The effect of calcium on myofibrillar turnover in primary chick leg skeletal muscle cultures was examined. Addition of the calcium ionophore A23187 at subcontraction threshold levels (0.38 #M) increased significantly rates of efflux of preloaded 45Ca +2 but had no effect on total protein accumulation. However, A23187 as well as ionomycin caused decreased accumulation of the myofibrillar proteins, myosin heavy chain (MHC), myosin light chain If (LClf), 2f (LC2f), a-actin (Ac), and tropomyosin (TM). A23187 increased the degradation rate of LClf, LC2f, and TM after 24 h. In contrast, the calcium ionophore caused decreased degradation of Ac and troponin-C and had no effect on the degradation of MHC, troponin-T, troponin-I, or o~,/~-desmin (Din). In addition, A23187 did not alter degradation of total myotube protein. The ionophore had little or no effect on the synthesis of total myotube proteins, but caused a marked decrease in the synthesis of MHC, LClf, LC2f, Ac, TM, and Dm after 48 h.The mechanisms involved in calcium-stimulated degradation of the myofibrillar proteins were also investigated. Increased proteolysis appeared to involve a lysosomal pathway, since the effect of the Ca ++ ionophore could be blocked by the protease inhibitor leupeptin and the lysosomotropic agents methylamine and chloroquine. The effects of A23187 occur in the presence of serum, a condition in which no lysosomal component of overall protein degradation is detected. The differential effect of A23187 on the degradative rates of the myofibrillar proteins suggests a dynamic structure for the contractile apparatus.

The effect of the carcinogen 3-methylcholanthrene (MCA) on protein accumulation in mouse tissues was examined. Administration of the hydrocarbon (250 mg/kg, intraperitoneally) to high-responder C57BL/6 (B6) mice resulted in the induction of five proteins in liver tissue. Quantitative analysis revealed that maximal induction of all five proteins occurred 2 days after MCA administration, with liver protein concentrations returning close to control values by 7 days after the treatment. No consistent effects on the concentrations of other liver proteins were seen. Cell-fractionation studies demonstrate that these proteins appear to be non-microsomal in origin. The induction of the five proteins was tissue-specific, since MCA had no effect on protein accumulation in the B6-mouse kidney, spleen or heart. In addition, their induction appeared to be correlated with the Ah locus, since MCA administration had no effect on the concentrations of the five proteins in the liver of the low-responder DBA/2 (D2) mouse strain. Comparing the extent and time course of this induction with that of previously characterized MCA-induced enzymes, we suggest that these five proteins may represent a new, previously unidentified, set of MCA-induced proteins.

We have previously demonstrated that cytochrome P45Od mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript.Exposure of most organisms to environmental pollutants results in the induction of sets of genes whose products act to metabolize these compounds. In rat liver exposed to aryl hydrocarbons such as 3-methylcholanthrene (MCA), a-napthaflovone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin, the hydrocarbon-metabolizing enzymes cytochromes P-450c and P-450d are the major induced proteins (12, 13). It has been determined that transcriptional regulation mediated by the cytosolic Ah receptor is primarily responsible for the hydrocarbon-induced accumulation of P-450c and its mouse homolog cytochrome P1-450 in intact liver (14, 26). However, it has been demonstrated that the Ah receptor-dependent 30-fold induction of P-450d mRNA and its mouse homolog P3-450 is mediated primarily at a posttranscriptional level (10,18,19). We and others (15,18,19) have also examined the MCA induction of the P-450c and P-450d genes in primary rat hepatocyte cultures grown in defined medium and have shown that both genes appear to be mainly posttranscriptionally regulated. Thus, the regulation of P-450d in cultured cells is identical with its regulation in liver cells, whereas P-450c loses much of its transcriptional inducibility and becomes regulated at a posttranscriptional step.To define the mechanism responsible for the hydrocarbon induction of P-450d mRNA, we have analyzed (i) the degradation rate, (ii) the relationship between protein synthesis and MCA induction, and (iii) the accumulation of nuclear mRNA processing intermediates in the presence and absence of MCA. These results show that the Ah receptormediated induction of P-450d mRNA is effected at an early step of processing of the nuclear precursor mRNA, perhaps at the process-versus-discard decision point, and not at the level of mature cytoplasmic mRNA turnover. Moreover, the inhibition of protein synthesis has no significant effect on the induction process, suggesting that the Ah receptor, which has been shown to enter the nucleus after hydrocarbon binding (22, 26), may act directly on an early event in the mRNA synthesis of P-450d.Degradati...

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