Endothelial Isoform Of Nitric Oxide Synthase Research Articles

Nitric Oxide-dependent Parasympathetic Signaling Is Due to Activation of Constitutive Endothelial (Type III) Nitric Oxide Synthase in Cardiac Myocytes

Nitric oxide synthase (NOS) isoforms are discovered in an increasing variety of cell types with different roles in signaling. The inducible NOS (i.e. iNOS or NOS II) is expressed in cardiac myocytes in response to specific cytokines. Independent of iNOS induction, however, receptor-dependent signaling is modulated by a constitutive nitric oxide (NO) synthase isoform in these cells (Balligand, J. L., Kelly, R.A., Marsden, P.A., Smith, T. W., and Michel, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 347-351). We now show that cardiac myocytes constitutively express the endothelial isoform of NO synthase (ecNOS or NOS III). Transcripts for NOS III were detected by Northern blot in myocyte extracts using as a probe a polymerase chain reaction-generated cDNA amplified with isoform and species-specific primers. In subcellular fractionation experiments, a calcium-sensitive NO synthase activity was present primarily in the particulate fraction, coinciding with the distribution of NOS III analyzed by protein immunoblotting. The localization of NOS III within cardiac myocytes was further demonstrated by immunohistochemistry. The functional role of NOS III was explored by analyzing the effects of NOS inhibitors on single myocyte L-type calcium current and contractility. Inhibition of NOS blocked the attenuation by carbamylcholine of the increases in both parameters induced by beta-adrenergic stimulation. We conclude that NO-dependent parasympathetic signaling is mediated by NOS III in cardiac myocytes.

L-thiocitrulline. A stereospecific, heme-binding inhibitor of nitric-oxide synthases

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (NO). The enzyme is inhibited by a variety of N omega-monosubstituted L-arginine analogs, and some of these compounds are useful in reversing pathologies associated with the overproduction of NO (e.g. the hypotension of septic shock). We report here that L-thiocitrulline (gamma-thioureido-L-norvaline) is a potent, stereospecific inhibitor of the constitutive brain and endothelial isoforms of NOS as well as the isoform induced in vascular smooth muscle cells by lipopolysaccharide and interferon-gamma. Steady state kinetic studies show L-thiocitrulline inhibition is competitive with L-arginine (Ki approximately 4-20% of KArgm), indicating that initial binding is as a substrate/product analog. In contrast to L-arginine and N omega-methyl-L-arginine, the prototypic NOS inhibitor, L-thiocitrulline binding elicits a "Type II" difference spectrum, indicating a high spin to low spin transition of the iron in the heme cofactor. This finding suggests that L-thiocitrulline is contributing the sixth ligand to heme iron, probably through the thioureido sulfur. Such interaction with heme iron neither stimulates nor inhibits the direct flavin-mediated cytochrome c reduction activity of the enzyme, but it does inhibit heme-dependent superoxide formation. In vivo, L-thiocitrulline is a potent pressor agent in both normal and endotoxemic rats, the latter finding suggesting utility in treating the hypotension of septic shock.

Immunohistochemical localization of nitric oxide synthase in the human placenta

We have studied the distribution of the endothelial isoform of nitric oxide synthase (NOS) through the term human umbilical cord and placenta by immunohistochemistry. Histochemistry with the NADPH diaphorase substrate nitroblue tetrazolium (NBT) has also been used to establish if other isoforms of NOS may be present in these tissues. Positive immunofluorescence for endothelial NOS was found in umbilical cord artery and vein endothelium, although positive staining was only found in approximately 50% of veins. The endothelium of stem villous vessels dissected from beneath the chorionic plate was also intensely immunostained. In the terminal villi punctate immunostaining was found at the basal aspect and around nuclei of syncytiotrophoblast, but was absent from stroma and endothelium of terminal villous vessels. A positive histochemical stain for NBT was found in cord artery and vein endothelium and stem villous vessel endothelium. Intense diffuse staining with NBT was found in syncytiotrophoblast, but no other cell types in the terminal villi stained with NBT. The endothelial NOS isoform appears to be localized in the resistance vasculature of the placenta, but not in the capillary endothelium of terminal villi where there is no underlying smooth muscle. It may contribute to the 'endothelial' function of syncytiotrophoblast if secreted towards the intervillous space or alternatively fulfil other signal transduction roles. The pattern of staining with NBT was similar to that with endothelial NOS and suggests that other isoforms of NOS are not present in the placental unit.

Purification and cDNA sequence of an inducible nitric oxide synthase from a human tumor cell line.

A combination of cytokines induced the expression of nitric oxide synthase (NOS) in a human colorectal adenocarcinoma cell line, DLD-1. We have purified the enzyme and examined some of its biochemical properties. An antiserum to an inducible NOS from murine macrophages cross-reacted with the DLD-1 NOS. The purified human and murine enzymes displayed a similar lack of dependence on exogenous calcium and calmodulin for activity, which contrasts with the requirement for calcium and calmodulin of purified brain and endothelial isoforms of NOS. We have also isolated a cDNA for a cytokine-induced NOS from DLD-1 cells. Sequence analysis of this cDNA and NOS cDNAs from human liver, smooth muscle, and macrophages suggests that, at the genetic level, there is a single isoform of human-inducible NOS.