Stimulated Pepsinogen Release Research Articles

PAR-2 modulates pepsinogen secretion from gastric-isolated chief cells.

In the present study, we investigated whether activation of protease-activated receptor type 2 (PAR-2) with SLIGRL (SL)NH2, a short mimetic agonistic peptide, directly stimulates pepsinogen secretion from gastric-isolated, pepsinogen-secreting (chief) cells. Immunostaining of gastric-dispersed chief cells with a specific anti-PAR-2 antibody demonstrated expression of PAR-2 receptors on membrane and cytoplasm. SL-NH2 and trypsin potently stimulated pepsinogen secretion (EC50 = 0.3 nM) and caused Ca2+ mobilization (EC50 = 0.6 nM). In contrast to SL-NH2, the scramble peptide LSIGRL-NH2 failed to stimulate pepsinogen release. Exposure to SL-NH2 also resulted in ERK1/2 phosphorylation and activation. Exposure of chief cells to phosphotyrosine kinase inhibitors and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one, a selective MEK inhibitor, significantly reduced secretion induced by SL-NH2. Pepsinogen secretion induced by SL-NH2 was desensitized by pretreating the cells with the mimetic peptide and trypsin, and exposure to SL-NH2 abrogates pepsinogen secretion induced by carbachol and CCK-8, but not secretion induced by secretin and vasointestinal peptide. Exposure to Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P) but not to calcitonin gene-related peptide increased pepsinogen release. The neurokinin-1 receptor antagonist, N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester, inhibited substance P-stimulated pepsinogen secretion, whereas it did not affect secretion induced by SL-NH2. Collectively, these data indicate that PAR-2 is expressed on gastric chief cells and that its activation causes a Ca2+-ERK-dependent stimulation of pepsinogen secretion.

The pepsinogen releasing effect of Helicobacter pylori lipopolysaccharide.

Helicobacter pylori lipopolysaccharide (LPS) affects pepsinogen release by a nontoxic mechanism. We hypothesized that this effect was characteristic of the organism and related to the clinical status of the strain. LPS was isolated from 11 H. pylori strains whose pathogenic profile was known and four other nongastric bacteria. The effects of luminal LPS on guinea pig gastric mucosal pepsinogen release was evaluated using the Ussing chamber technique. CCK-8 (10(-9)M) was used as a positive control. H. pylori LPS dose-dependently stimulated pepsinogen release with a maximal stimulation at 250 microg/ml (approximately 4500; p < .001 vs. control). LPS from other Helicobacter or Campylobacter species had no effect on pepsinogen release. ANOVA demonstrated significant differences in the efficacies of pepsinogen release between the 11 clinical H. pylori strains (p < .0001) despite the fact that they were all cagA+ and 90 had the cytotoxic vacA subtype s1. Physical and chemical disruption of the LPS suggested that both the structure and the carbohydrate composition of this molecule may play a critical role in pepsinogen release. Polymyxin B partly (p < .03) inhibited and dephosphorylation completely inhibited (p = .0002) LPS-stimulated pepsinogen release. Pepsinogen release is an innate property of all cagA+H. pylori LPS. The structure of the molecule and composition of side-chains are important in this response which appears to be partially lipid A driven.

Ammonia stimulates pepsinogen release from peptic glands via CA 2+ mediated pathway

Ammonia stimulates pepsinogen release from peptic glands via CA 2+ mediated pathway

Stimulation of pepsinogen release from chief cells byHelicobacter pylori: evidence for a role of calcium and calmodulin

To define the mechanisms by whichHelicobacter pyloristimulates pepsinogen secretion, thein vitrorelease of pepsinogen was studied using a preparation of pig chief cell monolayers.Helicobacter pyloriinduced a time- and concentration-dependent release of pepsinogen into the medium, with about a three-fold increase in pepsinogen secretion over controls found after 45 min of incubation. 3×107H. pylori produced 50% of the maximal response found at aH. pyloricount of 2×108. The action ofH. pyloridid not depend on the presence of the vacuolating toxin (vacA) and the cytotoxin-associated protein (cagA). Dibutyryl-cAMP and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also markedly stimulated pepsinogen secretion and enhanced the stimulatory effect ofH. pylori. Helicobacter pylori-stimulated pepsinogen release was inhibited by lanthanum and the calmodulin antagonist W-7, but not by the L-type Ca2+channel blocker nifedipine, TMB-8, an agent that blocks the release of Ca2+from intracellular stores, the protein kinase C inhibitor staurosporine and the protein kinase A inhibitor H-8. It is suggested thatH. pyloridirectly stimulates pepsinogen release from gastric chief cells and that this effect is mediated via the calcium/calmodulin messenger branch.

Recovery of Cholecystokinin Response of Porcine Gastric Chief Cells during Monolayer Culture

Cell isolation may impair secretory chief cell functions. To evaluate whether a monolayer culture results in a recovery, we compared the effects of cholecystokinin (CCK) octapeptide (CCK-8) on pepsinogen release from freshly isolated and from cultured porcine chief cells. CCK-8 had no significant effect on freshly isolated porcine chief cells but stimulated pepsinogen release from 36- and 60-hour cultured cells with EC50 values of 180 and 130 nmol/l, respectively. Maximal stimulation, achieved at a concentration of 1 micromol/l, amounted to 289 +/- 63 (p <0.01) and 401 +/- 64% (p <0.01) of the respective control value. In addition, the CCK-8 concentration-response curve for 60-hour, but not for 36-hour cultured chief cells displayed a second stimulatory peak at a CCK-8 concentration of 100 pmol/l (266 +/- 55% of control value, p < 0.05) with an EC50 value of 16 pmol/l. The CCKA-receptor antagonist devazepide (10 nmol/l) prevented the stimulatory effect of 1 micromol/l CCK-8 on pepsinogen release of 60-hour cultured cells. The adenylate cyclase activator forskolin (10 micromol/l) potentiated the low concentration CCK-8 effect, shifting the peak stimulation to a CCK-8 concentration of 10 pmol/l, and inhibited the high concentration CCK-8 effect on 60-hour cultured cells. These results indicate a time-dependent recovery of the CCK response of porcine gastric chief cells in monolayer culture and suggest that this model has an advantage over freshly isolated chief cells with regard to the pharmacological characterization of CCK effects.

Nitric oxide modulates pepsinogen secretion induced by calcium-mediated agonist in guinea pig gastric chief cells

Background & Aims : Nitric oxide, a putative cellular messenger synthesized from l-arginine, is a powerful modulator of gastric motility and secretions. The aim of this study was to investigate whether (1) guinea pig gastric chief cells express NO synthase, (2) NO modulates the pepsinogen secretion and guanosine 3′,5′-cyclic monophosphate (cGMP) generation induced by calcium (Ca 2+)-mediated agents, and (3) NO donors and cGMP analogues stimulate pepsinogen release. Methods : Chief cells were prepared by sequential digestion with collagenase and Ca 2+ chelation. NO generation was measured by determining the NO coproduct citrulline. Results : NO synthase immunoreactivities were constitutively expressed in approximately 70% chief cells. Carbachol (10 μmol/L) caused a 4–6-fold increase in pepsinogen release, citrulline generation, intracellular Ca 2+ concentration ([Ca 2+] i) and cGMP concentration. These effects were concentration dependently inhibited by N G -monomethyl- l-arginine ( l-NMMA). As gastrin, cholecystokinin, thapsigargin, and Ca 2+ ionophore increased NO generation, [Ca 2+] i seemed to regulate NO synthase activity. [Ca 2+] i chelator and calmodulin antagonist inhibited the carbachol-induced pepsinogen secretion and NO generation. Preincubating the cells with l-NMMA had no effect on carbachol-stimulated inositol triphosphate generation or [Ca 2+] i or Ca 2+-dependent adenosine triphosphatase levels. Nitrovasodilator agents and 8-bromo-cGMP stimulated pepsinogen release. Conclusions : Gastric chief cells express a Ca 2+/calmodulin-dependent NO synthase. NO modulates the stimulatory effect of Ca 2+-mediated agonists on pepsinogen release.

Bombesin does not stimulate pepsinogen release in isolated gastric chief cells.

Bombesin (BN)-related peptides, such as gastrin-releasing peptide (GRP), have been shown in vivo to stimulate release of pepsinogen. However, whether this is due to a direct interaction with chief cells is not clear. To clarify this we prepared isolated chief cells (> 90% pure) from guinea pig stomach. BN, GRP, or neuromedin B (NMB), at concentrations up to 1 microM, did not stimulate pepsinogen release or affect the stimulation caused by vasoactive intestinal peptide (VIP) (100 nM) or CCK-8 (10 nM), respectively. In addition, BN, GRP, or NMB at a concentration of 1 microM did not increase cAMP nor did they alter the increase in cAMP caused by VIP or secretin. BN (1 microM) did not alter basal cytosolic calcium [Ca2+]i or affect the increase in [Ca2+]i caused by CCK-8 (1 microM). Furthermore, BN, GRP, or NMB at a concentration of 1 microM did not increase the generation of inositol phosphates (IP) or alter the increase in [3H]IP1, [3H]IP2, or [3H]IP3, caused by CCK-8 (1 microM) or carbachol (1 mM). Binding studies demonstrated no saturable binding of either [125I][Tyr4]BN or [125I][D-Tyr0]NMB using experimental conditions where binding with other peptide ligands to other receptors on chief cells is seen. We conclude that BN-related peptides do not interact directly with specific receptors on chief cells to stimulate or alter stimulated pepsinogen secretion, increase the breakdown of inositol phosphates, or alter [Ca2+]i or cAMP.

Gastrin and CCK activate phospholipase C and stimulate pepsinogen release by interacting with two distinct receptors.

Both gastrin and cholecystokinin (CCK) can stimulate pepsinogen release from chief cells, but controversy exists about the receptors or intracellular mediators involved. In the present study, we prepared isolated chief cells from guinea pig stomach (> 90% pure) to investigate the ability of gastrin and CCK to alter cell function. The COOH-terminal octapeptide of CCK (CCK-8) caused an eightfold increase in pepsinogen release (EC50, 54 nM). Both CCK-8 and gastrin increased inositol phosphates, with CCK-8 (1 microM) and gastrin (3 microM) causing a 40- and 14-fold increase in [3H]IP1, 10- and 6-fold for [3H]IP2, and 8- and 4-fold for [3H]IP3. CCK-8 caused a half-maximal increase in [3H]IP3 at 2 nM, and the dose-response curve was monophasic, whereas with gastrin the curve was biphasic, with an EC50 of the initial component (20% maximal) at 38 nM and the second component at 10 microM. L-364,718 (0.1 microM) inhibited the secondary increase seen with gastrin concentrations > 10 nM. The CCK-A-selective agonist A-71378 was 85-90% as efficacious as CCK-8 and was equally potent. With 0.1 microM L-364,718, A-71378 caused no increase in [3H]inositol phosphates until > 10 nM, whereas CCK-8 caused 15% of maximal increase at concentrations > 0.3 nM. Similar results were obtained with cytosolic calcium measured using fura-2 or on CCK-8- or gastrin-stimulated pepsinogen release. These results demonstrate that gastrin and CCK-8 can alter chief cell function by interacting with either a CCK-A or CCK-B/gastrin receptor. Both receptors are coupled to phospholipase C and cause changes in inositol phosphates, cytosolic calcium, and pepsinogen release; however, the intracellular amplification differs between the two receptor subtypes. Activation by CCK-related peptides of the CCK-A receptor subtype accounts for 85-90% of the maximal changes in cellular function, and activation of the CCK-B/gastrin receptor accounts for 10-20% of maximal changes.

Chief cells possess a receptor with high affinity for PACAP and VIP that stimulates pepsinogen release.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the secretin-vasoactive intestinal peptide (VIP) family. To investigate whether PACAP alters chief cell function, we prepared isolated chief cells (> 90% pure) from guinea pig stomach. PACAP-38, PACAP-27, VIP, and secretin all caused a threefold increase in pepsinogen release. The dose-response curves of PACAP-38, PACAP-27, and VIP were biphasic, whereas with secretin it was not. The first phase comprised 40% of maximal release, and each of the three peptides (PACAP-38, PACAP-27, and VIP) were equipotent (EC50 0.1-0.3 nM). For the second phase, comprising 60% of maximal release, the relative potencies were PACAP-38 > PACAP-27 = VIP. 125I-labeled secretin, 125I-VIP, and 125I-PACAP-27 all demonstrated saturable binding to chief cells. Binding of both 125I-PACAP-27 and 125I-VIP was inhibited completely and with similar potencies by PACAP-38, PACAP-27, and VIP. Secretin had a > 500-fold lower affinity than PACAP-38 for displacing both 125I-PACAP-27 and 125I-VIP. With 125I-secretin, secretin was the most potent, and was 197 times more potent than PACAP-38, which was 6-8 times more potent than both PACAP-27 and VIP. We conclude that both PACAP-38 and PACAP-27 stimulate pepsinogen secretion from dispersed chief cells. In contrast to a number of other tissues, no evidence for a high-affinity receptor that interacted only with PACAP was found. PACAP and VIP interact with equal high affinity with a common receptor and with low affinity with the secretin receptor.

Both CCK-A and CCK-B/gastrin receptors mediate pepsinogen release in guinea pig gastric glands.

We evaluated the affinity of cholecystokinin octapeptide (CCK-8), gastrin, and subtype-selective CCK agonists for CCK/gastrin receptors and compared it with the ability of these peptides to stimulate phosphoinositide (PI) hydrolysis and pepsinogen release in guinea pig gastric glands. Competitive binding studies using 125I-labeled Bolton-Hunter-CCK-8 and 125I-gastrin showed the presence of CCK-B/gastrin receptors in gastric glands and dispersed chief cells. In contrast, the potency of peptides in stimulating PI hydrolysis in both gastric glands and dispersed chief cells displayed a profile similar to CCK-A receptors found in pancreatic acini, i.e., CCK-8 = A 71378 greater than A 71623 greater than A 70874 much greater than A 72962 = CCK-8 (desulfated) greater than gastrin II greater than gastrin I. In general, the rank order of potency of peptides for stimulation of PI hydrolysis correlated well with their ability to stimulate pepsinogen release. At concentrations greater than 10 microM, efficacies of gastrin I and II in stimulating pepsinogen release from gastric glands were near 90% of the maximal activity of CCK-8. The inhibitory potency of MK-329, a selective CCK-A receptor antagonist, was similar against either CCK-8 (10 nM) or gastrin I (10 microM), except that a minor portion (approximately 30-40%) of gastrin I-induced pepsinogen release was insensitive to MK-329. The MK-329-insensitive component was inhibited by CI-988, a potent and selective CCK-B/gastrin receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol stimulates pepsinogen release by opening a Ca2+ channel of guinea pig gastric chief cells

To better understand the mechanism by which ethanol causes gastric mucosal injury, the effects of ethanol on isolated guinea pig gastric chief cells were investigated in vitro. Ethanol at 300–900 mmol/L dose-dependently stimulated an increase in pepsinogen release without affecting chief cells' viability. Ethanol stimulated an increase in initial Ca2+ influx rate and intracellular Ca2+ concentration at the same concentrations as it stimulated pepsinogen release, whereas it did not stimulate cyclic adenosine monophosphate accumulation. Pepsinogen release stimulated by 900 mmol/L ethanol was almost equal to that stimulated by 10−8 mol/L COOH terminal octapeptide of cholecystokinin. Ethanol did not stimulate an increase in the accumulation of inositol trisphosphates and tetrakisphosphates in [3H]inositol-labeled chief cells, whereas cholecystokinin octapeptide caused a significant increase in inositol trisphosphates and tetrakisphosphates. Ethanol-stimulated pepsinogen release and increases in the initial Ca2+ influx rate and intracellular Ca2+ concentration were inhibited by 0.5 mmol/L La3+ or 1 mmol/L ethylene glycol tetraacetic acid but were not inhibited by nifedipine or verapamil. These results suggest that the effect of ethanol on pepsinogen release from the gastric chief cells may be due to its opening of a Ca2+ channel that is sensitive to La3+ and that the pepsinogen releasing action of ethanol may be one of factors for ethanol-induced gastric mucosal injury.

Gastrin-releasing peptide directly releases pepsinogen from guinea pig chief cells

Gastrin-releasing peptide (GRP) and bombesin can stimulate pepsinogen release by both gastrin-dependent and -independent mechanisms. Using isolated guinea pig gastric chief cells, we determined that GRP can act directly on the guinea pig chief cell to cause pepsinogen release. GRP and bombesin stimulated a 2.5- to 3-fold increase in pepsinogen release above basal release. Substance P also stimulated a small but significant increase in pepsinogen release. No gastrin immunoreactivity was detected in the supernatants of cells stimulated with up to 1 microM GRP or bombesin or 1 mM carbachol. GRP-stimulated pepsinogen release was completely inhibited by GRP/bombesin receptor agonists as well as substance P receptor antagonist but not by antagonists to receptors for gastrin, the octapeptide of cholecystokinin (CCK-8), secretin, vasoactive intestinal peptide (VIP), or muscarinic agents. Substance P-stimulated pepsinogen release was completely inhibited by substance P receptor antagonist but not by GRP/bombesin receptor antagonists. An additive effect on pepsinogen release was seen when GRP was combined with maximally effective concentrations of adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agents (VIP, secretin, 8-BrcAMP) but not with calcium-mediated agents (carbachol, CCK-8, gastrin). These results indicate that GRP can directly stimulate pepsinogen release from guinea pig chief cells by a specific GRP receptor that mobilizes intracellular calcium.

Characterization of cholinergic receptors mediating pepsinogen secretion from chief cells

By use of a highly enriched preparation of chief cells (greater than 90% pure) prepared from guinea pig stomach, the cholinergic receptors regulating pepsinogen secretion were studied. Each of five different muscarinic cholinergic agonists, but not the nicotinic cholinergic agonist nicotine, stimulated pepsinogen release. Carbamylcholine-stimulated pepsinogen release was inhibited by each of seven different cholinergic receptor antagonists with relative potencies of N-methylscopolamine greater than scopolamine = 4-diphenylacetoxy-N-methylpiperidine methiodide = atropine much greater than pirenzepine greater than AF-DX-116 much greater than tetraethylammonium. Binding of [N-methyl-3H]scopolamine ([3H]NMS) was time and temperature dependent, reversible, saturable, and specific. Analysis of [3H]NMS binding demonstrated a Kd of 1.3 nM for NMS with a binding capacity of 61 fmol/mg protein or 5,920 sites/chief cell. With the agonists carbamylcholine, acetylcholine, or muscarine, the receptor population could be divided into two classes of receptor sites: a class with high affinity (Kd, 12-53 microM) representing 73% of the binding sites and a class with low affinity (Kd, 2-5 mM) representing 27% of the binding sites. Each of the antagonists had equal affinity for both receptor populations. There was a close correlation between the ability of antagonists to inhibit [3H]NMS binding and carbamylcholine-stimulated pepsinogen release. Each agonist was 29- to 63-fold more potent at stimulating pepsinogen release than interacting with high-affinity receptors and 2,000- to 11,000-fold more potent than interacting with low-affinity receptors, suggesting spare receptors. Studies with the alkylating agent, propylbenzilylcholine mustard, demonstrated that there was a receptor reserve of 50-80%.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of Ca 2+ influx in F −-stimulated pepsinogen release from guinea pig gastric chief cells

In isolated guinea pig gastric chief cells, pepsinogen release was stimulated by NaF in a dose-dependent manner. Cholecystokinin (CCK) and Ca2+ ionophore A23187 had no additional effect on NaF-stimulated pepsinogen release. CCK caused a rapid increase in intracellular free Ca2+ concentration ([Ca2+]i) monitored by Quin-2 and markedly stimulated inositol phosphate accumulation in chief cells. By contrast, NaF did not cause any change in [Ca2+]i. NaF, even at a maximal concentration for pepsinogen release, appeared to be relatively ineffective on inositol phosphate accumulation. On the other hand, NaF markedly stimulated Ca2+ influx into chief cells. These results suggest that F- stimulates pepsinogen release probably by increasing Ca2+ influx into chief cells. Since F- is a well known activator of guanine nucleotide regulatory proteins (G proteins), it is proposed that there may exist a G protein regulating the opening of Ca2+ channel in gastric chief cells.

Adrenergic stimulation of pepsinogen release from rabbit isolated gastric glands.

Isolated gastric glands from the rabbit were used for studying the effect of catecholamines on the release of pepsinogen. Isoprenaline, adrenaline and noradrenaline stimulated pepsinogen release in a dose-dependent manner with similar maximal effects, but isoprenaline was significantly more potent than the other two agonists. The effect was mediated through beta-adrenoceptors, since the response was inhibited by the beta-adrenoceptor antagonist, propranolol, and since the alpha-adrenoceptor agonist, phenylephrine, was without effect in the concentration range 0.01 - 10 microM. Concentration-response curves for isoprenaline were shifted to the right in parallel by increasing doses of propranolol, and maximal response was not influenced by propranolol per se, which indicates a competitive type of antagonism. A Schild plot showed a pA2-value for propranolol of 7.70 and the slope of the regression line was 1.02. Studies with the beta 1-selective antagonist pafenolol and the beta 2-selective antagonist ICI 118.551 demonstrated that isoprenaline acted through beta 1-adrenoceptors. The results suggest an adrenergic component in the control of the peptic cells in rabbit gastric mucosa.

Pepsinogen release from isolated gastric glands.

The in vitro release of pepsinogen was studied using a preparation of isolated gastric glands from rabbits. The pepsinogen content of the glands was estimated to be about 700 U/mg dry wt. Spontaneous release of pepsinogen was found to be less than 1% of the total per hour and relatively constant for at least 2 h. Pepsinogen release was stimulated in a dose-dependent manner by both carbachol and isoproterenol. The cholinergic and beta-adrenergic stimulation was selectively inhibited by atropine and propranolol, respectively. Removal of external calcium inhibited the responses to both isoproterenol (partially) and carbachol (completely). Several agents, including histamine, prostaglandin (E2), and synthetic secretin, were found not to stimulate pepsinogen release. However, a crude secretin preparation (Boots) was found to produce significant stimulation. Dibutyryl cAMP increased pepsinogen release in a dose-dependent manner. Isoproterenol was found to increase the cAMP content of gastric glands and to stimulate adenylyl cyclase activity in homogenates. The beta-adrenergic stimulation of adenylyl cyclase was found to be selective for a population of gastric cells that was relatively depleted of parietal cells and distinct from the histamine-stimulated adenylyl cyclase activity. The results indicate that pepsinogen secretion by the gastric chief cell is regulated, in part, by separate cholinergic and beta-adrenergic mechanisms and that both calcium and cAMP play a role in this regulation.