Receptor binding of glucagon and adenosine 3′,5′-monophosphate accumulation in isolated rat fat cells

• The binding of glucagon and its effect on adenosine 3′,5′-monophosphate (cyclic AMP) accumulation and glycerol release was assessed in isolated rat fat cells at 37°C. • The dissociation constant of the displaceable (saturable, specific) binding was about 1.5 nM. The rate constant of dissociation ( k −1 ) varied from 8 8.6 · 10 −4 s −1 to 3.3 · 10 −3 s −1 , i.e. T 1 2 varied from 800 s to 200 s. Within an experiment, k −1 was the same when cells with prebound 125 I-labelled glucagon were suspended in medium containing either no glucagon or 1 μM of unlabelled glucagon. Negative homotropic cooperativity therefore seemed absent. The half-time of association of 70 pM 125 I-labelled glucagon was of the same order of magnitude as that of dissociation. • The conversion of prelabelled ATP to labelled cyclic AMP was enhanced by glucagon with a time lag of less than 20 s. The maximal accumulation was reached at 2–5 min both in the absence and in the presence of 2.5 mM theophylline. The concentration of glucagon causing half-maximal accumulation of cyclic AMP at 5 min was about 2 nM both in the absence and in the presence of theophylline. Glucagon stimulated glycerol release half-maximally at a concentration of about 2 nM in 1 h incubations. • The des-His 1 -glucagon exhibited about half of the maximal effect of glucagon on cyclic AMP accumulation even though it was able to inhibit binding of 125 I-labelled glucagon to the same extent as glucagon. Half-maximal displacement and half-maximal effect were obtained with about 200 nM of des-His 1 -glucagon. • The results are compatible with the following model for the action of glucagon on adipocytes. The adenylyl cyclase is stimulated in a dose-dependent fashion at the low receptor occupancies obtained 20 s after the addition of glucagon in submaximally stimulating concentrations. The increase in cyclic AMP concentration is antagonized so that the maximal accumulation with a given concentration of glucagon is obtained after a few minutes even though the receptor occupancy continues to increase. The histidine residue is necessary for maximal activation of the adenylyl cyclase.