Volume 115, Issue 4, Pages 919-928 (October 1998) Leukotriene D4–induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle  Nayoung Kim, Weibiao Cao, In Sung Song, Chung Yong Kim, Uy Dong Sohn, Karen M. Harnett, Piero Biancani  Gastroenterology  Volume 115, Issue 4, Pages 919-928 (October 1998) DOI: 10.1016/S0016-5085(98)70264-1 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 LTD4 causes a dose-dependent contraction of isolated esophageal (○) and LES (●) circular smooth muscle cells. Cells were contracted by exposure to the indicated concentration LTD4 for 30 seconds. Before adding LTD4, the cells were incubated for 7 minutes with 10 mmol/L L-cysteine to prevent the conversion of LTD4 to LTE4, and 10−5 mol/L indomethacin to avoid interference by endogenous prostanoids. Values are mean ± SEM of 5 animals, with 30 cells counted for each animal. Gastroenterology 1998 115, 919-928DOI: (10.1016/S0016-5085(98)70264-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 PTX inhibits the LTD4-induced contraction of (A) esophageal (P < 0.001, ANOVA) but not (B) LES circular smooth muscle cells. Intact muscle cells were incubated in the indicated concentration of LTD4 alone (○, control) or after a 60-minute pretreatment with 0.2 μg/mL PTX (●). Values are mean ± SEM of 3 animals, with 30 cells counted for each animal. Gastroenterology 1998 115, 919-928DOI: (10.1016/S0016-5085(98)70264-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 LTD4-induced contraction is mediated by Gq G proteins in LES cells and by Gi3 in esophageal cells. Muscle cells were permeabilized by brief exposure to saponin to allow diffusion of antibodies into the cytosolic side of the cell membrane. Cells were contracted with 10−9 mol/L LTD4 after a 60-minute preincubation in a cytosolic medium containing G-protein antibodies (1:200 dilution). LTD4-induced contraction of LES cells was significantly inhibited by Gq (Student t test, **P < 0.01) and not by Go, Gi1-Gi2, or Gi3 antibodies, whereas contraction of esophageal cells was inhibited by antibodies raised against Gi3 (Student t test, **P < 0.01) and not by Gq, Gi1-Gi2, or Go. Values are mean ± SEM of 3 animals, with 30 cells counted for each animal. Gastroenterology 1998 115, 919-928DOI: (10.1016/S0016-5085(98)70264-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 [35S]GTPγS binding to LTD4-activated membranes of the esophagus and the LES. Purified membranes were exposed to 10−6 mol/L LTD4 in the presence of [35S]GTPγS. LTD4-induced activation of specific G proteins was reflected by the amount of [35S]GTPγS bound to wells that were precoated with Gi3, Gq, or Gi1-Gi2 antibodies. G-protein activation was measured as a percentage increase in [35S]GTPγS binding in membranes exposed to LTD4 compared with unstimulated membranes. Exposure to 10−6 mol/L LTD4 caused significant activation of Gi3 (*P < 0.05, ANOVA) in esophageal muscle membranes. In the LES, LTD4 significantly activated Gq (#P < 0.001, ANOVA) and Gi3 (*P < 0.05, ANOVA). Values are mean ± SEM of 3 animals, with each measurement performed in triplicate. Gastroenterology 1998 115, 919-928DOI: (10.1016/S0016-5085(98)70264-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Different phospholipases mediate LTD4-induced contraction of LES and esophageal cells. Intact cells were contracted with 10−9 mol/L LTD4 alone (control) or after a 10-minute preincubation in a medium containing 10−6 mol/L of PI-PLC inhibitor U-73122, 10−4 mol/L of the PC-PLC inhibitor D609, or 10−4 mol/L of the PLD-dependent pathway inhibitor propranolol. In the esophagus, LTD4-induced contraction was significantly reduced by D609 and propranolol (#P < 0.001, ANOVA) and not affected by U-73122. LTD4-induced contraction of esophageal cells was abolished by a combination of D609 and propranolol. In contrast, in the LES, LTD4-induced contraction was significantly reduced by U-73122 (#P < 0.001, ANOVA) and D609 (**P < 0.01, ANOVA) and was not affected by propranolol. Values are mean ± SEM of 6 animals, with 30 cells counted for each animal. Gastroenterology 1998 115, 919-928DOI: (10.1016/S0016-5085(98)70264-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Heparin significantly reduced LTD4-induced contraction of LES (*P < 0.05, Student t test) but not of esophageal cells. Muscle cells were permeabilized by brief exposure to saponin to allow diffusion of heparin into the cytosol of the cell. Cells were contracted with 10−9 mol/L LTD4 alone or after a 1-minute preincubation in a cytosolic medium containing 0.1 mg/mL heparin. Values are mean ± SEM of 4 animals, with 30 cells counted for each animal. Gastroenterology 1998 115, 919-928DOI: (10.1016/S0016-5085(98)70264-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 LTD4-induced contraction is PKC dependent in the esophagus and calmodulin-dependent in the LES. Intact cells were contracted with 10−9 mol/L LTD4 alone (control) or after a 10-minute preincubation in a medium containing the PKC antagonist chelerythrine (10−5 mol/L) or the calmodulin antagonist W7 (10−5 mol/L). LTD4-induced contraction of esophageal cells was significantly inhibited by chelerythrine (#P < 0.001, ANOVA) and was not affected by W7. LES contraction was significantly inhibited by W7 (#P < 0.001, ANOVA). Values are mean ± SEM of 5 animals, with 30 cells counted for each animal. Gastroenterology 1998 115, 919-928DOI: (10.1016/S0016-5085(98)70264-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of W7 and chelerythrine on the contractile response of LES cells to low and high concentrations of LTD4. LES cells were incubated with the indicated concentrations of LTD4 in the absence (control) or presence of 10−5 mol/L chelerythrine or 10−5 mol/L W7. (A) The threshold response to 10−12 mol/L LTD4 was significantly reduced by the PKC antagonist chelerythrine (**P < 0.01, ANOVA), whereas the response to a maximally effective dose of LTD4 (10−9 mol/L) was significantly reduced by the calmodulin antagonist W7 (**P < 0.01, ANOVA). (B) Percent reduction of control contraction (in the absence of antagonists) induced by chelerythrine or W7. The effect of chelerythrine was largest at low LTD4 doses. W7 was more effective at high LTD4 doses. Values are means ± SEM of 5 and 4 animals, respectively, with 30 cells counted for each animal. Gastroenterology 1998 115, 919-928DOI: (10.1016/S0016-5085(98)70264-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions