Species specificity of cholecystokinin in gut and brain of several mammalian species.

Abstract

Immunoreactive intact cholecystokinin and its COOH-terminal octapeptide are found in brain as well as in extracts of gut of the monkey, dog, and pig, by using an antiserum with equivalent sensitivities for detecting the octapeptide in free form or incorporated in the intact molecule. The failure to detect intact cholecystokinin in extracts from monkey or dog by using an antiserum developed by immunization with porcine cholecystokinin is presume to be due to marked species differences in the NH2-terminal portion of the molecule. Tryptic digestion converted the intact cholecystokinin from all species to a peptide resembling the COOH-terminal octapeptide. The amount of cholecystokinin in the brain is comparable to that found in the gastrointestinal tract, the traditional site for this peptide.We have previously reported that a peptide resembling intact cholecystokinin (CCK) in size, charge, and immunologic specificity is found in extracts of the pig cerebral cortex (1). A peptide, first suggested to be gastrin-like (2) and later consid ered to resemble more closely the COOH-terminal octapeptide of CCK (CCK-8) (1,3,4), has been found in the brains of many animal species including not only mammals but also birds, fish, and amphibians (2). In this report we demonstrate that a CCK-like peptide is found in the brain as well as in the gut of the dog and monkey and that failure to detect it with an antiserum developed by immunization of a goat with porcine CCK (pCCK) is due to the marked species specificity of this antiserum which appears not to crossreact with CCK-8. Radioimmunoassay. 125I-Labeled synthetic human gastrin I and 125I-labeled CCK were prepared by our minor modification of the chloramine-T technique (5) using about 1.2 mCi of 125I (Amersham/Searle) for 1 ,tg of gastrin and 0.5 mCi of 125I for each 1 ,ug of CCK. Labeled gastrin was purified by starch gel electrophoresis and labeled CCK by adsorption to and elution from Quso G32 (5). MATERIALS AND METHODSThe production of the antisera used in this study has been described (1). One antiserum was prepared in a goat (goat 1) by immunization with pCCK. This antiserum does not crossreact with CCK-8 or heptadecapeptide gastrins at concentrations 1000-fold higher than the concentration of intact pCCK that decreases the ratio of antibody-bound to free labeled antigen to less than 10% of the ratio in the absence of unlabeled hormone. The other antiserum was prepared by immunization of a rabbit (rabbit B) with the COOH-terminal gastrin tetrapeptide amide. The antiserum used in the present study was obtained somewhat later in the course of immunization although from the same rabbit as reported previously (1). By using the later bleeding and '25I-labeled synthetic human gastrin as the tracer, the crossreactivities of pCCK and of CCK-8 were virtually identical on a molar basis. Heptadecapeptide gastrins reacted somewhat more strongly than the CCK peptides. Therefore, the gastrin content of all samples was also determined by using the guinea pig antiserum generally...

Characterization of a nontrypsin cholecystokinin converting enzyme in mammalian brain

“…The present study was undertaken because of the demonstration that the cerebral cortical tissues from several mammalian species contain CCK peptides in amounts comparable to those found in the gastrointestinal mucosa and that the fraction of the immunoreactivity in components smaller than CCK-33 is greater in the brain than in the gut (15). These observations suggested to us that brain extracts might contain an enzyme of high potency for the specific conversion of CCK-33 to the smaller hormonal forms.…”

“…In this report we characterize and partially purify 10,000 X g for 15 min. The supernatants were tested for ability to convert CCK-33 and gastrin-34 to smaller immunoreactive forms as described below.…”

Post-translational processing of cholecystokinin in pig brain and gut.

“…Thus, subsections of brain and gut differ only in the concentrations of CCK (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). The lack of agreement among laboratories is due in part to the unavailability of CCK33 or CCK39 from species other than the pig and the marked species specificity of antisera that are directed against the NH2-terminal portion ofthis molecule.…”

“…Among these, cholecystokinin (CCK) appears to be unique in that comparably high concentrations are found in neuronal and in mucosal tissues (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The various CCK-related peptides, immunochemically distinguishable by a variety of antisera (6,7,16), have not been shown to be extracted from tissues with equal efficiency by any single extractant. In this study we describe optimal methods for sequential extraction and radioimmunoassay (RIA) of the CCK peptides by using antisera specific for the NH2 terminus and COOH terminus, further characterize the properties of these peptides in different physicochemical systems, and determine whether, assuming a common precursor for all CCK forms, there are differences in the post-translational processing in the different tissues.…”

Pancreatic acinar cells: effects of microionophoretic polypeptide application on membrane potential and resistance

“…Short derivatives of CCK-PZ have recently been isolated from sheep brain (Dockray, Gregory & Hutchison, 1978) and indicated in hog intestine (Dockray, 1977;Straus & Yalow, 1978). Similarly, immunoreactivity to bombesin has been reported in the human gut (Polak, Bloom, Hobbs, Solcia & Pearse, 1976), sheep brain (Walsh & Dockray, personal communication, 1978) and lung (Wharton, Polak, Bloom, Ghatei, Solcia, Brown & Pearse, 1978).…”