Cellular cyclic AMP in dispersed mucosal cells from guinea pig stomach.

In dispersed mucosal cells from guinea pig stomach cyclic AMP was increased 4-fold by theophylline, 5-fold by prostaglandin E2, and 10- to 15-fold by histamine. Theophylline augmented the increase in cellular cyclic AMP caused by histamine or prostaglandin E1 and the actions of histamine and prostaglandin E1 were additive. Cellular cyclic AMP was not altered by carbachol, gastrin, secretin, vasoactive intestinal peptide, glucagon, insulin or the octapeptide of cholecystokinin. Metiamide or diphenhydramine but not atropine inhibited the increase in cellular cyclic AMP caused by histamine, but did not alter the concentration of cyclic AMP in control cells or in cells incubated with theophylline or prostaglandin E1.     

Secretion of intrinsic factor from dispersed mucosal cells isolated from guinea pig and rabbit stomach

In dispersed mucosal cells prepared from rabbit and guinea pig stomach, the secretion of intrinsic factor was constant (0.3–0.4%/min) for at least 30 min incubation at 37°C. Histamine or isobutyl methylxanthine increased cyclic AMP and intrinsic factor secretion in both cell preparations. Isobutyl methylxanthine potentiated and cimetidine competitively inhibited (K_i=5·10^?7 M) both effects of histamine. Dibutyryl cyclic AMP (1.0 mM), also caused a 3-fold increase in intrinsic factor secretion. These results suggest that in rabbit and guinea pig histamine interacts with H₂-receptors to increase cyclic AMP which mediates the rise in the rate of intrinsic factor secretion.     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Direct action of somatostatin on dispersed mucosal cells from guinea-pig stomach

In dispersed mucosal cells from guinea-pig stomach, somatostatin inhibited in a noncompetitive fashion (Ki, 2 x 10(-8) M) the increase in cellular cyclic AMP caused by histamine but not by prostaglandin E1 or phosphodiesterase inhibitors. Somatostatin also inhibited the increase in [14C]aminopyrine uptake caused by low concentrations of histamine probably by interfering with the synthesis of cellular cyclic AMP.     

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle

Summary Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca²⁺ uptake into and Ca²⁺ release from the membrane vesicles were studied by a rapid filtration method, and Ca²⁺ transport properties of the different regions of the stomach were compared. ATP-dependent Ca²⁺ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN₃. Oxalate affected Ca²⁺ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100mm NaCl and then diluted to 1/20 concentration with Na⁺-free medium had significantly higher ATP-independent Ca²⁺ uptake than vesicles preincubated in 100mm KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na⁺-dependent Ca²⁺ uptake, and NaCl enhanced Ca²⁺ efflux from fundus microsome vesicles. The halflife values of Ca²⁺ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca²⁺ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had ro effect on Ca²⁺ release from antrum microsome vesicles.Results suggest two distinct mechanisms of stomach membrane Ca²⁺ transport: (1) ATP-dependent Ca²⁺ uptake and (2) Na⁺–Ca²⁺ exchange; the latter in the fundus only.     

Gastric irritant-induced apoptosis in guinea pig gastric mucosal cells in primary culture

When the gastric mucosa is exposed to various irritants, apoptosis and subsequent gastric mucosal lesion can result in vivo. We here show that gastric irritants induced apoptosis in gastric mucosal cells in primary culture and examined its molecular mechanism. Ethanol, hydrogen peroxide, and hydrochloric acid all induced, in a dose-dependent manner, cell death, apoptotic DNA fragmentation, and chromatin condensation, suggesting that each of these gastric irritants induced apoptosis in vitro. Since each of these irritants decreased the mitochondrial membrane potential and stimulated the release of cytochrome c from mitochondria, gastric irritant-induced apoptosis seems to be mediated by mitochondrial dysfunction. Caspase-3, caspase-8, and caspase-9-like activities were all activated simultaneously by each of these irritants and the activation was concomitantly with cell death and apoptotic DNA fragmentation. Furthermore, pre-treatment of gastric mucosal cells with an inhibitor of caspase-8 suppressed the onset of cell death as well as the stimulation of caspase-3- and caspase-9-like activities caused by each of these gastric irritants. Based on these results, we consider that caspase-8, an initiator caspase, plays an important role in gastric irritant-induced apoptosis.     

Comparison of mammalian VIP bioactivities in dispersed acini from guinea pig pancreas

Mammalian VIP is identical in pig, cow, human, rat, dog and goat but differs in the guinea pig (GP) in positions 5, 9, 19, and 26. We now demonstrate that GP, goat, rat and synthetic mammalian VIP are indistinguishable in their inhibition of binding of 125I-labelled synthetic VIP to dispersed acini from GP pancreas and that GP, pig, dog, goat and synthetic VIP are also similar in their efficacy and potency in stimulating amylase release from these acini. Thus in spite of the differences in amino acid sequence, GP VIP appears to have full biologic potency in its action on dispersed acini from GP pancreas.     

The actions of tetraethylammonium on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas, replacing extracellular sodium by tetraethylammonium (1) abolished carbamylcholine-stimulated amylase secretion but did not alter the increase in amylase secretion caused by the C-terminal octapeptide of cholecystokinin, bombesin, ionophore A23187, vasoactive intestinal peptide or 8-bromoadenosine 3':5' monophosphate, (2) caused a parallel rightward shift in the dose-response curve for carbamylcholine-stimulated amylase secretion and (3) inhibited binding of N-[3H]methyl scopolamine to muscarinic cholinergic receptors. Detectable inhibition of carbamylcholine-stimulated amylase secretion and binding of N-[3H]methyl scopolamine occurred with 300 microM tetraethylammonium, and half-maximal inhibition of these functions occurred with 1-2 mM tetraethylammonium. Replacing extracellular sodium by Tris did not alter the stimulation of enzyme secretion caused by any secretagogue tested. These results indicate that the tetraethylammonium is a muscarinic cholinergic receptor antagonist and that enzyme secretion from pancreatic acini does not depend on extracellular sodium.     

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of ⁴⁵Ca or cellular cyclic AMP. Binding of ¹²⁵I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of ¹²⁵I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in ⁴⁵Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Potassium currents regulating secretion from Brunner's glands in guinea pig duodenum

This study examined the role of outward K(+) currents in the acinar cells underlying secretion from Brunner's glands in guinea pig duodenum. Intracellular recordings were made from single acinar cells in intact acini in in vitro submucosal preparations, and videomicroscopy was employed in the same preparation to correlate these measures with secretion. Mean resting membrane potential was -74 mV and was depolarized by high external K(+) (20 mM) and the K(+) channel blockers 4-aminopyridine (4-AP), quinine, and clotrimazole. The cholinergic agonist carbachol (60-2,000 nM; EC(50) = 200 nM) caused a concentration-dependent initial hyperpolarization of the membrane and an associated decrease in input resistance. This hyperpolarization was significantly decreased by 20 mM external K(+) or membrane hyperpolarization and increased by 1 mM external K(+) or membrane depolarization. It was blocked by the K(+) channel blockers tetraethylammonium (TEA), 4-AP, quinine, and clotrimazole but not iberiotoxin. When videomicroscopy was employed to measure dilation of acinar lumen in the same preparation, carbachol-evoked dilations were altered in a parallel fashion when external K(+) was altered. The dilations were also blocked by the K(+) channel blockers TEA, 4-AP, quinine, and clotrimazole but not iberiotoxin. These findings suggest that activation of outward K(+) currents is fundamental to the initiation of secretion from these glands, consistent with the model of K(+) efflux from the basolateral membrane providing the driving force for secretion. The pharmacological profile suggests that these K(+) channels belong to the intermediate conductance group.     

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.     

Helicobacter pylori lipopolysaccharide induces apoptosis of cultured guinea pig gastric mucosal cells

Helicobacter pylori lipopolysaccharide (LPS) is generally accepted as a low-toxicity virulence. Primary cultures of guinea pig gastric mucosal cells expressed the Toll-like receptor 4 and were sensitive to H. pylori LPS as well as Escherichia coli LPS. H. pylori LPS stimulated phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and c-Jun NH(2)-terminal kinase (JNK) 2. H. pylori LPS at >2.1 endotoxin unit/ml (>1 ng/ml) activated caspase-8, stimulated cytochrome c release from mitochondria, and subsequently activated caspases-9 and -3, leading to apoptosis. Epidermal growth factor blocked all of these apoptotic processes and inhibited apoptosis, whereas it did not modify the phosphorylation of TAK1, TAB1, and JNK2. A comparatively specific inhibitor of caspase-8 or -9 blocked apoptosis, whereas cytochrome c release was prevented only with a caspase-8-like inhibitor. Our results suggest that caspase-8 and mitochondria may play crucial roles in H. pylori LPS-induced apoptosis and that this accelerated apoptosis may be involved in abnormal cell turnover of H. pylori-infected gastric mucosa.     

Action of cholera toxin on dispersed acini from guinea pig pancreas.

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.     

Calcium transport in dispersed acinar cells from rat pancreas

The present studies were performed to attempt to elucidate the basis for the discrepancy between results of Kondo and Schulz (1976, Biochim. Biophys Acta 419, 76–92), who found that cholecystokinin and cholinergic agents increase uptake of ⁴⁵Ca by dispersed acinar cells from rat pancreas, and results of others (Matthews, E.K., Petersen, O.H. and Williams, J.A. (1973) J. Physiol. 234, 689–701; Chandler, D.E. and Williams, J.A. (1974) J. Physiol. 243, 831–846; Case, R.M. and Clausen, T. (1973) J. Physiol. 235, 75–102; Gardner, J.D., Conlon, T.P., Klaeveman, H.L., Adams, T.D. and Ondetti, M.A. (1975) J. Clin. Invest. 56, 366–375; Christophe, J.P., Frandsen, E.K., Conlon, T.P., Krishna, G. and Gardner, J.D. (1976) J. Biol. Chem. 251, 4640–4645; Shelby, H.T., Gross, L.P., Lichty, P. and Gardner, J.D. (1976) J. Clin. Invest. 58, 1482–1493 and Deschodt-Lanckman, M., Robberecht, P., de Neef, P., Lammens, M. and Christophe, J. (1976) J. Clin. Invest. 58, 891–898). They have reported that cholecystokinin and cholinergic agents do not alter or cause a slight decrease in uptake of ⁴⁵Ca by pancreatic acinar cells. Our present results indicate that increased uptake of ⁴⁵Ca by acinar cells incubated with cholecystokinin occurs only in cells washed with iced, 160 mM choline chloride and reflects increased cellular uptake of radioactivity from the wash solution but not from the incubation medium. We detected no effect of cholecystokinin on uptake of ⁴⁵Ca by cells washed with 160 mM choline chloride containing 5 mM ethylenediaminetetraacetate or by cells washed with Krebs-Ringer bicarbonate. Furthermore, cells washed with 160 mM choline chloride accumulated a substantial amount of ⁴⁵Ca from the wash solution and this accumulation was increased in cells that had been preincubated with cholecystokinin. Cells washed with Krebs-Ringer bicarbonate did not take up ⁴⁵Ca from the wash solution.     

Neuroimmune interactions in guinea pig stomach and small intestine

Enteric neuroimmune interactions in gastrointestinal hypersensitivity responses involve antigen detection by mast cells, mast cell degranulation, release of chemical mediators, and modulatory actions of the mediators on the enteric nervous system (ENS). Electrophysiological methods were used to investigate electrical and synaptic behavior of neurons in the stomach and small intestine during exposure to beta-lactoglobulin in guinea pigs sensitized to cow's milk. Application of beta-lactoglobulin to sensitized preparations depolarized the membrane potential and increased neuronal excitability in small intestinal neurons but not in gastric neurons. Effects on membrane potential and excitability in the small intestine were suppressed by the mast cell stabilizing drug ketotifen, the histamine H(2) receptor antagonist cimetidine, the cyclooxygenase inhibitor piroxicam, and the 5-lipoxygenase inhibitor caffeic acid. Unlike small intestinal ganglion cells, gastric myenteric neurons did not respond to histamine applied exogenously. Antigenic exposure suppressed noradrenergic inhibitory neurotransmission in the small intestinal submucosal plexus. The histamine H(3) receptor antagonist thioperamide and piroxicam, but not caffeic acid, prevented the allergic suppression of noradrenergic inhibitory neurotransmission. Antigenic stimulation of neuronal excitability and suppression of synaptic transmission occurred only in milk-sensitized animals. Results suggest that signaling between mast cells and the ENS underlies intestinal, but not gastric, anaphylactic responses associated with food allergies. Histamine, prostaglandins, and leukotrienes are paracrine signals in the communication pathway from mast cells to the small intestinal ENS.     

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.     

Cytosolic RAB3D is associated with RAB escort protein (REP), not RAB-GDP dissociation inhibitor (GDI), in dispersed chief cells from guinea pig stomach

Rab3D, a low-molecular-weight GTP-binding protein believed to be involved with regulated exocytosis, is associated with secretory granules in gastric chief cells. Although Rab3D is predominantly membrane associated, a significant fraction is cytosolic. Rab proteins are geranylgeranylated on their C-terminal cysteine motifs by geranylgeranyltransferase (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase and may accompany newly modified Rab proteins to their target membrane. In most tissues, cytosolic Rab proteins are complexed with rab-GDP dissociation inhibitor (rab-GDI). In the present study, we examined the interactions of Rab3D with cytosolic proteins in dispersed chief cells. Two REP isoforms and at least two GDI isoforms are present in chief cell and brain cytosol. When chief cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted with REP at >150 kDa, whereas rab-GDI eluted as a separate 65-kDa peak, suggesting that Rab3D exists as a complex with REP, but not with rab-GDI. In addition, a small fraction of Rab3D eluted as a monomer at 29 kDa. As has been demonstrated previously, in brain cytosol, Rab3 proteins co-elute with rab-GDI at approx. 90 kDa, suggesting that Rab3 proteins undergo active cycling between membrane and cytosolic compartments in this tissue. In vitro experiments revealed that Rab3D remains associated with REP after geranylgeranylation. Our findings suggest that, in gastric chief cells, Rab3D remains associated with REP after geranylgeranylation until it is presented to its target membrane.     

Na+ transport processes in isolated guinea pig nasal gland acinar cells

In the dispersed acinar cells of the submucosal nasal gland in the guinea pig, intracellular Na⁺ concentration ([Na⁺]i) was measured with a microfluorimetric imaging method and the cytosolic indicator dye, sodium-binding benzofuran isophthalate, under HCO₃^?-free conditions. In the unstimulated condition, the [Na⁺]i was averaged to 12.8 ± 5.2 mM. Addition of 100 μM ouabain or removal of external K⁺ caused an increase in [Na⁺]i. Replacement of external Cl^? with NO₃^? or addition of 0.5 mM furosemide reversibly decreased the [Na⁺]i. The recovery process from the reduced [Na⁺]i was inhibited by removal of either K⁺ or Cl^? in the bath solution. These findings indicate the presence of a continuous influx of Na⁺ coupled with K⁺ and Cl^? movement. Application of acetylcholine (ACh, 1 μM) caused an increase in [Na⁺]i by about 15–20 mM, which was completely inhibited by addition of 10 μM atropine. Increased cytosolic Na⁺ induced by ACh was extruded by the Na⁺-K⁺ pump. Removal of external Cl^? and addition of 50 μM dimethylamiloride inhibited ACh-induced increase in [Na⁺]i by about 66% and 19%, respectively. In both unstimulated and stimulated state, Na⁺-K⁺ pump, Na-K-Cl cotransport, and Na⁺-H⁺ exchange play a critical role in maintaining intracellular electrolyte environment and in controlling a continuous secretion of nasal fluids. © 1995 Wiley-Liss, Inc.