UBC Theses and Dissertations
The effects of a somatostatin monoclonal antibody on gastrin and insulin release Tang, Christine Louise
The effects of endogenous somatostatin on gastrin and insulin release were studied by passive immunization with a somatostatin monoclonal antibody, SOMA 10, in the isolated perfused rat stomach and pancreas, respectively. Previous passive immunization studies with somatostatin antiserum in the isolated perfused stomach have yielded conflicting results. The differences in results could be due to the differences in the binding characteristics of the antisera used, and the accessibility of the antiserum to the tissue. In previous studies in vivo and in isolated islets, somatostatin antiserum caused an increase in insulin release. Previous attempts at immunoneutralizing somatostatin in the isolated perfused pancreas of mammals have yielded negative results. However, the isolated perfused pancreas is an ideal model for passive immunization studies, because hormonal and central nervous system influences are eliminated, and the microanatomy of the islet is preserved. This study differed from previous studies in that a monoclonal antibody to somatostatin, which is more specific than somatostatin antiserum, was used in an attempt to neutralize endogenous gastric and pancreatic somatostatin. Fab fragments of SOMA 10 were made by papain digestion and purification on protein A-sepharose. These fragments are advantageous for passive immunization, since they contain the somatostatin binding site, and are much smaller than the intact antibody. Therefore they should more readily penetrate into the interstitium and neutralize endogenous somatostatin. SOMA 10 was purified by ammonium sulphate precipitation, in conjunction with hydroxylapatite chromatography. Purity was checked by gel filtration and affinity HPLC and determined to be 93%. Scatchard analysis calculated the binding capacity of SOMA 10 to be 8.3 μg/mg, and the dissociation constant to be 2.2 nM. Both SOMA 10 and the Fab fragments were shown to inhibit the effect of exogenously administered somatostatin in the isolated perfused stomach and in gastric fistula rats. Single passage of SOMA 10 in the isolated perfused stomach at 100 μg/ml caused a significant decrease in basal gastrin release. Recirculation of the antibody in the stomach caused an increase in cumulated gastrin release in comparison to controls in which perfusate without the antibody was recirculated. Infusion of the Fab fragment at 15 and 66 μg/ml caused an increase in basal gastrin release suggesting that somatostatin inhibits basal gastrin release. Immunocytochemical staining of the perfused stomachs revealed that the Fab fragments but not the intact antibodies had penetrated into the interstitium. In the pancreas, infusion of 45 μg/ml SOMA 10 and 30 μg/ml Fab fragments inhibited insulin secretion in response to 8.8 mM glucose. There are several explanations for the unexpected decrease in gastrin release and insulin release, observed when SOMA 10 was infused. The antibody and fragment could be binding to the somatostatin receptor and mimicking its effects on gastrin and insulin. A change in conformation of the somatostatin molecule due to binding of the antibody could change the affinity for the receptor. The increased secretion could be a result of neutralization of somatostatin, which inhibited a previously unknown inhibitor of gastrin and insulin release. Immunocytochemical staining revealed that both SOMA 10 and its Fab fragment had entered into the interstitium, implying that endocrine and paracrine effects of SOMA 10 could not be distinguished. In summary, the increase in gastrin release observed when Fab fragments were infused and when SOMA 10 was recirculated suggested that somatostatin exerts a continuous restraint on gastrin release. Both SOMA 10 and Fab fragments were found in the pancreatic interstitium implying that large molecules can pass through the vascular walls, and that endogenous and paracrine effects of the antibody cannot be distinguished in this model.