Duodenal administration of casein and oleate increased plasma levels from oral administration of a poorly water-soluble antiepileptic drug as compared to duodenal glucose and saline in a canine model. Pre-treatment with intravenous MK-329, benzodiazepine CCK A-receptor antagonist, blocked the duodenal oleate effect on drug plasma levels in a single dog preliminary study. In a follow-up study, oral drug co-administration with Intralipid increased drug plasma levels as compared to drug co-administration with a noncaloric equivalent-volume load in seven dogs. Pre-treatment with MK-329 reduced drug plasma levels from co-administration with Intralipid toward fasted-state values. While increased drug solubility in the lipid vehicle might have been projected to account for the fed-state effect in the oral studies, the gut peptide inhibitor studies suggest that biliary secretion plays a major role in promoting the dissolution and subsequent absorption of this lipophilic drug. The data also support the hypothesis that meal-enhanced pancreatic secretion provides a greater fluid volume for drug dissolution in the small intestine. An increase in the extent of drug dissolution in the stomach, as a result of meal prolongation of gastric residence time, does not appear to contribute substantially to fed-state increases in drug plasma levels from oral drug co-administration with a lipid meal.

Intestinal adaptation has been studied in rats with pancreatic atrophy induced by feeding a copper-deficient diet and penicillamine and in rats with carbohydrate maldigestion induced by feeding of an alpha-glucosidase inhibitor (acarbose). Pancreatic atrophy led to a significant increase of weight, protein, and DNA content as well as specific activities and total amounts of the enzymes sucrase and maltase in the distal but not in the proximal part of the small intestine. Plasma levels of CCK and GIP were significantly higher in rats with pancreatic atrophy, whereas plasma levels of gastrin and insulin were lower. Tissue concentrations of gastrin in the antrum and GIP in duodenum and jejunum were unchanged. Duodenal CCK and jejunal substance P, somatostatin, and VIP and ileal substance P and somatostatin were significantly decreased in rats with acinar atrophy. Glucosidase inhibition by acarbose feeding led to weight increase of the small intestine and cecum. This was more marked when acarbose was fed together with a fiber-free diet. Under these conditions the protein and DNA content also increased significantly in both gut segments and maltase and sucrase content predominantly in the distal part. Insulin plasma concentration decreased significantly in the acarbose-fed groups, whereas GIP, gastrin, and CCK plasma concentrations remained unchanged. After fiber-rich diet tissue concentrations of gastrin in the antrum and insulin in the pancreas were significantly higher and GIP concentrations in the duodenum and jejunum significantly lower than after fiber-free diet. Acarbose increased the pancreatic insulin concentration only in the fiber-free group and did not influence gastrin and GIP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

To study the effect of cholecystokinin octapeptide (CCK-8) on lipopolysaccharide (LPS)-stimulated pulmonary interstitial macrophages (PIM) in vitro.

METHODS

PIM were isolated and cultured in the presence or absence of LPS, CCK-8, proglumide (the antagonist of CCK receptors) and vehicle. The expression of membrane CD14 (mCD14) protein was assayed by flow cytometry and soluble CD14 (sCD14) in the supernatant was analyzed semi-quantitatively by Western blot. TNF-alpha in the supernatant was detected with ELISA.

RESULTS

CCK-8, at concentrations of 10(-7) mol/L and 10(-6) mol/L, significantly inhibited the expression of mCD14. Release of sCD14 and TNF-alpha in the supernatant was up-regulated by LPS (1 microg/ml) but reduced by CCK-8. The effect of CCK-8 was inhibited by proglumide.

CONCLUSIONS

CCK-8 negatively modulated several functions of LPS-stimulated PIM through CCK receptors. This may be one of the mechanisms for CCK-8 to alleviate inflammation in lung tissue during endotoxemia.

(-)-[3H]L364718 membrane binding assays were employed to localize and characterize cholecystokinin (CCK)-A binding sites in rat and cow brain. Specific binding was detected in all brain areas tested, but in all areas of rat brain and most areas of cow brain the level was too low to allow characterization of the ligand binding specificity of these sites. Membranes prepared from cow nucleus accumbens and striatum contained higher levels of (-)-[3H]L364718 specific binding which represented 55-70% of total binding. Characterization of the ligand binding properties of (-)-[3H]L364718 binding sites in cow nucleus accumbens revealed that these sites are similar to CCK-A sites found in pancreatic membranes. Binding of (-)-[3H]L364718 was saturable and had high affinity (Kd = 45 pm). Sites labeled by (-)-[3H]L364718 displayed stereospecificity for the stereoisomers of CR1409. The competition curve for CCKCCKCCK-A sites found in brain and peripheral tissues.

The possible effect of different cholecystokinin (CCK) receptor antagonists (MK-329 and L-365260) on the maintenance and reactivation of morphine conditioned place preference (CPP) were investigated in rats, respectively. The results show that the maintenance of morphine CPP could be induced by injection of morphine (10 mg/kg, s.c.) once for 3 days and this effects were significantly attenuated by pretreatment with 1 but not by 0.1 mg/kg L-365260. Furthermore, following a 28-day extinction, the morphine CPP disappeared and then reactivated again by a single injection of morphine (10 mg/kg). Pretreatment with L-365260 (1 and 0.1 mg/kg) significantly blocked this reactivation of morphine CPP. In contrast, pretreatment of MK-329 (1 and 0.1 mg/kg) failed to do so. The present study demonstrated that CCK-B receptor but not CCK-A receptor is involved in the maintenance and reactivation of morphine CPP. These findings suggest that CCK-B receptor antagonists might be of some value in the treatment and prevention of relapse to drug dependence long after detoxification.

We studied the aggressive behaviour induced by repeated treatment with apomorphine, a dopamine agonist (0.5 mg/kg s.c. twice daily, 10 days), in rats. The first signs of defensive aggressiveness appeared on the third day of apomorphine treatment and were generally seen on the 7th day. Aggressiveness induced by a challenge dose of apomorphine (0.5 mg/kg s.c.) on the 11th day was antagonized by haloperidol (0.05 and 0.1 mg/kg i.p.) and clozapine (10 mg/kg i.p.). An antagonist of N-methyl-D-aspartate (NMDA)-gated channels, dizocilpine (MK-801), also blocked the aggressive behaviour at 0.25 and 0.5 mg/kg i.p. but caused ataxia. When dizocilpine (0.25 mg/kg i.p.) and apomorphine were coadministered for 10 days, aggressive behaviour did not develop. At 0.025 mg/kg i.p., dizocilpine even accelerated the appearance of apomorphine-induced aggressive behaviour, which manifested on the 3rd day in all rats. In a separate study, a 7-day treatment with dizocilpine (0.25-1 mg/kg i.p.) of rats, sensitized by a prior 10-day apomorphine treatment, did not reverse the established aggressive behaviour. The coadministration of apomorphine and cholecystokinin (CCK) -A or -B antagonists, devazepide or L-365,260 (0.01-2.5 mg/kg i.p.) respectively, neither affected development of apomorphine-induced aggressive behaviour nor intensity of aggressiveness in the sensitized rats. In binding studies neither density nor affinity of striatal dopamine D2 receptors was changed by acute or chronic apomorphine treatment. The number of [3H]pCCK-8 binding sites in the frontal cortex increased already after a single injection of apomorphine.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent evidence supports a role for the serotonin-3 (5-HT3) receptors in the modulation of cholecystokinin (CCK)-induced satiation. Likewise, 5-HT's anorectic response has been linked to recruitment of peripheral CCK-A receptors. Evidence to date, however, does not elucidate whether there is a concomitant interaction between CCK-A and 5-HT3 receptors or whether each receptor functions independently in the negative feedback control of food intake elicited by CCK. In the present study, we used selective receptor antagonists to investigate the roles of CCK-A and 5-HT3 receptors in CCK-induced satiation. Intraperitoneal administration of CCK-8 reduced 30-min 15% sucrose intake in a dose-responsive manner. Prior treatment with ondansetron (1.0 mg/kg ip), a highly selective 5-HT3 receptor antagonist, attenuated CCK-induced suppression of food intake in a dose-responsive manner. Pretreatment with lorglumide (1.0 mg/kg ip), a selective CCK-A receptor antagonist, reversed CCK-induced inhibition of sucrose intake. Finally, simultaneous blockade of CCK-A and 5-HT3 receptors by lorglumide and ondansetron, as well as concomitant administration of the two antagonists with CCK, produced a significant synergistic increase in sucrose intake compared with intakes after administration of saline, CCK, or either antagonist alone. These findings support evidence that CCK-A and 5-HT3 receptors cooperate interdependently in control of short-term food intake. Most likely, this interconnection exists through a feed-forward parallel model arising from CCK-A and 5-HT3 receptors, where activation of one system engages the other to intensify the overall satiety signal.

The present study investigated (1) the pharmacological profile of cholecystokinin (CCK) receptor subtypes involved in the regulation of gastric pepsinogen secretion, (2) the influence of gastric acidity on peptic responses induced by CCK-8-sulfate (CCK-8S) or gastrin-I; and (3) the mechanisms accounting for the effects of CCK-like peptides on pepsinogen secretion. In anaesthetized rats, i.v. injection of CCK-8S or gastrin-I increased both pepsinogen and acid secretion. The pepsigogue effect of CCK-8S was higher than that of gastrin-I, whereas acid hypersecretion after CCK-8S was lower than that induced by gastrin-I. Peptic output following CCK-8S was partly blocked by i.v. injection of the CCKCCKCCK-8S was enhanced by devazepide (+84.5%) and blocked by L-365,260. In contrast, the gastric secretory actions of gastrin-I were insensitive to devazepide, but abolished by L-365,260. Excitatory effects of CCK-8S and gastrin-I were not modified by vagotomy or atropine, whereas cimetidine or alpha-fluoromethylhistidine (irreversible blocker of histidine decarboxylase) partly prevented acid hypersecretion induced by both peptides without affecting their pepsigogue effects. After pretreatment with omeprazole, both CCK-8S and gastrin-I failed to stimulate acid secretion, while they increased pepsinogen output. In rats with gastric perfusion of acid solutions, CCK-8S or gastrin-I increased peptic output in a pH-independent manner either with or without pretreatment with omeprazole. Ablation of capsaicin-sensitive sensory nerves as well as application of lidocaine to the gastric mucosa failed to modify the excitatory effects of CCK-8S or gastrin-I on pepsinogen and acid secretion. Blockade of the nitric oxide (NO) synthase pathway by N(G)-nitro-L-arginine-methyl ester prevented the pepsigogue actions of both CCK-8S and gastrin-I (-61.8% and -71.7%, respectively), without affecting the concomitant increase in acid output. In addition, both these peptides significantly increased the release of NO breakdown products into the gastric lumen. The present results suggest that: (1) both CCKCCKCCK-like peptides; (2) the excitatory inputs of CCK-8S and gastrin-I to chief cells are not driven through acid-dependent mechanisms or capsaicin-sensitive afferent sensory nerves; and (3) under in vivo conditions, the stimulant actions of CCK-like peptides on pepsinogen secretion are mediated, at least in part, by an increase in NO generation.

Cholecystokinin (CCK), a gastrointestinal (GI) hormone, is also present in structures of the central nervous system such as cortex, hippocampus, amygdala, olfactory tubercle and in regions involved in the regulation of the pituitary function. Although a number of studies have evaluated the effects of CCK on hypothalamic-pituitary-adrenal (HPA) axis function and on arginine vasopressin (AVP), prolactin (PRL) and growth hormone (GH) plasma levels in the laboratory animal, its role in humans has not been explored. Hence, we examined the effects of the exogenous administration of this GI hormone on corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cortisol, AVP, PRL and GH plasma levels in humans. To accomplish this, graded doses (0, 50, 140 and 420 ng/kg) of sulfated CCK octapeptide (CCK-8), the full biologically active peptide, were infused intravenously to healthy men in 30 min. Blood samples were collected 30 min and immediately before the infusion was started (baseline) and 15, 30, 45, 60 and 90 min thereafter. CRH, ACTH, and AVP were extracted from plasma proteins using cartridges of SepPak C18. These hormones and cortisol were measured by radioimmunoassay whereas PRL and GH were measured by immunoradiometric assay. CCK-8 increased plasma ACTH and cortisol levels only at the dose of 420 ng/kg, whereas it had no detectable effect on plasma CRH levels. It increased also plasma AVP levels at the doses of 140 and 420 ng/kg. However, this effect reached the statistical significance only at the highest dose tested. CCK-8 stimulated PRL and GH release in a dose-dependent fashion. The lowest stimulatory dose was 140 ng/kg for both hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

The CREM gene encodes both activators and repressors of cAMP-induced transcription. Inducible cAMP early repressor (ICER) isoforms are generated upon activation of an alternative, intronic promoter within the CREM gene. ICER is proposed to down-regulate both its own expression and the expression of other genes that contain cAMP-responsive elements such as a number of growth factors. Thus, ICER has been postulated to play a role in proliferation and differentiation. Here we show that ICER gene expression is induced by gastrin, cholecystokinin (CCK), and epidermal growth factor in AR42J cells. The time course of gastrin- and CCK-mediated ICER induction is rapid and transient, similar to forskolin- and phorbol 12-myristate 13-acetate-induced ICER expression. The specific CCK-B receptor antagonist L740,093 blocks the gastrin but not the CCK response, indicating that both the CCK-B and the CCK-A receptor can mediate ICER gene activation. Noteworthy, CREB is constitutively phosphorylated at Ser-133 in AR42J cells, and ICER induction proceeds in the absence of increased CREB Ser(P)-133. Gastrin-mediated ICER induction was not reduced in the presence of the protein kinase A inhibitor H-89, indicating a protein kinase A-independent mechanism. This is the first report on ICER inducibility via G(q)/G(11) protein-coupled receptors.

1. The ability of 5-HT2 and 5-HT4 receptor antagonists to modify the disinhibitory profile of diazepam and other agents was investigated in male BKW mice in the light/dark test box. 2. The 5-HT2A/2B/2C receptor antagonists ritanserin, MDL11939 and RP62203 and also methysergide, which failed to modify mouse behaviour when administered alone, caused dose-related enhancements (4 to 8 fold) in the potency of diazepam to disinhibit behavioural responding to the aversive situation of the test box. 3. Ritanserin was shown to enhance the disinhibitory potency of other benzodiazepines, chlordiazepoxide (4 fold), temazepam (10 fold) and lorazepam (10 fold), the 5-HT1A receptor ligands, 8-OH-DPAT (25 fold), buspirone (100 fold) and lesopitron (500 fold), the 5-HT3 receptor antagonists, ondansetron (100 fold) R(+)-zacopride (100 fold) and S(-)-zacopride (greater than a 1000 fold), the substituted benzamides, sulpiride (10 fold) and tiapride (5 to 10 fold) and the cholecystokinin (CCK)A receptor antagonist, devazepide (100 fold). It also reduced the onset of action of disinhibition following treatment with the 5-HT synthesis inhibitor parachlorophenylalanine. Ritanserin failed to enhance the disinhibitory effects of the CCKB receptor antagonist CI-988, the angiotensin AT1 receptor antagonist losarten or the angiotensin converting enzyme inhibitor ceranapril. 4. The 5-HT4 receptor antagonists SDZ205-557, GR113808 and SB204070 caused dose-related reductions in the disinhibitory effect of diazepam, returning values to those shown in vehicle treated controls. The antagonists failed to modify mouse behaviour when administered alone. 5. GR113808 was also shown to cause a dose-related antagonism of the disinhibitory effects of chlordiazepoxide, lorazepam, 8-OH-DPAT, buspirone, lesopitron, ondansetron, R(+)-zacopride, sulpiride, tiapride, devazepide, CI-988, losarten, ceranapril and parachlorophenylalanine. 6. It was concluded that in BKW mice (a) the failure of 5-HT2 and 5-HT4 receptor antagonists when administered alone to modify behaviour in the light/dark test indicates an absence of an endogenous 5-HT tone at the 5-HT2 and 5-HT4 receptors and (b) the enhancement by the 5-HT2 receptor antagonists and attenuation by the 5-HT4 receptor antagonists of drug-induced disinhibition indicates a plurality of 5-HT receptor involvement in the mediation of drug-induced disinhibitory profiles in the mouse.

Using acutely isolated rat substantia nigra neurons, our previous studies indicated that sulfated cholecystokinin octapeptide (CCK-8) excites substantia nigra dopaminergic neurons by increasing the cationic conductance and that pertussis toxin-insensitive G proteins mediate CCK-8 induction of cationic currents. G alpha q and G alpha 11 are expressed in various tissues, including the brain, and likely to mediate pertussis toxin-insensitive neural signal transductions. In the present study, two different experiments were performed to test the hypothesis that G alpha q/11 mediates CCK-8 enhancement of the cationic conductance. First, we investigated the expression of G alpha q and G alpha 11 mRNAs in CCK-8-responsive substantia nigra dopaminergic neurons by combining whole-cell patch-clamp recordings with a single-cell reverse transcriptase-polymerase chain reaction assay. After CCK-8-evoked cationic currents were recorded, cellular RNA was harvested from single neurons and used as a template for the subsequent reverse transcriptase-polymerase chain reaction analysis. G alpha q and G alpha 11 mRNAs were present in all substantia nigra dopaminergic neurons that responded to CCK-8. Substantia nigra dopaminergic neurons were also internally perfused with the antibody raised against the common C-terminus of G alpha q and G alpha 11 during whole-cell recordings. CCK-8 failed to induce cationic currents after dopaminergic neurons were dialyzed with the anti-G alpha q/11 antibody. Our studies suggest that CCK-8 activation of the cationic conductance in substantia nigra dopaminergic neurons is transduced by G alpha q and/or G alpha 11.

The neuropeptide cholecystokinin has been implicated in the actions of a number of central processes including anxiety and reward. For this reason, the aim of the present study was to compare the density of CCK-A and -B receptors and the mRNA encoding preproCCK throughout the brains of an alcohol-preferring (Fawn Hooded) rat strain with that of a non-alcohol-preferring (Wistar Kyoto) strain of rat. Our study revealed significant differences with regard to the central CCK system of the FH compared to the WKY rat, including differences in CCK-A receptor binding throughout the dorsal medulla, and altered CCK-B binding density throughout the cerebral cortex and reticular nucleus of the thalamus. The most striking result, given the altered behavioural phenotype of the FH rat, was the 33% lower density of CCKmRNA measured throughout the ventral tegmental area of the FH rat when compared to the WKY. This study also reports on a protocol to utilise a novel radioligand, [125I]-D-Tyr-Gly-A-71378, for autoradiographic detection of CCK-A receptors throughout the rat brain. As previously reported, CCK-A receptors were located throughout the area postrema, interpeduncular nucleus and nucleus tractus solitarii; however, binding to CCK-A receptors was also visualised throughout the medial pre-optic area, the arcuate nucleus and the circumventricular regions of the ventral hypothalamus, regions known to contain CCK-A receptors but which were previously undetectable using autoradiography in rat brain.

The frontal ganglion of the adult forms of the tobacco hornworm, Manduca sexta, was investigated immunocytochemically for the occurrence of the gastro-entero-pancreatic (GEP) neurohormonal peptides, namely insulin, nerve growth factor, epidermal growth factor, insulin C-peptide, somatostatin, glucagon, glicentin, pancreatic polypeptide (PP), polypeptide YY (PYY), secretin, vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), gastrin, cholecystokinin (CCK), enkephalin, alpha- and beta-endorphins, substance P, neurotensin, bombesin, motilin, ACTH, serotonin, and calcitonin. Among all the antisera tested, positive immunostaining was obtained with anti-insulin B-chain serum only. The insulin B-chain immunoreactivity was localized in 4-6 large (30-40 microns) neurons, in the neuropile, and in the recurrent nerve. It is speculated that the insulin-like immunoreactive material may be transported to the neurohaemal organ (corpora cardiaca) through the nervi cardiaco-somatogastrici.

This report reviews the effects of CCK on the pancreas and in particular analyzes recent studies in which CCK antagonists were used to evaluate the physiological role of CCK in modulating pancreatic function and morphology. CCK is released from endocrine cells of the small intestine in response to a meal. In various animal species there are CCK receptors on pancreatic acinar cells with two sites; occupation of the high affinity site is thought to mediate pancreatic secretion and growth, whereas occupation of the low affinity site by high CCK concentrations is thought to be responsible for supramaximal inhibition of secretion and pancreatitis. Recently, CCK receptors were also found on postganglionic cholinergic neurons in the gastrointestinal tract. Administration of CCK agonists stimulates pancreatic secretion and growth. Although in some previous studies CCK was given at doses that mimic its postprandial increase in plasma, these studies did not prove that the actions of exogenous CCK were physiologically important. In addition, it was unclear if CCK primarily acts as a true hormone or as a neurotransmitter. The development of specific CCK receptor antagonists made it possible to better evaluate the physiological role of CCK. In humans, CCK-A antagonists like loxiglumide or L-364,718 at doses that completely inhibited the action of supraphysiological doses of exogenous CCK reduced meal-stimulated pancreatic enzyme secretion only by approximately 50%. On the other hand, atropine abolished the postprandial increase in pancreatic secretion and in addition markedly reduced the increase in pancreatic secretion due to infusion of "physiological" doses of CCK (i.e., CCK doses that mimic its postprandial increase in plasma). The increase in pancreatic bicarbonate secretion was only slightly reduced by CCK blockade. CCK antagonists failed to reduce the postprandial increase in plasma insulin, but markedly reduced the postprandial PP release. CCK-A antagonists caused slight hypotrophy and hypoplasia of the exocrine pancreas. However, even after 9 months of effective blockade of the CCK-A-receptor, mice had normal body weight and an almost normal pancreas. CCK antagonists were unable to alter short-term changes in pancreatic growth due to feeding and fasting. In some species, CCK agonists induced development of pancreatic nodules and increased the growth of malignant tumors. Studies about the effects of CCK antagonists on induction and growth of pancreatic tumors showed controversial results. In conclusion, CCK may act on the pancreas by three pathways: (1) At low doses it serves as a neurotransmitter by acting on cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholecystokinin (CCK) is one of the most abundant neurotransmitter peptides in the brain. CCK appears to play an important role in the neurobiology of anxiety and panic disorders (PD) in both humans and animals. Recently, we reported that lack of CCKAR had a significant anxiogenic-like effect in rats. In this study, to investigate the role of CCKAR in PD, we compared the CCKAR gene in PD patients and normal controls. Subjects who fulfilled the DSM-IV criteria for PD were 17 males and 26 females. The sequence containing the Pst I polymorphic site in the boundary between intron 1 and exon 2 of the CCKAR gene was studied. Pst I digestion of the PCR products gave two individual alleles: A1 and A2. The A1 allele was the undigested fragment and the A2 allele was the digested one with two variant bands at 264 and 180 bp. Genotypic frequencies were 20.9% (A1-A1), 55.8% (A1-A2), and 41.7% (A2-A2) in patients, and 20.5% (A1-A1), 46.2% (A1-A2), and 33.3% (A2-A2) in controls. Allelic frequencies were 48.8% (A1) and 51.2% (A2) in patients, and 43.6% (A1) and 56.4% (A2) in controls. The chi-square test did not show a significant difference in either genotypic or allelic frequencies between patients and control subjects. The Pst polymorphism of CCKAR may not be associated with PD.

The pharmacological profile of FK480[(S)-(+)-N-<1-(2)-fluorophenyl)-3,4,6,7-tetra hydro-4-oxo-pyrrolo(3,2,1-jk) (1,4)benzodiaze-pine-3-yl>-1H-indole-2- carboxamide], a novel cholecystokinin type-A (CCK-A) receptor antagonist, was compared with that of the CCK-A receptor antagonist, loxiglumide. Both FK480 and loxiglumide inhibited 125I-labeled CCK-8 (125I-CCK-8) binding to rat pancreatic and guinea-pig gallbladder membranes with IC50 values of 0.40 +/- 0.04 and 0.06 +/- 0.02 nM for FK480 and 330 +/- 66 and 66 +/- 10 nM for loxiglumide, respectively. These two agents also inhibited 125I-CCK-8 binding to guinea-pig brain (cerebral cortex) receptors with respective IC50 values of 72 +/- 11 nM and>> 10 microM, indicating less affinity to central receptors. Intravenous administration of FK480 (ED50 = 18 micrograms/kg) was 2800 times more potent than that of loxiglumide (ED50 = 50 mg/kg) in inhibiting CCK-8-induced pancreatic amylase secretion in rats. Furthermore, FK480 had ED50 values of 10 and 8.4 micrograms/kg, respectively, in antagonizing CCK-8-induced inhibition of charcoal meal gastric emptying in mice when administered orally 1 or 5 hr before the CCK-8. Loxiglumide (ED50 = 23.5 mg/kg, when administered orally 1 hr before the CCK-8) also antagonized it, but its activity was 2400 times less than that of FK480. We conclude that FK480 is a potent, orally effective CCK-A receptor antagonist with long duration of action.

Nonsulfated CCK(58) [CCK(58)(ns)] has not been considered to be of biological importance because CCK(58)(ns) binds poorly to the CCK(A) receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK(58)(ns). CCK(58)(ns) is 14% as immunoreactive as sulfated CCK(8) [CCK(8)(s)]. Amino acid analysis demonstrated that CCK(58)(ns) was present at 50% the amount of CCK(58)(s). In addition, we found that CCK(58)(ns) does not potently displace an (125)I-labeled CCK(10) analog from the CCK(A) receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK(58)(ns) does bind to CCK(B) receptors and stimulates gastric acid secretion via this receptor. The presence of CCK(58)(ns) and its ability to selectively stimulate the CCK(B) receptor without stimulation of the CCK(A) receptor suggest that CCK(58)(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent.

Soluble and membrane phosphoinositide-specific phospholipases obtained separately from dispersed circular and longitudinal intestinal muscle cells were characterized for substrate specificity and G protein dependence using selective antibodies to various isoforms of phospholipase C (PLC) and G protein subunits. Western blot analysis disclosed the presence of the main PLC isozymes, PLC-gamma 1, PLC-delta 1, and PLC-beta 1. Soluble PLC from circular and longitudinal muscle was stimulated by guanosine 5'-O-(3-thiophosphate) and inhibited by PLC-beta 1 antibody (80-90%) and PLC-beta 3 antibody (approximately 25%) but not by G protein antibodies. Membrane PLC from circular and longitudinal muscle was stimulated by cholecystokinin octapeptide (CCK-8) and inhibited selectively by PLC-beta 1 antibody (85%), PLC-beta 3 antibody (15%), and G alpha q/11 antibody (90%). CCK-8-induced contraction in permeabilized circular muscle cells was also selectively inhibited by PLC-beta 1 antibody (76%), PLC-beta 3 antibody (24%), and G alpha q/11 antibody (86%). The combined effects of PLC-beta 1 and PLC-beta 3 antibodies on PLC activity and muscle contraction were additive, causing complete inhibition. Soluble and membrane PLC from circular and longitudinal muscle were immunologically similar but functionally different. The enzymes from circular muscle preferentially hydrolyzed endogenous and exogenous phosphatidylinositol 4,5-biphosphate (PIP2), confirming previous findings of preferential hydrolysis of PIP2 in dispersed intestinal circular muscle cells.

The effects of cholecystokinin sulfate octapeptide (CCK-8S) on [3H]gamma-aminobutyric acid (GABA) release have been studied in the anterior side of the rat nucleus accumbens on tissue punches exposed in superfusion to 30 mM KCl. CCK-8S in a concentration dependent manner (10-3000 nM) increased K+-evoked [3H]GABA release (EC50=192 nM). The increase caused by 1 microM CCK-8S ranged from 37% to 42%. CR 2945, (beta-[2-[[2-(8-azaspiro[4.5]dec-8-ylcarbonyl)-4,6-dimethylp henyl]-amino]-2-oxoethyl]-(R)-1-naphthalenepropanoic acid), a potent and selective nonpeptidergic CCK(B) antagonist, concentration-dependently blocked CCK-8S effect (IC50=2.16 nM). CCK-8S-induced increase in [3H]GABA overflow was completely blocked by 1 microM tetrodotoxin. Both the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) and the N-methyl-D-aspartic acid (NMDA) receptor antagonist dizocilpine (MK-801) antagonized the CCK-8S effect. By contrast, (+)-bicuculline, a GABA(A) receptor antagonist, was completely ineffective. Phaclofen, a selective GABA(B) antagonist, increased K+-evoked [3H]GABA release but did not affect the facilitative effect of CCK-8S. Moreover, tetrodotoxin failed to block AMPA-evoked [3H]GABA release but completely prevented the effect of NMDA (Mg2+ free conditions). The data presented suggest that CCK(B) receptors modulating [3H]GABA release from anterior accumbal punches may not be present on GABAergic terminals but could be located on glutamatergic interneurons.

In the preceding paper, by means of selective agonists to gastrin (HG-17) and cholecystokinin (CCK-39), we evidenced the existence of "gastrin-type" receptors that could regulate histamine release and "CCK-type" receptors that could stimulate somatostatin release in isolated rabbit fundic non-parietal cells (F1 cells). Furthermore, these receptors could induce phosphoinositide breakdown. To confirm the involvement of these receptor types in these biological and biochemical processes, we used selective antagonists, L-364,718 (3-(benzoylamino)-benzodiazepine) specific to "CCK-A-type" receptor and L-365,260 (3-(acylamino)-benzodiazepine) specific to "gastrin/CCK-B-type" receptor. Neither L-364,718 nor L-365,260 alone caused any significant stimulation of [3H]inositol phosphate ([3H]InsP) production and release of histamine or somatostatin-like immunoreactivity (SLI). Each analogue inhibited in a dose-dependent manner [125I]HG-17 or [125I]CCK-39 binding to F1 cells, [3H]InsP accumulation and histamine and SLI release stimulated by HG-17 or CCK-39. L-365,260 appeared to be 30-70 times more potent than L-364,718 in inhibiting [125I]HG-17 binding to F1 cells, as well as HG-17-induced [3H]InsP accumulation and HG-17-or CCK-39-enhanced histamine release (IC50 values: approximately 5-20 nM for L-365,260 and approximately 200-1500 nM for L-364,718). In contrast, L-364,718 was 200 to 400 times more potent than L-365,260 in inhibiting [125I]CCK-39 binding to F1 cells, CCK-39-induced [3H]-InsP accumulation and SLI release stimulated by CCK-39 or HG-17 (IC50 values: approximately 0.3-1 nM for L-364,718 and 100-200 nM for L-365,260). These results led to conclude: (i) the existence of a "gastrin-type" receptor related to histamine release: (ii) the existence of a "CCK-A-type" receptor related to somatostatin release; (iii) the existence of "gastrin type" and "CCK-A-type" receptors linked to the phosphoinositide breakdown pathway.

The mechanism of CCK action on gallbladder contractions in the physiological condition is unclear. Gallbladder contractions were monitored by means of chronically implanted force transducers in conscious dogs. Postprandial gallbladder contractions were partially inhibited by atropine and hexamethonium, and completely inhibited by devazepide. In vitro contractile response of canine gallbladder muscle strips to CCK-8 was also studied. CCK-8-induced muscle strip contraction was atropine and tetrodotoxin resistant, but was completely eliminated by devazepide. The existence of CCK receptors in the vagal nerve and gallbladder was examined by means of autoradiography. Forty-eight hours after ligation of the abdominal vagus, CCK-8 binding sites were found to accumulate in the subdiaphragmatic vagal nerve immediately proximal to the ligature, and similar binding sites were also found in the gallbladder smooth muscle layer. These binding sites were displaced by the addition of 10(-7) mol/1 unlabeled CCK-8 and devazepide, but L-365,260 had no effect. In conclusion, it is considerable that postprandial CCK-induced gallbladder contractions are controlled through CCK-A receptors both on the vagal nerve in stimulating endogenous release of acetylcholine and on the gallbladder directly to stimulate muscle contraction in the dog.

BACKGROUND

In humans, cholecystokinin (CCK) stimulates pancreatic secretion, and CCK-A receptor antagonists prevent it in vivo. However, the human pancreas has been reported to express mainly CCK-B receptors.

OBJECTIVE

To elucidate this discrepancy.

METHODS

We prepared dispersed acini from human pancreas and examined whether various doses of CCK stimulated the release of amylase, in comparison with the effects of neuromedin C, carbamylcholine, and secretin.

RESULTS

Human pancreatic acini did not respond to any dose of CCK or secretin. Amylase release was stimulated by carbamylcholine and neuromedin C dose-dependently and was inhibited by respective antagonists. The localizations of CCK receptors in the human duodenum were determined. High concentrations of CCK-A receptors were detected in the mucosa as well as in smooth muscle of the duodenum by microautoradiography.

CONCLUSIONS

In conclusion, human pancreatic acinar cells are responsible for carbamylcholine and neuromedin C but not for secretin. Neither CCK-A nor CCK-B receptor mediates amylase release from human pancreatic acini in vitro. Pancreatic secretion in humans in vivo may be regulated indirectly by CCK (via CCK-A receptors).

The sulfated octapeptide of cholecystokinin (CCK-8S) and CCK fragments were administered to mice to determine the subtype and central versus peripheral location of the CCK receptor that modulates dopamine release in the neostriatum. Dopamine release was decreased when unsulfated CCK (CCK-8U) or the butoxycarbonyl tetrapeptide of CCK (t-boc-CCK-4) was infused into the brain ventricles but not when injected subcutaneously. These CCK fragments bind to the brain-type (CCK-B) but not alimentary-type (CCK-A) receptor. Centrally or peripherally administered CCK-8S also lowered dopamine release and this action was not blocked by the selective CCK-A receptor antagonist, L 364,718. The increase in dopamine release following amphetamine administration was attenuated by central injections of t-boc-CCK-4, CCK-8U, or CCK-8S, and this action of CCK-8S was not prevented by L 364,718. These data are the first to demonstrate that CCK-B receptors in brain mediate the suppression of dopamine release by cholecystokinin, especially when release is augmented. CCK-B receptor agonists should be useful for the treatment of psychiatric conditions that result from hyperactive dopamine neurons.