Dysmorphogenic effects of nitric oxide (NO) and NO‐synthase inhibition: Studies with intra‐amniotic injections of sodium nitroprusside and NG‐monomethyl‐L‐arginine

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Abstract
Sodium nitroprusside (SNP), a chemical that is readily converted to nitric oxide (NO) in biological systems, was microinjected into the amniotic fluids of cultured whole rat conceptuses on day 10.5 of gestation and dysmorphogenic/embryotoxic effects were evaluated after a 24 hr incubation period. Injections of 217 ng/embryo (∼800 μM) resulted in whitened zones of dead cells in a discretely circumscribed region within the mesencephalon closely associated with the neural tube. These zones were observed with a high incidence after SNP microinjections and were referred to as “white caps” because of their microscopic appearance. At higher concentrations, the whitened zone extended into the rhombencephalon and occasionally appeared to extend the full length of the dorsal midline. The whitened zones of tissue separated readily from the apparently normal underlying tissues upon removal or disturbance of the amniotic membrane. Coinjection of ferrous hemoglobin with SNP selectively prevented the appearance of “white caps” but not other embryotoxic manifestations. Microinjections of the breakdown products of light-exposed SNP elicited generalized embryotoxicity but “white caps” were not observed. In separate experiments, we found that embryonic enzymes catalyzed significant conversion of arginine to citrulline, indicating expression of NO-synthase during organogenesis. NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO-synthase, was microinjected (50–150 ng/embryo; ∼200–600 μM) on day 10.5 of gestation and produced malformations that differed markedly from those elicited by SNP. Failure of anterior and posterior neural tube closure and profound underdevelopment of the hyoid arch and optic cup were observed at concentrations that produced no apparent growth deficit. 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 embryonic development.