Pii: s0928-4257(00)00154-6

J. Physiol. (Paris) 94 (2000) 135−138 2000 Elsevier Science Ltd. Published by Éditions scientifiques et médicales Elsevier SAS. All rights reservedS0928425700001546/FLA Modulation by GABA and delta opioid receptors
of neurally induced responses in isolated guinea-pig taenia coli
and human colonic circular muscle
Éva Makóa,b*, András Z Rónaia, György Ádámb, Gábor Juhászb, László Ritterc, Béla Lestárc, Vincenzo Crunellid aDepartment of Pharmacology, Semmelweis University of Medicine, P.O.B. 370, 1445 Budapest, Hungary bDepartment of Comparative Physiology, Eötvös University, Budapest, Hungary cDepartment of Surgery, Haynal Postgraduate University of Medicine, Budapest, Hungary dUniversity of Wales, College of Cardiff, Cardiff, UK Abstract — The GABA-ergic and opioid modulation of neurally induced muscle responses was studied in isolated guinea-pig taenia
coli and human colonic circular muscle, using identical field stimulation parameters (rectangular pulses of 0.5 ms duration, 9 V⋅cm–1
intensity, trains of 3 pulses at 0.5 Hz, repeated every 1/3/5 min).The stimulation-induced contractions were inhibited in both
preparations by GABA and baclofen; the IC
values in human colonic circular muscle were z100 and 31.0 µM, respectively. In guinea-pig taenia coli, the inhibition by 10–4 M GABA was dose-dependently reversed by 10–4–10–3 M of GABA receptor antagonist CGP 35348; antagonism by phaclofen was less effective in the same concentration range. In human colonic circular muscle, inhibitionby 3 × 10–5 M baclofen was fully reversed by 10–3 M CGP 35348. With the exception of caecum, the delta 2 opioid receptor agonistdeltorphin II was a potent inhibitor in human colonic circular muscle. 10–8 M Deltorphin caused a 74.4 ± 9.6% (n = 4) inhibition whichwas reversed by 10–6 M of delta receptor selective peptide antagonist BOC-Tyr-Pro-Gly-Phe-Leu-Thr(OtBu). Deltorphin II wasineffective in guinea-pig taenia coli even at 10–6 M; the same concentration caused an 84.3 ± 7.9 (n = 4) inhibition in humanpreparations. It is concluded that: 1) GABA-ergic modulatory mechanisms are present both in human colonic circular muscle andguinea-pig taenia coli; 2) the GABA receptors involved are of type B; and 3) delta opioid receptor-mediated modulation functions onlyin human colonic circular muscle in regions other than the caecum. 2000 Elsevier Science Ltd. Published by Éditions scientifiqueset médicales Elsevier SAS guinea-pig taenia coli / human colonic circular muscle / GABA receptors / delta opioid receptors
1. Introduction
lonic circular muscle ([9] and Makó in preparation). Inthe human colonic circular muscle, 5HT /H receptor mediated link is likely to be present in the stimulatory proenkephalinA/prodynorphin-derived opioid peptides neural network since cyproheptadine inhibited the are neurotransmitters of enteric interneurons and com- neural stimulation-induced contractions ([9] and Makó prise part of the intrinsic neural circuits regulating in preparation). To compare the opioid and GABA- peristalsis [1, 2, 5, 6, 8]. Our study was aimed at ergic modulatory mechanisms in the two isolated characterising the receptor types where these transmit- organs, the delta (delta 2) opioid receptor agonist ters exert their action in isolated, neurally stimulated deltorphin II (DT-II), the delta receptor selective pep- human colonic circular muscle and guinea-pig taenia tide antagonist BOC-Tyr-Pro-Gly-Phe-Leu-Thr(OtBu) coli. The common technical motif for both prepara- [BOC-YPGFLT(OtBu)] [12], GABA, the GABA re- tions was the choice of parameters of field neural stimulation. These parameters (short trains at a low antagonist CGP 35348 and phaclofen [3, 4, 7, 10] were frequency) were expected to facilitate the detection both of inhibitory and stimulatory actions of exog-enously administered receptor-specific agents. Thechosen field stimulation parameters elicited a mul- 2. Materials and methods
tiphasic or apparently monophasic muscle response Drugs used were as follows: (-) baclofen (Research with a dominant contractile component in both isolated Biochemicals International), CGP 35348 (Tocris organs ([9, 11] and Makó in preparation). The neural Neuramin), gamma-amino butyric acid (Sigma), circuitry involved in the contractile responses of the phaclofen (Tocris Neuramin). BOC-Tyr-Pro-Gly-Phe- two preparations is, naturally, quite different. The Leu-Thr(OtBu) was synthesised at the Research Group contractions were atropine-sensitive in guinea-pig tae- of Peptide Chemistry, Eötvös University, Budapest, as nia coli [11] whereas atropine-resistant in human co- described previously [12]. Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH ) was kindly supplied by G.
Tóth of Isotope Laboratory, Biological Research Cen- tre of Hungarian Academy of Sciences, Szeged. All theother substances were of analytical grade.
Male guinea-pigs, weighing 250–350 g, were used and the taenia coli segments were prepared andmounted as described previously [11]. In brief,5–8 mm long segments were mounted in Krebs’ solu-tion aerated with carbogen (O :CO = 95:5) at 37 °C.
An initial tension of 0.5 g was applied re-adjusted onceafter 5 min of 45–60 min equilibration period.
Subjects were 8 patients, 4 male and 4 female, age 28–76 years, undergoing colonic surgery for cancer (6)colitis ulcerosa (1) or slow colon (1). The preparationswere made from non-diseased tissue dissected accord-ing to the pre-set rules of disease-dependent surgical Figure 1. The inhibition of neural field stimulation-induced
protocols. Among the dissected tissues, the sigmoid contractions of human colonic circular muscle by baclofen colon (4), the rectum (4) and the caecum (1) were and the reversal of inhibition by CGP 35348. ·, 3 × 10–5 M represented (i.e. from one subject, two specimens were baclofen alone; [, 3 × 10–5 M baclofen + 10–3 M CGP35348 dissected). The postoperative use of tissue samples added later (arrow). Points represent the mean of two was approved by the Ethical Committee of the Uni- versity. The routinely used preoperative medicationwas metronidazole and aminoglycoside as antimicro-bials and benzodiazepine as sedative. Five operations 3. Results
were performed under general anesthesia; in threecases, epidural anesthesia was used.
Of the total of 65 human colonic circular muscle The specimens were transported in a special cham- strips, 61 responded to neural field stimulation. Only ber in thermostated (33 °C) carbogenated Krebs’ solu- those preparations were used for the pharmacological tion; the transportation took less than 20 min. The tests where an apparently monophasic contractile re- preparation was done at room temperature in carboge- sponse was present with or without spontaneous back- nated Krebs’ solution. After the removal of the mu- ground activity. Both GABA and baclofen inhibited cosal and serosal layer, 10–15 mm long, 2–2.5 mm the evoked contractions; in the case of the former, wide circularly oriented strips were cut from the there was a high propensity to develop tachyphylaxis.
intertaenial segment. The strips were mounted in At 10–4 M concentration, GABA exerted 55.4 ± 18.3% carbogenated Krebs’ solution at 37 °C. The muscles (mean ± SEM, n = 3) inhibition; at 10–3 M concentra- were left without applying any tension for 15 min, tion, the inhibition tended to be less than 50%.
washed once, then 1-g tension was applied. The Baclofen had a dose-dependent inhibitory effect with tension was re-adjusted after 10 min then the prepara- only a minor tendency for tachypylaxis; the IC tions were equilibrated for 60 min under resting con- was 31.0 µM (28.6, 33.4, n = 2). The inhibition caused ditions. The composition of Krebs’ solution was as by 3 × 10–5 M baclofen could be reversed by the follows (in mmol⋅L–1): NaCl 118.0, NaHCO 25.0, GABA receptor antagonist CGP 35348 (figure 1).
KCl 4.7, KH PO 1.2, CaCl 2.5, MgSO 1.2 (human) According to our previous findings in guinea-pig = 4.5 ± 1.0 µM, n = 5) but not mus- The parameters of field stimulation [11] were: rect- cimol (at 10–4 M) had inhibitory effect in preparations angular impulses of 0.5 ms duration, 9 V⋅c–1m (supra- stimulated neurally by parameters closely matching maximal) intensity; trains of 3 pulses at 0.5 Hz fre- the ones used presently. We characterised further this quency were repeated by 1 (guinea-pig) or 5 min inhibitory action by the reversal of the inhibition by (human). The contractions were measured under non- 10–4 M GABA by cumulatively administered GABA isometric conditions. From a single surgical specimen, receptor antagonists CGP 35348 and phaclofen (fig- 4–12 strips were prepared; they were randomly as- signed to the different pharmacological interventions.
Besides GABA and baclofen, the delta 2 opioid In one preparation, only a single drug or drug combi- receptor agonist deltorphin II was also a potent inhibi- tor in neurally stimulated human colonic circular For statistical comparisons ANOVA followed by muscle strips (figure 3) prepared from the sigmoid Student’s t-test or just Student’s t-test was used, colon (3 patients) or rectum (2 patients) but not in depending on the experimental paradigms.
preparations made from the caecum (1 patient).
Figure 4. The reversal of the inhibitory action of deltorphin
Figure 2. The inhibition of neural field stimulation-induced
II by BOC-Tyr-Pro-Gly-Phe-Leu-Thr(OtBu) in neurally contractions of guinea-pig taenia coli by GABA and the stimulated human colonic circular muscle. Bars and vertical reversal of inhibition by CGP 35348 or phaclofen. ·, lines represent the mean ± SEM of 4 independent experi- 10–4 M GABA alone; [, 10–4 M GABA + 10–4, 3 × 10–4 and 10–3 M CGP 35348 added in a cumulative manner (arrows); _, 10–4 M GABA + 10–4, 3 × 10–4 and 10–3 M phaclofen.
Points and vertical lines represent the mean ± SEM of by 10–8 M DT-II could be readily reversed by the delta 4 independent experiments. The reversal by CGP 35348 was opioid receptor selective peptide antagonist BOC- significant at 10–4 M and onward (ANOVA and Student’s t-test) whereas for phaclofen it became significant only at10–3 M.
In circular muscle strips prepared from the caecum, DT-II did not affect neural stimulation-induced con-tractions even at 10–6 M (n = 3, not shown); in sigmoid The washout was very slow and in most cases colon-derived strips obtained from the same patient, incomplete therefore in one preparation only a single DT-II was fully effective. Likewise, 10–6 M DT-II was concentration of DT-II could be tested. There was an ineffective in guinea-pig taenia coli (n = 4, not shown).
84.3 ± 7.9% (n = 4) inhibition at 10–6 M, 96.3 ± 3.0%(n = 3) at 10–7 M and 74.4 ± 9.6% (n = 4) at 10–8 M 4. Discussion
indicating that at these concentrations the effects fellinto the ‘plateau’ phase of the dose-response curve and Two types of inhibitory modulation was studied in must be below 10–8 M. The inhibition caused two isolated organ preparations where the field-stimulation induced contractions have been shown tobe either atropine-, (guinea-pig taenia coli, [11]) ortetrodotoxin muscle, [9] and Makó in preparation) indicating theinvolvement of neural elements. It was shown pres-ently that the GABA-ergic inhibitory modulation de-tected both in neurally stimulated human coloniccircular muscle and guinea-pig taenia coli is mediatedby B type of GABA receptors since 1.The action ofGABA is mimicked by the GABA receptor agonist baclofen; 2.the effect of either GABA or baclofencould be reversed by the GABA receptor antagonist CGP 35348. Of the GABA receptor antagonists we phaclofen. In the CNS the localisation of CGP 35348sensitive GABA receptors has been characterised as preferentially „hetero-presynaptic» relative to GABA- Figure 3. The inhibitory effect of deltorphin II on two types
ergic neurons [3, 10] i.e. they are present on the of neurally induced responses in human colonic circular terminals of non-GABA-ergic neurons. The presence muscle. Upper trace: inhibition of monophasic contractions of GABA in mammalian enteric neural elements is by deltorphin II (DT-II) and the reversal of inhibitionby BOC-Tyr-Pro-Gly-Phe-Leu-Thr(OtBu) (BOC-YPGFLT extensively documented (e.g. [6]); in a subpopulation (OtBu). Lower trace: inhibition of polyphasic contractions.
of human colonic neurons it has recently been de- scribed to colocalise with enkephalin or nitric oxide Froestl W., Mickel S.J., Von Sprecher G., Bittiger H., Olpe synthase [8]. If the enteric localisation of CGP 35348 sensitive receptors follows the CNS pattern (i.e.theyare hetero-presynaptic) we may assume either facilita- Hoyle C.H.V., Kamm M.A., Burnstock G., Lennard-Jones tory GABA receptors at nerve terminals releasing an J.E., Enkephalins modulate inhibitory neuromuscular trans- mission in circular muscle of human colon via δ-opioid inhibitory mediator, or inhibitory GABA receptors at receptors, J. Physiol. 431 (1990) 465–478.
nerve terminals releasing an excitatory mediator.
Deltorphin II was a potent inhibitor of neurally Jessen K.R., Hills J.M., Saffrey M.J., Immunohistochemical mediated contractions in circular muscle strips from demonstration of GABA-ergic neurons in the enteric nervoussystem, J. Neurosci. 6 (1986) 1628–1634.
human sigmoid colon or rectum but not from caecum;likewise, it was ineffective in guinea-pig taenia coli.
Kerr D.I., Ong J., Prager R.H., Gynther B.D., Curtis D.R., Delta and kappa opioid receptors are known to be Phaclofen: a peripheral and central baclofen antagonist, BrainRes. 405 (1987) 150–154.
present in human colon [1, 5]. Altough deltorphin II isa selective agonist of delta 2 subtype of opioid Krantis A., Nichols K., Staines W., Neurochemical character- receptors in CNS tests [13], from the present results ization and distribution of enteric GABA-ergic neurons andnerve fibres in the human colon, J. Auton. Nerv. Syst. 68 we cannot conclude that delta 2 receptors are present in human colonic circular muscle. First, hitherto wehave been unable to detect delta 1/delta 2 opioid Makó É., Rónai A.Z., Ádám Gy., Ritter L., Lestár B., In vitropharmacological analysis of the responses of human colonic circular muscle to neural stimulation, in: 6th Joint Meeting of nerve/smooth muscle preparations (Rónai, unpub- the Italian, Hungarian and Polish Pharmacological Societies, lished) nor are we aware of such successful differen- tiation from other sources. Second, detailed further [10] Raiteri M., Bonanno G., Paudice P., Cavazzani P., Schmid G., pharmacodynamic analysis is needed to make such a Human brain cholecystokinin: release of cholecystokinin-like declaration for the human colonic circular muscle; at immunoreactivity (CCK-LI) from isolated cortical nerve end- present the only valid statement is that these delta ings and its modulation through GABA(B) receptors, J.
receptors are highly responsive to the agonist action of Pharmacol. Exp. Ther. 278 (1996) 747–751.
[11] Rónai A.Z., Kardos J., Simonyi M., Potent inhibitory GABAB receptors in stimulated guinea-pig taenia coli, Neuropharma-cology 26 (1987) 1623–1628.
[12] Rónai A.Z., Magyar A., Orosz G., Borsodi A., Benyhe S., Tóth G., Makó É., Kátay E., Babka E., Medzihradszky K., Opioid Chamouard P., Klein A., Martin E., Adloff M., Angel F., antagonist properties of the highly δ-receptor selective BOC- Regulatory role of enteric kappa opioid receptors in human Tyr-Pro-Gly-Phe-Leu-Thr (OtBu) peptide and of its Phe1 and colonic motility, Life Sci. 53 (1993) 1149–1156.
Mel1 analogues, Arch. Int. Pharmacodyn. 330 (1995) Chamouard P., Rohr S., Meyer C., Baumann R., Angel F., Delta-opioid receptor agonists inhibit neuromuscular trans- [13] Zaki A.P., Bilsky E.J., Vanderah T.W., Lai J., Evans C.J., mission in human colon, Eur. J. Pharmacol. 262 (1994) 33–39.
Porreca F., Opioid receptor types and subtypes: The δ receptor Emri Z., Turner J.P., Crunelli V., Tonic activation of presyn- as a model, Annu. Rev. Pharmacol. Toxicol. 36 (1996) aptic GABA receptors on thalamic sensory afferents, Neu-

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