Region-specific Radioimmunoassays Research Articles

The anorexic hormone Peptide YY3 -36 is rapidly metabolized to inactive Peptide YY3 -34 in vivo

Peptide YY (PYY) is a 36 amino acid peptide hormone released from enteroendocrine cells. An N-terminally degraded metabolite, PYY3–36, has anorexigenic effects, which makes the PYY system a target for obesity treatment. However, little is known about the kinetics and degradation products of PYY. A related peptide, Neuropeptide Y (NPY), may be degraded from the C-terminus. We therefore investigated PYY degradation after in vitro incubations in porcine plasma and blood and in vivo by infusing PYY3–36 into multicatheterized pigs (n = 7) (2 pmol/kg/min). Plasma samples were analyzed by region-specific radioimmunoassays (RIA) and HPLC analysis. A metabolite, corresponding to PYY3–34 was formed after incubation in plasma and blood and during the infusion study. When taking the C-terminal degradation into account, the half-life (T½) of PYY in blood and plasma amounted to 3.4 ± 0.2 and 6.2 ± 0.2 h, respectively. After PYY3–36 infusion in pigs, the peptide was degraded with a T½ of 3.6 ± 0.5 min. Significant extraction (20.5 ± 8.0%) compatible with glomerular filtration was observed across the kidneys and significant C-terminal degradation (26.5 ± 4.8%) was observed across the liver. Net balances across the hind limb, splanchnic bed, and lungs were not significantly different from zero. PYY3–34 was unable to activate the Y2 receptor in a transfected cell line. In conclusion, PYY3–36 is extensively degraded to PYY3–34 in the pig, a degradation that renders the peptide inactive on the Y2 receptor. Currently used assays are unlikely to be able to detect this degradation and therefore measure falsely elevated levels of PYY3–36, leading to underestimation of its physiological effects.

The anorexic hormone Peptide YY3-36 is rapidly metabolized to inactive Peptide YY3-34 invivo.

Peptide YY (PYY) is a 36 amino acid peptide hormone released from enteroendocrine cells. An N-terminally degraded metabolite, PYY3–36, has anorexigenic effects, which makes the PYY system a target for obesity treatment. However, little is known about the kinetics and degradation products of PYY. A related peptide, Neuropeptide Y (NPY), may be degraded from the C-terminus. We therefore investigated PYY degradation after in vitro incubations in porcine plasma and blood and in vivo by infusing PYY3–36 into multicatheterized pigs (n = 7) (2 pmol/kg/min). Plasma samples were analyzed by region-specific radioimmunoassays (RIA) and HPLC analysis. A metabolite, corresponding to PYY3–34 was formed after incubation in plasma and blood and during the infusion study. When taking the C-terminal degradation into account, the half-life (T½) of PYY in blood and plasma amounted to 3.4 ± 0.2 and 6.2 ± 0.2 h, respectively. After PYY3–36 infusion in pigs, the peptide was degraded with a T½ of 3.6 ± 0.5 min. Significant extraction (20.5 ± 8.0%) compatible with glomerular filtration was observed across the kidneys and significant C-terminal degradation (26.5 ± 4.8%) was observed across the liver. Net balances across the hind limb, splanchnic bed, and lungs were not significantly different from zero. PYY3–34 was unable to activate the Y2 receptor in a transfected cell line. In conclusion, PYY3–36 is extensively degraded to PYY3–34 in the pig, a degradation that renders the peptide inactive on the Y2 receptor. Currently used assays are unlikely to be able to detect this degradation and therefore measure falsely elevated levels of PYY3–36, leading to underestimation of its physiological effects.

Chromogranins can be measured in samples from cats and dogs.

BackgroundMethods for objective evaluation of stress in animals are important, but clinically difficult. An alternative method to study the sympathetic activity may be to investigate Chromogranin A (CGA), Chromogranin B (CGB) and Secretogranin II (SG2). The aim of this study was to investigate the cross-reactivity of CGA, CGB and SG2 between man, cat and dog and to explore possibilities to measure these proteins in samples from cats and dogs.ResultsAdrenal gland extracts from feline and canine species were measured by region-specific radioimmunoassays in different dilution steps to explore possible inter species cross reactivity. High cross reactivity was found for cats in the CGA17-38, CGA324-337, CGA361-372, CGB and SG2 assays. High cross reactivity was found for dogs in the CGA17-38, CGA361-372, CGB and SN assays. The method measuring the intact CGA was not useful for measurements in cats and dogs.ConclusionsRegion-specific assays measuring defined parts of CGA, CGB and SG2 can be used for measurements in samples from cats and dogs. These results are promising and will allow for further studies of these proteins as possible clinical biomarkers in cats and dogs.

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Measurements of secretogranins II, III, V and proconvertases 1/3 and 2 in plasma from patients with neuroendocrine tumours

Introduction Chromogranin (Cg) and secretogranin (Sg) are members of the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In recent publications we have presented generation of region-specific antibodies against CgA and CgB and also development of several region-specific radioimmunoassays for measurements of specific parts of the Cgs. In this study we describe generation of antibodies against SgII, SgIII, SgV and the proconvertases PC1/3 and PC2 and development of radioimmunoassays for measurements of these proteins. Materials and methods Peptides homologous to defined parts of the secretogranin and proconvertase molecules were selected and synthesised. Antibodies were raised, radioimmunoassays were developed and circulating levels of the proteins in plasma samples from 22 patients with neuroendocrine tumours were measured in the assays. Results Increased plasma concentrations were recorded in 11, 4 and 3 of the patients with the SgII 154–165 (N-terminal secretoneurin), the SgII 172–186 (C-terminal Secretoneurin) and the SgII 225–242 assays respectively. The SgIII, SgV, PC1/3 and PC2 assays failed to detect increased concentrations in any of the patients. Conclusion Increased concentrations of SgII, especially the N-terminal part of secretoneurin could be measured in plasma from patients with endocrine pancreatic tumours and in this case this assay was quite comparable to measurements of CgA and CgB. Even though secretoneurin was not as frequently increased as CgA and CgB in patients with carcinoid tumours or pheochromocytoma it may be a useful marker for endocrine pancreatic tumours.

Measurements of chromogranin B can serve as a complement to chromogranin A

Objective CgA has been shown to be an excellent marker for neuroendocrine tumours. However, there are two major drawbacks with CgA measurements; elevated levels are common in patients with decreased renal function and in patients on treatment with proton pump inhibitors. These problems are not seen with CgB measurements. We have recently presented the development of 13 region-specific radioimmunoassays for measurements of CgB. A region-specific assay was identified, which measured higher concentrations of CgB than the other assays and seemed to be very useful as a marker for neuroendocrine tumours. The aim of the present study was therefore to further explore the diagnostic potential of this assay in the clinical management of patients with neuroendocrine tumours. Methods Measurements of CgB with two methods were compared with CgA in plasma samples from patients investigated for neuroendocrine tumours ( N = 86), patients with decreased renal function ( N = 35) and patients on treatment with proton pump inhibitors ( N = 29). Results The diagnostic sensitivity for the new CgB assay was almost as good as that for CgA. Furthermore, with CgB measurements we could avoid the falsely elevated levels of CgA found in patients with decreased renal function and treatment with proton pump inhibitors. Conclusions We conclude that the new CgB assay can serve as a complement to CgA measurements as an important tumour marker for neuroendocrine tumours.

Production, Secretion, and Biological Activity of the C-Terminal Flanking Peptide of Human Progastrin

Processing of progastrin, the 80-amino acid precursor of the hormone gastrin, generates a variety of peptides with distinct distributions and biological activities. However, little is known regarding the expression, secretion, and biological activity of the 6-amino acid C-terminal flanking peptide (CTFP) of progastrin. The objectives were to determine the concentration of CTFP in normal subjects and patients with gastrointestinal diseases and to investigate the biological activity of CTFP. CTFP, gastrin-amide (Gamide), glycine-extended gastrin (Ggly), and progastrin were measured using region-specific radioimmunoassay (RIA) in antral extracts and resected colorectal cancers (CRC) and in plasma from normal subjects (fasting and meal stimulated) and from patients with CRC, multiple endocrine neoplasia type 1 (MEN-1), or pernicious anemia. The effect of CTFP on proliferation, migration, and activation of the mitogen-activated protein kinase (MAPK) pathway in several types of gastrointestinal cell lines was determined. CTFP is by far the predominant progastrin-derived peptide found in the antrum (4-fold higher than Gamide), resected CRC, and circulation (60-fold higher than Gamide) and is released after meal stimulation. The hypergastrinemic patients (MEN-1, pernicious anemia) had elevated plasma Gamide but unaltered CTFP demonstrating differential secretion of these 2 progastrin-derived peptides. Finally, CTFP stimulated proliferation and migration and activated MAPK of cells in culture. The high and regulated expression of CTFP in healthy and diseased subjects combined with the evidence for biological activity of CTFP demonstrates that CTFP is not an inactive metabolite of progastrin processing but is a bioactive peptide with potential roles in the normal and diseased gastrointestinal tract.

Chromogranin A in gastric neuroendocrine tumours: an immunohistochemical and biochemical study with region-specific antibodies

The aim of the present study was to investigate ECLomas and enterochromaffin-like (ECL) cell hyperplasia in gastric human mucosa regarding the immunohistochemical expression of chromogranin A (CgA) epitopes and to measure the same CgA epitopes in plasma samples. Eight gastric biopsies from ECLomas, seven of type I and one of type III, and biopsies from one patient showing only ECL cell hyperplasia were included in the study. Our results revealed a varying expression of region-specific CgA epitopes in the ECLomas regarding both the frequency of immunoreactive cells and intensity of immunoreactivity. CgA284-301 (pancreastatin) was not revealed in any neoplasm, whereas CgA361-372 (catestatin) was expressed in all ECLomas. However, the number of immunoreactive cells to vesicular monoamino transporter 2 (VMAT 2) or the commercial monoclonal CgA (CgA250-284) antibodies were generally higher. The plasma concentrations of the region-specific CgA radioimmunoassays differed considerably, with highest concentrations of CgA1-17 and CgA116-130 epitopes and the lowest with the CgA17-37, CgA63-76, CgA238-247 and CgA441-424 epitopes. No relationship was found between tissue expression and plasma concentration of CgA epitopes. In conclusion, this study shows that VMAT 2 and the commercial CgA antibodies seem more useful for histopathological diagnosis of ECLomas than the antibodies to the other CgA regions.

A panel of 11 region-specific radioimmunoassays for measurements of human chromogranin A

Introduction: The primary structure of human chromogranin A (CgA) not only contains 10 pairs of basic amino acids, which are potential cleavage sites for specific endogenous proteases, but also other sites in the molecule can be subjected to cleavage. Several CgA-related peptides have been identified in tissue, and many of the biological effects attributed to CgA seem to be mediated by these peptides. Materials and Methods: Peptides homologous to defined parts of the human CgA molecule were selected and synthesised. Antibodies were raised, and 11 specific radioimmunoassays were developed. Plasma samples from 20 patients with neuroendocrine tumours were collected and measured in all assays. Results: All assays measured circulating levels of CgA-derived peptides. Only four of the assays measured concentrations that correlated with that of total CgA. However, concentrations of the individual CgA-related peptides were generally lower than the concentration of total CgA. Different neuroendocrine tumours seem to process CgA differently. The ratio between a given region-specific assay and total CgA is inversely correlated to tumour activity. Conclusion: The assays presented allow measurements of defined regions of CgA and will thus become important tools for further studies of processing of CgA.

Metabolism of recombinant progastrin in sheep.

Precursor forms of peptide hormones may be biologically active with effects distinct from the mature end product. Nonamidated progastrin-derived peptides stimulate growth of colonic epithelium and are elevated in the circulation of patients with colorectal carcinomas, whereas the amidated end product is the major regulator of gastric acidity. Using region-specific radioimmunoassays, we here compared the in vitro and in vivo metabolism of recombinant human progastrin-(6-80) and two other nonamidated gastrins, gastrin-17-Gly and Tyr(70)-progastrin-(71-80). Although progastrin-(6-80) was very stable in vitro, both progastrin-(6-80) and gastrin-17-Gly were degraded in vivo. The in vivo data were best fitted by a double-exponential decay curve, and the half-lives for progastrin-(6-80) (t1/2alpha = 5.1 +/- 1.1, t(1/2)beta = 42 +/- 11 min) were significantly (P < 0.05) longer than for gastrin-17-Gly (t(1/2)alpha = 2.2 +/- 0.6, t(1/2)beta = 13 +/- 1 min). Tyr(70)-progastrin-(71-80) was degraded more rapidly. Comparison with amidated gastrins suggests that peptide length, rather than sequence, is the critical determinant of clearance. Progastrin has the clearance characteristics to be considered a circulating hormone.

Characterisation of N-terminal chromogranin A and chromogranin B in mammals by region-specific radioimmunoassays and chromatographic separation methods.

Chromogranin A (CgA) and chromogranin B (CgB) are acidic proteins stored in and released from hormone granules in endocrine and neuroendocrine tissue. The chromogranins are postulated to serve as pro-hormones to generate biologically active peptides, which may influence hormonal release and vascular functions or have antibacterial functions. Although N-terminal and C-terminal regions show some species amino acid homology, the chromogranins as a whole display considerable interspecies differences, which prevents their use in comparative studies of biological functions. We present four new radioimmunoassays for the measurement of defined N-terminal regions of CgA and CgB. A new radioimmunoassay for measurement of intact bovine CgA has also been developed. With these assays and two previously published ones, we have compared the cross-reactivity of chromogranins from man, cattle, sheep, goat, pig and horse and compared adrenomedullar content and serum levels of CgA from these species. We have also studied the influence of peptide concentrations and the ionic strength of the mobile phase on molecular weight estimations. Assays with antibodies directed against the N-terminal parts of CgA and CgB showed sufficient interspecies cross-reactivity to allow comparative quantification of the circulating levels in man, cattle, sheep, goat, pig and horse. Assays measuring the intact human or bovine CgA were not suitable for comparative purposes in samples from sheep, goat, pig and horse. Molecular interactions between vasostatin immunoreactive material and intact bovine CgA were demonstrated in gel permeation studies, suggesting that conclusions about the degree of N-terminal processing from elution profiles should be made with caution. Reliable interspecies comparison of chromogranins is difficult, but measurements with region-specific assays may be helpful to study concentrations of chromogranins and chromogranin-related peptides.

Molecular forms of gastrin in the circulation of patients with achlorhydria.

To study circulating gastrin profile, both fasting and postprandially, in patients with achlorhydria due to auto-immune atrophic gastritis, comparing these with normal healthy controls. Circulating gastrins were measured using three region-specific radio-immunoassays: amidated gastrins (R98), N-terminal G34 (R526) and N-terminal G17 (GP168). Samples were analysed further using gel chromatography. Fasting gastrin concentrations were elevated in achlorhydria as measured using all three antisera: median 714 pmol/l (range 107-5176) in achlorhydria versus 12 pmol/l (2-33) in controls (R98), 343 pmol/l (45-4316) versus 10 pmol/l (5-41) (R526), and 720 pmol/l (14-6000) versus 2 pmol/l (1-10) (GP168). In patients, 47% of gastrin was amidated (95% in controls) and 30% was processed N-terminally only to G71 (4% in controls). Gastrin rose significantly postprandially: 1643 pmol/l (269-7142) in patients versus 24 pmol/l (5-142) in controls (R98), 432 pmol/l (113-4756) versus 15 pmol/l (7-45) (R526) and 2189 pmol/l (304-7150) versus 15 pmol/l (7-45) (GP168). Only 25% was amidated in the patient group (93.5% in controls) and 21% remained as component I (4% in controls). This abnormal gastrin profile associated with hypergastrinaemia secondary to achlorhydria is consistent with saturation of the enzymes involved in the processing of the pro-hormone, in particular amidation of the C-terminus.

Simultaneous elevation of serum parathyroid hormone(PTH) and parathyroid hormone-related protein(PTHrP) in a case of lung cancer with hypercalcemia

The parathyroid hormone related protein(PTHrP) is the most common causative peptide of humoral hypercalcemia of malignancy. In contrast, the serum level of parathyroid hormone(PTH) is low to undetectable in the majority of patients with malignancy associated hypercalcemia. Few cases exist in which the production and secretion of PTH by malignant nonparathyroid tumors have been authenticated. To our knowledge, there is very rare case in which a nonparathyroid tumor expressed simultaneously both the PTH and PTHrP. We report a case of squamous cell carcinoma of the lung with hypercalcemia which presented with simultaneous elevation of serum PTH and PTHrP. Severe hypercalcemia (serum calcium, 7.5mEq/L) was found in a 65-year-old man who had a squamous cell carcinoma of the lung without any body metastasis and detectable parathyroid abnormalities on isotope scintigraphy. The serum level of intact parathyroid hormone (PTH) concentration was markedly elevated as measured in two site radioimmunoreactive PTH assays (intact PTH 150pg/mL ; normal 9~55). The serum level of a PTHrP was also increased as measured in C-terminal region specific radioimmunoassay (PTHrP 99.1 pmol/L ; normal 13.8~55.3). There are no evidences of coincidental primary hyperparathyroidism in parathyroid MIBI scan and other imaging studies including neck ultrasonography and computed tomography. These results suggest that simultaneous elevation of serum PTH and PTHrP in this patient can be caused by production of both PTHrP and PTH in other nonparathyroid lesions such as squamous cell carcinoma.

Development of Region-specific Radioimmunoassays against Rat Prosecretin or Secretin and their Characterization

Preeclampsia is a multisystemic disorder specific to human pregnancy. It is common and major complication, characterized by hypertension, proteinuria and oedema. This clinical condition adversely affects the mother and the foetus. The causative factors remain poorly understood. Oxidative stress is implicated in preeclampsia which could provide the linkage between decreased placental perfusion and the maternal syndrome. Pregnancy has also been associated with depressed cell mediated immunity. The increase in activity of adenosine deaminase (ADA) in preeclamptic women compared to normotensive pregnant women is reported in earlier studies. The main objective of the present study was to correlate the adenosine deaminase activity with the antioxidant status in preeclamptic pa-tients. The study groups aged between 20 – 35 years included 20 normotensive primigravida wom-en and 20 primigravida diagnosed as preeclampsia in their last trimester. The control group included 20 healthy nonpregnant women volunteers of the same age group. The diagnosis of preeclampsia was made by a gynecologist at Lady Goshen Hospital. The adenosine deaminase activity and antioxidant status in the form of superoxide dismutase (SOD), glu-tathione (GSH) and total antioxidant activity (TAA) were measured. The extent of oxidative stress was estimated by measuring plasma malondialdehyde (MDA) levels. The comparison of MDA between controls and preeclamptic patients showed significance (p 0.000). The total antioxidant activity (TAA) between the controls and preeclamptic pa-tients showed significance (p 0.000). However, ADA activity did not show statistical signifi-cance among the different study groups. The ADA activity may not be significant due to other systemic causes such as anemia and malnutrition.

Expression and characterization of preproVIP derived peptides in the human male urogenital tract.

Expression of the gene sequence encoding vasoactive intestinal polypeptide (VIP) leads to the synthesis of a 170 amino acid precursor molecule which can be processed to five fragments: preproVIP 22-79, peptide histidine methionine (PHM), or peptide histidine valine (PHV), preproVIP 111-122, VIP and preproVIP 156-170. Using region specific radioimmunoassays and antisera against the functional domains of the VIP precursor in combination with immunocytochemistry and chromatography, the localization, distribution and identity of the preproVIP derived peptides within the human male urogenital tract were investigated. Postmortem as well as fresh tissue specimens were used. All the preproVIP derived peptides were expressed and could be demonstrated in nerve fibres throughout the urogenital tract in close relation to the epithelial lining and in vascular as well as non-vascular smooth muscle. The VIP-related peptide containing fibres were most abundant in the prostate parenchyma and the seminal vesicle. Using double immunostaining, co-localization of the various preproVIP derived peptides could be evidenced. The fact that all preproVIP derived peptides are present in the urogenital tract, should be taken into consideration when the regulatory aspects of neuropeptides in physiological and pathophysiological functions are discussed.

Chromogranin A Peptide-Specific Antisera and High-Performance Size Exclusion Chromatography Demonstrate Amino-Terminal and Carboxy-Terminal Fragments of the Native Molecule in Human Cell Lines

We have studied the pattern of amino-terminal and carboxy-terminal protein processing of chromogranin A (CgA) in five human cell lines, four derived from lung cancers: NCI-H478, NCI-H1011, NCI-H727, and BEN; and one derived from human medullary thyroid carcinoma: TT. This was accomplished by fractionation of cell extracts by high-performance size exclusion chromatography (HPSEC) and measurement of CgA in the fractions by radioimmunoassay (RIA). Three RIA's were used, one specific for the amino-terminus of CgA, one specific for the carboxy-terminal region, and a monoclonal antibody-based assay that recognizes only the native CgA molecule. We demonstrated the presence of different amino- and carboxy-terminal immunoreactive species of CgA in the different cell lines. The amino-terminal assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H1011 cell lines, and a similar peak in the TT cells. The amino-terminal assay did not recognize any distinct species in BEN and NCI-H727 cell lines. The carboxy-regional assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H101 cell lines and high-molecular-size species in the NCI-H727 and BEN cells. Our studies demonstrate with region-specific RIA's the presence of both amino- and carboxy- forms of CgA in human cells that secrete this protein. These results provide direct evidence that CgA-producing cells produce, probably through endoproteolytic processing of the native molecule, amino- and carboxy-terminal forms of the protein.

Quantitation and Chromatographic Characterisation of Neuropeptide F (NPF) Immunoreactivity in Molluscan Nervous Tissue Using Region-Specific Antisera

Neuropeptide F (NPF) immunoreactivity has been quantified in extracts of the central nervous system from four gastropod (Helix aspersa, Buccinum undatum, Littorina littorea, and Patella vulgaris) and two bivalve (Mytilus edulis and Pecten maximus) molluscs using two region-specific radioimmunoassays. The first employed an antiserum, NPF3, raised to a synthetic N-terminal fragment of H. aspersa NPF and the second employed an antiserum, PP221, raised to the synthetic C-terminal hexapeptide amide of mammalian pancreatic polypeptide which fully cross-reacts with the analogous region of H. aspersa NPF. NPF immunoreactivity was detected in acidified ethanolic brain extracts of the four gastropod molluscs by both antisera. However, only the C-terminally directed antiserum detected immunoreactivity in brain extracts of the two bivalve molluscs. Reverse-phase HPLC analysis of brain extracts from B. undatum and L. littorea resolved a single NPF immunoreactive peptide which was more hydrophobic than natural H. aspersa NPF chromatographed under the same conditions. Gel permeation chromatography of these NPF immunoreactive peptides indicated that they were of a similar molecular mass to that of H. aspersa NPF. These data suggest that NPF is widely distributed in molluscs with a high degree of structural conservation of N- and C-terminal regions within the gastropod molluscs but significant structural differences within the N-terminal regions of analogous peptides in bivalves.

Processing, release and metabolism of cholecystokinin in SK-N-MCIXC cells

The human cholinergic neuroepithelioma cell line SK-N-MCIXC, which expresses high levels of cholecystokinin (CCK) mRNA and secretes intact CCK into the media, was used to examine CCK processing and metabolism. Our data provide evidence for the existence of specific candidate processing enzymes in SK-N-MCIXC cells which may be involved in processing proCCK in the brain and indicate that SK-N-MCIXC cells provide a model system for studying the regulation of these enzymes. mRNAs for the intracellular processing enzymes, prohormone convertase 1 (PC1), PC2 and furin were present in SK-N-MCIXC cells. PC1 and/or PC2 and/or furin may cleave at the dibasic amino acid pairs Arg-Arg at the C-terminal part of proCCK, and Arg-X-X-Arg at the N-terminal of the CCK-58 sequence in proCCK. The SK-N-MCIXC cell line demonstrated spontaneous and regulated release of CCK and large amounts of CCK-precursors, as measured with region specific radioimmunoassays coupled to high performance liquid chromatography. Storage granules containing glycine-extended CCK were shown in SK-N-MCIXC cells using indirect immunofluorescence. The extracellularly localized CCK-metabolizing enzyme, neutral endopeptidase 24.11 (EC 3.4.24.11), was present in membranes from both SK-N-MCIXC cells and in intact slices of rat cerebral cortex. The rat cerebral cortex is a brain region known to be rich in CCK. The SK-N-MCIXC cell line provides an in vitro model to study the regulation of CCK synthesis and metabolism in neuronal systems since it contains the storage granules, mRNA, intact peptide, and complement of enzymes necessary for biosynthesis and metabolism of CCK.

Evidence against autocrine feedback regulation of cholecystokinin secretion in man

To determine whether exogenous cholecystokinin (CCK) inhibits endogenous CCK release, cholecystokinin-8S (CCK-8S) was infused intravenously during continuous intraduodenal stimulation of endogenous CCK by a meal. CCK was measured in plasma by 2 region-specific radioimmunoassays employing antibodies T204 and 1703. AB T204 binds to carboxy-terminal CCK peptides containing the sulphated tyrosyl region, including CCK-8S, and AB 1703 to carboxy-terminal CCK peptides containing at least 14 amino acid residues. Meal-stimulated plasma CCK concentrations remained elevated during the entire infusion period. CCK-8S infusion further increased meal-stimulated plasma CCK concentrations, when measured with AB T204, while meal-stimulated plasma CCK concentrations were not suppressed by CCK-8S infusion, when measured with AB 1703. It is concluded that meal-stimulated endogenous CCK release is not affected by the effects of intravenously administered CCK-8S. These data suggest that autocrine feedback regulation of CCK release is not operative in man.

A decade of experiences with radioimmunoassays for cholecystokinin in The Netherlands.

The radioimmunologic determination of cholecystokinin (CCK) has proved to be notoriously difficult. This is due to the specificity of antibodies, preparation of radiolabeled CCK and low CCK concentrations in human plasma. About 10 years ago we succeeded in developing two highly sensitive region-specific radioimmunoassays for CCK. Antibody T204 binds to the sulfated tyrosine region of CCK, while antibody 1703 reacts with biologically active molecular forms of CCK containing at least 14 amino acid residues. Both antibodies are devoid of significant cross-reaction with gastrin. By means of these two radio-immunoassays CCK concentrations were measured in both tissue and plasma of various species, including man. In addition, the molecular forms of CCK in tissue and plasma were characterized. These CCK assays were used to study the mechanism of CCK secretion. It appeared that digested rather than intact protein and fat stimulated CCK release from the small intestine. The physiologic and pathophysiologic role of CCK in humans was studied using CCK radioimmunoassays and specific CCK-receptor antagonists. CCK was found to play an important role in pancreatic enzyme secretion, gallbladder contraction, and gastrointestinal motility but possibly also in pancreatic carcinogenesis and regulation of satiety and satiation.