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.     

Synthesis of cyclic analogues of cholecystokinin highly selective for central receptors

Cyclic CCK analogues in which positions 28 and 31 have been replaced by lysine residues and whose side chains are bridged by a succinic moiety, were synthesized. They were tested for their ability to inhibit the binding of 125I-BH-CCK-8 to isolated rat pancreatic acini and to guinea pig brain membranes. These cyclic CCK-analogues were compared to the potent CCK analogue Boc-[Nle28,31]-CCK-7 and to Boc-Trp-Leu-Asp-Phe-NH2, analogue of CCK-4. These cyclic compounds appeared to be highly selective for central CCK receptors.     

Correlation between phospholipid breakdown, intracellular calcium mobilization and enzyme secretion in rat pancreatic acini treated with Boc-[Nle28, Nle31]-CCK-7 and JMV180, two cholecystokinin analogues.

In this work in vitro pharmacological profiles of two analogues of the C-terminal heptapeptide of cholecystokinin (CCK) were evaluated. The analogue Boc-[Nle28, Nle31]-CCK-7, a stable analogue of CCK-8, has the same activity profile as CCK-8, and was found to be very potent in stimulating amylase secretion, phospholipid breakdown and [Ca2+]i mobilization from rat pancreatic acini. It can be used as a probe for studying CCK-actions. The CCK-analogue Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester, (JMV180), which stimulates amylase secretion without inhibition at supramaximal concentrations, has different effects on phospholipid hydrolysis and [Ca2+]i mobilization, compared to CCK-8 and Boc-[Nle28, Nle31]-CCK-7. Compound JMV180 was unable to significantly promote phospholipid breakdown, and was only 50%-60% as efficacious as Boc-[Nle28, Nle31]-CCK-7 in promoting [Ca2+]i mobilization. These findings suggest that low affinity CCK-receptors might be responsible for the supra-maximal inhibition of amylase secretion, and are correlated with phospholipid breakdown and maximal [Ca2+]i mobilization.     

Characterization of cholecystokinin receptors in bullfrog (Rana catesbeiana) brain and pancreas

The binding of biologically active 125I-Bolton-Hunter-CCK-33 to bullfrog brain and pancreatic membrane particles was characterized. Both tissues exhibited time-dependent, saturable, reversible, and high affinity binding without evidence for cooperative interaction. Both bullfrog CCK receptors resembled their mammalian counterparts in having acidic pH optima for tracer binding and a Kd of about 0.5 nM. However, the receptors differed from their mammalian counterparts in that (1) the bullfrog brain membranes bound more tracer per mg protein than did the pancreatic membranes, (2) both bullfrog CCK receptors were relatively insensitive to dibutyryl cGMP, and (3) both bullfrog brain and pancreatic CCK receptors exhibited the same general specificity toward a variety of CCK and gastrin peptides. For both tissues, the relative order of receptor binding potency was CCK-8 greater than caerulein = CCK-33 greater than gastrin-17-II greater than CCK-8-ns = gastrin-17-I greater than caerulein-ns greater than gastrin-4 with the sulfated CCK peptides being 1000-fold more potent than their nonsulfated analogs. Sulfated gastrin was also relatively potent, being only 10-fold weaker than CCK-8. Gastrin-4 was 20 000-fold weaker than CCK-8 in interacting with the brain CCK receptor. The latter finding is in sharp contrast to the mammalian brain CCK receptor. We conclude that the bullfrog brain and pancreas contain similar CCK receptors of probable physiological significance and may represent an ancestral condition from which the two distinct CCK receptors present in mammalian brain and pancreas have evolved.     

Metal ion binding affinities of gastrin and CCK in membrane mimetic environments

The fully active gastrin and CCK analogues [Nle¹⁵]-gastrin- 17 and [Nle, Thr]-CCK-9 were analysed for their Ca²⁺ and Tb³⁺ affinities in various membrane mimetic conditions. In TFE both gastrin and CCK exhibited high affinities for calcium and terbium. At saturation level identical metal ion/peptide ratios were determined with Ca²⁺ and Tb³⁺, i.e. R = 3 for gastrin and R = 1 for CCK, confirming the very similar coordination properties of the two metal ions. The conformational effects of both metal ions were found to be very similar with a disordering effect in the case of gastrin and a conformational transition to β-turn type structure in the case of CCK. In order to mimic more properly physiological conditions, similar experiments were performed in the prsence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of mmolar Ca²⁺ concentrations. Induced lipid interaction via N-terminal lipodervatization of gastrin and CCK allowed to translocate quantitatively the two hormones into phospholipid bilayers and to examine the effect of extravesicular Ca²⁺ on the conformation of the peptide headgroups and on their display at the water/lipid interphase. The CCK moiety of the lipo-CCK inserted into phospholipid bilayers interacts with the lipid phase and addition of Ca²⁺ enhances the clustering of the peptide headgroups in a more β-sheet type conformation. Conversely, insertion of lipo-gastrin into the bilayers leads to full exposure of the gastrin headgroup to the bulk water in predominantly random coil structure. Again Ca²⁺ provokes aggregation. As the lipo-peptide/phospholipid system still represents only an artificial model, it remains hazardous to derive a biological relevance from these data. The significantly higher affinity of lanthanide ions than Ca²⁺ for the peptides could well play a role in the inhibibitory activity of lanthanum on the signal transduction of the CCK family of hormones.     

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.     

Gastrin/cholecystokinin-like immunoreactive peptides in the Dungeness crab, Cancer magister (Dana): immunochemical and biological characterization

The purpose of this investigation was to characterize a gastrin/cholecystokinin-like immunoreactant (G/CCK-LI) extractable from the crab, Cancer magister. G/CCK-LI was extracted best in boiling water and was found mainly in the stomach, hemolymph and carapace. A relatively large immunoreactive peptide in the stomach and apparently smaller forms in the hemolymph and carapace were separated by Sephadex G-50 fractionation. Anion-exchange chromatography further fractionated the stomach form into three major peaks. The crab material cross-reacted with three antisera specific for the common C-terminus of gastrin/CCK, but cross-reacted much less with three antisera directed against other portions of the gastrin molecule. Partially purified crab stomach G/CCK-LI inhibited the binding of labeled CCK to mouse brain G/CCK receptors but not to rat pancreatic CCK receptors. The crab peptide did not stimulate rat gastric acid or rat pancreatic amylase secretion. These results indicate that the crab peptides are structurally similar to, but distinguishable from, the bioactive C-terminal amino acid sequence common to gastrins and CCKs.     

Arginine 197 of the cholecystokinin-A receptor binding site interacts with the sulfate of the peptide agonist cholecystokinin

The knowledge of the binding sites of G protein-coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin-A receptor (CCK-AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three-dimensional model of the [CCK-AR-CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site-directed mutagenesis. Wild-type and mutated CCK-AR were transiently expressed in COS-7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I-BH-[Thr, Nle]-CCK-9; however, it bound the nonpeptide antagonist [3H]-SR27,897 as the wild-type receptor. The mutant was approximately 1,470- and 3,200-fold less potent than the wild-type CCK-AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500-fold lower potency and 800-fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild-type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK-AR, strongly support an interaction between Arg197 of the CCK-AR binding site and the sulfate of CCK. In addition, the mutated CCK-AR resembled the low affinity state of the wild-type CCK-AR, suggesting that Arg197-sulfate interaction regulates conformational changes of the CCK-AR that are required for its physiological activation.     

A synthetic peptide derivative that is a cholecystokinin receptor antagonist

So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.     

Identification and Characterization of a Specific Receptor for Cholecystokinin on Isolated Fundic Glands from Guinea Pig Gastric Mucosa Using a Biologically Active 125I-CCK-8 Probe

Abstract

Specific binding sites for cholecystokinin (CCK) have been identified and characterized in fundic glands isolated by collagenase treatment from guinea pig gastric mucosa using a biologically active ¹²⁵I-labeled derivative of the C-terminal octa-peptide of CCK (¹²⁵IIE-CCK-8). The time course of binding to these glands was rapid, temperature dependent and saturable. At 24, 30 and 37° C, half-maximal binding was reached at 5 min and full binding at 30 min. The addition of a large excess of CCK-8 after 15 and 30 min of binding at 24° C caused a prompt and rapid decline in radioligand bound showing that the interaction was reversible. There was a progressive decline in the amount of ¹²⁵IIE-CCK-8 bound to fundic glands with increasing concentrations of CCK-8 and other structurally related peptides. Gastrin II displaced 50% of the radioligand at 1.6nM, CCK-8 at 3.2nM, gastrin I at 16nM, and desulfated-CCK-8 and pentagastrin at 59nM. Secretin did not displace the radioligand from fundic glands at 1.0uM. The binding was also tissue specific as glands isolated from the antral mucosa did not contain specific binding sites for ¹²⁵IIE-CCK-8. This data provides evidence for specific receptors for CCK on gastric fundic glands that may be involved in the control of acid and pepsinogen secretion.     

Binding of Cholecystokinin-8 (CCK-8) Peptide Derivatives to CCKA and CCKB Receptors

Abstract: The structural requirements for the selective binding of cholecystokinin-8 (CCK-8)-related peptides to peripheral (CCK_A) receptors are not sufficiently understood. In this study, the interaction of a series of newly shortened analogues of CCK-8 with both receptor subtypes was analyzed by displacement studies using [³H]-CCK-8 and ¹²⁵l-Bolton-Hunter (BH)-CCK-8 as radioligands. The pentapeptide derivative of CCK-8, succinyl-Tyr (SO₃H)-Met-Gly-Trp-Met-phenethylamide, was found to bind selectively with high affinity to the CCK_A receptor. The replacement of Met²⁸ and/or Met³¹ by norleucine and of L-Trp³⁰ by its D-analogue had no significant effect on the binding properties of the peptide. Further C-terminal shortening resulted in a drastic loss of affinity and selectivity of the CCK receptor binding.     

Characterization of canine intestinal cholecystokinin-58 lacking its carboxyl-terminal nonapeptide. Evidence for similar post-translational processing in brain and gut.

An antibody raised against a synthetic cholecystokinin (CCK) analog, (1-27)-(CCK)-33, corresponding to the midregion of CCK-58, detected immunoreactivity in intestinal extracts which eluted between the positions of CCK-33/39 and CCK-58 on high performance liquid chromatography. This peak, lacking carboxyl-terminal cholecystokinin immunoreactivity, was purified by reverse phase and cation-exchange chromatographies. Amino acid, mass spectral, and microsequence analysis established that it was the amino-terminal desnonapeptide fragment of cholecystokinin-58, (1-49)-CCK-58. It was demonstrated further that CCK-58 has less biological activity than CCK-8, suggesting that the amino terminus either sterically hindered the ability of CCK-58 to exert its biological activity or that its amino terminus acted at another site to inhibit release of amylase from rat pancreatic acini. The desnonapeptide of CCK-58 by itself had no biological activity, nor did it affect CCK-8-stimulated amylase release from isolated rat pancreatic acini, suggesting that the amino terminus shields the carboxyl terminus from expressing its biological activity. Its presence in intestine suggests that it is released into the circulation where it could be detected by midregion antibodies. The presence of high proportions of (1-49)-CCK-58 indicates that most CCK-8 is directly derived from CCK-58. Its occurrence in brain and intestine indicates similar processing for procholecystokinin in both tissues.     

Valine-286 residue in the third intracellular loop of the cholecystokinin 2 receptor exerts a pivotal role in cholecystokinin 2 receptor mediated intracellular signal transduction in human colon cancer cells

Although expression of the gastrin/cholecystokinin-2 receptor (CCK2R) is widely reported in human colorectal cancer, little is known on its role in mediating mature amidated gastrin (gastrin-17 amide, G-17) induced intracellular signal transduction in colon cancer cells. The purpose of this study was to explore the intracellular events of colorectal cancer cells after gastrin binding to CCK2R. Meanwhile, the influence of a natural point mutation 286V-->F in the third intracellular loop of CCK2R on gastrin-envoked intracellular signal transduction was also investigated. Firstly, Colo320 cells were stably transfected with wild type (Colo320 WT) and mutant CCK2R (Colo320 M), respectively. The intracellular signal transduction events in response to gastrin were investigated in both Colo320 WT and Colo320 M cells. In Colo320 WT cells, G-17 induced formation of intracellular cyclic AMP and inositol 1,4,5-trisphosphate, and stimulated intracellular calcium mobilization. G-17 also stimulated tyrosine phosphorylation of ERKl/2, p38, FAK, and paxillin, and up-regulated the mRNA expression of early response gene c-Jun and c-Fos. However, G-17 inhibited proliferation and induced apoptosis in Colo320 WT cells. Mutation 286V-->F in the third intracellular loop of CCK2R blocked G-17 induced biological without affecting binding affinity of CCK2R to G-17. Our results suggest that activation of CCK2R by gastrin stimulates heterotrimeric G-protein Gq and G(12/13) mediated intracellular signal transduction pathway in colon cancer cells. The valine-287 residue in third intracellular loop of CCK2R plays a pivotal role in CCK2R mediated intracellular signal transduction.     

N-Carboxyacyl analogues of CCK with a substituted Gly: Interaction with pancreatic and gallbladder CCK receptors

It has been reported that certain N-carboxyacyl analogues of CCK-8 and of CCK-7 with a substituted Gly in position 3 or 4 of the peptide possess higher potencies at stimulating pancreatic enzyme secretion than at stimulating gallbladder contraction, suggesting that these analogues are able to differentiate subtypes of CCK_A receptors. However, no studies examined directly the interaction of these peptides with the CCK receptors in both tissues. In the present study, CCK-8 and various N-carboxyacyl analogues of CCK-7 and of CCK-8 were prepared by solid phase synthesis using Fmoc chemistry and were purified by HPLC; molecular weight and sufficient sulfation were determined by mass spectrometry. [¹²⁵I]Bolton-Hunter(BH)-CCK-8 binding to sections of the guinea pig pancreas and gallbladder was determined under identical conditions; amylase release from pancreatic acini and contraction of gallbladder muscle strips were measured in vitro. Each peptide stimulated amylase release (EC₅₀):

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). The same relative potencies were found for stimulation of gallbladder contraction, and for the inhibition of [¹²⁵I]BH-CCK-8 binding to pancreas and gallbladder sections. These data demonstrate that the CCK_A receptors in the pancreas and on gallbladder smooth muscle possess similar affinities for the various N-carboxyacyl analogues of CCK-7 and CCK-8 with a substituted Gly and provide further evidence that the CCK_A receptors in gallbladder and pancreas cannot be distinguished pharmacologically.     

Growth of mouse hepatocytes is stimulated by gastrin

Hepatocyte growth is regulated by various growth factors, including epidermal growth factor (EGF) and insulin. Recently, several additional peptide hormones have been shown to stimulate growth of hepatocyte only in the presence of EGF or insulin and are thus termed secondary mitogens. Gastrin regulates growth of normal and neoplastic gastrointestinal tissues, but the effect on growth of hepatocyte is unknown. We examined the effect of gastrin on growth of a normal mouse hepatocyte (NMH) line established in our laboratory. Effect of gastrin-17 (G-17) (10^?8 to 10^?6 M) on growth of NMH cells was examined in either the presence or absence of EGF in the culture medium. Growth of NMH cells was evaluated by incorporation of either bromodeoxyuridine (BrdU) or ³H-thymidine and by counting cells. Presence of a cell-surface receptor for G-17 was determined by Scatchard analysis using ¹²⁵I-G-17. In the presence of EGF, gastrin stimulated growth of NMH cells; in the absence of EGF, gastrin did not affect growth. The stimulatory effect of gastrin on NMH cells was blocked by JMV 320, a CCK-B type receptor antagonist. NMH cells possess a single, high affinity binding site for gastrin (K_d = 1.2 nM); EGF increased the gastrin binding capacity compared to non-treated cells (3.5 ± 0.4 vs. 2.2 ± 0.6 fmol/10⁶ cells). G-17 stimulated growth of NMH cells through a single high affinity receptor for G-17 which pharmcologically appears to be the CCK-B type only in the presence of EGF and thus can be considered a secondary mitogen. © 1995 Wiley-Liss, Inc.     

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.     

Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor

The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.     

Progastrin1-80 stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG(1-80)) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1-1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK(1) and CCK(2) receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK(1)-R and CCK(2)-R on IEC cells. High-affinity (K(d) = 0.5-1.0 nM) binding sites for (125)I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG >or= COOH-terminally extended G17 >or= G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.     

The first nonsulfated sulfakinin activity reported suggests nsDSK acts in gut biology

Invertebrate sulfakinins are structurally and functionally homologous to vertebrate cholecystokinin (CCK) and gastrin. To date, sulfakinins are reported to require a sulfated tyrosine for activity; sulfated and nonsulfated CCK and gastrin are active. This is the first nonsulfated sulfakinin activity reported. Nonsulfated Drosophila melanogaster sulfakinins or drosulfakinins (nsDSK I; PheAspAspTyrGlyHisMetArgPheNH2) and (nsDSK II; GlyGlyAspAspGlnPheAspAspTyrGlyHisMetArgPheNH2) decreased the frequency of contractions of adult D. melanogaster foregut (crop) in vivo. The EC50's for nsDSK I and nsDSK II were approximately 2 x 10(-9)M and approximately 3 x 10(-8)M, respectively. Nonsulfated DSK peptides also decreased the frequency of larval anterior midgut contractions. Sulfated DSK peptides decreased both adult and larval gut contractions. Whether sulfation is required for sulfakinin activity may depend on where the peptide is applied, what tissue is analyzed, or what preparation is used. D. melanogaster contains two sulfakinin receptors, DSK-R1 and DSK-R2; vertebrates contain two CCK receptors, CCK-1 and CCK-2. A sulfated DSK I analog, [Leu7] sDSK I, binds to expressed DSK-R1; the corresponding nonsulfated analog does not bind to DSK-R1. No DSK-R2 binding data are reported. Sulfated and nonsulfated CCK peptides preferentially bind to CCK-1 or CCK-2, respectively. Sulfated and nonsulfated sulfakinins may bind to DSK-R1 or DSK-R2, respectively. Sulfakinin activities, spatial and temporal distribution, and homology to CCK and gastrin suggest sulfated and nonsulfated DSK peptides act in diverse roles in the neural and gastrointestinal systems including gut emptying and satiety.     

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.