JBC, Vol. 251, Issue 22, 6915-6923, Nov, 1976
Identification of alpha-adrenergic receptors in uterine smooth muscle membranes by [3H]dihydroergocryptine binding
L. T. Williams, D. Mullikin and R. J. Lefkowitz
[3H]Dihydroergocryptine, a potent alpha-adrenergic antagonist, was used to
label smooth muscle membrane binding sites which have the characteristics
expected of alpha-adrenergic receptors. Binding of [3H]dihydroergocryptine
to rabbit uterine membranes was rapid and reversible with rate constants of
1.26 X 10(7) M-1 min-1 and 0.034 min-1 for the forward and reverse
reactions, respectively. [3H]Dihydroergocryptine binding was of high
affinity, with an equilibrium dissociation constant (KD) of 8 to 10 nM.
Binding was saturable with 0.14 to 0.17 pmol of [3H]dihydroergocryptine
bound/mg of protein at maximal occupancy of the sites. No cooperative
interactions among the sites were detected. The specificity of the binding
sites for a large number of adrenergic agonists and antagonists was
identical with the specificity of alpha-adrenergic responses to these
agents. The alpha-adrenergic agonist (-)-epinephrine competed for binding
with a KD of 0.23 muM. The order of potencies for several adrenergic
agonists in competing for the binding sites was (-)-epinephrine greater
than (-)-norepinephrine greater than (-)-phenylephrine greater than
(-)-isoproterenol in agreement with their alpha-adrenergic potencies. A
series of 19 phenylethylamine adrenergic agonists competed for binding in a
manner paralleling their potencies as alpha-adrenergic agonists.
alpha-Adrenergic antagonists such as phentolamine (KD = 15 nM) and
phenoxybenzamine (KD = 18 nM) potently competed for the binding sites. In
contrast, beta-adrenergic antagonists such as propranolol (KD = 27,000 nM)
and practolol (KD greater than 10(6) nM) did not have high affinity for the
binding sites. A series of ergot alkaloids competed for
[3H]dihydroergocryptine binding in a manner which paralleled their
potencies as alpha-adrenergic agents. Competition for binding sites by
alpha-adrenergic agonists and antagonists was a stereospecific process. The
(-)-stereoi somers of epinephrine, norepinephrine, and ergotamine were at
least 20- to 50-fold more potent than the corresponding (+)-stereoisomers.
Compounds devoid of significant alpha-adrenergic activity, such as
pyrocatechol, 3,4-dihydroxymandelic acid, normetanephrine, and D-lysergic
acid, did not effectively compete for [3H]dihydroergocryptine binding
sites. These rabbit uterine binding sites for [3H]dihydroergocryptine
appear to have characteristics indistinguishable from those of the
physiologically active alpha-adrenergic receptors.
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