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. 1996 Jul;16(4):499–515. doi: 10.1007/BF02150230

Neuronal isoform of nitric oxide synthase is expressed at low levels in human retina

Chang-Shin Park 1, Cristina Gianotti 1, Roy Park 1, Gopal Krishna 1
PMCID: PMC11563097  PMID: 8879752

Abstract

  1. The expression of neuronal isoform of nitric oxide synthase (nNOS) was studied in human retinal tissues. The cDNA sequence was cloned in human retinal poly (A)+ RNA by the RT-PCR method and encompassed an openreading frame of 4,302 bp encoding 1,434 amino acids. This sequence showed a possibility of genetic polymorphism in comparison to human brain form.

  2. Restriction fragment length polymorphism (RFLP) patterns of a partial cDNA fragment suggest that there is genetic polymorphism in the neuronal form of NOS. Important differences were observed in a certain region between human retinal and brain forms. This region is a result of frame shift by the addition of three cytidines. In this study, regions from human brain (cerebellum) and skeletal muscle as well as retina were sequenced to confirm the difference in this region. The sequences from these tissues were completely identical. This indicated that genetic polymorphism of nNOS gene was due to single base substitution and not frame shift phenomenon by addition or deletion of bases.

  3. The nNOS mRNA of ∼12 kb was detected by northern blot analysis. The lower level of the expression was distinguished in comparison to those of human brain and skeletal muscle. The cDNA transiently transfected into CHO-K1 cells expressed a protein which contained a significant level of NOS activity. The size of the nNOS was found to be ∼160 kDa by bothin vitro andin vivo translation systems. This NOS was calcium dependent and the Km for arginine was 4.4 μM.

  4. The Ca++, L-arginine and NADPH dependency along with the inhibitory effect of N-nitro-L-arginine on NOS activity were evaluated. The finding of a constitutive form of NOS in human retina, which is calcium-NADPH dependent, gives further credence to the possible role of nitric oxide in retinal function and neuronal diseases.

Key Words: neuronal NO synthase, human retina, RT-PCR, RFLP, genetic polymorphism, CHO-K1 cell

References

  1. Ahmad, I., and Barnstable, C. J. (1993). Differential laminar expression of particulate and soluble guanylate cyclase genes in rat retina.Exp. Eve Res.56:51–62. [DOI] [PubMed] [Google Scholar]
  2. Ahmad, I., Leinders-Zufall, T., Kocsis, J. D., Shepherd, G. M., Zufall, F., and Barnstable, C. J. (1994). Retinal ganglion cells express a cGMP-gated cation conductance activatable by nitric oxide donors.Neuron12:155–165. [DOI] [PubMed] [Google Scholar]
  3. Baylor, D. A. (1987). Photoreceptor signals and vision.Invest. Opthalmol. Vis. Sci.28:34–49. [PubMed] [Google Scholar]
  4. Bredt, D. S., and Snyder, S. H. (1990). Isolation of nitric oxide synthase a calmodulin-requiring enzyme.Proc. Natl. Acad. Sci. USA87:682–685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bredt, D. S., and Snyder, S. H. (1992). Nitric oxide, a novel neuronal messenger.Neuron8:3–11. [DOI] [PubMed] [Google Scholar]
  6. Bredt, D. S., Hwang, P. H., Glatt, C., Lowenstein, C., Reed, R. R., and Snyder, S. H. (1991). Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.Nature352:714–718. [DOI] [PubMed] [Google Scholar]
  7. Dawson, T. M., Dawson, V. L., and Snyder, S. H. (1992). A novel neuronal messenger molecule in brain: The free radical, nitric oxide.Ann. Neurol.32:297–311. [DOI] [PubMed] [Google Scholar]
  8. Dawson, V. L., Dawson, T. M., Bartley, D. A., Uhl, G. R., and Snyder, S. H. (1993). Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures.J. Neurosci.13:2651–2661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feelisch, M., and Noack, E. A. (1987). Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase.Eur. J. Pharmacol.139:19–30. [DOI] [PubMed] [Google Scholar]
  10. Ferrendelli, J. A. (1978). Distribution and regulation cyclic GMP in the central nervous system.Adv. Cyclic Nucleotide Res.9:453–464. [PubMed] [Google Scholar]
  11. Fujisawa, H., Ogura, T., Kurashima, Y., Yokoyama, T., Yamashita, J., and Esumi, H. (1994). Expression of two types of nitric oxide synthase mRNA in human neuroblastoma cell lines.J. Neurochem.63:140–145. [DOI] [PubMed] [Google Scholar]
  12. Galea, E., Feinstein, D. L., and Reis, D. J. (1992). Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures.Proc. Natl. Acad. Sci. USA89:10945–10949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garthwaite, J. (1991). Glutamate, nitrix oxide and cell-cell signalling in the nervous system.Trends Neurol. Sci.14:60–67. [DOI] [PubMed] [Google Scholar]
  14. Geller, D. A., Lowenstein, C. J., Shapiro, R. A., Nussler, A. K., Di Silvio, M., Wang, S. C. Nakayama, D. K., Simmons, R. L., Snyder, S. H., and Billiar, T. R. (1993). Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes.Proc. Natl. Acad. Sci. USA90:3491–3495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goureau, O., Lepoivre, M., Mascarelli, F., and Courtois, Y. (1992a). Nitric oxide synthase activity in bovine retina. InStructures and Functions of Retinal Proteins (L. L. Rigaud and INSERM, Eds.), J. Libbey Eurotext Ltd., London, pp. 395–398. [Google Scholar]
  16. Goureau, O., Lepoivre, M., and Courtois, Y. (1992b). Lipopolysaccharide and cytokines induce a macrophage type of nitric oxide synthase in bovine retinal pigmented epithelial cells.Biochem. Biophys. Res. Commun.189:854–859. [DOI] [PubMed] [Google Scholar]
  17. Goureau, O., Lepoivre, M., Becquet, F., and Courtois, Y. (1993). Differential regulation of inducible nitric oxide synthase by fibroblast growth factors and transforming growth factor β in bovine retinal pigmented epithelial cells.Proc. Natl. Acad. Sci. USA90:4276–4280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goureau, O., Hicks, D., Courtois, Y., and De Kozak, Y. (1994). Induction and regulation of nitric oxide synthase in retinal Muller glial cells.J. Neurochem.,63:310–317. [DOI] [PubMed] [Google Scholar]
  19. Gross, S. S., Stuehr, D. J., Aisaka, K., Jaffe, E. A., Levi, R., and Griffith, O. W. (1990). Macrophage and endothelial cell nitric oxide synthase: Cell-type selective inhibition by NG-nitroarginine and NG-methylarginine.Biochem. Biophys. Res. Commun.170:96–103. [DOI] [PubMed] [Google Scholar]
  20. Hope, B. T., Michael, G. J., Knigge, K. M., and Vincent, S. R. (1991). Neuronal NADPH diaphorase are identical nin brain and peripheral tissues.Proc. Natl. Acad. Sci. USA88:2811–2814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Imai, T., Hirata, Y., and Marumo, F. (1992). Expression of brain nitric oxide synthase mRNA in various tissues and cultured cells of rat.Biochem. Res.13:371–374. [Google Scholar]
  22. Janssens, S. P., Shimouchi, A., Quertermous, T., Bloch, D. B., and Bloch, K. D. (1992). Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase.J. Biol. Chem.,267:14519–14522. [PubMed] [Google Scholar]
  23. Knowles, R. G., Palacios, M., Palmer, R. M. J., and Moncada, S. (1989). Formation of nitric oxide from L-arginine in the central nervous system: A transduction system for stimulation of the soluble guanylate cyclase.Proc. Natl. Acad. Sci. USA86:5159–5162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Knowles, R. G., Merrerr, M., Salter, M., and Moncada, S. (1990). Differential induction of brain, lung and liver nitric oxide synthase by endotoxin in the rat.Biochem. J.270:833–836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krishna, G., Krishnan, N., Fletcher, R. T., and Chadr, G. (1976). Effects of light on cyclic GMP metabolism in retinal photoreceptors.J. Neurochem.27:717–722. [DOI] [PubMed] [Google Scholar]
  26. Kristensson, K., Aldskogius, M., Peng, Z.-C., Olsson, T., Aldskogius, H., and Bentivoglio, M. (1994). Co-induction of neuronal interferon-gamma and nitric oxide synthase in rat motor neurons after axotomy: A role in nerve repair or death.J. Neurocytol.23:453–459. [DOI] [PubMed] [Google Scholar]
  27. Lamas, S., Marsden, P. A., Li, G. K., Tempst, P., and Michel, T. (1992). Endothelial nitric oxide synthase: Molecular cloning and characterization of a distinct constitutive enzyme isoform.Proc. Natl. Acad. Sci. USA89:6348–6352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lambert, L. E., Whitten, J. P., Baron, B. M., Cheng, H. C., Doherty, N. S., and McDonald, I. A. (1991). Nitric oxide synthase in the CNS, endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues.Life Sci.48:69–75. [DOI] [PubMed] [Google Scholar]
  29. Lyons, C. R., Orloff, G. J., and Cunning, J. M. (1992). Molecular cloning and functional expression of an inducible nitroc oxide synthase from a murine macrophage cell line.J. Biol. Chem.267:6370–6374. [PubMed] [Google Scholar]
  30. Marsden, P. A., Schappert, K. T., Chen, H. S., Flowers, M., Sundell, C. L., Wilcox, J. N., Lamas, S., and Michel, T. (1992). Molecular cloning and characterization of human endothelial nitric oxide synthase.FEBS Lett.307:287–293. [DOI] [PubMed] [Google Scholar]
  31. Mollace, V., Colasanti, M., Rodino, P., Massoud, R., Lauro, G. M., and Nistico, G. (1993). Cytokine-induced nitric oxide generation by cultured astrocytoma cells involves a Ca2+-calmodulin-independent NO-synthase.Biochem. Biophys. Res. Commun.191:327–334. [DOI] [PubMed] [Google Scholar]
  32. Mollace, V., Salvemini, D., Anggard, E., and Vane, J. (1990). Cultured astrocytoma cells inhibit platelet aggregation by releasing a nitric oxide like factor.Biochem. Biophys. Res. Commun.172:564–569. [DOI] [PubMed] [Google Scholar]
  33. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991). Nitric oxide: Physiology, pathophysiology, and phamacology.Pharmacol. Rev.43:109–142. [PubMed] [Google Scholar]
  34. Nakane, M., Schmidt, H. H. H. W., Pollock, J. S., Forstermann, U., and Murad, F. (1993). Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle.FEBS Lett.316:175–180. [DOI] [PubMed] [Google Scholar]
  35. Park, C. S., Pardhasaradhi, K., Gianotti, C., Villegas, E., and Krishna, G. (1994). Human retina expresses both constitutive and inducible isoforms of nitric oxide synthase mRNA.Biochem. Biophys. Res. Commun.205:85–91. [DOI] [PubMed] [Google Scholar]
  36. Provis, J. M., and Mitofanis, J. (1990)., NADPH-diaphorase neurons of human retina have a uniform topographical distribution.Vis. Neurosci.4:619–623. [DOI] [PubMed] [Google Scholar]
  37. Simmons, M. L., and Murphy, S. (1992a). Cytokines regulate L-arginine-dependent cyclic GMP production in rat glial cells.Eur. J. Neurosci.5:825–831. [DOI] [PubMed] [Google Scholar]
  38. Simmons, M. L., and Murphy, S. (1992b). Induction of nitric oxide synthase in glial cells.J. Neurochem.59:897–905. [DOI] [PubMed] [Google Scholar]
  39. Snyder, S. H., and Bredt, D. S. (1991). Nitric oxide as a neuronal messenger.Trends Pharmacol. Sci.12:125–128. [DOI] [PubMed] [Google Scholar]
  40. Venturini, C. M., Knowles, R. G., Palmer, R. M. J., and Moncada, S. (1991). Synthesis of nitric oxide in the bovine retina.Biochem. Biophys. Res. Commun.180:920–925. [DOI] [PubMed] [Google Scholar]
  41. Vincent, S. R., and Kimura, H. (1992). Histochemical mapping of nitric oxide synthase in the rat retina.Neuroscience46:755–784. [DOI] [PubMed] [Google Scholar]
  42. Warren, K., Gianotti, C., Pardhasardhi, K., Park, C. S., and Krishna, G., (1994). Canavanine, a mixed type of inhibitor of brain nitric oxide synthase: The Jack bean connection.Can. J. Phisiol. Pharmacol.72:P14.1.2 (abstr.).
  43. Weiler, R., and Kewitz, B. (1993). The marker for nitric oxide synthase, NADPH diaphorase, colocalizes with GABA in horizontal cells and cells of the inner retina in the carp retina.Neurosci. Lett.158:151–154. [DOI] [PubMed] [Google Scholar]
  44. Wu, W., Liuzzi, F. J., Schinco, F. P., Depto, A. S., Li, Y., Mong, J. A., Dawson, T. M., and Snyder, S. H. (1994). Neuronal nitric oxide synthase in induced in spinal neurons by traumatic injury.Neuroscience61:719–726. [DOI] [PubMed] [Google Scholar]
  45. Yamamoto, R., Bredt, D. S., Snyder, S. H., and Stone, R. A. (1993). The localization of nitric oxide synthase in the rat eye and related cranial ganglia.Neuroscience54:189–200. [DOI] [PubMed] [Google Scholar]
  46. Zeevalk, G. D., and Nicklas, W. J. (1994). Nitric oxide in retina: Relation to excitatory amino acid and excototoxicity.Exp. Eye Res.58:343–350. [DOI] [PubMed] [Google Scholar]

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