DYSMORPHOGENIC EFFECTS OF NITRIC-OXIDE (NO) AND NO-SYNTHASE INHIBITION - STUDIES WITH INTRAAMNIOTIC INJECTIONS OF SODIUM-NITROPRUSSIDE AND N-G-MONOMETHYL-L-ARGININE

Sodium nitroprusside (SNP), a chemical that is readily converted to ni tric oxide (NO) in biological systems, was microinjected into the amni otic fluids of cultured whole rat conceptuses on day 10.5 of gestation and dysmorphogenic/embryotoxic effects were evaluated after a 24 hr i ncubation period. Injections of 217 ng/embryo (similar to 800 mu M) re sulted in whitened zones of dead cells in a discretely circumscribed r egion within the mesencephalon closely associated with the neural tube . These zones were observed with a high incidence after SNP microinjec tions and were referred to as ''white caps'' because of their microsco pic appearance. At higher concentrations, the whitened zone extended i nto the rhombencephalon and occasionally appeared to extend the full l ength of the dorsal midline. The whitened zones of tissue separated re adily from the apparently normal underlying tissues upon removal or di sturbance of the amniotic membrane. Coinjection of ferrous hemoglobin with SNP selectively prevented the appearance of ''white caps'' but no t other embryotoxic manifestations. Microinjections of the breakdown p roducts of light-exposed SNP elicited generalized embryotoxicity but ' 'white caps'' were not observed. In separate experiments, we found tha t embryonic enzymes catalyzed significant conversion of arginine to ci trulline, indicating expression of NO-synthase during organogenesis. N -G-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO-synthase , was microinjected (50-150 ng/embryo; similar to 200-600 mu M) On day 10.5 of gestation and produced malformations that differed markedly f rom those elicited by SNP. Failure of anterior and posterior neural tu be closure and profound underdevelopment of the hyoid arch and optic c up were observed at concentrations that produced no apparent growth de ficit. These studies with SNP and L-NMMA indicated that both an excess and a deficiency of NO can be embryotoxic/dysmorphogenic and suggest important roles for optimal levels of NO and NO synthases in normal em bryonic development. (C) 1994 Wiley-Liss, Inc.