TY - JOUR
T1 - A micromethod for the assay of cellular secretory physiology
T2 - Application to rabbit parietal cells
AU - Adrian, Thomas E.
AU - Goldenring, James R.
AU - Oddsdottir, Margret
AU - Zdon, Michael J.
AU - Zucker, Karl A.
AU - Lewis, Jonathan J.
AU - Modlin, Irvin M.
N1 - Funding Information:
This work was funded by grants from the Veterans Administration to T.E.A., I.M.M., K.A.Z., and M.J.Z. J.R.G. is the Twenty-sixth American College of Surgeons Scholar and recipient of an NRSA Postdoctoral Fellowship. The authors thank Nigel Cox of the Yale Medical Instrumentation Facility for his assistance in the design and production of the microtiter plate incubation chamber.
PY - 1989/11/1
Y1 - 1989/11/1
N2 - A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of [14C]-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of [14C]aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in [14C]aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes.
AB - A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of [14C]-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of [14C]aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in [14C]aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes.
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U2 - 10.1016/0003-2697(89)90606-4
DO - 10.1016/0003-2697(89)90606-4
M3 - Article
C2 - 2558591
AN - SCOPUS:0024457444
SN - 0003-2697
VL - 182
SP - 346
EP - 352
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -