Glucagon receptors on isolated hepatocytes and hepatocyte membrane vesicles. Discrete populations with ligand- and environment-dependent affinities

Analysis of glucagon and deshistidine glucagon binding to isolated canine hepatocytes and to hepatocyte membrane vesicles (formed by budding of hepatocytes in hypotonic medium) reveals two separate populations of hormone binding sites. Mathematical modeling further shows that the high affinity population represents 1% of the total in all four cases. Although calculated dissociation constants for hormone binding range from 0.2 to 400 nM, whether considering glucagon or deshistidine glucagon binding, or binding to the high affinity or low affinity receptor populations, receptor affinity increases 2- to 100-fold in the environment of the membrane vesicle; concomitant with this alteration in receptor affinity, receptor selectivity for the structure of the native hormone decreases 1.5- to 40-fold in hepatocyte-derived vesicles. Consideration of receptor affinity in relation to receptor number suggests that hepatocyte glucagon binding is distributed about equally between high and low affinity receptor populations at typical portal hormone levels. Nevertheless, consideration of receptor binding in relation to biological activity suggests that the activity of glucagon in inhibiting carbohydrate flux into glycogen is attributable to occupancy of the high affinity receptor population.     

Receptor binding and cell-mediated metabolism of [125I]monoiodoglucagon by isolated canine hepatocytes

We have developed a reverse-phase HPLC method to purify 125I-labeled products resulting from the chloramine-T-based iodination of glucagon and have used the products [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon) to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. The extent of binding of the three labeled glucagons to cell receptors differed at steady state (8.5, 11.9, and 12.6% of the three peptides, respectively, becoming cell-associated), but each of the labeled glucagons approached steady state binding at the same rate. Further, unlabeled glucagon competed for the binding of each of the labeled peptides in parallel under steady state conditions, and each of the peptides showed potent activity in inhibiting [14C]fructose incorporation into glycogen. Gel filtration of the acetic acid-extracted, cell-associated products of radiolabeled glucagon binding revealed 10-20% of the material as a shoulder on the descending limb of the peak of hormone for each of the three labeled peptides. Trypsin digestion of the lower molecular weight peptide derived from [(125I)iodoTyr13]glucagon resulted in a fragment containing residues 13 to 17 as the only detectable radiolabeled product. On the other hand, trypsin digestion of the analogous peptide derived from [(125I)iodoTyr10]glucagon revealed, in addition to the radiolabeled fragment containing residues 1 to 12, a major fragment identified by radiosequence analysis to contain residues 4 to 12 and a minor fragment identified to contain residues 7 to 12. We conclude that (a) notwithstanding apparent differences in affinities exhibited by [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon for binding to canine hepatocytes, the interactions of all three peptides with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH2-terminal region of the hormone has been modified by limited proteolytic cleavage.     

Analysis of glucagon-receptor interactions on isolated canine hepatocytes. Formation of reversibly and irreversibly cell-associated hormone

We have investigated the interactions of ligand with the canine hepatic glucagon receptor. Whereas time courses for radiolabeled glucagon binding to receptor and dissociation from receptor revealed fast and slow components at both 30 and 4 degrees C, time courses of ligand dissociation revealed a third component of irreversibly cell-associated (nondissociable) ligand only at the higher temperature. Related experiments identified that (a) the initial rate of formation of nondissociable ligand was slower than that of dissociably bound hormone; (b) the fraction of ligand bound to nondissociable sites achieved a plateau during extended incubations, whereas that bound to dissociable sites was seen to rise and then slowly to fall; (c) the kinetics of formation of a nondissociable ligand was consistent with linked, sequential reactions; (d) dissociable ligand-receptor complexes formed at 4 degrees C were converted to nondissociable complexes during subsequent incubation at 30 degrees C, and (e) nondissociable sites were filled by prior incubation of cells with unlabeled ligand. Analysis of receptor-bound hormone resulting from the incubation of cells with 125I-labeled glucagon and selected concentrations of either glucagon or [[127I]iodo-Tyr10]glucagon at steady state revealed in each case four components of receptor-bound ligand: those corresponding to high and low affinity components of dissociably bound ligand and to high and low affinity components of nondissociably bound ligand. Implications of these findings are considered in terms of mechanisms for the formation of irreversibly bound hormone and for the distribution of hormone among the various components of hepatic glucagon-binding sites.     

Reciprocal changes of insulin and glucagon receptors in primary cultured hepatocytes

The specific [125I]insulin binding to primary cultured hepatocytes was significantly greater than that to freshly isolated hepatocytes. Low affinity insulin binding sites in cultured cells were 6-fold greater in number than those of freshly isolated cells without a significant change in high affinity sites. However, both sensitivity (insulin concentration for half maximum stimulation) and responsiveness (% of increase above the basal level) to insulin for the stimulation of ODC activity were similar for isolated and cultured cells indicating an important role of high affinity sites in the insulin action. On the other hand, the specific [125I]glucagon binding to cultured cells was significantly decreased. Low affinity glucagon binding sites in cultured cells decreased by about 50% in cultured cells without a significant change in high affinity sites. Both sensitivity and responsiveness to glucagon for the stimulation of ketogenesis from palmitate also decreased as compared with those of isolated cells, indicating an important role of low affinity sites in the glucagon action. These results indicate that insulin and glucagon receptors were reciprocally changed in cultured cells, as compared with isolated cells.     

The relationship between glucagon receptors and metabolic profile in two strains of rats: Wistar-Furth and Sprague-Dawley

We studied glucagon and insulin binding to isolated hepatocyte receptors in Wistar-Furth (WF) and Sprague-Dawley (SD) rats, using 125I-labeled hormones. Hepatocytes from WF rats bound more glucagon than hepatocytes from SD rats. There were no differences in insulin binding. These observations prompted us to investigate other strain differences. Fasting and nonfasting serum glucose, glucagon, insulin, and growth hormone were measured. WF animals had a lower fasting glucose and higher fasting glucagon than SD animals, while SD rats had higher nonfasting insulin levels and a higher hepatic glycogen content. Total hepatic glucose production in response to glucagon (10(-8) M) was greater in WF than in SD rats, while glucagon-stimulated gluconeogenesis from alanine was the same in the two groups of animals. We concluded that the decreased glucagon binding does not play a significant role in the maintenance of serum glucose or in the gluconeogenetic response glucagon, and that neither these responses nor the serum glucagon levels appears to be correlated with the number of glucagon receptors. We conclude further that different animal strains of the same species may differ in their biologic responses.     

A quantitative analysis of glucagon binding to isolated intact neonatal and adult rat hepatocytes on the basis of two different binding models

The interaction of glucagon with specific receptors has been studied in isolated intact neonatal and adult rat hepatocytes. The hormone binding measured directly with ¹²⁵I-labelled glucagon was saturable and reversible. The ¹²⁵I-labelled glucagon binding was inhibited by unlabelled homologous hormone at concentrations ranging from 0.5 nM to 50 μM. Two different binding models were assumed to analyse the binding data by a nonlinear least-squares procedure: (I) a single class of independent sites and (II) two classes of independent sites. The comparison of the fitted theoretical curves reveals that both binding models are in fact compatible with these data. Adult hepatocytes have a considerably higher affinity for glucagon than neonatal hepatocytes; the binding capacity of neonatal liver cells from 1–7-days-old rats proved to be markedly reduced compared with the cells from adult rats. The glucagon-induced intracellular cyclic AMP production was measured at various hormone concentrations under conditions identical to those for the determination of extracellular hormone binding. The correlation of both parameters indicates a direct connection between receptor-occupancy and adenylate cyclase stimulation. These results suggest that a decrease receptor concentration in neonatal hepatocytes is responsible for the decreased cyclic AMP production.     

Epidermal growth factor (urogastrone)-stimulated gluconeogenesis in isolated mouse hepatocytes

In freshly isolated mouse hepatocytes obtained from fasted animals, we have studied the receptors for epidermal growth factor urogastrone (EGF-URO) in terms of the electrophoretic profile, ligand affinity, and numbers of EGF-URO receptors present on the cells, and also in terms of the ability of EGF-URO to stimulate gluconeogenesis, as reflected by the increased incorporation of [3-14C]pyruvate into glucose. The effects of EGF-URO were compared with those of glucagon. Ligand-binding studies revealed that the mouse hepatocytes possess an unusually high number of EGF-URO receptors (about 3 X 10(6) binding sites/cell), with a ligand dissociation constant of 4.4 nM. The binding of EGF-URO by mouse hepatocytes was more than 10-fold higher than the previously measured binding of EGF-URO by rat hepatocytes. Crosslink-labeling studies, coupled with gel electrophoretic analysis, demonstrated the presence of intact EGF-URO receptors, although some receptor processing had occurred during the isolation procedure. EGF-URO was able to stimulate the incorporation of 3-14C-labeled pyruvate into glucose; glucagon was unable to do so. In contrast, in rat hepatocytes isolated and assayed under identical conditions, glucagon (10 nM) caused a marked (250%) stimulation of the incorporation of pyruvate into glucose. Maximally, EGF-URO caused a 34% increase in the incorporation of [3-14C]pyruvate into glucose; a half-maximal effect was observed at a concentration of 2.5 nM EGF-URO. The stimulatory effect of EGF-URO was not dependent on the concentration of pyruvate, lactate, glucose, or calcium in the incubation medium. Although raising the concentration of pyruvate in the incubation medium increased the incorporation of [3-14C]pyruvate into glycogen, EGF-URO did not cause any change in the incorporation of radioactivity into glycogen. Overall, our data point to marked differences between rat and mouse liver preparations, in terms of the hormonal regulation of glucose metabolism, and our work documents a potential role for the remarkably high number of mouse hepatocyte EGF-URO receptors in terms of the modulation of gluconeogenesis in the mouse.     

Effect of insulin on glucagon binding to isolated rat epididymal adipocytes

Effect of insulin on glucagon binding to rat epididymal adipocytes was studied in vitro. [125I]iodoglucagon binding to isolated adipocytes was increased by preincubation of the cells with insulin. Maximal increase was observed with 7 X 10(-10) M insulin. In Scatchard analysis, [125I]iodoglucagon competition data generated one binding site with a single affinity for glucagon binding in the cells pretreated with buffer alone. Pretreatment of the cells with insulin increased the affinity without changes in the number of binding sites. [125I]iodoglucagon binding to isolated adipocytes was not affected by pretreatment of the cells with luteinizing hormone, follicle-stimulating hormone, growth hormone, or with prolactin. These results suggest that insulin stimulates glucagon binding to adipocytes.     

Identification of a Mg(2+)- and guanyl nucleotide-dependent glucagon receptor cycle by use of permeabilized canine hepatocytes.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.     

Effect of Escherichia coli lipopolysaccharide on the glucagon and insulin binding to isolated rat hepatocytes

Summary Number and affinity constant of low affinity binding sites of insulin and glucagon to isolated hepatocytes decreased when the cells were incubated with Escherichia coli 0111:B4 lipopolysaccharide. This effect agrees with a non-specific binding of lipopolysaccharide to hepatocytes, similar to the well-recognized non-specific binding of albumin. Also, binding of different lectins to their glycoprotein receptors did not affect the [¹⁴C]lipopolysaccharide interaction with the cell membrane surface. Endotoxin depresses gluconeogenesis from lactate when the precursor was incubated with the cells for short time intervals. The longer the preincubation interval with lipopolysaccharide, the higher the inhibition of gluconeogenesis in the absence and in the presence of glucagon.The effect of endotoxin was also studied on the glucagon-induced synthesis of cyclic AMP and the glucagon binding. Levels of cyclic AMP and hormone binding decreased with increasing both endotoxin concentrations and preincubation intervals at which cells were in contact with endotoxin.     

N-ethylmaleimide uncouples the glucagon receptor from the regulatory component of adenylyl cyclase

125I-Glucagon binding to rat liver plasma membranes was composed of high- and low-affinity components. N-Ethylmaleimide (NEM) and several other alkylating agents induced a dose-dependent loss of high-affinity sites. This diminished the apparent affinity of glucagon receptors for hormone without decreasing the binding capacity of membranes. Solubilized hormone-receptor complexes were fractionated as high molecular weight (Kav = 0.16) and low molecular weight (Kav = 0.46) species by gel filtration chromatography; NEM or guanosine 5'-triphosphate (GTP) diminished the fraction of high molecular weight complexes, suggesting that NEM uncouples glucagon receptor-N-protein complexes. Exposure of intact hepatocytes to the impermeable alkylating reagent p-(chloromercuri)benzenesulfonic acid failed to diminish the affinity of glucagon receptors on subsequently isolated plasma membranes, indicating that the thiol that affects receptor affinity is on the cytoplasmic side of the membrane. Hormone binding to plasma membranes was altered by NEM even after receptors were uncoupled from N proteins by GTP. These data suggest that a sensitive thiol group that affects hormone binding resides in the glucagon receptor, which may be a transmembrane protein. Alkylated membranes were fused with wild-type or cyc- S49 lymphoma cells to determine how alkylation affects the various components of the glucagon-adenylyl cyclase system. Stimulation of adenylyl cyclase with fluoride, guanylyl 5'-imidodiphosphate, glucagon, or isoproterenol was observed after fusion of cyc- S49 cells [which lack the stimulatory, guanine nucleotide binding, regulatory protein of adenylyl cyclase (Ns)] with liver membranes alkylated with 1.5 mM NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of glucagon receptors of rat hepatocytes: a synthetic peptide probe for the high affinity site

A glucagon analog with the following sequence has been synthesized: His- Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg -Leu-Gln-Glu-Phe-Leu-Gln-Trp-Ala-Leu-Gln-Thr. When interacting with rat hepatocytes, the analog mimics, in part, the activities of glucagon in receptor binding and inhibition of carbohydrate incorporation into glycogen. Comparison of the binding of the analog with that of glucagon demonstrates the existence of two distinct homogeneous populations of glucagon receptors. The synthetic analog acts as a specific probe for those receptors that have a high affinity for glucagon.     

Hormone receptors. 7. Characteristics of insulin receptors in a new line of cloned neonatal rat hepatocytes

1. A new line of cloned, differentiated rat hepatocytes (RL-PR-C) was evaluated for its usefulness as an in vitro system for studying the regulation of the insulin receptor. 2. Insulin rapidly reversibly and specifically bound to RL-PR-C hepatocytes. Binding of tracer 125I-labeled insulin, which was competitively inhibited by native insulin as well as by proinsulin and analogs of insulin and proinsulin in proportion to their biological activity, was not influenced by glucagon, corticotropin, or human growth hormone. Anti-insulin receptor serum from a patient with Acanthosis Nigricans Type B competed with 125I-labeled insulin for binding to cell surface sites. 3. Trypsinization destroyed insulin binding sites, but these were restored by incubation under growth conditions; a 75% restoration of binding sites was achieved by one cell population doubling. 4. RL-PR-C hepatocytes responded to insulin binding by an increase in glycogen synthesis from glucose. The insulin effect was maximal at 85 nM, but was detectable at lower, more physiological, concentrations. 5. Chronic exposure (for at least 3h) of hepatocytes to insulin (10(-10)--(10(-8) M) reduced by up to 60% the number of binding sites for insulin (down-regulation). Down-regulation was prevented by cycloheximide at concentration (10 micron) sufficient to inhibit markedly protein synthesis from tracer isoleucine. Recovery from down-regulation induced by native insulin at 10(-7 M or lower concentrations was complete by 18 h under growth conditions. 6. Although RL-PR-C hepatocytes spontaneously transform after about 90 population doublings, no significant differences between normal and transformed cells were observed in insulin binding characteristics and in interaction of cells with anti-insulin receptor serum. However, transformed cells exhibited a substantially reduced (maximum of 20%) down-regulation response to insulin. 7. RL-PR-C rat hepatocytes appear, for these reasons, to be a useful model system for studying the regulation of the insulin receptor.     

Uptake and transport of glucagon by rat liver: evidence for transcytotic pathway

Summary The binding of¹²⁵I-glucagon to the cell surface and the pathway of intracellular transport of this hormone by rat hepatocytes in vivo were studied by light and EM autoradiography. Radiolabeled glucagon injected into the blood stream was taken up predominantly by the hepatocytes. Negligible radioactivity was found to be associated with other cell types such as endothelial or Kupffer cells. Our results indicate that at early time points after injection glucagon has been preferentially interacting with the sinusoidal domain of the hepatocytes and found to be associated with coated pits and uncoated vesicles corresponding to endosomes. At 15–20 min time intervals glucagon grains were found within hepatocyte interior. Later, at 30 min after injection glucagon grains accumulate in the Golgi-lysosomal region of hepatocyte often in close proximity to the opening of the bile canaliculi. Accordingly a portion of internalized¹²⁵I-glucagon was found to be released into the bile thereby indicating that a transcytotic pathway may be involved in this peptide's clearance process.     

Studies of glucagon resistance in large rat adipocytes: 125I-labeled glucagon binding and lipolytic capacity

This study is concerned with potential modifications of large fat cells from adult rats (400-450 g) that make them resistant to stimulation by glucagon. The lipolytic capacity and (125)I-labeled glucagon-binding capability of these cells were compared with these properties of small glucagon-sensitive cells from young rats (130-160 g). As determined by maximal stimulation with theophylline, dibutyryl cAMP, or epinephrine, the lipolytic capacity of large cells was not markedly different from small cells, which suggests that an alteration contributing to glucagon insensitivity is not present in the enzymes involved with hormone-mediated lipolysis. Glucagon-binding studies did indicate a difference between the two cell types. Both large cells and particulate fractions from large cells bound less (125)I-labeled glucagon than small cells or small-cell particles. That diminished binding is not a consequence of glucagon degradation is indicated by the similar amounts of (125)I-labeled glucagon degraded by both cell types. The decrease in (125)I-labeled glucagon binding was not as marked as the decrease in lipolytic response to glucagon stimulation. This lack of correlation and the relationship between elevated phosphodiesterase levels and glucagon insensitivity described in the accompanying report suggest that diminished binding explains only in part the marked resistance to glucagon found in large cells.     

Receptor binding of selectively labeled (Tyr-10) and (Tyr-13)-mono-125I-glucagons and competition by homologous 127I-labeled isomers

Two monoiodinated derivatives of glucagon were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The purified (Tyr-10) and (Tyr-13)-mono-125I-labeled glucagon isomers were characterized and studied with respect to their binding to the receptors of isolated intact rat hepatocytes. The extent of steady-state binding to cellular receptor sites differed for the two labeled glucagon tracers at 37 degrees C as well as at 15 degrees C with (Tyr-10)-mono-125I-glucagon displaying higher receptor binding. The apparent equilibrium constants, Kd,app at 37 degrees C are 3.6 +/- 0.4 nM (mean +/- S.E. of three independent experiments) for the tyrosine-13-labeled tracer and 5.9 +/- 0.6 nM for the tyrosine-10-labeled glucagon with native glucagon as competitor. Since the observed Kd in the competition assay is a function of the true Kd values of the monoiodinated radioactive glucagon isomers and native glucagon, the dissociation constants were also measured with chemically identical tracer and competitor. Under these conditions, we obtained Kd values of 1.3 +/- 0.2 nM for the tyrosine-10-labeled analog and 2.0 +/- 0.2 nM for the tyrosine-13-labeled glucagon isomers confirming the higher receptor binding affinity of (Try-10)-mono-125I-glucagon. All competition curves fit the mathematical expression for a model of non-cooperative binding to a single class of receptors.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Quantitative analysis of internalization of glucagon by isolated hepatocytes

Biochemical methods have been used to quantitate total, acid-stable and acid-labile association of (mono[125I]iodoTyr10) glucagon with rat hepatocytes in suspension to evaluate internalization of glucagon and its receptors. Internalization is inhibited by low temperature, phenylarsine oxide, and by blocking receptor binding, consistent with receptor-mediated endocytosis. Approximately 30% of the total cell-associated hormone is internalized at 30 min of incubation. The rate declines until 90 min when the internalization of glucagon ceases, although the cells remain competent to internalize asialofetuin. From 90 min to 4 h, 27% of the maximum label internalized at 30 min remains within cells. The number of cell surface receptors decreases but the affinity of those remaining is unchanged. However, 1.7-2.7 surface receptors are lost to binding for each molecule of radiolabeled glucagon internalized. Uptake occurs according to a rate constant of 0.183 min-1 (t1/2 = 3.8 min). We conclude that (i) hepatocytes internalize a finite quantity of glucagon, implying the existence of undefined regulatory mechanisms; (ii) hormone is retained for greater than 2 h within cells and may play a physiological role within cells; and (iii) both occupied and unoccupied receptors become inaccessible to extracellular hormone as internalization proceeds; rapid recycling of receptors does not occur.     

Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon.

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.     

Glucagon binding by isolated chick hepatocytes

Studies have been made on the binding of 125I-glucagon by isolated chick hepatocytes. It was shown that pH and temperature dependence of the binding does not differ from that in rat hepatocytes. Optimum binding was observed at pH 7.6, the rate of binding being higher at 37 degrees C as compared to that at 20 degrees C, although the binding capacity increased with the decrease in the temperature. Unlabeled glucagon was able to compete with 125I-glucagon at the binding sites. Scatchard plot was found to be curvilinear revealing two classes of the binding sites with Kd values 10(-9) and 10(-7) M at temperatures 20 and 37 degrees C correspondingly. Earlier studies revealed in rats the binding sites of a sole class with Kd value 10(-9) M. Preincubation of cells with native glucagon results in changes of labeled glucagon binding, the effect being proportional to the concentration of native glucagon. Preincubation effect was observed at 37 degrees C, being absent at 20 degrees C; the effect was due to the decrease in the number of both high and low affinity binding sites. The presence of down-regulation of glucagon receptors in chick hepatocytes is suggested.