is downstream of AT1. Similarly, an increase in superoxide anion promoted by an equipotent dose of aldosterone (10 nmol l −1 ) was blocked by spironolactone or eplerenone, by preventing epidermal growth factor receptor transactivation, but not by inhibiting glucocorticoid receptors or protein synthesis, suggesting non-genomic MR effects. Combination of aldosterone plus endothelin-1 did not increase superoxide anion formation more than each agonist separately. We found that aldosterone increased phosphorylation of the redox-sensitive kinases ERK1/2-p90RSK and the NHE-1, effects that were eliminated by eplerenone or by preventing epidermal growth factor receptor transactivation. Finally, we provide evidence that the SFR is suppressed by MR blockade, by preventing epidermal growth factor receptor transactivation or by scavenging reactive oxygen specie...
Abstract-Myocardial stretch triggers an angiotensin II-dependent autocrine/paracrine loop of intracellular signals, leading to reactive oxygen species-mediated activation of redox-sensitive kinases. Based on pharmacological strategies, we previously proposed that mineralocorticoid receptor (MR) is necessary for this stretch-triggered mechanism. Now, we aimed to test the role of MR after stretch by using a molecular approach to avoid secondary effects of pharmacological MR blockers. Small hairpin interference RNA capable of specifically knocking down the MR was incorporated into a lentiviral vector (l-shMR) and injected into the left ventricular wall of Wistar rats. The same vector but expressing a nonsilencing sequence (scramble) was used as control. Lentivirus propagation through the left ventricle was evidenced by confocal microscopy. Myocardial MR expression, stretch-triggered activation of redoxsensitive kinases (ERK1/2-p90 RSK ), the consequent Na + /H + exchanger-mediated changes in pH i (HEPES-buffer), and its mechanical counterpart, the slow force response, were evaluated. Furthermore, reactive oxygen species production in response to a low concentration of angiotensin II (1.0 nmol/L) or an equipotent concentration of epidermal growth factor (0.1 μg/mL) was compared in myocardial tissue slices from both groups. Compared with scramble, animals transduced with l-shMR showed (1) reduced cardiac MR expression, (2) cancellation of angiotensin II-induced reactive oxygen species production but preservation of epidermal growth factor-induced reactive oxygen species production, (3) cancellation of stretch-triggered increase in ERK1/2-p90 RSK phosphorylation, (4)
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