Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike in rodents, suggesting that its anorexigenic action is mediated by alpha-melanocyte-stimulating hormone (alpha-MSH) but not corticotropin-releasing hormone. This led us to investigate whether MCH-containing neurons in the goldfish brain have direct inputs to alpha-MSH-containing neurons, using a confocal laser scanning microscope, and to examine whether the anorexigenic action of MCH is also mediated by other anorexigenic neuropeptides, such as cholecystokinin (CCK) and pituitary adenylate cyclase-activating polypeptide (PACAP), using their receptor antagonists. MCH- and alpha-MSH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. MCH-containing nerve fibers or endings lay in close apposition to alpha-MSH-containing neurons in the hypothalamus in the posterior part of the nucleus lateralis tuberis (NLTp). The inhibitory effect of ICV-injected MCH on food intake was not affected by treatment with a CCK A/CCK B receptor antagonist, proglumide, or a PACAP receptor (PAC(1) receptor) antagonist, PACAP((6-38)). ICV administration of MCH at a dose sufficient to inhibit food consumption also did not influence expression of the mRNAs encoding CCK and PACAP. These results strongly suggest that MCH-containing neurons provide direct input to alpha-MSH-containing neurons in the NLTp of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide via the alpha-MSH (melanocortin 4 receptor)-signaling pathway.

The influence of clonidine pretreatment on psychopathological, endocrine and respiratory effects of cholecystokinin tetrapeptide (CCK-4) was characterized. Patients with panic disorder (DSM-III-R) were given 50 micrograms CCK-4 i.v. at 1100 hours on 2 separate study days. In a randomized double-blind design they were additionally infused with 150 micrograms clonidine or placebo from 1040 to 1110 hours. After CCK-4 all patients experienced symptom attacks. No effects of clonidine on panic psychopathology or blood gas parameters were observed. After CCK-4, in the clonidine condition the pituitary release of adrenocorticotropin (ACTH) and prolactin was seemingly enhanced compared to placebo. Our results suggest that CCK-4-induced panic attacks are not suppressible by presynaptic alpha-2 receptor stimulation. Moreover, they point to a synergistic postsynaptic action of clonidine to CCK-4 upon pituitary hormone secretion. The diverging sites of action might possibly explain the discrepancies of psychopathological alterations and stress hormone secretion.

Diabetes mellitus (DM) is a major health problem at present affecting about 180 million people worldwide. DM is associated with many metabolic abnormalities in the body including the indigestion of carbohydrates leading to malnutrition and weight loss. In this article we investigate the cellular and molecular mechanisms of exocrine pancreatic insufficiency in streptozotocin (STZ, 60 mg kg(-1), i.p.)-induced DM in male rats compared to healthy age-matched controls. Either electrical field stimulation (EFS) or cholecystokinin octapeptide (CCK-8, 10(-8) M) can elicit large and significant (P < 0.05) increases in amylase output from pancreatic segments compared to basal secretion. Insulin (10(-6) M) alone has no significant effect on amylase output compared to basal but it enhanced the secretory responses to either EFS or CCK-8. When rats were rendered diabetic with STZ, either EFS or CCK-8-evoked amylase output was significantly (P < 0.01) decreased compared to the responses obtained with either EFS or CCK-8 alone in healthy age-matched control pancreas. In addition, CCK-8 can elicit large dose-dependent release of amylase in age-matched control and diabetic acinar cells with significantly (P < 0.05) reduced responses in diabetic acinar cells. CCK-8 evoked a large rapid increase in peak cytosolic free calcium concentration ([Ca2+]c) followed by a decrease to a plateau phase in age-matched control fura-2-loaded pancreatic acinar cells. These responses were significantly (P < 0.05) decreased in STZ-induced diabetic acinar cells. In the presence of 10(-6) M insulin, CCK-8 evoked a much larger increase in the Ca2+ transient compared to the response obtained with CCK-8 alone. These effects were significantly (P < 0.01) inhibited in STZ-induced diabetic acinar cells. Similarly, in zero extracellular Ca2+ [Ca2+]ĉ, the CCK-8-evoked [Ca2+]c was significantly (P < 0.05) reduced in both diabetic and age-matched control acinar cells, but with more pronounced reduction in diabetic acinar cells. CCK(A) receptor mRNA levels remained unchanged in diabetic rat acinar cells compared to age-matched healthy control. In contrast, amylase mRNA was significantly (P < 0.05) reduced in diabetic acinar cells compared to control. The results indicate that reduced amylase secretion in response to either EFS or CCK-8 in the diabetic pancreas may be due to reduced [Ca2+]c and gene expression for amylase and not to the gene expression of CCK(A) receptor in pancreatic acinar cells.

1. Long Evans rats were chronically instrumented with intravascular catheters and pulsed Doppler probes to assess changes in renal, mesenteric and hindquarters blood flows and vascular conductances in response to bombesin (2.5 micrograms kg-1, i.v.) and cholecystokinin (CCK) (0.5 and 5.0 micrograms kg-1, i.v.). 2. Bombesin caused an increase in heart rate and blood pressure, together with a transient renal vasoconstriction and prolonged mesenteric vasodilatation; there was an early hindquarters vasodilatation followed by vasoconstriction. 3. In the presence of phentolamine, bombesin caused a fall in blood pressure due to enhanced hindquarters vasodilatation; these effects were reversed by propranolol and hence were possibly due to circulating adrenaline acting on vasodilator beta 2-adrenoceptors. 4. During concurrent administration of phentolamine, propranolol and atropine, bombesin caused prolonged tachycardia and a rise in blood pressure. The renal vasoconstrictor and mesenteric vasodilator effects of bombesin were not reduced under these conditions and thus probably were direct and/or indirect non-adrenergic, non-cholinergic (NANC) effects. 5. CCK caused dose-dependent increases in blood pressure accompanied by renal, mesenteric and hindquarters vasoconstriction followed, after the higher dose, by vasodilatations. The lower dose of CCK increased heart rate but there was a bradycardia followed by a tachycardia after the higher dose. 6. Experiments with antagonists as described above indicated the pressor effect of CCK was mediated largely through alpha-adrenoceptors, as were the mesenteric and hindquarters vasoconstrictor effects; CCK exerted NANC negative chronotropic effects. 7. All the effects of CCK were markedly inhibited by L364,718. This observation, and the finding that L364,718 had no effect on the responses to bombesin, together with the dissimilarities in the regional haemodynamic effects of exogenous CCK and bombesin, indicate that the cardiovascular actions of the latter were not dependent on the release of endogenous CCK.

Glucocorticoids are potent anti-inflammatory drugs. The molecular mechanisms underlying these effects have not yet been fully revealed. The aim of the present study was to establish whether methylprednisolone pretreatment is beneficial and if it can block the pancreatic DNA binding of the transcription factor nuclear factor-kappaB (NF-kappaB) and proinflammatory cytokine synthesis during cholecystokinin-octapeptide (CCK)-induced acute pancreatitis in rats. Additionally, we set out to investigate the potential effects of methylprednisolone and CCK on pancreatic heat shock protein (HSP) synthesis. The dose-response (5-40 mg/kg) and time-course (6-72 h) curves of methylprednisolone on pancreatic HSP60 and HSP72 synthesis were evaluated following methylprednisolone treatment. We demonstrated that methylprednisolone specifically and dose-dependently induced HSP72 in the pancreas of rats, while it did not have a significant effect on HSP60 expression. The pancreatitis was induced near the peak level of HSP72 synthesis (2 x 30 mg/kg body weight [b.w.] methylprednisolone i.m. at an interval of 12 h, followed by a 12-h recovery period after the second injection of methylprednisolone) by administering 2 x 100 microg/kg CCK subcutaneously at an interval of 1 h. The injections of CCK in the vehicle-pretreated group significantly elevated the levels of pancreatic HSP60 and HSP72 2-4 h after the second CCK injection. Methylprednisolone pretreatment ameliorated many of the examined laboratory (the pancreatic weight/body weight [p.w./b.w.] ratio, the serum amylase activity, the plasma trypsinogen activation peptide concentration, the pancreatic levels of tumor necrosis factor-alpha and interleukin-6, the degree of lipid peroxidation, protein oxidation, nonprotein sulfhydryl group content and the pancreatic myeloperoxidase activity) and morphological parameters of the disease. Methylprednisolone pretreatment did not influence pancreatic NF-kappaB DNA binding, but decreased proinflammatory cytokine synthesis in this acute pancreatitis model. The findings suggest that the anti-inflammatory effect of large doses of methylprednisolone in secretagogue-induced pancreatitis occurs downstream of NF-kappaB DNA binding, and that increased pancreatic HSP72 synthesis may play a role in the protective effect of the drug.

1. Nerve growth factor (NGF), a powerful agent for the growth, differentiation and regeneration of lesioned cells of the central and peripheral nervous systems, has in recent years been indicated as a potential therapeutic agent capable of reversing the processes of cell damage in neurodegenerative events in man. Since NGF does not cross the blood-brain barrier and central NGF administration requires invasive surgical procedures, the discovery of substances modulating in vivo NGF synthesis in the brain will be extremely useful for a possible clinical use of NGF. 2. The aim of the present study to analyse if the content of NGF in the brain of adult mice can be affected by peripheral administration of cholecystokinin-8 (CCK-8), a well known neuropeptide which has stimulant actions on neurons in the brain and promotes a variety of neurobehavioural effects both in man and rodents. 3. The dose-response and time course effects of an i.p. injection of CCK-8 on the NGF concentrations in the hippocampus, cortex, hypothalamus and pituitary of adult male mice were analysed by use of a sensitive immunoenzymatic assay for NGF. The effects of pretreatment with selective CCK(A) and CCK(B) receptor antagonists and atropine on the NGF response to CCK injection were also studied. 4. The effects of CCK-8 were dose- and time-dependent and the injection of 8 nmol kg(-1) resulted in a 3 fold increase of NGF levels in the hypothalamus and pituitary, and about a 60% increase in the hippocampus. No effects were observed in the cortex. Pretreatment with a selective CCK(A) receptor antagonist blocked the CCK-induced NGF increase in the hypothalamus and pituitary. In the hippocampus the same effect was obtained with a CCK(B) receptor antagonist. Pretreatment with atropine suppressed the CCK-induced effects on NGF levels in all the brain regions examined. 5. Our results showing that i.p. injection with CCK-8 can modulate NGF levels in the brain through a mechanism which seems, in part, to be mediated via the vagal afferents, indicate that this neuropeptide may represent a useful pharmacological approach to enhance endogenous NGF levels in neuropathologies associated with a neurotrophin deficit.

A non-hydrolysable guanosine nucleotide analog, GTP[S] at 200 microM, stimulated amylase secretion which was inhibited by an anti-phospholipase A2 (PLA2) antibody in permeabilized pancreatic acini, indicating that the PLA2 pathway is linked to the GTP binding protein. A high affinity cholecystokinin (CCK) receptor agonist, CCK-OPE (10 microM), and a low affinity receptor agonist, CCK-8 (0.1 microM), both caused amylase secretion in permeabilized cells. The action of CCK-OPE was abolished by the G beta antibody but not by the G alpha-q,11 antibody, whereas the opposite was true of the CCK-8 response. Biscoclaurine alkaloid isotetrandrine (10 microM), a specific inhibitor of PLA2-coupled G proteins, abolished Ca2+ oscillations and amylase secretion induced by CCK-OPE (0.1-100 nM), but not by CCK-8 (10 pM) in intact acini. Gp antagonist-2A (10 microM), which inhibits the activation of Gq, also inhibited the actions of CCK-OPE (10 pM-1 microM) in intact acini. These observations indicate that the functional unit of the heterotrimeric G protein coupled to the high affinity CCK receptor appears to be different from that linked to the low affinity CCK receptor/Gq-alpha pathway. The regulatory site of this G protein coupled to the high affinity CCK receptor is on the beta subunit of Gq protein which elicits Ca2+ oscillations and monophasic amylase secretion via the PLA2 pathway.

Devazepide, a potent CCK-A receptor antagonist, and L-365,260, a selective CCK-B receptor antagonist, have been introduced as pharmacologic tools for differentiating the physiologic roles of CCK-A and CCK-B receptor subtypes. In the present study, we tested the effects of devazepide and L-365,260, on morphine antinociception in rats using the thermal sensorimotor tail flick test. Both devazepide and L-365,260 significantly enhanced the antinociceptive action of morphine, but only in rats that had not been acclimated to the laboratory environment or habituated to investigator handling. When tested with fully acclimated animals, devazepide and L-365,260 had no effect whatsoever; they neither enhanced nor attenuated morphine-induced antinociception. These observations indicate that the effects of devazepide and L-365,260, CCK antagonists, on morphine antinociception appear to be dependent on the animal's response to a new environment or to the stress induced by an unaccustomed experimental paradigm.

Many reports have emphasized the role of gastrin as a growth factor for normal gastrointestinal mucosa and gastrointestinal cancers. Recent studies have pointed out that this peptide acts also as a growth factor for the pancreatic cancer cell line AR42J. This effect is mediated by gastrin [cholecystokinin (CCK)-B] receptors. In the present study, we investigated gastrin (CCK-B) receptor expression in the azaserine-induced rat pancreatic carcinoma DSL-6, comparing it to normal rat pancreas, and we also characterized CCK receptor subtypes in this tumor. The results showed that there is extensive gastrin binding to the DSL-6 pancreatic carcinoma. No evidence of specific gastrin binding to normal pancreas was found. Analysis of the ability of gastrin-17-I to inhibit 125I-gastrin-I binding demonstrated that gastrin bound to a single class of receptors with a Kd of 0.21 +/- 0.04 nM and a binding capacity of 184 +/- 29 fmol/mg protein. 125I-Gastrin-I binding was inhibited by the specific CCK-B receptor antagonist L365,260 approximately 40 times more effectively than by the specific CCK-A receptor antagonist L364,718. Analysis of the ability of cholecystokinin octapeptide (CCK-8) to inhibit 125I-Bolton-Hunter-CCK-8 binding revealed two CCK binding sites, i.e., a high affinity site and a low affinity site. The observed binding affinities of CCK-8 were then introduced into the computer analysis of the dose-inhibition curve of the ability of gastrin-17-I to inhibit binding of 125I-Bolton-Hunter-CCK-8, which was significantly better fit by a three-site model than by a two-site model. The three sites meet the criteria for CCK-B, high affinity CCK-A, and low affinity CCK-A receptors. The binding capacity of CCK-B receptors constitutes 34% of the total high affinity CCK binding sites. This study demonstrated that DSL-6 pancreatic carcinoma expresses three subtypes of CCK receptors. Gastrin (CCK-B) receptors, which were not detected in normal rat pancreas, constitute about one third of the total high affinity CCK receptors. We suggest that novel expression of gastrin (CCK-B) receptors may be generated by gene mutation or amplification during carcinogenesis and may play an important role in promoting tumor growth.

BACKGROUND

Upregulation of matrix metalloproteinase-9 (MMP-9) induced by tumour necrosis factor-alpha (TNF-alpha) is reportedly involved in a variety of non-neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF-alpha-induced MMP-9 upregulation in cholangiocarcinoma (CC).

METHODS

We used two CC cell lines: HuCCT-1 and CCKS-1.

RESULTS

In an ex vivo study using HuCCT-1 and CCKS-1 cells, TNF-alpha treatment induced MMP-9 production and activation via interaction with TNF receptor-1 (TNF-R1) but not with TNF receptor-2 (TNF-R2), shown by zymography, and increased MMP-9 promoter activity (luciferase assay). As for the signalling pathway, TNF-alpha stimulation led to the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinase (p38MAPK) and translocation of nuclear factor kappaB (NF-kappaB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen-activated or extracellular signal-regulated protein kinase 1/2) and MG132 (inhibitor of NF-kappaB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF-kappaB was closely related to MMP-9 upregulation in both cell lines.

CONCLUSIONS

These data suggest that TNF-alpha/TNF-R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF-kappaB, which is closely associated with the production and activation of MMP-9 in cultured CC cells of HuCTT-1 and CCKS-1. Upregulation of MMP-9 with NF-kappaB activation may be involved in the tumour invasion of CC.

The review concentrates on practical applications of computer molecular modeling in peptide drug design. The examples of the predictions (successful or not) made by computational modeling before synthesis of peptide analogs, not the explanations provided after synthesis and biological testing of peptides, are discussed. The review spans over 20 years of predictions made by computer molecular modeling for bradykinin, angiotensin, thyrotropin-releasing factor, tuftsin, substance P, CCK-related peptides, luliberin, alpha-melanotropin and opioid peptides. The described examples are discussed in terms of finding the optimal way to use computer modeling for peptide design. The step-by-step 'technology' of peptide design is outlined in detail.

We evaluated the effects of a potent cholecystokinin (CCK)-B/gastrin receptor antagonist, L-365,260 (3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin - 3-yl)-N'-( 3-methylphenyl) urea); a selective CCK-A receptor antagonist, devazepide (L-364,718); and cimetidine on gastric acid secretion induced by pentagastrin, histamine and bethanechol in anesthetized rats. We also evaluated the effects of L-365,260 and cimetidine on acid secretion in pylorus-ligated rats. Intravenous administration of L-365,260, L-364,718 and cimetidine dose-dependently reduced acid secretion induced by pentagastrin (20 nmol/kg/hr), with ED50 values of 0.63, 19.1 and 2.5 mumol/kg, respectively. Of interest was the finding that L-365,260, like cimetidine, dose-dependently inhibited acid secretion induced by histamine (100 mumol/kg/hr) and bethanechol (5 mumol/kg/hr) with ED50 values of 5.9 and 4.3 mumol/kg, respectively. L-364,718, even at 30 mumol/kg, i.v., had only a slight effect on histamine- or bethanechol-induced acid secretion. Gastric acid secretion was suppressed by treatment with L-365,260 (3-100 mumol/kg, i.v.) and cimetidine (11.9-396.4 mumol/kg, i.v.) in pylorus-ligated rats, with ED50 values of 13.3 and 96.9 mumol/kg, respectively. These results indicate that L-365,260 suppresses acid secretion induced by histamine and bethanechol in rats and that the gastrin receptor plays an important role in acid secretion in pylorus-ligated rats.

Daily behaviors are strongly dominated by internally generated circadian rhythms, but the underlying mechanisms remain unclear. In mammals, photoentrainment of behaviors to light-dark cycles involves signaling from both intrinsically photosensitive retinal ganglion cells and classic photoreceptor pathways to the suprachiasmatic nucleus (SCN). How classic photoreceptor pathways work with the photosensitive ganglion cells, however, is not fully understood. Although cholecystokinin (CCK) peptide has been shown to be present in a variety of vertebrate retinas, its function at a systems level is also unknown. In the present study we examined a possible role of CCK-A receptors in photoentrainment using CCK-A receptor knockout mice. The lacZ reporter gene within a gene-knockout cassette revealed precise localization of CCK-A receptors in the circadian clock system. We demonstrated that CCK-A receptors were located predominately on glycinergic amacrine cells but were rarely found on SCN neurons. Moreover, Ca(2+) imaging analysis demonstrated that the CCK-A agonist, CCK-8 sulfate (CCK-8s), mobilized intracellular Ca(2+) in amacrine cells but not glutamate-receptive SCN neurons. Furthermore, light pulse-induced mPer1/mPer2 gene expression in SCN, behavioral phase shifts, and the pupillary reflex were significantly reduced in CCK-A receptor knockout mice. These data indicate a novel function of CCK-A receptors in the nonimage-forming photoreception presumably via amacrine cell-mediated signal transduction pathways.

Stimulus-secretion coupling in the pancreatic acinar cell is initiated by the secretagogues <em>CCK</em> and ACh and results in the secretion by exocytosis of the contents of zymogen granules. A key event in this pathway is the G protein-activated production of second messengers and the subsequent elevation of cytosolic-free Ca2+. The aim of this study was therefore to define the heterotrimeric G protein <em>alpha</em>-subunits present and participating in this pathway in rat pancreatic acinar cells. RT-PCR products were amplified from pancreatic acinar cell mRNA with primers specific for G<em>alpha</em>q, G<em>alpha</em>11, and G<em>alpha</em>14 but were not amplified with primers specific for G<em>alpha</em>15. The sequences of these PCR products confirmed them to be portions of the rat homologues of G<em>alpha</em>q, G<em>alpha</em>11, and G<em>alpha</em>14. The pancreatic-derived cell line AR42J similarly expressed G<em>alpha</em>q, G<em>alpha</em>11, and G<em>alpha</em>14; however, the Chinese hamster ovary (CHO) cell line only expressed G<em>alpha</em>11 and G<em>alpha</em>q. These data indicate that caution should be exercised when comparing signal transduction pathways between different cell types. The expression of these proteins in acinar cells was confirmed by immunoblotting samples of acinar membrane protein using specific antisera to the individual G protein <em>alpha</em>-subunits. The role of these proteins in Ca2+ signaling events was investigated by microinjecting a neutralizing antibody directed against a homologous sequence in G<em>alpha</em>q, G<em>alpha</em>11, and G<em>alpha</em>14 into acinar cells and CHO cells. Ca2+ signaling was inhibited in acinar cells and receptor-bearing CHO cells in response to both physiological and supermaximal concentrations of agonists. The inhibition was >75% in both cell types. These data indicate a role for G<em>alpha</em>q and/or G<em>alpha</em>11 in intracellular Ca2+ concentration signaling in CHO cells, and in addition to G<em>alpha</em>q and G<em>alpha</em>11, G<em>alpha</em>14 may also fulfill this role in rat pancreatic acinar cells.

Recent studies provide significant evidence that cholecystokinin (CCK) is involved in the induction and development of acute pancreatitis in experimental animals. However, the results obtained with specific CCK-A (peripheral) receptor antagonists are still controversial. The present studies were undertaken to evaluate the involvement of endogenous CCK and the CCK-A receptors in the development of severe acute pancreatitis induced in Otsuka Long-Evans Tokushima Fatty (OLETF) rats that have a selective defect in the CCK-A receptor. Three models of severe acute pancreatitis were induced by retrograde intraductal infusion of 4% sodium taurocholate, by the closed duodenal loop, or by a single intraperitoneal injection of 500 mg/100 g body weight of L-arginine in OLETF rats and control Long-Evans Tokushima Otsuka (LETO) rats. Plasma CCK levels rose up to 4- to 14-fold over the preloading values after the onset of acute pancreatitis in all three models in both groups of rats. However, histologic alterations as well as the magnitudes of increase in serum amylase and lipase activity and the pancreatic wet weight were significantly less in the OLETF rats than those in the LETO rats. In addition, 72 h after the onset of arginine pancreatitis, massive destruction of pancreatic parenchyma with a significant reduction in serum amylase and lipase activities and pancreatic wet weight was observed in the LETO rats, whereas these changes were not seen in OLETF rats. These results suggest that endogenous CCK and CCK-A receptors play a role in the development of severe acute pancreatitis in rats.

Acute and long-term changes of ornithine decarboxylase and polyamines during pancreatic adaptation in response to cholecystokinin administration (1 microgram kg-1 body wt every 8 h) were studied in rats. alpha-difluoromethylornithine, an irreversible and specific inhibitor of ornithine decarboxylase, was applied simultaneously to elucidate the essential role of polyamines in pancreatic growth. In the cholecystokinin-treated animals ornithine decarboxylase activity was increased after 2 h, reached a maximum after 8 h (444.6 pmol 14CO2 h-1 mg-1 DNA, about 65-fold greater than controls, P less than 0.001) followed by a significant increase of putrescine after 6 h and spermidine after 24 h while spermine remained unchanged. The trophic parameters increased in the following time sequence: thymidine kinase (12 h), DNA polymerase (24 h), pancreatic weight (2 days), protein (2 days) and DNA (5 days). alpha-difluoromethylornithine significantly delayed the increase in ornithine decarboxylase, putrescine and spermidine as well as all trophic parameters. Increases in ornithine decarboxylase, polyamines and all trophic parameters were completely inhibited by simultaneous application of the CCK receptor antagonist L-364,718. These data indicate an important role for ornithine decarboxylase and polyamines in cholecystokinin-induced pancreatic growth in rats.

In previous 2-year studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) conducted by the National Toxicology Program on female Harlan Sprague-Dawley rats, acinar-cell vacuolation, atrophy, inflammation, and arteritis developed at high incidence, and a rare occurrence of pancreatic acinar-cell adenomas and carcinomas was noted. In this investigation, we sought to identify the mechanism involved in the early formative stages of acinar-cell lesions. Pancreas from animals treated for 14 and 31 weeks with 100 ng TCDD/kg body weight or corn oil vehicle was examined immunohistochemically and/or morphometrically. Acinar-cell kinetics were analyzed using staining with hematoxylin and eosin and proliferating cell nuclear antigen. Expressions of cytochrome P450 (CYP) 1AAhR) were evaluated to assess direct effects of TCDD exposure. The cholecystokinin-A receptor (CCK-A receptor; CCKAR) and duodenal cholecystokinin 8 (CCK) revealed the associations of dioxin treatment with hormonal changes. Amylase localization showed acinar structural changes that could affect enzymatic production. Increased apoptotic activity in acinar cells occurred in 14- and 31-week-treated animals, with an increase in proliferative activity in the latter. Also in the latter, in the vacuolated acinar cells, CYP1AAhR, CCKAR, and amylase occurred. The intensity of CCKAR expression increased in nonvacuolated acinar cells; a decrease in the size of CCK-positive epithelial cells occurred in duodenum. Our findings indicate that dioxin-induced acinar-cell lesions might be related to a direct effect of TCDD on the pancreas. Increase in CYP1ACCKAR expressions in vacuolated acinar cells may be involved in the pathogenesis of pancreatic lesions. Changes in the expression of CYP or CCKAR may have induced the acinar-cell tumors by initiating proliferation.

This report deals with the effect of feeding inhibitors of pancreatic and brush border enzymes on pancreatic growth and enzyme composition and secretion. Raw soybean flour containing trypsin inhibitors caused pronounced growth of the pancreas which was accompanied by increased enzyme content and increased CCK and gastrin concentration in the plasma. Feeding of an amylase inhibitor to a starch-rich diet induced a marked fall in amylase content and secretion without changing growth parameters of the pancreas, indicating that not starch but glucose is the trigger for the maintenance of amylase content and secretion of the pancreas. The addition of an alpha-glucosidase inhibitor (acarbose) to a sucrose- or maltose-rich semisynthetic diet did not cause significant alteration in pancreatic growth or enzyme composition or secretion. In man pancreatic function was also unaltered by 8 weeks' intake of 3 X 200 mg acarbose.

We previously demonstrated that, in rat pancreatic acinar cells, the high-affinity cholecystokinin (<em>CCK</em>) receptor agonist JMV-180 utilizes the phospholipase <em>A</em>2 (PL<em>A</em>2) cascade to mediate Ca2+ oscillations and amylase secretion. In contrast, the low-affinity <em>CCK</em> receptor utilizes the phospholipase C beta 1 (PLC beta 1) pathway. We have investigated structural requirements of <em>CCK</em> analogues to activate different intracellular pathways. <em>CCK</em> analogues such as <em>CCK</em>-8 [Met28,31; half-maximal effective concentration (EC50) = 0.4 pM], <em>CCK</em>-7 (Met28,31; EC50 = 0.7 pM), and NON<em>A</em> (Thr28/Nle31; EC50 = 5 pM) caused a biphasic amylase secretion. Reduction of secretion occurred with high doses of these peptides >> 100 pM). In contrast, <em>CCK</em>-5 (Met31; EC50 = 20,000pM), JMV-180 (Nle28,31; 1,500 pM), and OPE (Nle28,31; 200 pM) caused a monophasic secretion. <em>CCK</em>-8, but not JMV-180, increased protein kinase C (PKC) activities. The PKC activator phorbol ester inhibited an increase in myo-inositol 1,4,5-trisphosphate levels induced by <em>CCK</em>-8 and abolished monophasic amylase secretion induced by OPE. <em>CCK</em>-8, <em>CCK</em>-7, and NON<em>A</em> caused Ca2+ oscillations (< 100 pM) or large Ca2+ transients >> 100 pM). In contrast, JMV-180 and OPE evoked Ca2+ oscillations, even in high doses. Ca(2+)-signaling modes induced by <em>CCK</em>-5 were intermediate types between <em>CCK</em>-8 and JMV-180. <em>CCK</em>-8- and <em>CCK</em>-7-stimulated Ca2+ spikes were inhibited by the PLC inhibitor U-73122, but not by the PL<em>A</em>2 inhibitor ONO-RS-082. The action of <em>CCK</em>-5 was only partially sensitive to the PLC inhibitor. In contrast, JMV-180- and OPE-stimulated Ca2+ oscillations were inhibited by the PL<em>A</em>2, but not by the PLC, inhibitor. NON<em>A</em> was sensitive to PLC and PL<em>A</em>2 inhibitors. <em>A</em>lthough JMV-180 differs from <em>CCK</em>-8 by having an <em>A</em>sp-2 phenylethylester, rather than an <em>A</em>sp-phenylalanine amide, it is unlikely that these differences in the carboxyl terminus are important in determining which second-messenger systems will be activated. This is because <em>CCK</em>-5 (Phe33-CONH2) causes monophasic amylase secretion and Ca2+ oscillation in a manner similar to those induced by JMV-180 (2-phenylethylester). Meanwhile NON<em>A</em> (Phe33-CONH2) appeared to activate PLC and PL<em>A</em>2 pathways. The actions of all <em>CCK</em> analogues were abolished by L-364,718, indicating mediation by <em>CCK</em>-<em>A</em> receptors. Therefore, depending on the agonists used, <em>CCK</em>-<em>A</em> receptor activation in pancreatic acini may result in differential involvement of second-messenger systems, Ca2+ signal transduction, and amylase secretion. On the basis of the amino acid sequence of the carboxy terminus of <em>CCK</em> analogues, it appears that key amino acids for this differentiation are Met28 (or Thr28) for PLC pathways and Nle28 for PL<em>A</em>2 pathways.

Members of the asperlicin family of fungal metabolites produced by Aspergillus alliaceus are known potent CCK(A) antagonists. Herein, we report the identification of the gene cluster responsible for directing their biosynthesis. We validate and probe the pathway by genetic manipulation, and provide the first biochemical characterization of the oxidative cyclization en route to the heptacyclic asperlicin E by reconstituting the activity of the FAD depend monooxygenase AspB. This report provides the first genetic characterization of a NRPS assembly line that efficiently activates two anthranilate building blocks and illustrates the remarkably efficient biosynthesis of the complex heptacyclic asperlicin E.

Canine jejunal epithelial cells were isolated and maintained in short-term culture to study cholecystokinin (CCK) release. Sequential digestion of jejunal mucosa with collagenase and ethylenediaminetetraacetic acid was followed by counterflow elutriation to enrich CCK-containing cells. After 40 hours in culture on collagen-coated plates, 8.4% of the initially seeded cells were attached; 8.7% of them stained positive with a C-terminal CCK/gastrin antibody and 2.5% stained positive with a gastrin-specific antibody. Basal release of CCK into the culture medium amounted to 1.3% of total cell content over 105 minutes. Receptor-independent stimulation of protein kinase C by the phorbol ester beta-phorbol-12-myristate-13-acetate caused significant CCK release. The inactive form, 4 alpha-phorbol-12-myristate-13-acetate, had no effect. Activation of adenylate cyclase by 10(-5) mol/L forskolin evoked a 2.5-fold increase in CCK concentrations, which was completely abolished by 10(-8) mol/L somatostatin. L-phenylalanine stimulated CCK release at 20 and 50 mmol/L, whereas D-phenylalanine caused significant hormone output only at 50 mmol/L. L-tryptophan had no effect. Cholecystokinin release stimulated by L-phenylalanine was not influenced by the addition of either somatostatin or somatostatin antibody. In conclusion, a system of isolated canine jejunal epithelial cells was developed in short-term culture. This preparation proved suitable for the study of CCK release on a cellular basis.

The mRNA levels for growth-associated protein 43 (GAP-43), acidic fibroblast growth factor (aFGF), alpha- and beta-calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and choline acetyltransferase (ChAT) in rat lumbar spinal motoneurons were studied by in situ hybridization 1, 5 and 21 days and 20 weeks following unilateral peripheral nerve sectioning, ventral rhizotomy or dorsal rhizotomy. Furthermore, CGRP- and aFGF-like immunoreactivities in the ventral horn were studied using immunohistochemistry. One to 21 days after axotomy, GAP-43 and alpha-CGRP mRNAs increased in lesioned motoneurons, while the aFGF mRNA levels were marginally higher in motoneurons on the lesion side as compared to the control side. beta-CGRP, CCK and ChAT mRNA levels, on the other hand, decreased during the short-term response (1-21 days) to axotomy. After ventral rhizotomy, but not peripheral axotomy, there was complete disappearance of aFGF-like immunoreactivity in the ventral root proximal to the lesion. In animals subjected to long-term survival (20 weeks) after peripheral axotomy, the expression of all studied substances had returned to normal levels. Unilateral dorsal rhizotomy did not induce any substantial short- or long-term shifts in the cellular expression of the GAP-43, aFGF, CGRP and CCK peptides or their mRNAs in motoneurons of lesioned segments. These results indicate that peptides/proteins in motoneurons are expressed differentially after axotomy. Whereas alpha-CGRP and GAP-43 are up-regulated, CCK and beta-CGRP become down-regulated and aFGF is largely unaffected.

Some data suggest that cholecystokinin (CCK) receptor agonists stimulate the growth of colon cancer. Melatonin, an endogenous indoleamine with strong antioxidant properties, displays antiproliferative and proapoptotic properties both in vivo or in vitro in several types of tumors. We used HT-29 human colon cancer cells, expressing CCK receptors, to test the antiproliferative effects of several antagonists of CCK-A and/or CCK-B and their possible synergism with melatonin. HT-29 cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37 degrees C. Cell proliferation was assessed by the incorporation of [3H]-thymidine into DNA. Annexin V-FITC plus propidium iodine were used for flow cytometry apoptosis/necrosis evaluation. The following drugs were tested: gastrin (CCK-B agonist); CCK-8s (CCK-A agonist); proglumide (CCK-A plus CCK-B antagonist); lorglumide (CCK-A antagonist); PD 135,158 (CCK-B antagonist and weak CCK-A agonist); devazepide or L 364,718 (CCK-A antagonist); L 365,260 (CCK-B antagonist), and melatonin. The results shown a lack of effects of gastrin on HT-29 cell proliferation, whereas CCK-8s induced proliferation at high doses. The order of the antiproliferative effect of the other drugs was devazepide>> lorglumide>> proglumide. These drugs produce cell death mainly inducing apoptosis. Melatonin showed strong antiproliferative effect at millimolar concentrations, and it induced apoptotic cell death. Melatonin generally enhanced the antiproliferative effects of devazepide, lorglumide and proglumide and increased the proglumide-induced apoptosis. These results suggest that melatonin and CCK-A antagonists are useful for controlling human colon cancer cell growth in culture and in combined therapy significantly increases their efficiency.

Peripheral administration of the unsulphated cholecystokinin octapeptide (CCK-8us) led to an anxiogenic-like action in the elevated plus-maze model of anxiety in rats. Devazepide and L-365,260 showed potent anxiolytic-like effects at similar doses. The fact that devazepide is 1000 times more potent as a CCK-A receptor antagonist than L-365,260, whereas the two compounds are nearly equipotent at the CCK-B receptor subtype, suggests that CCK-B rather than CCK-A receptors are involved in these effects. Similar results were obtained in mice using the two-compartment test. In the elevated plus-maze, the benzodiazepine antagonist, flumazenil, which was inactive when given alone, significantly antagonized the anxiogenic-like activity of CCK-8us and the anxiolytic-like effects of devazepide and L-365,260. These results suggest a complex interaction between benzodiazepine and CCK receptor mechanisms in the regulation of anxiety states.