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To elucidate the role of endogenous ghrelin in the regulation of energy homeostasis and gastric emptying, ghrelin knockout mice (ghrelin(-/-)) were generated. Body weight, food intake, respiratory quotient, and heat production (indirect calorimetry), and gastric emptying ((14)C breath test) were compared between ghrelin(+/+) and ghrelin(-/-) mice. In both strains, the effect of exogenous ghrelin on gastric emptying and food intake was determined. Ghrelin(-/-) mice showed some subtle phenotypic changes. Body weight gain and 24-h food intake were not affected, but interruption of the normal light/dark cycle triggered additional food intake in old ghrelin(+/+) but not in ghrelin(-/-) mice. Exogenous ghrelin increased food intake in both genotypes with a bell-shaped dose-response curve that was shifted to the left in ghrelin(-/-) mice. During the dark period, young ghrelin(-/-) mice had a lower respiratory quotient, whereas their heat production was higher than that of the wild-type littermates, inferring a leaner body composition of the ghrelin(-/-) mice. Absence of ghrelin did not affect gastric emptying, and the bell-shaped dose-response curves of the acceleration of gastric emptying by exogenous ghrelin were not shifted between both strains. In conclusion, ghrelin is not an essential regulator of food intake and gastric emptying, but its loss may be compensated by other redundant inputs. In old mice, meal initiation triggered by the light/dark cue may be related to ghrelin. In young animals, ghrelin seems to be involved in the selection of energy stores and in the partitioning of metabolizable energy between storage and dissipation as heat.

The contraction of longitudinal muscle strips of the rabbit duodenum in response to motilin and acetylcholine was investigated in normal and high K+-solutions in the presence and absence of external calcium, in order to demonstrate the existence of pharmaco-mechanical coupling for motilin and to examine whether the peptide mobilizes calcium from an intracellular store. In depolarized smooth muscle (140 mM K+), motilin (3.2×109 -1×10-7 M) and acetylcholine (1×10-5 M) were still capable of causing a considerable, transient, concentration-dependent contraction in the presence of Ca2+. The ‘extra’-contraction to motilin was not blocked by tetrodotoxin (1 µg/ml) nor by atropine (10-7 M), but acetylcholine (10-5 M) was blocked by atropine. Verapamil (10-7 M) could selectively block the K+ contraction without affecting the extra agonist contraction. Nitroprusside was ineffective up to 10-4 M in high K+-solutions, but in normal Hepes-buffer it caused a concentration-dependent rightward shift of the concentration-response curve of motilin and acetylcholine contractions. In a calcium-depleted medium, high K+-depolarized muscle strips were still responsive to motilin and acetylcholine, but higher concentrations (10-6 M) were needed than in the presence of calcium and the contractions reached only 57 +- 11% and 74 +- 9% respectively of the maximal contraction in 1.2 mM Ca2+ containing solutions. The response to motilin (10-6 M) was not only smaller than that to acetylcholine (10-5 M), it also faded more rapidly with time. The response to one agonist could not be repeated except by using a higher concentration of the same or the other agonist, and the magnitude of this second response depended upon the dose used in the first one. We conclude that pharmaco-mechanical coupling exists for motilin and that this peptide is able to elicit contractions by mobilization of calcium from an intracellular store. This store overlaps with the one used by acetylcholine. Our experiments also reinforce the hypothesis that in the rabbit motilin exerts a direct action upon smooth muscle cells.

The purpose of these experiments was to characterize the pattern of digestive myoelectric activity produced by four different test meals in dogs. A new approach for the analysis of the myoelectric activity of the small intestine was used. In contrast with methods dividing the tracings into present time periods, the present method respected the “motor entities,” hence the continuity, of the tracing. Two types of motor entities occurred: “activity units” which are groups of consecutive slow waves with spiking, and “rest units,” which are groups of consecutive slow waves without spiking. The frequency of occurrence and frequency of alternation of the various activity and rest units occurring during digestion was calculated. Digestive myoelectrical activity was characterized by the frequent occurrence and alternation of short activity and rest units. Three different patterns of digestive activity could be discriminated, ie, a fat, a protein and carbohydrate, and a canned food pattern.

In the present study, the motilin receptor was characterized by enzymatic digestion studies and by solubilization of the motilin-receptor complex from prelabeled membranes using the anionic detergent cholic acid. Motilin binding was significantly decreased by preincubation of membranes of rabbit antral tissue with trypsin, phospholipase A2, C, D, dithiothreitol and 2-mercaptoethanol but not by neuraminidase and β-galactosidase. Treatment of prelabeled membranes with 1% cholic acid resulted in solubilization of 24±5 % of the proteins and 65±3 % of the radioactivity. The latter was for 77±4 % due to the presence of the motilin-receptor complex as estimated with PEG-precipitation. Upon gelfiltration on Superose 6 the complex partially dissociated but 43±3 % eluted with macromolecular components in the void volume. This peak was not detected when membranes were first incubated with unlabeled motilin. Further disaggregation was accomplished by the addition of 0.5 M NaCl to the elution buffer. The chromatographic profile then showed a peak of about 370 kDa and a second one of 100kDa. The latter value probably reflects the molecular mass of a single 125I-motilin-receptor-complex.

We have tested the hypothesis that the imperfect correlation between the occurrence of plasma motilin peaks and the migrating motor complex (MMC) is related to the site of origin of the MMC. Gastric and small-intestinal motor activity was recorded manometrically in 16 normal subjects after an overnight fast. Blood samples were taken every 10 min and assayed for motilin. Fifty MMCs were recorded: 29 (58%) originated in the antrum and 21 (42%) in the small intestine. For gastric MMCs a motilin peak (45 ± 5 pM) was found 10 min before the occurrence of the MMC in the duodenum. For intestinal MMCs no peak could be demonstrated. On an individual basis 27 of the 29 gastric activity fronts were associated with a motilin peak, versus 5 out of 21 intestinal MMCs. It is concluded that in man only gastric activity fronts are associated with plasma motilin peaks. Apparently, motilin is only involved in the regulation of the MMCs originating from the stomach.

Intraduodenal pH, bicarbonate and amylase secretion, and gastric acid and pepsin output were studied in relation to the migrating motor complex in man. The occurrence of a motor complex in the duodenum was preceded by an increase in gastric acid and pepsin output and followed by a peak in bicarbonate and amylase secretion. It is concluded that the interdigestive phase in man is characterized by periodic activity complexes comprising both motor and secretory components. These observations may have important implications for the interpretation of currently used functional tests of gastrointestinal secretion.

In order to assess the possible role of the new candidate gut hormone, motilin, in cantrolling the interdigestive migrating motor complex (MMC) in man, 14 normal subjects were studied after an overnight fast by means of three pressure-recording catheters with orifices 25 cm apart in the upper small intestine. The typical aboral progressing bursts of pressure waves occurred at a mean interval of 137 minutes and were preceded by a peak motilin level 25 pmol/liter higher than the lowest level in the postactivity-front quiescent period. To study the effect of exogenous motilin, an infusion of pure porcine motilin at various dose levels was given to 16 normal volunteers shortly after the onset of the phase I quiescent period. Motilin infusion induced an activity front in 12 of the 16 subjects. The mean activity front interval was reduced to 46 min (P<0.001). This effect could be obtained tained even at the low dose level of 0.4 pmol/kg/min, which produced an increase in plasma motilin level of only 57 pmol/liter. These data suggest that a cyclic rise in plasma motilin levels is one of the factors involved in the production of the activity front of the migrating motor complex in man.

Recent studies suggested that certain erythromycin A (EM-A) derivatives are motilin receptor agonists. As proposed by Itoh they may be called “motilides”. We have investigated the Ca2+-dependence of contractions induced by two potent motilides, ME-34 [de(N-methyl) 8,9-anhydroeryhtromycin A 6,9-hemiacetal] and EM-523 [de(N-methyl)-N-ethyl-8,9-anhydro-erythromycin A 6,9-hemiacetal], in duodenal tissues and compared the results with those previously obtained with motilin.

In intact sheets of the guinea pig ileal longitudinal muscle, loaded with fura-2, both substance P (SP)- and K(+)-induced contractile responses are preceded by cytoplasmic free Ca2+ ([Ca2+]i) variations. In response to SP (10(-10)-10(-7) M), [Ca2+]i and force increased concentration dependently. From 10(-9) M on, the response was biphasic: an initial Ca2+ spike and force transient were followed by a tonic component. The [Ca2+]i and force vs. log [SP] curves were sigmoidal for the initial phasic component, while a homologous receptor desensitization caused a reduced tonic component of the [Ca2+]i and contractile response at higher concentrations of SP. Both intracellular Ca2+ release and Ca2+ influx play a role in the effect of the peptide. In depolarizing solutions (140 mM K+) and in Ca2(+)-free medium (2 mM EGTA), SP induced a transient increase in [Ca2+]i and force. The Ca2+ stores used by SP and acetylcholine (ACh) overlap. [Ca2+]i and force fell to base-line levels when the extracellular Ca2+ was reduced from 1.2 to 0.2 mM during stimulation with SP. Verapamil reduced the tonic response. We also studied the relation between [Ca2+]i and force for the peak and steady-state values after stimulation with increasing concentrations of SP and K+. The phasic force response was linearly related to log [Ca2+]i. During the sustained response to K+, the Ca2+ sensitivity of the contractile/regulatory proteins was decreased, whereas no changes were observed during prolonged stimulation with the peptide.

The long-acting somatostatin analogue SMS 201-995, also called Sandostatin®, is used in the treatment of acromegaly and peptide-secreting tumors. Little is known about its effect on gut motility, although such an effect might be expected considering the spectrum of activities of its parent molecule, somatostatin. We have studied the effect on the interdigestive motility of intravenous boluses of 0, 0.1, 0.5. 1.5, and 5.0 μg/kg of this analogue in 10 dogs with bipolar electrodes implanted along the entire small bowel. All doses induced, within 5 min of administration, premature phase-3 activity that was isolated to one segment, normally progressive, or simultaneous (stationary) in all channels. Only low doses induced isolated phase 3, whereas the frequency of the induction of stationary phase 3 increased with higher doses. In the next cycle of the migrating motor complex the duration of phase 1 was increased, although the duration of the whole cycle was not changed. This cycle ended with mostly ectopic phase-3 activity. During phase 2 of the following cycle ultra-rapid rushes of spiking activity progressing at a speed of 25 ± 3 cm/sec in the upper jejunum were regularly observed. Although this pattern occurs very rarely under control conditions, it was present in 20%, 80%, and 100% of the experiments after doses of 0.5, 1.5, and 5.0 μg/kg, respectively. We conclude that gastrointestinal side effects observed during administration of SMS 201-995 might be related to these motility effects, which warrant further investigation. SMS 201-995 may be a tool to study the mechanism of the induction of ultra-rapid rushes and of stationary phase 3.

The effect on the migrating myoelectric complex (MMC) of local nerve blockade in the jejunum was studied in five unanesthetized dogs. A silastic catheter was implanted in a terminal branch of a jejunal artery, perfusing a 5-to 10-cm segment, 45 cm below the ligament of Treitz. Small-bowel motor activity was studied electromyographically with implanted electrodes. Three different doses of tetrodotoxin (166, 333, 500 ng/ kg/h) were administered intra-arterially for 5 h. During the 333-and 500-ng but not during the 166-ng/kg/h perfusions ectopic activity fronts started just below the perfused segment. At this time no phase-3 activity was observed in the proximal bowel. In addition to ectopic fronts normal MMCs were observed during the perfusions. These observations show that local nerve blockade induces phase-3 activity, probably by inhibiting an inhibitory nerve action.

Basal fluctuations of motilin and their correlation to intestinal motor activity were studied in five hypophysectomized (anterior lobe resection) patients. A cyclic pattern of motor activity identical to the one described in normal volunteers was observed. The mean interval between two activity fronts (AF) was 115 min, which is not significantly different from the normal length. Motilin levels were within the normal range, and fluctuations correlated with the occurrence of an AF. The mean difference between motilin levels 15 min before the AF and at the time of the start of the AF was +15 pM (P < 0.05). For the levels 15 min after the AF the mean difference was - 9pM (P < 0.05). These values correlate well with those reported by us in normal volunteers, in whom the mean differences were +11 pM and -14pM, respectively. It is concluded that the adenohypophysis is not involved in the regulation of motilin levels or in the regulation of interdigestive motility in man.

Lysozyme was isolated from the small intestine of mice by combined ion-exchange and molecular sieve chromatography. This lysozyme differs from that isolated from the urine of mice with monocytoma in amino acid composition, and migration rate in cellulose acetate electrophoresis. As intestinal lysozyme originates at least in part from the Paneth cell, our results point towards the existence of isozymes of lysozymes in mice.