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Mycobacterium tuberculosis, the primary agent of tuberculosis, must acquire iron from the host to cause infection. To do so, it releases high-affinity iron-binding siderophores called exochelins. Exochelins are thought to transfer iron to another type of high-affinity iron-binding molecule in the bacterial cell wall, mycobactins, for subsequent utilization by the bacterium. In this paper, we describe the purification of exochelins of M. tuberculosis and their characterization by mass spectrometry. Exochelins comprise a family of molecules whose most abundant species range in mass from 744 to 800 Da in the neutral Fe(3+)-loaded state. The molecules form two 14-Da-increment series, one saturated and the other unsaturated, with the increments reflecting different numbers of CH2 groups on a side chain. These series further subdivide into serine- or threonine-containing species. The virulent M. tuberculosis Erdman strain and the avirulent M. tuberculosis H37Ra strain produce a similar set of exochelins. Based on a comparison of their tandem mass spectra, exochelins share a common core structure with mycobactins. However, exochelins are smaller than mycobactins due to a shorter alkyl side chain, and the side chain of exochelins terminates in a methyl ester. These differences render exochelins more polar than the lipophilic mycobactins and hence soluble in the aqueous extracellular milieu of the bacterium in which they bind iron in the host.

Rat and human very low density lipoproteins (VLDL) were fractionated by zonal ultracentrifugation, yielding sharply defined fractions with narrow sedimentation limits. Sedimentation coefficients for the individual fractions were determined at two densities with the analytical ultracentrifuge, and the results were analyzed to yield buoyant densities and molecular weights for the particles in each fraction. For the rat lipoproteins, the weight concentrations of triglycerides, cholesterol, phospholipid, and protein were determined for each fraction, and their molar concentrations of apolipoprotein B were measured with a radioimmunoassay. For the human lipoproteins the corresponding values were taken from Patsch et al. (Patsch, W., J. R. Patsch, G. M. Kostner, S. Sailer, and H. Braunsteiner. 1978. Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. J. Biol. Chem. 253:4911-4915). From these data, a ratio of the number of apoB peptides to the number of lipoprotein particles was calculated for each fraction. This ratio was close to 1 for all VLDL fractions, ranging in particle diameter from about 40 to 80 mm and 30 to 50 mm, respectively, for rat and human VLDL. The majority rat VLDL contain B-48 rather than B-100 as their (single) apoB peptide. Based on these data, we proposed that only a single copy of B-48 is required for VLDL assembly in rat liver, unless nascent hepatic VLDL contain additional apoB peptides which are uniformly lost from the plasma VLDL particles when they are analyzed.

Various molecular forms of CCK reduce food intake in rats. Although CCK-8 is the most studied form, we reported that CCK-58 is the only detectable endocrine peptide form in rats. We investigated the dark-phase rat chow intake pattern following injection of CCK-8 and CCK-58. Ad libitum-fed male Sprague-Dawley rats were intraperitoneally injected with CCK-8, CCK-58 (0.6, 1.8, and 5.2 nmol/kg), or vehicle. Food intake pattern was assessed during the dark phase using an automated weighing system that allowed continuous undisturbed monitoring of physiological eating behavior. Both CCK-8 and CCK-58 dose dependently reduced 1-h, dark-phase food intake, with an equimolar dose of 1.8 nmol being similarly effective (-49% and -44%). CCK-58 increased the latency to the first meal, whereas CCK-8 did not. The intermeal interval was reduced after CCK-8 (1.8 nmol/kg, -41%) but not after CCK-58. At this dose, CCK-8 increased the satiety ratio by 80% and CCK-58 by 160%, respectively, compared with vehicle. When behavior was assessed manually, CCK-8 reduced locomotor activity (-31%), whereas grooming behavior was increased (+59%). CCK-58 affected neither grooming nor locomotor activity. In conclusion, reduction of food intake by CCK-8 and CCK-58 is achieved by differential modulation of food intake microstructure and behavior. These data highlight the importance of studying the molecular forms of peptides that exist in vivo in tissue and circulation of the animal being studied. [ABSTRACT FROM AUTHOR]

Background: Corticotropin-releasing hormone (CRH) is expressed in the brain, immune cells and the gut, where gene expression is upregulated by lipopolysaccharide (LPS) 6 h after injection. Whether these changes are reflected by increased circulating levels of CRH and adrenocorticotropic hormone (ACTH) is unknown. Methods: LPS (100 μg/kg) was injected intraperitoneally in conscious rats, and blood processed for CRH using the new RAPID (reduced temperatures, acidification, protease inhibition, isotopic exogenous controls and dilution) method compared with EDTA blood with or without plasma methanol extraction. Hormone levels were measured by commercial radioimmunoassay. Results: The RAPID method improved blood recovery of 125I-CRH in vitro compared to EDTA only added to the blood without or with methanol extraction (90.8 ± 2.0 vs. 66.9 ± 2.6 and 47.5 ± 2.0%, respectively; p < 0.001 vs. RAPID). Basal CRH levels from blood processed by the RAPID method were 28.9 ± 2.8 pg/ml, and by other methods below the radioimmunoassay detection limit (<10 pg/ml). At 6 h after LPS, CRH plasma levels increased significantly by 2.9 times, and in the proximal colon tended to decrease (-27.6 ± 5.7%; p > 0.05), while circulating levels were unchanged at 3 or 4 h. ACTH levels rose compared to control rats (135.3 ± 13.8 vs. 101.4 ± 6.0 pg/ml; p < 0.05) 30 min after the increase in CRH, while at 3 or 6 h after LPS, the levels were not changed. Conclusion: Intraperitoneal LPS induces a delayed rise in plasma CRH levels associated with an elevation in ACTH plasma levels 30 min later, suggesting that under conditions of immune challenge, CRH of peripheral origin may also contribute to pituitary activation, as detected using the RAPID method of blood processing, which improves CRH recovery. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The gastrointestinal hormone CCK exists in various molecular forms, with differences in bioactivity between the well-characterized CCK-8 and larger CCK-58 previously reported. We have compared the effects of these peptides on cytosolic calcium concentration ([Ca2+]c), mitochondrial metabolism, enzyme secretion, and cell fate in murine isolated pancreatic acinar cells using fluorescence confocal microscopy and patch-clamp electrophysiology. CCK-58 (1-10 pM) induced transient, oscillatory increases of [Ca2+]c, which showed apical to basolateral progression and were associated with a rise of mitochondrial NAD(P)H. CCK-58 (10 pM) induced zymogen exocytosis in isolated cells and amylase secretion from isolated cells and whole tissues. Hyperstimulation with supraphysiological CCK-58 (5 nM) induced a single large increase of [Ca2+]c that declined to a plateau, which remained above the basal level 20 mm after application and was dependent on external Ca2+ entry. In cells dispersed from the same tissues, CCK-8 induced similar patterns of responses to those of CCK-58, with oscillatory increases of [Ca2+]c at lower (pM) concentrations and sustained responses at 5 nM. CCK-58 and CCK-8 exhibited similar profiles of action on cell death, with increases in necrosis at high CCK-58 and CCK-8 (10 nM) that were not significantly different between peptides. The present experiments indicate that CCK-8 and CCK-58 have essentially identical actions on the acinar cell at high and low agonist concentrations, suggesting an action via the same receptor and that the differences observed in an intact rat model may result from indirect effects of the peptides. Our data strengthen the argument that CCK-58 is an important physiological form of this gastrointestinal hormone. [ABSTRACT FROM AUTHOR]

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK[sub1] receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides. [ABSTRACT FROM AUTHOR]

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-δ and PKC-ε reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-δ and -ε, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-δ and -ε isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-ε redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-ε overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-δ and -ε isoforms translocate to specific acinar cell compartments and modulate zymogen activation. [ABSTRACT FROM AUTHOR]

Introduction: Several cholecystokinin (CCK) forms have been detected in plasma, but most studies on food intake investigated the effects of CCK-8 only. Recently, it has been demonstrated that CCK-58 is the only endocrine-active form of CCK in rats. Methods: CCK-58 was synthesized with a peptide synthesizer using FMOC chemistry and CCK-58 effects on food intake were compared to CCK-8 in rats. Results: Both CCK-58 and CCK-8 inhibited food intake in a dose-dependent manner and were equally potent at 30 min. CCK-58 showed a prolonged inhibition of food intake compared to CCK8 at the higher dose tested (7 nmol/kg), inhibiting food intake also at 60 min, and cumulative food intake was inhibited for up to 210 min by CCK-58. Conclusions: CCK-58 has the same potency in inhibiting food intake as CCK-8 in rats, but inhibits food intake longer. This might be due to its tertiary structure resulting in a delayed plasma degradation or a prolonged binding at the CCK receptor. As CCK-58 is the major CCK form in the gut wall and possibly in the circulating blood in humans, the effects of CCK on food intake might have been underestimated in the past. [ABSTRACT FROM AUTHOR]

In contrast to supramaximal CCK-8 or caerulein, acute or prolonged supraphysiological levels of endogenous CCK-58 do not cause pancreatitis. Compared with CCK-8, CCK-58 is a much stronger stimulant of pancreatic chloride and water secretion, equivalent to maximally effective secretin, but with a chloride-to-bicarbonate ratio characteristic of acinar fluid. Because supraphysiological endogenous CCK does not cause pancreatitis and because coadministration of secretin ameliorated caerulein- or CCK-8-induced pancreatitis, coincident with restoring pancreatic water secretion, we hypothesized that supramaximal CCK-58 would not induce pancreatitis. Conscious rats were infused intravenously with 2 or 4 nmol·kg-1·h-1 of CCK-8 or synthetic rat CCK-58 for 6 h, and pancreases were examined for morphological and biochemical indexes of acute pancreatitis. A second group was treated as above while monitoring pancreatic protein and water secretion. CCK-8 at 2 nmol·kg-1·h-1 caused severe edematous pancreatitis as evidenced by morphological and biochemical criteria. CCK-58 at this dose had minimal or no effect on these indexes. CCK-58 at 4 nmol·kg-1·h-1 increased some indexes of pancreatic damage but less than either the 2 or 4 nmol·kg-1·h-1 dose of CCK-8. Pancreatic water and protein secretion were nearly or completely abolished within 3 h of onset of CCK-8 infusion, whereas water and protein secretion were maintained near basal levels in CCK-58-treated rats. We hypothesize that supramaximal CCK-58 does not induce pancreatitis because it maintains pancreatic acinar chloride and water secretion, which are essential for exocytosis of activated zymogens. We conclude that CCK-58 may be a valuable tool for investigating events that trigger pancreatitis. [ABSTRACT FROM AUTHOR]

We hypothesized that endogenous CCK reduces food intake by activating the dorsal vagal complex (DVC) and the myenteric neurons of the gut. To test this hypothesis, adult rats were given camostat mesilate; a nonnutrient releaser of endogenous CCK, by orogastric gavage, and Fos-like immunoreactivity (Fos-LI) was quantified in the DVC and the myenteric plexus. The results for endogenous CCK were compared with those for exogenous CCK-8. Exogenous CCK-8 reduced food intake and stimulated Fos-LI in the DVC and in myenteric neurons of the duodenum and jejunum. In comparison, endogenous CCK reduced food intake and increased DVC Fos-LI but did not increase Fos-LI in the myenteric plexus. Similar to CCK-8, devazepide, a specific CCK1 receptor antagonist, and not L365,260, a specific CCK2 receptor antagonist, attenuated the reduction of food intake by camostat. In addition, Fos-LI in the DVC in response to both exogenous CCK-8 and carnostat administration was significantly attenuated by vagotomy, as well as by blocking CCK1 receptors. These results demonstrate for the first time that reduction of food intake in adult rats by endogenous CCK released by a nonnutrient mechanism requires CCK1 receptors, the vagus nerve, and activation of the DVC, but not the myenteric plexus. [ABSTRACT FROM AUTHOR]

We previously demonstrated the expression of bitter taste receptors of the type 2 family (T2R) and the α-subunits of the G protein gustducin (Gαgust) in the rodent gastrointestinal (GI) tract and in G1 endocrine cells. In this study, we characterized mechanisms of Ca2+ fluxes induced by two distinct T2R ligands: denatonium benzoate (DB) and phenylthiocarbamide (PTC), in mouse enteroendocrine cell line STC-1. Both DB and PTC induced a marked increase in intracellular [Ca2+] ([Ca2+]i) in a dose- and time-dependent manner. Chelating extracellular Ca2+ with EGTA blocked the increase in [Ca2+]i induced by either DB or PTC but, in contrast, did not prevent the effect induced by bombesin. Thapsigargin blocked the transient increase in [Ca2+]i induced by bombesin, but did not attenuate the [Ca2+]i increase elicited by DB or PTC. These results indicate that Ca2+ influx mediates the increase in [Ca2+]i induced by DB and PTC in STC-l cells. Preincubation with the L-type voltage-sensitive Ca2+ channel (L-type VSCC) blockers nitrendipine or diltiazem for 30 mm inhibited the increase in [Ca2+]i elicited by DB or PTC. Furthermore, exposure to the L-type VSCCs opener BAY K 8644 potentiated the increase in [Ca2+]i induced by DB and PTC. Stimulation with DB also induced a marked increase in the release of cholecystokinin from STC-1 cells, an effect also abrogated by prior exposure to EGTA or L-type VSCC blockers. Collectively, our results demonstrate that bitter tastants increase [Ca2+]i and cholecystokinin release through Ca2+ influx mediated by the opening of L-type VSCCs in enteroendocrine STC-1 cells. [ABSTRACT FROM AUTHOR]

Although ethanol abuse is the most common cause of pancreatitis, the mechanism of alcohol's effect on the pancreas is not well understood. Previously, we demonstrated that in vitro ethanol treatment of pancreatic acinar cells augmented the CCK-8-induced activation of NF-κB, a key signaling system involved in the inflammatory response of pancreatitis. In the present study, we determine the role for individual PKC isoforms in the sensitizing effect of ethanol on NF-κB activation. Dispersed rat pancreatic acini were treated with and without ethanol and then stimulated with CCK-8; 100 nM CCK-8 caused both NF-κB and PKC-δ, -∊, and -ζ activation, whereas 0.1 nM CCK-8 did not increase PKC-∊, PKC-ζ, or NF-κB activity. CCK-8 (0.1 nM) did activate PKC-δ. PKC-∊ activator alone did not cause NF-κB activation; however, together with 0.1 nM CCK-8, it caused NF-κB activation. Ethanol activated PKC-∊ without affecting other PKC isoforms or NF-κB activity. Of note, stimulation of acini with ethanol and 0.1 nM CCK-8 resulted in the activation of PKC-δ, PKC-∊, and NF-κB. The NF-κB activation to 0.1 nM CCK-8 in ethanol-pretreated acini was inhibited by both PKC-δ inhibitor and PKC-∊ inhibitor. Taken together, these results demonstrate the different modes of activation of PKC isoforms and NF-κB in acini stimulated with ethanol, high-dose CCK-8, and low-dose CCK-8, and furthermore suggest that activation of both PKC-∊ and -δ is required for NF-κB activation. These results suggest that ethanol enhances the CCK-8-induced NF-κB activation at least in part through its effects on PKC-∊. [ABSTRACT FROM AUTHOR]

The gut hormone peptide YY(3–36) [PYY(3–36)] decreases food intake when administered by intravenous infusion to lean and obese humans and rats. Whether chronic administration of PYY(3–36) produces a sustained reduction in food intake and adiposity is the subject of intense debate. Batterham et al. (R. L. Batterham, M. A. Cowley, C. J. Small, H. Herzog, M. A. Cohen, C. L. Dakin, A. M. Wren, A. E. Brynes, M. J. Low, M. A. Ghatei, R. D. Cone, and S. R. Bloom. Nature 418: 650–654, 2002) first reported that PYY(3–36) reduces food intake and weight gain in rats when injected into the peritoneal cavity twice daily for 7 days. Numerous laboratories have failed to confirm that daily injections of PYY(3–36) decrease body weight. Continuous subcutaneous administration of PYY(3–36) by osmotic minipump has been reported to reduce daily food intake in rodents but only during the first 3–4 days of administration. Here we show the effects of different daily patterns of intravenous infusion of PYY(3–36) on food intake, body weight, and adiposity in rats tethered via infusion swivels to computer-controlled pumps. Measurement of food bowl weight recorded by computer every 20 s permitted daily assessment of the instantaneous effects of PYY(3–36) administration on food intake and meal patterns. One-hour intravenous infusions of PYY(3–36) at 30 pmol·kg-1·min-1 every other hour for 10 days produced a sustained reduction in daily food intake of ∼20% and decreased body weight and adiposity by 7 and 35%, respectively. Thus dosage pattern is critical for producing a sustained effect of PYY(3–36) on food intake and adiposity. [ABSTRACT FROM AUTHOR]

Investigates the role for the enteric nervous system in the regulation of satiety via cholecystokinin-8 (CCK). CCK receptor or receptors involved in the activation; Role of the vagus nerve or small unmyelinated sensory neurons that participate in CCK- and nutrient-induced enteric activation; Subpopulation of enteric neurons activated by CCK and nutrients.

Pancreatic secretion of protein, water, chloride, and bicarbonate under basal conditions and in response to intravenous and intraduodenal stimuli were studied in awake rats fully recovered from surgery. During the basal phase of pancreatic secretion, protein output and water output were weakly correlated or uncorrelated, consistent with separate regulation and distinct cellular origin of enzyme (acinar cells) and water (duct cells), referred to as the two-component paradigm of pancreatic secretion. When pancreatic secretion was stimulated physiologically, water and protein output abruptly became strongly and significantly correlated, suggesting that protein secretion and water secretion are tightly coupled or that protein secretion is dependent on water secretion. The apparent function of this coupling is to resist or prevent increases in protein concentration as protein output increases. This pattern of secretion was reproduced by intravenous infusion of the CCK-58 form of cholecystokinin, which strongly stimulates pancreatic water and chloride secretion, but not by CCK-8, which only weakly stimulates water and chloride secretion in a non-dose-dependent manner. The remarkably tight association of water and protein secretion in food-stimulated and CCK-58-stimulated pancreatic secretion is consistent with a single cell type as the origin of both water and enzyme secretion, i.e., the acinar cell, and is not consistent with the two-component paradigm of pancreatic secretion. Because CCK-58 is the only detectable endocrine form of cholecystokinin in the rat and its bioactivity pattern is markedly and qualitatively different from CCK-8, actions previously recorded for CCK-8 should be reexamined. [ABSTRACT FROM AUTHOR]

Exogenously administered galanin inhibits cholinergic transmission to the longitudinal muscle and reduces peristaltic efficiency in the guinea pig ileum with a mechanism partially mediated by galanin receptor 1 (GAL-R1). We investigated the effect of exogenous galanin 1–16, which has high affinity for GAL-R1, on the ascending excitatory reflex of the circular muscle elicited by radial distension in isolated segments of guinea pig ileum. We used a three-compartment bath that allows dissecting the ascending pathway into the oral (site of excitatory motor neurons), intermediate (site of ascending interneurons) and caudal compartment (site of intrinsic primary afferent neurons). Galanin 1–16 (0.3–3 μmol L−1) applied to the oral compartment inhibited in a concentration-dependent manner the ascending excitatory reflex elicited by the wall distension in the caudal compartment. This effect was antagonized by the GAL-R1 antagonist, RWJ-57408 (1 and 10 μmol L−1). By contrast, galanin 1–16 was ineffective when added to the intermediate or caudal compartment up to 3 μmol L−1. GAL-R1 immunoreactive neurons did not contain neuron-specific nuclear protein, a marker for intrinsic primary afferent neurons. These findings indicate that GAL-R1s are present on motor neurons responsible for the ascending excitatory reflex, but not on ascending interneurons and intrinsic primary afferent neurons. [ABSTRACT FROM AUTHOR]

Protein kinase D (PKD) is a serine/threonine protein kinase activated by G protein-coupled receptor (GPCR) agonists through an incompletely characterized mechanism that includes its reversible plasma membrane translocation and activation loop phosphorylation via a protein kinase C (PKC)-dependent pathway. To gain a better understanding of the mechanism regulating the activation of PKD in response to GPCR stimulation, we investigated the role of its rapid plasma membrane translocation on its activation loop phosphorylation and identified the endogenous PKC isozyme that mediates that event in vivo. We had found that the activation loop of a PKD mutant, with reduced affinity for diacylglycerol and phorbol esters, was only phosphorylated upon its plasma membrane association. We also found that the activation loop phosphorylation and rapid plasma membrane dissociation of PKD were inhibited either by preventing the plasma membrane translocation of PKC∈, through abolition of its interaction with receptor for activated C kinase, or by suppressing the expression of PKC∈ via specific small interfering RNAs. Thus, this study demonstrates that the plasma membrane translocation of PKD, in response to GPCR stimulation, is necessary for the PKC∈-mediated phosphorylation of the activation loop of PKD and that this event requires the translocation of both kinases to the plasma membrane. Based on these and previous results, we propose a model of GPCR-mediated PKD regulation that integrates its changes in distribution, catalytic activity, and multisite phosphorylation. [ABSTRACT FROM AUTHOR]

Nonsulfated CCK58 [CCK58(ns)] has not been considered to be of biological importance because CCK58(ns) binds poorly to the CCKA 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 CCK58(ns). CCK58(ns) is 14% as immunoreactive as sulfated CCK8 [CCK8(s)]. Amino acid analysis demonstrated that CCK58(ns) was present at 50% the amount of CCK58(s). In addition, we found that CCK58(ns) does not potently displace an 125I-labeled CCK10 analog from the CCKA 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, CCK58(ns) does bind to CCKB receptors and stimulates gastric acid secretion via this receptor. The presence of CCK58(ns) and its ability to selectively stimulate the CCKB receptor without stimulation of the CCKA receptor suggest that CCK58(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent. [ABSTRACT FROM AUTHOR]

Galanin actions are mediated by distinct galanin receptors (GAL-R), GAL-R1, -R2 and -R3. We investigated the role of GAL-R1 in gastric motility and the expression of GAL-R1 in the rat stomach. In vivo, in urethane-anaesthetized rats, galanin (equipotent for all GAL-Rs) induced a short inhibition of gastric motility, followed by increase in tonic and phasic gastric motility; the latter was significantly reduced by the GAL-R1 antagonist, RWJ-57408. Galanin 1–16 (high affinity for GAL-R1 and -R2) induced a long-lasting decrease of intragastric pressure, which was not modified by RWJ-57408. In vitro, galanin and galanin 1–16 induced increase of intragastric pressure that was not affected by RWJ-57408. Tetrodotoxin (TTX) did not suppress the galanin excitatory effect, whereas the effect of galanin 1–16 on gastric contraction was increased by TTX- or N-nitro-L-arginine, an inhibitor of nitric oxide synthase. GAL–R1 immunoreactivity was localized to cholinergic and tachykinergic neurons and to neurons immunoreactive for nitric oxide synthase or vasoactive intestinal polypeptide. This study suggests that an extrinsic GAL-R1 pathway mediates the galanin excitatory action, whereas an extrinsic, non GAL-R1 pathway is likely to mediate the galanin inhibitory effect in vivo. GAL-R1 intrinsic neurons do not appear to play a major role in the control of gastric motility. [ABSTRACT FROM AUTHOR]

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCKA and CCKB receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCKA and CCKB receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity. [ABSTRACT FROM AUTHOR]

CCK-58 differs from CCK-8 in patterns of expression of pancreatic secretion of fluid and amylase and gallbladder contraction. These differences have physiological relevance only if CCK-58 release is stimulated by nutrients entering the intestine and if CCK-58 circulates in sizeable amounts. In this study, we report that when radio-labeled CCK-58 is added to rat blood and plasma is formed, there is extensive loss and degradation of the radioactive peptide. Therefore, a new method was developed to minimize loss and degradation of this label. This method recovered >85% of the label with no detectable degradation. Furthermore, the optimized method recovered all unlabeled exogenous cholecystokinin molecular forms in >80% yields. Blood from fasted rats and rats in which cholecystokinin release was stimulated by the trypsin inhibitor camostat contained only CCK-58 (3.5 ± 0.5 and 17 ± 1.5 fmol/ml, respectively). Because CCK-58 predominates in the blood, this molecular form should be used in studies on the physiology and pathophysiology of cholecystokinin. [ABSTRACT FROM AUTHOR]

The carboxyl terminal octapeptide of cholecystokinin (CCK-8) has been hypothesized to account for the bioactivity of all the molecular forms of cholecystokinin. However, the physiological relevance of CCK-58 has not been rigorously examined because of the lack of sufficient amounts of the peptide and concerns about inactivation of natural peptides during their purification. Therefore, canine-sulfated CCK-58 was synthesized and conditions determined for its unblocking and purification that preserved the sulfated tyrosine. Synthetic CCK-58 was indistinguishable from natural CCK-58 by amino acid analysis and by mass spectrometry. Synthetic CCK-58 and CCK-8 have different patterns of pancreatic stimulation: both caused a dose-related increase in amylase release, while only CCK-58 stimulated bile-pancreatic output volume. Thus, CCK-58 and CCK-8 are biased agonists at the CCK-A receptor (they have distinct patterns of action mediated by the same receptor). Previous work has demonstrated that the identical carboxyl termini of CCK-8 and CCK-58 have different solution conformations. Taken together, the physiological and structural results support the hypothesis that different carboxyl terminal conformations of CCK-58 and CCK-8 alter the expression of their biological activity. [Copyright &y& Elsevier]

Deals with a study which determined at the cellular level the critical secretory and electrophysiological events that are important for understanding how cholecystokinin (CCK) secretion is regulated by luminal CCK-releasing factor (LCRF). Direct effect of LCRF on human intestinal cells to stimulate CCK secretion; Effect of calcium-channel blockade on CCK release; Discussion of the results.

Presents evidence that the structure of cholecystokinin-58 (CCK-58) influences its biological activity. Purification of CCK-58; Importance of the amino terminus of CCK-58 to biological activity; Structure as shielding the bioactive carbonyl terminus.

Tests amino-terminal fragment LCRF-(1-35) for biological activity in vivo in rats and compares it with other putative cholecystokinin-releasing peptides, monitor peptide and gastrin-releasing peptide (GRP). Pancreatic secretion in response to intraduodenal administration of LCRF-(1-35); Effects of GRP and monitor peptide on pancreatic secretion.