Identification of adenosine receptor in rat pineal gland: evidence for A-2 selectivity.
We have examined the binding of the adenosine agonist radioligands [3H]cyclohexyladenosine [( 3H]CHA), R-N6-[3H]phenylisopropyladenosine [( 3H]R-PIA), and 5'-N-ethylcarboxamido[3H]adenosine [( 3H]NECA) to membranes prepared from rat pineal gland. The results showed that the A-1-selective ligands (CHA and R-PIA) had less than or equal to 10% specific binding. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 72% specific binding of the total binding. This specific binding was insensitive to cyclopentyladenosine (50 nM) or R-PIA (50 microM). To characterize this binding, we used the N-ethylmaleimide pretreatment method. Under these conditions, this binding was of high affinity with a KD of 51 +/- 10 nM and an apparent Bmax of 1,060 +/- 239 fmol/mg of protein. Specific binding was unaffected by the presence of MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM) (-25%), a result suggesting the involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. The rank of activity of adenosine analogues in displacing specific [3H]NECA binding was NECA greater than 2-chloroadenosine greater than S-adenosyl-L-homocysteine greater than CHA. Binding was also displaced by 3-isobutyl-1-methylxanthine (IC50 = 23.6 microM). These findings are consistent with the selective labeling by [3H]NECA of an A-2-type adenosine receptor in rat pineal membranes.
INSERM U 205, Laboratoire de Chimie Biologique, Villeurbanne, France.
N
N
Sarda
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A
Gharib
D
D
Reynaud
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L
Ou
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Pacheco
D015762:2-Chloroadenosine; D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008297:Male; D010660:Phenylisopropyladenosine / Q000378:metabolism; D010870:Pineal Gland / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011983:Receptors, Purinergic / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000378:metabolism
D011983:Receptors, Purinergic; D015762:2-Chloroadenosine; D010660:Phenylisopropyladenosine; D006165:Guanylyl Imidodiphosphate; D019830:Adenosine-5'-(N-ethylcarboxamide); C027513:N(6)-cyclohexyladenosine; D012435:S-Adenosylhomocysteine; D000241:Adenosine; D005033:Ethylmaleimide