A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy

1998 ◽  
Vol 274 (5) ◽  
pp. C1245-C1252 ◽  
Author(s):  
David J. Morris ◽  
Syed A. Latif ◽  
Michael D. Rokaw ◽  
Charles O. Watlington ◽  
John P. Johnson
Keyword(s):  
Glucocorticoid Receptors ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Mineralocorticoid Receptors ◽  
Hydroxylase Activity ◽  
A6 Cells ◽  
Ru 28318 ◽  
The One ◽  
Cytochrome P 450 ◽  
Significant Increment

We have confirmed that A6 cells (derived from kidney of Xenopus laevis), which contain both mineralocorticoid and glucocorticoid receptors, do not normally possess 11β-hydroxysteroid dehydroxgenase (11β-HSD1 or 11β-HSD2) enzymatic activity and so are without apparent “protective” enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6β-hydroxylase that transforms corticosterone to 6β-hydroxycorticosterone. This hydroxylase is cytochrome P-450 3A (CYP3A). We have now determined the effects of 3α,5β-tetrahydroprogesterone and chenodeoxycholic acid (both inhibitors of 11β-HSD1) and 11-dehydrocorticosterone and 11β-hydroxy-3α,5β-tetrahydroprogesterone (inhibitors of 11β-HSD2) and carbenoxalone, which inhibits both 11β-HSD1 and 11β-HSD2, on the actions and metabolism of corticosterone and active Na+ transport [short-circuit current ( I sc)] in A6 cells. All of these 11β-HSD inhibitory substances induced a significant increment in corticosterone-induced I sc, which was detectable within 2 h. However, none of these agents caused an increase in I sc when incubated by themselves with A6 cells. In all cases, the additional I sc was inhibited by the mineralocorticoid receptor (MR) antagonist, RU-28318, whereas the original I scelicited by corticosterone alone was inhibited by the glucocorticoid receptor antagonist, RU-38486. In separate experiments, each agent was shown to significantly inhibit metabolism of corticosterone to 6β-hydroxycorticosterone in A6 cells, and a linear relationship existed between 6β-hydroxylase inhibition and the MR-mediated increase in I scin the one inhibitor tested. Troleandomycin, a selective inhibitor of CYP3A, inhibited 6β-hydroxylase and also significantly enhanced corticosterone-induced I sc at 2 h. These experiments indicate that the enhanced MR-mediated I sc in A6 cells may be related to inhibition of 6β-hydroxylase activity in these cells and that this 6β-hydroxylase (CYP3A) may be protecting the expression of corticosterone-induced active Na+ transport in A6 cells by MR-mediated mechanism(s).

Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors

1993 ◽  
Vol 264 (4) ◽  
pp. C875-C884 ◽  
Author(s):  
T. J. Schmidt ◽  
R. F. Husted ◽  
J. B. Stokes
Keyword(s):  
Cell Line ◽  
Glucocorticoid Receptors ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Mineralocorticoid Receptors ◽  
Na Transport ◽  
Receptor Complexes ◽  
Binding Forms ◽  
Mineralocorticoid Antagonists

The A6 cell line derived from the toad kidney forms polarized, highly differentiated epithelial monolayers in culture and has been utilized as an experimental model for studying regulation of transepithelial Na+ transport by aldosterone. In the present study we evaluated the specific role(s) of glucocorticoid and mineralocorticoid receptors in mediating this enhanced electrogenic Na+ transport, which was measured experimentally as an increase in short-circuit current (Isc). Our data demonstrate that specific glucocorticoid agonists (100 nM), including RU 28362 and RU 26988, elicit “mineralocorticoid-like” increases in Isc that are blocked by the glucocorticoid antagonist RU 38486 but are unaffected by mineralocorticoid antagonists including RU 28318 and RU 26752. The stimulatory effects of aldosterone (100 nM) were also blocked by RU 38486 and not by mineralocorticoid antagonists. These data extend earlier studies suggesting that in this cell line aldosterone mediates its physiological effects via binding with relatively low affinity (dissociation constant Kd congruent to 25-50 nM) to glucocorticoid receptors, despite the presence of apparently normal mineralocorticoid receptors. Our in vitro biochemical studies also demonstrate that A6 glucocorticoid receptor complexes can be thermally activated or transformed to DNA binding forms which exhibitaltered elution profiles from anion-exchange resins. Thus, based on several criteria, these amphibian glucocorticoid receptors appear very similar to classical mammalian receptors and are capable of mediating all of the stimulatory effects of aldosterone on net Na+ transport.

Action of adenosine on chloride active transport of isolated frog cornea

1979 ◽  
Vol 237 (2) ◽  
pp. F121-F127
Author(s):  
B. S. Spinowitz ◽  
J. A. Zadunaisky
Keyword(s):  
Rana Catesbeiana ◽  
Chloride Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Adenyl Cyclase ◽  
Naturally Occurring ◽  
Chloride Pump ◽  
Net Flux ◽  
The One ◽  
Sustained Increase

Addition of adenosine (10–7 to 10–4 M) to the tear side of isolated corneas (Rana catesbeiana) produced a rapid, sustained increase in short-circuit current, potential difference, and radioisotopic chloride net flux. The increased net chloride flux accounted for the increased short-circuit current. Adenosine, a known activator of adenyl cyclase in other tissues, exerted its effects on chloride transport through a receptor different from the one described for epinephrine and prostaglandins in the corneal epithelium. Propranolol inhibited the epinephrine response but not the adenosine effect. Dipolyphloretin phosphate inhibited prostaglandin responses but did not affect the adenosine stimulation of chloride transport. Adenine and/or ribose, parts of the adenosine molecule, had no stimulatory effect, but 5'-AMP had a partial effect.The activation of the chloride pump with DBcAMP blocked the response to adenosine. Adenosine interacted with the effects of theophylline. Adenosine, a naturally occurring molecule, stimulated chloride transport by activation of adenyl cyclase through a separate membrane receptor in the corneal eqithelium.

Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

1986 ◽  
Vol 251 (2) ◽  
pp. C186-C190 ◽  
Author(s):  
J. P. Johnson ◽  
D. Jones ◽  
W. P. Wiesmann
Keyword(s):  
Enzyme Activity ◽  
Epithelial Cells ◽  
Atpase Activity ◽  
Hormonal Regulation ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Urinary Bladder ◽  
A6 Cells ◽  
Cultured Epithelial Cells ◽  
Stimulation Of

Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. We examined the effect of aldosterone and insulin on Na+-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (ISC) in TB6C cells. Aldosterone increases Na+-K+-ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in ISC, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase ISC in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na+-K+-ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na+ entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na+-K+-ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on ISC.

Photoelectrical and Photovoltaic Peroperties of n-ZnO/p-Si Heterojunction

2011 ◽  
Vol 399-401 ◽  
pp. 1477-1480
Author(s):  
Yan Li Xu ◽  
Jin Hua Li
Keyword(s):  
Solar Cell ◽  
Silicon Solar Cell ◽  
Ion Beam ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Open Circuit ◽  
Textured Surface ◽  
Si Solar Cell ◽  
The One ◽  
P Type

n-ZnO thin films doped In with 2 atm.% were deposited on p-type silicon wafer with textured surface by Ion Beam Enhanced Deposition method, after annealing and prepared front and back electrodes, the n-ZnO/p-Si heterojunction samples were fabricated. The photoelectric property of the sample were measured and compared with silicon solar cell. The result indicated the saturated photocurrent of n-ZnO/p-Si heterojunction was 20% greater than one of the Si solar cell. It means the ZnO/Si heterojunction has a higher ability of produce photoelectron then one of silicon solarcell. The result of the photovoltaic test of n-ZnO/p-Si heterojunction show The open circuit voltage and short-circuit current of the n-ZnO/p-Si heterojunction was 400mV and 5.5mA/cm2 respectively. It was much smaller than the one of silicon solar cells. The reason was discussed

Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells

2003 ◽  
Vol 284 (2) ◽  
pp. C404-C414 ◽  
Author(s):  
Diego Alvarez de la Rosa ◽  
Cecilia M. Canessa
Keyword(s):  
Sodium Channel ◽  
Hormonal Regulation ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Epithelial Sodium Channel ◽  
Hormonal Stimulation ◽  
A6 Cells ◽  
Epithelial Sodium Channel Enac

The purpose of this study was to examine the role of the serum- and glucocorticoid-induced kinase (SGK) in the activation of the epithelial sodium channel (ENaC) by aldosterone, arginine vasopressin (AVP), and insulin. We used a tetracycline-inducible system to control the expression of wild-type (SGK[Formula: see text]), constitutively active (S425D mutation; SGK[Formula: see text]), or inactive (K130M mutation; SGK[Formula: see text]) SGK in A6 cells independently of hormonal stimulation. The effect of SGK expression on ENaC activity was monitored by measuring transepithelial amiloride-sensitive short-circuit current ( I sc) of transfected A6 cell lines. Expression of SGK[Formula: see text] or SGK[Formula: see text] and aldosterone stimulation have additive effects on I sc. Although SGK could play some role in the aldosterone response, our results suggest that other mechanisms take place. SGK[Formula: see text] abrogates the responses to AVP and insulin; hence, in the signaling pathways of these hormones there is a shared step that is stimulated by SGK. Because AVP and insulin induce fusion of vesicles to the apical membrane, our results support the notion that SGK promotes incorporation of channels in the apical membrane.

On the natriuretic effect of verapamil: inhibition of ENaC and transepithelial sodium transport

2002 ◽  
Vol 283 (4) ◽  
pp. F765-F770 ◽  
Author(s):  
Alan S. Segal ◽  
John P. Hayslett ◽  
Gary V. Desir
Keyword(s):  
Short Circuit ◽  
Ussing Chamber ◽  
Surrogate Marker ◽  
Distal Tubule ◽  
Short Circuit Current ◽  
Channel Blockers ◽  
A6 Cells ◽  
Apical Side ◽  
Basolateral Side ◽  
Natriuretic Effect

The natriuretic effect of Ca2+ channel blockers has been attributed to hemodynamic changes and to poorly defined direct tubular effects. To test the possibility that verapamil may inhibit Na+ reabsorption at the distal tubule, its effect on transepithelial Na+transport in aldosterone-stimulated A6 cells was determined. Cells were grown on permeable supports, and short-circuit current ( I sc) measured in an Ussing chamber was used as a surrogate marker for transepithelial Na+ transport. Application of 300 μM verapamil to the apical side inhibited I sc by 77% and was nearly as potent as 100 μM amiloride, which inhibited I sc by 87%. Verapamil-induced inhibition of I sc was accompanied by a significant increase in transepithelial resistance, suggesting blockade of an apical conductance. Its action on transepithelial Na+ transport does not appear to occur through inhibition of L-type Ca2+ channels, since I sc was unaffected by removal of extracellular Ca2+. Verapamil also does not appear to inhibit I sc by modulating intracellular Ca2+stores, since it fails to inhibit transepithelial Na+transport when added to the basolateral side. The effect on Na+ transport is specific for verapamil, since nifedipine, Ba2+, 4-aminopyridine, and charybdotoxin do not significantly affect I sc. A direct effect of verapamil on the epithelial Na+ channel (ENaC) was tested using oocytes injected with the α-, β-, and γ-subunits. We conclude that verapamil inhibits transepithelial Na+transport in A6 cells by blocking ENaC and that the natriuresis observed with administration of verapamil may be due in part to its action on ENaC.

scholarly journals Epoxyeicosatrienoic acids affect electrolyte transport in renal tubular epithelial cells: dependence on cyclooxygenase and cell polarity

2007 ◽  
Vol 293 (1) ◽  
pp. F288-F298 ◽  
Author(s):  
Rolf M. Nüsing ◽  
Horst Schweer ◽  
Ingrid Fleming ◽  
Darryl C. Zeldin ◽  
Markus Wegmann
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Electrolyte Transport ◽  
Epoxyeicosatrienoic Acids ◽  
Tubular Cells ◽  
Luminal Side ◽  
Chloride Channel Blocker ◽  
Renal Tubular ◽  
Cytochrome P 450

We investigated the effects of epoxyeicosatrienoic acids (EETs) on ion transport in the polarized renal distal tubular cell line, Madin-Darby canine kidney (MDCK) C7. Of the four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) studied, only apical, but not basolateral, application of 5,6-EET increased short-circuit current ( Isc) with kinetics similar to those of arachidonic acid. The ion transport was blocked by preincubation with the cyclooxygenase inhibitor indomethacin or with the chloride channel blocker NPPB. Furthermore, both a Cl−-free bath solution and the Ca2+ antagonist verapamil blocked 5,6-EET-induced ion transport. Although the presence of the PGE2 receptors EP2, EP3, and EP4 was demonstrated, apically added PGE2 was ineffective and basolaterally added PGE2 caused a different kinetics in ion transport compared with 5,6-EET. Moreover, PGE2 sythesis in MDCK C7 cells was unaffected by 5,6-EET treatment. GC/MS/MS analysis of cell supernatants revealed the presence of the biologically inactive 5,6-dihydroxy-PGE1 in 5,6-EET-treated cells, but not in control cells. Indomethacin suppressed the formation of 5,6-dihydroxy-PGE1. 5,6-Epoxy-PGE1, the precursor of 5,6-dihydroxy-PGE1, caused a similar ion transport as 5,6-EET. Cytochrome P-450 enzymes homolog to human CYP2C8, CYP2C9, and CYP2J2 protein were detected immunologically in the MDCK C7 cells. Our findings suggest that 5,6-EET affects Cl− transport in renal distal tubular cells independent of PGE2 but by a mechanism, dependent on its conversion to 5,6-epoxy-PGE1 by cyclooxygenase. We suggest a role for this P450 epoxygenase product in the regulation of electrolyte transport, especially as a saluretic compound acting from the luminal side of tubular cells in the mammalian kidney.

Glucocorticoid regulation of genes in the amiloride-sensitive sodium transport pathway by semicircular canal duct epithelium of neonatal rat

2006 ◽  
Vol 24 (2) ◽  
pp. 114-123 ◽  
Author(s):  
Satyanarayana R. Pondugula ◽  
Nithya N. Raveendran ◽  
Zuhal Ergonul ◽  
Youping Deng ◽  
Jun Chen ◽  
...  
Keyword(s):  
Semicircular Canal ◽  
Glucocorticoid Receptors ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Neonatal Rat ◽  
Transcript Expression ◽  
Transport Pathway ◽  
Sulfonyl Urea ◽  
Inwardly Rectifying Potassium Channels ◽  
Inwardly Rectifying

The lumen of the inner ear has an unusually low concentration of endolymphatic Na+, which is important for transduction processes. We have recently shown that glucocorticoid receptors (GR) stimulate absorption of Na+ by semicircular canal duct (SCCD) epithelia. In the present study, we sought to determine the presence of genes involved in the control of the amiloride-sensitive Na+ transport pathway in rat SCCD epithelia and whether their level of expression was regulated by glucocorticoids using quantitative real-time RT-PCR. Transcripts were present for α-, β-, and γ-subunits of the epithelial sodium channel (ENaC); the α1-, α3-, β1-, and β3-isoforms of Na+-K+-ATPase; inwardly rectifying potassium channels [IC50 of short circuit current ( Isc) for Ba2+: 210 μM] Kir2.1, Kir2.2, Kir2.3, Kir2.4, Kir3.1, Kir3.3, Kir4.1, Kir4.2, Kir5.1, and Kir7.1; sulfonyl urea receptor 1 (SUR1); GR; mineralocorticoid receptor (MR); 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2; serum- and glucocorticoid-regulated kinase 1 (Sgk1); and neural precursor cell-expressed developmentally downregulated 4-2 (Nedd4-2). On the other hand, transcripts for the α4-subunit of Na+-K+-ATPase, Kir1.1, Kir3.2, Kir3.4, Kir6.1, Kir6.2, and SUR2 were found to be absent, and Isc was not inhibited by glibenclamide. Dexamethasone (100 nM for 24 h) not only upregulated the transcript expression of α-ENaC (∼4-fold), β2-subunit (∼2-fold) and β3-subunit (∼8-fold) of Na+-K+-ATPase, Kir2.1 (∼5-fold), Kir2.2 (∼9-fold), Kir2.4 (∼3-fold), Kir3.1 (∼ 3- fold), Kir3.3 (∼2-fold), Kir4.2 (∼3-fold ), Kir7.1 (∼2-fold), Sgk1 (∼4-fold), and Nedd4-2 (∼2-fold) but also downregulated GR (∼3-fold) and 11β-HSD1 (∼2-fold). Expression of GR and 11β-HSD1 was higher than MR and 11β-HSD2 in the absence of dexamethasone. Dexamethasone altered transcript expression levels (α-ENaC and Sgk1) by activation of GR but not MR. Proteins were present for the α-, β-, and γ-subunits of ENaC and Sgk1, and expression of α- and γ-ENaC was upregulated by dexamethasone. These findings are consistent with the genomic stimulation by glucocorticoids of Na+ absorption by SCCD and provide an understanding of the therapeutic action of glucocorticoids in the treatment of Meniere's disease.

Influence of filter supports on transport characteristics of cultured A6 kidney cells

1993 ◽  
Vol 265 (6) ◽  
pp. C1479-C1488 ◽  
Author(s):  
O. Candia ◽  
A. J. Mia ◽  
T. Yorio
Keyword(s):  
Amphotericin B ◽  
Water Flow ◽  
Cultured Cells ◽  
Short Circuit ◽  
Tritiated Water ◽  
Short Circuit Current ◽  
Kidney Cells ◽  
Scanning Electron Micrographs ◽  
Apical Surface ◽  
A6 Cells

Amphibian A6 kidney cells grown on Anocell filters developed a transepithelial potential difference of 37 mV, a short-circuit current (Isc) of 8 microA/cm2, and a resistance of 5 k omega.cm2. Other observations suggested a viable arginine vasopressin (AVP) V2 receptor-second messenger pathway in these cells: 1) AVP increased both an amiloride-sensitive Isc and adenosine 3',5'-cyclic monophosphate (cAMP) formation, and 2) scanning electron micrographs of A6 cells cultured on Anocell and ICN Cellagen filters demonstrated increased microvilli formation on the apical surface after AVP action. However, osmotic water flow (JV) across A6 cells on filter supports was not altered by either AVP or the permeable cAMP analogue dibutyryl cAMP (osmotic permeability coefficient = 2.5 x 10(-3) cm/s). Diffusional water flow (Jdw) measured across A6 cells on Anocell filters using tritiated water (THO) ranged from 6 to 8 microliters.min-1.cm-2. Neither AVP nor the membrane-permeabilizing agents amphotericin B and digitonin were able to enhance unidirectional THO fluxes, although amphotericin B increased the Isc. These results suggested that there was an unknown barrier in series with the A6 cells limiting water flow. THO fluxes across filter supports, without an associated cellular monolayer, gave Jdw values in the range 7-30 microliters.min-1.cm-2. Jv across the bare filter support was in the range of 0.3-1.5 microliters.min-1.cm-2, similar to that measured in the presence of an A6 monolayer. These observations suggest that the filter may be rate limiting for transepithelial water flow. Chloride fluxes across Anocell filters showed a stable value of 5 mu eq.h-1.cm-2. These observations exhibit the limitations of filter supports in the study of transport phenomena in cultured cells.

Analysis, Design and Prototyping of a Low-Speed High-Torque Six-Phase Fault-Tolerant Permanent Magnet Synchronous Machine for EVs

2013 ◽  
Vol 416-417 ◽  
pp. 66-72
Author(s):  
Ping Zheng ◽  
Fan Wu ◽  
Yi Sui ◽  
Peng Fei Wang ◽  
Bin Yu
Keyword(s):  
Permanent Magnet ◽  
Fault Tolerant ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Synchronous Machine ◽  
Permanent Magnet Synchronous Machine ◽  
Low Speed ◽  
Analysis Design ◽  
High Torque ◽  
The One

This paper deals with a low-speed high-torque six-phase fault-tolerant permanent magnet synchronous machine (PMSM) for wheel-driving electric vehicle (EV) applications. In machine design, winding arrangements and feasible slot/pole combinations are discussed and compared; a 24-slot/22-pole alternate-teeth-wound scheme is analyzed and designed. With reinforced slot-leakage component, the inductance of the machine is increased to restrain the one-phase short-circuit current to nearly 1.0 per unit preventing the machine from deteriorations in condition of that fault. The 24-slot/22-pole alternate-teeth-wound prototype machine is manufactured and the experimental verification is provided.

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