Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors Sorin Tunarua, Till F. Althoffa, Rolf M. Nüsingb, Martin Dienerc, and Stefan Offermannsa,d,1 aDepartment of Pharmacology, Max-Planck-Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany; dMedical Faculty, bInstitute for Clinical Pharmacology, J. W. Goethe University Frankfurt, 60590 Frankfurt, Germany; and cInstitute for Veterinary Physiology, Justus Liebig University, 35392 Giessen, Germany Edited by John H. Exton, Vanderbilt University School of Medicine, Nashville, TN, and approved April 25, 2012 (received for review January 30, 2012) Castor oil is one of the oldest drugs. When given orally, it has by the enteric nervous system or are direct effects on intestinal a laxative effect and induces labor in pregnant females. The smooth muscle remained unclear. effects of castor oil are mediated by ricinoleic acid, a hydroxylated The present study was undertaken to elucidate the molecular fatty acid released from castor oil by intestinal lipases. Despite the mechanism underlying the biological effect of castor oil-derived wide-spread use of castor oil in conventional and folk medicine, ricinoleic acid. Based on cellular signaling studies and an siRNA the molecular mechanism by which ricinoleic acid acts remains screening approach, we identified prostaglandin E2 receptors as unknown. Here we show that the EP3 prostanoid receptor is spe- targets of ricinoleic acid and show that the EP3 receptor medi- cifically activated by ricinoleic acid and that it mediates the phar- ates the effects of castor oil on the motility of the uterus and macological effects of castor oil. In mice lacking EP3 receptors, the the intestine. laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP3 recep- Results tor gene in intestinal epithelial cells did not affect castor oil-induced In a screen for potential receptor-mediated effects using a library 2+ diarrhea, mice lacking EP3 receptors only in smooth-muscle cells of biologically active lipids, we observed a Ca transient after were unresponsive to this drug. Thus, the castor oil metabolite exposure of various cell types to ricinoleic acid. The response ricinoleic acid activates intestinal and uterine smooth-muscle cells was strongest in the human megakaryocyte leukemia cell line fi via EP3 prostanoid receptors. These ndings identify the cellular MEG-01 (Fig. 1A). This effect was dose-dependent with an EC50 and molecular mechanism underlying the pharmacological effects of 5 μM (Fig. 1B) and could be blocked by pretreatment of cells of castor oil and indicate a role of the EP3 receptor as a target to with pertussis toxin (Fig. 1A). The biologically inactive trans induce laxative effects. isomer of ricinoleic acid, ricinelaidic acid [(9E,12R)-12-hydrox- yoctadec-9-enoic acid], as well as the nonhydroxylated homolog, G-protein coupled receptor | peristalsis | Ricinus communis | PGE2 oleic acid [(9Z)-octadec-9-enoic acid], were without effect (Fig. 1B). These data suggest that ricinoleic acid can specifically ac- astor oil, also known as Oleum Palmae Christi, is obtained tivate a G protein-coupled receptor (GPCR). To identify a pu- Cfrom the seeds of Ricinus communis and has been used tative GPCR activated by ricinoleic acid, we screened a small PHARMACOLOGY therapeutically for centuries (1, 2), being first described in the interfering RNA (siRNA) library targeting all known and pre- Ebers papyrus of ancient Egypt more than 3,500 y ago (3). Castor dicted nonolfactory human GPCRs for its ability to interfere with oil is a triglyceride characterized by a high content of the hy- activation of MEG-01 cells by ricinoleic acid. Fig. 1C shows that siRNA pools directed against mRNAs encoding EP3 and EP4 droxylated unsaturated fatty acid ricinoleic acid [(9Z,12R)-12- – hydroxyoctadec-9-enoic acid] (4). After oral ingestion of castor (20 22) strongly reduced ricinoleic acid effects in MEG-01 cells. We verified that EP and EP receptors are expressed in MEG- oil, ricinoleic acid is released by lipases in the intestinal lumen, 3 4 01 cells and that prostaglandin E (PGE ) has effects compara- and considerable amounts of ricinoleic acid are absorbed in the 2 2 ble to ricinoleic acid in these cells (Fig. S1). Consistent with intestine (5, 6). The released ricinoleic acid induces a strong a role of EP and EP receptors in mediating cellular effects of laxative effect (5, 7). There is also a well-documented labor-in- 3 4 ricinoleic acid, the selective antagonists of EP3 and EP4 recep- ducing effect of castor oil in pregnant females at term; however, tors, L-798,106 and L-161,982, respectively, at maximally active use of this drug for labor induction is not recommended because concentrations inhibited ricinoleic acid- and PGE2-induced cal- of unwanted effects, such as nausea (8). cium mobilization in MEG-01 cells (Fig. 1D). EP3/EP4-mediated The mechanisms underlying the pharmacological effects of effects of ricinoleic acid were not because of formation of PGE2 ricinoleic acid remain elusive. Castor oil is regarded as a stimu- in response to ricinoleic acid (Fig. S2 A and B). Consistent with lant and irritant laxative without known mechanism of action (9). this finding, ricinoleic acid effects were not affected by inhibition Several studies have shown that relatively high concentrations of of cyclooxygenase (COX)-1 and COX-2 (Fig. S2C). ricinoleic acid can cause ultrastructural alterations in the villous To further characterize the effects of ricinoleic acid on pros- tips of the intestinal mucosa (10, 11). Given the high concen- tanoid receptors, we heterologously expressed prostanoid recep- trations of ricinoleic acid used in these experiments, it is, how- tors together with the promiscuous G protein α-subunit Gα15 ever, not clear whether these unspecific morphological effects are relevant for the laxative effect of castor oil. In part, con- flicting data have been published with regard to the ability of Author contributions: S.T. and S.O. designed research; S.T., T.F.A., and M.D. performed research; R.M.N. contributed new reagents/analytic tools; S.T., T.F.A., M.D., and S.O. an- ricinoleic acid to induce procontractile effects on intestinal alyzed data; and S.O. wrote the paper. smooth muscle and to alter intestinal ion transport and water The authors declare no conflict of interest. flux. Although some groups observed an inhibition of water and This article is a PNAS Direct Submission. electrolyte absorption (12–14), others found an activation of ion Freely available online through the PNAS open access option. secretory processes by ricinoleid acid (15). In addition to effects 1 fl To whom correspondence should be addressed: E-mail: Stefan.Offermanns@mpi-bn. of ricinoleic acid on intestinal ion transport and water ux, evi- mpg.de. dence has been provided that ricinoleic acid can directly affect This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. intestinal motility (16–19). Whether these effects are mediated 1073/pnas.1201627109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1201627109 PNAS | June 5, 2012 | vol. 109 | no. 23 | 9179–9184 Downloaded by guest on September 28, 2021 30 2.5 isomer ricinelaidic acid was inactive (Fig. S3A). Whereas rici- A B ricinoleic acid 2.0 noleic acid was about one order-of-magnitude less potent than 20 PGE , the efficacy of ricinoleic acid to activate EP and EP 1.5 ricinelaidic acid 2 3 4 receptors was comparable with that of PGE2 (Fig. 1E). Ricino- RFU PTX AUC RA 1.0 10 oleic acid leic acid also activated murine EP3 and EP4 receptors (Fig. S3B). 0.5 None of the other prostanoid receptors, including IP, DP ,DP, +PTX 1 2 0 0 FP, and TP were activated by ricinoleic acid (Fig. 1F). In contrast 0 50 100 0 -6-7 -5 -4 3 time (seconds) agonist (log [M]) to oleic acid, ricinoleic acid was able to displace H-PGE2 from EP3 receptors expressed in CHO cells with an IC50 of 500 nM, C 2.0 but ricinelaidic acid hardly competed with PGE2 for binding 1.5 (Fig. 1G). Taken together, these data show that ricinoleic acid is 1.0 ratio a selective agonist of EP3 and EP4 receptors. 0.5 We then analyzed mice lacking EP or EP receptors (25–27) EP4 3 4 EP3 0 to test whether these receptors play a role in ricinoleic acid-in- 0 250 500 siRNA pools duced pharmacological effects in vivo. Mice lacking either EP3 −/− −/− −/− −/− (Ptger3 ;EP3 )orEP4 (Ptger4 ;EP4 ) showed normal 3 0.5 intestinal transit time (Fig. 2A). When given castor oil, wild-type D E EP1+PGE2 mock+PGE2 0.4 EP1+RA mock+RA mice responded with a strong diarrhea, starting about 30 min EP +PGE 2 2 2 0.3 EP2+RA after application. The laxative effect lasted for about 2 h. In- ** EP3+PGE2 fi AUC terestingly, EP3 receptor-de cient mice were completely un- AUC * 0.2 EP3+RA *** 1 ** EP4+PGE2 responsive, whereas mice lacking EP4 receptors responded like 0.1 EP +RA *** 4 ** wild-type mice (Fig. 2B). Similarly, ricinoleic acid given orally 0.0 0 also induced a strong laxative effect, which was abrogated in ricinol. acid +++ + 0 -7 -6 -5 -4 agonist (log [M]) mice lacking the EP receptor (Fig. 2C). Mice lacking EP PGE 2 + +++ 3 3 L798,106 + + + + receptors were, however, indistinguishable from wild-type mice L161,982 + + ++ with regard to the effect of other laxatives, such as 5-hydroxy- tryptophan or polyethylene glycol (PEG3000) (Fig. 2D). Because F 3.0 G 40000 2.0 ricinoleic acid has been reported to induce formation of PGE2 in 30000 the mammalian intestine (28), we tested the effect of COX in- 0.4 ** hibition on ricinoleic acid-induced diarrhea.