Relation of prostaglandin-induced increases in cellular cAMP to stimulation of pepsinogen secretion from dispersed chief cells

The actions of prostaglandins (PG) on cAMP in dispersed chief cells from guinea pig stomach were examined and compared to the actions of these agents on pepsinogen secretion. Maximal concentrations of A, B, or E prostaglandins caused a 2-5-fold increase in pepsinogen secretion and cellular cAMP. The relative order of potency for these actions was PGEs greater than PGAs greater than PGBs. Detection of prostaglandin-induced changes in cAMP was enhanced by adding a phosphodiesterase inhibitor to the incubation solution. The time courses for the effects of prostaglandins on pepsinogen secretion and cAMP were similar. With PGE1 an increase in cAMP and pepsinogen secretion was detected by 1 min and was maximal by 7.5 min. Although significant increases in cAMP were detected with a ten-fold lower concentration of PGEs than PGAs, a maximal increase in cAMP was observed with the same concentration, 30 microM, of either agent. These data indicate that prostaglandins that stimulate pepsinogen secretion increase cAMP in dispersed chief cells. However, comparison of the dose-response curves for the actions of prostaglandins on pepsinogen secretion and cAMP revealed that detectable increases in cAMP occurred with concentrations of these agents that were about ten-fold greater than those needed to stimulate pepsinogen secretion. Therefore, although the similarity in the kinetics and relative potencies of prostaglandin-induced changes in cAMP and enzyme secretion provides further evidence that changes in cAMP play a role in the mediation of prostaglandin-induced pepsinogen secretion, the present data suggest the involvement of a cellular messenger in addition to cAMP.     

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.     

Interaction between the calcium and adenylate cyclase messenger systems in dispersed chief cells from guinea pig stomach. Possible cellular mechanism for potentiation of pepsinogen secretion

To determine the role of the adenylate cyclase system in potentiation of enzyme secretion, we used cholera toxin to activate adenylate cyclase before examining the effects of agents on chief cell cAMP and pepsinogen secretion. Dispersed chief cells were obtained from guinea pig stomach by fractionation of mucosal cells on a Percoll gradient. Incubation of cells with 100 nM cholera toxin for 90 min and subsequent incubation with carbachol or cholecystokinin resulted in augmentation of cellular cAMP and potentiation of pepsinogen secretion. The rate of increase in cAMP with carbachol or cholecystokinin was similar to that for the potentiated secretory response. To determine the role of changes in cell calcium on these effects, we examined the actions of the ionophore A23187. In cells preincubated with cholera toxin, A23187 augmented cAMP and caused potentiation of pepsinogen secretion. The effects of A23187, carbachol, and cholecystokinin on cells preincubated with cholera toxin were abolished by removing extracellular calcium or by adding the calmodulin inhibitor trifluoperazine. These data indicate that in chief cells preincubated with cholera toxin, secretagogue-induced increases in cell calcium concentration activate calmodulin thereby augmenting levels of cAMP and causing potentiation of pepsinogen secretion. Modulation of adenylate cyclase by changes in chief cell calcium concentration appears to be one mechanism whereby secretagogue interaction can result in potentiation of pepsinogen secretion.     

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.     

Association of protein kinase A with AKAP150 facilitates pepsinogen secretion from gastric chief cells

Cross talk between signal transduction pathways augments pepsinogen secretion from gastric chief cells. A-kinase anchoring proteins (AKAPs) associate with regulatory subunits of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B) and localize this protein complex to specific cell compartments. We determined whether an AKAP-signaling protein complex exists in chief cells and whether this modulates secretion. In Western blots, we identified AKAP150, a rodent homologue of human AKAP79 that coimmunoprecipitates with PKA, PKC, and actin. The association of PKA and PP2B was demonstrated by affinity chromatography. Confocal microscopy revealed colocalized staining at the cell periphery for AKAP150 and PKC. Ht31, a peptide that competitively displaces PKA from the AKAP complex, but not Ht31P, a control peptide, inhibited 8-Br-cAMP-induced pepsinogen secretion. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and/or calcium. However, Ht31, but not Ht31P, inhibited carbachol- and A23187-stimulated augmentation of secretion from cells preincubated with cholera toxin. These data suggest the existence in chief cells of a protein complex that includes AKAP150, PKA, PKC, and PP2B. Disruption of the AKAP-PKA linkage impairs cAMP-mediated pepsinogen secretion and cross talk between signaling pathways.     

Cholecystokinin-stimulated pepsinogen secretion and cholecystokinin receptors on gastric chief cells in guinea pigs

We investigated cholecystokinin (CCK) receptors on isolated gastric chief cells from guinea pig. CCK stimulated pepsinogen secretion from chief cells at the same efficacy as that induced by carbamylcholine. Binding of 125I-labeled CCK-33 (125I-CCK) to chief cells was temperature-dependent, and was saturable and reversible at 37 degrees C. Hofstee plots of the ability of CCK-8 to inhibit binding of 125I-CCK showed a linear regression line, suggesting that CCK receptors possessed one binding site. The dissociation constant of the binding site was calculated to be 3.8 x 10(-10) M. The dose-response curve of CCK for pepsinogen secretion was superimposed on that for the binding to its receptors. These results indicated that gastric chief cells from the guinea pig possess CCK receptors that relate closely to the action of CCK involved in pepsinogen secretion.     

Cholinergic agonist-induced pepsinogen secretion from murine gastric chief cells is mediated by M1 and M3 muscarinic receptors

Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M3 receptor subtype (M3R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M1 receptor (M1R) in rat chief cells. We used mice that were deficient in either the M1 (M1R-/-) or M3 (M3R-/-) receptor or that lacked both receptors (M(1/3)R-/-) to determine the role of M1R and M3R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M1R-/- mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M3R-/- with WT glands. To explore the possibility that both M1R and M3R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M(1/3)R-/- double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from M(1/3)R-/- mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M1R and M3R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M1 and M3 receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.     

Endothelial cells stimulate ANF secretion from atrial myocytes in co-culture

Using a novel in vitro co-culture system, we investigated the possible influence of vascular endothelial cells on the secretion of atrial natriuretic factor (ANF) from atrial myocytes. Co-culture of bovine aortic endothelial cells grown on Cytodex-3 microcarrier beads with primary monolayer cultures of neonatal rat myocytes induced a 2.1-fold increase in immunoreactive ANF (irANF) in the medium, compared with irANF in medium from atrial cultures alone. This increase did not appear to be the result of processing of prohormone to more immunoreactive species, and could be inhibited by 47% with 10 microM acetylcholine. The endothelium-derived vasoconstrictor peptide, endothelin, elicited a dose-dependent increase in ANF secretion from atrial cultures, but, contrary to vasopressin, was incapable of further stimulating release from atrial-endothelial co-cultures. These experiments suggest that endothelium stimulates the release of ANF from myocytes, possibly by the action of the peptide endothelin.     

Leukotrienes stimulate acid secretion from isolated gastric parietal cells

Leukotrienes LTC4 and LTD4 display contractile effect on the stomach. The stimulation of acid secretion by LTC4, LTD4 and LTE4 was evidenced on a crude isolated cell preparation from rabbit gastric mucosa using the (14C)aminopyrine accumulation method. LTs were in the same order of potency. No potentiation with histamine, carbachol or IBMX was observed suggesting a specific mechanism for LTs on parietal cell.     

Phospholipases stimulate secretion in RBL mast cells

Roles for glycerophospholipids in exocytosis have been proposed, but remain controversial. Phospholipases are stimulated following the activation of the high-affinity receptor for immunoglobulin E (IgE) in mast cells. To study the biochemical sequelae that lead to degranulation, broken cell systems were employed. We demonstrate that the addition of three distinct types of exogenous phospholipases (i.e., bcPLC, scPLD, and tfPLA(2)), all of which hydrolyze phosphatidylcholine (PC), trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. Production of bioactive lipids by these phospholipases promotes release of granule contents through the plasma membrane and acts downstream of PKC, PIP(2), and Rho subfamily GTPases in regulated secretion. These exogenous phospholipase-induced degranulation pathways circumvent specific factors activated following stimulation of the IgE receptor as well as in ATP- and GTP-dependent intracellular pathways. Taken together, these results suggest that regulated secretion may be achieved in vitro in the absence of cytosolic factors via phospholipase activation and that products of PC hydrolysis can promote exocytosis in mast cells.     

Prostaglandins and enzyme secretion from dispersed rat pancreatic acinar cells

The role of prostaglandins in exocrine pancreatic enzyme secretion was studied. The effects of three inhibitors of prostaglandin and thromboxane syntheses, were evaluated on release of amylase from dispersed rat pancreatic acinar cells. Mepacrine inhibited, while indomethacin and imidazole had no effect on basal or carbachol or cholecystokinin stimulated enzyme release. Exogenous arachidonic acid or various prostaglandins (E₁, E₂, F_2α, I₂), also did not affect the secretory process. Acinar cells actively incorporated radioactive arachidonic acid, principally into phospholipids (especially phosphatidylcholine), however release of the free fatty acid and subsequent synthesis of radioactive endogenous prostaglandins was not stimulated by the presence of different pancreatic stimulants. Pancreatic microsomes were found to be lacking in cyclo-oxygenase, an enzyme involved in endegenous synthesis of prostaglandins. The data suggest that prostaglandins are not involved directly in excitation-secretion coupling in the exocrine pancreas.     

Porcine ileal polypeptide causes an increase in cytoplasmic Ca2+ in both parietal and chief cells resulting in acid and pepsinogen secretion

Porcine ileal polypeptide, an enterooxyntin isolated from distal small intestinal mucosal epithelium, has been observed to stimulate gastric acid secretion in vivo as well as in vitro (Wider, M.D. et al. (1984) Endocrinology 115, 1484-1491, Wider M.D. et al. (1986) Endocrinology 118, 1546-1550). We report here that porcine ileal polypeptide stimulates both acid (aminopyrine accumulation) and pepsinogen secretion in isolated, enriched populations of guinea pig parietal and chief cells in a dose-dependent manner. Further, 10(-9) M porcine ileal polypeptide caused an increase in cytoplasmic Ca2+ concentration in both parietal and chief cells similar in magnitude to that observed with gastrin-17 (10(-8) M) (as measured by both fura-2 and aequorin) and cholecystokinin octapeptide (CCK-OP) (10(-8) M), respectively. Porcine ileal polypeptide has been observed to cause no stimulation of cAMP production in gastric glands from guinea pigs (Gespach, C., personal communication) nor is there any effect of medium Ca2+ depletion on acid production observed with guinea pig gastric mucosal sections. It is concluded that porcine ileal polypeptide, at concentrations similar to circulating levels observed in plasma of normal pigs (5 x 10(-9) M), acts directly on the parietal and chief cells to cause the mobilization of intracellular Ca2+ from the stores resulting in acid and pepsinogen secretion. These experiments demonstrate that this peptide is a potent enterooxyntin and chief cell secretagogue which acts via the same signal transduction mechanisms as gastrin and cholecystokinin.     

Electron immunocytochemical localization of pepsinogen I (PgI) in chief cells, mucous-neck cells and transitional mucous-neck/chief cells of the human fundic mucosa

Specific PgI antibodies devoid of PgII cross reactivity have been applied to aldehyde-osmium fixed human, fundic-type, gastric mucosa investigated with the protein A-immunogold technique. PgI immunoreactivity has been detected in the homogeneous secretory granules of glandular chief cells, in bipartite granules of mucous-neck cells, in the granules of cells showing intermediate patterns and topography in between chief and mucous-neck cells (transitional cells), as well as in the granules of a few cells in the foveolar/mucous-neck boundary zone showing mixed foveolar/mucous-neck granule populations. The findings support progressive transformation of mucous-neck cells into chief cells.     

Electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of the calf fundic glands

The electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of calf fundic glands was studied using specific antisera for prochymosin and pepsinogen with a protein A-gold method. Prochymosin and pepsinogen immunoreactivities were detected in the same secretory granules of the chief, mucous neck and transitional cells, simultaneously using small and large colloidal gold particles. In chief cells, both immunoreactivities were distributed uniformly over the same zymogen granules showing a round, large, homogeneous and electron-dense appearance. In mucous neck cells, both immunoreactivities were found exclusively on the same electron-dense core located eccentrically in the mucous granule showing light or moderate electron density. In transitional mucous neck/chief cells, electron-dense cores became larger in size and some granules were occupied by the electron-dense core without a halo between the core and the limiting membrane. Both immunoreactivities were found uniformly over the electron-dense core. The granules having no halo in the transitional cells could not be distinguished from the typical zymogen granules in the chief cells.     

Endothelins stimulate aldosterone secretion from dispersed rat adrenal zona glomerulosa cells, acting through ETB receptors coupled with the phospholipase C-dependent signaling pathway

Compelling evidence indicates that endothelins (ETs) stimulates aldosterone secretion from rat zona glomerulosa (ZG) cells, acting through the ETB receptor subtype. We have investigated the mechanisms transducing the aldosterone secretagogue signal elicited by the pure activation of ETB receptors. Aldosterone response of dispersed rat ZG cells to the selective ETB-receptor agonist BQ-3020 was not affected by inhibitors of adenylate cyclase/protein kinase (PK)A, tyrosine kinase-, mitogen-activated PK-, cyclooxygenase- and lipoxygenase-dependent pathways. In contrast, the inhibitor of phospholipase C (PLC) U-73122 abrogated, and the inhibitors of PKC, phosphatidylinositol trisphosphate (IP(3))-kinase and calmodulin (calphostin-C, wortmannin and W-7, respectively) partially prevented aldosterone response to BQ-3020. When added together, calphostin-C and wortmannin or W-7 abolished the secretagogue effect of BQ-3020. BQ-3020 elicited a marked increase in the intracellular Ca2+ concentration ([Ca2+]i) in dispersed rat ZG cells, and the effect was abolished by the Ca(2+)-release inhibitor dantrolene. The Ca2+ channel blocker nifedipine affected neither aldosterone nor Ca2+ response to BQ-3020. Collectively, our findings suggest that (1) ETs stimulate aldosterone secretion from rat ZG cells through the activation of PLC-coupled ETB receptors; (2) PLC stimulation leads to the activation of PKC and to the rise in [Ca2+]i with the ensuing activation of calmodulin; and (3) the increase in [Ca2+] is exclusively dependent on the stimulation of IP(3)-dependent Ca2+ release from intracellular stores.     

Histamine secretion from permeabilized mast cells by calcium

A transient increase in the permeability of the mast cell membrane was caused by the exposure of the cells to low concentrations of saponin, 5 or 10 micrograms/ml. These concentrations had very little effect in the absence of calcium but caused 35 to 50% histamine release, having the character of a secretory response, when 0.25 mM or more calcium was added to the medium. The dose-response curve was steep between 25 microM and 250 microM calcium and tended to flatten with higher concentrations. The release was associated with a pronounced increase in calcium uptake, which was faster than the histamine release. The membrane changes were slight as indicated by only 7 to 12% leakage of lactate dehydrogenase and by the absence of any detectable change in the electron micrographs. The transient nature of the membrane change is shown by the following experiment. When the cells were first exposed to saponin in the absence of calcium, the amount of histamine released by the subsequent incubation with calcium varied inversely with the time interval that elapsed before calcium was added. If calcium was added after 15 minutes no histamine release occurred. When calcium uptake was studied in the same manner, the stimulation of calcium uptake in saponin-treated cells also declined progressively with increasing intervals after the exposure to saponin when calcium was added. Stimulation of both histamine release and calcium uptake was inhibited by antimycin A, the inhibition curves with 10(-9)M to 10(-7)M antimycin A being similar. The effect on the calcium uptake by itself could explain the inhibition of histamine release. But the release was also inhibited by the calmodulin antagonists, W-7 and mepacrine, suggesting that the influx of calcium in the permeabilized cells acts primarily through calmodulin-mediated enzyme activation.     

Secretory products from human adipocytes stimulate proinflammatory cytokine secretion from human endothelial cells

Hyperplasia and hypertrophy of fat cells can be found in obesity and increased adiposity is associated with endothelial dysfunction as an early event of atherosclerosis. However, it is unclear whether human adipocytes directly influence endothelial protein secretion. To study the crosstalk between fat and endothelial cells, human umbilical venous endothelial cells (HUVECs) were cultured in infranatants (Adipo) of primary differentiated human adipocytes. Interestingly, significantly increased secretion of 23 cytokines and chemokines from HUVECs was detected in four independent experiments after Adipo stimulation by protein array analysis detecting a total of 174 different proteins. Among those, time‐dependent Adipo‐induced upregulation of cytokine secretion in HUVECs was confirmed by ELISA for interleukin (IL)‐8, monokine induced by gamma interferon, macrophage inflammatory protein (MIP)‐1β, MIP‐3α, monocyte chemoattractant protein‐1, and IL‐6. Factors besides adiponectin, leptin, resistin, and tumor necrosis factor α appear to mediate these stimulatory effects. Our findings suggest that endothelial cell secretion is significantly influenced towards a proinflammatory pattern by adipocyte‐secreted factors. J. Cell. Biochem. 106: 729–737, 2009. © 2009 Wiley‐Liss, Inc.     

Electron immunocytochemical localization of pepsinogen I (PgI) in chief cells,mucous-neck cells and transitional mucous-neck/chief cells,of the human fundic mucosa

Summary Specific PgI antibodies devoid of PgII cross reactivity have been applied to aldehyde-osmium fixed human, fundic-type, gastric mucosa investigated with the protein A-immunogold technique. PgI immunoreactivity has been detected in the homogeneous secretory granules of glandular chief cells, in bipartite granules of mucous-neck cells, in the granules of cells showing intermediate patterns and topography in between chief and mucous-neck cells (transitional cells), as well as in the granules of a few cells in the foveolar/mucous-neck boundary zone showing mixed foveolar/mucous-neck granule populations. The findings support progressive transformation of mucous-neck cells into chief cells.     

Impaired aldosterone secretion from dispersed adrenal capsular cells of chronically alpha-MSH-treated rats

To investigate the chronic effects of alpha-melanocyte-stimulating hormone (alpha-MSH) on aldosterone secretion, synthetic alpha-MSH (8 micrograms/day) was infused in Sprague-Dawley rats by miniosmotic pumps for 5 days. Saline was infused in equivalent volume for 5 days using the same type of pumps in the control group of rats. Aldosterone secretion from the capsular cells of the two groups was examined in the basal state and in response to various stimuli of aldosterone secretion. Aldosterone secretion in vitro from the alpha-MSH-treated rats was significantly impaired in response to all stimuli tested including cyclic AMP, suggesting an intracellular defect in aldosterone synthesis in that group. These results are similar to those observed after chronic adrenocorticotropin administration.