Protective effect of CR 1409 (cholecystokinin antagonist) on experimental pancreatitis in rats and mice

CR 1409, a glutaramic acid derivative with competitive cholecystokinin-antagonistic activity, was administered IP and evaluated in comparison with proglumide (the model CCK-receptor antagonist), gabexate (protease inhibitor) and PGE₂ (cytoprotective) on two different models of experimental pancreatitis. Acute pancreatitis was induced in mice by six IP injections of 50 μg/kg caerulein at hourly intervals. The drugs were administered 30 minutes before each caerulein administration. Blood samples and pancreata were collected 3 hours after the last caerulein injection. In the second experiment, pancreatitis was induced in rats by injecting 0.3 ml 6% sodium taurocholate interstitially into the pancreas. The drugs were administered twice, 30 minutes before and 3 hours after taurocholate. The animals were killed 6 hours after laparotomy and blood samples and pancreata were collected. CR 1409 exhibited on both pancreatitis models a protective effect in a dose range of 0.3–10 mg/kg. Proglumide exhibited a protective activity at higher doses (200–400 mg/kg). Gabexate and PGE₂ were effective only in pancreatitis induced by taurocholate in a dose range of 30–60 mg/kg and 60–130 μg/kg respectively. These results, showing a high protective effect of CR 1409 on different models of acute pancreatitis, suggest an important role of CCK in the pathogenesis of pancreatitis.     

Potency of L-364,718 as an antagonist of the behavioral effects of peripherally administered cholecystokinin

A new antagonist of the peripheral cholecystokinin receptor, L-364,718, was found to block the reductions in food intake and exploratory activity induced by intraperitoneal administration of cholecystokinin octapeptide sulfate. L-364,718 significantly reversed the cholecystokinin-induced reduction in feeding at doses of 10 micrograms/kg - 10 mg/kg i.p. L-364,718 significantly reversed the cholecystokinin-induced reduction in exploratory activity at doses of 500 ng/kg - 10 mg/kg i.p. The time course of antagonist activity of L-364,718 was immediate to 90 minutes after intraperitoneal administration. L-364,718 had no significant effect on food intake or exploratory activity when administered alone, over the dose range of 100 ng/kg-10 mg/kg i.p. This compound appears to be at least one hundred times more potent than proglumide or benzotript as an antagonist of the behavioral effects of peripherally administered cholecystokinin.     

Blocking of cholecystokinin octapeptide behavioral effects by proglumide

An open field apparatus was used to assess the effect of proglumide, a selective antagonist of cholecystokinin octapeptide (CCK-8), to block the behavioral effect of CCK-8 in rats. Intracerebroventricular (ICV) injection of CCK-8 (0.5 to 2 micrograms) was effective in suppressing general exploratory activities and this effect was blocked by proglumide at doses of 2 to 5 micrograms administered ICV or 1 mg/kg administered subcutaneously. The effect of peripherally administered CCK-8 (10 micrograms/kg) was blocked by peripherally administered proglumide at a dose of 2 mg/kg but not by centrally administered proglumide at a dose of 5 micrograms/rat. The behavioral effect of CCK-8 was thus clearly blocked by proglumide.     

Effect of proglumide, a cholecystokinin receptor antagonist, on caerulein-stimulated pancreatic enzyme secretion and pancreatic polypeptide release in the dog

In five conscious dogs we studied the effect of proglumide, a cholecystokinin (CCK) antagonist, on caerulein-stimulated pancreatic secretion and release of pancreatic polypeptide (PP). Graded doses of caerulein (15-240 ng/kg per h) were infused intravenously. Experiments were repeated with a fixed infusion of proglumide (40 mg/kg per h). Release of PP following increasing doses of caerulein was significantly inhibited by proglumide (P less than 0.01). However, proglumide did not significantly affect caerulein-stimulated pancreatic protein secretion. Proglumide might be useful in defining the physiological role of CCK.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and 1/20 of that of bradykinin (BK) on a weight basis. The activity of PGF₁ and PGF₂ was only 1/20 of that of PGE₁ or PGE₂.

In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only 1/3–1/8 of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the anti-histamine, pyrilamine (2.5 mg/kg, i.v.).     

The cholecystokinin receptor antagonist CR1409 increases plasma cholecystokinin in rats

This study was undertaken to determine whether plasma cholecystokinin (CCK) levels are affected by the administration of the CCK-receptor antagonist CR1409 to rats. Infusion of 0.19, 0.94 and 4.75 mg/kg.h CR1409 for 30 min each into 6 conscious rats increased (P less than 0.05) plasma CCK from 1.3 +/- 0.5 to 6.0 +/- 1.2, 5.4 +/- 1.2, and 5.4 +/- 1.0 pM, respectively. In a subsequent study infusion of stepwise increasing lower doses of 0.3, 1.5, 7.5, 37.5 and 187.5 micrograms/kg.h CR1409 for 30 min each into 6 other rats dose-dependently increased (P less than 0.05) plasma CCK from 1.4 +/- 0.3 to 3.1 +/- 0.6, 4.1 +/- 0.8, 5.4 +/- 1.0, 5.9 +/- 0.8 and 7.1 +/- 1.1 pM, while infusion of saline did not affect plasma CCK concentrations. We therefore conclude that the CCK-receptor antagonist CR1409 increases plasma CCK in the rat.     

Different peripheral and central antagonistic activity of new glutaramic acid derivatives on satiety induced by cholecystokinin in rats

New glutaramic acid derivatives with cholecystokinin antagonistic activity were evaluated for their capacity to inhibit the satiety effect induced in the rat by intraperitoneal (i.p.) injection of cholecystokinin octapeptide (CCK-8). The most active compound, CR 1409, is about 4000 times more potent than proglumide when injected peripherally (i.p.). This compound competitively inhibits the action of CCK-8 at the receptor responsible for the satiety effect. In contrast, CR 1409, i.p. or intracerebroventricularly (i.c.v.) injected does not exhibit antagonistic effects when CCK-8 is administered i.c.v., confirming the existence of at least two different populations of CCK receptors.     

MK-329 blocks the inhibition of alcohol intake by CCK-8

Peripheral administration of sulfated cholecystokinin octapeptide (CCK-8) potently reduces alcohol intake, preference, and blood levels in rats. MK-329 (L-364,718 or Devazepide) acts at peripheral cholecystokinin (CCK_A) receptors to antagonize CCK-8's physiological and behavioral effects, such as pancreatic stimulation and inhibition of feeding. We determined whether CCK_A receptor blockade would also prevent CCK-8's alcohol satiety effect. Water-deprived female and male rats (n = 7 for each) received randomized combinations of intraperitoneal injections of MK-329 (0, 100, 200, or 400 μg/kg) followed by CCK-8 (0 or 4 μg/kg). Rats were then given access to 5% w/v ethanol for 30 min, followed by 30-min access to water, with food ad lib. MK-329 at all doses significantly (p < 0.05) reduced the suppression of alcohol intake and food intake by CCK-8. MK-329 alone increased alcohol intake at 400 μg/kg, and increased food intake, in females and males at 100 and 200 μg/kg, respectively. We concluded that CCK-8's alcohol and food satiation effects depend on specific, peripheral CCK_A receptors, and satiation of alcohol consumption and drinking-associated feeding reflect an endogenous functional interaction of CCK-8 with CCK_A receptors.     

C-terminal deletion analogs of a crustacean pigment-dispersing hormone

This study deals with the effect of deamidation and C-terminal truncation on the potency of an octadecapeptide pigment-dispersing hormone (PDH: Asn- Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-Arg-Val-Met-Thr-Glu-Ala-NH₂), first described as light-adapting distal retinal pigment hormone (DRPH) from Pandalus borealis. Bioassay of synthetic analogs for melanophore pigment dispersion in destalked fiddler crabs (Uca pugilator) showed that deamidation causes a 300-fold decrease in potency. The analogs 1–17-NH₂ and 1–16-NH₂ were about 3 times more potent than 1–18-OH. Further truncation led to decreases in potency, with the peptide 1–9-NH₂ being the smallest C-terminal deletion analog to display activity (0.001% potency). Smaller analogs (1–8-NH₂, 1–6-NH₂ and 1–4-NH₂) were inactive when tested in doses as high as 500 nmoles/crab. On the basis of our earlier work on N-terminal deletion analogs and the present findings the residues 6 to 9 seem to be important for PDH action.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

FMRFamide receptors in Helix aspersa

An in vitro receptor binding assay and an isolated heart bioassay were developed and used to characterize the structure-activity relations (SAR) of FMRFamide receptors in a land snail, Helix aspersa. In the radioreceptor assay, binding of ¹²⁵I-desaminoTyr-Phe-norLeu-Arg-Phe-amide (¹²⁵I-daYFnLRFamide) at 0°C to Helix brain membranes was reversible, saturable, and specific, with a K_D of 14 nM and a B_max of 85 fmol/mg brain. A lower affinity site was also observed (K_D=245 nM; B_max=575 fmol/mg brain). In the heart bioassay, daYFnLRFamide and other FMRFamide analogs increased myocardial contraction force. The SAR of cardiostimulation correlated with the specificity of high affinity ¹²⁵I-daYFnLRFamide binding to brain and heart receptors. The SAR was also similar to that described for other molluscan FMRFamide bioassays, except for a marked preference for N-blocked analogs. Peptides with N-terminal extensions of desaminoTyr, Tyr, Tyr-Gly-Gly, and acetyl, exhibited the highest potency in both radioligand displacement and cardiostimulation. The endogenous Helix heptapeptide analogs of FLRFamide (pQDP-, NDP-, and SDP-FLRFamide) were stimulatory on the heart at low doses, but were inhibitory at moderate to high doses. These peptides were 20 times weaker than FMRFamide in both the brain and heart receptor binding assays, with IC₅₀s about 10 μM. The results suggest that the effects of FMRFamide in Helix are receptor-mediated, and that the heptapeptides do not interact at FMRFamide receptors.     

Receptor type for cholecystokinin on isolated intestinal muscle cells of the guinea pig

Smooth muscle cells isolated from the longitudinal muscle layer of guinea pig ileum were used to determine the presence and type of cholecystokinin/gastrin receptor mediating contraction. This was accomplished with a series of cholecystokinin and gastrin agonists (CCK-8, des(SO3)CCK-8, gastrin-17, des(SO3)gastrin-17 and pentagastrin) and antagonists (glutaramic acid derivatives CR 1392, CR 1409, CR 1505 and proglumide). The order of potency of agonists based on EC50 values derived from concentration-response curves was: CCK-8 greater than des(SO3)CCK-8 greater than gastrin-17 greater than des(SO3)gastrin-17. The inhibitory dissociation constant (Ki) for the antagonist CR 1505 derived from Schild plots was the same whether sulfated CCK-8 or desulfated gastrin-17 was used as agonist (4.47 +/- 0.76 versus 4.68 +/- 0.78 nM). Pentagastrin acted as a partial agonist and inhibited partially the response to CCK-8. The Ki values determined for all antagonists with pentagastrin as agonist were similar to those with CCK-8 as agonist. The order of potency of agonists and the independence of Ki values from the type of agonist used implied that CCK and gastrin interact with one receptor type; the receptor is more sensitive to CCK-8 but is minimally influenced by sulfation of the tyrosine residue. In this respect, the receptor appears to be distinct from the CCK receptor on gallbladder muscle cells and pancreatic acinar cells.     

Parathyroid hormone binding sites in the brain

Parathyroid hormone (PTH) has been shown to have actions within the brain, suggesting the presence of central PTH receptors. This possibility was examined by determining the binding of ¹²⁵I-labeled [Nle^8,18,Tyr³⁴]bovine PTH to the plasma membranes of rat and rabbit brains. Specific binding of the tracer to membranes of the whole brain was time and tissue dependent, and was greater with membranes from the hypothalamus than with membranes from the cerebellum, cerebrum, or brain stem. The binding of the tracer to rat hypothalamic membranes was saturable and competitively displaced by unlabeled PTH(1–34), PTH(3–34), [Nle^8,18,Tyr³⁴]PTH(1–34), and by PTH-related protein, indicating the presence of a single class of high-affinity (dissociation constant = 2–5 nM), low-capacity (maximum binding capacity, B_max = 110–250 fmol/mg protein) binding site. The binding of radiolabeled PTH to these sites was not displaced by unrelated peptides of comparable molecular size (calcitonin, calcitonin-gene related peptide, adrenocorticotropin). The binding of PTH to these sites did not, however, appear to stimulate adenylate cyclase activity, as in peripheral PTH target sites. Thus, although these results indicate the presence of PTH receptors in the brain, these binding sites have a lower affinity than those in peripheral tissues and may utilize a different signal transduction system.     

The participation of CCK-8-ergic mechanisms in the regulation of emotional behavior in rodents

Effects of CCK-8 receptor agonists caerulein and pentagastrin and CCK-8 receptor antagonist proglumide on exploratory and locomotor activity of mice and rats were studied. Systemic administration of caerulein (500 ng/kg 1 mcg/kg) decreased significantly the exploratory activity of mice in elevated plus-maze. This anxiogenic-like action of caerulein was attenuated by acute pretreatment with proglumide (1 and 15 mg/kg) but not with diazepam (up to 0.75 mg/kg). Proglumide slightly increased the exploratory activity of rats in plus-maze; on the other hand, caerulein and pentagastrin potently decreased the measures of exploration in this test. Caerulein (10-100 mcg/kg) and proglumide (1 and 15 mg/kg) inhibited 3H-pentagastrin binding in mice brain in in vivo experiments. The data obtained indicate that CCK-8-ergic mechanisms in brain play an important role in the generation of anxiety states in rodents.     

Purine enzyme inhibition fails to alter benzodiazepine receptor binding in brain

Binding of [³H]flunitrazepam to benzodiazepine receptors in brain from several species, including human, was measured in vitro in the presence and absence of purine-metabolizing enzyme inhibitors. Incubation with potent inhibitors of either adenosine deaminase (2′-deoxycoformycin and erythro-9-(2-hydroxy-3-nonyl)-adenine) or guanine deaminase (5-amino-4-imidazole carboxamide) failed to alter [³H]flunitrazepam binding in homogenates of several different regions of human, rabbit, rat or guinea pig brain. These findings are in contrast to those of Norstrand et al. [Enzyme 29, 61–65 (1983)] who reported substantial alterations in [³H]flunitrazepam binding to human brain membranes in the presence of erythro-9-(2-hydroxy-3-nonyl)-adenine (increase) and 5-amino-4-imidazole carboxamide (decrease). In our studies, [³H]flunitrazepam binding was also unaltered in more anatomically intact brain sections following treatment with purine enzyme inhibitors. Furthermore, in vivo administration of erythro-9-(2-hydroxy-3-nonyl)-adenine to mice at a dose (200 mg/kg, i.p.) known to almost totally inhibit central adenosine deaminase activity also failed to alter brain [³H]flunitrazepam binding measured ex vivo, 30–120 min post injection.

While previous studies have shown that purines such as inosine interact with benzodiazepine receptors, our results raise some questions about the role of endogenous purines in regulating benzodiazepine receptors, at least in vitro and also acutely _vivo following purine enzyme inhibitor administration.     

Modulation of dopamine D1 receptor binding by neurotensin in the rat striatum

The effect of neurotensin on binding characteristics of dopamine D1 receptors was examined in the rat striatal membranes through radioreceptor assay. Neurotensin or its analogs were added to incubation medium of[³H]SCH 23390 saturation or dopamine/[³H]SCH 23390 inhibition experimental systems. Neurotensin did not modulate D1 antagonist binding but converted a part of D1 agonist high affinity binding sites to a low affinity state. Neurotensin_8–13 had the same potency as neurotensin itself, whereas neurotensin_1–8 had only weak activity in modulating D1 agonist binding. GTP and neurotensin had the same effect on D1 agonist binding. However, when both neurotensin and GTP were added, the result was the same as with either alone.

These data suggest that neurotensin modulates the functional state of D1 receptors probably via a GTP binding protein in the rat striatum.     

Modulating effect of cerulein on benzodiazepine receptors

Subcutaneous administration of caerulein (100-500 micrograms/kg) significantly reduced the development of picrotoxin (8 mg/kg) seizures in male mice. The same doses of caerulein inhibited 3H-flunitrazepam binding in in vivo experiments. Proglumide, an antagonist of cholecystokinin receptors, in low dose (5 mg/kg) potentiated the effects of caerulein (100 micrograms/kg), whereas the administration of proglumide in high dose (25 mg/kg) reduced the action of caerulein on 3H-flunitrazepam binding and picrotoxin seizures. Caerulein (5-1000 nM) decreased 3H-flunitrazepam binding in in vitro experiments only after supplementation of the binding medium with 120 mM NaCl and 5mM KCl. The results suggest the possible interaction of caerulein with chloride ionophor. It seems probable that the direct interaction of caerulein with chloride ionophor in involved in the inhibitory effect of caerulein on picrotoxin seizures and 3H-flunitrazepam binding.     

Histamine H1-Receptors Modulate Somatostatin Receptors Coupled to the Inhibition of Adenylyl Cyclase in the Rat Frontoparietal Cortex

Puebla, L., A. OcaÑa and E. Arilla. Histamine H₁-receptors modulate somatostatin receptors coupled to the inhibition of adenylyl cyclase in the rat frontoparietal cortex. Peptides 18(10) 1569–1576, 1997.—Since exogenous histamine has been previously shown to increase the somatostatin (SS) receptor-effector system in the rat frontoparietal cortex and both histamine H₁-receptor agonists and SS modulate higher nervous activity and have anticonvulsive properties, it was of interest to determine the participation of the H₁-histaminergic system in this response. The intracerebroventricular (i.c.v.) administration of the specific histamine H₁-receptor agonist 2-pyridylethylamine (PEA) (10 μg) to rats 2 h before decapitation increased the number of SS receptors (599 ± 40 vs 401 ± 31 femtomoles/mg protein, p< 0.01) and decreased their apparent affinity for SS (0.41 ± 0.03 vs 0.26 ± 0.02 nM, p < 0.01) in rat frontoparietal cortical membranes. No significant differences were seen for the basal and forskolin (FK)-stimulated adenylyl cyclase (AC) activities in the frontoparietal cortex of PEA-treated rats when compared to the control group. In the PEA group, however, the capacity of SS (10⁻⁴ M) to inhibit basal and FK (10⁻⁵ M)-stimulated AC activity in frontoparietal cortical membranes was significantly higher than in the control group (34 ± 1% vs 20 ± 2%, p < 0.001). The ability of low concentrations of the stable GTP analogue 5′-guanylylimidodiphosphate [Gpp(NH)p] to inhibit FK-stimulated AC activity in frontoparietal cortical membranes was similar in the PEA-treated and control animals. These results suggest that the increased SS-mediated inhibition of AC activity in the frontoparietal cortex of PEA-treated rats may be due to the increase of the number of SS receptors induced by PEA. Pretreatment with the H₁-receptor antagonist mepyramine (30 mg/kg, intraperitoneally (IP) prevented the PEA-induced changes in SS binding and SS-mediated inhibition of AC activity. Mepyramine (30 mg/kg, IP) alone had no observable effect on the somatostatinergic system. The in vitro addition of PEA or mepyramine to frontoparietal cortical membranes obtained from untreated rats did not affect the SS binding parameters. Altogether, these results suggest that the H₁-histaminergic system modulates the somatostatinergic system in the rat frontoparietal cortex.     

Chronic systemic treatment with epidermal growth factor in the rat increases the mucosal surface of the small intestine

We examined the effects of treatment with human recombinant epidermal growth factor (EGF) on the functioning small intestine in the rat. Male Wistar rats, 7–8 weeks old, were treated with EGF administered subcutaneously in doses of 0 (n = 7) or 150 μg/kg/day (n = 8) for 4 weeks. The histological composition and mucosal surface area of the perfusion-fixed small intestine was quantified with stereological principles. The length of the gut remained unchanged. The amount of tissue and surface area per length of gut (median (ranges)) were increased from 117 (101–131) mg/cm and 2.6 (2.1–3.5) cm²/cm in the controls to 146 (138–152) mg/cm and 3.5 (2.5–3.8) cm²/cm for the complete small intestine (both comparisons p < 0.02). The weight increase was due to mucosal growth in all parts of the intestine, whereas the surface area was only increased in proximal and middle parts. It is concluded that EGF treatment in rats increases the mucosal weight and surface area of the functioning small intestine.     

Antiallergic effect of ardisiaquinone A, a potent 5-lipoxygenase inhibitor

The antiallergic effects of ardisiaquinone A, a potent 5-lipoxygenase inhibitor, were examined. Pretreatment with ardisiaquinone A (0.1–10 μM) significantly inhibited compound 48/80-induced production of cysteinyl-leukotrienes (cys-LTs; LTC₄, LTD₄ and LTE₄) in rat peritoneal mast cells, but not histamine release. The IC₅₀ value was 5.56 μM. Pre-administration with ardisiaquinone A (0.1–1 mg/kg, s.c.) dose-dependently inhibited rat homologous passive cutaneous anaphylaxis (PCA) and the maximal inhibitory ratio was 22.3 ± 3.9% at the dose of 1 mg/kg. Ardisiaquinone A (1–5 mg/kg, s.c.) dose-dependently prevented the allergen-induced increase of tracheal pressure in ovalbumin-sensitized guinea pigs, especially during the late phase.

In conclusion, the findings of this study show that 5-lipoxygenase inhibitor ardisiaquinone A partially attenuates the allergen-induced increases of vascular permeability and tracheal pressure via the inhibition of cys-LTs production in mast cells.