Releaseof contractile agents from rat vas deferens by clonidine

Releaseof contractile agents from rat vas deferens by clonidine

Life Sciences, Vol. 49, pp. 1643-1649 Printed in the U.S.A. Pergamon Press RELEASE OF CONTRACTILE AGENTS FROM RAT VAS DEFERENS BY CLONIDINE Neide H...

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Life Sciences, Vol. 49, pp. 1643-1649 Printed in the U.S.A.

Pergamon Press

RELEASE OF CONTRACTILE AGENTS FROM RAT VAS DEFERENS BY CLONIDINE

Neide H. Jurkiewiez & Aron Jurkiewicz

Department of Pharmacology, Escola Paulista de Medieina, Caixa Postal 20372, S~o Paulo 04034, Brazil (Received in final form September 23, 1991)

SUMMARY Clonidine induces contractile effects on the isolated rat vas deferens, but not on rat uterus or guinea-pig ileum. However, we have observed that if donidine is incubated for about 10 rain with a nutrient solution containing an isolated rat vas deferens, the resulting solution can contract an isolated rat uterus, or guinea-pig ileum indicating the involvement of a substance released from the vas. This contractile effect was partially reduced by naloxone and by serotonin antagonists, and by using a denervated vas, indicating that opioids, serotonin and eventually other substances released from nerve tissue of the vas can be involved. Clonidine has been used as a therapeutic agent for different pathological conditions, as for instance in the treatment of hypertension (1), in acceleration of growth in children (2), in short-term analgesia (3), to diminish the symptoms of opiate withdrawal syndrome (4), and as an aid in smoking cessation (5), which suggests that this substance has multiple mechanisms of action. From the pharmacological standpoint clonidine has been classified as a sympathomimetic agent. Thus, in the rat vas deferens, clonidine induces either inhibitory effects, which have been ascribed to an interaction with presynaptic alpha2-adrenoceptors (6), or contractions, due to interactions with either alpha1 or alpha2 postsynaptic adrenoceptors (6-7). Our objective is to present some results indicating that in rat vas deferens donidine might have an additional mechanism of action, releasing endogenous agents. The outflow of these agents was detected in the nutrient solution, which was able to contract other smooth muscle preparations such as the rat uterus and guinea-pig ileum. Experiments were performed to provide an answer to two main questions: (a) if the releasing mechanism depends, or not, on an interaction of donidine with alpha-adrenoceptors, and Co) if non-adrenergic substances, as acetylcholine, serotonin, opioids, prostaglandim or purines, are linked to this contractile activity, since elonidine or other sympathomimetic drugs can be involved with receptors interacting with these endogenous substances, in a number of tissues (8-13).

0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc

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METHODS Male adult Wistar rats from our own colony, BAW-2, weighting 300-350g (5-6-month old), were killed by an overdose of ether. The vas deferens was carefully mounted in a 10-ml organ bath, in aerated nutrient solution, at 30 ° C, as previously described (7). Uteri were obtained from female rats (1-2-month old, 100-150g) pretreated with a single subcutaneous dose of stilboestrol (15 g/100g body weight, 24h before) and killed with an overdose of ether. Isolated uterus preparations were mounted in 10ml organ chambers in a nutrient solution with the same composition as that used for the vas (7) at 30° C, with lg load. Contractile activity was measured as isotonic contractions with 6 times amplification. Clonidine (3x10"3 M) was incubated during 5-10min with an isolated vas deferens, in 10ml nutrient solution. This solution was then transferred at once to the uterus chamber, to be tested for its contractile activity, in comparison with treated preparations. Only one control plus one treated incubate were tested in a given experiment, to avoid desensitization Treatment, either of the vas deferens, of the incubation medium, or of the uterus, was performed in eight groups of experiments, as an attempt to exclude several factors that might be involved in the effect of donidine: in experiment 1, a 7-day denervated vas deferens was used instead of a normal vas. In experiment 2, the alpha-adrenoceptor antagonist yohimbine (10"4M) was preincubated in the vas chamber for 15 min before adding donidine, and left in incubation medium throughout the experiment, to verify for the involvement of alpha-adrenoceptors. In experiment 3, the synthesis of prostaglandins was blocked by means of indomethaein (10-6M), that was used as described above for yohimbine. In experiment 4, the enzyme apyrase (250 #g) was added immediately after removing the incubation medium from the vas chamber and maintained for 15rain before addition of the medium to the uterus chamber; this treatment was able to abolish the contractile activity of a control solution of ATP in uterus. For experiment 5, a dose of atropine (106M) able to completely block a control effect of acetylcholine was preincubated in the uterus chamber 15 min before adding the incubate, and maintained thereafter. In experiments 6 and 7, a single dose of ketauserin (6x10-SM) or cyproheptadine (10TM) that was able to block a control effect of serotonin, was used as described for atropine. In experiment 8 a dose of naloxone (3x10"8), able to block a control effect of [met]enkephalin was used as described for atropine. The following drugs were used: donidine hydrochloride (Boehringer, Germany); yohimbine hydroehloride, atropine sulfate, acetylcholine hydrochloride, naloxone hydrochloride, and potato purified apyrase (all from Sigma, USA), ketanserin (Sandoz, Switzerland), adenosine-5'-triphosphate (ATP, Boehringer, Germany); indomethacin and cyproheptadine hydrochloride were kindly supplied by Dr. B.B. Vargaftig, I. Pasteur, France); [Met]5-enkephalin was synthesized by Prof. L. Juliano, Dept. of Biophysics, Escola Paulista de Medieina, Brazil). RESULTS

Clonidine (usually 3x 10-3 M) was added to an isolated vas deferens preparation. After 5-10min~ the vas was withdrawn, and the whole liquid content transferred to another 10-ml organ bath, in which a rat uterus or guinea-pig ileum, was mounted for recording isotonic

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(a) Contraction, rat uterus

Clonidine + vas

Clonidine W

(b) 80 N 6O o

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~ Clonidine

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vas deferens

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Nutrient solution

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FIG. 1 Contractile effect of don/dine incubates in rat isolated uterus. (a) Typical experiment, showing the sustained contraction induced by a nutrient solution (10ml) in which clon/dine (3x10"3) was incubated for 10 rain in the presence of a vas deferens (clon/dine + vas), that was removed shortly before adding the incubate to the uterus chamber. In contrast, a vas deferens-free incubate (clonidine) caused a negligible contraction which returned to the baseline after about 1rain. Before adding the second incubate the uterus chamber was washed out (W), and a 30rain interval allowed. (13) Mean contractions from 3 groups of experiments similar to (a), measured at the 10th minute after adding don/dine incubates. Open bars are controls, using complete incubates (clon/dine + vas + nutrient solution), while striped bars show contractions obtained in the same uterus when one or two components, were missing (- sign) during incubation. The three stripped bars were significantly lower than controls. Each pair of bars is a mean of at least 12 experiments. Vertical lines show one S.E.M.

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contractions. The following results refer to the uterus, essentially similar.

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though the effects in ileum were

The clonidine incubate induced a fast sustained contraction of the uterus, corresponding to about 50% of a maximal contraction induced by acetylcholine (10 -4 M). Only negligible effects were observed when incubates were obtained from experiments in which clonidine or the vas deferens were missing (fig 1), indicating that clonidine-induced contractile activity is most likely due to one or more substances released from the vas. Incubations could be repeated up to three times using the same vas deferens, at 30min intervals, without significant tachyphylaxis. A slight desensitization was noticed (10-20%), usually after the fourth uterine contraction. Smaller contractions were obtained with incubates in which lower concentrations of clonidine were used; however these contractions were inconsistent or practically absent, when compared with controls, if the concentrations of clonldine were lower than 10-4 M. To verify the involvement of alpha adrenoceptors, the vas deferens was preincubated with the competitive antagonist yohimbine. This treatment with yohimbine did not prevent the uterine contraction to occur. In additional experiments, contractile effects could not be TABLE I Decrease of Contractile Activity of Clonidine Incubates by Some Treatments.

Rat uterus contraction a (ram +_S.E.M.)

Expt Treatment 1 Denervation 2 Yohimbine 3 Indomethacin 4 Apyrase 5 Atropine 6 Ketanserin 7 Cyproheptadine 8 Naloxone

n 8 4 4 4 4 6 5 5

Untreated b 59.2 __.7.5 62.0 +_6.6 67.2 +_7.7 48.2 +_4.4 60.0 +_2.9 63.8 +_6.8 74.8 +_10.3 66.0 +_9.3

Treated c 9.2 __.4.3 47.0 +_6.0 52.0 +_12.0. 43.0 +_8.6 52.8 +_5.9 22.6 +_10.2 18.5 +_6.9 13.2 +_5.9

% decrease 85% 23% 25% 11% 12% 65% 75% 80%

pd 0.01 NS NS NS NS 0.01 0.01 0.01

aContractile (i.e. oxytocic) activity was measured as isotonic contractions of rat isolated uterus preparations, as shown in rigA. The maximum effect in this bPreparationwas 111.0 +__12.3mm (cumulative acetylcholine, 10"6M, n=4~. Untreated preparations were mounted as shown in fig.la: clonidine (3x10 "~ M), was incubated during 10min with an isolated vas deferens, in 10ml nutrient solution (7). The vas deferens was removed, and the incubate then added to the uterus chamber, during 10min, to be compared for its contractile activity, in relation to treated preparations. CTreatment consisted in repeating the procedure described above for untreated preparations, except that denervation or drug addition was performed. Denervation was made according to Kasuya et al. (14). dDegree of significance of differences between treated and untreated group.

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obtained when other adrenergic agonists as noradrenaline (n = 5), amphetamine (n = 4), or an imidazoline structurally related to donidine, oxymetazoline (n = 4), were incubated instead of donidine. Therefore, a liaison between the oxytocic effect and adrenoceptors could not be demonstrated. In relation to a direct effect of clonidine in receptors other than the adrenergic ones, or indirect, by release of endogenous substances, several possibilities were investigated (Table I): acetylcholine receptors, which were reported to interact with donidine (9) are not involved, since the effect was not inhibited by the muscarinic antagonist atropine. Purines, mainly ATP, that are known to be released from nerve terminals of the vas (10), were also not involved since the oxytocic activity was not destroyed by adding the enzyme apyrase to the incubation medium. The release of prostaglandins was also checked because it has been reported that adrenergic stimulation increases prostaglandin synthesis and release in rat vas (11); indomethacin, an inhibitor of prostagiandin synthesis, did not block the contractile effect, indicating that prostaglandins are not involved. Although clonidine might interact with histamine receptors (12), antihistamines were not tested because histamine is known to cause uterus relaxation. Yet, a dear reduction of the oxytoeic effect was observed in three different experiments: (a) when denervated vasa were used, showing that the oxytoeic activity depends on the integrity of the autonomic nerve terminals, which densely innervate the organ; (b) by the use of the opioid antagonist naloxone, and (c) by the use of serotonin antagonists, indicating that opioids and serotonin may be involved in the contractile effects. DISCUSSION Our results indicate that donidine induces a neural outflow of endogenous agents from the vas deferens. The possibility that more than one substance is released is given by at least two experimental evidences: (a) the effect was reduced by two different types of drugs such as serotonin and opioid antagonists, and (b) even high doses of these antagonists were not able to completely block the contractile effects, indicating that the respective residual effects are due to a second agent. This hypothesis is corroborated by the fact that the substances here advanced as candidates for such a release are known to be present in the vas, and have been described to be released by donidine in other systems. However, an exact account of the number of substances in the incubate, and their interplay to induce the oxytocic effect, is out of the scope of the present publication and cannot be given before additional experiments are performed. A multiple release of endogenous substances, including opioids and serotonin, has been previously reported for clonidine, in some/n vivo experiments: clonidine-induced analgesia in rats is inhibited by naloxone and by depletors of serotonin nervous pathways (13). Clonidine diuretic activity in rats is also partially inhibited by naloxone, and is probably mediated by a peptide, the Atrial Natriuretic Factor (15). In addition, it has been reported that clonidine releases beta-endorphin in rat brain (16), neuropeptide Y in guinea-pig cardiac nerves (17), dynorphin in rat spinal cord (18), and growth hormone in various species, including man (2). Furthermore, a low molecular weight agent, the recently described endogenous Clonidine Displacing Substance, competes for the binding of clonidine to its receptors, and has a contractile activity in smooth muscle (19). In relation to the vas deferens, although an indirect

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effect of clonldine has not been reported to our knowledge, it is known that serotonin is also present in this organ (20) and that neuropeptides as [met]enkephalin (21), neuropeptide Y (10), and Caieitonin Gene Related Peptide (22) are present in vas nerve terminals. Since our main purpose was to detect the presence of released substances in the nutrient solution, a high dose of elonidine was used. However,. the fact that an outflow could not be detected when using doses of clonidine lower than 10-4 M does not necessarily mean that endogenous substances are not released by these doses of clonidine, for two main reasons: (a) the outflow into the nutrient solution depends not only on the release but also on the diffusion through the organ wall, and therefore it is possible that pan of the outflow is precluded by the diffusion process; (b) the released substances are immediately diluted into the nutrient solution, as soon as crossing the external surface of the vas; since the average volume of a vas ( less than 100 mm3) is at least 100 times smaller than that of the nutrient solution (10,000 nam3), it can be concluded that the released substances are at least 100 times more diluted in nutrient solution than when freed inside the wall of the vas, assuming that the distribution in the organ is homogeneous. It is clear that additional experiments are necessary to obtain a quantitative picture about this issue. In conclusion, our results show at least two outstanding aspects that might contribute to a better understanding of the pharmacological spectrum of activity of clonidine: (a) practically all previous demonstrations of the capacity of clonldine to release endogenous substances were performed in vivo while the results in the vas were obtained in vitro, with fewer variables involved; (b) our method allows the pool of released substances to be easily collected in a small volume of nutrient solution, for further chemical isolation and identification. Consequently, the clonidine incubate can be a useful tool for the identification of putative neurotransmitters, co-transmitters, or modulators in vas. It is conceivable that some of these substances might be released in other tissues, associated with the therapeutic properties of clonidine on the different clinical situations listed above (1-5). ACKNOWLEDGMENTS We thank Haydee Renter for technical assistance, and the representatives of Boehringer and Sandoz (Brazil) for the gifts of donidine and ketanserin, respectively. This work was supported in part by grants from FAPESP, Finep, and CNPq (Brazil).

8$2LFd~2LC,F~ 1. 2. 3. 4. 5. 6. 7. 8. 9.

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