1. Volume 122, Issue 5, Pages 1442-1454 (May 2002)
Constitutive expression and function of cyclooxygenase-2 in murine gastric muscles 
Christophe Porcher, Burton Horowitz, Orline Bayguinov, Sean M. Ward, Kenton M. Sanders 
Gastroenterology 
Volume 122, Issue 5, Pages (May 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
(A–F) COX-2–LI is expressed in a subpopulation of enteric neurons and ICC. (A and D) COX-2–LI (green) and (B and E) nuclear staining (propidium iodide; red) in the same transverse section of the (A–C) murine fundus, and the (D–F) antrum. (C and F) Double-labeling of COX-2–LI and nuclear staining are shown in the right columns. Nuclei were negative for COX-2–LI.Yellow regions in merged image result from COX-2–LI in regions above and below nuclei. COX-2–LI was observed in a subpopulation of cells in the longitudinal (LM) and circular (CM) muscle layers and in all myenteric ganglia (MG). Note also the presence of COX-2–LI in septae (arrow). (G and J) COX-2–LI (green) and (H and K) NOS-LI (red) in the same optical section at the level of the (G–I) myenteric plexus. (G and H) COX-2–LI and NOS-LI were observed in a subpopulation of myenteric neurons, respectively. (I) Combined immunofluorescences of COX-2–LI and NOS-LI, shown in the right column, revealed that a population of COX-2–LI were also NOS-LI (arrows; yellow). Other neurons were COX-2–LI but were immunonegative for NOS, and a third population were NOS-LI but were immunonegative for COX-2. (J–L) COX-2–LI and NOS-LI were also found in the same varicose nerve fiber trunks within the circular muscle layer (arrows). (L) Some nerve fibers were also immunopositive for COX-2 but immunonegative for NOS, and other fibers displayed immunoreactivity for NOS but not for COX-2. (M–O) COX-2–LI (M, green) and vimentin-like immunoreactivity (Vim-LI; N, red) in the same optical section in the circular muscle layer in the gastric antrum. (O) Combined immunofluorescences of COX-2–LI and Vim-LI are shown in the right column. (N) Vim-LI reveals IC-IM (arrow) within the circular muscle layer. (O) COX-2–LI was found within cell bodies of IC-IM and within the bipolar lateral processes of IC-IM within the circular muscle layer (arrow; yellow).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

3. Fig. 2
(A) Molecular expression of COX-2 in murine gastric myocytes. Qualitative RT-PCR on total RNA prepared from murine myocytes. Total RNA was prepared from murine antrum and fundus smooth muscle cells. cDNA was prepared from the RNA, and COX-1– and COX-2–specific primers were used to determine qualitative levels of transcriptional expression in these myocytes. COX-1 amplicon = 111 bp; COX-2 amplicon = 101 bp. β-actin expression was used to assure the quality of the RNA preparations. β-actin intron spanning amplicon = 498 bp. The amplified products (5 μL) were separated by electrophoresis on a 4% agarose/1X Tris–acetic acid–ethylenediaminetetraacetic acid gel, and the DNA bands were visualized by ethidium bromide staining. RT lanes show controls lacking reverse transcriptase. (B) Relative expression of COX-1 and COX-2 in gastric muscles. Quantitative real-time RT-PCR on total RNA prepared from murine antrum and fundus tissues. The bar graphs display values for COX-1 and COX-2 steady-state transcripts relative to β-actin in the same RNA preparation. The results are expressed as means ± standard error. *Significant difference between COX-1/COX-2 ratios in fundus- and antrum-derived RNA (n = 3, P < 0.05).
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Effects of PG synthesis inhibitors in fundus muscles. (A) Excerpts of a record testing the effects of indomethacin on fundus tone. In the first trace, after tone developed, SNP (1 μmol/L) was added to show maximal relaxation. Then the effects of SNP were washed out, tone was restored (this portion of the record was omitted), and 1 μmol/L indomethacin was added. Indomethacin caused significant relaxation of tone. (B) A similar experiment testing the effects of GR253035X (1 μmol/L). This compound also reduced fundus tone; however, the degree of relaxation was significantly less than that caused by an equivalent amount of indomethacin. (C) Summary of 15 experiments in which concentrations of either indomethacin or GR253035X were added in a cumulative manner and the percentage of maximal relaxation is plotted as a function of COX inhibitor concentration. Note that the concentration-response curve for the COX-2 inhibitor is shifted to the right of the concentration response for indomethacin.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Effects of PG synthesis inhibitors on circular muscle contractions of the murine gastric antrum. (A) ACh (1 μmol/L) increased the amplitude and frequency of spontaneous contractions. These actions were reversible after washout of the drug. (B) The effects of indomethacin (1 μmol/L) on spontaneous contractile activity and the effects of ACh in the presence of indomethacin. Indomethacin increased the amplitude but decreased the frequency of circular muscle contractions. These effects of indomethacin developed over a 1-hour incubation period of the drug (slashed bars represent a 1-hour interval). ACh (1 μmol/L) in the presence of indomethacin produced a large increase in amplitude and slight potentiation in the frequency of circular muscle contractions that were reversible on washout. (C) The effects of GR253035X (1 μmol/L) on circular muscle activity under control conditions and after ACh stimulation. GR253035X increased the amplitude but decreased the frequency of circular muscle contractions over a 1-hour incubation period (represented by the parallel slashed bars). In the continued presence of GR253035X, ACh (1 μmol/L) produced a large increase in the amplitude and potentiated in the frequency of the circular muscle contractions.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Summary of the effects of PG synthesis inhibitors in antral muscles. (A) A summary of the changes in mechanical force produced by the circular muscle of the gastric antrum in response to the exogenous application of indomethacin (1 μmol/L), plotted as a percentage change in the maximal contraction. Indomethacin caused on average a 192% increase in force of spontaneous contractions. (B) The changes in mechanical force produced by the circular muscle of the gastric antrum in response to the exogenous application of GR253035X (1 μmol/L), plotted as a percentage change in the maximal contraction. GR253035X caused on average a 56% increase in the force of spontaneous contractions of the circular muscle layer. (C) The change in spontaneous mechanical activity in response to ACh (1 μmol/L) under control conditions and 1 hour after the addition of indomethacin (1 μmol/L). In the presence of indomethacin, ACh produced a 153% increase in the force of spontaneous contractions compared with ACh added under control conditions. (D) The changes in spontaneous mechanical activity in response to ACh (1 μmol/L) under control conditions and 1 hour after the addition of GR253035X (1 μmol/L). In the presence of GR253035X, ACh produced a 58% increase in the force of spontaneous contractions compared with ACh alone. All changes above control were significant to at least P < 0.05 (asterisks).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Specific COX-2 inhibitor was without effect on fundic tone in COX-2 −/− mice. (A) Excerpts of a record testing the effects of indomethacin on fundus tone from a C57/BL6 mouse, a strain-matched control for COX-2 −/− mice. Indomethacin caused a relaxation similar to that produced for Balb/C mice. (B) The effects of indomethacin on a gastric fundus from a COX-2 −/− mouse. The degree of relaxation to indomethacin in the COX-2 −/− tissues was significantly reduced compared with controls. (C) The fundus relaxation to the specific COX-2 inhibitor, GR253035X, in a C57/BL6 mouse and (D) the lack of relaxation response to GR253035X in a COX-2 −/− fundus. (E) The fundus relaxation responses to GR253035X and indomethacin in C57/BL6 controls and (F) the fundus relaxations for COX-2 −/− mice to GR253035X and indomethacin. In COX-2 −/− mice, the degree of fundus relaxations to both GR253035X and indomethacin was significantly attenuated. (E and F) The changes shown are significant (P < 0.002). (G) The genotyping profile of COX-2 knockout mice and controls. A 280-bp fragment of the Neo gene was amplified in homozygote −/− or heterozygote +/− mice, whereas the COX-2–specific primers amplified an 857-bp product from the age-matched homozygote +/+ and heterozygote +/− mice.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Specific COX-2 inhibitor was without effect on gastric antral contractions of COX-2 −/− mice. (A) ACh (1 μmol/L) increased the amplitude and frequency of spontaneous contractions in control C57/BL6 mice in a reversible manner. (B) ACh also increased the amplitude and frequency of contractions in antral tissues from COX-2 −/− mice. (C) The effects of indomethacin on spontaneous contractions and the effects of ACh in the continued presence of indomethacin in a C57/BL6 mouse. (D) The effects of a similar treatment in a COX-2 −/− mouse. Indomethacin had a decreased effect on spontaneous contractions in the COX-2 −/− mouse compared with age-matched +/+ and +/− mice. (E) The increase of contractile activity of an antral muscle from a C57/BL6 mouse to GR253035X. In the continued presence of GR253035X, ACh further potentiated spontaneous contractions. (F) In comparison, GR253035X produced no increase in the force of contractions in COX-2 −/− mice. ACh in the presence of GR253035X increased the frequency and slightly increased the amplitude of contractions.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
Summary of the effects of PG synthesis inhibitors in COX-2 −/− and control antral muscles. (A and B) The increase in amplitude of contractions in response to the exogenous application of indomethacin (1 μmol/L), plotted as a percentage of maximal contraction, for controls and COX-2 −/− mice, respectively (n = 6 animals). Indomethacin produced an 809% increase in amplitude of contractions in controls and only a 105% in COX-2 −/− mice. (C and D) The response to GR253035X (1 μmol/L) for control and COX-2 −/− tissues, respectively. In control tissues there was a 55% increase in force of contractions compared with a 9% decrease in contractile force in COX-2 −/− mice (D, *P > 0.05). (E and F) The effects of ACh (1 μmol/L) in the presence of indomethacin for controls and COX-2 −/− tissues, respectively. In the presence of indomethacin, ACh produced a 159% increase in amplitude of contractions in controls and a 145% in COX-2 −/− mice. (G and H) The response to ACh (1 μmol/L) in the presence of GR253035X for controls and COX-2 −/− tissues, respectively. In the presence of GR253035X, ACh produced a 53% increase in force of contractions compared with 12% in COX-2 −/− mice. Responses in all panels, except D and H, showed significant increases in contractile activity (P < 0.05; asterisks).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions