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Aim This study aims to determine, based on existing data, whether the mechanism resulting in liver dysfunction in HELLP syndrome resembles that in Sinusoidal Obstruction Syndrome (SOS). Background HELLP syndrome is a serious pregnancy disorder with high maternal and perinatal morbidity and mortality rates. Because of poor insight in its pathophysiology, particularly that of the liver involvement, clinical management is limited to symptomatic treatment, often followed by termination of pregnancy. SOS is a rare, potentially life-threatening complication of radio and/ or chemotherapy in the preparation of hematopoietic cell transplantation. The etiology of liver dysfunction in SOS is - unlike that in HELLP syndrome - better-understood and seems to be initiated by direct toxic damage and demise of endothelial cells, causing hepatic sinusoidal obstruction and ischemia. Methods We searched Pubmed, Embase and Cochrane for reports on the etiology of HELLP and SOS. This yielded 73 articles, with 14 additional reports from the references listed in these articles. Results The dysfunctional placenta in women developing HELLP initiates a cascade of events that eventually results in liver dysfunction. The placenta releases, besides anti-angiogenetic factors, also necrotic debris and cell-free DNA, a mixture that not only induces systemic endothelial dysfunction as in preeclampsia, but also a systemic inflammatory response. The latter aggravates the endothelio-toxic effects in the systemic cardiovascular bed, amplifying the already increased pro-thrombotic conditions. Particularly in microcirculations with extremely low shear forces, such as in the hepatic sinusoids, this will facilitate microthrombi formation and fibrin deposition eventually resulting in obstruction of the sinusoids similar as in SOS. The latter causes ischemic damage and progressive demise of hepatocytes. Conclusion The available information supports the concept that the liver damage in HELLP and SOS results from sinusoidal ischemia, presumably resulting from partially overlapping pathophysiological mechanisms.

The indoor environmental quality in an operating room (OR) affects not only the patient health but also the well-being of the surgical staff. The main requirement in the OR is to reduce the risk of surgical site infection. To reduce this risk, the indoor air quality must be controlled, which requires technical standards on heating, ventilation and air conditioning (HVAC). Although the technical standards state thermal comfort must be achieved, significant discrepancies between these standards and the thermal comfort standard exist. To evaluate the thermal comfort, we have analyzed these discrepancies and we have performed a survey in 4 major Belgian hospitals. This paper summarizes the technical standards and compares them to the thermal comfort standard and to data gathered on site. Significant discrepancies between the HVAC standards and thermal comfort standard are indicated. The survey shows a similarity between the comfort standard and the actual situation in an OR. The current technical standards cannot lead to acceptable thermal conditions for all members of the surgical team due to their different needs to achieve thermal comfort. The study of such discrepancies can serve as useful input to revise the current HVAC standards and the design of ventilation and luminaire systems.

Heat is transferred through building envelopes by conduction, radiation, and convection. Of these, convective heat transfer is often the weakest ‘link’ in the overall heat transfer model. While conduction and radiation heat transfer are supported by well-established analytical and numerical models, the treatment of convection is much less rigorous. Convection modeling involves solution of fluid dynamics problems in which the complexity of an enclosed space's geometry and the diversity of indoor airflow patterns require many fit-for-purpose convection correlations. These must take into account specifics of the space and of the heating ventilation and air-conditioning (HVAC) system. This paper provides an overview of the current knowledge on the modeling of convective heat transfer in load calculation and building energy simulation programs. The paper also discusses various issues related to the robustness of convection coefficient correlations, and reports on new experiments conducted to test the sensitivity of existing convection correlations. The discussed issues relate to: (1) the non-uniformity of the indoor temperature field; (2) the selection of a proper characteristic dimension; (3) the effect of adiabatic and non-adiabatic obstructions in the zone; and (4) the impact of airflow disturbance.

The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro , MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA 2 value of 9.17 ± 0.01 ( n = 5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 μM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [ 125 I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The p K i of MA-2029 was 8.58 ± 0.04 in the rabbit colon homogenate ( n = 4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo , orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 μg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 μg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.

Backgrounds & aims The motilin receptor (MTLR) is an important therapeutic target for treatment of hypomotility disorders. The negative outcome in clinical trials with the motilin agonist, ABT-229, indicated that desensitization may limit the therapeutic usefulness of motilides. We therefore compared the mechanisms involved in the intracellular trafficking of the MTLR after stimulation with motilin, erythromycin-A (EM-A) or ABT-229. Methods Desensitization was studied by measuring changes in Ca2+ rises and by receptor binding studies in CHO cells co-expressing the Ca2+ indicator apoaequorin and the MTLR, C-terminally tagged with EGFP. Receptor phosphorylation was studied by immunoprecipitation. MTLR-EGFP trafficking to organelles and translocation of β-arrestins were visualized by fluorescence microscopy. Results Agonist-induced desensitization of the MTLR was due to receptor internalization with potencies (p-int50) in the order of: ABT-229 (8.3) > motilin (7.86) > EM-A (4.77) but with no differences in the internalization kinetics (t1/2: ∼25 min). The percentage cell surface receptor loss was more profound after exposure to ABT-229 (88 ± 1%) than to motilin (63 ± 10%) or EM-A (34 ± 2%). For motilin and EM-A MTLR phosphorylation probably occurs via G protein-coupled receptor kinases while for ABT-229 phosphorylation was also protein kinase C dependent. All agonists translocated cytosolic β-arrestin-2 with greater affinity to the plasma membrane than β-arrestin-1. After internalization the MTLR co-localized with transferrin but not with cathepsin D. After stimulation with motilin and EM-A the t1/2 for MTLR resensitization was 3 h and 1 h, respectively but amounted 26 h for ABT-229. Conclusion Our results suggest that the resensitization kinetics determine the desensitization properties of the motilin agonists.

Background & Aims: The G-protein–coupled receptor GPR39 is a member of a family that includes the receptors for ghrelin and motilin. Recently the peptide obestatin was identified as a natural ligand for GPR39. The objective of this study was to gain insight into the biological function of the GPR39 receptor. Methods: GPR39 −/− mice were generated and analyzed. Results: Endogenous GPR39 expression was detected in the brain (septum-amygdala) and the gastrointestinal system (parietal cells, enterocytes, neurons, and pancreas). Gastric emptying of a solid meal (measured by the 14 C octanoic breath test) in GPR39 −/− mice was accelerated significantly with a gastric half-emptying time of 49.5 ± 2.2 minutes compared with 86.9 ± 8.4 minutes in GPR39 +/+ mice. A more effective expulsion of distally located pellets (30%–75% of length) was observed in the colon of GPR39 −/− mice. Four hours after pylorus ligation, the volume of gastric secretion was increased significantly (GPR39 −/− : 638 ± 336 μL; GPR39 +/+ : 225 ± 170 μL), but gastric acid secretion was unchanged. The mature body weight and body fat composition of GPR39 −/− mice was significantly higher compared with GPR39 +/+ mice, but this was not related to hyperphagia because 24-hour food intake did not differ between both genotypes. In contrast, deficiency of the GPR39 receptor led to reduced hyperphagia after fasting. The cholesterol levels were increased significantly in the GPR39 −/− mice. Conclusions: Our data partially confirm and extend the described in vivo effects of obestatin and suggest that this peptide plays a functional role in the regulation of gastrointestinal and metabolic function through interaction with the GPR39 receptor.

The motilin receptor (MTLR) represents a clinically useful pharmacological target, as agonists binding to the MTLR have gastroprokinetic properties. In order to compare the molecular basis for interaction of the MTLR with motilin and with the non-peptide motilin agonist, erythromycin-A (EM-A), the negatively charged E 119 located in the third transmembrane (TM 3 ) region was mutated to D (E119D) and Q (E119Q), respectively, and changes in activity of the mutant receptors were verified. Methods: Each mutant receptor was stably transfected in CHO-cells containing the Ca 2+ indicator apo-aequorin. Receptor activation in response to motilin, EM-A and their analogues was assessed by Ca 2+ -luminescense. Results: In the E119Q mutant, the Ca 2+ response to motilin and EM-A was abolished while in the E119D mutant it was reduced with 62% (motilin) and 81% (EM-A). The pEC 50 values were shifted from 9.65 ± 0.03 to 7.41 ± 0.09 (motilin) and from 6.63 ± 0.12 to 4.60 ± 0.07 (EM-A). Acetylation of the N-terminal amine group as in [ N -acetyl-Phe] 1 mot (1–14), decreased the potency 6.3-fold (WT-MTLR) and 148-fold (E119D). Acetylation of EM-A enol ether induced a more pronounced shift in potency: 7943-fold (WT-MTLR) and 1413-fold (E119D). Conclusion: The comparable loss of affinity of the mutant receptors for motilin and EM-A indicate that these agonists both interact with the TM 3 domain of the MTLR. The results with acetylated derivatives support an ionic interaction between E 119 of the MTLR with the N + of the desosamine sugar in EM-A, but not with the N + of the free amine group in motilin.

Ghrelin and motilin form a new family of structurally related peptides. We compared the gastroprokinetic effects of ghrelin, the ghrelin receptor agonist, growth hormone releasing peptide 6 (GHRP-6), and motilin in rats in vivo and in vitro. Methods Ghrelin, GHRP-6 or motilin (10–150 μg/kg) were injected i.p. and the effects on gastric emptying and transit were measured after intragastric application of Evans blue. In antral and fundic strips the effect of motilin, ghrelin or GHRP-6 was studied during electrical field stimulation (EFS) in the absence and presence of NG-nitro- l -arginine methyl ester hydrochloride ( l -NAME) (300 μM). Results Ghrelin and GHRP-6 but not motilin accelerated gastric emptying and transit in rats. Ghrelin was more potent than GHRP-6 and the dose–response relationship for ghrelin but not for GHRP-6 was bell-shaped. In fundic or antral strips, neural responses to EFS consisted of an on-relaxation that was reversed into a cholinergically mediated contraction by addition of the nitric oxide (NO)-synthase blocker, l -NAME. The post-stimulus off-contraction was cholinergically mediated. Under normal conditions, the ghrelin agonists reduced the on-relaxations in fundic strips and increased the cholinergic off-contractions in antral and fundic strips. The concentration response curves in muscle strips of the fundus were bell-shaped with maximal effects for ghrelin at 1.2 μM (on-responses) and 0.66 μM (off-responses) and for GHRP-6 at 0.50 μM (on-responses) and 0.26 μM (off-responses). No effects were observed with motilin between 1 nM and 0.1 μM. Studies in the presence of l -NAME confirmed the effect of the ghrelin agonists on cholinergic excitatory motor responses. No effects were observed with motilin under the different experimental conditions. The presence of growth hormone secretagogue receptor 1a transcripts in the strip preparations was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Conclusion Ghrelin and GHRP-6 but not motilin accelerate gastric emptying and transit by activating cholinergic excitatory pathways in the enteric nervous system in addition to the known vagal pathways.

Intestinal motilin is known to stimulate gastrointestinal motility. Recently, it was shown that the motilin gene and the motilin receptor are expressed in various regions of the brain. We studied whether motilin can activate pathways in the rat hippocampus to stimulate gastric motility. Gastric motility was monitored in conscious rats, whereas extracellular electrical activity recordings of the hippocampus were performed on anaesthetized rats to measure the influence of microinjection of motilin and CCK-8 into the hippocampus and into the cerebral ventricles. We found that neurons in the CA3 region of the hippocampus are sensitive to gastric distension, and that injection of motilin into the hippocampus increased the amplitude of gastric contractions by 35.3+/-6.8%, while CCK-8 injection inhibited motility by -27.3+/-6.8%. The hippocampal motilin-induced stimulation of gastric motility (30.6+/-5.5%) was completely abolished by subdiaphragmal vagotomy (-2.8+/-4.4%) but unaffected by the intravenously applied receptor blockers atropine, phentolamine and propranolol. In vivo extracellular recordings of gastric distension-responsive CA3 neurons revealed that intracerebroventricular administration of motilin increased firing while CCK-8 inhibited firing. These opposite effects of motilin and CCK-8 fit with the nature of the actions of these gut-brain peptides on gastric motility. Our findings suggest that the stimulation of gastric motility by motilin administered in the hippocampus reflects the existence of a functional interaction between the hippocampus and a vago-vagus reflex running via a noncholinergic and nonadrenergic efferent pathway.

Due to motilin’s relation to the migrating motor complex (MMC), the physiology of motilin has been mostly studied in man and dog. The cat does not have an MMC pattern, and little is known about cat motilin. Therefore we identified the cat motilin precursor (GenBank accession no. AF127917 ) and developed a quantitative polymerase chain reaction (PCR) to explore its distribution in the gastrointestinal tract and in the central nervous system (CNS). The precursor is closely related to the dog precursor and consists of an open reading frame of 348 bp encoding the signal peptide (25 amino acids), the motilin sequence (22 amino acids) and the motilin associated peptide (69 amino acids). One amino acid of the signal peptide was subject to gene polymorphism. Quantification of motilin messenger RNA (mRNA) was for the first time achieved. It is most abundant in the gastrointestinal tract, with the highest concentration in the duodenum, the lowest in the colon and is not detectable in the corpus. However an important expression was also observed in several regions of the CNS, except the striatum and cerebral cortex. The highest level was in the hypothalamus (although 23-fold lower than in the duodenum), the lowest level in the pons. Moderate levels were found in the thyroid. These data suggest that the physiological role of motilin may extend beyond its effect on gastrointestinal motility.

Recombinant human interleukin-11 (rhIL-11) normalizes depressed smooth muscle tension generation towards motilin and substance P (SP) in rabbits with colitis. The aim of this paper was to evaluate the effect of rhIL-11 treatment on motilin and SP release which could have an effect on the contractility changes. Rabbits received 4, 40, 72 or 720 microg/kg rhIL-11 s.c. or saline, 1 h later a continuous s.c. administration of rhIL-11 was started with or without the induction of colitis (135 mg/kg TNBS) for 5 days. Motilin and SP levels were measured by RIA, motilin mRNA expression by RT-PCR. TNBS-colitis did not affect plasma motilin levels but increased the motilin content of the duodenal mucosa 1.7-fold. rhIL-11 treatment dose-dependently increased plasma motilin levels (720 microg/kg day: 3.5-fold) and the motilin content of the duodenal mucosa (720 microg/kg day: 3.0-fold). The effects of rhIL-11 were similar in normal rabbits and were accompanied by an increased motilin mRNA expression. TNBS-colitis decreased plasma SP levels 2.7-fold and the SP content in the colonic muscle layer 7.1-fold. The decrease in the muscle layer, but not in the plasma, was normalized by rhIL-11 treatment. In normal rabbits, rhIL-11 caused a decrease in plasma SP levels, but had no effect on the tissue content of SP. In conclusion, treatment of inflamed or normal rabbits with rhIL-11 increases plasma and tissue levels of motilin in the duodenal mucosa via an increased expression of motilin in the endocrine cells and induces the release of SP from extrinsic neurons. These changes do not explain the beneficial effect of rhIL-11 on the lowered contractility in inflamed rabbits although a change in balance of neuropeptides may influence gastro-intestinal inflammation.

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 32P-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 Lys23-Lys24. 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’.

Background and Aim: Motilin stimulates gastrointestinal motility, but in vitro studies reveal a direct smooth muscle effect, whereas in vivo studies reveal a neurally mediated effect. The aim of the present study was to determine if motilin binds to microsomes (smooth muscle) and/or synaptosomes (neurons). Methods: Subcellular fractions were prepared from tissue of rabbit gastric antrum and colon by differential centrifugation and density gradient centrifugation and characterized by determining motilin binding and the presence of membrane markers. Results: The purified microsomal fraction, enriched in the smooth muscle marker 5′-nucleotidase, was found to have the highest specific motilin binding in both antrum and colon. In the antrum, but not in the colon, the mitochondrial fraction also showed enrichment of [ 3 H]-saxitoxin binding (marker for synaptosomes) and motilin binding, although the latter was much lower than in the microsomal fraction. Two receptor binding sites were characterized in both the antral mitochondrial/synaptosomal and colonic microsomal fraction (antrum: p K d,1 9.89±0.19, p K d,2 8.18±0.11, colon: p K d,1 9.72±0.31, p K d,2 8.39±0.58). Conclusion: Motilin binding is predominantly associated with smooth muscle membranes in both antrum and colon of the rabbit. In both organs two motilin binding sites are present with comparable affinities, but the density in the colon is much higher for both sites. Whether they represent neural and smooth muscle receptors will require studies with isolated smooth muscle cells and neurons.

The presence of motilin in human milk and the influence of human milk on the degradation of [ 125 I][Nle 13 ] porcine motilin by gastric and duodenal fluids were investigated. Milk and plasma samples were collected from 14 mothers, and motilin was measured by radioimmunoassay. Plasma levels were 416 ± 37 pg/mL. In 8 defatted samples the motilin level was 105 ± 14 pg/mL, in the six others levels were above 1000 pg/mL but dilution curves were non-linear. After solid-phase extraction milk levels were 108 ± 21 pg/mL in 13 samples, in 1 sample the dilution curve was still non-linear. The stability of motilin after ingestion was studied in vitro by incubating [ 125 I][Nle 13 ] porcine motilin with gastric and intestinal juices obtained from newborns (10 times diluted). Incubations were performed at 37 °C at pH 1.8, 3.2 and 5.8 for the gastric fluid and at pH 7.4 for the duodenal fluid. After different times of intervais (5, 10, 20 and 30 minutes) intact motilin was precipitated with trichloroacetic acid and the radioactivity of the supernatant was determined. Motilin was rapidly degraded by gastric juice. The breakdown was greatest at pH 3.2 (74 % after 30 minutes) and lowest at pH 5.8 (29 %), the pH after milk feeding in neonates. Degradation by intestinal juice at pH 7.4 was also very rapid (77 % after 30 minutes). Human milk and BSA inhibited partially the gastric digestion at pH 3.2 (17 and 29 %, respectively). Digestion by intestinal juice was not affected by human milk and BSA. These results suggest that digestion of motilin in the stomach may be sufficiently retarded by human milk in the newborn to exert a biological role.

Copyright (c) 1997 Elsevier Science B.V. All rights reserved. We previously reported the existence of motilin receptors in the cerebellum of the rabbit. We now explored the existence of motilin receptors in other brain regions and determined their association with neurons by subcellular fractionation studies. Autoradiographic studies with [ 125 I]nle 13 -porcine motilin on rabbit coronal brain sections revealed specific binding sites in the hippocampus, thalamus, hypothalamus and amygdaloid body. Receptor binding studies allowed the identification of two binding sites. In all regions the density of the high-affinity binding site was lower than in the cerebellum, but its affinity was the same, except for the hypothalamus. No differences were found for affinity or density of the low-affinity receptor site. Homogenates of rabbit cerebellum were subjected to differential centrifugation. The highest motilin binding (10-times more than in the postnuclear supernatant) was found in the fraction which also showed maximal enrichment of [11- 3 H]saxitoxin binding (selective marker for voltage-sensitive Na + channels), 6.9-fold, and cytochrome c oxidase activity (mitochondrial marker), 2.4-fold. In discontinuous sucrose density gradient centrifugation the motilin and saxitoxin binding both peaked in the 0.85-1 M layer, while cytochrome c oxidase was maximal in the 1.2 M layer. In conclusion, motilin receptors exist in several regions of the rabbit brain and are probably associated with synaptosomes. These findings further support a neurotransmitter role for motilin in the brain. © 1997 Elsevier Science B.V.

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.

Vasoactive intestinal polypeptide (VIP) is reported to exert an autocrine control on neuroblastoma cell tumours: VIP is produced by the tumour and stimulates cell differentiation. This study tested the hypothesis that the parent peptide; the pituitary adenylate cyclase activating polypeptide (PACAP) may have a similar role. It was found that PACAP mRNA and PACAP were expressed in 12/12 tumours; it was also observed that PACAP receptor mRNA and functional PACAP receptors were expressed in 12/12 and 5/9 tumours, respectively. VIP mRNA and VIP were detected in 9/12 tumours. VIP receptor mRNA was expressed in 5/12 tumours and functional VIP receptors were never demonstrated. The tumours having the highest VIP levels also had the highest PACAP contents and were associated with a watery diarrhoea syndrome due to activation of intestinal VIP receptors. As PACAP recognizes the PACAP receptors and the VIP receptors with the same high affinity it may contribute to the syndrome and is a likely candidate for an autocrine control of neuroblastoma cell growth and differentiation.

In man, rabbit and cat, the effects of motilin and motilides are neurally mediated in vivo, whereas in vitro binding and contractility studies suggest the presence of a smooth muscular receptor. The aim of this study was to investigate in vitro interactions of motilin with the enteric excitatory neurotransmission in the gastric antrum of the rabbit. Circular muscle strips from the pre-pyloric antrum were subjected to electrical field stimulation (1 ms, 1-32 Hz, 10 s train) and muscle twitch responses were recorded isometrically. Induced twitch responses were frequency dependent (1-32 Hz) and entirely neurogenic (tetrodotoxin sensitive). [Nle(13)]motilin dose-dependently (10(-9)-10(-8) M) enhanced the amplitude of, atropine sensitive, evoked contractions. At 4 Hz the response, expressed as a % of the response to 32 Hz, increased from 15.5 +/- 4.1% (control) to 28.1 +/- 5.8% (motiiin 10(-9) M), and to 45.8 +/- 3.6% (motilin 10(-8.5) M) (P < 0.05). This effect was not inhibited by hexamethonium (10(-3.3) M) but was abolished by the motilin receptor antagonist GM-109 (10(-5) M). In unstimulated strips, motilin induced phasic-tonic contractions with a threshold concentration of 10(-8) M and an pEC(50) of 7.48, which were also inhibited by GM-109 (10(-5) M) but not by tetrodotoxin (10(-5.5) M). The maximal tension, frequency and dose-dependency of carbachol-induced contractions were not influenced by motilin (pEC(50), carbachol: 6.48 +/- 0.06 (control), 6.49 +/- 0.07 (motilin)). In conclusion, motilin enhances contractions induced by electrical field stimulation in the rabbit antrum by a post-ganglionic interaction with the cholinergic neurotransmission in vitro at low doses and interacts directly with antral smooth muscle at high doses. This model is an accurate reflection of the in vivo effects of motilin and provides a tool to study neurogenic and myogenic actions of motilin and motilides in vitro. (C) 1997 Elsevier Science B.V.

The data regarding the identity of motilin-like immunoreactivity in the central nervous system are controversial. The aim of the present study was to clarify whether motilin mRNA is present in the brain of rabbit and man. Total RNA, prepared from several regions of the rabbit brain, was subjected to RT-PCR aimed at amplifying a 294 bp cDNA fragment of the rabbit motilin precursor. The amplified product was subcloned and sequenced. The sequence showed 7 differences compared to the one reported for the duodenal precursor (1). However the duodenal precursor from the rabbit used in the present study revealed identical substitutions. One of these, involving amino acid -11 of the signal peptide, was shown to be due to gene polymorphism, as has also been described at this site in man. By radioimmunoassay the highest concentration of motilin (fmol/mg protein) was detected in the hippocampus (4788 +/- 295), the lowest in the telencephalon (2127 +/- 221). Using a similar approach, but starting from commercial human brain mRNA, the sequence of a comparable cDNA fragment of the human brain motilin precursor was obtained. Its sequence was identical with the one published for the human intestinal precursor (41). Our study demonstrates that motilin mRNA is present in the brain of man and rabbit. Together with our recent findings of central motilin receptors, they suggest a central role for motilin.

Motilin was isolated from acid extracts of the small intestine of chickens by a combination of gel filtration chromatography, ion-exchange, and reverse-phase HPLC. The purification was monitored using a radioreceptor assay. The sequence of chicken motilin is FVPFFTQSDIQKMQEKERNKGQ. Although the six residues differing from porcine motilin (4, 7–10, and 12) are mostly in the pharmacophore of porcine motilin, the affinity of chicken motilin and of the (1–14) fragment of chicken motilin for the motilin receptor of rabbit antral smooth muscle is not much reduced (p K d s of 8.90 and 8.45), compared with the affinity of [Nle 13 ]porcine motilin (p K d 9.12). With smooth muscle tissue of the chicken, however, receptors could not be demonstrated with binding studies. In the tissue bath chicken motilin induced a dose-dependent tonic contraction, which was most pronounced with muscle strips prepared from chicken jejunum. This response was blocked by the Ca 2+ antagonist verapamil, but atropine, TTX, l -NNA, guanethidine, prazosin, and yohimbine had no effect. The pEC 50 for chicken motilin in the chicken jejunum was 7.41. Motilins from other species had lower potencies, and [Phe 3 , Leu 13 ]porcine motilin, an antagonist in the rabbit, was an agonist in the chicken. The motilin agonists erythromycin A and EM-523 were almost without effect. Tested against rabbit duodenum, chicken motilin had a smaller potency than mammalian motilins. Thus, chicken motilin and the chicken motilin receptor differ from their mammalian counterparts.

The present study was undertaken to explore motilin's transduction pathway in the rabbit. Guanine nucleotides inhibited 125I-motilin binding in rabbit antral tissue and increased the dissociation of motilin from its receptor. Motilin, the motilin agonist erythromycin A enol ether (EM-201) and carbachol (taken as control) increased the production of inositol phosphates in rabbit duodenal smooth muscle strips labeled with myo-[2-3H]inositol. The effect of carbachol was blocked by atropine. Dose-response curves revealed that 50% of this effect was obtained with 3.9 nM motilin, 170 nM EM-201, 0.54 microM carbachol. Chromatographic separation of the inositol phosphate metabolites showed significant increases in the levels of [3H]inositol bisphosphate and of [3H]inositol trisphosphate. The three substances were without effect upon the metabolism of cAMP, nor did they modulate the rise in cAMP induced by GTP. We propose that motilin's transduction pathway uses a G protein that causes an increase in inositol trisphosphate which is rapidly metabolized, and which may release calcium from intracellular stores.

The acid extract of a liver metastasis from a patient with elevated plasma motilin levels contained large quantities of motilin (3.37 micrograms/ml). The extract was concentrated on a C18-column and motilin was isolated by gel chromatography (Sephadex G-50) followed by cation ion exchange chromatography (HR5/5 Mono-S) and three successive steps of reverse phase chromatography (Nucleosil 300-5 C18). The pure peptide was sequenced and the identity of porcine and human motilin was confirmed. This is the first report of a tumor containing large amounts of motilin.

Objectives The KDIGO 2012 criteria emphasize the independent role of glomerular filtration rate (GFR) and albuminuria to assess the prognosis of chronic kidney disease (CKD), as both relate independently to all-cause mortality and cardiovascular morbidity/mortality. In this study we want to determine the prevalence of CKD in women with a recent history of preeclampsia. Methods We included 602 primiparous women with a history of preeclampsia. They were all at least 4 months postpartum and had no history of pre-existent hypertension, diabetes or kidney disease. We estimated GFR by the CKD-epi-equation, and quantified urinary protein loss (micro-albuminuria: >3.0 mg/mmol creatinine; macro-albuminuria: proteinuria >300 mg/24 h). We categorized women on the basis of the KDIGO 2012 criteria. Results Median time postpartum was 9 (6–18) months. Table 1 shows the different GFR and albuminuria categories. 419 (70%) women had a normal GFR and no albuminuria. Based on the KDIGO guideline, 77 (13%) women with a moderately increased risk would require yearly monitoring, in over 85% of the cases because of excessive urinary protein loss. Only 21 (3.5%) women were classified to be at high risk and would require referral to a nephrologist for twice yearly monitoring. Conclusions Follow-up of kidney function is relevant for about 16% of all women with a history of preeclampsia. Kidney function should be part of cardiovascular risk assessment after preeclampsia, with special emphasis to be directed on the postpartum disappearance of the preeclampsia-induced albuminuria. Disclosures J. Spaan: None. V. Lopez van Balen: None. L. Peeters: None. M. Spaanderman: None.

Motilin was isolated from cat small intestine by a series of chromatographic steps. Using a radioreceptor assay, based upon binding of iodinated porcine motilin to rabbit antral smooth muscle membranes, it was shown that cat duodenal mucosa contains about 495 ng/g tissue, the jejunal mucosa 161 ng/g tissue and the ileal mucosa 95 ng/g tissue motilin. The duodenal mucosa was extracted with 6% acetic acid and concentrated on a cation exchange Whatman CM-52 gel. After lyophilization the material was further purified by gel filtration (Sephadex G-50), followed by reverse phase (C 18 ), cation exchange HPLC (Mono S) and three runs on a reverse phase HPLC (Nucleosil 300-5C 18 ) column. The UV absorbance and the radioreceptor assay were used to monitor the purification. After Mono S chromatography two forms of motilin were detected. The major peak corresponded to a 22 amino acid peptide, which differed only from canine motilin at position 12, where Lys is replaced by Arg. The smaller peak probably corresponds to a deamidated form of this peptide. The sequence homology between cat and porcine/human motilin or cat and rabbit motilin is 81.8% and 72.7%, respectively. The conservation of the first six amino acids in all five species studied is striking, confirming that the biological acitivity of the peptide resides in the N-terminal part.

We demonstrate binding of [125I][Nle13-po]motilin to homogenates of cat gastric and small intestinal, but not to colonic smooth muscle tissue. The density was ( B max in fmol/mg protein): 0 (fundus); 12 ± 2 (corpus); 22 ± 3 (antrum); 55 ± 12 (duodenum); 44 ± 10 (jejunum); 17 ± 1 (ileum); 0 (colon). A significant ( p ) difference was found between the dissociation constant for motilin in the stomach ( pK d = 8.84 ± 0.06 ) and in the small intestine ( pK d = 8.58 ± 0.08 ). The motilides erythromycin-A (EM-A), EM-523, and EM-A N- oxide displaced labeled [Nle13-po]motilin bound to cat duodenal receptor with potencies ( pK d ) of 5.47 ± 0.23 , 7.60 ± 0.24 , and N- terminus of motilin interacts with the receptor. In the tissue bath, duodenal strips mounted in the longitudinal direction responded to motilin, EM-523, and EM-A (pEC50: 8.29 ± 0.08 ; 7.12 ± 0.12 ; 5.99 ± 0.15 ). The compounds had a comparable intrinsic activity ( 83 ± 3% ; 80 ± 5% ; 82 ± 5% of the response to ACh), which was unaffected by atropine, TTX, hexamethonium, and zacopride but reduced by verapamil and calcium-free medium. Cat stomach and small intestine possess smooth muscle motilin receptors, which have comparable properties as those found in man and in rabbit.

The effect of three doses of erythromycin on interdigestive gastrointestinal motility and on plasma motilin levels was studied in healthy volunteers and patients with diabetic gastroparesis. Abnormalities of interdigestive motility were observed in 40% of the patients. In healthy volunteers, 40 mg erythromycin elicited a premature phase 3 that started in the stomach. In contrast to the spontaneous gastric phase 3, this erythromycin-induced phase 3 was not accompanied by a motilin peak. In patients with diabetic gastroparesis, 40 mg erythromycin induced a premature phase 3 in three patients, no response in one patient, and a burst of antral contractions in another patient. Doses of 200 and 350 mg erythromycin elicited a burst of antral phase-3-like contractions in both volunteers and patients, which was not accompanied by a motilin peak. This phase-3-like activity did not migrate to the small intestine and was not followed by a phase 1, but by a prolonged period of antral contractile activity. The number and amplitude of antral contractions after 200 or 350 mg erythromycin were significantly higher than after 40 mg. The motor patterns induced by different doses of erythromycin offer potential therapeutic applications.

Erythromycin has been shown to act as a motilin agonist by binding to motilin receptors on gastrointestinal smooth muscle and to improve the severely impaired gastric emptying in patients with diabetic gastroparesis. To elucidate the motor pattern that accounts for this accelerated emptying, the effect of 200 mg erythromycin vs. placebo on postprandial motility of the stomach and the upper small intestine was examined in 13 normal subjects. Erythromycin significantly increased the amplitude of the antral contractions during the 2-hour postprandial study period (maximal difference in mean amplitude of distal antral contractions between erythromycin and placebo recorded from 80 to 90 minutes after meal: 123 +/- 17 vs. 44 +/- 12 mm Hg; P less than 0.005). The total number of antral contractions was not affected, but the contractions could be recorded manometrically higher up in the stomach after erythromycin than after placebo (9-12 vs. 3-6 cm above the pylorus). Antroduodenal coordination was significantly improved during the first postprandial hour, and the first normal phase 3 of the migrating motor complex, indicating the reappearance of fasting motility, occurred earlier after erythromycin than after placebo (128.3 +/- 14.3 vs. 173.4 +/- 16.1 minutes; P less than 0.05). These changes in postprandial motility induced by erythromycin may well account for its accelerating effect on gastric emptying.

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.

Introduction About 7 years after preeclampsia, the prevalence of microalbuminuria is reported to be around 30% [1] . As proteinuria resolves in most cases within two years of post partum, [2] this raises the question of whether microalbuminuria first regresses after preeclampsia or persists. Objectives This cross-sectional study describes the prevalence of microalbuminuria in the years following preeclampsia. Methods Women with a history of preeclampsia (n = 823) were evaluated 4–180 months after delivery. Women with pre-existing hypertension, renal disease or diabetes mellitus were excluded (n = 65). In a 24 h urine collection we determined total protein (pyrogallol red), microalbuminuria (turbidometry, Cobas Bio and since 2007 nephelometry, BN ProSpec) and creatinine (Jaffe method). Microalbuminuria was considered to be present when there was ⩾3.5 mg/mmol of creatinine. Proteinuria was defined as a total protein level of ⩾300 mg/24 h. Results Microalbuminuria was present in 12.0%, CI 9.7–14.6 and proteinuria in 2.7%, CI 1.7–4.1 of all cases. Microalbuminuria decreased up to 40 months postpartum, but increased after that period of time (p = 0.01 compared with 24–40 months). Conclusion This study suggests a U-shaped microalbuminuria pattern over time. Longitudinal research is necessary to confirm this.

Introduction Several studies have shown that the risk of premature cardiovascular disease (CVD) is increased after maternal placental syndromes (MPS), including hypertensive disorders and placental abruption. Although a high prevalence of CVD risk factors has been observed for women with a history of preeclampsia and pregnancy-induced hypertension, it is unclear whether patients with previous placental abruption exhibit the same cardiovascular risk profile. Objectives To investigate the association of placental abruption with the presence of modifiable CVD risk factors that may be of potential use for prevention programs. Methods We performed a case–control study of 75 women with a history of placental abruption and a control group of 79 women with uneventful pregnancies. At 6–9 months postpartum we measured the following CVD risk factors: blood pressure, body-mass index (BMI), fasting blood glucose levels, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides and CRP. Baseline variables in the two groups with and without a previous abruption were expressed as means and standard deviations (SD). Where appropriate, means were adjusted for potential confounders using a generalized linear model. Data were further stratified for women with or without additional MPS-related complications, i.e. preeclampsia, gestational hypertension and intrauterine growth restriction. Results Women who experienced placental abruption had a significantly higher systolic and diastolic blood pressures, BMI, fasting blood glucose levels, CRP, total cholesterol, HDL-cholesterol, LDL-cholesterol and cholesterol/HDL ratio, as compared to controls. Characteristics Reference group n = 79 Placental abruption n = 75 P-value Systolic BP 114 (13) 122 (13) Diastolic BP 75 (7) 79 (11) 0.022 CRP 0.65 (1.03) 1.89 (3.04) 0.008 Total cholesterol 143.7 (55.4) 194.3 (36.3) HDL 45.6 (16.8) 54.2 (13.9) 0.001 LDL 83.4 (42.9) 117.6 (35.4) Triglycerides 83.5 (41.0) 94 (46.1) 0.134 Cholesterol/HDL ratio 3 (1.54) 3.54 (1.16) 0.02 Fasting glucose 3.89 (1.30) 5.09 (0.35) These associations remained significant in women with previous placental abruption without concomitant other MPS only for plasma lipid profile, BMI and fasting blood glucose levels, but not for diastolic and systolic blood pressure. Conclusion A history of placental abruption is independently associated with increased BMI, fasting blood glucose levels, total cholesterol and LDL-cholesterol postpartum. Early detection of CVD risk factors in women with previous placental abruption offers an attractive opportunity for primary and secondary prevention.