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In the course of adaptation of the rat kidney collecting duct cells to hypo-osmotic medium, the organic anion transporter inhibitor probenecid reduced significantly the regulatory cell volume decrease in response to a hypotonic shock. Both probenecid and hypotonic shock delayed significantly the entry into a cell of the fluorescent dye calcein, which exists as anion at neutral pH. Thus, the organic osmolyte transport plays an important role in the regulatory decrease of the principal cell volume under the hypo-osmotic conditions.

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Senescence-associated alterations in structure and function of cornea make it more sensitive to traumas and disease. Trauma of cornea leads to edema and vision impairment and only corneal transplantation remains the effective for vision correction. Role of aquaporins for cornea endothelium function, as well as age-related changes of their activity, are not entirely understood. Herein, we studied changes with age the water permeability (Pf) of the plasma membranes of the corneal endothelial cells and the level of expression in them of the mRNA genes of the water channels of the aquaporins AQP1 and AQP3 in Wistar and senescence-accelerated OXYS rats. From the age of 3 to 18 months, Pf in Wistar rats increased, in OXYS - decreased and was twice lower than in Wistar rats. The expression of aqp1 mRNA (studied by RT-PCR) in the endothelium was the same in Wistar and OXYS rats at the age of 3 months. By the age of 18 months, it increased only in Wistar rats and became twice higher than in OXYS rats. The expression of aqp3 mRNA in the endothelium of 3-month-old OXYS rats was half that of Wistar rats and did not change with age, while in Wistar rats it decreased and at 18 months was 4 times lower than in 3 months. We supposed that increased water permeability of endothelial cells in Wistar rats is adaptive and compensates for the decrease in endothelial cell density with age, while the accelerated aging of OXYS rats abolishes this compensation.

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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.

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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.

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This paper presents the new priority data on the effect of genetically determined deficiency of endogenous vasopressin on the functioning of V2 receptor in the plasma membrane of the principal cells of the collection ducts of Brattleboro rats. It was found that 2-day desmopressin administration results in the activation of V2 receptor gene expression in Brattleboro rat, whereas in Wistar rats the content of mRNA did not change. In addition, short-term exposure of the collection ducts of the kidneys to the hormone led to V2 receptor internalization from the plasma membrane in Wistar rats, but had no effect on the protein content in Brattleboro rats.

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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.

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The regulatory decrease in the volume of principal cells of collecting ducts to hypoosmotic shock has been investigated experimentally and using the mathematical modeling. A mathematical model of the response of collecting duct principal cells to hypotonic shock has been constructed on the basis of the experimental time course of changes in cell volume measured by the fluorescent dye Calcein. It was shown that the regulatory decrease in volume under hypotonic conditions occurs via a marked release of osmolytes and is accompanied by a decrease in water permeability of the cell membrane. The mathematical modeling of transmembrane transport processes allowed us to quantitatively estimate the changes in membrane water permeability, which decreased tenfold, from 2 x 10(-1) cm/s to 2 x 10(-2) cm/s. It was also shown that the effective regulatory decrease in the volume of collecting duct principal cells in hypotonic medium results from a significant increase in membrane permeability for K+, Cl-, and organic anions.

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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.

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The kidney of immaturely born mammals in early postnatal development is insensitive to the effect of the antidiuretic hormone, vasopressin. It has been demonstrated that water permeability of the epithelial cells in the collecting ducts of a rat kidney increases during development; in this process, the response to desmopressin, an agonist of vasopressin V2 receptors, appears at the age of 20 days. The observed increase in water permeability is connected with an increased content of the water channel's proteins aquaporins AQP2 and AQP3 in the plasma membrane. The calcium-dependent protein kinase C isoforms are the likely components of the vasopressin signal's transduction and are possibly involved in the mechanisms underlying the maturation of sensitivity to this hormone. The contents of three protein kinase C isoforms (alpha, delta, and zeta) in rats at different periods of their postnatal development were estimated using Western blot hybridization. It has been shown that the contents of protein kinase C isoforms alpha and delta increase with development, whereas the content of isoform zeta remains constant. The most likely participant of the mechanism providing for maturation of the cell's hormonal competence for vasopressin is the calcium-dependent protein kinase Ca, because it's content in the plasma membrane is maximal on days 20-24, which coincides with the time when the vasopressin action appears.

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The effect of plasma membrane water permeability on the rate of changes in the volume of principal cells of collecting ducts of the outer substantia medullaris under conditions of hypoosmotic shock has been studied. Changes in cell volume were studied by the fluorescent method. It was shown that the hypotonic shock induced a rapid increase in the cell volume with the characteristic time that depended on plasma membrane water permeability. The decrease in volume occurred much more slowly, and the rate of volume decrease directly correlated with the rate of swelling. The inhibition of potassium transport by barium chloride decreased the rate of volume restoration, without affecting substantially the duration of the swelling phase. The inhibition of mercury-sensitive water channels by mercury caused a significant increase in the time of both cell swelling and volume restoration. It was concluded that the state of water channels largely determines the rate of the regulatory response of epithelial cells of collecting ducts to hypoosmotic shock and affects the exchange of cell osmolites.

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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.

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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.

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Principal mechanism of the transepithelial water permeability increase in the kidney collecting ducts in response to vasopressin involves insertion of aquaporin 2 (AQP2) into the apical membrane. Previously we have shown that water permeability of the basolateral membrane also may be increased with stimulation of V2-receptors. It is known that inhibition of G(i)-proteins with pertussis toxin blocks redistribution of AQP2 into the apical membrane following the application of vasopressin or forskolin. The aim of the present study was to investigate potential involvement of G(i)-proteins in regulation of basolateral membrane water permeability. Effect of pertussis toxin on the ability of desmopressin to increase the basolateral membrane osmotic water permeability was investigated, and the expression of Galpha(i)2 and Galpha(i)3 genes under normal conditions and after 2 days of water deprivation were evaluated. We demonstrated that dehydration leds to a 30% increase of Galpha(i)3 mRNA content while the Galpha(i)2 mRNA level remains unchanged. In control experiments, basolateral membrane water permeability increased in response to desmopressin from 59.2 +/- 6.61 to 70.6 +/- 9.2 microm/s (p < 0.05, paired t-test). Pertussis toxin completely blocked this reaction (53.5 +/- 5.18 vs 50.1 +/- 6.50 microm/s, respectively). We conclude that G(i)-proteins participate in the mechanism of the basolateral membrane water permeability increase in response to stimulation of V2-receptors. Clarification of the G(i)-proteins role in this process requires further investigation, but most likely they are involved in regulation of aquaporin transport and insertion into the cell membrane.

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In the study, the role of PKC and Ca++ in vasopressin regulation of the plasma membrane water permeability was studied in the cells of the mouse kidney collecting duct. Coefficient of osmotic water permeability of total cell surface (Pf) was calculated from the initial rate of cell swelling following the osmotic shock caused by changing the medium osmolarity from isotonic to hypotonic (300 mOsm to 200 mOsm). Desmopressin (dDAVP 1 nM) increased the Pf in hydrated mice from 168.4 +/- 11.8 microm/s up to 231.3 +/- 14.7 microm/s. The Ca++ chelator BAPTA prevented the desmopressin-induced increase in water permeability. Inhibition of PKC (Ro-31-8220 0.1 microM) also abolished the desmopressin-stimulated increase of plasma membrane water permeability, whereas inhibitor of PKC alone did not suppress the stimulation of the water permeability by db-cAMP. The PKC activity and calciumdependent second messengers seem to be important for regulation of water permeability by vasopressin.

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Intracellular sodium concentration in CCD fragments micro-dissected from the kidneys of 10-day and adult rats using fluorescent dye Na+ Green, was studied. The steady state level of intracellular sodium was lower in fragments from amiloride kidney (24.50 +/- 1.3 and 35.3 +/- 4.9 mM, n = 8, respectively). Amiloride reduced this parameter but, in the epithelia cells from immature kidney, the effect of imaloride was less pronounced. Fast nongenomic action of aldosterone on intracellular sodium concentration was found in both groups. Aldosterone significantly raised the steady state sodium level in epithelial cells in conditions of low sodium concentration (14 mM) in outer medium in both type of CCD fragments: from 10-day pups and adult animals (4.0 +/- 1, 5.5 +/- 0.5 and 5.1 +/- 0.2, 7.9 +/- 0.2 mM, n = 8). We suggest that, along with the well-known mineral-corticoid effect, aldosterone takes part in regulation of cell volume in CCD rat kidney from the earliest stages of postnatal ontogenesis.

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