Guinea Pig Chief Cells Research Articles

Regulatory mechanism of pepsinogen gene expression in monolayer cultured guinea pig gastric chief cells.

We have investigated the effects of dibutyryl cAMP, forskolin, carbamylcholine chloride (carbachol), ionomycin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of guinea pig pepsinogen mRNA in monolayer cultured gastric chief cells. After exposure of the cells to each of these compounds for 4 to 24 hr, and at 48 hr after primary culture, total cellular RNA was isolated using acid guanidium-phenol-chloroform and then was reverse transcribed to cDNA. Obtained cDNA was amplified by polymerase chain reaction (PCR) using primers detecting guinea pig pepsinogen mRNA and human beta-actin mRNA as an internal standard. The PCR products were separated and quantified using capillary electrophoresis. Dibutyryl cAMP and forskolin significantly increased pepsinogen mRNA, but carbachol, ionomycin, and TPA failed to increase that. These findings suggested that pepsinogen gene expression was up-regulated by intracellular cAMP, but not by intracellular calcium or protein kinase C in guinea pig chief cells.

Calcineurin Mediates Calcium-induced Potentiation of Adenylyl Cyclase Activity in Dispersed Chief Cells from Guinea Pig Stomach: FURTHER EVIDENCE FOR CROSS-TALK BETWEEN SIGNAL TRANSDUCTION PATHWAYS THAT REGULATE PEPSINOGEN SECRETION

In cholera toxin-treated gastric chief cells, incubation with a cholinergic agonist (carbamylcholine), a regulatory peptide (cholecystokinin), or a calcium ionophore (A23187) causes a dose- and time-dependent potentiation of cAMP levels. Because this augmented response is calcium/calmodulin-dependent, we hypothesized that it was mediated by calcineurin (protein phosphatase 2B). To test this hypothesis, we examined the actions of calcineurin inhibitors on secretagogue-induced potentiation of cAMP levels in guinea pig chief cells. Preincubation of cells with 0.1 microM FK-506 completely prevented carbachol-induced augmentation of cAMP levels and pepsinogen secretion from cholera toxin-treated cells. Cyclosporin-A, another calcineurin inhibitor, also prevented the augmented cAMP response. FK-506 and cyclosporin inhibited augmentation of cAMP levels following treatment with cholecystokinin(26-33) and A23187, but not the smaller increase in cAMP following treatment with a phorbol ester that activates protein kinase C. Hence, the actions of calcineurin inhibitors were limited to secretagogues that increase cellular calcium. Rapamycin, an agent that competes with FK-506 for the immunophilin, FK binding protein 12, does not inhibit calcineurin. In the present study, preincubation with rapamycin did not prevent carbachol-induced augmentation of cAMP levels in cholera toxin-treated chief cells. However, a molar excess of rapamycin reversed the inhibitory actions of FK-506. These experiments provide further evidence that the actions of FK-506 on cholera toxin-treated gastric chief cells are caused by its inhibitory actions on calcineurin. FK-506 also inhibited potentiation of cAMP levels when carbachol was added to cells that were preincubated with forskolin, an agent that directly activates adenylyl cyclase. We conclude that, in gastric chief cells, calcineurin mediates cross-talk between the calcium/calmodulin and adenylyl cyclase signaling pathways.

Characterization of CCKA receptor mediated pepsinogen secretion in porcine chief cells.

Cholecystokinin octapeptide (CCK-8s) is an endogenous stimulus of gastric pepsinogen secretion. Previous studies with isolated guinea pig chief cells indicated that this process is mediated through the CCKA receptor subtype, with an additional contribution from CCKB receptors. For comparison, we examined the mechanism of CCK-8s stimulated pepsinogen secretion in a larger nonrodent species, using highly enriched porcine chief cells as a functional in vitro model. Porcine chief cells responded weakly to stimulation by CCK-8s alone, but the efficacy was markedly enhanced in the presence of 10 mumol l-1 forskolin. Under these conditions, pepsinogen secretion was potently stimulated by CCK-8s and the CCKA receptor selective heptapeptide, A-71,378 (EC50 = 4.7 and 33 nmol l-1), but not by CCKB receptor selective agonists. The prototype CCKA receptor selective antagonist L-364,718 blocked pepsinogen secretion with approximately 2,000-fold higher affinity than the CCKB receptor selective analogue, L-365,260. This functional profile was consistent with the affinity rank order of all tested compounds at CCKA-receptor-like [125I]-BH-CCK-8s binding sites in the porcine gastric mucosa. Comparison with cloned CCKA receptors from other species revealed that the receptors mediating pepsinogen secretion in the pig have similar pharmacology, possibly with slight differences in agonist potencies. In contrast to the guinea pig, porcine CCKB receptors appear to have no direct role in pepsinogen secretion.

Mediation of pepsinogen secretion from guinea pig chief cells by Ca 2+/calmodulin-dependent protein kinase II

In the presence of Ca 2+ bound to calmodulin, Ca 2+/calmodulin-dependent protein kinase II (CaMK II) exhibits an intramolecular autophosphorylation and modulates many cell functions. In this study, the role of CaMK II in pepsinogen secretion was investigated in cultured guinea pig chief cells by using a specific CaMK II inhibitor, 1-[N,O- Bis(5- isoquinolinesulfonyl)-N- methyl- l-tyrosyl ]-4- phenyl-piperazine (KN-62), and an antibody for the Thr-286-autophosphorylated α subunit of CaMK II which specifically recognized the autophosphorylated form of CaMK II. KN-62 inhibited the pepsinogen secretion stimulated by carbamylcholine chloride, cholecystokinin octapeptide, and ionomycin in a dose-dependent manner without affecting intracellular Ca 2+ concentrations, but had no effect on the secretion by 12- O-tetradecanoyl phorbol-13-acetate (TPA) and forskolin. Heavy staining with the antibody for autophosphorylated CaMK II was observed in the cytoplasm of chief cells treated with carbamylcholine chloride or ionomycin, but only light staining was seen in cells treated with TPA or forskolin. Thus, CaMK II and its autophosphorylation may be a critical step in the intracellular pathway by which Ca 2+ causes pepsinogen secretion from guinea pig chief cells.

Effects of dibutyryl guanosine 3′,5′-cyclic monophosphate and sodium nitroprusside in pepsinogen secretion from guinea pig chief cells with respect to intracellular Ca 2+

Both Ca 2+ and adenosine 3′,5′-cyclic monophosphate act as intracellular second messengers in pepsinogen secretion from chief cells. However, the role of intracellular guanosine 3',5'-cyclic monophosphate (cGMP) in this process has not been defined. Although dibutyryl cGMP (dbcGMP), a membrane-permeable derivative of cGMP, has been shown to inhibit pepsinogen secretion only stimulated by cholecystokinin (CCK), the intracellular mechanism of this effect remains unclear. We evaluated the role of intracellular cGMP in pepsinogen secretion from monolayer cultured guinea pig chief cells using dbcGMP and sodium nitroprusside, both of which increase intracellular cGMP. Dibutyryl cGMP and sodium nitroprusside have now been shown to inhibit pepsinogen secretion induced by not only CCK octapeptide but also carbamylcholine chloride and ionomycin in a dose-dependent manner. Furthermore, dbcGMP reduced the increase in intracellular free Cat+ concentration induced by carbamylcholine chloride, CCK octapeptide, and ionomycin. These results suggest that intracellular cGMP may inhibit pepsinogen secretion by reducing the intracellular free Ca 2+ concentration.

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.

Mediation of pepsinogen secretion from guinea pig chief cells by Ca 2+/calmodulin-dependent protein kinase II and its autophosphorylation

Mediation of pepsinogen secretion from guinea pig chief cells by Ca 2+/calmodulin-dependent protein kinase II and its autophosphorylation

Thapsigargin defines roles of Ca2+ in initial, sustained, and potentiated stimulation of pepsinogen secretion

The roles of Ca2+ in agonist-induced pepsinogen secretion from guinea pig chief cells remain unclear. We used cholecystokinin octapeptide (CCK-8) or secretin alone or with thapsigargin (TG) to clarify these roles. TG releases Ca2+ from intracellular stores by inhibiting microsomal Ca(2+)-adenosinetriphosphatase (ATPase), thereby depleting intracellular Ca2+ (Cai2+) stores. In most cells TG also causes Ca2+ influx. In the present study, with an extracellular Ca2+ concentration ([Ca2+]o) of 1.5 mM, CCK-8 (0.1 microM) caused a rapid increase in pepsinogen secretion; however, the rate decreased with time. With [Ca2+]o = 0, the initial increase was similar but later secretion was abolished, suggesting that Ca2+ influx was important for sustained secretion. With [Ca2+]o = 1.5 mM, TG (0.1 microM) caused a 2.7-fold sustained increase in in Cai2+ concentration ([Ca2+]i) and a ninefold sustained increase in pepsinogen secretion. With [Ca2+]o = 0, TG caused a transient 66% increase in [Ca2+]i and a 50% increase in pepsinogen secretion. The time course of TG-induced pepsinogen secretion correlated with the time course of TG-induced increases in [Ca2+]i. These data demonstrated that Ca2+ influx itself was a potent stimulant of pepsinogen secretion. We further focused on the roles of increasing [Ca2+]i from Cai2+ stores. With or without extracellular Ca2+ (Cao2+) present, addition of CCK-8 (0.1 microM) 10 min after TG caused no further increase in [Ca2+]i, demonstrating depletion of the inositol 1,4,5-trisphosphate-sensitive pool. The Ca(2+)-mobilizing agent CCK-8 caused no pepsinogen secretion 10 min after TG preincubation, demonstrating that mobilization of Ca2+ from intracellular stores was important in the rapid initial phase stimulation of pepsinogen secretion caused by CCK-8. In contrast, preincubation with TG had no effect on pepsinogen secretion by secretin, an agent that increases adenosine 3',5'-cyclic monophosphate. A 6-min preincubation with TG potentiated the subsequent stimulation of pepsinogen secretion caused by secretin in the presence of Cao2+ where [Ca2+]i remained elevated. However, TG-induced potentiations of secretin-stimulated pepsinogen secretion was abolished once [Ca2+]i had returned to the basal level in the absence of Cao2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Distinguishing multiple CCK receptor subtypes. Studies with guinea pig chief cells and transfected human CCK receptors.

Distinguishing multiple CCK receptor subtypes. Studies with guinea pig chief cells and transfected human CCK receptors.

Y2 receptors for peptide YY and neuropeptide Y on dispersed chief cells from guinea pig stomach.

Peptide YY (PYY) and neuropeptide Y (NPY) inhibit agonist-induced adenosine 3',5'-cyclic monophosphate (cAMP) production and pepsinogen secretion from chief cells. We used radiolabeled PYY and NPY to characterize receptors on chief cells from guinea pig stomach. Binding of 125I-labeled PYY was rapid (70% maximal within 10 min) and specific (not inhibited by secretin, vasoactive intestinal peptide, cholecystokinin, carbachol, prostaglandin E2, forskolin, or cholera toxin). Measurement of the ability of PYY to inhibit binding of 125I-PYY indicated the presence of 1.8 x 10(3) high-affinity [dissociation constant (Kd) = 1.7 nM] and 5.1 x 10(4) low-affinity (Kd = 83.3 nM) sites/cell. Internalization of bound 125I-PYY was suggested by slow and incomplete dissociation in the presence of unlabeled PYY (50% after 2 h) and was examined further by measuring residual binding after washing with acetic acid (pH 2.5), glycine (pH 10.5), or trypsin. After 30 min at 37 degrees C, internalization of radioligand was evidenced by the failure of washing with these solutions to remove 50-65% of bound radioactivity. At 4 degrees C, internalization of 125I-PYY was nearly abolished. Binding of 125I-PYY and 125I-NPY was inhibited by NPY-(13-36) but not by [Leu31,Pro34]NPY indicating that these are Y2 receptors. In guinea pig chief cells, PYY and NPY modulate cAMP-mediated pepsinogen secretion by interacting with specific high-affinity Y2 receptors.

Prostaglandin protects isolated guinea pig chief cells against ethanol injury via an increase in diacylglycerol.

We studied cellular processes activated by prostaglandins (PG) that are involved in the protection of gastric chief cell injury estimated in terms of dye exclusion test, release of lactate dehydrogenase (LDH), or 51Cr from prelabeled chief cells. Pretreatment of chief cells with 3 x 10(-6) M PGE2 or PGE1 at 37 degrees C and pH 7.4 for 15 min maximally reduced not only ethanol- but also taurocholic acid-caused LDH release from chief cells. PGs equipotently stimulated increases in the accumulation of diacylglycerol and cyclic AMP without elevating intracellular Ca2+ concentrations in gastric chief cells. The rank order of the potency was equal to that of PGs to reduce the injury. Pretreatment of chief cells with synthetic 1-oleoyl-2-acetyl-sn-glycerol (OAG) or 12-o-tetradecanoyl phorbol 13-acetate (TPA) reduced the injury of chief cells, while 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, failed to reduce the injury and 1-(5-isouinolinylsulfonyl)-2-methylpiperazine (H7) blocked the protective action of PGE2. On the other hand, forskolin and dbcAMP had no effect on ethanol-caused LDH release and diacylglycerol formation in chief cells. These results suggest that PGE2 and PGE1 possess the direct protective action against ethanol- or taurocholic acid-caused injury in chief cells, presumably through the activation of the diacylglycerol/protein kinase C signaling pathway.

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.

Cholinergic desensitization of pepsinogen secretion and calcium mobilization of dispersed guinea pig chief cells

When dispersed chief cells from guinea pig stomach were first incubated with carbachol, washed, and then reincubated with carbachol in fresh incubation solution, the stimulation of pepsinogen secretion and the rise in intracellular calcium concentration during the second incubation were reduced. Carbachol did not cause residual enzyme secretion, but the same range of concentrations that causes enzyme secretion caused desensitization that was rapid, temperature dependent, and reversible with time. Preincubation with carbachol caused approximately a 65% reduction in enzyme secretion stimulated during a subsequent incubation with this agonist, but the potency of carbachol was unaffected. Prior exposure to carbachol also reduced subsequent stimulation caused by cholecystokinin (CCK-8), gastrin I, ionophore A23187, or 12-O-tetradecanoylphorbol 13-acetate but did not alter stimulation by any agonist that increases cellular cAMP. Carbachol pretreatment of Fura-loaded chief cells caused a threefold increase in the EC50 for carbachol-stimulated [Ca2+]i and approximately a 30% reduction in the maximal rise in [Ca2+]i in response to carbachol or CCK-8. Inhibition of [N-methyl-3H] scopolamine binding by carbachol following carbachol pretreatment indicated that modulation of receptor affinity or number did not account for functional desensitization. These data indicate that carbachol causes heterologous desensitization of pepsinogen secretion stimulated by agonists that mobilize cellular Ca2+ or activate protein kinase C through a postreceptor action and suggest that an attenuated rise in chief cell calcium is one mechanism mediating the desensitization of enzyme secretion.

Protein kinase C modulates effects of prostanoids on cyclic adenosine monophosphate in guinea pig chief cells

In the course of examining the role of protein kinase C in signal transduction in dispersed chief cells from guinea pig stomach, we observed that phorbol esters inhibit prostaglandin (PG)-stimulated increases in cyclic adenosine monophosphate (cAMP). Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, decreased maximal levels of PGE2-stimulated cAMP by 40%. This dose-dependent effect was observed within 30 sec and was maximal by 1 min of incubation at 37 degrees C. Phorbols that do not activate protein kinase C did not have this effect. Adding H7, a protein kinase C inhibitor, abolished the inhibitory effects of PMA, indicating that these effects are not caused by activation of cyclic nucleotide phosphodiesterases. PMA did not alter the increase in cellular cAMP caused by cholera toxin, forskolin, secretin, or vasoactive intestinal peptide. Hence the site of these prostanoid-specific actions of protein kinase C does not appear to be stimulatory or inhibitory guanine nucleotide binding proteins or the catalytic component of the adenylyl cyclase system. In dispersed chief cells, activation of protein kinase C may inhibit prostanoid-induced stimulation of the adenylyl cyclase system by a direct effect on prostaglandin receptors.

The cholecystokinin-induced Ca 2+ shuttle from the inositol trisphosphate-sensitive and ATP-dependent pool, and initial pepsinogen release connected with cytoskeleton of the chief cell

In guinea pig chief cells, inositol 1,4,5-triphosphate (IP 3) caused release of Ca 2+, which was accumulated by ATP, from an endoplasmic reticulum-enriched fraction in both the permeable system and the cell-free system. This was mimicked with the Ca 2+ ionophores A23187 and ionomycin on a large scale since an IP 3-sensitive Ca 2+ pool might be a subset of the Ca 2+ ionophore-sensitive Ca 2+ pool. The permeable chief cells, but not the cell-free system, retained the ability to react to synthetic cholecystokinin octapeptide (CCK-OP) with Ca 2+ release from an IP 3-sensitive pool due to of the non-additive but constant effect in exerting Ca 2+ release from the store(s) induced by the combination with IP 3 and CCK-OP. The increase in the cytosolic free Ca 2+ concentration of intact chief cells responding to CCK-OP or the Ca 2+ ionophore, ionomycin, comprised two components, namely, that by the Ca 2+ entry from the extracellular space, and that by the Ca 2+ release from the intracellular space(s) (as measured by fura-2). When CCK-OP or ionomycin was added, there was a biphasic response of pepsinogen secretion. An initial but transient response reaching a peak in 5 min was followed by a sustained response reaching a peak in 30 min. The initial pepsinogen release was independent of medium Ca 2+, whereas the sustained one was dependent on medium Ca 2+. The results suggest that the intracellular Ca 2+ release from the store(s), presumably endoplasmic reticulum, may trigger the initial pepsinogen release, whereas the sustained pepsinogen secretion may be caused by acting in concert with the initial response and external Ca 2+ entry. On the other hand, the disruption of the microtubular-microfilamentous system by cholchicine or cytochalasin D failed to cause the Ca 2+ release evoked by either IP 3, CCK-OP or Ca 2+ ionophores and to cause the CCK-OP- or ionomycin-induced initial pepsinogen release. These findings suggest that the IP 3-sensitive pool is the same Ca 2+ store which is completely or partially sensitive to CCK-OP and Ca 2+ ionophores, respectively, and that the assembly of the cytoskeletal system is involved in initial intracellular Ca 2+ metabolism and the following initial pepsinogen release. The assembly of the cytoskeletal system may be an early event in mediating the CCK-OP-induced initial pepsinogen release, perhaps by causing the Ca 2+ release from an IP 3-sensitive pool of the chief cell. The translocation or attachment of the IP 3-sensitive pool brought about by cytoskeletal system might be necessary to cause Ca 2+ release after the cell stimulation with CCK-OP.