Binding of cholecystokinin to high affinity receptors on isolated rat pancreatic acini

The binding of cholecystokinin (CCK) to its receptors on isolated rat pancreatic acini was investigated employing high specific activity, radioiodinated CCK (125I-BH-CCK), prepared by the conjugation of 125I-Bolton-Hunter reagent (125I-BH) to CCK. Binding was specific, time-dependent, reversible, and linearly related to the acinar protein concentration. After incubation for 30 min at 37 degrees C, the 125I-BH-CCK both in the incubation medium and bound to acini remained intact, as judged by gel filtration and trichloroacetic acid precipitation studies. Scatchard analysis was compatible with two classes of binding sites on acini: a very high affinity site (Kd, 64 pM) and a lower affinity site (Kd, 21 nM). 125I-BH-CCK binding to acini was competitively inhibited by CCK and four of its analogues in proportion to their biological potencies but not by unrelated hormones. Stimulation of amylase secretion by CCK and inhibition of 125I-BH-CCK binding by the same analogues carried out under identical conditions revealed a correlation (r = 0.99) between binding potency and amylase secretion. Stimulation of amylase secretion by CCK closely paralleled the occupancy of the high affinity CCK binding sites. It is concluded that the high affinity CCK binding sites most likely are the receptors mediating the stimulation of amylase secretion by CCK.     

Down-regulation and recycling of high affinity cholecystokinin receptors on pancreatic acinar cells

First incubating dispersed acini from rat pancreas with monensin, a cation ionophore that can inhibit recycling of receptors, inhibited binding of 125I-cholecystokinin 8 (125I-CCK-8) measured during a second incubation by as much as 50%. A maximal effect of monensin required 90 min of first incubation. Detectable inhibition of binding of 125I-CCK-8 occurred with 300 nM monensin, and inhibition increased progressively with concentrations of monensin up to 25 microM. Pancreatic acini possess two classes of receptors that bind 125I-CCK-8. One class has a high affinity (Kd = 461 pM) and a low capacity for CCK (512 fmol/mg DNA); the other class has a low affinity (Kd = 47 nM) and a high capacity for CCK (18 pmol/mg DNA). First incubating acini with monensin caused an 84% decrease in the number of high affinity CCK receptors with no change in the number of low affinity CCK receptors or the values of Kd for either class of receptors indicating that there is recycling of high affinity CCK receptors but not low affinity CCK receptors. First incubating acini with monensin did not alter CCK-stimulated amylase secretion indicating that in contrast to previous conclusions, occupation of low affinity CCK receptors mediates CCK-stimulated enzyme secretion. Moreover, the biphasic dose-response curve for CCK-stimulated enzyme secretion from monensin-treated acini suggests that pancreatic acini also possess a third, previously unrecognized class of very low affinity CCK receptors.     

Low-affinity CCK-1 receptors inhibit bombesin-stimulated secretion in rat pancreatic acini--implication of the actin cytoskeleton

EXPERIMENTAL OBJECTIVES: Stimulation of low-affinity CCK-1 receptors on pancreatic acini leads to inhibition of enzyme secretion. We studied signal transduction mechanisms to identify potential causes for the reduced secretion. RESULTS: Co-stimulation experiments with CCK, CCK-JMV-180, and bombesin revealed an inhibition of bombesin-stimulated enzyme secretion by low-affinity CCK-1 receptors. Binding of 125I-gastrin-releasing peptide (the mammalian analogue of bombesin) to acini after CCK preincubation was not altered. After a short preincubation of acini with high concentrations of CCK, intracellular calcium remained responsive to bombesin. In contrast to bombesin or CCK at concentrations of 10(-10) M or lower, high concentrations of CCK caused a strong activation of p125 focal adhesion kinase (p125(FAK)) and a marked reorganisation of the actin cytoskeleton. CONCLUSIONS: Inhibitory mechanisms triggered by low-affinity CCK-1 receptors interrupt enzyme secretion from pancreatic acini at late stages in the signal transduction cascades since bombesin receptor binding and early signalling events remained intact after CCK preincubation. A reorganisation of the actin cytoskeleton is suggested to be the mechanism by which low-affinity CCK-1 receptors actively interrupt enzyme secretion stimulated by other receptors.     

Cholecystokinin receptor occupation and cholecystokinin-induced calcium mobilization in the early phase in rat pancreatic acini.

We examined receptor occupation, calcium mobilization and amylase release for cholecystokinin octapeptide (CCK-8) within a 3-min incubation period at 37 degrees C using dispersed acini from rat pancreas. Analysis of competitive binding inhibition data obtained after a 3-min incubation revealed the presence of only a single class of CCK receptors, while two classes of CCK receptor, i.e., high-affinity and low-affinity CCK receptors, were detected when binding reached a steady-state after a 60-min incubation. The IC50 of CCK receptors calculated from the 3-min binding data was 19.0 +/- 0.5 nM (mean +/- S.D.), close to the Kd of the low-affinity CCK receptors determined by equilibrium binding studies. Exposure of fura-2-loaded acini to 10-1000 pM CCK-8 caused an immediate and dose-dependent increase in [Ca2+]i followed by a gradual decrease in [Ca2+]i. The CCK-stimulated amylase release after 3 min of incubation was biphasic; amylase release increased over the dose range of 3-300 pM CCK-8, peaked at 300 pM CCK-8 and decreased with supramaximal concentrations of CCK-8. Our data suggest that occupation of the low-affinity, but not the high-affinity, CCK receptors is more directly associated with calcium mobilization and subsequent stimulation of amylase release in rat pancreatic acini.     

Importance of sulfation of gastrin or cholecystokinin (CCK) on affinity for gastrin and CCK receptors

We investigated the importance of sulfation of gastrin or cholecystokinin (CCK) on influencing their affinity for gastrin or CCK receptors by comparing the abilities of sulfated gastrin-17 (gastrin-17-II), desulfated gastrin-17 (gastrin-17-I), CCK-8 and desulfated CCK-8 [des(SO3)CCK-8] to interact with CCK or gastrin receptors on guinea pig pancreatic acini. For inhibiting binding of 125I-gastrin to gastrin receptors, gastrin-17-II (Kd 0.08 nM) greater than CCK-8 (Kd 0.4 nM) greater than gastrin-17-I (Kd 1.5 nM) greater than des(SO3)CCK-8 (Kd 28 nM). For inhibiting binding of 125I-Bolton Hunter-labeled CCK-8 to CCK receptors the relative potencies were: CCK-8 much greater than des(SO3)CCK-8 = gastrin-17-II greater than gastrin-17-I. Each peptide interacted with both high and low affinity CCK binding sites. The relative abilities of each peptide to interact with high affinity CCK receptors showed a close correlation with their abilities to cause half-maximal stimulation of enzyme secretion. These results demonstrate that, in contrast to older studies, sulfation of both CCK and gastrin increase their affinities for both gastrin and CCK receptors. Moreover, the gastrin receptor is relatively insensitive to the position of the sulfate moiety, whereas the CCK receptor is extremely sensitive to both the presence and exact position of the sulfate moiety.     

Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.     

Mutual dependence of VIP/PACAP and CCK receptor signaling for a physiological role in duck exocrine pancreatic secretion

Unlike in rodents, CCK has not been established as a physiological regulator in avian exocrine pancreatic secretion. In the isolated duck pancreatic acini, 1 nM CCK was required for stimulation of amylase secretion, maximal effect being achieved at 10 nM; picomolar CCK was without effect. Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 (10(-12)-10(-7) M) alone had no effect, but made picomolar CCK effective. VPAC agonist VIP 10(-10)-10(-7) M stimulated amylase secretion marginally, but made CCK 10(-12)-10(-10) M effective also. PACAP-27 and VIP both shifted the maximal CCK concentration from 10(-8) to 10(-9) M. This sensitizing effect was mimicked by forskolin. CCK dose dependently induced intracellular Ca2+ concentration ([Ca2+]i) oscillations. PACAP-38 (1 nM), PACAP-27 (1 nM), VIP (10 nM), or forskolin (10 microM) alone did not stimulate [Ca2+]i increase, neither did they modulate CCK (1 nM)-induced oscillations; but when they were added to cells simultaneously exposed to subthreshold CCK (10 pM), calcium spikes emerged. Amylase secretion induced by the simultaneous presence of 10 pM CCK and VPAC agonists was completely blocked by removing extracellular calcium, but the protein kinase C inhibitor staurosporine (1 microM) was without effect. CCK (10 nM)-induced secretion was inhibited by CCK1 receptor antagonist FK480 (1 microM). Gastrin from 10(-12) to 10(-6) M did not stimulate amylase secretion nor did it (100 nM) induce [Ca2+]i increase. The above data suggest that duck pancreatic acini possess both CCK1 and VPAC receptors; simultaneous activation of both is required for each to play a physiological role.     

Protein malnutrition and response of pancreatic acinar cells to stimulation by cholecystokinin

Pancreatic lobules were isolated from 2 groups of male Wistar rats after 23 days of diet. A control group (C) fed on a 20% protein diet (16% gluten + 4% casein) and an experimental group (E) on a 5% protein diet (4% gluten + 1% casein). After isolation, lobules were preincubated 10 min with 10 muCi [3H]-leucine, washed, then incubate within Krebs Ringer bicarbonate Hepes. Basal secretion, then stimulated secretion (50 pM of cholecystokinin (CCK] of radioactive and non-radioactive protein and amylase outputs were measured. During basal secretion, in (E) group, lobules secreted more proteins than (C) one, the same outputs of amylase and radioactive protein were observed in both groups. The stimulated secretion by CCK increased the outputs of non-radioactive protein and amylase of lobules (T) (2-3 fold), but was without effect on lobule (E) outputs. Therefore, a low-protein diet involved a decrease of CCK sensibility on acinar cells, this fact might be mediated by a decreasing number and/or affinity of their CCK receptors.     

Cholecystokinin stimulates the recruitment of the Src-RhoA-phosphoinositide 3-kinase pathway by Vav-2 downstream of G(alpha13) in pancreatic acini

In isolated rat pancreatic acini, Src, RhoA, PI3-K, Vav-2, G(alpha12), and G(alpha13) were detected by immunoblotting. CCK enhanced the levels of these proteins, and the levels of Src and RhoA were reduced by the Src inhibitor herbimycin A and the Rho inhibitor pravastatin. The PI3-K inhibitor wortmannin reduced the level of PI3-K. These inhibitors also decreased amylase secretion in CCK-treated pancreatic acini without altering basal secretion. Immunoprecipitation studies indicated that CCK caused Src to associate with Vav-2, RhoA, and PI3-K and RhoA and Src to associate with Vav-2. Ras, RasGAP, and SOS did not coimmunoprecipitate with Vav-2, and RasGAP and SOS did not coimmunoprecipitate with RhoA. CCK also enhanced Vav-2 and RhoA to coimmunoprecipitate with G(alpha13). We conclude that CCK stimulates the recruitment of the Src-RhoA-PI3-K signaling pathway by Vav-2 downstream of G(alpha13) in pancreatic acini.     

Glucocorticoids increase cholecystokinin receptors and amylase secretion in pancreatic acinar AR42J cells

We recently reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase (Logsdon, C.D., Moessner, A., Williams, J.A., and Goldfine, I.D. (1985) J. Cell Biol. 100, 1200-1208). In addition, in these cells glucocorticoids increased the volume density of secretory granules and rough endoplasmic reticulum. In the present study we investigate the effects of glucocorticoids on the receptor binding and biological effects of cholecystokinin (CCK) on AR42J cells. Treatment with 10 nM dexamethasone for 48 h increased the specific binding of 125I-CCK. This increase in binding was time-dependent, with maximal effects occurring after 48 h, and dose-dependent, with a one-half maximal effect elicited by 1 nM dexamethasone. Other steroid analogs were also effective and their potencies paralleled their relative effectiveness as glucocorticoids. Analyses of competitive binding experiments conducted at 4 degrees C to minimize hormone internalization and degradation revealed the presence of a single class of CCK binding sites with a Kd of approximately 6 nM and indicated that dexamethasone treatment nearly tripled the number of CCK receptors/cell with little change in receptor affinity. Treatment with 10 nM dexamethasone increased both basal amylase secretion and the amylase released in response to CCK stimulation. In addition, dexamethasone increased the sensitivity of the cells to CCK. The glucocorticoid decreased the concentration of CCK required for one half-maximal stimulation of amylase secretion from 35 +/- 6 to 8 +/- 1 pM. These data indicate, therefore, that glucocorticoids induce an increase in the number of CCK receptors in AR42J cells, and this increase leads to enhanced sensitivity to CCK.     

Receptors for cholecystokinin and gastrin peptides display specific binding properties and are structurally different in guinea-pig and dog pancreas

In the light of the strong potency of gastrin-related peptides on pancreatic exocrine secretion in dog, we analyzed the binding properties of peptides related to cholecystokinin (CCK) and gastrin on dog pancreatic acini compared to guinea-pig acini. Moreover, we determined apparent molecular masses of photoaffinity labelled CCK/gastrin receptors in the two models. Using the CCK radioligand, receptor selectivity towards CCK/gastrin agonists and antagonists was found to be lower in dog acini than in guinea-pig acini. Performing the binding with CCK and gastrin radioligands in combination with N2,O2'-dibutyryl-guanosine 3',5'-monophosphate, revealed that in dog acini there exist two different sub-classes of CCK/gastrin receptors having high and low selectivity, the latter ones being able to bind gastrin with high affinity (Kd = 2.1 nM). SDS-PAGE analysis of covalently cross-linked receptors using several photosensitive CCK and gastrin probes of different peptide chain lengths demonstrated that in guinea-pig, CCK peptides bound to a 84-kDa component whereas in dog pancreas, CCK and gastrin peptides bound to three distinct molecular species (Mr approximately equal to 78,000, 45,000, 28,000). Performing cross-linking in the presence of 1 microM CCK indicated that a 45-kDa protein is the putative CCK/gastrin receptor in dog pancreas. Our results support the concept of heterogeneity of CCK/gastrin receptors.     

Characterization of interactions between CCK-33 and CCK receptors in isolated dispersed pancreatic acini.

In isolated dispersed pancreatic acini, we have characterized the interactions between cholecystokinin (CCK) and CCK receptors by simultaneously measuring CCK-33 immunoreactivity and CCK bioactivity. Incubation of acinar cells with CCK-33 at cell density of 0.2-0.3 mg acinar protein per ml resulted in stimulation of amylase release concomitant with significant and time-dependent decrease of the immunoreactive CCK. With L-364,718 (0.1 microM), a specific CCK receptor antagonist, immunoreactive CCK levels in the media were not significantly altered during incubation; however, CCK-stimulated amylase release was almost completely abolished (94% inhibition). Vasoactive intestinal peptide (1 nM) significantly potentiated CCK stimulated amylase release without affecting immunoreactive CCK in the media. Insulin (167 nM) did not affect the CCK stimulated amylase release or immunoreactive CCK in the media. Incubation of acinar cells with CCK-33 at 4 degrees C did not affect the levels of immunoreactive CCK; however, a significant change in levels of immunoreactive CCK were found at 37 degrees C at 90 min. Incubation of cell free medium with CCK-33 in the presence or absence of secreted enzymes revealed no changes in CCK immunoreactivity in the medium at 90 min. Addition of bacitracin in the incubation media did not affect the CCK immunoreactivity or bioactivity. These findings indicate that in isolated rat pancreatic acini, CCK-33 stimulates amylase release through a receptor that is specifically blocked by L-364,718. Specificity of the interactions of CCK-33 with acinar cells in the media appears to be receptor-mediated and time- and temperature-dependent.     

Effect of AA861, a 5-lipoxygenase inhibitor,on amylase secretion from rat pancreatic acini

We have examined the effects of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of 5-lipoxygenase, on the action of cholecystokinin (CCK) and other secretagogues in the stimulation of amylase secretion from dispersed rat pancreatic acini. AA861 inhibited amylase secretion caused by CCK, carbamylcholine (carbachol), bombesin or calcium ionophore A23187 but failed to affect amylase secretion by vasoactive intestinal peptide or 12-O-tetradecanoyl-phorbol 13-acetate. Inhibition by AA861 of CCK or carbachol-induced amylase secretion was confined to the relatively lower concentrations of these secretagogues. AA861 did not inhibit receptor binding of CCK or alter the cellular calcium mobilization induced by CCK. In kinetic studies, AA861 was effective only on amylase secretion from pancreatic acini incubated with CCK for more than 5 min. Indomethacin, a known inhibitor of cyclooxygenase, did not affect the amylase secretion caused by all secretagogues used. These results indicate that the 5-lipoxygenase pathway of arachidonate metabolism may be involved in the actions of calcium-dependent secretagogues of amylase secretion in rat dispersed pancreatic acini, especially for sustaining stimulation of amylase secretion by CCK.     

Relationship of CCK/gastrin receptor binding to amylase release in dog pancreatic acini

Effects of synthetic peptides belonging to the CCK/gastrin family (CCK-39, CCK-8, G/CCK-4, G-17ns) on amylase release in dog pancreatic acini have been measured and correlated with binding of three radio-labelled CCK/gastrin peptides: ¹²⁵I-BH-(Thr,Nle)-CCK-9, ¹²⁵I-BH-(2–17)G-17ns and ¹²⁵I-BH-G/CCK-4 prepared by conjugation of the peptides to iodinated Bolton-Hunter reagent and purified by reverse-phase-HPLC. All the CCK/gastrin peptides produced the same maximal amylase release response. Half-maximal responses (D₅₀) were obtained with 2 · 10^?10 M CCK-8; 6 · 10^?10 M CCK-39; 10^?7 M G.17 ns and 2 · 10^?6 M G/CCK-4. Dose-response curves for G-17 ns and G/CCK-4 were similar in configuration but not parallel with those for CCK-8 and CCK-39.Binding studies with ¹²⁵I-BH(Thr,Nle)-CCK-9 demonstrated the presence of specific CCK receptors on dog pancreatic acini. There was a good correlation between receptor occupancy by CCK-8 and CCK-39 and amylase stimulation since maximal amylase stimulation was achieved when 40–50% of high affinity receptors were occupied. In contrast, a saturation of these receptors was required for maximal stimulation by G-17 ns and G/CCK-4 suggesting the existence of a fraction of receptors that can be occupied by G-17 ns and G/CCK-4 without stimulation of amylase release. Binding studies with labelled (2–17)-G-17 ns and G/CCK-4 confirmed the presence of high affinity sites for G-17 ns and G/CCK-4. These sites were not related to amylase release.This study points out a possible species specificity of biological action of gastrin/CCK peptides on pancreatic exocrine secretion in higher mammals.     

C-terminal peptides of calcitonin gene-related peptide act as agonists at the cholecystokinin receptor

We have previously described that [Tyr⁰]CGRP(28–37) acts as a receptor antagonist of rat CGRP in guinea pig pancreatic acini. We therefore examined other C-terminal peptides of CGRP for such activity. CGRP-acetyl(28–37) acetate did act as a rat CGRP antagonist. However, C-terminal CGRP peptides of 4 to 8 amino acid residues did not antagonize the actions of rat CGRP but stimulated amylase secretion. In pancreatic acini, a maximally effective concentration of rat CGRP (100 nM) caused a 2.1-fold increase in amylase secretion. When the C-terminal peptides of CGRP were tested in at 100 μM, CGRP(34–37) caused a 1.8-fold increase in amylase secretion, CGRP(33–37) a 2.8-fold increase, CGRP(32–37) a 9.2-fold increase, CGRP(31–37) a 4.1-fold increase, and CGRP(30–37) a 5.1-fold increase. Further studies with the most effective peptide, CGRP(32–37), demonstrated that it did not cause release of lactate dehydrogenase, and thus did not cause amylase release by cell damage. Unlike rat CGRP, CGRP(32–37) did not increase cellular cyclic AMP, but did stimulate outflux of ⁴⁵Ca. CGRP(32–37)-stimulated amylase release was not inhibited by the substance P receptor antagonist, spantide, by the bombesin receptor antagonist, [D-Phe⁶]bombesin(6–13) propylamide, or by the muscarinic receptor antagonist, atropine, but was inhibited by the CCK receptor antagonist L364,718. C-terminal peptides of CGRP inhibited binding of ¹²⁵I-BH-CCK-8, with the relative potencies of the peptides being the same as their relative potencies for stimulating amylase secretion. The present data demonstrate that C-terminal peptides of CGRP, although they have only 2 amino acid residues in common with CCK(26–33), act exclusively at CCK receptors on pancreatic acini to stimulate amylase secretion.     

Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.

A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCK8) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988) Am. J. Physiol. 254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCK8 on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCK8 was reduced. Measurement of 45Ca2+ efflux and cytosolic free calcium concentration ([Ca2+]i) by the fluorescence of fura-2-loaded acini in a stirred cuvette showed that JMV-180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCK8. When [Ca2+]i of individual fura-2-loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 microM) induced repetitive transient [Ca2+]i spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCK8 (1 nM) caused a large increase in [CA2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 microM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+]i and 1,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV-180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCK8. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phosphorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCK8. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional and molecular characterization of CCK receptors in the rat pancreatic acinar cell line AR 4-2J.

Competitive inhibition binding studies on membranes from the rat pancreatic AR 4-2J cell line revealed the predominance (80%) of low selectivity CCK receptors (KD of 1 nM and 4 nM for, respectively, CCK-8 and gastrin-17I (G-17I] over selective receptors (20% with a KD of 1 nM and 1 microM for, respectively, CCK-8 and G-17I). Amylase secretion was stimulated by low concentrations of CCK-8, G-17I and CCK-4. G-17I-induced amylase secretion was unaffected by 100 nM of the selective peripheral CCK-A receptor antagonist L-364,718, suggesting that amylase hypersecretion followed non-selective CCK receptor activation, a function normally assumed by selective CCK-A receptors in rat pancreatic acini. Direct ultraviolet irradiation of AR 4-2J cell membranes preloaded with 125I-BH-CCK-33 or 125I(Leu)G(2-17)I resulted in covalent cross-linking with, respectively, a 90 kDa protein and a 106 kDa protein, both distinct from the 81 kDa CCK binding species revealed in normal rat pancreatic membranes. Gpp[NH]p increased the dissociation rate of CCK-8 and G-17I from AR 4-2J cell membranes, indicating a coupling of receptors with guanyl nucleotide regulatory protein(s) G. [32P]ADP-ribosylation of AR 4-2J cell membranes allowed to detect the presence of two Gs alpha (the 50 kDa form predominating over the 45 kDa form) and one Gi alpha (41 kDa). However, Gi and Gs may not be involved in gastrin stimulation of amylase secretion, as Bordetella pertussis toxin and cholera toxin pretreatment of cells did not suppress G-17I-dependent amylase secretion.     

Preincubation with Substance P Induces Substance P-Stimulated Phosphatidylinositol Turnover in Cultured Cerebellar Astrocytes

In this study we have investigated the effects of preincubation of cultured astrocytes with substance P (SP) on subsequent 125I-Bolton-Hunter conjugated SP (125I-BHSP) receptor binding, and SP-stimulated phosphatidyl-inositol (PI) accumulation. Spinal cord astrocytes preincubated for up to 96 h with SP (0.001-1,000 nM) suffered a dose-dependent decrease in both subsequent 125I-BHSP and SP-stimulated PI turnover. In contrast, preincubation of cerebellar astrocytes with SP resulted in an increase in SP-stimulated PI turnover, with no change in 125I-BHSP receptor binding. SP-induced PI turnover in cerebellar astrocytes was maximal after 72 h of preincubation with 0.1 nM SP. These data suggest that increased coupling between receptor and second messenger occurs in response to chronic exposure to SP.     

Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.     

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.