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Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation

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Abstract

In this study, we wished to clarify the distribution and co-localization of nitric oxide synthase and NADPH-diaphorase (NADPH-d) in nerve cells, nerve fibres and parenchymal cells in exocrine and endocrine pancreas, and to assess the influence of fixation on the staining pattern obtained. For this purpose, we applied nitric oxide synthase immunocytochemistry and NADPH-d histochemistry to rat and human pancreas under different fixation conditions. Antibodies to neuronal and endothelial nitric oxide synthase were similarly applied. We found complete co-localization of neuronal nitric oxide synthase and NADPH-d in ganglion cells, and in nerve fibres around acini, excretory ducts, blood vessels and in islets of Langerhans of rat and human pancreas. Immunoreactivity for endothelial nitric oxide synthase was co-localized with NADPH-d in endothelial cells. However, in NADPH-d reactive islet and ductal epithelial cells we could detect neither brain nor endothelial nitric oxide synthase immunoreactivity with any fixation protocol applied. There were marked differences in NADPH-d staining of both neurons and parenchymal cells under different fixation conditions. These results indicate the existence of different types of NADPH-d, which are associated or not associated with nitric oxide synthase(s), and which are differently influenced by various fixation procedures in rat and human pancreas.

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References

  • Aimi Y, Fujimura M, Vincent SR, Kimura H (1991) Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat. J Comp Neurol 306:382–392

    Google Scholar 

  • Alm P, Larsson B, Ekblad E, Sundler F, Andersson K-E (1993) Immunohistochemical localization of peripheral nitric oxide synthase-containing nerves using antibodies raised against synthesized C- and N-terminal fragments of a cloned enzyme from rat brain. Acta Physiol Scand 148:421–429

    Google Scholar 

  • Belai A, Schmidt HHHW, Hoyle CHV, Hassel CJS, Saffrey MJ, Moss J, Förstermann U, Murad F, Burnstock G (1992) Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut. Neurosci Lett 143:60–64

    Google Scholar 

  • Berthoud HR, Powley TL (1991) Morphology and distribution of efferent vagal innervation of rat pancreas as revealed with anterograde transport of Dil. Brain Res 553:336–341

    Google Scholar 

  • Berthoud HR, Niijima A, Sauter FJ, Jeanrenaud B (1983) Evidence for a role of the gastric, coeliac and hepatic branches in vagally stimulated insulin secretion in the rat. J Auton Nerv Syst 7:97–110

    Google Scholar 

  • Bouwens L, Klöppel G (1994) Cytochemical localization of NADPH-diaphorase in the four types of pancreatic islet cell. Histochemistry 101:209–214

    Google Scholar 

  • Cervero F, McRitchie HA (1982) Neonatal capsaicin does not affect unmyelinated efferent fibers of the autonomic nervous system: functional evidence. Brain Res 239:283–288

    Google Scholar 

  • Chey WY (1991) Regulation of pancreatic exocrine secretion. Int J Pancreatol 9:7–20

    Google Scholar 

  • Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci USA 88:7797–7801

    Google Scholar 

  • Dawson TM, Steiner JP, Mong JA, Snyder SH (1993) Multiple isoforms of neuronal nitric oxide synthase. Soc Neurosci Abstr 19:905A

    Google Scholar 

  • De Groat WC, Vizzard MA, Erdman SL, Erickson VL, Stewart RJ, Roppolo JR, Forstermann U (1993) Differential distribution of NADPH-diaphorase and nitric oxide synthase immunoreactivity in the thoracolumbar-sacral spinal cord of the cat. Soc Neurosci Abstr 19:510A

    Google Scholar 

  • Dinerman JL, Dawson TM, Schell MJ, Snowman A, Snyder SH (1994) Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc Natl Acad Sci USA 91:4214–4218

    Google Scholar 

  • Dun NJ, Dun SL, Forstermann U, Tseng LF (1992) Nitric oxide synthase immunoreactivity in rat spinal cord. Neurosci Lett 147:217–220

    Google Scholar 

  • Gossrau R (1994) Further studies on the usefulness of the NADPH-tetrazolium salt system for the visualization of nitric oxide synthase (NOS). Ann Anat [Suppl] 176:91

    Google Scholar 

  • Grozdanovic Z, Baumgarten HG, Brüning G (1992) Histochemistry of NADPH-diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse. Neuroscience 48:225–235

    Google Scholar 

  • Hope BT, Michael GJ, Knigge KM, Vincent SR (1991) Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 88:2811–2814

    Google Scholar 

  • Kirchgessner AL, Gershon MD (1990) Innervation of the pancreas by neurons in the gut. J Neurosci 10:1626–1642

    Google Scholar 

  • Kolb H, Kolb-Bachofen V (1992) Type 1 (insulin-dependent) diabetes mellitus and nitric oxide. Diabetologia 35:796–797

    Google Scholar 

  • Matsumoto T, Nakane M, Pollock JS, Kuk JE, Förstermann U (1993) A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue fixative. Neurosci Lett 155:61–64

    Google Scholar 

  • Mayer B, John M, Heinzel B, Werner ER, Wachter H, Schultz G, Böhme E (1991) Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxide-reductase. FEBS Lett 288:187–191

    Google Scholar 

  • Miller RE (1981) Pancreatic neuroendocrinology: peripheral neural mechanisms in the regulation of the islets of Langerhans. Endocrine Rev 4:471–494

    Google Scholar 

  • Neuhuber WL (1989) Vagal afferent fibers almost exclusively innervate islets in the rat pancreas as demonstrated by anterograde tracing. J Auton Nerv Syst 29:13–18

    Google Scholar 

  • Neuhuber WL, Irminger D (1990) Visceral afferent projections to the thoracolumbar spinal cord in normal and capsaicin-treated rats. In: Zenker W, Neuhuber WL (eds) The primary afferent neuron. Plenum Press, New York, pp 189–199

    Google Scholar 

  • Neuhuber WL, Wörl J, Berthoud HR, Conte B (1994) NADPH-diaphorase-positive nerve fibres associated with motor endplates in the rat esophagus: new evidence for co-innervation of striated muscle by enteric neurons. Cell Tissue Res 276:23–30

    Google Scholar 

  • Radke R (1990) Innervation des exokrinen und endokrinen Pankreas. Schwer-Verlag, Stuttgart

    Google Scholar 

  • Santer RM, Symons D (1993) Distribution of NADPH-diaphorase activity in rat paravertebral, prevertebral and pelvic sympathetic ganglia. Cell Tissue Res 271:115–121

    Google Scholar 

  • Scherer-Singler U, Vincent SR, Kimura H, McGeer EG (1983) Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. J Neurosci Methods 9:229–234

    Google Scholar 

  • Schmidt HHHW, Gagne GD, Nakane M, Pollock JS, Miller MF, Murad F (1992a) 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. J Histochem Cytochem 40:1439–1456

    Google Scholar 

  • Schmidt HHHW, Warner TD, Ishii K, Sheng H, Murad F (1992b) Insulin secretion from pancreatic cells caused by l-argininederived nitrogen oxides. Science 255:721–723

    Google Scholar 

  • Sharkey KA, Williams RG, Dockray GJ (1984) Sensory substance P innervation of the stomach and pancreas. Gastroenterology 87:914–921

    Google Scholar 

  • Shimosegawa T, Abe T, Satoh A, Asakura T, Yoshida K, Koizumi M, Toyota T (1992) Histochemical demonstration of NADPH-diaphorase activity, a marker for nitric oxide synthase, in neurons of the rat pancreas. Neurosci Lett 148:67–70

    Google Scholar 

  • Shimosegawa T, Abe T, Satoh A, Abe R, Kikuchi Y, Koizumi M, Toyota T (1993) NADPH-diaphorase activity in neurons of the mammalian pancreas: coexpression with vasoactive intestinal polypeptide. Gastroenterology 105:999–1008

    Google Scholar 

  • Tay SSW, Moules EW, Burnstock G (1993) Localization of NADPH-diaphorase (nitric oxide synthase) in the guinea pig pancreas in situ and in culture. Soc Neurosci Abstr 19:510A

    Google Scholar 

  • Vincent SR (1992) Nitric oxide and arginine-evoked insulin secretion. Science 258:1376

    Google Scholar 

  • Vincent SR, Kimura H (1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46:755–784

    Google Scholar 

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Wörl, J., Wiesand, M., Neuhuber, W.L. et al. Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation. Histochemistry 102, 353–364 (1994). https://doi.org/10.1007/BF00268906

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