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FABAD J. Pharm. Sci., 29, 127-132, 2004 RESEARCH ARTICLE

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Bilgen BAfiGUT*°, Feyza ADEMO⁄LU*, Gülden GÜRSOY*, ‹clal ÇAKICI**, Nurettin ABACIO⁄LU*

Effect of Melatonin on the Isolated Rat Anococcygeus Muscle

Summary

The aim of the present study was to investigate the effect of mela- tonin on the isolated rat anococcygeus muscle (RAM). The RAM was isolated as described by Gillespie 1971. The muscles were mounted in an organ bath containing modified Krebs-Henseleit solution at 37°C and, changes in the tension due to the precon- tractile agents and melatonin were measured by isometric force displacement transducers. Although cumulative addition of mela- tonin (10^-10-10^-3M) produced no effect on the basal tone of the anococcygeus muscle, it caused an inhibitory action on the muscle pre-contracted by KCl (70 mM) and phenylephrine (1.7x10^-6M). In the sodium fluoride (NaF) (3x10^-3M)-precontracted muscles, melatonin (10^-5-10^-3M)-induced maximum relaxation was found higher than that obtained in phenylephrine or KCl- precontracted RAM. Incubation with phorbol myristate acetate (PMA (10^-10-10^-4M) an activator of protein kinase C) produced no effect on the resting or precontracted muscle. In conclusion, the inhibitory effect of melatonin might be related to the G-protein mediated pathways, but not to protein kinase C.

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Keeyy WWoorrddss :: Melatonin, phenylephrine, KCl, NaF, rat anococcygeus muscle

Received : 2.5.2005 Revised : 28.7.2005 Accepted : 29.7.2005

Melatoninin ‹zole S›çan Anokoksigeus Kas› Üzerindeki Etkisi

Özet

Bu çal›flman›n amac› s›çan anokoksigeus kas›nda melatoninin etkisinin incelenmesidir. S›çan anokoksigeus kas› 1971’de Gil- lespie taraf›ndan tan›mlanan yönteme gore izole edilmifl ve mo- difiye Krebs-Henseleit solüsyonu içerisinde 37°C’lik izole organ banyosuna as›lm›flt›r. Prekontraktil ajanlar ve melatoninle olu- flan gerim de¤ifliklikleri izometrik gerim ileticisi yard›m› ile kay- dedilmifltir. Melatoninin kümülatif olarak uygulanmas› kas›n dinlenme gerimini de¤ifltirmemesine ra¤men KCl ve fenilefrinle prekontrakte edilmifl dokuda inhibitor etkiye yol açm›flt›r. Sodi- um florür (NaF) ile prekontrakte dokuda melatonin KCl ve fe- nilefrinle prekontrakte edilmifl s›çan anokoksigeus kas›na gore daha fazla gevflemeye neden olmufltur. Forbol miristat asetat (protein kinaz C aktivatörü) ile inkübasyon hem kas›n dinlenme gerimini hem de prekontrakte kas› etkilememifltir. Sonuç olarak melatoninin inhibitor etkisi protein kinaz C’den ba¤›ms›z olarak G-proteini arac›l› yolaklarla iliflkili olabilir.

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Annaahhttaarr KKeelliimmeelleerr :: Melatonin, fenilefrin, KCl, NaF, s›çan anokoksigeus kas›

* Gazi University, Faculty of Pharmacy, Department of Pharmacology, 06330 Etiler, Ankara - TURKEY

* Yeditepe University, Faculty of Pharmacy, Department of Pharmacology, 34755, ‹çerenköy, ‹stanbul - TURKEY

° Corresponding author e-mail: bilgenh@yahoo.com IINNTTRROODDUUCCTTIIOONN

Melatonin, N-acetyl-5-methoxytryptamine, is a sec-

retory product of the pineal gland. High concentrations of melatonin (10 mM- 1 mM) have been reported to inhibit vasoconstrictor responses in blood ves-

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Animals were housed in a 12-hr light-dark cycle (lights on at 08.00 lights off at 20.00) with free access to food and water. Rats were anesthetized by thi- opental sodium injection (60 mg/kg, i.p.) 1-2 hrs after lights on and, the anococcygeus muscles were isolated as described previously¹¹. The twin muscles were mounted individually under a resting tension of 750 mg in 15 ml organ baths containing modified Krebs-Henseleit solution gassed with a mixture of 95% O₂and 5% CO₂at 37°C and exposed to the same experimental procedures. Tissues were allowed to equilibrate for at least 30 min with frequent was- hings before start of the experiment. Changes in tension were measured by the force displacement transducers (Commat, Turkey) coupled to a polyg- raph (Tumel, Turkey).

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Exxppeerriimmeennttaall PPrroottooccooll

All drugs were added in their final concentration in the isolated organ bath and exposed to the tissue until a stable response was achieved. After equilibration, effects of melatonin (10^-10-10^-3M) and the vehicle (dimethylsulfoxide, DMSO– final concentration 8%) were tested on the resting tension of the isolated RAM. Tissues were then precontracted with KCl (70 mM) or phenylephrine (1.7x10^-6M). Upon attainment of a contraction plateau, melatonin (10^-5-10^-3 M) or nifedipine (Ca⁺²channel blocker, 10^-12-10^-8M) or vehicle (DMSO, final concentration 3%) was given cumulatively. In another set of experiments, the effect of melatonin (10^-4-10^-3M) on the sodium fluoride (NaF) (3x10^-3M), G-protein activator, precontracted RAM was examined with a similar experimental protocol. Before the precontraction by NaF, the muscles were incubated with 10 mM AlCl₃for 5 min, for the stabilisation of NaF-induced contraction. RAM was also incubated with phorbol myristate acetate (PMA) (10^-10-10^-4M) for 45 min to test whether or not it was possible to induce a precontraction via protein kinase C activation. PMA was also tested on KCl (35 mM)-preactivated tissues.

Baflgut, Ademo¤lu, Gürsoy, Çak›c›, Abac›o¤lu

sels from a variety of species. In the rabbit basilar artery, melatonin (10^-5-10^-3M) has an inhibitory effect on KCl-induced contractions¹and, melatonin-induced relaxation was also observed in the rat^2,3 and rabbit aorta⁴. In the earlier in vitro studies, it has been shown that melatonin has an inhibitory action on the response to serotonin in rat uterus⁵, rat duodenum⁶, and cat trachea⁷. Isolated rat papillary muscles⁸, guinea-pig isolated proximal colon⁹ and, rat gastric fundus¹⁰are among the nonvascular smooth muscle preparations on which melatonin-induced effects have been investigated. Although vascular and nonvascular effects of melatonin have been demonstrated in various tissues, there is no investigation on the effect and mechanism of melatonin on the isolated rat anococcygeus muscle (RAM).

The isolated RAM was originally described by Gillespie¹¹and introduced as a convenient smooth muscle preparation which should be useful both for teaching and research¹². It is easily isolated and its additional advantages have been cited as: The ab- sence of spontaneous tone providing a constant ba- se-line for measuring responses; obtaining readily and reproducibly direct and indirect sympathomi- metic effects due to the dense adrenergic innervation; providing a useful control for the selectivity on adrenergic and antiadrenergic responses, and bilate- rality of the muscle permitting both the control and test preparations to be isolated from the same ani- mal. Although the exact physiological function is unknown, the RAM might play a role in the control of the rectum and scrotum¹³.

The aim of the present study was to investigate the muscular effects and the possible mechanism of the effects of melatonin on the isolated RAM.

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MAATTEERRIIAALLSS aanndd MMEETTHHOODDSS

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Tiissssuuee PPrreeppaarraattiioonn

Local bred male albino rats (250-300g) were used in the experiments according to the proposals of the Declaration of Helsinki and the European Commu- nity Guidelines for the use of experimental animals.

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FABAD J. Pharm. Sci., 29, 127-132, 2004 D

Drruuggss aanndd SSoolluuttiioonnss

The composition of Krebs-Henseleit solution (mM) was as follows : NaCl 118.1, KCl 4.8, NaHCO₃ 25.0, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 2.5, glucose 11.1.

Phenylephrine hydrochloride, melatonin, NaF, PMA and nifedipine were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). KCl and AlCl3 were obtained from Merck (Darmstadt, Germany). Drugs were dissolved in distilled water and diluted with Krebs-Henseleit solution. Melatonin and nifedipine were dissolved in DMSO. All concentrations were given as the final concentration in bath solution.

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Daattaa aannaallyyssiiss

Relaxations were expressed as the percentages of the contractions achieved by KCl, phenylephrine or NaF. Vehicle (DMSO) produced concentration-dependent relaxations on KCl-, phenylephrine- and NaF- precontracted RAM so that melatonin and nifedipine responses were expressed as ˘ values indi- cating the vehicle value alone subtracted from the values of either melatonin or nifedipine. The results are given as the means± SEM with the number of experiments (n). Emax is the expression of the maxi- mal inhibition. IC₅₀values of the relaxant effect of melatonin were determined by probit regression analysis and expressed as pIC₅₀(-log[M]) in Table 1.

Differences between pIC₅₀values and also between E_maxvalues of melatonin and nifedipine were analy- sed by using Student’s t test. Values of p<0.05 were taken to indicate statistical significance.

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REESSUULLTTSS

After equilibration, cumulative administration of melatonin (10^-10-10^-3M) did not produce any effect on the basal tonus of isolated RAM (n=4). These data suggest that either melatonin receptors may not be present in the muscle examined or that melatonin can combine with its receptors to result in an effect only if the tissue is in preactivated state.

Melatonin (10^-5-10^-3M) produced concentration-dependent relaxations on KCl (70 mM), phenylephrine (1.7x10^-6 M) and NaF (3x10^-3 M) precontracted RAM (Fig.1) (n=6). pIC₅₀and E_maxvalues of melatonin are shown in Table 1. Melatonin produced signi- ficantly higher inhibitions in NaF-precontracted

FFiigguurree 11.. Effects of melatonin (10^-5-10^-3M) on KCl (70 mM), phenylephrine (1.7x10^-6M) or NaF (3x10^-8M) precontracted rat anococcygeus muscle (RAM) (n=6).

Data represent the means ± S.E.M.

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Taabbllee 11.. pIC₅₀and Emax Values of Inhibitory Effect of Melatonin and Nifedipine on KCl- (70 mM), Pheny- lephrine- (1.7x10^-6M) or NaF- (3x10^-8M) Precontracted Rat Anococcygeus Muscle

Data represent the means ± SEM. * p<0.05 compared to those obtained in KCl-precontracted rat anococcygeus muscle. + p<0.05 compared to those obtained in phenylephrine-precontracted rat anococcygeus muscle.

%% RReellaaxxaattiioonn

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ports related to the melatonin levels of human plasma during the circadian rhythm¹⁵.

Melatonin can activate or inhibit signal transduction cascades independent of receptors or through receptors. The ability of melatonin to act independently from its receptors is attributed to its small and high- ly lipophilic nature and/or due to an activated upta- ke mechanism. Several studies demonstrated that le- ukocytes¹⁶and hamster skin cultures¹⁷have an ability to synthesize melatonin, and these findings ra- ised the possibility that melatonin might have a pa- racrine action to reduce the increased smooth muscle tone. In many cases, melatonin-induced effects are inhibitory and require previous activation of the cell by a stimulatory agent¹⁸. For example, in KCl-precontracted vascular tissues, melatonin (10^-5-10^-3M) has been reported to produce concentration-dependent inhibitions^1,2,3,19. In these studies, relaxant effects of melatonin have been proposed to be due to the inhibition of voltage-dependent Ca⁺²channels. It has also been shown that porcine arterial smooth muscle relaxes in response to high concentrations of melatonin and other related melatonin receptor li- gands; melatonin-induced responses have not been shown to be associated with the inhibition of cyclic- GMP(guanosine monophosphate)-specific phospho- diesterase activity²⁰. The mechanism of the relaxant effect of melatonin may involve the alteration of transmembranous Ca⁺² influx, phosphoinositide turnover or cyclic GMP. In the present study, inhibitions observed with nifedipine (an L-type Ca⁺²channel blocker) confirmed the presence of voltage-ope- rated Ca⁺²channels in the isolated RAM. Inhibition of KCl-induced contractions by melatonin in a concentration-dependent manner like nifedipine, impli- cated that melatonin-induced inhibitions might be caused by the blockage of voltage-dependent Ca⁺² channels, since the pharmacologic effect of KCl on smooth muscle mainly involves influx of Ca⁺² via voltage-dependent Ca⁺²-channels.

Previous studies showed that melatonin has an antiadrenergic effect⁸. Melatonin-induced inhibition of RAM than those obtained in KCl- or phenylephrine-

precontracted RAM.

No signs of contraction were obtained in the presence of PMA (10^-10-10^-4M) in isolated RAM. Lengthe- ning the incubation period of PMA up to 45 min, did not produce any significant effect. In the tissues preactivated by low concentration of KCl (35 mM), PMA did not cause any effect.

Nifedipine (10^-12-10^-8 M) produced concentration- dependent relaxations on KCl (70 mM)-and phenylephrine (1.7x10^-6 M) precontracted RAM (Fig.2) (n=7). pIC₅₀ and E_max values of nifedipine are shown in Table 1. Nifedipine appeared to be more

efficacious and potent than melatonin causing re- ductions in KCl-induced precontraction. Nifedipine produced an inhibitor effect smaller than that of melatonin on the phenylephrine-precontracted tissue, and at higher concentrations (10^-7-10^-5 M), did not further reduce the phenylephrine-induced precontraction of the RAM.

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DIISSCCUUSSSSIIOONN

The present study demonstrates for the first time that melatonin inhibits the contractile responses induced by KCl-, phenylephrine-, or NaF on the RAM.

Although this action of melatonin occurred at the concentrations in excess of that found in the plasma, these results are in agreement with the previous re-

FFiigguurree 22.. Concentration response curves of nifedipine (10^-12-10^-8 M) on KCl (70 mM) or phenylephrine (1.7x10^-6M) precontracted rat anococcygeus muscle (RAM) (n=6-7).

Data represent the means ± S.E.M.

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FABAD J. Pharm. Sci., 29, 127-132, 2004

contractions caused by metoxamine and clonidine has been shown suggesting that these effects are occurred via inositoltriphosphate (IP₃) pathway³. In another study, Girouard and Champlain²¹showed that melatonin inhibited the contractile responses induced by phenylephrine in mesenteric beds from spontaneously hypertensive rats and Wistar-Kyoto rats. Melatonin exerted its inhibitory effect on α- adrenergic induced vasoconstriction of mesenteric ar- teries through a low-affinity membrane receptor coupled to inhibit of IP₃formation and independent of its antioxidant scavenging properties. In the present study, we showed that melatonin causes inhibitions on phenylephrine–precontracted RAM. Since melatonin-induced inhibitions on the mobilization of Ca⁺²from intracellular stores as well as the influx through the voltage-sensitive channels have been reported in several tissues¹⁸, inhibitions on phenylephrine precontracted RAM might also have occurred via an inhibitor effect on the signal transduction mechanisms activated by phenylephrine (e.g.

phospholipid hydrolysis)²².

It is well-known that breakdown of inositol phospho- lipids to inositol triphosphate mobilizes intracellular Ca⁺² and diacylglycerol. Subsequently, diacylglycerol-induced activation of protein kinase C is thought to be a secondary event in excitation-contraction co- upling after G-protein activation^23,24. On the basis of the knowledge mentioned above, we used NaF as a precontractile agent to investigate whether or not melatonin interacts with the G-protein activated contractile mechanisms. The mechanism of fluoride (F -) induced activation of G-proteins has been intensively investigated. In the presence of Al⁺³ , F- forms a complex AlF^-4, which has a similar structure to PO₄^–, and is able to interact with the guanosine 5’-diphosp- hate situated on the alpha-subunit of the G-proteins, resulting in activation by mimicking guanosine 5’–triphosphate (GTP) at its binding site²⁵. In the study of Zeng et al.²⁶, F- has also been shown to have the ability to stimulate G-proteins mediating the gating of calcium channels. Ayotunde et al.²⁷ sugges- ted that F- might have a role in increasing intracellular Ca⁺²as a result of activation of phospholipase C

and accumulation of IP₃; opening of Ca⁺² channels in plasma membrane and also protein kinase C activation. NaF also has a contractile effect in smooth muscle via Ca⁺²mobilization, which is mediated by a G-protein activation as well as by a G-protein-independent mechanism involving activation of membranal Ca⁺²channels^14,28. In the present study, we have first demonstrated that NaF-induced contraction of the anococcygeus muscle might be related to the opening of membranal Ca⁺²channels and/or the resul- tant increase in intracellular Ca⁺² . The incubation with PMA did not induce any contraction on the isolated RAM, although PMA is an activator of protein kinase C. Therefore, we assumed that the NaF-induced response was not mediated by the activation of protein kinase C, but related to the interaction between melatonin and another signal transduction mechanism responsible for the contractile effect of NaF.

In summary, we have demonstrated in the present study that, in isolated RAM, melatonin inhibited KCl-, phenylephrine- or NaF-induced contractions.

The inhibitory action of melatonin may be related to voltage-gated Ca⁺²channels and, at least in part, via an accumulation of IP₃and an increase in intracellular Ca⁺², but not by activation of protein kinase C. In order to clarify the mechanisms of the observed inhibitory effect of melatonin and the physiological significance of this effect, further studies are neces- sary using specific agents which modify the above- mentioned signal transduction pathways.

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Acckknnoowwlleeddggeemmeennttss

This study was supported by the Research Founda- tion of Gazi University (Project Code=SBE 11/99-11) Preliminary results of the present study were pre- sented at the XV. National Congress of Turkish Pharmacological Society in Antalya, Turkey (November 1-5, 1999)

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REEFFEERREENNCCEESS

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