Volume 119, Issue 4, Pages 1054-1063 (October 2000) Orphanin FQ causes contractions via inhibiting purinergic pathway in the rat colon  Toku Takahashi, Didier Bagnol, David Schneider, Yohei Mizuta, Tadashi Ishiguchi, Kathy LePard, James J. Galligan, Stanley J. Watson, Chung Owyang  Gastroenterology  Volume 119, Issue 4, Pages 1054-1063 (October 2000) DOI: 10.1053/gast.2000.18154 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) Contractions of the circular muscle of distal colon induced by OFQ (10−10 to 10−7 mol/L). (B) Dose-response curve of muscle contractions induced by OFQ (10−10 to 10−7 mol/L) and [Phe1ψ(CH2-NH)Gly2]NC(1-13)NH2(10−10 to 10−7 mol/L). Data are expressed as percent of carbachol (10−6 mol/L)-induced contractions. OFQ and [Phe1ψ(CH2-NH)Gly2]NC(1-13)NH2 (10−9 to 10−7 mol/L) caused significant colonic contractions in a dose-dependent manner. EC50s of OFQ and [Phe1ψ(CH2-NH)Gly2]NC(1-13)NH2 were 0.28 ± 0.03 × 10−9 mol/L and 0.76 ± 0.08 × 10−9 mol/L, respectively (n = 6). Gastroenterology 2000 119, 1054-1063DOI: (10.1053/gast.2000.18154) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Effects of (A) TTX (10−7 mol/L), (B)L-NAME (10−4 mol/L), and (C) suramin (10−4 mol/L) on OFQ (10−8 mol/L)-induced contractions of the circular muscle of the distal colon. TTX itself enhanced spontaneous contractions in the circular muscles of the colon because of the removal of inhibitory neural influence on the smooth muscle cells. In the presence of TTX, OFQ (10−8 mol/L) failed to elicit additional contractions in both the proximal and distal colon. L-NAME (10−4 mol/L) and suramin (10−4 mol/L) significantly enhanced spontaneous contractions. OFQ-induced contractions were preserved in the presence of L-NAME, whereas OFQ (10−8 mol/L)-induced contractions were significantly reduced in the presence of suramin in the circular muscle of the distal colon. The pattern of suramin-induced contractions was similar to that of OFQ (10−8 mol/L)-induced contractions of the distal colon. Results were reproducible from 5 muscle strips from 5 rats. Gastroenterology 2000 119, 1054-1063DOI: (10.1053/gast.2000.18154) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effects of (A) reactive blue 2 (3 × 10−5 mol/L) and (B) PPADS (3 × 10−5 mol/L) on OFQ (10−8 mol/L)-induced contractions of the circular muscle of the distal colon. Contractions induced by OFQ (10−8 mol/L) were almost completely eliminated by reactive blue 2 but not by PPADS. Effects of (C) reactive blue 2 (3 × 10−5 mol/L) and (D) PPADS (3 × 10−5 mol/L) on the dose-response curve of OFQ (10−9 to 10−7 mol/L)-induced contractions of the distal colon (n = 5). Gastroenterology 2000 119, 1054-1063DOI: (10.1053/gast.2000.18154) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Characterization of electrically induced IJPs recorded from circular smooth muscle cells of the distal colon. (A) Focal electric field stimulation (V) of an interganglionic nerve fiber tract induces a biphasic IJP. The peak hyperpolarization is inhibited by 10−7 mol/L apamin and 3 × 10−5 mol/L reactive blue 2, whereas the slow sustained hyperpolarization is eliminated by 10−4 mol/L NLA. (A) The residual hyperpolarization present after the addition of NLA was abolished by 3 × 10−7 mol/L TTX. The inhibitory effects of (B) apamin and (C) reactive blue 2 on the peak amplitude of the IJP were concentration dependent. Gastroenterology 2000 119, 1054-1063DOI: (10.1053/gast.2000.18154) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 (A–C) Effect of OFQ on electrically induced IJPs recorded from circular smooth muscle cells of the distal colon. (A) OFQ (10−7 mol/L) reversibly inhibits the peak amplitude of IJPs induced by focal electrical stimulation. (B) The inhibitory effect of OFQ on the peak amplitude of the IJP was concentration dependent but was altered by 10−6 mol/L naloxone. (C) In contrast, 10−6 mol/L naloxone significantly shifted the concentration-response curve for IJP inhibition caused by the μ-receptor agonist, DAGO (n = 5; *P < 0.05 by 2-tailed, unpaired t test). Gastroenterology 2000 119, 1054-1063DOI: (10.1053/gast.2000.18154) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Effects of OFQ and suramin on ATP-induced hyperpolarization. In the presence of TTX (3 × 10−7 mol/L), microinjection of ATP resulted in a transient membrane hyperpolarization of the circular smooth muscle. (B) The response to ATP was not affected by OFQ (3 × 10−7 mol/L) but was significantly inhibited by suramin (10−4 mol/L) (n = 3–5; *P < 0.01 by paired t test). Gastroenterology 2000 119, 1054-1063DOI: (10.1053/gast.2000.18154) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 In situ hybridization of mRNA of OFQ receptors in the brain and colon in rats. (A) Dark-field autoradiograms and (B) dark-field and (C and D) bright-field photomicrographs illustrating the distribution of OFQ receptor mRNA in transverse sections of the rat colon. (A) Numerous cells (arrows) at the edge of sections (dotted line) expressed OFQ receptor mRNA. These cells (arrowheads in B–D) were distributed in the myenteric plexus (dotted line, MP), located between the longitudinal (LM) and the circular muscle (CM). C is a magnification of D and allows better visualization of the silver grains that were distinguished in the myenteric plexus (arrowheads in C). No cells in the smooth muscle layers displayed OFQ receptor mRNA. (A, B, and D, bar = 50 μm; C, bar = 20 μm). Gastroenterology 2000 119, 1054-1063DOI: (10.1053/gast.2000.18154) Copyright © 2000 American Gastroenterological Association Terms and Conditions