EXENDIN-4 AND EXENDIN-(9-39)NH2 - AGONIST AND ANTAGONIST, RESPECTIVELY, AT THE RAT PARIETAL-CELL RECEPTOR FOR GLUCAGON-LIKE PEPTIDE-1-(7-36)NH2

Exendin-4 is a novel peptide from Heloderma suspectum venom which is 53% homologous with glucagon-like peptide-1 GLP-1-(7-36)NH2, a stimulant of cAMP-dependent H+ production in rat parietal cells. It was the aim of the present study to determine whether this effect of GLP-1-(7-36)NH2 is shared by exendin-4, and whether the responses to either peptide are blocked by exendin-(9-39)NH2, a competitive specific exendin receptor antagonist. In enriched rat parietal cells H+ production was measured indirectly by [C-14]aminopyrine accumulation. cAMP production was determined by radioimmunoassay. [I-125]GLP-1-(7-36)NH2 was prepared using chloramine T followed by high pressure liquid chromatography (HPLC) purification. Exendin-4 (10(-12)-10(-8) M) stimulated [C-14]aminopyrine accumulation in a concentration-dependent manner (EC(50) = 7.6 x 10(-11) M). At the maximally effective concentration (10(-9) M) exendin-4 was as effective as GLP-1-(7-36)NH2 reaching 70-80% of the response to 10(-4) M histamine. Likewise, exendin-4 (10(-11)-10(-7) M) stimulated parietal cell cAMP production up to 2.8-fold. Maximal stimulation by exendin-4 of [C-14]aminopyrine accumulation was not affected by ranitidine (10(-4) M), but was concentration-dependently reduced by exendin-(9-39)NH2 (10(-11)-10(-7) M). At the maximal concentration, exendin-(9-39)NH2 completely abolished the responses to 10(-9) M exendin-4 and to 10(-9) M GLP-1-(7-36)NH2 while not altering stimulation by 10(-4) M histamine. Binding of [I-125]GLP-1-(7-36)NH2 to enriched parietal cells was displaced by exendin-4 (K-i = 4.6 X 10(-10) M) as well as by exendin-(9-39)NH2 (K-i = 4.0 X 10(-9) M). Covalent cross-linking of [I-125]GLP-1-(7-36)NH2 to parietal cell membranes revealed a single band at 59 kDa which was abolished by 10(-7) M unlabeled GLP-1-(7-36)NH2, exendin-4 or exendin-(9-39)NH2. In rat parietal cells exendin-4 and GLP-1-(7-36)NH2 are equipotent and equieffective stimuli of cAMP-dependent H+-production, apparently via identical 59 kDa membrane receptors. Inhibition of [I-125]GLP-1-(7-36)NH2 binding requires about 1 log unit higher concentrations of exendin-4 than stimulation of [C-14]aminopyrine accumulation suggesting the presence of spare receptors. Exendin-(9-39)NH2 is a useful pharmacological tool to specifically block the parietal cell responses to exendin-4 as well as to GLP-1-(7-36)NH2.