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• W1965244669 abstract "We demonstrated that ginsenosides, the active ingredient of Panax ginseng, enhance endogenous Ca2+-activated Cl– currents via Gαq/11-phospholipase C-β3 pathway in Xenopus laevis oocytes. Moreover, prolonged treatment of ginsenosides induced Cl– channel desensitization. However, the molecular mechanisms involved in ginsenoside-induced Cl– channel desensitization have not yet been determined precisely. To provide answers to these questions, we investigated the changes in ginsenoside-induced Cl– channel desensitization after intraoocyte injection of inositol hexakisphosphate (InsP6), which is known to bind β-arrestins and interfere with β-arrestin-induced receptor down-regulation, and cRNAs coding β-arrestin I/II and G-protein receptor kinase 2 (GRK2), which is known to phosphorylate G protein-coupled receptors and attenuate agonist stimulations. When control oocytes were stimulated with ginsenosides, the second, third, and fourth responses to ginsenosides were 69.6 ± 4.1, 9.2 ± 2.3, and 2.6 ± 2.2% of the first responses, respectively. Preintraoocyte injection of InsP6 before ginsenoside treatment restored ginsenoside effect to initial response levels in a concentration-, time-, and structurally specific manner, in that inositol hexasulfate had no effect. The EC50 was 13.9 ± 8.7 μM. Injection of cRNA coding β-arrestin I but not β-arrestin II blocked InsP6 effect on prevention of ginsenoside-induced Cl– channel desensitization. Injection of cRNA coding GRK2 abolished ginsenoside effect enhancing Cl– current. However, the GRK2-caused loss of ginsenoside effect on Cl– current was prevented by coinjection of GRK2 with GRK2-K220R, a dominant-negative mutant of GRK. These results indicate that ginsenoside-induced Cl– channel desensitization is mediated via activation of GRK2 and β-arrestin I. We demonstrated that ginsenosides, the active ingredient of Panax ginseng, enhance endogenous Ca2+-activated Cl– currents via Gαq/11-phospholipase C-β3 pathway in Xenopus laevis oocytes. Moreover, prolonged treatment of ginsenosides induced Cl– channel desensitization. However, the molecular mechanisms involved in ginsenoside-induced Cl– channel desensitization have not yet been determined precisely. To provide answers to these questions, we investigated the changes in ginsenoside-induced Cl– channel desensitization after intraoocyte injection of inositol hexakisphosphate (InsP6), which is known to bind β-arrestins and interfere with β-arrestin-induced receptor down-regulation, and cRNAs coding β-arrestin I/II and G-protein receptor kinase 2 (GRK2), which is known to phosphorylate G protein-coupled receptors and attenuate agonist stimulations. When control oocytes were stimulated with ginsenosides, the second, third, and fourth responses to ginsenosides were 69.6 ± 4.1, 9.2 ± 2.3, and 2.6 ± 2.2% of the first responses, respectively. Preintraoocyte injection of InsP6 before ginsenoside treatment restored ginsenoside effect to initial response levels in a concentration-, time-, and structurally specific manner, in that inositol hexasulfate had no effect. The EC50 was 13.9 ± 8.7 μM. Injection of cRNA coding β-arrestin I but not β-arrestin II blocked InsP6 effect on prevention of ginsenoside-induced Cl– channel desensitization. Injection of cRNA coding GRK2 abolished ginsenoside effect enhancing Cl– current. However, the GRK2-caused loss of ginsenoside effect on Cl– current was prevented by coinjection of GRK2 with GRK2-K220R, a dominant-negative mutant of GRK. These results indicate that ginsenoside-induced Cl– channel desensitization is mediated via activation of GRK2 and β-arrestin I. Ginseng, the root of Panax ginseng C. A. Meyer, has been used as a representative tonic for several hundreds years in such countries as Korea, China, and Japan. Currently, ginseng is one of the most famous and precious herbal medicines consumed in around the world (1Tyler V.E. J. Pharm. Technol. 1995; 11: 214-220Crossref Scopus (19) Google Scholar, 2Nah S.Y. Kor. J. Ginseng Sci. 1997; 21: 1-12Google Scholar). Recent accumulating evidence shows that ginsenosides are the pharmacologically active ingredient of ginseng. Ginsenoside is one of the derivatives of triterpenoid dammarane, consisting of 30 carbon atoms. Recent studies have suggested that G proteins mediate some ginsenoside effects. For example, Nah and McCleskey (3Nah S.Y. McCleskey E.W. J. Ethnopharmacol. 1994; 42: 45-51Crossref PubMed Scopus (74) Google Scholar) and Nah et al. (4Nah S.Y. Park H.J. McCleskey E.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8739-8743Crossref PubMed Scopus (131) Google Scholar) showed in neuronal cells that ginsenosides inhibit voltage-dependent Ca2+ current in sensory neurons through the activation of pertussis toxin-sensitive G protein. Choi et al. (5Choi S. Rho S.H. Jung S.Y. Kim S.C. Park C.S. Nah S.Y. Br. J. Pharmacol. 2001; 132: 641-648Crossref PubMed Scopus (44) Google Scholar, 6Choi S. Kim H.J. Ko Y.S. Jeong S.W. Kim Y.I. Simonds W.F. Oh J.W. Nah S.Y. J. Biol. Chem. 2001; 276: 48797-48802Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) demonstrated that ginsenoside treatment increased Ca2+-activated Cl– current through the signaling pathway that activates pertussis toxin-insensitive Gαq/11 proteins coupled to PLC-β3 in Xenopus laevis oocytes. Choi et al. (5Choi S. Rho S.H. Jung S.Y. Kim S.C. Park C.S. Nah S.Y. Br. J. Pharmacol. 2001; 132: 641-648Crossref PubMed Scopus (44) Google Scholar) also showed that Ca2+-activated Cl– current produced by ginsenoside treatment diminished spontaneously after reaching peak amplitude, even in the continued presence of ginsenosides in X. laevis oocytes. However, very little is known about molecular mechanism(s) involved in Cl– channel desensitization induced by ginsenoside treatment. Desensitization of a receptor is defined as the diminished or abolished response to an agonist after repeated stimulation. The desensitizing process of G protein-coupled receptors (GPCRs), 1The abbreviations used are: GPCR, G protein coupled receptor; GRK, G protein coupled receptor kinase; InsP6, inositol hexakisphosphate; InsS6, inositol hexasulfate; mAChR, muscarinic acetylcholine receptor; ACh, acetylcholine; PMA, phorbol 12-myristate 13-acetate; PLC, phospholipase C; PKC, protein kinase C; GTS, ginsenosides.1The abbreviations used are: GPCR, G protein coupled receptor; GRK, G protein coupled receptor kinase; InsP6, inositol hexakisphosphate; InsS6, inositol hexasulfate; mAChR, muscarinic acetylcholine receptor; ACh, acetylcholine; PMA, phorbol 12-myristate 13-acetate; PLC, phospholipase C; PKC, protein kinase C; GTS, ginsenosides. which are mainly coupled to Gαi/o-, Gαs-adenylate cyclase, or Gαq/11-phospholipase C, is well characterized (7Kwatra M.M. Schwinn D.A. Blank J.L. Kim C.M. Benovic J.L. Krause J.E. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 9161-9164Abstract Full Text PDF PubMed Google Scholar, 8Appleyard S.M. Celver J. Pineda V. Kovoor A. Wayman G.A. Chavkin C. J. Biol. Chem. 1999; 274: 23802-23807Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 9Oakely R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar). The main specialized regulatory proteins for homologous GPCR desensitization process are GRKs and β-arrestins (10Ferguson S.S.G. Caron M.G. Cell Dev. Biol. 1998; 9: 119-127Crossref PubMed Scopus (160) Google Scholar, 11Pierce K.L. Lefkowitz R.J. Nat. Rev. 2001; 2: 727-733Crossref Scopus (375) Google Scholar). GRKs mediate the phosphorylation of the receptors that are occupied by agonists. The phosphorylated receptors create a binding site for regulatory proteins, β-arrestins, which are involved in the endocytosis of desensitized receptors and bindings of β-arrestins to the phosphorylated receptor facilitate to uncouple it from its target G proteins for termination of effector stimulation (10Ferguson S.S.G. Caron M.G. Cell Dev. Biol. 1998; 9: 119-127Crossref PubMed Scopus (160) Google Scholar, 12Sasakawa N. Ferguson J.E. Sharif M. Hanley M.R. Mol. Pharmacol. 1994; 46: 380-385PubMed Google Scholar, 13McConalogue K. Corvera C.U. Gamp P.D. Grady E.F. Bunnet N.W. Mol. Biol. Cell. 1998; 9: 2305-2324Crossref PubMed Scopus (76) Google Scholar, 14Sullivan Hanley N.R. Hensler J.G. J. Pharmacol. Exp. Ther. 2002; 300: 468-477Crossref PubMed Scopus (53) Google Scholar). On the other hand, recent reports showed that InsP6 blocks visual arrestin interaction with phosphorylated rhodopsin by a direct binding to visual arrestin, resulting in elimination of light-induced inactivation of rhodopsin (15Palczewski K. Rispoli G. Detwiler P.B. Neuron. 1992; 8: 117-126Abstract Full Text PDF PubMed Scopus (90) Google Scholar, 16Palczewski K. Pulvermuller A. Buczylko J. Gutmann C. Hoffman K.P. FEBS Lett. 1991; 295: 195-199Crossref PubMed Scopus (58) Google Scholar). In the present study, to further characterize the molecular mechanism(s) underlying ginsenoside-induced Ca2+-activated Cl– channel desensitization, we studied the regulation of the coupling between short- and long-term ginsenoside treatment and Ca2+-activated Cl– channel activity in X. laevis oocytes. To this aim, we first examined the changes in ginsenoside-induced desensitization on Ca2+-activated Cl– current after intraoocyte injection of InsP6, which is known to bind β-arrestins and block β-arrestin-dependent receptor trafficking. We further examined the effects of GRK2 after intraoocyte injections of cRNAs coding GRK2. Herein, we present results suggesting that ginsenoside-induced Cl– channel desensitization involves the activation of GRK2/β-arrestin I in X. laevis oocytes. Materials—cDNAs coding GRK2 and GRK2-K220R and β-arrestin I and II proteins were kindly provided by Dr. Robert Lefkowitz (Duke University, Durham, NC). m1 muscarinic acetylcholine receptor was purchased from Guthrie Research Institute (Sayre, PA) Ginsenosides were provided by Korea Ginseng and Tobacco Research Institute (Taejon, Korea). The stock solution of ginsenosides was prepared and used as described in previous experiment (5Choi S. Rho S.H. Jung S.Y. Kim S.C. Park C.S. Nah S.Y. Br. J. Pharmacol. 2001; 132: 641-648Crossref PubMed Scopus (44) Google Scholar). InsP6 was obtained from Calbiochem, and all other agents were obtained from Sigma. In Vitro Synthesis of cRNA—Recombinant plasmids containing cDNA inserts for GRK2, GRK2-K220R, β-arrestin I/II, or m1 mAChR proteins were linearized by digestion with appropriate restriction enzymes. The cRNAs from linearized templates were obtained with an in vitro transcription kit (mMessage mMachine; Ambion, Austin, TX) using an SP6, T3, or T7 RNA polymerase. The RNA was dissolved in RNase-free water at 1 μg/μl, divided into aliquots, and stored at –70 °C until used. Oocyte Preparation—X. laevis frogs were obtained from Xenopus I (Ann Arbor, MI). Their care and handling were in accordance with the highest standards of institutional guidelines. To isolate oocytes, frogs were operated on under anesthesia with an aerated solution of 3-aminobenzoic acid ethyl ester. Oocytes were separated by treatment with collagenase and agitation for 2 h in a Ca2+-free medium containing 82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5 mm HEPES, 2.5 mm sodium pyruvate, 100 units/ml penicillin and 100 μg/ml streptomycin. Stage V–VI oocytes were collected and stored in ND96 (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, and 5 mm HEPES, pH 7.5) supplemented with 0.5 mm theophylline and 50 μg/ml gentamicin. This oocyte-containing solution was maintained at 18 °C with continuous gentle shaking and changed every day. Oocyte Treatment before Electrophysiological Recordings—In the first series of experiments, InsP6 or InsS6 (10 nl each), freshly dissolved in distilled water, was injected into cytosol, and electrophysiological recordings were performed 20 min after injection except as indicated otherwise. In the second series of experiments, the oocytes were injected with cRNA(s) at 40 ng/oocyte (or concentrations as indicated), and electrophysiological recordings were performed 2 days later. Control oocytes were injected with 40 nl of H2O. Electrophysiological Recording—Two-electrode voltage-clamp recordings were obtained from individual oocytes placed in a small Plexiglas net chamber (0.5 ml), which was continuously superfused with the bathing medium (i.e. ND96). The microelectrodes were filled with 3 m KCl and had a resistance of 0.2–0.7 MΩ. The electrophysiological experiments were performed at room temperature using an Oocyte Clamp amplifier (OC-725C; Warner Instrument, Hamden, CT). Linear leak and capacitance currents were corrected with a leak subtraction procedure. Ginsenosides were applied to oocytes by bath perfusion (17Choi S. Lee J.H. Kim Y.I. Kang M.J. Rhim H. Lee S.K. Nah S.Y. Eur. J. Pharmacol. 2003; 468: 83-92Crossref PubMed Scopus (13) Google Scholar). Data Analysis—To obtain the concentration-response curve in the presence of InsP6, the observed peak amplitudes were normalized and plotted and then fitted to the Hill equation below using Origin software (Northampton, MA): y/ymax = [A]nH /([A]nH + [EC50]nH), where y is the percentage inhibition at a given concentration of InsP6, ymax is the maximal peak current, EC50 is the concentration of InsP6 producing half-maximum effect of the control response to ginsenosides, [A] is the concentration of InsP6, and nH is the interaction coefficient. All values are presented as means ± S.E. The differences between means of control and ginsenoside treatment data were analyzed using unpaired Student’s t test. A value of p < 0.05 was considered statistically significant. Rapid Desensitization of Ginsenoside- or ACh-induced Cl– Current Responses after Repeated Application of Ginsenosides or ACh—In the present study, we examined the changes in ginsenoside effect on the Cl– current after repeated application of ginsenosides. As shown in Fig. 1A, treatment of ginsenosides induced Cl– current enhancement in a voltage-dependent manner. The reversal potential was near –20 mV, indicating that ginsenosides activate endogenous Ca2+-activated Cl– currents in X. laevis oocytes (5Choi S. Rho S.H. Jung S.Y. Kim S.C. Park C.S. Nah S.Y. Br. J. Pharmacol. 2001; 132: 641-648Crossref PubMed Scopus (44) Google Scholar, 6Choi S. Kim H.J. Ko Y.S. Jeong S.W. Kim Y.I. Simonds W.F. Oh J.W. Nah S.Y. J. Biol. Chem. 2001; 276: 48797-48802Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Oocytes first stimulated with ginsenosides were washed with ND96 for 3–5 min until the basal current was recovered again and then restimulated with ginsenosides. The second, third, or fourth Cl– current responses for ginsenosides were dramatically diminished, and the magnitudes of Cl– current were 69.6 ± 4.1, 9.2 ± 2.3, and 2.6 ± 2.2%, respectively, of the first responses of ginsenosides (n = 15 oocytes each from three different batches of donors) (Fig. 1B, inset). As a positive control, cRNA coding m1 muscarinic acetylcholine receptors (mAChRs) was injected into oocytes; receptor activation also enhances Cl– currents via Gαq/11-PLC-inositol trisphosphate pathway (18Hill J.J. Peralta E.G. J. Biol. Chem. 2001; 276: 5505-5510Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Repeated treatment of oocytes expressing m1 mAChR with ACh resulted in a gradual attenuation of the magnitude of Cl– current. The second, third, or fourth Cl– current responses for ginsenosides were diminished, and the magnitudes of Cl– current were 14.2 ± 7.1, 8.3 ± 6.3, and 4.1 ± 3.2%, respectively, of the first responses of ACh (n = 13 oocytes each from three different batches of donors) (Figs. 3C and 5B).Fig. 3Effect of InsP6 after GTS- or ACh-induced short- or long-term Cl– channel desensitization. A and B, injection of InsP6 into oocytes that were desensitized by short- or long-term treatment with ginsenosides failed to induce GTS response on Ca2+-activated Cl– current. A, inset, the traces of the current recorded at the time points 1 and 2 are superimposed. Histograms show that peak outward currents (mean ± S.E.; n = 14–15 oocytes each) recorded in oocytes injected with H2O (first treatment with GTS) and InsP6 (fifth treatment of GTS). B, inset, histograms show that ginsenoside induced peak outward currents (mean ± S.E.; n = 12–14 each) recorded in oocytes before (0 h) and after (16 h) ginsenoside treatment. C and D, injection of InsP6 into oocytes expressing m1 mAChRs that were desensitized by short- or long-term treatment with ACh failed to induce ACh response on Ca2+-activated Cl– current. C, inset, the traces of the current recorded at the time points 1 and 2 are superimposed. Histograms show that peak outward currents (mean ± S.E.; n = 13–14 oocytes each) recorded in oocytes injected with H2O (first treatment with ACh) and InsP6 (fifth treatment of ACh). D, inset, histograms show that ACh-induced peak outward currents (mean ± S.E.; n = 10–12 each) recorded in oocytes before treatment of ACh (0 h) and after ACh treatment (16 h). Bars denote bath application of GTS (50 μg/ml) or ACh (100 μM) and arrow indicates intraoocyte injection of InsP6.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5β-Arrestin I but not β-arrestin II blocks InsP6 effect on ginsenoside- and m1 mAChR-induced Cl– channel desensitization. A, H2O or InsP6 were injected into oocytes that were previously injected with cRNA coding β-arrestin I or II and incubated for 20 min in ND96. GTS was applied repeatedly by bathing. Histograms of peak outward currents after repeated treatment of GTS. B,H2O or InsP6 was injected into oocytes that were previously injected with cRNAs coding m1 mAChR alone, β-arrestin I and m1 mAChR or β-arrestin II and m1 mAChR and incubated for 20 min in ND96. Histograms of peak outward currents after repeated treatment of ACh. *, p < 0.01; significantly different from β-arrestin II injected oocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of InsP6 on the Rapid Desensitization of Ginsenoside- or ACh-induced Cl– Current Responses—Because preintraoocyte injection of InsP6 prevents the desensitization on Ca2+-activated Cl– currents induced by repeated treatment of substance P or lysophosphatidic acid in X. laevis oocytes expressing substance P receptors or lysophosphatidic acid receptors (12Sasakawa N. Ferguson J.E. Sharif M. Hanley M.R. Mol. Pharmacol. 1994; 46: 380-385PubMed Google Scholar), the above results prompted us to test the possibility that InsP6 might also prevent ginsenoside-induced Cl– channel desensitization. As the first step toward testing this hypothesis, we investigated the effect of InsP6 on the rapid loss of Cl– current responses after short-term treatment with ginsenosides or ACh. We injected InsP6 (100 μm intracellular concentration) into oocytes 20 min before treatment with ginsenosides or ACh. Preintraoocyte injection of InsP6 neither changes basal membrane current compared with H2O-injected oocytes (H2O/InsP6 = 0.24 ± 0.05/0.22 ± 0.07 μA, n = 15 each) nor affects ginsenoside- or ACh-induced Cl– current responses (Figs. 1D, inset, and 5B). However, as shown in Fig. 1, C and D, preintraoocyte injection of InsP6 not only prevented the loss of ginsenoside- or ACh-induced Cl– current response but also slightly enhanced ginsenoside- or ACh-induced Cl– current response (Fig. 1D, inset and Fig. 5B). The second, third, or fourth Cl– current response for ginsenosides was maintained, and the magnitudes of the current were 106.6 ± 3.1, 134.8 ± 5.3, and 138.7 ± 7.2%, respectively, of the first responses of ginsenosides (n = 12 oocytes each; from three different batches of donors) (Fig. 1D, inset). For m1 mAChR, the second, third, or fourth Cl– current response for ACh was also maintained, and the magnitudes of the current were 102.4 ± 10.1, 94.8 ± 12.3, and 114.7 ± 13.2%, respectively, of the first responses of ACh (n = 12 oocytes each; from three different batches of donors) (Fig. 5B). Concentration- and Time-dependent Effects of InsP6 on Ginsenoside-induced Cl– Channel Desensitization—Fig. 2A shows the concentration-dependent response curve for prevention by InsP6 on the loss of ginsenoside-induced Cl– current responses. InsP6 prevented the loss of ginsenoside-induced Cl– current responses in a dose-dependent manner. The EC50 was 13.9 ± 8.7 μm. These inhibitory effects on desensitization were not simply a result of anionic charge, because the InsP6 analog-like inactive InsS6 had no effect, indicating that the attenuating effect on ginsenoside-induced desensitization of Ca2+ activated Cl– current is specific to InsP6 (Fig. 2A, inset). Fig. 2B shows the time course of onset of the inhibitory effect of InsP6 on ginsenoside-induced Cl– channel desensitization. The inhibitory effect reached a maximum level 15 min after intraoocyte injection, and the effect was persistent for at least 6 h. Effect of InsP6 on Restimulation of Ginsenosides or ACh In Oocytes Undergoing Short- or Long-Term Treatment with Ginsenosides or ACh—As a next step, we examined whether intraoocyte injection of InsP6 to oocytes that have been desensitized by several repeated ginsenoside treatments or oocytes expressing m1 mAChRs that have also been desensitized by several repeated ACh treatments could prevent ginsenoside- or ACh-induced Cl– channel desensitization. As shown in Fig. 3, InsP6-injected oocytes that were desensitized previously by short-term treatment with ginsenosides or ACh failed to respond to ginsenoside or ACh treatment. We also tested whether intraoocyte injection of InsP6 in oocytes that have been undergone prolonged treatment with ginsenosides (100 μg/ml, 16 h) or ACh (100 μM, 16 h) could affect ginsenoside- or ACh-induced Cl– current responses, because long-term treatment of ginsenosides or ACh induced the loss of ginsenoside- or ACh-induced Cl– current responses (Fig. 3, B and D, inset). Initial treatment of ginsenosides or ACh in oocytes that have been undergone prolonged treatment with ginsenosides or ACh failed to enhance Ca2+-activated Cl– current, indicating that long-term treatment of ginsenosides or ACh induces Cl– channel desensitization. Moreover, in oocytes that have been undergone prolonged treatment with ginsenosides or ACh, the loss of Cl– current response to ginsenosides or ACh was not restored by intraoocyte injection of the InsP6. Time Course of Recovery of Ginsenoside- or ACh-induced Cl– Channel Desensitization—To characterize recovery from Cl– channel desensitization at longer times, we employed the following procedure: ginsenosides or ACh response was recorded in 13–14 oocytes (control group), and the mean control response was calculated. Test group oocytes were exposed to ginsenosides (100 μg/ml) or ACh (100 μm) for 30 min, completely washed several times with ND96, and then the response to the second application of ginsenosides or ACh was measured in these oocytes during the indicated times. As shown in Fig. 4, initial recovery of ginsenoside-induced Cl– current response during the first 5–60 min remained about 5–30% of control and was followed by 60% recovery over 2–4 h, whereas initial recovery of ACh-induced Cl– current response during the first 5–120 min remained about 1–3% of control and was followed by 10–45% recovery over 4–8 h. Thus, recovery time on ACh-induced Cl– response from desensitization was slower than that of ginsenoside-induced Cl– response; the half-recovery time to control level from desensitization was about 145.8 ± 63 and 619.5 ± 26.9 min for ginsenosides and m1 mAChRs, respectively. However, ginsenoside- and ACh-induced Cl– current responses had nearly recovered to initial control levels after 24 h. Overexpression of β-Arrestin I but Not β-Arrestin II Blocks InsP6 Effect on Ginsenoside- and ACh-induced Cl– Channel Desensitization—As a next step, we tested whether or not overexpression of β-arrestins related with receptor desensitization could affect ginsenosides or ACh effect on Cl– current. We also tested whether or not overexpression of these proteins could affect InsP6-induced attenuation on ginsenoside- or ACh-induced Cl– channel desensitization. As illustrated in Fig. 5A, intraoocyte injection of cRNA coding β-arrestin I or β-arrestin II did not affect either basal Cl– current (H2O/β-arrestin I = 0.22 ± 0.04:0.21 ± 0.06 μA; H2O/β-arrestin II = 0.20 ± 0.04: 0.23 ± 0.03 μA; n = 15 each) or the Cl– current evoked by initial ginsenoside treatment (H2O/β-arrestin I = 3.5 ± 0.2: 3.2 ± 0.5 μA; H2O/β-arrestin II = 3.5 ± 0.2:3.1 ± 0.1 μA; n = 15 each) (Fig. 5). However, intraoocyte injection of cRNA coding β-arrestin I significantly blocked the preventive effect of InsP6 on ginsenoside-induced Cl– channel desensitization in the fourth treatment of ginsenosides (H2O + InsP6/β-arrestin I + InsP6 = 4.8 ± 0.8:1.3 ± 0.4 μA; p < 0.01; measured at a Vc value of +60 mV, n = 15 oocytes each, from three different batches of frogs), whereas intraoocyte injection of cRNA coding β-arrestin II failed to block the preventive effect of InsP6 (H2O + InsP6/β-arrestin II + InsP6 = 4.8 ± 0.8:3.9 ± 0.7 μA). Similarly, we could also observe that preintraoocyte injection of InsP6 prevented the loss of ACh-induced Cl– current response in oocytes expressing m1 mAChRs and that injection of cRNA coding β-arrestin I (ACh + InsP6/β-arrestin I + InsP6 = 4.6 ± 0.8/1.7 ± 0.4 μA; p < 0.01; measured at a Vc value of +60 mV, n = 14 oocytes each, from three different batches of frogs) but not β-arrestin II (ACh + InsP6/β-arrestin II + InsP6 = 4.8 ± 0.8:4.0 ± 0.6 μA) blocked the effect of InsP6 on prevention of ACh-induced Cl– channel desensitization (Fig. 5B). Overexpression of GRK2 Inhibits the Effect of Ginsenoside or ACh on Cl– Current Enhancement—Because the results above showed the possibility that β-arrestin I might participate the desensitization signaling pathway on a loss of ginsenoside- or ACh-induced Cl– current responses, we investigated the roles of GRK in the effect of ginsenoside or ACh on Cl– current enhancement. We first injected cRNA coding GRK2 alone or coinjected cRNAs coding m1 mAChR and GRK2 into oocytes and observed the effect of ginsenoside or ACh on Cl– current. As summarized in Fig. 6, A and B, ginsenosides or ACh applied to H2O-injected vehicle oocytes enhanced the Cl– current. In contrast, ginsenosides or ACh failed to enhance the Cl– current in cells injected with cRNA coding GRK2 or cRNAs coding m1 mAChR and GRK2 (Fig. 6, A and B). However, intraoocyte injection of cRNA coding GRK2-K220R, which is a dominant-negative mutant of GRK2 and lacks kinase activity, had no effect on ginsenoside- or ACh-induced Cl– current enhancement. Moreover, coexpression of GRK2 and GRK2-K220R abolished the inhibitory effect of GRK2 on ginsenoside- or ACh-induced Cl– current enhancement. The extent of the blockade of the ginsenoside effect on Cl– current by GRK2 cRNA injection was proportional to the amount of cRNA injected (Fig. 6C), and the time required for the injected cRNAs to work was >16 h (Fig. 6D). Repeated treatment of ginsenosides or ACh in oocytes expressing m1 mAChR induced a loss of Cl– current response in oocytes injected with cRNAs coding GRK2-K220R alone and cRNAs coding GRK2 and GRK2-K220R, respectively (Fig. 6, A and B). Effects of InsP6 on PMA-evoked Loss of Ginsenoside-induced Cl– Current Responses—The activation of PLC produces lipidsoluble 1,2-diacylglycerol, an endogenous protein kinase C (PKC) activator. Previous reports showed that activation of PKC by treatment with PMA, an active PKC activator, causes receptor phosphorylation and receptor uncoupling from PLC-mediated inositol phospholipid metabolism, resulting in a loss of Cl– current responses by agonist stimulations (19Leeb-Lundberg L.M. Cotecchia F.S. Lomasney J.W. Debernaardis J.F. Lefkowitz R.J. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5651-5655Crossref PubMed Scopus (286) Google Scholar, 20Orellana S. Solski P.A. Brown J.H. J. Biol. Chem. 1987; 262: 1638-1643Abstract Full Text PDF PubMed Google Scholar, 21Moran O. Dascal N. Mol. Brain Res. 1989; 5: 193-202Crossref PubMed Scopus (52) Google Scholar, 22Lupu-Meiri M. Shapira H. Oron Y. Pfluegers Arch. Eur. J. Physiol. 1989; 413: 498-504Crossref PubMed Scopus (22) Google Scholar, 23Singer D. Boton R. Moran O. Dascal N. Pfluegers Arch. Eur. J. Physiol. 1990; 416: 7-16Crossref PubMed Scopus (20) Google Scholar). In the present study, we first examined the effects of the PKC activator PMA on the ginsenoside-induced Cl– current enhancements. As shown in Fig. 7, treatment with PMA, but not 4α-phorbol 12,13-didecanoate, an inactive PKC activator, induced a loss of ginsenoside-induced Cl– current enhancement but PKA activators had no effect on ginsenoside-induced Cl– current responses (data not shown). The inhibitory effect of PMA was dose-dependent, and the IC50 was 35.6 ± 4.7 nm (Fig. 7B, inset). But cotreatment of the PKC inhibitor staurosporine with PMA prevented PMA-induced inhibition of the ginsenoside effect on Cl– currents, indicating that PMA-evoked loss of ginsenoside-induced Cl– current responses is specific to PKC activation. We also tested whether preintraoocyte injection of InsP6 prevent PMA-induced inhibition on ginsenoside-evoked Cl– current. However, as shown in Fig. 7B, preintraoocyte injection of InsP6 had no effect on PMA-induced loss of ginsenoside-evoked Cl– current responses. Although ginsenosides, the active ingredient of Panax ginseng, have been widely used as pharmacological agents for a long time, few reports have related ginsenoside signal transduction until now. In previous studies, we demonstrated that activation of membrane components by ginsenosides at the extracellular side enhanced a Ca2+-activated Cl– current via a Gαq/11-phospholipase C-β3 pathway. We showed also that prolonged treatment with ginsenosides induced a desensitization of Cl– current response after reaching peak amplitude in X. laevis oocytes (5Choi S. Rho S.H. Jung S.Y. Kim S.C. Park C.S. Nah S.Y. Br. J. Pharmacol. 2001; 132: 641-648Crossref PubMed Scopus (44) Google Scholar, 6Choi S. Kim H.J. Ko Y.S. Jeong S.W. Kim Y.I. Simonds W.F. Oh J.W. Nah S.Y. J. Biol. Chem. 2001; 276: 48797-48802Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The present study was performed to further characterize the desensitization signaling pathway of a loss of ginsenoside-induced Cl– current response. In this study, we have provided three principal findings affirming that GRK2 and β-arrestin I are involved in the main molecular mechanisms mediating ginsenoside-induced Cl– channel desensitization in X. laevis oocytes. First, we showed that short- or long-term treatment with ginsenosides induced a complete loss of ginsenoside effect on Cl– current enhancement, whereas preintraoocyte injection of InsP6, but not InsS6 (an inactive analog of InsP6), prevented the desensitization of Cl– current induced by repeated treatment with ginsenosides. These results support the concept that the attenuating effect on ginsenoside-induced Cl– channel desensitization is specific to InsP6. The EC50 (13.9 ± 8.7 μm) obtained from the present study was relatively high and suggests that InsP6 interacts with β-arrestin I with low affinity. However, there was structural specificity for InsP6 but not InsS6, and the potency of the Cl– channel desensitization-blocking action of InsP6 was very similar to the concentration of InsP6 binding to arrestins (16Palczewski K. Pulvermuller A. Buczylko J. Gutmann C. Hoffman K.P. FEBS Lett. 1991; 295: 195-199Crossref PubMed Scopus (58) Google Scholar). Moreover, because the concentration of InsP6 in variety of cells is in the range of 1–20 μm, the EC50 values for this pharmacological action might be physiologically significant (24Heslop J.P. Irvine R.F. Tashjian A.H. Berridge M.J. J. Exp. Biol. 1985; 119: 395-401Crossref PubMed Google Scholar, 25Szwergold B.S. Graham R.A. Brown T.R. Biochem. Biophys. Res. Commun. 1987; 14: 874-881Crossref Scopus (111) Google Scholar, 26Shears S.B. Biochem. J. 1989; 260: 313-324Crossref PubMed Scopus (225) Google Scholar). Interestingly, we observed that intraoocyte injection of InsP6 did not restore ginsenoside or ACh responses on Ca2+-activated Cl– channels after short- or long-term Cl– channel desensitization caused by ginsenoside or ACh treatment had already occurred (Fig. 3). The present study further suggested that membrane component(s) or m1 mAChR that might be interacting with ginsenosides or ACh could be down-regulated after short- or long-term treatment with ginsenosides or ACh and that intraoocyte injected-InsP6 was no more helpful for the down-regulated membrane components or m1 mAChRs. As for the second piece of evidence for the role of β-arrestins in ginsenoside-induced Cl– channel desensitization, we demonstrated that overexpression of β-arrestin I cRNA, but not β-arrestin II cRNA, induced ginsenoside- or ACh-induced Cl– channel desensitization in oocytes or in oocytes expressing m1 mAChRs that were preinjected with InsP6 before ginsenoside or ACh treatment. These results suggest that extra copies of β-arrestin I produced by injection of cRNAs coding β-arrestin I might bind with InsP6 and sequestrate free InsP6 by forming complexes with InsP6 and β-arrestin I. Therefore, the results of the experiments performed with β-arrestin I further suggest that β-arrestin I mediates ginsenoside- and m1 mAChR-induced Cl– channel desensitization. The third and final piece of evidence suggesting a role for GRK2 in ginsenoside- and m1 mAChR-induced Cl– current desensitization comes from the experiments involving injections of cRNAs coding GRK2, which is known to phosphorylate GPCRs after agonist stimulation and facilitate β-arrestin binding to phosphorylated receptors for the receptor down-regulation process. The results of these experiments (i.e. blocking of the ginsenoside or ACh effect that enhances Ca2+-activated Cl– current by GRK2 cRNA injection but not by dominant-negative GRK2-K220R, which lacks kinase activity, and blocking of GRK action by cRNA coinjection of GRK2 and dominant-negative GRK2-K220R) suggest that the kinase activity of GRK2 is a key role for the inhibitory effect on ginsenoside- and m1 mAChR-induced Cl– current enhancement and that the kinase activity of GRK2 is also involved in signaling pathway of ginsenoside- or m1 mAChR-induced Cl– channel desensitization. Moreover, these results further suggest that phosphorylation of membrane receptors by GRK2 that might interact with ginsenosides or phosphorylation of m1 mAChR by GRK2 could be enough for ginsenoside- or m1 mAChR-induced Cl– channel desensitization (Fig. 6). Recent studies have indicated that β-arrestin I as well as β-arrestin II are involved in GPCR desensitization after their respective agonist stimulations (10Ferguson S.S.G. Caron M.G. Cell Dev. Biol. 1998; 9: 119-127Crossref PubMed Scopus (160) Google Scholar, 11Pierce K.L. Lefkowitz R.J. Nat. Rev. 2001; 2: 727-733Crossref Scopus (375) Google Scholar). However, the present study does not support the idea that β-arrestin II functions as a direct mediator of ginsenoside- or m1 mAChR-induced Cl– channel desensitization. Most of the GPCRs that couple to the activation of pertussis toxin-insensitive G protein and PLC and the release of Ca2+ from inositol trisphosphate-sensitive intracellular stores showed indistinguishable affinity to both β-arrestin isoforms and formed a stable complexes with β-arrestin I or II even after 1 h (9Oakely R.H. Laporte S.A. Holt J.A. Caron M.G. Barak L.S. J. Biol. Chem. 2000; 275: 17201-17210Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar, 12Sasakawa N. Ferguson J.E. Sharif M. Hanley M.R. Mol. Pharmacol. 1994; 46: 380-385PubMed Google Scholar, 13McConalogue K. Corvera C.U. Gamp P.D. Grady E.F. Bunnet N.W. Mol. Biol. Cell. 1998; 9: 2305-2324Crossref PubMed Scopus (76) Google Scholar). In present study, we also observed that once a loss on ginsenoside- or ACh-induced Cl– current responses by short-term and repeated treatment with ginsenoside or ACh was initiated, the desensitization lasted for up to 8 h before complete recovery (Fig. 4). However, at present, we can offer no satisfactory explanation for the lack of effect of β-arrestin II cRNA injection on ginsenoside-induced Cl– channel desensitization. To clarify the exact role of β-arrestins in ginsenoside-induced Cl– channel desensitization, further studies will be needed. We also tested the role of second messenger-dependent protein kinase (i.e. PKC) activation on ginsenoside-induced Cl– channel desensitization. As shown in Fig. 7, it is likely that the desensitization pathway via PKC activation on ginsenoside-induced Cl– current enhancement might be mediated via a different pathway compared with β-arrestin I-mediated Cl– current desensitization, because preintraoocyte injection of InsP6 did not reverse PMA-evoked loss of ginsenoside-induced Cl– current responses (27Kato K.I. Kaneko S. Nomura Y. J. Neurochem. 1988; 50: 766-773Crossref PubMed Scopus (24) Google Scholar, 28Barish M.E. J. Physiol. (Lond.). 1983; 342: 309-325Crossref Scopus (490) Google Scholar, 29Ito I. Hirono C. Yamagishi S. Nomura Y. Kaneko S. Sugiyama H. J. Cell. Physiol. 1988; 134: 155-160Crossref PubMed Scopus (29) Google Scholar). What is the mechanism of desensitization by PKC if it does not involve β-arrestins? Previous reports demonstrated that treatment with a PKC activator (i.e. 12-O-tetradecanoylphorbol-13-acetate) not only attenuated ACh- or 5-HT-induced Ca2+-activated Cl– current enhancement in X. laevis oocytes injected with rat brain mRNA but also increased phosphorylation of oocyte membrane proteins (27Kato K.I. Kaneko S. Nomura Y. J. Neurochem. 1988; 50: 766-773Crossref PubMed Scopus (24) Google Scholar). But treatment with 12-O-tetradecanoylphorbol-13-acetate had no effect on Cl– current enhancement evoked by intraoocyte injection of inositol trisphosphate or Ca2+ (28Barish M.E. J. Physiol. (Lond.). 1983; 342: 309-325Crossref Scopus (490) Google Scholar, 29Ito I. Hirono C. Yamagishi S. Nomura Y. Kaneko S. Sugiyama H. J. Cell. Physiol. 1988; 134: 155-160Crossref PubMed Scopus (29) Google Scholar). In the present study, PMA-induced desensitization was also not affected by intraoocyte-injected InsP6. These results suggest that the main role of PKC activation in terminating ginsenoside action on Cl– current enhancement might be the phosphorylation of membrane proteins that interact with ginsenosides or other proteins involved in the ginsenoside signaling pathway, such as the Gαq family, rather than β-arrestins (30Aragay A.M. Quick M.W. J. Biol. Chem. 1999; 274: 4807-4815Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Similarly, intraoocyte injection of InsP6 did not reverse 12-O-tetradecanoylphorbol-13-acetate-caused loss of substance P-induced Cl– current responses (12Sasakawa N. Ferguson J.E. Sharif M. Hanley M.R. Mol. Pharmacol. 1994; 46: 380-385PubMed Google Scholar). Because we could not explain exactly the mechanism of PMA-induced desensitization on ginsenoside-induced Cl– current response, further studies will be needed to clarify the role of PKC activation in ginsenoside-induced Cl– channel desensitization We also investigated recovery time kinetics from Cl– channel desensitization in both ginsenosides and m1 mAChRs in X. laevis oocytes. The half-recovery time to control level from desensitization was about 145.8 ± 63 and 619.5 ± 26.9 min for ginsenosides and m1 mAChRs, respectively. Interestingly, in myenteric neurons, substance P stimulation of neurokinin receptor-1, which is also coupled to the Gαq/11-PLC-inositol trisphosphate pathway, induced intracellular Ca2+ mobilization, and half-recovery time to control levels from desensitization induced by repeated treatment of substance P was about 20 min (13McConalogue K. Corvera C.U. Gamp P.D. Grady E.F. Bunnet N.W. Mol. Biol. Cell. 1998; 9: 2305-2324Crossref PubMed Scopus (76) Google Scholar). Thus, it seems that the time course for resensitization on ginsenosides and m1 mAChR from Cl– channel desensitization in X. laevis oocytes was much slower compared with neurokinin receptor-1 in neurons. However, recent reports also showed that various other GPCRs expressed in X. laevis oocytes did not recover as rapidly to control levels from desensitization as neurokinin receptor-1 in neurons did. For example, in oocytes expressing the endothelinA receptor, Ca2+-activated Cl– current response to endothelin1 from desensitization was not completely recovered even after 90 min (31Cyr C.T. Rudy B. Kris R.M. J. Biol. Chem. 1993; 268: 26071-26074Abstract Full Text PDF PubMed Google Scholar). Moreover, in oocytes injected with rat brain mRNAs, Ca2+-activated Cl– current responses to ACh and serotonin from desensitization recovered partially after 60 min and recovered completely only after 240 min, respectively (23Singer D. Boton R. Moran O. Dascal N. Pfluegers Arch. Eur. J. Physiol. 1990; 416: 7-16Crossref PubMed Scopus (20) Google Scholar, 32Dascal N. Ifune C. Hopkins R. Snutch T.P. Lubbert H. Davidson N. Simon M.I. Lester H.A. Mol. Brain Res. 1986; 1: 201-209Crossref Scopus (83) Google Scholar). These different recovery times might be caused by different types of cells, and further studies will be needed to clarify the recovery time discrepancy between X. laevis oocytes and neurons. In summary, using an X. laevis oocyte model system for explanation of ginsenoside signaling pathway that allows various foreign gene expressions, we obtained further results suggesting that GRK2 and β-arrestin I mediate ginsenosideinduced Ca2+-activated Cl– channel desensitization. This may be one of the desensitization signaling pathways that underlies ginseng action." @default.
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• W1965244669 title "Prevention of Ginsenoside-induced Desensitization of Ca2+-activated Cl– Current by Microinjection of Inositol Hexakisphosphate in Xenopus laevis Oocytes" @default.
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