M. Nakane

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.

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.

Isoforms of Nitric Oxide Synthase and the Nitric Oxide-Cyclic Gmp Signal Transduction System

From the work in our laboratory and subsequently other laboratories, it has been known for many years that cyclic GMP induces the relaxation of numerous smooth muscle preparations including vascular, airway and intestinal smooth muscle (Katsuki and Murad, 1977; Katsukiet al., 1977; Murad et al., 1978; Muradet al., 1978; Rapaport and Murad, 1983; Murad, 1986). Smooth muscle relaxation was the first physiological function clearly related to cyclic GMP synthesis. The proposed functions of cyclic GMP have expanded considerably since, as briefly discussed below.

Bioassay for EDRF/NO by accumulation of cyclic GMP in RFL-6 fetal rat lung fibroblasts

Abstract We describe the use of fetal rat lung fibroblasts (RFL-6 cells) as a bioassay for endothelium-derived relaxing factor or nitric oxide (EDRF/NO). In these cells grown to confluency in 6-well plates, authentic NO, at concentrations as low as 2 nM, or EDRF/NO, basally released from as few as 1–2 × 10 6 endothelial cells, causes accumulation of guanosine 3′,5′-cyclic monophosphate. If cells are grown in 48-well plates (well area 1 10 that of 6-well plates) this gives a detection limit of 100–200 fmol NO and the possibility of detecting the basal EDRF/NO release from 1–2 × 10 5 endothelial cells. Thus, this method is more sensitive than any other currently available. In addition, RFL-6 cells may be used to detect the activity of EDRF/NO synthase in cell homogenates and column fractions during purification, making them an invaluable aid in purification and characterization of EDRF/NO synthase from brain and neuronal cells.