Showing papers by "David S. Bredt published in 1991"

Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.

Abstract: Nitric oxide is a messenger molecule, mediating the effect of endothelium-derived relaxing factor in blood vessels and the cytotoxic actions of macrophages, and playing a part in neuronal communication in the brain. Cloning of a complementary DNA for brain nitric oxide synthase reveals recognition sites for NADPH, FAD, flavin mononucleotide and calmodulin as well as phosphorylation sites, indicating that the synthase is regulated by many different factors. The only known mammalian enzyme with close homology is cytochrome P-450 reductase.

Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.

Abstract: Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors in cerebellar slices. Nitric oxide synthase (EC 1.14.23.-) immunoreactivity is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults. We show that the nitric oxide synthase inhibitors, N omega-nitro-L-arginine (EC50 = 20 microM) and N omega-monomethyl-L-arginine (EC50 = 170 microM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids. This effect is competitively reversed by L-arginine. Depletion of the culture medium of arginine by arginase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity. Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation. Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside. These data establish that NO mediates the neurotoxicity of glutamate.

Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues.

Abstract: NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 11423-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity

Inositol trisphosphate receptor: phosphorylation by protein kinase C and calcium calmodulin-dependent protein kinases in reconstituted lipid vesicles.

Abstract: We have previously demonstrated that the inositol 1,4,5-trisphosphate (IP3) receptor is phosphorylated by cyclic AMP-dependent protein kinase (PKA). In the present study, phosphorylation of IP3 receptors has been examined with purified receptor protein reconstituted in liposomes to remove detergent that can inhibit protein kinases. The IP3 receptor is stoichiometrically phosphorylated by protein kinase C (PKC) and Ca2+ calmodulin-dependent protein kinase II (CaM kinase II) as well as by PKA. Phosphorylation by the three enzymes is additive and involves different peptide sequences. Phosphorylation by PKC, which is stimulated by Ca2+ and diacylglycerol, and by CaM kinase II, which requires Ca2+, provides means whereby Ca2+ and diacylglycerol, formed during inositol phospholipid turnover, may regulate IP3 receptor physiology.

Purification and molecular cloning of nitric oxide synthase

Abstract: A method of purifying calmodulin-dependent nitric oxide synthase provides a homogeneous preparation of the enzyme. The enzyme is used to raise antibodies which are a useful immunohistochemical reagent. The antibodies localize calmodulin-dependent nitric oxide synthase to a number of anatomical sites, including retina, intestine, adrenal gland, and vasculature. However, activated macrophages, which are known to possess a nitric oxide producing activity, do not display an immunoreactive protein of appropriate size on Western blots using the antibodies. Nucleotide sequences encoding calmodulin-dependent nitric oxide synthase indicate a novel sequence with a flavin binding site consensus sequence.