α1-Adrenergic Receptors of A Smooth Muscle Cell Line: Guanine Nucleotides Do Not Regulate Agonist Affinities

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Abstract
Using [3H]-dihydroergocryptine, we have identified in membranes prepared from the DDT1 MF-2 smooth muscle cell line a binding site with characteristics of the α1-adrenergic receptor. Specific binding (90–95% of total binding) was saturable with a binding site concentration of 197±44 fmol/mg protein and was of high affinity with a dissociation constant of 1.7±0.4 nM. The order of agonist competition for the binding site was epinephrine (Ki=2.3±0.5μM) ≥ norepinephrine (Ki=4.4±1.3μM) ≫ isoproterenol (Ki=195.5±27.6μM), consistent with an α-adrenergic interaction. Computer modelling of competition curves obtained with prazosin (α1-selective) and yohimbine (α2-selective) indicated that the DDT1 cell αa-adrenergic receptor was predominantly (>95%) of the α1-subtype. Guanine nucleotides, either GTP or 5'-guanylylimidodiphosphate, did not reduce the affinity of either epinephrine of phenylephrine for the [3H]-dihydroergocryptine binding site.