Ferid Murad

Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction.

Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.

Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle

Complementary DNA clones corresponding to human brain nitric oxide (NO) synthase have been isolated. The deduced amino acid sequence revealed an overall identity with rat brain NO synthase of about 93% and contained all suggested consensus sites for binding of the co‐factors. The cDNA transfected COS‐1 cells showed significant NO synthase activity with the typical co‐factor requirements. Unexpectedly, messenger RNA levels of this isoform of NO synthase was more abundant in human skeletal muscle than human brain. Moreover, we detected high NO synthase activity and the expressed protein in human skeletal muscle by Western blot analysis, indicating a possible novel function of NO in skeletal muscle.

Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase

Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation.

Regional distribution of EDRF/NO-synthesizing enzyme(s) in rat brain.

Stimulation of soluble guanylyl cyclase and increase in cyclic GMP in rat fetal lung fibroblasts (RFL-6 cells) was used as a bioassay to detect EDRF/NO formation. The cytosolic fraction of whole rat brain synthesized an EDRF/NO-like material in a process dependent on L-arginine and NADPH. The enzymatic activity was destroyed by boiling and inhibited by N omega-nitro-L-arginine. Hemoglobin and methylene blue blocked the effect of EDRF/NO. When different brain regions were analyzed in the presence of L-arginine and NADPH, the cytosolic fraction from cerebellum showed the highest EDRF/NO-forming activity (2-3 times higher than whole brain). Activity similar to whole brain was found in hypothalamus and midbrain. Enzymatic activities in striatum, hippocampus and cerebral cortex were about two thirds of whole brain. The lowest activity (less than half of whole brain) was found in the medulla oblongata.

Ca2+/calmodulin-regulated nitric oxide synthases.

NO synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and nitric oxide (NO) or a NO-releasing compound. At least three isoforms of NOS exist (types I-III). The activities of the type I isoform purified from brain and the type III isoform purified from endothelial cells are regulated by the intracellular free calcium concentration ([Ca2+]i) and the Ca(2+)-binding protein calmodulin. At resting [Ca2+]i, both isozymes are inactive; they become fully active at [Ca2+]i greater than or equal to 500 nM Ca2+. Longer lasting increases in [Ca2+]i may downregulate NO formation, for in vitro phosphorylation by Ca2+/calmodulin protein kinase II decreases the Vmax of NOS. Besides the conversion of L-arginine, type I NOS, Ca2+/calmodulin dependently, generates H2O2 and reduces cytochrome c/P450. Other redox activities, i.e. the reduction of nitroblue tetrazolium to diformazan (NADPH-diaphorase) or of quinoid-dihydrobiopterin to tetrahydrobiopterin, by NOS appear to be Ca2+/calmodulin-independent.

Characterization of nitric oxide synthases in non-adrenergic non-cholinergic nerve containing tissue from the rat anococcygeus muscle.

Tissue homogenates prepared from rat anococcygeus muscle converted l‐arginine to l‐citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in crude and partially‐purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8‐tetrahydrobiopterin and calcium, and inhibited by NG‐nitro‐l‐arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat vas deferens, which does not contain NO releasing non‐adrenergic non‐cholinergic neurones, had no NO synthase activity.

Characterization and localization of nitric oxide synthase in non-adrenergic non-cholinergic nerves from bovine retractor penis muscles

1 Partially purified soluble nitric oxide (NO) synthase was isolated from the bovine retractor penis muscle (BRP), a tissue in which the inhibitory response to non‐adrenergic non‐cholinergic nerve (NANC) stimulation appears to be mediated by NO or NO‐like material. 2 NO synthase from BRP used l‐arginine as a substrate, required NADPH, tetrahydrobiopterin, and FAD as co‐factors and was Ca2+/calmodulin‐dependent. The activity of NO synthase was inhibited by NG‐methyl‐l‐arginine and NG‐nitro‐l‐arginine, and haemoglobin blocked the effect of NO formed by the enzyme. 3 On reducing SDS polyacrylamide gel electrophoresis the apparent molecular mass of NO synthase from BRP was 160 ± 2 kDa, which is similar to that of the cerebellar NO synthase. Protein immunoblot and immunoprecipitation showed that NO synthase from BRP cross‐reacted with the selective antiserum to neuronal NO synthase from rat cerebellum. 4 Immunohistochemistry using the same antiserum demonstrated that NO synthase in BRP was located exclusively within nerve fibres. Thus, autonomic nerves synthesizing the NANC neurotransmitter seem to contain an isoform of NO synthase which is similar to that from rat cerebellum.

Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-ß

RAW 264.7 macrophages induced with lipopolysaccharide and interferon-gamma expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by NG-nitro-L-arginine and NG-methyl-L-arginine. Transforming growth factor-beta 1 prevented the induction of both enzymes.

Nitric oxide synthase in bovine superior cervical ganglion.

Abstract: We investigated the mechanism of increases in cyclic GMP levels in bovine superior cervical ganglion (SCG) in response to muscarinic receptor stimulation. Acetylcholine increased cyclic GMP levels in SCG. This increase was inhibited by NG‐methyl‐L‐arginine (NMA), and the inhibition was reversed by L‐arginine. Soluble nitric oxide (NO) synthase was partially purified from bovine SCG using 2′,5′‐ADP Sepharose affinity chromatography. The resulting enzyme activity was Ca2+/calmodulin dependent and required NADPH and tetrahydrobiopterin as co‐factors. Superoxide dismutase protected and oxyhemo‐globin blocked the effect of NO formed by the enzyme. NMA inhibited the activity of the NO synthase. In western blots, an antibody generated against rat brain NO synthase specifically recognized the NO synthase from SCG as a 155‐kDa protein band. Immunohisto chemistry using the same antibody demonstrated that NO synthase was localized in postganglionic neuronal cell bodies of the SCG. Immunofluorescent labeling showed that some of the cells staining positive for dopamine‐β‐hydroxylase also contained NO synthase. Thus, NO is synthesized in specific cells within bovine SCG, including sympathetic neurons, and mediates the acetylcholine‐induced stimulation of soluble guanylyl cyclase.

Endothelial cells have a particulate enzyme system responsible for EDRF formation: Measurement by vascular relaxation

Endothelium-derived relaxing factor (EDRF) released from endothelial cells (EC) has been shown to be nitric oxide (NO) or a closely related molecule. In cultured EC, the enzyme responsible for the formation of EDRF, EDRF-synthase, was initially described as being cytosolic, but more recently we have found it to be predominantly particulate. In view of this discrepancy we have investigated the EDRF synthesizing activity of cytosolic and particulate fractions isolated from native bovine aortic EC. EDRF was measured by cGMP formation in rat fetal lung cultured fibroblasts (RFL-6) and by the ability of cell fractions to relax endothelium-denuded, preconstricted rabbit aortic strips. Cytosolic fractions from native EC (100 micrograms) had no effect on the tone of rabbit aortic strips and little effect on cGMP levels in RFL-6 cells in the presence of L-arginine and NADPH (100 microM). However, under the same conditions the 100,000 x g pellet fractions relaxed rabbit aortic strips and increased cGMP levels in RFL-6 cells. Thus EDRF synthase from native EC, like those grown in culture, is located mainly in the particulate fraction.