Release Of Cholecystokinin-like Immunoreactivity Research Articles

The HIV-1 coat protein gp120 and some of its fragments potently activate native cerebral NMDA receptors mediating neuropeptide release.

The objective of this study was to investigate the effects of the HIV-1 envelope protein gp120 and its peptide fragments on the function of N-methyl-D-aspartate (NMDA) receptors mediating release of cholecystokinin (CCK) and somatostatin (SRIF). These are nonconventional NMDA receptors recently found to be activated by glycine or D-serine 'only'. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mM K+ from superfused rat neocortex synaptosomes was potently increased by gp120, its cyclic V3 loop and the linear V3 sequence BRU-C-34-A, but not by RP-135 (a central portion of BRU-C-34-A). The EC50 values of gp120 were 0.02 nM (CCK-LI release) and 0.01 nM (SRIF-LI release). The releasing effect of gp120 was prevented by blocking the glycine site or the ion channel of NMDA receptors, but not the glutamate recognition site; in addition, the gp120 effect was strongly inhibited by nanomolar concentrations of Zn2+ ions and by low micromolar concentrations of ifenprodil. It is concluded that gp120 acts as a very potent agonist at the glycine site of NMDA receptors sited on CCK- and SRIF-releasing nerve endings; the protein is able to activate the receptor channel in the absence of glutamate. Gp120 activates the receptors through its V3 loop as peptide fragments related to V3 retain near-maximal activity. The sensitivity of the gp120 effect to both Zn2+ and ifenprodil would not be incompatible with the idea that these NMDA receptors contain the triple subunit combination NR1/NR2A/NR2B.

Evidence for functional native NMDA receptors activated by glycine or D-serine alone in the absence of glutamatergic coagonist.

In this study we have examined the effects of N-methyl-D-aspartate (NMDA) receptor activation on the release of cholecystokinin and somatostatin from rat neocortical nerve endings. The release of cholecystokinin-like immunoreactivity (CCK-LI) and of somatostatin-like immunoreactivity (SRIF-LI) elicited by 12 mM K+ from superfused synaptosomes, but not the spontaneous release, was increased by NMDA in a concentration-dependent manner. The effects of NMDA could be prevented by antagonists selective for the glutamate recognition site, the receptor channel and the glycine site of the NMDA receptor. In the absence of NMDA, glycine increased on its own and in a concentration-dependent manner the depolarization-evoked release of both CCK-LI and SRIF-LI. This effect of glycine was strychnine-insensitive and could be mimicked by D-serine, a stereoselective agonist at the NMDA receptor glycine site. Antagonists selective for the glycine site or for the NMDA receptor channel prevented the effects of glycine/D-serine; these effects were, however, insensitive to blockade of the glutamate recognition site of the NMDA receptor, suggesting that glutamate released from synaptosomes or present as contaminant was not involved. The neuropeptide release elicited by D-serine was strongly inhibited by ifenprodil (0.3 microM) and by Zn2+ ions (50 nM), selective ligands at the NR2B and NR2A subunits of NMDA receptors, respectively. It is concluded that nerve terminals of CCK- and SRIF-releasing neurons possess non-conventional NMDA receptors whose channels can be operated by glycine or D-serine without apparent activation of the glutamatergic coagonist site. These receptors may display the triple subunit combination NR1/NR2A/NR2B.

Release of cholecystokinin-like immunoreactivity in the frontal cortex of conscious rats as assessed by transcerebral microdialysis: effects of different depolarizing stimuli.

The release of cholecystokinin-like immunoreactivity (CCK-LI) from the frontal cortex of freely moving rats has been studied using a transcerebral microdialysis technique coupled to a radioimmunoassay procedure. Basal levels of CCK-LI in the dialysate were above detection limits (2.4 +/- 0.7 pg/20 min; n = 8). High-K+ media evoked CCK-LI overflow in a concentration-dependent manner. The threshold concentration was 50 mM KCl. The peak overflow evoked by 100 mM K+ amounted to 42.7 +/- 2.8 pg/20 min (n = 6); it was totally Ca2+ dependent but insensitive to 1 microM tetrodotoxin. Infusion of 4-aminopyridine (1 mM; 20 min) evoked an overflow of CCK-LI (32 +/- 2.3 pg/20 min; n = 4), which was totally Ca2+ dependent and tetrodotoxin sensitive. Depolarization with 100 micrograms/ml of veratrine (20 min) provoked a CCK-LI overflow (62.2 +/- 10 pg/20 min; n = 6), which was also blocked by tetrodotoxin or by the absence of Ca2+ ions. The CCK-LI material collected under basal conditions or during veratrine infusion consisted essentially of CCK octapeptide sulfate. The veratrine-induced CCK-LI overflow did not change significantly when the infusion time was prolonged to 100 min. A second 20-min stimulus with 100 micrograms/ml of veratrine applied 200 min after a first 20-min stimulus evoked a barely significant CCK-LI overflow. These data suggest that one single 20-min stimulus with 100 micrograms/ml of veratrine may be sufficient to deplete the CCK-LI releasable stores and that > 200 min are required to replenish the depleted CCK-containing vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of cyclic nucleotides and calcium in the nutrient-induced release of cholecystokinin-like immunoreactivity in rats.

1. This study was undertaken with an isolated vascularly perfused rat duodenojejunal preparation to investigate the mechanisms of the release of cholecystokinin measured by immunoassay (cholecystokinin-like immunoreactivity, CCK-LI). 2. Intra-arterial infusion of forskolin (2-20 microM) evoked a prompt and well-sustained secretion of CCK-LI which was increased to a mean value of 600% of basal with the highest dose tested. 3-Isobutyl-1-methylxanthine (IBMX) (10(-6)-10(-4) M) stimulated the secretion of CCK-LI (maximal increase of 400% of basal at 10(-4) M). 3. Intra-arterial infusion of beta-phorbol 12-myristate 13-acetate (5 x 10(-7)-5 x 10(-6) M) and calcium ionophore A23187 (10(-6)-10(-5) M) alone or in combination provoked only a transient increase in the release of CCK-LI. 4. Luminal infusion of a 5% ovalbumin hydrolysate solution produced an immediate release of CCK-LI followed by a well-sustained secretion at 580% of basal. Intra-arterial infusion of IBMX (10(-5) or 10(-4) M) in combination with luminal peptone induced a release of CCK-LI which was equal to the sum of the CCK responses evoked by IBMX and peptone given separately. 5. Intra-arterial infusion of EGTA (2 mM) abolished the forskolin- and peptone-induced CCK secretion while luminal EGTA (2 mM) had no inhibitory effect. Verapamil (5 x 10(-5)-10(-4) M) or nifedipine (10(-5)-5 x 10(-5) M) inhibited the peptone-evoked CCK secretion. A high concentration of trifluoperazine (10(-4) M) strongly reduced the release of CCK-LI induced by intraluminal peptone while 10(-5) M was ineffective. 6. It is concluded that the peptone-induced secretion of CCK-LI involves a cyclic AMP-dependent mechanism and the activation of calcium channels possibly located at the basolateral side of the CCK cell.

Stimulation of receptors of gamma-aminobutyric acid modulates the release of cholecystokinin-like immunoreactivity from slices of rat neostriatum.

Slices of rat dorsal neostriatum were incubated in Krebs-Henseleit medium and the release of cholecystokinin-like immunoreactivity (CCK-IR) was induced by veratridine or high concentrations of K+. It was investigated whether drugs which act at receptors for gamma-aminobutyric acid (GABA) affected the release. The GABAA-receptor agonists muscimol and isoguvacine enhanced the veratridine-induced release of CCK-IR. This effect was abolished by the GABAA-receptor antagonist, bicuculline. When used alone, bicuculline decreased the release. The GABAB-receptor agonist, (-)-baclofen, decreased the veratridine-induced release of CCK-IR. The stereoisomer (+)-baclofen, which has low intrinsic activity, had no effect when used alone, but antagonized the effect of (-)-baclofen as did delta-amino-n-valeric acid, another antagonist at GABAB-receptors. When the release of CCK-IR was stimulated by K+ (40 mM) in the presence of tetrodotoxin, it was no longer affected by GABAA-receptor agonists or antagonists. Thus, their sites of action were probably not in the immediate vicinity of the nerve-endings which release CCK-IR. Under these conditions, stimulation of GABAB-receptors still reduced the release of CCK-IR. Therefore, it is concluded that these receptors are in the immediate vicinity of or even on the terminals which release CCK-IR.

An intragastric meal releases the putative satiety factor cholecystokinin from hypothalamic neurons in cats

The release of cholecystokinin-like immunoreactivity (CCK-LI) from feline hypothalamus was studied in relation to a meal by use of the push-pull perfusion technique. While levels of CCK-LI in the perfusate of overnight-fasted anesthetized cats were below assay sensitivity (<7pg/30min), intragastric administration of a carbohydrate-amino acid meal elicited a 3-fold increase in CCK-LI, identified by high-performance liquid chromatography as the C-terminal octapeptide of CCK (CCK-8). Examination of an extrahypothalamic site showed no comparable release in CCK-LI. Intravenous infusions of CCK-8 at doses imitating physiological blood levels up to 400 times higher than those seen after physiological stimulation demonstrated the existence of a CCK blood-brain barrier in the lateral hypothalamus. These observations provide support that CCK may play a physiological role in termination of feeding behavior.

Effects of selective dopamine D 2-receptor agonists on the release of cholecystokinin-like immunoreactivity from rat neostriatum

It was investigated whether or not the release of cholecystokinin-like immunoreactivity from slices of rat caudatoputamen could be modulated via dopamine D 2-receptors. The release of immunoreactivity was stimulated by high concentrations of K + (35 to 55 mM). The effects of two D 2-selective dopaminergic agonists, RU 24926 and LY 141865, were tested on the K +-induced release. Both agents enhanced the release of immunoreactivity in a concentration-dependent manner. The effect of RU 24926 was blocked by (−)-sulpiride and domperidone, two selective D 2-antagonists. It is concluded that stimulation of dopamine receptors of the D 2-subtype enhances the release of cholecystokinin-like immunoreactivity from nerve endings in rat neostriatum.