PACAP Regulation of Secretion and Proliferation of Pure Populations of Gastric ECL Cells

Abstract

The gastric enterochromaffin-like (ECL) cell plays a major role in the regulation of gastric acid secretion. We have previously described that Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is present on myenteric neurons in the rat and colocalizes with its high-affinity receptor, PAC1, expressed on the surface of gastric ECL cells. The study of ECL cell physiology has been hampered by the inability to isolate and purify ECL cells to homogeneity. Density gradient elutriation alone yields only 65-70% purity of ECL cells. In the present study, we used fluorescence-activated cell sorting (FACS) with a novel fluorescent ligand, Fluor-PACAP-38, for isolating pure ECL cells. FACS was used to isolate ECL cells based on their relatively small size, low density, and ability to bind the fluorescent ligand Fluor-PACAP-38. The sorted cells were unambiguously identified as ECL cells by immunohistochemical analysis using anti-PACAP type-I (PAC1), anti-histidine decarboxylase (HDC), and anti-somatostatin antibodies. Further confocal microscopy demonstrated that Fluor-PACAP-38, a ligand with a higher affinity for PAC1, bound to extracellular receptors of these FACS-purified cells. FACS yielded an average of 2 million ECL cells/4 rat stomachs, and >99% of the sorted cells were positive for PAC1 receptor and HDC expression. The absence of immunohistochemical staining for somatostatin indicated lack of contamination by gastric D cells, which are similar in size and shape to the ECL cells. Internalization of PACAP receptors and a rapid Ca2+ response in purified ECL cells were observed upon PACAP activation, suggesting that these cells are viable and biologically active. These ECL cells demonstrated a dose-dependent stimulation of proliferation in response to PACAP, with a maximum of 30% proliferation at a concentration of 10-7 M. Microarray studies were perfor med to confirm the expression of genes specific for ECL cells. These results demonstrate that rat gastric ECL cells can be isolated to homogeneity by using a combination of density gradient centrifugation, followed by cell sorting using Fluor-PACAP. These techniques now allow microarray studies to be performed in ECL cells to characterize their functional gene expression and will facilitate pharmacological, biochemical, and molecular studies on ECL cell function.

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…In support of this hypothesis, PACAP elevates [Ca 2ϩ ] i in ECL cells and adjacent parietal cells in rabbit gastric glands, whereas histamine receptor antagonists abolish the Ca 2ϩ response in adjacent parietal cells (Athmann et al, 2000). PACAP also stimulates proliferation of gastric ECL cells through activation of the PKA, protein tyrosine kinase, and MAPK pathways (Lä uffer et al, 1999;Oh et al, 2005). Intracerebroventricular injection of PACAP stimulates gastric acid secretion (Ozawa et al, 1997), sug-324 gesting that PACAP may act centrally to regulate gastric acid release.…”

Isolation, functional characterization, and transcriptome of Mastomys ileal enterochromaffin cells

“…Initially, PACAP-positive Mastomys ileal EC cells could not be identified using the same concentrations and conditions that were used to isolate gastric ECL cells (38). Increasing the fluoro-PACAP concentration 100-fold resulted in identification of a discrete sortable cell population.…”

“…The fluoro-PACAP-labeling strategy has been previously utilized to isolate gastric ECL cells (38). Using the same conditions [fluoro-PACAP (0.1 M); 30 min of pre-FACS incubation], we were unable to identify PACAP-positive Mastomys ileal EC cells (Fig.…”

“…We considered that the ability to collect cells using an increased fluoro-PACAP concentration might reflect different levels of expression of the PACAP receptor subtypes on the EC cell compared with ECL cells and subsequently confirmed this by RT-PCR (21). Nevertheless, we considered fluoro-PACAP labeling (although successful; produced 96% pure EC cells) a suboptimal sorting strategy because it induces ECL cell function (secretion and proliferation) (38). Thus a fluoro-PACAP-sorted cell population would represent a prestimulated EC cell cohort and limit the ability to establish "true" baseline (nonstimulated) parameters of EC cell function.…”

“…More recently, a fluorescence-activated cell sorting (FACS) approach has been utilized to obtain homogeneous (ϳ99% pure) parietal and ECL cell populations from the rat gastric mucosa (29,38). This methodology has not been applied to ileal cell preparations.…”