Neural and muscular receptors for motilin in the rabbit colon

Motilin receptors were classically recognized in the gastroduodenal area, where they help to regulate interdigestive motility. More recently, motilin receptors were identified in the colon where their biologic significance remains unclear. We aimed here to characterize the motilin receptors of the rabbit colon. Distal colon and duodenum were obtained from sacrificed rabbits. Tissues homogenized by Polytron were submitted to differential centrifugation to obtain neural synaptosomes or smooth muscle plasma membranes enriched solutions. Motilin binding to these membranes was determined by the displacement of (125)I MOT by the native peptide MOT 1-22, or by peptide analogues MOT 1-12 [CH(2)NH](10-11) or GM-109 and by erythromycin derivative GM-611. Motilin binding capacity was maximum in colon nerves (49.5 +/- 6.5 fmol/mg protein vs. 19.9 +/- 2.5 in colon muscles or 9.4 +/- 2.8 and 6.6 +/- 1.2 in duodenal muscles and antral nerves respectively); all tissues expressed similar affinity for MOT 1-22, and the motilin agonist GM-611 bound equally to neural or muscle tissues from the rabbit colon; the synthetic antagonist MOT 1-12 [CH(2)NH](10-11) showed greater affinity for colon nerves than for colon muscles (plC50: 7.23 +/- 0.07 vs. 6.75 +/- 0.03). Similar results were obtained with the peptide antagonist GM-109; receptor affinity toward MOT 1-12 [CH(2)NH(10-11)] was always five times superior in neural tissues, whether they came from the colon or the antrum, than in muscle tissues, whether they were obtained from colon or from duodenum. Motilin receptors are found in very high concentration in nerves and in muscles from rabbit colon; specific motilin receptor subtypes are identified in nerves (N) and muscles (M) of the rabbit colon; N and M receptor subtypes seem independent of the organ location.     

Synthesis and in vitro evaluation of [Leu13]porcine motilin fragments.

Several peptide fragments representing N-terminal, C-terminal, and internal sequences of [Leu13]porcine motilin ([Leu13]pMOT) were synthesized using Fmoc solid phase methodology. Peptides were assayed for motilin receptor binding activity in a rabbit antrum smooth muscle preparation and for stimulation of contractile activity in segments of rabbit duodenum. In vitro activity was directly correlated with motilin receptor binding affinity for all [Leu13]pMOT fragments examined. N-Terminal fragments of just over half the length of the native peptide are nearly equipotent as full-length motilin. These results suggest that the N-terminal segment, together with residues from the mid-portion of the molecule, constitutes the bioactive portion of pMOT. The C-terminal segment, in contrast, contributes little to receptor binding affinity or in vitro activity.     

下丘脑室旁核胃动素对消化功能影响的研究

目的:研究下丘脑室旁核注入胃动素及其拮抗剂对大鼠消化功能和体重增长的研究。方法:将剂量为0.005-5nmol的motilin和GM109注入大鼠下丘脑室旁核,1小时后可观察到大鼠摄食量显著增加并持续到两小时后。进食量的计算是通过预先称量好的鼠粮和应用药物20分钟、1小时、两小时后剩余数量比较而得出。实验持续一周。将实验组和对照组的进食量和体重进行比较。结果:室旁核注入胃动素5nmol的实验组和合并应用GM1090.005nmol的实验组在应用药物后1小时和2小时,可观察到摄食量显著增加(p<0.01),一周后体重也增加(p>0.05),然而摄食量的增加有显著性差异,体重的增加并无显著性差异。其他实验组也没有观察到显著性差异。结论:胃动素有调节消化运动,促进胃肠排空,促进食欲的作用。可能由于胃肠排空是频繁的,没有充裕的时间消化吸收,从而体重增加无显著性差异。     

下丘脑室旁核胃动素对消化功能影响的研究

目的：研究下丘脑室旁核注入胃动素及其拮抗剂对大鼠消化功能和体重增长的研究。方法：将剂量为0.005-5nmol的motilin和GM109注入大鼠下丘脑室旁核,1小时后可观察到大鼠摄食量显著增加并持续到两小时后。进食量的计算是通过预先称量好的鼠粮和应用药物20分钟、1小时、两小时后剩余数量比较而得出。实验持续一周。将实验组和对照组的进食量和体重进行比较。结果：室旁核注入胃动素5nmol的实验组和合并应用GM1090.005nmol的实验组在应用药物后1小时和2小时,可观察到摄食量显著增加（p〈0.01）,一周后体重也增加（p〉0.05）,然而摄食量的增加有显著性差异,体重的增加并无显著性差异。其他实验组也没有观察到显著性差异。结论：胃动素有调节消化运动,促进胃肠排空,促进食欲的作用。可能由于胃肠排空是频繁的,没有充裕的时间消化吸收,从而体重增加无显著性差异。     

Motilin activates neurons in the rat amygdala and increases gastric motility

Motilin and motilin receptors have been found in most regions of the brain, including the amygdala, one of the most important parts of the limbic system. Our previous study found that administration of motilin in the hippocampus stimulates gastric motility. We now explore the effect of motilin in the amygdala on gastric motility. In conscious rats, gastric motility was recorded after microinjection of motilin, motilin receptor antagonist (GM-109) or a mixture of the two into the basomedial amygdala nucleus (BMA). In anesthetized rats the changes of spontaneous discharges of gastric distention sensitive neurons (GDSN) in the BMA were recorded after intracerebroventricular (i.c.v.) microinjection of motilin or GM-109. In conscious rats the amplitude of gastric contractions increased dose-dependently after microinjection of motilin in the BMA, and decreased after microinjection of GM-109. The excitatory or inhibitory effects induced by motilin or GM-109 alone, were weakened by microinjection of a mixture solution of both. The spontaneous discharge frequency of gastric distention excitatory neuron (GDEN) was mainly inhibited by i.c.v. microinjection of motilin but excited by GM-109. In contrast, the spontaneous discharge frequency of gastric distention inhibitory neuron (GDIN) was mainly excited by motilin, but inhibited by GM-109. Our findings suggest that motilin may regulate gastric motility by modulating neural pathways in the BMA.     

Antagonistic effect of human alpha-CGRP [8-37] on the in vivo regional haemodynamic actions of human alpha-CGRP

In conscious rats, infusion of human alpha-CGRP [8-37] (30 nmol/kg/min) caused small, reversible reductions in hindquarters flow and vascular conductance only, whereas at a dose of 300 nmol/kg/min there was a tachycardia and an increase in mean arterial blood pressure, together with renal, mesenteric and hindquarters vasoconstrictions. Human alpha-CGRP (0.03 nmol/kg/min) caused tachycardia, hypotension, and transient renal, but sustained hindquarters, vasodilatation; these changes were accompanied by mesenteric vasoconstriction. Infusion of human alpha-CGRP [8-37] (30 nmol/kg/min) during administration of human alpha-CGRP (0.03 nmol/kg/min) abolished the effects of the latter but these re-appeared when the human alpha-CGRP [8-37] infusion was stopped. This dose of human alpha-CGRP [8-37] did not affect cardiovascular responses to isoprenaline. These results indicate that human alpha-CGRP [8-37] is an effective antagonist of the cardiovascular actions of human alpha-CGRP in vivo.     

Neuropeptide Y Y1 receptor mediated mesenteric vasoconstriction in the pig in vivo

The object of the present study was to investigate the effects of the sympathetic cotransmitter neuropeptide Y (NPY), and the closely related gut hormone peptide YY (PYY), on splanchnic blood flow regulation in the anaesthetized pig in vivo. Systemic injections of NPY, PYY and the NPY Y(1) receptor agonist [Leu(31)Pro(34)]NPY (470 pmol kg(-1) each) evoked pressor and mesenteric vasoconstrictor responses that were largely abolished by the selective NPY Y(1) receptor antagonist H 409/22 (60 nmol kg(-1) min(-1)). In contrast, the NPY Y(2) receptor agonist N-acetyl[Leu(28)Leu(31)]NPY(24-36) (1.1 nmol kg(-1)), a dose of which potently evoked splenic NPY Y(2) receptor mediated (not affected by H 409/22) vasoconstriction, did not evoke any mesenteric vascular response. Mesenteric vascular responses to angiotensin II (10 pmol kg(-1)), alpha,beta-methylene ATP (10 nmol kg(-1)) and the alpha(1)-adrenoceptor agonist phenylephrine (15 nmol kg(-1)), were not inhibited by H 409/22. It is concluded that NPY and PYY evokes porcine mesenteric vasoconstriction mediated by the NPY Y(1) receptor subtype, as demonstrated by selective and specific inhibition exerted by the NPY Y(1) receptor antagonist H 409/22, in vivo.     

Cholinergic role on release and action of motilin

In conscious dogs with gastric fistula and platinum electrodes on the antrum, duodenum and jejunum, IV atropine 100 micrograms/kg/hr and hexamethonium 10 mg/kg/hr, blocked cyclic increases in fasting plasma motilin concentration (PMC) and spontaneous migrating myoelectric complexes (MMCs) of both antrum and duodenum. The two drugs also blocked occurrence of premature MMCs produced by synthetic porcine motilin. In anesthetized dogs, electrical stimulation of cervical vagi with stimulation parameters: 9 V, 10 c/s, 5 msec, caused a significant increase in both portal and femoral venous PMC which was blocked by atropine. Fractionations of vagus nerve extracts by gel filtration using Sephadex G-50 superfine column revealed most of motilin-like immunoreactivity (MLI) with the same mobility as pure porcine motilin. Studies suggest that cholinergic influence plays a significant role on release of motilin.     

Comparison of motilin binding to crude homogenates of human and canine gastrointestinal smooth muscle tissue

Pharmacological studies indicate that in man and in rabbit, but not in dog, motilin has a direct influence upon gastrointestinal smooth muscle. In accordance with this hypothesis we have presented direct biochemical evidence for the presence of motilin receptors on rabbit smooth muscle tissue. We have now extended our studies to human and canine tissue. Tissue homogenates were studied in binding experiments with iodinated porcine [Leu13]motilin and iodinated canine motilin. It was ascertained that the iodination procedure had little effect on the biological activity of the porcine analogue. In the human antrum specific binding of the iodinated porcine analogue was only found in the smooth muscle layer. It was absent in mucosal or serosal preparations. At 30 degrees C and pH 8.0, binding was maximal after 60 min of incubation, and was reversed by the addition of unlabeled porcine motilin. Binding was enhanced in the presence of calcium and magnesium ions. At a concentration of 10 mM MgCl2, binding was 220% of the binding observed in its absence. Displacement studies with synthetic porcine [Leu13]motilin or synthetic natural porcine motilin indicated a dissociation constant (Kd) of 3.6 +/- 1.6 nM and a maximal binding capacity (Bmax) of 77 +/- 9 fmol per mg protein. Canine motilin displaced iodinated porcine motilin with an apparent Kd of 2.2 +/- 0.9 nM. Compared to antral binding, receptor density decreased aborally and orally, and was absent in jejunum and ileum. In dog specific binding could not be demonstrated in antral and duodenal tissue, neither with labeled porcine nor with labeled canine motilin. However, labeled canine motilin was equipotent to labeled porcine motilin in binding studies with human tissue: the dissociation constant was 0.9 +/- 0.6 nM. The present studies therefore demonstrate the existence of a specific motilin receptor in the antroduodenal region of the human gut. Apparently, such receptors are not present in the canine gut. Our data support the hypothesis that in the human gastrointestinal tract, the gastroduodenal area is motilin's target region.     

Sexual differences of the inhibitory effect of ethanol on gastrointestinal motility: in vivo and in vitro studies

Female brain is more sensitive to the acute exposure of ethanol. This study aimed to investigate the sexual difference of the ethanol-induced inhibition of gastrointestinal motility. Wistar rats were fasted and allowed drinking water only 12 - 18 h before the experiments. In the in vivo experiments, by using an oral radiochromium motility marker, the liquid gastric emptying and intestinal transit were [corrected] measured 30 min after ethanol treatment. In the in vitro study, strips of stomach and duodenum smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were also examined. Systemic administration of ethanol (2 g/kg, i.p.) significantly inhibited the gastric emptying and intestinal transit, and the effect on female rats turned out to be greater than that on the male rats (P < 0.05). In an in vitro study, ethanol (0.38 x 10(-3) M - 1.34 x 10(-3) M) inhibited the motility of gastric antrum and duodenum in rats of both sexes, but there was no sexual difference in the inhibitory effect of ethanol on muscle strips. We concluded that sexual difference of the ethanol-induced inhibition of gastrointestinal motility was not resulted from the smooth muscle itself.     

Centrally administered neuropeptide Y delays gastric emptying via Y2 receptors in rats

It has been shown that centrally administered neuropeptide Y (NPY) delays gastric emptying. To determine the receptor subtypes of NPY mediating the inhibitory effects on gastric emptying, effects of intracerebroventricular injection of NPY, [Leu31,Pro34]NPY (a Y1 agonist) and NPY-(3-36) (a Y2 agonist) on solid gastric emptying and postprandial antropyloric motility were studied in conscious rats. Intracerebroventricular injection of NPY and NPY-(3-36), but not [Leu31,Pro34] NPY, delayed solid gastric emptying in a dose-dependent manner (0.03-3 nmol). After the feeding (40 min), contractions with low frequency and high amplitude of the antrum were frequently observed, and the peak contraction of the antrum occurred most often 3-6 s before the peak contraction of the pylorus. Intracerebroventricular injection of NPY and NPY-(3-36) (3 nmol), but not [Leu31,Pro34]NPY, significantly reduced antral contractions and the number of antropyloric coordination events. It is suggested that centrally administered NPY impairs postprandial antral contractions and antropyloric coordination via Y2 receptors, resulting in delayed gastric emptying.     

Anxiolytic effect of motilin and reversal with GM-109, a motilin antagonist, in mice

There have been few reports on the effects of the brain-gut peptide motilin on the central nervous system (CNS). We administered motilin intracerebroventricularly to mice and investigated the effect of motilin on anxiety using an elevated plus-maze. Motilin produced a significant decrease in anxiety with an inverted U-shaped dose response. To determine whether the anxiolytic effect of motilin was mediated via motilin receptors in the brain, the effect of GM-109, a novel motilin receptor antagonist, was investigated. GM-109 showed a significant and dose-dependent antagonism on the motilin-induced anxiolytic effect. GM-109 administered alone had no effect on anxiety. These results suggest that motilin receptors are present in the brain and may have a role in anxiety and emotion.     

Distribution and characterization of motilin receptors in the cat

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 < 0.05) 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 < 4.3, respectively. Studies with [Leu¹³-po]motilin fragments showed that the 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.     

Plasma extravasation mediated by lipopolysaccharide-induction of kinin B1 receptors in rat tissues.

The present study was performed to: (a) evaluate the effects of kinin B1 (Sar[D-Phe8]-des-Arg9-BK; 10 nmol/kg) and B2 (bradykinin (BK); 10 nmol/kg) receptor agonists on plasma extravasation in selected rat tissues; (b) determine the contribution of a lipopolysaccharide (LPS) (100 microg/kg) to the effects triggered by B1 and B2 agonists; and (c) characterize the selectivity of B1 ([Leu8]desArg9-BK; 10 nmol/kg) and B2 (HOE 140; 10 nmol/kg) antagonists as inhibitors of this kinin-induced phenomenon. B1 and B2 agonists were shown to increase plasma extravasation in the duodenum, ileum and also in the urinary bladder of the rat. LPS pretreatment enhanced the plasma extravasation mediated only by the B1 agonist in the duodenum, ileum, trachea, main and segmentar bronchi. These effects were prevented by the B1. but not the B2 antagonist. In normal rats, the B2 antagonist inhibited the effect of B2 agonist in all the tissues analyzed. However, in LPS-treated rats, the B2 antagonist was ineffective in the urinary bladder. These results indicate that kinins induce plasma extravasation in selected rat tissues through activation of B1 and B2 receptors, and that LPS selectively enhances the kinin effect on the B1 receptor in the duodenum, ileum, trachea and main and segmentar bronchi, and may increase B1 receptor expression in these tissues.     

Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220-300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40-90 mg x kg(-1) x h(-1) iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 microm fluorescently labeled microspheres. N/OFQ (0.6-60 nmol/kg iv) caused hypotension (SAP, baseline: 154 +/- 11 mmHg, 15 nmol/kg N/OFQ: 112 +/- 10 mmHg, P = 0.009), vasodilation (venules: 23.9 +/- 1.2 microm, 26.7 +/- 1.2 microm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 +/- 3.4, 123.5 +/- 11.8, P = 0.009), and leukocyte adhesion (2.0 +/- 0.9, 15.2 +/- 0.9/100 microm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 +/- 370 microm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 +/- 0.13 vs. 2.06 +/- 0.38, P < 0.05). The NOP antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101; 60 and 150 nmol/kg iv), H(1) and H(2)antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg x kg(-1) x min(-1)) also abolished vasodilation and macromolecular leak to N/OFQ in vivo (P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (approximately 200 microm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10(-5) M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: -6.9 +/- 3.8%; N/OFQ: 32.6 +/- 8.4%; pyrilamine: 31.5 +/- 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.     

The actions of neurokinins and substance P in canine pylorus, antrum and duodenum

Analogues highly selective for receptors for substance P [beta-Ala4,Sar9,Met(02)11]-SP(4-11), for neurokinin A, [Nle10]-NKA(4-10), and for neurokinin B, [beta-Asp4,MePhe7]-NKB(4-10), were administered intraarterially before and after atropine or tetrodotoxin, to characterize the locations on nerve and muscle of the different receptor subtypes in the canine antrum, pylorus and duodenum. Circular muscle strips from each region were also studied in vitro. The NK-2 receptors in the antrum and the pylorus were located postsynaptically on smooth muscle. The NK-3 receptors, on the other hand, were located on neuronal sites in the antrum and duodenum. NK-1 receptors were located on neuronal and nonneuronal sites in the antrum, pylorus and duodenum. Only nonneural receptors could be activated in vitro.     

Nphe1]nociceptin(1-13)-NH2 antagonizes nociceptin-induced hypotension, bradycardia, and hindquarters vasodilation in the anesthetized rat

The purpose of this study was to investigate the effects of [Nphe1]nociceptin(1-13)-NH2 on nociceptin-induced decreases in mean arterial pressure (MAP), heart rate (HR), and hindquarters vascular bed resistance (HVBR) of the anesthetized rat. The results showed that i.c.v. or i.v. [Nphe1]nociceptin(1-13)-NH2 (1.5-12 nmol/kg and 5-120 nmol/kg, respectively) could antagonize the depressor effects of i.c.v. or i.v. nociceptin (3 and 30 nmol/kg, respectively) on MAP and HR. Furthermore, [Nphe1]nociceptin(1-13)-NH2 (5-120 nmol/kg) could reverse nociceptin (30 nmol/kg)-induced decrease of HVBR. However, [Nphe1]nociceptin(1-13)-NH2 had no significant effects on similar effects induced by morphine. Our results suggest that [Nphe1]nociceptin(1-13)-NH2 acts as a selective antagonist of the nociceptin receptor in the cardiovascular system of the rat.     

Motilin receptors in the human antrum

Motilin is an intestinal peptide that stimulates contraction of gut smooth muscle. The motilin receptor has not been cloned yet, but motilin-receptor agonists appear to be potent prokinetic agents for the treatment of dysmotility disorders. The aim of this study was to determine neural or muscular localization of motilin receptors in human upper gastrointestinal tract and to investigate their pharmacological characteristics. The binding of (125)I-labeled motilin to tissue membranes prepared from human stomach and duodenum was studied; rabbit tissues were used for comparison. Solutions enriched in neural synaptosomes or in smooth muscle plasma membranes were obtained. Various motilin analogs were used to displace the motilin radioligand from the various tissue membranes. The highest concentration of human motilin receptors was found in the antrum, predominantly in the neural preparation. Human motilin receptors were sensitive to the NH(2)-terminal portion of the motilin molecule, but comparison with rabbit showed that both species had specific affinities for various motilin analogs [i.e., Mot-(1-9), Mot-(1-12), Mot-(1-12) (CH(2)NH)(10-11), and erythromycin]. Motilin receptors obtained from synaptosomes or muscular plasma membranes of human antrum expressed different affinity for two motilin-receptor agonists, Mot-(1-12) and Mot-(1-12) (CH(2)NH)(10-11), suggesting that they correspond to specific receptor subtypes. We conclude that human motilin receptors are located predominantly in nerves of the antral wall, are functionally (and probably structurally) different from those found in other species such as the rabbit, and express specific functional (and probably structural) characteristics dependent on their localization on antral nerves or muscles, suggesting the existence of specific receptor subtypes, potentially of significant physiological or pharmacological relevance.     

High and low affinity receptors mediate growth effects of gastrin and gastrin-Gly on DLD-1 human colonic carcinoma cells

Gastrin (G17) and N-carboxymethylgastrin (G17-Gly) have been shown to stimulate the growth of colon cancer cells both in vivo and in vitro. The identity of the receptor mediating these effects is controversial. A recent study demonstrated the presence of a low affinity binding site for G17 and G17-Gly on the DLD-1 human colon cancer cell line. The goal of the current study was to further investigate the role of this receptor in mediating the growth-promoting effects of gastrin peptides. Binding of [Leu(15)]G17 and [Leu(15)]G17-Gly to DLD-1 cell membranes in competition with [(3)H]G17-Gly was examined. Binding of [(3)H]cholecystokinin-8 (CCK8) to DLD-1 cell membranes was also assessed. Whole cell binding experiments were carried out using [(125)I-Tyr(12),Leu(15)]G17-Gly. In addition, the ability of [Leu(15)]G17 and [Leu(15)]G17-Gly to stimulate cell growth, as determined by cell counting, was tested. [Leu(15)]G17 and [Leu(15)]G17-Gly competed with [(3)H]G17-Gly at both a high and a low affinity site on DLD-1 membranes. The IC(50) values for [Leu(15)]G17 were 6.0 x 10(-8) M and 6.9 x 10(-6) M while those for [Leu(15)]G17-Gly were 3.2 x 10(-9) M and 4.9 x 10(-6) M. [(3)H]CCK8 did not bind to either site. [Leu(15)]G17-Gly also competed with [(125)I-Tyr(12),Leu(15)]G17-Gly at both a high and a low affinity site on DLD-1 cells with similar affinities as observed with membranes. [Leu(15)]G17 and [Leu(15)]G17-Gly significantly stimulated the growth of DLD-1 cells in a dose-dependent and biphasic manner. The binding profiles of the peptides tested suggest that these sites are different from previously identified wild-type and mutant CCK(1) or CCK(2) receptors.     

Effect of enteral glutamine on leucine, phenylalanine and glutamine metabolism in hypercortisolemic subjects

The effect of enteral Gln on protein and Gln metabolism was investigated during experimental hypercortisolemia. Four groups of subjects that had received corticosteroids and either enteral Gln (0.5 g x kg(-1) x day(-1) for 2 days) or isonitrogenous Ala-Gly were studied in a fasted or in a fed state. In either state, enteral Gln, compared with Ala-Gly, induced no statistically significant change in the endogenous rate of Leu appearance, an index of proteolysis, Leu oxidation, and nonoxidative Leu disposal, an index of protein synthesis, as studied by kinetics of [1-(13)C]Leu. Similar data were obtained from kinetics of [(2)H(5)]Phe, resulting in an unchanged protein balance in both cases. In contrast, enteral Gln significantly decreased the endogenous rate of Gln appearance and Gln de novo synthesis in the fed state (P < 0.05) as estimated by the kinetics of [(15)N]Gln. This decrease in Gln de novo synthesis induced by Gln could contribute to spare amino acids in hypercatabolic patients.