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CELLULAR AND MOLECULAR
Gut Hormone Lab, Center for Gastroenterological Research, Department of Pathophysiology, Katholieke Universiteit Leuven, Leuven, Belgium (L.T., I.D., T.T., T.L.P.); Department of Biological Chemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium (J.P., P.R.); Euroscreen, Brussels, Belgium (E.B.); and Kosan Biosciences, Hayward, California (Y.L., C.C.)
Received November 30, 2004; accepted March 9, 2005.
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Abstract |
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) became clinically relevant when it was discovered that EM-A accelerated delayed gastric emptying in patients with diabetic gastroparesis (Janssens et al., 1990). As a result, more potent motor-stimulating EM derivatives were developed without antibiotic activity: the motilides (Omura et al., 1987). Recently the clinical development of the most promising motilide ABT-229 was stopped because of its failure to improve symptoms in patients with functional dyspepsia with normal or delayed gastric emptying (Talley et al., 2000) and in patients with type 1 diabetes mellitus (Talley et al., 2001). The authors of the studies with ABT-229 concluded that there was no future for drugs with the prokinetic profile of the motilides, but others have argued that ABT-229 had undesirable properties that may be avoided in new motilides (Tack and Peeters, 2001; Camilleri, 2002). The most important property in this respect is the development of tachyphylaxis. Previous studies described a decreased contractile response of rabbit colonic myocytes due to down-regulation of motilin receptors after chronic oral treatment (Bologna et al., 1993) and after short-term intravenous treatment with erythromycin A (Depoortere et al., 1991), the parent compound from which ABT-229 was derived. One-month treatment of rabbits with ABT-229 also resulted in a down-regulation of motilin receptors and a reduced contractile response to ABT-229 and motilin (Depoortere et al., 1999). Tachyphylaxis was also observed after treatment of healthy subjects with ABT-229. A single dose of ABT-229 increased gastric emptying after the first meal, but emptying of a second meal was not affected, although ABT-229 plasma levels were still high (Verhagen et al., 1997).
Recently, we have shown that the Ca2+ fluxes induced by peptidyl and nonpeptidyl motilin agonists in a Chinese hamster ovary (CHO) cell line expressing the cloned human motilin receptor and the Ca2+ indicator apoaequorin (CHO-MTLR) correlate strongly with the contractile response of these compounds in rabbit duodenal strips (Thielemans et al., 2002). In the present study, we used the aequorin-based luminescence assay to study the desensitization of the motilin receptor by ABT-229 in more detail. To clarify whether desensitization is a common characteristic of all motilides, the desensitizing properties of several motilides with different structural features were compared. To determine the mechanism underlying desensitization, receptor internalization following application of ABT-229 was quantified by receptor binding studies and visualized by using an enhanced green fluorescent protein (EGFP)-tagged motilin receptor (MTLR-EGFP).
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Materials and Methods |
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; Abbott Laboratories, Abbott Park, IL). Macrolide KOS1326 was synthesized by Kosan Biosciences, Inc. (Hayward, CA).
Cell Culture
A CHO-K1 cell line stably expressing the human motilin receptor and the mitochondrially targeted apoaequorin was obtained from Euroscreen SA (Brussels, Belgium). The cells were cultured in Ham's F-12 containing 10% fetal bovine serum, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin B (Invitrogen, Carlsbad, CA), 400 µg/ml G418, and 5 µg/ml puromycin (Sigma-Aldrich, St. Louis, MO) and spliced once a week with 5 mM EDTA in PBS.
Calcium Measurements: Aequorin Luminescence Assay
Suspended cells (5 x 106 cells/ml) were loaded with coelenterazine h (5 µM) (Molecular Probes, Leiden, The Netherlands) at room temperature for at least 4 h to reconstitute active aequorin. Cells were then diluted 10-fold with BSA medium (Dulbecco's modified Eagle's medium/Ham's F-12 with Hepes, without phenol red, 0.1% BSA, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B) and 100 µl/well was injected (50,000 cells) into a 96-well plate containing increasing concentrations of the test compound diluted in BSA medium.
Immediately after injection of cells, the emitted light was measured using the Microlumat plus luminometer (Berthold Technologies, Bad Wildbad, Germany) for 20 s. The intensity of the emitted light was integrated using the Winglow software (Berthold Technologies), yielding for each well one value representative of the emitted light and, hence, of the stimulation of the motilin receptor by the agonist present in the well. This value was expressed as a percentage of maximal stimulation obtained with Triton X-100 (0.9%). All values were corrected for background by subtracting the blanc value (= BSA medium). The negative logarithm of the concentration producing half of the maximal response (pEC50) was calculated from the dose-response curves by linear interpolation. All experiments were performed in duplicate, and each compound was tested at least two times. Results are represented as mean ± standard error of the mean (S.E.M.).
To assess desensitization, cells were pretreated for at least 1 h with different concentrations of agonist during loading, washed, and centrifuged before dose-response curves were established with motilin. The effect of pretreatment on the maximal response was assigned as the effect of desensitization. The average of the maximal plateau values of the dose-response curve to motilin under desensitizing conditions was expressed as a percentage of maximal response under control conditions. The potency of a compound to induce desensitization was calculated after plotting the remaining maximal responses as a function of the concentration of the compound used to desensitize. A sigmoid concentration-response curve was fitted through the data by nonlinear regression analysis using the Graph-Pad Prism software (GraphPad Software Inc., San Diego, CA), and a pDC50 value and its standard error of the best-fit value (negative logarithm of the concentration producing half-maximal desensitization) was calculated. For each compound, 
values were calculated by subtraction of pEC50 values with pDC50 values. The square root of the sum of squares of the standard deviation (S.E.M. for pEC50) and the standard error of the best-fit value (S.E. for pDC50) resulted in the standard deviation of these values. A pooled standard deviation (sp) was derived from which a t value was calculated for the comparison of two values, with n1 + n2 - 2 degrees of freedom using:
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Receptor Binding Studies
Membrane Preparation. CHO-K1 cells expressing the MTLR were cultured until 90% confluence was reached. After removal of medium, cells were scraped from the plates in Ca2+/Mg2+-free PBS. After centrifugation for 3 min at 1500 g, pellets were resuspended in a buffer containing 15 mM Tris-HCl pH 7.5, 2 mM MgCl2, 0.3 mM EDTA, and 1 mM EGTA and homogenized in a glass homogenizer. The crude membrane fraction was collected by two consecutive centrifugation steps at 40,000g for 25 min separated by a washing step in the same buffer. The final pellet was resuspended in a buffer containing 7.5 mM Tris-HCl pH 7.5, 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, and 250 mM sucrose and flash frozen in liquid nitrogen. The protein content was determined by the Folin method (Lowry et al., 1951).
Cell Membrane Receptor Binding. Competition binding assays were performed by incubating CHO-MTLR membranes (8 µg of protein/tube) in a final volume of 0.1 ml with 125I-motilin (0.3 nM) for 60 min at 31°C. Unlabeled motilin, motilin fragments, and motilides were used as competitors at concentrations ranging from 10-12 to 10-4 M in binding buffer (25 mM Hepes pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA). After incubation, the samples were filtered on GF/B filters, washed, and counted in a gamma counter. All values were corrected for nonspecific binding determined by addition of an excess (10-5 M) of unlabeled motilin. IC50 values were determined by nonlinear regression using a single-site model (PRISM; GraphPad Software Inc.). Each compound was tested three times in duplicate.
Construction and Expression of the MTLR Coding Sequence as an N-Terminal Fusion Protein with the EGFP
The cDNA for the full coding sequence inserted in the pcDNA3.1 vector (provided by Euroscreen SA) was amplified by PCR using the proof reading Taq polymerase Pfu Turbo (Stratagene, La Jolla, CA). The 5' primer contained the XhoI restriction site followed by a Kozak consensus sequence preceding the ATG initiation codon and 18 gene-specific nucleotides. The 3' primer contained a 5' EcoRI restriction site followed by the 26 terminal nucleotides of the coding sequence, the TGA stop codon being mutated to remove the translation termination signal and provide an in phase sequence with the EGFP coding sequence that follows immediately the multiple cloning site in the pEGFP plasmid (BD Biosciences Clontech, Palo Alto, CA). The PCR product was digested by DpnI restriction enzyme to remove parental target DNA, purified on QIAquick PCR purification columns (QIAGEN GmbH, Hilden, Germany), and digested by XhoI and EcoRI. The pEGFP plasmid was digested in the same manner. The digestion products were purified on QIAquick PCR purification columns and an aliquot of the resulting 50 µl was run on a 0.8% Tris acetate EDTA agarose gel. A fraction of the digestion products, i.e., the motilin coding sequence and the pEFGP vector, were mixed and dried in a speed vacuum concentrator. The dried DNA was resuspended, ligated with T4 DNA ligase (Promega, Madison, WI), and transformed into TOP10 One Shot Ultracompetent Escherichia coli bacterial host cells according to manufacturer's instructions (Invitrogen, Carlsbad, CA). Several colonies were picked; miniprep DNA was prepared using the GFX Micro Plasmid Preparation kit (Amersham Biosciences Inc., Piscataway, NJ), and the presence of an insert was verified by gel electrophoresis. Three clones were checked by automated DNA sequencing on an ABI machine using BigDye Terminator chemistry. One clone was then amplified using the GenElute Endotoxin-Free Midi Prep kit (Sigma-Aldrich). Transfection of 20 µg of purified plasmid DNA was carried out by electroporation (Electroporator II; Invitrogen) according to the manufacturer's instructions into CHO-PAM28 cells—CHO cells stably expressing the aequorine gene (Euroscreen SA). After selection by G418 (Invitrogen), 24 individual clones were picked and further amplified for functional characterization.
Visualization of Endocytosis of the MTLR-EGFP
CHO cells containing the MTLR-EGFP were grown on four-well coverglass chambers until 80% confluency. After washes with ice-cold BSA medium, digital pictures were made with an Olympus camera on an inverted Nikon microscope (40x oil) equipped with a fluorescence unit and filtered with filtercube B-2A (EX BP540–580, DM RK595, EM BA 600–610). BSA medium containing 10-5 M compound of interest was added, after which the cells were gently shaken at 4°C for 1 h for equilibration binding, followed by 1 h incubation at 37°C. Internalization was stopped by washing the cells with ice-cold PBS. Pictures of the same cells were taken. For quantification, the fluorescent grayscale pictures were converted to black and white. The percentage black and white at the membrane and in the cytosol was calculated for 10 cells per condition before and after stimulation using Scion Image (Scion Corporation, Frederick, MD) and compared with control. Each condition was evaluated in three different experiments. Means were compared using an unpaired t test (PRISM; GraphPad Software Inc.).
Quantification of MTLR-EGFP Internalization
The capacity of different compounds to promote internalization was examined by adding the compound of interest (10-5 M) to the CHO cells containing the MTLR-EGFP in 12-well plates for 60 min at 37°C. Surface-bound ligands were removed with a gentle acid wash (50 mM sodium citrate, 0.2 mM sodium phosphate, 90 mM NaCl, and 0.1% bovine serum albumin, pH 5.0; 10 min, 4°C), which does not affect subsequent receptor binding, and then a radioreceptor binding assay was performed (125I-motilin, 0.3 nM, 4 h at 4°C) to measure receptors remaining at the cell surface. Internalized receptors were expressed as a percentage loss of cell surface binding compared with cells not exposed to the compound of interest. Means ± S.E.M. were compared using an unpaired t test (PRISM; GraphPad Software Inc.).
Contractility Studies
Integral rabbit duodenal segments (1.5–2 cm) were vertically suspended in tissue baths containing Hepes buffer pH 7.4 (11.6 mM Hepes, 11.5 mM glucose, 137 mM NaCl, 5.9 mM KCl, 1.2 mM CaCl2, and 1.2 mM MgCl2) continuously gassed with 100% O2 and kept at 37°C. Contractions were recorded isotonically using HP 7DCDT-1000 transducers from Hewlett Packard (Palo Alto, CA) and a displacement control unit obtained from Janssen Scientific Instrument Division (Beerse, Belgium). Signal output was connected to a recorder and to a computer and sampled for digital analysis using the WinDaq data acquisition system and a DI-2000 PGH card (Dataq Instruments, Akron, OH).
Strips were equilibrated in the tissue bath until a stable response to acetylcholine (ACh, 0.1 mM) was obtained. Strips were then stimulated with either ABT-229 (69 nM), KOS1326 (0.17 µM), EM-A (14.1 µM), ME4 (0.44 µM), or EM-523 (66 nM) at a concentration corresponding to five times their potency to induce contractions. After 4 min, strips were washed out two times and washed again 2', 6', and 10' after the first wash out. The washing procedure was then repeated every 10 min until 1 h after the application of the agonists. Strips were then stimulated for a second time with the compound of interest and washed again according to the procedure described above. After the third application, strips were not washed out but a supramaximal dose of motilin (1 µM) followed by a supramaximal dose of ACh (0.1 mM) was applied. Desensitization was expressed as a percentage of the response of the agonists obtained after the first application. The response to motilin at the end of the experiment was expressed relative to the response to ACh. All experiments were approved by the Ethical Committee of the University of Leuven.
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Results |
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To investigate which structural elements underlie the stronger desensitizing potency of ABT-229 in comparison to EM-A, several EM-A derivatives were tested, containing one or two ABT-229-like modifications (Fig. 2). The modifications include the presence of an enol configuration (ME4, ME67, EM-523, and KOS1326), the presence of an ethyl group at the amine group of the desosamine sugar (ME36, EM-523), and the lack of a hydroxyl at position 12 of the lactone ring (ME67) or at position 4'' of the cladinose sugar (KOS1326).
Figure 3 shows that compounds with an enol configuration such as ABT-229, ME4, EM-523, ME67, and KOS1326 induced a more profound desensitization at 10-5 M than EM-A. In contrast, desensitization was seemingly absent with ME36, which compared with EM-A, contains only a modification at the amine group of the desosamine sugar.
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; Thielemans et al., 2002). Therefore, although the pretreatment concentration was sufficient to induce a maximal response for all compounds tested, one could argue that less potent compounds may require higher pretreatment concentrations. To fully discriminate between potency and ability to desensitize, we therefore established a "concentration response curve of desensitization" for each motilide. Figure 4 shows the effect of pretreatment of the CHO-MTLR cells with increasing concentrations of motilin, EM-A, or ABT-229 on the maximal response to a subsequent concentration response curve to motilin.
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Although ABT-229 is 8.5 times less potent than motilin in activating the cells (pEC50: 8.46 ± 0.08 versus 9.39 ± 0.03), it is 10.2 times more potent in inducing desensitization (pDC50: 8.78 ± 0.03 versus 7.77 ± 0.08). Thus, potency does not seem to solely determine the ability to desensitize. To further illustrate this point, Table 1 shows the difference between the pDC50 and pEC50 values, referred to as value.
Five compounds have a similar value (motilin, 1.62 ± 0.15; EM-A, 2.33 ± 0.28; ME4, 2.18 ± 0.42; ME67, 2.09 ± 0.25; and EM-523, 2.20 ± 0.28) indicating that there is a relationship between potency and desensitizing ability (Pearson's r = 0.9054). However, for ABT-229 and KOS1326, the value is significantly lower than in these compounds (p < 0.01) and for ME36, it is significantly higher (p < 0.01).
Binding of Motilides with the MTLR. Because both activation and desensitization result from the interaction of the ligand with its receptor, we investigated whether the higher than expected ability of ABT-229 and KOS1326 to desensitize could be explained by a higher affinity for the motilin receptor. The pIC50 values (Table 1) calculated from the displacement curves did not reveal an increased affinity for ABT-229 or KOS1326.
Comparison of MTLR-EGFP and Wild-Type Receptor. Because desensitization involves receptor internalization, an EGFP-tagged motilin receptor was generated and stably expressed in CHO cells to follow receptor trafficking. The cells also expressed the Ca2+ indicator apoaequorin (MTLR-EGFP), which allowed us to determine whether GFP interfered with signal transduction and desensitization.
For the wild-type receptor and the MTLR-EGFP, the order of potency to induce Ca2+ release is: motilin>ABT-229>EM-A (Fig. 5A). The absolute potency (pEC50) of all compounds is 9.17 ± 1.14 (motilin), 8.12 ± 0.17 (ABT-229), and 6.45 ± 0.08 (EM-A) in the MTLR-EGFP and is somewhat lower than in the MTLR.
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Internalization of the EGFP-Tagged MTLR. In cells expressing the EGFP-tagged receptor, changes in the fluorescence distribution after stimulation with the ligands at 10-5 M were studied by fluorescence microscopy. Images obtained before and after stimulation are shown in Fig. 6A.
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The images were analyzed and the percentage change in fluorescence in the cytosol and in the membrane owing to stimulation was calculated (Fig. 6B). All agonists induced a significant cytosolic increase and a significant decrease of membrane fluorescence compared with control (p < 0.005). The cytosolic increases were similar for motilin, ABT-229, and EM-A (22 ± 2, 25 ± 2, and 20 ± 2%, respectively, p < 0.0001 versus control). In contrast, ABT-229 (25 ± 2%) decreased membrane fluorescence significantly more compared with motilin (16 ± 2%, p < 0.01), which in turn induced a more significant decrease compared with EM-A (8 ± 2%, p < 0.01).
Radioligand Binding with the EGFP-Tagged MTLR. To validate the semiquantitative data of the analysis of the fluorescence distribution, the more quantitative method of receptor binding was used to measure ligand-induced endocytosis. Radioligand binding with 125I-motilin was performed after pretreatment of MTLR-EGFP with 10-5 M motilin, ABT-229, or EM-A for 20 and 60 min at 37°C (Fig. 7). Residual binding after 20-min prestimulation was 44.9 ± 2.3% (motilin), 30.8 ± 3.8% (ABT-229), and 124.3 ± 2.0% (EM-A) and was further reduced to 30.8 ± 3.3% (p = 0.0029), 21.3 ± 0.8% (p < 0.0001), and 96.1 ± 4.4% (p < 0.0001), respectively, after prestimulation for 60 min.
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Contractility Studies. The desensitizing properties of ABT-229, KOS1326, EM-A, ME4, and EM-523 were also compared in vitro in the tissue bath by determining the contractile response of segments of rabbit duodenum to three applications with a 1-h interval. Figure 8 shows that for all compounds the contractile response decreases, but the effect was most pronounced with ABT-229; the third application resulted in a contraction that was only 8.92 ± 0.96% of its first application. EM-A is at the other end with 86.50 ± 4.22% (n = 3). Compounds EM-523 and ME4 showed a comparable decrease after the third application (56.58 ± 4.71 and 55.20 ± 8.86%, respectively, n = 4).
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Discussion |
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As a general rule, ligands cause both receptor-mediated signaling and signal desensitization. In the case of G protein-coupled receptors, a ligand signals by activating its receptor, which in turn activates a second messenger (G protein) inside the cell. G protein-coupled receptor desensitization involves the uncoupling of receptors from their heterotrimeric G proteins, resulting in the internalization (endocytosis) of receptors to endosomes and down-regulation (Ferguson, 2001).
The events of activation and desensitization have been linked in many receptor systems, among which the 
adrenoceptor has been characterized best (Benovic et al., 1988). Our data confirm that there is indeed a correlation between the ability to activate and desensitize, e.g., EM-A is less potent than the enol ether derivatives ME4, EM-523, and ME67 and also induces less desensitization. However, the results with ABT-229 suggest a decoupling of activation and desensitization. Such discrepancies have also been described for the µ-opioid receptor, which is related to the motilin receptor, in the sense that both receptors can be stimulated by a peptide (motilin versus enkephalin) and by a naturally occurring nonpeptide (motilides versus morphine, methadone, etorphine, etc.). The desensitization of the µ-opioid receptor mediated by methadone and L-
-acetyl methadone was more pronounced than for morphine and was disproportionate to their efficacies (Yu et al., 1997). Furthermore, functional desensitization of the monkey D1A dopamine receptor cannot be predicted reliably from the agonist potency for stimulating adenylate cyclase (Lewis et al., 1998).
The general paradigm that activation and internalization are coupled is based on studies on 2-adrenergic and muscarinic receptors demonstrating that partial agonists cause less internalization than full agonists and the amount of receptor internalization caused by an agonist generally correlates with coupling efficiency to G proteins (Toews and Perkins, 1984; Thompson and Fisher, 1990; Szekeres et al., 1998). Again, exceptions have been described (Mahan et al., 1985; Barak et al., 1994). Perhaps the best evidence that G protein coupling is not required for receptor endocytosis is provided by the observation that in S49 murine lymphoma cell lines, which either lack Gs or have point mutations preventing receptor/G protein interactions, 2-adrenergic internalization in response to agonist stimulation is normal (Mahan et al., 1985).
In our data, the discrepancy between activation and desensitization is translated into a discrepancy between activation and internalization. Similarly, observations with the - and µ-opioid receptor in transfected cells and enteric neurons in intact animals show that both peptidyl (enkephalin) and nonpeptidyl (etorphine) agonists stimulate receptor endocytosis in the expected manner. Remarkably, the high affinity µ-opioid receptor agonist morphine does not cause detectable internalization, even at concentrations that strongly inhibit adenylate cyclase (Keith et al., 1996; Sternini et al., 1996).
Our results indicate that desensitization and internalization of the motilin receptor are coupled in line with the paradigm. However, recently it was shown in the same cellular context that morphine does desensitize the µ-opioid receptor without causing internalization (Borgland et al., 2003). Therefore, it has been suggested that receptor endocytosis is a mechanism for resensitization and down-regulation rather than desensitization (Tsao et al., 2001). As the motilin receptor in our study is stimulated for 1 h to cause desensitization, it cannot be excluded that the activity that is still measured is the sum of desensitization, internalization, resensitization, and down-regulation.
All of these apparently diverse effects can be accounted for in terms of the "ensemble theory" (Kenakin, 2002a,b). This theory states that a receptor can adopt numerous conformations, each unfolding and exposing different regions to the intracellular apparatus and each associated with specific functions (activation, desensitization, internalization, etc.). These conformations may differ, even for ligands with similar affinity and related structures, and may lead, for example, to different desensitizing properties despite similar activation potencies. Apparently our data illustrate this point because ABT-229, structurally closely related to EM-523, ME4, and ME67 and with a comparable potency, causes stronger desensitization. Our data also suggest that the presence of an ethyl at the amine group of the desosamine sugar, as in ME36, reduces the desensitizing ability, whereas removal of the hydroxyl at position 4'' of the cladinose sugar, as in ABT-229 and KOS1326 but not at position 12 of the lactone ring as in ME-67, causes an increase.
Internalization was also measured by receptor binding studies. Prestimulation with EM-A induced receptor up-regulation. An increase in receptor density after ligand stimulation has been demonstrated for A1 adenosine receptors after antagonist treatment (Ciruela et al., 1997), and inverse agonists can up-regulate histamine H2 receptors (Alewijnse et al., 1998), cannabinoid receptors (Bouaboula et al., 1999), and opioid receptors (Zaki et al., 2001). Both agonist and antagonist treatments have been shown to up-regulate dopamine receptors (Filtz et al., 1994; Zhang et al., 1994; Geurts et al., 1999).
Our data support the hypothesis that tachyphylaxis might underlie the disappointing outcome of motilides in clinical trials. Internalization of the motilin receptor might explain why the high blood levels of ABT-229 were not able to induce gastric contractions and, hence, accelerate gastric emptying of a second meal in healthy subjects (Verhagen et al., 1997). However, the effects of ABT-229 have not just been described as nonexistent; moreover a worsening of symptoms has been reported by patients with functional dyspepsia (Talley et al., 2000) and diabetic gastroparesis (Talley et al., 2001). This might lead to the conclusion that ABT-229 is still active (Camilleri, 2002) after a 4-week treatment, apparently contradicting the occurrence of tachyphylaxis. This paradox might be resolved if one considers the long-term action of ABT-229 as the induction of desensitization rather than activation. Indeed, when motilin receptors are not present on the cell surface, even endogenous motilin will not be able to induce contractions. The observation that less antral contractions after a second meal occur in healthy volunteers treated with ABT-229 compared with placebo (Verhagen et al., 1997) is consistent with this hypothesis. A down-regulation of motilin receptors has also been shown in rabbits that had been treated with ABT-229 for 4 weeks (Depoortere et al., 1999). Whether this underlies the worsening of symptoms is still to be proven, as gastric emptying and antral contractility have not been measured at the end of both studies by Talley et al. (2000, 2001). Besides gastric emptying, impaired accommodation and hypersensitivity are also considered as underlying pathophysiological mechanisms that can be targeted for treatment of dyspeptic patients. A higher tone, increased intensity, and frequency of spontaneous contractions in the proximal stomach and a reduction of meal-induced relaxation have been described for erythromycin (Bruley des Varannes et al., 1995; Distrutti et al., 1999; Piessevaux et al., 2001). It has therefore been pointed out that the worsening of symptoms might also be due to an effect of ABT-229 on gastric fundus relaxation (Tack and Peeters, 2001). Combining both hypotheses, the adverse effects might be caused by (acute) spatial nonspecific action of ABT-229 in decreasing fundus relaxation and by (chronic) functional nonspecific action in decreasing antral motility by tachyphylaxis.
In conclusion, our data suggest that it should be possible to develop motilides with high potency and less desensitizing ability. Therefore, it is indicated that before attempting large-scale clinical trials, the desensitizing potency of a motilide should be evaluated.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EM-A, erythromycin-A; CHO, Chinese hamster ovary; MTLR, motilin receptor; EGFP, enhanced green fluorescent protein; ABT-229, N-ethyl, N-methyl 4'' deoxy EM-B enolether; MTLR-EGFP, EGFP-tagged motilin receptor; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PCR, polymerase chain reaction; ACh, acetylcholine; ME4, EM-A enolether; ME36, N-ethyl, N-methyl EM-A; ME67, EM-B enolether; EM523, N-ethyl, N-methyl EM-A enolether; KOS1326, 4'' deoxy EM-A enolether.
Address correspondence to: T. L. Peeters, Gut Hormone Lab, Gasthuisberg O & N, B-3000 Leuven, Belgium. E-mail: theo.peeters{at}med.kuleuven.ac.be
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) and EGFP-tagged MTLR (
) by motilin and motilides. The response to motilin after pretreatment with a ligand at 10-5 M is expressed as percentage versus control (no pretreatment). Values are mean ± S.E.M. of three experiments in duplicate.
