Enzymes Synthesizing Guanylate Cyclase-Activating Factors in Endothelial Cells, Neuroblastoma Cells, and Rat Brain

Summary: Endothelium-derived relaxing factor (EDRF)/nitric oxide (NO)-like material synthesized by bovine aortic endothelial cells (BAE cells), N1E-115 neuroblastoma cells, and rat brain were assayed by their stimulating effect on soluble guanylate cyclase of rat fetal lung fibroblasts (RFL-6 cells). Intact BAE cells and N1E-115 cells released a guanylate cyclase-activating factor (GAF). GAF release was stimulated by bradykinin and neurotensin, respectively, and was abolished in Ca2+-free medium. When subcellular fractions were analyzed, some GAF-synthesizing activity was found in the cytosolic fraction of BAE cells, but most of the activity was associated with the particulate fraction. In N1E-115 cells and rat brain, however, all GAF synthase activity was localized in the cytosolic fraction. All enzyme activities required l-arginine and nicotinamide adenine nucleotide phosphate (NADPH) for GAF synthesis, all were inhibited by NG-nitro-l-arginine and NG-methyl-l-arginine, and hemoglobin or methylene blue abolished the effect of the GAF produced by all enzymes. All enzymes were sensitive to Ca2+, with the major increase in activity occurring between 100 and 500 nM free Ca2+. The activity of all crude enzyme preparations was independent of exogenous calmodulin. Partial purification of the cytosolic and detergent-solubilized particulate endothelial enzymes by affinity chromatography on 2′,5′-ADP-Sepharose resulted in EDRF/NO synthase activities dependent on exogenous calmodulin. Similarly, GAF synthase from N1E-115 cells and rat brain partially purified by DE52 anion exchange chromatography was dependent on exogenous calmodulin. High concentrations of the calmodulin antagonists calmidazolium and trifluoperazine partially inhibited GAF synthesis by the cytosolic and particulate enzymes from BAE cells. Both inhibitors were more potent against GAF synthases from N1E-115 cells and rat brain. We conclude that N1E-115 neuroblastoma cells and rat brain contain only cytosolic Ca2+/calmodulin-regulated GAF synthase whereas BAE cells contain also a particulate Ca2+/calmodulin-dependent isoenzymes.