Binding of agonists and antagonists to β-adrenoceptors in rat vas deferens: relationship to functional response

TY - JOUR

T1 - Binding of agonists and antagonists to β-adrenoceptors in rat vas deferens

T2 - relationship to functional response

AU - May, John Marvin

AU - Abel, Peter W.

AU - Minneman, Kenneth P.

PY - 1985/12

Y1 - 1985/12

N2 - The properties of β-adrenoceptors in rat vas deferens were examined using radioligand binding assays of 125I-pindolol (125IPIN) and inhibition of electrically-evoked contractions of vas deferens in vitro. 125IPIN labelled a single class of high affinity binding sites with apparently mass action kinetics in membrane preparations of vas deferens with properties consistent with an essentially homogeneous population of β2-adrenoceptors. Isoprenaline inhibited electrically evoked (60 V, 1.0 ms, 0.1 Hz) contractions of vas deferens with an EC50 of 18.0±2.1 nM. KB values for antagonists in competitively antagonizing this response correlated well (r2=0.99) with the KD values for inhibition of 125IPIN binding. Inhibition of 125IPIN binding by isoprenaline, adrenaline, noradrenaline and salbutamol was determined under conditions designed to produce high and low affinity agonist binding. In the presence of 10 mM MgCl2, agonists inhibited specific 125IPIN binding with a relatively high potency and low Hill slope, while in the presence of 154 mM NaCl and 300 μM guanosine-5′-triphosphate, agonists inhibited specific 125IPIN binding with a lower potency and an apparent Hill slope closer to 1. To determine which affinity state was relevant to functional receptor stimulation, receptor density was decreased with bromoacetylalprenololmenthane (BAAM). Treatment of membrane preparations with 0.3 μM BAAM produced a 45% decrease in the Bmax for 125IPIN with no change in the apparent KD. Treatment of intact vasa deferentia with increasing concentrations of BAAM resulted in a progressive rightward shift in the dose-response curve to isoprenaline or salbutamol folowed by a decreased maximum response. KA values for isoprenaline and salbutamol in activating the functional β-adrenoceptors were compared with KI values for agonist inhibition of specific 125IPIN binding. The KA values for both agonists were not significantly different from the low affinity KI values, but were significantly different from the high affinity KI values. These data suggest that 1) a homogeneous population of β2-adrenoceptors inhibiting contraction of rat vas deferens can be labelled with 125IPIN, 2) there is a substantial β-adrenoceptor reserve in rat vas deferens; and 3) the initial event in signal transduction by β-adrenoceptors in rat vas deferens is the binding of agonists to the low affinity form of the receptor which is not complexed with the guanine nucleotide binding protein.

AB - The properties of β-adrenoceptors in rat vas deferens were examined using radioligand binding assays of 125I-pindolol (125IPIN) and inhibition of electrically-evoked contractions of vas deferens in vitro. 125IPIN labelled a single class of high affinity binding sites with apparently mass action kinetics in membrane preparations of vas deferens with properties consistent with an essentially homogeneous population of β2-adrenoceptors. Isoprenaline inhibited electrically evoked (60 V, 1.0 ms, 0.1 Hz) contractions of vas deferens with an EC50 of 18.0±2.1 nM. KB values for antagonists in competitively antagonizing this response correlated well (r2=0.99) with the KD values for inhibition of 125IPIN binding. Inhibition of 125IPIN binding by isoprenaline, adrenaline, noradrenaline and salbutamol was determined under conditions designed to produce high and low affinity agonist binding. In the presence of 10 mM MgCl2, agonists inhibited specific 125IPIN binding with a relatively high potency and low Hill slope, while in the presence of 154 mM NaCl and 300 μM guanosine-5′-triphosphate, agonists inhibited specific 125IPIN binding with a lower potency and an apparent Hill slope closer to 1. To determine which affinity state was relevant to functional receptor stimulation, receptor density was decreased with bromoacetylalprenololmenthane (BAAM). Treatment of membrane preparations with 0.3 μM BAAM produced a 45% decrease in the Bmax for 125IPIN with no change in the apparent KD. Treatment of intact vasa deferentia with increasing concentrations of BAAM resulted in a progressive rightward shift in the dose-response curve to isoprenaline or salbutamol folowed by a decreased maximum response. KA values for isoprenaline and salbutamol in activating the functional β-adrenoceptors were compared with KI values for agonist inhibition of specific 125IPIN binding. The KA values for both agonists were not significantly different from the low affinity KI values, but were significantly different from the high affinity KI values. These data suggest that 1) a homogeneous population of β2-adrenoceptors inhibiting contraction of rat vas deferens can be labelled with 125IPIN, 2) there is a substantial β-adrenoceptor reserve in rat vas deferens; and 3) the initial event in signal transduction by β-adrenoceptors in rat vas deferens is the binding of agonists to the low affinity form of the receptor which is not complexed with the guanine nucleotide binding protein.

UR - http://www.scopus.com/inward/record.url?scp=0022399583&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022399583&partnerID=8YFLogxK

U2 - 10.1007/BF00500814

DO - 10.1007/BF00500814

M3 - Article

C2 - 2869414

AN - SCOPUS:0022399583

VL - 331

SP - 324

EP - 333

JO - Naunyn-Schmiedeberg's Archives of Pharmacology

JF - Naunyn-Schmiedeberg's Archives of Pharmacology

SN - 0028-1298

IS - 4

ER -