Ningxin Zeng

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.

Local pH elevation mediated by the intrabacterial urease of Helicobacter pylori cocultured with gastric cells.

Helicobacter pylori resists gastric acidity by modulating the proton-gated urea channel UreI, allowing for pH(out)-dependent regulation of urea access to intrabacterial urease. We employed pH- and Ca(2+)-sensitive fluorescent dyes and confocal microscopy to determine the location, rate, and magnitude of pH changes in an H. pylori-AGS cell coculture model, comparing wild-type bacteria with nonpolar ureI-deletion strains (ureI-ve). Addition of urea at pH 5.5 to the coculture resulted first in elevation of bacterial periplasmic pH, followed by an increase of medium pH and then pH in AGS cells. No change in periplasmic pH occurred in ureI-deletion mutants, which also induced a slower increase in the pH of the medium. Pretreatment of the mutant bacteria with the detergent C(12)E(8) before adding urea resulted in rapid elevation of bacterial cytoplasmic pH and medium pH. UreI-dependent NH(3) generation by intrabacterial urease buffers the bacterial periplasm, enabling acid resistance at the low urea concentrations found in gastric juice. Perfusion of AGS cells with urea-containing medium from coculture at pH 5.5 did not elevate pH(in) or [Ca(2+)](in), unless the conditioned medium was first neutralized to elevate the NH(3)/NH(4)(+) ratio. Therefore, cellular effects of intrabacterial ammonia generation under acidic conditions are indirect and not through a type IV secretory complex. The pH(in) and [Ca(2+)](in) elevation that causes the NH(3)/NH(4)(+) ratio to increase after neutralization of infected gastric juice may contribute to the gastritis seen with H. pylori infection.

The Pituitary Adenylate Cyclase Activating Polypeptide Type 1 Receptor (PAC1‐R) Is Expressed on Gastric ECL Cells: Evidence by Immunocytochemistry and RT‐PCR

Abstract: 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 (∼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.

Galanin inhibition of enterochromaffin-like cell function

BACKGROUND & AIMS Galanin, a 29-amino acid neuropeptide found in the gastric mucosa, inhibits basal and pentagastrin-stimulated acid secretion. Its cellular target is unknown. The aim of this study was to determine whether galanin inhibits Ca2+ signaling and histamine release in enterochromaffin-like (ECL) cells. METHODS Isolated rat ECL cells were purified to 85% homogeneity by a combination of elutriation, density gradient centrifugation, and 48-hour culture. Intracellular calcium concentration ([Ca2+]i) was determined using video imaging with Fura-2 in a 37 degreesC superfusion chamber. Histamine was measured by radioimmunoassay. RESULTS Reverse-transciption polymerase chain reaction of the ECL cell RNA showed a galanin type I receptor subtype. Galanin inhibited gastrin, Bay K8644, and K+ depolarization-induced calcium mobilization and entry as well as reduced basal calcium levels. Pretreatment with pertussis toxin decreased the effect of galanin. Galanin inhibited basal and gastrin-stimulated histamine release by approximately 60% with a median effective concentration of 1.10(-10) mol/L. The inhibitory actions of galanin on histamine release and Ca2+ influx could be reduced by a galanin antagonist, galantide. CONCLUSIONS Galanins inhibition of acid secretion can be explained in part by inhibition of calcium signaling and histamine release from the ECL cells due to activation of a Gi,o protein-coupled receptor.

Peptide YY inhibition of rat gastric enterochromaffin-like cell function

BACKGROUND & AIMS The cellular target for peptide YY (PYY) inhibition of gastric acid secretion is unknown. The aim of this study was to determine whether PYY inhibits histamine release from isolated enterochromaffin-like (ECL) cells by stimulation of a selective Y receptor. METHODS Isolated rat gastric ECL cells were analyzed in short-term culture for histamine release and for changes in intracellular calcium concentration using video imaging. RESULTS Gastrin-stimulated histamine release was inhibited with a 50% inhibiting concentration of 2.10(-9) mol/L. Inhibition of histamine release and of calcium entry by PYY and [Pro34]PYY and no effect of PYY(3-36) characterizes an inhibitory Y1 receptor subtype. Reverse-transcription polymerase chain reaction of ECL cell RNA showed that the receptor was the nontruncated Y1 isoform. The inhibitory action of PYY and related peptides on gastrin-stimulated histamine release and calcium signaling was eliminated by pretreatment with 200 ng/mL pertussis toxin. Additive but not synergistic inhibitory effects of PYY and somatostatin on gastrin-stimulated histamine release were observed. CONCLUSIONS Activation of a Y1 inhibitory receptor subtype present on the gastric ECL cell that inhibits gastrin-induced ECL cell histamine release and Ca2+ entry by activation of a Gi or G(o) class of protein may account for inhibition of gastric acid secretion by PYY released from the small intestine.

Role of neuropeptide-sensitive L-type Ca2+ channels in histamine release in gastric enterochromaffin-like cells

Peptides release histamine from enterochromaffin-like (ECL) cells because of elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) by either receptor-operated or voltage-dependent Ca(2+) channels (VDCC). To determine whether VDCCs contribute to histamine release stimulated by gastrin or pituitary adenylate cyclase-activating polypeptide (PACAP), the presence of VDCCs and their possible modulation by peptides was investigated in a 48-h cultured rat gastric cell population containing 85% ECL cells. Video imaging of fura 2-loaded cells was used to measure [Ca(2+)](i), and histamine was assayed by RIA. Cells were depolarized by increasing extracellular K(+) concentrations or by 20 mM tetraethylammonium (TEA(+)). Cell depolarization increased transient and steady-state [Ca(2+)](i) and resulted in histamine release, dependent on extracellular Ca(2+). These K(+)- or TEA(+)-dependent effects on histamine release from ECL cells were coupled to activation of parietal cells in intact rabbit gastric glands, and L-type channel blockade by 2 microM nifedipine inhibited 50% of [Ca(2+)](i) elevation and histamine release. N-type channel blockade by 1 microM omega-conotoxin GVIA inhibited 25% of [Ca(2+)](i) elevation and 14% of histamine release. Inhibition was additive. The effects of 20 mM TEA(+) were fully inhibited by 2 microM nifedipine. Both classes of Ca(2+) channels were found in ECL cells, but not in parietal cells, by RT-PCR. Nifedipine reduced PACAP-induced (but not gastrin-stimulated) Ca(2+) entry and histamine release by 40%. Somatostatin, peptide YY (PYY), and galanin dose dependently inhibited L-type Ca(2+) channels via a pertussis toxin-sensitive pathway. L-type VDCCs play a role in PACAP but not gastrin stimulation of histamine release from ECL cells, and the channel opening is inhibited by somatostatin, PYY, and galanin by interaction with a G(i) or G(o) protein.Peptides release histamine from enterochromaffin-like (ECL) cells because of elevation of intracellular Ca2+concentration ([Ca2+]i) by either receptor-operated or voltage-dependent Ca2+ channels (VDCC). To determine whether VDCCs contribute to histamine release stimulated by gastrin or pituitary adenylate cyclase-activating polypeptide (PACAP), the presence of VDCCs and their possible modulation by peptides was investigated in a 48-h cultured rat gastric cell population containing 85% ECL cells. Video imaging of fura 2-loaded cells was used to measure [Ca2+]i, and histamine was assayed by RIA. Cells were depolarized by increasing extracellular K+ concentrations or by 20 mM tetraethylammonium (TEA+). Cell depolarization increased transient and steady-state [Ca2+]iand resulted in histamine release, dependent on extracellular Ca2+. These K+- or TEA+-dependent effects on histamine release from ECL cells were coupled to activation of parietal cells in intact rabbit gastric glands, and L-type channel blockade by 2 μM nifedipine inhibited 50% of [Ca2+]i elevation and histamine release. N-type channel blockade by 1 μM ω-conotoxin GVIA inhibited 25% of [Ca2+]i elevation and 14% of histamine release. Inhibition was additive. The effects of 20 mM TEA+ were fully inhibited by 2 μM nifedipine. Both classes of Ca2+ channels were found in ECL cells, but not in parietal cells, by RT-PCR. Nifedipine reduced PACAP-induced (but not gastrin-stimulated) Ca2+ entry and histamine release by 40%. Somatostatin, peptide YY (PYY), and galanin dose dependently inhibited L-type Ca2+ channels via a pertussis toxin-sensitive pathway. L-type VDCCs play a role in PACAP but not gastrin stimulation of histamine release from ECL cells, and the channel opening is inhibited by somatostatin, PYY, and galanin by interaction with a Gi or Go protein.

Role of PACAP1 Receptor in Regulation of ECL Cells and Gastric Acid Secretion by Pituitary Adenylate Cyclase Activating Peptide

Abstract: 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 PAC1m 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.