FUNCTIONAL-CHARACTERIZATION OF A MUSCARINIC RECEPTOR STIMULATING GASTRIN-RELEASE FROM RABBIT ANTRAL G-CELLS IN PRIMARY CULTURE

In previous studies carbachol-induced stimulation of gastrin release f rom antral G-cells in primary culture suggested the presence of muscar inic acetylcholine receptors on this cell type. Therefore, we attempte d to pharmacologically characterize the muscarinic acetylcholine recep tor subtype involved. Enzymatically isolated rabbit antral mucosal cel ls (0.8% G-cells) were separated by counterflow elutriation yielding a fraction (1.7% G-cells) that was placed in culture on collagen-coated well plates. After 24-36 h of culture 13.0 +/- 2.4% of total adherent cells were immunoreactive for gastrin as shown by immunocytochemical staining using the avidin-biotin complex method. In this preparation b asal gastrin release ranged from 3.3 +/- 0.3 to 4.1 +/- 0.3% of total cellular content. Maximal gastrin release in response to the acetylcho line receptor agonist carbachol (10(-4) M) or the selective muscarinic receptor agonist arecaidine propargyl ester (10(-4) M) was 8.5 +/- 0. 4% and 7.6 +/- 0.4% of total cellular content, respectively. The EC(50 ) values were 3.7 +/- 0.5 X 10(-6) M carbachol and 1.8 +/- 0.4 X 10(-6 ) M arecaidine propargyl ester. At a concentration of 10(-6) M the non -selective muscarinic receptor antagonist atropine and the muscarinic M(3) receptor preferring antagonist hexahydro-sila-difenidol (HHSiD; M (3) greater than or equal to M(1) > M(2)) completely inhibited gastrin release in response to carbachol (K-i values: 52 X 10(-9) M atropine and 29 X 10(-9) M HHSiD) and arecaidine propargyl ester (K-i values: 1 1 X 10(-9) M atropine and 13 X 10(-9) M HHSiD). In contrast the muscar inic M(1) receptor preferring antagonist pirenzepine (M(1) > M31 M(2)) even at high concentrations (10(-6)-10(-5) M) inhibited the carbachol - and arecaidine propargyl ester-induced gastrin release by only about 40%, while the muscarinic M(2)/M(4) receptor antagonist 1-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepin-6-one hydrochloride (AQ-RA 741; M(2) = M(4) > M(1) > M(3)) was ineffective at all concentrations tested (10( -9)-10(-5) M). We conclude that muscarinic receptor agents may directl y stimulate gastrin release from antral G-cells via activation of musc arinic M(3) receptors most probably residing on the G-cells themselves .