An improved method to evaluate secretory activity of isolated gastric glands and cells.
The accumulation of [14C]aminopyrine (AP) is a valuable and widely used method to probe acid secretion of gastric glands and parietal cells. Usually, the dry weight of glands is used to normalize the AP accumulation ratio, and since the nonhomogeneity of the suspension makes it impossible to evenly distribute glands by simple pipetting, it is necessary to scrupulously dry and weigh each and every experimental sample. Thus, massive, time-consuming procedures of tube drying and weighing are involved. Moreover, the weighing of approximately 1 mg dried gland samples in a 1-g Eppendorf tube introduces considerable sample variance. Here, we present a modified protocol to simplify the AP accumulation method by introducing a generic 3H labeling of protein for normalization. Freshly isolated glands were treated with high specific activity 3H-labeled succinimidyl propionate (3H-succ, 60 Ci/mmol) for 10 min at room temperature during the normal isolation/washing procedure. This reagent reacts with primary amines, and even at normal cell pH the efficiency of reaction (25-30%) is more than adequate. The 3H-labeled glands are then processed normally with simultaneous monitoring of 3H (representing gland amount) and AP (representing the extent of acid accumulation) in separate energy windows of a liquid scintillation counter. Dose- and time-dependent efficiency of 3H labeling were evaluated. The relations between labeling and gland protein and dry weight were linear. No detrimental effects of reagent were noted in the useful range of 1-3 nM 3H-succ. Although some limited sample weighing or protein determination must be made for each batch of 3H-labeled glands, this method avoids massive tube weighings and provides the convenience of double label counting with a highly reproducible method for normalizing data.
Rong
Q
Q
Department of Molecular & Cell Biology, University of California, Berkeley, USA.
Bastaki
S M A
SM
Forte
J G
JG
D000632:Aminopyrine; D000818:Animals; D002250:Carbon Radioisotopes; D005744:Gastric Acid / Q000378:metabolism*; D005753:Gastric Mucosa / Q000378:metabolism*; D066298:In Vitro Techniques; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D011422:Propionates; D011506:Proteins / Q000032:analysis; D011817:Rabbits; D019275:Radiopharmaceuticals; D014316:Tritium
D002250:Carbon Radioisotopes; D011422:Propionates; D011506:Proteins; D019275:Radiopharmaceuticals; D000632:Aminopyrine; D014316:Tritium