The analysis of expression of CCK and IP3 receptors in gallstones patients with type 2 diabetes mellitus.

BACKGROUND/AIMS: Type 2 diabetes mellitus (T2DM) is correlated with gallbladder diseases. This study aimed to investigate the expression of CCK and IP3 receptors in patients with gallbladder stones and T2DM and its correlation with the hypomotility of the gallbladder. METHODOLOGY: 26 patients with gallstones and T2DM (Group 1) and 24 gallstones patients without T2DM (Group 2) were enrolled in this study. The emptying function of the gallbladder was measured by ultrasonography. The activity of CCK-R was analyzed by radioligand method and the IP3-R antibody was used to detect the IP3-R from patients in both groups. RESULTS: Gallbladder ejection volume (EV) ((11.6±5.1) ml3 vs (21.5±7.8) ml3) and gallbladder ejection fraction (GBEF2)(%)((17.2±11.3) ml3 vs (52.8±12.9) ml3) were significantly lower (P<0.01) in patients with gallstones and T2DM. The amount of CCK-R and the activity of CCK-R in Group 1 were significantly lower than that in Group 2 (P<0.01). And IP3-R in Group 1 was much lower than that in Group 2, as well (P<0.01). CONCLUSION: The expression of CCK-R and IP3-R in gallstones patients with T2DM was much lower in such patients, leading to impaired gallbladder emptying function and the formation of gallstones.

Structural basis of cholecystokinin receptor binding and regulation.

Two structurally-related guanine nucleotide-binding protein-coupled receptors for two related peptides, cholecystokinin (CCK) and gastrin, have evolved to exhibit substantial diversity in specificity of ligand recognition, in their molecular basis of binding these ligands, and in their mechanisms of biochemical and cellular regulation. Consistent with this, the CCK1 and CCK2 receptors also play unique and distinct roles in physiology and pathophysiology. The paradigms for ligand recognition and receptor regulation and function are reviewed in this article, and should be broadly applicable to many members of this remarkable receptor superfamily. This degree of specialization is instructive and provides an encouraging basis for the diversity of potential drugs targeting these receptors and their actions that can be developed.

Clinical significance of gastrin receptors in human colon cancers.

We have measured gastrin receptors (GR) in surgical specimens from 67 patients with primary colon cancers in order to determine the clinical significance of GR in colon cancer. GR analysis was performed on these specimens, and 22 cancers (32.8%) had no detectable GR. Thirty-eight cancers (56.7%) had high-affinity (Kd less than 1.0 nM) levels of GR. Seven cancers (10.4%) had only low-affinity GR (Kd greater than 1.0 nM). Twenty patients (29.9%) had cancers with GR greater than 10 fmol/mg protein. Mean GR content was significantly greater (11.8 +/- 2.9 fmol/mg protein) in Dukes' Stage A and B cancers when compared to Stage C and D cancers (6.2 +/- 1.6 fmol/mg protein). A significantly greater percentage (52.4%) of patients in the early stages (A and B) had tumors with greater than 10 fmol/mg protein compared to patients with more advanced (C and D) cancers (19.6%). GR content did not correlate with histological differentiation, patient age, or preoperative carcinoembryonic antigen levels. No difference in the GR content was noted between left and right colon cancers or in patients of different sex or race. GR content of normal colon mucosa correlated with the GR content of colon cancers from the same surgical specimen, suggesting that these tumors maintain their normal complement of GR. In the early period of follow-up, 12 of 43 (28%) Stage C and D patients with GR less than 10 fmol/mg protein have died, whereas all 8 Stage C and D patients with GR greater than 10 fmol/mg protein are alive. GR content of colon cancers may have prognostic significance and may identify a group of patients with colon cancer that may benefit from hormonal therapy with antigastrin drugs.

Novel expression of gastrin (cholecystokinin-B) receptors in azaserine-induced rat pancreatic carcinoma: receptor determination and characterization.

Many reports have emphasized the role of gastrin as a growth factor for normal gastrointestinal mucosa and gastrointestinal cancers. Recent studies have pointed out that this peptide acts also as a growth factor for the pancreatic cancer cell line AR42J. This effect is mediated by gastrin [cholecystokinin (CCK)-B] receptors. In the present study, we investigated gastrin (CCK-B) receptor expression in the azaserine-induced rat pancreatic carcinoma DSL-6, comparing it to normal rat pancreas, and we also characterized CCK receptor subtypes in this tumor. The results showed that there is extensive gastrin binding to the DSL-6 pancreatic carcinoma. No evidence of specific gastrin binding to normal pancreas was found. Analysis of the ability of gastrin-17-I to inhibit 125I-gastrin-I binding demonstrated that gastrin bound to a single class of receptors with a Kd of 0.21 +/- 0.04 nM and a binding capacity of 184 +/- 29 fmol/mg protein. 125I-Gastrin-I binding was inhibited by the specific CCK-B receptor antagonist L365,260 approximately 40 times more effectively than by the specific CCK-A receptor antagonist L364,718. Analysis of the ability of cholecystokinin octapeptide (CCK-8) to inhibit 125I-Bolton-Hunter-CCK-8 binding revealed two CCK binding sites, i.e., a high affinity site and a low affinity site. The observed binding affinities of CCK-8 were then introduced into the computer analysis of the dose-inhibition curve of the ability of gastrin-17-I to inhibit binding of 125I-Bolton-Hunter-CCK-8, which was significantly better fit by a three-site model than by a two-site model. The three sites meet the criteria for CCK-B, high affinity CCK-A, and low affinity CCK-A receptors. The binding capacity of CCK-B receptors constitutes 34% of the total high affinity CCK binding sites. This study demonstrated that DSL-6 pancreatic carcinoma expresses three subtypes of CCK receptors. Gastrin (CCK-B) receptors, which were not detected in normal rat pancreas, constitute about one third of the total high affinity CCK receptors. We suggest that novel expression of gastrin (CCK-B) receptors may be generated by gene mutation or amplification during carcinogenesis and may play an important role in promoting tumor growth.

Lorglumide and loxiglumide inhibit gastrin-stimulated DNA synthesis in a rat tumoral acinar pancreatic cell line (AR42J).

Many reports emphasized the role of gastrin as growth factor on normal gastrointestinal mucosa and pancreas. In the present study, we analyzed the proliferative effects of cholecystokinin (CCK) and gastrin peptides on a rat tumoral pancreatic cell line, AR42J, which possesses both CCKA and CCKB receptor subtypes. The results showed a good correlation between the binding of gastrin to CCKB receptor [Kd 1.125 +/- 0.3 (SD) nM] and its ability to either induce ornithine decarboxylase activity [50% effective concentration, 0.6 +/- 0.3 nM] and [3H]-thymidine incorporation [50% effective concentration, 2 +/- 0.4 nM]. Furthermore, the ability of different cholecystokinin and gastrin antagonists such as proglumide and asperlicin derivatives (respectively, CR1409, CR1505, and L364,718) were tested. We found that all antagonists displaced 125I-labeled gastrin binding, with the following order of potencies: L364,718 greater than CR1409 greater than CR1505 greater than proglumide. Furthermore, the 50% inhibitory concentration of CR1409 and CR1505 to inhibit gastrin stimulated ornithine decarboxylase activity (an early event involved in cell proliferation) and [3H]thymidine incorporation were in agreement with their constants of inhibition (Ki) on gastrin binding. The L364,718 compound, at a concentration which fully occupied the CCKA without affecting the CCKB, had no effect on gastrin stimulated ornithine decarboxylase activity and [3H]thymidine incorporation. In addition, this compound appeared to be a full agonist on CCKB receptor. These results confirm the implication of the CCKB receptor in the proliferative response of AR42J cells to gastrin.

Inhibition of human gastric adenocarcinoma xenograft growth in nude mice by alpha-difluoromethylornithine.

We studied the effect of inhibition of polyamine biosynthesis by alpha-difluoromethylornithine on the growth of a human gastric adenocarcinoma (CLEES) xenotransplanted in nude mice. CLEES is a well-differentiated gastric adenocarcinoma of the intestinal type. The doubling time has ranged from 7 to 10 days through 11 passages. Electron microscopic and immunohistochemical studies comparing the original tumor and xenotransplants showed similar structure and similar amounts of carcinoembryonic antigen. Polyamine biosynthesis is required for cell division. alpha-Difluoromethylornithine inhibits ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. In this study, 48 athymic mice were used in two experiments. In the first experiment, two groups of 12 mice each were inoculated with CLEES tumor cells and received either tap water or a 3% alpha-difluoromethylornithine solution as drinking water. Tumor size was measured twice weekly. Tumor size was significantly decreased from controls by the fourth week of treatment and at all points of analysis thereafter for 7 wk. In the second experiment, alpha-difluoromethylornithine significantly reduced tumor concentrations of the polyamines putrescine and spermidine. In addition, the tumor content of DNA was significantly reduced in treated mice (0.64 +/- 0.16 mg) compared to controls (4.76 +/- 0.92 mg). Our data suggest that inhibition of polyamine biosynthesis may be a useful component of multidrug chemotherapy for human gastric adenocarcinoma. Establishment of tumor lines such as this gastric adenocarcinoma will facilitate further studies on the biological behavior of human gastric cancer and its response to chemotherapeutic manipulation in vivo.

Cholecystokinin-B/gastrin receptor: a novel molecular probe for human small cell lung cancer.

The brain-gut hormones, gastrin and cholecystokinin, have a trophic effect on the gastrointestinal mucosa in vivo and promote the growth of several neoplastic cell lines. In this study, cholecystokinin-B/gastrin receptor has been demonstrated to provide a novel molecular marker for the diagnosis of small cell lung cancer by using biopsy specimens. Physiological expression of the receptor mRNA is detectable in particular areas of the human brain, stomach, and pancreas but not in the lung. The receptor mRNA was detected selectively in all small cell lung cancer (10 cases) with a RT-PCR assay. By contrast, it was detectable in only 1 of 13 squamous cell carcinomas or 21 adenocarcinomas of the lung. Thus, the cholecystokinin-B/gastrin receptor could be an attractive therapeutic target for small cell lung cancer.

Common or distinct receptors for gastrin and cholecystokinin in gastric mucosa?

The differentiation between gastrin (HG) and cholecystokinin (CCK) receptors in gastric mucosa was examined on isolated parietal (F3) and non-parietal (F1) cells from rabbit fundic mucosa separated by elutriation. Direct binding assays on enriched cell populations were performed using 125I-labeled HG-17, 125I-labeled CCK-8 and 125I-labeled CCK-39 as probes. (1) On F1 cells, the dissociation constants (Kd) for the two labeled CCKs were nearly the same (62 pM for CCK-8 and 74 pM for CCK-39) but the binding capacity for CCK-8 was 2-times higher than for CCK-39. HG-17 also bound to this cell population, but its Kd value as about 2-times higher (110 pM) than that of CCK. The presence of two distinct classes of sites on F1 cells can be suggested from competition studies: one more specific for CCK, which bound CCK-8 and CCK-39 with the same affinity, and another class more specific for gastrin, which bound CCK-8 and HG-17 with the same affinity and CCK-39 with a low affinity. (2) On F3 cells, CCK-8 and HG-17 bound with similar affinities (Kd values 81 pM for CCK-8 and 87 pM for HG-17), but CCK-39 did not specifically bind to this cell population. The presence of a binding site more specific for HG than for CCK on F3 cells was confirmed by competition studies in which CCK-33 competed for binding with labeled HG-17 and labeled CCK-8 with a 50-times lower affinity than the other peptides.

Cholecystokinin-B/gastrin receptor binding peptides: preclinical development and evaluation of their diagnostic and therapeutic potential.

The high sensitivity of pentagastrin stimulation in detecting primary or metastatic medullary thyroid cancer (MTC) suggests widespread expression of the corresponding receptor type on human MTC. Indeed, autoradiographic studies demonstrated cholecystokinin (CCK)-B/gastrin receptors not only in >90% of MTCs but in a high percentage of small cell lung cancers and potentially a variety of gastrointestinal adenocarcinomas. In a pilot study, we have demonstrated the feasibility of radiolabeled gastrin-I to target CCK-B receptor-expressing tissues in vivo in animals and patients (T. M. Behr et al., Eur. J. Nucl. Med., 25: 424-430, 1998). The aim of the present study was to systematically optimize, in a preclinical model, suitable radioligands for targeting CCK-B receptors in vivo. For this purpose, a variety of CCK/gastrin-related peptides, all having in common the COOH-terminal CCK-receptor binding tetrapeptide sequence Trp-Met-Asp-PheNH2 or derivatives thereof, were studied. They were radioiodinated by the Iodogen or Bolton-Hunter procedures. The peptides tested were members of the gastrin- or cholecystokinin families or possessed characteristics of both, which differ by the intramolecular position of a tyrosyl moiety (occurring in native or sulfated form). Their stability and affinity were studied in vitro and in vivo; their biodistribution and therapeutic efficacy were tested in nude mice bearing s.c. human MTC xenografts. Diethylene-triamine-pentaacetate derivatives of suitable peptides were synthesized, evaluated, and labeled with (111)In. All members of the CCK or gastrin family were stable in serum (with t(1/2)s of several hours at 37 degrees C); nevertheless, the stability of those peptides was highest that bore the NH2-terminal pGlu residues (e.g., big gastrin, gastrin-I, caerulein, and others) or D-amino acids. In accordance to their comparably low affinity, nonsulfated members of the CCK family showed fairly low uptake in the tumor and other CCK-B receptor-expressing tissues (e.g., the stomach). Sulfated CCK derivatives performed significantly better but additionally displayed a high uptake in normal, CCK-A receptor-expressing tissues (such as the liver/gallbladder, pancreas, and bowel). Best tumor uptake and tumor:nontumor ratios were obtained with members of the gastrin family, probably because of their selectivity and affinity for the CCK-B receptor subtype. Pilot therapy experiments in MTC bearing animals showed significant antitumor efficacy as compared with untreated controls. (111)In-Labeled diethylene-triamine-pentaacetate derivatives of minigastrin showed excellent targeting of CCK-B receptor-expressing tissues in animals and a normal human volunteer. These data suggest that CCK/gastrin analogues may be a useful new class of receptor binding peptides for diagnosis and therapy of CCK-B receptor-expressing tumors, such as MTC or small cell lung cancer. Nonsulfated gastrin derivatives may be preferable because of their CCK-B receptor selectivity, and hence, lower accretion in normal CCK-A receptor-expressing organs. Further preclinical as well as clinical studies are ongoing.

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells.

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.

Molecular identification and characterization of the gastrin receptor in guinea pig gastric glands.

The binding characteristics of 125I-labeled Leu15-gastrin and the molecular identification of the gastrin receptor was investigated in dispersed guinea pig gastric glands. The binding of [125I]gastrin to gastric glands was temperature dependent, saturable, and specific. At 37 C, the binding was rapid, became maximal within 10 min, and declined after 30 min; at 24 C, binding reached a steady state between 30 and 60 min. The dissociation of specifically bound gastrin was also rapid, with 35% of the radioligand dissociating in 5 min at 37 C. Gastrin displaced [125I]gastrin in a dose-dependent manner, with 50% displacement at 4.4 nM. Scatchard analysis of the saturation curve was best described by a one-site binding model with a Kd of 2.3 nM and maximum binding of 6.0 x 10(-10) M/mg DNA. A significant reduction of [125I]gastrin binding to glands occurred in the presence of GTP (0.1 mM), (Bu)2-cGMP (1.0 mM), and protease inhibitors. Chemical cross-linking studies using the cross-linking reagent disuccinimidyl suberate and sodium dodecyl sulfate-gel electrophoresis identified a major band with a mol wt of 78K and several other lower mol wt bands in the range of 60K, 48K, and 38K. Identical electrophoretic patterns were obtained when glands were bound and solubilized in the presence and absence of dithiothreitol, showing the lack of disulfide bonds in the gastrin receptor subunit structure. Since dithiothreitol significantly enhanced radioligand binding when present during binding, its observed actions are most likely in the intracellular processing of the radioligand and not at the receptor level.

High gastrin and cholecystokinin (CCK) gene expression in human neuronal, renal, and myogenic stem cell tumors: comparison with CCK-A and CCK-B receptor contents.

Gastrin and cholecystokinin (CCK) are two major regulatory peptides synthesized by human gut and brain tissues as well as by selected tumors, in particular gastrin-producing neuroendocrine tumors. In the present study we have evaluated gastrin and CCK gene expression in a group of primary human tumors, including neuronal, renal, and myogenic stem cell tumors, using in situ hybridization techniques. In addition, CCK-A and CCK-B receptors were evaluated in the same group of tumors with receptor autoradiography. Most tumors had gastrin messenger ribonucleic acid (mRNA): 10 of 11 medulloblastomas, 5 of 5 central primitive neuroectodermal tumors, 11 of 11 Ewing sarcomas, 8 of 10 neuroblastomas, 4 of 4 Wilms' tumors, 5 of 5 rhabdomyosarcomas, and 10 of 10 leiomyosarcomas. CCK mRNA was restricted predominantly to Ewing sarcomas (9 of 11) and leiomyosarcomas (5 of 10). CCK-A and CCK-B receptors were not frequently found in these tumors, except for leiomyosarcomas. These data suggest that gastrin and CCK may play a previously unrecognized role in this group of human stem cell tumors. If the increased gastrin mRNA indeed translates into increased gastrin production, measurement of gastrinemia may have a diagnostic significance in the early detection of these tumors. As these two hormones have been reported to act as potent growth factors, they may be of pathophysiological relevance for patients with such stem cell tumors.

Localization of cholecystokinin binding sites in the adult and developing Brazilian opossum brain.

Cholecystokinin (CCK) is now recognized as one of the most abundant peptides in the mammalian central nervous system. We have previously used immunohistochemistry to localize CCK in the adult and developing Brazilian opossum brain. However, little is known about the distribution of CCK binding sites in the developing mammalian brain. Therefore, to further our knowledge of the sites of action for CCK during development, we initiated a series of studies to localize CCK binding sites in the adult and developing Brazilian opossum. This species was chosen because pups are born in a fetus-like state. Receptor autoradiography was performed on coronally sectioned brains of 1 to 60 day postnatal (PN) animals and adults with 125I-Bolton Hunter-CCK-8 as the radioligand. Binding is evident in the 1PN opossum brainstem and is observed in the developing forebrain by 5PN. Region-specific binding increases during development, and binding in the 35PN brain resembles the adult pattern. Binding is evident prior to the detection of CCK-like immunoreactivity in many areas. The facial motor nucleus is identifiable and exhibits high levels of binding in Brazilian opossum pups of 10 to 35 days of age. However, binding is undetectable in the facial motor nucleus of 45 and 60PN pups. In general, the binding patterns for CCK in the adult opossum resemble those of other mammals and likely mediate similar physiological functions. However, some cholecystokininergic pathways appear to be unique to neonatal mammals.

Age-related changes in gallbladder contractility and gallbladder cholecystokinin receptor population in the guinea pig.

We have examined the effects of aging on guinea pig biliary motility both in vitro and in vivo. The first experiment compared contractile tension of gallbladder strips from young adult (6-12 months old) and 3-year-old guinea pigs in vitro. Contraction of gallbladder strips from the young guinea pigs was twice as forceful and was more sensitive to octapeptide of cholecystokinin (CCK-8) stimulation than the gallbladder strips from the older guinea pigs. The two groups were also studied in vivo by measuring changes in the intraluminal pressure of the gallbladder in response to exogenously administered doses of CCK-8. Young adult guinea pigs were more sensitive to CCK-8 at the lower doses tested and demonstrated gallbladder contractions that were more forceful than that of the old guinea pigs. CCK receptors were measured on gallbladder muscularis membranes from young adult and old guinea pigs. The number of receptors on gallbladder membranes decreased with age: 65.0 +/- 17.7 fmoles/mg protein on membranes from 1 year old; 7.9 +/- 2.0 fmoles/mg protein on membranes from 3 years old. The binding affinity of CCK receptors on gallbladder muscularis membranes for binding to CCK-8 was not significantly different in the two age groups studied. We conclude that age-related decreases in gallbladder responses to CCK-8 may be due to decreased concentrations of CCK receptors on gallbladder muscle cells.

Development and age-related changes in pancreatic cholecystokinin receptors and duodenal cholecystokinin in guinea pigs.

We have investigated the changes associated with development and aging on the interrelationships between cholecystokinin (CCK) and the pancreas in the guinea pig. Three groups (1 month old, 1 year old, and 3 years old) of male guinea pigs were sacrificed while feeding in order to measure food-stimulated levels of CCK in blood and in duodenal mucosa by radioimmunoassay (RIA), as well as the pancreatic concentrations of CCK receptors. Systemic blood concentrations of CCK did not change with age. However, the concentration and content of CCK in duodenal mucosa increased more than 3-fold with age. A single class of high-affinity (KD less than or equal to 0.1 nM) CCK-receptor was found on the pancreatic membranes. The concentration (fmol/mg protein) of these receptors significantly diminished by one-half with increasing age. We also found an apparently similar fall in the receptor-binding affinity, but the difference was not significant. We conclude that in the guinea pig, duodenal content of CCK increases so as to compensate for the decreasing concentration of pancreatic CCK receptors, or, perhaps, vice versa. The diminished exocrine function of the pancreas, seen with increasing age, may well reflect both the diminished number of CCK-receptors and the reduction of pancreatic acinar cells.

Design of nonpeptidal ligands for a peptide receptor: cholecystokinin antagonists.

A series of 3-substituted 5-phenyl-1,4-benzodiazepines, nonpeptidal antagonists of the peptide hormone cholecystokinin (CCK), have been synthesized. Designed on the basis of facts regarding CCK, its natural-product antagonist asperlicin (3), and the antianxiety agent diazepam (4), these compounds represent a significant departure from existing CCK antagonists. They also constitute perhaps the first examples of simple, nonpeptidal ligands for a peptide receptor to arise by design rather than by screening. These compounds serve to illuminate the distinction between central and peripheral CCK receptors, as well as to provide orally effective CCK antagonists of potential pharmacological or therapeutic utility. One rationale for their receptor affinity has possible applications in the design of nonpeptidal ligands for other receptors, peptidal as well as nonpeptidal.

Investigation of peripheral cholecystokinin receptor heterogeneity by cyclic and related linear analogues of CCK26-33: synthesis and biological properties.

A possible heterogeneity of peripheral receptors for CCK26-33 [Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] (CCK8) was investigated by replacement of the flexible Gly29 residue, reported to be crucially involved in the CCK8 folding, by a D-Lys residue in Boc[Nle28,31]CCK27-33, a derivative as active as CCK8. The linear peptide Boc-Asp-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2 was cyclized through amide bond formation between the side chains of Asp26 and D-Lys29 to give the peptide Boc-Asp-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2. Analogues 1 and 2 were shown to stimulate secretion of amylase from rat pancreas with a potency that was respectively 40 and 80 times lower than that of CCK8. In contrast, both peptides acted as weak antagonists (EC50 approximately 10(-5) M) of the CCK8-induced contractions of guinea pig ileum. Peptides 3 and 4 obtained by removal of the phenylalanine from 1 and 2 were inactive in all bioassays despite amidification of their C-terminal Asp32 residue, a modification known to induce antagonist properties in CCK7. Cyclization between residues 28 and 31 in Boc[Asp28,Lys31]CCK27-33 gave compound Boc-Tyr(SO3H)-Asp-Gly-Trp-Lys-Asp-Phe-NH2, which was inactive in all bioassays. The pharmacological properties of these first described cyclic analogues of CCK8 were in agreement with their binding affinity to brain and pancreas receptors, suggesting the existence of a heterogeneity of peripheral receptors and emphasizing the usefulness of cyclic peptides in structure-activity studies.

Cholecystokinin, dopamine D2 and N-methyl-D-aspartate binding sites in the nucleus of the solitary tract of the rat: possible relationship to ingestive behavior.

Receptor autoradiography was used to investigate the distribution of brainstem binding sites for cholecystokinin, dopamine and N-methyl-D-aspartate with particular reference to the nucleus of the solitary tract of the rat, an area involved in the control of ingestive behavior. Binding sites for the A and B subtypes of the cholecystokinin receptor, labeled with [(125)I]cholecystokinin octapeptide sulfate in the presence or absence of antagonists for the devazepide (A) or L-365,260 (B) receptor, were present throughout the caudal rostral extent of the nucleus of the solitary tract, the A type predominating in the commissural, medial and gelatinous part and the B type in the lateral part. In the most rostral part of the medial nucleus of the solitary tract, both A and B receptors were present. Dopamine D2 receptors, labeled with [(125)I]NCQ-298, were found in all parts of the nucleus of the solitary tract. No binding to the dopamine D1 receptor, labeled with [(125)I]SCH-23982, was found in the brainstem. N-Methyl-D-aspartate receptors, labeled with [(3)H]dizocilpine maleate, were also present in the entire caudorostral extent of the nucleus of the solitary tract. Binding to cholecystokinin A receptors was co-distributed with [(125)I]NCQ-298 and [(3)H]dizocilpine maleate binding in the caudal and rostral parts of the nucleus of the solitary tract, and binding to cholecystokinin B receptors overlapped with [(125)I]NCQ-298 and [(3)H]dizocilpine maleate binding in the rostral nucleus of the solitary tract. These results are consistent with the hypothesis that cholecystokinin, dopamine and glutamate interact in the nucleus of the solitary tract in the control of ingestive behavior.

Localization and quantification of cholecystokinin receptors in rat brain with storage phosphor autoradiography.

The present study evaluated a new imaging technique that demonstrated the application of storage phosphor autoradiography in the localization and quantification of cholecystokinin receptors in rat brains and compared the results with film autoradiography. Cryostat sections were incubated with [125I]Bolton-Hunter-labeled sulfated cholecystokinin octapeptide followed by exposure to a storage phosphor-imaging screen and suitable autoradiography film. To obtain satisfactory images, it took 6 days with film autoradiography vs. 15 hours with the storage phosphor technique. Both film and storage phosphor autoradiograms showed the same cholecystokinin receptor distribution in brain sections; however, the film imaged more details. To reach the lowest possible response ratio between low and high receptor density regions in rat brains, storage phosphor autoradiography was about 240-fold faster than film. In addition, the new technique presented a considerably larger exposure time range for maintaining that ratio. The binding per area showed a linear relationship with the thickness of sections between 5 and 14 microns. In the linear response range, the quantitative results of both methods are comparable. In conclusion, storage phosphor autoradiography is a faster technique for localizing and quantifying peptide receptors in tissue sections but slightly compromised in resolution when compared with film autoradiography.

Preclinical and initial clinical evaluation of 111In-labeled nonsulfated CCK8 analog: a peptide for CCK-B receptor-targeted scintigraphy and radionuclide therapy.

UNLABELLED: The presence of cholecystokinin (CCK)-B (gastrin) receptors has been shown in more than 90% of medullary thyroid cancers (MTCs) and in a high percentage of small cell lung cancers, stromal ovarium cancers and several other tumor types. METHODS: The aim of this study was to evaluate in vitro and in vivo whether 111In-labeled CCK-B receptor-specific CCK8 analog [D-Asp26,Nle28,31]CCK26-33 (D-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2) is suitable for CCK-B receptor scintigraphy based on the finding that unlabeled nonsulfated diethylenetriamine pentaacidic acid [DTPA0]CCK8 and tetraazacyclododecanetetraacetic acid [DOTA0]CCK8 analogs show high and specific binding for CCK-B receptors in human tumors. Fifty percent inhibitory concentrations were in the low nanomolar range. RESULTS: In vitro, [111In-DOTA0]CCK8 showed specific internalization in CCK-B receptor-positive rat pancreatic tumor cells AR42J. Internalization of the analog appeared to be time and temperature dependent and receptor specific. From the data obtained with [111In-DOTA0]CCK8 and (125I)I-gastrin, the latter being a specific ligand for the CCK-B receptor, the rat pancreatic cell line CA20948 also appeared to be CCK-B receptor positive. This provides an in vitro and in vivo rat tumor model because this cell line can be grown to solid tumors in Lewis rats. In vivo biodistribution experiments in CA20948 tumor-bearing Lewis rats showed rapid clearance of [111In-DOTA0]CCK8, and specific uptake was found in the CCK-B receptor-expressing stomach and tumor. Furthermore, comparing [111In-DOTA0]CCK8 with the radioiodinated nonsulfated CCK10 analog (D-Tyr-Gly-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2), both ligands having high affinity for the CCK-B receptor, tumor-to-blood ratios were significantly higher for [111In-DOTA0]CCK8 than for 125I-CCK10, analogous to the findings with radioiodinated and 111In-labeled octreotide. The study in humans with [111In-DTPA0]CCK8 showed receptor-specific uptake in the CCK-B receptor-positive stomach and in metastases in the neck region up to 48 h after injection. CONCLUSION: [111In-DOTA0]CCK8 is most promising for scintigraphy and, after coupling to therapeutic radionuclides, for radionuclide therapy of human CCK-B receptor-positive tumors such as MTC and small cell lung cancer.