The intracellular distribution of neuronal nitric oxide synthase (nNOS) was studied in the caudal neurosecretory system of a teleost, Oreochromis niloticus (Cichlids), by means of post-embedding immunogold labeling with a polyclonal antibody directed against nNOS of human origin. Ultrastructural examination demonstrated that neuronal NOS-like molecules are distributed within the Dahlgren cell perikarya, the neurosecretory axons, and the urophysial axon terminals. In the neurosecretory somata, gold particles for nNOS were mainly cytosolic, whereas in the neurosecretory axons and axon terminals they were associated with the membrane and/or the dense core of neurosecretory granules. Double immunogold labelings for nNOS/urotensin I (UI) and nNOS/urotensin II (UII) demonstrated that nNOS-like molecules are colocalized with UI and/or UII in the neurosecretory granules contained within the urophysial terminals. The present findings suggest that both a soluble cytosolic and a particulate neuronal NOS are expressed in the caudal neurosecretory neurons. They confirm previous biochemical data on the same species.

Keywords:
Neuronal NO synthase, Gold immunoelectron microscopy, Caudal neurosecretory system, Urotensin I, Urotensin II, Oreochromis niloticus, Teleost
1.
Anken, R.H., and H. Rahmann (1996) An atlas of the distribution of NADPH-diaphorase in the brain of the highly derived swordtail fish Xiphophorus helleri (Atherinoformes: Teleostei). Brain Res., 37: 421–449.
2.
Aoki, C., S. Fenstemaker, M. Lubin, and C.G. Go (1993) Nitric oxide synthase in the visual cortex of monocular monkeys as revealed by light and electron microscopic immunocytochemistry. Brain Res., 620: 97–113.
3.
Arevalo, R., J.R. Alonso, E. Garcia-Ojeda, J.G. Brinon, C. Crespo, and J. Aijon (1995) NADPH-diaphorase in the central nervous system of the tench (Tinca tinca L., 1758). J. Comp. Neurol., 352: 398–420.
4.
Arevalo, R., F. Sanchez, J.R. Alonso, J. Carretero, R. Vazquez, and J. Aijon (1992) NADPH-diaphorase activity in the hypothalamic magnocellular neurosecretory nuclei of the rat. Brain Res. Bull., 28: 599–603.
5.
Bendayan, M. (1982) Double immunocytochemical labelling applying the protein A-gold technique. J. Histochem. Cytochem., 30: 81–85.
6.
Bern, H.A., D. Pearson, B.A. Larson, and R.S. Nishioka (1985) Neurohormones from fish tails: the caudal neurosecretory system. I. ‘Urophysiology’ and the caudal neurosecretory system of fishes. Recent Prog. Horm. Res., 41: 533–552.
7.
Bredt, D.S., and S.H. Snyder (1992) Nitric oxide, a novel neuronal messenger. Neuron, 8: 3–11.
8.
Bredt, D.S., P.M. Hwang, and S.H. Snyder (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature, 347: 768–770.
9.
Brenman, J.E., D.S. Chao, S.H. Gee, A.W. McGee, S.E. Craven, D.R. Santillano, Z. Wu, F. Huang, H. Xia, M.F. Peters, S.C. Froehner, and D.S. Bredt (1996) Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell, 84: 757–767.
10.
Bruning, G., R. Katzbach, and B. Mayer (1995) Histochemical and immunocytochemical localization of nitric oxide synthase in the central nervous system of the foldfish, Carassius auratus. J. Comp. Neurol., 358: 353–382.
11.
Calka, J., G. Wolf, and M. Brosz (1994) Ultrastructural demonstration of NADPH-diaphorase histochemical activity in the supraoptic nucleus of normal and dehydrated rats. Brain Res. Bull., 34: 301–308.
12.
Ceccatelli, S. (1997) Expression and plasticity of NO synthase in the neuroendocrine system. Brain Res. Bull., 44: 533–538.
13.
Ceccatelli, S., A.L. Hulting, X. Zhang, L. Gustafsson, M. Villar, and T. Hökfelt (1993) Nitric oxide synthase in the rat anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion. Proc. Natl. Acad. Sci. USA, 90: 11292–11296.
14.
Ceccatelli, S., J.M. Lundberg, J. Fahrenkrug, D.S. Bredt, S. Snyder, and T. Hökfelt (1992) Evidence for involvement of nitric oxide in the regulation of hypothalamic portal blood flow. Neuroscience, 51: 769–772.
15.
Cioni, C., N. Francia, C. Fabrizi, M. Colasanti, and G. Venturini (1998) Partial biochemical characterization of nitric oxide synthase in the caudal spinal cord of the teleost Oreochromis niloticus. Neurosci. Lett., 253: 68–70.
16.
Cioni, C., A. Greco, A. Pepe, L. De Vito, and M. Colasanti (1997) Nitric oxide synthase in the caudal neurosecretory system of the teleost Oreochromis niloticus. Neurosci. Lett., 238: 57–60.
17.
Costa, A., P. Trainer, M. Besser, and A. Grossman (1993) Nitric oxide modulates the release of corticotropin-releasing hormone from the rat hypothalamus in vitro. Brain Res., 605: 187– 192.
18.
Domoto, T., M. Teramoto, K. Tamura, and Y. Yasui (1994) Ultrastructural study on NOS-immunoreactive nerve terminals in the rat coeliac ganglion. NeuroReport, 6: 169–172.
19.
Faber-Zuschratter, H., and G. Wolf (1994) Ultrastructural distribution of NADPH-diaphorase in cortical synapses. NeuroReport, 5: 2029– 2032.
20.
Fukuto, J.M., and B. Mayer (1996) The enzymology of nitric oxide synthase. In Methods in Nitric Oxide Research (ed. by M. Feelisch and J.S. Stamler), Wiley, Mew York, pp. 147–160.
21.
Hecker, M., A. Mulsch, and R. Busse (1994) Subcellular localization and characterization of neuronal nitric oxide synthase. J. Neurochem., 62: 1524–1529.
22.
Hökfelt, T., S. Ceccatelli, L. Gustafsson, A.L. Hulting, V. Verge, M. Villar, X.J. Xu, Z.Q. Xu, Z. Wiesenfeld-Hallin, and X. Zhang (1994) Plasticity of NO synthase expression in the nervous and endocrine systems. Neuropharmacology, 33: 1221–1227.
23.
Holmqvist, B., and P. Ekstrom (1997) Subcellular localization of neuronal nitric oxide synthase in the brain of a teleost; an immunoelectron and confocal microscopical study. Brain Res., 745: 67–82.
24.
Holmqvist, B.I., T. Ostholm, P. Alm, and P. Ekstrom (1994) Nitric oxide synthase in the brain of a teleost. Neurosci. Lett., 171: 205–208.
25.
Kato, M. (1992) Involvement of nitric oxide in growth hormone (GH)-releasing hormone-induced GH secretion in rat pituitary cells. Endocrinology, 131: 2133–2138.
26.
Lederis, K., J.N. Fryer, and C.R. Yulis (1985) The fish neuropeptide urotensin I: its physiology and pharmacology. Peptides, 6: 353–361.
27.
Llewellyn-Smith, I.J., Z.M. Song, M. Costa, D.S. Bredt, and S.H. Snyder (1992) Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons. Brain Res., 577: 337–342.
28.
Loesch, A., and G. Burnstock (1998) Perivascular nerve fibers and endothelial cells of the rat basilar artery: immuno-gold labelling of antigenic sites for type I and type III nitric oxide synthase. J. Neurocytol., 27: 197–204.
29.
Loesch, A., A. Belai, and G. Burnstock (1994) An ultrastructural study of NADPH-diaphorase and nitric oxide synthase in the perivascular nerves and vascular endothelium of the rat basilar artery. J. Neurocytol., 23: 49–59.
30.
Lutz-Bucher, B., and B. Koch (1994) Evidence for an inhibitory effect of nitric oxides on neuropeptide secretion from isolated neural lobe of the rat pituitary gland. Neurosci. Lett., 165: 48–50.
31.
Morello, M., A. Reiner, G. Sancesario, E.J. Karle, and G. Bernardi (1997) Ultrastructural study of nitric oxide synthase-containing striatal neurons and their relationship with parvalbumin-containing neurons in rats. Brain Res., 776: 30–39.
32.
Morot Gaudry-Talarmain, Y., N. Moulian, F.A. Meunier, B. Blanchard, D. Angaut-Petit, L. Faille, and C. Ducrocq (1997) Nitric oxide and peroxynitrite affect differently acetylcholine release, choline acetyltransferase activity, synthesis, and compartmentation of newly formed acetylcholine in Torpedo marmorata synaptosomes. Nitric Oxide, 1: 330–345.
33.
Ota, M., J.T. Crofton, G.T. Festavan, and L. Share (1993) Evidence that nitric oxide can act centrally to stimulate vasopressin release. Neuroendocrinology, 57: 955–959.
34.
Perez, S.E., F. Adrio, M.A. Rodriguez, R. Anadon, and I. Rodriguez-Moldes (1995) The nitric oxide synthase (NOS)-like immunoreactive extrahypophysial projections of the neurosecretory preoptic nucleus of the electric ray (Elasmobranchs) suggest a neuroregulatory role for this nucleus. Neurosci. Lett., 195: 85– 88.
35.
Rettori, V., N. Belova, W.L. Dees, C.L. Nyberg, M. Gimeno, and S.M. McCann (1993) Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro. Proc. Natl. Acad. Sci. USA, 90: 10130–10134.
36.
Saeij, J.P., R.J. Stet, A. Groeneveld, L.B. Verburg-van Kemenade, W.B. van Muiswinkel, and G.F. Wiegertjes (2000) Molecular and functional characterization of a fish inducible-type nitric oxide synthase. Immunogenetics, 51: 339–346.
37.
Sagar, S.M., and D.M. Ferriero (1987) NADPH diaphorase activity in the posterior pituitary: relation to neuronal function. Brain Res., 400: 348–352.
38.
Schober, A., C.R. Maltz, and D.L. Meyer (1993) Enzymehistochemical demonstration of nitric oxide synthase in the diencephalon of the rainbow trout (Oncorhynchus mickiss). Neurosci. Lett., 151: 67–70.
39.
Snyder, S.H. (1992) Nitric oxide: first in a new class of neurotransmitters. Science, 257: 494–496.
40.
Villani, L. (1999) Development of NADPH-diaphorase activity in the central nervous system of the cichlid fish, Tilapia mariae. Brain Behav. Evol., 54: 147–158.
41.
Villani, L., and T. Guarnieri (1995a) Localization of NADPH-diaphorase in the goldfish brain. Brain Res., 679: 261–266.
42.
Villani, L., and T. Guarnieri (1995b) Ultrastructural localization of NADPH-diaphorase in the goldfish brain. Brain Res., 705: 332–336.
43.
Vincent, S.R. (1994) Nitric oxide: a radical neurotransmitter in the central nervous system. Prog. Neurobiol., 42: 129–160.
44.
Vincent, S.R., and H. Kimura (1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience, 46: 755–784.
45.
Wolf, G., S. Würdig, and G. Schünzel (1992) Nitric oxide synthase in rat brain is predominantly located at neuronal endoplasmic reticulum: an electron microscopic demonstration of NADPH-diaphorase activity. Neurosci. Lett., 147: 63–66.
© 2000 S. Karger AG, Basel
2000
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.