A Functional in Vitro Model to Examine Signaling Mechanisms in Gastrin-Mediated Human Cell Growth

Hong Jin Kim, B. Mark Evers, Nitesh A. Banker, Mark R. Hellmich, Courtney M. Towsend

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Gastrin (G-17) is a trophic hormone with a high affinity for the cholecystokinin-B receptor (CCK-BR); the mechanisms linking receptor binding and activation of downstream events to cell growth are not known, and these studies have been hampered by the lack of a cell model. We have established a pancreatic carcinoid cell line, BON, which produces a number of gut hormones; however, these cells lack native CCK-BR. The purpose of our study was to develop a model cell line containing the CCK-BR and to characterize the cellular mechanisms involved in gastrin regulation of human cell growth. BON cells were transfected with an expression plasmid containing the human CCK-BR, and stable clones were selected using G418. Functional CCK-BR was confirmed by reverse transcriptase-polymerase chain reaction, 125 I-gastrin binding, and mobilization of intracellular calcium ([Ca 2+ ] i ) in response to G-17. Stable transfectants were treated with G-17 (±) the CCK-BR antagonist, L365,260 (L-60); growth was assessed using a Coulter counter. G-17 stimulated the growth of the stable clones, whereas the selective CCK-BR antagonist, L-60, abolished this G-17-mediated trophic effect. We have shown that G-17, acting through the CCK-BR, mobilizes [Ca 2+ ] i as a second messenger and stimulates cell growth. Our unique BON cell line, stably transfected with the human CCK-BR, provides a novel paradigm to further delineate signaling mechanisms in gastrin regulation of human cell growth.

Original languageEnglish
Pages (from-to)69-77
Number of pages9
JournalJournal of Gastrointestinal Surgery
Volume1
Issue number1
DOIs
StatePublished - 1997

Bibliographical note

Funding Information:
Gastrin and cholecystokinin (CCK) are structurally related peptides that share an identical carboxyl pentapeptide, which appears to be involved in the physiologic action of both hormones. The receptors for gastrin/CCK are cell surface receptors, structurally related to the larger superfamily of seven transmem- brane-spanning, G-protein-coupled receptors. Three subtypes of gastrin/CCK receptors have been identified based on pharmacologic and structural differences: the CCK-preferring CCK-A receptor (CCK-AR), the gastrin-preferring CCK-B receptor (CCK-BR), and the less well-characterized CCK-C receptor. The CCK-BR has been detected in gastric parietal cells, pancreatic acinar cells, gut smooth muscle cells, and in numerous gut neoplasms. 11,12 Gastrin binds to the CCK-BR, activates G-proteins, and initiates a complex cascade of downstream intracellular events including activation of adenylyl cyclase, hydrolysis of phosphatidyl inositol, mobilization of intracellular calcium, activation of protein kinases, and induction of certain nuclear transcription factors (e.g., members of the AP-1 gene family). 13-15 L365,260 (L-60), a nonpeptide antagonist specific for CCK-BR, ef- Supported by grants from the National Institutes of Health (PO1 DK35608, RO1 DK48345, and T32 DK07639) and the James E. Thompson Molecular BiologyL aboratoryf or Surgical Research. Presented at the Thirty-SeventhA nnualM eetingo fThe Societyf or Surgeryo f the AlimentaryT ract, San Francisco,C alif.,M ay 19-22, 1996. Reprint requests: Courmey M. Townsend,J r., M.D., Department of Surgery,T he Universityo f Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0527.

ASJC Scopus subject areas

  • Surgery
  • Gastroenterology

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