Nitric oxide and nitric oxide synthase activity in plants

Research on NO in plants has gained considerable attention in recent years mainly due to its function in plant growth and development and as a key signalling molecule in different intracellular processes in plants. The NO emission from plants is known since the 1970s, and now there is abundant information on the multiple effects of exogenously applied NO on different physiological and biochemical processes of plants. The physiological function of NO in plants mainly involves the induction of different processes, including the expression of defence-related genes against pathogens and apoptosis/programmed cell death (PCD), maturation and senescence, stomatal closure, seed germination, root development and the induction of ethylene emission. NO can be produced in plants by non-enzymatic and enzymatic systems. The NO-producing enzymes identified in plants are nitrate reductase, and several nitric oxide synthase-like activities, including one localized in peroxisomes which has been biochemically characterized. Recently, two genes of plant proteins with NOS activity have been isolated and characterized for the first time, and both proteins do not have sequence similarities to any mammalian NOS isoform. However, different evidence available indicate that there are other potential enzymatic sources of NO in plants, including xanthine oxidoreductase, peroxidase, cytochrome P450, and some hemeproteins. In plants, the enzymatic production of the signal molecule NO, either constitutive or induced by different biotic/abiotic stresses, may be a much more common event than was initially thought.     

New aspects of the location of neuronal nitric oxide synthase in the skeletal muscle: a light and electron microscopic study.

The action of nitric oxide (NO) synthesized by NO synthases (NOS) is spatially restricted. Hence, the intracellular location of NOS might play an important role for the functional interactions of NO with its target molecules. In the skeletal muscle the neuronal NOS (nNOS) is considered to be the predominant isoform expressed as a muscle specific elongated splice variant. There are only a few and highly discrepant reports of the subcellular distribution of nNOS, which prompted us to re-examine the distribution of nNOS in the skeletal muscle of rat and mouse applying immunocytochemistry and NADPH-diaphorase (NADPH-d) histochemistry. Light microscopically, the sarcolemma, areas beneath the sarcolemma, areas around the nuclei, and the cross striation were labeled by antibodies and by the NADPH-d reaction as well. Ultrastructurally, nNOS visualized immunocytochemically or by the histochemical BSPT-reaction, was associated discretely with extrajunctional portions of the sarcolemma. Both reaction products were additionally observed in the vicinity of endoplasmic reticulum and mitochondria, or associated with their outer membranes. In the neuromuscular junction (NMJ)-region NOS was localized to the cytoplasm of nerve terminals and terminal Schwann cells. In contrast to the commonly accepted assumption, the enzyme was found in association with the presynaptic, and not with the postsynaptic membrane. Cytosolic NADPH-d was exhibited especially between mitochondria accumulated in the postsynaptic region of the NMJ. Surprisingly, in nNOS-/--mice the skeletal muscle showed patterns of significant nNOS-immunoreactivity and NADPH-d activity possibly due to alternative nNOS-splice isoforms, which might be up-regulated to compensate for decreased NO formation.     

Proximal effects in the modulation of nitric oxide synthase reactivity: a QM-MM study

Nitric oxide synthases (NOS) are heme proteins that have a cysteine residue as axial ligand, which generates nitric oxide (NO). The proximal environment, specifically H-bonding between tryptophan (Trp) 178 and thiolate, has been proposed to play a fundamental role in the modulation of NOS activity. We analyzed the molecular basis of this modulation by performing electronic structure calculations on isolated model systems and hybrid quantum-classical computations of the active sites in the protein environment for wild-type and mutant (Trp 178 × Gly) proteins. Our results show that in the ferrous proteins NO exhibits a considerable trans effect. We also showed that in the ferrous (Fe⁺²) mutant NOS the absence of Trp, experimentally associated to a protonated cysteine, weakens the Fe–S bond and yields five coordinate complexes. In the ferric (Fe⁺³) state, the NO dissociation energy is shown to be slightly smaller in the mutant NOS, implying that the Fe⁺³–NO complex has a shorter half-life. We found computational evidence suggesting that ferrous NOS is favored in wild-type NOS when compared to the Trp mutant, consistently with the fact that Trp mutants have been shown to accumulate less Fe⁺²–NO dead end species. We also found that the heme macrocycle showed a significant distortion in the wild-type protein, due to the presence of the nearby Trp 178. This may also play a role in the subtle tuning of the electronic structure of the heme moiety.     

Neuronal nitric oxide synthase proteolysis limits the involvement of nitric oxide in kainate-induced neurotoxicity in hippocampal neurons

In this work, we investigated the role of nitric oxide (NO) in neurotoxicity triggered by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in cultured hippocampal neurons. In the presence of cyclothiazide (CTZ), short-term exposures to kainate (KA; 5 and 15 min, followed by 24-h recovery) decreased cell viability. Both NBQX and d-AP-5 decreased the neurotoxicity caused by KA plus CTZ. Long-term exposures to KA plus CTZ (24 h) resulted in increased toxicity. In short-, but not in long-term exposures, the presence of NO synthase (NOS) inhibitors (l-NAME and 7-NI) decreased the toxicity induced by KA plus CTZ. We also found that KA plus CTZ (15-min exposure) significantly increased cGMP levels. Furthermore, short-term exposures lead to decreased intracellular ATP levels, which was prevented by NBQX, d-AP-5 and NOS inhibitors. Immunoblot analysis revealed that KA induced neuronal NOS (nNOS) proteolysis, gradually lowering the levels of nNOS according to the time of exposure. Calpain, but not caspase-3 inhibitors, prevented this effect. Overall, these results show that NO is involved in the neurotoxicity caused by activation of non-desensitizing AMPA receptors, although to a limited extent, since AMPA receptor activation triggers mechanisms that lead to nNOS proteolysis by calpains, preventing a further contribution of NO to the neurotoxic process.     

GABA-immunoreactive neurons and terminals in the cat periaqueductal gray matter: A light and electron microscopic study

Immunocytochemical and electron microscopic methods were used to study the GABAergic innervation in adult cat periaqueductal gray matter (PAG). A mouse monoclonal antibody against γ -aminobutyric acid (GABA) was used to visualize the inhibitory neuronal system of PAG. At light microscopy, GABA-immunopositive (GABA_IP) neurons formed two longitudinally oriented columns in the dorsolateral and ventrolateral PAG that accounted for 36% of the neuronal population of both PAG columns; their perikaryal cross-sectional area was smaller than that of unlabeled (UNL) neurons found in the same PAG subdivisions. At electron microscopic level, patches of GABA immunoreactivity were readily detected in neuronal cell bodies, proximal and distal dendrites, axons and axon terminals. Approximately 35–36% of all terminals were GABA_IP; they established symmetric synapses with dendrites (84.72% of the sample in the dorsolateral PAG and 86.09% of the sample in the ventrolateral PAG) or with cell bodies (7–10% of the sample). Moreover, 49.15% of GABA_IP axon terminals in the dorsolateral and 52.16% in the ventrolateral PAG established symmetric synapses with GABA_IP dendrites. Immunopositive axon terminals and unlabeled terminals were also involved in the formation of a complex synaptic arrangment, i.e. clusters of synaptic terminals in close contact between them that were often observed in the PAG neuropil. Moreover, a fair number of axo-axonic synapses between GABA_IP and/or UNL axon terminals were present in both PAG subdivisions. Several dendro-dendritic synapses between labeled and unlabeled dendrites were also observed in both PAG subdivisions. These results suggest that in the cat PAG there exist at least two classes of GABArgic neurons. The first class could exert a tonic control on PAG projecting neurons, the second could act on those GABAergic neurons that in turn keep PAG projecting neurons under tonic inhibition. The functional implications of this type of GABAergic synapse organization are discussed in relation to the dishinibitory processes that take place in the PAG.     

Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in the cat spinal cord

The distributions of neuronal nitric oxide synthase immunoreactivity (NOS-IR) and NADPH-diaphorase (NADPH-d) activity were compared in the cat spinal cord. NOS-IR in neurons around the central canal, in superficial laminae (I and II) of the dorsal horn, in the dorsal commissure, and in fibers in the superficial dorsal horn was observed at all levels of the spinal cord. In these regions, NOS-IR paralleled NADPH-d activity. The sympathetic autonomic nucleus in the rostral lumbar and thoracic segments exhibited prominent NOS-IR and NADPH-d activity, whereas the parasympathetic nucleus in the sacral segments did not exhibit NOS-IR or NADPH-d activity. Within the region of the sympathetic autonomic nucleus, fewer NOS-IR cells were identified compared with NADPH-d cells. The most prominent NADPH-d activity in the sacral segments occurred in fibers within and extending from Lissauer's tract in laminae I and V along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These afferent projections did not exhibit NOS-IR; however, NOS-IR and NADPH-d activity were demonstrated in dorsal root ganglion cells (L7-S2). The results of this study demonstrate that NADPH-d activity is not always a specific histochemical marker for NO-containing neural structures.     

The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.     

HRP-labeled masticatory neurons in the rat trigeminal mesencephalic nucleus: a light and electron microscopic study

The neurons innervating the muscles of mastication were labeled retrogradely with horseradish peroxidase (HRP) which was injected into each muscle of mastication of the rats. The TMB-HRP labeled neurons were for light microscopic and DAB-HRP labeled neurons for electron microscopic study. Many HRP-labeled mesencephalic neurons were observed in the trigeminal mesencephalic nucleus (TMEN) after HRP injection in jaw-closing muscles (JCM). On the other hand, no labeled neurons were found following the application of HRP to the lateral pterygoid and the anterior belly of the digastric muscles, with the exception of a very few from the mylohyoid muscle. The latter three muscles were jaw-opening muscles (JOM). The mesencephalic neurons of each JCM in the TMEN were rather randomly distributed, although they were concentrated more in the caudal region of this nucleus. These neurons were typically unipolar, with spherical to oval perikarya. Each neuron had a single process which coursed caudolaterally to join the mesencephalic tract of the trigeminal nerve. Ultrastructurally, mesencephalic masticatory neurons had a rather regular nucleus locating either centrally or eccentrically in the perikaryon, which is rather plump. The cytoplasm was endowed with very well developed Golgi apparatus and rough endoplasmic reticulum. Neurofilaments, varying in number, intermingled mostly with the Golgi apparatus in the cytoplasm. Somatic spines were frequently observed; however, synapses abutting upon the soma were few. Macula adherens-like structures were occassionally encountered in the contact zone between two cells.     

Deletion of the autoregulatory insert modulates intraprotein electron transfer in rat neuronal nitric oxide synthase

Comparative CO photolysis kinetics studies on wild-type and autoregulatory (AR) insert-deletion mutant of rat nNOS holoenzyme were conducted to directly investigate the role of the unique AR insert in the catalytically significant FMN-heme intraprotein electron transfer (IET). Although the amplitude of the IET kinetic traces was decreased two- to three-fold, the AR deletion did not change the rate constant for the calmodulin-controlled IET. This suggests that the rate-limiting conversion of the electron-accepting state to a new electron-donating (output) state does not involve interactions with the AR insert, but that AR may stabilize the output state once it is formed.     

Light and electron microscopic structure of golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure)

Summary After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguishable by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pieces of tissue left in a 1 % K₂Cr₂O₇ solution for 13 h before impregnation in a 0.5 % AgNO₃ solution for 2h. Thick sections (30–50 m) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.     

The protective effect of taurine pretreatment on carbon tetrachloride-induced hepatic damage--a light and electron microscopic study

Summary.  The results regarding taurine pretreatment on CCl₄-induced hepatic injury are controversial. To assess the therapeutic efficacy of taurine on rat liver injury, hepatic malondialdehyde, glutathione, and hydroxyproline levels together with morphologic alterations in the liver following CCl₄ administration were investigated. The rats were divided into three groups. Taurine-treated animals received 15 ml/kg/day of a 5% taurine solution by a gastric tube for 5 days before administering CCl₄ (2 ml/kg, intraperitoneally, in a single dose). CCl₄-treated rats received the same amount of saline solution. Control animals received no treatment. The increase of hepatic malondialdehyde formation in the CCl₄-treated group was partially prevented by taurine pretreatment, but taurine had no significant effect on the glutathione and hydroxyproline content in the CCl₄-treated rats. Taurine pretreatment induced a marked beneficial effect regarding the prevention of hepatocellular necrosis and atrophy as demonstrated morphologically. In conclusion, these results suggest that taurine pretreatment might not significantly change the biochemical parameters, but prevents the morphologic damage caused by CCl₄ in the early stages. Received March 17, 2001 Accepted July 18, 2001     

A light and electron microscopic study of the ciliated urn of Phascolosoma agassizii (sipunculida)

Summary The free swimming ciliated urn found in the coelomic fluid of Phascolosoma agassizii has been studied by electron microscopy. The urn is a multicellular structure composed of three cell types: (a) ciliated cells which possibly function in capturing cell debris and foreign particles; (b) cupola cells which are capable of phagocytozing latex particles; and (c) lobe cells which are capable of phagocytozing carbon particles. The lobes are separated from the ciliated cells by a semilunar area, with mucoprotein staining characteristics, containing fibrils which appear to be the structural support for the urn. Ciliated cells and lobes are attached to the semilunar area by hemidesmosomes.This work is based on a thesis submitted in partial fulfillment of the requirements for the degree Master of Arts in the Department of Biology, San Francisco State University, San Francisco, California, U.S.A.I wish to thank Dr. John C. Lee, formerly of the Department of Pathology, University of California Medical Center, San Francisco, for encouragement and use of his electron microscope facilities     

The dorsal strand of Polyandrocarpa misakiensis (Protochordata: Ascidiacea): a light and electron microscopic study

The dorsal strand of a budding ascidian, Polyandrocarpa misakiensis , was studied by light and electron microscopy. The length and morphology of this organ vary even among zooids in the same colony. After curving between the paired posterior nerve trunks, the strand turns left and terminates in a dead end. The dorsal strand of this species is one of the simplest types among ascidians, since there is no branching, no extensive nerve plexus, and no neuronal or non-neuronal endocrine cells surrounding it. The strand mainly consists of a simple cuboidal epithelium, but has several variations, such as multilayered regions, and protrusions of a cellular chain. The strand cells usually have mitochondria, free ribosomes and a Golgi complex of dictyosomal type. There are many mitotic cells in the strand epithelium. The presence, between the cerebral ganglion and dorsal strand epithelium, of cells with morphology intermediate between the strand and neuronal cells suggests that the dorsal strand might supply cellular components, such as neurones, to the cerebral ganglion in adult zooids. At the caudal end, the strand cells show ultrastructural features suggesting active protein synthesis and secretion. These cells appear to be liberated from the epithelium to release the content of their granules.