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Mediating events in the action of aldosterone
Journal of Swroid Biochemistry. Vol. 12. pp. 219 to 224 Pcrpmon Press Ltd 1980. Printedm Great Britain
EVENTS IN THE ACTION ALDOSTERONE*
MEDIATING
OF
I. s EDELMAN and DIANA MARVER Department of Biochemistry, College of Physicians and Surgeons of Columbia University, New York, NY 10032. and Department of Internal Medicine, Southwestern Medical School, University of Texas Health Science Center, Dallas TX 75235. U.S.A. SUMMARY Aldosterone action is mediated by induction of the synthesis of proteins (AIP) in target ceils, initiated by binding to a cytoplasmic receptor and subsequent attachment of the complex to chromatin. The participation of DNA in chromatin “acceptor activity” is indicated by the following findings: DNase I impairs acceptor activity of rat kidney chromatin by -75%. when the DNA/RNA ratios were reduced by -45%. In addition, the DNA specific, non-destructive probes. ethidium bromide and proflavine sulfate inhibited acceptor activity completely. while netropsin and actinomycin D reduced the number of chromatin acceptor sites by 60 and 207,. respectively. At the concentrations used. all four probes inhibited RNA synthesis completely, measured in the presence of E. coli polymerase. Evidence has also been obtained that implicates induction of mRNA [poly A( +)-RNA] in augmentation of specific proteins. Three possible functions for AIPs have been postulated. The “sodium pump” theory contends that AIP stimulates the activity of the sodium pump (on the serosal side of the cell) directly. The “metabolic theory” suggests that AIP regulates the supply of ATP. The “permease theory” posits that AIP enhances the permeability to Na+ of the luminal mucosal membrane. The “sodium pump” theory has not been validated in that aldosterone has no effect on the K, or the V,,,,, for ATP of toad bladder Na/K-ATPase (the enzymatic equivalent of the Na+ pump). Aldosterone has little or no effect on Na + transport across the toad bladder in the absence of substrate, and the subsequent addition of pyruvate enhances Na’ transport with no latent period. Both citrate synthase and malate dehydrogenase activities are increased by aldosterone. Consequently, the metabolic pathway may be one of the key mechanisms. Apical membrane permeability to Na+ is also stimulated by aldosterone. Studies with amiloride, and noise-frequency analysis indicate an effect of aldosterone on apical conductance. This effect, however, appears to depend on energy metabolism. Thus, both the passive apical entry of Na+, and the active extrusion of Na+ across the basal-lateral boundary may depend on the induction of enzymes that modulate energy metabolism.
1. INTRODUCDON Edelman, Bogoroch son[2] independently
and
Porter[l]
and
William-
proposed that aldosterone augments transepithelial Na* transport (I,.,.) by induction of RNA and protein synthesis. Subsequent studies have inquired into the properties of the receptors for aldosterone, characterization of the induced RNA and proteins, and the physiological roles played by the aldosterone induced proteins (AIP). The present review concerns recent results on the first and la+ steps, i.e. binding of aldosterone-receptor complexes to the genome and the role of energetic and permeability factors in augmentation of INa. 2. CHROMATIN-BINDING OF RECEPTOR COMPLEXES High-affinity
nuclear
mineralocorticoid
receptors
Reprint requests and all correspondence to be sent to Dr. I. S. Edelman. Department of Biochemistry, College of Physicians and Surgeons of Columbia University, 630 West 168 Street, New York, N.Y. 10032, U.S.A. l Financial support was provided by U.S.P.H.S. Program Project Grant No. HL-06585 (National Heart, Lung and Blood Institute).
were first revealed in radioautographs of [3H]-aldosterone in toad bladder epithelium [I, 33. The recep tors appear to be resident in the cytoplasm (in the absence of aldosterone) but on binding of aldosterone, the resultant complex attaches to the chromatin r4,51. The validity of this concept was supported by studies with spirolactone ([‘HI-SC-26304) a mineralocorticoid antagonist, in that [3H]-SC-26304receptor complexes had little or no affinity for renal chromatin in uiuo or in oitro [6]. In addition, the magnitude of the increase in transepithelial Na+ transport correlates with the binding of aldosterone receptor complexes to chromatin, and with the increases in the synthesis of polyA( + )-RNA (putatively mRNA) and of proteins [7-lo]. Very little is known, however, of the determinants of chromatin acceptor activity. The induction hypothesis implies an interaction (direct or indirect) between the steroid-receptor complex and the DNA coding for specific mRNAs. The DNA backbone has beeen implicated in nuclear or chromatin acceptor activity in two types of studies: steroidreceptor complexes bind to deproteinized DNA and to DNA-cellulose columns, and pretreatment of nuclei or chromatin with DNase but not RNase mark219
220
I. S.
EDELMANand DIANA MARVER
edly
reduces acceptor activity [S, 11, 121. The role of DNA in acceptor activity, however, has not been confirmed in all systems. For example, pretreatment of immature rat uterus nuclei with DNase impaired acceptor activity for [3H]-dexamethasone-receptor complexes but not that of [3H]-estradiol-17fl-receptar-complexes [ 131. Our re-examination of the effects of DNase I on acceptor activity of nuclei and chromatin from kidney, for [3H]-aldosterone-receptor complexes, confirmed the inhibitory consequences of pretreatment with this agent. This effect was CaZ+ dependent and not simulated by RNase (unpublished observations). The finding that DNase I is more effective in hydrolyzing DNA at transcriptionally active than at inactive segments of the genome suggests that template-active DNA may coaribute significantly to acceptor activity [ 14). It is possible, however, that hydrolysis of segments of DNA released closely associated chromosomal proteins essential to acceptor activity. If acceptor sites are associated with template active regions of the genome pretreatment of nuclei or chromatin with inhibitors of RNA synthesis might impair attachment of the complexes to these sites. The inhibitors of transcription that were used to explore this possibility included: (1) actinomycin D, an intercalator which binds specifically to G:C base pairs and protrudes into the minor groove of DNA [15,16]; (2) netropsin, which binds with a high affinity to clusters of A:T pairs from the minor groove and does not intercalate [17. 181; and (3) ethidium bromide and proflavine sulfate, both major-groove intercalatois, with some base pair preference [ 19-22). Netropsin also binds to secondary sites, not as yet clearly defined, with lower affinity [17, 18.231. In addition, recent evidence suggests that ethidium bromide may also bind from the minor groove [24,25-J. To analyze the basis for the effects of the probes on acceptor activity, two reference standards were used; inhibition of RNA synthesis in the presence of E. coli RNA polymerase and the density of probes bound per mg DNA. Labeled donor fractions were prepared by adding C3H]-aldosterone (OS-l.0 x 10-s M) + 10 x dexamethasone + 200x unlabeled d-aldosterone to rat renal cytosol-glycerol mixtures (0°C) 30 min before addition of isolated renal chromatin. Following incubation the chromatin fractions were extracted with 0.4 M KCl. All of the probes inhibited template activity effectively (i.e. C3H]-CTP incorporation into RNA) (Fig. 1). Ethidium bromide and proflavin sulfate were highly effective in blocking acceptor activity. In fact, acceptor activity was more sensitive than C3H]-CTP incorporation, to proflavin sulfate. In contrast, actinomycin D inhibited only 20% of acceptor activity and netropsin required IO-fold higher concentrations to inhibit acceptor activity than RNA synthesis. These differences may reflect differences in the number of probe molecules bound to exposed DNA. The dependence of the degree of inhibition of
a
Acfinomycin
D
NCtWSin
Probe Conccntrotion
( x I(? M1
Fig. 1. The effects of various probes on RNA synthesis and binding of [‘HI-aldosterone-receptor complexes to rat kidney chromatin. Purified chromatin from rat kidneys were incubated with the probes for 10min at 25°C and then for 20min at 25°C with rat renal cytosol prelabeled with 5 x 10e9 M [3H]-aldosterone + 10 x dexamethasone + 200 x d-aldosterone. Acceptor activity was assessed by extraction of the chromatin with 0.4 M KC1 (O---O, and RNA synthesis with E. coli polymerase (O---O). The results are expressed as ‘4 of the diluent controls. Each point is a mean of 4-5 experiments.
0’
“‘n” a005
0.05
001
0.1
I
r
Fig. 2. Inhibition of chromatin acceptor activity (+a) or RNA synthesis (O--O) as a function of probe molecules bound/DNA PO, (r). The equilibrium dissociation constants were determined for each probe and the observed inhibition of acceptor and RNA synthesis with increasing probe concentrations were used to determine the inhibition as a function of the number of molecules bound/DNA PO,.
Action of aldosterone acceptor activity on the number of probe molecules bound per DNA PO,+ (T) indicate that the order of sensitivity of [3H]-CTP incorporation to the probes is actinomycin D > netropsin > ethidium bromide > progavine sulfate (Fig. 2). The potential of actinomycin D to inhibit acceptor activity was explored up to DNA occupancy 35fold greater than that needed to inhibit 100% of [3H]-CT’P incorporation. At the highest density of actinomycin D binding to DNA, 30% inhibition of acceptor activity was obtained. With netropsin, ethidium bromide and proflavine sulfate, 50% inhibition of acceptor activity was obtained at probe densities ‘of 18, 30, and 38 mol/mg DNA, respectively. A series of control studies with ethidium bromide indicated that the probes do not bind to nor disrupt the donor aldosterone-receptor complexes and do not release chromosomal proteins. While a clearer understanding of the nature of the mineralocorticoid acceptor site awaits further study, these results indicate that acceptor sites are inhibited with DNA-specific probes and that the degree of inhibition is related to the site-specificity of the probe, as well as the amount of a given probe bound; thereby impli~ting the DNA backbone in the process. 3. ROLE OF AlP IN MlNERALOCORTlCOlD ACTION The two-barrier model of Koefoed, Johnsen and UssingE26] implies that the AIPs may play three distinct. but not necessarily independent, roles: (1) increased Na’ pump activity as a result either of activation of preexisting pumps or an increase in the number of pumps; (2) augmentation of the energy supply, presumably by enhanced mitochondrial oxidative phospho~~tion; and (3) facihtation of passive Na’ entry across the apical piasma membrane. If aldosterone increased Nap-ATPase activity in proportion to the effect on Na+ transport, serious consideration would have to be given to the Na+ pump hypothesis. Recently, Chun-Sik Park (unpublished observations) re-examined this issue in the isolated toad bladder by evaluating the partial reactions, i.e. Na’, Mg’+ dependent phosphbrylation of the enzyme by AT”P(y). In crude epithelial homogenates activated with deoxycholate, incubation in aldosterone (5 x 10-s M) for S-6 h had no effect on the KY for (AT’*Pf (3.8 i 0.2 x iO-sM vs 3.4 + 0.4 x IO- a M). Moreover, the total number of enzyme sites was only marginally increased by the hormone (84 + 10 vs 71 + 8, in pmol of 3zP/mg protein). Thus, convincing evidence is not yet available that enhanced Na’ pump activity plays a significant role in the action of aldosterone on transepithelial Na+ transport. Studies with a variety of metabolic inhibitors implicated induction of mitochondrial enzymes in mineralocorticoid action [27.28] which was further supported by the findings of aldosterone-dependent increases in the activities of mitochondrial citrate syn-
221
thase. glutamate dehydrogenase. malate dehydrogenase and glutamic-oxaloacetic transaminase [29-311. The effect on citrate synthase (the most prominent responder) included: (a) a linear correlation with the increase in tran~pithelial Nat transport; (b) no dependence on NaC uptake into the epithelium; (c) coincidence in time with the effect on Na+ transport: and (d) abolition of the effects on the enzyme and on Na+ transport by actinomycin D and puromycin. In addition, aldosterone increased renal mitochondrial NADH/NAD+ ratio concurrently with the increases in citrate synthase activity and the urinary K+,Na+ ratio, in the adrenalectomized rat [32]. Evidence was obtained indicating that aldosterone recently increases the synthesis of citrate synthase to the same extent as the increase in enzyme activity [S). The increase in citrate synthase activity is maximal in the renal medulla, the zone that is richest in the nephron segments responsive to aldosterone. The relevance to mineralocorticoid action was supported by the findings that agents the block this action, namely. actinomycin D. and spirolactone (K-26304), abolished the aldosterone-dependent increase in enzyme activity and in ratiomethionine inco~ration into citrate synthase. Moreover, equimolar doses of dexamethasone (a potent glucocorticoid) had no effect on either the activity or synthesis of this enzyme. In addition, Saito, Essig and Caplan[33] measured Na* transport and oxygen consumption under various physiological conditions and reported estimates of two-fold increases in the metabolic driving force (in proportion to the increase in Na+ transport across the frog skin) in response to aldosterone. These results implicate modulation of energy metabolism in mineralocorticoid action. These effects, however, may be coordinate with effects on the apical membrane Na+ conductance pathway. The “permease theory” proposed by Crabbe and de Weer[34] and Sharp et a!.[353 has been supported by Civan and Hoffman[36] who noted that in the toad bladder, aldosterone elicited small (- 20%) but significant increases in transepithelial electrical conductance. Moreover, Saito and Essig[37] found that aldosterone increased the conductance of the active transport pathway [K J but had no effect on the paracellular passive pathway (K&, and estimated that apical Na+ conductance contributes about half of K,. These results are compatible with a significant effect of aldosterone on apical permeability to Na+ (P&. This view has also been supported recently in studies on the rabbit urinary bladder [38] and the rabbit colon [39]. Spooner and Edelman[40] used amiloride to block apical Na’ conductance selectively, in the toad bladder. During the first - 3 h of the response to aldosterone, the change in short-circuit current (AI,) correlated linearly with the change in total conductance (AK). After 3 h however AI, increased out of proportion to the increase in AK. These results imply bipolar effects, involving both the apical and basallateral plasma membrane surfaces. The possibility
222
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EDEL.MANand DIANA MARVER
that these bipolar effects are mediated by common precursors was raised by the finding that both AI, and AK depended on the metabolic state of the epithelium [40]. Chun-Sik Park (unpublished observations) also used amiloride to probe the effect of aldosterone on the apical surface of the toad bladder Titration curves revealed that a mucosal Na’ concentration of 7.5 mEqfi., half-maximum inhibition (Ki) was obtained at amiloride concentrations of 1.6 f 0.4 x lo-‘M (control) and 3.1 & lo-’ M (aldosterone, 5 x lo-‘M); P < 0.05. At a mucosal Nat concentrations of 115 mEq/l., the corresponding Kis were 2.3 & 0.3 x IO-’ M (control and 6.3 + 0.8 x lo-’ M (aldosterone 5 x IO-‘M); P c 0.001. These results indicate that aldosterone modified the apical surface as evidence by a significant shift upward in the Ki for amiloride. Thus, indirect evidence was obtained of modulation of the apical surface by aldosterone. The non-invasive electrophysiological methods of Lindemann et al.[41,42], were used in recent studies to explore the effects of aldosterone on PNa and Na, (intracellular Na+ concentration) in the toad bladder (L. G. Palmer et al., unpublished observations). Timecourse studies showed that during the first 4 h of the response to aldosterone (5 x lo-‘M on the serosal side only), INp and Pr,. increased in proportion; P < 0.001, r = 0.97. The average fractional changes at 4 h were 2.63 (INa), 2.61 (PNJ and 0.49 (Na,). These results indicate an early change in PNa, as a determinant in the action of aldosterone. The possibility (as suggested in earlier studies on substrate depletion followed by substrate repletion) of metabolic regulation of PN., was explored by modifying ATP/ADP content with the inhibitor 2-deoxyglucose: At 5 mM, both INa and PNs were depressed by 63% with only an insignificant (16%) fall in Na,. The striking effects of 2deoxyglucose on PNa, prompted a study of the role of metabolic pathways in the effects of aldosterone on the properties of the apical boundary. Hemibladders were depleted of endogenous substrate by incubation in substrate-free media for 15-20 h. One of each pair was exposed to aldosterone (5 x lo-‘M) for the entire period of incubation, and both were then challenged with Na pyruvate (5 mM). Addition of the pyruvate to the serosal medium stimulated INa by 113”,/,, PNa by 101% and lowered Na, by lSD/ after 1.5 h of exposure to the substrate. No significant changes in INaor Pt.,, were recorded in the control hemibladders, but Na, fell by 23%. On the basis of these studies we postulated that there exists in the apical membrane, a population of Na+ specific channels that are modified by aldosterone (which may exert its effect through metabolic pathways). Further support for this conclusion was obtained by fluctuation (noise frequency) analysis which was used to estimate the total channel number and the single channel currents in the apical boundary. Amiloride at various submaximal doses served to identify the Na+-specific channels. After 6 h, aldosterone elicited a 2.4-fold increase
in INa and a corresponding
2.2-fold increase in the number of Na’-specific apical channels. In contrast single channel Na+ conductance was independent of hormonal status. The involvement of modifications in Na’ permeability of the apical surface in hormone action. raises the problem of the nature of the biochemical events that determine these responses. Energy-dependent activation of Na channel proteins could result from phosphorylation or dephosphorylation at a crucial site [43]. Evidence has also been presented of considerable stimulation of turnover of membrane phospholipids by aldosterone [44,45]. Moreover, 2methyl-2-[p-(1,2,3,4-tetrahydro-l-naphthyl)phenoxy] proprionic acid (TIPA) an inhibitor of acetyl CoA carboxylase blocks the aldosterone dependent increment in Na+ transport. An interesting recent finding is the inhibition of aldosterone-dependent amino acid incorporation into membrane proteins by TIPA, in toad bladder [46]. Thus, ongoing fatty acid synthesis may be required for the recruitment of Na* channel proteins into the apical membrane, a process that may be energy dependent. A variety of other mechanisms may also play a role in the process, including synthesis of proteolipids or changes in divalent ion content of the apical membrane. REFERENCES 1.
Edelman I. S., Bogoroch R. and Porter G. A.: On the
mechanism of action of aldosterone on sodium transport: the role of protein synthesis. Proc. nam. Ad. Sci. SO(1963) 1169-I 177. 2. Williamson H. E.: Mechanism of antinatriuretic action of aldosterone. Biochem. Pharm. 12 (1963) 1449-1450. 3 Porter G. A., Bogoroch B. and Edelman I. S.: On the mechanism of action of aldosterone on sodium transport: the role of RNA synthesis. Proc. natn. Acad. Sci. 52 (1964) 13261333. 4. Herman T. S., Fimognari G. M. and Edelman I. S.: Studies on renal aldosterone binding proteins. J. biol. Gem. 243 (1968) 3849-3856. 5. Marver D., Goodman D. and Edelman I. S.: Relationships between renal cytoplasmic and nuclear aldosterone receptors. Kidney Internatl. 1 (1972) 210-223. 6. Marver D., Stewart J., Funder J. W., Feldman D. and Edelman I. S.: Renal aldosterone receptors: studies with [‘HI-aldosterone and the anti-mineralocorticoid C3H]-spirolactone (SC-26304). Proc. natn. Acad. Sci. 71 (1974) 1431-1435. 7. Rossier B. C., Wilce P. A. and Edelman I. S.: Kinetics of RNA labeling in toad bladder epithelium: effects of aldosterone and related steroids. Proc. natn. Ad. Sci. 71 (1974) 3101-3105. 8. Law P. Y. and Edelman I. S.: Induction of citrate synthase by aldosterone in the rat kidhey. J. Mem. Biol. 41 (1978) 41-64. 9. Kusch M., Farman N. and Edelman I.S.: Binding of aldosterone to cytoplasmic and nuclear receptors of the urinary bladder epithelium of Bufo marinus. Am. J. Phy. 4 (1978) C82-C89.
10. Farman N.. Kusch M. and Edelman I. S.: Aldosterone receptor occupancy and sodium transport in the urinary bladder of Bufo marinus. Am. J. Phy. 4 (1978) C90-C96.
11. Rousseau G., Higgins S. J., Baxter J. D.. Gelfand D.. and Tomkins G. M.: Binding of the glucocorticoid receptors to DNA. J. biol. Chem. 250 (1975) 6015-6021.
Action of aldosterone 12. Eisen H. J. and Glinsman W.: Partial purification of the glucocorticoid receptor from rat liver: a rapid, twostep procedure using DNA-cellulose. Biochem. biophys. Res. Comnnm. 70 (1976) 367-372. 13. Higgins S. J.. Rousseau G. G., Baxter J. D. and Tomp kins G. M.: Nature of nuclear acceptor sites for glucocorticoid- and estrogen-receptor complexes. J. biol. Chem. 248 (1973) 5873-5879.
14. Weintraub H. and Groudine M.: Chromosomal subunits in active genes have an altered conformation. Science 193 (1976) 848-856. 15. Jain S. C. and Sobcll H. M.: Stereochemistry of actinomycin binding to DNA-I. Refinement and further structural details of the actinomycin-deoxyguanosine crystalline complex. J. molec. Biol. 68 (1972) l-20. 16. Sobell H. M. and Jain S. C.: Stereochemistry of actinomycin binding to DNA-II. Detailed molecular model of actinomycin-DNA complex and its implications. J. molec. Biol. 68 (1972) 21-34. 17. Reinert K. E.: Adenosine. Thymidine cluster-specific elongation and stiffening of DNA induced by the oligopeptide antibiotic netropsin. J. molec. Eiol. 72 (1972) 593-607. 18. Wartell R. M., Larson J. E. and Wells R. D.: Netrop sin. A specific probe for A-T regions of duplex deoxyribonucleic acid. J. biol. Chem. 249 (1974) 6719-6731. 19. Dagleish D. G., Peacocke A. R., Fey G. and Harvey C.: The circular dichroism in the ultraviolet of aminoacridines and ethidium bromide bound to DNA. Biopoly. 10 (1971) 1853-1863. 20. Ramstein J.. Dourlet M. and Leng M.: Interaction between proflavine and deoxyribonucleic acid influence of DNA base composihon. Biochem biophys. Res. Commun. 47 11972) 874-882.
21. Krugh T. R. and Reinhardt C. G.: Evidence for sequence preferences in the intercalative binding of ethidium bromide to dinucleoside monophosphates. J. molec. Biol. 97 (1975) 133-162. 22. Olmstead J. and Keams D. R.: Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. Biochemistry 16 (1977) 3647-3654. 23. Zimmer C., Reinert K. E., Luck G., Wahnert U., Lober G. and Thrum H.: Interaction of the ohgopeptide antibiotics netropsin and distamycin A with nucleic acids. J. molec. Biol. 58 (1971) 329-348. 24. Tsai C. C., Jain S. C. and Sobell H. M.: X-ray crystallography visualization of drug-nucleic acid intercalative binding structure of an ethidium dinucleoside monophosphate crystalline complex, ethidium 5-iodouridylyl-(3’,5’)-adenosine. Proc. natn. Acad. Sci. 72 (1975) 628-632. 25. Doenecke D.: Ethidium bromide (EB) binding to nucleosomal DNA effects on DNA cleavage patterns. Exptl. cell Res. 109 (1977) 309-315. 26. Koeford-Johns-en V. and Ussing H. H.: The nature of the frog skin potential. Acta Physiol. Stand. 42 (1958) 298-308.
21. Fimognari G. M., Porter G. A. and Edelman I. S.: The role of the tricarboxylic acid cycle in the action of aldosterone on sodium transport. B&him. biophys. Acta 135 (1967) 89-99.
28. Edelman I. S. and Fanestil D. D.: Mineralocorticoids. In Biochemical Actions of Hormones (Edited by G. Litwack). Academic Press. New York, Vol. I (1970) Chap. 8. pp. 321-364.
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29. Kinne R. and Kirsten R.: Der Einfluss von aldosteron auf die aktivitaf mitochondrialer und cytoplasmatischer enzyme in der rattenniere. PJugers Arch. 300 (1968) 244-254. 30. Kirsten E., Kirsten P., Leaf A. and Sharp G. W. G.: Increased activity of enzymes of the tricarboxylic acid cycle in response to aldosterone in the toad bladder. Pfhqers Arch. Jo0 (1968) 213-225. 31. Kirsten E.. Kirsten R. and Sharp G. W. G.: Effects of sodium transport stimulating substrances on enzyme activities in the toad bladder. PJugers Arch. 316 (1970) 26-33. 32. Kirsten R. and Kirsten E.: Redox state of pyridine nucleotides in renal response to aldosterone. Am. J. Physiol. 223 (1972) 229-235.
33. Saito T.. Essig A. and Caplan S. R.: The effect of aldosterone on the energetics of sodium transport in the frog skin. Biochim. biophys. Acta 318 (1973) 371-382. 34. Crabbe J. and de Weer P.: Action of aldosterone and vasopressin on active transport of sodium by the isolated toad bladder. J. Physiol. 180 (1965) 560. 35. Sharp G. W. G., Coggins C. H., Lichtenstein N. S. and Leaf A.: Evidena for a mucosal effect of aldosterone on sodium transport in the toad bladder. J. clin. Invest. 45 ( 1966) 1640-l647. 36. Civan M. M. and Hoffman R. E.: Effect of aldosterone on electrical resistance of toad bladder. Am. J. Physiol. 2fo (1971) 324-328. 37. Saito T. and Essig A.: Affect of aldosterone on active and passive conductance and EN, in the toad bladder. J. memb. Biol. 13 (1973) 1-18. 38. Lewis S. A., Eaton D. C. and Diamond J. M.: The mechanism of Na+ transport by rabbit urinary bladder. J. memb. Biol. 28 (1976) 41-70. 39. Frizzell R. A. and Schultz S. G.: Effect of aldosterone on ion transport by rabbit colon. in vitro. J. memb. Biol. 39 (1978) l-26. 40. Spooner P. M. and Edelman I. S.: Further studies on the effect of aldosterone on electrical resistance of toad bladder. Biochim. biophys. Acta 406 (1975) 304-314. 41. Fuchs W., Hviid-Larsen E. and Lindemann B. : Estimation of intraallular Na+ activity and of Na+ permeability from current-voltage curves of Na’ channels in frog skin. Pjugers Arch. 355 (1975) R71. 42. Van Driessche W. and Lindemann B.: Fluctuations of Na-current through the Na-selective membrane of frog skin. Pjugers Arch. 362 (1976) R28. 43. Liu A. Y.-C. and Greengard P.: Aldosterone-induced increase in protein phosphatase activity of toad bladder. Proc. natn. Acad. Sci., U.S.A. 71 (1974) 3869-3873. 44. Goodman D. B. P., Allen J. E. and Rasmussen
H.: Studies on the mechanism of action of aldosterone: Hormone-induced changes in lipid metabolism. Biochemistry 10 (1971) 3825-3831. 45. Lien E. L., Goodman D. B. P. and Rasmussen H.: Effects of an acetyl-coenzyme A carboxylase inhibitor and a sodium-sparing diuretic on aldosterone-stimulated sodium transport. lipid synthesis, and phospholipid fatty acid composition in the toad urinary bladder. Biochemistry 16( 1975) 2749-2754. _ 46. Scott W. N.. Reich I M. and Goodman D. B. P.: Inhibition of fatty acid synthesis prevents incorporation of aldosterone-induced proteins into membranes. J. biol. Chem. 254
(1979)4957-4959.
DISCUSSION Dunl. 1 wonder whether your effect of aldosterone on passive permeability could not be due to an effect of the steroid, for example, on lipid metabolism? As you know. Rasmussen demonstrated several years ago that aldoster-
one could affect phosphohpid metabolism. Therefore, if aldosterone induces a change in lipid metabolism which is an energy requiring process, the membrane passive permeability will be modified in an energy-dependent way.
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I. S. EDELMAN and DIANAMARVER
Edelnmn. That is certainly one of the possible explanations for energy dependent regulation of apical sodium permeability. We are now considering three possible mechanisms including phospholipid turnover. As you mentioned Goodman and Rassmussen found that aldosterone changes membrane phospholipid acid synthesis in toad bladder epithelium. This effect is blocked by inhibitors of RNA and protein synthesis. Thus a change in the phospholipid constituents of the apical membrane could effect the opening or closing of sodium channels, even though the channels are made up of proteins, because of lipid-protein interactions. Crab& I enjoyed this because I’ve been biased all the way, but there is one comment I would like to make and then two short questions. First we’ve tried also to get a clear view of the electrical profile of those sodium tranc porting epithelial cells treated with aldosterone and actually the only parameter that was accessible experimentally, in our hands, among those examined, and that changed significantly with aldosterone treatment was the apical conductor for sodium go from that standpoint we see eye to eye. Now two short questions. First, if I understood you correctly, you’ve mentioned early in your talk for mammalian tissue sensitive to aldosterone and transporting sodium such as a colon and the structures in the outer medulla, there was some evidence that aldosterone could increase the amount of ATP-ase that could be recovered from the tissue. Now could this be demonstrated only through manipulation of the availability of sodium to the animal? In other words, does one need drastic conditions to demonstrate this affect or could this be correlated with physiological events ascribed to aldosterone. The second question is, you’ve laid emphasis on aerobic metabolism as playing a key role for expression of the aldosterone effect. Do you see how to reconcile this with the observations that have been made indicating that the effect of aldosterone on short-circuit current in amphibian epithelia
could still be demonstrated when aerobic metabolism is inhibited or blocked? Edelman. Peter Spooner and I found a small but significant effect of aldosterone on short-circuit current in the completely anaerobic state. The effect was very much smaller than had been reported by Handler and did not reflect an increase in sodium transport because net sodium flux measured isotopically was nil. The residual short circuit current under those circumstances is probably a diffusion current and has nothing to do with transepithelial sodium transport. Now with respect to the first question, the problem of whether aldosterone indicates Na/KATPase via a mineralocorticoid pathway is vexing and very unsatisfactory. With the exception of Schmidt and Dubach (who use very special techniques) no-one has found an effect of aldosterone on kidney Na/K-ATPase within the period of the maximum e&t on urinary sodium-potassium excretion. In addition, the effect is also evoked by glucocorticoids. The concentrations of aldosterone used in these and similar experiments is sufficient to evoke the entire effect through the glucocorticoid pathway. Aldosterone, in the rat, has an affinity for the glucocorticoid receptor which is only a fifth of that of the most potent glucocorticoids. M-k. You implied that ATP is the metabolic mediator of the effects. Ar there measurable changes in the ATP levels or ATP/ADP ratios that correspond with the time course? Edehmm. The aldosterone invoked change in apical sodium permeability appears to depend on the energy state of the cell. Measurements of ATP/ADP ratios led to onlv great confusion. Kasbaker, working in my laboratories found an increase, the group in Leaps laboratory found no change, and Handler found a decrease. This measurement is complicated by a variety of technical problems, in terms of satisfactory yields, and the problem of compartmentalization.
EVENTS IN THE ACTION ALDOSTERONE*
MEDIATING
OF
I. s EDELMAN and DIANA MARVER Department of Biochemistry, College of Physicians and Surgeons of Columbia University, New York, NY 10032. and Department of Internal Medicine, Southwestern Medical School, University of Texas Health Science Center, Dallas TX 75235. U.S.A. SUMMARY Aldosterone action is mediated by induction of the synthesis of proteins (AIP) in target ceils, initiated by binding to a cytoplasmic receptor and subsequent attachment of the complex to chromatin. The participation of DNA in chromatin “acceptor activity” is indicated by the following findings: DNase I impairs acceptor activity of rat kidney chromatin by -75%. when the DNA/RNA ratios were reduced by -45%. In addition, the DNA specific, non-destructive probes. ethidium bromide and proflavine sulfate inhibited acceptor activity completely. while netropsin and actinomycin D reduced the number of chromatin acceptor sites by 60 and 207,. respectively. At the concentrations used. all four probes inhibited RNA synthesis completely, measured in the presence of E. coli polymerase. Evidence has also been obtained that implicates induction of mRNA [poly A( +)-RNA] in augmentation of specific proteins. Three possible functions for AIPs have been postulated. The “sodium pump” theory contends that AIP stimulates the activity of the sodium pump (on the serosal side of the cell) directly. The “metabolic theory” suggests that AIP regulates the supply of ATP. The “permease theory” posits that AIP enhances the permeability to Na+ of the luminal mucosal membrane. The “sodium pump” theory has not been validated in that aldosterone has no effect on the K, or the V,,,,, for ATP of toad bladder Na/K-ATPase (the enzymatic equivalent of the Na+ pump). Aldosterone has little or no effect on Na + transport across the toad bladder in the absence of substrate, and the subsequent addition of pyruvate enhances Na’ transport with no latent period. Both citrate synthase and malate dehydrogenase activities are increased by aldosterone. Consequently, the metabolic pathway may be one of the key mechanisms. Apical membrane permeability to Na+ is also stimulated by aldosterone. Studies with amiloride, and noise-frequency analysis indicate an effect of aldosterone on apical conductance. This effect, however, appears to depend on energy metabolism. Thus, both the passive apical entry of Na+, and the active extrusion of Na+ across the basal-lateral boundary may depend on the induction of enzymes that modulate energy metabolism.
1. INTRODUCDON Edelman, Bogoroch son[2] independently
and
Porter[l]
and
William-
proposed that aldosterone augments transepithelial Na* transport (I,.,.) by induction of RNA and protein synthesis. Subsequent studies have inquired into the properties of the receptors for aldosterone, characterization of the induced RNA and proteins, and the physiological roles played by the aldosterone induced proteins (AIP). The present review concerns recent results on the first and la+ steps, i.e. binding of aldosterone-receptor complexes to the genome and the role of energetic and permeability factors in augmentation of INa. 2. CHROMATIN-BINDING OF RECEPTOR COMPLEXES High-affinity
nuclear
mineralocorticoid
receptors
Reprint requests and all correspondence to be sent to Dr. I. S. Edelman. Department of Biochemistry, College of Physicians and Surgeons of Columbia University, 630 West 168 Street, New York, N.Y. 10032, U.S.A. l Financial support was provided by U.S.P.H.S. Program Project Grant No. HL-06585 (National Heart, Lung and Blood Institute).
were first revealed in radioautographs of [3H]-aldosterone in toad bladder epithelium [I, 33. The recep tors appear to be resident in the cytoplasm (in the absence of aldosterone) but on binding of aldosterone, the resultant complex attaches to the chromatin r4,51. The validity of this concept was supported by studies with spirolactone ([‘HI-SC-26304) a mineralocorticoid antagonist, in that [3H]-SC-26304receptor complexes had little or no affinity for renal chromatin in uiuo or in oitro [6]. In addition, the magnitude of the increase in transepithelial Na+ transport correlates with the binding of aldosterone receptor complexes to chromatin, and with the increases in the synthesis of polyA( + )-RNA (putatively mRNA) and of proteins [7-lo]. Very little is known, however, of the determinants of chromatin acceptor activity. The induction hypothesis implies an interaction (direct or indirect) between the steroid-receptor complex and the DNA coding for specific mRNAs. The DNA backbone has beeen implicated in nuclear or chromatin acceptor activity in two types of studies: steroidreceptor complexes bind to deproteinized DNA and to DNA-cellulose columns, and pretreatment of nuclei or chromatin with DNase but not RNase mark219
220
I. S.
EDELMANand DIANA MARVER
edly
reduces acceptor activity [S, 11, 121. The role of DNA in acceptor activity, however, has not been confirmed in all systems. For example, pretreatment of immature rat uterus nuclei with DNase impaired acceptor activity for [3H]-dexamethasone-receptor complexes but not that of [3H]-estradiol-17fl-receptar-complexes [ 131. Our re-examination of the effects of DNase I on acceptor activity of nuclei and chromatin from kidney, for [3H]-aldosterone-receptor complexes, confirmed the inhibitory consequences of pretreatment with this agent. This effect was CaZ+ dependent and not simulated by RNase (unpublished observations). The finding that DNase I is more effective in hydrolyzing DNA at transcriptionally active than at inactive segments of the genome suggests that template-active DNA may coaribute significantly to acceptor activity [ 14). It is possible, however, that hydrolysis of segments of DNA released closely associated chromosomal proteins essential to acceptor activity. If acceptor sites are associated with template active regions of the genome pretreatment of nuclei or chromatin with inhibitors of RNA synthesis might impair attachment of the complexes to these sites. The inhibitors of transcription that were used to explore this possibility included: (1) actinomycin D, an intercalator which binds specifically to G:C base pairs and protrudes into the minor groove of DNA [15,16]; (2) netropsin, which binds with a high affinity to clusters of A:T pairs from the minor groove and does not intercalate [17. 181; and (3) ethidium bromide and proflavine sulfate, both major-groove intercalatois, with some base pair preference [ 19-22). Netropsin also binds to secondary sites, not as yet clearly defined, with lower affinity [17, 18.231. In addition, recent evidence suggests that ethidium bromide may also bind from the minor groove [24,25-J. To analyze the basis for the effects of the probes on acceptor activity, two reference standards were used; inhibition of RNA synthesis in the presence of E. coli RNA polymerase and the density of probes bound per mg DNA. Labeled donor fractions were prepared by adding C3H]-aldosterone (OS-l.0 x 10-s M) + 10 x dexamethasone + 200x unlabeled d-aldosterone to rat renal cytosol-glycerol mixtures (0°C) 30 min before addition of isolated renal chromatin. Following incubation the chromatin fractions were extracted with 0.4 M KCl. All of the probes inhibited template activity effectively (i.e. C3H]-CTP incorporation into RNA) (Fig. 1). Ethidium bromide and proflavin sulfate were highly effective in blocking acceptor activity. In fact, acceptor activity was more sensitive than C3H]-CTP incorporation, to proflavin sulfate. In contrast, actinomycin D inhibited only 20% of acceptor activity and netropsin required IO-fold higher concentrations to inhibit acceptor activity than RNA synthesis. These differences may reflect differences in the number of probe molecules bound to exposed DNA. The dependence of the degree of inhibition of
a
Acfinomycin
D
NCtWSin
Probe Conccntrotion
( x I(? M1
Fig. 1. The effects of various probes on RNA synthesis and binding of [‘HI-aldosterone-receptor complexes to rat kidney chromatin. Purified chromatin from rat kidneys were incubated with the probes for 10min at 25°C and then for 20min at 25°C with rat renal cytosol prelabeled with 5 x 10e9 M [3H]-aldosterone + 10 x dexamethasone + 200 x d-aldosterone. Acceptor activity was assessed by extraction of the chromatin with 0.4 M KC1 (O---O, and RNA synthesis with E. coli polymerase (O---O). The results are expressed as ‘4 of the diluent controls. Each point is a mean of 4-5 experiments.
0’
“‘n” a005
0.05
001
0.1
I
r
Fig. 2. Inhibition of chromatin acceptor activity (+a) or RNA synthesis (O--O) as a function of probe molecules bound/DNA PO, (r). The equilibrium dissociation constants were determined for each probe and the observed inhibition of acceptor and RNA synthesis with increasing probe concentrations were used to determine the inhibition as a function of the number of molecules bound/DNA PO,.
Action of aldosterone acceptor activity on the number of probe molecules bound per DNA PO,+ (T) indicate that the order of sensitivity of [3H]-CTP incorporation to the probes is actinomycin D > netropsin > ethidium bromide > progavine sulfate (Fig. 2). The potential of actinomycin D to inhibit acceptor activity was explored up to DNA occupancy 35fold greater than that needed to inhibit 100% of [3H]-CT’P incorporation. At the highest density of actinomycin D binding to DNA, 30% inhibition of acceptor activity was obtained. With netropsin, ethidium bromide and proflavine sulfate, 50% inhibition of acceptor activity was obtained at probe densities ‘of 18, 30, and 38 mol/mg DNA, respectively. A series of control studies with ethidium bromide indicated that the probes do not bind to nor disrupt the donor aldosterone-receptor complexes and do not release chromosomal proteins. While a clearer understanding of the nature of the mineralocorticoid acceptor site awaits further study, these results indicate that acceptor sites are inhibited with DNA-specific probes and that the degree of inhibition is related to the site-specificity of the probe, as well as the amount of a given probe bound; thereby impli~ting the DNA backbone in the process. 3. ROLE OF AlP IN MlNERALOCORTlCOlD ACTION The two-barrier model of Koefoed, Johnsen and UssingE26] implies that the AIPs may play three distinct. but not necessarily independent, roles: (1) increased Na’ pump activity as a result either of activation of preexisting pumps or an increase in the number of pumps; (2) augmentation of the energy supply, presumably by enhanced mitochondrial oxidative phospho~~tion; and (3) facihtation of passive Na’ entry across the apical piasma membrane. If aldosterone increased Nap-ATPase activity in proportion to the effect on Na+ transport, serious consideration would have to be given to the Na+ pump hypothesis. Recently, Chun-Sik Park (unpublished observations) re-examined this issue in the isolated toad bladder by evaluating the partial reactions, i.e. Na’, Mg’+ dependent phosphbrylation of the enzyme by AT”P(y). In crude epithelial homogenates activated with deoxycholate, incubation in aldosterone (5 x 10-s M) for S-6 h had no effect on the KY for (AT’*Pf (3.8 i 0.2 x iO-sM vs 3.4 + 0.4 x IO- a M). Moreover, the total number of enzyme sites was only marginally increased by the hormone (84 + 10 vs 71 + 8, in pmol of 3zP/mg protein). Thus, convincing evidence is not yet available that enhanced Na’ pump activity plays a significant role in the action of aldosterone on transepithelial Na+ transport. Studies with a variety of metabolic inhibitors implicated induction of mitochondrial enzymes in mineralocorticoid action [27.28] which was further supported by the findings of aldosterone-dependent increases in the activities of mitochondrial citrate syn-
221
thase. glutamate dehydrogenase. malate dehydrogenase and glutamic-oxaloacetic transaminase [29-311. The effect on citrate synthase (the most prominent responder) included: (a) a linear correlation with the increase in tran~pithelial Nat transport; (b) no dependence on NaC uptake into the epithelium; (c) coincidence in time with the effect on Na+ transport: and (d) abolition of the effects on the enzyme and on Na+ transport by actinomycin D and puromycin. In addition, aldosterone increased renal mitochondrial NADH/NAD+ ratio concurrently with the increases in citrate synthase activity and the urinary K+,Na+ ratio, in the adrenalectomized rat [32]. Evidence was obtained indicating that aldosterone recently increases the synthesis of citrate synthase to the same extent as the increase in enzyme activity [S). The increase in citrate synthase activity is maximal in the renal medulla, the zone that is richest in the nephron segments responsive to aldosterone. The relevance to mineralocorticoid action was supported by the findings that agents the block this action, namely. actinomycin D. and spirolactone (K-26304), abolished the aldosterone-dependent increase in enzyme activity and in ratiomethionine inco~ration into citrate synthase. Moreover, equimolar doses of dexamethasone (a potent glucocorticoid) had no effect on either the activity or synthesis of this enzyme. In addition, Saito, Essig and Caplan[33] measured Na* transport and oxygen consumption under various physiological conditions and reported estimates of two-fold increases in the metabolic driving force (in proportion to the increase in Na+ transport across the frog skin) in response to aldosterone. These results implicate modulation of energy metabolism in mineralocorticoid action. These effects, however, may be coordinate with effects on the apical membrane Na+ conductance pathway. The “permease theory” proposed by Crabbe and de Weer[34] and Sharp et a!.[353 has been supported by Civan and Hoffman[36] who noted that in the toad bladder, aldosterone elicited small (- 20%) but significant increases in transepithelial electrical conductance. Moreover, Saito and Essig[37] found that aldosterone increased the conductance of the active transport pathway [K J but had no effect on the paracellular passive pathway (K&, and estimated that apical Na+ conductance contributes about half of K,. These results are compatible with a significant effect of aldosterone on apical permeability to Na+ (P&. This view has also been supported recently in studies on the rabbit urinary bladder [38] and the rabbit colon [39]. Spooner and Edelman[40] used amiloride to block apical Na’ conductance selectively, in the toad bladder. During the first - 3 h of the response to aldosterone, the change in short-circuit current (AI,) correlated linearly with the change in total conductance (AK). After 3 h however AI, increased out of proportion to the increase in AK. These results imply bipolar effects, involving both the apical and basallateral plasma membrane surfaces. The possibility
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EDEL.MANand DIANA MARVER
that these bipolar effects are mediated by common precursors was raised by the finding that both AI, and AK depended on the metabolic state of the epithelium [40]. Chun-Sik Park (unpublished observations) also used amiloride to probe the effect of aldosterone on the apical surface of the toad bladder Titration curves revealed that a mucosal Na’ concentration of 7.5 mEqfi., half-maximum inhibition (Ki) was obtained at amiloride concentrations of 1.6 f 0.4 x lo-‘M (control) and 3.1 & lo-’ M (aldosterone, 5 x lo-‘M); P < 0.05. At a mucosal Nat concentrations of 115 mEq/l., the corresponding Kis were 2.3 & 0.3 x IO-’ M (control and 6.3 + 0.8 x lo-’ M (aldosterone 5 x IO-‘M); P c 0.001. These results indicate that aldosterone modified the apical surface as evidence by a significant shift upward in the Ki for amiloride. Thus, indirect evidence was obtained of modulation of the apical surface by aldosterone. The non-invasive electrophysiological methods of Lindemann et al.[41,42], were used in recent studies to explore the effects of aldosterone on PNa and Na, (intracellular Na+ concentration) in the toad bladder (L. G. Palmer et al., unpublished observations). Timecourse studies showed that during the first 4 h of the response to aldosterone (5 x lo-‘M on the serosal side only), INp and Pr,. increased in proportion; P < 0.001, r = 0.97. The average fractional changes at 4 h were 2.63 (INa), 2.61 (PNJ and 0.49 (Na,). These results indicate an early change in PNa, as a determinant in the action of aldosterone. The possibility (as suggested in earlier studies on substrate depletion followed by substrate repletion) of metabolic regulation of PN., was explored by modifying ATP/ADP content with the inhibitor 2-deoxyglucose: At 5 mM, both INa and PNs were depressed by 63% with only an insignificant (16%) fall in Na,. The striking effects of 2deoxyglucose on PNa, prompted a study of the role of metabolic pathways in the effects of aldosterone on the properties of the apical boundary. Hemibladders were depleted of endogenous substrate by incubation in substrate-free media for 15-20 h. One of each pair was exposed to aldosterone (5 x lo-‘M) for the entire period of incubation, and both were then challenged with Na pyruvate (5 mM). Addition of the pyruvate to the serosal medium stimulated INa by 113”,/,, PNa by 101% and lowered Na, by lSD/ after 1.5 h of exposure to the substrate. No significant changes in INaor Pt.,, were recorded in the control hemibladders, but Na, fell by 23%. On the basis of these studies we postulated that there exists in the apical membrane, a population of Na+ specific channels that are modified by aldosterone (which may exert its effect through metabolic pathways). Further support for this conclusion was obtained by fluctuation (noise frequency) analysis which was used to estimate the total channel number and the single channel currents in the apical boundary. Amiloride at various submaximal doses served to identify the Na+-specific channels. After 6 h, aldosterone elicited a 2.4-fold increase
in INa and a corresponding
2.2-fold increase in the number of Na’-specific apical channels. In contrast single channel Na+ conductance was independent of hormonal status. The involvement of modifications in Na’ permeability of the apical surface in hormone action. raises the problem of the nature of the biochemical events that determine these responses. Energy-dependent activation of Na channel proteins could result from phosphorylation or dephosphorylation at a crucial site [43]. Evidence has also been presented of considerable stimulation of turnover of membrane phospholipids by aldosterone [44,45]. Moreover, 2methyl-2-[p-(1,2,3,4-tetrahydro-l-naphthyl)phenoxy] proprionic acid (TIPA) an inhibitor of acetyl CoA carboxylase blocks the aldosterone dependent increment in Na+ transport. An interesting recent finding is the inhibition of aldosterone-dependent amino acid incorporation into membrane proteins by TIPA, in toad bladder [46]. Thus, ongoing fatty acid synthesis may be required for the recruitment of Na* channel proteins into the apical membrane, a process that may be energy dependent. A variety of other mechanisms may also play a role in the process, including synthesis of proteolipids or changes in divalent ion content of the apical membrane. REFERENCES 1.
Edelman I. S., Bogoroch R. and Porter G. A.: On the
mechanism of action of aldosterone on sodium transport: the role of protein synthesis. Proc. nam. Ad. Sci. SO(1963) 1169-I 177. 2. Williamson H. E.: Mechanism of antinatriuretic action of aldosterone. Biochem. Pharm. 12 (1963) 1449-1450. 3 Porter G. A., Bogoroch B. and Edelman I. S.: On the mechanism of action of aldosterone on sodium transport: the role of RNA synthesis. Proc. natn. Acad. Sci. 52 (1964) 13261333. 4. Herman T. S., Fimognari G. M. and Edelman I. S.: Studies on renal aldosterone binding proteins. J. biol. Gem. 243 (1968) 3849-3856. 5. Marver D., Goodman D. and Edelman I. S.: Relationships between renal cytoplasmic and nuclear aldosterone receptors. Kidney Internatl. 1 (1972) 210-223. 6. Marver D., Stewart J., Funder J. W., Feldman D. and Edelman I. S.: Renal aldosterone receptors: studies with [‘HI-aldosterone and the anti-mineralocorticoid C3H]-spirolactone (SC-26304). Proc. natn. Acad. Sci. 71 (1974) 1431-1435. 7. Rossier B. C., Wilce P. A. and Edelman I. S.: Kinetics of RNA labeling in toad bladder epithelium: effects of aldosterone and related steroids. Proc. natn. Ad. Sci. 71 (1974) 3101-3105. 8. Law P. Y. and Edelman I. S.: Induction of citrate synthase by aldosterone in the rat kidhey. J. Mem. Biol. 41 (1978) 41-64. 9. Kusch M., Farman N. and Edelman I.S.: Binding of aldosterone to cytoplasmic and nuclear receptors of the urinary bladder epithelium of Bufo marinus. Am. J. Phy. 4 (1978) C82-C89.
10. Farman N.. Kusch M. and Edelman I. S.: Aldosterone receptor occupancy and sodium transport in the urinary bladder of Bufo marinus. Am. J. Phy. 4 (1978) C90-C96.
11. Rousseau G., Higgins S. J., Baxter J. D.. Gelfand D.. and Tomkins G. M.: Binding of the glucocorticoid receptors to DNA. J. biol. Chem. 250 (1975) 6015-6021.
Action of aldosterone 12. Eisen H. J. and Glinsman W.: Partial purification of the glucocorticoid receptor from rat liver: a rapid, twostep procedure using DNA-cellulose. Biochem. biophys. Res. Comnnm. 70 (1976) 367-372. 13. Higgins S. J.. Rousseau G. G., Baxter J. D. and Tomp kins G. M.: Nature of nuclear acceptor sites for glucocorticoid- and estrogen-receptor complexes. J. biol. Chem. 248 (1973) 5873-5879.
14. Weintraub H. and Groudine M.: Chromosomal subunits in active genes have an altered conformation. Science 193 (1976) 848-856. 15. Jain S. C. and Sobcll H. M.: Stereochemistry of actinomycin binding to DNA-I. Refinement and further structural details of the actinomycin-deoxyguanosine crystalline complex. J. molec. Biol. 68 (1972) l-20. 16. Sobell H. M. and Jain S. C.: Stereochemistry of actinomycin binding to DNA-II. Detailed molecular model of actinomycin-DNA complex and its implications. J. molec. Biol. 68 (1972) 21-34. 17. Reinert K. E.: Adenosine. Thymidine cluster-specific elongation and stiffening of DNA induced by the oligopeptide antibiotic netropsin. J. molec. Eiol. 72 (1972) 593-607. 18. Wartell R. M., Larson J. E. and Wells R. D.: Netrop sin. A specific probe for A-T regions of duplex deoxyribonucleic acid. J. biol. Chem. 249 (1974) 6719-6731. 19. Dagleish D. G., Peacocke A. R., Fey G. and Harvey C.: The circular dichroism in the ultraviolet of aminoacridines and ethidium bromide bound to DNA. Biopoly. 10 (1971) 1853-1863. 20. Ramstein J.. Dourlet M. and Leng M.: Interaction between proflavine and deoxyribonucleic acid influence of DNA base composihon. Biochem biophys. Res. Commun. 47 11972) 874-882.
21. Krugh T. R. and Reinhardt C. G.: Evidence for sequence preferences in the intercalative binding of ethidium bromide to dinucleoside monophosphates. J. molec. Biol. 97 (1975) 133-162. 22. Olmstead J. and Keams D. R.: Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. Biochemistry 16 (1977) 3647-3654. 23. Zimmer C., Reinert K. E., Luck G., Wahnert U., Lober G. and Thrum H.: Interaction of the ohgopeptide antibiotics netropsin and distamycin A with nucleic acids. J. molec. Biol. 58 (1971) 329-348. 24. Tsai C. C., Jain S. C. and Sobell H. M.: X-ray crystallography visualization of drug-nucleic acid intercalative binding structure of an ethidium dinucleoside monophosphate crystalline complex, ethidium 5-iodouridylyl-(3’,5’)-adenosine. Proc. natn. Acad. Sci. 72 (1975) 628-632. 25. Doenecke D.: Ethidium bromide (EB) binding to nucleosomal DNA effects on DNA cleavage patterns. Exptl. cell Res. 109 (1977) 309-315. 26. Koeford-Johns-en V. and Ussing H. H.: The nature of the frog skin potential. Acta Physiol. Stand. 42 (1958) 298-308.
21. Fimognari G. M., Porter G. A. and Edelman I. S.: The role of the tricarboxylic acid cycle in the action of aldosterone on sodium transport. B&him. biophys. Acta 135 (1967) 89-99.
28. Edelman I. S. and Fanestil D. D.: Mineralocorticoids. In Biochemical Actions of Hormones (Edited by G. Litwack). Academic Press. New York, Vol. I (1970) Chap. 8. pp. 321-364.
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29. Kinne R. and Kirsten R.: Der Einfluss von aldosteron auf die aktivitaf mitochondrialer und cytoplasmatischer enzyme in der rattenniere. PJugers Arch. 300 (1968) 244-254. 30. Kirsten E., Kirsten P., Leaf A. and Sharp G. W. G.: Increased activity of enzymes of the tricarboxylic acid cycle in response to aldosterone in the toad bladder. Pfhqers Arch. Jo0 (1968) 213-225. 31. Kirsten E.. Kirsten R. and Sharp G. W. G.: Effects of sodium transport stimulating substrances on enzyme activities in the toad bladder. PJugers Arch. 316 (1970) 26-33. 32. Kirsten R. and Kirsten E.: Redox state of pyridine nucleotides in renal response to aldosterone. Am. J. Physiol. 223 (1972) 229-235.
33. Saito T.. Essig A. and Caplan S. R.: The effect of aldosterone on the energetics of sodium transport in the frog skin. Biochim. biophys. Acta 318 (1973) 371-382. 34. Crabbe J. and de Weer P.: Action of aldosterone and vasopressin on active transport of sodium by the isolated toad bladder. J. Physiol. 180 (1965) 560. 35. Sharp G. W. G., Coggins C. H., Lichtenstein N. S. and Leaf A.: Evidena for a mucosal effect of aldosterone on sodium transport in the toad bladder. J. clin. Invest. 45 ( 1966) 1640-l647. 36. Civan M. M. and Hoffman R. E.: Effect of aldosterone on electrical resistance of toad bladder. Am. J. Physiol. 2fo (1971) 324-328. 37. Saito T. and Essig A.: Affect of aldosterone on active and passive conductance and EN, in the toad bladder. J. memb. Biol. 13 (1973) 1-18. 38. Lewis S. A., Eaton D. C. and Diamond J. M.: The mechanism of Na+ transport by rabbit urinary bladder. J. memb. Biol. 28 (1976) 41-70. 39. Frizzell R. A. and Schultz S. G.: Effect of aldosterone on ion transport by rabbit colon. in vitro. J. memb. Biol. 39 (1978) l-26. 40. Spooner P. M. and Edelman I. S.: Further studies on the effect of aldosterone on electrical resistance of toad bladder. Biochim. biophys. Acta 406 (1975) 304-314. 41. Fuchs W., Hviid-Larsen E. and Lindemann B. : Estimation of intraallular Na+ activity and of Na+ permeability from current-voltage curves of Na’ channels in frog skin. Pjugers Arch. 355 (1975) R71. 42. Van Driessche W. and Lindemann B.: Fluctuations of Na-current through the Na-selective membrane of frog skin. Pjugers Arch. 362 (1976) R28. 43. Liu A. Y.-C. and Greengard P.: Aldosterone-induced increase in protein phosphatase activity of toad bladder. Proc. natn. Acad. Sci., U.S.A. 71 (1974) 3869-3873. 44. Goodman D. B. P., Allen J. E. and Rasmussen
H.: Studies on the mechanism of action of aldosterone: Hormone-induced changes in lipid metabolism. Biochemistry 10 (1971) 3825-3831. 45. Lien E. L., Goodman D. B. P. and Rasmussen H.: Effects of an acetyl-coenzyme A carboxylase inhibitor and a sodium-sparing diuretic on aldosterone-stimulated sodium transport. lipid synthesis, and phospholipid fatty acid composition in the toad urinary bladder. Biochemistry 16( 1975) 2749-2754. _ 46. Scott W. N.. Reich I M. and Goodman D. B. P.: Inhibition of fatty acid synthesis prevents incorporation of aldosterone-induced proteins into membranes. J. biol. Chem. 254
(1979)4957-4959.
DISCUSSION Dunl. 1 wonder whether your effect of aldosterone on passive permeability could not be due to an effect of the steroid, for example, on lipid metabolism? As you know. Rasmussen demonstrated several years ago that aldoster-
one could affect phosphohpid metabolism. Therefore, if aldosterone induces a change in lipid metabolism which is an energy requiring process, the membrane passive permeability will be modified in an energy-dependent way.
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I. S. EDELMAN and DIANAMARVER
Edelnmn. That is certainly one of the possible explanations for energy dependent regulation of apical sodium permeability. We are now considering three possible mechanisms including phospholipid turnover. As you mentioned Goodman and Rassmussen found that aldosterone changes membrane phospholipid acid synthesis in toad bladder epithelium. This effect is blocked by inhibitors of RNA and protein synthesis. Thus a change in the phospholipid constituents of the apical membrane could effect the opening or closing of sodium channels, even though the channels are made up of proteins, because of lipid-protein interactions. Crab& I enjoyed this because I’ve been biased all the way, but there is one comment I would like to make and then two short questions. First we’ve tried also to get a clear view of the electrical profile of those sodium tranc porting epithelial cells treated with aldosterone and actually the only parameter that was accessible experimentally, in our hands, among those examined, and that changed significantly with aldosterone treatment was the apical conductor for sodium go from that standpoint we see eye to eye. Now two short questions. First, if I understood you correctly, you’ve mentioned early in your talk for mammalian tissue sensitive to aldosterone and transporting sodium such as a colon and the structures in the outer medulla, there was some evidence that aldosterone could increase the amount of ATP-ase that could be recovered from the tissue. Now could this be demonstrated only through manipulation of the availability of sodium to the animal? In other words, does one need drastic conditions to demonstrate this affect or could this be correlated with physiological events ascribed to aldosterone. The second question is, you’ve laid emphasis on aerobic metabolism as playing a key role for expression of the aldosterone effect. Do you see how to reconcile this with the observations that have been made indicating that the effect of aldosterone on short-circuit current in amphibian epithelia
could still be demonstrated when aerobic metabolism is inhibited or blocked? Edelman. Peter Spooner and I found a small but significant effect of aldosterone on short-circuit current in the completely anaerobic state. The effect was very much smaller than had been reported by Handler and did not reflect an increase in sodium transport because net sodium flux measured isotopically was nil. The residual short circuit current under those circumstances is probably a diffusion current and has nothing to do with transepithelial sodium transport. Now with respect to the first question, the problem of whether aldosterone indicates Na/KATPase via a mineralocorticoid pathway is vexing and very unsatisfactory. With the exception of Schmidt and Dubach (who use very special techniques) no-one has found an effect of aldosterone on kidney Na/K-ATPase within the period of the maximum e&t on urinary sodium-potassium excretion. In addition, the effect is also evoked by glucocorticoids. The concentrations of aldosterone used in these and similar experiments is sufficient to evoke the entire effect through the glucocorticoid pathway. Aldosterone, in the rat, has an affinity for the glucocorticoid receptor which is only a fifth of that of the most potent glucocorticoids. M-k. You implied that ATP is the metabolic mediator of the effects. Ar there measurable changes in the ATP levels or ATP/ADP ratios that correspond with the time course? Edehmm. The aldosterone invoked change in apical sodium permeability appears to depend on the energy state of the cell. Measurements of ATP/ADP ratios led to onlv great confusion. Kasbaker, working in my laboratories found an increase, the group in Leaps laboratory found no change, and Handler found a decrease. This measurement is complicated by a variety of technical problems, in terms of satisfactory yields, and the problem of compartmentalization.