Effects of alpha-amanitin and cycloheximide on 1,25-dihydroxyvitamin D3-dependent calbindin-D28K and its mRNA in vitamin D3-replete chick intestine

• Published in: The Journal of biological chemistry 1986-06, Vol.261 (16), p.7311-7315
• Main Authors: Theofan, G, Norman, A W
• Format: Article
• Language: English
• Subjects: Amanitins - pharmacology; Analytical, structural and metabolic biochemistry; Animals; Binding and carrier proteins; Biological and medical sciences; Calcitriol - pharmacology; Calcium-Binding Proteins - analysis; Chickens; Cycloheximide - pharmacology; Fundamental and applied biological sciences. Psychology; Intestines - analysis; Male; Protein Biosynthesis; Proteins; RNA, Messenger - analysis; S100 Calcium Binding Protein G - analysis; S100 Calcium Binding Protein G - genetics; Transcription, Genetic - drug effects; Uridine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)38392-8

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.