Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 208, No. 1, April 15, pp. l-10, 1981

Inhibition

by Heparin and Dextran Sulfate of Stimulated Rat Pancreatic Adenylate Cyclase

M. DESCHODT-LANCKMAN,

P. ROBBERECHT,

AND J. CHRISTOPHE

Department of Biochemistry and Nutrition, Medical School, Universitc.5 Libre de Bruxelles, Brussels, Belgium Received June 3, 1980 Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 pg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 pg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 pg/ml. Besides, heparin inhibited (I, = 6 pg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent Kd value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.

The adenylate cyclase system present in rat pancreatic plasma membranes consists of distinct hormone receptors, a common catalytic unit, and guanyl nucleotide sites which allow the transduction of the hormone-generated signal and the activity of the catalytic unit. This system is activated by the synergetic action of GTP and of a peptide belonging to either the secretin-vasoactive intestinal peptide family (1, 2) or to the cholecystokinin-pancreozymin (CCK)’ family (3,4). The combined action of the hormone and the nonhydrolyzable analog Gpp(NH)p leads to a fully activated, nonreversible “holocatalytical

state” (5-7). In this state, the coupling of the receptor and the catalytical unit is not required any more for full enzyme activity (8). The existence of distinct hormone receptors interacting with a unique effector subunit provides a good tool to study whether inhibitors of adenylate cyclase activity act already at a specific hormone receptor level or on later events. We have previously shown that the inhibition exerted by wheat germ agglutinin on the CCK-stimulated adenylate cyclase activity of rat pancreatic plasma membranes could be explained in part by the competitive inhibition exerted by the lectin on the binding of CCK (9). Recent reports from Salomon et al. (10) and Amsterdam et al. (11) indicate that heparin is a potent inhibitor of rat ovarian adenylate cyclase and reduces the binding of chorionic gonadotropin (10). In this paper, we present evidence that heparin and dextran sulfate inhibit rat

i Abbreviations used: CCK, cholecystokinin-pancreozymin; CCK-8, C-terminal octapeptide of CCK; VIP, vasoactive intestinal peptide: EDTA, ethylenediamine tetraacetic acid; Hepes, N-2-hydroxyethylpiperasine-hr’-2-ethanesulfonic acid; GPP(NH)P, guanosine 5’-(&r-imido)triphosphate; Irn, concentration provoking 50% inhibition of adenylate cyclase activity. 1

0003-9861/81/050001-10$02.00/O Copyright All rights

Q 1981 hy Academic Press. Inc. of reproduction in any form reserved.

DESCHODT-LANCKMAN,

2

ROBBERECHT,

pancreatic adenylate cyclase. For the enzyme stimulated by secretin and vasoactive intestinal peptide, the inhibition was weak and of a noncompetitive type. For the CCK-&stimulated enzyme, the major inhibition observed was of a mixed type. The activity of adenylate cyclase after dansylation of pancreatic plasma membranes suggested that lysine residues in CCK receptors can be involved not only in the binding of the hormone (3,4) but also in an electrostatic interaction with the highly negatively charged heparin, comparable to that between antithrombin and heparin (12). MATERIALS

AND

METHODS

Rat pancreatic plasma membranes. The procedure for preparing semipurified rat pancreatic plasma membranes and the main characteristics of this fraction were described previously (6). Briefly, Wistar albino rats were sacrificed by decapitation, their pancreases (12-14 g) were dissected free of fat and homogenized at 2-4°C in 5 vol of a buffer (pH 7.4) made of 20 mM Tris-HCl, 0.3 M sucrose, 5 mM 2-mercaptoethanol, 1 mM EDTA, 2 mM MgClr, Trasylol (500 kallikrein inhibitor units/ml), and an hepatic extract of mixed phospholipids (1 mg/ml). The homogenate was further diluted to 10% (v/v) with the same buffer, filtered on medical gauze, and centrifuged for 10 min at 1809. The supernatant was centrifuged for 15 min at 15,OOOg and the resulting 18Og to 15,000g pellet, rehomogenized in the same buffer, was layered over a discontinuous sucrose gradient adjusted to pH 7.8 at 22°C and centrifuged for 3 h at 90,OOOg. The plasma membrane-rich fraction, collected at the 2735% (w/w) sucrose interface of the gradient, was centrifuged at 100,OOOgfor 20 min and rehomogenized in 20 mM Tris-HCl buffer (pH 7.4) containing 5 mM 2-mercaptoethanol, Trasylol(500 kallikrein inhibitor units/ml), mixed phospholipids (1 mg/ml), and 0.5 mg/ml bovine serum albumin. This membrane suspension was stored in liquid nitrogen until use. Dansylation of pancreatic plasma membranes. Pancreatic plasma membranes (3 mg protein/ml) suspended in 20 mM Tris-HCI buffer (pH 7.4) containing Trasylol (500 kallikrein inhibitor units/ml), 5 mM 2-mercaptoethanol, and phospholipids (1 mg/ ml) but without bovine serum albumin were reacted for 10 min at 20°C with the dansyl chloride reagent in a volume ratio of 1:l. Water-soluble dansyl chloride was prepared as follows (13): 3 mg of cycloheptaamylose dansyl chloride was mixed with 30 mg of Schardinger-&dextrin in 1.5 ml water. After 10 min at 22°C the mixture was filtered and immediately used. Such pretreated membranes and their un-

AND

CHRISTOPHE

treated controls were centrifuged at 4°C for 7 min at 30,OOOg in a Beckman Centrifuge (Model J-21 B). The supernatant containing most of the reagent was discarded. The membrane pellet was rehomogenized in 20 mM Tris-HCl buffer (pH 7.4) containing Trasylol(500 kallikrein inhibitor units/ml), 5 mM 2-mercaptoethanol, phospholipids (1 mg/ml), and 0.1% bovine serum albumin. Adenylate cyclase assay. The activity of adenylate cyclase was determined by measuring the amount of cyclic [“PIAMP formed from [cx-~‘P]ATP. The basic assay medium contained 30 mM Tris-HCl, 5 mM MgClr, 0.5 mM EGTA, 1 mM theophylline, Trasylol (83 kallikrein inhibitor units/ml), 0.93 mM 2-mercaptoethanol, 1 mM cyclic AMP, 0.5 mM [a-=P]ATP (1.106 cpm), and an ATP-regenerating system consisting of 10 mM phospho(enol)pyruvate and pyruvate kinase (30 pg/ml). The pH was adjusted to 7.4 at 25°C. When an activating peptide was present, the medium was also enriched with 10 pM GTP. The reaction was initiated by the addition of 10 ~1 of the membrane suspension (equivalent to 12 rg protein) to 50 ~1 of the reaction mixture. The reaction was terminated after 7 min at 37°C by adding 0.5 ml of a 2% sodium dodecyl sulfate solution containing 1 mM ATP, 0.5 mM cyclic AMP, and cyclic [8-3H]AMP (approx + 20,000 cpm for determination of the recovery). The separation of cyclic AMP from ATP was achieved by the procedure of Salomon et al. (14) with the use of Dowex 5OW-X4 and neutral alumina columns. Persistent activation of rat pancreatic adenylate cyc2ase. The detailed procedure has been previously described (8). Briefly, rat pancreatic plasma membranes (l-2 mg protein) were incubated for 5 min at 30°C in the combined presence of 30 qM Gpp(NH)p and of 0.3 pM CCK in 2.4 ml of a medium containing 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgClc, 5 mM dithioerythritol, and mixed hepatic phospholipids (0.2 mg/ml). The incubation was stopped by adding 7 ml of ice-cold washing buffer composed of 20 mM Hepes-Tris (pH 7.4) with 0.1 mM MgC12, 30 mM NaCl, and 0.1 mM dithioerythritol. After immediate centrifugation at 50,OOOg for 5 min at O”C, the pellet was rehomogenized in the washing buffer and centrifuged again. This procedure was repeated three times. Plasma membranes were finally resuspended in the washing buffer, sonicated (3 X 0.5 s), and stored in liquid nitrogen. Adenylate cyclase activity was assayed in the standard medium with or without the simultaneous presence of Gpp(NH)p and CCK-8. Radioiodinatkm of CCK. Highly purified porcine CCK was labeled by conjugating ‘%I-N-succinimidyl3-(4-hydroxyphenyl)propionate to the free NH* groups as described by Bolton and Hunter (15). CCK (5 rg) in 5 pl of 0.05 M acetic acid was mixed with 20 pl of 0.1 M sodium borate buffer (pH 8.5). The mixture was added to 1 mCi of dried ‘%I-N-succinimidyl-3-(4-hy-

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droxyphenyl)propionate. After 30 min vigorous stirring at 0°C 0.2 M glycine in 250 ~1 of 0.05 M borate buffer (pH 8.5) was added to the reaction mixture to remove excess unreacted Bolton-Hunter reagent. After 5 min at O”C, the solution was mixed with 1.5 ml of 0.1 M sodium phosphate buffer (pH 7.2) containing 0.2% gelatin. The ‘?-labeled hormone was separated from the other radioactive peaks by gel filtration on superfine Sephadex G-50 column (100 x 1 em) according to Rehfeld (16). The sample was eluted at 4°C with 0.1 M sodium phosphate buffer (pH 7.2) containing 0.2% gelatin, at a flow rate of 5 ml/h. Fractions of 1.0 ml were collected at 4°C. The peak containing unaggregated ‘251-CCK was aliquoted, stored at -20°C. and used within 2 weeks.

Assay of “‘I-CCK

binding to rat pancreatic plasma

membranes. The binding assay was conducted at 37°C in a 20 mM Tris-HCl buffer (pH 7.0) containing 0.2% bovine serum albumin, Trasylol (500 kallikrein inhibitor units/ml), 0.5 mg/ml bacitracin, and ‘%ICCK (approx 30,006 cpm per assay) in a final volume of 120 ~1. The reaction was initiated by the addition of a 20-~1 aliquot of plasma membranes containing 20-30 fig protein. Separation of membrane-bound and free hormone was achieved by adding 2 ml of ice-cold 0.1 M sodium phosphate buffer (pH 7.2) enriched with 0.2% albumin followed by immediate filtration of the mixture through EHWP cellulose acetate filters of 0.45~pm pore size (Millipore Corp., Belford, Mass.) The radioactivity remaining on the filters was counted with a Packard Model 5220 autogamma scintillation counter. All assays were performed in duplicate. Nonspecific binding was determined by the binding which occurred in the presence of 2 pM unlabeled hormone, and specific binding was calculated by subtracting this nonspecific binding from total binding. Protein determination. Protein determination was performed according to Lowry et al. (17) using bovine serum albumin as a standard. Materials. Cyclic [8-3H]AMP (27 Ci/mmol), [a32P]ATP (50 to 10 Ci/mmol), and ‘251-N-succinimidyl3-(4-hydroxy, 5-iodophenyl) propionate (>1600 Ci/ mmol) were obtained from the Radiochemical Centre (Amersham). Heparin from porcine intestinal mucosa (sodium salt, grade I, 170 U/mg, Af, 6-25,000), dextran sulfate (sodium salt, weight average Af, 40,000), chondroitin4-sulfate (from whale cartilage), and chondroitin-6sulfate (from shark cartilage) were obtained from Sigma Chemicals Company (St. Louis, MO.). Phospho(enol)pyruvate (trisodium salt), pyruvate kinase in cristalline suspension, cyclic AMP, ATP, (sodium salt, grade I), GTP, and Gpp(NH)p were also purchased from Sigma. Water-soluble dansyl chloride (cycloheptaamylose-dansyl chloride complex and Schardinger-fl-dextrin) was obtained from Pierce (Rockford, III.). Highly purified porcine CCK (99% pure, 33 amino

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3

acid hormone) was a generous gift of Dr. V. Mutt (Karolinska Institutet, Stockholm, Sweden). Essentially pure porcine vasoaetive intestinal peptide was also prepared by Dr. Mutt and supplied by the National Institutes of Health (Bethesda, Md.). Synthetic secretin was a generous gift from Dr. W. Kiinig (Hoechst Aktiengesllschaft, Frankfurt, West Germany). CCK-8 was donated by Dr. Lucania (Squibb Institute for Medical Research, Princeton, N. J.) All other reagents were of the highest grade available. RESULTS

Inhibition of Stimulated Pancreatic Adenylate Cyclase by Sulfated Glycosaminoglycans Control data mentioned in the legend of Fig. 1 show, in line with those previously published (7), that the activity of adenylate cyclase was stimulated less efficiently by 10 PM Gpp(NH)p alone than by the synergetic action of 10 PM GTP and 0.1 /*M secretin, 1 PM VIP or 0.1 PM CCK-8, or by 10 mM NaF. The hormone concentrations tested allowed nearly maximal activation (7). Fifty percent inhibition of CCK&stimulated adenylate cyclase activity was observed at a heparin concentration (I& of 10 pg/ml (Fig. 1). By contrast, at the same heparin concentration, fluoride- and VIPsensitive enzyme showed no inhibition, and the stimulations by secretin and Gpp(NH)p were reduced by 20 and 10% only. Maximal inhibitions observed at a 50 pg/ml heparin concentration corresponded to 93, 48, 20, 18 and O%, respectively, in the presence of CCK-8, Gpp(NH)p, secretin, VIP, and fluoride. The enzyme activity stimulated by CCK8 was also more susceptible to inhibition by dextran sulfate when compared to other stimulants (Fig. 2). At a 4 pg/ml dextran sulfate concentration, which provoked half-maximal inhibition of CCK-8stimulated adenylate cyclase activity, the stimulations by secretin and fluoride were unaffected, and those by Gpp(NH)p and VIP were reduced by 20 and 7% only. Maximal inhibitions observed at a 50 pg/ml dextran sulfate concentration corresponded to 100, 82, 78, 76, and 48%, respectively, in the presence of CCK-8, VIP, secretin, Gpp(NH)p, and fluoride.

4

DESCHODT-LANCKMAN,

ROBBERECHT,

AND

CHRISTOPHE

ity, up to a concentration (data not shown).

of 100 pg/ml

Rapid Inhibitory Effects of SuEfated Glycosaminoglycans on the Time Course of Adenylate Cyclase Activation

Heparin

concentration

in iJg/ml

FIG. 1. Dose-effect curve of heparin on stimulated pancreatic adenylate cyclase. Adenylate cyclase activity was determined after 7 min incubation at 3’7°C as described under Materials and Methods. In the absence of heparin, velocities of 809, 665, 620, 542, and 265 pmol cyclic AMP formed X min-’ X mg membrane protein-’ were observed with, respectively, 0.1 gM CCK-8 and 10 pM GTP (@), 0.1 PM secretin and 10 FM GTP (0), 1 pM VIP and 10 pM GTP (A), 10 mM NaF (+), and 10 pM Gpp(NH)p (0). Inhibition by heparin was expressed in percentage of these maximal control values. Means of two experiments performed in duplicate.

By contrast with heparin and dextran sulfate, chondroitin-4-sulfate and chondroitin-6-sulfate exerted no effect on stimulated pancreatic adenylate cyclase activ-

0Dextran

50 suItat.

concentration

inpg/ml

FIG. 2. Dose-effect curve of dextran sulfate on stimulated pancreatic adenylate cyclase. Adenylate cyclase activity was determined after ‘7 min incubation at 37°C as described under Materials and Methods. The velocities of adenylate cyclase activity in the presence of 0.1 pM CCK-8 and 10 PM GTP (O), 0.1 pM secretin and 10 MM GTP (0), 1 HIM VIP and 10 pM GTP (A), 10 mM NaF (+), and 10 pM Gpp(NH)p (0) are mentioned in the legend of Fig. 1. The inhibition exerted by dextran sulfate was expressed in percentage of the maximal control values. Means of two experiments performed in duplicate.

The inhibitory effect of 50 pg/ml heparin on the activity of adenylate cyclase in the presence of CCK-8 occurred within 1 min, the shortest interval tested (Fig. 3A) suggesting that the effect of heparin was rapid if not instantaneous. On the other hand, the 2-min lag period observed when the enzyme was stimulated by Gpp(NH)p alone was not prolonged by the presence of 50 pg/ml heparin (Fig. 3B). Type of Inhibition Exerted by Heparin and Dextran Sulfate on HormoneStimulated Adenylate Cyclase

Figure 4 is a Hofstee’s representation of dose-effect curves of adenylate cyclase activation by CCK-8 in the absence or in the presence of heparin. At low heparin concentration (10 pg/ml), the apparent K, of the hormone-stimulated enzyme was increased but maximal velocity was unaffected while at higher heparin concentration (20-40 pg/ml) both the apparent K, and maximal velocity were affected. Similar results were observed with dextran sulfate: low concentrations increased the K, of the octapeptide-stimulated enzyme and were without effect on maximal velocity. The inhibitory effects became partly noncompetitive when the concentration of dextran sulfate exceeded 4 pg/ ml (data not shown). When adenylate cyclase was activated via the secretin receptors, the inhibitory effects exerted by heparin in the concentration range of 20-100 pg/ml were moderate and noncompetitive (Fig. 5). Similar data were obtained with dextran sulfate (data not shown). Effect of Heparin on Persistently Cyclase

and Dextran Sulfate Activated Adenylate

As previously shown (8), when adenylate cyclase was persistently activated, the

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5

A

0

5 Tlmo

IO (mln

)

5

0 Time

10 (min

)

FIG. 3. Effect of sulfated glycosaminoglycans on the time course of adenylate cyclase activation. Rat pancreatic membranes were added at 3’7°C to a preformed adenylate cyclase medium already enriched with all the other components. The results are expressed as pmol cyclic AMP formed X mg protein-‘. The basal activity was not subtracted. Results in panels A and B are the means of two experiments performed in duplicate. Panel A: Activation by 0.1 pM CCK-8 and 10 nM GTP in the absence (0) or presence (0) of 50 rg/ml heparin. Panel B: Activation by 10 PM Gpp(NH)p in the absence (0) or presence of 50 rg/ml heparin (0) or 50 rig/ml dextran sulfate (A).

kinetics of cyclic AMP production were identical in the absence of any stimulant or in the presence of both 1.0 PM CCK-8 and 10 ~.JM Gpp(NH)p (Fig. 6A). Heparin used at a high 20 pg/ml concentration inhibited the velocity of this catalytic activity by 3-10% (Figs. 6A and B). The inhibition observed in the presence of 20 pg/ ml dextran sulfate was more pronounced (-55%). It thus appears that dextran sulfate was able to inhibit directly the maximal velocity of adenylate cyclase while heparin was almost without effect. Neither of the sulfated glycosaminoglycans was able to modify the apparent K, of the persistently activated adenylate cyclase for ATP-Me (data not shown).

Inhibition Heparin

of ‘251-CCK Binding by and Dextran Sulfate

Considering the preferential inhibitory effect of heparin and dextran sulfate on the adenylate cyclase activity stimulated by CCK-8, it was of interest to examine whether these glycosaminoglycans were able to interfere with the specific binding of the hormone. In line with data previously obtained (3) with [3H]caerulein, a peptide structurally related to CCK (18), an apparent steady-state of binding of ‘%ICCK to plasma membranes was observed at 37°C after lo-15 min (data not shown).

This binding equilibrium was inhibited by increasing concentrations of CCK-8 with a DW of 20 nM (Fig. 7A). The binding of ‘251-CCK was also tested in presence of increasing concentrations of glycosaminoglycans (Fig. 7A). Heparin and dextran sulfate inhibited the binding of lzI-CCK by 50% at concentrations of 14 and 6 pg/ ml, respectively, and by almost 100% at 20-50 rg/ml. The dose-effect curves of glycosaminoglycans on tracer ‘251-CCK binding developed in a narrower concentration range than that obtained with CCK-8. Figure 7B illustrates that the apparent Kd value of inhibition of lzI-CCK binding by unlabeled CCK-8 was not modified while the apparent number of binding sites was decreased.

Effects of Dansylation Plasma Membranes Cyclase Stimulation Secretin

of Pancreatic on Adenylate by CCK-8 and by

The binding of heparin to several plasma components has been shown to implicate lysine residues (12,19). Having previously shown that the binding of the CCK analog [3H]caerulein was markedly reduced after pretreatment of pancreatic plasma membranes with dansyl chloride, citraconic anhydride, and acetic anhydride, three reagents of free amino groups (3, 4), it was

DESCHODT-LANCKMAN,

ROBBERECHT,

AND

I 0 cyclic 160 260 cyclic AMP lormcd/CCK-I3 concentration ( pmol . min-‘. mg protein-t. IOnM-’

1

FIG. 4. Hofstee’s representation of the dose-effect curves of adenylate cyclase activation by CCK-8 in the presence of increasing concentrations of heparin. Adenylate cyclase was determined after 7 min incubation at 37°C with 10 FM GTP and increasing concentrations of CCK-8 without (0) or with heparin at concentrations of 10 rg/ml (0), 20 pg/ml (O), and 40 wg/ml (M). Results are expressed as pmol of cyclic AMP formed X min-’ X mg membrane protein-‘, after subtraction of the value observed with GTP alone. Means of two experiments performed in duplicate.

tempting to postulate that the competitive effect of heparin on CCK-g-stimulated adenylate cyclase activity was due to its interaction with lysine residues in or near specific CCK receptors. The inhibitory effect exerted by the dansylation procedure on adenylate cyclase activity was found to be of a mixed type when activated via CCK receptors (Fig. 8A), and noncompetitive when the enzyme was stimulated by secretin (Fig. 8B). The stimulation by fluoride was unaffected by the dansylation procedure (data not shown). DISCUSSION

Inhibitory effects of sulfated glycosaminoglycans have been observed on the adenylate cyclase activity from ovary, thyroid, liver, ileum mucosa, and platelets.

CHRISTOPHE

100 200 AMP formed /Secretln concentration (pm01 min-‘. mg prokIn-‘. IOnM-’

7

300 )

FIG. 5. Hofstee’s representation of the dose-effect curves of adenylate cyclase activation by secretin in the presence of increasing concentrations of heparin. Adenylate cyclase activity was determined after ‘7 min incubation at 37°C with 10 FM GTP and increasing concentrations of secretin without (0) or with heparin at concentrations of 20 fig/ml (A), 40 pgg/ml (A), and 100 kg/ml (0). Results are expressed as pmol of cyclic AMP formed X min-’ X mg membrane protein-‘, after subtraction of the value observed with GTP alone. Means of two experiments performed in duplicate.

This general phenomenon was documented in the presence of hormones as diverse as gonadotropins, thyroid-stimulating hormone, glucagon, catecholamines, and prostaglandins (10, 11, 20). Maximal inhibitions attainable were however different for each tissue. In this study, we demonstrate that stimulated pancreatic adenylate cyclase activity was inhibited by heparin and by dextran sulfate, and that stimulation by CCK-8 was more sensitive to the inhibitory effects than the other stimulations tested. Heparin and dextran sulfate conceivably exerted their effects at three levels: (a) the catalytic subunit; (b) the coupling between several occupied hormone receptors and the catalytic subunit; and (c) hormone receptors. The effects of sulfated glycosaminogly-

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.-;; z; 75 m> E?p ;z 50 ;; L?s

25

:.E s0

5 Time (min)

IO

lI!!!s 0 IO 20 30 40 50 Sulfated Glycosamincglycan concentraton (inpg/ml )

FIG. 6. Effects of heparin and dextran sulfate on persistently activated adenylate cyclase in rat pancreatic plasma membranes. Rat plasma membranes were preactivated by incubation at 30°C in the combined presence of 30 PM Gpp(NH)p and of 0.3 PM CCK-8 and washed extensively as described under Materials and Methods. Results in panels A and Bare the means of two experiments performed in duplicate. Panel A: Effects of heparin and dextran sulfate on the time course of persistent activity. The control activity was determined in the standard adenylate cyclase medium at 37”C, in the absence of any stimulant (O), or in the presence of 10 PM Gpp(NH)p plus 1 PM CCK8 (0): the similarity of the data indicated that the adenylate cyclase system in pretreated membranes was fully activated. The effects of 20 Ng/ml heparin (0) or 7 pg/ml (A) and 20 pg/ml (A) dextran sulfate were tested on persistent adenylate cyclase activity in the absence of any added stimulant. Panel B: Dose-effect curves of heparin and dextran sulfate. The persistent activity was determined after 7 min incubation at 3’7°C in the standard adenylate cyclase medium, and in the absence or presence of increasing concentrations of heparin (0) or dextran sulfate (A). Results are expressed in percentage of the value found in absence of added glycosaminoglycans.

cans on the catalytic subunit were measured directly on the persistently activated state of the enzyme (taking advantage of the fact that the catalytic effector and hormone receptors are not coupled any more in this state (8)) as well as on native membranes stimulated by fluoride or by Gpp(NH)p. The maximal inhibitory effect obtained with heparin (-15%) was much lower than that obtained with dextran sulfate (-69%: Fig. 6B) in the persistently active state. The inhibitory effects obtained with the same glycosaminoglycans on native membranes stimulated by fluoride were similar (-0 and -48%, respectively) while those observed in native membranes activated by Gpp(NH)p were higher (-48 and -76%, respectively, Figs. 1 and 2). These data indicate that the activity of the catalytic subunit itself, including guanyl regulatory sites, was inhibited to various extent at high concentrations of the polyanions. The inhibitory effects exerted by sulfated glycosaminoglycans at the outer face of plasma membranes were tested in the

presence of three hormones. The inhibitory effects obtained with heparin (Fig. 1) and dextran sulfate (Fig. 2) on secretinand VIP-stimulated adenylate cyclase were similar in magnitude. Moreover, the inhibition exerted by these glycosaminoglycans on secretin-activated cyclase was essentially noncompetitive (Fig. 5), suggesting that the number of catalytic subunits effectively activated by the hormone was reduced with no alteration in the appK, of hormone activation. The mechanism of this effect might be “nonspecific,” consisting for instance in a restriction of the lateral mobility of secretin receptors and/or in a lower availability of secretin receptors as compared to CCK receptors in pancreatic plasma membranes (see below). Of greater interest was the interaction of glycosaminoglycans with peptides of the CCK family because of its relative specificity. The use of ‘%I-CCK allowed a direct study of the inhibition exerted on hormone binding to specific receptors. The estimated I50 values of heparin and dex-

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ROBBERECHT,

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FIG. 7. (A) Inhibition of izI-CCK binding to rat pancreatic plasma membranes by unlabeled CCK-8, heparin, and dextran sulfate. iasI-CCK (approx 30,000 cpm-’ per assay) was incubated at 37°C with pancreatic plasma membranes for 15 min, in the absence (A) or presence of increasing concentrations of unlabeled CCK-8 (A), dextran sulfate (O), or heparin (0). The radioactivity specifically bound was determined as described under Materials and Methods. The results are expressed in percentage of the radioactivity bound in presence of tracer only and are the means of two experiments performed in duplicate. (B) Effects of heparin on competition curves of specific binding of ‘%I-CCK in the presence of increasing concentrations of unlabeled CCK-8. ‘251-CCK (approx 30,000 cpm-’ per assay) was incubated at 37°C with pancreatic plasma membranes for 15 min in the absence (A) or presence of increasing concentrations of unlabeled CCK-8, and in the absence (A) or presence of 10 pg/ml (0) or 14 fig/ml (+) heparin. The radioactivity specifically bound was determined as described under Materials and Methods. The results are expressed in percentage of the total radioactivity offered and are the means of two experiments performed in duplicate.

tran sulfate for inhibition of ‘251-CCK binding (14 and 6 pg/ml, respectively: Fig. 7A) and the apparent Ki values of the same glycosaminoglycans for CCK-&stim-

ulated adenylate cyclase activity (10 and 4 rcg/ml, respectively) were similar despite variations in the incubation conditions. It is unlikely that heparin exerted its

B

FIG. 8. Hofstee’s representation of dose-effect curves of adenylate cyclase activation by CCK-8 and by secretin in dansylated pancreatic plasma membranes. Adenylate cyclase activity was determined after 7 min incubation at 37°C as described under Materials and Methods, in the presence of 10 pM GTP in the assay medium. Adenylate cyclase activity was expressed as pmol of cyclic AMP formed X min-’ X mg membrane protein-‘, after subtraction of the value observed with GTP alone. Increasing concentrations of CCK-8 (Panel A) or of secretin (Panel B) were incubated in the presence of untreated (0) or dansylated membranes (A) as described under Materials and Methods.

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inhibitory effects by complexing directly with CCK-8 since both molecules are highly negatively charged: heparin contains two to three sulfate groups per glycosyl residue (21) and CCK-8 contains two aspartic acid residues in addition to a sulfated tyrosine (22). The present data indirectly support the existence of “spare” CCK receptors. There is already some evidence that CCK receptors are in excess over catalytic subunits in rat pancreatic plasma membranes (6, 23). We have shown that lo-14 pg/ml heparin reduced the number of specific CCK binding sites without affecting the apparent Kd value of binding, which remained about 30 nM (Fig. 7B). This noncompetitive inhibition exerted by heparin on the binding of ‘251-CCK might reflect the slow reversibility of heparin binding to or near hormone receptors. The increase in the apparent Km of CCK-&activated adenylate cyclase with no alteration in maximal velocity, in the presence of a low (10 fig/ ml) heparin concentration (Fig. 4), was not in contradiction with hormone binding data provided ‘%pare” receptors were present. Indeed, the progressive neutralization of spare receptors by heparin would at first increase the apparent Km of activation of adenylate cyclase. Were all spare receptors neutralized, any further reduction in the number of binding sites would then affect these receptors which are stoichiometrically able to bind to the catalytic subunits of adenylate cyclase, so that a decreased maximal velocity of adenylate cyclase activity would follow (Figs. 4 and 7B) as shown by Freychet (24). The fact that secretin stimulation of adenylate cyclase activity was inhibited noncompetitively at all concentrations of heparin (Fig. 5), suggests by contrast that there were no spare receptors for secretin present in the plasma membrane preparations. Three considerations support the hypothesis that the specific interaction of heparin and dextran sulfate with CCK receptors involved lysine residue(s) located in or near receptor sites: (i) the specific binding of heparin to antithrombin (12) and to platelet factor 4 (19) involves

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9

lysine residues; (ii) the binding of [3H]caerulein is impaired when pancreatic plasma membranes are pretreated with free amino group blocking reagents such as dansyl chloride, acetic anhydride, and citraconic anhydride (3, 4); (iii) in dansylated pancreatic plasma membranes, where reactive amino groups (probably c-amino groups on lysine residues) were modified by the large dansyl group, the inhibition of CCK-8 activated adenylate cyclase was of a mixed type (Fig. 8, Panel A). The biological significance of the present data is unknown. The heparin analog heparan sulfate is a general constituent of the external surface of mammalian cells (25). Its distribution in acinar cells from the rat, mouse, and guinea pig pancreas is complex (26-28), involving Golgi elements, zymogen granules, and the discharged secretion. The persistence of sulfated components in basal nonsecretory structures indicates that they are not exclusively involved in the discharge of secretion material. Considering that plasmalemma glycoproteins on the whole show no clear preferential localization to the secretory pole of acinar cells (29), it is conceivable but not proven that sulfated glycosaminoglycans located in the basolatera1 zone might interfere in vivo with initial events in stimulus-secretion coupling by limiting the binding of CCK to cell surface receptors. ACKNOWLEDGMENTS Aided by Grant 20,403 from the Fonds de la Recherche Scientifique Medicale (Belgium) and by Grant RO-IAM-1’7010 from the National Institutes of Health. We are grateful to Mrs. J. Ballinckx for the typescript of the manuscript. We thank Drs. M. Waelbroeck and M. Svoboda for helpful discussion.

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