Group I secreted PLA2 in the maintenance of human lower esophageal sphincter tone  Weibiao Cao, Karen M. Harnett, Jose Behar, Piero Biancani  Gastroenterology  Volume 119, Issue 5, Pages 1243-1252 (November 2000) DOI: 10.1053/gast.2000.19581 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Identification of group I sPLA2 by Western blot. Solubilized LES circular smooth muscle samples were loaded into an SDS–polyacrylamide gel electrophoresis system, transferred to nitrocellulose membrane, and incubated with mouse anti-human sPLA2-I antibody at a 1:500 concentration for 1 hour with shaking. Two samples are shown. Human pancreatic sPLA2-I was detected as a thick band in LES circular smooth muscle (14.3 kilodaltons). Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of exogenous group I sPLA2 on LES basal tone. LES circular muscle strips were incubated in the indicated concentration of group I sPLA2 purified from Naja naja venom. Group I sPLA2 caused a concentration-dependent increase of in vitro LES tone (P < 0.01, ANOVA). Values are means ± SE of 4 samples. Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of PLA2 inhibitors on LES basal tone. LES circular muscle strips were incubated in the indicated concentration of phospholipase A2 inhibitors. MJ33, the inhibitor selective for group I sPLA2, caused a concentration-dependent reduction of in vitro LES tone (P < 0.001, ANOVA), whereas the cytosolic PLA2 inhibitor AACOCF3 and the group II sPLA2 inhibitor MJ45 had no effect. Values are means ± SE of 3–4 samples. Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Effect of cyclooxygenase or lipoxygenase inhibitors on LES basal tone. LES circular muscle strips were incubated in the indicated concentration of NDGA, indomethacin (Indom), or aspirin. Cyclooxygenase inhibitors indomethacin and aspirin caused a concentration-dependent reduction in LES basal tone (P < 0.001, ANOVA). The lipoxygenase inhibitor NDGA had no effect. These data suggest that prostaglandins and thromboxanes, but not leukotrienes, play a role in LES basal tone. Values are mean ± SE of 3–4 samples. Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of the lipoxygenase inhibitor NDGA or indomethacin on the contraction of isolated smooth muscle cells from the LES. Cells were contracted with a maximal dose (0.1 U/mL) of group I sPLA2 purified from Naja naja venom in the presence of NDGA or indomethacin. sPLA2-induced contraction of isolated LES cells was significantly reduced by indomethacin (P < 0.001, ANOVA) and unaffected by NDGA. These data suggest that prostaglandins and thromboxanes mediate sPLA2-induced contraction of LES smooth muscle cells. Values are mean ± SE of 3 samples. Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Effect of PTX on the contraction of isolated smooth muscle cells from the LES. Cells were contracted with group I sPLA2 purified from Naja naja venom in the absence (control) or presence of PTX (0.2 μg/mL). LES cells contracted in a concentration-dependent manner in response to sPLA2-I. PTX reduced the contractile response at each concentration tested (P < 0.001, ANOVA), suggesting that sPLA2-induced contraction is mediated by PTX-sensitive G proteins. Values are mean ± SE of 3 samples. Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 sPLA2-I–induced contraction of permeabilized smooth muscle cells from the LES is inhibited by Gi3 antibodies. LES 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 sPLA2-I (0.1 U/mL) alone (control) or after 60 minutes'; preincubation in cytosolic medium containing G-protein antibodies (1:200 dilution). sPLA2-I–induced contraction of LES cells was significantly inhibited by Gi3 (**P < 0.001, ANOVA) and not by Gq, Gi1/i2, or Go antibodies. Values are the means ± SE of 3 human samples with each sample performed in triplicate. Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 PKC and PC-PLC inhibitors reduce human LES tone. LES circular muscle strips were incubated in phosphatidylinositol-specific phospholipase C inhibitor U-73122 (10−6 mol/L), the phosphatidylcholine-specific phospholipase C inhibitor D609 (10−4 mol/L), or the PKC inhibitor chelerythrine (10−4 mol/L). LES tone was significantly reduced by D609 and chelerythrine (**P < 0.001, ANOVA), suggesting that human LES tone is mediated by activation of PC-PLC and a PKC-dependent pathway. Values are mean ± SE of 5–6 samples. Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 PKC and PC-PLC inhibitors reduce sPLA2-I-induced contraction of isolated smooth muscle cells from the LES. Cells were contracted with a maximally effective dose of sPLA2-I (0.1 U/mL) in the absence (control) or presence of the phosphatidylinositol-specific phospholipase C inhibitor U-73122 (10−6 mol/L), the phosphatidylcholine-specific phospholipase C inhibitor D609 (10−4 mol/L), an inhibitor of a phospholipase D–dependent pathway propranolol (10−4 mol/L), the PKC inhibitor chelerythrine (10−5 mol/L), or the calmodulin inhibitor CGS9343B (10−5 mol/L). sPLA2-I–induced contraction was significantly reduced by D609 and chelerythrine (**P < 0.001, ANOVA). Values are the means ± SE of 3 samples with 30 cells counted at random for each data point. Gastroenterology 2000 119, 1243-1252DOI: (10.1053/gast.2000.19581) Copyright © 2000 American Gastroenterological Association Terms and Conditions