In vivo effects of transcriptional and translational inhibitors on duodenal vitamin D-dependent calcium-binding protein messenger ribonucleic acid stimulation by 1,25-dihydroxycholecalciferol.
The in vivo stimulation of vitamin D-dependent calcium-binding protein (9 K CaBP) synthesis by 1,25-dihydroxycholecalciferol [1,25(OH)2D3] in the rat duodenum has been analyzed using a specific [32P]complementary DNA probe for rat 9 K CaBP and inhibitors of RNA transcription (actinomycin D, alpha-amanitin) or protein synthesis (cycloheximide). The relative amounts of 9 K CaBP messenger RNA (mRNA) were assayed by dot-blot hybridization and the relative amounts of 9 K CaBP by RIA. Both inhibitors were injected at doses which significantly inhibited by 80-95% [35S]methionine or [3H]uridine incorporation into protein and RNA, respectively. In vitamin D-deficient rats, a single 1,25(OH)2D3 injection (650 pmol/100 g BW) resulted in a rapid production of 9 K CaBP mRNA which was significantly detectable as early as 3 h, and was followed by an increase of 9 K CaBP levels. Injection of actinomycin D (25 micrograms/100 g BW) 1 h before 1,25(OH)2D3 treatment and repeated every 4 h did not prevent the hormone-induced elevation of duodenal CaBP mRNA, even when the actinomycin dose was doubled and given 2 h before hormonal treatment. alpha-Amanitin (2 micrograms/100 g BW) also failed to block the hormonal stimulation. The protein synthesis inhibitor cycloheximide (25 micrograms/100 g BW) did not cause any change in the 1,25(OH)2D3-induced CaBP mRNA but blocked the CaBP increase after hormone injection. Thus, transcription inhibitors did not prevent the in vivo hormone-induced elevation of 9 K CaBP mRNA, which suggests that 1,25(OH)2D3 increases 9 K CaBP synthesis by increasing 9 K CaBP gene expression at one or more posttranscriptional steps. More precise data will be obtained by measuring the rate of 9 K CaBP gene transcription on isolated nuclei from rat duodenum.
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000235:genetics*; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004386:Duodenum / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D008297:Male; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014158:Transcription, Genetic / Q000187:drug effects
D000546:Amanitins; D002135:Calcium-Binding Proteins; D012333:RNA, Messenger; D003609:Dactinomycin; D003513:Cycloheximide; D002117:Calcitriol