Gen. Pharmac. Vol. 22, No. 1, pp. 93-97, 1991 0306-3623/91 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1991 Pergamon Press plc

STUDY OF THE CONTRACTION INDUCED BY NOREPINEPHRINE A N D CLONIDINE IN THE ISOLATED

GUINEA-PIG ILEUM

C. C. A. REIS

Laboratory of Pharmacology, School of Pharmaceutical Sciences, UNESP, Araraquara, SP, Brazil

(Received 18 April 1990)

Abstract--1. Norepinephrine (NE) and clonidine produce a phasic, dose-dependent contraction of the isolated guinea-pig terminal ileum.

2. The effect of NE was blocked by prazosin which produced a parallel rightward shift of the concentration-effect curve to NE, with a significant depression of maximum effects.

3. Yohimbine and indomethacin noncompetitively blocked, whereas practolol potentiated, the contractile effect of NE.

4. The contractile effect of clonidine was not antagonized by indomethacin or atropine. 5. These results suggest that the isolated guinea-pig terminal ileum has excitatory receptors sensitive

to clonidine stimulation and excitatory alpha receptors sensitive to blockade by prazosin, and that the activation of the latter may be related to the activation of endogenous prostaglandin synthesis.

INTRODUCTION

Despite the classical concept that alpha and beta adrenergic receptors have inhibitory effects on gastro- intestinal smooth muscle, there are reports about the occurrence of excitatory smooth muscle responses mediated by adrenergic receptors (Newman and Thienes, 1933; Munro, 1951, 1952, 1953; Reynolds et al., 1967; Bricola et al., 1982).

When administered at concentrations higher than 10-tmoi/1, epinephrine can produce contraction of the entire ileum, an effect that is more evident in the terminal portion, whereas the inhibitory effect pre- dominates when the drug is administered at low concentrations (Munro, 1953). In addition to epi- nephrine, norepinephrine and neosynephrine produce motor responses in the terminal segment of the guinea-pig ileum (Munro, 1952).

Bauer (1982) noted that norepinephrine and epi- nephrine can contract the terminal port ion of the guinea-pig ileum, whereas phenylephrine has an excitatory effect throughout the ileum. The stimu- lating effect was abolished by phentolamine, but yohimbine had no antagonistic effect on the alpha excitatory receptors.

Norepinephrine can also stimulate the conversion of arachidonic acid to prostaglandins in rabbit muscle and iris microsomes. The fact that phentol- amine prevents this effect suggests an involvement of alpha adrenoceptors in this event (Abdel-Latif et al., 1983).

In view of the above considerations, the objective of the present investigation was to determine which adrenergic receptors are implicated in the excitatory effect of norepinephrine on the isolated guinea-pig terminal ileum and whether prostaglandins are also involved in this event.

MATERIALS AND METHODS

Male albino guinea-pigs (Cavia porcellus) weighing 300-550g were killed by cerebral concussion and exsan- guinated by the cervical veins. After laparotomy, the distal portion of the ileum was excised and transferred to aerated nutrient Tyrode solution maintained at 37°C. The proximal end of the ileum was fixed to a lever to record the contrac- tions on a smoked drum coupled to an electric kymograph.

The preparations were allowed to equilibrate under a resting load of 1 g for I hr, with washing at 15 rain intervals. After this time, two cumulative concentration-effect curves (CEC) to norepinephrine and clonidine were obtained starting from 10-tmol/l to maximum response doses. The agonists were administered at 3 rain intervals and the period of stabilization between the first and second CEC was 1 hr.

After the period of stabilization following the first curve, a blocker was added to the preparation and left in contact with the ileum for 20 min, except for indomethacin, which was left in contact for 30 min. The second CEC was then obtained in the presence of the blocker.

For the groups in which NE was used as agonist, the following blockers were used: yohimbine (10-Smol/l and 5 x 10-tmol/1), prazosin (5 x 10-Stool/I), practolol (10-4tool/l) and indomethacin (3 x 10-6tool/l). For the groups in which clonidine was used as agonist, the following blockers were used: atropine (10 -s tool/l) and indomethacin (3 x 10 -6 tool/l).

The variations obtained for the different groups were studied in terms of CEs0 (the molar concentration of the agonist causing an effect equal to 50% of the maximum effect in individual experiments) and of maximum effects.

The CE~ and maximum individual effects of each agonist were analysed using Student's t-test for paired data when the results belonged to the same group, or the t-test for independent samples to analyse intergroups (Snedecor and Cochran, 1980). The level of significance was set at 0.05.

The affinity of each agunist for the receptor site was estimated by calculating the pD 2 (Arunlakshana and Schild, 1959).

93

94 C.C.A. REIS

Drugs and solutions L-Norepinephrine bitartrate and yohimbine hydrochloride

(Sigma Chemical Company), clonidine hydrochloride (Catapressan R, Boehringer Ingelheim and Cia Ltd), prac- tolol hydrochloride (ICI Farma Ltd), indomethacin (Merck Sharp and Dohme), prazosin (Pfizer Quimica), atropine sulfate (E. Merck A. G. Darmstadt).

Each drug solution was prepared with Tyrode solution just prior to use. The expressed concentrations refer to the final concentration in the organ bath.

RESULTS

Group I--Control

The addition of increasing doses of N E to the nutrient medium produced a dose-dependent con- traction of the isolated guinea-pig terminal ileum.

The CEs0 and maximum values obtained from the second CEC to N E did not differ significantly from those obtained from the first CEC (P > 0.05) (Table 1). These results indicate that there was no wear of the terminal ileum preparat ion after the first CEC to NE, permitting us to consider the first CEC to N E as the control of the second CEC.

Group H--Effect of yohimbine on norepinephrine- induced contraction

The addition of yohimbine (5 x 10 -6 mol/1) to the preparat ion did not produce a significant decrease of the maximum effect of N E and caused no significant changes in the CEso of the agonist (Table 1).

However, the maximum effects induced by norepi- nephrine in the presence of yohimbine (10 -5 mol/l) were significantly lower (P < 0.001) than those ob- tained for the control curve. No significant difference was obtained in the CEs0 values. (Table 1, Fig. 1).

Group Ill--Effect of prazosin on norepinephrine- induced contraction

Figure 3 shows that the CEC to N E carried out in the presence of prazosin (5 x 10 -8 mol/l) is shifted to the right in relation to the control CEC. The hori- zontal shift o f the curve was significant (P < 0.005) at the CEs0 level. The maximum effects obtained in the presence of the antagonist were significantly lower (P < 0.005) than those obtained for the control curve (Table i, Fig. 1).

Group IV--Effect of practolol on norepinephrine- induced contraction

The addition of practolol to the nutrient medium caused a leftward horizontal shift of the CEC to N E which was significant at the CEs0 level (P < 0.05). No significant difference was observed in the maximum effects induced by norepinephrine in the presence and in the absence of practolol (Table 1, Fig. 1).

Group V--Effect of indomethacin on norepinephrine- induced contraction

Indomethacin (3 x 10 -6 mol/1) caused a change in the CEC to NE, i.e. the CEs0 values obtained in the presence of this drug were significantly lower than the CE50 values obtained for the control curve (P < 0.05). Comparison of the maximum effects showed that indomethacin produced a significant decrease in this parameter (P < 0.001) in relation to control values (Table 1, Fig. 1).

Group VI--Effect of clonidine on the isolated guinea- pig ileum

The addit ion of increasing clonidine doses to the nutrient medium produced a dose-dependent con- traction of the isolated terminal ileum, the CEs0 and maximum effects obtained from the second CEC to clonidine did not differ significantly from those obtained from the first CEC (Table 2, Fig. 2).

Group VII--Effect of indomethacin on clonidine- induced contraction

Table 2 shows that the addition of indomethacin (3 x 10-6 mol/l) to the organ bath did not produce a significant change in the in the CEC to clonidine in terms of CE50 and maximum effects, indicating that prostaglandins are not related to smooth muscle contraction in the isolated ileum caused by stimu- lation of clonidine-sensitive receptors (Table 2,

Fig. 2).

Group VIII--Effect of atropine on clonidine-induced contraction

The second CEC to clonidine carried out in the presence of atropine (10 -8 mol/1) did not show a significant horizontal shift in relation to the control CEC. The maximum effects of clonidine did not differ

Table 1. Mean geometrical CEs0 values (molar concentration of the agonist that causes an effect equal to 50% of the maximum effect in individual experiments) and confidence limits, and mean ( _+ SEM) maximum effects produced

by norepinephrine (NE) in the isolated guinea-pig ileum

CEs0 x 10 -5 mol/I (confidence limits) Maximum effects _+ SEM

Group Ia.CEC 2a.CEC Ia.CEC 2a.CEC N Control 6.43 (1.01-40.0) 5.36 (0.49-58.2) Ns 5.46 ___ 1.15 5.20 +_ 1.28 Ns 5 Yohimbine 29.8 (21.5~1.1) 20.0 (10.2-40.1) Ns 3.07 + 0.33 2.86 _ 0.37 Ns 5

(5× 10 6mol/l) Yohimbine 46.7 (39.9-54.6) 37.0 (24.7-61.3) Ns 2.64 + 0.29 0.68 +_ 0.13"* 6

(10 5 mol/l) Prazosin 46. I (44.1-48.2) 60.4 (50.3-68.8)* 3.99 + 0.36 2.89 + 0.28* 6

(5×10 Smol/i) Practolol 2.27 (0.78-5.69) 0.57 (0.26-1.22)* 5.22 _+ 0.86 5.15 + 0.87 Ns 6

(10-4 mol/I) lndomethacin 2.43 (1.51-3.91) 1.12 (0.55-2.27)* 3.89 + 0.57 1.38 + 0.55** 6

(3 x 10 -6 mol/l)

Two full concentration--effect curves (CEC) to NE were carried out for each preparation under different experimental conditions. The blockers were added to the organ bath before the beginning of the second CEC, except for indomethacin that was added 30 min before. *P < 0.05; **P < 0.001. N = Number of experiments. NS = Not significant.

Effects of NE and clonidine on ileum 95

Table 2. Mean geometrical CEs0 values (molar concentration of the agonist that causes an effect equal to 50% of the maximum effect in individual experiments) and confidence limits, and mean (_+ SEM) maximum effects produced

by clonidine in the isolated guinea-pig terminal ileum

CEs0 x 10 -5 mol/I (confidence limits) Maximum effects + SEM

Group la.CEC 2a.CEC Ia.CEC 2a.CEC N

Control 7.06 (6.11-8.17) 9.22 (5.90-14.3) Ns 7.43 _+ 0.60 7.17 _+ 0.95 Ns 6 Indomethacin 24.5 (6.41-92.1) 17.0 (3.57-81.6) Ns 5.13 _+ 0.76 5.25 -I- 0.68 Ns 6

(3 x 10-6tool/I) Atropine 15.9 (5.92-42.4) 13.8 (5.26-36.4) ss 5.80 + 0.27 5.94 -t- 0.36 ss 5

(3 x 10 8 tool/I)

Two full concentration-effect curves (CEC) to clonidin¢ were carried out for each preparation under different experimental conditions. Atropine and indomethacin were added to the organ bath 20 and 30 rain before the second CEC, respectively

NS = Not significant. N = Number of experiments.

s ignif icantly in the p resence a n d absence o f a t rop ine (Table 2, Fig. 2).

DISCUSSION

The a dd i t i on o f increas ing doses o f n o r e p i n e p h r i n e to the nu t r i en t m e d i u m p r o d u c e d a phas ic dose- d e p e n d e n t c o n t r a c t i o n o f the isolated gu inea-p ig

100

50

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0 6.0 5.5 5.0 4.5 4.0 3.5

t e rmina l i leum s ta r t ing f r o m the 10 -6 mol / l concen - t ra t ion . It shou ld be p o i n t e d ou t t ha t the sensi t ivi ty o f the i leum to the cont rac t i l e effect o f n o r e p i n e p h r i n e var ied t h r o u g h o u t the year , be ing sharp ly h igher du r ing the pe r i od f r o m F e b r u a r y to May .

Prazos in , a selective b locker o f alpha~ ad ren - ergic r ecep to r s (Ber thelsen a n d Pet t inger , 1977;

I I

l

/ J 3 ~ / Prazosln

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Fig. 1. Mean concentration-effect curves (CEC) for the contractile effect o f norepinephrine (NE) obtained for the isolated guinea-pig terminal ileum maintained in nutrient Tyrode solution at 37°C. Two CEC were obtained for each preparation, one of them in the absence (Q Q) and the other in the presence (& A ) of an antagonist. Each point represents the mean of 6 experiments and the vertical bars

represent the SEM.

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C. C. A. REIS

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r3x lo- M]

i~r t I I J 5.0 5.5 4.0 4.5 3.0

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Fig. 2. Mean concentration-effect curves (CEC) for the contractile effect of clonidine obtained for the isolated guinea-pig terminal ileum maintained in nutrient Tyrode solution at 37°C. Two CEC were obtained for each preparation, one of them in the absence (O O) and the other in the presence of an antagonist (A A). Each point represents the mean of 6 experiments and the vertical bars represent

the SEM.

Timmermans et al., 1980; Kobinger and Pichler, 1981), when administered at the 5 x 10 -8 mol/1 con- centration caused a rightward horizontal shift of the CEC to NE and significantly decreased the maximum effect obtained. An antagonist that induces a fight- ward horizontal shift of the CEC to an agonist and depresses the maximum responses obtained combines the characteristics of a competitive antagonist with those of a noncompetitive antagonist. Van den Brink and Lien (1977) called this type of antagonism com- petitive and metactoid. The present results indicate the existence of excitatory alpha~ adrenergic receptors in the isolated guinea-pig terminal ileum.

The participation of alpha2 adrenergic receptors in the contractile effect of norepinephrine was evalu- ated using yohimbine, a blocker of these receptors (Berthelsen and Pettinger, 1977; Timmermans et aL, 1980; Langer and Pimoule, 1982; Kobinger and Pichler, 1981). At the concentration of 5 x 10 -6 mol/i, yohimbine did not alter the contractile effect of norepinephrine in a significant manner, but at the concentration of 10-Smol/l it antagonized the contractile effect of the catecholamine in a non- competitive manner. These results suggest that the alpha: receptors may not be directly related to the contractile effect of norepinephrine.

However, clonidine, a selective agonist at the alpha 2 receptor level (Berthelsen and Pettinger, 1977; Starke, 1981; Nakaki et al., 1983), produced a phasic dose-dependent contraction of the isolated guinea-pig terminal ileum.

Contraction was not blocked by atropine, demon- strating that the release of acetylcholine from post- ganglionary fibers of the parasympathetic nervous system is not related to this effect.

The mean pD 2 values calculated for norepinephrine and clonidine did not differ significantly from one another. However, the mean value of the maximum effects of norepinephrine was significantly lower than

the mean value of the maximum effects of clonidine. On this basis, it is clear that an excitatory functional unit exists in the isolated guinea-pig terminal ileum which is activated in a more potent manner by clonidine than by norepinephrine, although its specific NE-binding sites have no affinity for yohimbine.

Practolol, a betal adrenergic receptor blocker, potentiated the contractile effect of NE, indicatin~ that the activation of these receptors is related to the relaxation of intestinal smooth muscle.

As mentioned earlier, the activation of adrenergic receptors may result in stimulation of prostaglandin synthesis in several tissues (Davies et aL, 1968; Gilmore et al., 1968; Wennmalm and Brundin, 1978; Seregi et al., 1982; Bhattacherjee et al., 1979; Abdel- Latiff and Smith, 1982). In the present experiments, indomethacin, a prostaglandin synthesis inhibitor (Vane, 1971; Ferreira et al., 1971), significantly reduced the excitatory response evoked by NE. This fact indicates that the contractile effect of NE is related, at least in part, to the release of endogenous prostaglandins. On the other hand, indomethacin did not interfere with the excitatory response to clonidine, indicating that the stimulation of the clonidine-sensitive functional unit is not related to the release of endogenous prostaglandins.

The stimulation of prostaglandin synthesis due to the activation of alpha adrenergic receptors has been reported by Olson et al. (1983), who noted that the increased renin release due to stimulation of alpha receptors is related to the activation of prostaglandin synthesis.

Another hypothesis that may explain the indomethacin-induced decrease in the contractile effect of norepinephrine is based on data reported by Horrobyn et al. (1974), who postulated that prostaglandins may be necessary for norepinephrine interaction with adrenergic receptor sites or for

Effects of NE and

the activation of some link in the chain of events occurring between receptor stimulation and muscle contraction. Thus, basal prostaglandin release may provide an opt imum condition for the activation of the contractile machinery. Blockade of prostaglandin synthesis would then lead to a reduced efficiency of the effects that culminate in the excitatory response.

However, Forster and Ponicke (1983), after studying the action of several catecholamines on cyclo-oxygenase, concluded that there was no clear correlation between the chemical structure of phenylethanolamine derivatives and their ability to activate this enzyme. From this viewpoint, stimu- lation of prostaglandin synthesis may not depend on the activation of adrenergic receptors, and the sympathomimetic amine may function as a cofactor for the cyclo-oxygcnase reaction of arachidonic acid.

The large number of studies that have demon- stratcd the ability of catecholamines to stimulate prostaglandin synthesis and have reported increased prostaglandin synthesis due to adrenoccptor activation in several tissues, taken together with the present results, indicate that the activation of excitatory alpha receptors that are sensitive to blockade by prazosin in the isolated guinea-pig terminal ileum leads to activation of endogenous prostaglandin synthesis.

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