Characterization of bombesin receptors on canine antral gastrin cells.
Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Vigna
S R
SR
Center for Ulcer Research and Education, V.A. Wadsworth Medical Center, Los Angeles, CA 90073.
Giraud
A S
AS
Mantyh
P W
PW
Soll
A H
AH
Walsh
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001839:Bombesin / Q000378:metabolism; D002469:Cell Separation; D004285:Dogs; D005455:Fluorescent Antibody Technique; D019886:Gastrin-Releasing Peptide; D005755:Gastrins / Q000378:metabolism; D008969:Molecular Sequence Data; D009092:Mucous Membrane; D010455:Peptides / Q000378:metabolism; D011706:Pyloric Antrum / Q000166:cytology / Q000378:metabolism*; D011869:Radioligand Assay; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013004:Somatostatin / Q000378:metabolism
D005755:Gastrins; D010455:Peptides; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D013004:Somatostatin; D019886:Gastrin-Releasing Peptide; D001839:Bombesin