Molecular analysis of [beta]-adrenergic receptor subtypes in the rat
collecting duct. effects on cell camp and ca2+ levels..
Mandon, Beatrice, Esteban Siga, Agnes Champigneulle, Martine Imbert
-Teboul, and Jean-Marc Elalouf.
Departement de Biologie Cellulaire et Moleculaire, Service de
Biologie Cellulaire, CEA Saclay, 91191 Gif sur Yvette cedex, France ;
College de France, Laboratoire de Physiologie Cellulaire, 11 place
Marcelin Berthelot, 75231 Paris cedex 05, France
APStracts 2:0010F, 1995.
Expression and functional properties of [beta]-adrenergic receptors ([beta]-
ARs) were studied in rat collecting tubules isolated by microdissection.
Reverse transcription-polymerase chain reaction (RT-PCR) experiments
demonstrated that the [beta]1 and [beta]2-AR mRNAs, but not the [beta]3
subtype, are expressed in the cortical collecting tubule (CCD). Quantitation
of mRNAs, carried out using mutant RNAs as internal standards, further
showed that [beta]1 and [beta]2-ARs transcripts are present at comparable
amounts in CCD (3,000-4,000 copies /mm of tubular length), but reach
6-8 times lower levels in the outer medullary collecting tubule
([beta]1:480+/-180; [beta]2: 590+/-110). Functional studies, carried out in
CCD, corroborated the expression of these two receptor subtypes. The rank
order of potency of [beta]-agonists for stimulating cAMP accumulation was
isoproterenol > noerepinephrine= epinephrine, and similar
efficiencies were found for a [beta]1 and a [beta]2 antagonist to inhibit
isoproterenol-dependent cAMP formation. Fura-2 fluorescence
measurements revealed that isoproterenol (10[mu]M) induces a biphasic
rise of intracellular free Ca concentration ([Ca2+]i),
consisting of an initial fast increase (_Ca=122nM) followed by a
plateau phase (_Ca=58nM). In the absence of basolateral Ca, the
initial peak was still observed, suggesting intracellular Ca2+
release. Norepinephrine, epinephrine as well as selective [beta]1 and [beta]2
-agonists also increased [Ca2+]i in CCD. Only slight
[Ca2+]i variations were produced by isoproterenol in the outer
medullary collecting tubule(_Ca=21nM) and the cortical thick
ascending limb (_Ca=25nM). These results show that both [beta]1 and [beta]2-
ARs are expressed in the collecting tubule and that they predominate in
the CCD. The two receptor subtypes contribute to cAMP accumulation
induced by [beta]-agonists. They also trigger [Ca2+]i variations,
indicating their possible coupling to several transduction pathways
in the rat CCD.
Received 16 September 1994; accepted in final form 21 December
1994.
APS Manuscript Number F334-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 February 1995.