Characterization of prazosin-sensitive alpha 2 B-adrenoceptors expressed by cultured rat IMCD cells.
alpha 2-Adrenoceptor subtype expression was investigated in cultured rat inner medullary collecting duct (IMCD) cells using radioligand binding studies, Northern blot analysis, and adenosine 3',5'-cyclic monophosphate (cAMP) assays. [3H]rauwolscine bound to a single class of alpha 2-adrenoceptors with high affinity [Kd = 1.7 +/- 0.3 nM, maximum binding (Bmax) = 45.2 +/- 10.8 fmol/mg protein]. alpha 2-Adrenoceptor ligands inhibited [3H]rauwolscine binding with a rank order of potency characteristic of interaction with the alpha 2B-adrenoceptor [inhibitory constant (Ki) values (in nM) rauwolscine (1.95) greater than ARC-239 (8.52) greater than prazosin (237) greater than oxymetazoline (30,000)]. Northern blot analysis was performed using poly(A)+ RNA isolated from 90% confluent rat IMCD cells and probes derived from alpha 2-adrenoceptor DNA sequences from the rat nonglycosylated alpha 2B-adrenoceptor and the human alpha 2A-adrenoceptor. The alpha 2B probe hybridized to a 4.2-kb band under high stringency conditions, but the alpha 2A-adrenoceptor probe did not hybridize to this band. In functional studies, the full alpha 2-adrenoceptor agonists epinephrine and UK-14,304 potently inhibited vasopressin-stimulated cAMP accumulation by 50 to 70% [half-maximal response (EC50) (in nM) epinephrine = 11.2, UK-14,304 = 6.4]. Guanabenz and clonidine were partial agonists, inhibiting cAMP accumulation by 30 to 40% and were less potent than the full agonists [EC50 (in nM) 56.0 guanabenz and 94.5 clonidine]. Epinephrine-induced inhibition of cAMP accumulation was blocked by rauwolscine, prazosin, and ARC-239 but not by the alpha 1-adrenoceptor antagonist corynanthine. We conclude that rat IMCD cells in primary culture express functional alpha 2-adrenoceptors of the alpha 2B-subtype.
Yasuda
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G
Department of Medicine, Creighton University School of Medicine, Omaha, Nebraska 68131.
Sun
L
L
Umemura
S
S
Pettinger
W A
WA
Jeffries
W B
WB
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D015152:Blotting, Northern; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D007679:Kidney Medulla / Q000187:drug effects / Q000502:physiology*; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008297:Male; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011224:Prazosin / Q000494:pharmacology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014667:Vasopressins / Q000494:pharmacology; D015016:Yohimbine / Q000378:metabolism / Q000494:pharmacology
D000316:Adrenergic alpha-Agonists; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D014667:Vasopressins; D011061:Poly A; D015016:Yohimbine; D012313:RNA; D000242:Cyclic AMP; D015232:Dinoprostone; D007545:Isoproterenol; D011224:Prazosin; D004837:Epinephrine