Xylazine-induced delay of small intestinal transit in mice

European Journal of Pharmacology, 83 (1982) 55-60

55

Elsevier BiomedicalPress

XYLAZINE-INDUCED DELAY OF SMALL INTESTINAL TRANSIT IN MICE WALTER H. HSU Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011, U.S.A.

Received 10 February 1982, revised MS received25 May 1982, accepted 16 June 1982

W.H. HSU, Xylazine-induced delay of small intestinal transit in mice, European J. Pharmacol. 83 (! 982) 55-60. Subcutaneous injection of xylazine (0.1-3.0 mg/kg) produced a dose-dependent delay of small intestinal transit without affecting gastric emptying in the conscious mice. The xylazine-induced delay of small intestinal transit was antagonized by a2-adrenoceptor antagonists, e.g., yohimbine, piperoxan and tolazoline. The antagonism of xylazine activity by yohimbine was dose-dependent, and the maximal antagonistic effect was seen at 1 mg/kg. Other adrenoceptor antagonists with only al-blocking activity, e.g., thymoxamine, prazosin and phenoxybenzamine at the doses studied did not reduce the depressant effect of xylazine on small intestinal transit. A fl-adrenergic antagonist, propranolol was not effective in reducing xylazine activity. The opioid antagonist, naloxone did not reduce the effective of xylazine, nor did yohimbine antagonize the morphine-induced delay of small intestinal transit. The xylazine-induced delay of small intestinal transit was not altered by atropine, hexamethonium, haloperidol, methysergide, chlorpheniramine or cimetidine. Pretreatment of mice with reserpine and a-methyl-p-tyrosine or 6-hydroxydopamine failed to reduce the intestinal effect of xylazine. These results suggest that xylazine-induced delay of small intestinal transit is mediated by postjunctional a2-adrenoceptors and appears not to involve activation of opioid, cholinergic, dopaminergic, histaminergic, or serotonergic receptors. Xylazine

Smallintestinal transit

a2-Adrenoceptors

1. Introduction Xylazine (Rompun, Bayer 1470, 2-(2,6-dimethylphenylamino)-4-H-5,6-dihydro-l,3-thiazine) is a well-known sedative in biological research and in veterinary medicine. When injected alone or concurrently with other anesthetics, xylazine produced depression, analgesia and muscle relaxation via its action on the CNS (Burns and McMullan, 1972; Clark and Hall, 1969; Moye et al., 1973). A1though xylazine has been used clinically for over 10 years, its mechanism of action is still not wellunderstood. It has also been suggested that xylazine is an a2-adrenergic agonist (Berthelsen and Pettinger, 1977). For example, xylazine-induced acute hypertension is antagonized by phentolamine (Schmitt et al., 1970), xylazine-induced analgesia is antagonized by yohimbine and piperoxan (Schmitt et al., 1974). Xylaxine induced CNS depression is antagonized by yohimbine, 0014-2999/82/0000-0000/$02.75 © 1982 ElsevierBiomedicalPress

piperoxan, phentolamine and tolazoline (Hsu, 1981). All of these antagonists are effective in blocking a2-adrenoceptors (Drew, 1978; Fain and Garcia-S/finz, 1980; Hoffman and Lefkowitz, 1980). Current evidence strongly suggests that activation of a2-adrenoceptors located at the Auerbach's plexus inhibits acetylcholine release (Vizi, 1969 and 1979; Vizi and Knoll, 1971), thereby decreasing the small intestinal contractions (Drew, 1978; Andr6jak e t al., 1980). For example, it was reported that clonidine delayed the small intestinal transit which was blocked by a2-adrenoceptor antagonists (Ruwart et al., 1980). The present study was undertaken to investigate xylazine-induced delay of small intestinal transit, and to determine whether this is mediated by activation of a2-adrenoceptors , and whether this effect involves other neural pathways.

56 2. Materials and methods 2.1. Animals Male white mice (Biolab, Minneapolis, MN, U.S.A.) weighing 3 0 - 4 0 g were used in the present study. All mice in each experiment were from a single shipment. Food was withheld 20-24 h before each experiment, but the animals were allowed access to water. Water was withheld during the experiment. 2.2. Injection and experimental procedures Unless indicated, injections were made subcutaneously with a vol of 0.1 m l / 1 0 g of body weight, For determination of small intestinal transit (SIT) and gastric emptying (GE), 0.1 ml of black ink (Pelikan, G M B H D300, Hanover, Germany) with or without NaS~CrO4 (10000 cpm, New England Nuclear, Boston, MA, U.S.A.), was administered intragastrically 20 min after s.c. injection of xylazine or morphine. When neostigmine was studied, it was injected immediately before the ink gavage. Mice were anaesthetized lightly with ethyl ether for gavage. Thirty min after gavage, the animals were killed by cervical dislocation and the gastrointestinal tract was excised and the farthest distance traveled by the ink was divided by the total length of the small intestine and multiplied by 100 to yield SIT. The stomach and intestine were counted separated in a gamma counter (Biogamma, Beckman Instruments, Fullerton, CA, U.S.A.) for 51Cr activity and G E was expressed as the percentage of total 51Cr in the gut found in the small intestine. In the experiment where animals were pretreated with reserpine and a-methyl-ptyrosine, SIT was determined 15 min after the ink gavage. The present method is a modification of Ruwart, Klepper and Rush used in rats (Ruwart et al., 1980). 2.3. Reserpine and a-methyl-p-tyrosine pretreatment as well as chemical sympathectomy Mice were injected with reserpine (5 m g / k g i.p. 24 h before xylazine injection) and a-methyl-ptyrosine (200 m g / k g × 2 i.p. at 24 and 12 h before

xylazine injection). It was reported that with this method, the brain monoamines were depleted (Azzaro et al., 1971). In chemical sympathectomy, mice were injected with 6-hydroxydopamine HC1 (250 m g / k g i.v. 24 h before xylazine injection). It was reported that with this method, the sympathetic nerve endings were totally destroyed (Furness et al., 1970). 2.4. Test preparations Drugs studies and their sources were as follows: xylazine HCI (Bayvet, Shawnee, KS); morphine sulfate (Eli Lilly, Indianapolis, IN, U.S.A.); piperoxan HC1 (May and Baker, Dagenham, Essex, England); tolazoline HC1 (Ciba-Geigy, Summit, N J, U.S.A.); thymoxamine HC1 (WarnerLambert, Ann Arbor, MI, U.S.A.); prazosin HCl (Pfizer, New York, NY, U.S.A.); chlorpheniramine maleate (Elkins-Sinn, Cherry Hill, NJ, U.S.A.); hexamethonium chloride (K and K, Plainview, NY, U.S.A.); phenoxybenzamine HC1 and cimetidine HC1 (Smith, Kline, and French, Philadelphia, PA, U.S.A.); haloperidol (McNeil, Fort Washington, PA, U.S.A.); methysergide maleate (Sandoz, East Hanover, N J, USA); naloxone HCI (Endo, Garden City, NY, U.S.A.); yohimbine HC1, reserpine, a-methyl-p-tyrosine methylester, 6-hydroxydopamine HC1, dl-propranolol HC1, neostigmine sulfate and atropine methylnitrate (Sigma, St. Louis, MO, U.S.A.). All drugs were dissolved in distilled water, except that haloperidol and phenoxybenzamine HC1 were dissolved in a small amount of 85% lactic acid then diluted with distilled water. Reserpine was dissolved in a small anount of glacial acetic acid, then was diluted with distilled water, and 6-hydroxydopamine HCI was dissolved in 0.2% ascorbic acid ( w / v in 0.15 M NaC1). Unless indicated, all doses were calculated on the basis of weight of drug base. 2.5. Statistical analyses The data were subjected to analysis of variance. Standard error was calculated from the residual error term of the appropriate analysis of variance. Differences between mean values of the control

57 a n d pretreated groups were evaluated using the

TABLE1

two-tailed S t u d e n t ' s t-test.

Influence of adrenergic blocking agents on xylazine-induced delay of small intestinal transit (SIT). Potential antagonists were given 10 min before xylazine (1 mg/kg). Mean SIT (see Methods) and S.E.M. are indicated for each group of 7-10 animals. *** P<0.001 when compared to the control group that received only xylazine.

3. Results

3.1. Effects of xylazine on small intestinal transit (SIT) and gastric emptying (GE)

Pretreatment

I n fasted mice, s.c. injection of xylazine (0.1-3.0 m g / k g ) caused a delay of SIT in a d o s e - d e p e n d e n t m a n n e r (fig. 1). However, xylazine at all doses studied did n o t significantly affect GE.

3.2. Effects of adrenergic blocking agents on xylazine-induced delay of SIT Mice treated with xylazine (1 m g / k g ) alone showed a depression of SIT (table 1). Piperoxan a n d tolazoline (1 m g / k g each) were able to reduce the effect of xylazine on SIT. Y o h i m b i n e antagonized the effect of xylazine o n SIT in a dose-dep e n d e n t m a n n e r . A t b o t h 1 a n d 3 m g / k g , yohimb i n e completely abolished the effect of xylazine (1 m g / k g ) . I n contrast, 3 other a-blockers, thymoxa m i n e (3 m g / k g ) , prazosin a n d p h e n o x y b e n z a m i n e (1 m g / k g each) did n o t antagonize the effect

60 t u,J o

~

~

t

~

t

-

~

.

~

Primary type of

Dose (mg/kg)

SIT

0.03 O.l 0.3

antagonism None (control) Vohimbine

ot2

Ot I -}- ~t 2

1

a 1-'}-~t2 aI

1 3

27+2.6 27-+2.2 2 8 - 2.1 50-+4.3 *** 69-+4.6 *** 70-+4.1 *** 55-+5.9 *** 50 -+8.0 *** 29*-2.5

aI aI fl

1 1 3

30*- 1.2 27 *- 1.7 31.-2.3

1

3 Piperoxan Tolazoline Thymoxamine Phenoxybenzamine Prazosin Propranolol

of xylazine. A fl-blocker p r o p r a n o l o l (3 m g / k g ) did n o t reduce the xylazine-induced delay of SIT. T h e doses of antagonists used in the study were usually the m a x i m a l doses that did n o t signific a n t l y change SIT when given alone.

40 1 20 0 80 "I

~ " ' ~ i

TABLE 2 ' ~ . ~ , , , ~ . ~ . i

60

--

Influence of dopaminergic, histaminergic, serotonergic or ganglionic blocking agent on xylazine-induced delay of small intestinal transit (SIT). Potential antagonists were given 10 min before xylazine (0.3 mg/kg). Mean SIT (see Methods) and S.E.M. are indicated for each group of 7-10 animals.

40 20-

0

,

0.03

0.1

Pretreatment

Dose (mg/kg)

SIT

None (control) Haloperidol Chlorpheniramine Cimetidine Methysergide Hexamethonium

3 3 1 5 5

43 -+2.4 42 *- 3.0 44*-2.9 50*-6.2 36.- 3.6 35--+3.1

,

0.3

1

3

Xylazine,mglkg Fig. 1. Effect of different doses of xylazine (s.c.) on GE and SIT in mice. Mean GE and SIT (see Methods) with S.E.M. are shown for each group of 7-10 animals. * P<0.05 and *** P< 0.001 when compared to the control group.

58 80 604O

Xyl

Morph

Nalox + Morph

Nalox + Xyl

Yohim + Morph

Fig. 2. Effect of naloxone on xylazine- and morphine-induced delay of SIT. Naloxone or yohimbine (3 mg/kg s.c.) were given 10 min before xylazine or morphine (1 mg/kg s.c.). The ink gavage of 0.1 ml was performed 20 min after ×ylazine or morphine injection. Mean SIT (see Methods) and S.E.M. are indicated for each group of 7-10 animals. *** P<0.001 when compared to the group receiving morphine alone. Xyl=Xylazine, Morph--Morphine, Nalox=Naloxone, Yohim-Yohimbine. Ordinate: SIT.

3.3. Failure of naloxone in blocking xylazine-induced delay of S I T

p r e s e n t study, n a l o x o n e a n d y o h i m b i n e a l o n e at 3 m g / k g d i d n o t affect SIT.

M o r p h i n e a n d x y l a z i n e (1 m g / k g e a c h ) d e l a y e d S I T (fig. 2). N a l o x o n e (3 m g / k g ) a n t a g o n i z e d the effect of morphine but did not affect xylazinei n d u c e d d e l a y of SIT. Y o h i m b i n e at the d o s e (3 m g / k g ) t h a t w a s p r e v i o u s l y s h o w n to c o m p l e t e l y a b o l i s h t h e e f f e c t of x y l a z i n e , d i d n o t red u c e the m o r p h i n e - i n d u c e d d e l a y of S I T . I n the

3.4. Interactions of xylazine with cholinergic, dopaminergic, serotonergic and histaminergic antagonists in mice

100 -

***

806O-

A s s h o w n in fig. 3, x y l a z i n e (0.3 m g / k g ) c a u s e d a d e l a y o f SIT. A t r o p i n e (1 m g / k g ) a n t a g o n i z e d t h e s t i m u l a t o r y effect o f n e o s t i g m i n e (0.1 m g / k g )

,

I

-~

[1

I

40-

,o.

i Con

Atrop

Neost

Atrop + Neost

Xyl

Atrop + Xyl

Fig. 3. Influence of atropine on the intestinal transit effect of xylazine or neostigmine. Atropine (l mg/kg s.c.) was given 10 min before xylazine (0.3 mg/kg s.c.) or 30 min before neostigmine (0.1 mg/kg s.c.). The ink garage of 0.1 ml was performed 20 min after xylazine or immediately after neostigmine injection. Mean SIT (see Methods) and S.E.M. are indicated for each group of 7 10 animals. *** P < 0.001 when compared to the control group. Con = Control, Atrop = Atropine, Neost = Neostigmine, Xyl = Xylazine.

59 TABLE3 Xylazine activity in mice pretreated with 6-hydroxydopamine

(6-OHDA). 6-HydroxydopamineHCI (250 mg/kg i.v.) was given 24 h before xylazine(1 mg/kg). Mean SIT (see Methods) and S.E.M. are indicated for each group of 6 animals, Treatment

SIT

P

(A) None (B) 6-OHDA (c) 6-OHDA+xylazine

63-+3.5 40-+5.2 18± 1.8

B vs. A, < 0 . 0 1 C vs. B, <0.01

on SIT, but failed to change the xylazine-induced delay of SIT. Results in table2 show that hexamethonium, haloperidol, methysergide, chlorpheniramine and cimetidine at the doses studied did not affect SIT, nor did they alter the response to xylazine. Hexamethonium alone at doses greater than 5 m g / k g delayed SIT.

3.5. Xylazine activity in mice pretreated with 6-hydroxydopamine or reserpine and a-methyl-p-tyrosine In mice pretreated with 6-hydroxydopamine (250 m g / k g i.v.), there was no significant change of behaviour. Results in table 3 show that 6-hydroxydopamine alone significantly delayed SIT. In the animals pretreated with 6-hydroxydopamine, xylazine still caused a 45% delay of SIT when compared to the animals which received only 6-hydroxydopamine, In mice pretreated with reserpine (5 m g / k g i.p.) and a-methyl-p-tyrosine (200 m g / k g × 2 i.p.), there was a marked sedation and diarrhea. In these animals, SIT was determined at 15 min after the ink gavage. Xylazine (1 mg/kg) reduced SIT from 57 ± 5.2 to 26 ± 1.6 (n = 12, P<0.001).

4. Discussion The present findings show that xylazine caused a delay of small intestinal transit (SIT) without affecting gastric emptying. This effect was dosedependent. The data suggest that the effect of xylazine on SIT is mediated by az-adrenoceptors, because (1) adrenoceptor antagonists with a 2blocking activity, e.g., yohimbine, piperoxan and

tolazoline (Drew, 1978; Fain and Garcia-S~iinz, 1980; Hoffman and Lefkowitz, 1980) significantly reduced or abolished xylazine-induced delay of SIT and (2) adrenoceptor antagonists without c~2blocking activity, e.g., prazosin, phenoxybenzamine and thymoxamine (Drew, 1978; Hoffman and Lefkowitz, 1980) did not antagonize this effect of xylazine. The results further support the hypothesis that activation of a2-adrenoceptors induces delay of SIT. For example, an a2-adrenoce ptor agonist, clonidine was reported to delay SIT in rats, which was antagonized by a2-adrenoceptor blockers, but was not by a~-adrenoceptor blockers (Ruwart et al., 1980). In the present study, the fl-adrenoceptor blocker, propranolol did not reduce the effect of xylazine on SIT suggesting that this effect of xylazine is not mediated by fl-adrenergic receptors. The present findings suggest the xylazine-induced delay of SIT does not involve dopaminergic, histaminergic or serotonergic receptors, because haloperidol, chlorpheniramine, cimetidine, methysergide at the doses studied failed to reduce the effect of xylazine. Recent reports indicate that a2-adrenergic agonists may affect the opioid system. For exampie, xylazine and an analog of clonidine have been shown to possess opioid agonistic activity (Atlas and Sabol, 1980; Laurence and Livingston, 1980). In addition, clonidine was reported to release flendorphin (Pettibone and Mueller, 1981) and to inhibit diarrhea in morphine-dependent animals (Nakaki et al., 1981). Based on these observations, it is possible that xylazine may delay SIT by activating the opioid system. However, in the present study, the effect of xylazine was not antagonized by naloxone, nor was the effect of morphine antagonized by yohimbine indicating that the a2-adrenergic and opioid systems are two parallel systems with regard to the effect on SIT. It is well established that the ct2-agonists act presynaptically on the Auerbach's plexus to inhibit acetylcholine release (Vizi, 1969 and 1979; Vizi and Knoll, 1971), thus xylazine may act in the same manner. However, it was also reported that in rats, the classical a2-agonist, clonidine acted directly (not through the cholinergic p a t h w a y ) t o inhibit the smooth muscle contractions of the small

60 i n t e s t i n e ( R u w a r t et al., 1980). I t is n o t k n o w n t h a t

in mice, whether xylazine directly acts on the a2-receptors

in the smooth

muscle or indirectly

inhibit acetylcholine release. If xylazine activates a2-adrenoceptors to delay SIT, this aca c t s to

t i o n is m o s t l i k e l y p o s t j u n c t i o n a l t o t h e a d r e n e r g i c terminal, since in animals depleted of noradrenaline, xylazine caused a delay of SIT similar to that occurred in animals with normal tissue noradrena-

line. The xylazine-induced delay of the small intestinal transit may be clinically important. It was found in this laboratory that xylazine caused a

delay of the gastrointestinal transit in dogs which was more remarkable than that produced by atro-

pine (unpublished data), In summary,

the present

study suggests that transit is d u e t o a c t i v a t i o n o f p o s t j u n c t i o n a l a z - a d r e n -

xylazine-induced delay of the small intestinal oceptors

i n t h e gut. T h i s e f f e c t a p p e a r s

not

to

involve fl-adrenergic, dopaminergic, histaminergic, serotonergic or opioid pathways and does not appear

to b e d u e

to i n t e r a c t i o n s

with cholinergic

receptors.

Acknowledgements I am grateful to Mr. Paul Ryan for technical assistance, Mr. Arthur Anderson for statistical analysis, and Drs. Franklin A. Ahrens and Donald C. Dyer for a helpful discussion. The following companies are also acknowledged for their donation of drugs used in the study: Bayvet (xylazine); May and Baker (piperoxan); Warner-Lambert (thymoxamine); Pfizer (prazosin); Smith, Kline and French (Phenoxybenzamine); McNeil (haloperidol).

References Adr6jak, M., Y. Pommier and H. Schmitt, 1980, Effects of some a-adrenoceptor agonists and antagonists on the guinea pig ileum. Naunyn-Schmiedeb. Arch. Pharmacol. 314, 83. Atlas, D. and S.L. Sabol, 1980, A novel analogue of clonidine with opiate receptor agonist activity, Biochem. Biophys. Res. Commun. 94, 924. Azzaro, A.J., G.R. Wenger, C.R. Craig and R.E. Stitzel, 1971, Reserpine-induced alterations in brain amines and their relationship to changes in the incidence of minimal electro-

shock seizures in mice, J. Pharmacol. Exp. Ther. 180, 558. Berthelsen, S. and W.A. Pettinger, 1977, A functional basis for classification of a-adrenergic receptors. Life Sci. 21,596. Burns, S. and W.C. McMullan, 1972, Clinical application of Bay Va 1470 in the horse, VM SAC 67, 79. Clark, K.W. and L.W. Hall, 1969, Xylazine a new sedative for horses and cattle, Vet. Rec. 85, 512. Drew, G.M., 1978, Pharmacological characterization of the presynaptic a-adrenoceptors regulating cholinergic activity in the guinea pig ileum, J. Pharmacol. (Paris) 64, 293. Fain, J.N. and J.A. Garcia-SSinz, 1980, Role of phosphatidylinositol turnover in alpha I and of adenylate cyclase inhibition in alpha 2 effects of catecholamines, Life Sci. 26, 1183. Furness, J.B., G.R. Campbell, S.M. Gillard, T. Malmfors, J.L.S. Cobb and G. Burnstock, 1970, Cellular studies of sympathetic denervation produced by 6-hydroxydopamine in the vas deferens, J. Pharmacol. Exp. Ther. 174, 111. Hoffman, B.B. and R.J. Lefkowitz, 1980, Alpha-adrenergic receptor subtypes, N. Engl. J. Med. 302, 1390. Hsu, W.H., 1981, Xylazine-induced depression and its antagonism by alpha-adrenergic blocking agents, J. Pharmacol. Exp. Ther. 218, 188. Laurence, D. and A. Livingston, 1980, Interaction of xylazine with opiate receptors of the mouse vas deferens, J. Physiol. (London) 317, 90p(Abstr.). Moye, R.J., A. Pailet and M.W. Smith, 1973, Clinical use of xylazine in dogs and cats, VM SAC 68, 236. Nakaki, T., P.C.J. Chang, Y. Tokunaga and R. Kato, 1981, Ctz-adrenoceptors modulating diarrhea in morphine-dependent rats, J. Pharm. Pharmacol. 33, 397. Pettibone, D.J. and G.P. Mueller, 1981, Clonidine releases immunoreactive/3-endorphine from rat pars distalis, Brain Res. 221,409. Ruwart, M.J., M.S. Klepper and B.D. Rush, 1980, Clonidine delays small intestinal transit in the rat, J. Pharmacol. Exp. Ther. 212, 487. Schmitt, H., G. Fournadjev and H. Schmitt, 1970, Central and peripheral effects of 2-(2,6-dimethylphenyl-amino-4-H-5,6dihydro-3-thiazin) (Bayer 1470) on the sympathetic system, European J. Pharmacol. 10, 230. Schmitt, H., J.C. Le Douarec and N. Petillot, 1974, Antagonism of the antinociceptive action of xylazine, an a-sympathomimetic agent, by adrenoceptor and cholinoceptor blocking agents, Neuropharmacology 13, 295. Vizi, E.S., 1969, The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip, Br. J. Pharmacol. 35, 10. Vizi, E.S., 1979, Presynaptic modulation of neurochemical transmission, Prog. Neurobiol. 12, 181. Vizi, E.S. and J. Knoll, 1971, The effect of sympathetic nerve stimulation and guanethidine on parasympathetic neuroeffector transmission; the inhibition of acetylcholine release, J. Pharm. Pharmacol. 23, 918.