Anomalous Increase in Nitric Oxide Synthase Activity by Certain Nitric Oxide‐Generating Compounds in Intact Neuronal Cells

Abstract: It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO‐generating compounds S‐nitroso‐N‐acetylpenicillamine (SNAP), 3‐morpholinosydnonimine (SIN‐1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E‐115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of l‐[³H]arginine into l‐[³H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN‐1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN‐1 and SNP markedly stimulated l‐[³H]citrulline formation in a time‐ and concentration‐dependent manner when cells were incubated with the compounds for >1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E‐115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN‐1 or SNP in intact cells was dependent on the presence of extracellular Ca²⁺, suggesting that it might be due to increased Ca²⁺ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN‐1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken‐cell preparations and in intact cells. Thus, modulation of NOS activity by NO‐releasing compounds is not dependent on cyclic GMP formation and might not be related in a simple fashion to NO generation. Alternatively, activation of guanylate cyclase and stimulation of NOS activity might require different redox species of NO. Our present findings might be of clinical relevance in relation to long‐term use of NO‐generating compounds as therapeutic agents.