ECL-cell Histamine Research Articles

Commentary: netazepide for gastric acid suppression – another kid on the block?

Just when it seemed all over, the manuscript by Boyce et al.1 examining the pharmacokinetics and pharmacodynamics of single dose administration of netazepide, a peripheral CCK-B (gastrin) receptor antagonist, in normal volunteers will most certainly reinvigorate interest in gastric acid secretion and its pharmacological inhibition. Netazepide appears to be a potent inhibitor of gastric acid secretion with rapid onset and a prolonged duration of action.1 If it can be shown to be both safe and effective as maintenance therapy, it may well give proton pump inhibitors (PPIs) a real run for their money. The PPIs, which irreversibly block the final common path of gastric acid production,2 have been the dominant class of antisecretory drugs for over two decades and their continuing supremacy was all but assured with the demise of the potassium competitive acid blockers (PCABs) (although there may be a comeback for this class of agents too3). In addition, concerns have surfaced recently about potential adverse effects from prolonged PPI use4 which has opened the door to other novel compounds such as netazepide. Of course, much work remains to be done with this agent and there are, as yet, many unanswered questions (some of which the authors may well address in two upcoming manuscripts concerning repetitive dosing of netazepide). For example, will tachyphylaxis develop after repeated administration of this competitive receptor antagonist, a phenomenon that has been well described with histamine H2 receptor antagonists, which act one step further along the gastric acid secretory pathway?5 In addition, the endocrine, neurocrine and paracrine networks that control gastric acid secretion and its feedback are redundant.6 It remains to be seen whether upregulation of other pathways could occur leading to additional loss of efficacy (tolerance) with time. Alternatively, will the drug, which appears to have a long secondary half-life, accumulate after repeated dosing leading to unexpected side effects? Moreover, little is known about the metabolism of this agent which has only been administered to a small cadre of normal volunteers. Will the effect of this agent be predictable in patients or will alterations in metabolism lead to ‘slow’ and ‘fast’ metabolisers and unpredictable antisecretory effects? Finally, will potent ECL-cell inhibition by netazepide lead to G-cell hyperplasia and hypergastrinaemia, with the potential for rebound ECL-cell histamine release and excessive gastric acid production4 on withdrawal of the drug? This possibility is of some interest given current concerns regarding rebound gastric acid hypersecretion in double-blind randomised trials of PPI use vs. placebo in normal volunteers.7, 8 On the other hand, judicious use of netazepide in concert with PPIs may lead to more precise titration of individual antisecretory effects, thereby mitigating ECL-cell hyperplasia and parietal cell hypertrophy. This is an exciting and provocative first-in-human study of a novel compound with significant potential. I fully agree with the authors' conclusions that ‘studies of repeated doses of netazepide are justified’.1 Declaration of personal interests: David Metz has served as a consultant and advisory board member for Novartis, as a consultant for AstraZeneca and Takeda, and has received research funding from Fercica. Declaration of funding interests: None

The ECL Cell: Relay Station for Gastric Integrity

The term "enterochromaffin cell" was introduced more than 100 years ago. The cells that are morphologically similar to the enterochromaffin cells have been referred to as "enterochromaffin-like cells". One of the enterochromaffin-like cell populations in the oxyntic mucosa of stomach is known to produce and store histamine and chromogranin A, and referred to as ECL cells. The biology and the functional morphology and topology of the ECL cells have been extensively studied, since they were discovered 45 years ago. ECL-cell histamine plays an important role in the regulation of gastric acid secretion, particularly in response to gastrin stimulation. The time-course responses of ECL cells to gastrin include mobilization of histamine, hypertrophy, hyperplasia, dysplasia and formation of ECL-cell carcinoids. The ECL cells are controlled by a complex regulatory system involving endocrine, paracrine and neural pathways. The physiological significance of ECL cells reflects the nature of their products such as histamine, chromogranin A-derived peptides, Reg protein and yet-unknown hormone.

Ischemia mobilizes histamine but not pancreastatin from ECL cells of rat stomach: evidence for a cytosolic histamine compartment

Histamine in the rat stomach resides in enterochromaffin-like (ECL) cells and mast cells. The ECL cells are peptide-hormone-producing endocrine cells known to release histamine and chromogranin-A-derived peptides (such as pancreastatin) in response to gastrin. Ischemia (induced by clamping of the celiac artery or by gastric submucosal microinfusion of the vasoconstrictor endothelin) mobilizes large amounts of ECL-cell histamine in a burst-like manner. This report examines the ECL-cell response to ischemia and compares it with that induced by gastrin in rats. Arterial clamping (30 min) and gastric submucosal microinfusion (3 h) of endothelin, vasopressin, or adrenaline caused ischemia, manifested as a raised lactate/pyruvate ratio and mucosal damage. Whereas microinfusion of gastrin released both histamine and pancreastatin, ischemia mobilized histamine only. The mucosal concentrations of histamine and pancreastatin, the number and immunostaining intensity of the ECL cells, and the ultrastructure of the ECL cells were unchanged following ischemia. The long-term effects of ischemia and reperfusion (60-90 min) on gastric mucosa were examined in rats treated with the proton pump inhibitor omeprazole for 4 days. The activity of the ECL cells was suppressed (reflected in low histamine-forming capacity) but returned to normal within 1 week, illustrating the ability of the ECL cells to recover. We suggest that ischemia mobilizes cytosolic ECL-cell histamine without affecting the storage of histamine (and pancreastatin) in the secretory organelles and without causing lasting ECL-cell impairment.

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 50s of 1.25 nM and 1.3 × 10 − 11 M respectively, values 10–1000× more potent than L365,260. Lanreotide inhibited secretion and proliferation (2.2 nM, 1.9 × 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 × 10 − 10 M) as did the novel dopamine/somatostatin chimera BIM23A760 (22 nM, 4.9 × 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 10–1000× 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.

The vagus regulates histamine mobilization from rat stomach ECL cells by controlling their sensitivity to gastrin

The ECL cells in the oxyntic mucosa secrete histamine in response to gastrin, stimulating parietal cells to produce acid. Do they also operate under nervous control? The present study examines histamine mobilization from rat stomach ECL cells in situ in response to acute vagal excitation and to food or gastrin following vagal or sympathetic denervation. Applying the technique of microdialysis, we monitored the release of histamine by radioimmunoassay. Microdialysis probes were placed in the submucosa on either side of the stomach, 3 days before experiments. The rats were awake during microdialysis except when subjected to electrical vagal stimulation. One-sided electrical vagal stimulation raised serum gastrin and mobilized gastric histamine. However, gastrin receptor blockade prevented the histamine mobilization, indicating that circulating gastrin accounts for the response. Vagal excitation by hypoglycaemia (insulin) or pylorus ligation did not mobilize either gastrin or histamine. The histamine response to food was almost abolished by gastrin receptor blockade, and it was halved on the denervated side after unilateral subdiaphragmatic vagotomy. While the histamine response to a near-maximally effective dose of gastrin was unaffected by vagotomy, the response to low gastrin doses was reduced significantly. Abdominal ganglionic sympathectomy failed to affect the histamine response to either food or gastrin. In conclusion, gastrin is responsible for most of the food-evoked mobilization of ECL-cell histamine. The histamine response to electrical vagal stimulation reflects the effect of circulating gastrin rather than a direct action of the vagus on the ECL cells. Vagal denervation was accompanied by an impaired histamine response to food intake, probably reflecting the right-ward shift of the serum gastrin concentration-histamine response curve. The results suggest that the vagus controls the sensitivity of the ECL cells to gastrin.

Plasma ghrelin is increased following gastrin stimulation

Background: Ghrelin is primarily secreted from the gastric mucosa and has been shown to increase appetite and weight in humans. Plasma concentrations are increased pre-meals but reduced in the fed state. We therefore examined the effects of gastrin stimulation on peripheral plasma ghrelin concentrations in normal human subjects. Methods: Eight normal, H. pylori-negative volunteers (4 male; mean age 26.9 (21–54) years) were studied. Ethical approval for this study was granted by the Ethical Committee of Queen's University of Belfast. Each underwent basal and maximal acid secretory studies before, on completion, and 28 days following a 28-day course of the proton pump inhibitor, esomeprazole 40mg daily. For each acid secretory test, basal blood samples were collected 50 and 40 minutes prior to commencing a 60-minute intravenous infusion of pentagastrin (0.6µg/kg/hr) with further blood sampling at 45, 90 and 240 minutes after commencing the infusion. Plasma gastrin and ghrelin concentrations were assessed by radioimmunoassay. Statistical analysis employed the Wilcoxon Signed Ranks test. Results: Plasma ghrelin was increased by pentagastrin infusion before (p<0.01), on completion (p<0.05) and 28 days following (p<0.01) esomeprazole (165.2 to 220.1 pmol/l, 237.7 to 266.5 pmol/l, 181.9 to 201.0 pmol/l, respectively). The ghrelin increase occurred at 45min and remained elevated at 240min in all three studies. Mean basal plasma gastrin and ghrelin was also increased (25.0ng/l to 51.3 ng/l (p<0.05) and 165.2 pmol/l to 237.7 pmol/l (p<0.01) respectively) following 28 days of esomeprazole. By day-28 post-esomeprazole, both plasma gastrin and ghrelin concentrations had returned to pre-treatment values. Conclusions: Plasma ghrelin was significantly increased by infusion of pentagastrin at a dose inducing maximal gastric acid secretion. This increase occurs immediately and may imply linkage with ECL cell histamine release. Treatment with esomeprazole also causes a significant rise in plasma ghrelin probably by causing an endogenous hypergastrinaemia. This study demonstrates that gastrin plays a key role in ghrelin activity and release. It also demonstrates that the mode of cellular communication amongst oxyntic and antral endocrine cell types requires further examination and additional evaluation of pharmacological intervention is necessary. Acknowledgements: PM Lynch was supported by a United Hospitals Trust Clinical Research Fellowship.

ECL cell histamine mobilization studied by gastric submucosal microdialysis in awake rats: methodological considerations.

The ECL cells are endocrine/paracrine cells in the acid-producing part of the stomach. They secrete histamine in response to circulating gastrin. Gastric submucosal microdialysis has been used to study ECL-cell histamine mobilization in awake rats. In the present study we assess the usefulness and limitations of the technique. Microdialysis probes were implanted in the gastric submucosa. Histological analysis of the stomach wall around the probe revealed a moderate, local inflammatory reaction 1-2 days after implantation; the inflammation persisted for at least 10 days. Experiments were conducted 3 days after the implantation. The "true" submucosal histamine concentration was determined by perfusing at different rates (the zero flow method) or with different concentrations of histamine at a constant rate (the no-net-flux method): in fasted rats it was calculated to be 87+/-5 (means+/-S.E.M.) nmol/l and 76+/-9 nmol/l, respectively. The corresponding histamine concentrations in fed rats were 93+/-5 and 102+/-8 nmol/l, respectively. With a perfusion rate of 74 microl/hr the recovery of submucosal histamine was 49%, at 34 microl/hr the recovery increased to 83%. At a perfusion rate below 20 microl/hr the microdialysate histamine concentration was close to the actual concentration in the submucosa. The ECL-cell histamine mobilization was independent of the concentrations of Ca2+ in the perfusion medium (0-3.4 mmol/l Ca2+). In one experiment, histamine mobilization in response to gastrin (10 nmol/kg/hr subcutaneously) was monitored in rats pretreated with prednisolone (60 mg/kg) or indomethacin (15 mg/kg). The two antiinflammatory agents failed to affect the concentration of histamine in the microdialysate either before or during the gastrin challenge, which was in accord with the observation that the inflammatory reaction was modest and that inflammatory cells were relatively few around the probe and in the wall of the probe. In another experiment, rats were given aminoguanidine (10 mg/kg) or metoprine (10 mg/kg) 4 hr before the start of gastrin infusion (5 nmol/kg/hr intravenously). Metoprine (inhibitor of histamine N-methyl transferase) did not affect the microdialysate histamine concentration, while aminoguanidine (inhibitor of diamine oxidase) raised both basal and gastrin-stimulated histamine concentrations. We conclude that microdialysis can be used to monitor changes in the concentration of histamine in the submucosa of the stomach, and that the inflammatory reaction to the probe is moderate and does not affect the submucosal histamine mobilization.

Role of gastrin in the development of gastric mucosa, ECL cells and A-like cells in newborn and young rats.

Histamine-producing ECL cells and ghrelin-producing A-like cells are endocrine/paracrine cell populations in the acid-producing part of the rat stomach. While the A-like cells operate independently of gastrin, the ECL cells respond to gastrin with mobilization of histamine and chromogranin A (CGA)-derived peptides, such as pancreastatin. Gastrin is often assumed to be the driving force behind the postnatal development of the gastric mucosa in general and the ECL cells in particular. We tested this assumption by examining the oxyntic mucosa (with ECL cells and A-like cells) in developing rats under the influence of YF476, a cholecystokinin-2 (CCK(2)) receptor antagonist. The drug was administered by weekly subcutaneous injections starting at birth. The body weight gain was not affected. Weaning occurred at days 15-22 in both YF476-treated and age-matched control rats. Circulating gastrin was low at birth and reached adult levels 2 weeks after birth. During and after weaning (but not before), YF476 greatly raised the serum gastrin concentration (because of abolished acid feedback inhibition of gastrin release). The weight of the stomach was unaffected by YF476 during the first 2-3 weeks after birth. From 4 to 5 weeks of age, the weight and thickness of the gastric mucosa were lower in YF476-treated rats than in controls. Pancreastatin-immunoreactive cells (i.e. all endocrine cells in the stomach) and ghrelin-immunoreactive cells (A-like cells) were few at birth and increased gradually in number until 6-8 weeks of age (control rats). At first, YF476 did not affect the development of the pancreastatin-immunoreactive cells, but a few weeks after weaning, the cells were fewer in the YF476 rats. The ECL-cell parameters (oxyntic mucosal histamine and pancreastatin concentrations, the histidine decarboxylase (HDC) activity, the HDC mRNA levels and serum pancreastatin concentration) increased slowly until weaning in both YF476-treated and control rats. From then on, there was a further increase in the ECL-cell parameters in control rats but not in YF476 rats. The postnatal development of the ghrelin cells (i.e. the A-like cells) and of the A-like cell parameters (the oxyntic mucosal ghrelin concentration and the serum ghrelin concentrations) was not affected by YF476 at any point. We conclude that gastrin affects neither the oxyntic mucosa nor the endocrine cells before weaning. After weaning, CCK(2) receptor blockade is associated with a somewhat impaired development of the oxyntic mucosa and the ECL cells. While gastrin stimulation is of crucial importance for the onset of acid secretion during weaning and for the activation of ECL-cell histamine formation and secretion, the mucosal and ECL-cell growth at this stage is only partly gastrin-dependent. In contrast, the development of the A-like cells is independent of gastrin at all stages.

Differentiation of gastric ECL cells is altered in CCK2 receptor–deficient mice

Background & Aims: Gastrin stimulation of the type 2 cholecystokinin (CCK2) receptor results in ECL cell proliferation and histamine secretion. This report describes the effects of targeted disruption of the CCK2 receptor gene on ECL cell morphology and function. Methods: The ECL cells in the oxyntic mucosa of CCK2 receptor–deficient (knockout [KO]) vs. wild-type (WT) mice were investigated by immunocytochemical and biochemical approaches, as well as by electron microscopy. Results: Immunocytochemistry demonstrates similar numbers (cells per millimeter of horizontal length of mucosa) of pancreastatin- or vesicle monoamine transporter-2 (VMAT-2)–immunoreactive cells in WT mice and KO mice. However, only WT mice harbor histamine-immunoreactive ECL cells. The mucosal histamine content in KO mice (likely originating from mast cells) is only a minute fraction of that present in WT animals. The activity of the histamine forming enzyme, histidine decarboxylase (a marker of ECL cells), was undetectable in the oxyntic mucosa of KO mice yet was readily apparent in the mucosa from WT animals. Electron microscopy revealed numerous ECL cells in WT mice. In KO animals, these cells were replaced by an “ECL-like” cell type, characterized by a lack of secretory vesicles (a hallmark feature of normal ECL cells) and the presence of dense-core granules and microvesicles in numbers comparable to those found in WT ECL cells. Based on ultrastructural features, the ECL-like cells in KO mice can be readily distinguished from other gastric endocrine cells, including A-like cells and D cells. Conclusions: Absence of a single gene product, the CCK2 receptor, alters the differentiation and function of gastric ECL cells.GASTROENTEROLOGY 2002;123:577-585

ECL-cell histamine mobilization in conscious rats: effects of locally applied regulatory peptides, candidate neurotransmitters and inflammatory mediators.

1. The ECL cells control gastric acid secretion by mobilizing histamine in response to circulating gastrin. In addition, the ECL cells are thought to operate under nervous control and to be influenced by local inflammatory processes. 2. The purpose of the present study was to monitor histamine mobilization from ECL cells in conscious rats in response to locally applied regulatory peptides, candidate neurotransmitters and inflammatory mediators. 3. Microdialysis probes were implanted in the submucosa of the acid-producing part of the rat stomach. Three days later, the agents to be tested were administered via the microdialysis probe and their effects on basal (48 h fast) and stimulated (intravenous infusion of gastrin-17, 3 nmol kg(-1) h(-1)) mobilization of ECL-cell histamine was monitored by continuous measurement of histamine in the perfusate (radioimmunoassay). 4. Locally administered gastrin-17 and sulfated cholecystokinin-8 mobilized histamine as did pituitary adenylate cyclase-activating peptide-27, vasoactive intestinal peptide, peptide YY, met-enkephalin, endothelin and noradrenaline, adrenaline and isoprenaline. 5. While gastrin, sulfated-cholecystokinin-8, met-enkephalin and isoprenaline induced a sustained elevation of the submucosal histamine concentration, endothelin, peptide YY, pituitary adenylate cyclase activating peptide, vasoactive intestinal peptide, noradrenaline and adrenaline induced a transient elevation. 6. Calcitonin gene-related peptide, galanin, somatostatin and the prostanoid misoprostol inhibited gastrin-stimulated histamine mobilization. 7. The gut hormones neurotensin and secretin and the neuropeptides gastrin-releasing peptide, neuropeptide Y and substance P failed to affect ECL-cell histamine mobilization, while motilin and neuromedin U-25 had weak stimulatory effects. Also acetylcholine, carbachol, serotonin and the amino acid neurotransmitters aspartate, gamma-aminobutyric acid, glutamate and glycine were inactive or weakly active as was bradykinin. 8. In summary, a range of circulating hormones, local hormones, catecholamines, neuropeptides and inflammatory mediators participate in controlling the activity of rat stomach ECL cells in situ.

Role of ECL-cell histamine in acute gastric mucosal damage induced by ischemia-reperfusion

Role of ECL-cell histamine in acute gastric mucosal damage induced by ischemia-reperfusion

Role of ECL-cell histamine in acute gastric mucosal damage induced by ischemia-reperfusion

Role of ECL-cell histamine in acute gastric mucosal damage induced by ischemia-reperfusion

Mobilization of rat stomach ECL-cell histamine in response to omeprazole and/or YF476: An in vivo microdialysis study

Mobilization of rat stomach ECL-cell histamine in response to omeprazole and/or YF476: An in vivo microdialysis study

Mobilization of rat stomach ECL-cell histamine in response to omeprazole and/or YF476: An in vivo microdialysis study

Mobilization of rat stomach ECL-cell histamine in response to omeprazole and/or YF476: An in vivo microdialysis study

Isolated rat stomach ECL cells generate prostaglandin E 2 in response to interleukin-1β, tumor necrosis factor-α and bradykinin

The ECL cells control parietal cells by releasing histamine in their immediate vicinity. Gastrin and pituitary adenylate cyclase-activating peptide (PACAP) stimulate histamine secretion from isolated ECL cells, while somatostatin and galanin inhibit stimulated secretion. Prostaglandin E 2 and related prostaglandins likewise suppress ECL-cell histamine secretion. Conceivably, that is how they inhibit acid secretion. In the present study, we examined if prostaglandin E 2 can be generated by isolated ECL cells. Rat stomach ECL cells were purified (>90% purity) by counterflow elutriation and gradient centrifugation and cultured for 48 h. ECL cell stimulants (gastrin and PACAP) and inflammatory agents (interleukin-1β, tumor necrosis factor-α and bradykinin) were tested for their ability to induce prostaglandin E 2 accumulation (24-h incubation), measured by radioimmunoassay. Gastrin and PACAP did not affect prostaglandin E 2 accumulation but interleukin-1β (300 pg/ml), tumor necrosis factor-α (10 ng/ml) and bradykinin (1 μM) induced a 2- to 3-fold increase in the amount of prostaglandin E 2 accumulated. While the combination of interleukin-1β and bradykinin induced a 9-fold increase, the combination interleukin-1β+tumor necrosis factor-α and bradykinin+tumor necrosis factor-α induced additive effects only. The combination of interleukin-1β+tumor necrosis factor-α+bradykinin did not induce a greater effect than interleukin-1β+bradykinin. The effect of interleukin-1β+bradykinin was abolished by adding 10 nM hydrocortisone (suppressing phospholipase A 2 and cyclooxygenase) or 1 μM indomethacin (inhibiting cyclooxygenase). Incubating ECL cells in the presence of interleukin-1β+bradykinin for 24 h reduced their ability to secrete histamine in response to gastrin. The inhibitory effect was reversed by 1 μM indomethacin. Also, increasing the concentrations of hydrocortisone in the medium resulted in an enhanced gastrin-stimulated histamine secretion. Hence, the previously described acid-inhibiting effect of inflammatory agents may be explained by inhibition of ECL-cell histamine mobilization, consequent to enhanced formation of prostaglandin E 2 by cells in the oxyntic mucosa, including the ECL cells themselves.

Control of secretion from rat stomach ECL cells in situ and in primary culture

The acid-producing part of the stomach is rich in peptide-hormone-producing endocrine/paracrine cells of different types. In birds and all mammals studied, ECL cells constitute the quantitatively predominant endocrine cell population in this location. They produce histamine and an as yet unidentified peptide hormone. The paracrine action of the ECL cells is to provide histamine to mediate the stimulating effect of gastrin on the acid-secreting parietal cells: the gastrin-ECL cell-parietal cell axis. Secretion of histamine from the ECL cells was studied in intact conscious rats subjected to gastric submucosal microdialysis and using isolated cells in primary culture. The microdialysis experiments revealed that ECL-cell histamine can be mobilized by the local infusion of gastrin, pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), peptide YY (PYY), met-enkephalin, endothelin and noradrenaline/adrenaline. While gastrin and met-enkephalin induced a sustained elevation of the submucosal histamine concentration, endothelin, PYY, PACAP, VIP, and noradrenaline/ adrenaline induced a transient elevation. Somatostatin, galanin and the prostanoid, misoprostol, inhibited gastrin-stimulated histamine mobilization. Studies of isolated ECL cells (80-90% purity) showed gastrin, PACAP and VIP to stimulate histamine secretion and somatostatin, galanin and misoprostol to inhibit gastrin-stimulated secretion. At present, it seems unlikely that metenkephalin, endothelin, adrenaline and PYY act directly on the ECL cells in situ since the effects could not be reproduced with isolated ECL cells. Clearly, the ECL cells operate under the multifactorial control of circulating hormones, local hormones, catecholamines, neuropeptides and inflammatory mediators.

α-Fluoromethylhistidine depletes histamine from secreting but not from non-secreting rat stomach ECL cells

Histamine in the oxyntic mucosa of the rat stomach occurs in mast cells (10%) and ECL cells (90%). Unlike the mast cells, the ECL cells operate under the control of gastrin. α-Fluoromethylhistidine, an irreversible inhibitor of the histamine-forming enzyme, histidine decarboxylase depletes ECL-cell but not mast-cell histamine. This report shows that the effectiveness by which histidine decarboxylase inhibition depletes ECL-cell histamine depends on the rate of histamine secretion. Rats received α-fluoromethylhistidine by continuous subcutaneous infusion for 24 h. Maximally effective doses (≥3 mg/kg/h) inhibited histidine decarboxylase and reduced oxyntic mucosal histamine in fed rats by 80–90%. In fasted rats, the reduction was 50%. α-Fluoromethylhistidine greatly reduced the number of histamine-immunoreactive ECL cells (immunocytochemistry) and of secretory vesicles in the ECL cells (electron microscopy) in fed but not in fasted rats. The half-life of oxyntic mucosal histamine (determined upon histidine decarboxylase inhibition) was 2.6 h in fed rats and 19.4 h in fasted rats. The amount of histamine secreted in response to gastrin (monitored by gastric submucosal microdialysis) was greatly reduced by α-fluoromethylhistidine in fed rats but not in fasted rats. ECL cells were isolated from rat stomach by elutriation (80% purity). Their histamine content was determined after culture, with or without α-fluoromethylhistidine, in the presence of varying concentrations of gastrin. In a medium containing 10 nM gastrin, ECL cells responded to a maximally effective concentration of α-fluoromethylhistidine (0.1 nM) with 80% reduction in histamine content. In the absence of gastrin, ECL cells responded to α-fluoromethylhistidine with 45% reduction of histamine; the releasable histamine pool was unaffected. In conclusion, the combination of histidine decarboxylase inhibition and a high rate of histamine secretion will promptly exhaust the ECL-cell histamine pool, while histidine decarboxylase inhibition and a low secretion rate will affect the histamine pool much less.

Histamine depletion does not affect pancreastatin secretion from isolated rat stomach ECL cells

ECL cells co-secrete histamine and pancreastatin, a chromogranin A-derived peptide, in response to gastrin. The aim of the study was to explore possible ways to deplete ECL cells of histamine without affecting pancreastatin and to examine how histamine depletion affects pancreastatin secretion. Isolated rat stomach ECL cells (80–85% purity), prepared by counter-flow elutriation, were cultured for 48 h in the presence of α-fluoromethylhistidine (histidine decarboxylase inhibitor), bafilomycin A 1 (inhibitor of vacuolar-type proton-translocating ATPase) or reserpine (inhibitor of vesicular monoamine transporter). At this stage, the cells were challenged with 10 nM (EC 100) gastrin-17 for 30 min. Histamine and pancreastatin were determined by radioimmunoassay. Maximally effective concentrations of α-fluoromethylhistidine, bafilomycin A 1 and reserpine were found to lower ECL-cell histamine (by 60%, 78% and 80%, respectively) without affecting pancreastatin. Basal histamine secretion was reduced in a dose-dependent manner by all three drugs. Gastrin-evoked histamine secretion was reduced greatly by the three agents, while pancreastatin secretion was unaffected. The results show that histamine can be depleted not only by inhibiting its formation (α-fluoromethylhistidine), but also (and more effectively) by inhibiting histamine vesicular uptake, directly (reserpine) or indirectly (bafilomycin A 1). The results also indicate that although histamine is co-stored with pancreastatin, it is not required for either storage or secretion of pancreastatin.

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 μl 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 α-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 α-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.

Secretory organelles in ECL cells of the rat stomach: an immunohistochemical and electron-microscopic study

ECL cells are numerous in the rat stomach. They produce and store histamine and chromogranin-A (CGA)-derived peptides such as pancreastatin and respond to gastrin with secretion of these products. Numerous electron-lucent vesicles of varying size and a few small, dense-cored granules are found in the cytoplasm. Using confocal and electron microscopy, we examined these organelles and their metamorphosis as they underwent intracellular transport from the Golgi area to the cell periphery. ECL-cell histamine was found to occur in both cytosol and secretory vesicles. Histidine decarboxylase, the histamine-forming enzyme, was in the cytosol, while pancreastatin (and possibly other peptide products) was confined to the dense cores of granules and secretory vesicles. Dense-cored granules and small, clear microvesicles were more numerous in the Golgi area than in the docking zone, i.e. close to the plasma membrane. Secretory vesicles were numerous in both Golgi area and docking zone, where they were sometimes seen to be attached to the plasma membrane. Upon acute gastrin stimulation, histamine was mobilized and the compartment size (volume density) of secretory vesicles in the docking zone was decreased, while the compartment size of microvesicles was increased. Based on these findings, we propose the following life cycle of secretory organelles in ECL cells: small, electron-lucent microvesicles (pro-granules) bud off the trans Golgi network, carrying proteins and secretory peptide precursors (such as CGA and an anticipated prohormone). They are transformed into dense-cored granules (approximate profile diameter 100 nm) while still in the trans Golgi area. Pro-granules and granules accumulate histamine, which leads to their metamorphosis into dense-cored secretory vesicles. In the Golgi area the secretory vesicles have an approximate profile diameter of 150 nm. By the time they reach their destination in the docking zone, their profile diameter is between 200 and 500 nm. Exocytosis is coupled with endocytosis (membrane retrieval), and microvesicles in the docking zone are likely to represent membrane retrieval vesicles (endocytotic vesicles).