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Examination of the mechanisms by which extracellular calcium stimulates gastrin release from human antral G cells

University of British Columbia

In the stomach, luminal Ca²⁺ concentrations stimulate gastrin release. The objective of these studies was to determine whether voltage-dependent calcium channels (VDCCs) and the calcium receptor (CaR) mediate calcium-stimulated gastrin release from human antral gastrin (G) cells. The presence of L-type VDCCs in our preparation of G cell-enriched human antral cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis of extracted mRNA using oligonucleotide primers to human class C or D, L-type α₁ subunit sequences. Products of approximately the expected size were obtained, and sequence analysis confirmed a 100% homology to previously published sequences. L-type VDCCs were localized to the G cell via immunocytochemistry. Nitrendipine (1 μM), an L-type VDCC antagonist, significantly reduced Ca²⁺ (3.6 mM) or terbutaline (10⁻⁸-10⁻⁵ M) stimulated gastrin release. These findings suggest that Ca²⁺ influx through L-type VDCCs is involved in Ca²⁺- and β-adrenergic stimulated gastrin release. The presence of the CaR, was examined by RT-PCR using primers to the human parathyroid CaR sequence. One product of approximately the expected size was obtained, with a 100% sequence identity to that previously published. Immunocytochemistry localized the CaR to gastrin, but not somatostatin-containing cells. Gastrin release was stimulated by two known agonists of the CaR, Ca²⁺ (3.6-9 mM) and spermine (100 μM and 1 mM), and was reduced by 1 μM nitrendipine. These findings suggest that the CaR is present in G cells, and activation by Ca²⁺ and/or spermine activates L-type channels and stimulates gastrin release. G cells exhibit an extended dose response, therefore, the involvement of a CaR variant was examined by RT-PCR using primers spanning the entire CaR coding region. Two products approximately 306 (varl) and 100 (var2) base pairs (bps) shorter were obtained. Sequence analysis of product varl revealed a cDNA with a deletion corresponding to exon two; product var2 has yet to be cloned or sequenced. It is possible that the antral CaR splice variants have a decreased affinity for Ca²⁺. These studies suggest that Ca²⁺ stimulates gastrin release by activating L-type VDCCs, and/or the CaR. In antral cells the CaR mRNA is alternatively spliced deleting the region normally encoding exon 2.

Thesis/Dissertation

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2009-02-17

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http://hdl.handle.net/2429/4720

Physiology

University of British Columbia

UBCV

DSpace