TEE JOURNAL OF RI~LOGICAI. CHEMISTRY Vol. 247, So. 4, Issue of February 25, pp. 1211-1218, 1972

Printed ir., U.S.A.

Glucagon Receptors in ~-Cells

RIXDING OF l”“I-GJXJChGON 24-K-D ACTIVATION OF ADENYLATE CYCLASE

(Received for publication, September 1, 1971)

IRA D. GOLJ>FINE, JESSE ROTH, AND LUTZ BIRNBAUMER

From the Diabetes Section. Clinical Endocrinology Branch and Membrane Regulation Section, Laboratory of Xukitioti and Encloa-inology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Ma~~ylancl $0014

SUMMARY

Glucagon-sensitive, insulin-secreting tumors of the Syrian (golden) hamster were homogenized in 1 mM NaHC03 and subjected to differential centrifugation. The 10,000 x g particles were used for both binding of 1251-glucagon and ac- tivation of adenylate cyclase. Glucagon was the only hor- mone that activated adenylate cyclase. 1251-Glucagon bound to receptors rapidly and was competitively displaced by 1 pg per ml or less of unlabeled hormone; other polypeptide hor- mone were without effect. The glucagon concentration that produced half-maximal activation of adenylate cyclase was 10 ng per ml and half-maximal displacement of lz51- glucagon was 5 ng per ml; 2-29 glucagon, missing only the NH2-terminal histidine, bound to receptors but did not acti- vate adenylate cyclase. When 2-29 glucagon was added to native glucagon, it blocked activation of adenylate cyclase. Extracts of porcine gut that contained glucagon immunoreac- tivity also activated adenylate cyclase but were only one- tenth as potent as pancreatic glucagon; 2-29 glucagon in- hibited the effect of “gut glucagon.”

Glucagon is a direct potent stimulator of insulin release (1). Glucagon-stimulated insulin release, unlike the insulin release induced by glucose, is mediated by activation of adenylate cyclase and elevation of adenosine cyclic 3’) 5’-monophosphate (2-5). Although there are several excellent in vitro techniques for study of pancreatic P-cell function, none of these provide large amounts of tissue to study hormone receptor-adenylate cyclase interactions. In hamsters transplantable &cell tumors occur which synthesize and secrete insulin in viva and cause hypoglycemia in the host animals (6). Recently, studies from our laboratory have shown that slices of these tumors secrete insulin in response to glucagon in vitro (7). In the present study, using particulate fractions of these tumors, we measured directly the binding of glucagon to its receptor and correlated binding with glucagon-stimulated adenylate cyclase activity. Particular attention was paid to those features previously described for glucagon’s action on the liver (8-l 1).

MATERIALS AND METHODS

[32P]ATP (1 to 5 Ci per mM) was purchased from International Chemical and Nuclear, cyclic [3H]AMP1 (12 Ci per m&f) from Schwarz BioResearch, ACTH from Sigma, human albumin from Pentex, and DL-arginine and n-glucose from Nutritional Bio- chemicals. The following materials were generously provided as gifts: purified beef-pork glucagon (containing the entire 29 amino acid sequence) from Lilly; a fragment of glucagon con- taining the NHz-terminal 1-21 portion from Dr. W. W. Bromer, Lilly; a COOH-terminal portion of glucagon (amino acids 20 to 29) from Dr. K. Lubke, Schering; glucagon with the NHt-ter- minal histidine removed (2-29 glucagon) from Dr. F. Sundby, Novo; synthetic secretin from Ayerst; chromatographically pure pork insulin from Dr. P. Freychet (12) ; “gut glucagon,” an ex- tract of porcine gut which contained 600 ng of glucagon immuno- reactivity per mg of protein, from L. Heding, Novo; malignant islet cell tumors from Professor Hadley Kirkman; purified heart phosphodiesterase from Dr. G. Aurbach; and purified liver mem- branes from Dr. D. M. Neville.

Preparation of Tumors-The tumors were serially transplanted subcutaneously in Syrian hamsters (Mesocricetus auratus). Under the light microscope (Fig. 1) the tumor tissue was com- posed of cords and sheets of homogeneous small cuboidal cells which had small centrally located round or oval nuclei. They were pierced occasionally by capillaries that were surrounded by a single layer of columnar cells that had the same kind of nuclei except that they were located basally. Connective tissue was extremely sparse, and only occasional mitotic figures were ob- served. B-Granules and or-granules were not detected on stain- ing with aldehyde fuchsin and chrome- alum - hematoxylin phloxine, respectively.

When the tumors had reached 2 to 5 cm in diameter, they were excised, trimmed, and homogenized by 10 brisk strokes in a Dounce homogenizer with an A pestle at 4” in 10 volumes of 1 mM NaHC03. After filtering through two layers of cheesecloth, the homogenate was centrifuged at 600 X g for 10 min. The supernatant was centrifuged again for 10 min at 10,000 X g. The 10,000 X g pellets were washed and resuspended in 1 mM NaHC03 and either stored at -60” or lyophilized and stored at -20”.

1 The abbreviations used are: cyclic hVP, adenosine cyclic 3’,5’-monophosphate; ACTH, adrenocorticotropic hormone.

1211

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Glucagon Receptors in p-Cells Vol. 247, i?so. 4

FIG. 1. Photomicrograph of islet cell tumor. A tumor was ex- cised from a hamster 1 month after implantation, fixed in Bouin’s solution, and stained with hematoxylin and eosin. This section (X 188 magnification) illustrates the cordlike arrangement of uniform cuboidal cells. The capillary at the lower right, which is cut longitudinally, is lined by a single layer of cells that differ slightly in morphology from the other tumor cells. (See “Ma- terials and Methods” for more detailed description of the morph- ology.)

Assay of Adenylate Cyclase-Assay of adenylate cyclase was performed by an adaptation of the method of Krishna, Weiss, and Brodie (13). Unless otherwise stated, the incubation solu- tion contained, in a 60-~1 volume, 50 mM Tris-HCI (pH 7.8), 3.3 mg per ml of human serum albumin, 2.5 mM ATP, 3 to 6 X lo6 cpm of a-labeled [“‘P]ATP, 10.0 mM theophylline, 20 m&r creatine phosphate (Calbiochem), 1 mg per ml of phosphocreatine kinase (Sigma), and about 40 pg of 10,000 x g particles that had been stored frozen. Reactions were started with addition of enzyme protein. All incubations were at 30”. Incubations were ter- minated at 15 min by the addition of 100 ~1 of a solution that contained unlabeled cyclic AMP (12.5 mM) and ATP (35 mM), cyclic rH]AMP (25,000 cpm), and lauryl sulfate (1 g per 100 ml) ; the mixture was then boiled.

To separate the cyclic AMP from ATP and other nucleotides, the mixtures were applied to columns of Dowex 50-X2 (H+, 200- 400 mesh, BioRad) ; the appropriate effluent fractions were ad- sorbed twice with ZnSOd and Ba(OH)s, and the 3H- and 32P- labeled cyclic AMP in the supernatants were counted by liquid scintillation. Recoveries of cyclic [3H]AMP were about 50%.

To show that the 32P-labeled product of our adenylate cyclase assay was cyclic [32P]AMP, it was mixed with cyclic [3H]AMP and treated with purified heart phosphodiesterase as described by Marcus and Aurbach (14). After 1 hour, 93% of the 32P- labeled product and 92yo of the 3H-labeled cyclic AMP were hydrolyzed. Further, on Dowex 50 chromatography all of the a2P-labeled product eluted as cyclic AMP.

Iodination of Glucagon-Glucagon that has an average of 1 iodine atom per molecule has been shown to have the full biologi- cal activity of native hormone (8). To prepare 1251-glucagon at high specific radioactivity with minimum alterations, the con- centrations of all reactants were kept relatively high, and the amount of chloramine T, the oxidizing agent, was reduced markedly below that usually used to iodinate for radioimmuno-

TABLE I Adenylate cyclase activity in freshly prepared whole tumor

homogenates, 600 X g particles, 10,000 X g particles, and 10,000 X g supernatants

In this experiment tumors were homogenized, fractionated, and used fresh. Each tissue preparation was incubated with and with- out glucagon (10 fig per ml) for 15 min at 30” in a total volume of 60 ~1 (see under “Materials and Methods”). Results, which represent the mean i S.D. for triplicate determinations, are ex- pressed as picomoles of cyclic AMP produced per mg of protein per 15 min.

Preparation Protein

w/ml

Homogenate. . . 0.96 600 X g particles.. 0.134 10,000 X g particles. . . . 0.234 10,000 X g supernatant. 0.67

- I Cyclic AMP produced

Glucagon absent

gicomoles/mg protein/l5 min

314 f 36 589 f 20 126 f 3 294 f 49 611 f 93 1312 f 51

88 f 36 78 f 27

assay; the hormone, iodide, and chloramine were all present at about 5 x 10m5 M. Typically, the following were added in suc- cession: 10 ~1 of glucagon (1 mg per ml in 0.1 N NaOH) ; 20 ,ul of 0.3 M sodium phosphate buffer, pH 7.4; 4 to 5 mCi of i2?I- sodium (Union Carbide) in 20 ~1 of 0.1 N NaOH; 10 ~1 of chlor- amine T (60 pg per ml in phosphate buffer) ; 5 ~1 of sodium metabisulfite (400 pg per ml in phosphate buffer), and 100 ~1 of human serum albumin (50 mg per ml in phosphate buffer). About 50% of the radioactivity was incorporated into the hor- mone, measured as radioactivity precipitated in 5% trichloro- acetic acid.

To purify the r251-labeled product, the iodination solution was applied to a l-ml column packed with powdered cellulose (What- man and Reeve Angel Company) that had been washed with Verona1 buffer, 0.1 M, pH 8.5. Washing the column with Verona1 buffer eluted about 60% of the applied radioactivity, which represented unreacted iodide and degraded hormone. When the radioactivity in the effluent reached low levels, the i251-gluca- gon was eluted by the application of 10 ml of human serum al- bumin, 50 mg per ml in Verona1 buffer. About 10% of the applied radioactivity, which was equivalent to about 25% of the intact 1251-glucagon that had been applied to the column, was recovered in the albumin effluent. This material which theoretically had 0.5 atom of iodine per molecule of glucagon, or about 250 PCi per pg was used for the binding studies; 90% or more of the radioactivity was bound by a high concentration of highly purified liver plasma membranes (12), absorbed to the origin on chromatoelectrophoresis on Whatman No. 3MM paper, absorbed to 50 mg of talc tablets (Gold Leaf Pharmacal Co.), and was precipitated by 5% trichloroacetic acid.

Binding of r25I-Glucagon-Assays were performed in 300 ~1 of Tris buffer (50 mM, pH 7.8) that contained 20 mg per ml of human serum albumin, 100 pg per ml (3 X lo-l1 M) of 1251-glu- cagon, and 300 1.18 of 10,000 x g particles that had been stored frozen. All incubations were at 30”. After 15 min, 200 ~1 were transferred to plastic micro test tubes. The tubes were then centrifuged in a Beckman microcentrifuge for 5 min at 4”. The supernatants, removed by aspiration with a 20-gauge needle attached to a vacuum, were discarded. The tubes which con- tained the hormone bound to the particles were counted. This

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Glucogon l/i/l

TOTAL PROTEIN cpg)

MINUTES OF INCUBATION

FIN. 2. Top, glucagon-stimulated adenylate cyclase as a func- tion of protein content; 10,000 X g particles were incubated in a total volume of 60 bl in the presence and absence of glucagon (10 pg per ml) for 1.5 min at 30” (see “Materials and Methods”). Cyclic AMP production, expressed as picomoles of cyclic AMP produced per 15 minis plotted as a function of the amount of tissue protein, expressed as micrograms in the incubation tube. Each point represents the mean for triplicate determinations. Bottom, glucagon-stimulated adenylate cyclase as a function of duration of incubation. Conditions are as above: the tissue Drotein was 40 rg per tube. Cyclic AMP production, expressed as total pico- moles of cyclic AMP produced per mg of protein is plotted as a function of the duration of the incubation. Each point is the mean of triplicate determinations.

separation procedure is a modification of the method of Rodbell et al. (8). Bound and free hormone were also separated on Oxoid filters (Amersham) and similar results were obtained. When assays were performed in the presence of an excess of un- labeled glucagon (35 pg per ml), 2 to 4% of the total lzsI radio- activity was “bound” and presumably represented “nonspecific” adsorption.

RESULTS

Glucagon-sensitive adenylate cyclase activity was detected in fresh tumor homogenates as well as in freshly prepared 600 x g

= Glucagon + GTP 0

z Li

c

IOOl 10-C

FIG. 3. The effect of ATP and GTP on adenylate cyclase ac- tivity. In this experiment 10,000 X g particles (115 rg of tissue protein per tube) were incubated in 60 ~1 total volume for 2+ min at 30” in the presence of ATP (1.3 pM to 1 mM) in the presence and absence of glucagon (IO pg per ml), fluoride (10 mM), and GTP (10 PM). The ATP regenerating system and the Mg2+ concentra- tion (5 mM) were constant (see “Materials and Methods”). At each ATP concentration the stimulation of cyclic AMP production is expressed as a percentage of that obtained in the presence of ATP alone (“control”). Results are the mean of duplicates.

and 10,000 x g particles (Table I). The small amount of adenylate cyclase activity present in the 10,000 X g supernatant was unaffected by the addition of glucagon. Since the 10,000 x g particles had the highest content of enzyme per mg of pro- tein and were stable with storage up to 4 months, we used 10,000 x g particles for all subsequent experiments. Both in the pres- ence and absence of glucagon, the production of cyclic AMP was linear up to 40 pg of protein and up to 15 min of incubation (Fig. 2). Elimination of either the ATP regenerating system or theophylline caused a marked fall in cyclic AMP accumula- tion. Halving or doubling the Mg*:ATP ratio had little effect on adenylate cyclase activity.

Because it had been reported with liver membranes that ATP and GTP affect both glucagon and fluoride stimulation of ade- nylate cyclase (lo), the effects of these nucleotides were studied in @-cell particles (Fig. 3). With ATP at low concentrations, glucagon stimulation of adenylate cyclase was only slightly above the control. The ratio of glucagon to control activit.y increased from slightly more than 1 with ATP at 1.3 x lop6 M

to greater than 2 with ATP at 1O-3 M. In contrast, fluoride- control activity was about 4 at the low concentration of ATP and fell to less than 2 at the higher concentration of ATP (Fig. 3). GTP at 1O-5 M maintained a high glucagon-control activity which was independent of alterations in the ATP concentration. GTP, in the absence of glucagon, had no effect. Thus, in the &cell as in the liver, ATP or GTP appears to be necessary for glucagon activation of adenylate cyclase. Further, ATP inhib- its fluoride-stimulated activity in the P-cell in a manner similar to that of GTP in the liver.

Glucagon was effective at 1 ng per ml (Fig. 4) (approximately 3 x lo-lo M), which is equivalent to glucagon concentrations in the pancreatic vein (15) ; presumably the p-cells, which are nor- mally adjacent to the glucagon-secreting cells, are exposed to

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Glucagon Receptors in p-Cells

GLUCAGON-SENSITIVE ADENYL CYCLASE ACTIVITY IN BETA CELL PARTICLES

I ,,,‘ I ‘I”“‘! ““” “‘T 0 I IO 100 1000 10,000

GLUCAGON nghl

FIG. 4. The effect of glucagon and other hormones on adenylate cyclase activity. In this experiment 10,000 X g particles (40 pg of protein per tube) were incubated in a total volume 60 ~1 in the absence and presence of glucagon or other hormones for 15 min at 30” (see “Materials and Methods”). Cyclic AMP production ex- pressed as picomoles per mg per 15 min is plotted as a function of glucagon concentration. Other hormones, when present, were at 10,000 ng per ml. Results are the mean of triplicate determina- tions.

TABLE II

FIG. 5. Binding of ‘251-glucagon and displacement by glucagon and other hormones. In this experiment 10,000 X g particles (300 pg of protein per tube) were incubated in a total volume of 300 ~1 with 12SI-glucagon (100 pg per ml) in the absence and presence of unlabeled glucagon or other hormones for 15 min at 30” (see “Materials and Methods”). Other hormones, when present, were at 1,000 ng per ml. The percentage of total radioactivity re- covered in the pellet (see “Materials and Methods”) is plotted as a function of hormone concentration. Note that 80% of the bound radioactivity was displaced by glucagon at 1,000 ng per ml. Points represent the mean of duplicates.

‘25I-Glucagon binding in freshly prepared whole tumor homogenates, 600 X g particles, and in 10,000 X g supernatants COMPARISON OF 9 GLUCAGON BlNDlNG END GLVCAGON-SENSITIVE

In this experiment, tumors were homogenized, fractionated, and used fresh. 1251-Glucagon (6200 cpm equivalent to 30 pg) was incubated with each tissue fraction for 15 min in a total vol- ume of 300 ~1 (see under “Materials and Methods”) in the pres- ence and absence of excess unlabeled glucagon (16 pg per ml). Total binding is radioactivity recovered in the pellet in the ab- sence of unlabeled hormone. Specific binding equals total bind- ing minus radioactivity in the pellet in the presence of an excess of unlabeled hormone. Results which are expressed as counts per minute per mg of protein are the mean of duplicate determi- nations.

Preparation Protein

m&T/ml

Homogenate. 0.96 600 X g particles. 0.134 10,000 X g particles. 0.234 10,000 X g supernatant.. 0.67

Binding of I’W-glucagon

Specific

cpm/mg firotein 306 1,449 21

1,690 4,451 38 2,799 6,280 44

0 700 0

much higher levels. Maximal activation was seen at 1 pg per

ml. Two other substances that also stimulate insulin release, glucose (16 mM) and arginine (16 mM), as well as insulin itself (10 pg per ml), did not alter adenylate cyclase activity either in the absence or presence of glucagon at half-maximal stimulatory

concentrations (30 ng per ml). ACTH, growth hormone, se- cretin, and pentagastrin also failed to stimulate adenylate cy-

clase activity (Fig. 4).

Vol. 247, so. 4

BINDING OF ‘=I GLUCAGON TO BETA CELL PARTiCLES

L

0 I IO 100 1000

GLUCAGON “g/ml

d I IO 100 IO

GLUCAGON w/ml

00

FIG. 6. Comparison of glucagon stimulation of adenylate cy-

clase and glucagon inhibition of %L-glucagon binding. Data from Figs. 4 and 5 were replotted for comparison. The 0% and 100% represent binding and adenylate cyclase activation in the absence of unlabeled glucagon and in the presence of unlabeled glucagon at 1000 ng per ml, respectively.

rz51-Glucagon was bound to whole tumor homogenates as well as to 600 x g and 10,000 x g particles; the binding of rz51-gluca- gon was displaced by unlabeled glucagon (Table II). Specific binding was highest in the 10,000 x g particles.

As shown in Tables I and II, the whole homogenate had relatively low specific binding of i251-glucagon but had relatively

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I I I I I J I 5 IO 16 20 30

MINUTES OF INCUBATION

FIG. 7. The time dependence of binding and dissociation of ‘251-glucagon. In this experiment three tubes were prepared, each of which contained 1251-glucagon (100 pg per ml) and 10,000 X g particles (1,000 pg of protein per ml). An excess of unlabeled glucagon (35 fig per ml), in a negligible volume, was added to one tube at zero time and to one tube at 15 min; one tube never re- ceived unlabeled hormone. Incubations were at 30”, and at the designated time points, duplicate 200-4 aliquots were removed for analysis (see “Materials and Methods”). The solid line and solid points indicate binding in the absence of unlabeled glucagon; the dashed line and open circles indicate results obtained when unlabeled glucagon was added after 15 min of incubation. Zero per cent binding, which represents the binding obtained when excess unlabeled glucagon was present from time = 0, was 2.5% of the total radioactivity. One hundred per cent binding, which represents the binding observed after 10 min of incubation in the absence of unlabeled glucagon, was 10.9% of the total radioactiv- ity.

high adenylate cyclase activity. Conversely, the 600 x g particles had low adenylate cyclase activity but had increased specific lz51-glucagon binding. The lack of correlation of bind- ing and adenylate cyclase activity in these two fractions may reflect differences in the concentration of degradative enzymes among the fractions. The 10,000 x g particles had the highest specific binding per mg of protein and were stable for months on storage. The remainder of the binding experiments were performed with 10,000 X g particles that had been stored frozen. It will be noted also that the nonspecific binding of radioactivity to the 10,000 X g particles was much less with frozen than with fresh particles.

Unlabeled glucagon displaced 1251-glucagon from P-cell par- ticles (Fig. 5) ; displacement was detected with glucagon at 1 ng per ml or less, and maximum displacement was observed at 1 pg per ml. ACTH, growth hormone, secretin, pentagastrin, and insulin were without effect. When glucagon binding and glu- cagon activation of adenylate cyclase were compared (Fig. 6), similar curves were obtained that differed only slightly in their sensitivity to unlabeled glucagon; the half-maximal effect on binding was detected at 5 ng per ml and for activation of ade- nylate cyclase at 10 ng per ml. These data suggest a close rela- tionship between the two functions.

The binding of glucagon to the P-cell particles was rapid; with 1251-glucagon at 100 pg per ml (about 3 X lC@ M), over 80% of

‘*‘I GLUCAGON

‘-1 GLUCAGON +

UNLABELEDGLUCAGON

n 0

Y, , I I I I 0 300 600 900

TOTAL PROTEIN (pg)

FIG. 8. Binding of x251-glucagon as a function of tissue protein In this experiment 1261-glucagon (100 pg per ml) and increasing amounts of 10,000 X g particles (0 to 900 pg of protein per tube) were incubated in a total volume of 300 ~1 for 15 min at 30” (see “Materials and Methods”) in the presence and absence of un- labeled glucagon (35 pg per ml). Results are plotted as bound/ free 12SI-glucagon as a function of protein content, expressed as micrograms per tube. Points represent the mean of duplicate determinations.

binding occurred within 1 min and maximal binding was ob- served at 5 min or less (Fig. 7). The addition of unlabeled glu- cagon produced a prompt dissociation of the 1251-glucagon from the receptor (Fig. 7). The binding of 1261-glucagon increased as the concentration of protein was increased (Fig. 8). At the highest protein concentration used, more than 25yo of the 1251- glucagon was bound to receptor.

As in the liver system, glucagon was degraded rapidly by the P-cell particles. After incubation with 10,000 X g particles at 30” for 15 min more than half of the unbound 129-glucagon failed to adsorb to talc,2 two-thirds was soluble in trichloroacetic acid and greater than 80% had lost its capacity to bind to a fresh aliquot of B-cell particles. This rapid degradation obscures the actual sensitivity of the receptor to low concentrations of glucagon and prevents measurements of the total concentration of binding sites as well as equilibrium and kinetic constants for this system.

A glucagon analog that lacks only the NHS-terminal histidine, 2-29 glucagon, inhibited the binding of 1251-glucagon; it was about one-third as potent as native l-29 glucagon in displacing the labeled hormone from the receptor (Fig. 9). By itself, 2-29 glucagon had no effect on adenylate cyclase activity (Table III). With glucagon, at 0.03 pg per ml, a concentration that gives about half of the maximal effect on adenylate cyclase activity,

2 Purified iodinated glucagon (like insulin and other polypeptide hormones) readily adsorbs to talc. Hormone that is degraded or damaged will adsorb poorly or not at all.

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1216 G&wagon Receptors in P-Cells Vol. 247, No. 4

INHIBITION BY 2-29 GLUCAGON OF l-29 GLUCAGON

STIMULATION OF ADENYL CYCLASE

EFFECT OF GLUCAGONDERIVATIVES ON ‘=I GLUCAGON BINDING

600

I c ) l-29 mcagon IO ,wg/mi

\

2-29 Glucogor

: l-29 Glucagon 003pg/ml

I IO 100 1000 W/ml OF GLUCAGON DERIVATIVE

FIG. 9. The effects of glucagon derivatives on binding of r261- glucagon. In these experiments W-glucagon (100 pg per ml) and 10,000 X g particles (300 pg per tube) were incubated in a 300~~1 incubation volume with and without glucagon derivatives for 15 min at 30” (see “Materials and Methods”). Each point is the mean of duplicates; results of two experiments were combined. The 0% binding, which represents Is61 radioactivity bound in the presence of an excess of unlabeled l-29 glucagon (35 pg per ml), represented 1.6% of the total radioactivity in both experiments. The 100% binding, which represents iZ51-glucagon radioactivity bound in the absence of unlabeled glucagon, was 9.5% and 11.3% in the two experiments.

-t I T 1 .I .3 T!z- 3.0 10.0 L

pg/ml 2-29 GLUCAGON

FIG. 10. The inhibitory effect of 2-29 glucagon on adenylate cyclase activation by l-29 glucagon. In this experiment a series of tubes that contained 10,000 X g particles (37 pg of protein per tube) were incubated with and without l-29 and 2-29 glucagon for 15 min at 30” in a total volume of 60 ~1 (see “Materials and Meth- ods”). Results are expressed as picomoles of cyclic AMP pro- duced per 15 min per mg of protein as a function of the concentra- tion of 2-29 glucagon; each point is the mean of triplicates. The open circles (along the vertical axis) are results obtained in the absence of 2-29 glucagon. The solid line and solid points indicate results in which a fixed concentration of l-29 glucagon (0.03 pg per ml) was incubated with a variabIe concentration of 2-29 glucagon (0.03 to 10 pg per ml).

TABLE III Effect of l-29 glucagon and glucagon derivatives

on adenylate cyclase activity In this experiment, 10,000 X g particles (37 pg of protein) were

incubated in a total volume of 60 J.J in the presence and absence of glucagon (0 to 1.0 pg per ml) or glucagon derivatives (1 pg per ml) (or both) for 15 min at 30’ (see under “Materials and Meth- ods”). Results which represent the mean f S.D. for triplicate determinations are expressed as picomoles of cyclic AMP pro- duced per mg of protein per 15 min of incubation.

TABLE IV Effect of 1-29 glucagon, 2-29 glucagon, fluoride, and

methanol on adenylate cyclase activity

In this experiment, 10,000 X g particles (37 EL@; of protein) were incubated in a total volume of 60 bl with or without the above substances for 15 min at 30” (see under “Materials andMethods”). Results, which represent the mean f S. D. for triplicate deter- minations, are expressed as picomoles of cyclic AMP produced

l-29 Glucagon Glucagon derivative added

0 1 0.03 1 1.0

adml

None....................... 253 f S 405 f 14 533 f 45 2-29 Glucagon. . . 229 f 25 202 f 24 1-21 Glucagon. _. 221 f 1 415 f 50 20-29 Glucagon. 247 f 6 407 f 30

the addition of 2-29 glucagon produced progressive inhibition of the glucagon-stimulated adenylate cyclase (Fig. 10). At 0.1 pg per ml Z-29, glucagon obliterated one-half of the effect of native hormone; at 1.0 1.18 the effect of l-29 glucagon on adenyl- ate cyclase was reduced to that seen in the absence of any hor- mone. The inhibition by 2-29 glucagon was due to binding of the analog to glucagon receptor and not to a nonspecific effect on the enzyme; 2-29 glucagon was without effect on fluoride activation of the adenylate cyclase (Table IV). Glucagon se- quences 1-21 and 20-29, which had little or no effect on 125I- glucagon binding, did not affect control adenylate cyclase ac-

per mg of protein per 15 min.

Substance Concentration

-

Cyclic AMP produced

l.Orgperml 10 fig per ml

10.0 rnM

10 pg per ml 10 rnM

14.3 mgper ml

None. . l-29 Glucagon. Fluoride. . 2-29 Glucagon. + Fluoride.. . . . Methanol.. . .

179 f 4 582 rt 127 551 f 48 523 f 33

. . 2,135 f 35

tivity (Fig. 9; Table III). Secretin, whose amino acid sequence is very similar to that of glucagon, neither activated adenylate cyclase nor displaced i251-glucagon from its receptor (Figs. 4, and 5).

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EFFECT OF “GUT GLUCAGON” ON ADENYL CYCLASE ACTIVITY

IOOQ

1

0 = 600

5 1

I I I I , I I I I I I I I ,

0 I 1 , I ,111,

IO 100 1000 “GUT GLUCAGON” ng-eqv/ml

FIG. 11. The effect of gut glucagon on adenylate cyclase. In

this experiment, 10,000 X g particles (40 pg of protein per tube) were incubated in a total volume of 60 ~1 with increasing amounts of gut glucagon for 15 min at 30” (see “Materials and Methods”). Results are expressed as picomoles of cyclic AMP produced per 15 min per mg of protein as a function of gut glucagon concentration, expressed as nanograms of glucagon immunoreactivity determined with a pancreatic glucagon standard. Pancreatic glucagon (1.0 pg per ml) in this experiment produced 999 pmoles of cyclic AMP. Points represent the mean of triplicate determinations,

TABLE V Effect of guf glucagon on adenylate cyclase

In this experiment, 10,000 X g particles (40 pg of protein) were incubated in a total volume of 60 ~1 with l-29 glucagon, 2-29 glucagon, or gut glucagon for 15 min at 30” (see under “Materials and Methods”). Results, which represent the mean f S.D. for triplicate determinations, are expressed as picomoles of cyclic AMP produced per mg of protein per 15 min.

Substance COllC.5P tra tion

Cyclic AMP produced

None .................................. Pancreatic glucagon. ................. Pancreatic glucagon. .................. 2-29 Glucagon ......................... Gutglucagon .......................... Pancreatic glucagon ................... + Gut glucagon. ..................... Pancreatic glucagon. .................. + 2-29 Glucagon ...................... Gutglucagon .......................... + 2-29 Glucagon ......................

1.0 0.03

10.0 0.5 1.0 0.5 0.03

10.0 0.5

10.0

296 f 20 792 z!z 65 701 f 45 311 32 128 470 f 49 781 i 113

224 zt 49

270 f 27

Extracts of the gut contain materials that stimulate insulin release in vitro and these extracts also contain immunoreactivity that cross-reacts with antisera to pancreatic glucagon (15, 16). We examined the effects of gut glucagon on adenylate cyclase and glucagon-receptor interactions. Gut material that con- tained glucagon immunoreactivity stimulated adenylate cyclase (Fig. 11). The gut material, whose glucagon content was ex- pressed as nanograms of immunoreactivity equivalent to pan- creatic glucagon, appeared to be about IO-fold less potent than

the pancreatic glucagon in stimulating adenylate cyclase when one-half maximal concentrations were compared (Figs. 4, and 11). However, gut glucagon produced a maximal effect that was near that of pancreatic glucagon (see legend to Fig. 11). In the presence of 1 pg per ml of pancreatic glucagon, a dose that produces a maximal effect, the addition of gut glucagon produced no further effects (Table V), although the adenylate cyclase system was capable of further stimulation by methanol (17) (Table IV). Further, the effect of gut glucagon on adenyl- ate cyclase was obliterated in the presence of 2-29 glucagon (Table V), suggesting that gut glucagon and pancreatic glucagon shared similar receptors and affected the same adenylate cyclase.

DISCUSSION

The first step in the action of protein hormones is rapid re- versible binding of the hormone to a specific receptor on the plasma membrane of the target cell. With 1251-labeled hormones of high specific radioactivity, hormone-receptor interaction has been studied directly in several systems (8, 12, 18-27). An early subsequent step in the action of many hormones is the activation of adenylate cyclase, with an increase in the intra- cellular concentration of cyclic AMP, the nucleotide that medi- ates the actions of many hormones. Direct measurements of hormone binding have been correlated with hormone-activated adenylate cyclase in a few of these systems: ACTH in the adrenal (18), angiotensin in the adrenal and kidney (19), and glucagon and epinephrine in the liver (S-11, 20, 21).

The present study indicates that the relationship between glucagon binding and glucagon activation of adenylate cyclase can be studied in tissue from the insulin-secreting tumor of Syrian (golden) hamster. Since these tumors are pancreatic in origin (6), contain and secrete insulin in viva (6), and secrete insulin in response to glucagon in vitro (7), it is likely their adenylate cyclase system is similar to that of the normal P-cell.

Since the liver and pancreas are both derived from the endo- dermal epithelium of the foregut, it is not surprising that there are similarities between our studies with P-cell particles and those of Rodbell and coworkers (8-11) with liver membranes. In the P-cell, one-half maximal glucagon concentrations for displacement of 1251-glucagon binding (5 ng per ml) and glucagon activation of adenylate cyclase (10 ng per ml) were close to those seen for both functions in the liver (14 ng per ml). In both systems, l-21 glucagon, 20-29 glucagon, and secretin had little or no activity, whereas 2-29 glucagon bound to receptors but did not activate adenylate cyclase. In contrast to the liver, where rapid binding and dissociation of lz51-glucagon requires the addition of ATP or GTP, binding and dissociation in the B-cell occur very rapidly without addition of nucleotides.

Glucagon immunoreactivity has been found in the intestine of several species (28), confirming earlier studies which had de- tected the presence of glucagon-like bioactivity (29, 30). The oral intake of glucose results in higher plasma insulin levels than intravenous infusion of glucose (31). Fractions of intes- tine containing gut glucagon immunoreactivity stimulate insulin release (15, 16, 32), and plasma gut glucagon immunoreactivity is elevated by oral glucose (15). Thus, it is possible that a major role of gut glucagon is to promote insulin release. Al- though gut glucagon has not been purified, it appears to differ from pancreatic glucagon in several properties. Gut and pan- creatic glucagon react differently with antibodies to pancreatic glucagon (28, 33). Further, gut glucagon is probably hetero-

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1318 Glucagon Receptors in B-Cells Vol. 247, No. 4

geneous. On gel filtration it yields two peaks of immunoreac- tivity, only one of which is associated with glycogenolytic activity in the liver (32). On ion exchange chromatography, multiple peaks of immunoreactivity are recovered that differ in their insulinogenic potencies in vitro (16).

The extract of intestine that we used contained not only gut glucagon but probably also contained secretin, gastrin, and pancreozymin, all of which stimulate insulin release (34). How- ever, it is unlikely that these contaminants stimulated adenylate cyclase, since synthetic secretin and pentagastrin (which con- tains t,he active tetrapeptide of gastrin and pancreozymin) were without effect. Furthermore, 2-29 glucagon inhibited the effect of gut glucagon. In preliminary studies, gut glucagon inhibited 129-glucagon binding to receptors, but supplies were exhausted before detailed experiments could be performed. The role of gut glucagon in &JO needs further clarification; this hamster tumor preparation appears to be a useful tool for this purpose.

Acknowledgments-We thank Dr. ,Martin Rodbell and Dr. Stephen Pohl for communicating results of their studies prior to publication, Professor Hadley Kirkman for his gift of the hamster tumors, Mr. William Hardy for technical assistance, and Dr. Alan Rabson for microscopic examination of the tumors.

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Ira D. Goldfine, Jesse Roth and Lutz BirnbaumerACTIVATION OF ADENYLATE CYCLASE

-Cells: BINDING OF 125I-GLUCAGON ANDβGlucagon Receptors in

1972, 247:1211-1218.J. Biol. Chem. 

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