ANP32A represses Wnt signaling across tissues thereby protecting against osteoarthritis and heart disease.

OBJECTIVES: To investigate how ANP32A, previously linked to the antioxidant response, regulates Wnt signaling as unraveled by transcriptome analysis of Anp32a-deficient mouse articular cartilage, and its implications for osteoarthritis (OA) and diseases beyond the joint. METHODS: Anp32a knockdown chondrogenic ATDC5 cells were cultured in micromasses. Wnt target genes, differentiation markers and matrix deposition were quantified. Wnt target genes were determined in articular cartilage from Anp32a-deficient mice and primary human articular chondrocytes upon ANP32A silencing, using qPCR, luciferase assays and immunohistochemistry. Co-immunoprecipitation, immunofluorescence and chromatin-immunoprecipitation quantitative PCR probed the molecular mechanism via which ANP32A regulates Wnt signaling. Anp32a-deficient mice were subjected to the destabilization of the medial meniscus (DMM) OA model and treated with a Wnt inhibitor and an antioxidant. Severity of OA was assessed by cartilage damage and osteophyte formation. Human Protein Atlas data analysis identified additional organs where ANP32A may regulate Wnt signaling. Wnt target genes were determined in heart and hippocampus from Anp32a-deficient mice, and cardiac hypertrophy and fibrosis quantified. RESULTS: Anp32a loss triggered Wnt signaling hyper-activation in articular cartilage. Mechanistically, ANP32A inhibited target gene expression via histone acetylation masking. Wnt antagonist treatment reduced OA severity in Anp32a-deficient mice by preventing osteophyte formation but not cartilage degradation, contrasting with antioxidant treatment. Dual therapy ameliorated more OA features than individual treatments. Anp32a-deficient mice also showed Wnt hyper-activation in the heart, potentially explaining the cardiac hypertrophy phenotype found. CONCLUSIONS: ANP32A is a novel translationally relevant repressor of Wnt signaling impacting osteoarthritis and cardiac disease.

[Flemish guideline for the prevention and use of seclusion and restraint]. / Vlaamse richtlijn voor de preventie en het gebruik van afzondering en fixatie.

BACKGROUND: International consensus states that seclusion and restraint should only be applied as briefly and as little as possible. However, audits by the Care Inspectorate show that this is not always the case in Flemish mental health care (MHC). AIM: To describe the development of a multidisciplinary guideline for the prevention and application of seclusion and restraint in inpatient MHC, underpinned by both clinical-scientific and legal evidence. METHOD: The GRADE method formed the basis for the development of the guideline. To integrate both types of evidence, two research phases were added. This article provides an overview of the challenges involved in implementing this interdisciplinary method. RESULTS: There are gaps in both clinical-scientific and legal evidence. Nevertheless, the study resulted in a comprehensive guideline because we underpinned the recommendations with practice- and experience-based expertise of the Flemish stakeholders, and integrated the clinical-scientific and legal evidence. CONCLUSION: Focus on implementation research and a clear legal framework for Flanders are necessary to safeguard the (human) rights of MHC users, also in the event of aggression and escalation.

Electronic visual analogue scales for pain, fatigue, anxiety and quality of life in people with multiple sclerosis using smartphone and tablet: a reliability and feasibility study.

OBJECTIVE: To explore the reliability and feasibility of electronic visual analogue scales in people with multiple sclerosis (MS) and healthy individuals. DESIGN: Cross-sectional observational study Setting: Clinical setting Subjects: Convenience sample of 52 people with MS and 52 matched healthy controls Interventions: NA Main measures: Participants scored 15 statements assessing fatigue, pain, anxiety and quality of life on an electronic visual analogue scale (eVAS), either using a smartphone or a tablet (randomly allocated). To check for test-retest reliability, statements were administered in two separate randomly ordered groups. Subjects completed a feasibility questionnaire. RESULTS: Mean (SD) eVAS scores ranged from 35 (28.1) to 80 (22.1) in MS group, and from 57 (28.0) to 86 (13.2) in controls. Intra Class Correlations ranged from 0.73 to 0.95 in MS sample; 0.61 to 0.92 in controls. For most statements, Bland-Altman plots indicated no systematic error, but relatively large random error of the eVAS scores (exceeding 20mm). Considerable ceiling effects (i.e. better health) were found in healthy controls. Similar reliability was found among smartphone or tablet, different demographic groups and the experience-groups. CONCLUSION: Electronic visual analogue scales are reliable and useful for people with MS to register fatigue, pain, anxiety and quality of life.

Effect of peripheral obestatin on food intake and gastric emptying in ghrelin-knockout mice.

BACKGROUND AND PURPOSE: The finding that obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's stimulatory effect on food intake and gastric emptying has been questioned. The effect of obestatin has been mostly investigated in fasted rodents, a condition associated with high blood levels of ghrelin which may mask the effect of obestatin. We therefore investigated the effect of obestatin on food intake, gastric emptying and gastric contractility in ghrelin knockout mice. EXPERIMENTAL APPROACH: The effect of obestatin on 6-h cumulative food intake was studied in fasted wildtype (ghrelin+/+) and ghrelin knockout (ghrelin-/-) mice. In both genotypes, the effect of obestatin and/or ghrelin was studied in vivo on gastric emptying measured with the (14)C-octanoic acid breath test and in vitro on neural responses elicited by electrical field stimulation (EFS) of fundic smooth muscle strips. KEY RESULTS: Administration of obestatin did not influence fasting-induced hyperphagia or gastric emptying in both genotypes. Injection of ghrelin accelerated gastric emptying in ghrelin+/+ and ghrelin-/- mice but the effect was not reversed by co-injection with obestatin. In fundic strips from ghrelin+/+ and ghrelin-/- mice, ghrelin increased EFS-induced contractions, but obestatin was without effect. However, co-administration with obestatin tended to reduce the excitatory effect of ghrelin in both genotypes. CONCLUSIONS AND IMPLICATIONS: In ghrelin-/- mice, obestatin failed to affect food intake and gastric motility. These results suggest that endogenous ghrelin does not mask the effect of obestatin and confirm that obestatin administered peripherally is not a major regulator of satiety signalling or gut motility.

Effect of peripheral obestatin on gastric emptying and intestinal contractility in rodents.

Obestatin has recently been discovered in the rat stomach. It is encoded by the ghrelin gene and has been claimed to be a functional opponent of ghrelin and to be the natural ligand of the GPR39 receptor. The latter could not be confirmed by Holst et al. (Endocrinology, 2006). Yet, in GPR39 knockout mice, gastric emptying is accelerated. We verified the effects of obestatin on gastric emptying and intestinal contractility in rodents. Gastric emptying was measured with the (14)C octanoic breath test in mice. In vitro, the effect of obestatin was studied on electrically stimulated and non-stimulated strips from the fundus and small intestine of mice and rats. Obestatin (60, 125, 250 nmol kg(-1)) did not affect gastric emptying parameters (T(half) and T(lag)) and did not inhibit the prokinetic effects of ghrelin. Mouse and rat intestinal and fundic smooth muscle strips did not respond to obestatin either in the absence or in the presence of electrical field stimulation. Obestatin (125 nmol kg(-1)) did not inhibit fasting-induced hyperphagia. Our results suggest that peripheral obestatin is not a satiety signal that plays a role in the regulation of gastric emptying and do not support the concept that obestatin is a physiological opponent of ghrelin.

Evolution of vocal fold nodules from childhood to adolescence.

SUMMARY: Bilateral (quasi) symmetrical lesions of the anterior third of the vocal folds, commonly called vocal fold nodules (VFNs) are the most frequent vocal fold lesions in childhood caused by vocal abuse and hyperfunction. This study evaluates their long-term genesis with or without surgery and voice therapy. A group of 91 postmutational adolescents (mean age, 16 years), in whom VFNs were diagnosed in childhood, were questioned to analyze the evolution of their complaints. Thirty four of them could be clinically reexamined by means of the European Laryngological Society-protocol, including a complete laryngological investigation and voice assessment. A total of 21% of the questioned group (n=91) had voice complaints persisting into postpubescence with a statistically significant difference (P

Role of endogenous ghrelin in the hyperphagia of mice with streptozotocin-induced diabetes.

Ghrelin is an orexigenic peptide involved in the regulation of energy homeostasis. To investigate the role of ghrelin in the hyperphagia associated with uncontrolled streptozotocin-induced diabetes, food intake was followed in diabetic ghrelin knockout (ghrelin(-/-)) and control wild-type (ghrelin(+/+)) mice and diabetic Naval Medical Research Institute noninbred Swiss mice treated with either saline or the ghrelin receptor antagonist, D-Lys3-GH-releasing peptide-6 (D-Lys3-GHRP-6) for 5 d. In diabetic ghrelin(-/-) mice, hyperphagia was attenuated, and the maximal increase in food intake was 50% lower in mutant than in wild-type mice. The increased food intake observed during the light period (1000-1200 h) in ghrelin(+/+) mice was abolished in mutant mice. Diabetic ghrelin(-/-) mice lost 12.4% more body weight than ghrelin(+/+) mice. In diabetic ghrelin(+/+) mice, but not in ghrelin(-/-) mice, the number of neuropeptide Y (NPY)-immunoreactive neurons was significantly increased. Diabetic Naval Medical Research Institute noninbred Swiss mice were hyperphagic and had increased plasma ghrelin levels. Treatment with D-Lys3-GHRP-6 reduced daily food intake by 23% and reversed the increased food intake observed during the light period. The change in the number of NPY- (2.4-fold increase) and alpha-MSH (1.7-fold decrease)-immunoreactive hypothalamic neurons induced by diabetes was normalized by D-Lys3-GHRP-6 treatment. Our results suggest that enhanced NPY and reduced alpha-MSH expression are secondary to the release of ghrelin, which should be considered the underlying trigger of hyperphagia associated with uncontrolled diabetes.

Induction of secondary structure in the peptide hormone motilin by interaction with phospholipid vesicles.

Motilin is an intestinal peptide hormone that binds to a membrane bound receptor located in the gut tissue. Circular dichroism (CD) was used to study the interaction between either porcine or rabbit motilin or a 1-16 fragment of porcine motilin, with model systems of lipid membranes: sodium dodecyl sulphate (SDS), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The CD measurements show significant induction of secondary structure in both motilins and the fragment when negatively charged vesicles (DOPG) or negatively charged micelles (SDS) were present. In contrast, neutral DOPC vesicles did not induce any change in the secondary structure compared to water, in which a random-like secondary structure dominates. The induced secondary structure in the presence of DOPG vesicles is very close to that induced by a mixed aqueous solution containing 30% hexafluoroisopropanol, in which previous NMR-studies have resulted in a three-dimensional solution structure of porcine motilin. In both porcine and rabbit motilin the alpha-helix content is about 50%. This is in agreement with the presence of an amphipathic helix in the C-terminal half of motilin interacting with phospholipid membranes. The interaction appears to be mainly electrostatic in nature, and does not induce any significant alterations in the vesicle, as monitored by EPR studies of spin labels located at the fifth carbon atom of the backbone in a stearic acid molecule. In the 1-16 fragment the alpha-helical content induced by DOPG and SDS is only about 20%.

Influence of ghrelin on gastric emptying and meal-related symptoms in idiopathic gastroparesis.

BACKGROUND: Ghrelin, the endogenous ligand of the growth hormone secretagogue receptor, is released from the stomach. Animal studies suggest that ghrelin stimulates gastrointestinal motor activity. AIM: To investigate the influence of ghrelin on gastric emptying rate and meal-related symptoms in idiopathic gastroparesis. METHODS: In six patients with idiopathic gastroparesis, a breath test was used to measure gastric emptying rates (t(1/2)) for solids and liquids after administration of saline or ghrelin 40 microg/30 min in a double-blind, randomized fashion. At each breath sampling, the patient was asked to grade the intensity of six different symptoms (epigastric pain, bloating, postprandial fullness, nausea, belching and epigastric burning) and these were added to obtain meal-related symptom severity score. RESULTS: Ghrelin significantly enhanced liquid emptying (t(1/2): 86 +/- 7 vs. 53 +/- 6 min, P = 0.02) and tended to enhance solid emptying (144 +/- 45 vs. 98 +/- 15 min, P = 0.06). Ghrelin pre-treatment significantly decreased cumulative meal-related symptom score (196 +/- 30 vs. 136 +/- 23, P = 0.04) and individual scores for fullness (55 +/- 8 vs. 39 +/- 8, P = 0.02), and for pain (40 +/- 8 vs. 16 +/- 5, P < 0.05). CONCLUSIONS: In idiopathic gastroparesis, administration of ghrelin enhances gastric emptying and improves meal-related symptoms. These observations suggest a potential for ghrelin receptor agonists in the treatment of gastroparesis.

Initial staging of malignant melanoma by positron emission tomography and sentinel node biopsy.

UNLABELLED: By a retrospective study and literature review we aimed to evaluate the accuracy of Sentinel Node Biopsy (SNB) and F-18-fluorodeoxyglucose positron emission tomography (PET) for early detection of lymph node metastases. MATERIAL AND METHODS: Every patient presenting with a malignant melanoma without clinical lymph node involvement and a Breslow index over 1 mm or a recurrence was subjected to a preoperative PET scan and a sentinel node biopsy. Over a period of 10 months, 5 patients were included. They were submitted to conventional staging techniques, PET and SNB. RESULTS: In none of the patients the PET scan showed signs of lymph node involvement or distant metastases. However, two patients, both with a Breslow index of 1.4, had micrometastases in the sentinel node. CONCLUSION: Already in this small group of patients, PET scanning missed two metastases (40%). This is confirmed by several recent publications, stating that the resolution of positron emission tomography is about 5 mm and thus insufficient to detect micrometastases. Several larger series showed a sensitivity of PET to detect lymph node involvement of 15-50%. Therefore we conclude that PET is of limited use in these patients without palpable lymph nodes. Sentinel node biopsy however proves to be a useful tool and should be considered in the initial staging of malignant melanoma without palpable lymph node or distant metastases.

Somatostatin analog octreotide (SMS 201-995) prevents the decrease in blood pressure after oral glucose loading in the elderly.

In elderly subjects blood pressure (BP) may fall after a meal. The mechanism of this phenomenon is unknown, but it has been suggested that it may be mediated by insulin and/or vasoactive gut hormones. We studied in normo- and hypertensive elderly subjects the effects of the synthetic long-acting somatostatin analog octreotide (SMS 201-995) on the BP reduction that follows oral glucose administration in subjects who are recumbent and on their postglucose plasma vasoactive intestinal polypeptide (VIP) and insulin concentrations. After placebo treatment, mean arterial pressure fell by 15 +/- 1 mm Hg (P less than 0.001) in the 10 hypertensive subjects and by 7 +/- 2 mm Hg (P less than 0.01) in the 10 normotensive subjects. In contrast, when 50 micrograms octreotide were given sc, BP did not change significantly in either group. Oral glucose did not induce a rise in plasma VIP after either octreotide or placebo administration. The postglucose rises in plasma glucose concentrations were similar after octreotide and placebo treatments in both groups. After placebo administration the postglucose plasma insulin levels increased from 79 to 519 pmol/L in the hypertensive subjects and from 63 to 464 pmol/L in the normotensive subjects, whereas after octreotide treatment plasma insulin increased little in either group. These data indicate that treatment with octreotide holds promise for patients with symptomatic postprandial hypotension, and that VIP does not seem to play a role in this phenomenon.

Isolation and sequencing of the cDNA encoding the motilin precursor from sheep intestine.

The nucleotide sequence of sheep prepromotilin has been determined from cDNA clones. The nucleotide sequence revealed an open reading frame of 345 nucleotides encoding 115 amino acids. The amino acid sequence deduced from the nucleotide sequence consists of a 25 amino acid signal peptide, followed by the 22 amino acid motilin sequence, an endoproteinase cleavage site (Lys23-Lys24) and a 66 amino acid motilin associated peptide (MAP). Compared with human and pig motilin we observed two substitutions at positions 10 (Leu-->Val) and 19 (Asn-->Tyr). The second one may explain the poor cross-reactivity of ovine motilin with C-terminally directed antibodies against porcine motilin. The sheep motilin precursor exhibits the same structure as the motilin precursors from rabbit, pig and man. However, while there is considerable identity in the amino acid sequences as well as in the nucleotide sequences of the signal peptide and motilin, the MAP strongly differs between the species. This may be a result of 'mosaic evolution' at the molecular level.

Sequence and characterization of cDNA encoding the motilin precursor from chicken, dog, cow and horse. Evidence of mosaic evolution in prepromotilin.

Motilin is involved in the regulation of the fasting motility pattern in man and in dog, but may have a different role in other species. Immunoreactive motilin has been demonstrated in several species, but the sequence is mostly unknown. The aim of this study was to isolate and sequence the cDNA encoding the motilin precursor from several mammalian species and from chicken. Total RNA was isolated from the duodenal mucosa of the chicken, dog, cow and horse. In each case single stranded cDNA was synthesized. Motilin cDNA fragments were amplified by PCR, ligated into a plasmid and cloned. Clones which were positive after screening with an appropriate (32)P-labeled probe were sequenced. The 5'- and 3'-ends were determined by the rapid amplification of cDNA ends (RACE) method. Analysis of the cDNAs revealed an open reading frame coding for 115 (chicken and cow), or 117 (dog and horse) amino acids. It consists of a 25 amino acid signal peptide, motilin itself, and a 68 (chicken and cow) or 70 (dog and horse) amino acid motilin associated peptide (MAP). As in all motilin precursors already sequenced (man, monkey, pig and rabbit), an endoproteinase cleavage site is present at Lys(23)-Lys(24). Comparison of all known sequences shows considerable identity in amino acid and nucleotide sequence of the signal peptide and motilin. However, the MAPs differ not only in length but also, more strongly, in amino acid and nucleotide sequence. Our study demonstrates that the N- and C-terminal regions of the motilin precursor have evolved at different rates, which is evidence for 'mosaic evolution'.

Isolation and sequence of cDNA encoding the motilin precursor from monkey intestine. Demonstration of the motilin precursor in the monkey brain.

The motilin precursor cDNA has been isolated and sequenced from a cDNA library prepared from monkey small intestine. The sequence indicates a 345 bp open reading frame, a 63 bp 5' untranslated region and a 154 bp 3' untranslated region. The sequence encodes a 115 amino acid motilin precursor composed of a 25 amino acid signal peptide, the 22 amino acid motilin peptide and a 68 amino acid motilin associated peptide (MAP). Compared with the human motilin precursor cDNA, there are two amino acid substitutions in the signal peptide, one in motilin and four in the MAP. The presence of the motilin precursor in hypothalamus, hippocampus and cerebellum was demonstrated by RT-PCR.

Identification and expression of the motilin precursor in the guinea pig.

Motilin has never been isolated from rodents, the most frequently used laboratory animals, despite several attempts. We have isolated and sequenced the motilin precursor from duodenal mucosa of guinea pig (GenBank accession number AF323752) and studied its expression in several tissues. The percent homology with human motilin is the lowest yet observed due to several unique substitutions in the C-terminal end. As expected, the precursor was present in the gut mucosa with the exception of the gastric corpus. It was also present in medulla oblongata, nucleus of the solitary tract, hypophysis, spinal cord, hypothalamus, and cerebellum but not in the cerebral cortex. For the first time we demonstrated motilin expression in the thyroid.

Intravenous erythromycin dramatically accelerates gastric emptying in gastroparesis diabeticorum and normals and abolishes the emptying discrimination between solids and liquids.

Erythromycin, a macrolide antibiotic, has recently been shown to have a motilin like effect on gastrointestinal muscle strips. In this study, we have evaluated the effect of erythromycin on patients with delayed gastric emptying and healthy subjects using the dual radionuclide technique. Twelve patients with gastroparesis diabeticorum and ten healthy age- and sex-matched controls were studied. Gastric emptying of solids and liquids was determined using 99mTc-SC scrambled egg and 111In-DTPA in water. Following a baseline study and on a separate day, each patient and control received a 15-min i.v. perfusion of erythromycin starting at meal ingestion. Eleven out of the 12 patients were restudied after a 3-wk oral administration. In patients and controls, i.v. erythromycin dramatically accelerated gastric emptying of both solids and liquids which were emptied at the same rate. After chronic oral administration, solid and liquid emptying remained significantly accelerated. Erythromycin appears to be a very powerful gastrokinetic drug. Derived compounds with the gastrokinetic effect and without the antibiotic activity could be useful in dyspeptic patients with delayed gastric emptying.

Rubinstein-Taybi syndrome caused by a De Novo reciprocal translocation t(2;16)(q36.3;p13.3).

Rubinstein-Taybi syndrome (RTS) is a multiple congenital anomalies and mental retardation syndrome characterized by facial abnormalities, broad thumbs, and broad big toes. We have shown previously that disruption of the human CREB-binding protein (CBP) gene, either by gross chromosomal rearrangements or by point mutations, leads to RTS. Translocations and inversions involving chromosome band 16p13.3 form the minority of CBP mutations, whereas microdeletions occur more frequently (approximately 10%). Breakpoints of six translocations and inversions in RTS patients described thus far were found clustered in a 13-kb intronic region at the 5' end of the CBP gene and could theoretically only result in proteins containing the extreme N-terminal region of CBP. In contrast, in one patient with a translocation t(2;16)(q36.3;p13.3) we show by using fiber FISH and Southern blot analysis that the chromosome 16 breakpoint lies about 100 kb downstream of this breakpoint cluster. In this patient, Western blot analysis of extracts prepared from lymphoblasts showed both a normal and an abnormal shorter protein lacking the C-terminal domain, indicating expression of both the normal and the mutant allele. The results suggest that the loss of C-terminal domains of CBP is sufficient to cause RTS. Furthermore, these data indicate the potential utility of Western blot analysis as an inexpensive and fast approach for screening RTS mutations.

GM-611 (Chugai Pharmaceutical).

GM-611 is an erythromycin derivative that acts as an agonist at the motilin receptor. It is being developed by Chugai as a potential treatment for gastric motility disorder [169036], as well as reflux esophagitis, non-ulcer dyspepsia and diabetic gastroparesis [347963]. GM-611 is in phase II trials in the US for reflux esophagitis [322624], [347955], [399349]. GM-611 acts by a novel mechanism whereby it stimulates and promotes peristalsis in the stomach and other segments of the gastrointestinal tract [334994]. The drug was shown to produce a dose-dependent sustained depolarization of rabbit duodenal smooth muscle. Depolarization appeared to be associated with activation of monovalent cation-selective channels [273336]. In December 2000, Credit Suisse First Boston predicted that successful development of GM-611 could lead to sales over $500 million [400228].

The influence of food temperature on postprandial blood pressure reduction and its relation to substance-P in healthy elderly subjects.

Blood pressure (BP) in the elderly may decrease after a meal or oral glucose loading. The mechanism of this phenomenon is still unclear. In addition, the effect of the temperature of a meal on postprandial BP is unknown. However, it has been suggested that vasoactive gastrointestinal peptides are involved in the etiology of postprandial BP reduction. Therefore, we studied the effects of a cold and a warm glucose solution on BP, heart rate, plasma glucose, insulin, and substance-P levels in 15 healthy elderly subjects with a mean age of 74 +/- 3 (SD) years. With an interval of at least 2 days, a warm (50 degrees C) and a cold (5 degrees C) solution (75 g glucose/300 mL water) were given in random order. After the cold glucose loading mean arterial pressure increased by a maximum of 3.9 +/- 1.3 mmHg (P less than 0.01). In contrast, BP decreased after the warm solution by a maximum of 8.0 +/- 1.1 mmHg (P less than 0.001). Neither test had an influence on plasma substance-P levels. Our data suggest that postprandial blood pressure reduction in the elderly is dependent on food temperature. Substance-P does not seem to play a role in this phenomenon.

Motilin receptors of the rabbit colon.

Binding studies with iodinated motilin revealed that in the small intestine motilin receptor density decreased aborally, disappeared in the caecum but returned in the colon and rectum. The highest density was in the distal colon (112 +/-/11 fmol/mg protein). The dissociation constant was the same in all regions (overall mean 1.10 +/- 0.22 nM). The ability of erythromycin-A (EM-A) and of two derivatives, EM-A N-oxide and EM-523, to displace motilin showed no difference between the tissues studied. Their order of potency was: motilin greater than EM-523 greater than EM-A greater than EM-A N-oxide. Proximal circular colonic smooth muscle strips showed maximal contractile responses towards motilin, EM-523 and EM-A of, respectively, 80 +/- 3%, 78 +/- 4% and 84 +/- 2% relative to the maximum obtained with acetylcholine. In proximal longitudinal muscle only a response of +/- 20% was obtained. Similar responses were obtained in the distal colon. The order of potency to induce contractions as reflected in the pED50 values was: motilin (8.03 +/- 0.1) greater than EM-523 (7.55 +/- 0.03) greater than EM-A (5.84 +/- 0.04) in proximal circular colon. The responses were not blocked by TTX (10(-6) M) or atropine (10(-6) M), but were reduced by verapamil (10(-6)M). The abundance of motilin receptors in colonic smooth muscle, if applicable to other species, opens new perspectives for the therapeutic applications of macrolides with motilin agonist properties.