Fast Nongenomic Effect of Aldosterone on the Volume of Principal Cells in Collecting Tube and Genetic Heterogeneity of Epithelial Sodium Channel in the Postnatal Ontogenesis of Rat Kidney.

The effects of amiloride, epithelial sodium pump inhibitor, on the fast nongenomic effect of aldosterone in principal cells of an isolated segment of the distal portion of renal collecting tubes were studied in 10-day-old and adult rats. Fluorescent staining with Calcein AM showed various effects of amiloride (10(-5) M) on the stabilizing effect of aldosterone (10 nM) in hypotonic shock (280/140 mOsm/kg). Amiloride attenuated by 30% the effect of aldosterone on the amplitude of principal cell swelling in adult animals and almost completely abolished this effect in 10-day rats (p<0.05). These age-specific differences in the contribution of the distal portion of the collecting tube to the nongenomic effect of aldosterone did not depend on genetic heterogeneity of its α-subunit.

[THE ROLE OF THE PI3-KINASE IN THE FAST NONGENOMIC ALDOSTERONE EFFECTS IN THE PRINCIPAL CELLS OF THE CORTICAL COLLECTING DUCT OF THE RAT KIDNEY IN THE POSTNATAL ONTOGENESIS].

The involvement of wortmannin (10 -5 M), phosphatidyl inositol 3-kinase (PI3K) blocker, in the implementation of the rapid nongenomic aldosterone (10 nM) effects on the intracellular sodium (Na i +) and the principal cell volume of cortical collecting duct (CCD) of 10-day and adult rat kidney CCD was studied. Using fluorescence microscopy with intracellular dye Na Green and Calcein we found that wortmannin weakened the effect of aldosterone on the Na i + at low sodium in the extracellular medium (14 mM NaCl), and slowed the rate of reduction of the principal cell volume in the presence of aldosterone at the hypoosmotic shock (240/140 mOsm) since 10 days of age. The findings suggest the participation of phosphatidylinositol pathway in the fast nongenomic aldosterone effects (seconds and minutes) at the early stage of postnatal ontogenesis.

Role of epithelial sodium channel in the realization of homeostatic effects of aldosterone on the volume of principal cells of cortical collecting ducts in rats after hypoosmotic stress.

The mechanism of aldosterone effects (10 nM) on the volume of the principal cells of cortical collecting ducts after hypoosmotic stress (280/140 mOsm/kg) was studied using fluorescence microscopy. The experiments with intracellular fluorescent dye calcein showed that aldosterone significantly decreases the amplitude and rate of increase in principal cell volume during hypoosmotic stress. Epithelial sodium channel blocker amiloride (10(-5)M) significantly attenuated the effects of aldosterone on the amplitude and rate of changes in cell volume. The obtained data attest to the contribution of epithelial sodium channel to the realization of rapid non-genomic effects of aldosterone on the amplitude and rate of changes in volume of the principal cells of cortical collecting ducts in rat kidney after hypoosmotic stress.

Investigation on genetic heterogeneity of α-subunit of the epithelial sodium channel in rat renal cortex.

Genotypic variability of αENaC mRNA in adult rat kidney caused by deletion at the 3'-end of α-subunit DNA was studied by real-time PCR using specific probes with a fluorescent dye (TaqMan). It was found that mRNA deletion forms (a and b), products of alternative splicing of aENaC gene, are absent in the cortex of adult rat kidney.

[Fast nongenomic aldosterone effect on the kinetics of intracellular sodium and cell volume in the cortical part of collecting ducts of the rat kidney].

The fast nongenomic aldosterone effect on intracellular sodium ([Na+]i) and cell volume was studied by the fluorescent microscopy in isolated cortical part of collecting duct of the rat kidney (CCD). It was shown that aldosterone (10 nM) raised [Na+]i in hyposodium outer medium (14 mM). The rate of [Na+]i changes in response to external sodium shift (137-14 mM) twice as low in the presence of aldosterone (p < 0.05). Corticosterone (100 nM) was unable to simulate aldosterone effect. Similarly to sodium channel blocker amiloride (10(-5) M), protein kinase C (PKC) inhibitor RO-31-8220 (10(-7) M) abolished aldosterone effect. Aldosterone (10 nM) significantly decreased the amplitude and increased the characteristic time of the cell volume restoration in hypotonic medium of the rat principle cells (p < 0.001). Corticosterone (50 nM) was also unable to reproduce aldosterone effect. Amiloride (10(-5) M) did not significantly influence either the amplitude or the characteristic time of cell volume restoration during hypoosmotic challenge (p > 0.05). For the first time the specificity and important role of Ca(2+)-dependent kinase in the nongenomic aldosterone effects on ENaC activity and cell volume regulation in rat CCD were demonstrated.

Effect of aldosterone on kinetics of intracellular sodium in cortical portion of collecting ducts in rat kidney.

We studied the effect of protein kinase C inhibitor RO-31-8220 (10(-7) M) on rapid nongenomic effect of aldosterone in cells of isolated segment of distal region of collecting duct in rat kidney. Experiments with fluorescent dye Na-Green showed that the inhibitor abolished the modulating effect of aldosterone (10 nM) on intracellular sodium concentration at external sodium concentration of 14 mM. Aldosterone decreased by half the initial rate of the changes in internal sodium concentration in both 10-day and mature rats (p<0.05). Similarly to sodium channel blocker amiloride (10(-5) M), RO-31-8220 abolished rapid nongenomic effect of aldosterone on the rate of the changes in internal sodium concentration.

[Aldosterone: the mechanisms of molecular action].

Aldosterone: a steroid hormone of adrenal cortex, has recently attracted much interest not only due to its great importance in regulation of salt and water balance, but also because of its key role in therapy of cardiovascular and renal pathology. The classical genomic mechanism of molecular action of aldosterone is mediated through interaction with mineral-corticoid receptors. Fast nongenomic pathway of cell signal transduction begins with interaction with hypothetic membrane receptors and includes activation of different kinase cascades. Interference of these two pathways of signal transduction assures abroad spectrum of aldosterone effects depending on the cell type, and also secures multycomponent regulation depending on the need of specific functional and stress situation. This review is dedicated to modern views of mechanisms of aldosterone molecular action, mostly of the level of aldosterone-sensitive segment of kidney nephron.

[Developmental pecularuties of aldosterone regulation of rat epithelial Na channel expression and functional activity].

Developmental changes of mRNA alfa-subunit ENaC abundance in renal cortex of 10-day old and in adult rats and aldosterone (10 nM) influence on it were studied. The mRNA level was lower in young rat renal cortex and there was no aldosterone effect on it within 5 hours and 30 minutes, in contrast to adult rats. Intracellular sodium concentration [Na+ i] measured by fluorescent dye Na+ Green in CCD fragments micro-dissected from the kidneys was lower in fragments from immature kidneys, whereas fast nongenomic of aldosterone (10 nM) was the same at both ages. Aldosterone significantly raised [Na+ i] by 40 and 50% in conditions of low sodium concentration (14 mM) in outer medium in epithelial cells of both type of CCD fragments from 10-day old and adult animals, respectively (p < 0.05). We assume that heterochrony of fast and long time genomic effect takes place in aldosterone molecular mechanism maturation. Fast nongenomic effect on the [Na+ i] appeared earlier compared to delayed genomic effect of aldosterone mediated through the changes of the mRNA alpha-subunit ENaC abundance.

[Age-related specifics of aldosterone reception in distal segments of the rat nephrons and of Na(+), K(+)-ATPase expression induction]. / Vozrastnye osobennosti retseptsii al'dosterona v distal'nykh segmentakh nefronov krys i induktsii ékspressii Na(+), K(+)-ATFazy.

The reason of unresponsiveness of young 10-day rats kidney to aldosterone was explored. The aldosterone binding in distal segments of renal nephrons and the influence of hormonal induction on the mRNA of the alpha and beta subunits of the Na+, K(+)-ATPase in 10-day and 2-month old rats were investigated. There was no age related difference in the aldosterone specific binding in presence of RU-38486 (10(-7) M; Russel Uclaf) in the cortical collecting tubules: 0.26 +/- 0.04 (n = 9) and 0.22 +/- 0.03 (n = 8) mMol/mm of tubule lengths in 10 day and adult rats, respectively. By Nozern blot analysis and RT-PCR more then three and two fold increase of the mRNA abundance of both subunits was found in young and adult renal cortex compare to the adrenalectomized control after aldosterone induction (5 micrograms/100 g. v. b. w. 4 times i/p injections in 3 hour interval between injections) (p < 0.01). By RT-PCR no expression of the alpha 2, alpha 3 and beta 2 isoforms has been observed in all experimental conditions. The age difference was discovered when aldosterone was injected together with spironolactone (5 micrograms and 12 mg per 100 g. b. w. respectively). It was shown, that spironolactone inhibits the effect of aldosterone in adults whereas the latter was unaffected in young rats. The scheme of the age-related differences in the aldosterone regulation of the sodium pump induction in the target cell of the distal part of the rat nephrons is presented.

[The effect of the phosphorylation of the Na+,K(+)-ATPase alpha subunit in rat kidneys by protein kinase C on the activity of the enzyme]. / Vliianie fosforilirovaniia al'fa-sub''edinitsy Na+,K(+)-ATFazy pochek krys proteinkinazoi C na aktivnost' fermenta.

Phosphorylation was shown to lead to a change in the conformational equilibrium toward E1 form associated with a decrease in apparent affinity for the K+ in alpha-1 subunit of the rat kidney Na+, K(+)-ATPase. Rate of transition from E2 to E1 was apparently unaffected by phosphorylation. ATP hydrolysis by the protein kinase C-phosphorylated Na+, K(+)-ATPase shows a decrease in the Vmax and Km for K+.

Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.

[The age-related characteristics of the hormonal regulation of Na+, K(+)-ATPase activity in the kidney cortex of rats]. / Vozrastnye osobennosti gormonal'noi reguliatsii aktivnosti Na+, K(+)-ATFazy v kore pochek krys.

The aldosterone binding in isolated distal convoluted and cortical collecting tubules of renal nephrons and the influence of hormonal induction on the Na, K-ATPase activity in membrane fraction of kidney cortex were studied in 10-day- and 2-month-old rats. No reliable difference in aldosterone-specific binding was revealed (0.26 +/- 0.04 and 0.22 +/- 0.03 fmol/mm of tubule length, respectively, at the age of 10 days and 2 months). It was found that Na, K-ATPase activity increased with age from 0.39 +/- 0.06 to 0.72 +/- 0.10 mumol Pi/mg of protein.1 hour.100 microliters. Aldosterone induction caused approximately a 3-fold increase of the enzyme activity in both age groups comparing to the control level. Co-induction of aldosterone and spironolactone resulted in a 50% decrease of Na, K-ATPase activity in adult rats, but did not influence that in young rats. The revealed age-related differences in the mechanism of hormonal Na, K-ATPase regulation are supposed to underlie the absence of physiological reaction of the kidney to aldosterone in early postnatal ontogenesis.

[The effect of aldosterone on the second messenger systems in the rat kidney]. / Vliianie al'dosterona na sistemy vtorichnykh posrednikov v pochke krys.

The effect of aldosterone on the cAMP level, the state of inositol-phosphate pool and the intracellular free Ca2+ activity in the rat kidney, were studied. The cAMP level in the kidney cortex homogenate was not changed after i.p. injection of aldosterone. Reliable changes in the content of inositol-phosphates in the kidney cell suspension was not revealed after aldosterone treatment. A short-term enhancement of the intracellular Ca2+ activity in the isolated distal convoluted and cortical collecting tubules under the influence of aldosterone, was revealed. The participation of the second messenger systems in the realisation of aldosterone effects in kidney and the role of Ca2+ in the mechanism of aldosterone action, are discussed.

[The hormonal regulation of mRNA Na,K-ATPase expression in rat kidneys in postnatal ontogeny]. / Gormonal'naia reguliatsiia ékspressii mRNK Na,K-ATFazy v pochkakh v postnatal'nom ontogeneze.

A study was made of effects of aldosterone, aldosterone+dexamethasone, and aldosterone+spironolactone on Na,K-ATPase mRNA expression in renal cortex of adult and 10 day old rats, when kidney is not sensitive to the hormone injection. It is shown that hormonal induction of synthesis of Na,K-pump mRNA occurs in the early postnatal period apart from mineral corticoid receptors. It seems probable that aldosterone exerts its action in 10 day old rats by interaction with glucocorticoid receptors inducing synthesis of different amounts of alpha- and beta-subunits of Na,K-ATPase.

[Aldosterone reception by a kidney membrane fraction and cytosol from rats in postnatal ontogeny]. / Retseptsiia al'dosterona fraktsiei membran i tsitozolem pochek krys v postnatal'nom ontogeneze.

Specific binding of aldosterone by crude membrane fraction, the affinity and maximal concentration of cytosolic receptors of aldosterone were studied in the kidney cortex of the Wistar rats as well as the plasma level and adrenal biosynthesis of aldosterone. The specific binding of aldosterone was found to increase in membrane fraction and to decrease in cytosol during kidney maturation. The affinity of the cytosol receptors to aldosterone did not change in the same period. The level of biosynthesis in adrenals and plasma concentration of aldosterone was higher in adult rather than in young rats. The membrane binding of aldosterone seems to take part in the mechanism of cell sensitivity to aldosterone.

[Changes in the receptor link during the development of renal sensitivity to aldosterone and antidiuretic hormone]. / Izmeneniia retseptoronogo zvena v protsesse razvitiia chuvstvitel'nosti pochki k al'dosteronu i antidiureticheskomu gormonu.

The aldosterone, cAMP and ADH receptors were studied in kidneys of 10-14- and 60-day old rats. Concentrations of aldosterone and cAMP receptors were reduced, while ADH binding was increased during the period of maturation of kidney functions. The investigation of the affinity and molecular weights of cAMP and ADH receptors suggests their complex nature and altering of their individual properties during ontogenesis. Aldosterone and cAMP with their receptors seem to participate in the regulation of gene activity in ontogenesis and ADH seems to participate in the formation of its own receptors.