Section of IUPHAR GI pharmacology symposium with hungarian academy of sciences on biochemical pharmacology as an approach to gastrointestinal disorders (basic science to clinical perspectives)

D~gesth,e Diseases and Sciences, Vo£ 41, No. 2 (Febra~ 1996), pp. 440-451

Section of IUPHAR GI Pharmacology Symposium with Hungarian Academy of Sciences on

Biochemical Pharmacology as an Approach to Gastrointestinal Disorders

(Basic Science to Clinical Perspectives) P6cs, Hungary, October 12-14, 1995

The Gastrointestinal Section of International Union of Pharmacology (IUPHAR) was established in 1994 in Montreal, Canada. The establishment of the GI Section recognizes the international progress of gastrointestinal pharmacology, including basic and human studies.

The Gastrointestinal Section will actively participate in the main congress (IUPHAR). Smaller sym- posia will be organized for times between the main IUPHAR congresses.

The Gastrointestinal Section of IUPHAR has organized the first symposium as "Biochemical Pharma- cology as an Approach to Gastrointestinal Diseases:

from Basic Science to Clinical Perspectives" in Octo- ber 10-12, 1995. P6cs, Hungary.

The main topics are (1) Gastrointestinal secretory and excretory functions, (2) Gastrointestinal motility, (3) Biochemical-pharmacological mechanisms in neural and hormonal actions involved in GI functions, (4) Main normal and pathological biochemical mechanisms in GI functions, (5) GI muscosal injury and protection, (6) Molecular mechanisms of premalignant and malignant diseases in GI tract, (7) Use of isolated cells and cell cultures in biochemical- pharmacological studies to approach GI diseases. For more information contact either of the organizers.

Timothy S. GagineHa, Ph.D. University of Wisconsin School of Pharmacy 425 N. Charter St. Madison, WI 53706, USA Phone: 608-249-6740 Fax: 608-249-6795

Gyula M6zsik, M.D. Se.D.(med). First Department of Medicine, University Medical School of P&,.s, Ifjfis~ig u. 13. H-7643 P6cs, HUNGARY Phone: (36) 72-324-122 Fax: (36) 72-327-660

440

International Scientific Committee F. Capasso (Naples, Italy) M.O. Christen (Suresnes, France) E.E. Daniel (Canada) M. Diener (Zurich, Switzerland) P. Holzer (Graz, Austria) S. Okabe (Kyoto, Japan) S. Szab6 (Long Beach, USA)

(A-12)

Local Organizing Committee

O. Abdel Salam (P6cs, Hungary) B. B6dis (P6cs, Hungary) O. Karfidi (P6cs, Hungary) /k. Kinily (P&:s, Hungary) L Nag), (P~a:s, Hungary) A. Pfir (P6cs, Hungary) G. Siit6 (P6cs, Hungary) /k. Vinczc (Pks, Hungary)

Digeaive Dimues and Sden~, Vol 41, No. 2 (February 1996) 0 1 6 3 - 2 1 1 ~ . 5 0 / 0 © 1996 Plenum Publhhin$ Corporation

ABSTRACTS ON IUPHAR GI PHARMACOLOGY

MEDIATING ROI.E OF NEUROPEPTIDES AND NITRIC OXIDE IN GUT MOVEMENTS. L lilaah0. Depa,qment of Phaonacology, University Medical School P~cs. H-7643 Pdes, Hungary

A better understanding oflbe mechanisms of coordinated intestinal movements is of potential use for the management of of movement disorders such as paralytic liens or dysmotility associated with infiamreatory bowel disease.

Besides aeetylcholine (ACh) which is probably the most important excitatury ncurotransminer in the gut, neuropeptides can also participate in the regulation of intestinal smooth muscle movements as non-adrenergic, non-cholinergic (NANC) fleurotransmitters.

Tachykinins, namely substance P (SP) and neurokinin A ('NKA) appear in both ext~sic (afferent) and intrinsic neurons in the intestine, where, es calcitonin gene- related peptide (CGRP) is mainly located in the exlrinsic neurons. Stimulation of extrinsic neurons by the sensory stimulant capsaicin (CAP) causes both excitatory and inhibitory motor effecls in this tissue. The inhibitury action is probably mediated by CGRP acting directly on the smooth muscles The mechanism of the excitatory action of CAP seems more complex. CAP-sensitive nerve endings release one or more unknown mediator(s) which in turn excite(s) myenteric neurons. The final transmitter~ of the response are ACh and tachykinins. In the effecl of the inner, both NKI, NK2 and NK~ receptors seem to be involved.

A range of nsuropeptides appear in the intrinsic, myenteric neurons of the gut. Tachykinins are probable excitatory neuroWansmitlers. SP acting through NK~ receptors seems more important for the longitudinal, while both SP and NKA (the latter preferring NK2 receptors) probably mediate NANC excitation in the circular muscle in response to intraiuminal pressure stimuli or electrical field stimulation,

Endogenous CCK-like peptides seem to mediate a CAP-resistant motor response due to mesentenc nerve stimulation. While CCK-8 potently stimulates peristalsis, we could not find evidcoce for the involvement of endogenous CCK in this rcflex.

The neuropeptide VIP and the guscous free radical nillic oxide (NO), as well as ATP are the most likely candidates for NANC inhibitory neurolransminers acting in the gasl~ointestin~ smooth muscle,VIP and the related peptide PACAP relax the intestinal smooth muscle directly bat excite myenteric neurons, thereby releasing ACh and tachykinins. NO has a similar dual site of action in the guinea-pig ileum, while it causes primary contraction, relaxation and afrer-contraetion in the rat ileum, in a tetrodotoxin-resistanl fashion. NO seems to be involved in the peristaltic reflex in the guinea-pig and rat small intestine; for VIP and PACAP, more s, peeifin antagonists are still needed to establish their roles in coordinated reflexes.

Work in the author's laboratory has been supporled by Hungarian ETT and OTKA grants and by the Anstrian-Hungarian Foundation.

NITRIC OXIDE AS A MODULATOR OF FLUID ABSORPTION IN RAT JEJUNUM IN VIVO. E. Beubler. A. Sehirgi-D~gen. Dept. o f Experimental & Clinical Pharmacology, University of Graz, Austria

The role of nitric oxide ('NO) as a regulator o f intestinal fluid transport is still controversial. In part, the experimental work describes secretory properties of NO in vitro and in vivo, other investigators, however, demonstrate a proabsorptive role of NO.

In the present study we present data to support the latter, namely the hypothesis of an absorptive effect of NO. We tested the effects of the NO-synthase inhibitor L-NAME and of NO-donors L-arginine and sodium nitroprusside on basal fluid transport and on intestinal fluid secretion stimulated by 5-HT, P O ~ , E. eoli STa and cholera toxin.The experiments were performed in the tied off loop of the anaesthetized rat in vivo. L-NAME was infused intravenously in the absence or presence of L-arginine or sodium nitroprusside. 5-HT (0.16 l.tg.min "s, 30 rain), and PGF_~ (0.16 l~g.min "t, 30 rain) were infused close intraarterially and E. colt STa (10 U.ml "~, 30 rain) and ehotera toxin (0.5 ~tg ml q, 4 h) were administered intraluminally. Net fluid transport was determined gravimetrically.

The inhibitor of NO synthetase, L-NAME, dose dependently (0.25 - 25 mg kg "1 i.v.) reversed net fluid absorption to net fluid secretion. L- N A M E (25 mg kg "t) significantly enhanced fluid secretion stimulated by 5-HT, PGF-a, E. eoli STa- and cholera toxin.

L-arginine (400 mg kg "l) and sodium nitroprusside (1 mg kg "t) slightly, though not significantly enhanced fluid absorption in controls but significantly inhibited 5-HT, PGFa-, E. coti STa- and cholera toxin- induced fluid secretion.

The results indicate that exogenously administered or endogenously formed NO exerts a proabsorptive action in the intestine. This effect may also counteract the secretory response to secretagogues. NO thus may attenuate pathological secretory conditions in the intestine.

MECHANISM OF CHOLERA. I~. Beubler. Dept. of Experimental & Clinical Pharmacology, University of Graz, Austria

The effect o f cholera toxin on intestinal fluid secretion is commonly considered to be mediated by cyclic adenosine monophosphate. In the meanwhile, also 5-hydroxytryptamine (5-HT), prostaglandin E~ (PGE~) and the function of neuronal structures have been implicated in the

pathogenesis of cholera. To elucidate the role of 5-HT and PGE2 in mediating cholera

secretion, experiments were performed in the rat jejunum in vivo. Cholera toxin was administered intraluminally (0. I - 0.5 ~tg ml "l, 1-5 h). 5-HT receptor antagonists and indomethacin were administered subcutaneously and Ca-antagonists intraarterially. 5-HT was measured by HPLC and PGF_~ by radioimxnunoassay.

Cholera toxin dose-dependently and time-dependently increased mean net fluid secretion with a maximum at 4 h. It also dose dependently caused 5-HT and PGF_~ release. The 5-HT2 receptor antagonist ketanserin and the eyclooxygenase inhibitor indomethacin both partially reduced cholera toxin-induced fluid secretion but not 5- HT release. The 5-HT~ receptor antagonists tropisetron, ondansetron and granisetron dose-dependently reduced and at higher doses totally blocked cholera toxin-induced secretion. The most potent blocker turned out to be granisetron. Both nifedipine and verapamil also dose- dependently inhibited eholera toxin-induced secretion.

In conclusion, our results provide evidence for a predominant role of 5-HT in cholera toxin-induced secretion. Our data suggest a model in which cholera toxin may activate the adenylate cyclase-cAMP system in enterochromaffine cells, resulting in 5-HT release. 5-HT then aetivates 5-HT z receptors, which cause P G ~ formation and 5-HT3 receptors on neuronal structures. Stimulation of 5-HTz and 5-HTj receptors leads to the profuse secretion Which may use Ca as a final

mediator.

CONTENT COMPENSATION POSSIBILITY OF EPIDERMAL GROWTH FACTOROF SALIVA IN RAT. J.Blazsck, K.Offenm011er, G.Varga. K.Birkk MWenc-A. T.Zel!es Dept. of Oral Biology, Semraeh~is Medical University, Budapest, Hungary

T ic intact surface of mucous membrane is one of the basic conditions of the physiological function of Gnstro intestinal Tract (GI). Epidermal Growth Factor (EOF) plays an important role in vital functions and regulation of GI cells with many favoumblc effects.

In the present study we report how the salivary and glandular EGF level depends on the increased functional activity of salivary gland, in the intact (Control) or both side SubMandibalar + SubLingual (SM+SL) exeisionated (Ablatio) animals. For the increase of functional activity of salivary glands used the gustatoric stimulation effect of the 0,5% Citric acid content drinking water ( Zelles et at. 1984 ). The intact and ablationated animals we divided, "top watur"concerulng "citric acid water" drinking groups (.marked. C, Ab. Cit. Ab+Cit rcsp.) On the 7 o ̀day under narcosis collected the Pilocarpm sttmulated (0.25mgl100gbw) saliva from the every second animals. The salivary gland v, as excised and homogeulsed. We n'a~ttred the EGF level by modified radio- receptor assay and the protein content by Bio-Rad Protein Assay.

Protein concentration of salivary glands decreased in control animals in 30 minutes after stimulation (Parotid-90*/,, SM-10%). Protein co. both before and after stimulation in all of treated groups is near the same as after stimulation level of control group. EGF ec. (ng/mg tissue) increases about 40% in SM tissues of C/t-group animals, and decreases the parotid tissues of all treated groups tab-88%, C/t-89°/0, Ab+Cit-95%). We can measure dec- tease of EGF cc. after Pilecarpin stimulation to a great extent in all salivary tissues (SM:C-3%0, C/t-38%0, Parotid:C-49%0,Ab-62%, Ot-7%0,Ab+Clt-27%). Protein content in the samples of saliva was significant higher than the started level in all treated groups tab +240%, Cit +80*/0, Ab+Ot+ 350%). Change of level of protein in control saliva is lower than 10./*. EGF ec.of saliva decreases in ablation group (-10*A), while in the other treated animals increases (Cit +20%, Ab+Cit+ 200%). Change of EGF ec. of saliva is tower than 10%o in control group. Under experimental conditions and results the following conlnsions can be drawn:

- EGF level of saliva can increase by the higher activity of salivary glands - EGF content of saliva doesn't present exclusively one type of salivary gland - C o m p t e m e n t e r salivary glandacan compenzation of EGF content of saliva,

when the other main gland has pathological state.

(Aq3) Digestive Diseases and Science, VoL 41, No, 2 (February 1996) 4 4 1

T H E EFFECt" O F HISTAMINE IN T H E W H O L E S T O M ACH A N D MIXED ISOLATED GASTRIC M U C O S A L CELLS IN RATS. B. B6dis~ O. Kar~di, O.M.E. Abdel-Salam, R. Faludi, L Nakrv, Gv. M6zsik. First Depar tment of Medicine, Medical University School of P6cs, Pdcs, Hungary

Histamine is a namely aggressive factor in the stomach due to stimulation of H~ receptors and gastric acid secretion. The a im of this study was to compare the cytoprotective effect of low doses of histamine in vivo and in vitro. In vivo studies the gastric mucosal damage was produced by intragastric administration of 1 ml 96 % ethanol (EtOH) in Sprague-Dawley rats. The animals were sacrificed at 1 hr after E tOH administration, when the gastric mucosal damage was measured, Histamine (Peremin, Chinoin, Hungary) were given s.c. at 30 min before administration of E tOH without and with PGI2Na (5 pg/kg s.c.). The values of pD2 and pA~ were determined. Mixed population of rat gastric mucosal cells (GMCs) was isolated by the method of Nagy et al. (Gastroenterology 107, 907-914, 1994). Cells were preincubated for 60 min with histamine (10e-10" M) without or with PGI2Na (10 ' M). At the end of this incubation cells were treated by 15 % EtOH without or with indomethacin (IND, in doses of 104-10 ~ M) for 5 min. Cell viability was tested by trypan blue exclusion test and succinic dehydrogenase activity. Results: 1) Histamine stimulated the PGI2-induced gastric cytoprotection in vivo (pDz value 4.7, pAz value 3.75) (M6zsik et al. Gastroenterology 108, A171, 1995); 2) There was no measurable acid secretion by our method in isolated GMCs after incubation of 10~-10 ~ M histamine; 3) PGI2Na (I0" M) protected the pretreated cells against the combined effect of E tOH (15%) and IND (10 3 M); 4) Histamine preincubation did not prevent the EtOH-induced cell injury. Conclusions: 1) Freshly isolated rat GMCs cannot be stimulated by low doses of histamine; 2) The cytoprotective effect of histamine failured in vitro; 3) The mechanisms of histamine- induced cytoprotection differ in vitro and in vivo. This studywas supportedbythe MungarianNational ResearchFound (OTKAT020098)and the Ministr2~f Welfareand Health (~--vr-03660/93).

A B S T R A C T S O N I U P H A R G I P H A R M A C O L O G Y

NEW PHARMACOLOGICAL INSIGHTS FOR THE USE OF A GASTROINTESTINAL SELECTIVE CALCIUM ANTAGONIST IN IRRITABLE BOWEL SYNDROME (IBS) H.O. Christen. Solvay Pharma, Suresnes 92151. France.

Irritable Bowel Syndrome (IBS) i s a m u l t l f a e t o r i a l gastrointestinal disorder, the etiology of which has been intensively investigated. Nowadays in addition to gastrointestinal hypermotility generally recognized, a recent new hypothesis concerning the mechanism implicated, focuses on the role of visceral efferents which mediate the hypersensitivity of the gut. Besides the drugs typically developed for the t r ea tment of IBS, a new therapeutic approach is based on modulation of calcium-influx via an interaction with the L-type voltage-dependent calcium channels. It is well established that this mechanism ~ a r t i c i p a t e s in the c o n t r a c t i o n - r e l a x a t i o n coupl ing and i s Involved in the control Of gastrointestinal motility. Calcium antagonists known to act when the cell membrane is depolarized following electrical stimulation, have been shown in the recent years to interfere with gastrointestinal hormone- or mediator-induced phenomena at the intestinal level. Such an effect has been demonstrated both in vitro and in vivo, In vitro Pinaverium bromide (Dicetel ®) a selective gastrointestinal calcium antagonist, inhibits CCK-, gastrin- and substance P-induced contractlon in single isolated intestinal smooth muscle cells (I). This effect was confirmed in vivo by showing the inhibitory effect of this drug orally administered on the colonxc response to eating in rats, which is controlled by a mechanism involving CCK (2). A decrease in the density of substance P innnunoreactive fibers, was also observed usin~ confocal laser microscopy technique following incubation ot circular intestinal muscle layers with Pinaverlum (3). These data provide rationale for the role of a selective calcium antagonist on hormone-induced intestinal reactions implicated in hypersensitivity of the gut and supports its beneficial effect in IBS, a multifactorial disease. Taken together these new findings open new perspectives for the use of a selective calcium antagonist in functional gastrointestinal disorders from a research and clinical standpoint.

(1) BOBO MH., MAGOUS R., CHRISTEN MO. & BALI JP., Life Sciences, 54, 1947-1954, 1994 (2) FIORAMONTI J., CHRISTEN 140., DUPRE L., BUENO L., Gastroenterology, 108, N°4, A598, 1995 (3) JULE Y., CHRISTEN MO., Gastroenterology, 108 N°4, A624, 1995

SECRETORY PATHWAYS IN HISTAMINE-CONTAINING ENTEROCHROMAFFIN-LIKE CELLS OF RAT S'I~MACH. D. Chen, C-M, Zh~o, K An0¢rsson, R, HLkanson. Dept. of Pharmacology, University of Lund, Lund, Sweden

Histamine-producing enterochromaffin-like (F__,CL) cells are numerous in the oxyntic mucosa of the rat stomach. They respond to gastrin by secretory activation, hypertrophy and hyperplasia. They contain cytoplasmic granules and vesicles, ot-Fluoromethyl- histidine (ot-FMI-I) depletes histamine from the ECL cells by inhibiting the histamine-forming enzyme histidine decarboxylase. Long- term hypergastrinemia increases the ECL-cell histamine concentration. The present study adresses the questions of how histamine is stored in the ECL cells and how i r i s released upon st imulation with gastrin. Sprague-Dawley rats were treated with a - F M H , omeprazole, u-FMH+omeprazote, and gastrin infusion. ECL celt profiles in electron mierographs were analyzed planimetrieally. Based on ultrastructural observations, we characterized and classified the granules/vesicles in the ECL cells into granules (median profile diameter 120 nm), secretory vesieles (180 nm), mierovesi- d e s (70 nm), and vacuoles (more than 500 nm). The granules were unaffected by a-FMH, reduced in number by long-term hypergastrinemia but increased in number after cessation of the hypergastrinemia. Secretory vesicles were markedly reduced in number by a- FMH and significantly reducedby hy~rgastrinemia. Microvesicles were reduced in number by a - F M H but increased in number in response to gastrin infusion. Vacuoles were observed in response to the hypcrgastrinemia. Accordingly, F.,CL cells respond to long- standing gastrin stimulation by hypertrophy and by the develop- merit of vacuoles. The findings support the view that histamine xs stored in secretory vesicles and vacuoles, perhaps also in granules and mierovesieles. Granules develop into soeretory vesicles by actively taking up preformed histamine from the cytoplasm. Gas- t r in acce lera tes severa l o f the s teps that control the bir th and subsequent maturation of the storage/secretory organelles, namely the formation o f granules, their transformation into secretory, vest- t ies, the process o f excytosis and eadoeytosis (resulting in nuerov- esicles), and the fusion o f several secretory vesicles with each other (resulting in vacuoles).

HAS THE AIDS TREATMENT EFFECT ON THE WHOLE GASTROINTESTINAL TRACT IN HEAT SHOCK PROTEIN LEVEL? P. Csere, G. Vfirbir6, B. Stimegi*, Gy. M6zsik First Department of Medicine and Department of Biochemistry*, Medical University of P~.cs, Hungary

AIDS has several manifestations in the gastrointestinal tract - dominantly in the oral cavity. The nucleotide analogue, 2",3'-dideoxyeitidine (ddC) as an anti-AIDS drug produce also some effects on the alimentary tract (like as inflammation). The analysis of the expression of the heat shock proteins (HSP-60, HSP-70) is a new marker of the inflammation caused by external stress. HSP-60 is located mainly in the mitochundria, HSP-70 is found in both tile cytoplasm and in the mitochondria. Our aim of recent study was to reveal whether ddC can produce any expressive biochemical changes in tile oral cavity or generally in the gastrointestinal tract. Methods: The rats were treated with intraperitoneal administration of 1 mg ddC/day for two weeks. The animals were killed by cervical dislocation, then the bueeal, lingual, oesophageal, gastric, duodenal and colon tissues (containing the mucosa and muscle) were removed, and frozen in liquid nitrogen. Protein extract was obtained and separated by SDS-PAGE, followed by the Western blotting of the proteins to the nitro-cellulose membrane. After binding the first antibody (mouse-derived anti-heat shock protein), a second antibody was administrated (anti-mouse IgG, marked with pernxidase). The antigen antibody reaction was detected by 3-3 diaminobenzidine). Results: 1. The HSP-60 monoclonal antibody binding was visible in the control and in tile treated group of oesophageal, gastric, duodenal and colon tissues, quite the contrary as in the bueeal and lingual samples where it was not detectable in the treated group. 2. The HSP-70 was detectable in every tissue of both treated and non-treated group. 3. To verify whether the cause was oxidative damage the NADH-dehidrogenase (Complex I) and citochrome-oxidase (Complex 11I), members of the respiratory chain were examined, but no alterations were found. Our results lndteate that the disappearance of HSP-60 in the oral cavity is a unique phenomenon following ddC treatment. The interaction between the ddC and the HSP-60 might cause this damage, however the proper mechanism is not yet known. This study was supported by Hungarian National Research Fund (OTKA No. T020098) and Ministry of Health and Welfare (ETT-03 660/93)

( A - 1 4 )

4 4 2 ~ D i ~ a.d ScOnces, Iiol. 4l, No. 2 (February 1006)


MECHANISMS AND REGULATION OF CL- SECRETION IN THE LARGE INTESTINE: STUDIES WITH THE RAT DISTAL COLON. M. Diener. Inst. for Veterinary Physiology, Univ. Zfirich, CH-8057 Z~ich, Switzerland

The regulation of CI- secretion in rat distal colon by different second messenger pathways, the cAMP-, the Ca 2 +- and the cGMP- pathway, was studted with the Ussing chamber technique and with whole-cell recordings at isolated colonic crypts. Agonists increasing the intracellular cAMP concentration like forskolin or vasoactive intestinal peptide induce a depolarization of the membrane by increasing the apical CI- conductance. Their effect can be inhibited by a protein kinase A inhibitor suggesting a phosphorylation of apical CI" channels. In addition, forskolin stimulates bumetanide-sensitive S6Rb*-uptake mediated by the basolateral Na+-K+-CI -- cotransporter.

In contrast, carbachol, an agonist o f the Ca 2 +-pathway, causes a hype. rpolarization of the membrane. This effect is exclusively due to an mcrease in a Ba 2 *-sensitive K* conductance. There is no evidence for a direct activation of Cl" channels by intracellular Ca 2 + in native colonic epithelium. Experiments with the intact mucosa in the Ussing chamber revealed an indirect dependence of the ability of carbachol to induce CI- secretion from the spontaneous production of c A M P Only when the mucosal cAMP production is stimulated by the continuous release of prostaglandins or neurotransmitters, the tissue can respond to carbachol with CI- secretion. These results strongly suggest that the primary second messenger controlling the activity of apical CI- channets is cAMP, while Ca 2+ determines the dr iving force for CI- secretion by controlling K* conductance.

Also the cGMP-pathway shows an indirect dependence on the cAMP-pathway in the rat distal colon. In this tissue, the effect of heat-stable E.coli toxin (STa) is inhibited by indomethacin. Inhibition can be overcome by prostaglandin E2 or forskolin, but not by a hydrolysis-resistant cAMP-analogue. The effect of STa is mimicked by inhibitors of ¢GMP-sensitive phosphodiesterases, which inhibit the action of subsequent administration of STa.

Consequently, there is a complex interaction of all three pathways responsible for the control o f colonic Cl" secretion with a central role of cAMP as the second messenger, which regulates apical CI- channels.

CHANGES OF CAPSAICIN-SENSITIVE FIBERS NEUROPEPTIDES IN EXPERIMENTAL GASTROINTESTINAL DISEASES. S. Evangelista, D. Renzi* Pharmacol. Dept., Menarini Pharm., *Universi- ty of Florence, Clin. Pathophysiol. Dept., Firemze, I ta- ly.

Catcitonin gene-related peptide (CGRP) is a marker of afferent f ibers in the upper gastrointestinal tract being almost completely depleted following the treatment with the selective neurotoxin capsaicin. Decreased levels of gastric CGRP-like immunoreactivity ( l i ) were observed during acetic ac id- , cysteamine-, concentrated ethanol- or water immersion stress-ulcers. The ulcerogens did not a f fect t issue content of other peptides suggesting that reduction in gastr ic CGRP-li cannot be a t t r ibuted only to a damage of the t issue and restorat ion of CGRP-li was observed in animals bearing ulcers in healing status. These findings together with the observation that CGRP could be released during increased acid-back diffusion suggest the involvement of CGRP during gastric ulcers. Similar results were obtained in duodenal ulcers produced by cysteamine, dulcerozine or mepirizole. Decrease in CGRP-Ii and SP-li was associated with the development of gastroduodenal ulcers. In a rat model of co l i t i s such as that induced by TNB, decreased levels of CGRP-, NKA- and SP-li were observed. These lat ter peptides were reduced during the acute pha- ses of co l i t i s and were related to an upregulation of B-PPT mRNA expression. These findings show that sensory neuropeptides were involved in several experimental diseases and might play a signif icant role in their pathogenesis.

THE ROLE OF MOLECULAR EPIDEMIOLOGY IN THE CONTROL OF GASTRO-INTESTINAL CANCERS

lstv~tn Ember~ Jinos Sindor Institute of Preventive Medicine, University Medical School of P&s

!]-7643 P&:s Szigcti st. 12. Hungary

The advances of molecular biology in describing the basic alterations underlying the base of the uncontrolled cell proliferation have made it possible to improve the effectivity of cancer control. The application of molecular biological tools improves the validity of known biomarkers and determines new targets for cancer related investigations. The application of the oucogcne related markers in traditional epidemiolngical studies revealed the etiological background of the hepatecellular carcinomas. Due to these results using the mutational spectrometry it is possible to differ the smoking related cervix cancers from HPV induced malignancies, or the HBV induced liver cancers from aflatuxln induced one's. The occupational and environmental medicine can apply the investigation of selected oncogenes' expression in lymphocytes to describe the personal cancer-risk of subjects exposed to carcinogenic chemicals. It is known that there are some rare Ra-ras alleles which increase the personal susceptibility to environmental agents growing the probability of breast cancer developmenL To make an established therapeutical decision in the case of breast cancer, some data on the erbB2 oncogene status is required. The MDR-I gone status determines the cancer sensitivity to anticancer drugs. Because Ore p$3 mutation and deletion is a definite sign of the malignant character in colon pelyposis, the investigation of p53 status in tissue samples from colon polyps may be a routine diagoostical task in the near future. The determination of am23 gone status is very informative parameter for predicting the metastatic abilities of breast cancers. Some oocogene products (erbB2, p53) appear in bloodstream. These proteins are useful markers to control the effect of therapeutical processes and results. The progression of colon polypesis was the first carcinogenic process which was described by genetic markers. Because there are some other proved premalignant lesions of colon it seems to be a promising field to apply the molecular cpideminlogy to elucidate the background of these colon diseases

_and~o im p=eva'..-the-adequP-cy ~'24'- -'liagatgic, al..and~'herapeutieal-P roce~es--

OXIDATIVE STRESS AND ITS PREVENTION IN TOXIC LIVER LESIONS. J.Feh~r, A.BI~zovios, G.Lena>el. 2nd Dept.of Medicine Semmelweis University, Buda~ pest, Hungary.

The yearly intake of alcoholic beverages in the population of Hungary is very high, It is about 12 litres/year/capita.The mortality rate of micronodulare liver cirrhosis is 43/lO0.OO0/year. The cumulative survival time of these patients is 50 percent within 5 years in total of patients involving all the treated and untreated ones.In the mechanism by which the alcohol produces toxic effect on the liver,is the oxidative stress. The different diseases are:fatty liver,alcoholic hepatitis,liver cirrhosis.Much evidence is presented that nutritional antioxidants e.g.vitamin E and certain drugs,namely flavonolignanes from Silybum marianum exert protective effect on several immune dysfunction.The toxic reactions of the liver can be investigated by different methods.In clinical and experimental studies silymarin and silibinln exerted favourable effect on acute and chronic hepatltis,liver cirrhosis and toxic hepatic inJury.Iat has been shown that vitamin E is able to protect membranes from oxidative damage as an electron donor and to break free radicals initiated chain reaction among the polyunsaturated fatty acids in membranes. Our studies show that in hyperlipldemic rats and in patients with chronic alcoholic liver disease the extra-hepaticaly detectable oxidative stress state were favourable influenced by silymarin treatment.Applied therapy decreased lipid peroxidation and improved the antioxidant protection of patients.If the animals were treated with ~itamln E or silibinin the values were better, than the two antioxidants were added together.

(A-15) Digestive Diseases and 5c~u~, VoL 41, No. 2 (February 1996) 4 4 3

A B S T R A C T S O N I U P H A R G I P H A R M A C O L O G Y

REACTIVE OXYGEN AND NITROGEN METABOLITES IN MUCOSAL INJURY AND PATHOPHYSIOLOGY:AN OVERVIEW. ~ _ Q a g i l l g ~ . Madison, Wl, USA.

Inflammatmy bowel disease (IBD) - Crohn's disease of the colon and ulcetalive colitis - are a~omlmnied by the pmcluetion of a complex array of mediames of inflammation and factors that attempt to maintain

of muoosal structure and function. Granulocyte generated oxidants and the reactive nitrogen membolite nitric oxide (NO), or their melabolie intermediates and products, are implicated as mediators of the intestinal secretion and diarrhea associated with IBD. One of the m ~ potent oxidants thought to be present in the inflamed mucosa is monochloramine (MCL), which is formed from the reacdon between HOCI and ammonia. MCL (5 pM to 50 pM) evokes chloride-dependent increases in short-circuit current in the rat colon (EC50 3.2 gM) /n v/fro and in cultured human T u eeUs (ECS0 20 /d~). The mechanism of MCL's action involves prostaglandins and is partially dependent on enteric nerves and calcium. Similarly, sodium nitroprusside and NO stimulate chloride secretion in the rat colon (EC50's aplxox. 80 and 8 ~M, respectively). The secretion is partially dependent upon the intrinsic innervafion. The oxidative activation by NO of guanylin, an endogenous stimulant of particulate guanylate cyelase and chloride secretion in the rat oolon, may initiate the secretory ~ . NO produced physiologically by constitutive NO synthase in nerves and vascular eodothelium is beneficial to the inlegrity of the intestinal mucosa, and under certain circumstances NO enhances fluid absorplion. Thus, at low concentrations produced constitutively (< riM), NO offers mucosal protective effects and may be a physiologic regatator of water absorption; higher concentrations generated during inflammation by the inducible isoform of NO synthase (iNOS) lead to pathology and diarrhea as a pathophysiologic consequence. Intensive ~ h is being focused on idendfiying selective inhibitors ofiNOS as primary or adjunctive therapy in IBD.

MODIFICATION OF THE PATHOBIOLOGICAL EVENTS BY POTENTIAL HEPATOPHARMACOLOGICAL AGENTS. A. Jeney, F. Timdr, Zs. Sehaff, A. Zalatnal, K. Lapis, J. Oldh. A. Divald. I. Inst. Pathol. Exp. Cancer Res. Semmelweis Univ. Med. Budapest, Hungary

The elucidation of the pathobiological events in chronic liver diseases, - such as cellular injury, cell proliferation, remodelling of extracellular matrix, - has major importance for establishing the rational bases of hepatopharmacology. To follow this concept the molecular-pathological features of chemically induced liver damage in rats were characterized and modulated by potential hepatopharmaeologieal compounds. Among the 55 chemical compounds tested acut liver damage could be most effectively abolished by prostacyclin (PGI-2) and thiazolidine compounds. In addition prosmeyclins were also able to prevent the formation of cirrhosis in experimental animals. The mode of actions of the prostacyclins were investigated in short term hepatocyte culture. The hepatotoxie induced metabolic alterations (reduced glueoneogenesis and protein synthesis, lipid peroxidation etc) could be restored by prostacyelins. It was shown that PGI-2 could circumvent the augmented catabolic rate of 4.5-phosphatidyl inositol-diphosphate (PIP2) in CCL 4 induced hepatocyte injury. In addition the increased inWacellular calcium concentration in the injured cells was also normalized by POI-2. Thus PIP 2 metabolism appears to be a critical process in the mechanism of hepatocyte damage and its protection. Interestingly PGI-2 showed effectivity at the advanced stage of liver injury, whereas thiazolidines were only active when administered before the application of the hepatotoxic agent. The formation of extracelhilar matrix components; collagen and proteoglycans could be reduced by amino-imidazol- carboxamide, silymarin and 5-bexil-2-dcoxiuridin resp. The presented data provide further evidences that compounds with various targets are required in hepatopharmacology. References: Exp.Mol.Pathol. 42:163-166, 1985; Tokai J.Exp.Clin. Med. l 1:135-145, 1986; Biocbem. Pharmacol. 40:1477-1483, 1990

DISTRIBUTION OF MUSCARINIC RECEPTOR mRNAs IN THE GASTROINTESTINAL TRACT OF NORMAL OR STRESSED RATS. B. Hunvadv*'. l~. Meze~. K. Pacak t. G~,. Hatta t. M. Palkovits t. National Institute of Mental Health t. National Institute of Neurological Disorders and Stroke l, NIH, Bethesda, MD, USA; First Deptartment of Internal Medicine. Medical University of P6cs, Hungary'.

Functional roles of the vagal innervation of the gastrointestinal/GF system and the effect of musearinic receptor agonists and antagonists have been widely reported in several studies. Five subtypes of cholinergic muscarinic receptors (M1-M5) have been characterized by pharmacological and molecular biological methods. All of them have been cloned in the past decade. The M3 receptor subtype was demonstrated in acid secreting cells both by functional and binding assays, while the M 1 subtype is reported in the intramural ganglionic cells. In the recent study the distribution of M1-M5 musearinie receptor subtypes was mapped by in situ hybridization method in the rat GI tract. Twelve ~m frozen sections from the non treated or immobilized (two hours) rats (3-5 rats/group) were hybridized with saLUTP labeled ribonucleotides (400-500 nucleotide each) directed against mRNAs of the M1-M5 muscarinic receptor subtypes. MI and M3 receptor mRNAs were present in different locations and in different densities in the GI tract of rats, while there was a completo lack in detection of mRNAs of M2, M4 or M5 subtypes. M1 subtyp~ mRNA was found over the myenterie ganglionic cells all over the stomach and duodenum, over some deep glandular cells and tunica propria ceils of the prepyloric area. No labeling was seen over the fundic or cardiao mucosa (over epithelial or tunica propria cells). The distribution of MI receptor subtype mR.NA was not changed in immobilized rats. M3 receptor subtype mRNA was detected unevenly over the parietal cell rich portion of the cardiac, fundic or prepylorie mueosa and in a very high density over the duodenal surface epithelial cells. No labeling was seen over the mucin secreting cell layer or over the tunica museularis mucosee in the stomach. A large number of tuniea propria cells were also labeled in the stomach and the duodenum. Some of these cells were in close contact with th~ epithelial cells. The density of the labeling was markedly increased in stressed rats, both over the epithelial and tunica pmptia cells. These data confirmed the data obtained by pharmacological and radio ligand binding assays on M1 and M3 receptors in the stomach and support the existence of a non edaitheliat cell regulation system i . the GI trac, L

444

EFFECTS OF ERYTHROMYCIN ON THE PROPULSIVE MOTILITY OF GASTROINTESTINAL TRACT IN RATS O. Karfidi, B. Bddis, Gy. M6zsik First Department of Medicine, Medical University of Pdcs, Pdcs, Hungary

The frequent gastrointestinal (GI) side-effects of erythromycin (ERYTH) are known (nausea, vomit, diarrhoea). Recently, small doses of ERYTH have been examined like prokinetic agent supposed its motilin agonism. The measuring of postprandial migrating motor complexes showed different effects of small-, medium- and large doses of ERYTH on the GI motility in animal models. The aim of our study was to examine the effects of different doses of ERYTH on the propulsive movement of intraluminal content in rats. Materials and methods: The observations were carried out on Sprague-Dawley rats weighing 250-300 g of body weight. The animals were fasted for 24 hours before the experiments but water was given freely. The animals received 1.5 ml 1.5% methylcellulose (MC) painted with 0.05% phenol-red intragastrically. ERYTH lactobionate was given intravenously in 0.1; 0.25; 1.0; 5.0; 10.0 and 20.0 mg/kg doses at 15 rain before the MC administration. The animals were sacrificed at 0, 20, 60 and 120 min after MC administration, when the phenol-red content of the stomach and six equal parts of small intestine was measured spectrophotometrically. The distance between the front of painted bulk and the pylorus was expressed in percent values of total length of small intestine. The gastric emptying was calculated from the rate of the measured total and intestinal phenol-red.* Results: 1. Small doses of ERYTH (0.1; 0.25) increased the gastric emptying and the propulsive motility of upper small intestine after 20 rain. 2. Medium doses of ERYTH (1.0; 5.0) decreased the GI motility after 20 rain. 3. Large doses of ERYTH (10.0; 20.0) had no significant effects on the GI motility. 4. The significant effects of ERYTH on the G[ motility exist only in the first 60 rain. after its application. Conclusions: Small doses of ERYTH may be suitable to use like prokinetic drug, but in rats it has short effect-t£me and small distance to the inhibitory doses. This study was supported by the grant of Hungarian National Research Fund (OTKA T-020098).

(A-16)

Dig~ive Disease..# and Science, VoL 41, No. 2 (February 1996)

ABSTRAC'I~ ON IUPHAR GI PHARMACOLOGY

GASTRIC HYPEREMIC RESPONSE TO CENTRAL VAGAL ACTIVATION: INVOLVEMENT OF NITRIC OXIDE AND "EFFERENT ~UNCTI~N" OF C~PSAICIN SENSITIVE AFFERENT NEURONS IN RATS A, Kiralv. G. Sut6. E. H. Livin2ston. P.H. Guth. S. St. Pierre* and Y, Me~. cCURE/UCLA Gastroenteric Biology Center, VA Wadsworth

t r . and Dept 0£ Medicine and Brain Research I n s t i t u t e . Univers i ty of Cal i forn ia Los Angeles, Los Angeles, * I n s t i t u t e Natlo~al de la Recherche ~c ien t i f lque-San te , Univers i ty of Quebec, Polnte Cla i re , Quebec, H9R IG6

Thyrotropin r e l eas ing hormone (TRH) in the medulla is involved in the vagal regula t ion of gas t r i c funct ions . TRH or s tab le TRN analog, RX 77368. i n jec ted into the dorsal vagal complex or i n t r a c i s t e r n a l l y ( i . e . ) a c t i va t e s vagal e f f e r en t discharges leading to the s t imula t ion of ga s t r i c acid sec re t ion , motor funct ion and mucosal blood flow (GMBF), as well as the re lease of histamine, se ro ton in , PGE 2 and nitric oxide through muscarinio dependent pathways. Splanchnic capsalcin sensitive afferent fibers contained ca l c l t on in gene re la ted peptide (CGRP) which can be released upon stimulation of afferents and excerts potent vasod i l a to ry effect. In the present study the per iphera l t r a n s m i t t e r s mediating gastric mucosal hyperemia fn response to i.c. injection of TRH analog were investigated in ure thane-anes the t ized r a t s . The GMBF was measured by using the hydrogen gas-clearance technique and blood p ressu re was monitored throuRhout the experlment.

RX 77368 ~30 rig, i . e . ) increased by 129% OMBF within 30 mln post injection and decreased by 42 % the vascular resistance. Indomethacin (5 mg/k~, i.p.), pyrilamine (I mg/kg i.v. bolus + 2 mg/kg/R I.~. infusion), the VIP receptor antagonist, (4 CI-~-Phe°Leu')VIP, (6.7 mg/kg/h i.v. infusion), and the substance P receptor antagonist, CP- 96,345, (3 mg/kg, i.v.) did not influence the mucosal hYoeremia induce6by i.c. in~ection of TPdlanalog, while the CGRP antagonist, hCGRPs~ z (15 pg/kg i.v. bolus + 3 ~g/kg[h i.v. infusion) prevented the increase in GMBF. None of the antagonists influenced basal GMBF while blocking the gastric hyperemic response to exogenous agonists (VIP, a-CGRP). In capsa i c in -p re t r ea t ed r a t s , the 142% increase in GMBF induced by TRB analog was not a l t e y e d b ~ hCORPs.s7 and blocked hy L- NAME (10 mg/kg, ivj and L-Nea~ actxon was reverses o y ~- a rg ln ine (5D0 mg/kg, ~v). Bethanechol (150 pg/kg/h) in~used close arterially to the stomach elevated the GMBF by I02%. The ga s t r i c hxperemic response to bethanechol was inh ib i ted by 73% by administration of hCGRPs_~r.

These results indicate that the gastric hyperemic response to central vagal activation induced by intracisternal TRil analog involves per iphera l muscarinic- mediated release of CGRP from capsaicln sensitive afferent fibers. In addition there is capsaicin-independent pathways that involves nitric oxide. Such crosstalk between vagal efferents and CGRP containing sensory afferent fibers .may generate local as well as reflex modulating gastric response to vagal efferent stimulation.

GASTRIC MOTOR EFFECTS OF ENDOTHELIN IN THE LOWER BRAINSTEM OF THE RAT. Z.K. Krow/cki and P.J. Homby. Dept. of Pharmacology, LSU Medical Center, New Orleans, LA, USA

Specific binding sites for endoth¢lin (ET) am present in the doted vagal complex (DVC) of the rat and cardiovascular effects of ET in the DVC are well-defint~i These findings prompted us to investigate the effects of two isoforms of ET, ET-I and ET-3, on gastric, tone and motility afar their application on the surface of the area ~ (AP; 16 and 160 pmol in 5 ~tl) or microinjection into the DVC (0.1-100 pmol, 30 nl) of ct-chloraloseJxylazine anesthetized rats. Changes in intragsstfic pressure were quantified by measuring the peak response (PRIGP; cm HzO) and the area of the response (ARIGP; ca2). Changes in pylotic motility (PMMI) were quantified with minute motility index. All the data, including mean medal pressu~ (MAP; mmHg) and heart rate (HR; bpm), are shown as means • SE for (N) animals.

PKIGP AKIGP PMMI MAP HR 0 0 0 0 -3±3

2.3+__0.8 1.4+__0.2" 3.5+2.7 84+_10" 217+-64"

AP N Vehic~ (4) ET-; 160pmoI (4) before vag~omy ET-I 160 pmol (4) ~ vagotomy DVC Vehicle (8) ET-1 l pmol (7) ET-I 10 pmol (5) Ve~e (5) ET-3 ] pmol (3) ET-310opmot (s)

0 0 ÷ 0.1-+0.3 6±$ + 15±12 ÷

0 0 0.4+-0.3 0 0 1.6+-0 4" 0.4±0.2 0.2+-0.6 -t I +-6 4±3 1.9+0.2" 3.3±1.4" 1.0+ 1.0 24+31 j 16:k5

0 0 -0.1+_.0.7 0 0 1.2-+1.2 0 .1+0.I 0.8±0.6 0(1) 22t2 2.0±0.8" 0A+-0.1" 2.1+|.2 -2+-20 O) -3+-3

"P<O,05 vs. corresponding vehide and ~P<0.05 vs, ET-I effect before vagotomy by ANOVA with subsequent Student-Newman-Keuls or Dunn's tests

These results demonstrate that ET-1-evoked increases in gastric tone, after its application on the surface of the AP, are vagally mediated and are comparable to the effects of ET-I or ET-3 microinjected into the DVC. Supported by the LSU Neuroscience Center Incentive Grant (ZKK) and PHS grant DK42714 (PJH).

BICUCULLINE BLinKS THE INHIBITORY EFFECTS OF SUBSTANCE P BUT NOT VASOACTIVE INTESTINAL POLYPEPTIDE ON GASTRIC MOTOR FUNCTION IN THE NUCLEUS RAPHE OBSCURUS (nROb) OF THE RAT. Z.K. Krowiold and P.J. Homby. Dopt. of Pharmacology, LSU Medical Center, New Orleans, LA, USA

Substance P (SP) and vasoactive intestinal polypoptid¢ (VIP), microinjectexi into the nROb of the rat decrease gastric tone and inhibit gastric motility and SP-evoked gastric inhibition is mediated by nitric oxide (NO)-containing pathways (Snc. Neurosci. Abstl. 1993, 19: 960; Gnstroenterology 1995, 108: A983). Since A-GABAergic control of gastric relaxant motor activity specifically targets the NO mediated r e s p o ~ (Kmntis et al., Gasn'oenterology 1995,108: A632), we tested the hypothesis that gastric inhibitory effects of SP and VIP in the nROb, could be abolished by bicuculline methiodide (BMI), a GABAA receptor antagonist. Substance P 035 pmol) and VIP (100 pmol) were microinjected into the nROb of ot-chloralose-anesthctized rats before and 60 ruin after BMI (0.4 mgxkg "t, sc). Changes in intragsstric pr~mre were quantified by measuring tim ~ response (PRIGP; cm HxO) mad the area of the response (ARIGP; cm ). Changes in pyloric (PMMI) and greater curvature (GCMMI) motilities were quantified with minute

motility indexes. The data are shown as means :L SE for 5-6 animals. PRIGP ARIGP PMMI GCMMI

Vehicle 0. I _+0.1 0 0.4+0.2 0.2+_0.2 SP b~om BMI -1.2_+0. l" - 1.2_+0.1" -5.0+_ 1.0" -0.5+_0.4 SP a~cr BMI -0.4+-0.3 + -0.4+_0.3* .l.5+_I.g + 0.1+-0.! Vcbide -0. I -+0. I 0 0.2±0.3 0.3±0.2 VIP before BMt -1.5+-0.3" -2.3+0.6" -5A+-1.5" -0.9-+.0.g VIP aft~ BMI -2.3±0.5" -3.4±0.8 ° -3.9f 1.6 -I.2+-0.6 ~'P<0.05 ~ eddie and ~P<O.05 w. SP ¢tf~t before BM1 bY ANOVA with mb~Nem Stude, t-New,mn-Ke,As test

These findings show that SP--evoked gastric relaxations in the nROb are mediated through the A-GABAergic pathway. However, the mechanism of VIP-evoked gasmc inhibition is unclear. Supported by PHS grant DK42714 (PJH) and the LSU Neuroscience Center of

Excellence (ZKK).

Digestive Dise~es and Selene, Pot 41, No. 2 (Febraa~ 1996)

PATHOLOGIC BASIS OF ESOPHAGEAL MOTILITY DISORDERS J. Lonovics*, LA. Simon**, 1st Dept. of Medicine, Albert Szent- Gy6rgyi Medical University, Szeged, Hungary*, 2nd Dept. of Medicine, County Hospital, Szcksz,~rd, Hungary**

Among esophageal motility disorders gastroesophageal reflux disease (GERD) affects a substantial number of population, whereas other motility disturbances including achalasia, hypertensive lower esophageal sphincter (LES), diffuse esophageal spasm, and nutracker esophagus occur relatively rarely.

GERD is a complex motility disorder affecting the upper gut, which results in a pathologic reflux of gastric and intestinal contents to the esophagus. Hypomotility changes involve both the esophagus and the LES, as well as the stomach. Pathologic basis of gastroesophageal motility disorder is not clearly understood, an improper release of acetylcholine or a vagally-mediated nonnholinergic nonadrenergie inhibitory mechanism [candidate neurotransmitters are vasoactive intestinal peptide (VIP) and nitric oxide (NO)] is suspected. Failure of the motility defense mechanisms and tissue resistance of the esophagus will lead to the development of symptoms and morphological alterations, e.g. reflux esophagitis. In the development of atypical symptoms such as non cardiac chest pain and respiratory complications local factors, aspiration of the refluxed material and a vagally mediated coronary or bronchial spasm may be involved.

Achalasia or cardiospasm is characterized by lack of relaxation of LES and loss of peristalsis in the lower two third of the esophagus. Motility disorder is caused mainly by denervation of postganglionic nou-cbolinergic non-adrenergic inhibitory neurons resulting a marked impairment of both acetylcholine and VIP or NO release. On the contrary hypertensive LES is characterized by increased resting LES pressure associated with normal sphincter relaxation and esophageal peristalsis. In diffuse esophageal spasm simultaneous or repetitive non- propagating contractions are recorded, while in nutracker esophagus manometry shows a pettem of high-amplitude but peristaltic contractions. In spastic motility disorders neural dysfunction may involve both afferent (sensory) and efferent fibers resulting an exaggerated response of the esophagus to cholinergic stimulation.

Nonspecific esophageal motility disorders are associated with broad spectrum of manometric abnormalities including frequent nontransmitted contractions, retrograde contxactions, prolonged duration of peristaltic waves, and isolated incomplete LES relaxation but pathologic basis of these abnormalities is unknown.

(A-I~) 445

DISTRIBUTION OF HISTAMINE2 RECEPTOR mRNA IN THE RAT GASTROINTESTINAL TRACT. Eva Mezev. K.Pacak,M,P~lkovits* NIH,NINDS,CNB,*NIMH,LCB, Bethesda, Md 20892

Histamine 2 receptor blockers have long been used in ulcer treatment, but their exact target site is still not fully understood. Earlier we have reported the presence of H2 receptor mRNA in the lamina propria of the stomach, but were unable to detect the mRNA in epithelial cells. To inrease the sensit ivi ty of the detection in the present study we used complementary ribonucleic acid (cRNA) probes to map the distribution of the histamine 2 receptor mRNA using in situ hybridization histochemistry. We have found that cells of the lamina propria were positive for the H2 mRNA in the entire length of the GI tract. In the stomach epithelium, the H2 mRNA was localized to the middle portion of the parietal cells (as confirmed with a specific antibody that recognizes the proton pump, a gift from Dr. Adam Smolka), as well as the surface mucuoe cells. Along the small and large intestinal epithelium, goblet cells, which correspond to mucus cells in the stomach, were labelled. Immobil ization stress caused a significant increase in the amount of H2 receptor mRNA in both the lamina propria and in many epithelial cells in the stomach as well as the rest of the G1 tract.

Our results suggest that the H2 receptor blocking drugs exert their effect at three target sites in the GI tract: they may affect acid secretion by a portion of the parietal cells; they may affect immune function of cells in the lamina propria, and they may increase the mucus secretion by mucus cels in the stomach and goblet ceils in the intestine.


BIOCHEMICAL ENERGETICAL BACKGROUNDS AND rlu~tl¢. REGULATIONS IN THE GASTRIC MUCOSA IN PATIENTS WITH DIIFF~P, JEINT GASTRIC SECR]WgORY RESPONSES. AN OVERVIEW. Gy. M6zslk, L Jut'icskay, O. Kat~di. L NaL=,V. First Dept. of Mad., Mad. Univ. P&:s, P6cs, Hungary.

The gastric acid secretion is a result of lot of sources of active metabolic complex responses. The aims of this study were: 1) to evaluate the correlations between fundlc mucosal biochemistry (adenine-adenosine, AMP, ADP, ATP and calculated values of adenylate pool [ATP+ADP+AMP], "energy charge" [(ATP+0.SADP)/(ATP+ADP+AMP)] and gastric acid secretion (Basal a d d output=BAO; maximal acid output=MAO); 2) to study the correlations among tissue level of ATP - membrane ATPase . tissue level of ADP and gastric acid secretion (BAO, MAO). Materials and Methods: The observations were carried out on gastric fundtc mucosa of patients who underwent gastric partial resection. The adenlne-adenosine, adenosine mono- (AMP), adenosine dl-(ADP) adenosine trtphosphate (ATP), r tbonudeic ~-~NA) and deoxyribonucleic (DNA) on the other hand, the MgZ÷-,MgZ~*Na'r-K T- dependent and Na+-K+-dependent ATP-ases were prepared and their quantities (enzyme activities) were measured. The biochemical gradients were calculated In accordance to I mgDNA, the enzyme activities in pMol/mg mucosal protein/h. The correlations of these parameters were analyzed.

RESULTS: Na~K.~DE PEN DENT ATP-ASE

BAO + ~.m ~ t ~'~ +

i l - .J- : " - 'l N~K-~DEPENDE!h~ ATP-ASE

M A ~ ' ~ e _ - ATP ADP ++

* - mmole I hour (rmen = SEl@ ; 4-~- mnoate~ I mgONA (mere t $EM) ; ~-~ - ,,-' -~,~ ~ p, / ra~ memo. ixcx. x ho~r (raeea ,SEt@

Conclusion: The gastric acid secretory responses depend on not only the number of parietal ceils ba t on their biochemical building up. This study was supported by OTKA No. T 020098 and ETI'-03 660/93.

A NEW BIOCIIEMICAL EXPLANATION FOR TIlE DEVELOPMENT AND THE LOCATION OF "GENUINE" GASTRIC, DUODENAL, AND JEJUNAL ULCERS IN PATIENTS. AN OVERVIEW. Gy. M6zsik, I. Juricskay, L. NAGY. First Department of Medicine, Medical University of P(~cs, Hungary.

The aims of this study were to evaluate biochemically the building up of the "genuine" gastric, duodenal and jejunal ulcers and their mucosae and muscular layers on the tissue specimens of human patients who underwent partial gastric resection because of "genuine" peptic ulcer. Methods: The gastric (BAO and MAO) secretory responses were determined before surgery. The tissue specimens from the stomach, duodenum or je junum were obtained from the patients a t the surgery, when the mucosa and the muscular layers were separated and put Into liquid nitrogen. The tissues were homogenized in 0.5 M ice-cold perchloHc acid. Thereafter, ATP, ADP, AMP, adenine- adenosine were separated. The aden)late pool (ATP+ADP+AMP) and the energy charge [(ATP+0.5 ADP)/(ATP+ADP+AMP)] were calculated. The measured and the calculated biochemical parameters were calculated to I mg DNA. Results: (l) Adenine-adenosine, ATP, ADP, AMP, aden)late pool and the "energy charge" of fundlc mucosa significantly increased by Increased gastric secretory responses (GSR); (2) ADP and ATP increased in gastric antral mucosa, while no change was observed In biochemical parameters in duodenal and jejunal mucosa by the increase in GSR; (3) No significant changes (increase or decrease) in the different biochemical parameters of gastric, duodenal or jejunal musculature were found by the increase of GSR; (4) Significant energitical (adenine-adenosine, AMP, ADP, ATP) gradient exists between fundic vs unreal, ant ra l vs duodenal (jejunal) and fundlc vs duodenal (jejunal) mucosa in patients with increased GSIL Conclusions: L The ulcer area (site) Indicate biochemicaUy a signifieantly increased biomechanisms around the ulcer;, 2. The Increased metabolic adaptation is mostly suitable in fundic mucusa of patients with increased GSR, it is less in antra l and duodenal mucosae In these patients. 3. The positive adaptation is much more less in the whole stomach in patients with normal or decreased GSR; 4. The most vulnerable tissue ts the duodenal and jejunal morose; 5. The localization of genuine gastric, duodenal and jejunal ulcers is determined by the energetical and biochemical building up of their mncosal tissues. This study was supported by the grant of OTKA No. T 020098 and E T r -03 66O/93.

446

PHARMACOLOGIC STUDIES ON ISOLATED GASTRIC MUCOSAL CELLS: A MODEL OF ACUTE INJURY AND PROTECTION. L. Naqy, S. Szabo, Gy. MSzsik. 1st Department of Medicine, University Medical School of Pdcs, H-7643 P~cs, Hungary, "Departments of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA Background/Aims: The mechanisms of chemically induced gastric mucosal injury are poorly understood. The main purpose of this method-oriented presentation is to provide an overview of the techniques of the preparation of freshly isolated gastric mucosal ceils {GMCs) developed in our laboratories. The biochemical-morphologic methods to test the degree of cell injury and protection at subcellular (i.e., plasma membrane, mitochondria and nuclei) levels are also reviewed. Methods: Unfractionated GMCs were prepared from rat glandular stomach with two sequential 30 rain incubations with pronase (0,5 mg/ml) and EGTA (10"~mol/I). Initial cell viability and membrane receptor sensibility were tested by stimulation of pepsinogen secretion from chief cells by carbachol or 16,16-dimethyl prostaglandin E z (dmPGE 2) and HCI production in parietal cells by histamine and 3-isobutyl-l-metylxanthin. Plasma membrane damage (trypan blue exclusion, LDH leakage) and integrity of mitochondria (succinic dehydrogenase activity) and nuclei (ethidium bromide-DNA fluorescence) were tested to distinguish between mild (reversible) and severe (irreversible) cell injury. Viable GMCs (1.5 x 107) dispersed in albumin containing physiological buffer and selected ethanol concentration ( t5%) were used for direct cytoprotection after preincubation with wide concentration (10 .2-108 M) of g astroprotective agents, i.e. dmPGE z, cysteamine (CEA), N-acetyl-l-cysteine {NAC), taurine (TA), histamine (H), ganglioside GM~, sucrose octasulfate (SOS), nitecapone (N), glycine or L-arginine for 60 min. Results: The incubation of GMCs with 0-25% ethanol produced dose-dependent reversible or irreversible cell damage. Only a partial cytoprotection (up to 21%) was detected after at least 60 rain preincubation with dmPGE 2, CEA, SOS, NAC, TA, and low concentration of histamine. Conclusions: 1. High yield of viable unfractionated rat GMCs can be isolated by sequential incubations with pronase and EGTA. 2. Sensitive biochemical-morphotogic procedures permit the distinction between mild and severe cell damage by parallel monitoring plasma membrane, mitochondrial and nuclear integrity. 3. Only a slight protection against ethanol was detected by dmPGE2, thiol compounds and sucralfate. 4. Freshly isolated mixed GMCs are proposed for acute pathogenetic and pharmacologic studies because of the elimination of the influence of local vascular and neuroendocrine factors. (Supported by ETT I".03 646/93 and OTKA T 016727/.

(A-IS) Dige~ve Diseases and Sciences, VoL 41, No. 2 (February 1996)

A B S T R A C T S ON I U P H A R GI P H A R M A C O L O G Y

ALTERATION OF VASCULAR PERMEABILITY DURING ULCER HEALING: EFFECT OF bFGF AND AUTONOMIC REGENERATION. M. Nakamura*, M.Kitajima**. *Dept. of Internal Medicine, **Dept. of Surgery, School of Medicine, Keio University, Tokyo 160, Japan

The microvascular network of the gastrointestinal tract is characterized by the rich distribution of the perivascular autonomic nerves. These nerve fibers have been classified into cholinergic, adrenergic, peptidergic and nitroxergic nerves by the histochemical methods but the electron microscopic observation revealed that each nerve fiber contains several kinds of syaaptic vesicles having various kinds of neurotransrnitters. The receptor localization of these nerves were visualized by the specific binding of radiolabeled receptor antagonists. The localization of the chollnergic, peptidergic and nRroxergic nerves was studied by the binding sites of tritiated qulnuclidinyl benzilate (QNB), tz~-VIP, nsI-CGRP and trifiated L- arginine, respectively. By these experiments, the binding sites of QNB were mostly seen on the endothelial cells of the microcirculatory components as well as on the epithelial cells. VIP and CGRP binding sites were also accumulated on the endothelial cells of the micmvascular system.

During the healing process of the acetic acid-induced gastric ulcer healing, the increase of the vascular permeability was shown in these regenerated vessels by the intraaortic infusion of horseradish peroxidase. The number of these nerves and their receptors was markedly reduced on the regenerated micmvascular network. The administration of CS23, acid-stable human recombinant br-'GF, decreased the increase of the vascular permeability and accelerated the regeneration of these autonomic nerves and microvescular network revealed by the immunoreactivity of E-selecfin and CD36.

In conclusion, the autonomic nervous regulation of the microvascular network was suggested to play an important role in the maintenance of the vascular physiology in the healthy and regenerating gastric mucosa, bFGF was shown to stimulate the microvascular regeneration as well as autonomic reinnervation.

CYTOPROTECTIVE EFFECT OF VARIOUS DRUGS ON INDOMETHACIN-INDUCED DAMAGE OF RABBIT GASTRIC MUCOSAL CELLS. S. Okabe. S. T~kahashi. Dept. Appl. Pharmacol, Kyoto Pharmaceutical Univ, Kyoto 607, Japan

We examined whether leminoprazole, omeprazole, sucrallate and 16,16-dmPGE2 protect the gastric mucosal cells against indomethacin (IM) -induced damage. <Methods> Gastric mucosal cells were isolated from rabbit stomachs, and the viability was assessed by MTT and dye exclusion methods. {35S] methionine- labelled synthesized proteins were detected by autoradiography following sodium dodecylsufate-polyacrylamide (10%) gel electrophoresis. <Results> Exposure o! gastric mucosal cells to IM for 4 h apparently reduced viability in a dose-related manner. Pretreatment with leminoprazole and sucralfate for 4 h significantly prevented the reduction of cell viability by 50 ,aM IM, although they did not prevent severe cell damage induced by 500 pM IM Omeprazole had no effect on cell protrection. 16,16-dmPGE2 significantly prevented the cell damage induced by IM both at 50 and 500 pM All drugs alone did not affect cell viability. The cytoprotection by leminoprazole was expressed with a 2-h lag period. Leminoprazole did not promote PGE2 synthesis by the cells, but apparently induced the synthesis of 83 kDa-, 72 kDa-, 52 kDa- and 35 kDa-proteins. Sucraltate and 16, 16-dmPGE2 had no elfect on the induction of such proteins. Both cytoprotection and induction of such protein synthesis in reposne to leminoprazole were abolished by cycloheximide and actinomycin D. < Conclusion> These results indicate that leminoprazole directly protects gastric mucosal cells against mild damage induced by IM and that its cytoprotective effect might be mediated through de novo synthesized proteins.

C U R R E N T PROBLEMS AND RESULTS OF T U M O R I M M U N O L O G Y . P.Nemeth and Timea Berki. Immuno- logical and Biotechnological Laboratories, University Medical School of P6cs, HUNGARY

Tumor immunology is one of the most intensively studied field of the medical/biological sciences in our century. However, the clinical problems of the tumor patients have not been completely solved so far. Immunologic control of the tumor growth has well been investigated and different escape mechanisms for tumor cells have been discovered recently. In spite of promising results in details no appropri- ate generally accepted strategy has been developed. Both in the diagnostic approaches and therapeutic efforts different routes are used parallely.

Practical applications of monoclonal antibodies for diagnostic use were developed in our institute. During a retrospective study remarkable prognostic relevances were defined with transforming growth factor alpha (TGF-a) and tumor necrosis factor alpha (TNF-c0 on breast cancer patients. Similar analysis of the malignant tumors of the gastro-intestinal system is under elaboration.

In an other model experiment series a new photo- immuno-targeting method was developed for selective destruction of unwanted cell population including the tumor cells in vitro and (experimentally) in vivo. Gastro-intestinal adenocarcinomas transplanted into nude mice were targeted successfully by this technique. The lecture reviews the main aspects of the current tumor immunology using some of our own results.

(A-19)

Digestive Diseases and Sciences, VoL 41, No. 2 (February 1996)

CRITICAL EVALUATION OF ACINAR, DUCTAL AND INTESTINAL FACTORS INFLUENCING PANCREATIC CYTOPROTECTION. M. Papp. Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary

The author gives a brief survey on the glandular, acinar and ductal factors of the pancreas, furthermore on the intrajejunal events of the digestion which affect pancreatic cytoprotection in health and under pathological condition. He stresses the importance of the unimpaired excretory ductal system of the gland and free outflow of the juice in maintenance of this organ-preserving mechanism.

447

PROSTAGLANDINSAND LEUKOTRIENES IN GI DISEASES AND MUCOSAL PROTECTION. K.D. _Ra~_ford. Div. of Siomed. Sciences, Sheffield Hallam University, Sheffield, U.K.

Prostaglandins (PGs) and leukotrienes (LTs) play a central role in mucosal defence being involved in regulating blood flow, vascular integrity, as well as the production of acid, pepsin and mucus, and cellular regeneration. It is now recognized that there is also a complex interplay between systems regulating and other mucosal defensive processes among them nitric oxide (NO) production. The molecular biology of enzymes regulating production of PGs and LTs is now well advanced though the molecular events underlying the mode of action of ulcerogenic and anti-ulcer drugs with the various enzyme systems is only just being discovered, as too, the complex receptor-mediated events from interaction of the PGs and LTs with their respective recptors, the intracellular transductional pathways so activated, and physiological consequences of these differing actions. Research is now poised to use all the molecular and cellular biological tools we now have, as well as teachings from the molecular anbd cellular processes, to understand the reasons for the coming and going of ulcers and inflammatory bowel diseases in patients and why they may suddenly develop life-threatening GI ulcers, heamorrhage and malignancies, after being exposed to states or drugs for such variable periods of time such that it is not possible to predict when they will develop these conditions.


TIlE EFFECT OF RESINIFERATOX1N ON EXPERIMENTAL GASTRIC ULCER IN RATS. O.M.E. Abdel Salam, *J. Szolcsfinyi, Gy. Mrzsik. First Department of Medicine and*Department of Pharmacology, Medical University of P~es, If~fisfig fit, H-7643, Pres, Hungary.

Capsaicin sensitive sensory nerves are involved in the modulation of gastric mucosal integrity. The present study aimed to evaluate the effect of the capsaicin analogue resiniferatoxin (RTX) on the gastric mucosal damage produced by different noxious agents in the rat. Methods: Sprague-Dawley strain rats, of both sexes, 180-200 gm of body weight were used. Animals were deprived of food for 24h before the experiments, but allowed water ad libitum. Gastric mucosal damage was evoked in pylorus-ligated rats by the administration of intragastric (ig) HCI (2 ml of 0.6 N), ig ethanol (2 ml of 50% in water vA' or 96%), ig acidified aspirin (200 mg/kg dissolved in 2 ml of 0.15 N HCI), subcutaneous (sc) aspirin (200 mg/kg plus ig 2 ml of 0.15 N HCI) and sc indomethacin (20 mg/kg). Animals were sacrificed at different time intervals after administration of the above ulcerogens, when gastric secretory responses, the number and severity of mucosal lesions were noted. Res,lts: resiniferatoxin (applied ig in the dose-range of 0.4-1.8 lag/kg based on studies with laser Doppler flowmetry and found to produce a pronounced increase in gastric mucosal blood flux in the rat) protected against the development of gastric mueosal injury produced by the above ulcerogenic agents in pylorns-ligated rats. Resiniferatoxin administered in fixed volume of 2 mt of physiological saline significantly inhibited gastric acid secretion in the lh pylorus-ligated rats relative to vehicle-treated controls. Resiniferatoxin by itself did not produce visible gastric mucosal damage in the saline-treated controls. Conclusions: The capsalcln analogue resiniferatoxin exerts gastroprotective effects in different experimental ulcer models. The protective action of RTX is likely to involve both an enhancement of the microcirculation in addition to an inhibition of gastric acid secretion. This study was supported by the grants of Hungarian National Research Fund (OTKA N ~ T 020098 & T 016945) and Ministry of Health and Welfare (ETT-03 660/93 & ETr-T 563).

ORGAN CULTURE OF PIG GASTRIC MUCOSA: SYSTEM FOR STUDY OF ENZYMES REGULATING PROSTA- GLANDIN PRODUCTION BY NSAIDS. K.D. Rainsford, S. Tsang. N. Aliehani. Div. Biomed. Sci. & Health Res. Inst. Sheffield Hallam University, Sheffield, U.K.

Most in vitro culture system developed to study gastric mucosal cellular and biochemical events regulated by prostaglandins (PGs) and the actions of non-steroidal anti-imflammatory drugs (NSAIDs) have used isolated cells. The integrity and phenotypic stability of these cellular systems, as well as the complex interrelationships between the cell types in the mucosa is qestionable. We, therefore, devised organ culture systems for culturing pig fundic mucosa; (the pig mucosa being representative of that in humans) and used this to study the regulation of PG enzymes and PG production by NSAIDs. Methods: Explants (1-2 em square) of sterile excised fundic mucosa were pre-cultured overnight in DMEM + 5% FCS at 37 "C in 95% CO~/5% O2 to initially achieve stability. The media was changed. NSAIDs or solvent controls were added with or without interleukin-1 (to induce PG production), fresh media and the mucosa cultured for 24 to 96 hours in DMEM. Immunohisto- chemistry was used to localise PGHS (=COX) isoenzymes TxB~ and PGIz synthases; the media was collected for assay of PGs. Results: The mucosa was stable and maintained structural integrity up to 11 days in culture. Inhibition of PG production was achieved by all the NSAIDs. Aspirin (100, 500 t~M) blocked basal and IL-la-induced PGHS-2 parallel with this; this being the predominant isoform present in the smooth muscle, endothelial and few mucous cells in the mucosa. Other COX inhibitors, e.g. oxicams maintained exhibited differential effects by inhibiting PG production without affecting PGHS-2 production. Conclusions: Pig mucosa can successfully be maintained in organ culture for long periods (days) and used to sludy PG production and its regulation by NSAIDs.

INVESTIGATION OF THE MECHANISM OF PEPTIC ULCER

HEALING USING A PRIMARY CULTURED GASTRIC

EPITHELIAL CELL MODEL,

Nobuhiro Sato

Department of Gastroenterology, Juntendo University, Tokyo Japan

The pathogenesis and mechanism of healing of peptic ulcer have

been studied extensively using in vivo animal models and in vitro

culture cells. However, the detail cellular and molecular mechanism of

gastric ulcer healing is still unclear. Recently, we have established a

new wound repair model of ulcers using primary cultured gastric

epithelial cells ( J Gastroenterol Hepatol 9:325:1994, BBRC

199:799:1994, BBRC 199:1453:1994, BBRC 202:285:1994). In this

model, the wound healing involves the initial cell migration followed by

proliferation of the cells surrounding the wound. The acto-myosin

system and Ca2+-calmodulin system play a key role of the healing

process. We have investigated various factors that modulate the

process of healing using this systemAccumulating evidence reveals

that growth factors and extracellular matrix play an important role on

gastric epithelial restoration in this culture cell model. EGF, Insulin,

HGF, PDGF accelerated the restoration and extracellular matrix

modulated this process. In another investigation, aggressive agents,

such as ethanol, bile acid, ammonia and a water extract ofH. Pylori

inhibited epithelial restoration in this system. From these results,

apparemly this gastric culture cell model is useful toot in biochemical-

pharmacological studies to approach gastrointestinal diseases.

(A.20) 4 4 8 D~stive Diseases and Sciences, VoL 4l, No. 2 (February 1996)


PENTADECAPEPEPTIDE BPC 157 AND CHRONIC GASTRIC ULCER. P. Sikiri~s S. Seiwertl h Z. Grabarevid~ R. Ru~man, M. Petek~ S. Mii~ 1. Rotkvid~ B. Turkavid~ V. Jagid, M. Hanleva~Jd, J. Separovi~ P. Konievoda, Lj. Jurina~ D. Liubanovi~. M. Bratuli~ B. Artukovi~, M. TF:lia G D. Peravi~, G. Buliat, M. Giura~ir h P. MildlY.. CDD, Medical and Veterinary Faculty, Unh,ersity of ZagPeb, Zagreb, Croatia The protection of stomach and duodenum in conjecture with attti- inflammatory effect was demonstrated for a novel gastric juice peptide, BPC. BPC ( i.p/i.g.) was investigated in rats m comparison with several reference standards hi three experimental ulcer models (48 b-restraint stress, subcutaneous cysteamine, intragastrical 96% ethanol ulcer tests) (pre-/co-/post-treatment). Only BPC regunens were consistently effective in all of die tested models. On the other hand, bromoeriptine, amantadine, famotidine, cimetidine and somatostatin were ineffective (restraint stress). A dose-dependent protection (cysteamine) and/or partial positive effect (related to treatmont conditions) (ethanol), was obtained with glueagon, NPY and seeretin whereas CCK /26-301 was not effective. Based on Monastral blue studies BPC beneficial effeet appears to be related to a strong endothelial protection (Life Set 1994, JPh(Paris) 1993).Consequently, the possibility that a similar beneficial effect could be seen also m chronic lesions, such as acetic acid ulcers in rats, appears in the focus of the present study. Methods/Results. Chronic gastric ulcers were induced in rats by submucosal injection of 50pl of 20% acetic acid. One day thereafter, the medication (per kg b .w ) ~ith BPC- 157 ( 10 lag or 10 ng/kg b.w.), ranitidine (50 mg/kg) or saline (5.0 ml/kg) was imtiated, given as once daily admimstration till die end of the experiments (last applieatiou 24 hours before euil~anasia). The gastric lesions were assessed by nsive obsetwers as described before (means:eSD. ms- ' ) (Calabro et al.. Digestion 1904) utnnediately prior the initiation of the therapy (69±25), and after 10 days (control: 274+133. BPC 157 p.g 32.2--'20. BPC 157 ,o 34±18, ranitldme 46.8±27. P<0.05 vs.comrol, at least). Conclusion. Therefore. it seems likely that BPC 157, besides a st;t'nlg sal;lial3, effect on acute mucosal lesions, could be markedly effective also m promoting chrome gastric ulcc~" healing.

PENTADECAPEPEPTIDE BPC 157 AND NO-SYSTEM: MUCOSAL INTEGRITY AND BLOOD PRESSURE. p. Sikiri~ S. Seiwerth, Grabarevi~ R. Ru~man~ NL Petdq S. Mi~e, L Rolkvi~ B. Turkovi~ V. Jagi~ M. Han~eva~[ki~ J. Separovi~ P. Konievoda, Li. Jurina, D. Liubanovi/. M. Bratuli~ B. A.r'tukoyi~ 1~ Tis~liar. CDD, Medical and Veterinary Faculty, University o f Zagreb, Zagreb, Croatia

A dear protection of gastrointestinal tract and an evident anti-inflammatory effect were shown for a pentadeeapeptide BPC 157 (a fragment of organoprotective gagric juice peptide, coded BPC) (i.p./i.g) in comparison with several reference standards in various ulcer models (1-6) (pre-/ce-/posttreatment). On the other hand, a crucial role for NO-system was also postulated. Therefore, a possible interaction of pentadecapeptide BPC 157 with NO-system in mucesal protection (a) and blood pressure (BP) maintenance 03) was investigated. Methods/Results. Male Albino Wi.ctar rats, 250 g b.w., were used for the experiments, a. Gastnc lesions (means~SD, mm ~) (4) were induced by 96% ethanol (i.g 1 ml/rat, euthamsia 1 h thereafter). The beneficial effect of BPC 157 (/ks i.p.) (10 p.g (147~32), 10 og (157±42)), given simultaneously with ethanol (control: 488±106), was reversed toward the control values (236.+.30 (pg), 245-+-34 (ng)) by NO- antagonist, L-NAME (5 mg/kg i.v.) applied 5 s in before (402::t:125). b, In BP studies, the earotic artery was cannulated and BP measured (means~SD, mmHg). BPC 157 (10 ~g or 10 ng/kg i.v.) or saline (I ml/kg i.v.) were given either 15 min before (i), or 10 s i n afl.er (it) L-NAME (5 mg/kg i.v.), 6) L- NAME produced an immediate BP-increase, a maximum already following I0 sin, with raised values for entiere 90 sin-test pealed. In pretJ'eatment, a rather conststent beneficial activity was observe: in/ag- group a dear effect, 10-65 s in -,dt.er L-NAME (140~ 16/119±26 vs. 181:t:21/150±2.2 control), and in rig--group an effect between 5 and 40 mm alter L-NAME (143+15/146±10 vs. 168:H9/167:L--6 in controls), (it) In posttreatment BPC regimen, an obvious salutary effect (10.-45 sin following ~g BPC medication /134+45/124±33 vs. control 181±21/163:L--6/, 5-20 min alter ng BPC application /143±9/134~25 vs. control 176:~23/178±25/) was consistently noted. Conclusion. Together, these data strca~gly indicate that BPC-system significantly contributes in NO-system effects, particularly those connected with the maintenance of mucosal integrity and BP. References. P Sikirie et al, 1. Exp Clin Gastroenterol 1, 15, 1991, 2. J Physiol (Pzris) 87, 313, 1993, 3. Life Sci 53, PL 291, 1993, 4. Life Sci 54, PL 63. 1994. 5 Inflantmopharmacology 2, 121, 1993, 6. Digestion 55, 58, 1994

Digestive Diseases and Sciences, VoL 4L No. 2 (February 1996)

CLINICAL L~D PHAI~IACEUTICAL APPROACH OF THE ESOPHAGEAL MOTILITY DISORDERS. L . A . S i m o n a n d J . L o n o v i c s . D e p t . o f G a s t r o e n t e r o l o g y , T o l n a C o u n t y T e a c h i n g H o s p i t a l , S z e k s z d r d a n d l s t . D e p t of I n t e r n a l Medicine, S z e n t - G y g r g y i Albert Univ. Medical School, Szeged, Hungary

The authors g i v e an overvlew on the recent development of clinical diagnosis and pharmacological management of esophageal motility disorders, relating on details the following topics: I. Hild clinical forms of achalasia should and can be diagnosed in their early phase.While in adva;Iced forms endoscopic pneumatic dilatation is the preferred method of treatment, in these early stages considerable improvement in qualit~ of life can be reached using long-acting nxtrate or Ca-channel blocker therapy. 2. Diffuse esophageal spasm and "nutcracker" esophagus are more frequent in the background of angina-like chest pain as expected earlier. In their pharmacological treatment the modern Ca - channel blockers (e.g.diltiazem) may play an important role. 3. Aspecific motility d i s t u r b a n c e s : elderly patients frequently present esophageal symptoms due to dismotility. Diagnostic explanation and reassurence are important, but the drug treatment is generally a trial. However, our recent study showed that in cases of weak peristaltic contr- actlons a stimulating strategy (i.e.prokinetics like cisapride) might be very useful. 4. The primary pathogenetical cause of gastroesophageal reflux disease (GERD) is the viciou~ circle of incompetent lower esophageal sphincted (LES) - pathological reflux - esophagitxs. In spite of that, therapeutic efforts are mostly focused on the inhibition of gastric acid sec - retion, in mild GERD cases, in particularely mixed with dyspeptic symptoms due to delayed

~ astric emptying, the use of prokinetic drugs i.e. cisapride) is inherently more logical.

HYPOTHALAMIC CRF RELEASE MEDIATES THE INHIBITION OF GASTRIC EMPTYING INDUCED BY INTERLEUKIN-IB IN RATS G. Silt0, A Kir~ily, V. Plourde, Y. Tachd CURE/UCLA Gastroentcric Biology Center, Veterans Administration, Wadsworth Medical Center, Bldg. 115, Wilshire and Sawtelle Birds., Los Angeles, California, 90073

However IL-1B was originally described as a key mediator of immune response, it may influence the function of non-immune organ-systems including the gastrointestinal (GI) tract. The modulation of the GI functions by IL-1B may be responsible for gastrointestinal symptoms induced by the activation of immune system. A series of experiments were designed to clarify the pathways which mediate IL-113-induced inhibition of gastric emptying of non-caloric meal in rats. in particular, the role of specific IL-1 receptors and corticotropin releasing factor (CRF) originating from the hypothalamus was examined.

The gastric emptying was estimated by measuring the emptying rate of 1.5% methylcellulose + 0.05% phenol red solution during 20 s in period= IL-1B was given intravenously (i.v.) or intracisternally (Lc.) at doses of 0. t, 1, 3 or 10 ng/rat 30 s in before the gastric emptying test except in one experiment where the EDs0 dose of IL-1B (3 ng/rat i.v. or 0.1 ng/rat Lc.) was given 5, 60, 180 or 480 s in before the gastric emptying measurement. In a separate group of rats, IL-1 receptor antagonist (IL-1RA) (3 pg/rat i.v. or 100 rig/rat i.c.) or CRF antagonist (20/Jg/rat i.e.) was injected at the same time as the delivery of IL-113. In a third group of rats, indomethacin (5 mg/kg, i.p.) was injected 60 min before II..-113 (3 ng, ix. or 0.1 ng i.c).

IL-111 injected either i.v. or i.e. dose dependently decreased gastric emptying up to 90%. The onset of action was rapid (5 min after i.v. or i.e. injection). The inhibitory effect was maintained over 180 s in after i.e. injection and shorter lasting (less than 60 s in) after i.v. administration. The inhibition of gastric emptying induced by IL-1B i.v. or i.e., was prevented by coinjection of IL-1RA. IL-1RA injected i.e. could also partially antagonize the delayed gastric emptying induced by Lv. IL-lg. Indomethacin abolished the inhibition of gastric emptying after beth i.c, or i.v. administration of IL-lfl. CRF antagonist given i.e. reversed the action of central IL-tB by 52% and that of i.v. IL-lfl by 100%.

These data indicate that II..-113 injected toy or i.e. potently inhibits gastric emptying. This action is mediated by specific IL-1 receptors and central prostaglandin dependent CRF pathways which are known to influence vagal outflow to the stomach. These findings provide evidence that the interaction between the immune and neuroendocrine systems may have implications in the gastric stasis associated with immune challenges.

(A-21) 4 4 9

ABSTRACFS ON IUPHAR GI PHARMACOLOGY

COMPARATIVE STUDIES ON ISOLATED GASTRIC MUCOSAL MIXED CELLS AND HEPATOMA, MYELOMA CELL LINES WITH ETHANOL,

INDOMENTHACIN AND THEIR COMBINATION I. Szab6, B. B6dis, *P. Ndmeth and Gy. M6~ik First De4)artmout nf Medicine, *Immunological and Bintechnnlogical Laboratory, University Medical School ofP6cs, P6cs, Hungary.

Ethanol (EtoH) and indomethaein (IND) are widely used agents in vivo gastrointestinal studies. The aims of our study were: L to analyze the toxic effect of 5 rain EtoH lrealment in vitro on stable cultured cells; 2. to evaluate the differences bctwsen the Sp-2/0-Agl4 and Hep G2 cells; 3. to analyze the damaging effect of EtoH on acutely isolated gastric mucosal cells (GMC): 4. to evaluate the effect of IND on these types of cells; 5. to analyze the effect of combination of EtoH and IND. Methods : Sp.-2/0-Agl4 is a non-secreting mouse myeloma (CRL 1581), while Hep G2 is a human hepatocelhitar carcinoma cell (FIB 8065). 105 cells were used for each experiment. GMC ceils from 1-2 unfastcd Sprague-Dawley mrs were isolated by the method of Nag), et al (Gastrcentcrnlogy, 1994). The segments of glandular stomach without the blood vessels and the surrounding connective tissue were incubated in a physiologic solutions containing pmnase E (0.5 mg/ml) and EGTA (10"3M). ARer several washing of cells the final cell peUct was resuspended in an alhumin containing (0.2% w/v) solution (pH 7.4). Cells were incubated with EtnH (1-5-10-15-20% v/v), IND (10"8-10"31vl), and their combination for 5 rain in shaking bath 37°C. Viability was tested with Trypan blue exclusion test at several times (5, 60 rain; 4, 24 hr) after each trestment. Results: I. EtoH concentratlun-dcpendantly decreased the viability in both cell lineS. 2. The lower concentratinn of Etch did not decrease the viability in Hep G2 cells, but them was a great fall between 15-20"/,, otherwise the viability curve was the same in all counting times. 3. Any concentratinn nf EtoH decreased more potently the viability of GMC cells than it did in the ease nf Sp2/0-Agl4 ceils. 4. IND had no effect on the viability of the cultured cells, in the case of GMC only the high dose (10"3M) nf [ND decreased significantly the number of viable cells. 5. Using combined these agents IND aggravated the EtoH-induced nell injury specially the 10"3M dnse. Conclusions: 1. Our results indicate that GMC cells arc more vulnerable than cultured ceils, this difference derives rather from the isolation procodum than their types. 2. In our in vitro study when IND was applied without other aggressive factor such as EtoFL it was not toxic for the cells. Probably, as the result of combined treatment shows, the decrease of level of endogenous prostaglandins might have a role in the enhanced toxic effesl of EtoI-L This study was supported by the Hungarian National Research Found (OTKA "I"020098) and the Minislry of Welfare and Health (ET-03 660t93).

PEPTIDE RECEPTOR ANTAGONISTS AND MONOCLONAL ANTIBODIES RAISED AGAINST PEPTIDES: TOOLS TO STUDY PHYSIOLOGICAL REGULATION OF PANCREATIC FUNCTION. G. Varga. [nst. o f Experimental Med., Hung. Acad. Sci., Budapest, Hungary

It is now well established tl~at cholecystokinin (CCK) exerts a major role in the physiological regulation of pancreatic enzyme secretion, but the rote of other putative peptide regulators, such as gastrin, bombesin-like peptides, somatostatin and galanin, is less clear. A specific approach to evaluate the importance of bioaetive peptides in the physiological regulation of pancreatic exocrine function is that specific blockade of the binding of the peptide to its receptor should lessen or abolish the biological function to endogenous stimulants thought m act through the release of the given peptide, lmmunoneutralization (i.e. administration of specific, high affinity antibodies) is one approach to this, but the use of a specific and competitive receptor blocker is generally better. Unfortunately, clear definition of the role nf hioaetive peptides in the physiology nf pancreatic enzyme secretion has been long hampered by the lack of specific and potent receptor antagonists. The availability of such compounds has stimulated a broad array of investigations into the physiological actions of bioactive substances and tn examine their putative role in certain diseases. In a series of experiments, selective receptor blockade by CCK/gastrin, bombesin, and galanin receptor antagonists, as well as immunonsutralization of circulating CCK and sematnstatin were achieved, while pancreatic enzyme secretion was evaluated on different in vivn and in vitro test systems. Data from these experiments suggest that cimulating CCK, indeed, is a primary factor in regulating pancreatic enzyme secretion, acting on CCK-A receptors in rats. Bombesin-like peptides have a minor role in mediating pancreatic function under physinlngical conditions, although gastrin-releasing peptide- preferring receptors are located on pancreatic acini. Gastrin does not seem to play a physiological role in pancreatic enzyme secretion. Immunoneutralization of cimulating snmatostatin led to an increased pancreatic secretory response to CCK stimulation suggesting an inhibitory role of endogenous somatostatin. Rseeptnr antagonists that antagonise the actions of galanin in CNS, behaved as partial agonists of the peptide on the exocrine pancreas. Therefore, these antagonists are not suitable to evaluate the physiological role of galanin in this organ. In conclusion, regulation of the exocrine function of the pancreas is complex involving several bioactive peptides, and this control is not completely understood.

NITRIC OXIDE. CAPSAICIN-SENSITIVE SENSORY NEURONS AND MUCOSAL MAST CELLS IN REGULATION OF ACID SECRETION IN RAT STOMACHS AFTER DAMAGE. K. Takeuchi, S. Kato, K. Takehara. Department of Pharmacology & Experimental Therapeutics, Kyoto Pharmaceutical University, Yamashins, Kyoto 607, Japan

The gastric mucosa responds to damaging agents by significantly decreasing acid secretion. This acid inhibitory mechanism in the damaged stomach involves endogenous nitric oxide (NO) in addition to prostaglandins. We recently found that such acid responses after damage are changed from "inhibitory" into "stimulatory" in the presence of N°-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO biosynthesis. The present study was therefore performed to investigate the mechanism of acid stimulatory response to L-NAME in the rat stomach after damage, Methods: Male SD rats (230-260 g) were used after 18 h fasting. Under urethane anesthesia, a rat stomach was mounted in an ~-vivo chamber, perfusexl with saline, and the potential difference (PD), luminal pH. and acid secretion were measured before and after application of 20 mM taurocholate (TC) for 30 rain, with or without pretreatmeat with L- NAME ( 10 mg/kg, i,v.). Results: Exposure of the stomach to TC caused a PD reduction, an increase of lumiual pH, and a decrease of acid secretion. However, in the animals pmtreated with L-NAME, the acid secretion was not reduced but actually increased significantly after TC treatment, without any change in the PD response. The acid stimulator)' effect of L-NAME in the damaged stomach was significantly antagonized by the simultaneous administration of L-argiaine but not that of D-arginine(200 mgtkg,i.v.). The enhanced acid secretory response to TC in the presence of L-NAME was also significantly inhibited by prior administration of cimetidine. FPL-52694 (a mast cell stabilizer), spantide (a substance P antagonist) or functional ablation of capsaicin-sensitive sensory neurons. The luminal appearance of bistamine increased (--4 times greater than basal levels) following TC treatment, but this response was significantly mitigated by spantide or sensory deafferentation. Conclusion: These results suggest that 1) damage in the stomach activates the acid stimulator)' pathway in addition to the NO-dependent inhibitory mechanism, 2) the acid stimulation may be mediated by histamine mluased from the mast cells through capsaicin-sensitive sensory neurons, and 3) L-NAME unmasks the acid stimulatory response by suppressing

the inhibitory pathway.

ELISA AND WESTERN BLOT STUDIES WITH BASIC FIBROBLAST GROWTH FACTOR (bFGF) AND PLATELET- DERIVED G R O W T H FACTOR (PDGF) IN EXPERIMENTAL DUODENAL ULCER DEVELOPMENT AND HEALING. A. Vincze. M. Nagara. Zs. Sandor. S. Szabo. Dept. of Pathology, Brigham & W o m e n ' s Hosp., Harvard Med. Sch., Boston, MA, and Dept.s. o f Pathology and Pharmacology, Univ. of California, Irvine, VA Med. Cent., Long Beach, CA, USA

Cys tean~e causes severe duodenal ulcers with perforation within 24- 48 h after its administration in rats. Because of its simplicity and mprodudbil i ty, this animal model offers a good opportunity to examine the biochemical changes in the duodenal mucosa during the early, pre- dcerogenie as well as the later, healing phase. Time-dependent changes of duodenal and gastric mucosal levels of basic fibmblast growth factor CoFGF) and platelct-derived growth factor were (PDGF) examined in rats after oral arlmirtistration of the duodenal ulcerogen cysteamine-HCl (25 rag/100 g, x.3 at 4 hr intervals). The animals were killed at 12, 24, 48 ha', 7 or 14 days after the first dose of cystcamine, and the two largest dian'm.ters o f duodenal dee r s were measured and the d e e r area was calculated. The gastric and duodenal mucosa was scraped, homogenized for Western blot and ELISA studies. The duodenal ulcer formation was macroseopically detectable at t 2 hr, while the most severe and largest deers were seen at 48 hr. ParaleLly, the duodenal mucosal concentration ofbR3Fincreased at 12 and 24 hr compared to the controls and reached its maximum at 48 hr. The P D G F concentration was slightly elevated at 12 and 48 hr, and a more than 3-fold peak was seen 24 hr after the first dose o f cysteamine. After 48 hr the duodenal mucosal bFGF level was sin'a'lar to the controls, wl-,ge that o f PDGF was slightly lower. The gastric maeosat level o f IxFGF and PDGF did not change during the development and healing of duodenal ulcers. ~ 1. The early (24--48 hr) elevation of duodenal mueosal Iff:GF and PDGF might be a tissue-specific response to duodenal ulceration. 2. These high endogenous levels of g rowth factors are not sufficient to prevent the ulcer formation and are not maintained in the spontaneous healing phase (7-14 days). 3. Thus, bFGF and PDGF may have a role in the natural history of duodenal ulcer disease.

(A-22) 450 o ~ i w Diseases and Sciences, Vol. 41, No. 2 (February 1996)

A B S T R A C T S O N I U P H A R GI P H A R M A C O L O G Y

MODULATION OF GASTRIC EPITHELIAL AND MESENCHYMAL RESTORATION BY GROWTH FACTORS. Watanabe S, Wang XE, Hirose M, Miyazaki A, Sato N Dept,of Gastroenterology, Juntendo Univ., Tokyo, Japan

A direct cellular pharmacological treatment using several growth factors is now attempting to result in a superior quality of ulcer healing However, the precise cellular mechanism of growth factors is still controversial. Therefore, in this study, we investigated the role of basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) on gastric mucosal restoration using primary cultured rabbit gastric epithelial cells and fibroblasts. METHOD: Isolated rabbit gastric epithelial cells and fibroblasts were prepared from identical rabbit and cultured in F-12 medium. Inoculated epithelial cells (92% mucous cell) formed complete monolayer in 4gh and fibroblasts formed complete monolayer in 4 weeks. A wound with cell-free area of constant size was created by mechanical cell denudation using rotating silicon tip. The restoration process was monitored by measuring wound size every 12 h using a time-lapse video system The proliferative cells were detected by serial staining for BrdU, Effects of bFGF (I-100ng/ml) and HGF (l-10ng/ml) were assessed. RESULT: HGF accelerated the gastric epithelial restoration with the promotion of cell migration and proliferation, however, it did not show any effects on fibroblasts bFGF accelerated the gastric mesenchymal restoration but not for epithelial restoration. These effects of HGF and bFGF were dose depandent, CONCLUSION: The favorable effects of HGF for gastric restoration is mainly on epithelial cells and bFGF has effects on mesenchymal cells The difference of target ceils of growth factor might modulate gastric ulcer healing and the integrity of growth factor network during the process of ulcer healing is essential for high quality ulcer healing

PROSTAGLANDIN F~: DIFFEREaNT EFFECTS ON THE CIRCULAR AND IZ)NGITUDINAL CONTRACTIONS OF THE COLON. T. Wittmann Albert Szent-Gy6rgyi Medical University, Ist. Department of Medicine, Szeged, Hungary.

Researches on the Prostaglandins (PG) provided large series of information concerning their effects on the gastrointestinal mucosa, on the intestinal secretion and absorption. E types of PGs are believed to play a physiological role in the regulation of the GI smooth muscle activity, but the clear mechanism of their regulatory effect remained still unknown.

Aim: To examine the effect of PGE 2 on the circular and l-~itudinal colonic contractions in chronic'aogs.

Methods: A mechanical transducer capable of recording ~ e o u s l y the variations of the circular and longitudinal smooth muscle movements was implanted on the canine colon. After a period of 6 postoperative days the colonic motility was recorded in fasting state two hours before (control period) and two hours after the i.v. perfusion of PGE 9. Three different doses of PGE, 2 were used-O, lllllOpglkg-and fo~'r dogs underwent two recording s~sions with each dose. The variation in the duration, amplitude and frequency of the circular and longitudinal contractions were determined.

: PGE 2 reduces significantly the duration and amplitude of the contractions (-38 to-64%) but it does not modify the same

mdlm3~uceseters o f the longitudinal contractions. Each dose of PGF.o stronge premature bursts of the longitudinal coloniE

contractions, while this effect is absent or moderate at the level of the circular contractions.

Conclusion: PG.E 2 has a regulatory effect on the colonic motility, ~ a l l doses. PGE 2 acts differently on the circular and longitudinal colonic muscles. This special motor effect makes more comprehensible the role of PGE2 in the genesis of the colonic motility disorders leading to diarrhea.

(A-23) D~ge~ive Diseases and Science~, Vol 41, No. 2 (February 1996) 4 5 1

Documents

Section of IUPHAR GI pharmacology symposium with hungarian academy of sciences on biochemical pharmacology as an approach to gastrointestinal disorders (basic science to clinical perspectives)