Non-parietal Cells Research Articles

Production of Ectopic Gastric Intrinsic Factor in Gastric Mucosa of Humans with Chronic Gastritis

Ectopic expression of gastric intrinsic factor (IF) has been described in rodent models of chronic gastritis. The current study undertook to determine if ectopic IF was also present in chronic gastritis in humans and might identify the process of ectopic protein expression as part of the response to chronic injury. Archived biopsies from mid-body, angularis and prepylorus of 9 patients with and without chronic gastritis and food-cobalamin malabsorption were examined in a blinded fashion by immunocytochemistry as were biopsies from 5 normal subjects. Cells with ectopic IF were further examined with antibodies against pepsin or with Griffonia simplicifolia II (GSII) to identity cells in the mucous neck cell compartment. Ectopic IF production in non-parietal cells was identified in cells that were H(+),K(+)-ATPase-negative but IF-positive in 7 of the 9 patients (6/9 in the angularis and/or prepylorus biopsies and 1/9 only in the mid-body). These included 5 of the 6 H. pylori-infected patients and all 5 patients with severe food-cobalamin malabsorption. No normal control subjects demonstrated ectopic IF. The cells with ectopic IF were pepsinogen-positive peptic cells and were not GSII-positive. Expression was most extensive in patients and gastric regions with inflammation. In all but one sample, ectopic IF was observed near anatomical mucosal junctions, such as antral/body and prepylorus/duodenum junctions. These data in humans with and without gastritis are consistent with the hypothesis that local factors influence ectopic gastric IF expression, arising from either the anatomical location, the focal inflammation, or both.

Cell proliferation in the gastric epithelium of the ulcer rat

Objective. Cell division is brisk in the ulcer margin and many of the new cells will migrate over and cover the ulcer bed. The aim of this study was to determine how agents that promote or delay gastric ulcer healing influence cell proliferation in the gastric epithelium. Material and methods. Acetic acid ulcers were produced in the rat gastric corpus; non-ulcer rats served as controls. All rats were given a continuous infusion of 3H-thymidine. Some rats were also given gastrin or indomethacin, or infected with Helicobacter pylori. The rats were killed after 1, 2, 6 or 13 days, and the ulcer margin and undamaged corpus were excised for determination of labeling index (LI) by autoradiography. Antrum, duodenum and colon were also studied. Silver grain counting was carried out in some groups. Results. LI in the ulcer margin grew exponentially, reaching 84% after 6 days; gastrin increased, and indomethacin decreased LI significantly. In 6-day ulcer rats that were given 3H-thymidine only during the first day LI was 5%, while in those given 3H-thymidine only during the last day LI was 27%. LI and silver grain counting results indicated that during the first 6 days of healing the epithelial cells in the ulcer margin divide twice. In the undamaged epithelium of the 1-day ulcer rats LI was <one-third, and in the antral epithelium one-half of that in the non-ulcer controls. Conclusions. During the first 6 days of healing, the LI of the non-parietal epithelial cells increases exponentially in the ulcer margin; this is enhanced by gastrin. The unexpected finding in the ulcer rats of decreased LI in undamaged oxyntic and antral epithelium may be caused by a block of the G1 restriction point in the cell cycle. The reason for this remains obscure.

Possible role of histamine chloramines in delayed termination of gastric acid secretion

The presence of H, pylori is associated with a mucosal inflammatory reaction that consists of a large number of polymorphonuclear inflammatory cells. Histamine plays an important role in the control of gastric acid secretion. Recently, chlorinated derivatives of histamine that is produced by chemical reaction with neutrophil metabolite, hypochlorous acid, have been identified as having multiple effects on the intestinal tract. We compared the effects of histamine and histamine chloramines on the H1 and H2 receptors, using guinea pigs' ileum (HI) and atrium (H2) in vitro,and on the gastric acid secretion in rive in rats, and studied binding to H2 receptors in vitro. With respect tothe effects on HI and H2 receptors, histamine monochloramine (HisCl) showed agonlst effects similar to those seen with histamine, Histamine dichloramine (HisCI2) had effects on H1 receptors similar to those seen with histamine, but the agonist effects on H2 receptors were of about 1/3 of histamine. However, unlike histamine, the H2 agonist effects of HisC1 and HisC12 did not disappear after twice repeated wash out and exchanging of the nutrient solution. With respect to the effect on gastric acid secretion in vivo, the effect of HisC1 was similar to that of histamine, while the effect of HisCl2 was 38% of histamine. Binding by H2 receptors of HisCl and HisCl2 was observed in H2 receptor binding assay. Conclusion: The time to return to the basal secretory level after completion of stimulation by HisCl or HisC12 was significantly prolonged compared with histamine. These results suggest that histamine chloramines, which strongly bind with H2 receptors, delay the termination of gastric acid secretion. A TRANSCRIPTIONAL REPRF~SOR MEDIATES CELL TYPESPECIFIC EXPRESSION OF THE CANINE H+,K+-ATPase aSUBUNIT GENE. A. Muraoka, M. Kaise, J. Yamada, Y.-J. Guo, I. Song, T. Yamada. Dept. int. Med., U. Mich. Meal. Ctr., Arm Arbor, MI. We have reported previously that the nuclear transcription factor Spl is critically imlxn'tant for transcriptional activity of the canine H+,K+-ATPase wsubunit gene promoter as elimination of Spl binding diminishes basal transcriptional activity by 80%. The observation that Spl is ubiquitously expressed while the expression of H+,K+-ATPase is restricted in a tissue specific manner to gastric parietal ceils suggests that in non-parietal cells Spl-conferred transcriptional activity is attenuated by one or more negative cis-regulatory elements. In order to explore this possibility, we examined H+,K+-ATPase tx-subunit gene promoter activity in both cultured canine parietal ceils and in non-parietal cell lines employing a chimeric construct using the luciferase gene as a reporter. As before, potent Spl-driven basal transcriptional activity was detected between bases -54 to -45 relative to the transcription initiation site in all cell types examined. In parietal cells, addition of 5'-upstreana segments up to 4kb in length consistently increased transcriptional activity. However, in non-parietal cells, transcriptional activity was repressed by addition of 5'-upstream segments beyond base -69. For example, the promoter activity observed in MDCK (kidney) cells with a segment extending to base -111 was only 30% of that observed with a segment extending only to base -69. In order to confin'n represser activity in this region, we utilized luciferase constructs comprised of the putative represser element (-111 to -69) ligated either to the native H÷,K+-ATPase mimimal promoter (-54 to +34 relative to the Cap site) or to the thymidine kinase promoter. In parietal cells, luciferase activity was not repressed with either construct but in non-parietal cells, repression was noted with both. To characterize the putative represser element, gel mobility shift assays were performed by co-incubating the 32P-labeled DNA segment in question (bases -111 to -69) with nuclear extracts from various cell types and a distinctive protein was bound by the probe, although the sizes of the proteins from the parietal and non-parietal cells were different. Gel shift assays using sequentially mutated unlabeled oligonuclcotides as competitors disclosed a single represser binding element residing between bases -102 to -94 (5'-GAGGTCACT-3') which was recognized by nuclear proteins from both parietal and non-parietal cells. These results indicate that attenuation of Spl-driven transcription through the binding of a represser protein to a negative cis-regulatory element in the gene promoter may account, at least in part, for cell type-specific expression of the H+,K+-ATPase ~t-subunit gene. The degree of repression may depend on the characteristics of the represser protein which binds to this sequence.

Gastrin action on aminopyrine accumulation in isolated pig parietal cells requires cAMP

The mechanism of action of gastrin on pig parietal cells was investigated. The aminopyrine accumulation technique was used to estimate acid production in gastric mucosal cells, containing 10–20% parietal cells, and in enriched parietal cells, containing 65–95% parietal cells. The gastrin analogue pentagastrin stimulated aminopyrine accumulation in a dose-dependent fashion irrespective of the proportion of non-parietal cells present. The apparent EC 50 for pentagastrin was 5 nM and the maximally effective concentration was 100 nM. The histamine H 2-receptor antagonist ranitidine did not affect the action of pentagastrin. The stimulatory effects of various doses of histamine on aminopyrine accumulation in highly enriched parietal cells were potentiated by the inclusion of 100 nM pentagastrin in the incubation medium. In another series of experiments using mucosal cells, the action of effective doses of pentagastrin were potentiated by the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX), which alone elicited an aminopyrine accumulation equal to 50% of that obtained by 100 μM histamine. When ranitidine (100 μM) was included, the action of IBMX was almost completely abolished. However, the dose-response curve for pentagastrin in the presence of ranitidine plus IBMX was similar to that obtained in the absence of IBMX. Dibutyryl-cAMP (DBcAMP, 1 mM) in the presence of ranitidine (100 μM) also potentiated the action of all effective doses of pentagastrin on mucosal cells. The protein kinase A inhibitor Rp-cAMPS, present at 500 μM in the incubation medium, significantly reduced the action of each effective concentration of pentagastrin on aminopyrine accumulation in enriched parietal cells. These results in pig parietal cells were interpreted as indicative of: (i) an action of gastrin exerted directly on the parietal cells; (ii) elevation of intracellular cAMP having a permissive role in the action of gastrin on aminopyrine accumulation.

Biological effects of human gastrin I and II chemically modified at the [formula omitted] tetrapeptide amide

Binding to gastrin receptors and gastric acid secretion experiments were performed with gastrin derivatives modified at the C- terminal tetrapeptide amide from HG-13 sequence. 1. When the ultimate phenylalanine amide was replaced by a phenethylester or a phenetylamide moiety, the resulting compound bound to gastrin receptors ( K d ≈ 10 nM ) and exhibited antagonist activity on gastrin-induced acid secretion in the anesthetized rat. 2. Changing the peptide bond between Trp and Leu residues to a -Ψ(CH 2-NH)- bond resulted in a compound which also bound to gastrin receptors ( K d ≈ 10 nM ) but presented agonist activity on acid secretion in the rat. In contrast, when the peptide bond between Leu and Asp residues was replaced by a -Ψ(CH 2-NH)- bond, the resulting compound was devoid of any affinity for gastrin receptor ( K d > 10 −6 M ) and of any biological activity. 3. The HG-13 derivatives were synthesized in sulfated and unsulfated forms: O-sulfation of the HG-13 tyrosine residue did not change its intrinsic in vivo activity but enhanced its affinity for gastrin receptors ( K d ≈ 0.3 nM ). On the contrary, O-sulfation of the various chemically modified HG-13 had no significant effect in either in vitro or in vivo experiments. 4. Finally, no significant difference between binding on parietal (F3) and nonparietal (F1) cells was observed, in agreement with the presence of a gastrin-type receptor in these two cell populations.

Effects of histamine and activators of the cyclic AMP system on protein synthesis in and release of high molecular weight glycoproteins from isolated gastric non‐parietal cells

1. Glycoprotein and protein synthesis in and release from pig isolated, enriched gastric mucous cells were measured by the incorporation of N-acetyl-[14C]-D-glucosamine and [3H]-L-leucine, respectively, into cellular and released acid precipitable material. 2. Histamine and activators of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) system maximally stimulated total protein and glycoprotein synthesis in and release from the cells at concentrations of histamine (10 microM), forskolin (10-100 microM), 3-isobutyl-1-methylxanthine (100 microM), and dibutyryl cyclic AMP (1-3 mM), respectively. In the presence of 3-isobutyl-1-methylxanthine (30 microM) histamine stimulation was enhanced. 3. As shown by gel chromatography, stimulation by histamine (100 microM), forskolin (10 microM), 3-isobutyl-1-methylxanthine (100 microM) and dibutyryl cyclic AMP (1 mM) resulted in a release of high molecular weight (approximately 2 x 10(6) daltons) glycoproteins from the cells. The histamine H2-receptor antagonist, ranitidine (100 microM), blocked the effect of histamine. 4. We conclude that cyclic AMP-dependent processes are involved in the regulation of protein and glycoprotein synthesis in and the release of high molecular weight (mucous) glycoproteins from pig gastric non-parietal cells and that histamine may be a physiological activator of this system.

“Gastrin” and “CCK” receptors on histamine-and somatostatin-containing cells from rabbit fundic mucosa —I: Characterization by means of agonists

A previous study has suggested the presence of two distinct binding sites for gasrin and cholecystokinin (CCK) in isolated non-parietal cells from rabbit gastric mucosa: a receptor which binds CCK-8 and CCK-39 with a high affinity and a receptor which binds gastrin and CCK-8 with the same high affinity and CCK-39 with a lower affinity. To characterize these receptors, their ability to induce phosphoinositide breakdown was investigated. Gastrin (HG-17), CCK-39 and CCK-8 induced [ 3H]-inositol phosphate ([ 3H]InsP) accumulation from [ 3H]inositol prelabelled cells with a high potency ( ec 50: 0.3–2.7 nM) but CCK-8 exhibited a higher efficacy than HG-17 or CCK-39. HG-17, CCK-8 and CCK-39 induced a rapid accumulation of [ 3H]inositol monophosphate ([ 3H]InsP 1), [ 3H]inositol bisphosphate ([ 3H]InsP 2) and [ 3H]inositol trisphosphate ([ 3H]InsP 3) but CCK-8 caused a two times higher accumulation than HG-17 or CCK-39. Histamine- and somatostatin-containing cells appeared to be located in this non-parietal cells population. HG-17, CCK-8 and CCK-39 dose-dependently induced histamine release with the following order of potency: HG-17 = CCK-8 ( ec 50 ≈ 0.2 nM) > CCK-39 ( ec 50 ≈ 4 nM) . In addition, HG-17 exhibited the highest efficacy. HG-17, CCK-8 and CCK-39 enhanced somatostatin-like immunoreactivity (SLI) release with the following order of potency: CCK-8 ( ec 50 ≈ 0.1 nM) = CCK-39 > HG-17 ( ec 50 ≈ 10 nM) ; CCK-8 and CCK-39 exhibited the highest efficacy. These results led us to the following conclusions: (i) existence of a “gastrin-type” and of a “CCK-type” receptor mediating phosphoinositide breakdown in these gastric non-parietal cells. CCK-8 interacts with both receptor-types with the same affinity; (ii) the release of histamine from histaminecontaining cells could be induced following “gastrin-type” receptors activation; (iii) somatostatin release from D-cells present in this non-parietal cells population could be induced following “CCK-type” receptors activation.

“Gastrin” and “CCK” receptors on histamine-and somatostatin-containing cells from rabbit fundic mucosa—II: Characterization by means of selective antagonists (L-364, 718 and L-365, 260)

In the preceding paper, by means of selective agonists to gastrin (HG-17) and cholecystokinin (CCK-39), we evidenced the existence of “gastrin-type” receptors that could regulate histamine release and “CCK-type” receptors that could stimulate somatostatin release in isolated rabbit fundic nonparietal cells (F1 cells). Furthermore, these receptors could induce phosphoinositide breakdown. To confirm the involvement of these receptor types in these biological and biochemical processes, we used selective antagonists, L-364,718 (3-(benzoylamino)-benzodiazepine) specific to “CCK-A-type” receptor and L-365,260 (3-(acylamino)-benzodiazepine) specific to “gastrin/CCK-B-type” receptor. Neither L-364,718 nor L-365,260 alone caused any significant stimulation of [ 3H]inositol phosphate ([ 3H] InsP) production and release of histamine or somatostatin-like immunoreactivity (SLI). Each analogue inhibited in a dose-dependent manner [ 125I] HG-17 or [ 125I]CCK-39 binding to F1 cells, [ 3H]InsP accumulation and histamine and SLI release stimulated by HG-17 or CCK-39. L-365, 260 appeared to be 30–70 times more potent than L-364,718 in inhibiting [ 125I]HG-17 binding to F1 cells, as well as HG-17-induced [ 3H]InsP accumulation and HG-17- or CCK-39-enhanced histamine release ( ic 50 values: ≈5–20 nM for L-365,260 and ≈200–1500 nM for L-364,718 ). In contrast, L-364,718 was 200 to 400 times more potent than L-365,260 in inhibiting [ 125I]CCK-39 binding to F1 cells, CCK-39-induced [ 3H]-InsP accumulation and SLI release stimulated by CCK-39 or HG-17 ( ic 50 values: ≈0.3–1 nM for L-364, 718 and 100–200 nM for L-365,260 ). These results led to conclude: (i) the existence of a “gastrintype” receptor related to histamine release; (ii) the existence of a “CCK-A-type” receptor related to somatostatin release; (iii) the existence of “gastrin type” and “CCK-A-type” receptors linked to the phosphoinositide breakdown pathway.

Aspects of the Role of Prostaglandins in Gastrin Histamine Regulation of Gastric Acid Secretion

There is convincing evidence from in vitro studies that prostaglandins interfere with the gastrin histamine regulation of gastric acid secretion in an opposing manner. They can inhibit the action of histamine on the parietal cell when given as single-dose treatment. Conversely, when given at high doses, they liberate endogenous histamine, probably from nonparietal cells. In in vivo experiments prostaglandins are potent inhibitors of acid secretion when studied with single-dose treatment but are capable of stimulating basal acid secretion when chronically applied to rats. Studies performed in whole animal preparations on the effect of cyclooxygenase inhibitors have not fully clarified which of these two histamine-modulating effects of prostaglandins is of prime physiologic significance but favour suppression of histamine activity with subsequent acid inhibition. It appears that somatostatin release is the mechanism through which prostaglandin can simultaneously inhibit acid secretion and release plasma gastrin. At least in the rat, the pattern of plasma gastrin and somatostatin release is, similarly to acid secretion, partly reversed during prolonged prostaglandin treatment.

Effects of PGE2, carbenoxolone, cholecystokinin, and the thromboxane‐mimetic u46619 on protein and glycoprotein production by isolated pig gastric mucosal cells

We studied the effects of prostaglandin (PG) E2, carbenoxolone, cholecystokinin-octapeptide (CCK-OP), and the thromboxane-mimetic U46619, all known to stimulate gastric mucus secretion in vivo, on protein and glycoprotein synthesis in and release from isolated and enriched pig gastric mucous cells, as measured by the incorporation of [3H]L-leucine and N-acetyl-[14C]D-glucosamine respectively into cellular and released acid insoluble material. PGE2 stimulated glycoprotein and protein synthesis (EC50 7 and 30 nmol/L, respectively) and release (EC50 50 and 140 nmol/L, respectively) in a concentration-dependent manner, whereas carbenoxolone, CCK-OP and U46619 failed to enhance the incorporation of the tracers. We conclude that stimulation of mucus secretion by PGE2 is related to direct effects on protein and glycoprotein production of gastric non-parietal cells, whereas indirect effects may be involved in the stimulation by carbenoxolone, CCK-OP, and U46619.

Intracellular coupling of prostaglandin inhibition of acid secretion in isolated rabbit gastric parietal cells

Acid secretion from isolated rabbit gastric parietal cells can be stimulated by gastric secretagogues, histamine (cyclic-AMP pathway) and carbachol (inositol phosphate pathway). Prostaglandins (PG) from E series are potent inhibitors of acid secretion. The intracellular mechanism of this inhibition was examined by using a stable PGE1-analogue, misoprostol. Aminopyrine (AP) accumulations due to histamine, IBMX and forskolin were dose-dependently inhibited by misoprostol, whereas a weak but significant biphasic effect on carbachol-induced AP accumulation was observed. The cyclic-AMP formation induced by histamine and IBMX were also inhibited by misoprostol in a non-competitive way. The potent effect of forskolin on cyclic-AMP levels was not modified by misoprostol in parietal cells, whereas it was potentiated in non-parietal cells. The inhibitory effect of misoprostol on AP accumulation was reduced by incubation of parietal cells with Bordetella pertussis toxin (IAP) but not with Cholera toxin (CT). Pretreatment of the cells with IAP did not alter cyclic-AMP levels of resting and histamine-stimulated parietal cells but abolished the inhibitory effect of misoprostol. Treatment with CT increased basal and histamine-stimulated cyclic-AMP levels and masked the inhibitory effect of misoprostol. The biphasic effect of misoprostol on carbachol-stimulated AP accumulation in parietal cells was confirmed on carbachol-stimulated phospholipase C activity and on [Ca 2+]i stimulated by carbachol. These data confirm a direct and specific effect of the prostanoid on the Gi-subunit of the adenylate cyclase coupled to the histamine H 2-receptor, and a biphasic effect on the phospholipase C pathway of the parietal cells.

Binding of cholecystokinin and somatostatin to isolated porcine gastric mucosal cells and effects on aminopyrine uptake

Mucosal cells were prepared by enzymatic digestion of porcine gastric mucosa with pronase and collagenase. The resulting cell suspension contained 10-15% parietal cells, which responded to histamine stimulation by an up to 20-fold increase in [14C]aminopyrine accumulation over control levels. Cholecystokinin-8 (CCK-8) evoked a more moderate stimulation of [14C]aminopyrine accumulation, whereas somatostatin inhibited histamine-stimulated accumulation. Parietal cells were enriched by elutriation and isopycnic centrifugation on density gradients of Percoll. A fraction with 60% parietal cells bound approximately three times more iodinated CCK-8 than a fraction containing 70% non-parietal cells. Binding of [125I]BH-CCK-8 to preparations containing 30-60% parietal cells was specifically inhibited to about 50% by 10(-9) M unlabelled CCK-8 but not by bombesin. Cell fractions containing about 30% parietal cells also bound [125I]somatostatin. Unlabelled somatostatin at 10(-9) M inhibited tracer binding by about 50%, while CCK-8 did not affect somatostatin binding to such a preparation. The results suggest the existence of specific receptors for CCK and somatostatin on porcine parietal cells exerting a regulatory influence on acid secretion.

Effects of Histamine on Protein and Glycoprotein Production of Isolated Pig Gastric Mucosal Cells

The production of glycoprotein and protein by isolated pig gastric non-parietal cells was measured by incorporation of N-acetyl-[14C]-D-glucosamine [( 14C]GlcNAc) and [3H]-L-leucine [( 3H]Leu), respectively, into acid insoluble material (AIM). Histamine enhanced incorporation of the tracers into cellular and released AIM in a concentration-dependent manner. The H2 receptor antagonist ranitidine completely blocked the effects of histamine (100 mumol/l) on [3H]Leu incorporation into cellular and released AIM (IC50 37 and 32 mumol/l, respectively) but had no inhibitory effect on the 16,16-dimethylprostaglandin-E2 - and forskolin-stimulated tracer incorporation. The H1 receptor antagonist mepyramine did not inhibit the histamine effect. We conclude that histamine is a stimulant of protein, via H2 receptors, and glycoprotein production of isolated pig gastric non-parietal cells.

Common or distinct receptors for gastrin and cholecystokinin in gastric mucosa?

The differentiation between gastrin (HG) and cholecystokinin (CCK) receptors in gastric mucosa was examined on isolated parietal (F3) and non-parietal (F1) cells from rabbit fundic mucosa separated by elutriation. Direct binding assays on enriched cell populations were performed using 125I-labeled HG-17, 125I-labeled CCK-8 and 125I-labeled CCK-39 as probes. (1) On F1 cells, the dissociation constants ( K d) for the two labeled CCKs were nearly the same (62 pM for CCK-8 and 74 pM for CCK-39) but the binding capacity for CCK-8 was 2-times higher than for CCK-39. HG-17 also bound to this cell population, but its K d value was about 2-times higher (110 pM) than that of CCK. The presence of two distinct classes of sites on F1 cells can be suggested from competition studies: one more specific for CCK, which bound CCK-8 and CCK-39 with the same affinity, and another class more specific for gastrin, which bound CCK-8 and HG-17 with the same affinity and CCK-39 with a low affinity. (2) On F3 cells, CCK-8 and HG-17 bound with similar affinities ( K d values 81 pM for CCK-8 and 87 pM for HG-17), but CCK-39 did not specifically bind to this cell population. The presence of a binding site more specific for HG than for CCK on F3 cells was confirmed by competition studies in which CCK-33 competed for binding with labeled HG-17 and labeled CCK-8 with a 50-times lower affinity than the other peptides.

Roles of endogenous leukotrienes in damage caused by ethanol in isolated rat gastric cells.

The activity of prostaglandin (PG) and leukotriene (LT) synthesis and the role of endogenous LT in cellular resistance were assessed in isolated parietal and nonparietal cells of rats. Rat gastric cells were isolated and the fractions rich (F1) and poor (F2) in parietal cells were obtained. In F1, more PGE2, PGI2 (measured as the stable metabolite 6-keto-PGF1 alpha), and sulfidopeptide (SP)-LTs and less thromboxane A2 (TXA2, measured as the stable metabolite TXB2) were synthesized than in F2. AA861, an inhibitor of 5-lipoxygenase, inhibited the synthesis of SP-LTs in both fractions in a dose-related way. AA861 alone at any concentrations used did not affect the viability of the cells, but prevented the cell damage caused by ethanol in both fractions, the effect of AA861 being inhibited by indomethacin. These results suggest that in rat stomach, PGs and LTs, except TX, are synthesized mainly in parietal cells rather than in nonparietal cells. Endogenous LT may play a crucial role in the development of cellular damage caused by ethanol independently of systemic and extracellular factors. The balance of PG and LT may be important for regulation of cellular resistance.

Prostaglandin E 2 output is greater by isolated rat gastric parietal cells than non-parietal cells

We appreciate the opportunity to respond to the insightful comments of Dr. Gerber (1) on our manuscript (2). We agree with Dr. Gerber's review on the significance of prostaglandins in the maintenance of gastric mucosal integrity and inhibition of gastric acid secretion

Prostaglandin E 2 output by isolated rat gastric parietal cells and non-parietal epithelial cells

Prostaglandins have acid antisecretory and cytoprotective effects in gastric mucosa when given exogenously. This study's purpose was to isolate preparations of parietal and non-parietal cells from rat stomachs and to compare prostaglandin output by these cells. Gastric epithelial cells were isolated from rat stomachs using pronase. Cells from different incubation times were collected separately and enriched by discontinuous Percoll gradient. Cell types were identified by hematoxylin and eosin stain, succinic dehydrogenase activity (parietal cells), periodic acid Schiff staining (mucous cells), Bowie staining (chief cells) and electron microscopy. Prostaglandin E 2 activity was measured by radioimmunoassay. Parietal cells were purified to over 90% while the non-parietal preparation contained 67% chief cells and over 31% mucous cells. By electron-microscopy, cell integrity was seen to be maintained. The parietal cell enriched fraction contained two and one-half times the amount of prostaglandin E 2 that the non-parietal chief cell enriched fraction did, p<0.01. These results raise the question as to whether output of PGE 2 by parietal cells could play a role in modulating gastric acid secretion directly by parietal cells as well as in protecting the deeper layers of gastric mucosa against damaging agents in-vivo.

Binding and biological actions of prostaglandin E2 and I2 in cells isolated from rabbit gastric mucosa.

1. We have investigated the binding of the tritiated forms of prostaglandin E2 (PGE2) and a stable analogue of prostacyclin (Iloprost) to isolated cells of rabbit oxyntic mucosa. 2. The highest degree of specific [3H]PGE2 binding occurred in a cellular fraction enriched in parietal cells. [3H]Iloprost binding occurred predominantly in cells identified as mucous cells. 3. PGE2 binding to the parietal cell fraction was associated with its ability to inhibit histamine-stimulated aminopyrine accumulation by these cells. Iloprost binding did not correlate with a biological action on the parietal cells. 4. PGE2 and Iloprost reduced Trypan Blue staining in cells exposed to 10% (w/v) ethanol. Iloprost (10(-8) to 10(-6) M) reduced Trypan Blue staining in cells identified as mucous and parietal cells. PGE2 (10(-8) M) significantly reduced Trypan Blue staining in parietal cell-enriched fractions. 5. Cyclic AMP stimulation in response to either prostanoid occurred most potently on non-parietal cell fractions. However PGE2 or Iloprost binding affinities did not correlate with cyclic AMP formation. 6. These data provide evidence for true PGE2 receptors on oxyntic mucosal cells. The receptors appear to mediate inhibition of acid secretion. Iloprost binds to sites which might mediate cellular protection.

Calcium, calmodulin, and cyclic adenosine monophosphate modulate prostaglandin E2 release from isolated human gastric mucosal cells.

We studied prostaglandin E2 (PGE2) release from isolated cells of the human gastric mucosa. Mucosal cells were enzymatically isolated from biopsy specimens of human fundic mucosa. The results from these crude cell preparations were compared to those obtained in fractions with enriched (65-80%) or depleted parietal cell content (3-7%) which were prepared from gastric mucosa obtained at surgery. PGE2 release in the enriched parietal cell fractions exceeded that from crude or parietal cell depleted preparations 3- and 13-fold, respectively. However, despite this quantitative difference, all preparations responded similarly to the test agents. Newly synthesized PGE2 was not stored intracellularly but was released into the incubation medium. Release increased linearly for 30 min. Addition of the calcium ionophore A23187 enhanced PGE2 release 4- to 5-fold. The effect of A23187 required the presence of extracellular Ca2+ (10(-3) mol/liter). Assuming that A23187 alters Ca2+ flux in gastric cells as it does in other cell systems our data indicate that increased Ca2+ influx enhances PGE2 release. Since calmodulin is of importance for intracellular Ca2+ action, the calmodulin antagonists trifluoperazine and W7 were tested. Both antagonists inhibited PGE2 release by 65-85%, trifluoperazine being slightly more effective. Activation of the adenylate cyclase system by forskolin or direct addition of (Bu)2cAMP, a stable cAMP-analog, also inhibited PGE2 release. We conclude that PGE2 is released from parietal and from nonparietal cells of the human gastric mucosa, although the major quantity is released from the light density fraction that is enriched in parietal cells. In parietal and nonparietal cells Ca2+ is of importance in the regulation of gastric mucosal PGE2 release and calmodulin seems to mediate this intracellular action of Ca2+. cAMP inhibits PGE2-release from gastric cells.

Mechanisms of action of antisecretory drugs. Studies on isolated canine fundic mucosal cells.

Cellular mechanisms underlying the actions of antisecretory agents were studied with dispersed canine fundic cells; aminopyrine accumulation monitored parietal cell (PC) function. Canine PC have pharmacologically typical histamine (H) H2 and muscarinic (M) receptors. PC also have gastrin (G) receptors, which were selectively blocked by gastrin/CCK antagonists. Potentiating interactions occurred between secretagogues, one of the components of the interdependency between regulatory pathways. Prostaglandins (PG) E2 inhibited H-stimulated PC function. Treatment of PC with pertussis toxin (PT), which inactivates the inhibitory GTP-binding protein of adenylate cyclase (Gi), markedly reduced PG inhibition, indicating PG action via Gi. PC function can also be directly inhibited by H+/K+-ATPase inhibitors, such as omeprazole. When canine mucosal cells were studied, stimulatory G and inhibitory M receptors were present on fundic somatostatin (S) cells. Histamine was localized to canine fundic mast cells, which lacked G or M receptors, a conclusion that may not pertain to fundic histamine cells in other species. Nonparietal cell receptors may be important modulators of the regulation of acid secretion.