Release Of CCK-LI Research Articles

In vivo release of cholecystokinin-like immunoreactivity in rat frontal cortex under freely moving conditions

Release of cholecystokinin-like immunoreactivity (CCK-LI) was measured in rat medial prefrontal cortex in vivo by brain dialysis and enzyme immunoassay under freely moving condition. Perfusion of 50 mM K + resulted in the increase of CCK-LI in the dialysate. The data on high-performance liquid chromatography (HPLC) of the dialysate showed that the increase of the CCK-LI was mainly due to the increase of CCK octapeptide sulfate itself. In the preliminary experiments, we applied this brain dialysis method for determination of CCK-LI release with drug treatment. After treatment with sulpiride (i.p.), a D 2 dopamine receptor antagonist, a significant increase of CCK-LI was observed, indicating that this brain dialysis technique is applicable to detect change in the level of CCK-LI release after a certain drug treatment.

Cholecystokinin: Corelease with dopamine from nigrostriatal neurons in the cat.

Halothane-anaesthetized cats were implanted with push-pull cannulae to demonstrate the in vivo release of cholecystokinin-like immunoreactivity (CCK-LI) in the substantia nigra and the ipsilateral caudate nucleus. The spontaneous and the calcium-dependent potassium-evoked release of CCK-LI were observed in both structures. In addition, the local application of tetrodotoxin (10-6 M) reduced the spontaneous release of the peptide. 6-OHDA lesions made in the substantia nigra pars compacta led to a complete destruction of nigrostriatal dopaminergic neurons. CCK-LI levels were not affected in the caudate nucleus but were reduced substantially in the substantia nigra. The activation of dopaminergic cells induced by the nigral application of alpha-methyl-para-tyrosine (10-4 M) stimulated the release of CCK-LI and dopamine in the ipsilateral caudate nucleus, whilst opposite effects were seen in the substantia nigra. Similar results were obtained when dopaminergic transmission was blocked in the caudate nucleus suggesting that the evoked release of CCK-LI by the alpha-methyl-para-tyrosine treatment originates from dopaminergic nerve terminals and not from other CCK-LI containing fibres in response to released dopamine. Dopamine (10-7 M) as well as the D1 agonist SKF 38393 (10-5 M) stimulated CCK-LI release when applied into the caudate nucleus while the D2 agonist, LY 171555 (10-6 M) slightly reduced peptide release. The local application of cholecystokinin-8 sulfate (CCK-8S) (10-8 M, for 30 min) into the substantia nigra pars compacta increased the firing rate of dopaminergic cells and stimulated the release of newly synthesized 3H-dopamine from dendrites and nerve terminals. These results suggest, but do not definitively prove, that, in the cat, CCK-LI and dopamine are coreleased from nigrostriatal mixed dopaminergic/CCK-LI neurons and that CCK-LI released from dendrites is, like dopamine, involved in the regulation of the activity of these cells.

Neuronal cholecystokinin-like immunoreactivity is postprandially released from primate hypothalamus

By use of the push-pull perfusion technique, release of neuronal cholecystokinin-like immunoreactivity (CCK-LI) from hypothalamus of owl monkeys was investigated in relation to an intragastric meal. In overnight fasted, halothane-anesthetized owl monkeys, levels of CCK-LI in the hypothalamic push-pull perfusate were below assay sensitivity (<4pg/30min). After intragastric administration of a carbohydrate/amino acid meal, however, a 10-fold increase in CCK-LI release (51 ± 7pg/30min) was observed in 5 out of 15 perfusion sites during the first postprandial 30 min. During the subsequent two 30-min intervals, release of CCK-LI was still increased with 32 ± 5pg/30min and 15 ± 6pg/30min, respectively. Thereafter, CCK-LI release was below assay sensitivity again. Addition of 40 mM potassium chloride (KCl) to the perfusion solution, which causes neuronal depolarization, resulted in a second increase in CCK-LI release of 56 ± 7pg/30min which was comparable to the meal-induced release. All sites that exhibited an increase in CCK-LI were located in the anterolateral aspect of the hypothalamus. In experiments without meal-induced release, KCl did not have any effect on CCK-LI in perfusate, suggesting that these particular sites did not contain CCK-releasing terminals. High performance liquid chromatography (HPLC) identified the C-terminal octapeptide of CCK (CCK-8) as the predominant molecular form of CCK within the owl monkey hypothalamus. No gastrin-17 was present. This study extends previously reported findings in cats to a primate species that (1) neurons which release CCK in hypothalamus are activated by stimuli generated by a gastric meal load, and (2) further supports the hypothesis that neuronal CCK within the hypothalamus may play a physiological role which correlates with the termination of feeding.

Blockade of gamma-aminobutyric acid-receptors of the B-subtype inhibits the dopamine-induced enhancement of the release of cholecystokinin-like immunoreactivity from slices of rat dorsal caudatoputamen.

When slices of rat dorsal caudatoputamen (= neostriatum) are incubated in vitro, cholecystokinin-like immunoreactivity (CCK-LI) is released upon addition of veratridine (3.75 mumol/l). This release is affected by dopamine and by gamma-aminobutyric acid (GABA)-receptor agonists. Dopamine enhances the release by stimulating dopamine D2-receptors and decreases it via D1-receptors. GABAA-receptor agonists enhance the veratridine-induced release of CCK-LI, while GABAB-receptor agonists decrease it. In the present investigation, it was examined whether GABA-receptors are involved in the effect which dopamine exerts via D2-receptors. The GABAA-receptor antagonist bicuculline (10 mumol/l) and the blocker of the GABAA-receptor ionophore picrotoxin (1 mumol/l) did not affect the dopamine (0.1 mumol/l)-induced increase in the release of CCK-LI. However, the GABAA-receptor agonist muscimol (1 mumol/l) not only enhanced the release of CCK-LI, but also prevented a further enhancement by dopamine (0.1 mumol/l). This effect of muscimol was blocked by bicuculline (10 mumol/l). In the presence of delta-amino-n-valeric acid (0.1 mmol/l), which has been described to block GABAB-receptors, dopamine no longer enhanced the veratridine-induced release of CCK-LI. delta-Amino-n-valeric acid also inhibited the pronounced enhancement of the release of CCK-LI caused by dopamine (0.1 mumol/l and 1 mumol/l in the presence of the preferential D1-receptor antagonist SCH 23390. The effect of delta-amino-n-valeric acid persisted in the presence of bicuculline (10 mumol/l and 100 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Release of cholecystokinin from rat cerebral cortex in vivo: role of GABA and glutamate receptor systems

Using cortical cups in chloralose-urethanized rats, the in vivo release of cholecystokinin-like immunoreactivity (CCK-LI) from cerebral cortex was examined. Resting levels of cholecystokinin-like immunoreactivity ranged from 20 to 30 pg/20 min sample. The addition of potassium (40 mM) in excess, resulted in a highly significant elevation in the levels of CCK-LI in the cortical superfusate. Deletion of calcium and the substitution of cobalt (10 mM), resulted in a significant reduction in both resting release and the release otherwise evoked by the addition of potassium. Focal electrical stimulation of the cortex (20 Hz), resulted in a significant (1.9 ± 0.2-fold, n = 8) increase in the levels of CCK-LI. The addition of glutamate (10 −6−10 −4 M) of kainic acid (10 −8−10 −6 M), also resulted in significant elevations in the levels of CCK-LI. The co-administration of a putative glutamate receptor antagonist, kynurenic acid (10 −4 M) resulted in a significant reduction in the levels of release otherwise evoked by the addition of glutamate, but not electrical stimulation. The addition of GABA (10 −5−10 −3 M) resulted in a dose-dependent decrease in the resting release of CCK-LI, and the release evoked by glutamate. Picrotoxin (10 −6−10 −4 M), resulted in a highly significant increase in the levels of CCK-LI in the cortical effluent. These results are consistent with a tonic GABAergic inhibition of CCK-releasing neurons. The treatment of the animal with diazepam (30 mg/kg, i.p.) also resulted in a significant reduction in resting release and the release otherwise evoked by focal cortical stimulation.

Modulation of cholecystokinin release from posterior nucleus accumbens by D-2 dopamine receptor

The effect of specific D-2 dopamine (DA) receptor agonists and antagonists on potassium (55 mM)-evoked release of cholecystokinin-like immunoreactivity (CCK-LI) was studied in tissue slices of the rat posterior nucleus accumbens (NAc). Incubating the tissue slices in 100 nM or 1 μM of LY-141865, a specific D-2 DA receptor agonist, reduced the release of CCK-LI as indicated by a significant decrease in the S 2/S 1 ratio. Addition of 10 μM of (−)-sulpiride, a specific D-2 DA receptor antagonist, blocked the inhibitory effect of 100 nM of LY-141865 on the release of CCK-LI. In contrast, 10 μM of the specific D-1 DA receptor antagonist SCH 23390 was unable to attenuate the decrease in release of CCK-LI caused by 100 nM of LY-141865. Furthermore, the active isomer of LY-141865, LY-171555 at 0.1 to 50 nM, also decreased the release of CCK-LI from the tissue slices, while the inactive isomer, LY-181990 at 1 nM, did not affect CCK-LI release. The inhibitory effect of LY-171555 on the release of CCK-LI was lost when its concentration was increased to 100 nM, thus revealing a biphasic effect of D-2 DA receptor stimulation on the release of CCK-LI. These results demonstrate that stimulation of D-2 DA receptor can modulate the release of CCK from in vitro slices of the rat posterior NAc.

Molecular heterogeneity of canine cholecystokinin in portal and peripheral plasma

The release of molecular forms of cholecystokinin (CCK) into the portal and peripheral blood in response to an intraduodenal perfusion of sodium oleate (9 mmol · h −1) was studied in six conscious dogs with chronic portal vein catheters. Immunoreactive CCK as concentrated from 20 ml plasma by C18 SEP PAK cartridges and the pattern of molecular forms of CCK were studied by G50 gel filtration. CCK-like immunoreactivity (CCK-LI) was measured in the column eluates with antibody 5135, which measures gastrin and CCK equally and requires the intact carboxyl-terminus for full recognition. Gastrin was measured specifically with antibody 1611. Intraduodenal perfusion with oleate did not alter basal gastrin release. Release of CCK-LI by intraduodenal oleate was calculated by the increments of the integrated CCK-LI peaks over basal. Total CCK-like immunoreactivity (CCK-LI), calculated by integration of all CCK-LI peaks in gel filtration eluates, increased over basal by 12 fmol/ml in the portal and by 6 fmol/ml in the peripheral plasma after intraduodenal perfusion with sodium oleate. The main molecular forms eluted on gel filtration in positions of CCK33,39 and of CCK8. The pattern of CCK in the peripheral plasma was similar to that in the portal plasma except that in the peripheral plasma large molecular forms were more abundant than small forms. This finding was confirmed when CCK39 and CCK8 were infused either into the portal vein or into the peripheral vein and peripheral plasma CCK levels were measured. Elimination of CCK8 after portal vein infusion compared to peripheral vein infusion was about 3 times higher than that of CCK39. The abundance of large molecular forms of CCK in the circulating blood which are similar in potency to small forms, underlines their role in the physiology of CCK.

In vivo studies on the basal and evoked release of cholecystokinin and vasoactive intestinal polypeptide from cat cerebral cortex and periventricular structures

In cat, radioimmunoassay revealed high concentrations of vasoactive intestinal polypeptide (VIP) and cholecystokinin (CCK)_(approximately 10–90 ng/g wet weight tissue) in various regions of the cerebral cortex and in periventricular structures. Superfusion of cat cerebral cortex or perfusion from the lateral ventricle to the cisterna magna with artificial cerebrospinal fluid (CSF) was carried out in cats anesthetized with chloralose-urethane. The superfusate was assayed for VIP-and CCK-like immunoreactivity (VIP-LI and CCK-LI) simultaneously. Basal efflux of VIP-LI was 13.8 ± 41 fmol/30 min/cm 2 cortex and 31.6 ± 5.0 fmol/30 min in ventriculo-cisternal superfusate. Basal efflux of CCK-LI, assayed in the same superfusate sample, was 10.7 ± 2.5 fmol/30/min/cm 2 cortex in cortical cup superfusate and 25.6 ± 4.4 fmol/30 min in ventriculo-cisternal superfusate. The addition of potassium (50 mM) and veratridine (7.5 × 10 −5M) to the superfusate produced significant increases in the levels of VIP-LI in both ventricular and cortical effluents. The potassium evoked release of VIP-LI and CCK-LI was inhibited by the substitution of cobalt (4 mM) for the calcium ion in the perfusion media; basal efflux was not affected. Separation of immunoreactivity by gel filtration chromatography demonstrated the CCK-LI in cat cortical tissue co-chromatographed with the COOH-terminal 4,8,33 and 39 amino acid peptide fragments of CCK and with a larger molecule. In contrast, the immunoreactivity in cortical and ventricular superfusate obtained during basal and evoked release co-migrated with authentic CCK octapeptide. For VIP, all immunoreactivity in tissue extract of cerebral cortex existed as a single peak which co-migrated with authentic VIP-28. The molecular pattern in cortical superfusate corresponded to that found in tissue extract.