Localization of cholecystokinin-like immunoreactivity in isolated nerve terminals.

Abstract

Subcellular fractionation of the rat cerebral cortex demonstrated the presence of immunoreactive cholecystokinin in the pellet identified by electron microscopy as containing a high proportion of synaptic vesicles. The recovery in this pellet of 40% of the total immunoreactivity in the initial cortical extract is quite comparable to the recovery of other peptides such as vasoactive intestinal polypeptide and somatostatin, which are also located in synaptosomes and for which roles as neuroregulators or transmitters have been suggested. The evidence of concentration of cholecystokinin-like peptides in the synaptosomal pellet is consistent with our earlier demonstration by immunohistochemical techniques of cholecystokinin's presence in rabbit cerebral cortical neurons. These observations and the evidence for diminished concentration of cholecystokinin-like peptides in the brains of hyperphagic mice are consistent with cholecystokinin's suggested role as a neuroregulator for appetite. There are several likenesses in the biologic properties of cholecystokinin (CCK) and vasoactive intestinal polypeptide (VIP). Each was originally isolated from gastrointestinal mucosa (1-4) and was shown subsequently to be present in the brain (5-8). There do appear to be some differences in that there is not uniform agreement as to the hormonal form(s) of each of these peptides in each tissue site in which it has been found. Some have reported that VIP immunoreactivity in brain and gut was accounted for by a single component resembling intact VIP (6, 7), while another group has detected a minor component of smaller molecular size in the colonic mucosa (9). However, in the case of CCK, COGH-terminal fragments appear to be quite prominent. Several laboratories have reported that the COGH-terminal fragments, including the octapeptide (CCK-8), may account for virtually all the recoverable activity in the brain (10,11) or in the gut (11). We have found both intact CCK and CCK-8 in the brain and gut, the apparent relative concentrations depending on the site and the extraction technique employed (8,12 Generally subcellular fractionation was carried out according to the method of Whittaker (16). Portions of the extracts were centrifuged at 40C for 10 min at 1000 X g. The resulting pellet was resuspended in the same volume of 0.32 M sucrose and centrifuged again under the same conditions. The pellet (P1) was saved for assay. The two supernatants were pooled (Si), a portion was saved for assay, and the remainder was centrifuged in a Sorvall RC2B for 55 min at 17,000 X g. The supernatant (S2) was saved for assay. The pellet (P2) was resuspended in 3 ml of cold 0.32 M sucrose, 1 ml was saved for assay, and the remainder was applied to a discontinuous gradient. Two milliliters of the suspension was mixed with 5 ml of 1.4 M sucrose and placed at the bottom of the tube (mean concentration 1.2 M sucrose). Then 5 ml of 0.8 M and 2 ml of 0.32 M sucrose were layered above and the tube was centrifuged for 110 min at 55,000 X g. The fractions fl...

Cholecystokinin release mediated by 5‐HT3 receptors in rat cerebral cortex and nucleus accumbens

“…The origin of the released CCK-LI is most probably neuronal since CCK-LI has been reported to be concentrated in CNS nerve terminals (Pinget et al, 1978). Possible 'gliasomal' contamination of our nerve ending preparations should not contribute significantly to the released peptide inasmuch as CCK has not been detected in glial cells (Loren et al, 1979).…”

Brain cholecystokinin and nutritional status in rats and mice.

“…The effect of chronic undernutrition in early life was examined by placing newborn rats in artificially enlarged litters of 22 pups each. Animals reared in this manner suffer significant early malnutrition and remain stunted as adults (12)(13)(14).…”

Reduction of food intake by central administration of cholecystokinin octapeptide in the rat is dependent upon inhibition of brain peptidases

“…Of the several different fragments of CCK, the octapeptide (CCK-8) is believed to be the predominant form in the brain (Rehfeld, 1978;Larsson & Rehfeld, 1979). CCK-8 has been found in cell bodies, neurones and nerve terminals (Pinget et al, 1978;Innis et al, 1979;Vanderhaeghen et al, 1980), and specific CCK-receptors have been demonstrated in discrete areas of the brain (Zarbin et al, 1983;Van Dijk et al, 1984;Day et al, 1986) including a number of regions that are considered to be involved 'Author for correspondence. in the regulation of feeding behaviour.…”