Reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria.

Gastric acid exerts a feedback inhibition on the secretion of gastrin from antral G cells. This study examines whether gastrin gene expression is also regulated by changes in gastric pH. Achlorhydria was induced in rats by the gastric H+/K+ ATPase inhibitor, omeprazole (100 mumol/kg). This resulted in fourfold increases in both serum gastrin (within 2 h) and gastrin mRNA levels (after 24 h). Antral somatostatin D cells probably act as chemoreceptors for gastric acid to mediate a paracrine inhibition on gastrin secretion from adjacent G cells. Omeprazole-induced achlorhydria reduced D-cell activity as shown by a threefold decrease in antral somatostatin mRNA levels that began after 24 h. Exogenous administration of the somatostatin analogue SMS 201-995 (10 micrograms/kg) prevented both the hypergastrinemia and the increase in gastrin mRNA levels caused by omeprazole-induced achlorhydria. Exogenous somatostatin, however, did not influence the decrease in antral somatostatin mRNA levels seen with achlorhydria. These data, therefore, support the hypothesis that antral D cells act as chemoreceptors for changes in gastric pH, and modulates somatostatin secretion and synthesis to mediate a paracrine inhibition on gastrin gene expression in adjacent G cells.     

Augmentation of release of gastric somatostatin-like immunoreactivity by adenosine, adenosine triphosphate and their analogs

The effect of adenosine, adenosine 5'-triphosphate (ATP) and ATP analogs on basal gastric somatostatin-like immunoreactivity (SLI) release was studied using the vascularly perfused rat stomach. The release of gastric SLI was stimulated by adenosine (0.6-60 microM) concentration dependently, while the release of immunoreactive gastrin was inhibited by 1 and 10 microM adenosine. The stimulatory action of adenosine was probably mediated by adenosine receptors because the receptor antagonist, 8-phenyltheophylline, abolished the action of adenosine. In addition, the adenosine-induced release of SLI was not mediated by a cholinergic or beta-adrenergic mechanism, since atropine, hexamethonium or propranolol did not block the action of adenosine. Dipyridamole enhanced the adenosine-stimulated, but not 2-chloroadenosine-induced SLI release. Since the adenosine analog, 2-chloroadenosine, is resistant to the adenosine uptake mechanism, it is likely that adenosine-stimulated release of gastric SLI is due to the activation of extracellular receptors. ATP also stimulated gastric SLI release. The analogs alpha,beta-methyleneadenosine triphosphate and diphosphate, which are resistant to metabolic breakdown, did not stimulate basal SLI release, while gamma,beta-methyleneadenosine triphosphate, which can be metabolized, increased the release of SLI. In addition, the ATP-induced release of SLI was abolished by 8-phenyltheophylline. Therefore, the action of ATP is likely to be a result of its metabolism to adenosine.     

Role of circulating somatostatin in regulation of gastric acid secretion, gastrin release, and islet cell function. Studies in healthy subjects and duodenal ulcer patients.

Studies were designed (a) to determine whether somatostatin is released into the circulation after meals in sufficient amounts to regulate gastric or pancreatic islet function in humans and (b) to investigate the possible role of somatostatin in the pathogenesis of duodenal ulcer disease. Mean plasma somatostatin-like immunoreactivity (SLI) increased from 6.2 +/- 1.5 pg/ml to a peak level of 13.8 +/- 1.3 pg/ml in eight healthy subjects after a 1,440-cal steak meal (P less than 0.005). When somatostatin-14 was infused intravenously, basal and food-stimulated gastric acid secretion and also basal and food-simulated plasma insulin and glucagon concentrations were reduced significantly at mean plasma SLI concentrations within the range seen after a meal. Thus, the amount of somatostatin reaching the systemic circulation after a steak meal was sufficient to inhibit gastric acid secretion and islet cell function. On the other hand, basal and food-stimulated plasma gastrin concentrations were reduced by intravenous somatostatin only at plasma SLI concentrations that were several-fold greater than post-prandial SLI concentrations. Although duodenal ulcer patients had significantly higher basal, food-stimulated, and peak pentagastrin-stimulated gastric acid secretion rates than healthy controls, duodenal ulcer patients and controls had nearly identical basal and food-stimulated SLI concentrations. Moreover, food-stimulated gastric acid secretion and gastrin release were inhibited by intravenous somatostatin to the same extent in ulcer patients and controls. These studies suggest that duodenal ulcer patients release normal amounts of somatostatin into the circulation and that target cells controlling acid secretion and gastrin release are normally sensitive to somatostatin in these patients.     

Inhibitory actions of tachykinins and neurokinins on release of somatostatin-like immunoreactivity from the isolated perfused rat stomach

The present experiments were designed to test the effect of tachykinins and neurokinins on release of somatostatin-like immunoreactivity (SLI) from the isolated perfused stomach. Physalaemin, substance P, kassinin, eledoisin, neurokinin A and neurokinin B inhibited basal SLI release in a dose-dependent manner (1 X 10(-9) to 1 X 10(-7) M). Among all the peptides, physalaemin and substance P were the least potent, whereas kassinin, eledoisin, neurokinin A and B were the more potent compounds in suppressing SLI release. It is proposed that this inhibitory action of tachykinins and neurokinins was mediated by the substance P-K or neurokinin-2 receptor subtype in which kassinin, neurokinin A and eledoisin have been shown to be the more potent agonists. The present study also showed that the effect of neurokinin A, neurokinin B and kassinin was not due to an action involving release of acetylcholine as cholinergic antagonists did not block the SLI inhibitory effect. This suggests that these peptides acted directly on somatostatin containing D-cells.     

Increased visualization of antral gastrin-producing G-cells after acute stimulation of gastrin release in the rat

The effect of acute stimulation of gastrin release on the number of antral gastrin-producing G-cells stained by conventional immunohistology was investigated in two experimental models. The substituted benzimidazole derivate BY 308 was administered at a dosage that induces complete achlorhydria and thereby led to marked hypergastrinaemia. Two hours after drug administration, antral G-cell density was increased by 14% and 35% in 12-h and 48-h fasted rats, respectively. Both serum gastrin levels and G-cell density further increased after 3 and 8 days' treatment with BY 308 whereas the somatostatin (D)-cell count did not change prior to 8 days' administration. In the isolated, vascularly perfused rat stomach, acetylcholine was perfused for 36 min; this regimen increased gastrin release four-fold and enhanced the G-cell count by 24% whereas 8 min acetylcholine perfusion did not alter G-cell density significantly but stimulated gastrin output. The results of this study suggest that acute changes in the secretory activity of the gastrin cell are accompanied by an alteration of the staining characteristics thus indicating that an increase in the antral G-cell count does not solely depend upon formation of new cells.     

Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation

The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.     

Cholecystokinin is a physiological regulator of gastric acid secretion in man

Abstract. CCK8 is a poor stimulant of gastric acid secretion in vivo , but is equipotent to gastrin-17 (G17) in in vitro systems. To further evaluate the role of cholecystokinin (CCK) in regulating acid output in humans, dose-response curves were constructed to CCK8 or G17 (6·4–800 pmol kg^-1 per h) with and without a specific CCK-A receptor antagonist (loxiglumide). During loxiglumide infusion, G17-stimulated acid output was unchanged, whereas CCK8-stimulated secretion increased significantly. Gastric somatostatin-14 release increased fivefold with CCK8 alone, but was blocked with loxiglumide administration. These data suggest that CCK8 directly stimulates acid secretion by binding to a CCK-B/gastrin receptor on parietal cells, but at the same time inhibits acid responses by stimulating gastric somatostatin release to a CCK-A receptor-mediated pathway. To test which action of CCK is relevant under physiological circumstances, the effect of loxiglumide on fasting and post-prandial acidity was measured through continuous pH-metry. After eating, gastrin levels increased fourfold compared to controls with concomitant increases in acid secretion. These results suggest that post cibum, CCK is an inhibitor of acid secretion by regulating gastrin through local somatostatin; they support the hypothesis that CCK acts as an enterogastrone.     

Somatostatin-gastrin interactions in the rat stomach

Low concentrations of somatostatin and gastrin within or slightly above the range of physiologically circulating levels were perfused in the isolated, vascularly perfused rat stomach preparation. Somatostatin at 10 and 50 pg/ml significantly inhibited acetylcholine-stimulated gastrin secretion by 26% and 45%, respectively, whereas perfusion of 50 and 500 pg/ml exogenous gastrin did not modify gastric somatostatin secretion. Perfusion of somatostatin-antiserum significantly increased gastrin release by 235%. It is concluded that (1) somatostatin is a powerful inhibitor of the gastrin cell under in vitro conditions; the data are in accordance with a concept that endogenous somatostatin could act as a true hormone; (2) the secretory activity of the somatostatin cell is not significantly affected by circulating gastrin.     

Inhibition of gastrin release by secretin is mediated by somatostatin in cultured rat antral mucosa.

Somatostatin-containing cells have been shown to be in close anatomic proximity to gastrin-producing cells in rat antral mucosa. The present studies were directed to examine the effect of secretin on carbachol-stimulated gastrin release and to assess the potential role of somatostatin in mediating this effect. Rat antral mucosa was cultured at 37 degrees C in Krebs-Henseleit buffer, pH 7.4, gassed with 95% O2-5% CO2. After 1 h the culture medium was decanted and mucosal gastrin and somatostatin were extracted. Carbachol (2.5 X 10(-6) M) in the culture medium increased gastrin level in the medium from 14.1 +/- 2.5 to 26.9 +/- 3.0 ng/mg tissue protein (P less than 0.02), and decreased somatostatin-like immunoreactivity in the medium from 1.91 +/- 0.28 to 0.62 +/- 0.12 ng/mg (P less than 0.01) and extracted mucosal somatostatin-like immunoreactivity from 2.60 +/- 0.30 to 1.52 +/- 0.16 ng/mg (P less than 0.001). Rat antral mucosa was then cultured in the presence of secretin to determine its effect on carbachol-stimulated gastrin release. Inclusion of secretin (10(-9)-10(-7) M) inhibited significantly carbachol-stimulated gastrin release into the medium, decreasing gastrin from 26.9 +/- 3.0 to 13.6 +/- 3.2 ng/mg (10(-9) M secretin) (P less than 0.05), to 11.9 +/- 1.7 ng/mg (10(-8) secretin) (P less than 0.02), and to 10.8 +/- 4.0 ng/mg (10(-7) M secretin) (P less than 0.02). Secretin (10(-7) and 10(-8) M) also increased concomitantly culture medium somatostatin concentration. To determine whether secretion inhibition of carbachol-stimulated gastrin release was mediated by somatostatin, antral mucosa was cultured with carbachol, secretin (10(-9)-10(-7) M), and antibodies to somatostatin. Inclusion of somatostatin antibodies in the culture medium abolished the capacity of secretin (10(-7) and 10(-8) M) to inhibit carbachol-stimulated gastrin release. Results of these studies indicate (a) that secretin inhibits carbachol-stimulated gastrin release and (b) that under the conditions of these experiments secretin inhibition of gastrin release is mediated, at least in part, locally through release of antral somatostatin.     

A1- and A2-selective adenosine antagonists: in vivo characterization of cardiovascular effects

Caffeine, a nonselective adenosine receptor antagonist, 7-methyl-1,3-dipropylxanthine, a purported A2 selective antagonist and a 1,3-dipropyl-8-phenylxanthine amine congener (XAC), an A1 selective antagonist, were evaluated for their in vivo selectivity at A1 vs. A2 adenosine receptors. Blockade of the negative chronotropic effect of bolus i.v. injections of 2-chloroadenosine, R-phenylisopropyladenosine and N-ethylcarboxamidoadenosine was utilized as an index of antagonism at A1 receptors; blockade of the hypotensive effect of the same series of adenosine agonists was used as an index of activity at A2 receptors. In addition, blockade of the potentiating effect of adenosine on the hypertensive and chronotropic effects of nicotine was studied to assess further the role of A1 and A2 adenosine receptors in this response. The potent antagonist XAC displayed considerable A1 selectivity as demonstrated by blockade of adenosine receptor-mediated bradycardia at doses 5- to 10-fold lower than those antagonizing adenosine receptor-mediated hypotension. XAC also selectively blocked potentiation by adenosine of the positive chronotropic effect of nicotine, at doses which had minimal effects on the enhancement of the hypertensive effect of nicotine. The caffeine homolog 7-methyl-1,3-dipropylxanthine exhibited A2 selectivity as demonstrated by prevention of adenosine receptor-mediated hypotension at doses which only minimally attenuated the bradycardiac effect of adenosine analogs. Caffeine displayed no selectivity for A1 vs. A2 adenosine receptors. The results indicate that selective analogs such as XAC and F-methyl-1,3-dipropylxanthine will be useful probes for investigation of receptors involved in the physiological functions of adenosine.     

Gastric antisecretory drugs induce leukocyte-endothelial cell interactions through gastrin release and activation of CCK-2 receptors

Antisecretory drugs are effective antiulcer agents, but its chronic use generates hypergastrinemia and accelerates the development of atrophic gastritis in Helicobacter pylori-positive patients. We have recently shown that gastrin exerts a proinflammatory effect in rats through CCK-2 receptor activation that contributes to the inflammation induced by H. pylori. The present study was designed to examine whether gastrin hypersecretion in response to treatment with antisecretory drugs induces an inflammatory response that could promote mucosal atrophy. The effects of omeprazole or famotidine on leukocyte/endothelial cell interactions in vivo were analyzed in rat mesenteric venules using intravital microscopy. Administration of a single dose of omeprazole or famotidine acutely increased gastrinemia and leukocyte rolling and adhesion, but not emigration into the interstitium. Daily treatment with omeprazole for a short period (3 days) induced a similar response, but when this treatment was extended to 14 days and a steady hyper-gastrinemic state was established, increased leukocyte rolling, adhesion, and emigration was observed. Pretreatment with the CCK-2 receptor antagonist proglumide prevented these inflammatory events in all cases. Leukocytes from rats treated with omeprazole showed increased expression of CD11b/CD18 initially in granulocytes (3-day protocol) and later in monocytes and lymphocytes (14-day protocol). These changes were not observed in animals pretreated with proglumide, and they were not reproduced by incubation of leukocytes from untreated animals in vitro with gastrin. Thus, hypergastrinemia induced by chronic treatment with antisecretory drugs may promote inflammation, which could partly explain their worsening effect in corpus gastritis observed in H. pylori-infected patients.     

Action of somatostatin analogue (SMS 201–995) on the growth-promoting effect resulting from sustained achlorhydria in rat gastric mucosa, with special reference to endocrine cell behaviour

We investigated whether a new long-acting somatostatin analogue (SMS 201-995) could antagonize the trophic effect induced by hypergastrinaemia, resulting from chronic omeprazole treatment, on the rat gastric mucosa and particularly on endocrine cell growth. SMS was administered concomitantly with omeprazole for 70 days. Gastric morphometric and cell proliferative parameters, gastric acid secretion and plasma gastrin levels were examined. New findings with omeprazole pointed out: (i) a trophic effect on the antral mucosa and (ii) that the increase observed in gastrin cell number was not due to stimulation of gastrin cell production by omeprazole but more likely to a prolongation of the gastrin cells' life span. As compared to omeprazole alone, simultaneous SMS administration significantly decreased the parietal cell (P less than 0.05) and gastrin cell (P less than 0.01) labelling indices, mucosal height of total glandular stomach (P less than 0.05) and antral mucosal height (P less than 0.05). It tended to lower fundic mucosal height and fundic argyrophil cell density (P less than 0.2 and P less than 0.1, respectively). SMS, in our conditions, did not accentuate the inhibitory effect of omeprazole on gastric acid secretion nor reduce high plasma gastrin levels. We conclude that SMS modestly counteracts the growth-promoting effect observed in rat gastric mucosa after prolonged omeprazole treatment.     

Nicotine and ethanol activate protein kinase A synergistically via G(i) betagamma subunits in nucleus accumbens/ventral tegmental cocultures: the role of dopamine D(1)/D(2) and adenosine A(2A) receptors

Tobacco and alcohol are the most commonly used drugs of abuse and show the most serious comorbidity. The mesolimbic dopamine system contributes significantly to nicotine and ethanol reinforcement, but the underlying cellular signaling mechanisms are poorly understood. Nicotinic acetylcholine (nACh) receptors are highly expressed on ventral tegmental area (VTA) dopamine neurons, with relatively low expression in nucleus accumbens (NAcb) neurons. Because dopamine receptors D(1) and D(2) are highly expressed on NAcb neurons, nicotine could influence NAcb neurons indirectly by activating VTA neurons to release dopamine in the NAcb. To investigate this possibility in vitro, we established primary cultures containing neurons from VTA or NAcb separately or in cocultures. Nicotine increased cAMP response element-mediated gene expression only in cocultures; this increase was blocked by nACh or dopamine D(1) or D(2) receptor antagonists. Furthermore, subthreshold concentrations of nicotine with ethanol increased gene expression in cocultures, and this increase was blocked by nACh, D(2) or adenosine A(2A) receptor antagonists, Gbetagamma or protein kinase A (PKA) inhibitors, and adenosine deaminase. These results suggest that nicotine activated VTA neurons, causing the release of dopamine, which in turn stimulated both D(1) and D(2) receptors on NAcb neurons. In addition, subthreshold concentrations of nicotine and ethanol in combination also activated NAcb neurons through synergy between D(2) and A(2A) receptors. These data provide a novel cellular mechanism, involving Gbetagamma subunits, A(2A) receptors, and PKA, whereby combined use of tobacco and alcohol could enhance the reinforcing effect in humans as well as facilitate long-term neuroadaptations, increasing the risk for developing coaddiction.     

Desensitization of adenosine and dopamine receptors in rat brain after treatment with adenosine analogs

Maximally tolerated doses of N6-[(R)-1-methyl-2-phenylethyl] adenosine (0.50 nmol/hr/2 wk), 5'-N-ethylcarboxamide adenosine (NECA, 0.04 nmol/hr/2 wk) or deoxycoformycin (5 nmol/hr/1 wk) were administered i.c.v. to rats using mini-osmotic pumps. Adenosine receptor function was subsequently assayed using both ligand binding and adenylate cyclase assays. Binding to A1 receptors was quantitated using [3H]N6-[(R)-1-methyl-2-phenylethyl]adenosine, a selective agonist ligand at A1 receptors. Differences in the binding of this ligand and that of [3H]NECA, which binds to A1 and A2 receptors with similar affinities, were used to quantitate A2 receptors. None of the treatments affected A1 receptor function as assessed by both ligand binding and adenylate cyclase assays. A2 receptor binding and A2 receptor-mediated stimulation of adenylate cyclase were blunted in striatal membranes from NECA- and deoxycoformycin-treated rats but unaffected in striatal membranes from N6-[(R]-1-methyl-2-phenylethyl]adenosine-treated rats. All three pretreatments attenuated D1 dopamine receptor-mediated stimulation of adenylate cyclase in striatal membranes. These results suggest that 1) the A2 adenosine receptor system is susceptible to desensitization and 2) different mechanisms are involved in the NECA- and deoxycoformycin-induced desensitization of A2 adenosine receptor and D1 dopamine receptor systems. It is suggested that the D1 dopamine receptor desensitization is, in fact, due to the tonic stimulation of adenosine A1 receptors.