Yonsei Med J.  2006 Apr;47(2):223-229. 10.3349/ymj.2006.47.2.223.

Generation of Nitric Oxide in the Opossum Lower Esophageal Sphincter during Physiological Experimentation

• Lee SJ
• Park H
• Chang JH
• Conklin JL

• KMID: 1110746
• DOI: http://doi.org/10.3349/ymj.2006.47.2.223

Abstract

Lipopolysaccharide (LPS), given in vivo, modulates opossum esophageal motor functions by inducing the inducible nitric oxide synthase (iNOS), which increases nitric oxide (NO) production. Superoxide, a NO scavenger, is generated during this endotoxemia. Superoxide is cleared by superoxide dismutase (SOD) and catalase (CAT) to protect the physiological function of NO. This study examined whether lower esophageal sphincter (LES) motility, NO release, and iNOS and nitrotyrosine accumulation in the LES are affected by LPS in vitro. Muscle strips from the opossum LES were placed in tissue baths containing oxygenated Krebs buffer. NO release was measured with a chemiluminescence NOx analyzer, and Western blots were performed to analyze iNOS and nitrotyrosine production. The percent change in resting LES tone after a 6-hour exposure to LPS was significantly increased compared to pretreatment values. The percent LES relaxation upon electrical stimulation was significantly decreased in the control group at 6 hours, indicating that the LPS treatment had an effect. The NO concentration in the tissue bath of LPS-treated muscle without nerve stimulation was significantly less than that of LPS treatment combined with SOD/CAT or SOD/CAT alone. iNOS and nitrotyrosine were detectable and increased over time in the LES muscle of both the control and LPS-treated groups. Antioxidant enzymes may play a role in regulating NO-mediated neuromuscular functions in the LES.

Keyword

Nitric oxide; lower esophageal sphincter

MeSH Terms

Tyrosine/analogs & derivatives/chemistry
Time Factors
Superoxide Dismutase/metabolism
Opossums
Nitric Oxide Synthase Type II/metabolism
Nitric Oxide/*chemistry/metabolism
Muscles/metabolism
Male
Luminescence
Lipopolysaccharides/chemistry/metabolism
Female
Esophageal Sphincter, Upper/*anatomy & histology/metabolism
Esophageal Sphincter, Lower/*anatomy & histology/metabolism
Catalase/metabolism
Blotting, Western
Antioxidants/chemistry/metabolism
Animals

Figure

• 

  Fig. 1 The percent change in LES resting tone after exposure to LPS and/or SOD/CAT. The percent change in LES tone is on the Y-axis. The change in tension after 6 hours was significantly increased to 133.0 ± 13.7% in the LPS (1 µg/mL)-treated group, compared with its pretreatment values. Group I, control; group II, LPS; group III, LPS with SOD/CAT 25 units/mL; group IV, LPS with SOD/CAT 100 units/mL, group V, SOD/CAT 100 units/mL. LPS, lipopolysaccharide; SOD, superoxide dismutase; CAT, catalase. Filled bar: pretreatment group, empty bar: posttreatment group. Values are mean ± SE. *p<0.05 vs pretreatment group.

• 

  Fig. 2 Effect of LPS and/or SOD/CAT on EFS-induced relaxation of the LES. Percent relaxation is on the Y-axis. The percent relaxation of the LES by EFS was significantly decreased after 6 hours in the control group (n = 12; PreTx., 77.0 ± 5.0%; PostTx, 63.9 ± 5.3%, p<0.05). Group I, control; group II, LPS (1 µg/mL); group III, LPS with SOD/CAT 25 units/mL; group IV, LPS with SOD/CAT 100 units/mL, group V, SOD/CAT 100 units/mL. EFS, electrical field stimulation (4 sec trains of 1 msec, 35 V square wave pulses at 3 Hz); LPS, lipopolysaccharide; LSC, LPS with SOD/CAT. Filled bar: pretreatment group, empty bar: posttreatment group. Values are mean ± SE. *p<0.05 vs. pretreatment group.

• 

  Fig. 3 The effect of LPS and/or SOD/CAT on levels of NO without nerve stimulation. The percent change in NO level in the tissue bath (against pretreatment level) without EFS is on the Y-axis. The NO levels were increased in all groups after 6 hours. The NO level in the LPS (1 µg/mL)-treated group (n = 10; 290.5 ± 75.4%) was significantly less than that of the LPS with SOD/CAT group (n = 8; 100 units/mL, 716.2 ± 142.3%) or the SOD/CAT alone group (n = 5; 100 units/mL, 805.2 ± 123.8%). Group I, control; group II, LPS; group III, LPS with SOD/CAT 25 units/mL; group IV, LPS with SOD/CAT 100 units/mL, group V, SOD/CAT 100 units/mL. EFS, electrical field stimulation (4 sec trains of 1 msec, 35 V square wave pulses at 3 Hz). LPS, lipopolysaccharide; SOD, superoxide dismutase; CAT, catalase. Values are mean ± SE. *p<0.05, compared with LPS with SOD/CAT 100 units/mL.

• 

  Fig. 4 The effect of LPS and/or SOD/CAT on nerve-stimulated levels of NO. The EFS-induced release of NO in the controls (n = 10) was decreased compared with its pretreatment values (p = 0.083). Group I, control; group II, LPS; group III, LPS with SOD/CAT 25 units/mL; group IV, LPS with SOD/CAT 100 units/mL, group V, SOD/CAT 100 units/mL. EFS, electrical field stimulation (4 sec trains of 1 msec, 35 V square wave pulses at 3 Hz). LPS, lipopolysaccharide; LSC, LPS with SOD/CAT; SOD, superoxide dismutase; CAT, catalase. Filled bar: pretreatment group, empty bar: posttreatment group. Values are mean ± SE.

• 

  Fig. 5 Accumulation of nitrotyrosine proteins in the LES. The density ratio of band against that of LES muscle immediately after harvesting the tissue (not exposed to an in vitro organ bath) (a, n = 3) was significantly increased in the control (b, n = 7), LPS-(c, n = 8), or LPS with SOD/CAT (100 units/mL)-treated (d, n = 4) groups. *p<0.05 vs in vitro organ bath.

Reference

1. Park H, Cullen JJ, Clark E, Conklin JL. Effect of inhibiting iNOS on endotoxin-induced changes in esophageal motor function. Neurogastroenterol Motil. 1999. 11:278A.

2. Park H, Clark E, Cullen JJ, Conklin JL. Effect of endotoxin on opossum esophageal motor function. Neurogastroenterol Motil. 2000. 12:215–221.

3. Brovkovych V, Patton S, Brovkovych S, Kiechle F, Huk I, Malinski T. In situ measurement of nitric oxide, superoxide and peroxynitrite during endotoxemia. J Physiol Pharmacol. 1997. 48:633–644.

4. Leichus LS, Thomas RM, Murray JA, Conklin JL. Effect of oxygen radicals and radical scavenging on opossum lower esophageal sphincter. Dig Dis Sci. 1997. 42:592–596.

5. Thomas RM, Fang S, Leichus LS, Oberley LW, Christensen J, Murray JA, et al. Antioxidant enzymes in intramural nerves of the opossum esophagus. Am J Physiol. 1996. 270(1 Pt 1):G136–G142.

6. Arkovitz MS, Wispe JR, Garcia VF, Szabo C. Selective inhibition of the inducible isoform of nitric oxide synthase prevents pulmonary transvascular flux during acute endotoxemia. J Pediatr Surg. 1996. 31:1009–1015.

7. Hamilton CA, Berg G, Mcintyre M, Mcphaden AR, Reid JL, Dominiczak AF. Effects of nitric oxide and superoxide on relaxation in human artery and vein. Atherosclerosis. 1997. 133:77–86.

8. Muijsers RB, Folkerts G, Henricks PA, Sadeghi-Hashjin G, Nijkamp FP. Peroxynitrite: a two-faced metabolite of nitric oxide. Life Sci. 1997. 60:1833–1845.

9. Miller MJ, Thompson JH, Zhang XJ, Sadowska-Krowicka H, Kakkis JL, Munshi UK, et al. Role of inducible nitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis. Gastroenterology. 1995. 109:1475–1483.

10. Flesch M, Kilter H, Cremers B, Laufs U, Sudkamp M, Ortmann M, et al. Effects of endotoxin on human myocardial contractility involvement of nitric oxide and peroxynitrite. J Am Coll Cardiol. 1999. 33:1062–1070.

11. Takakura K, Goto Y, Muramatsu I. Nitric oxide synthase induction and relaxation in lipopolysaccharide-treated gastric fundus muscle of rats. Life Sci. 1996. 58:9–17.

12. Brune B, Schmidt KU, Ullrich V. Activation of soluble guanylate cyclase by carbon monoxide and inhibition by superoxide anion. Eur J Bioochem. 1990. 192:683–688.

13. Murray JA, Du C, Ledlow A, Manternach PL, Conklin JL. Guanyl cyclase inhibitors: effect on tone, relaxation and cGMP content of the lower esophageal sphincter. Am J Physiol. 1992. 263:G97–G101.

14. Uc A, Murray JA, Kooy N, Conklin JL. Effect of peroxynitrite on motor functions of the opossum esophagus. Dig Dis Sci. 2001. 46:30–37.

15. Boota A, Zar H, Kim YM, Johnson B, Pitt B, Davies P. IL-1 stimulates superoxide and delayed peroxynitrite production by pulmonary vascular smooth muscle cells. Am J Physiol. 1996. 271(6 Pt 1):L932–L938.

16. Yang CC, Alvarez RB, Engel WK, Askanas V. Increase of nitric oxide synthases and nitrotyrosine in inclusion-body myositis. Neuroreport. 1996. 8:153–158.