Characterization of alpha 2-adrenoceptors in a plasma membrane enriched fraction from the insulin-secreting cell line RINm5F.
A plasma membrane enriched fraction from the insulin-secreting cell line RINm5F was used to characterize [3H]clonidine binding. After a single self-generating Percoll gradient, the specific activity of 5'-nucleotidase (a plasma membrane marker) of the membrane fraction was enriched about 8-fold over that of the homogenate and nearly 30% of the total amount was recovered. The fraction was essentially free of mitochondria and secretory granules. [3H]clonidine binding to this membrane fraction revealed a single, high affinity binding site with a Kd of 2.3 nmol/l. The binding was competitively inhibited by adrenergic agonists in the following order of potency: clonidine greater than epinephrine greater than phenylephrine greater than isoproterenol, and by antagonists in the order of potency: idazoxan greater than yohimbine greater than propranolol greater than prazosin. Pertussis toxin pretreatment of the cells did not alter the inhibition of [3H]clonidine binding by epinephrine and clonidine nor the estimated receptor number for [3H]clonidine. In conclusion, the pharmacologic characteristics of [3H]clonidine binding sites on a plasma membrane enriched fraction from insulin-secreting RINm5F cells demonstrate that the receptor is of the alpha 2-adrenergic subtype.
Ullrich
S
S
Institut de Biochimie Clinique, Centre Médical Universitaire, Geneve, Switzerland.
Wollheim
C B
CB
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003000:Clonidine / Q000378:metabolism; D054755:Imidazoline Receptors; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D018341:Receptors, Adrenergic, alpha-2
D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D054755:Imidazoline Receptors; D007328:Insulin; D011942:Receptors, Adrenergic, alpha; D018341:Receptors, Adrenergic, alpha-2; D003000:Clonidine