Prostaglandin E2 stimulates rat and human colonic mucin exocytosis via the EP4 receptor  Adam Belley, Kris Chadee  Gastroenterology  Volume 117, Issue 6, Pages 1352-1362 (December 1999) DOI: 10.1016/S0016-5085(99)70285-4 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 RT-PCR analysis of prostaglandin EP-receptor mRNAs expressed by LS174T cells. Total RNA was extracted from LS174T cells, and RT-PCR was performed using EP-specific primers as described in Materials and Methods. Panels show the PCR analysis for EP2, EP3, and EP4, respectively. Lane 1 in each panel shows the molecular size marker where the 1018-bp and 506/517-bp fragments are indicated on the left. Lanes 2 and 3 in each panel show RT-negative controls and the PCR product of expected size from LS174T mRNA, respectively. Lane 4 in each panel shows the PCR products from positive control cDNAs: EP2, 419 bp; EP3, 397 bp; and EP4, 434 bp. EP1-receptor mRNA was not detected in LS17T cells. PCR product identities were confirmed by DNA sequencing. Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 S4B chromatography of secreted mucins by LS174T cells. (A) Mucins were metabolically labeled with [3H]glucosamine and isolated by S4B chromatography from cells stimulated for 12 hours with media + vehicle (●), 10 μmol/L PMA (□), 50 μmol/L forskolin (▵), or 100 nmol/L PGE2 (▾). The counts per minute (cpm) in each fraction were monitored by liquid scintillation counting. The Vo (fractions 6–10) of the column is indicated by blue dextran (BD; Mr > 2 × 106), and the included fractions are indicated by thyroglobulin (TG; 669,000) and bovine serum albumin (BSA; 67,000). (B) Fluorograph of the pooled [3H]S4B Vo mucins (30,000 cpm) isolated from above and resolved on 7% SDS-PAGE. Note that the radiolabeled mucins were retained in the stacking gel. The arrowhead indicates the border between the stacking and separating gel. (C) Immunoblot of secreted LS174T S4B Vo mucins. CsCl-purified mucins (1 μg) from LS174T cells were used as a control (lane 1). The secreted high-molecular-weight glycoproteins (50 μg/lane) from unstimulated (lane 2), PMA- (lane 3), forskolin- (lane 4), or PGE2- (lane 5) stimulated cells collected in the S4B Vo fractions were retained in the stacking gel and were identified as mucins by immunoblotting using an anti–CsCl-purified LS174T mucin rabbit immune serum.20 The arrowhead indicates the border between the stacking and separating gel. (D) Partitioning of secreted radiolabeled S4B Vo mucins from unstimulated (●) and PGE2-stimulated (▾) LS174T cells by cesium chloride density gradient centrifugation. Greater than 80% of the recovered radiolabeled mucins were in the high-density fractions (fractions 6–8; density > 1.44 g/mL). Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 S4B chromatography of secreted mucins by LS174T cells. (A) Mucins were metabolically labeled with [3H]glucosamine and isolated by S4B chromatography from cells stimulated for 12 hours with media + vehicle (●), 10 μmol/L PMA (□), 50 μmol/L forskolin (▵), or 100 nmol/L PGE2 (▾). The counts per minute (cpm) in each fraction were monitored by liquid scintillation counting. The Vo (fractions 6–10) of the column is indicated by blue dextran (BD; Mr > 2 × 106), and the included fractions are indicated by thyroglobulin (TG; 669,000) and bovine serum albumin (BSA; 67,000). (B) Fluorograph of the pooled [3H]S4B Vo mucins (30,000 cpm) isolated from above and resolved on 7% SDS-PAGE. Note that the radiolabeled mucins were retained in the stacking gel. The arrowhead indicates the border between the stacking and separating gel. (C) Immunoblot of secreted LS174T S4B Vo mucins. CsCl-purified mucins (1 μg) from LS174T cells were used as a control (lane 1). The secreted high-molecular-weight glycoproteins (50 μg/lane) from unstimulated (lane 2), PMA- (lane 3), forskolin- (lane 4), or PGE2- (lane 5) stimulated cells collected in the S4B Vo fractions were retained in the stacking gel and were identified as mucins by immunoblotting using an anti–CsCl-purified LS174T mucin rabbit immune serum.20 The arrowhead indicates the border between the stacking and separating gel. (D) Partitioning of secreted radiolabeled S4B Vo mucins from unstimulated (●) and PGE2-stimulated (▾) LS174T cells by cesium chloride density gradient centrifugation. Greater than 80% of the recovered radiolabeled mucins were in the high-density fractions (fractions 6–8; density > 1.44 g/mL). Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 S4B chromatography of secreted mucins by LS174T cells. (A) Mucins were metabolically labeled with [3H]glucosamine and isolated by S4B chromatography from cells stimulated for 12 hours with media + vehicle (●), 10 μmol/L PMA (□), 50 μmol/L forskolin (▵), or 100 nmol/L PGE2 (▾). The counts per minute (cpm) in each fraction were monitored by liquid scintillation counting. The Vo (fractions 6–10) of the column is indicated by blue dextran (BD; Mr > 2 × 106), and the included fractions are indicated by thyroglobulin (TG; 669,000) and bovine serum albumin (BSA; 67,000). (B) Fluorograph of the pooled [3H]S4B Vo mucins (30,000 cpm) isolated from above and resolved on 7% SDS-PAGE. Note that the radiolabeled mucins were retained in the stacking gel. The arrowhead indicates the border between the stacking and separating gel. (C) Immunoblot of secreted LS174T S4B Vo mucins. CsCl-purified mucins (1 μg) from LS174T cells were used as a control (lane 1). The secreted high-molecular-weight glycoproteins (50 μg/lane) from unstimulated (lane 2), PMA- (lane 3), forskolin- (lane 4), or PGE2- (lane 5) stimulated cells collected in the S4B Vo fractions were retained in the stacking gel and were identified as mucins by immunoblotting using an anti–CsCl-purified LS174T mucin rabbit immune serum.20 The arrowhead indicates the border between the stacking and separating gel. (D) Partitioning of secreted radiolabeled S4B Vo mucins from unstimulated (●) and PGE2-stimulated (▾) LS174T cells by cesium chloride density gradient centrifugation. Greater than 80% of the recovered radiolabeled mucins were in the high-density fractions (fractions 6–8; density > 1.44 g/mL). Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 S4B chromatography of secreted mucins by LS174T cells. (A) Mucins were metabolically labeled with [3H]glucosamine and isolated by S4B chromatography from cells stimulated for 12 hours with media + vehicle (●), 10 μmol/L PMA (□), 50 μmol/L forskolin (▵), or 100 nmol/L PGE2 (▾). The counts per minute (cpm) in each fraction were monitored by liquid scintillation counting. The Vo (fractions 6–10) of the column is indicated by blue dextran (BD; Mr > 2 × 106), and the included fractions are indicated by thyroglobulin (TG; 669,000) and bovine serum albumin (BSA; 67,000). (B) Fluorograph of the pooled [3H]S4B Vo mucins (30,000 cpm) isolated from above and resolved on 7% SDS-PAGE. Note that the radiolabeled mucins were retained in the stacking gel. The arrowhead indicates the border between the stacking and separating gel. (C) Immunoblot of secreted LS174T S4B Vo mucins. CsCl-purified mucins (1 μg) from LS174T cells were used as a control (lane 1). The secreted high-molecular-weight glycoproteins (50 μg/lane) from unstimulated (lane 2), PMA- (lane 3), forskolin- (lane 4), or PGE2- (lane 5) stimulated cells collected in the S4B Vo fractions were retained in the stacking gel and were identified as mucins by immunoblotting using an anti–CsCl-purified LS174T mucin rabbit immune serum.20 The arrowhead indicates the border between the stacking and separating gel. (D) Partitioning of secreted radiolabeled S4B Vo mucins from unstimulated (●) and PGE2-stimulated (▾) LS174T cells by cesium chloride density gradient centrifugation. Greater than 80% of the recovered radiolabeled mucins were in the high-density fractions (fractions 6–8; density > 1.44 g/mL). Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Secretion of 3H-labeled mucins in response to PGs and EP-receptor agonists. LS174T cells (80% confluence) were labeled for 48 hours with [3H]glucosamine and stimulated for 12 hours at 37°C with PGs or EP-receptor agonists. Secreted glycoproteins were analyzed by S4B chromatography, and each point depicts the percent increase in S4B Vo cpm (mucins) above control. (A) PGs: PGE2 (■), PGF2α (○), iloprost (□), and 13,14-dihydro-15-keto-PGE2 (▴). (B) EP-receptor agonists: butaprost (EP2; ▴), sulprostone (EP3/EP1; □); M&B28767 (EP3/EP4; ○), and 1-OH-PGE1 (EP4; ■). Data represent the means ± SEM of 3–5 experiments. Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Secretion of 3H-labeled mucins in response to PGs and EP-receptor agonists. LS174T cells (80% confluence) were labeled for 48 hours with [3H]glucosamine and stimulated for 12 hours at 37°C with PGs or EP-receptor agonists. Secreted glycoproteins were analyzed by S4B chromatography, and each point depicts the percent increase in S4B Vo cpm (mucins) above control. (A) PGs: PGE2 (■), PGF2α (○), iloprost (□), and 13,14-dihydro-15-keto-PGE2 (▴). (B) EP-receptor agonists: butaprost (EP2; ▴), sulprostone (EP3/EP1; □); M&B28767 (EP3/EP4; ○), and 1-OH-PGE1 (EP4; ■). Data represent the means ± SEM of 3–5 experiments. Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Time course of [3H]-glucosamine–labeled mucin secretion by LS174T cells stimulated with secretagogues. Cells were stimulated for 0.5, 1, 3, 6, and 12 hours with 10 μmol/L PMA (■), 50 μmol/L forskolin (●), or 250 nmol/L PGE2 (▴), and the secreted mucins that accumulated in the supernatants were quantified by S4B chromatography as described in Materials and Methods. Each point depicts the percent of maximal secretion above control. Data represent the means ± SEM of 3 experiments. Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Secretion of 3H-labeled mucins and nonmucin glycoproteins in rat colon. Rats were injected intraperitoneally with [3H]glucosamine for 4 hours to metabolically label mucins. Colonic loops were made and injected with ethanol vehicle or 5 μmol/L of either PGE2, 1-OH-PGE1, 13,14-dihydro-15-keto-PGE2, sulprostone, or 10 μmol/L butaprost for 2 hours. Secreted mucins were isolated and quantified by S4B chromatography and liquid scintillation counting. (A) PGE2 and the EP4 agonist 1-OH-PGE1 caused a significant (P < 0.05 compared with vehicle control by the Student t test) increase in mucin secretion (measured as S4B Vo mucins), whereas the other agonists had no effect (n = 3 or 4 loops per condition except butaprost, in which n = 2). (B) S4B chromatography profiles of 3H-labeled glycoproteins secreted in response to vehicle (•), 5 μmol/L PGE2 (▾), or 5 μmol/L 1-OH-PGE1 (▵) in rat colon. The Vo (fractions 6–8) of the column is indicated by blue dextran (BD; Mr > 2 × 106), and the included fractions are indicated by thyroglobulin (TG; 669,000) and bovine serum albumin (BSA; 67,000). Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Secretion of 3H-labeled mucins and nonmucin glycoproteins in rat colon. Rats were injected intraperitoneally with [3H]glucosamine for 4 hours to metabolically label mucins. Colonic loops were made and injected with ethanol vehicle or 5 μmol/L of either PGE2, 1-OH-PGE1, 13,14-dihydro-15-keto-PGE2, sulprostone, or 10 μmol/L butaprost for 2 hours. Secreted mucins were isolated and quantified by S4B chromatography and liquid scintillation counting. (A) PGE2 and the EP4 agonist 1-OH-PGE1 caused a significant (P < 0.05 compared with vehicle control by the Student t test) increase in mucin secretion (measured as S4B Vo mucins), whereas the other agonists had no effect (n = 3 or 4 loops per condition except butaprost, in which n = 2). (B) S4B chromatography profiles of 3H-labeled glycoproteins secreted in response to vehicle (•), 5 μmol/L PGE2 (▾), or 5 μmol/L 1-OH-PGE1 (▵) in rat colon. The Vo (fractions 6–8) of the column is indicated by blue dextran (BD; Mr > 2 × 106), and the included fractions are indicated by thyroglobulin (TG; 669,000) and bovine serum albumin (BSA; 67,000). Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Competition of [3H]PGE2-specific binding to LS174T cell membranes by PGs and EP-receptor agonists. Membranes (50 μg) from LS174T cells were incubated with 3 nmol/L [3H]PGE2 and various concentrations of PGs and EP-receptor agonists for 1 hour at room temperature. The suspensions were applied to filters by rapid vacuum filtration, and binding was quantified by liquid scintillation counting. Specific binding was determined in parallel with 10 μmol/L PGE2. (A) PGs: PGE2 (■), PGF2α (○), iloprost (□), and 13,14-dihydro-15-keto-PGE2 (▴). (B) EP-receptor agonists: butaprost (EP2; ▴), sulprostone (EP3/EP1; □), M&B28767 (EP3/EP4; ○), and 1-OH-PGE1 (EP4; ■). The data represent the mean of duplicate determinations from an experiment that was repeated 3 times with similar results. Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 Competition of [3H]PGE2-specific binding to LS174T cell membranes by PGs and EP-receptor agonists. Membranes (50 μg) from LS174T cells were incubated with 3 nmol/L [3H]PGE2 and various concentrations of PGs and EP-receptor agonists for 1 hour at room temperature. The suspensions were applied to filters by rapid vacuum filtration, and binding was quantified by liquid scintillation counting. Specific binding was determined in parallel with 10 μmol/L PGE2. (A) PGs: PGE2 (■), PGF2α (○), iloprost (□), and 13,14-dihydro-15-keto-PGE2 (▴). (B) EP-receptor agonists: butaprost (EP2; ▴), sulprostone (EP3/EP1; □), M&B28767 (EP3/EP4; ○), and 1-OH-PGE1 (EP4; ■). The data represent the mean of duplicate determinations from an experiment that was repeated 3 times with similar results. Gastroenterology 1999 117, 1352-1362DOI: (10.1016/S0016-5085(99)70285-4) Copyright © 1999 American Gastroenterological Association Terms and Conditions