Biosynthesis of the catalytic subunit of (Na+, K+)-ATPase in toad kidney and toad bladder epithelial cells.
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Animals↗
Biomedical subjects
B C Rossier
Publications and source records attributed to B C Rossier.
Immunochemical evidence for a transmembrane orientation of both the (Na+, K+)-ATPase subunits.
Antibodies were raised against the large catalytic subunit (apparent Mr 96000) and the glycoprotein (apparent Mr 60000) of the sodium- and potassium-dependent adenosine triphosphatase [(Na+, K+)-ATPase] from Bufo marinus. The specificity of each antiserum was assessed by two-dimensional immunoelectrophoresis using toad kidney microsomes or the purified holoenzyme as a source of antigen and by indirect immunoprecipitation of detergent-solubilized (Na+, K+)-ATPase subunits from radioiodinated or biosynthetically labeled kidney holoenzyme, microsomes, or postnuclear supernatant. The anticatalytic subunit serum reacted exclusively with a 96000-dalton protein. The antiserum to the glycoprotein was rendered specific to this subunit by absorption with purified catalytic subunit. The two antisera were agglutinating and lytic in the presence of complement when toad erythrocytes were used as targets, indicating that antigenic determinants of both subunits were exposed on the cell surface. The specific reactivities with surface-exposed antigenic determinants of both subunits could be absorbed with toad red blood cells. Such absorbed antisera still reacted with detergent-treated or untreated kidney microsomes, revealing the presence of cytoplasmic and/or intramembranous antigenic sites. Our immunochemical data demonstrate that the glycoprotein subunit of (Na+, K+)-ATPase spans the lipid bilayer and confirm the transmembrane orientation of the catalytic subunit postulated from functional studies.
Animals↗
Thyroid hormone-aldosterone antagonism on Na+ transport in toad bladder. Evidence for a triiodothyronine nuclear receptor.
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Aldosterone↗
Inhibition of the anti-natriuretic action of aldosterone by thyroid hormone in the rat.
Urinary sodium excretion was measured in adrenalectomized, euthyroid rats, within 4 hours after injection of aldosterone and/or thyroid hormone (T3). Rats which received both hormones excreted significantly more Na than rats injected with aldosterone alone. Reverse-T3 did not affect the antinatriuretic action of aldosterone. Natriuresis following injection of T3 alone was similar to that measured in the absence of exogenous hormones. These observations suggest an interaction between aldosterone and T3 on renal Na excretion.
Aldosterone↗
Testosterone: a specific competitive antagonist of aldosterone in the toad bladder.
Testosterone (100 nM to 40 microM) antagonized the effect of aldosterone (10 nM) on Na+ transport in the toad bladder measured in vitro as short-circuit current (SCC). Half-maximal inhibition occurred at an antagonist-agonist molar ratio of 150:1. The antagonist action of testosterone was reversed by addition of more aldosterone. The antagonism was specific in the sense that testosterone (20 microM) did not inhibit the response of the SCC to oxytocin (50 mU/ml). By itself, testosterone (up to 20 microM) had no agonist activity on base-line SCC. Finally, testosterone (500 nM to 20 microM) specifically displaced [3H]aldosterone (5 nm) from its cytoplasmic and nuclear binding sites in bladders incubated in vitro at 25 or 0 degrees C and labeled at steady state. There was a significant linear correlation between the effect of testosterone on the aldosterone-dependent SCC and its effect on [3H]aldosterone binding sites in the cytoplasm and in the nucleus. We conclude that 1) testosterone is a specific competitive antagonist of aldosterone, and 2) [3H]aldosterone nuclear and cytoplasmic binding sites could be mineralocorticoid receptors, mediating the action of aldosterone on Na+ transport.
Aldosterone↗
Isolation and separation of toad bladder epithelial cells.
The epithelium of the urinary bladder of Bufo marinus is composed of 5 cell types, i.e., granular (Gr), mitochondria-rich (MR) and goblet (G) cells which face the urinary lumen, microfilament-rich (MFR) and undifferentiated cells (Un) located basally. The epithelium was dissociated by collagenase and EGTA treatment. Fractionation of dispersed cells by isopycnic centrifugation on dense serum albumin solutions yielded 4 fractions: (i) a very light fraction (p approximately equal to 1.025) enriched in MR and MFR cells; (ii) a light fraction (p approximately equal to 1.045) enriched in vacuolated Gr cells; (iii) a heavy fraction (p approximately equal to 1.065) composed essentially of aggregated Gr cells, and (iv) a pellet (p approximately equal to 1.085) enriched in G and undifferentiated cells. Recoveries were based on cell counts and DNA measurements. DNA content per cell was 13.2 pg +/- 0.9 (n = 37). From 1 g fresh tissue, 62 +/- 5 x 10(6) (n = 10) cells were recovered before isopycnic centrifugation of which about 70% excluded Trypan blue. After centrifugation, 90 to 95% of the cells excluded the vital dye and approximately 3(9) x 10(6) cells were recovered from the gradient. Cell metabolism in each fraction was estimated by oxygen consumption measurements in absence or presence of ouabain, acetazolamide, and dinitrophenol. The consumption measurements in absence or presence of ouabain, acetazolamide, and dinitrophenol. The consumption was threefold higher in the very light and light fractions when compared to the heavy and pellet fractions. Ouabain sensitive oxygen consumption (QO2) represented 12 to 35% of the total O2 consumption depending on the cell fraction, and acetazolamide sensitive QO2 varied from -0.8% in the heavy fractions to 20% in the lighter fractions. DNP increased QO2 in all fractions by 20 to 50%. Finally, the cells were able to reaggregate and form junctional complexes upon addition of calcium to the medium.
Animals↗
Microfilament-rich cells in the toad bladder epithelium.
Basal cells of the bladder epithelium of Bufo marinus have been found heterogenous and consist of microfilament-rich cells (MFR-cell) and undifferentiated cells (Un-cell). The MFR-cell, which represents approximately 20% of the epithelial cell population, lies between the epithelial layer lining the urinary space and the basement membrane; it extends under several epithelial cells by processes of varying widths and lengths which contact, via desmosomes, other MFR-cells, as well as cells in the superficial layer, i.e., granular and mitochondria-rich cells. The cytoplasm of MFR-cell is filled with intermediate filaments arranged in bundles which run parallel to the plane of the epithelium and no dense granules, typical of granular cells, have been detected. Strong immunofluorescence for actin is associated with cells which occupy the same basal position as MFR-cells. Undifferentiated cells have no contact via desmosomes with adjacent cells and their cytoplasm is filled with free ribosomes; they lack bundles of intermediate filaments and possess no specialized organelles. After a 4-hr pulse of 3H-thymidine, 1.5% of epithelial cells incorporate thymidine into nuclear DNA, out of which 3/4 are basally and 1/4 are apically located. Identification of cell types by electron microscopy reveals that approximately 10% of undifferentiated basal cells are labeled, whereas less than 0.1% of granular cells and no MFR-cells incorporate 3H-thymidine into DNA. When dissociated from the epithelium and separated by isopycnic centrifugation, MFR-cells possess a mean buoyant density of approximately 1.025, cosediment with mitochondria-rich cells and exhibit a strong immunofluorescence for actin. The function of MFR-cells remains unknown; however, they may play a role in cell coupling and responses to hormonal and physical factors.
Actins↗
Purification and characterization of (Na+ + K+)-ATPase from toad kidney.
This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], Jørgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.
Animals↗
Thyroxine and Na+ transport in toad: role in transition from poikilo- to homeothermy.
The effects of thyroxine (T4) on Na+ transport, oxygen consumption (QO2), and Na+-K+-ATPase activity were studied in the urinary bladder and liver of the toad Bufo marinus. In the bladder, T4 in vitro (10(-8) to 10(-6) M) had no significant effect on these parameters during 15 h of incubation. When injected intraperitoneally (approximately 20 microgram/(kg body wt.day) for 6 days), T4 lowered base-line, short-circuit current by 62% (P less than 0.0025) and potential difference by 37% (P less than 0.001), increasing tissue resistance by 40% (P less than 0.02). T4 depressed QO2/DNA (-25%, P less than 0.05) with no significant effect on Na+-K+-ATPase activity. In liver, T4 increased the recovery per cell DNA of mitochondrial proteins by 32% (P less than 0.025), corresponding to an increased QO2 (stage IV) of isolated mitochondria per cell DNA (+54%, P less than 0.01). There was no significant effect on Na+-K+-ATPase activity. These results suggest that, unlike its function in the rat, T4 in the toad does not regulate cellular thermogenesis by inducing Na+-K+-ATPase. This major difference could account at least in part for the transition from poikilothermy to homeothermy. In addition, T4 has a distinct inhibitory effect on Na+ transport in the urinary bladder, which suggests an antagonism to the action of aldosterone.
Animals↗
Thyroid hormone-aldosterone interaction on Na+ transport in toad bladder.
Repeated administration of thyroxine (T4) in vivo (2 microgram/100 g body wt for 6 days) lowered by 2.3 times (P less than 0.025, df = 18) the basal rate of Na+ transport measured by the short-circuit current (SCC) in vitro in the urinary bladder of the toad (Bufo marinus). This difference was not accounted for by a change in the plasma aldosterone concentration. Moreover the response of the SCC to aldosterone in vitro was markedly inhibited in bladders from T4-treated animals (P less than 0.001, df = 18). These findings raised the possibility of a direct interaction between thyroid hormone and aldosterone in the target cell. The effects of L-triiodothyronine (T3) and aldosterone were examined in vitro. T3 alone (60 nM) had no significant effect on the base-line SCC (deltamuA = -14 +/- 11 (SE) muA per hemibladder; P greater than 0.3, n = 10). By contrast, T3 (60 nM) inhibited the response of the SCC to aldosterone from 6 to 8 h after its addition (deltamuA = -98 +/- 19 muA per hemibladder; P less than 0.001, n = 10). The inhibition by T3 was present at 6 nM (P less than 0.01, n = 10) and became not significant at 0.6 nM. T3 had no significant effect on base-line or aldosterone-stimulated H+ transport. Thyroid hormone might therefore regulate the late response of the SCC to aldosterone at the level of its target cell.
Aldosterone↗
Effects of 3'deoxyadenosine and actinomycin D on RNA synthesis in toad bladder: analysis of response to aldosterone.
Previous studies have shown that aldosterone increases transepithelial active Na+ transport after a latent period of about 60 min and incorporation of 3H-uridine into polyadenylated RNA (poly(A)(+)RNA) (putatively poly(A)(+)mRNA) as early as 30 min after aldosterone addition. To assess the physiological importance of this pathway, the effects of 3'deoxyadenosine and actinomycin D were compared in studies on the urinary bladder of the toad Bufo marinus. 3'deoxyadenosine (30 microgram/ml) only partially, though significantly, inhibited the aldosterone-dependent increase in Na+ transport measured as short-circuit current (scc). The incorporation of 3H-uridine into poly(A) (+)RNA was inhibited by 70 to 80%. In contrast, Actinomycin D (2 microgram/ml) totally inhibited the aldosterone-dependent increase in scc, and the incorporation of 3H-uridine into poly(A)(+)RNA by 68 to 75%. 3'deoxyadenosine or actinomycin D alone had no significant effects on baseline scc, while inhibiting poly(A)(+)RNA to the same extent. The differential effects of deoxyadenosine and actinomycin on aldosterone-dpendent Na+ transport may be related to their different sites of action on RNA synthesis: both drugs inhibited, to a similar extent, cytoplasmic poly(A)(+)mRNA: however, 3'deoxyadenosine, in contrast to Actinomycin D, failed to inhibit poly(A)(-)RNA, sedimenting between 4S and 18S (putatively poly(A)(-)mRNA). We conclude that the mineralocorticoid action of aldosterone during the first three hours depends on the synthesis of both poly(A)(+)mRNA and poly(A)(-)mRNA.
Aldosterone↗
Role of RNA in the action of aldosterone on Na+ transport.
Recent data describing the effects of aldosterone on the induction of messenger RNA (= mRNA) and ribosomal RNA (= rRNA) are reviewed. In the urinary bladder of the toad, aldosterone induces a few specific polyadenylated mRNAs (= poly(A)(+)mRNA) during the latent period, i.e., 30 to 60 min after hormone addition. Later i.e., 90 to 240 min after aldosterone addition, 18S and 28S cytoplasmic rRNA subunits are also induced. The effect of poly(A)(+)mRNA is mineralocorticoid-specific and correlates well with the aldosterone-dependent Na+ transport. Actinomycin D which inhibits both poly(A)(+)mRNA and nonpolyadenylated mRNA (= POLY(A)(-)mRNA) totally abolishes the response to aldosterone on Na+ transport. 3'deoxyadenosine (cordycepin), which inhibits poly(A)(+)mRNA but not poly(A)(-)mRNA, only inhibits 50 to 60% of the physiological response. These differential effects suggest that an intact poly(A)(-)mRNA pathway is also an important factor in mediating the action of aldosterone. In contrast, 3'deoxycytidine, which inhibits rRNA but not mRNA, does not impair the mineralocorticoid response, at least during the first 3 hr of aldosterone action.
Aldosterone↗
Multiplicity of steroid hormone receptors. Proceedings of a satellite workshop of the XI Acta Endocrinologica congress, Lausanne, 23 June, 1977.
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Animals↗
Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium.
In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with 3H-uridine (30 min pulse - 140 min chase), with or without aldosterone (3.5 x 10(-8) M, 7 X 10(-8) M) in the presence or absence of SC-9420 (7 X 10(-6) M, 2.5 X 10(-5) M) at molar ratios of 200:1 to 280:1. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5 -- 20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of 3H-uridine (30 min pulse -- 150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change in scc correlated linearly with the fractional change in 3H-uridine of 12S cytoplasmic RNA (r=0.95, p less than 0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of 3H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na+ transport.
Aldosterone↗
Effects of 3'-deoxycytidine on rRNA synthesis in toad bladder: analysis of response to aldosterone.
Previous studies showed that aldosterone augments transepithelial active Na+ transport and the incorporation of [3H]uridine into polyadenylated RNA (poly(A)(+)-RNA) (putatively mRNA) early in the latent period. Soon thereafter, incorporation of [methyl-14C] groups, as well as [3H]uridine into rRNA is also increased. To evaluate the role of rRNA in mineralocorticoid action, the inhibitor 3'-deoxycytidine was used in studies on the urinary bladder of the toad Bufo marinus. 3'-deoxycytidine suppressed the incorporation of [methyl-14C] and [3H]uridine into nuclear precursors of rRNA and subunits of cytoplasmic rRNA. In contrast, 3'-deoxycytidine inhibited incorporation of ]3H]uridine into cytoplasmic poly(A)(+)-RNA minimally. In control experiments, 3'-deoxycytidine had no significant effect on Na+ transport, measured as the short-circuit current (scc), when given alone. 3'-Deoxycytidine also had no significant effect on the aldosterone-dependent increase in scc. In the presence of 3'-deoxycytidine, aldosterone enhanced both the scc and the incorporation of [3H]uridine into poly(A)(+)-RNA significantly. We conclude that during the first 3 h, the mineralocorticoid action of aldosterone is not sensitive to inhibition of rRNA synthesis. Previous studies, however, implicate mRNA synthesis in this early response.
Aldosterone↗
Actions of aldosterone on polyadenylated ribonucleic acid and Na+ transport in the toad bladder.
Polyadenylated ribonucleic acid [poly(A)(+)-RNA] has been isolated from the cytoplasm of the epithelium of the urinary bladder of the toad (Bufo marinus) by oligo-(deoxythymidylate)cellulose chromatography. Aldosterone increased the incorporation of [3H]uridine, and of [3H]uridine and [3H]adenosine (given in combination) into 7S, 12S, and 18S poly(A)(+)-RNA during the first 30 min of the action of the hormone, as defined by either a pulse or pulse-chase sequence. The quantity of cytoplasmic poly(A)(+)-RNA that hybridized to [3H]poly(uridine) was also increased by aldosterone. These results are consistent with the inference of induction of messenger RNA synthesis. This effect was most marked during the first 30 min of the action of the hormone.
Aldosterone↗
Actions of aldosterone on rRNA and Na+ transport in the toad bladder.
Aldosterone increased methylation by [methyl-3H]methionine of nuclear ribosomal ribonucleic acid (rRNA) sedimenting at 18S, 28S, and 40S within 90 min and of 28S cytoplasmic RNA within 240 min of continuous exposure to the precursor and the hormone, in the toad bladder. In addition, incorporation of [14C]uridine into cytoplasmic 4S transfer RNA, and 18S and 28S rRNA was enhanced after 240 min of continuous exposure to the precursor and the hormone. Aldosterone had minimal effects on the 3H or 14C-labeled, acid-soluble pools. These results suggest that aldosterone augments the synthesis of rRNA at the transcriptional level. Ribosomes isolated 240 min after treatment of the toad bladder with aldosterone showed increased incorporation of [3H]-phenylalanine into peptides with both endogenous messenger RNA (mRNA) and exogenous mRNA (i.e., poly(uridylic acid) in an in vitro assay. Inhibition of reinitiation with NaF or poly (AUG) reduced the rate of amino acid polymerization by 45% but the aldosterone to control ratio remained significantly high. These results imply an increase in active ribosomes and perhaps in endogenous mRNA content. Our findings, however, do not distinguish between a steroid-dependent increase in the total number of active ribosomes and an increase in translational activity per ribosome.
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