Effects of alpha-amanitin and cycloheximide on 1,25-dihydroxyvitamin D3-dependent calbindin-D28K and its mRNA in vitamin D3-replete chick intestine.
We have examined the effects of the transcriptional inhibitor alpha-amanitin and the translational inhibitor cycloheximide on levels of calbindin-D28K (28-kDa calcium binding protein, CaBP) and CaBP-mRNA in the vitamin D-replete chick intestine. Chicks were raised on one of four diets: "normal" (1% calcium, 0.6% phosphorus); high calcium (3.3% calcium, 0.5% phosphorus); low calcium (0.3% calcium, 0.6% phosphorus); or low phosphorus (1% calcium, 0.09% phosphorus). Chicks were then treated either with alpha-amanitin (20 micrograms/chick) or cycloheximide (600 micrograms/chick) 2 h prior to a dose of 6.5 nmol of 1,25-dihydroxyvitamin D3. Duodenal mucosa was collected from 0 to 120 min afterward and assayed for CaBP-mRNA by dot blot hybridization and for CaBP using an enzyme-linked immunosorbent assay. In the absence of inhibitor, CaBP levels were depressed by high calcium and elevated by low calcium or low phosphorus, as expected. These changes occurred, however, without a change in CaBP-mRNA levels. alpha-Amanitin had no effect on CaBP or on CaBP-mRNA levels in chicks raised on any of the diets. Cycloheximide inhibited CaBP levels, and surprisingly also inhibited CaBP-mRNA levels in all four dietary groups. These results indicate that continual protein synthesis is necessary for the expression of CaBP-mRNA, suggesting the existence of a rapidly turned over protein that may be required for stabilization or for processing of the chick intestinal CaBP messenger RNA.
D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000032:analysis*; D002645:Chickens; D003513:Cycloheximide / Q000494:pharmacology*; D007422:Intestines / Q000032:analysis*; D008297:Male; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis*; D064030:S100 Calcium Binding Protein G / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014529:Uridine / Q000378:metabolism
D000546:Amanitins; D002135:Calcium-Binding Proteins; D012333:RNA, Messenger; D064030:S100 Calcium Binding Protein G; D003513:Cycloheximide; D002117:Calcitriol; D014529:Uridine