alpha-Amanitin, a toxic substance from the mushroom Amanita phalloides, is a potent inhibitor of DNA-dependent RNA polymerase II (the nucleoplasmic form) from sea urchin, rat liver, and calf thymus. This compound exerts no effect on the activity of polymerase I (nucleolar form) or polymerase III (also nucleoplasmic). The inhibition is due to a specific interaction with polymerase II or with a complex of DNA and polymerase II.

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la,25-dihydroxyvitamin D3 was examined for its ability to affect the DNA-dependent RNA polymerases (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of rachitic chick intestinal cell nuclei in vivo. Nucleoplasmic (form II) RNA polymerase activity was stimulated 2-fold (P < 0.05) within 2-3 hr after an oral dose of 0.27 sAg (0.65 nmol) of la,25-dihydroxyvitamin D3 to rachitic chicks. The form II polymerase activity returned to control values by 5-9 hr after dosing with the sterol. In contrast, the nucleolar (form I) RNA polymerase was not increased within this period. Solubilization of nuclear protein and resolution of the two RNA polymerases on DEAE-Sephadex also revealed that there was an increase in polymerase II activity when assayed on exogenous DNA template. This evidence suggests that la,25-dihydroxyvitamin D3 acts at the level of the enzymology of intestinal cell transcription and that increased mRNA synthesis after administration of this hormone cannot be due solely to a change in chromatin template activity.It is now well established that vitamin D3 is converted to 25-hydroxyvitamin D3 (25-OH-D3) in several tissues (1, 2) and that this metabolite is again hydroxylated to la,25-dihydroxyvitamin D3 [la, 25-(OH)2-D] in the kidney (3). la,25-(OH)2-D3 has been shown to be the most active metabolite of vitamin D3 in stimulating intestinal calcium absorption (4) and bone calcium resorption (5, 6), and it is considered to be the physiologically active form of vitamin D3. Studies using radioactive vitamin D3 have shown that the active metabolite functions by localizing in the nucleus of target cells (7,8). More recently, it has been shown that la,25-(OH)2-D3 binds initially to a specific cytosol receptor in the intestine (9) and is then transported to the nucleus where it becomes associated with the chromatin (10-13). Furthermore, the la,25-(OH)rDr mediated stimulation of intestinal calcium absorption is inhibited by actinomycin D in vivo (14) and in vitro (15). Recent evidence suggests that vitamin D3 and its active metabolite may modulate the synthesis of a specific mRNA which codes for the de novo synthesis of calcium-binding protein (16), a functional mediator of calcium translocation in the intestine (17). This evidence indicates that la,25-(OH)rD3 may be functioning at the level of gene transcription, similar to the steroid hormones (18,19).Further support for the view that la,25-(OH)rD3 stimulates calcium absorption by regulating specific mRNA synthesis has been demonstrated in vitro using a-amanitin (15), the specific inhibitor of nucleoplasmic DNA-dependent RNA polymerase II (20,21). Since a-amanitin inhibits the induction of a number of enzyme activities in eukaryotic systems (22-24), we decided to determine the effect of la,25-(OH)2-D3 in vivo on the transcriptive enzymes of mucosal nuclei as assayed in vitro. Evidence is presented in this report that la, 25-(OH)2-D3 significantly stimulates RNA polymerase II activity in the target tissue after saturation of mucosal cell nuclei ...

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