Effects of the active constituents of Catha edulis on the neuromuscular junction

NeuropharmacologyVol. 26, No. 5, pp. 401405, 1987 Printed in Great Britain. All rights reserved

0028-3908/87 $3.00 + 0.00 Copyright 0 1987 Pergamon Journals Ltd

EFFECTS

OF THE ACTIVE CONSTITUENTS OF CATHA EDULIS ON THE NEUROMUSCULAR JUNCTION A. N. GUANTAI, J. W. MWANGI, GICHURU MURIUKI and K. A. M. KURIA

Mitishamba Drug Research Centre, Department of Pharmacy, College of Health Sciences, University of Nairobi, P.O. Box 30197, Nairobi, Kenya (Accepted 17 February 1986)

-)-Cathinone and d-norpseudoephedrine (DNE) in the dose range 0.2-1.2 mg/ml produced a reduction in contractions of skeletal muscle, evoked by direct and indirect electrical stimulation and antagonised the facilitatory action of physostigmine on the neuromuscular junction; but failed to antagonise a partial blockade induced by d-tubocurarine (dTb) as occurs with norepinephrine or enineohrine. The local anaesthetic actions of (- l-cathinone and DNE were found to be almost eauivalent _ _ , to that of lignocaine. These results indicate that (-)-cathinone and DNE may have a direct blocking action on the neuromuscular junction, which is independent of cholinergic and adrenergic transmission. Summary-(

Key words: Catha edulis, active constituents, neuromuscular junction.

Catha edulis Forsk (Khat or Miraa) is an evergreen shrub which grows along the Eastern Coast of Africa. It is well known for its stimulant effects on the central nervous system for which it is abused. (-)-Cathinone and d-norpseudoephedrine (DNE) are two phenylalkylamines that have been isolated

from the young shoots of the khat plant (United Nations Document, 1975; Wolfes, 1930, Guantai, 1982). (-)-Cathinone is the major alkaloid in the tips of the juicy young shoots, while DNE is present in smaller amounts in the shoots and is widespread in the whole plant (Guantai and Maitai, 1982). These two alkaloids are structurally similar to ephedrine and their pharmacological profiles would be expected to be qualitatively similar. On the fast contracting anterior tibialis muscle of the cat, ephedrine in 2 mg or larger doses depresses the contractions of the muscle when stimulated at a frequency 0.2 Hz, while smaller doses (500 pg) cause a small, consistent facilitation of contraction which is converted to a depressant effect when the stimulus is increased to 5 Hz. This effect is not modified by prior treatment of the tissue with CLand/or p adrenoceptor blockers (Gallagher and Shinnick-Gallagher, 1979). Norepinephrine and epinephrine antagonise the partial blockade induced by d-tubocurarine (Bowman and Nott, 1969). However, the indirectly-acting sympathomimetic agent ephedrine does not antagonise the partial paralysis induced by d-tubocurarine even at doses (6Opg) that would cause a small facilitation when injected close intra-arterially (Gallagher and Shinnick-Gallagher, 1979). This effect is not altered by pretreatment with adrenoceptor blockers. In the presence of neostigmine, ephedrine does not enhance the contractions of the anterior tibialis muscle, unlike the combination of epinephrine-

neostigmine or norepinephrine-neostigmine. It is evident that the actions of ephedrine at the neuromuscular junction are not due to its indirect sympathomimetic effect (Gallagher and ShinnickGallagher, 1979). (-)-Cathinone and DNE are reported as having a releasing effect on transmitter at the noradrenergic nerve endings of the same magnitude as (+) amphetamine (Kalix, 1983a,b) and their peripheral actions are summarised as being mainly sympathomimetic (Kalix, 1984). A literature survey has not revealed any published work on the actions of (-)-cathinone and DNE at the neuromuscular junction. The present study was undertaken in order to investigate the effects of the two alkaloids at the neuromuscular junction.

401 N.P.26,5--A

METHODS Materials

Rats, weighing 200-300 g and bred by the National Public Health Laboratory (NPHL), Kenya, were used. (-)-Cathinone HCI was a generous donation by the United Nations Narcotics Laboratory. Tubocurarine was obtained from Asta Werke (Germany), DNE from Knoll Fine Chemical Inc. (New York), ephedrine from E. T. Monks and Co. Ltd (Nairobi) and physostigmine from BDH, Poole (U.K.). Lignocaine HCl was obtained from Pharmaceutical Manufacturing Company (Kenya); atropine sulphate from E. Merck Darmstadt, propranolol from Sigma Chemical Co. Ltd U.S.A. and phenoxybenzamine from Smith Kline and French. The Krebs solution used was prepared according to the method described by Krebs and Hensleit (1932).

402

A. N.

GIJANTAI et

Phrenic nerve-diaphragm preparation White albino rats, weighing 200-300 g, were killed by a blow to the head and exsanguination. The

carotid arteries were cut and the animals bled as much as possible to clear the chest cavity. The phrenic nerve was carefully dissected out with the hemi-diaphragm muscle attached to it. The nerve was attached to an electrode (814-80950-O Palmer and George Washington Bioscience), mounted in a 50 ml double-walled organ bath, filled with the Krebs solution (Krebs and Hensleit, 1932) maintained at 37” &-1°C and continuously aerated with carbogen (95% oxygen 5% CO& The tissue was allowed to stabilise for 30min and then electrically stimulated through the phrenic nerve with 10 V maximum at the rate of 1 pulse/set for 20 set, using a scientific and Research Instruments Ltd stimulator. Electricallyinduced muscular contractions were recorded using isometric transducers connected to a Devices recorder fitted with an amplifier and write-out mechanism. Different concentrations of (-)-cathinone (0.2, 0.4, 0.6 mg/ml) in the bath were tested, allowing one minute tissue-drug contact time before indirectly stimulating the diaphragm muscle through the nerve. The change in contractile response was noted. The same procedure was repeated using DNE (0.2, 0.4, 0.6 mg/ml bath concentrations). Modzjication by tubocurarine

d-Tubocurarine 40 pg/ml, was tested on the phrenic nerve preparation pre-treated with known concentrations of the test drug for 1 min. The mixture of drugs was left in contact with the tissue for 2 min and the change in electrically-induced contractile response noted. The recovery time allowed was 5 min.

al.

Efect of (-)-cathinone

and DNE on the Sciatic nerve

in situ A frog was decapitated and eviscerated. The front legs were pinned on a vertical cork board, leaving the hind legs hanging freely. The feet were immersed in 0.05 N hydrochloric acid and the brisk reflex withdrawal of the feet was observed within the first 2 sec. To avoid damage of the cells of the skin by acid, the feet of the frogs were quickly washed with water and the muscles allowed to relax. A known concentration of the test drug was placed in the abdominal sac until the sciatic nerves were completely immersed. The withdrawal reflex of the foot was tested after every min, always washing off the acid after each test. When the response was no longer obtained with the smaller concentration of acid the next larger concentration was tried, limiting the contact time to 10 set for 0.2 N HCl to avoid damage to the tissues. Concentrations of drugs were chosen such that the withdrawal reflex was blocked within 20 min because after 20 min the sensitivity of the feet to the acid decreased even in absence of a local anaesthesia. A frog was used to test only one concentration of drug. RESULTS

(-)-Cathinone, DNE and ephedrine caused a dose-dependent inhibition of the contractile response of the intact phrenic-nerve diaphragm preparation (Fig. 1). (-)-Cathinone, however, was slightly more potent than both DNE and ephedrine. 0

ModiJcation by physostigmine Physostigmine (1.4 pg/ml) was left in contact with the phrenic nerve diaphragm preparation for 1 min while recording the effect. Without washing, a known concentration of either (-)-cathione, DNE or ephedrine was added into the bath and the change in the contractile response noted. Alternatively, physostigmine (1.4 pg/ml) was tested on the tissue after pretreatment with a known concentration of the drugs. Effects of (-)-cathinone, DNE and ephedrine on the directly-stimulated muscle

To investigate

whether (-)-cathinone, DNE and ephedrine have a direct effect on the skeletal muscle which is not mediated through the nerve, the diaphragm muscle was stimulated directly and the effect of known concentrations of the test drugs was investigated. The results were compared to those obtained by the use of d-tubocurarine 0.02 mg/ml and lignoCaine 0.1 mg/ml.

5

-50

-

; 4 g,

-60

-

-70

-

-80

-

-90

-

B E 8 $ a s s

-100

L--L02

0.0

0.4

06

08

10

12

Bath cancentratlon (mg / ml )

Fig. 1. The blocking e&et of (-)-cathinone

(C), d-norpseudoephedrine (DNE), and ephedrine (E) on the phrenic nerve-diaphragm preparation in the rat. Each value represents the mean of 8 separate experiments.

Effects of Catha edulis on the neuromuscular

The stimulatory action of physostigmine on the neuromuscular junction was completely blocked by ephedrine, (-)-cathinone and DNE. Conversely, physostigmine enhanced the blockade of the neuromuscular junction induced by (-)-cathinone, DNE and ephedrine (Fig. 2). The same dose of (-)-cathinone DNE and ephedrine caused a greater inhibition of muscle contraction on the directlystimulated muscle than the indirectly-stimulated muscle (Table 1). Phenoxybenzamine (20 pg/ml) and propranolol (20pg/ml) had no effect on the actions of either (-)-cathinone or DNE, unlike ephedrine. The results from experiments using the frog sciatic nerve revealed that DNE and (-)-cathinone had local anaesthetic effects, equivalent to those produced by lignocaine (Table 2).

5

t

a i? % e t Y a”

403

junction

-60

-

-70

-

-80

-

DISCUSSION -90

-

-100 1 00

I

I

02

0.b

\ 06

08

I

10

Concentration (*g/ml

12

)

Fig. 2. Lack of antagonism of the blocking effect of (-)-cathinone (C) d-norpseudoephedrine (DNE) and Ephedrine (E) on the neuromuscular junction by physostigmine (Phy). x---x C B---B C + Phy. a--_O DNE 0-0 DNe + Phy. O---O E x---x E + Phy. Note that Figure 1 is superimposed on Figure 2 to show clearly the lack of antagonism and the evident potentiation of blockade of neuromuscular transmission by physostigmine. Each value is the mean of 8 separate experiments. Table 1. Effect of (-)-cathinone,

The in vivo actions of ephedrine at the neuromuscular junction were first investigated by Bulbring and Burn (1942), after reports that ephedrine could relieve the hypotonia of myasthenia gravis, either alone (Edgeworth, 1930), or in combination with other drugs (Viets and Schwab, 1939). It was then suggested that this action of ephedrine was related to the indirect release of norepinephrine and epinephrine (Bulbring and Burn, 1942). Since epinephrine auguments the contractions of skeletal muscles (Breckenridge, Burn and Matachinsky, 1967), it would be expected that ephedrine would

o-norpseudocphedrine and ephedrine on the directly-stimulated diaphragm muscle of the rat

Final bath concentrationa

Drug

1 min

% Reduction in contractile response 2 min 3 min

Ephedrine

0.60(8)

48.75* 1.77

75 + 0.60

DNE

0.60 (6) 0.48 (6) 0.30 (5) 0.40 (8) 0.02 (3) 0.1 (3)

51.6 +_0.90 30.33 k 1.31 23.56 + 1.5 40.61 k 0.74 No change 66. I5 & 4.98

Block 60.45 f 1.45 38.00 + 2.7 65.49 + 1.61 No change Block

Cathinone Tubocurarine Lignocaine

4 min

Block 80.00 f 0.5 61.5 f 3.6 Block

Block

Toncentration in bath as in mg ml-‘. Number in parentheses indicates the number of tissue preparations tested and on which the mean f SE is based with respect to the control (100%). Table 2. Effect of cathinone. DNE and eohedrine on the sciatic nerve of frog in uiuo

Drug DNE Cathinone Ephedrine Lignocaine Physostigmine Curare Atropine

Concentration 5 10 5 IO 5 10 2.5 5 0.3 I 100

Time (min) taken for the disappearance of the withdrawal reflex in HCI 0.05 N 0.1 N 0.2 N II 8 13 II 16 I2 10 7 13 -

(-) No anaesthesia was detected within the allowed 20 min. k&d-foot contact time 0.05 N HCI 0.10 N HCI -2osec 0.20 N HCI’IO sec.

15 10 15 12 17 13 13 8 15 -

18 12 I8 13 20 16 15 IO 16 -

A. N. GUANTAIet al.

404

Both ~~entratlon

(mg/ml

f

Fig. 3. The potentiation of the blocking effect of cathinone (C) d-norpseudoephedrine (DNE) and ephedrine (E) on the neuromuscular junction by d-tubocurarine (dTb) 40 yg/ml. x-x E + dTb 0-0 DNE +dTb l ---. C+dTb. Each value is the mean of 6-S separate experiments obtained after drug-tissue contact time of 1min. Compare with the results in Figure 1.

potentiate the facilitatory action of neostigmine and/or aminophylline. However it has been shown that ephedrine does not potentiate the actions of neosti~ine at the neuromuscular junction but has an antagonistic effect (Gallagher and ShinnickGallagher, 1979), indicating that ephedrine does not behave like epinephrine or norepinephrine at the muscular junction. A direct mechanism of action was therefore suggested (Gallagher and ShinnickGallagher, 1979). (-)-Cathinone and DNE bear a close structural similarity to the indirectly-acting phylalkylamines, amphetamine and ephedrine, and their pharmacological profiles would be expected to be qualitatively similar. The present results indicate that (-)-cathinone and DNE do not behave like epinephrine and norepinephrine at the neuromuscular junction. Unlike epinephrine, that augments the contractions of skeletal muscle (Breckenridge et al., 1967), (-)-cathinone and DNE caused a dosedependent blockade of transmission across the phrenic nerve diaphragm junction at all contractions employed (-)-Cathinone (0.2-l .2 mg/ml). (0.45 mgiml), DNE (0.5 mg/ml) and ephedrine (0.75 mg/ml) caused a 50% contractile inhibition. Larger doses of (-)-cathinone (0.8 mg/ml), DNE (2 mg/ml) and ephedrine (1 mg/ml) caused total blockade of transmission.

(-)-Cathinone and DNE did not potentiate, but antagonised the stimulatory action of neostigmine on the neuromuscular. A similar action has been observed with ephedrine (Gallagher and ShinnickGallagher, 1979) and confirmed in the present study. These findings, coupled with the inability of phenoxybenzamine (CI-blocker) and propranolol (/?-blocker) to alter any of the actions of (-)-cathinone and DNE, do not support the concept of an indirect sympathomimetic action of (-)cathinone and DNE. These compounds could be acting at sites other than the adrenoceptors that may be present at the neuromuscular junction (Bowman and Raper, 1967). (-)-Cathinone, DNE and ephedrine, enhanced the blockade of the neuromuscular junction induced by ~-tu~curarine (Fig. 3). This action is unlike that of norepinephrine or epinephrine which antagonise a partial blockade induced by d-tubocurarine (Maddock, Rankin and Youman, 1948). The present results show that (-)-cathinone and DNE have pronounced a local anaesthetic action (Table 2). However, it was noted that the doses required to induce local anaesthesia were about 20 times greater than those required to cause blockade of transmission at the neuromuscular junction. It is therefore unlikely that the blockade of the neuromuscular junction, induced by (-)-cathinone and DNE, is entirely due to their local anaesthetic eff.&s. This concurs with the finding that no significant change in either the resting membrane potential or electrical characteristics of the membrane occurs with doses of ephedrine that cause depression of neuromuscular transmission (Shinnick-Gallagher and Gallagher, 1979). On the erectly-stimulated diaphragm muscle, dtubocurarine did not cause any contractile inhibition, as its pharmacological action is exerted on the postjunctional cholinergic receptors. However, (-)cathinone and DNE not only inhibited the contractile properties of the directly-stimulated diaphragm muscle of the rat, but the rate and the magnitude of the blockade were also increased (Table 1). The potentiation of blockade of transmission at the neuromuscular junction induced by d-tubocurarine, by (-)-cathinone and DNE, coupled with the inability of physostigmine to antagonise the blockade, implies that the blockade is not ~holiner~caIly mediated. It is also evident that the actions of (- )-cathinone and DNE of the neuromuscular junction are similar to those of ephedrine and are not mediated through adrenergic mechanisms. The local anaesthetic actions of DNE and (-)-cathinone do not fully explain the muscle paralysis caused by these two substances. It is suggested that (-)-~athinone and DNE exert their actions directly on the diaphragm muscle at sites other than the classical adrenergic and cholinergic receptors. Research in progress in this laboratory will seek to elucidate the mechanism of action of (-)-cathinone and DNE.

Effects of Carha edulis on the neuromuscular junction REFERENCES

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