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Glucagon and prostaglandin E 1 stimulate adenylate cyclase in Madin-Darby canine kidney cells with an approximate EC 50 of 3 ∗ 10 −8 and 1 ∗ 10 −7 M respectively. The rise in cAMP is accompanied by a transient rise in intracellular Ca ++ measured with the fluorescent calcium indicator Indo-1. A comparable increase in intracellular Ca 2+ without a rise in cAMP occurs with the cholinergic agonist carbamylcholine. Stimulation of adenylate cyclase by the β-adrenergic agonist isoproterenol or directly by forskolin has no effect on intracellular Ca ++ . By all criteria studied the rise in intracellular Ca ++ and the increase in cAMP are independent from each other.

When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, the amount of degradation was negligible. At 30 degrees C, acid-washing did not remove specifically bound ligand; thus, it was assumed that the ligand was internalised at this temperature, since some of the specifically bound ligand could be washed off at lower temperatures. This was confirmed in experiments when monolayer cultures of hepatocytes were incubated with fluorescein-labelled derivatives of glucagon. The distribution of specific binding on the cell surface was studied at both 30 and 4 degrees C using video intensification microscopic techniques. In keeping with studies using radiolabelled glucagon, more fluorescence was detected following incubation at 4 degrees C than at 30 degrees C and it could be removed by washing the cells. Video intensification microscopy indicated that at the lower temperature, the bound ligand was distributed all over the cell surface. At the higher temperature, ligand-derived fluorescence could only be detected in mobile intracellular vesicles.

Anti-peptide antibodies, raised against the N-terminal sequence (amino acids 2-10) of the turkey beta 1-adrenoceptor [Yarden et al., Proc. Natl. Acad. Sci. USA (1986) 83, 6795-6799] recognized the 50 kDa- but not the 40 kDa-form of the receptor, thus confirming the previous assumption that the N-terminus of the 50 kDa form is lost during its conversion to the 40 kDa-form [Jür beta, R., Hekman, M.Helmreich, E.J.M. (1985) Biochemistry 24, 3349-3354]. By in situ proteolysis small amounts of receptor fragments were formed, which could be recognized by the N-terminus specific antibody. Therefore, although the production of the stable 40 kDa receptor species by proteolytic removal of a portion of the N-terminal appears to be the predominant route, there exists an additional pathway of degradation which must involve the initial cleavage of the carboxyl terminal.

The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC 50 values of 6.8·10 −9 , 1.7·10 −8 , 1.8·10 −8 and 5.4·10 −9 M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp 25 -glucagon and native glucagon, respectively. From the IC 50 values K d values of 2.16·10 −9 , 4·10 −9 , 2·10 −9 and 1.72·10 −9 M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp 25 -glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp 25 -glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp 25 -glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp 25 -glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp 25 -glucagon and 2-thiolTrp 25 -glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp 25 -glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.