PACAP type I receptor activation regulates ECL cells and gastric acid secretion.

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in gastric nerves, and PACAP receptors (PAC1) are found on gastric enterochromaffin-like (ECL) cells. Expression of PAC1 splice variants in purified ECL cells was determined by RT-PCR. PACAP effects on ECL cells were analyzed by video imaging of [Ca(2+)](i) and histamine release; its effects on gastric glands were examined by confocal microscopy of [Ca(2+)](i) in ECL and parietal cells. PACAP action on D cells was measured by [Ca(2+)](i) and radioimmunoassay. PACAP effects on acid secretion were determined in fistula rats with or without neutralizing anti-somatostatin antibodies. All splice variants of PAC1 were found, but vasoactive intestinal polypeptide (VIP) receptor (VPAC) products were absent. PACAP-27 and -38 dose-dependently raise [Ca(2+)](i) in ECL cells, and stimulated histamine release. VIP had a much lower affinity, which demonstrates the presence of PAC1 but not VPAC. PACAP elevated [Ca(2+)](i) in ECL and parietal cells of superfused gastric glands, but only the parietal cell signal was inhibited by ranitidine, showing the absence of PAC1 on parietal cells, and demonstrating functional coupling between the cell types. PACAP and VIP stimulated calcium signaling and somatostatin release from D cells with almost equal efficacy. Acid secretion was stimulated after intravenous injection of PACAP into rats treated with somatostatin antibody. PACAP is a candidate as a mediator of neural regulation of acid secretion.

Gastrointestinal dysfunction in mice with a targeted mutation in the gene encoding vasoactive intestinal polypeptide: a model for the study of intestinal ileus and Hirschsprung's disease.

In 1970, Drs. Said and Mutt isolated a novel peptide from porcine intestinal extracts with powerful vasoactive properties, and named it vasoactive intestinal peptide (VIP). Since then, the biological actions of VIP in the gut as well as its signal transduction pathways have been extensively studied. A variety of in vitro and in vivo studies have indicated that VIP, expressed in intrinsic non-adrenergic non-cholinergic (NANC) neurons, is a potent regulator of gastrointestinal (GI) motility, water absorption and ion flux, mucus secretion and immune homeostasis. These VIP actions are believed to be mediated mainly by interactions with highly expressed VPAC(1) receptors and the production of nitric oxide (NO). Furthermore, VIP has been implicated in numerous physiopathological conditions affecting the human gut, including pancreatic endocrine tumors secreting VIP (VIPomas), insulin-dependent diabetes, Hirschsprung's disease, and inflammatory bowel syndromes such as Crohn's disease and ulcerative colitis. To further understand the physiological roles of VIP on the GI tract, we have begun to analyze the anatomical and physiological phenotype of C57BL/6 mice lacking the VIP gene. Herein, we demonstrate that the overall intestinal morphology and light microscopic structure is significantly altered in VIP(-/-) mice. Macroscopically there is an overall increase in weight, and decrease in length of the bowel compared to wild type (WT) controls. Microscopically, the phenotype was characterized by thickening of smooth muscle layers, increased villi length, and higher abundance of goblet cells. Alcian blue staining indicated that the latter cells were deficient in mucus secretion in VIP(-/-) mice. The differences became more pronounced from the duodenum to the distal jejunum or ileum of the small bowel but, became much less apparent or absent in the colon with the exception of mucus secretion defects. Further examination of the small intestine revealed larger axonal trunks and unusual unstained patches in myenteric plexus. Physiologically, the VIP(-/-) mice showed an impairment in intestinal transit. Moreover, unlike WT C57BL/6 mice, a significant percentage of VIP(-/-) mice died in the first postnatal year with overt stenosis of the gut.

Three-year oral pantoprazole administration is effective for patients with Zollinger-Ellison syndrome and other hypersecretory conditions.

BACKGROUND: Zollinger-Ellison syndrome and idiopathic hypersecretion are gastrointestinal hypersecretory conditions requiring long-term maintenance. AIMS: The safety and efficacy data for short-term (6-month) treatment of Zollinger-Ellison syndrome and idiopathic hypersecretion with oral pantoprazole were previously published. This study extends the initial observations to 3 years. METHODS: The primary efficacy end point for this report was the control of gastric acid secretion in the last hour before the next dose of oral pantoprazole (acid output of <10 mmol/h; <5 mmol/h in subjects with prior acid-reducing surgery). Dose titration was permitted to a maximum of 240 mg per 24 h. RESULTS: Twenty-four subjects completed the study. The acid output of 28 of 34 subjects was controlled at initial enrolment. The mean acid output rates were <10 mmol/h throughout the 36 months of treatment for 90-100% of the patients. The majority of the patients were controlled with b.d. doses of 40 or 80 mg pantoprazole at 36 months (acid output was controlled in 24 of 24 subjects). Pantoprazole was generally well tolerated with minimal adverse events reported. CONCLUSIONS: Maintenance oral pantoprazole therapy up to 3 years at dosages of 40-120 mg b.d. was effective and well tolerated in patients with Zollinger-Ellison syndrome and other hypersecretory conditions.

Pituitary adenylate cyclase activating peptide receptors regulate the growth of non-small cell lung cancer cells.

We have identified pituitary adenylate cyclase activating peptide (PACAP) receptors on small cell lung cancer cell line NCI-N417 in a previous study. In this study, the role of PACAP in the growth and signal transduction of non-small cell lung cancer cells was investigated. Northern blot analysis with a full-length human PACAP receptor cDNA probe revealed a major 7.5-kb hybridizing transcript when total RNA extracted from NCI-H838 cells was used. PACAP bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 14,000/cell) when NCI-H838 cells were used. Specific 125I-labeled PACAP binding was inhibited with high affinity by PACAP-27 and PACAP-38, with moderate affinity by PACAP(6-38), and with low affinity by vasoactive intestinal polypeptide, PACAP(28-38), and PACAP(16-38). PACAP-27 elevated cAMP in a dose-dependent manner, and the increase in cAMP caused by PACAP was reversed by PACAP(6-38). PACAP-27, but not vasoactive intestinal polypeptide, elevated cytosolic Ca2+ in individual NCI-H838 cells. PACAP-27 stimulated arachidonic acid release, and the increase caused by PACAP was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in NCI-H838 cells, whereas the PACAP antagonist PACAP(6-38) reduced colony formation in the absence or presence of exogenous PACAP-27. In nude mice bearing NCI-H838 xenografts, PACAP(6-38) slowed tumor growth significantly. These data suggest that biologically active type 1 PACAP receptors are present on human non-small cell lung cancer cells, which exhibit dual signal transduction pathways and regulate cell proliferation.

Prospective study of the need for long-term antisecretory therapy in patients with Zollinger-Ellison syndrome following successful curative gastrinoma resection.

A long-term cure is now possible in more than 30% of selected patients with Zollinger-Ellison syndrome who undergo gastrinoma resection. The need, however, for continued gastric acid antisecretory therapy in these patients remains controversial. The current study was designed to determine whether post-operative antisecretory therapy is needed in patients who have undergone successful gastrinoma resection and, if so, to attempt to define criteria with which to identify patients who require therapy. Twenty-eight consecutive patients who had previously undergone curative gastrinoma resection were prospectively studied. When antisecretory therapy was discontinued, 43% (12/28) of these patients developed gastro-oesophageal reflux, diarrhoea, acid-peptic symptoms or endoscopic evidence of acid-peptic disease within 2 weeks and were deemed to have failed a trial of antisecretory drug withdrawal. The remaining 57% (16/28) of patients who successfully discontinued antisecretory therapy were followed for a mean time of 31 months after withdrawal of therapy. Analysis of acid output studies pre-operatively, as well as at the time of drug withdrawal, demonstrated that patients who were unable to discontinue antisecretory therapy exhibited higher pre-operative maximal acid output values and higher basal acid output values at the time of attempted drug withdrawal than patients who were able to discontinue therapy. Despite these findings, there was significant overlap in acid output values between groups so that it was not possible to define specific acid output criteria for successful drug withdrawal. Pre-operative clinical characteristics, such as the presence or absence of gastro-esophageal reflux or acid-peptic disease, or post-operative laboratory values, such as the fasting serum gastrin level, did not correlate with the ability to discontinue antisecretory therapy. We conclude that following successful curative gastrinoma resection, 40% of patients still require antisecretory therapy and that both symptom evaluation as well as upper endoscopy should be used to guide attempted drug withdrawal. Although patients who are not able to discontinue therapy have significantly higher acid output measurements than those who are able to discontinue therapy, neither acid output criteria nor any other laboratory or clinical characteristics are able to predict the need for continued antisecretory therapy in these patients.

Rabeprazole is superior to omeprazole for the inhibition of peptone meal-stimulated gastric acid secretion in Helicobacter pylori-negative subjects.

BACKGROUND: Peptone meal-stimulated gastric acid output is considered to be a reliable means to evaluate drug-mediated inhibition of stimulated gastric acid output, an important measure of the efficacy of the agents--such as proton pump inhibitors--used to treat acid-related disorders. AIM: To compare the initial and overall inhibitory effects on peptone meal-stimulated gastric acid secretion of rabeprazole and omeprazole, 20 mg, in Helicobacter pylori-negative subjects on the first and eighth days of treatment. METHODS: Healthy volunteers (n = 27) were randomized in a single-centre, double-blind, double-dummy, 2 x 2 cross-over study. Subjects received an oral dose of rabeprazole or omeprazole, 20 mg once daily, for 8 days. After a 2-4-week washout period, subjects were crossed over to receive the other medication for 8 days. Peptone meal-stimulated gastric acid secretion was measured at hours 11 and 23 at baseline and on days 1 and 8 of treatment. RESULTS: On days 1 and 8, rabeprazole demonstrated a significantly greater inhibition of peptone meal-stimulated gastric acid secretion compared with omeprazole at all time points (P < 0.03). Median values of steady-state inhibition on day 1 were statistically significant at hour 23 (rabeprazole 100% vs. omeprazole 74%, P < 0.02). CONCLUSIONS: Rabeprazole, 20 mg, demonstrated superior control of peptone meal-stimulated gastric acid secretion compared with omeprazole, 20 mg, after the first dose and after the eighth daily dose. Rabeprazole achieved a more rapid onset of acid inhibition and a greater steady-state reduction in peptone meal-stimulated gastric acid secretion.

Essential structural motif in the C-terminus of the PACAP type I receptor for signal transduction and internalization.

The objectives of the study reported here were to identify amino acid residues of the C-terminus that are critical for intracellular signaling. A total of nine amino acid substitution and truncation mutants were constructed by PCR and confirmed by sequencing. Mutant and wildtype receptors were stably transfected into NIH/3T3 fibroblasts and studied for their ability to bind PACAP-27 and activate phospholipase C (PLC) and adenylyl cyclase (AC). Receptor affinity of 125I-PACAP-27 for the wildtype and mutants were similar (Kd = 0.6-1.5 nM). However, truncation of the entire 63 amino acids of the hPAC1 resulted in no signaling to either AC or IP. Addition of the proximal 10 amino acids of the C-terminus failed to restore AC or IP signaling, whereas addition of the proximal 27 amino acids of the C-terminus resulted in reconstitution of complete AC and IP responses, identical to the WT. Point mutations within this 17 amino acid region identified specific amino acids involved in PAC1 signaling. These results indicate that a structural motif within the proximal region of the carboxyl terminus is critical for G protein coupling.

Molecular cloning, functional expression, and chromosomal localization of the human cholecystokinin type A receptor.

The results presented here describe for the first time the molecular cloning of the human CCKA-R. Expression of the recombinant receptor shows the expected subtype pharmacology and coupling to phosphoinositide hydrolysis reported for the native human CCKA-R. This knowledge will enhance our understanding of its distribution, pharmacology, and structure and will improve our understanding of its physiological role in the gastrointestinal and nervous systems in humans. Ultimately, this should hasten the understanding and therapy of gastrointestinal and neuropsychiatric disorders.

Differential signaling and immediate-early gene activation by four splice variants of the human pituitary adenylate cyclase-activating polypeptide receptor (hPACAP-R).

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belonging to the VIP/secretin/glucagon family, is present in the hypothalamus, anterior pituitary, and adrenal gland where it regulates hormone release, in the GI tract where it modulates motility, and in human tumoral cell lines where it shows a growth-promoting effect. It is now appreciated that alternative splicing of two exons of the rat PACAP-R gene generate four major rPACAP-R splice variants that are differentially expressed in tissues and variably coupled to intracellular second messengers. Because of the potential implications of these findings in human physiology, we cloned the hPACAP-R gene. Similar to the rat, two exons (SV-1 and SV-2) are alternatively spliced to account for four major hPACAP-R receptor splice variants. These splice variants (hPACAP-R-null, hPACAP-R-SV1, hPACAP-R-SV2, hPACAP-R-SV-3) were cloned from a human frontal cortex cDNA library, stably transfected in NIH/ 3T3 cells and each characterized for ligand affinity, stimulation of adenylate cyclase (AC) and phospholipase C (PLC), and ligand-induced expression of the proto-oncogenes, c-fos, and c-myc. Stably transfected NIH/3T3 cells expressing similar numbers of receptors of the four splice variants showed nearly identical responses for ligand affinity and potency for P-38- and P-27-stimulated increases in cAMP and total inositol phosphates. However, each receptor splice variant differed in their ligand-stimulated efficacy for total inositol phosphate stimulation. The hPACAP-R-SV2 showed the greatest efficacy for stimulating phospholipase C that was approximately seven-fold greater than the hPACAP-R-SV1, twofold greater than the hPACAP-R-Null, and 1.5-fold greater than the hPACAP-R-SV-3 splice variants. To determine whether the splice variants also differ in their ability to stimulate immediate early gene expression, c-fos and c-myc transcripts were assayed by Northern blot and quantified by densitometry. PACAP-38 increased c-fos and c-myc expression for all four of the receptor splice variants that paralleled the efficacy for PLC stimulation, with the the SV-2 splice variant showing the greatest stimulation. These results show that the hPACAP-R-SV2 exhibits enhanced efficacy for coupling to both PLC and activation of the protooncogenes, c-fos and c-myc suggesting a novel and potentially important mechanism for differentially activating signal transduction pathways that influence cellular growth and differentiation.

Cholecystokinin receptor family. Molecular cloning, structure, and functional expression in rat, guinea pig, and human.

A review of the literature encompassing numerous pharmacological, physiological, and biochemical studies indicates the presence of at least four CCK receptor types, CCKA, CCKB, gastrin, and CG-4 receptors. Multiple subtypes of the CCKAR have been postulated to account for the differences in pharmacology or affinity cross-linking of CCKARs between pancreas and gallbladder and the presence of high and low affinity CCKARs on pancreatic acini. Multiple subtypes of the CCKBR have been postulated to explain the differences in pharmacology and physiology between gastric and gallbladder smooth muscle CCKBRs. We recently cloned and functionally expressed both the CCKAR and the CCKBR from rat, guinea pig, and human. The CCKAR and CCKBR are 48% homologous and constitute a family of receptors within the guanine nucleotide-binding regulatory protein-coupled superfamily of receptors. Each receptor is highly conserved between species. A single cDNA encoding a single protein is present in both pancreas and gallbladder and can account for both high and low affinity CCKARs found on pancreatic acini when transfected into COS-7 cells. A single cDNA encoding a single CCKBR protein is present in both the central nervous system and the periphery including the gastrointestinal system. Therefore, the gastrin receptor is actually a CCKBR present on parietal cells. Genomic and cDNA library hybridization as well as Northern and Southern hybridization studies among rat, guinea pig, and human species identifies only two members of the CCK receptor family, CCKAR and CCKBR. Although these studies do not identify other closely related members of the CCK receptor family, they do not rule out the existence of other less closely related members. Furthermore, differences in tissue and species-specific posttranslational processing, receptor coupling, and associated membrane protein and lipid heterogeneity may be among some of the other factors that may account for the phenotypic expression of more receptor subtypes than molecular studies would predict.

Role of PACAP1 receptor in regulation of ECL cells and gastric acid secretion by pituitary adenylate cyclase activating peptide.

We previously reported that PAC1 is expressed on ECL cells resulting in stimulation of [Ca2+]i, histamine and acid secretion. The study reported here characterized the signaling by PAC1 on ECL cells; determined the effects of PACAP on the gastric acid secretion in vivo, and determined the effects of chronic administration of PACAP-27 on ECL cell proliferation. PACAP-27 dose dependently stimulated ECL cell Ca2+ and AC with detectable stimulation at 1 nM and maximal stimulation at 100 nM (six-fold). In rats PACAP-27 administration (10 pmol/kg/h) increased the rate of gastric acid secretion when an antisomatostatin antibody was co-administered. Chronic administration of PACAP (10 pmol/h for seven days) via osmotic pump resulted in a more than twofold increase in BrdU incorporation into ECL cells. PACAP acting at the PAC1 results in dual signaling responses to both [Ca2+]i. AC in ECL cells stimulates gastric acid secretion via the actions of histamine acting at the parietal cell and in whole animals leads to proliferation of ECL cells when administered chronically.

The pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1-R) is expressed on gastric ECL cells: evidence by immunocytochemistry and RT-PCR.

The current study was undertaken to determine the presence and distribution of PAC1-Rs within the gastric mucosa. Polyclonal antibodies to the carboxyl terminus of the rat PAC1-R were generated and shown to be specific against the PAC1-R expressed in NIH 3T3 cells. Western blot analysis using isolated (approximately 85% pure) ECL cell membranes identified a 48 kD protein consistent with the calculated molecular mass of the cloned PAC1-R. RT/PCR performed using specific primers for the PAC1-R confirmed the presence of splice variants of the rat PAC1-R, but not VPAC1-R or VPAC2-R. These data provide the first direct evidence for the existence of functional PACAP Type I receptors on ECL cells of the gastric mucosa and suggest a potential role for PACAP in the stimulation of gastric acid secretion and in the regulation of the growth of ECL cells.

Characterization of the pharmacology, signal transduction and internalization of the fluorescent PACAP ligand, fluor-PACAP, on NIH/3T3 cells expressing PAC1.

Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.

PACAP hybrid: a new PACAP receptor antagonist.

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) hybrid, a synthetic antagonist, was investigated on NIH/3T3 cells containing PACAP receptor (R) splice variants (SVs). PACAPhybrid inhibited 125I-PACAP-27 binding to NIH/3T3 cells stably expressing PACAP-R basic, SV-1, SV-2 or SV-3 with an IC50 of 1000 nM. PACAPhybrid antagonized the ability of PACAP-27 to elevate cAMP regardless of the PACAP-R SV used. PACAP was more efficacious at increasing cytosolic Ca2+ in NIH/3T3 cells containing PACAP-R SV-2 than PACAP-R basic, SV-1 or SV-3. PACAPhybrid antagonized the increase in cytosolic Ca2+ caused by PACAP-27 regardless of the PACAP-R SV used. PACAP was more potent at elevating c-fos mRNA using NIH/3T3 cells transfected with PACAP-R SV-2 than PACAP-R basic, SV-1 or SV-3. PACAPhybrid antagonized the increase in c-fos mRNA caused by PACAP-27. These data suggest that PACAPhybrid is a useful PACAP receptor antagonist for PACAP-R SVs.

GERD and its complications. The pathogenic relationship between symptoms and disease progression.

Chronic daytime and noctural acid reflux causes mucosal damage, heartburn, and other symptoms of GERD. The esophageal complications of GERD result from long-term exposure of the esophagus to acid. Extraesophageal GERD complications, such as laryngitis and asthma, develop when some of the acidic refluxate in the proximal esophagus enters the lower or upper airways because of comprised defense mechanisms. A growing body of evidence suggests that nocturnal reflux is more important to the development of severe complications than is daytime reflux. Defects in the lower esophageal sphincter and clearance mechanisms are related in part to recumbency during sleep; they may explain why transient episodes of nocturnal reflux outnumber daytime episodes in patients with GERD. Currently, the hope is that management strategies aimed at control of nocturnal symptoms can prevent, reduce the severity of, or resolve the long-term complications of chronic GERD.

Intravenous esomeprazole 40 mg vs. intravenous lansoprazole 30 mg for controlling intragastric acidity in healthy adults.

BACKGROUND: Intravenous (IV) formulations of proton pump inhibitors are effective for patients in whom oral therapy is not appropriate. AIM: To compare IV esomeprazole and IV lansoprazole for the control of intragastric pH. METHODS: In this open-label crossover study, healthy, Helicobacter pylori-negative adults were randomized to one of two treatment sequences, each consisting of two 5-day dosing periods of IV esomeprazole 40 mg or IV lansoprazole 30 mg. Twenty-four-hour intragastric pH monitoring was conducted on days 1 and 5 of each dosing period. RESULTS: On days 1 and 5, intragastric pH was >4.0 significantly longer with esomeprazole than lansoprazole (least-squares means: day 1, 40.0% vs. 33.6%; day 5, 61.9% vs. 45.4%; both P < 0.0001). During the first 4 h of pH monitoring, intragastric pH was >4.0 significantly longer on days 1 and 5 with esomeprazole than lansoprazole (P < 0.0001). Kaplan-Meier estimates of median hours to stable pH >4.0 were 4.92 for esomeprazole and 5.75 for lansoprazole (P = 0.0014 for test on Gehan scores). CONCLUSION: In healthy adults, IV esomeprazole 40 mg controlled intragastric acidity faster and more effectively than IV lansoprazole 30 mg.

Switching between intravenous and oral pantoprazole.

Proton pump inhibitors (PPIs) are the most effective antisecretory drugs available for controlling gastric acid acidity and volume. They are the drugs of choice in the treatment of moderate-to-severe gastroesophageal reflux disease, hypersecretory disorders, and peptic ulcers. Currently in the United States, they are only available in an oral formulation. However, pantoprazole will soon be available in an intravenous formulation and will extend the power of PPIs to inpatient hospital settings. Intravenous pantoprazole has been shown to be effective and safe in clinical trials. Intravenous pantoprazole is indicated for the treatment of patients who require PPI therapy but who are unable to take oral medication. Intravenous pantoprazole has been shown to maintain acid suppression in patients switched from oral PPIs, so no change in dosage is required when switching from one formulation to the other. Potential hospital-based uses for intravenous PPI therapy include perioperative use as prophylaxis for acid aspiration syndrome during induction of anesthesia, prophylaxis for stress-related mucosal disease, and management of gastrointestinal bleeding from stress or acid peptic disease.

The effect of Zollinger-Ellison syndrome and neuropeptide-secreting tumors on the stomach.

Zollinger-Ellison syndrome (ZES) is caused by a tumor that secretes gastrin and is the most common of the malignant islet cell tumors. ZES leads to hypergastrinemia, which, in turn, causes an overproduction of gastric acid and results in complications of peptic ulcer disease. Of all the islet cell tumors, gastrinoma tumors have undergone the most extensive study, providing a model of tumor management. Increased awareness and improved biochemical and radiologic techniques mean that these disorders are being recognized in more patients. Advances in the management of gastric acid secretion and new localization methods have significantly reduced the morbidity and mortality associated with ZES. The use of intravenous proton pump inhibitors such as pantoprazole will make surgical and perioperative management more favorable for patients. Radiologic and nuclear medicine studies permit the detection of the majority of islet cell tumors and improve the ability for surgical resection. With the recent cloning of the gene for multiple endocrine neoplasia type I (MEN-I) and the recognition of tumor markers associated with the development of islet cell tumors, early detection of these tumors may someday be possible.