Characterization of bombesin receptors on canine antral gastrin cells

Steven R. Vigna, Andrew S. Giraud, Patrick W Mantyh, Andrew H. Soll, John H. Walsh

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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°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 > [Tyr4]-bombesin = hGRP-27 > GRP-10 > ranatensin > neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 μM did not inhibit bombesin binding. These results suggest that canine antral gastrin cells express a saturable binding site for bombesin with the functional properties of a bombesin receptor. We propose that this receptor mediates the stimulatory action of bombesin/GRP on gastrin secretion by gastrin cells.

Original languageEnglish (US)
Pages (from-to)259-264
Number of pages6
JournalPeptides
Volume11
Issue number2
DOIs
StatePublished - 1990
Externally publishedYes

Fingerprint

Bombesin Receptors
Bombesin
Gastrin-Secreting Cells
Gastrins
Canidae
Kassinin
Antral
Binding Sites
Cholecystokinin B Receptor
Neurokinin A
Density Gradient Centrifugation
Centrifugation
Cell Separation
Cholecystokinin
Collagenases
Somatostatin
Edetic Acid
Inhibitory Concentration 50
Digestion

Keywords

  • Gastrin
  • Gastrin cells
  • Gastrin releasing peptide (GRP)
  • Neuropeptides
  • Receptor autoradiography
  • Stomach

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Physiology
  • Cellular and Molecular Neuroscience

Cite this

Characterization of bombesin receptors on canine antral gastrin cells. / Vigna, Steven R.; Giraud, Andrew S.; Mantyh, Patrick W; Soll, Andrew H.; Walsh, John H.

In: Peptides, Vol. 11, No. 2, 1990, p. 259-264.

Research output: Contribution to journalArticle

Vigna, Steven R. ; Giraud, Andrew S. ; Mantyh, Patrick W ; Soll, Andrew H. ; Walsh, John H. / Characterization of bombesin receptors on canine antral gastrin cells. In: Peptides. 1990 ; Vol. 11, No. 2. pp. 259-264.
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AU - Walsh, John H.

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N2 - 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°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 > [Tyr4]-bombesin = hGRP-27 > GRP-10 > ranatensin > neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 μM did not inhibit bombesin binding. These results suggest that canine antral gastrin cells express a saturable binding site for bombesin with the functional properties of a bombesin receptor. We propose that this receptor mediates the stimulatory action of bombesin/GRP on gastrin secretion by gastrin cells.

AB - 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°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 > [Tyr4]-bombesin = hGRP-27 > GRP-10 > ranatensin > neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 μM did not inhibit bombesin binding. These results suggest that canine antral gastrin cells express a saturable binding site for bombesin with the functional properties of a bombesin receptor. We propose that this receptor mediates the stimulatory action of bombesin/GRP on gastrin secretion by gastrin cells.

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KW - Neuropeptides

KW - Receptor autoradiography

KW - Stomach

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