(1/379) Proliferative effects of cholecystokinin in GH3 pituitary cells mediated by CCK2 receptors and potentiated by insulin.

1. Proliferative effects of CCK peptides have been examined in rat anterior pituitary GH3 cells, which express CCK2 receptors. 2. CCK-8s, gastrin(1-17) and its glycine-extended precursor G(1-17)-Gly, previously reported to cause proliferation via putative novel sites on AR4-2J and Swiss 3T3 cells, elicited significant dose dependent increases of similar magnitude in [3H]thymidine incorporation over 3 days in serum-free medium of 39 +/- 10% (P < 0.01, n = 20), 37 +/- 8% (P < 0.01, n = 27) and 41 +/- 6% (P < 0.01, n = 36) respectively. 3. CCK-8s and gastrin potentially stimulated mitogenesis (EC50 values 0.12 nM and 3.0 nM respectively), whilst G-Gly displayed similar efficacy but markedly lower potency. L-365,260 consistently blocked each peptide. The CCK2 receptor affinity of G-Gly in GH3 cells was 1.09 microM (1.01;1.17, n = 6) and 5.53 microM (3.71;5.99, n = 4) in guinea-pig cortex. 4. 1 microM G-Gly weakly stimulated Ca2+ increase, eliciting a 104 +/- 21% increase over basal Ca2+ levels, and was blocked by 1 microM L-365,260 whilst CCK-8s (100 nM) produced a much larger Ca2+ response (331 +/- 14%). 5. Insulin dose dependently enhanced proliferative effects of CCK-8s with a maximal leftwards shift of the CCK-8s curve at 100 ng ml(-1) (17 nM) (EC50 decreased 500 fold, from 0.1 nM to 0.2 pM; P < 0.0001). 10 microg ml(-1) insulin was supramaximal reducing the EC50 to 5 pM (P = 0.027) whilst 1 ng ml(-1) insulin was ineffective. Insulin weakly displaced [125I]BHCCK binding to GH3 CCK2 receptors (IC50 3.6 microM). 6. Results are consistent with mediation of G-Gly effects via CCK2 receptors in GH3 cells and reinforce the role of CCK2 receptors in control of cell growth. Effects of insulin in enhancing CCK proliferative potency may suggest that CCK2 and insulin receptors converge on common intracellular targets and indicates that mitogenic stimuli are influenced by the combination of extracellular factors present.  (+info)

(2/379) Benzodiazepine receptor agonists modulate thymocyte apoptosis through reduction of the mitochondrial transmembrane potential.

Peripheral-type benzodiazepines have been shown to exert immunological effects. In this study, we examined the effects of the peripheral-type benzodiazepines on murine thymocytes. Murine thymocytes that were incubated with the peripheral-type benzodiazepines underwent apoptosis associated with the collapse of mitochondrial transmembrane potential (delta psi(m)). The drugs stimulated dexamethasone- and etoposide-induced apoptosis with the enhanced collapse of delta psi(m). The central-type benzodiazepines had no effect on either the delta psi(m) or apoptosis. The reduction of delta psi(m) depended on protein synthesis and protein phosphorylation. These results suggest that the immunomodulating effect of benzodiazepines is in part due to the modulation of thymocyte apoptosis associated with the collapse of delta psi(m).  (+info)

(3/379) Analysis of the behaviour of selected CCKB/gastrin receptor antagonists in radioligand binding assays performed in mouse and rat cerebral cortex.

1. The previously described complex behaviour of the CCKB/gastrin receptor antagonist, L-365,260, in radioligand binding assays could be explained by a variable population of two binding sites. We have investigated whether other CCKB/gastrin receptor ligands (PD134,308, PD140,376, YM022 and JB93182) can distinguish between these sites. 2. In the mouse cortex assay, Hill slopes were not different from unity and the ligand pKI values did not differ when either [125I]-BH-CCK-8S or [3H]-PD140,376 was used as label as expected for a single site (G2). 3. In the rat cortex, where previous analysis of replicate (n=48) L-365,260 data indicated the presence of two CCKB/gastrin sites (G1 and G2), the competition data for PD134,308, PD140,376, YM022 and JB93182 could be explained by a homogeneous population of CCKB/gastrin sites because the Hill slope estimates were not significantly different from unity. However, the estimated affinity values for JB93182 and YM022 were significantly higher and that for PD134,308 was significantly lower than those obtained in the mouse cortex when the same radioligand was used. In view of our previous data obtained with L-365,260, the rat cortex data were also interpreted using a two-site model. In this analysis, SR27897 expressed approximately 9 fold, PD134,308 approximately 13 fold and PD140,376 approximately 11 fold selectivity for the G2 site. In contrast, JB93182 expressed approximately 23 fold and YM022 approximately 4 fold selectivity for the G1 site. If the two-site interpretation of the data is valid then, because of its reverse selectivity to L-365,260, JB93182 has been identified as a compound which if radiolabelled could provide a test of this receptor subdivision.  (+info)

(4/379) Characterization of the binding of a novel radioligand to CCKB/gastrin receptors in membranes from rat cerebral cortex.

1. We have investigated the binding of a novel radiolabelled CCKB/gastrin receptor ligand, [3H]-JB93182 (5[[[(1S)-[[(3,5-dicarboxyphenyl)amino]carbonyl]-2-phenylethyla mino]-carbonyl]-6-[[(1-adamantylmethyl) amino]carbonyl]-indole), to sites in rat cortex membranes. 2. The [3H]-JB93182 was 97% radiochemically pure as assessed by reverse-phase HPLC (RP-HPLC) and was not degraded by incubation (150 min) with rat cortex membranes. 3. Saturation analysis indicated that [3H]-JB93182 labelled a homogeneous population of receptors in rat cortex membranes (pKD=9.48+/-0.08, Bmax=3.61+/-0.65 pmol g(-1) tissue, nH=0.97+/-0.02, n=5). The pKD was not significantly different when estimated by association-dissociation analysis (pKD=9.73+/-0.11; n=10). 4. In competition studies, the low affinity of the CCKA receptor antagonists, L-364,718; SR27897 and 2-NAP, suggest that, under the assay conditions employed, [3H]-JB93182 (0.3 nM) does not label CCKA receptors in the rat cortex. 5. The affinity estimates obtained for reference CCKB/gastrin receptor antagonists were indistinguishable from one of the affinity values obtained when a two site model was used to interpret [125I]-BH-CCK8S competition curves obtained in the same tissue (Harper et al., 1999). 6. This study provides further evidence for the existence of two CCKB/gastrin sites in rat cortex. [3H]-JB93182 appears to label selectively sites previously designated as gastrin-G1 and therefore it may be a useful compound for the further discrimination and characterization of these putative receptor subtypes.  (+info)

(5/379) L-365,260 inhibits in vitro acid secretion by interacting with a PKA pathway.

The aim of this study was to analyse the antisecretory mechanism of L-365,260 in vitro in isolated rabbit gastric glands. We showed that compound L-365,260, described as a non-peptide specific competitive CCK-B receptor antagonist, was able to dose-dependently inhibit [14C]-aminopyrine accumulation induced by histamine (10(-4) M), carbachol (5x10(-5) M), 3-isobutyl-1-methyl-xanthine (IBMX) (5x10(-6) M) and forskolin (5x10(-7) M) with similar IC50 values respectively of 1.1+/-0.6x10(-7) M, 1.9+/-1.2x10(-7) M, 4.2+/-2.0x10(-7) M and 4.0+/-2.8x10(-7) M. We showed that L-365,260 acted beyond receptor activation and production of intracellular second messengers and that it had no action on the H+/K+ -ATPase. We found that L-365,260 inhibited cyclic AMP-induced [14C]-aminopyrine accumulation in digitonin-permeabilized rabbit gastric glands, suggesting that this compound acted, at least in part, as an inhibitor of the cyclic AMP-dependent protein kinase (PKA) pathway.  (+info)

(6/379) Central and peripheral benzodiazepine ligands prevent mitochondrial damage induced by noise exposure in the rat myocardium: an ultrastructural study.

Noise represents an environmental stress factor affecting several organs and apparatuses, including the cardiovascular system. In experimental animals undergoing noise exposure, subcellular myocardial changes have been reported, especially at the mitochondrial level. In previous studies we found that diazepam, acting at both central and peripheral benzodiazepine receptors, prevented the onset of this myocardial damage. In the present study, we investigated the specific role played by central and/or peripheral benzodiazepine receptors in preventing noise-induced myocardial alterations. In particular, the effect of clonazepam as a selective ligand for central sites, in comparison with the efficacy of ligands selective for peripheral sites, such as Ro 5-4864 and PK-11195, was evaluated. Rats were pretreated with the test drugs 30 min before exposure to noise for 6 or 12 hr and then sacrificed. After fixing, samples of right atrium and ventricle were taken and processed for either transmission or scanning electron microscopy. After 6 hr of noise exposure, only the atrium exhibited significant mitochondrial alterations, whereas after 12 hr both atrium and ventricle were damaged. As expected, diazepam prevented noise-induced mitochondrial injury at both 6 and 12 hr. By contrast, clonazepam was effective only after 6 hr. The peripheral ligand PK-11195 attenuated mitochondrial damage at both 6 and 12 hr, whereas Ro 5-4864 was effective only after 12 hr. In the present study, we confirm that noise exposure induces mitochondrial damage in the rat myocardium. Drugs acting at both central and peripheral benzodiazepine receptors significantly prevent this damage. Differences in the amount and in the duration of the protective effect might depend on variability in the potency and in the pharmacokinetics of the specific drugs.  (+info)

(7/379) Rat hippocampal neurons are critically involved in physiological improvement of memory processes induced by cholecystokinin-B receptor stimulation.

The involvement in memory processes of the neuropeptide cholecystokinin (CCK) through its interaction with the CCK-B receptors was studied. The two-trial recognition memory task was used. Control animals showed recognition memory after a 2 hr time interval but not after a 6 hr time interval between the two trials. The improving effect of a selective CCK-B agonist, BC 264, intraperitoneally administered (0.3 microgram/kg) in the retrieval phase of the task (6 hr time interval), was also observed after its injection (1 pmol/0.5 microliter) in the dorsal subiculum/CA1 of the hippocampus but not in the caudate/putamen nucleus or in the prefrontal cortex of rats. The CCK-B antagonist L-365,260 injected (10 ng/0.5 microliter) into this region of the hippocampus abolished the improving effect of BC 264 injected intraperitoneally. Furthermore, L-365,260 injected in the hippocampus suppressed the recognition of the novel arm normally found in the controls (2 hr time interval) when it was injected before the acquisition or the retrieval phase of the task. In addition, an increase of the extracellular levels of CCK-like immunoreactivity in the hippocampus of rats during the acquisition and retention phase of the task was observed. Finally, CCK-B receptor-deficient mice have an impairment of performance in the memory task (2 hr time interval). Together, these results support the physiological involvement of the CCKergic system through its interaction with CCK-B receptors in the hippocampus to improve performance of rodents in the spatial recognition memory test.  (+info)

(8/379) Apparent pA2 values of benzodiazepine antagonists and partial agonists in monkeys.

Drugs that bind to benzodiazepine recognition sites of gamma-aminobutyric acid type A receptor complexes may function as agonists in some behavioral assays and as antagonists in other behavioral assays. The present studies compared the effects of the benzodiazepines midazolam, flumazenil, bretazenil, Ro 41-7812, and Ro 42-8773 and the beta-carboline, beta-carboline-3-carboxylate-t-butyl ester (beta-CCt) under two different types of schedule-controlled responding in squirrel monkeys. One group of monkeys responded under a fixed-ratio schedule of stimulus-shock termination, and a second group of monkeys responded under a multiple fixed-ratio schedule of food presentation involving suppressed and nonsuppressed behavior. Under the schedule of stimulus-shock termination, midazolam produced dose-related decreases in response rate, and these effects were surmountably antagonized by flumazenil, bretazenil, Ro 41-7812, Ro 42-8773, and beta-CCt. Schild plot analysis of these data revealed the following mean pA(2) values: flumazenil, 7.18; bretazenil, 7.62; Ro 41-7812, 7. 06; Ro 42-8773, 6.95. Apparent pA(2) values were not calculated for beta-CCt because the CL of the slope of the Schild plot included positive values. Under the multiple schedule, midazolam, bretazenil, and Ro 42-8773 dose-dependently increased rates of suppressed responding, whereas flumazenil, Ro 41-7812, and beta-CCt had no significant rate-altering effects. Flumazenil antagonized the antisuppressant effects of midazolam and bretazenil; however, individual variability in these effects prohibited the determination of apparent pA(2) values. These results indicate that in vivo pA(2) values may be determined for benzodiazepine-site ligands. These results further demonstrate that some benzodiazepine-site ligands, e. g., bretazenil and Ro 42-8773, may function as both agonists and as competitive antagonists in vivo.  (+info)