Masaki Nakane

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.

A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue to fixative

In histochemical studies using fixed brain tissue, NADPH-diaphorase has been found to be colocalized with soluble nitric oxide synthase. In the present study, using fresh tissues from eight different regions of rat brain, NADPH-diaphorase activity was found mostly in the particulate fraction, whereas most of the nitric oxide synthase activity was located to the cytosolic fraction. Also, the distribution of NADPH-diaphorase activity among brain regions was different from that of nitric oxide synthase. Pretreatment of the fractions with paraformaldehyde virtually abolished the NADPH-diaphorase activity in the particulate fraction, whereas 40-60% of the NADPH-diaphorase activity remained intact in the cytosolic fraction. These results suggest that during fixation most NADPH-diaphorase activity is inactivated and only some of the NADPH-diaphorase activity associated with soluble nitric oxide synthase remains intact.

Neurons in rat cerebral cortex that synthesize nitric oxide: NADPH diaphorase histochemistry, NOS immunocytochemistry, and colocalization with GABA

Neurons that stain for NADPH diaphorase, which colocalizes with nitric oxide synthase (NOS), are scattered uniformly across neocortex, and denser in entorhinal cortex. In the primary sensorimotor cortex, 0.5-2% of neurons contain NOS. These are most numerous in layers II-III, whereas NOS-positive fibers are concentrated in layers IV and VI. Most stained neurons are aspiny bipolar cells. Some in deep layers are multipolar; very few are pyramidal-shaped. In layer IV, NOS-positive neurons and their dendrites are confined to the septa between barrels. Retrograde tracing experiments demonstrate that NOS-positive cells are local circuit neurons. Double staining demonstrates that NOS-positive neurons also contain GABA.

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.

Light and electron microscopic demonstration of guanylate cyclase in rat brain

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC4.6.1.2.) was histochemically demonstrated in rat brain with light and electron microscopes by using a specific monoclonal antibody to soluble guanylate cyclase from rat brain. Under a light microscope, intense reactions were seen in caudate-putamen complex, neocortex and cerebellar cortex. Immunoreactive cells were mainly some types of neurons such as small neurons in caudate-putamen complex, Purkinje cells in cerebellar cortex and pyramidal cells in neocortex. Some astroglial cells were also stained. Not all neurons or glial cells exhibited the positive guanylate cyclase reactivity. Electron microscopic examination revealed that guanylate cyclase was localized within postsynaptic components (perikaryon and dendrites) in neurons and in the cytoplasm and plasma membrane of astroglia. Presynaptic terminals were free of reaction. The observation supports a possibility that cyclic GMP is involved in the postsynaptic events of neuronal transmission and the regulation of intracellular processes.

Expression of soluble guanylate cyclase activity requires both enzyme subunits.

Soluble guanylate cyclase purified from rat lung exists as a heterodimer of two subunits (70 kDa and 82 kDa). Recent cloning and sequencing of both subunit entities have revealed their primary structures. Transient expression in COS-7 cells by transfection with expression vectors containing the coding regions of the 70 kDa or the 82 kDa subunit cDNA showed no guanylate cyclase activity when cells were transfected with either subunit cDNA alone. However, a marked enzymatic activity was found after transfection with both subunits that was activated by sodium nitroprusside. The combination of separately expressed guanylate cyclase subunits could not reconstitute enzymatic activity in vitro. Furthermore, cotransfection with antisense oligonucleotides against the 70 kDa subunit or the 82 kDa subunit mRNA inhibited the guanylate cyclase activity. These data indicate that both the 70 kDa and the 82 kDa subunits must be present and interactive with each other in order to see basal guanylate cyclase activity and activation with sodium nitroprusside.

Molecular cloning of a cDNA coding for 70 kilodalton subunit of soluble guanylate cyclase from rat lung

A complementary DNA clone corresponding to the 70 kDa subunit of soluble guanylate cyclase (EC 4.6.1.2) of rat lung has been isolated. The primary structure of the cDNA consisted of 3063 nucleotides including a 1857-nucleotide coding region for 619 amino acids, and the calculated molecular weight was 70476. Blot hybridization of total poly(A)+RNAs from rat tissues detected a mRNA of about 3.4 kilobases. The amount of mRNA was abundant in lung, cerebrum and cerebellum, moderate in heart and kidney, and low in liver and muscle. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated the presence of one gene in the rat haploid genome. The amino acid sequence of the 70 kDa subunit has partial homology with particulate guanylate cyclase from sea-urchin sperm, and protein phosphatase inhibitor I.

Developmental changes of cytosolic and particulate nitric oxide synthase in rat brain

In the presence of added flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), both cytosolic and particulate nitric oxide synthase (NOS) activities can be detected in rat brain. Developmental changes of the cytosolic and particulate NOS in rat cerebellum and cerebrum were determined biochemically and immunochemically. Particulate NOS activity in the cerebrum increased during the first week of development, but then decreased and became almost undetectable in adult rats. In contrast, the cytosolic NOS in cerebellum showed low activity in newborns, but then constantly increased reaching an 8-fold higher level in adult rats. The activities of cerebellar particulate and cerebral cytosolic NOS also increased slightly during maturation. Western blot analysis using a polyclonal antibody raised against rat cerebellar cytosolic NOS revealed that the particulate and cytosolic fractions of 1-week-old and adult rat brains contained the same 160 kDa NOS protein. The relative content of the NOS protein correlated well with the relative amount of NOS activity in all brain fractions. These results indicate that distribution of NOS in the rat brain changes during maturation, but the same NOS protein is likely to be responsible for activities in immature and mature brains, cytosolic and particulate fractions. We suggest that nitric oxide might play a role in functional differentiation of the brain.

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.

Differential effects of vitamin D receptor activators on aortic calcification and pulse wave velocity in uraemic rats

BACKGROUND Vascular calcification is associated with an increase in cardiovascular mortality in stage 5 chronic kidney disease. To determine if vitamin D receptor activators (VDRAs) have differential effects in the pathogenesis of aortic calcification, we assessed the effects of paricalcitol and doxercalciferol in vivo using 5/6 nephrectomized (NX) rats. To quantify the functional consequences of vascular calcification, pulse wave velocity (PWV), an aortic compliance index, was measured. METHODS NX rats were fed a diet containing 0.9% phosphorous and 0.6% calcium 4 weeks prior to and throughout the study. On Day 0, rats received vehicle or VDRA (0.083, 0.167 and 0.333 microg/kg, i.p.) three times per week for 6 weeks. At Day 0 and Weeks 2 and 6, blood was drawn and PWV was measured by Doppler ultrasound. RESULTS VDRAs (0.167 and 0.333 microg/kg) consistently lowered PTH at Weeks 2 and 6. All doses of paricalcitol increased serum calcium at Week 6 but not at Week 2, while the two higher doses of doxercalciferol increased serum calcium at both Weeks 2 and 6. Treatment with paricalcitol (0.333 microg/kg) increased serum phosphorus at Weeks 2 and 6; these changes were not different from those observed in 5/6 NX rats. All doses of doxercalciferol increased serum phosphorus at Week 6. Paricalcitol had no effect on Ca x P; however, the two highest doses of doxercalciferol increased Ca x P at Weeks 2 and 6 above that observed in the 5/6 NX vehicle-treated group. There were no differences in aortic calcium and phosphorus contents at the end of 6 weeks among SHAM-, 5/6 NX- and paricalcitol-treated rats. However, treatment with the two higher doses of doxercalciferol caused a significant elevation in aortic calcium and phosphorus contents. Measurements of PWV demonstrated differential effects of VDRAs on vascular compliance. Paricalcitol produced no effects on PWV, while the two highest doses of doxercalciferol increased PWV at Week 6. CONCLUSIONS In uraemic rats with established secondary hyperparathyroidism, we demonstrate differential effects of paricalcitol and doxercalciferol on aortic calcification and PWV, independent of serum Ca, P and Ca x P, suggesting different mechanisms of action between VDRAs.