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. 2003 Oct 22:3:13.
doi: 10.1186/1472-6793-3-13.

Regulation of amylin release from cultured rabbit gastric fundic mucosal cells

Ian L P Beales  1 , John Calam
Affiliations

Regulation of amylin release from cultured rabbit gastric fundic mucosal cells

Ian L P Beales et al. BMC Physiol. .

Abstract

Background: Amylin (islet amyloid polypeptide) is a hormone with suggested roles in the regulation of glucose homeostasis, gastric motor and secretory function and gastroprotection. In the gastric mucosa amylin is found co-localised with somatostatin in D-cells. The factors regulating gastric amylin release are unknown. In this study we have investigated the regulation of amylin release from gastric mucosal cells in primary culture. Rabbit fundic mucosal cells enriched for D-cells by counterflow elutriation were cultured for 40 hours. Amylin and somatostatin release over 2 hours in response to agonists were assessed.

Results: Amylin release was significantly enhanced by activation of protein kinase C with phorbol-12-myristate-13-acetate, adenylate cyclase with forskolin and elevation of intracellular calcium with A23187. Cholecystokinin (CCK), epinephrine and glucagon-like peptide-1 (GLP-1) each stimulated amylin release in a dose-dependent manner. Maximal CCK-stimulated release was greater than either epinephrine or GLP-1, even when the effects of the latter two were enhanced by isobutylmethylxanthine. Stimulated amylin release was significantly inhibited by carbachol (by 51-59%) and octreotide (by 33-42%). Somatostatin release paralleled that of amylin.

Conclusions: The cultured D-cell model provides a means of studying amylin release. Amylin secretion is stimulated by receptor-dependent and -independent activation of Ca2+/protein kinase C and adenylate cyclase pathways. Inhibition involves activation of muscarinic receptors and auto-regulation by somatostatin.

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Figures

Figure 1
Figure 1
Receptor-independent stimulation of amylin (top) and somatostatin (bottom) from cultured D-cells. Cells were stimulated with phorbol-12-myristate-13-acetate 100 nM (PMA), forskolin 10 μM (FSK) and A23187 1 μM for 2 hours. Results expressed as % of cell content of peptide released, mean ± SEM, ** P < 0.01 vs. control, * P < 0.05 vs. control.
Figure 2
Figure 2
CCK-stimulated amylin (top) and somatostatin (bottom) release from cultured D-cells. Cultured mucosal cells were stimulated with CCK for 2 hours. Results expressed as % of cell content of peptide released, mean ± SEM, * P < 0.05 vs. control.
Figure 3
Figure 3
Effect of epinephrine on amylin (top) and somatostatin (bottom) release from cultured D-cells. Cells were stimulated with increasing concentrations of epinephrine (EPI) in the presence or absence of isobutylmethylxanthine 100 μM (IBMX) for 2 hours. Results expressed as % of cell content of peptide released, mean ± SEM, * P < 0.05 vs. control.
Figure 4
Figure 4
Effect of glucagon-like peptide-1 on amylin (top) and somatostatin (bottom) release from cultured D-cells. Cells were stimulated with increasing concentrations of glucagon-like peptide-1 (GLP) in the presence or absence of isobutylmethylxanthine 100 μM (IBMX) for 2 hours. Results expressed as % of cell content of peptide released, mean ± SEM, * P < 0.05 vs. control.
Figure 5
Figure 5
Effect of octreotide and carbachol on basal amylin and somatostatin release from cultured D-cells. D-cells were stimulated with either octreotide 10 nM (OCT) or carbachol 100 μM (CBH) for 2 hours and release of amylin and somatostatin assessed. Results expressed as % of cell content of peptide released, mean ± SEM.
Figure 6
Figure 6
Inhibitory effects of octreotide and carbachol on agonist-stimulated amylin (top) and somatostatin (bottom) release. D-cells were stimulated with either CCK 10 nM or epinephrine with isobutylmethylxanthine both 100 μM (EPI) in combination with octreotide 10 nM (OCT) or carbachol 100 μM (CBH) for 2 hours. Results expressed as % of cell content of peptide released, mean ± SEM, * P < 0.05 vs. relevant agonist-stimulated release.
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