A comparison of the effects of gastrin, somatostatin and dopamine receptor ligands on rat gastric enterochromaffin-like cell secretion and proliferation

Gastrin regulates ECL cell histamine release and is a critical determinant of acid secretion. ECL cell secretion and proliferation is inhibited by gastrin antagonists and somatostatin but little is known about the role of dopamine agonists in this process. Since the ECL cell exhibits all three classes of receptor we evaluated and compared the effects of the gastrin receptor antagonist, (YF476), lanreotide (SST agonist) and novel dopaminergic agents (BIM53061 and BIM27A760) on ECL cell histamine secretion and proliferation. Highly enriched (>98%) ECL cell preparations prepared from rat gastric mucosa using a FACS approach were studied. Real-time PCR confirmed presence of the CCK2, SS2 and SS5 and D1 receptors on ECL cells. YF476 inhibited histamine secretion and proliferation with IC(50)s of 1.25 nM and 1.3 x 10(-11) M respectively, values 10-1000x more potent than L365,260. Lanreotide inhibited secretion and proliferation (2.2 nM, 1.9 x 10(-10) M) and increased YF476-inhibited proliferation a further 5-fold. The dopamine agonist, BIM53061, inhibited gastrin-mediated ECL cell secretion and proliferation (17 nM, 6 x 10(-10) M) as did the novel dopamine/somatostatin chimera BIM23A760 (22 nM, 4.9 x 10(-10) M). Our studies demonstrate that the gastrin receptor antagonist, YF476, is the most potent inhibitor of ECL cell histamine secretion and proliferation. Lanreotide, a dopamine agonist and a dopamine/somatostatin chimera inhibited ECL cell function but were 10-1000x less potent than YF476. Agents that selectively target the CCK2 receptor may provide alternative therapeutic strategies for gastrin-mediated gastrointestinal cell secretion and proliferation such as evident in the hypergastrinemic gastric carcinoids associated with low acid states.     

Physiological significance of ECL-cell histamine.

In the oxyntic mucosa of the mammalian stomach, histamine is stored in ECL cells and in mucosal mast cells. In the rat, at least 80 percent of oxyntic mucosal histamine resides in the ECL cells. Histamine is a key factor in the regulation of gastric acid secretion. Following depletion of ECL-cell histamine by treatment with alpha-fluoromethylhistidine (alpha-FMH), basal acid secretion was reduced, and gastrin-stimulated acid secretion was abolished. Vagally-induced acid secretion (by insulin injection or pylorus ligation) was unaffected by alpha-FMH treatment but inhibited by an H2 antagonist. These results suggest that gastrin stimulates acid secretion via release of ECL-cell histamine, whereas vagally-induced acid secretion--although histamine-dependent--does not rely on ECL-cell histamine. Gastrin is known to have a trophic effect on the oxyntic mucosa. By combining long-term hypergastrinemia with continuous infusion of alpha-FMH, we were able to show that gastrin-evoked trophic effects in the stomach do not depend on ECL-cell histamine.     

CCK2 receptor antagonists: pharmacological tools to study the gastrin-ECL cell-parietal cell axis

Gastrin-recognizing CCK2 receptors are expressed in parietal cells and in so-called ECL cells in the acid-producing part of the stomach. ECL cells are endocrine/paracrine cells that produce and store histamine and chromogranin A (CGA)-derived peptides, such as pancreastatin. The ECL cells are the principal cellular transducer of the gastrin-acid signal. Activation of the CCK2 receptor results in mobilization of histamine (and pancreastatin) from the ECL cells with consequent activation of the parietal cell histamine H2 receptor. Thus, release of ECL-cell histamine is a key event in the process of gastrin-stimulated acid secretion. The oxyntic mucosal histidine decarboxylase (HDC) activity and the serum pancreastatin concentration are useful markers for the activity of the gastrin-ECL cell axis. Powerful and selective CCK2 receptor antagonits have been developed from a series of benzodiazepine compounds. These agents are useful tools to study how gastrin controls the ECL cells. Conversely, the close control of ECL cells by gastrin makes the gastrin-ECL cell axis well suited for evaluating the antagonistic potential of CCK2 receptor antagonists with the ECL-cell HDC activity as a notably sensitive and reliable parameter. The CCK2 receptor antagonists YF476, YM022, RP73870, JB93182 and AG041R were found to cause prompt inhibition of ECL-cell histamine and pancreastatin secretion and synthesis. The circulating pancreastatin concentration is raised, was lowered when the action of gastrin on the ECL cells was blocked by the CCK2 receptor antagonists. These effects were associated with inhibition of gastrin-stimulated acid secretion. In addition, sustained receptor blockade was manifested in permanently decreased oxyntic mucosal HDC activity, histamine concentration and HDC mRNA and CGA mRNA concentrations. CCK2 receptor blockade also induced hypergastrinemia, which probably reflects the impaired gastric acid secretion (no acid feedback inhibition of gastrin release). Upon withdrawal of the CCK2 receptor antagonists, their effects on the ECL cells were readily reversible. In conclusion, gastrin mobilizes histamine from the ECL cells, thereby provoking the parietal cells to secrete acid. While CCK2 receptor blockade prevents gastrin from evoking acid secretion, it is without effect on basal and vagally stimulated acid secretion. We conclude that specific and potent CCK2 receptor antagonists represent powerful tools to explore the functional significance of the ECL cells.     

Differentiation of the gastric mucosa. I. Role of histamine in control of function and integrity of oxyntic mucosa: understanding gastric physiology through disruption of targeted genes

Many physiological functions of the stomach depend on an intact mucosal integrity; function reflects structure and vice versa. Histamine in the stomach is synthesized by histidine decarboxylase (HDC), stored in enterochromaffin-like (ECL) cells, and released in response to gastrin, acting on CCK(2) receptors on the ECL cells. Mobilized ECL cell histamine stimulates histamine H(2) receptors on the parietal cells, resulting in acid secretion. The parietal cells express H(2), M(3), and CCK(2) receptors and somatostatin sst(2) receptors. This review discusses the consequences of disrupting genes that are important for ECL cell histamine release and synthesis (HDC, gastrin, and CCK(2) receptor genes) and genes that are important for "cross-talk" between H(2) receptors and other receptors on the parietal cell (CCK(2), M(3), and sst(2) receptors). Such analysis may provide insight into the functional significance of gastric histamine.     

PACAP stimulates gastric acid secretion in the rat by inducing histamine release

Previous studies have shown that pituitary adenylate cyclase-activating peptide (PACAP) stimulates enterochromaffin-like (ECL) cell histamine release, but its role in the regulation of gastric acid secretion is disputed. This work examines the effect of PACAP-38 on aminopyrine uptake in enriched rat parietal cells and on histamine release and acid secretion in the isolated vascularly perfused rat stomach and the role of PACAP in vagally (2-deoxyglucose) stimulated acid secretion in the awake rat. PACAP has no direct effect on the isolated parietal cell as assessed by aminopyrine uptake. PACAP induces a concentration-dependent histamine release and acid secretion in the isolated stomach, and its effect on histamine release is additive to gastrin. The histamine H2 antagonist ranitidine potently inhibits PACAP-stimulated acid secretion without affecting histamine release. Vagally stimulated acid secretion is partially inhibited by a PACAP antagonist. The results from the present study strongly suggest that PACAP plays an important role in the neurohumoral regulation of gastric acid secretion. Its effect seems to be mediated by the release of ECL cell histamine.     

Molecular cloning and characterization of a new human histamine receptor, HH4R

A new histamine receptor, HH4R, was cloned from human leukocyte cDNA. The deduced amino acid sequence showed about 40% identity to that of the human histamine H3 receptor, HH3R. HH4R-expressing cells responded to histamine, inhibiting forskolin-induced cAMP accumulation. An H3 agonist, N-alpha-methylhistamine (NAMHA), bound specifically to HH4R, while another H3 agonist, R(-)-alpha-methylhistamine (RAMHA), and the H3 antagonist, thioperamide, competed with this binding. RAMHA, NAMHA, and imetit inhibited forskolin-induced cAMP accumulation in HH4R-expressing cells. However, the binding affinities and agonistic activities of H3 agonists to HH4R were weaker than those to HH3R. Low expression of HH4R was detected in a wide variety of peripheral tissues by RT-PCR; however, in contrast with HH3R, expression was not detected in the brain. These observations indicate that the clone is a distinct histamine receptor from HH3R, and thus is named HH4R.     

Intracellular signal transduction during gastrin-induced histamine secretion in rat gastric ECL cells

Activation of G_qprotein-coupled receptors usually causes a biphasic increase inintracellular calcium concentration ([Ca²⁺]_i)that is crucial for secretion in nonexcitable cells. In gastric enterochromaffin-like (ECL) cells, stimulation with gastrin leads to aprompt biphasic calcium response followed by histamine secretion. Thisstudy investigates the underlying signaling events in this neuroendocrine cell type. In ECL cells, RT-PCR suggested the presence of inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes1-3. The IP₃R antagonist 2-aminoethoxydiphenyl borateabolished both gastrin-induced elevation of[Ca²⁺]_i and histamine release. Thapsigarginincreased [Ca²⁺]_i, however, without inducinghistamine secretion. In thapsigargin-pretreated cells, gastrinincreased [Ca²⁺]_i through calcium influxacross the plasma membrane. Both nimodipine and SKF-96365 inhibitedgastrin-induced histamine release. The protein kinase C (PKC) activatorphorbol 12-myristate 13-acetate induced histamine secretion, an effectthat was prevented by nimodipine. In summary, gastrin-stimulatedhistamine release depends on IP₃R activation andplasmalemmal calcium entry. Gastrin-induced calcium influx wasmediated by dihydropyridine-sensitive calcium channels that appear tobe L-type channels activated through a pathway involving activation of PKC.

     

The effect of somatostatin on baseline and stimulated acid secretion and vascular histamine release from the totally isolated vascularly perfused rat stomach

The effect of somatostatin-(1-14) (S1-14) on the gastrin- and histamine-induced acid secretion and gastrin-evoked vascular histamine release was studied in isolated vascularly perfused rat stomachs being continuously perfused by a gassed buffer containing 10% ovine erythrocytes and 50 microM isobutyl methylxanthine (IMX). Concentrations of gastrin (520 pM) and histamine, (0.5 microM) were chosen to give acid secretion in the same range (61.5 +/- 7.0 and 49.4 +/- 9.4 mumol/60 min). S1-14 induced a concentration-dependent decrease in acid secretion stimulated by both gastrin and histamine. Even at the lowest concentration examined (0.1 nM) somatostatin gave a significant inhibition of both gastrin- and histamine-stimulated acid secretion. The inhibitory effect was, however, most marked for gastrin-stimulated acid secretion (P less than 0.05 at 1 nM concentration of S1-14). Gastrin gave an immediate and marked vascular histamine release which was inhibited by somatostatin in the higher concentrations (1.0 and 5.0 nM). Somatostatin at the lowest concentration tested (0.1 nM) did not inhibit the gastrin-induced vascular histamine release although it did inhibit acid secretion. Furthermore, baseline histamine release was not affected by somatostatin. This study suggests that somatostatin inhibits acid secretion both via a direct effect of the parietal cell and by inhibiting gastrin-induced histamine release. Baseline histamine release is regulated by a mechanism not sensitive to somatostatin.     

Gastrin effects on isolated rat enterochromaffin-like cells following long-term hypergastrinaemia in vivo.

The enterochromaffin-like (ECL) cells play an important role in the regulation of gastric acid secretion. They respond to gastrin by a prompt increase in histamine secretion, an effect which is mediated by the CCK-(B)/gastrin receptor acting through the IP(3)/DAG pathway. In the rat, long-term treatment with acid secretion inhibitors induces hypergastrinaemia which, in turn, results in ECL cell hypertrophy and hyperplasia. The aim of the present study was to evaluate various functional parameters in acutely isolated rat ECL cells, following long-term hypergastrinaemia in vivo. Rats were treated with vehicle or a supramaximal daily dose of omeprazole for more than 10 weeks to ensure ECL cell hyperplasia. ECL cells were isolated from vehicle-treated animals and 24, 72 and 120 h after the last dose of omeprazole. The functional activity of the acutely isolated ECL cells was determined by measuring gastrin-and forskolin-induced histamine secretion. Changes in cytosolic free calcium upon gastrin stimulation were monitored by digital video imaging. ECL cells successively regained their ability to respond to gastrin following long-term hypergastrinaemia, reaching close to vehicle-treated levels 120 h after the last dose of omeprazole. In the rat, the response pattern of the ECL cells appears to normalise in parallel with the normalisation of plasma gastrin levels.     

Purification of a histamine H3 receptor negatively coupled to phosphoinositide turnover in the human gastric cell line HGT1.

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.     

Priming interactions between platelet activating factor and histamine in the in vivo microcirculation.

To elucidate whether priming exists between platelet-activating factor (PAF) and histamine in the microcirculation, we measured the clearance of FITC-dextran 150 in response to the topical applications of substimulatory concentrations of PAF and histamine. Maximal priming by PAF was observed when a 5-min interval separated the applications of 10(-9) M PAF and 10(-6) M histamine. The mean (+/- SEM) clearance resulting from this sequence of agonist administration was 7529 +/- 659 nl.2 h-1.g-1, representing a 4.5-fold enhancement in FITC-dextran 150 clearance compared with that evoked by 10(-6) M histamine alone (1664 +/- 397 nl.2 h-1.g-1). Lowering the PAF priming dose to 10(-11) M, or reversing the order of agonist addition to the microcirculation, resulted in diminished but significant responses of 3545 +/- 1143 and 4467 +/- 1170 nl.2 hr-1.g-1, respectively. Coapplication of PAF and histamine or increasing the time interval between the agonists to 15 min greatly reduced the responses to 1906 +/- 678 and 2770 +/- 837, respectively. The PAF receptor antagonist WEB 2086 (2 mg/kg i.v.), the H1 blocker pyrilamine (10 mg/kg i.v.), and leukocyte depletion with cyclophosphamide (150 mg/kg i.p.) completely abolished the PAF priming effect. In addition, the 5-lipoxygenase inhibitor RG 5901 (1 or 10 mg/kg i.v.) produced a two-thirds attenuation in PAF priming. We conclude that 1) PAF has the ability to prime the in vivo microvascular actions of histamine in both a concentration and time-dependent fashion; 2) this primed response is receptor mediated; and 3) histamine can prime the microcirculation for enhanced responses to PAF. Our data also demonstrate that leukocytes and the release of leukotrienes participate in PAF priming.     

Properties of isolated gastric enterochromaffin-like cells.

The gastric enterochromaffin-like cell (ECL) has been studied in gastric fundic glands by confocal microscopy and as a purified cell preparation by video imaging of calcium signaling and measurements of histamine release. Regulation of gastric acid secretion is largely due to alterations of histamine activation of the H2 receptor on the parietal cell and can be divided into central neural regulation, with direct actions of neuronally released mediators and into peripheral regulation by substances released from other endocrine cells. Gastric neuronal stimulation of acid secretion by alteration of ECL cell function is probably mediated by pituitary adenylate cyclase activating peptide (PACAP) receptors on the ECL cell, which activate calcium signaling and histamine release. Peripheral stimulation of acid secretion via the ECL cell is largely mediated by gastrin stimulation of calcium signaling and histamine release. Gastric neuronal inhibition of ECL cell function is probably mediated by galanin inhibition of calcium signaling, and histamine release and peripheral inhibition of ECL cell function is mainly due to somatostatin release from D cells.     

Gastric submucosal microdialysis: a method to study gastrin- and food-evoked mobilization of ECL-cell histamine in conscious rats

Rat stomach ECL cells are rich in histamine and chromogranin A-derived peptides, such as pancreastatin. Gastrin causes the parietal cells to secrete acid by flooding them with histamine from the ECL cells. In the past, gastric histamine release has been studied using anaesthetized, surgically manipulated animals or isolated gastric mucosa, glands or ECL cells. We monitored gastric histamine mobilization in intact conscious rats by subjecting them to gastric submucosal microdialysis. A microdialysis probe was implanted into the submucosa of the acid-producing part of the stomach (day 1). The rats had access to food and water or were deprived of food (48 h), starting on day 2 after implantation of the probe. On day 4, the rats received food or gastrin (intravenous infusion), and sampling of microdialysate commenced. Samples (flow rate 1.2 microl min(-1)) were collected every 20 or 60 min, and the histamine and pancreastatin concentrations were determined. The serum gastrin concentration was determined in tail vein blood. Exogenous gastrin (4-h infusion) raised microdialysate histamine and pancreastatin dose-dependently. This effect was prevented by gastrin receptor blockade (YM022). Depletion of ECL-cell histamine by alpha-fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, suppressed the gastrin-evoked release of histamine but not that of pancreastatin. Fasting lowered serum gastrin and microdialysate histamine by 50%, while refeeding raised serum gastrin and microdialysate histamine and pancreastatin 3-fold. We conclude that histamine mobilized by gastrin and food intake derives from ECL cells because: 1) Histamine and pancreastatin were released concomitantly, 2) histamine mobilization following gastrin or food intake was prevented by gastrin receptor blockade, and 3) mobilization of histamine (but not pancreastatin) was abolished by alpha-fluoromethylhistidine. Hence, gastric submucosal microdialysis allows us to monitor the mobilization of ECL-cell histamine in intact conscious rats under various experimental conditions not previously accessible to study. While gastrin receptor blockade lowered post-prandial release of ECL-cell histamine by about 80%, unilateral vagotomy reduced post-prandial mobilization of ECL-cell histamine by about 50%. Hence, both gastrin and vagal excitation contribute to the post-prandial release of ECL-cell histamine.     

The mechanism of histamine secretion from gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells play a pivotal role in theperipheral regulation of gastric acid secretion as they respond to thefunctionally important gastrointestinal hormones gastrin andsomatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the keystimulus of histamine release from ECL cells in vivo and in vitro.Voltage-gated K⁺ andCa²⁺ channels have been detectedon isolated ECL cells. Exocytosis of histamine following gastrinstimulation and Ca²⁺ entry acrossthe plasma membrane is catalyzed by synaptobrevin andsynaptosomal-associated protein of 25 kDa, both characterized as asoluble N-ethylmaleimide-sensitivefactor attachment protein receptor protein. Histamine release occursfrom different cellular pools: preexisting vacuolar histamineimmediately released by Ca²⁺ entryor newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized bycytoplasmic HDC and accumulated in secretory vesicles byproton-histamine countertransport via the vesicular monoaminetransporter subtype 2 (VMAT-2). The promoter region of HDC containsCa²⁺-, cAMP-, and protein kinaseC-responsive elements. The gene promoter for VMAT-2, however, lacksTATA boxes but contains regulatory elements for the hormones glucagonand somatostatin. Histamine secretion from ECL cells is thereby under acomplex regulation of hormonal signals and can be targeted at severalsteps during the process of exocytosis.

     

Mechanisms of histamine-induced relaxation in bovine small adrenal cortical arteries

Adrenal steroidogenesis is closely correlated with increases in adrenal blood flow. Many reports have studied the regulation of adrenal blood flow in vivo and in perfused glands, but until recently few studies have been conducted on isolated adrenal arteries. The present study examined vasomotor responses of isolated bovine small adrenal cortical arteries to histamine, an endogenous vasoactive compound, and its mechanism of action. In U-46619-precontracted arteries, histamine (10(-9)-5 x 10(-6) M) elicited concentration-dependent relaxations. The relaxations were blocked by the H(1) receptor antagonists diphenhydramine (10 microM) or mepyramine (1 microM) (maximal relaxations of 18 +/- 6 and 22 +/- 6%, respectively, vs. 55 +/- 5% of control) but only partially inhibited by the H(2) receptor antagonist cimetidine (10 microM) and the H(3) receptor antagonist thioperamide (1 microM). Histamine-induced relaxations were also blocked by the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA, 30 microM; maximal relaxation of 13 +/- 7%) and eliminated by endothelial removal or L-NA combined with the cyclooxgenase inhibitor indomethacin (10 microM). In the presence of adrenal zona glomerulosa (ZG) cells, histamine did not induce further relaxations compared with histamine alone. Histamine (10(-7)-10(-5) M) concentration-dependently increased aldosterone production by adrenal ZG cells. Compound 48/80 (10 microg/ml), a mast cell degranulator, induced significant relaxations (93 +/- 0.6%), which were blocked by L-NA plus indomethacin or endothelium removal, partially inhibited by the combination of the H(1), H(2), and H(3) receptor antagonists, but not affected by the mast cell stabilizer sodium cromoglycate (1 mM). These results demonstrate that histamine causes direct relaxation of small adrenal cortical arteries, which is largely mediated by endothelial NO and prostaglandins via H(1) receptors. The potential role of histamine in linking adrenal vascular events and steroid secretion requires further investigation.     

Chronic Helicobacter pylori infection results in gastric hypoacidity and hypergastrinemia in wild-type mice but vagally induced hypersecretion in gastrin-deficient mice

Helicobacter pylori infection is a causal factor of gastric cancer (which is associated with low gastric acid secretion) or duodenal ulcer (high acid secretion). Parietal cells and ECL cells in the stomach are controlled by gastrin, which plays a crucial role in the regulation of acid secretion. The present study was undertaken to identify a possible role of gastrin in determining the different responses of the parietal cells and ECL cells to chronic H. pylori infection. Wild-type (C57BL/6J) gastrin(+/+) mice and gastrin(-/-) knockout mice, generated through targeted gene disruption and backcrossed eight times to C57BL/6J, were infected with H. pylori for 9 months. The acid output was measured 4 h after pylorus ligation (known to cause vagal excitation). The gastric mucosa was examined by immunocytochemistry with antisera to alpha-subunit of H+/K(+)-ATPase for the parietal cells, and to histamine and vesicle monoamine transporter-2 for the ECL cells, and by quantitative electron microscopy. In infected gastrin(+/+) mice, the acid output and the percentage of secreting parietal cells (freely fed state) were 20-30% of the values in uninfected controls, while the density and ultrastructure of parietal cells were normal. The infected mice had hypergastrinemia and displayed hypertrophy and hyperplasia of ECL cells. Although uninfected gastrin(-/-) mice had lower the acid output than uninfected gastrin(+/+) mice, there was a higher acid output (approximately 3 times) in infected gastrin(-/-) mice than their uninfected homologues. The numbers of parietal cells and ECL cells remained unchanged in infected gastrin(-/-) mice. In conclusion, chronic H. pylori infection results to impaired parietal-cell function (acid hyposecretion), hypergastrinemia and hyperplasia of ECL cells in wild-type mice but leads to vagally induced hypersecretion in gastrin-deficient mice.     

Selective stimulation of histamine H3 autoreceptors

A simple derivative of histamine, alpha-methylhistamine i.e. 4-(2-aminopropyl)-imidazole, was shown to potently inhibit the K+-induced release of [3H] histamine from slices of rat cerebral cortex previously incubated in the presence of [3H] histidine. The maximal inhibition elicited by alpha-methylhistamine was of about 60% i.e. similar to that elicited by exogenous histamine. The effect occurred with an EC50 value of 4.3 +/- 1.1 X 10(-9) M about 10 times lower than that of histamine and was reversed by a H3-receptor antagonist. Since alpha-methylhistamine is known to display negligible potency at H1- and H2-receptors, this compound appears to be the first highly potent and selective H3-receptor agonist to be identified.     

Neurohormonal regulation of secretion from isolated rat stomach ECL cells: a critical reappraisal

ECL cells are endocrine/paracrine cells in the oxyntic mucosa. They produce, store and secrete histamine and chromogranin A-derived peptides such as pancreastatin. The regulation of ECL-cell secretion has been studied by several groups using purified ECL cells, isolated from rat stomachs. Reports from different laboratories often disagree. The purpose of the present study was to re-evaluate the discrepancies by studying histamine (or pancreastatin) secretion from standardized preparations of pure, well-functioning ECL cells. Cells from rat oxyntic mucosa were dispersed by pronase digestion, purified by repeated counter-flow elutriation and subjected to density gradient centrifugation. The final preparation consisted of more than 90% ECL cells (verified by histamine and/or histidine decarboxylase immunocytochemistry). They were maintained in primary culture for 48 h before they were exposed to candidate stimulants and inhibitors for 30 min after which the medium was collected for determination of mobilized histamine (or pancreastatin). Gastrin-17 and sulphated cholecystokinin octapeptide (CCK-8s) raised histamine secretion 4-fold, the EC(50) for both peptides being around 100 pM. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP-27) (5-fold increase) and the related neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) (3-fold increase) mobilized histamine with similar potency (EC(50) ranging from 80 to 140 pM). Adrenaline, isoprenaline and terbutaline stimulated secretion by activating a beta2 receptor subtype, while acetylcholine and carbachol were without effect. Secretion experiments were invariably run in parallel with a gastrin standard curve. Somatostatin, prostaglandin E2 (PGE2) and the PGE1 congener misoprostol inhibited PACAP- and gastrin-stimulated secretion by more than 90%, with IC(50) values ranging from 90-720 (somatostatin) to 40-200 (misoprostol) pM. The neuropeptide galanin inhibited secretion by 60-70% with a potency similar to that of somatostatin. Proposed inhibitors such as peptide YY, neuropeptide Y and the cytokines interleukin 1-beta and tumor necrosis factor alpha induced at best a moderate inhibition of gastrin- or PACAP-stimulated secretion at high concentrations, while calcitonin gene-related peptide, pancreatic polypeptide and histamine itself were without effect. Inhibition of gastrin- or PACAP-stimulated secretion was routinely compared to a somatostatin standard curve. In conclusion, gastrin, PACAP, VIP/PHI and adrenaline stimulated secretion. Somatostatin and PGE2 were powerful inhibitors of both gastrin- and PACAP-stimulated secretion; although equally potent, galanin was less effective than somatostatin and PGE2.     

Cardiovascular effects of histamine administered intracerebroventricularly in critical haemorrhagic hypotension in rats.

The study was designed to determine the cardiovascular effects of histamine administered intracerebroventricularly (icv) in a rat model of volume-controlled haemorrhagic shock. The withdrawal of approximately 50% of total blood volume resulted in the death of all control saline icv treated animals within 30 min. Icv injection of histamine produced a prompt dose-dependent (0.1-100 nmol) and long-lasting (10-100 nmol) increase in mean arterial pressure (MAP), pulse pressure (PP) and heart rate (HR), with a 100% survival of 2h after treatment (100 nmol). The increase in MAP and HR after histamine administration in bled rats in comparison to the normovolaemic animals was 2.7-3.3- and 1.3-3.6-fold higher, respectively. Pretreatment with chlorpheniramine (50 nmol icv), H1 receptor antagonist, inhibited the increase in MAP, PP, HR and survival rate produced by histamine, while chlorpheniramine given alone had no effect. Neither ranitidine (50 nmol icv), H2 histamine receptor antagonist, nor thioperamide (50 nmol icv), H3 receptor blocker, influenced the histamine action, however, when given alone, both evoked the pressor effect with elongation of survival time. It can be concluded that histamine administered icv reverses the haemorrhagic shock conditions, and histamine H1 receptors are involved.     

Stimulatory effects of endogenous and exogenous nitric oxide on gastric acid secretion in anesthetized rats

We previously reported the stimulatory effect of endogenous nitric oxide (NO) on gastric acid secretion in the isolated mouse whole stomach and histamine release from gastric histamine-containing cells. In the present study, we investigated the effects of endogenous and exogenous NO on gastric acid secretion in urethane-anesthetized rats. Acid secretion was studied in gastric-cannulated rats stimulated with several secretagogues under urethane anesthesia. The acid secretory response to the muscarinic receptor agonist bethanechol (2 mg/kg, s.c.), the cholecystokinin(2) receptor agonist pentagastrin (20 microg/kg, s.c.) or the centrally acting secretagogue 2-deoxy-D-glucose (200 mg/kg, i.v.) was dose-dependently inhibited by the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA, 10 or 50 mg/kg, i.v.). This inhibitory effect of L-NNA was reversed by a substrate of NO synthase, L-arginine (200 mg/kg, i.v.), but not by D-arginine. The histamine H(2) receptor antagonist famotidine (1 mg/kg, i.v.) completely inhibited the acid secretory response to bethanechol, pentagastrin or 2-deoxy-D-glucose, showing that all of these secretagogues induced gastric acid secretion mainly through histamine release from gastric enterochromaffin-like cells (ECL cells). On the other hand, histamine (10 mg/kg, s.c.)-induced gastric acid secretion was not inhibited by pretreatment with L-NNA. The NO donor sodium nitroprusside (0.3-3 mg/kg, i.v.) also dose-dependently induced an increase in acid secretion. The sodium nitroprusside-induced gastric acid secretion was significantly inhibited by famotidine or by the soluble guanylate cyclase inhibitor methylene blue (50 mg/kg, i.v.). These results suggest that NO is involved in the gastric acid secretion mediated by histamine release from gastric ECL cells.