Neuroendocrine mechanism of gastric acid secretion: Historical perspectives and recent developments in physiology and pharmacology

Abstract

The physiology of gastric acid secretion is one of the earliest subjects in medical literature and has been continuously studied since 1833. Starting with the notion that neural stimulation alone drives acid secretion, progress in understanding the physiology and pathophysiology of this process has led to the development of therapeutic strategies for patients with acid-related diseases. For instance, understanding the physiology of parietal cells led to the developments of histamine 2 receptor blockers, proton pump inhibitors, and recently, potassium-competitive acid blockers. Furthermore, understanding the physiology and pathophysiology of gastrin has led to the development of gastrin/CCK2 receptor antagonists. The need for refinement of existing drugs in patients have led to 2^nd and 3^rd generation drugs with better efficacy at blocking acid secretion. Further understanding of the mechanism of acid secretion by gene targeting in mice has enabled us to dissect the unique role for each regulator to leverage and justify the development of new targeted therapeutics for acid-related disorders. Further research on the mechanism of stimulation of gastric acid secretion and the physiological significances of gastric acidity in gut microbiome is needed in the future.

Graphical abstract

In the stomach, endocrine pathways include G cell-ECL cell and G cell-parietal cells, paracrine pathways include ECL cell-parietal cell, D cell-ECL cell, D cell-G cell and D cell-parietal cell, neuronal pathways include nerve- all cell types, particularly ECL cell, and luminal pathways include HCl-D cell and food-G cell (not shown). Note: pharmacological targets on parietal cells and ECL cells.

Perspectives from history

In 1833, William Beaumont (1785 – 1853) published a book entitled Experiments and Observations on the Gastric Juice and the Physiology of Digestion, which was based on the observations of a patient named Alexis St. Martin who was accidentally shot by a gun in 1822 and survived with a permeant gastric fistula (1, 2). Subsequently, William Osler (1849–1919), known as the father of modern medicine, wrote an article entitled William Beaumont. A pioneer American physiologist highlighting the important contributions of Beaumont including (a) a more accurate and complete description of the components of gastric juice; (b) confirmation of previous observations that hydrochloric acid was the acid-type contained in gastric juice; (c) recognition that gastric juice and gastric mucus were separate entities secreted by the stomach; and (d) that digestion was dependent on muscular motions of the stomach (3–5).

Neural regulation of gastric acid secretion was demonstrated by Ivan Petrovich Pavlov (1849 –1936). In 1897, Pavlov summarized his work in a book entitled Lectures on the Work of the Principal Digestive Glands (6), which is one of the most important books in neurobiology. Using canine models of gastric fistula and gastric pouches, Pavlov demonstrated the neural control of salivary, gastric, and pancreatic secretions. In describing gastric secretion, Pavlov suggested a role for vagal stimulation in the process of acid secretion, which was verified experimentally by severing vagal nerve innervation to the stomach (Fig. 1). Pavlov also proposed the concept that conditional reflexes regulate digestion; the novel idea that psychology can regulate physiology. In 1904, Pavlov received the Nobel Prize in Physiology or Medicine for his ground-breaking experimental work in neurophysiology (7, 8).

Figure. 1:

The neuroendocrine mechanism of gastric acid secretion (HCl). Gastric wall is rich in blood supply and innervation by parasympathetic nerves from the dorsal motor nucleus of the vagus nerve (DMV), particular Cck neuron and its subtype of acetylcholine-contained Chat+ neurons, and by intrinsic neurons containing pituitary adenylate cyclase-activating polypeptide (PACAP) and galanin. Ingested food (Food) in the gastric lumen stimulates G cells to release gastrin (aminated gastrin) and gly-gastrin into the blood circulation. Gastrin stimulates CCK₂ receptors (CCK₂R) which are expressed on ECL cells and parietal cells. Activation of CCK₂R on the ECL cells induces histamine formation by stimulation of histidine decarboxylase (HDC) and histamine secretion. Through paracrine mechanisms, the released histamine stimulates H₂ receptors (H₂R) which are located on parietal cells. Activation of H₂R triggers parietal cells to secret HCl via H⁺,K⁺-ATPase in cooperation with K⁺ and Ca²⁺ channels and Na⁺/H⁺ and Cl⁻/HCO3⁻ exchangers. ECL cells are also under neural control, particularly stimulation by PACAP on PAC₁ receptor (PAC₁R) and inhibition by galanin on Gal₁ receptor (Gal₁R) and under paracrine control by somatostatin on somatostatin subtype 2 receptor (SSTR₂). Parietal cells are also under endocrine (e.g. gly-gastrin), paracrine (histamine, somatostatin) and neural control (acetylcholine released from vagal nerve-chat+ neuron) on muscarinic acetylcholine receptor subtype 3 (M₃) receptor. G cells are under neural control (acetylcholine on M₃R and galanin on Gal1 receptor) and paracrine control (somatostatin on SSTR₂). D cells in the stomach consist of open-type in the antrum and close-type in the oxyntic mucosa and are under control of PACAP on PAC₁R. The negative feedback loop between circulating gastrin and gastric luminal HCl is mediated by the opened D cells which are under neural control via PAC₁R and M₄R.

Once the vagal neurotransmitter acetylcholine was validated as an agonist of gastric acid secretion, other regulators were soon uncovered. For this, the endocrine regulation of gastric acid secretion was proposed by John Sydney Edkins (1863 – 1940). In 1905, Edkins proposed the novel concept in stomach that hormones, like secretin that was discovered by W.M. Bayliss and E.H. Starling (9), could also regulate acid secretion. To test this experimentally, an extract from the stomach pylorus (but not fundus) injected into the jugular vein resulted in gastric acid and pepsin secretion in anesthetized cats (10). It was subsequently discovered that the compound stimulating acid secretion was a hormone, produced specifically in the pyloric mucosa, and was named “gastrin” (11, 12). This original hypothesis was validated by H.J. Tracy and R.A. Gregory in 1964 by demonstrating that a series of synthetic peptides structurally related to gastrin also had agonist properties for acid secretion (13) (Fig. 1).

Paracrine regulation of gastric acid secretion was derived from an initial observation by Leon Popielski (1866 – 1920) who found that gastric acid secretion was induced by histamine when given subcutaneously but not intravenously in the canine model of gastric fistula (14, 15). Endogenous histamine was believed to be secreted by mast cells located in the gastric mucosa until gastric enterochromaffin-like (ECL) cells were identified by Rolf Håkanson. In 1966, Håkanson observed dopamine-containing epithelial cells in the oxyntic mucosa of rats that had been injected with L-DOPA and this cell population was morphologically indistinguishable from 5-hydroxytryptamine-containing EC cells (16). A year later, Håkanson found that the ECL cells contained histamine (17–20) (Fig. 1).

Once the major effectors of acid secretion were uncovered, studies by John G. Forte (1934–2012) and George Sachs (1935–2019), in particular, interrogated the intracellular mechanisms that regulate acid secretion including the structure of parietal cells (21, 22), the physiology and cell biology of parietal cells (23, 24), the physiology of proton pump functions (25, 26), and the location and function of ion pumps that are essential to support acid secretion (27) (Fig. 1). For timely reviews see (28–30).

Based on the understanding of the neural regulation of gastric acid secretion, Lester Dragstedt (1893–1975) demonstrated the therapeutic effects of vagotomy for duodenal ulcer in 1947, and as such, truncal, selective, and highly selective vagotomy were once commonly performed to treat peptic ulcer disease (31–33) (Fig. 1). In 1972 James Black (1924 – 2010) discovered that the effect of histamine on acid secretion was mediated by histamine H2 receptors (H2R), which led to the development of the selective H2R antagonists (H2RA) metiamide (34, 35) and then cimetidine (36), which was launched in 1976 as Tagamet (34, 36–38) (Fig. 1). In 1988 Black, with Ellion and Hitchings, received the Nobel Prize in Physiology or Medicine for pioneering research that led to new classes of drugs: importantly histamine H2RA to address acid hypersecretion-induced gastric and duodenal ulcer diseases. Cimetidine and other H2RAs that followed, such as famotidine, nizatidine, and ranitidine, provided effective medical treatment for acid-related disorders. However, patients began to relapse despite continuing H2RA treatment. Eventually it was realized that H2RA-induced acid suppression results in an increase in serum gastrin (hypergastrinemia) and tolerance to H2RA treatment (39).

The gastrin receptor was cloned and characterized by Alan S. Kopin and co-workers in 1992 using isolated canine parietal cells (40), and was shown to be one of the receptors that bind cholecystokinin (CCK), a hormone secreted by the L cells in duodenum. Two types of CCK receptors have been identified, type A, “alimentary,” and type B, “brain”. Based on recommendations of the International Union of Pharmacology (IUPHAR) committee regarding receptor nomenclature and drug classification, the CCK-A receptor was renamed CCK1 receptor (CCK1R), and the CCK-B receptor was renamed CCK2 receptor (CCK2R). CCK1R binds and responds to sulfated CCK with a 500- to 1,000-fold higher affinity or potency than sulfated gastrin or nonsulfated CCK. The CCK2R binds and responds to gastrin or CCK with almost the same affinity or potency but discriminates poorly between sulfated and nonsulfated peptides (41).

The CCK2R is localized to the ECL cells and the neck zone proliferating cells of the stomach. Physiologically, gastrin stimulates acid secretion by acting directly on the parietal cells and/or by mobilizing histamine release from the ECL cells, which then induces acid secretion by binding to the H2 receptors located on parietal cells (Fig. 1). Gastrin is also a growth factor, particularly for the oxyntic mucosa of the stomach, where hypergastrinemia stimulates proliferations of CCK2R+ progenitors, resulting in increased parietal- and ECL-cell mass. With unchecked gastrin-mediated proliferation, ECLoma and carcinoid tumors develop via the activation of extracellular signal-regulated kinase/mitogen-activated protein kinase signaling (42–45).

With the success of H2RA, pharmaceutical companies such as Merck, Lilly, and Wyeth, became interested in researching CCK2R antagonists (CCK2RAs). Many inhibitors were synthesized, but problems with potency, selectivity for the CCK2R, agonist activity, and oral bioavailability hampered their efficacy. Some CCK2RAs were tested in healthy subjects and patients, but none proved worthy of further development (46–49). In fact, the interest in CCK2RAs waned when substituted benzimadazoles, like omeprazole, were shown to inhibit the H⁺, K⁺-ATPase (50, 51), which is the proton pump on gastric parietal cells (52). After decades of investigation about the properties of a pump that secretes acid, Sachs and colleagues (53–55) described the nature of this pump, which facilitated the development of inhibitors to block its activity. Proton pump inhibitors (PPIs) proved superior to H2RAs in healing acid-related conditions and, although PPIs also induce hypergastrinemia, drug tolerance does not develop because of the mechanism of drug action (56). The finding that H. pylori causes most cases of peptic ulcer disease (57), and can be eradicated by a short course of PPI treatment and a cocktail of antibiotics, was likely another reason for disinterest in researching and developing a CCK2RA for clinical use. After H. pylori eradication had been introduced, the need for maintenance therapy for peptic ulcer disease was largely eliminated and gastro-esophageal reflux disease (GERD) became the main indication for prolonged inhibition of gastric acid. Many millions of patients have taken PPI since omeprazole was marketed in the 1980s. Although in recent years there has been a resurgence of interest in the physiology and pathology of gastrin, especially the trophic effects of hypergastrinemia on the stomach, pancreas and colon, there remains no CCK2RA available for clinical use (58, 59). Thus, novel therapeutic strategies are needed to inhibit gastric acid secretion and prevent the trophic/tumor-promoting effects of gastrin when using CCK2RA, as is described below.

Control of gastric acid secretion by gastrin/CCK₂ receptor antagonists

Physiology

Gastrin is a peptide hormone produced by a single gene that is synthesised as pre-progastrin, and is processed into progastrin and amidated gastrin peptide fragments, mostly G17 and G34 (60). Both gastrin fragments have a C-terminal amidated tetrapeptide that bind to CCK2R in the stomach, and in the central and peripheral nervous systems.

G cells in the gastric antrum produce gastrin, which stimulates gastric acid secretion, proliferation, migration, differentiation, and have anti-apoptotic effects on gastric epithelial cells. Gastrin also regulates various genes in the gastric mucosa and stimulates paracrine cascades, including cytokines and growth factors (60, 61). Gastrin activates CCK2R on ECL cells in the oxyntic mucosa of the stomach to secrete histamine, which in turn stimulates histamine H₂-receptors on adjacent parietal cells to secrete acid via the H⁺/K⁺-ATPase (Fig. 1).

The ingestion of food causes secretion of gastrin into the systemic circulation. Initially, the buffering capacity of food neutralises gastric acid, but as acid secretion continues and digestion proceeds and the gastric contents move into the duodenum, the buffering capacity of the food bolus diminishes and intragastric pH falls. This acidic pH stimulates D cells in the gastric antrum to secrete somatostatin, a hormone that switches-off gastrin secretion. Secretion of gastric acid is also under the control of vagal stimulation, which leads to gastrin release. The vagus also controls the release of somatostatin. Experiments in healthy human subjects, in which food or intravenous gastrin 17 was used to stimulate gastric acid secretion, indicate that gastrin accounts for about 90% of acid secreted after a meal (62) (Fig. 1).

Pathophysiology

Ingestion of food at mealtimes leads to physiological increases and decreases in circulating gastrin throughout the day. However, chronic reduction in the acid output by disease, such as autoimmune chronic atrophic gastritis (CAG), genetic mutations of the proton pump, H. pylori infection of the oxyntic mucosa, or by an acid suppressant, such as a PPI or a potassium-competitive acid blocker (P-CAB), causes hypergastrinemia (62), which is the persistent elevation of circulating gastrin, which can be friend or foe depending on the circumstances, as follows:

Hypergastrinemia in patients with CAG stimulates ECL-cell growth and, in some patients, may cause gastric neuroendocrine tumours (g-NETs), which have malignant potential (63, 64). CAG patients also had a seven-fold increase in the risk of gastric cancer in one study (65) and 200-fold in another study (66). Mutations of KCNQ1 or KCNE1 genes, which are potassium channels in parietal cells (67), or mutation of the ATP4A gene, which encodes the α-subunit of the proton pump (68), cause achlorhydria and hypergastrinemia, which leads to g-NETs and gastric carcinoma in some patients. Hypergastrinemia induced by long-term PPI therapy is associated with a 3-fold increased risk of gastric carcinoma (69), even after H. pylori-eradication (70).

Patients with Zollinger–Ellison syndrome (ZES) have hypergastrinemia due to ectopic secretion of gastrin from a NETs (usually in the pancreas/duodenum), which is associated with ECL-cell growth and greater malignant potential than g-NETs in CAG patients (71). In order to control ZES, the majority of ZES patients require lifelong treatment for the marked gastric acid hypersecretion induced by hypergastrinemia, with potent gastric acid anti-secretory agents (72).

Gastrin is known to induce miR-222 overexpression (73), which has been reported to be upregulated in many types of cancer (74–77). Higher levels of miR-222 are associated with more advanced disease and reduced 5year survival (78, 79). miR-222 expression is also increased in gastric cancer tissue-derived mesenchymal stem cells and in the stomach of H. pylori infected individuals (80, 81). The CCK2RA netazepide reduced overexpressed miR-222 in the serum and gastric corpus mucosa of patients with CAG, hypergastrinemia and g-NETs, and miR-222 returned to normal levels after stopping netazepide treatment (73).

The Mongolian gerbil has been used as an efficient, robust, and cost-effective rodent model that recapitulates many features of H. pylori-induced gastric inflammation and carcinogenesis in humans (82). Netazepide prevented oxyntic mucosal inflammation induced by H. pylori infection in Mongolian gerbils, indicating that gastrin may also have inflammatory activity (59).

Preclinical studies

Several benzodiazepine-derived CCK2RA, such as L-364,718 (devazepide), L-365,260, CI-988, YM022, Z-360 and YF476 (netazepide) have been invented (83, 84). Two new classes have recently been synthesized, JB95008 is a substituted imidazole (85) and JNJ-26070109 is a benzamide derivative (86). JNJ-26070109 caused dose-dependent inhibition of pentagastrin-stimulated acid secretion in rats and dogs and prevented rebound hyper-responsiveness of acid secretion to pentagastrin after stopping omeprazole in rats. YM022 also prevented rebound hyper-responsiveness of acid secretion to pentagastrin after stopping omeprazole (87). However, most other CCK2RAs have had problems with potency, selectivity for the CCK2 versus CCK1 receptor, solubility or oral bioavailability. Netazepide, a benzodiazepine derivative, has been the most studied CCK2RA (88–90) with main results as follows:

In in vitro studies, netazepide caused a concentration-dependent inhibition of specific binding of the ligand [¹²⁵I] CCK-8 to rat and cloned human CCK2R in vitro. The affinity of netazepide for rat gastrin receptors was 264 and 70 times higher than that of two other CCK2RA, L-365,26020 and CI-988, respectively, and 4100 times its affinity for rat pancreatic CCK1 receptors. Netazepide had very little affinity for various other receptors, such as histamine, muscarinic and benzodiazepine receptors.

In anaesthetised rats, intravenous netazepide caused dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion, with an ED₅₀ of 0.0086 mmol/kg, compared with an ED₅₀ of 0.013 mmol/kg for intravenous famotidine.

In Heidenhain pouch dogs, intravenous and oral netazepide each caused dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion, with ED₅₀ values of 0.018 and 0.020 mmol/kg, respectively. ED₅₀ for intravenous and oral famotidine was 0.078 and 0.092 μmol/kg, respectively

In dogs with a gastric fistula, intravenous netazepide dose-dependently inhibited pentagastrin-induced gastric acid secretion, with ED₅₀ 0.0023 mmol/kg. Also in gastric fistula dogs, oral netazepide, famotidine, and the PPI, omeprazole, each dose-dependently inhibited peptone-induced gastric acid secretion, with ED₅₀ of 0.11, 0.76 and 4.28 mmol/kg, respectively. Therefore, netazepide was about 7 and 40 times more potent than famotidine and omeprazole, respectively. Comparison of ED₅₀ for intravenous and oral netazepide indicates that the oral bioavailability was 26–28% in rats and 27–50% dogs.

In rats, netazepide prevented the increases in ECL-cell activity and density, oxyntic mucosal thickness, mucosal histamine decarboxylase (HDC) activity and serum pancreastatin caused by PPI-induced hypergastrinemia (91). It should be noted that the ECL cells make up the larger part (60–70%) of the endocrine cells of the stomach. They are rich in pancreastatin/chromogranin (CGA)-related peptides which shared with most other peptide hormone-producing cells (92–94). Surgical removal of the acid-producing part of the stomach eliminates most pancreastatin (70–80%) from the blood stream, and the synthesis and release of pancreastatin from the ECL cells is known to be via the exocytotic proteins under neurohormonal regulation, including gastrin (95–100). Thus, pancreastatin is a biomarker of ECL-cell activity (101–107). ECL cells contain vesicular monoamine transporter 2 (VMAT2) to accumulate histamine into the secretory vesicles (108–110), while EC cells contain VMAT1 (and therefore accumulate serotonin)(111). Reserpine, once-used clinical drug, inhibited histamine secretion from the ECL cells (110, 112).

Thus, these preclinical studies have shown netazepide to be a potent, highly selective, competitive and orally active CCK2RA. Indeed, netazepide has been described as the “gold standard” CCK2RA (83) and has been used in many other experimental animal-based studies, to assess the physiology and pathology of gastrin.

Clinical studies

In early clinical studies, L-365,260 produced modest and short-lasting inhibition of gastrin-stimulated acid secretion (113). Since then, netazepide has been the only CCK2RA used in studies to assess the effect of a CCK2RA on gastric acid production in humans. To date, there have been no studies of a CCK2RA in patients with acid-related diseases. However, the clinical pharmacology of oral netazepide has been characterised in a series of studies in healthy subjects.

In a first-in-human, single-dose study in which netazepide was compared with the H2RA, ranitidine, both increased 24-h gastric pH. The onset of activity was similarly rapid for both, but activity of ranitidine lasted only about 12 h whereas that of netazepide exceeded 24 h. Median t_max and t_1/2 were about 1 and 7 h, respectively, and the pharmacokinetics were dose proportional (114). Recent human CCK2R binding studies have shown that netazepide is an insurmountable (non-competitive) CCK2RA, not a competitive antagonist as suggested by the preclinical studies (unpublished data). That would explain the long duration of activity of netazepide in raising 24-h gastric acidity.

Single doses of netazepide caused dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion, which persisted after repeated doses (115). Repeated doses also caused hypergastrinemia, which is consistent with persistent acid suppression.

Netazepide and the PPI, rabeprazole, alone and combined once daily for 6 weeks were compared with respect to their effect on basal and pentagastrin-induced gastric acid secretion and 24-h circulating gastrin and CgA at baseline, start and end of treatment, and to determine if netazepide can prevent the trophic effects of PPI-induced hypergastrinemia (116). Single doses of netazepide alone and rabeprazole alone caused dose-dependent inhibition of pentagastrin-stimulated gastric acid secretion, which persisted after repeated doses. Rabeprazole alone and netazepide alone for 6 weeks were similarly effective in reducing acid and increasing serum gastrin. A combination of rabeprazole and netazepide increased serum gastrin and reduced basal acid secretion more than either treatment alone, suggesting more effective acid suppression. Rabeprazole alone increased plasma CgA – a sign of increased ECL-cell activity - whereas netazepide alone reduced plasma CgA - a sign of reduced ECL-cell activity. When combined with rabeprazole, netazepide prevented the increase in CgA resulting from rabeprazole-induced hypergastrinemia, which is consistent with blockade of CCK2R on ECL cells. Thus, netazepide suppressed pentagastrin-stimulated gastric acid secretion as effectively as rabeprazole; the reduction in basal acid secretion and greater increase in serum gastrin by the combination is consistent with more effective acid suppression. The increase in plasma CgA after rabeprazole is consistent with a trophic effect on ECL cells, which netazepide prevented. Therefore, netazepide with or without a PPI, is a potential treatment for acid-related conditions, and netazepide is a potential treatment for the trophic effects of hypergastrinemia.

In a study to assess whether netazepide can prevent the trophic effect of the PPI, esomeprazole, dosed for 28 days, esomeprazole increased circulating gastrin and CgA, whereas netazepide increased gastrin but not CgA, and inhibited dose-dependently the CgA response to esomeprazole. Gastrin and CgA returned to baseline within 2–3 days of esomeprazole withdrawal; netazepide did not shorten that time. There was no rebound dyspepsia after PPI withdrawal that others have reported (117). Thus, netazepide can prevent the trophic effects of hypergastrinemia, but a study of netazepide in patients on long-term PPI therapy is required to establish whether PPI withdrawal really can lead to rebound hyperacidity, and whether netazepide can prevent it.

Although PPIs are safe medicines, PPI-induced hypergastrinemia is associated with: ECL-cell and parietal-cell hyperplasia; fundic gland polyps; increased risk of gastric cancer and of bone fractures; and rebound hyperacidity and dyspepsia after PPI withdrawal. A CCK2RA should be free of those effects and should prevent them if co-administered with a PPI (118).

Ceclazepide is a new benzodiazepine-derived CCK2RA and prodrug that has advantages over netazepide in terms of selectivity, solubility and bioavailability, and is in early clinical development for acid-related conditions (119).

Control of gastric acid secretion by potassium-competitive acid blockers (P-CABs)

Studies aimed to understand H⁺ transport by the H⁺, K⁺-ATPase and the development of acid pump blockers were accompanied by similar efforts to understand potassium (K⁺)-transport and making K⁺-binding site blockers. Potassium transport by the H⁺, K⁺-ATPase occurs from the gland lumen to the cytoplasmic face of the apical surface of parietal cells and is required for pump activity. With the development of PPIs moving forward at a rapid pace, the development of P-CABs followed suit and the first successful chemistry was reported in 1981 by Long and colleagues as SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo[1 ,2-a]pyrindine-3-acetonitrile)(120). There were subsequently a number of P-CABs in development, but SCH 28080 moved rapidly to investigations in numerous acid secretion models including in human patients (121–123). SCH 28080 was shown to be a competitive but reversible inhibitor of H⁺, K⁺-ATPase activity, was highly selective for the H⁺, K⁺-ATPase pump, and had a rapid onset of action (121, 124, 125). The original P-CABs had unwanted side-effects in human clinical trials that hindered further development until Takeda Pharmaceuticals, under the scientific leadership of Tadataka Yamada (1945–2021), resurrected this class of drugs. P-CAB development was re-initiated to respond to several unmet needs that emerged from PPI usage, including suppression of night-time acid secretion and rapid symptom relief.

Takeda synthesized a new pyrrole derivative in 2010, 1-[5-(2-fluorophenyl)-1-(pyridin-3- ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine monofumarate ((126), TAK-438 or vonoprazan fumarate), that was a completely different formulation from other P-CABs including SCH 28080 (127). In 2011 this drug was fully characterized and found to be K⁺-competitive, accumulated more effectively than SCH 28080 in parietal cells, had a slow disassociation rate and very high affinity for the H⁺, K⁺-ATPase pump, thus providing faster onset and longer-lasting inhibition of acid secretion compared to SCH 28080 (128) In many clinical trials, TAK-438 has proven to be effective at maintaining a high night-time pH with a good safety profile (129–131), and is effective or superior to PPI treatment with a similar safety profile for treating ulcer diseases, erosive esophagitis, H. pylori infection, and GERD (132–134). Greater acid suppression results in much higher levels of serum gastrin and the associated risks compared with PPIs (135). Tegoprazan and fexuprazan are the latest generation of P-CABs, with similar efficacy profiles to TAK-438 (136–138). A recent study demonstrated the molecular structure of the H⁺, K⁺-ATP with TAK-438 bound (vonoprazan)(139). P-CAB therapy for the treatment of acid-related disorders has been reviewed recently by others (140–142).

Control of gastric acid secretion revealed through genetic dissection

The stomach is primarily innervated by parasympathetic nerves from the dorsal motor nucleus of the vagus nerve (DMV, Fig. 1). Studies using immunohistochemistry showed that the preganglionic vagal nerve terminals act at cholinergic ganglia in the stomach, to then stimulate intrinsic cholinergic neurons to fire (via nicotinic receptor activation) and release acetylcholine at muscarinic receptors on cell targets such as parietal cells and G cells (143). Both pituitary adenylate cyclase-activating polypeptide (PACAP)-containing fibers and galanin-containing fibers were found in the gastric wall (Fig. 1), including mucosa, submucosa, myenteric plexus and muscularis (144–147).

A recent study using single-cell transcriptomics, recombinase-expressing mouse lines and recombinase-dependent antegrade neural projection tracing techniques showed that DMV-Cck neurons and DMV-Pdyn neurons exclusively innervate the glandular stomach (Fig. 1), targeting distinct enteric neuron subtypes through acetylcholine, respectively. The enteric neuron subtypes were Chat+, which release acetylcholine to induce gastric contraction, and Nos1+, which release nitric oxide to induce gastric relaxation (148)(Fig. 1).

Utilization of various mouse gene knockout (KO) models have enabled us to better understand the functional role of each regulatory peptide, neurotransmitter, neuroendocrine-mediated receptor, pump, or transporter in the process of gastric acid secretion (Fig. 1-inset).

Gastrin KO in mice led to impaired gastric acid secretion at basal level as well as in response to stimuli such as gastrin, histamine, or carbachol that mimics the effect of acetylcholine on both the muscarinic and nicotinic receptors (149, 150). Glycine-extended gastrin synergized with amidated gastrin 17 to stimulate parietal cells in gastrin KO mice (151). Furthermore, chronic H. pylori infection impaired parietal-cell function with low acid output, hypergastrinemia and hyperplasia of ECL cells in wild-type mice but led to vagally-induced hypersecretion of acid in gastrin KO mice, providing a partial explanation that H. pylori infection is a causal factor for either gastric cancer (which is associated with low gastric acid secretion) or duodenal ulcer (high acid secretion)(152).

CCK2R (but not CCK1R) KO led to altered differentiation of ECL cells that were characterized by a lack of secretory vesicles and histamine formation, markedly impaired gastric acid secretion, atrophy of the oxyntic mucosa and hypergastrinemia (153–156). CCK1+2R double KO mice exhibited impaired acid secretion in response to vagal stimulation but partially normalized the ECL cells, likely by a compensative mechanism via up-regulating PACAP type 1 receptor (PAC1) on the ECL cells (156).

Acid secretion stimulated directly by vagal neurons, which mostly secrete acetylcholine in the gastric body, is mediated by muscarinic receptor subtypes 3 and 5 (M3R and M5R) in mice, probably via Chat+ neurons (157, 158). M3R KO mice had an impaired gastric secretion and lack of trophic responses to hypergastrinemia (159). However, it was also shown in M4R KO mice that M3R activation mediated vagal stimulation of acid secretion in concert with M4R, which indirectly modulated the rate of acid secretion after stimulation (160). These results suggested that the activation of M4R after vagal stimulation inhibited somatostatin release from D cells, thus potentiating the rate of acid secretion occurring via M3R activation (160).

Parietal cells secrete HCl by a mechanism involving at the least H⁺,K⁺-ATPase, voltage-gated potassium (Kv) channel, Ca⁺⁺ channels, and Cl⁻/HCO₃⁻ and Na⁺/H⁺ exchangers. Achlorhydria was found in mice lacking H⁺,K⁺-ATPase (161), the KCNE2 potassium channel (162), Ca2+ channel (163), Cl⁻/HCO3⁻ exchangers (Slc4a2, or Ae2) (164, 165), and NHE4 Na⁺/H⁺ exchangers (166).

G cells, ECL cells, and parietal cells express somatostatin receptor type 2 (SSTR2). SSTR2 KO mice showed a shift from endocrine/paracrine to neural pathways in regulation of gastric acid secretion, i.e., i) a shift in the regulation of the ECL cells from a paracrine somatostatin-SSTR2-dependent pathway to a neural dependent pathway, with galanin-GalR1 pathway being a strong candidate; and ii) a shift in the stimulatory regulation of the parietal cells from the gastrin-ECL cell axis to an enhancement of the direct action of gastrin and vagal pathways (167).

It is known from studies of isolated ECL cells that PACAP stimulates histamine secretion via PAC1R, whereas galanin suppressed histamine via Gal1R (98). However, studies using SSTR2 KO mice showed that peripheral PACAP inhibited gastric acid secretion, probably by activation of PAC1R on gastric D cells to induce somatostatin release which inhibits both ECL and parietal cells, whereas galanin inhibited gastric acid secretion through a somatostatin-independent mechanism (168, 169). Furthermore, PAC1 receptor KO mice had an increased basal gastric acid output which was associated with increased number of parietal cells (170). Taken together, these results suggest that a dynamic interaction exists between PACAP-ECL cells pathway and PACAP-D cells in controlling gastric acid secretion.

In comparison with wild-type mice, HDC KO mice exhibited impaired acid secretion with secondary hypergastrinemia, whereas mast cell-deficient mice had unchanged acid secretion (171). H2R KO mice had impaired acid secretion in response to histamine and gastrin but not carbachol, suggesting an independent cholinergic signaling pathway in controlling the parietal cells (172, 173). This phenotype can be explained by the known mechanism of secretory cells in general and parietal cells in particular, i.e., the crosstalk between intracellular Ca⁺⁺ level and cAMP signaling system (174–176)(Fig. 1).

Conclusions

1. There is a rich history of studies that link acid secretion to neural innervation of the stomach via vagal afferents that secrete acetylcholine. The stomach has a complicated circuit of cells that are innervated by the vagus nerve, each of which contribute to acid secretion by providing negative (somatostatin) or positive (acetylcholine, gastrin, histamine) regulators.

2. Once it was determined that acid hypersecretion was the cause of morbidity and mortality in human patients, surgical vagotomy was used to block neural innervation to the stomach thus blocking acid secretion. With numerous side effects arising from such an intervention, more selective vagal blockade was done to cut vagal innervation as close to parietal cells as possible. Doing so reduced acid secretion while also reducing some side effects of the surgery.

3. Drugs that serve to block vagal innervation were not possible due to the widespread localization of vagal afferent neurons through the human body. However, this problem resulted in the development of specific and targeted therapeutics that were developed to unique receptors on parietal cells, which have been used successfully since about 1980. These include H2 receptor antagonists to block histamine H2 receptors and PPIs to block the H⁺, K⁺-ATPase proton pump.

4. New classes of drugs are being developed regularly to fill-in needed gaps in efficacy of current therapeutics. For this, potassium-competitive K+-channel blockers (P-CABs) like TAK-438 and gastrin CCK2 receptor antagonists, like netazeptide and ceclazepide, are in the pipeline.

5. In using various KO mice, the unique role for each regulator and the mechanisms of physiological compensation has been uncovered. This feature has been used to leverage and justify the development of new targeted therapeutics for gastric acid-related disorders.

Perspectives for future

Today, clinical management of gastric acid-related disorders has been revolutionized by the understanding of the mechanism of acid secretion and accordingly, the introduction of potent antisecretory medications (28). However, the statement by William Beaumont in 1838 that “the quantity of gastric juice secreted by the stomach for the solution of its contents does not depend upon the quantity of food introduced into the cavity, but upon the general requirements of the system” (1), should be readdressed.

The stomach, particularly gastric acid, has been thought to play an important role in the regulation of bone metabolism and mineral homeostasis. It was reported that impaired gastric acidification negatively affects calcium homeostasis and bone mass, based on the bone phenotype of CCK2R KO mice (177). However, this was at odds with the bone phenotype of HDC KO mice that displayed the same impaired acidification as CCK2R KO mice but had an increased bone formation and reduced bone resorption (178) Thus, it is unlikely that a lack of gastric acid production per se causes bone loss (179)(see also a separate article by Chen D et al. in this SI). But on the other hand, studies are needed in the future to evidently demonstrate that an adequate gastric acid production is important for bone metabolism and mineral homeostasis, particularly in connection with long-term use of PPIs and aging.

The gut microbiome/microbiota has been demonstrated to play an important role in physiology and diseases, including metabolism, immune system, CNS system, circulation system, and diseases such as obesity, diabetes, inflammatory bowel disease, dementia, chronic fatigue syndrome, several types of cancer, autoimmune diseases, asthma, and mental disorders/diseases (180–185). Studies are needed in the future to demonstrate the importance of gastric acid production upon the requirements and maintenance of a healthy gut microbiome.

While mechanism that regulate gastric acid secretion have been used effectively to inhibit acid secretion, resulting in effective treatments for acid-related disorders/diseases, the physiological mechanisms whereby the activation of brain ganglia by food, metabolic hormones, gastric luminal contents, and elevated pH stimulate acid secretion are poorly understood. In addition to the cells aforementioned, such as opened-type G cells and D cells, tuft cells have been proposed to be a potential chemo-sensor for the acid secretion. Tuft cells have been implicated in muscarinic responses, producing acetyl choline themselves, and chemosensing via taste receptors. The functional significance of Tuft cells is largely unknown and would thus be an important cell to interrogate in future studies (30).