Aldosterone-induced proteins in toad urinary bladders. Identification and characterization using two-dimensional polyacrylamide gel electrophoresis.

M Geheb,G Huber,E Hercker,M Cox

doi:10.1016/s0021-9258(19)68465-6

Abstract

Aldosterone-stimulated Na+ transport is mediated by new protein synthesis, but the identification of specific aldosterone-induced proteins (AIPs) has proven difficult and the cellular function of such proteins is unknown. Using high resolution two-dimensional polyacrylamide gel electrophoresis and autoradiography we have identified AIPs of similar isoelectric points (5.8 to 6.4) and molecular weights (70,000 to 80,000) in membrane-rich and cytosolic subcellular fractions of epithelial cells derived from single toad urinary bladders. The ability of actinomycin D to inhibit both AIP synthesis and aldosterone-induced Na+ transport is consistent with a role for these proteins in the natriferic action of aldosterone. In addition, since non-natriferic concentrations of cortisol did not induce similar proteins, AIP synthesis appears to be mineralocorticoid-specific. The relationship of AIP synthesis to Na+ transport was also studied. Since amiloride, which blocks Na+ transport in high resistance epithelia, did not affect the synthesis of these proteins, Na+ transport is not required for their synthesis. In addition, similar proteins were not induced when Na+ transport was stimulated by antidiuretic hormone and theophylline. Consequently, AIP synthesis is not merely a nonspecific consequence of the cellular metabolic changes associated with Na+ transport.

Highlights

Michael Geheb$, Gary Huber, Eileen Hercker, and Malcolm Cox8 From the Renal-ElectrolyteSection, Medical Service, Philadelphia Veterans AdministrationMedical Center, and Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
The toad urinary bladder is a well characterized twalatbaironlbraiadatd6litholn.cii4soteaet)yna-llelardsrsoinorcofddosohneatlfecreecma.oirtvinoIfrnoeontndtdloicrehesamyc-oedtfiulsnyordldeocsdaiioimtprudwnilsrociDieconennitds,gogetusNhtoliibenntaiicstscnn+oee(ihda7nltnlrid0uubtoa,chl0innaetu0e-srr0nbpsinatoinofmoattrrarhta8triirlfc0yAaietf,ir0IriesobPi0rcnp0lciaccsos)rdyoionnanndeotscepctemiftshierinitAtneseoehsI.ms-nn,ePiTst--ohfetaisccrhteuaerenlcanilscatytleal;yawsvet,nspheafrioiiuoetlsnlhabblibevAynelliiieoInfuadPuogmesrstc,hmeichanacoenogdtnaaiidlnccesmegousgfmrooiiArlnnireotcIgcuaePunpslNtsaulsiorycaymfh+nwmptsiehlontteiurenvgadsaiehnitnistsnoestdrosiptegf.oosdaae1rHtdtn2oeos,rlti0iewuhsnt0ciriev0sti(rvn.e8opea)prrtre,ehhypcyauahsdbnrinbltoaaiietlqldoieruodngeenei--s,rs synthesis appears to be mineralocorticoid-specific.The Weinitially attempted to identify aldosterone-induced proteins (AIPs) in total cellular relationship of AIP synthesis to Na+ transportwas homogenates of epithelial cells derived from single toad uristudied
Putative AIPswere identified in the cytosolic (120,000 X g supernatant) and in one of the membrane-rich fraction(s120,000X g pellet); these proteins had molecular weights of-70,000 and -15,000, respectively [14].No AIPswere evident in the other membranerich (15,000X g pellet) fraction [14].the resolution achieved with one-dimensional electrophoresis was not sufficientlyprecisefor this procedure to be used in an extensive series of studies correlating thephysiological and biochemical effects of aldosterone

Summary

Introduction

Sion’’model of steroid hormone action (1,A2)l.dosterone (and We employed subcellular fractionation and one-dimenother adrenocortical steroids) bind to a single cytosolic and two nuclear receptors, and occupation of t h e high affinity nuclear receptor correlates with the natriferic acotifvtihtyese hormones [3,4].Aldosterone stimulates the synthesiosf RNA withcharacteristics of messenger-RNA; RNAsynthesis is inhibited by specific mineralocorticoid antagonists (spirolactones) and is not stimulatebdy inactive analogues of aldosterone (l7a-isoaldosterone) or by non-natriferic concentrations of glucocorticoids (cortisol) (’46).Most of the older evidence that the natriferic effect of aldosterone is mediated by new protein synthesis rests on inhibitor stud(ie7s). Putative AIPswere identified in the cytosolic (120,000 X g supernatant) and in one of the membrane-rich fraction(s120,000X g pellet); these proteins had molecular weights of-70,000 and -15,000, respectively [14].No AIPswere evident in the other membranerich (15,000X g pellet) fraction [14].the resolution achieved with one-dimensional electrophoresis ( of high molecular weight proteins in the membrane-rich fractions) was not sufficientlyprecisefor this procedure to be used in an extensive series of studies correlating thephysiological and biochemical effects of aldosterone

Methods

Results

Conclusion