అనాటమీ & ఫిజియాలజీ: ప్రస్తుత పరిశోధన
అందరికి ప్రవేశం
ISSN: 2161-0940
ISSN: 2161-0940
Sakara Tunsophon and Ilka Nemere
Protein kinase C (PKC) is involved in the rapid 1,25(OH) 2 D 3 -mediated extrusion of calcium in chick intestinal epithelial cells. A previous report demonstrated that PKC a and PKC b redistribution corresponded to the dose- response curve for calcium extrusion. We investigated the role of both PKC a and PKC b in hormone-stimulated calcium extrusion by using siRNA in primary cultures of intestinal cells. The results indicated that there was no change in calcium extrusion in cells transfected with siRNA to PKC a , whereas the siRNA to PKC b significantly decreased calcium extrusion in 1,25(OH) 2 D 3 -treated cells when compared with the corresponding hormone-treated cells in transfected and nontransfected cells ( P < 0.01). Similar results were also found using chemical inhibitors of PKC a (safingol) and PKC b ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione]) in intestinal cell suspension. The results demonstrated that PKC b inhibited the extrusion of calcium (resulting in increased calcium uptake) from intestinal cells treated with 1,25(OH) 2 D 3 when compared with vehicle controls ( P < 0.001), the PKC a blocker did not show any difference in calcium extrusion among the treatment groups. Using confocal microscopy, we reduced the hormone exposure to 30 sec in order to view redistribution of PKC a and PKC b . Rapid redistribution of PKC a was found to significantly increase in fluorescence in the apical membrane region after a 30 sec exposure of cells to 300- or 650 pM 1,25(OH) 2 D 3 ( P < 0.01 and 0.001, respectively). By comparison, PKC b immunofluorescence was found to increase significantly in the basal lateral region of the cells, relative to controls after the exposure of cells to 300- ( P < 0.01) or 650 pM ( P < 0.05) seco-steroid. The results suggest that PKC a is the PKC isotype involved in 1,25(OH) 2 D 3 -mediated calcium efflux in intestinal epithelial cells.