calcimycin and Neural-Tube-Defects

Neural tube (NT) closure is a multifactorial process that involves yet unresolved molecular mechanisms. It had been shown previously that high levels of nitric oxide (NO) block the process of NT closure in the chick embryo by inhibiting methionine synthase (MS). The MS inhibition and its effect on NT closure could be alleviated by folic acid, suggesting the involvement of the folate-methionine pathway in the process. Here we test the hypothesis that endogenous nitric oxide synthase (NOS) activity regulates the MS activity required in the process of NT closure. The experiments described here reveal that NOS activity per se, is indeed critical for NT closure in the chick embryo. Inhibition of NOS activity with either 2,4-diamino-6-hydroxypyrimidine (DAHP), which blocks biosynthesis of the NOS co-factor tetrahydrobiopterin (BH4), or with calmidazolium, which blocks calcium-calmodulin binding to NOS, resulted in reduced MS activity and consequently ablated NT closure. Addition of BH4 or the calcium ionophore A23187 restored NOS and MS activities, resulting in NT closure. The results described here imply that NOS and MS activities can serve as functional markers in this developmental process as they are essential in the process of NT closure.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Animals; Biopterins; Calcimycin; Calmodulin; Chick Embryo; Enzyme Inhibitors; Fatty Acids; Fluorescein; Fluorescent Dyes; Imidazoles; Indicators and Reagents; Neural Tube Defects; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitroprusside; Vitamin B 12

The effects of local anesthetics (ketamine HCl, lidocaine HCl, procaine HCl, and tetracaine HCl) on stage 8 (four-somite) chick embryos were investigated. In general, embryos responded to drug treatment in a dose-related manner during the first 6 hr of incubation. Concentrations of 500 micrograms/ml (ca. 2 mM) or higher were embryolethal, whereas 100-200 micrograms/ml (0.1-0.8 mM) preferentially inhibited elevation of neural folds. The latter effect was detectable within 3 hr of treatment and was readily reversible. Tetracaine was the most potent among the four local anesthetics tested at any given dose. Compared to controls, cells in the defective neuroepithelium were less elongated and exhibited smoother apical (luminal) surfaces, thinner microfilament bundles, and less intense actin-specific fluorescence. Furthermore, the effects of local anesthetics (100-200 micrograms/ml) on stage 8 chick embryos were not identical to those of cytochalasin D (0.05 micrograms/ml), colchicine (1 microgram/ml), or ionophore A23187 (25 micrograms/ml), although all treatments produced neural tube defects. Overall results suggest that local anesthetics inhibit closure of the neural tube through their disruptive action on the organization and function of microfilaments in developing neuroepithelial cells.

Topics: Anesthetics, Local; Animals; Calcimycin; Chick Embryo; Colchicine; Cytochalasin B; Ketamine; Lidocaine; Microscopy, Electron; Neural Tube Defects; Procaine; Tetracaine; Time Factors

Topics: Animals; Calcimycin; Calcium; Central Nervous System; Female; Mice; Neural Tube Defects; Papaverine; Pregnancy

Papaverine (50 micrograms/ml) preferentially inhibited uplifting of neural folds in explanted stage 8 chick embryos. Affected neuroepithelial cells often lost their wedge-shaped and elongated appearance. Also, luminal surfaces of most affected cells were smoother than usual as evidenced by the marked decrease in the number of cytoplasmic extensions, but the integrity of other structures (including cytoskeletal components) was not noticeably affected. The observed changes in cell surface topography were due, at least in part, to the imparied ability of apical microfilaments to contract and their eventual relaxation. The "relaxing" effect of papaverine on neural folds could be reversed by subsequent treatment with ionophore A23187. Since papaverine and ionophore A23187 are known to alter the normal distribution of intracellular Ca2+ and changes in cell surface topography are correlated with contractile activities of apical microfilaments, papaverine elicits neural tube closure defects by lowering intracellular free Ca2+ levels, thereby relaxing contracted apical microfilaments in neuroepithelial cells.

Topics: Abnormalities, Drug-Induced; Animals; Calcimycin; Chick Embryo; Microscopy, Electron; Microscopy, Electron, Scanning; Neural Tube Defects; Papaverine; Teratogens; Time Factors