DEMONSTRATION OF BETA-2 ADRENERGIC-RECEPTORS OF HIGH COUPLING EFFICIENCY IN HUMAN NEUTROPHIL SONICATES

  • 1 January 1980
    • journal article
    • research article
    • Vol. 96  (1) , 15-23
Abstract
Using highly purified (> 95%) neutrophil (PMN [polymorphonuclear leukocyte]) sonicates, .beta. adrenergic (.beta.-1/.beta.-2) pattern of adenylate cyclase activation after agonist stimulation and the coupling characteristics of the .beta. adrenergic receptor to the adenylate cyclase enzyme were studied. Adenylate cyclase was highly responsive to agonist activation, with peak isoproterenol (100 .mu.M) stimulation causing the generation of 119 .+-. 9.5 (mean .+-. SEM [SE of mean]) pmol/mg per min cyclic[c]AMP (224% above basal levels) compared to 171.7 .+-. 8.6 following NaF (10 mM) stimulation. The agonist pattern of adenylate cyclase activation suggested the presence of .beta.-2 adrenergic receptors, as isoproterenol with a Kact [activation constant] of 0.7 .mu.M was more potent than epinephrine (Kact = 8.5 .mu.M) or norepinephrine (Kact = 90 .mu.M). Butoxamine (.beta.-2 antagonist) was .apprx. 25 times more potent than practolol (.beta.-1 antagonist), with Kds of 0.75 and 17.5 .mu.M, respectively. Receptor coupling efficiency was determined by measuring isoproterenol binding and adenylate cyclase activation with the same PMN sonicates and incubation conditions for each assay. The apparent Kd for isoproterenol binding was 2.82 .+-. 0.53 .mu.M, the Kact was 0.47 .+-. 0.05 .mu.M and the mean Kd/Kact ratio was 6.5. Highly coupled receptor-enzyme systems have ratios .gtoreq. 1. Using sucrose gradient-purified PMN sonicates, isoproterenol required the guanine nucleotides GTP or Gpp(NH)p [5''-guanylyl-imidodiphosphate] to activate adenylate cyclase. The GTP effect on adenylate cyclase responsiveness to isoproterenol was associated with a 10-fold decrease in the isoproterenol binding affinity. Apparently the human PMN has .beta.-2 adrenergic receptors which are highly coupled to the adenylate cyclase enzyme. PMN sonicates a suitable model for the study of .beta.-2 adrenergic receptors and the mechanism of hormone-induced adenylate cyclase activation in man.