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PubMed1993

Distinct receptors for cholecystokinin and gastrin on canine fundic D-cells.

Despite the extensive amino acid homology between gastrin and cholecystokinin (CCK) at the biologically active carboxyl terminus, the receptors through which these peptides exert their action are heterogeneous. In previous studies, we have examined the biological activity of gastrin/CCK peptides on isolated canine fundic D-cells and observed that CCK is a more potent and efficacious stimulant of somatostatin release than gastrin. We performed the present studies to distinguish between distinct CCK (CCK-A subtype) and gastrin (CCK-B/gastrin subtype) receptors on canine D-cells. Consistent with this observation was our finding that the CCK-A receptor selective antagonist L-364,718 dose dependently (10(-11)-10(-7) M) inhibited CCK-mediated somatostatin release but at the same doses did not alter the effect of gastrin. CCK and gastrin exhibited similar potency in displacing bound 125I-labeled Leu15 gastrin-17 from D-cells. However, when 125I-CCK octapeptide (CCK-8) was used as the radioligand, a fraction of the bound label could not be displaced with gastrin, but this fraction was completely displaced with CCK-8. In D-cells pretreated with high concentrations of gastrin, L-364,718 was able to inhibit the gastrin-resistant fraction of 125I-CCK-8 binding, but the CCK-B/gastrin receptor selective antagonist (PD 134308) was unable to influence this fraction of binding in doses as high as 10(-6) M. These studies delineate the presence of distinct CCK-A and CCK-B/gastrin receptors on canine fundic D-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Pancreatic acini possess endothelin receptors whose internalization is regulated by PLC-activating agents.

Endothelin-1 (ET-1) and ET-3 mRNA have been found in the pancreas. We investigated the ability of ET-1, ET-2, and ET-3 to interact with and alter dispersed rat pancreatic acinar cell function. Radiolabeled ETs bound in a time- and temperature-dependent fashion, which was specific and saturable. Analysis demonstrated two classes of receptors, one class (ETA receptor) had a high affinity for ET-1 but a low affinity for ET-3, and the other class (ETB receptor) had equally high affinities for ET-1 and ET-3. No specific receptor for ET-2 was identified. Pancreatic secretagogues that activate phospholipase C (PLC) inhibited binding of 125I-labeled ET-1 (125I-ET-1) or 125I-ET-3, whereas agents that act through adenosine 3',5'-cyclic monophosphate (cAMP) did not. A23187 had no effect on 125I-ET-1 or 125I-ET-3 binding, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate reduced binding. The effect of cholecystokinin octapeptide (CCK-8) was mediated through its own receptor. Stripping of surface bound ligand studies demonstrated that both 125I-labeled ET-1 and 125I-labeled ET-3 were rapidly internalized. CCK-8 decreased the internalization but did not change the amount of surface bound ligand. Endothelins neither stimulate nor alter changes in enzyme secretion, intracellular calcium, cAMP, or [3H]inositol trisphosphate (IP3). This study demonstrates the presence of ETA and ETB receptors on rat pancreatic acini; occupation of both receptors resulted in rapid internalization, which is regulated by PLC-activating secretagogues. Occupation of either ET receptor did not alter intracellular calcium, cAMP, IP3, or stimulate amylase release.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Central and peripheral effects of CCK receptor antagonists on satiety in dogs.

The role of cholecystokinin (CCK) as a central and peripheral satiety factor was studied using the CCK-B (L-365,260) and CCK-A (MK-329) receptor antagonists in esophageal-fistula dogs. Suppression of feeding was induced by either balloon gastric distension or small bowel nutrient infusion and was measured as the volume sham fed. Intracerebroventricular L-365,260 abolished satiety behavior from gastric distension (volume sham fed 2,667 +/- 211 ml vs. vehicle alone 1,217 +/- 446 ml, P < 0.05) but not small bowel infusion. Intravenous MK-329 abolished satiety behavior from small bowel infusion (volume sham fed 1,900 +/- 521 ml vs. vehicle alone 210 +/- 198 ml, P < 0.05) but not from gastric distension. The volume sham fed after intracerebroventricular MK-329 with balloon gastric distension or small bowel infusion did not differ from control. These results suggest that, in the brain, CCK is a physiological mediator of satiety behavior from gastric distension but not small bowel nutrients and, in the periphery, CCK is a physiological mediator of satiety behavior from small bowel nutrients but not gastric distension.

Animals↗
PubMed1993

Partial agonism by gastrin for a cholecystokinin receptor mediating pepsinogen secretion.

Isolated gastric glands from rabbit were used to characterize the functional cholecystokinin (CCK)-like peptide receptors that mediate pepsinogen secretion. Pepsinogen secretion was stimulated by both CCK octapeptide sulfate (CCK-8) and A-71378, a selective CCK-A-type receptor agonist, with similar mean effective doses (1.0 and 0.8 nM, respectively). Compared with CCK-8, gastrin-17 (G-17-I) showed reduced potency and only partial efficacy for stimulation of pepsinogen secretion while inhibiting the maximal CCK-8-stimulated response. The nonpeptide inhibitors, asperlicin and L-364,718, inhibited pepsinogen secretion with identical pA2 values for antagonism of both CCK and gastrin, indicating that both peptides interact with the same functional receptor. Specific binding of [3H]CCK-8 to isolated chief cell membranes was displaced fully by both CCK and gastrin, indicating full receptor occupancy by both peptides. A novel synthetic peptide analogue, pseudogastrin [(Glu)5-Ala-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH2], was used to investigate the structural basis for the lower potency and efficacy of G-17-I. The potency of CCK and gastrin analogues for pepsinogen secretion was found to be dependent on both sulfation of a tyrosine residue and the position of the tyrosine residue relative to the COOH-terminal phenylalanine amide. The efficacy appears to be determined partially by the extended NH2-terminal sequence of G-17-I. The results of the present study are interpreted to show that pepsinogen secretion is mediated by a CCK-A-type receptor and gastrin acts at the same receptor as a partial agonist.

Amino Acid Sequence↗
PubMed1994

Nucleoside diphosphate kinase associated with rat pancreatic membranes regulates CCK receptor affinity.

We have previously demonstrated in permeabilized rat pancreatic acini that the existence of two affinity states of the pancreatic cholecystokinin (CCK) receptor seen in intact cells depends on the presence of ATP. In the present study, we demonstrate that this effect of ATP is mediated by the enzyme nucleoside diphosphate kinase (NDPK). Northern blot hybridization analysis demonstrated NDPK mRNA in pancreas. Furthermore, pancreatic membranes possessed NDPK activity, which transferred high-energy phosphate groups to [8-3H]GDP. This enzyme also utilized UTP and ITP as a source of gamma-phosphate for GTP formation while guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was formed in the presence of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). However, adenylyl (beta, gamma-methylene)-diphosphate (AMP-PCP) did not serve as a substrate for NDPK. Analysis of 125I-Bolton-Hunter-labeled CCK octapeptide ([125I]BH-CCK-8) binding data in the absence of nucleotides was consistent with a single affinity state with dissociation constant (Kd) equal to 80 pM and maximal binding equal to 50.8 fmol/mg. ATP, UTP, ITP, ATP gamma S, and GTP gamma S all induced two CCK binding affinity states, which in the presence of 1 mM ATP were Kd = 74 pM for high-affinity sites and Kd = 4.3 nM for low-affinity sites: AMP-PCP did not induce two affinity states. GDP at 10 microM had no effect on CCK binding but potentiated the effect of ATP. GTP gamma S, in addition to inducing high- and low-affinity states, also elicited a significant concentration-dependent reduction in the total number of measurable CCK receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Induction of postprandial intestinal motility and release of cholecystokinin by polyamines in rats.

Polyamines are known to play a major role in postprandial adaptation of the digestive tract. Experiments were designed to determine whether ingested polyamines induce change in intestinal motility associated with a cholecystokinin (CCK) release and whether endogenous polyamines are involved in the intestinal and colonic motor response to a meal. Intestinal and colonic motility was assessed in rats equipped with intestinal electrodes, and plasma CCK was determined using a bioassay. Orogastric administration of putrescine, spermidine, or spermine (20 mumol) disrupted intestinal migrating myoelectric complexes (MMCs) and increased the frequency of colonic spike bursts. After a 6-day treatment with the ornithine decarboxylase inhibitor alpha-difluoromethylornithine, the duration of postprandial disruption of MMCs, but not the stimulation of colonic motility, induced by a 3-g meal was significantly reduced. The duration of MMC disruption and the increase in colonic spike burst frequency after spermidine administration (20 mumol) were significantly reduced by CCK-A and CCK-B antagonists. Eight minutes after saline administration plasma CCK concentration was 0.9 +/- 0.4 pM; it rose to 4.7 +/- 2.8 pM, 8 min after spermidine (20 mumol). These results indicate that exogenous polyamines disrupt intestinal MMCs and stimulate colonic motility through a release of CCK acting at CCK-A and CCK-B receptors and suggest that endogenous polyamines are involved in the postprandial control of intestinal motility.

Animals↗
PubMed1995

Stability of circadian and minor cycles of exocrine pancreatic secretion in atropine- and MK-329-infused rats.

We previously demonstrated the existence of a circadian rhythm of pancreatic secretion of fluid and digestive enzymes, which was superimposed on by a regular 1.84-h minor cycle of exocrine pancreatic secretion of fluid and total protein, amylase, and chymotrypsinogen (25). Direct control of these daily and hourly rhythms of pancreatic secretory function has not been addressed. Cholinergic and cholecystokinin (CCK)-associated influences on these two rhythms of exocrine pancreatic secretion were investigated in rats provided with pancreatic, biliary, duodenal, and jugular vein cannulas, allowing separate drainage of bile and pure pancreatic juice, as well as intravenous infusions of atropine sulfate and/or MK-329. Rats were kept in restraint cages under controlled temperature and humidity, with a regular 12-h light cycle, and divided into five groups. The first group of fed rats was constantly infused with 200 micrograms.kg-1.h-1 atropine, the second with 0.5 mg.kg-1.h-1 MK-329, and the third with both. In the group where both drugs were simultaneously infused, 500 micrograms.kg-1.h-1 atropine was intraperitoneally administered, whereas MK-329 was infused by intravenous cannula. Two groups consisted of fasted rats, of which one was also given atropine (100 micrograms.kg-1.h-1). Three-day experiments were performed separately with fed rats, and 2-day experiments were performed with fasted rats; atropine and/or MK-329 infusion, starting on day 2, was constant over 48 h in both fed and fasted rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Identification of CCK-A receptors on chief cells with use of a novel, highly selective ligand.

Functional studies suggest that guinea pig chief cells have both cholecystokinin-A (CCK-A) and CCK-B receptors (CCK-A-R and CCK-B-R, respectively). However, all efforts to directly characterize the specific CCK-A-R using binding have been unsuccessful. Recent studies describe specific CCK-A-R agonists such as A-71378 ([desamino-Nle28,31-(N-methyl)Asp32]CCK heptapeptide]. In the present study, [D-Tyr-Gly]A-71378 was synthesized, which has > 300-fold selectivity for CCK-A-R and can be iodinated. [D-Tyr-Gly]A-71378 was equipotent to A-71378 in stimulating pepsinogen release from purified guinea pig chief cells. Binding of 125I-labeled [D-Tyr-Gly]A-71378 was saturable and specific. Potencies for inhibiting binding were as follows: [D-Tyr-Gly]A-71378 = A-71378 = 4x CCK octapeptide (CCK-8) > 1,000x des(SO4)-CCK-8, gastrin. In contrast, for 125I-gastrin binding they were CCK-8 > gastrin-17-I > des(SO4)-CCK-8 >> A-71378 or [D-Tyr-Gly]A-71378. Binding of [D-Tyr-Gly]A-71378 was best fitted by a two-site model. In contrast, 125I-gastrin binding was fitted with a single-site model. For inhibiting binding of 125I-[D-Tyr-Gly]A-71378, the CCK antagonists had relative affinities of L-364,718 >> L-365,260, and the reverse was true with 125I-gastrin. Correlation of binding with changes in biological activity suggested low-affinity CCK-A-R were mediating these changes. These results demonstrate directly for the first time that guinea pig chief cells possess CCK-A-R and CCK-B-R. The pharmacology of these CCK-A-R resembles those on other tissues. This novel, highly selective CCK-A ligand should be useful because it will identify CCK-A-R when they make up as little as 0.2% of the total CCK receptor number.

Animals↗
PubMed1996

Effect of ethanol on cholecystokinin-stimulated zymogen conversion in pancreatic acinar cells.

Exocrine pancreatic zymogens are proteolytically processed to active forms after they are secreted into the small intestine. However, intracellular conversion of zymogens to active forms can be stimulated by treating pancreatic acinar cells with high doses of cholecystokinin (0.1 microM) or carbamylcholine (0.1 mM). The high doses of cholecystokinin are unlikely to be achieved physiologically. The ability of ethanol to sensitize the acinar cell to zymogen conversion Induced by cholecystokinin or carbamylcholine was examined. Ethanol (10-200 mM) had no effect alone or when combined with carbamylcholine. However, ethanol (25 mM) added with low-dose cholecystokinin (0.1 nM) generated zymogen conversion that was 1) sixfold higher than cholecystokinin alone and 2) equivalent to that generated by highdose cholecystokinin (10 microM). The ability of ethanol to enhance cholecystokinin-induced zymogen conversion was dependent on the dose of ethanol and the duration of ethanol treatment. The cholecystokinin receptor antagonist, L-364,718, blocked the conversion stimulated by the addition of ethanol with cholecystokinin. This effect of ethanol did not change the affinity or number of cholecystokinin receptors, suggesting an effect more distal in the stimulus-activation cascade. These findings demonstrate that ethanol selectively sensitizes the pancreatic acinar cell to cholecystokinin-stimulated zymogen proteolysis.

Animals↗
PubMed1996

CK-independent increases in pancreatic secretion induced by dietary protein in chronic BPJ-diverted rats.

Previously, we demonstrated that, in rats with chronic bile-pancreatic juice (BPJ) diversion, pancreatic enzyme secretion was increased after feeding animals a 25% casein fat-free diet. We determined whether cholecystokinin (CCK) or the cholinergic pathway is associated with the response of pancreatic secretion after protein ingestion in the diverted rats, using a potent CCK antagonist, MK-329 or FK-480, and a cholinergic blocker, atropine. Secretion rates of chymotrypsin and trypsin in the fasting state were very high 7 days after a BPJ diversion, and the hypersecretion of the proteases was markedly reduced with an injection of MK-329, FK-480, or atropine and was further reduced by combined injection of FK-480 and atropine. The lowered secretion of the proteases in CCK-antagonized rats was increased after oral feeding of a protein diet and after a duodenal instillation of some protein sources, especially hydrolysate of guanidinated casein (HGC). The CCK-independent increases by HGC instillation are completely depressed by atropine. In rats treated with only atropine, the lowered secretion tended to be increased by a duodenal instillation of HGC. Increases in secretion after an administration of the protein source in CCK-antagonized rats were not affected by bestatin, an inhibitor of brush-border peptidases. We conclude that the stimulatory effects of dietary protein on the pancreatic enzyme secretion partially do not depend on CCK in chronic BPJ-diverted rats and that the CCK-independent increase is atropine sensitive.

Animals↗
PubMed1997

Endogenous CCK disrupts the MMC pattern via capsaicin-sensitive vagal afferent fibers in the rat.

A meal disrupts migrating motor complexes (MMC) in the rat intestine through stimulation of peripheral cholecystokinin (CCK)-B and central CCK-A receptors. The aim of this study was to determine pathways implicated in postprandial disruption of the MMC mediated by CCK. Sprague-Dawley rats were prepared with electrodes for electromyography in the small intestine, and ablation of vagal afferent C-fibers by capsaicin was carried out. Endogenous release of CCK was induced by oral administration of soybean trypsin inhibitor (SBTI). In control rats SBTI disrupted MMC and generated an irregular spiking activity that lasted longer than 3 h. Intravenous infusion of L-365,260 (2 x 10(-7) mol/kg) but not of L-364,718 (3 x 10(-9) mol/kg) restored the MMC pattern. In capsaicin-treated rats, SBTI did not modify fasting activity. Infusion of CCK octapeptide (CCK-8) at 3 x 10(-9) mol.kg-1.h-1 disrupted the MMC, although the response was quantitatively and qualitatively different from SBTI. The effect was reversed by intravenous infusion of L-364,718 or L-365,260 and intracerebroventricular infusion of L-364,718. In capsaicin-treated rats, the intracerebroventricular or intravenous infusion of L-364,718 inhibited CCK-8 effects. However, the intravenous infusion of L-365,260 did not reverse the MMC pattern. These results suggest that the disruption of the MMC mediated by CCK is due to stimulation of peripheral CCK-B receptors located in vagal afferent fibers. This initiates a reflex including stimulation of central CCK-A receptors. Exogenous CCK also stimulates peripheral CCK-A receptors not located in capsaicin-sensitive vagal afferent fibers.

Afferent Pathways↗
PubMed1997

Both free and complexed trypsin inhibitors stimulate pancreatic secretion and change duodenal enzyme levels.

Secretion of pancreatic digestive enzymes was measured in pancreatic cannulated rats after duodenal stimulation with Kunitz or Bowman-Birk protease inhibitors or their complexes with trypsin and/or chymotrypsin. Free and complexed inhibitors were bound by the duodenal epithelium, stimulated the discharge of cholecystokinin, and significantly increased secretion rates of alpha-amylase, trypsinogen, and chymotrypsinogen. Inasmuch as secretion rates returned to basal levels with cholecystokinin-A receptor antagonists, the stimulation was likely to be mediated by cholecystokinin. Soya factors also influenced the duodenal concentration of pancreatic enzymes under simulated feeding conditions. Thus the level of alpha-amylase increased while the trypsin concentration decreased in rats gavaged with free or complexed inhibitors. The same was true for chymotrypsin when the Bowman-Birk inhibitor was used, but the Kunitz inhibitor and its trypsin complex actually raised the luminal concentration of chymotrypsin. Accordingly, because soya inhibitors remained effective in stimulating pancreatic secretion after elimination of their inhibitory activity by complex formation, it is questionable whether the signal for cholecystokinin secretion was solely due to lowering of duodenal protease levels.

Animals↗
PubMed1997

Effects of telenzepine and L-364,718 on canine pancreatic secretion before and after vagotomy.

In six conscious dogs we compared the action of the M1-receptor antagonist telenzepine (20.25-81.0 nmol.kg-1.h-1), the cholecystokinin (CCK) antagonist L-364,718 (0.025-0.1 mg.kg-1.h-1), and combinations of both on the pancreatic secretory response to intraduodenal tryptophan, given against a secretin background before and after truncal vagotomy. Before vagotomy, the higher doses of telenzepine and of L-364,718 significantly (P < 0.05) decreased the protein response to tryptophan by up to 97%. After vagotomy, all doses of L-364,718 abolished the protein response, whereas telenzepine had no further effect. Before and after vagotomy, all combinations abolished the protein response. The plasma CCK-like immunoreactivity basally, during secretin, and in response to tryptophan was not altered by vagotomy, telenzepine, and/or L-364,718. These findings indicate that in dogs 1) potentiation exists between M1 receptors and CCK for stimulation of the pancreatic enzyme response to intraduodenal tryptophan, 2) the cholinergic fibers of the enteropancreatic reflex activated by tryptophan run within the vagus nerves and end at least in part on M1 receptors, 3) CCK acts in part independently of the vagal nerves, and 4) the CCK release by intestinal tryptophan is not influenced by vagotomy, telenzepine, and/or L-364,718.

Animals↗
PubMed1997

High-affinity CCK-A receptors on the vagus nerve mediate CCK-stimulated pancreatic secretion in rats.

Cholecystokinin (CCK) receptors are found on vagal afferent fibers. In pancreatic acini, CCK receptors exist in high- and low-affinity states. The aim of this study was to identify the vagal CCK-A receptor affinity state that mediates the effect of CCK on pancreatic protein secretion. Using a rat model with a pancreatic-biliary cannula, we studied the effects of CCK-JMV-180 on exocrine pancreatic function. CCK-JMV-180 acts as an agonist on high-affinity CCK receptors and as an antagonist on low-affinity CCK receptors. Infusion of CCK-JMV-180 (22-88 micrograms.kg-1.h-1) caused dose-dependent increases in pancreatic protein secretion, which were blocked by the CCK-A receptor antagonist L-364,718. Acute vagotomy in anesthetized rats and perivagal application of capsaicin in conscious rats abolished pancreatic responses to CCK-JMV-180 at 22 and 44 micrograms.kg-1.h-1. CCK-JMV-180 did not reduce pancreatic responses to CCK octapeptide infusion at 20 and 40 pmol.kg-1.h-1. To demonstrate that endogenously released CCK also acts on high-affinity CCK-A receptors, we showed that in conscious rats intraduodenal infusion of 18% casein produced a threefold increase in protein secretion and elevated plasma CCK levels from 0.7 to 8.4 pM. Infusion of CCK-JMV-180 at 44 micrograms.kg-1.h-1 failed to inhibit pancreatic responses to casein. In separate studies, perivagal application of 1% capsaicin inhibited 95% and 90% of the pancreatic responses to casein and casein combined with intravenous CCK-JMV-180, respectively. The neurotoxic effect of capsaicin on small-diameter sensory vagal fibers was verified by immunohistochemical and retrograde tracing studies. In conclusion, we demonstrated that in contrast to their effect on satiety, which is mediated by vagal low-affinity CCK-A receptors, exogenous CCK and endogenous CCK under physiological conditions act through high-affinity CCK-A receptors to mediate pancreatic protein secretion. These findings suggest that different affinity states of the vagal CCK receptors mediate different digestive functions.

Animals↗
PubMed1997

An amino-terminal fragment of LCRF, LCRF-(1-35), has the same activity as the natural peptide.

A cholecystokinin (CCK)-releasing peptide, luminal CCK-releasing factor (LCRF), has been purified from rat jejunal secretion. Amino acid analysis and mass spectral analysis showed that the purified peptide is composed of 70-75 amino acid residues and has a mass of 8,136 Da. Microsequence analysis of LCRF yielded an amino acid sequence for the amino-terminal 41 residues. To determine the biologically active region of the molecule, a peptide was synthesized consisting of the amino-terminal 35 amino acids of LCRF. In this study, intraduodenal infusion of LCRF-(1-35) significantly stimulated pancreatic secretion in conscious rats. The dose-response curves to LCRF-(1-35) and to monitor peptide were similar and biphasic, with higher doses producing submaximal pancreatic secretory responses. The CCK-A receptor antagonist MK-329 abolished the pancreatic secretory response to intraduodenally infused LCRF-(1-35). These results demonstrate that LCRF biological activity is contained within the amino-terminal 35-amino acid portion of LCRF, and this fragment may be useful for investigating the role of LCRF in gastrointestinal function.

Animals↗
PubMed1997

Molecular mechanisms for the growth factor action of gastrin.

We have previously observed that gastrin has a cholecystokinin B (CCK-B) receptor-mediated growth-promoting effect on the AR42J rat pancreatic acinar cell line and that this effect is paralleled by induction of expression of the early response gene c-fos. We undertook these experiments to elucidate the mechanism for induction of c-fos and the linkage of this action to the trophic effects of gastrin. Gastrin (0.1-10 nM) dose dependently induced luciferase activity in AR42J cells transfected with a construct consisting of a luciferase reporter gene coupled to the serum response element (SRE) of the c-fos promoter. This effect was blocked by the specific CCK-B receptor antagonist D2 but not by the specific CCK-A receptor antagonist L-364,718 or by pertussis toxin, indicating that gastrin targets the SRE via specific CCK-B receptors through a mechanism independent of Gi. Inhibition of protein kinase C (PKC) either by prolonged (24 h) exposure of the cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (100 nM) or by incubation with the selective inhibitor GF-109203X (3.5 microM) resulted in an 80% reduction in luciferase activity. Similar results were observed in the presence of the specific extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD-98059 (50 microM). We measured ERK2 activity in AR42J cells via in-gel kinase assays and observed that gastrin (1 pM-100 nM) induced ERK2 enzyme activity in a dose-dependent manner. Addition of GF-109203X and PD-98059, either alone or in combination, produced, respectively, partial and total inhibition of gastrin-induced ERK2 activity. Gastrin induction of ERK2 activity also resulted in a threefold increase in the transcriptional activity of Elk-1, a factor known to bind to the c-fos SRE and to be phosphorylated and activated by ERK2. PD-98059 blocked the growth-promoting effect of gastrin on the AR42J cells, demonstrating that this effect depends on activation of MEK. Our data lead us to conclude that the trophic actions of gastrin are mediated by ERK2-induced c-fos gene expression via PKC-dependent and -independent pathways.

Animals↗
PubMed1999

CCK receptor dysfunction in muscle membranes from human gallbladders with cholesterol stones.

Human gallbladders with cholesterol stones exhibit impaired muscle contraction induced by agonists that act on transmembrane receptors, increased membrane cholesterol content, and abnormal cholesterol-to-phospholipid ratio compared with those with pigment stones. The present study was designed to investigate the functions of the CCK receptor of gallbladder muscle membranes by radioreceptor assay and cross-linking. 125I-labeled CCK-8 binding was time-dependent, competitive, and specific. Scatchard analysis showed that the maximum specific binding (Bmax) was significantly decreased in cholesterol compared with pigment stone gallbladders (0.18 +/- 0. 07 vs. 0.38 +/- 0.05 pmol/mg protein, P < 0.05). In contrast, the affinity for CCK was higher in cholesterol than pigment stone gallbladders (0.18 +/- 0.06 vs. 1.2 +/- 0.23 nM). Similar results were observed in binding studies with the CCK-A receptor antagonist [3H]L-364,718. Cross-linking and saturation binding studies also showed significantly less CCK binding in gallbladders with cholesterol stones. These abnormalities were reversible after incubation with cholesterol-free liposomes. The Bmax increased (P < 0.01) and the dissociation constant decreased (P < 0.001) after incubation with cholesterol-free liposomes. In conclusion, human gallbladders with cholesterol stones have impaired CCK receptor binding compared with those with pigment stones. These changes are reversed by removal of the excess membrane cholesterol. These receptor alterations may contribute to the defective contractility of the gallbladder muscle in patients with cholesterol stones.

Bile Pigments↗
PubMed2001

Acid inhibition by intestinal nutrients mediated by CCK-A receptors but not plasma CCK.

We examined the role of CCK-A receptors in acid inhibition by intestinal nutrients. Gastric acid and plasma CCK and gastrin levels were measured in rats with gastric and duodenal fistulas during intragastric 8% peptone and duodenal perfusion with saline, complete liquid diet (CLD; 20% carbohydrate, 6% fat, and 5% protein), and the individual components of CLD. Acid output was significantly inhibited (50-60%) by CLD, lipid, and dextrose. Plasma CCK was significantly increased by CLD (from 2.6 +/- 0.3 to 4.8 +/- 0.5 pM) and lipid (4.6 +/- 0.5 pM). CCK levels 50-fold higher (218 +/- 33 pM) were required to achieve similar acid inhibition by exogenous CCK-8 (10 nmol x kg(-1) x h(-1) iv). Intestinal soybean trypsin inhibitor elevated CCK (10.9 +/- 2.5 pM) without inhibiting acid secretion. The CCK-A antagonist MK-329 (1 mg/kg iv) reversed acid inhibition caused by CLD, lipid, and dextrose. Peptone-stimulated gastrin (21.7 +/- 1.9 pM) was significantly inhibited by CLD (14.5 +/- 3.6 pM), lipid (12.3 +/- 2.2 pM), and dextrose (11.9 +/- 1.5 pM). Lipid and carbohydrate inhibit acid secretion by activating CCK-A receptors but not by altering plasma CCK concentrations.

Animals↗