The effect of fasting and cholecystokinin stimulation on normal and neoplastic tissue in the rat pancreas.

Pancreatic nodules were produced in rats by either feeding raw soya flour alone or by injection of azaserine plus raw soya flour feeding. The resulting nodules were studied to determine whether there was any functional difference between this tissue and the relatively normal internodular pancreas. Tissue DNA and trypsin content were significantly elevated in nodules compared to the adjacent tissue. With fasting, protein and enzyme content increased significantly and equally in both nodular and internodular tissues. RNA levels fell significantly and the decrease was more pronounced in nodular tissue. The responsiveness of the multinodular pancreas to cholecystokinin was examined by measuring pancreatic secretion basally and in response to cholecystokinin. Both the volume and protein content secreted by the multinodular pancreas were greatly elevated above control levels. When corrected for pancreatic weight, the difference remained significant and appeared to be due to increased basal secretion by the nodular pancreas. These studies demonstrate that azaserine-raw soya flour induced nodules are functionally efficient. Furthermore, the secretory response to cholecystokinin of these nodules is equal to or higher than that of normal tissue.     

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.     

The effect of cholecystokinin octapeptide on pituitary-adrenal hormone secretion

The purpose of this investigation was to determine the influence of cholecystokinin octapeptide (CCK-OP) on pituitary-adrenal hormone secretion. CCK-OP at a dose of 5 μg/kg (i.p.) elevated plasma corticosterone from 27 to 43 μg/100 ml in one experiment and from 12 to 50 μg/100 ml in a second experiment: Lower doses of CCK-OP (0.5 μg/kg) elevated corticosterone from 12 μg/100 ml to 20 μg/100 ml. CCK-OP (1, 10, and 100 ng/ml) had no effect on ACTH-induced corticosterone released by isolated adrenal cells in vitro when tested in the presence of 50 pg of ACTH_1?24. 100 and 500 ng of CCK-OP resulted in an increased pituitary ACTH release equal to 123% (n.s.) and a 206% (P < 0.05) of control, respectively. In comparison, a $\text{3}\text{5}$ hypothalamic stalk median eminence equivalent increased ACTH release to 313% of control (P < 0.05). The exact mechanism of this CCK effect on pituitary ACTH release is unknown. Although it is likely that the direct effects on the pituitary in vitro represent a pharmacologic and not a physiologic effect of this peptide, in vivo doses are between doses used for pancreatic effects and satiety effects suggesting that there may be a physiologic stimulating action of this peptide on the hypothalamic-pituitary-adrenal axis but at a level above the adrenal and pituitary.     

Characterization of cholecystokinin receptor subunits on pancreatic plasma membranes

To determine the size and subunit structure of the pancreatic cholecystokinin (CCK) receptor, 125I-CCK33 was covalently cross-linked to its receptor on mouse pancreatic acinar plasma membranes utilizing the bifunctional cross-linker disuccinimidyl suberate. When CCK was cross-linked at pH 7.4 to either purified plasma membranes or to isolated pancreatic acini and then followed by preparation of plasma membranes, the major labeled protein band revealed by polyacrylamide gel electrophoresis was Mr = 120,000 in the absence of reducing agent and Mr = 80,000 in the presence of reducing agent. A similar banding pattern was also observed when different cross-linkers, ethylene glycol bis(succinimidyl succinate) or dithiobis (succinimidyl propionate), were employed. At pH 6.0, where CCK binding to its receptors is optimal, the labeling pattern was similar to that seen at 7.4, although the two bands were more heavily labeled. Both the binding of CCK to its receptors on plasma membranes and the appearance of the two cross-linked proteins on gels were inhibited in a parallel manner by increasing concentrations of unlabeled CCK8; similar results were observed with dibutyryl cyclic GMP, a competitive inhibitor of CCK binding and action. The data indicate, therefore, that the CCK receptor possesses subunit structure whereby an Mr = 76,000 binding subunit is linked to an Mr = 40,000 nonbinding subunit by a disulfide bond.     

Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester.

Nuclear factor kappa-B (NF-kappa B) has been shown to play an important role in LPS-mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP-dependent protein kinase in the regulation of NF-kappa B activity. In this study we have investigated the mechanism of NF-kappa B induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF-alpha NF-kappa B element was transfected into the RAW264 macrophage-like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8-bromo cAMP. To further study NF-kappa B induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF-kappa B binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA-binding activity confirmed these results and indicated that LPS induced a 55 KD DNA-binding protein. Induction of this NF-kappa B binding activity was not inhibited by pretreatment with the PKC inhibitor H-7. H-7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down-regulates PKC, had no effect on LPS induction of NF-kappa B activity in these cells. These results suggest that the induction of NF-kappa B in macrophages by LPS is independent of PKC.     

Localization of the murine cholecystokinin A and B receptor genes

We have determined the chromosomal locations of the two cholecystokinin (CCK) receptor genes in the mouse. Genetic localization utilized an interspecific backcross panel formed from the cross (C57BL/6J x Mus spretus) F₁ x Mus spretus. Genomic DNAs from 94 individuals in the backcross were analyzed by Southern hybridization with rat CCK_A and CCK_B receptor cDNA probes. Unique map positions were determined by haplotype analysis with 650 previously mapped loci in the mouse backcross. The CCK_A receptor gene (Cckar) mapped to mouse Chromosome (Chr) 5, in tight linkage with the DNA marker D5Bir8. The CCK_B receptor gene (Cckbr) mapped to mouse Chr 7, tightly linked to the -hemoglobin locus (Hbb). This localization places Cckbr in the same region as the mouse obesity mutation tubby (tub), which also maps near Hbb (2.4±1.4 cM). Since CCK can function as a satiety factor when administered to rodents, localization of Cckbr near the tub mutation identifies this receptor as a possible candidate gene for this obesity mutation.     

Molecular heterogeneity of canine cholecystokinin in portal and peripheral plasma

The release of molecular forms of cholecystokinin (CCK) into the portal and peripheral blood in response to an intraduodenal perfusion of sodium oleate (9 mmol X h-1) was studied in six conscious dogs with chronic portal vein catheters. Immunoreactive CCK as concentrated from 20 ml plasma by C18 SEP PAK cartridges and the pattern of molecular forms of CCK were studied by G50 gel filtration. CCK-like immunoreactivity (CCK-LI) was measured in the column eluates with antibody 5135, which measures gastrin and CCK equally and requires the intact carboxyl-terminus for full recognition. Gastrin was measured specifically with antibody 1611. Intraduodenal perfusion with oleate did not alter basal gastrin release. Release of CCK-LI by intraduodenal oleate was calculated by the increments of the integrated CCK-LI peaks over basal. Total CCK-like immunoreactivity (CCK-LI), calculated by integration of all CCK-LI peaks in gel filtration eluates, increased over basal by 12 fmol/ml in the portal and by 6 fmol/ml in the peripheral plasma after intraduodenal perfusion with sodium oleate. The main molecular forms eluted on gel filtration in positions of CCK33,39 and of CCK8. The pattern of CCK in the peripheral plasma was similar to that in the portal plasma except that in the peripheral plasma large molecular forms were more abundant than small forms. This finding was confirmed when CCK39 and CCK8 were infused either into the portal vein or into the peripheral vein and peripheral plasma CCK levels were measured. Elimination of CCK8 after portal vein infusion compared to peripheral vein infusion was about 3 times higher than that of CCK39. The abundance of large molecular forms of CCK in the circulating blood which are similar in potency to small forms, underlines their role in the physiology of CCK.     

The effect of reduced salinity on tissue and plasma composition of the dogfish,Squalus acanthias

• 1.1. Dogfish (Squalus acanthias) were acclimated to reduced salinities and their plasma, muscle tissue and erythrocytes subsequently analysed.
• 2.2. Decrease in the osmolarity of the plasma was principally due to a fall in urea concentration and a significant fall in the concentrations of sodium and chloride.
• 3.3. Changes in the muscle and erythrocytes in dilute media were a decrease in urea, potassium, sodium and chloride concentrations.
• 4.4. The concentrations of the free amino acids in the muscle and the red blood cells decreased more than would be expected by the movements of water only.
• 5.5. The results were discussed in relation to the regulation of cellular volume and the involvement of the free amino acid pool of the tissues in this process.

     

Synergistic effect on blastogenesis in murine spleen cells of lipopolysaccharide, lipid A, and acid-degraded polysaccharide from Fusobacterium nucleatum

Interchangeable combinations of Fusobacterium nucleatum Fev1 lipopolysaccharide (LPS) with its split products by acetic acid hydrolysis, i.e. lipid A (LA) and degraded polysaccharide (PS), amplified the blastogenic response in murine spleen cell cultures as measured by [3H]thymidine uptake. Athymic murine spleen cells precultured with LPS-Fev1 for 48 h (stage 1), washed twice and cultured together with fresh cells and either LA or PS for 72 h (stage 2) gave a synergistic response over that found in spleen cell cultures of thymic mice. Spleen cells pre-cultured with LA or PS and with fresh cells and LPS-Fev1 added to stage 2 cultures gave less significant amplification compared with precultures of LPS and either LA or PS together with fresh cells added to stage 2. Precultures with LA, PS or LPS-Fev1 and with pokeweed mitogen (PWM) and fresh cells added produced an additional increment of synergy which was most pronounced in spleen cell cultures of normal mice.     

The effect of cholecystokinin in controlling appetite and food intake in humans.

The present review of the satiating effect of cholecystokinin in humans has revealed that cholecystokinin is a physiological satiety factor in humans. The results demonstrate the efficacy of the satiating actions of exogenous and endogenous CCK in humans. The therapeutic potential of CCK analogues cannot be estimated until further studies are performed that demonstrate the efficacy of CCK analogues for decreasing body weight, and the safety of CCK when administered repetitively for prolonged periods.     

The effect of cyclodextrin on lipopolysaccharide production in cultures of Bordetella pertussis

The effect of adding 500 micrograms of (2,6-0-dimethyl) beta-cyclodextrin (Me-beta-CD) per ml of Stainer-Scholte (SS) medium in two-day shaker flask cultures of Bordetella pertussis on the production of lipopolysaccharide (LPS) was investigated. The amount of LPS per 10(9) cells found in the supernatants of these cultures was either somewhat reduced or unaffected by comparison with the amounts in cultures grown in SS-medium alone. In addition, the time course of LPS release from cultures of B. pertussis strain 3843 cells during a 96-h growth period in normal and Me-beta-CD-enriched SS medium is described. By using the enriched medium bacterial growth, the production of filamentous haemagglutinin (FHA) and of pertussis toxin (Pt) and the levels of haemagglutination and lymphocytosis-promoting activity were enhanced to various degrees. Measurements made on sedimented whole and on sonicated B. pertussis cells grown in the two media showed no differences in LPS content. The reasons for the reduced/unaffected LPS production are discussed. It has been suggested that an interaction between hydrophobic cavities of the Me-beta-CD molecules and the 'lipid A' part of LPS reduces the reactivity of LPS in the Limulus Amoebocyte Lysate (LAL) assay. This possibility, however, was rejected as the reactivity of Me-beta-CD-spiked purified B. pertussis strain 3803 LPS, compared with unspiked samples, remained unchanged.     

Visualization of the molecular dynamics of lipopolysaccharide on the plasma membrane of murine macrophages by total internal reflection fluorescence microscopy

The molecular action of Alexa 594-labeled lipopolysaccharide (LPS) from Escherichia coli was examined on living peritoneal macrophages of C57BL/6 mice by total internal reflection fluorescence microscope (TIRFM), and the molecular kinetics of LPS was analyzed. TIRFM visualization of the action of fluorescence-labeled LPS revealed an increase in the mean fluorescence intensity of LPS on the plasma membrane of wild type macrophages at 60 min after administration, indicating the oligomerization of LPS after binding to the macrophages. Additionally, a time-dependent sharp decrease in the mean diffusion coefficient of LPS was observed. On the other hand, both mean fluorescence intensity and diffusion coefficient of LPS in cases of TLR4(-/-), MD2(-/-), MyD88(-/-), and TRIF(-/-) macrophages were significantly different from the corresponding values of wild type macrophage, whereas differences were also noticed among these molecule-deficient macrophages. Furthermore, statistical analysis indicated the major role of receptors (TLR4 and MD2) and intracellular signaling molecules (MyD88 and TRIF) in oligomerization and lowering of the diffusion rate of LPS on the plasma membrane of murine macrophages, respectively.     

Preventive effect of cholecystokinin octapeptide on experimental amnesia in rats

The effect of intracerebroventricular (ICV) administration of cholecystokinin octapeptide (CCK-8) on electroconvulsive shock (ECS)-induced amnesia in passive avoidance response was studied in rats. In normal rats, CCK-8 in doses from 1 ng to 1 microgram had no effect on the response when injected before the training trials, immediately after foot shock or before the first retention test. However, proglumide, a CCK-8 receptor blocker, induced marked amnesia when injected in doses from 0.1 to 10 micrograms before the training trials and in doses of 1 and 10 micrograms before the first retention test, though not subsequent to foot shock. ECS given immediately after the foot shock caused amnesia in the 24 hr and 48 hr retention tests, which could have been prevented by CCK-8 injected in doses of 10 ng to 1 microgram prior to the training trials, of 10 ng to 1 microgram following ECS and of 0.1 and 1 microgram before the first retention test. In addition, the effects of CCK-8 and proglumide became pronounced following chronic ICV infusion, using an osmotic minipump, for 7 days at a dose of 1 ng/day and 10 ng/day, respectively. The amnesia induced by proglumide was not affected by arginine vasopressin (AVP), while AVP in doses of 10 ng and 100 ng given 30 min before the training trials prevented ECS-induced amnesia. The antiamnesic effect of AVP was abolished by simultaneous administration of proglumide. On the other hand, AVP-antiserum produced marked amnesia which could be antagonized by CCK-8. However, the antiamnesic effect of CCK-8 was not suppressed by AVP-antiserum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia modulates lipopolysaccharide induced TNF-alpha expression in murine macrophages

The pro-inflammatory activity of Tumor necrosis factor-alpha (TNF-alpha) together with tissue hypoxia determine the clinical outcome in sepsis and septic shock. p38 MAPKinase is the primary intracellular signaling pathway that regulates lipopolysaccharide (LPS)-induced TNF-alpha biosynthesis, however, the effect of hypoxia on LPS mediated activation of p38 is not known. Here we report that SB203580, a specific p38 MAPK inhibitor, which completely abolished LPS-induced TNF-alpha expression by the mouse macrophage cell RAW264.7 in normoxic conditions, lost the inhibitory effect in hypoxic conditions. Hypoxia did not modulate expression of p38 MAPK, but increased that of p-MK2, a downstream target of p38 MAPK. In LPS induced endotoxemia mice model SB203580 had no inhibitory effect on the serum levels of TNF-alpha. Furthermore, hypoxia inducible factor-1alpha (HIF-1alpha) was detected in vivo after LPS administration but its expression was not affected by SB203580. Our data indicate that LPS induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-alpha secretion. Furthermore, the induction of HIF-1alpha in mice with endotoxemia suggested a synergistic effect on p38 mediated TNF-alpha expression. These findings provide new insights on the pathophysiological effects of hypoxia in sepsis and septic shock.