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

Arginine-vasopressin (AVP) induces the translocation of water channels(aquaporin-2, AQP2) from intracellular vesicles mainly into the apical membranes of renal collecting duct principal cells [shuttle hypothesis (for reviews, see Wade et al.,1981; Klussmann et al.,2000; Nielsen et al.,2002)]. The AQP2 shuttle constitutes the molecular basis of AVP-regulated antidiuresis. It is initiated by binding of AVP to vasopressin V2 receptors (V2 receptors), mainly located on the basolateral surface of principal cells. The agonist-occupied V2 receptor activates adenylyl cyclase via G_s (Birnbaumer et al.,1992). The resulting increase in cAMP leads to activation of protein kinase A (PKA), phosphorylating, among other substrates, AQP2. The phosphorylation by PKA and also the tethering of PKA by protein kinase A anchoring proteins (AKAP) to unknown compartments are prerequisites for the shuttle (Klussmann et al.,1999; Klussmann et al.,2000; Klussmann et al., 2001a; Katsura et al., 1997; Nielsen et al., 2002). Another prerequisite for the AVP-induced translocation of AQP2 appears to be the inactivation of the small GTP-binding protein Rho and the depolymerization of F-actin (Klussmann et al.,2001b; Valenti et al.,2000; Tamma et al.,2001).

Prostaglandins (PGs) act as autocrine and paracrine lipid mediators,controlling many physiological processes(Funk, 2001; Breyer and Breyer, 2000). The effects of prostaglandin E₂ (PGE₂) on the osmotic water permeability of the collecting duct have been investigated by using various animal models. In the absence of AVP, basolaterally or luminally administered PGE₂ increases basal osmotic water permeability in rabbit cortical collecting ducts, most likely by stimulation of cAMP synthesis via G_s/adenylyl cylase (Sakairi et al., 1995). In contrast, in AVP-stimulated rabbit cortical collecting ducts, basolaterally administered PGE₂ inhibits osmotic water permeability. The effect has been ascribed to inhibition of cAMP synthesis via G_i/adenylyl cyclase (Sonnenburg et al., 1988). In addition, PGE₂ induces elevation of cytosolic Ca²⁺ in rabbit collecting ducts by stimulating both a release from intracellular stores and influx from the extracellular medium. Elevation of cytosolic Ca²⁺, via the G protein G_q, has also been suggested to contribute to the diuretic effect of PGE₂(Hebert et al., 1990; Hebert et al., 1993; Hebert, 1994). In the rat terminal inner medullary collecting duct, PGE₂ alone has no effect on basal osmotic water permeability, but attenuates AVP-induced increases in osmotic water permeability. Again, this was suggested to be due to elevation of cytosolic Ca²⁺ (Nadler et al., 1992).

PGE₂ interacts with four different G protein-coupled receptors designated EP₁, EP₂, EP₃ and EP₄(Coleman et al., 1994; Narumiya et al., 1999; Breyer and Breyer, 2001; Namba et al., 1993; Hatae et al., 2002). The EP receptor subtypes expressed by principal cells have not been identified, but the inhibitory effect of PGE₂ on the AVP-induced increase in osmotic water permeability is likely to be mediated by EP₁ and/or EP₃ receptors, which are coupled to the G_q/phospholipase C (PLC) and G_i/adenylyl cyclase system, respectively(Sakairi et al., 1995). This assumption is supported by the finding that the stable PGE₂analogue, sulprostone, a selective EP₁/EP₃ receptor agonist, inhibits the AVP-induced increases in osmotic water permeability in the rabbit cortical collecting duct(Hebert et al., 1993; Hebert, 1994). However, the underlying signal transduction pathways are not understood. EP₃receptors, in addition to coupling to the G_i/adenylyl cyclase system, mediate Rho activation, most likely through the G proteins G_12/13, and subsequently the formation of stress fibers(Negishi et al., 1995; Hasegawa et al., 1997; Aoki et al., 1999). Activated Rho poses a block to the AVP-induced AQP2 shuttle in principal cells(Klussmann et al., 2001b; Tamma et al., 2001). Thus, we hypothesized that PGE₂ exerts its diuretic effect through activation of Rho via EP₃ receptors (see above). To investigate this possibility, the effects of sulprostone, combined with an EP₁ receptor antagonist, SC19220, on the cellular localization of AQP2, on Rho activity and on F-actin were analyzed using primary cultured rat inner medullary collecting duct (IMCD) cells. In addition, levels of cytosolic Ca²⁺ and adenylyl cyclase activity were determined. Our data suggest that activation of the G_12/13/Rho pathway rather than inhibition of adenylyl cyclase or stimulation of PLC (increase in cytosolic Ca²⁺) underlies the EP₃ receptor-mediated diuretic action of PGE₂ observed in the presence of AVP. In addition, they provide strong evidence for a central role of Rho in both diuretic and antidiuretic responses.

AVP was synthesized by Dr M. Beyermann, Forschungsinstitut für Molekulare Pharmakologie, Berlin. Sulprostone and SC19220 were purchased from Cayman Chemical, Alexis Biochemicals, Grünberg, Germany, and carbachol,PGE₂, forskolin, dibuyryl cAMP (Bt₂cAMP) and thapsigargin from Sigma, Deisenhofen, Germany. [³²P]cAMP and myo-[2-³H]inositol were purchased from Amersham Pharmacia Biotech,Freiburg, Germany. Clostridium difficile toxin B was kindly provided by Dr K. Aktories, Albert-Ludwigs-Universität, Freiburg, Germany. The plasmid encoding a fusion protein consisting of glutathion S-transferase (GST)and the Rho-binding domain of the Rho effector Rhotekin was kindly provided by Dr M. A. Schwartz (The Scripps Research Institute, La Jolla, CA, USA).

IMCD cells were obtained from rat renal inner medullae and cultured on cover slips as described previously (Maric et al., 1998). Bt₂cAMP was added to the culture medium for maintenance of AQP2 expression. Bt₂cAMP was removed 16 hours prior to experiments, which were performed 6 days after seeding. AQP2 was detected by confocal laser scanning microscopy (LSM 410; Carl Zeiss, Jena,Germany) using specific antibodies raised against the C terminus of AQP2 and Cy3-conjugated anti-rabbit secondary antibodies(Klussmann et al., 1999; Klussmann et al., 2001b; Maric et al., 1998). F-actin was detected after staining with tetramethyl-rhodamine isothiocyanate(TRITC)-conjugated phalloidin by confocal laser scanning microscopy [LSM 410(Klussmann et al.,2001b)].

For quantification of the effects of AVP, forskolin, Bt₂cAMP,sulprostone and SC19220 on AQP2 localization, the ratio of intracellular/plasma membrane fluorescence intensities was calculated as described previously (Klussmann et al.,1999; Klussmann et al.,2001b). For all groups, mean and standard error values were calculated. Statistical analyses were performed using the Student's t-test and one-way analysis of variance(Klussmann et al., 1999; Klussmann et al., 2001b).

The pull-down of active GTP-bound Rho from IMCD cells was essentially carried out as described previously (Ren and Schwartz, 2000). In brief, IMCD cells were grown in 60 mm dishes and incubated with agonists as indicated. GTP-Rho was precipitated from lysates derived from 7 confluent dishes of IMCD cells using the GST-Rhotekin fusion protein (20-30 μg) coupled to glutathion Sepharose 4B. GTP-Rho was eluted by boiling the precipitate in Laemmli buffer (10 minutes) containing DTT (40 mM). Total RhoA in IMCD cell lysates and precipitated GTP-RhoA were detected by western blot analysis using commercially available anti-RhoA monoclonal antibodies (Santa Cruz Biotechnology, Heidelberg, Germany) and peroxidase-conjugated anti-mouse secondary antibodies. Signals were visualized using the Lumi-light western blot detection system and a Lumi-Imager F1 (Roche Diagnostics, Mannheim, Germany). To quantify the amount of active RhoA, signal densities were determined and related to the signal densities obtained for total RhoA. Ratios obtained for the various experimental conditions were normalized to those ratios obtained for control cells. Statistical analysis was carried out using the Newman-Keuls multiple comparison test.

Preparation of crude membrane fractions and the adenylyl cyclase assay were carried out as described previously(Oksche et al., 1996; Schülein et al., 1996).[³²P]cAMP was isolated using the two column method(Salomon et al., 1974). Statistical analysis was carried out using the unpaired t-test for independent single assay results.

IMCD cells were grown in 24-well plates. Five days after seeding, the culture medium was replaced by Bt₂cAMP-free medium containing 74 kBq/ml myo-[2-³H]inositol (specific activity 37 MBq/ml). For the uptake of myo-[2-³H]inositol, the cells were grown for 20 hours at 37°C. The cells were washed with Bt₂cAMP-free culture medium containing 10 mM LiCl and further incubated in Bt₂cAMP-free medium containing agonists as indicated. Total inositolphosphates were assayed as described previously (Kirk et al.,1990). Changes in the content of total inositolphosphates are largely due to the formation of inositol-1,4,5-trisphosphate(InsP₃) and therefore described as changes in InsP₃ content.

Measurements of cytosolic Ca²⁺ were essentially performed as described previously (Schaefer et al.,2000). In brief, IMCD cells were grown to confluency on glass coverslips. Fura-2-AM (Molecular Probes, Leiden, The Netherlands) loading was carried out by incubation of the cells in Hepes-buffered saline (128 mM NaCl,6 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5.5 mM glucose, 0.5%bovine serum albumine, 10 mM Hepes, pH 7.4, 10 μM fura-2-AM; 30 minutes,37°C). Coverslips were mounted in a custom-made chamber, overlaid with HBS buffer and placed on an inverted epifluorescence microscope (Axiovert 100,Carl Zeiss, Jena, Germany) equipped with a monochromator (Polychrome II,TILL-Photonics, Martinsried, Germany). Agonists were added as indicated. Thapsigargin was applied at the end of each experiment to prevent the internal Ca²⁺ stores limiting the responses to sequentially applied agonists. Fura-2 was alternatively excited at 340, 358, and 380 nm. Emitted light was filtered through a 505-nm long-pass filter and recorded with a 12 bit cooled CCD camera (Imago, TILL-Photonics). Cytosolic Ca²⁺concentrations were calculated as described(Grynkiewicz et al., 1985). In each imaging experiment, data from 30-80 individual cells were collected.

Primary cultured rat IMCD cells were used to determine the effect of EP₃ receptor stimulation on the cellular localization of AQP2. This model system preserves key features of principal cells in situ. In particular,IMCD cells contain the molecular machinery required for the AVP-induced AQP2 translocation. However, for unknown reasons AQP2 predominantly inserts into the basolateral plasma membranes of IMCD cells in response to AVP(Klussmann et al., 1999; Klussmann et al., 2001b; Maric et al., 1998; Maric et al., 2001; Lorenz et al., 2003). Fig. 1 (laser scanning microscopy) and Fig. 2[quantitative and statistical analysis(Klussmann et al., 1999;(Klussmann et al., 2001b)]show a mainly intracellular distribution of AQP2 under control conditions. AVP induced the translocation of AQP2 to the plasma membrane. The EP₁/EP₃ receptor agonist sulprostone, the EP₁receptor antagonist SC19220 or a combination of both did not modify the intracellular localization of AQP2 in non-stimulated IMCD cells. Likewise,incubation of the cells with SC19220 alone had no effect on the AVP-induced translocation of AQP2. However, sulprostone alone or combined with SC19220 reduced or abolished, respectively, the AVP-induced AQP2 translocation. The results indicate that stimulation of EP₃ receptors abolishes AVP-induced AQP2 translocation in IMCD cells. This inhibitory effect appears to be attenuated by EP₁ receptor stimulation.

EP₃ receptor-mediated activation of Rho and subsequent formation of stress fibers has recently been reported(Hasegawa et al., 1997). In IMCD cells, activation of Rho is accompanied by formation of stress fibers and prevents AQP2 translocation in response to elevation of cAMP(Klussmann et al., 2001b). Therefore, the ability of sulprostone to induce the formation of stress fibers in IMCD cells was examined. Stress fibers were detected by staining F-actin with TRITC-conjugated phalloidin and visualization by laser scanning microscopy (Fig. 3). As reported, AVP caused a decrease of stress fibers(Klussmann et al., 2001b). In contrast, sulprostone alone or combined with SC19220 induced the formation of stress fibers under both conditions, i.e. in the presence or absence of AVP. SC19220 alone did not influence the content of stress fibers in non-stimulated cells, nor did it inhibit the AVP-induced depolymerization of stress fibers. The data suggest that stimulation of the V2 and EP₃ receptor by AVP and sulprostone/SC19220, respectively, has opposing effects on the F-actin cytoskeleton in IMCD cells.

We have previously shown that AVP and C. difficile toxin B induce a depolymerization of F-actin in IMCD cells(Klussmann et al., 2001b)(Fig. 3). Here, we determined the effects of AVP, toxin B and of sulprostone on RhoA activity(Fig. 4A,C). Active (GTP-bound)RhoA was quantitatively analyzed by pull-down assays, using the Rho-binding domain of Rhotekin fused to GST (Ren and Schwartz, 2000). AVP and toxin B caused a decrease in the amount of active RhoA compared to control cells(Fig. 4A,C). In contrast,sulprostone, alone or combined with SC19220, increased RhoA activity(Fig. 4B,C). AVP, added to cells preincubated with sulprostone alone, decreased RhoA activity but the level of RhoA activity observed in cells incubated with AVP alone was not reached (Fig. 4C). In cells incubated with sulprostone, SC19220 and AVP, RhoA activity was similar to that of control cells, indicating that selective EP₃ receptor stimulation abolished the AVP-mediated inhibition of RhoA(Fig. 4B,C). The data reveal a bidirectional control of RhoA activity in IMCD cells with the antidiuretic agent AVP inhibiting and the diuretic agent sulprostone (combined with SC19220) stimulating it. In addition, the data suggest that in the presence of AVP the EP₃ receptor-induced stimulation of RhoA is attenuated by EP₁ receptor activation (see above; Figs 1 and 2).

Our results indicate that Rho activation is the cellular mechanism underlying EP₃ receptor-mediated diuresis. However, previous studies suggested that inhibition of the G_i/adenylyl cyclase system contributes to the diuretic effect of PGE₂ (Sonnenburg et al.,1988; Breyer and Breyer, 2001). Therefore, the effect of sulprostone on AVP-stimulated adenylyl cyclase activity was determined in IMCD cell membrane preparations(Fig. 5). Sulprostone did not stimulate adenylyl cyclase activity but reduced the AVP-stimulated adenylyl cyclase activity by about 18%.

To test whether EP₃ receptor-mediated inhibition of adenylyl cyclase is relevant for the inhibition of AVP-induced AQP2 translocation, the effects of sulprostone and SC19220 on the AQP2 shuttle were determined in IMCD cells exposed to either high levels of dibutyryl cyclic adenosine monophosphate (Bt₂cAMP, 500 μM) or forskolin (100 μM), a strong, direct activator of adenylyl cyclase. Fig. 6 shows that even under these conditions, sulprostone alone or combined with SC19220, maintained its ability to inhibit the AQP2 shuttle, indicating that the inhibition of the AQP2 shuttle through EP₃ receptors is independent of cellular cAMP levels. Thus, as in many other systems, the receptor-mediated inhibition of adenylyl cyclase does apparently not contribute to the cellular response. A quantitative analysis of the cellular distribution of AQP2 is shown in Fig. 7 [compare Fig. 2(Klussmann et al., 1999; Klussmann et al., 2001b)].

Elevation of cytosolic Ca²⁺ in response to PGE₂/sulprostone stimulation of rabbit cortical collecting ducts has also been suggested to contribute to the inhibitory effect of PGE₂ on AVP-induced increases in osmotic water permeability(Hebert, 1994). We, therefore,investigated the effects of sulprostone on the formation of InsP₃ and cytosolic Ca²⁺ levels in IMCD cells. Fig. 8A shows that AVP and the muscarinic receptor/G_q-stimulating agonist carbachol induced statistically significant 1.4- and 2.4-fold increases in InsP₃ respectively; in contrast, sulprostone failed to induce InsP₃ formation in IMCD cells. Cytosolic Ca²⁺ was imaged in single, fura-2-loaded IMCD cells (data not shown). In agreement with other reports(Nasrallah et al., 2001; Lorenz et al., 2003), AVP,sulprostone and PGE₂ induced a small rise in cytosolic Ca²⁺ (from about 50 nM to less than 200 nM) in a small number of IMCD cells tested (2.4, 1.4 and 1.4%, respectively; Fig. 8B). Preincubation of cells with SC19220 invariably abolished the elevation of cytosolic Ca²⁺ in response to sulprostone or PGE₂, indicating that the cytosolic Ca²⁺ signals resulted from EP₁ receptor stimulation (see Introduction). The data suggest that EP₃ receptor stimulation does not induce the formation of InsP₃ or an increase in cytosolic Ca²⁺ in IMCD cells. Thus, Ca²⁺ is not involved in EP₃ receptor-mediated stimulation of Rho and inhibition of the AVP-induced AQP2 shuttle.

AVP regulates antidiuresis by inducing the translocation of AQP2 into the apical plasma membranes of collecting duct principal cells. This step is prevented by activation of RhoA (Klussmann et al., 2001b; Tamma et al.,2001). The action of AVP is antagonized by several diuretic agents including endothelin-1, bradykinin and PGE₂. The molecular mechanism is not known. We show that the synthetic PGE₂ analogue sulprostone, through EP₃ receptors, stimulates RhoA in IMCD cells in a cAMP- and Ca²⁺-independent manner and antagonizes both the AVP-induced (V2 receptor-mediated) inhibition of RhoA and the AVP-induced AQP2 translocation. These findings point to a central role of Rho in the diuretic response. The presence of EP₃ receptors in the human collecting duct (Breyer and Breyer, 2000; Breyer et al., 1996a; Breyer et al., 1996b; Morath et al., 1999) suggests that the mechanism we characterized is also operable in the human kidney. A model of the proposed mechanism is depicted in Fig. 9.

The EP₃ receptor-induced activation of RhoA in IMCD cells is,most likely, mediated by the G proteins G_12/13(Namba et al., 1993; Hasegawa et al., 1997; Katoh et al., 1996; Nakamura et al., 1998; Yamaguchi et al., 2000; Hatae et al., 2002). G_12/13 directly activate Rho guanine nucleotide exchange factors(RhoGEFs, e.g. p115) which in turn activate RhoA(Wells et al., 2002). Neither cAMP nor Ca²⁺ are involved in this pathway. In contrast, the inhibition of RhoA through V2 receptors most likely involves cAMP. AVP stimulates cAMP synthesis and subsequent activation of PKA which may phosphorylate Rho (Lang et al.,1996; Forget et al.,2002). We have recently shown that the forskolin-induced AQP2 shuttle in CD8 cells (see Introduction) is accompanied by RhoA phosphorylation, a decrease in RhoA activity, and an increased interaction of RhoA with Rho guanine nucleotide dissociation inhibitor [RhoGDI(Tamma et al., 2003)], the protein that terminats Rho activity(Forget et al., 2002). Accordingly, activation of RhoA via G_12/13 following the stimulation of EP₃ receptors and inhibition of RhoA by cAMP-dependent phosphorylation, following the stimulation of V2 receptors, are most likely the pathways for the bidirectional control of RhoA in IMCD cells.

The mechanism by which activated Rho and its effectors inhibit AVP-induced AQP2 translocation apparently involves the F-actin cytoskeleton. F-actin, in particular subapical F-actin, also referred to as the terminal web, has a barrier function in many exocytic processes. Its disintegration is considered a prerequisite for exocytosis in various cell types including chromaffin cells, mast cells and pancreatic acinar cells(Valentijn et al., 1999). Similarly, the F-actin network (stress fibers) in IMCD cells may function as a physical barrier which hinders AQP2-bearing vesicles reaching the plasma membrane. Several lines of evidence support this view. Activation of RhoA via EP₃ receptors or expression of constitutively active RhoA lead to the formation of stress fibers and inhibited AVP-induced AQP2 translocation in IMCD cells [see above and Klussmann et al.(Klussmann et al., 2001b)]. Effectors of activated Rho that promote the formation of stress fibers are the Rho kinases (Tapon and Hall,1997). Inhibition of Rho kinases with the inhibitor Y-27632 reduces the content of stress fibers in IMCD and CD8 cells and induces translocation of AQP2 independently of cAMP elevation(Klussmann et al., 2001b; Tamma et al., 2001). Similarly, depolymerization of stress fibers induced by cytochalasin D allows AQP2 translocation without elevation of cAMP in IMCD and CD8 cells(Klussmann et al., 2001b; Tamma et al., 2001). In addition to its barrier function, F-actin may directly interact with AQP2(Brown et al., 1998; Umenishi et al., 2000).

The inhibitory effect of PGE₂via EP₃receptors on AVP-induced increases in osmotic water permeability has been ascribed to an inhibition of adenylyl cyclase activity(Hebert, 1994; Breyer and Breyer, 2001). However, pertussis toxin, an inhibitor of G_i, does not prevent PGE₂ antagonizing the effects of AVP in cultured rabbit cortical collecting duct cells or in isolated rat outer medullary collecting ducts(Noland et al., 1992; Aarab et al., 1993; Aarab et al., 1999). In IMCD and CD8 cells, pertussis toxin inhibits the AQP2 shuttle, presumably by inhibition of G_i3 located on AQP2-bearing vesicles(Valenti et al., 1998). Therefore, it cannot be used to test the involvement of adenylyl cyclase in the inhibitory effect of sulprostone on the AQP2 shuttle. However, we show here that EP₃ receptor activation leads to inhibition of the AQP2 shuttle despite high levels of cAMP (Figs 6 and 7), strongly suggesting that inhibition of adenylyl cyclase does not contribute to the diuretic effect.

Endothelin-1 and bradykinin antagonize AVP-induced antidiuresis by stimulation of their cognate receptors (ET_B- and B₂receptors respectively) located on principal cells (for a review, see Klussmann et al., 2000). ET_B receptors couple to both the G_q/PLC system and G_i/adenylyl cyclase; B₂ receptors activate the G_q/PLC system. Therefore, inhibition of cAMP synthesis and elevation of cytosolic Ca²⁺ have been suggested to contribute to the diuretic effects of these agents. However, both receptors also mediate Rho activation (Gohla et al.,1999; Kitamura et al.,1999). In analogy to the EP₃ receptor signaling, we propose that diuretic receptors like ET_B and B₂receptors exert their diuretic effects by activating the G_12/13/Rho pathway.

The identification of Rho and its effectors, the Rho kinases, as central regulators of water reabsorption opens the door to new therapeutic concepts for the treatment of diseases characterized by disturbed water homeostasis,e.g. nephrogenic diabetes insipidus (NDI) or other diuretic states. Congenital NDI is mainly caused by mutations in the V2 receptor(Oksche and Rosenthal, 1998). The inactivation of Rho or Rho kinases may induce the translocation of AQP2 independently of functional V2 receptors and thus reduce the loss of water. Inhibitors of Rho kinases (Y-27632 and hydroxyfasudil) are currently being tested or approved for the treatment of several diseases(Wettschurek and Offermanns,2002). The use of these substances is limited owing to the fact that they cannot be applied in a tissue-specific manner. An alternative approach is the retrograde transfer of genes into the tubular system of the kidney (Moullier et al.,1994). Candidate genes are dominant negative mutants of Rho or Rho kinases under control of an inducible version of the collecting duct-specific AQP2 promotor.

Our data suggest that the EP₁ receptor antagonist SC19220 augments the inhibitory effects of sulprostone on AQP2 translocation and Rho activity, implying that activation of the EP₁ receptor reduces the EP₃ receptor-induced diuresis. EP₁ receptors couple to the G_q/PLC system; their activation results in the formation of InsP₃ with subsequent elevation of cytosolic Ca²⁺ and generation of diacylglycerol (DAG) which in turn activates PKC. Recently, it was shown that PKC causes F-actin disassembly through activation of Src kinase, which in turn stimulates the Rho-specific GTPase-activating protein p190, thereby inactivating Rho(Brandt et al., 2002). In addition, PKC phosphorylates G₁₂, thereby attenuating its activity(Fields et al., 1997). However, PKC activation via the PLC pathway is unlikely to account for the EP₁ receptor effect in IMCD cells, since sulprostone does not induce the formation of InsP₃ to a detectable degree and mediates an increase of cytosolic Ca²⁺ in only a minority of cells. It is possible that stimulation of other phospholipases through EP₁ receptors leads to activation of PKC. For example, activation of phospholipase D results in the formation of phosphatidic acid which is converted to DAG in a subsequent step(Newton, 1995; Newton, 1997).

In summary, our data indicate that the signal transduction pathway underlying the diuretic effect of PGE₂ and possibly that of other diuretic agents includes Rho activation without the involvement of a cAMP- or Ca²⁺-dependent step. In addition, the data suggest that the pharmacological interference with the Rho pathway in principal cells is a strategy suitable for the treating diseases characterized by a disturbed water homeostasis.

This work was supported by grants from Deutsche Forschungsgemeinschaft (Ro 597/6), the European Union (QLK-CT-2001-00987), Deutscher Akademischer Austauschdienst (DAAD; Vigoni program) and Fond der Chemischen Industrie. We thank M. Beyermann for synthesis of AVP, M. A. Schwartz (The Scripps Research Institute, La Jolla, CA, USA) for providing the plasmid encoding the GST-Rhotekin fusion protein and G. Schultz for critical reading of the manuscript. We are grateful to A. Geelhaar, B. Oczko, J. Eichhorst and N. Albrecht for excellent technical assistance.