Role of medullary thyrotropin-releasing hormone (TRH) in vagally-mediated adaptive cytoprotection in rats

A980

AGA

ABSTRACTS

GASTROENTEROLOGY,

• ROLE OF MEDULLARY THYROTROPIN-RELEASING HORMONE (TRH) IN VAGALLY-MEDIATED ADAPTIVE CYTOPROTECTION IN RATS. /-[ Kaneko, L. Wang, K. Kato, Y. Tach6. CURE/Gastroenteric Biology Center, West Los Angeles VAMC, Dept. of Med. and Brain Res. Inst., UCLA, Los Angeles, CA 90073. There is growin~ evidence that medullary TRH exerts a vagal-dependent eytoprotection against ethanol. However, the central mechanism by which vagally-mediated adaptive cytoprotection occurs is still unknown. Aim: To investigate (1) the effect of immunoneutralization of medullary 'TRH on adaptive cytoprotection in urethane-anesthetized rats, and (2) medullary sites actrvated by mtragastrie administration of mild/strong irmant in conscious rats. Methods: Rats underwent either bilateral cervical vagotomy or sham opera~h after, rats received intragastrically (ig, 1 ml) either saline or 0.35N-HCI and 15 min later 0.6N-HC1. In other groups, rats were injected intracisternally with control,, TRH (#8964)-, or peptide YY (PYY: #9153), antibody (260 pg/20 td), and 15 min later, rats received either saline or 0.35N-HCI followed by 0.6N-HCI. Percentage of corpus mucosal lesions [UI(%)] and Fos immunoreactivity ;in medullary nuclei was determined i h after the strong irritant. Results: Intragastric 0.35N-HCI significantly reduced by 65.4% the g a s t r i ~ a l injury induced by subsequent exposure to 0.6N-HC1 (UI: saline: 29.7 ± 1.5%; 0.35N-HCI: 10.6 ± 2.3%, P<0.01, n=6). Vagotomy completely abolished the protective effect of 0.35N-HCI (UI: saline: 30.8 4- 3.5%; 0.35N-HCI: 30.9 a: 1.1%, n=6). In control- o r PYY-antibody-pretreated rats, adaptive cytoprotective effect of 0.35N-HCI was not affected. The TRH-antibody aggravated 0.6N HCl-indncedgastric mueosal damage by 42.1% and completely blocked the protective effect of 0.35N-HCI. Intragastric administration of 1.0N-HCI in control-antibody pretreated rats induced gastric lesions of similar magnitude (37.8 * 3.3%, n=7) that observed in TRH-antibody-pretreated rats receiving 0.6N-HCI (42.9 q- 5.6%, n=6). Gastric lesions in control-antibody group were reduced to 18.7 a: 2.5% by 0.35N-HCI given 15 min before 1.0N-HCI (P<0.01, n=6). Fos ~ositivecell count (nb/15 sections) in the brain stem was: treat saline+ saline 0.35N + saline saline+ 0.6N 0.35N + 0.6N DMN

3.3±2.6

5.3±4.0

11.5±2.8

23.54-4.3**#

j NTS 254±32 803±123" 1939+272"* 1725±101'* (DMN: dorsal motor nucleus of the vagus, NTS: nucleus tractus s01itarius, • P<0.05, ** P<0.01, compared With saline + saline ig treated group, # P<0.05~ compared With saline + 0.6N-HCI ig treated group, ANOVA, n=6) Conclusion: These results indicate that (1) medullary TRH is involved in ~ d e n t adaptive gastric eytoprotection and confers resistance of the mucosa to gastric injury against HCI, and that (2) dorsal vagal complex, especially the DMN, is activated during adaptive cytoprotection.

• THE ROLE OF THE N-TERMINAL REGION OF THE HUMAN CCKA RECEPTOR IN LIGAND BINDING K. Kennedy, C. Escrieut, M. Dufresne, P. Clerc, N. Vaysse, D. Fourmy. INSERM U151, Institut Louis Bugnard, CHU Rangueil, Toulouse, FRANCE. The existence of different isoforms of specific G-protein coupled receptors is well documented. A truncated form of the CCKA receptor (CCKA-R) has been identified in pancreatic membranes by photoaffinity labeling using the peptide antagonist JMV179 (Molecular Pharmacology, 1994, 45(4), 599). Peptide mapping of the labeled receptor suggested that the receptor was truncated in the region of its N-terminus. To date, structure/function studies on CCK/gastrin receptors have concentrated On the involvement of the transmembrane domains in the interactions with non-peptide antagonists. The discovery of this truncated form of the CCKA-R has led us to study the effects of truncation on the pharmacology of the human CCKA-R, with respect to various agonists a n d antagonists, peptidic and non-peptidic, in order to assess the involvement of the N-terminal region of the receptor in ligand= binding. Using PCR we have therefore constructed a series of human CCKA-Rs truncated to different degrees in their N-terminal and first transmembrane domain. The cDNA coding for these receptors was transiently expressed in COS-7 cells and binding experiments carried out on membranes prepared from these ceils. The receptor proteins were identified by SDS-PAGE analysis after photoaffinity labeling. The functionality of the truncated receptors was tested by measurement of mobilization of [Ca2+]i or phospholipid hydrolysis in intact cells. Removal of the N-terminal residues 1-37 resulted in a receptor showing no significant difference in affinity and selectivity, for the agonists and antagonists tested, from that of the wild-type receptor, however, the kinetics of binding for CCK9 and JMV179 were greatly affected. Binding studies on the CCKA-Rs truncated to a greater degree suggest an important role for the amino acid residues in the upper part of the first tmnsmembrane domain in the interactions of both CCK9 and peptide antagonists with the receptor~

Vol. 1 0 8 ,

No. 4

INCRETIN RELEASETO ORAL AND DUODENAL GLUCOSELOADS IN MAN. M..Katschlnski J. Schirra, C. Weidmann, T. Sch~fer, R. Arnold, B. Giike. Dept. of Gastroenterology, University Hospital of Marburg, Germany. Truncated glucagon-like peptide-1 (GLP-1) and glucose-dependentinsulinotropic polypeptide (GIP} are the major incretins in man, GIP is released from the duodenum depending on the rate of glucose absorption and not the mere presence of nutrients in the gut. The mechanism ruling GLP-1 secretion must be different since it is mainly released from the distal gut which is not exposed to luminal contact with nutrients during peak GLP-1 release. This study compared time course and magnitude of incretin release to oral and duodenal glucose loads to better characterize mechanisms of release. METHODS: 8 healthy male volunteers were studied on 4 separate days. In each subject, 45 g and 90 g glucose dissolvedin 360 ml water were orally administered. Moreover, a 12.5% glucose solution (0.5 kcal/ml; 773 mosmol/kg) was duodonally perfused at 2 ml/min (1 kcal/min) and 4 ml/min (2 kcal/min) for 180 rain delivering total glucose loads of 45 g and 90 g, respectively. In each experiment, blood was regularly sampled to determine glucose, IR=Cpeptide, IR-Insulin,IR-I;IP and IR-GLP-1 (antibody GA 1178} over 180 min. RESULTS: Time course: Duodenal perfusion of glucoseelicited a constant, load-dependentGIP release with the plateau being achieved after 30 (1: kcal/min) end 15 (2 kcal/mio) min, respectively. The lower duodenal glucose load failed to increase GLP-1 plasma levels whereas the higher load brought about a steady State of GLP-1 release after 15 min. Oral administration of the same glucose loads induced n dose-dependent GIP release peaking at 30 rain. Afterwards, IR-GIP gradually declined returning to basal level at 150 min with 45 g and remaining'elevated throughout"the Study period with 90 g. GLP-1 release to oral glucose was load-dependent, peaked at 15 min with 45 g and at 30 rain with 90 g. Basal levels reappeared at 75 min with 45 g and at 180 min with 90 g glucose. AUC ovetbasal" 180 rain;D: duodenal,O: oral load Parameter D/45 g D/90 g 0/45 g 0/90 g Glucose(retool/I) 143±3.9 25.4_+3.0* 15.3_+2.4 25,8±3.8"* IR-GIP(pg/I) 1.3_+0.2 3,1 -+0.5** 1.6_+0.5 3.8±0.6***# IR-GLP-1(pmngl) 0.1_+2.5 15.9_+3.3"* 15.0_+2.8## 32.3-+3.2**## IR-Insnlin{mUll) 154.2±25.6 420.1±59.0"*'266.9±39.7###594.4_+72.8"**# IR-C-peptideO/g/I) 12.5-+1.2 23.5±1.7"** 14.9-+1.0# 26A ±2.5"*# *: p < 0.05, **: p< 0.01, * **: p< 0.001 vs. 45 g glucose,samerouteof administration,#: p < 0.05, ##: p < 0.01, ###: p < 0.001 vs. identicalglucoseloadwith duodenalperfosion. CONCLUSIONS: A comparable GIP release was induced by oral administration and duodenal perfusion of 45 g glucose. With 90 g, the oral route released more GIP probably reflecting the initial rapid gastric emptying exceeding a duodenal delivery of 2 kcal/min. Oral administration of identical glucose loads yielded a markedly higher GLP-1 release than the duodenal route. This finding would reflect higher duodenal delivery of glucose associated with the early phase of gastric emptying. By contrast with GIP, a threshold delivery of 1 kcal/min into the duodenum must be exceeded to release GLP-I. The mere luminal presence or absorption of glucose are insufficient to induce secretion of GLP-I. We suggestthat a thresholdrate of duodenalnutrient flow and/or nutrient absorptionwould initiate a neural and/or hormonalsignal to the distal gut releasing GLP-L The incretin effect in responseto low duodenalglucoseloads would be mainly mediated by GiP. The markedly higher insulin release b~ oral as compared to duodenal admini, stration of glucose would be explained by distinctly hzgher releases of GLP-1 Ilower glucose load) and GLP-1 and GIP (higher Ioad).

1,25-DIHYDROXYVITAMIN D3 CAUSES ACTIVATION OF c-SRC, AND TYROSINE PHOSPHORYLATIONS OF SEVERAL OTHER PROTEINS INCLUDING ERK-1 AND -2, IN RAT COLONOCYTES. S. Khare, H. Roy, M. Bissonnette, R. Wall, B. Aquino, M. D. Sitrin and T. A. Brasitus. Dept. of Medicine, University of Chicago, Chicago, IL. While 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is known to influence cell growth and stimulate PKC-dependent serine and threonine phosphorylations in rat colonocytes, the effect of this secosteroid on tyrosine phosphorylations has not been examined. Moreover, recent studies have implicated both c-src, a tyrosine kinase, and ERK-1 and -2, serine/threonine kinases activated by tyrosine phosphorylation, in regulating cell growth. In the present studies it was, therefore, of interest to examine the effect of 1,25(OH)2D3 on these kinases. Methods: Cells were isolated using a modified Weiser technique and treated with 1,25(OH)2D3 for the indicated times and concentrations. After cell lysis, proteins were pi'obed by Western blotting with antiphosphotyrosine antibodies. The tyrosine kinase, c-src, was immunoprecipitated with monoclonal antibodies, and utilized for src kinase assays with in vitro phosphorylation of the exogenous substrate, a CDC-2 derived Pe2Pltide, or was studied by src autophosphorylation in the presence of [3,3 P]ATP. ERK-1 and ERK-2 Were immunoprecipitated using polyclonal antibodies, and Western blots were subsequently probed with antiphosphotyrosine antibodies. Results: Increases in tyrosine phosphorylation (2-3 fold) of several proteins, ranging from 29 kDa to 64 kDa, were observed when rat colonocytes were treated with 1,25(OH)2D3 (10 uM final concentration) for times ranging from 15 sec to 12 re.in. The addition of 1,25(OH)2D3 to colonocytes rapidly activated c-src tyrosine kinase activity, as measured by CDC-2 phosphorylation, in a time and concentration-dependentmanner, Incubation for 30 sec with 10 nM 1,25(OH)2D3 caused a maximal increase in phosphorylation (2-fold). This secosteroid was found to cause a biphasic activation of c src, as measured by autophosphorylation, with peaks at 60 sec and 9 rain, respectively. Tyrosine phosphorylation of ERK- 1 (44 kDa) and ERK-2 (42 kDa) were found to peak at 9 min following 1,25(OH)2D3treatment. Summary: In isolated rat colonocytes, 1,25(OH)2D3 rapidly stimulates tyrosine phosphorylations of several proteins including ERK-1 and ERK-2 and causes biphasic activation of ppGOc=src. The time course for c-src activation is consistent with possible ERK activation by src, and may be involved in the known actions of 1,25(OH)2D3 o n important cellular events.