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First published online 26 June 2003
doi: 10.1242/jcs.00640

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The prostaglandin E₂ analogue sulprostone antagonizes vasopressin-induced antidiuresis through activation of Rho

¹ Forschungsinstitut für Molekulare Pharmakologie, Campus Berlin-Buch, Robert-Rössle-Strasse 10, 13125 Berlin, Germany
² Universita de Bari, Dipartimento di Fisiologia Generale e Ambientale, Via Amendola 165/A, 70126 Bari, Italy
³ Freie Universität Berlin, Institut für Pharmakologie, Thielallee 67-73, 14195 Berlin, Germany

View larger version (81K): [in a new window]   Fig. 1. The effect of EP3 receptor stimulation on the localization of AQP2 in IMCD cells. IMCD cells were left untreated (control) or incubated with AVP (100 nM; 15 minutes), the EP1/EP3 receptor agonist sulprostone (1 µM; 30 minutes), or the EP1 receptor antagonist SC19220 (10 µM; 40 minutes). If cells were incubated with both sulprostone and SC19220, the SC19220 (10 µM) was added 10 minutes prior to sulprostone (1 µM). AVP was present during the final 15 minutes. After completion of incubations, cells were fixed, permeabilized, and incubated with rabbit anti-AQP2 antibody and secondary Cy3-conjugated anti-rabbit antibodies. AQP2 immunofluorescence was detected by laser scanning microscopy. Scale bars: 20 µm.

View larger version (20K): [in a new window]   Fig. 2. Quantitative analysis of the effect of EP3 receptor stimulation on the localization of AQP2 in IMCD cells. IMCD cells were treated as indicated in the legend to Fig. 1. AQP2 immunofluorescence signals were detected by laser scanning microscopy. The intracellular and plasma membrane fluorescence signal intensities were determined and related to nuclear signal intensities. Ratios of intracellular/plasma membrane signal intensities were calculated (n20 cells for each condition tested; mean ± s.e.; three independent experiments). Ratios >1 indicate a mainly intracellular distribution of AQP2, and ratios <1 indicate a predominant localization at the plasma membrane. *, values significantly different from untreated control cells (P<0.001).

View larger version (78K): [in a new window]   Fig. 3. The effect of EP3 receptor stimulation on the F-actin cytoskeleton in IMCD cells. IMCD cells were incubated with the indicated agents as described in the legend to Fig. 1. After completion of the incubations, cells were fixed, permeabilized and incubated with TRITC-conjugated phalloidin (0.1 mg/ml). Phalloidin fluorescence was detected by laser scanning microscopy. Shown are single representative cells from at least three independent experiments for each condition.

View larger version (12K): [in a new window]   Fig. 4. Bidirectional control of RhoA activity through V2 and EP3 receptor stimulation in IMCD cells. IMCD cells were incubated as indicated in the legend to Fig. 1. Subsequently, GTP-bound RhoA was precipitated and detected by western blotting (for details see Materials and Methods). (A) Cells were either left untreated, incubated with AVP (100 nM; 15 minutes) or with C. difficile toxin B, which inhibits all proteins of the Rho family (4 µg/ml; 4 hours). (B) Cells were either left untreated, incubated with AVP or with both sulprostone and SC19220 in the absence or presence of AVP. (C) For quantification, immunoblots were densitometrically analyzed (for details see Materials and Methods). RhoA activity is indicated by the amount of GTP-bound RhoA related to the amount of total RhoA in the whole cell lysates. Values represent amounts of RhoA normalized to untreated control cells. Results are means ± s.e. of at least three independent experiments per condition (n=3-6). *, statistically different from untreated control cells (for untreated control cells versus sulprostone-treated cells P<0.01, for all other comparisons P<0.001); #, statistically different from AVP-stimulated cells (for AVP-stimulated cells versus + AVP + sulprostone P<0.05, for all other comparisons P<0.001).

View larger version (18K): [in a new window]   Fig. 5. Determination of adenylyl cyclase activity in membrane preparations of IMCD cells. Crude membrane fractions of IMCD cells were prepared, and adenylyl cyclase activity was determined (30 µg protein/reaction). Membranes were left untreated, incubated with AVP (100 nM; 15 minutes), sulprostone (1 µM; 30 minutes) or sulprostone (1 µM; 30 minutes) with AVP (100 nM) added for the final 15 minutes. Data represent the mean ± s.e. of three independent experiments and are calculated as the percentage of AVP-stimulated activity. Absolute values for adenylyl cyclase activity (pmol/mg protein/minute) in the three independent experiments were: basal 15.1, 11.3 and 6.82; AVP: 76.7, 35.7 and 32.8; sulprostone: 21.0, 7.7 and 7.9; sulprostone and AVP: 66.5, 28.8 and 25.3. *, values significantly different from AVP-stimulated control (P<0.05).

View larger version (108K): [in a new window]   Fig. 6. The effect of EP3 receptor stimulation on the localization of AQP2 in IMCD cells in the presence of high levels of cAMP. IMCD cells were left untreated (control) or incubated with AVP (100 nM; 15 minutes), Bt2cAMP (500 µM; 15 minutes), forskolin (100 µM; 15 minutes), a strong, direct activator of adenylyl cyclase, and with or without sulprostone (1 µM; 30 minutes). If cells were incubated with both sulprostone and SC19220, SC19220 (10 µM) was added 10 minutes prior to sulprostone. AVP, Bt2cAMP or forskolin were present during the final 15 minutes. After completion of the incubations, cells were fixed, permeabilized, and incubated with rabbit anti-AQP2 antibody and secondary Cy3-conjugated anti-rabbit antibodies. AQP2 immunofluorescence was detected by laser scanning microscopy. Scale bars: 20 µm.

View larger version (22K): [in a new window]   Fig. 7. Quantitative analysis of the effect of EP3 receptor stimulation on the localization of AQP2 in IMCD cells in the presence of high levels of cAMP. IMCD cells were treated as indicated in Fig. 6. AQP2 immunofluorescence signals were detected by laser scanning microscopy and the ratios of intracellular/plasma membrane signal intensities were determined as described in the legend to Fig. 2 (n=20 cells for each condition tested; mean ± s.e.; three independent experiments). *, ** and #, values significantly different from AVP-, Bt2cAMP- and forskolin-stimulated cells respectively (P<0.001).

View larger version (12K): [in a new window]   Fig. 8. Inositol-1,4,5-trisphosphate (InsP3) formation in and Ca2+-responsiveness of IMCD cells. (A) IMCD cells were incubated with myo-[2-3H]inositol for 20 hours at 37°C in culture medium without Bt2cAMP. Thereafter, cells were incubated with AVP (100 nM), sulprostone (1 µM) or carbachol (100 µM; 60 minutes for each agonist). Subsequently, radioactive InsP3 was isolated as described in Materials and Methods. One out of three independent experiments yielding similar results is shown. InsP3 is expressed as a percentage of total [2-3H]inositol incorporated by IMCD cells. *, statistically different from untreated control cells (P<0.05). (B) Cytosolic Ca2+ was determined in IMCD cells (for details see Materials and Methods) and the percentage of cells responding to stimulation with AVP (100 nM), sulprostone (1 µM) or PGE2 (1 µM) by elevation of cytosolic Ca2+ was determined. Control cells were left untreated. If indicated, the EP1 receptor antagonist SC19220 (10 µM) was added 10 minutes prior to the experiment. The cytosolic Ca2+ concentration in untreated cells was about 50 nM; stimulation by AVP, sulprostone or PGE2 induced an elevation to 200 nM in the responding cells.

View larger version (101K): [in a new window]   Fig. 9. A model for the involvement of Rho in prostaglandin E2 (PGE2)-antagonized vasopressin-mediated water reabsorption. Vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells by binding to vasopressin V2 receptors (V2R). The agonist-occupied V2R activates adenylyl cyclase (AC) via the G protein Gs. The resulting increase in cAMP leads to activation of protein kinase A (PKA), phosphorylating AQP2 and Rho. Rho phosphorylation decreases its activity, resulting in the depolymerisation of F-actin and facilitating the insertion of AQP2 predominantly into the apical plasma membrane. Stimulation of EP3 receptors by PGE2, achieved by incubation of IMCD cells with a combination of the PGE2 analogue sulprostone, an EP1/EP3 receptor agonist and the EP1 receptor antagonist SC19220, induces the activation of Rho, most probably via the G proteins G12/13. Rho activation is independent of increases in cAMP and cytosolic Ca2+. It promotes the formation F-actin which hinders AQP2-bearing vesicles reaching the plasma membrane by acting as a physical barrier. EP3 receptor-mediated activation of the G protein Gi, inhibiting adenylyl cyclase, is unlikely to contribute to the diuretic effect of PGE2. In the presence of AVP, the EP3 receptor-induced Rho activation and inhibition of AQP2 translocation are attenuated by EP1 receptor stimulation; the underlying signaling pathway is not known.