Digestive Diseases and Sciences

, Volume 40, Issue 11, pp 2390–2397 | Cite as

Effect of nitroblue tetrazolium on NO synthase and motor function of opossum esophagus

  • Jeffrey L. Conklin
  • Brian W. O'Meara
  • Joseph A. Murray
  • Sheng Yun Fang
  • Eugene D. Clark
  • James Christensen
Motility, Nerve-Gut Interactions, Hormones, And Receptors


Nitric oxide mediates neuromuscular events in the opossum esophagus. The NADPH diaphorase stain is used to localize nitric oxide synthase-containing enteric neurons. Cells stain by the NADPH diaphorase technique because they reduce nitroblue tetrazolium to the visible formazan. The effects of nitroblue tetrazolium on neuromuscular function and nitric oxide synthase of esophageal muscle were studied. The NADPH diaphorase stain was performed. Nitroblue tetrazolium inhibited lower esophageal sphincter relaxation, abolished the latency gradient of the off response, and inhibited nitric oxide synthase. The NADPH diaphorase technique stained myenteric plexus nerve cell bodies and nerve processes. Nitroblue tetrazolium is not a nonspecific muscle or nerve toxin, as nerve-mediated cholinergic responses, responses to exogenous nitric oxide, and responses to myogenic stimulation were maintained after nitroblue tetrazolium abolished the off response and lower esophageal sphincter relaxation. Nitroblue tetrazolium inhibits nitric oxide-mediated events and nitric oxide synthase. It stains neurons in the esophageal myenteric plexus.

Key words

nitric oxide enteric nervous system nonadrenergic noncholinergic nerves autonomic nervous system gastrointestinal motility NADPH diaphorase 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Christensen J, Lund GF: Esophageal responses to distension and electrical stimulation. J Clin Invest 48:408–419, 1969PubMedGoogle Scholar
  2. 2.
    Murray J, Du C, Ledlow A, Bates JN, Conklin JL: Nitric oxide: Mediator of nonadrenergic noncholinergic responses of opossum esophageal muscle. Am J Physiol 261:G401-G406, 1991PubMedGoogle Scholar
  3. 3.
    Weisbrodt NW, Christensen J: Gradients of contractions in the opossum esophagus. Gastroenterology 62:1159–1166, 1972PubMedGoogle Scholar
  4. 4.
    Yamato S, Spechler SJ, Goyal RK: Role of nitric oxide in esophageal peristalsis in the opossum. Gastroenterology 103:197–204, 1992PubMedGoogle Scholar
  5. 5.
    Tottrup A, Svane D, Forman A: Nitric oxide mediating NANC inhibition in opossum lower esophageal sphincter. Am J Physiol 260:G385-G389, 1991PubMedGoogle Scholar
  6. 6.
    Conklin JL, Du C, Murray J, Bates JN: Characterization and mediation of inhibitory junction potentials from the opossum lower esophageal sphincter. Gastroenterology 104:1439–1444, 1993PubMedGoogle Scholar
  7. 7.
    Du C, Murray J, Bates JN, Conklin JL: Nitric oxide: Mediator of nonadrenergic noncholinergic hyperpolarization of opossum esophageal muscle. Am J Physiol 261:G1012-G1016, 1991PubMedGoogle Scholar
  8. 8.
    Alm P, Larsson B, Andersson KE: NADPH diaphorase activity and non-adrenergic non-cholinergic relaxation of the human gastrointestinal tract. Acta Physiol Scand 146:285–287, 1992PubMedGoogle Scholar
  9. 9.
    Belia A, Schmidt HHHW, Hoyle CHV, Hassall CJS, Saffrey MJ, Moss J, Forstermann U, Murad F, Burnstock G: Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut. Neurosci Lett 143:60–64, 1992PubMedGoogle Scholar
  10. 10.
    Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH: Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci USA 88:7797–7801, 1991PubMedGoogle Scholar
  11. 11.
    Grozdanovic Z, Baumgarten HC, Bruning G: Histochemistry of NADPH diaphorase, a marker for neuronal nitric oxide synthase, in the peripheral autonomic nervous system of the mouse. Neuroscience 48:225–235, 1992PubMedGoogle Scholar
  12. 12.
    Hassall CJS, Saffrey MJ, Belia A, Hogle CHV, Moules EW, Moss J, Schmidt HHHW, Murad F, Forstermann U, Burnstock G. Nitric oxide synthase immunoreactivity and NADPH-diophorase activity in a subpopulation of intrinsic neurons of the guinea pig heart. Neurosci Lett 143:65–68, 1992PubMedGoogle Scholar
  13. 13.
    Hope BT, Micheal GT, Knigge KM, Vincent SR: Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 88:2811–2814, 1991PubMedGoogle Scholar
  14. 14.
    Keast JR: A possible neural source of nitric oxide in the rat penis. Neurosci Lett 143:69–73, 1992PubMedGoogle Scholar
  15. 15.
    Scherer-Singler U, Vincent SR, Kimura H, McGeer EG: Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. J Neurosci Methods 9:229–234, 1983PubMedGoogle Scholar
  16. 16.
    Ward SM, Xue C, Shuttleworth CW, Bredt DS, Snyder SH, Sanders KM: NADPH diaphorase and nitric oxide synthase colocalization in enteric neurons of canine proximal colon. Am J Physiol 263:G277-G284, 1992PubMedGoogle Scholar
  17. 17.
    Young HM, Furness JB, Shuttleworth CWR, Bredt DS, Snyder SH: Colocalization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine. Histochemistry 97:375–378, 1992PubMedGoogle Scholar
  18. 18.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC: Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85, 1985PubMedGoogle Scholar
  19. 19.
    Murray J, Clark ED: Characterization of nitric oxide synthase in the opossum esophagus. 106:1444–1450, 1994Google Scholar
  20. 20.
    Stuehr DJ, Griffith OW: Mammalian nitric oxide synthases. Adv Enzymol 65:287–345, 1992PubMedGoogle Scholar
  21. 21.
    Schmidt HHHW, Lohmann SM, Walter U: The nitric oxide and cGMP signal transduction system. Regulation and mechanism of action. Biochim Biophys Acta 1178:153–175, 1993PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Jeffrey L. Conklin
    • 1
  • Brian W. O'Meara
    • 1
  • Joseph A. Murray
    • 1
  • Sheng Yun Fang
    • 1
  • Eugene D. Clark
    • 1
  • James Christensen
    • 1
  1. 1.From the Department of Internal Medicine, Division of GastroenterologyUniversity of Iowa College of Medicine and Department of Veterans Affairs HospitalIowa City

Personalised recommendations