Species difference in stereoselective involvement of CYP3A in the mono-N-dealkylation of disopyramide.

1. To determine which CYP isoenzyme is involved in the N-dealkylation of disopyramide (DP) metabolism in human and dog, and to determine the stereoselectivity of DP metabolism with human CYP and dog CYP isoenzymes, the following in vitro metabolism studies of DP were conducted: correlation between human CYP isoenzyme activities and DP metabolism with human liver microsomes; inhibition of DP metabolism in human and dog liver microsomes with chemical inhibitors of CYP isoenzymes; inhibition of DP metabolism in human microsomes with human CYP antibodies; inhibition of DP metabolism in dog liver microsomes with human and dog CYP antibodies; metabolism of DP with human (CYP3A4) and dog (CYP3A12) cDNA-expressed isoenzymes; determination of Km and Vmax of DP enantiomers by using cDNA-expressed CYP3A4 and CYP3A12. 2. In human liver microsomes, the formation of the mono-N-dealkylated disopyramide (MNDP) metabolite was best correlated with CYP3A4 activities. DP metabolism was substantially inhibited by ketoconazole, troleandomycin (TA) and human CYP3A4 antibody. DP was metabolized by cDNA-expressed CYP3A isoenzymes. In dog liver microsomes, DP metabolism was inhibited by ketoconazole, TA and dog anti-CYP3A12. DP was also metabolized by cDNA-expressed CYP3A12. 3. CYP3A4 and CYP3A12 are the principal isoenzymes involved in DP metabolism in human and dog respectively. There was no stereoselectivity in N-dealkylation of DP by human CYP3A4. However, there was notable stereoselectivity in the N-dealkylation by dog CYP3A12.

Morphological characterization of actively fusing L6 myoblasts.

We have characterized morphologically the surface of L6 myoblasts at the time of active cell fusion using transmission electron microscopy. Two subclones of the L6 line were used in these studies: the L6Cl55 line that fuses to form multinucleated syncytia and the NF44 non-fusing variant. Ultrastructural analysis revealed an electron-opaque material at localized points of cell-cell apposition in actively fusing L6Cl55 cells. This material may be transported by and secreted from smooth-surfaced cytoplasmic vesicles with an electron-dense core. In contrast to L6Cl55 cells, the electron-dense plaques were seen infrequently in cultures of the NF44 non-fusing variant. This previously unidentified substance may be associated with cell-cell recognition or adhesion, both necessary prerequisites for myoblast membrane fusion. Alternatively, the electron-dense plaques may be directly involved in the fusion event.

Temperature-sensitive non-fusing myoblast variant and spontaneous revertant: isolation and characterization.

A stable, temperature-sensitive, non-fusing variant of the L6 rat myoblast cell line has been isolated following mild EMS-induced mutagenesis. At the permissive temperature (37 degrees C), the growth characteristics and developmental pattern of the tsA1 variant are essentially identical to those of the parental L6D0 line at either 37 degrees C or 40 degrees C. At the nonpermissive temperature (40 degrees C), the tsA1 variant grows normally but does not align, fuse, or synthesize detectable amounts of beta-tropomyosin or myosin LC2. A peptide corresponding to myosin LClemb is barely detectable. The temperature-sensitive period spans the interval from 4 to 72 h post-plating with a midpoint at approximately 40 h. Under standard culture conditions, commitment to terminal differentiation occurs between days 3 and 4, and alignment and fusion begin on days 4 and 5, respectively. Thus, the temperature-sensitive event occurs very early in the L6 developmental program. A spontaneous revertant of the temperature-sensitive phenotype (tsA1 [R3]) exhibits recovery of the capacities to align, fuse, and synthesize the repertoire of muscle-specific proteins, suggesting that a single pleiotropic mutation in the tsA1 variant may regulate several stages in L6 myogenesis.