Kayoko Tateishi

Production of rabbit antibody specific for amino-terminal residues of cholecystokinin octapeptide (CCK-8) by selective suppression of cross-reactive antibody response

Antibody specific for the amino-terminal region of cholecystokinin octapeptide (CCK-8) was generated in a highly reproducible way in New Zealand white rabbits by a novel immunization procedure which involves immunization with CCK-8 peptide conjugate coupled with keyhole limpet hemocyanin (KLH) and inhibiting cross-reacting antibody formation by treatment of the animals with a potent tolerogenic conjugate of beta-alanyl-tetragastrin and a copolymer of D-glutamic acid and D-lysine (D-GL). The antisera thus produced specifically react with an amino-terminal region of CCK-8 but not with the non-sulfate form of CCK-8, nor with the carboxy-terminal region which shares a cross-reactive determinant among gastrin and cholecystokinin-related peptides (caerulein, CCK-4, CCK-8, CCK-33 and CCK-39). The antisera produced by this method allowed us to measure specifically CCK in extracts from tissue such as duodenum containing gastrin and CCK at comparable levels.

The distribution of cholecystokinin octapeptide-like structures in the lower brain stem of the rat: an immunohistochemical analysis.

The distribution of immunoreactive cholecystokininoctapeptide (CCK-8)-like structures in the lower brain stem of the rat was investigated using indirect immunofluorescence. In addition to the well known immunoreactive CCK-8-like containing cell groups such as those in the ventral tegmental area, substantia grisea centralis of the mesencephalon, and n. linealis rostralis, the present study demonstrated a much wider distribution of immunoreactive CCK-8-like cells in the lower brain stem, i.e. those in the inferior colliculus, n. parabrachialis colliculi posterioris, lateral lemniscus, lateral parabrachial area, n. centralis superior, nucleus of group O, pontine substantia grisea centralis, n. tractus solitarii, area postrema, n. tractus spinalis nervi trigemini and reticular formation just dorsal to the inferior olivary complex. We also demonstrated an extensive network of immunoreactive CCK-8-like fibers in various areas of the lower brain stem, including the auditory system, visual system, viscerosensory area, parabrachial nucleus, dorsal and ventral tegmental nuclei, and interpeduncular nucleus. The possible importance of CCK is briefly discussed.

An aspect of the organizational principle of the γ-aminobutyric acidergic system in the cerebral cortex

The possibility of the colocalization of a Ca2+-binding protein, parvalbumin (PV), with peptides, cholecystokinin (CCK) and somatostatin (SS), all of which are known to be contained gamma-aminobutyric acidergic neurons in the cerebral cortex, was tested in the rat visual cortex by means of immunohistochemistry using the adjacent section technique. No PV immunoreactive (PV-I) neurons showed CCK-like immunoreactivity, and almost none (932 out of 934 PV-I neurons examined) showed SS-like immunoreactivity. These observations suggest that the GABAergic system of the cerebral cortex may consist of some distinct subsystems with different chemical and presumably physiological characteristics.

The occurrence of cholecystokinin-like immunoreactive neurons in the rat neostriatum: light and electron microscopic analysis

The present study demonstrates the existence of cholecystokinin-like immunoreactive (CCKI) neurons in the rat neostriatum. Light and electron microscopic findings suggest that these CCKI cells correspond to medium-size aspiny neurons classified by Golgi studies.

Ontogeny of cholecystokinin-8 containing neuron system of the rat: an immunohistochemical analysis--II. Lower brain stem.

The ontogeny of the cholecystokinin-8 neuron system in the lower brain stem of the rat was investigated by means of indirect immunofluorescence. Cholecystokinin-8-like immunoreactive structures first appeared in the primordium of the ventral tegmental area, ventral tegmental nucleus of Gudden and the area lateral to the superior central nucleus of the fetus on gestational day 17. Although cholecystokinin-8-like immunoreactive structures appeared gradually in various lower brain stem areas from this day, and developed markedly from postnatal day 6, thereafter, the ontogeny of cells and fibers was different. In general, the fibers increased in number as the rats grew and reached the maximum in adulthood, whereas the cells reached a maximum number between postnatal days 6-12, after which they decreased in number. Colchicine treatment, however, demonstrated the presence of cholecystokinin-8-like immunoreactive cells in various areas of the lower brain stem. These findings suggest that the cholecystokinin-containing neuron system is active in the adult and that cholecystokinin-containing cell somata cannot store a sufficient amount of the peptide for visualization in the absence of colchicine. Thus, it might be concluded that cholecystokinin may play an important role in the brain function of the adult as a neurotransmitter or neuromodulator, though its exact function is now open to discussion.

A novel immunization procedure for production of anti-cholecystokinin-specific antiserum of low cross-reactivity

An anti-cholecystokinin octapeptide (CCK-8) antibody without cross-reactivity with pentagastrin, whose amino acid sequence is identical with the carboxy-terminal portion of CCK-8, was produced by immunization with CCK-8-keyhole limpet hemocyanin and injection of a conjugate of pentagastrin and a copolymer of D-glutamic acid and D-lysine to inhibit production of antibody cross-reacting with pentagastrin.

Cholecystokinin-8-like immunoreactivity in the pre- and post-central gyri of the human cerebral cortex

Cellular localization of cholecystokinin-8-like immunoreactive (CCK-IR) structures in the pre- and post-central gyri of the human cerebral cortex was examined by indirect immunofluorescence. CCK-IR was localized mostly in perikarya of non-pyramidal cells such as multipolar, bitufted and bipolar cells, and to a lesser extent in medium and small pyramidal cells. CCK-IR fibers were also observed in these gyri, predominantly in layers I and III.

CCK pathway from supramammillary region to the nucleus anterior ventralis thalami of the young rats

The existence of cholecystokinin-8-like immunoreactive (CCKI) pathway from supramammillary region (SUM) to the nucleus anterior ventralis thalami (AVT) was demonstrated in this study by means of experimental immunohistochemical manipulations in very young rats, because destruction of unilateral SUM, which contains CCKI neurons, resulted in a disappearance of CCKI fibers in the ipsilateral AVT.

High plasma cholecystokinin response following ingestion of test meal by patients with non-insulin dependent diabetes mellitus.

Postprandial responses of plasma cholecystokinin (CCK) in patients with non-insulin dependent diabetes mellitus (NIDDM) were studied with a CCK specific radioimmunoassay. After the ingestion of a liquid test meal, plasma CCK levels increased from the basal level of 9.8 +/- 1.1 pg/ml to a peak of 19.4 +/- 1.8 pg/ml at 20 min in healthy subjects (n = 10). The ingestion of a test meal in patients with NIDDM (n = 10) resulted in a significantly greater increase of plasma CCK than in healthy subjects and a significant increase of plasma CCK from a basal level of 14.2 +/- 4.4 pg/ml to a peak of 47.4 +/- 12.4 pg/ml at 10 min.

Cholecystokinin-8-like immunoreactive neuron pathway from the supramammillary region to the ventral tegmental nucleus of Gudden of the rat

The existence of a pathway from the supramammillary region (SM) to the ventral tegmental nucleus of Gudden (NTV) containing cholecystokinin-8 (CCK)-like immunoreactive (CCKI) structures was demonstrated by experimental manipulations in the rat. Destruction of the SM resulted in the disappearance of CCKI fibers in the NTV.