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Dipyrone and gastric emptyingBrazilian Journal of Medical and Biological Research (2005) 38: 99-104ISSN 0100-879X

Effect of the GABAB agonist baclofenon dipyrone-induced delayed gastricemptying in rats

Departamento de Pediatria, Núcleo de Medicina e Cirurgia Experimental andCentro de Investigação em Pediatria, Faculdade de Ciências Médicas,Universidade Estadual de Campinas, Campinas, SP, Brasil

E.F. Collaresand A.M. Vinagre

Abstract

Dipyrone administered intravenously (iv) or intracerebroventricularly(icv) delays gastric emptying (GE) in rats. Gamma-aminobutyric acid(GABA) is the most potent inhibitory neurotransmitter of the centralnervous system. The objective of the present study was to determinethe effect of icv baclofen, a GABAB receptor agonist, on delayed GEinduced by dipyrone. Adult male Wistar rats received a saline test mealcontaining phenol red as a marker. GE was indirectly evaluated bydetermining the percent of gastric retention (%GR) of the meal 10 minafter orogastric administration. In the first experiment, the animalswere injected iv with vehicle (Civ) or 80 mg/kg (240 µmol/kg) dipy-rone (Dpiv), followed by icv injection of 10 µl vehicle (bac0), or 0.5(bac0.5), 1 (bac1) or 2 µg (bac2) baclofen. In the second experiment,the animals were injected icv with 5 µl vehicle (Cicv) or an equalvolume of a solution containing 4 µmol (1333.2 µg) dipyrone (Dpicv),followed by 5 µl vehicle (bac0) or 1 µg baclofen (bac1). GE wasdetermined 10 min after icv injection. There was no significantdifference between control animals from one experiment to anotherconcerning GR values. Baclofen at the doses of 1 and 2 µg significant-ly reduced mean %GR induced by iv dipyrone (Dpivbac1 = 35.9% andDpivbac2 = 26.9% vs Dpivbac0 = 51.8%). Similarly, baclofen signifi-cantly reduced the effect of dipyrone injected icv (mean %GR: Dpicvbac1= 30.4% vs Dpicvbac0 = 54.2%). The present results suggest thatdipyrone induces delayed GE through a route in the central nervoussystem that is blocked by the activation of GABAB receptors.

CorrespondenceE.F. Collares

Departamento de Pediatria

Faculdade de Ciências Médicas

UNICAMP

13083-970 Campinas, SP

Brasil

Fax: +55-19-3788-7322

E-mail: pediat@fcm.unicamp.br

Publication supported by FAPESP.

Received July 14, 2003

Accepted September 16, 2004

Key words• Gastric emptying• Dipyrone• GABA• GABAB receptors• Baclofen

Introduction

Gastric emptying consists of the transferof gastric content to the small bowel as aresult of the action of stimulating and inhib-itory mechanisms that control the motor ac-tivity of the stomach, pylorus and duode-num. Under physiological conditions, gas-tric emptying occurs in an appropriate man-

ner for the conditions of digestion and ab-sorption present in the small bowel (1,2).

Intravenous (iv) and intracerebroventricu-lar (icv) administration of dipyrone to ratsleads to a delay in gastric emptying of a liquidmeal (saline). This phenomenon is more in-tense during the first hour after iv administra-tion and is abolished by subdiaphragmatic va-gotomy and electrolytic lesion of the hypotha-

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lamic paraventricular nucleus (3). These ob-servations suggest that dipyrone influencesgastric emptying through the central nervoussystem (CNS), leading to a predominance ofinhibitory mechanisms conveyed by the vagusnerve on the motor activity of the stomach,pylorus and/or duodenum. However, the neu-rotransmitters possibly involved in the media-tion of this effect are unknown.

Gamma-aminobutyric acid (GABA) isthe most important inhibitory neurotrans-mitter in the CNS, where three differentreceptors have been identified: GABAA,GABAB and GABAC (4-7). GABAA recep-tors show a predominantly postsynaptic lo-calization and, when activated, lead to rapidmembrane hyperpolarization due to an in-crease in Cl- channel conductance (4,5).GABAC receptors are also associated withCl- channels, but differ from GABAA recep-tors in terms of pharmacological, structural,functional and genetic aspects and cellularlocalization (6-8). Activation of GABAB re-ceptors through Gi/Go membrane protein sig-naling leads to the inhibition of adenylatecyclase, increases K+ channel conductance,and reduces Ca2+ channel conductance (5).

Studies using agonists and antagonists ofGABA receptors located in neurons of thedorsal vagal complex (DVC) have providedevidence that this acid is involved in thecontrol of gastrointestinal motility (9-13). Inparticular, baclofen (ß-p-chlorophenyl-GABA), a lipophilic derivative of GABA,binds as a specific agonist to GABAB recep-tors which, when activated, induce stimula-tion of the gastric acid secretion and motoractivity of the stomach through the vagusnerve (11,14-16).

The objective of the present study was totest the hypothesis that activation of GABAB

receptors with icv baclofen inhibits delayedgastric emptying induced by dipyrone in rats.

Material and Methods

Male Wistar rats weighing 250-300 g,

adapted to laboratory conditions for 2 weeks,were used. The experimental protocols ap-plied in the present study followed the rec-ommendations of the Brazilian College ofAnimal Experimentation. For surgical pro-cedures, the animals were sedated by intra-peritoneal injection of 75 mg/kg thiopental.After the procedures, the animals were keptin individual cages with ration and wateravailable ad libitum.

Solutions of dipyrone or baclofen (Sigma,St. Louis, MO, USA) were prepared justbefore administration using sterile salinesolution as vehicle, with dipyrone being pro-tected from light. The iv or icv doses ofdipyrone have been described elsewhere (3).Equivalent volumes of the vehicle were usedas control.

Eight days before the study, a 15-mm 21-G stainless steel cannula was implanted intothe right lateral ventricle of each animal andfixed to the skull with two screws, self-polymerizing acrylic and instant adhesive(cyanoacrylate ester). The cannulae wereimplanted at the following coordinates inrelation to the bregma according to the atlasof Groot (17): anteroposterior = -0.2 mm,right lateral = 1.5 mm, vertical = 4.2 mm. Formicroinjection, a 28-G internal cannula wasconnected through polyethylene tubing to a25-µl Hamilton syringe.

In the first experiment, animals were in-jected iv through a caudal vein with vehicle(Civ) or 80 mg/kg (240 µmol/kg) dipyronesolution (Dpiv), followed by icv injection of10 µl vehicle (bac0) or an equal volume of asolution containing 0.5, 1 or 2 µg baclofen(bac0.5, 1 and 2, respectively) over a periodof 30 s, with the animal remaining connectedto the system for an additional 30 s. Gastricemptying was determined 10 min after re-moval of the internal cannula.

In the second experiment, animals re-ceived 5 µl of the vehicle (Cicv) or an equalvolume of a solution containing 4 µmol(1333.2 µg) dipyrone (Dpicv) icv over a pe-riod of 30 s, with the animal remaining con-

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nected to the system for an additional 30 s.Using another injection system, the animalsreceived 5 µl vehicle (bac0) or an equalvolume of 1 µg baclofen solution (bac1)over a period of 30 s, with the animal re-maining connected to the system for an addi-tional 30 s. Gastric emptying was deter-mined 10 min after removal of the internalcannula.

Gastric emptying was evaluated in theanimals between 14:00 and 17:00 h after a24-h fast during which only water was avail-able, and which was withdrawn at the time ofthe test. The test meal consisted of 2 ml/100g body weight of an aqueous 0.9% NaClsolution containing 60 µg/ml phenol red asmarker. In all experiments, gastric emptyingwas indirectly evaluated in non-sedated ani-mals, except for the time of sacrifice, bydetermining the percent of gastric retention(%GR) of the phenol red-containing test mealrecovered within the gastric content 10 minafter orogastric administration of the meal(18). Phenol red concentrations were meas-ured with a spectrophotometer at 560 nm.Higher icv doses of baclofen (≥1.5 µg/kg) inrats have induced exploratory behavioral ef-fects like grooming, gnawing and, in somecases, rigid posture with extended limbs andarched back (19). In the present study, thebaclofen doses ranged from 2 to ~8 µg/kgand induced the reactions described above.However, these reactions did not interferewith the measurement of gastric emptying invivo.

At the end of the study, all animals weresacrificed and injection into the lateral ven-tricle was confirmed by administering 10 µlof a 1% Evans blue solution through thecannula. The brains were then removed andfixed in 10% formalin for 24 h. Specimenswere then cut into coronal sections and icvinjection was confirmed when the dye wasdetected in the fourth ventricle.

The gastric retention results are reportedas means ± SEM. ANOVA was used forstatistical analysis and pairs were compared

by the Tukey test (α = 0.05). The dose-response effect of baclofen was determinedby calculation of the correlation coefficientfor ordered pairs (r).

Results

Figure 1 shows the results of the firstexperiment in which iv administration ofdipyrone caused a significant increase in thegastric retention of animals receiving ve-hicle icv (bac0) compared to controls (mean%GR: Dpivbac0 = 51.8 vs Civbac0 = 33.2%).In contrast, no significant difference in gas-tric retention was observed between the groupof animals receiving iv dipyrone followed by1 and 2 µg icv baclofen (bac1 and bac2) andits respective control group (mean %GR:Dpivbac1 = 35.9 vs Civbac1 = 24.2% andDpivbac2 = 26.9 vs Civbac2 = 19.0%). There-

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**

**

*

C Dp C Dp C Dp C Dpbac0 bac0.5 bac1 bac2

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Figure 1. Gastric retention of a saline test meal 10 min after orogastric administration torats. Eight days before the experiment, all animals were implanted with a stainless steelcannula into the right lateral ventricle. The animals were injected iv with vehicle (C) or 80mg/kg (240 µmol/kg) dipyrone (Dp), followed by icv injection through an internal cannula of10 µl vehicle (bac0) or an equal volume of solution containing 0.5, 1 or 2 µg baclofen(bac0.5, bac1 and bac2, respectively). Gastric retention, reported as mean ± SEM inpercent, was determined 10 min after removal of the internal cannula. The number ofanimals in each group is given in parentheses. *P < 0.05 (Tukey test).

(9) (7)

(8)(7)

(9)

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(9)

(8)

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fore, 1 and 2 µg doses of baclofen signifi-cantly reduced gastric retention induced byiv dipyrone (Dpivbac1 and Dpivbac2 vsDpivbac0). A strong negative correlation wasobserved between the dose of baclofen andgastric retention in animals receiving dipy-rone (r = -0.97). Control animals injected ivwith vehicle also showed a negative correla-tion between the dose of the GABAB agonistand gastric retention (r = -0.92), with thereduction in gastric retention being nonsig-nificant, except for bac0.5 vs bac2 controls(mean %GR: Civbac0.5 = 34.7 vs Civbac2 =19.0%).

In the second experiment (Figure 2), icvadministration of 1 µg baclofen also signifi-cantly reduced the effect of previous icvinjection of 4 µmol (1333.2 µg) dipyrone ongastric retention of the test meal (mean %GR:Dpicvbac1 = 30.4 vs Dpicvbac0 = 54.2%). Nosignificant difference in gastric retention wasobserved between the control animals in-jected icv with vehicle or baclofen (mean%GR: Cicvbac1 = 26.3 vs Cicvbac0 = 29.5%).

Discussion

The mechanisms involved in the phe-nomenon of delayed gastric emptying in-

duced by dipyrone in rats, or the site(s) ofaction of the drug in the CNS, are unknown.However, in the present study we showedthat icv administration of baclofen, a specif-ic GABAB receptor agonist, abolished thiseffect.

GABAA and GABAB receptors are dis-tributed within the rat CNS at equivalentconcentrations in some areas, with a pre-dominance of the former in most brain re-gions and of the latter in some others (20,21).

In the CNS, activation of GABAB recep-tors by baclofen results in the hyperpolariza-tion of postsynaptic membranes or inhibi-tion of the release of the neurotransmitter atpresynaptic terminals (5). Activation of post-synaptic GABAB receptors leads to a pro-longed increase in K+ channel conductance,which is responsible for the generation ofslow inhibitory GABA-ergic events in theCNS (4,5,22), including the modulation ofrhythmic hippocampal activity (23). Presyn-aptic GABAB receptors are primarily in-volved in the regulation of neurotransmitterrelease, since the main effect of baclofenconsists of reducing the release of excitatoryand inhibitory synaptic transmitters (4,5).This action of baclofen has been observed ina variety of synapses, with the drug reducingthe release of noradrenaline, dopamine, ace-tylcholine, serotonin, glutamate, and aspar-tate. Presynaptic GABAB receptors may alsofunction as autoreceptors, reducing the re-lease of GABA and thus diminishing thepostsynaptic inhibition mediated by the acti-vation of GABAA receptors (22).

Since in a previous study (3) the effect ofdipyrone on gastric emptying could be abol-ished by subdiaphragmatic vagotomy, wemay assume that the DVC is primarily in-volved in this phenomenon. The DVC isformed by the nucleus tractus solitarii whoseneurons receive information through the af-ferent route and by the dorsal nucleus of thevagus nerve, where cholinergic stimulatingand non-cholinergic, non-adrenergic inhibi-tory motoneurons are located, whose axons

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n (%

)

60

40

20

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C Dp C Dpbac0 bac1

icvicv

Figure 2. Gastric retention of asaline test meal 10 min afterorogastric administration to rats.Eight days before the experi-ment, all animals were im-planted with a stainless steelcannula into the right lateral ven-tricle. The animals were injectedicv through an internal cannulawith 5 µl vehicle (C) or an equalvolume of solution containing 4µmol (1333.2 µg) dipyrone (Dp),followed by injection of 5 µl ve-hicle (bac0) or an equal volumeof solution containing 1 µgbaclofen (bac1). Gastric reten-tion, reported as mean ± SEM in% for N = 8 animals, was deter-mined 10 min after removal ofthe internal cannula. *P < 0.01(Tukey test).

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correspond to the efferent route of the vagusnerve (2).

In the rat, GABAB receptors are locatedpredominantly at presynaptic afferent termi-nals of the vagus nerve, which project intothe nucleus tractus solitarii, although evi-dence also indicates a postsynaptic locationin this nucleus (24).

One may speculate that the phenomenonof delayed gastric emptying induced by di-pyrone is the result of activation of a routethat stimulates inhibitory motoneurons and/or inhibits excitatory motoneurons of thevagus nerve. Since there is strong evidencethat GABA plays a role in the control ofgastric motility at the level of the DVC (9,11-13), activation of presynaptic GABAB re-ceptors by baclofen that leads to the block-ade of excitation of a non-cholinergic, non-adrenergic route and/or blockade of the inhi-bition of a cholinergic route might explainthe results observed in the present study.

However, in addition to the possible in-volvement of the DVC which also influ-ences gastric motility, a role of GABA inother nearby nuclei such as the nucleusambiguus and nucleus raphe obscurus hasbeen demonstrated (9,25). GABA receptorshave also been identified in the hypothalam-ic paraventricular nucleus (26,27), whichshows connections with the DVC and, whenlesioned electrolytically, blocks the effect ofdipyrone (3). In addition, the DVC is underthe influence of other regions of the CNSthat interfere with gastric motility, such asthe central nucleus of the amygdala and theinsular cortex, where GABA receptors havealso been identified (20,21,28-31).

Based on the evidence showing a moreextensive effect of dipyrone on the CNS, ananticonvulsant effect of this drug has beendemonstrated in various experimental ratmodels of epilepsy. Different hypotheses toexplain this effect have been proposed, suchas the inhibition of prostaglandin produc-tion, blockade of the inactivation of adeno-sine (a potent inhibitory neuromodulator),

activation of GABAA receptors, and anantiglutamatergic effect (32,33). Finally, anextrasynaptic presence of GABAB receptorshas been proposed, although for these recep-tors to be activated continuously, elevatedconcentrations of GABA in the extracellularfluid are necessary or its concentration needsto be increased close to these receptorsthrough the release from various neurons(4,23,34).

Thus, it is possible that various CNSstructures and mediators are involved which,when activated, express the effect of dipy-rone on gastric emptying, in addition to sitesfor the blockade of this effect by baclofen.

In the first experiment, although no sta-tistically significant difference was observedbetween Civbac0 and the other baclofen con-trols, gastric emptying tended to be decreasedfollowing this agonist. This observation sug-gests that icv baclofen may have an effect onthis gastric function per se. Nevertheless,this drug had a marked and statistically sig-nificant inhibitory effect on the dipyrone-induced delay of gastric emptying. The re-duction of the effect of iv dipyrone adminis-tration followed by 1 and 2 µg icv baclofenwas slightly higher (a 31% reduction in meanGR in the Dpivbac1 group and a 48% reduc-tion in the Dpivbac2 group compared to theDpivbac0 group) than that observed in therespective controls (a 27% reduction in meanGR in the Civbac1 group and a 42% reduc-tion in the Civbac2 group compared to theCivbac0 group). Taken together, these resultssuggest that these two drugs have oppositeeffects on the CNS through independentmechanisms.

The results of the second experiment sup-ported more convincingly the view thatbaclofen blocks a route that is required forthe effect of dipyrone in the CNS. Baclofeninduced a 4-fold reduction in the effect ofdipyrone on gastric retention (a 44% reduc-tion in mean GR for the Dpicvbac1 groupcompared to the Dpicvbac0 group) comparedto that observed in the respective control (an

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11% reduction in mean GR for the Cicvbac1group compared to the Cicvbac0 group).

The present results suggest that dipy-

rone induces delayed gastric emptyingthrough a route in the CNS that is blocked bythe activation of GABAB receptors.

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