European Journal of Pharmacology 30 (1975) 29-35 © North-Holland Publishing Company

DUAL ACTION OF CHLOROQUINE

ON FROG'S SKELETAL MUSCLE

CONTRACTION Edward AYITEY.SMITH and Guenrikh A. VARTANIAN Departments o f Pharmacology and Physiology, University of Ghana Medical School, P O. Box 4236, A ccra, Ghana

Received 14 May 1974, accepted 2 ffeptember 1974 E. AYITEY-SMITH and G.A. VARTANIAN, Dual action of chloroquine on frog's skeletal muscle contraction, European J. Pharmacol. 30 (1975) 29-35. Studies on the effect of chloroquine on frog's rectus abdominis muscle have been performed. Relatively low concentrations of chloroquine, 5 x 10-s and 2 X 10--4 g/ml inhibited cholinesterase activity and potentiated acetylcholine (ACh)-induced contractions but antagonized carbachol and caffeine contractions, as well as ACh-induced contractions of eserinized muscle. High concentrations (5 x 10-4 and 2 X 10 -3 g/ml) non-competitively antagonized contractions to ACh, carbachol, caffeine and potassium. It was suggested that the blocking action of chloroquine was due to its local anaesthetic property and interference with intracellular calcium movements. Frog's rectus abdominis muscle Non-competitive antagonism Calcium

Isotonic contraction

1. Introduction The prolonged administration of chloroquine has been reported to cause severe muscle weakness associated with necrosis and vacuolation of the muscle fibres (Loftus, 1963; Rewcastle and Humphrey, 1965; Smith and O'Grady, 1966; and Macdonald and Engel, 1970). This chronic aspect of chloroquine's action on skeletal muscle is more extensively documented than its acute pharmacological effect. So far, only one group of workers has presented evidence for a blocking action of chloroquine on neuromuscular transmission (Chinyanga et al., 1972; Vartanian and Chinyanga, 1972). The purpose of this study is to investigate in more detail the action of chloroquine on skeletal muscle contraction.

2. Materials and methods 2.1. Isolated frog rectus abdominis muscle preparation

Experiments were performed on rectus abdominis

Chloroquine

Anticholinesterase action

muscle from frogs (Rana occipitalis) weighing 7 0 - 1 0 0 g. The rectus abdominis muscle was isolated, divided at the midline and the pair suspended separately under 2 g tension in frog's Ringer solution of pH 7.4 (composition (g/l): NaC16.5; KCI 0.14; CaC12 0.12; NaHCO3 0.20; NaH2PO4 0.01) in two organ baths (40 ml capacity) (Perry, 1968). The Ringer solution was aerated with 95% oxygen and 5% carbon dioxide and the experiment was conducted at room temperature (27 to 29°C). 45 min was allowed for the muscles to equilibrate before drugs were tested and isotonic contractions recorded on a kymograph. During the experiment, cumulative dose-response curves of ACh, carbachol, potassium and caffeine were determined. Then the drugs were washed out 3 times from the muscle preparations, 10 rain later chloroquine was added to the organ bath and further 15 rain later the dose-response curves o f the drugs were repeated. In part of the experiment with ACh, 2 hr instead of 15 min were allowed after adding chloroquine and then ACh dose-response curve was repeated. Further details have been given in the resuits.

30

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Fig. 1. A, Effect of v~Irious concentrations of chloroquine (CQ in g/ml) on contractions of rectus abdominis muscles to ACh. The different concentrations of CQ were obtained by addition of the drug to the bath medium 15 min before addition of ACh. Control is ACh curve in the absence of CQ. B, AOa curves 15 min after 3 wash-outs of chioroquine from muscle. The concentrations of C(~ shown by each curve were those before the wash-out. 6-10 muscle preparations were used for the determination of each curve. The vertical bars represent standard errors.

2.2. Serum cholinesterase activity The effect o f chloroquine on cholinesterase was studied in vitro using rat serum as a source of the enzyme (Perry, 1968). Serum was diluted 1 - 5 with frog Ringer solution. 6 test tubes (A, B, C, D, E and F), each containing 125/ag acetylcholine (ACh) in 0.5 ml were prepared. Diluted serum was added to ACh in each tube to make up 5 ml, and 0.8 ml o f the solution was added to the organ bath giving a final concentration o f 5 × 10 -~ g/rnl ACh. Serum was added to ACh in tube A, a control, and 0.8 ml o f the solution was immediately withdrawn and added to the organ bath, so that ACh was not hydrolysed within that short period. However, ACh in the serum contained in the remaining 5 tubes was allowed to stand for 5 min before 0.8 ml o f each solution was added to the organ bath. Serum for tubes C, D, E and F was pretreated for 5 min with either physostigmine or chloroquine giving concentrations o f 1 X 10 -4 g/ml physostigmine and 5 × 10 -s, 2 × 10 -~ and 5 × 10 -4 g/ml chloroquine respectively. After adding 0.8 ml aliquots of physostigmine in serum from tube C and chloroquine in serum from D, E and F to the organ bath, the final concentrations became 2 × 10 -6,

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E. A yite y-Smith, G.A. Vartanian, A ction o f chloroquine on muscle contraction

2.3. Drugs Drugs used were chloroquine sulphate (May and Baker); caffeine (Hopkin and Williams); acetylcholine chloride, carbachol chloride and physostigmine sulphate (Sigma Chemical Company). All drugs were dissolved in Ringer solution and all concentrations refer to the base of the salts, except for physostigmine.

3. Results

3.1. Effect of chloroquine 3.1.1. On acetylcholine contractions Chloroquine exhibited a dual action on ACHinduced contractions of the rectus abdominis muscle (fig. 1A). At relatively low concentrations (5 X 10-s and 2 X 10-4 g/ml) chloroquine enhanced the contractions while at high concentrations (5 × 10-4 and 2 × 10 -3 g/ml) it depressed them. When chloroquine was washed out from the muscle, its depressant effect on ACh contractions was

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partially reversed (fig. 1B). The enhanced contractions produced by 5 X 10-s g/ml chloroquine were abolished but those of 2 × 10-4 g/ml were still slightly above those of control. Exposure of the ,muscle to chloroquine for', 2 hr appeared not to intensify its blocking effect but modified its enhancing effect (fig. 2). It was also found that antagonism between ACh and chloroquine was non-competitive (fig. 7A).

3.1.2. On physostigmine (Eserine) Fig. 3 shows the effect of chloroquine on AChinduced contractions of muscles suspended in Ringer solution containing 1 × 10-4 g/mt physostigmine. When cholinesterase was inhibited with physostigmine, low concentrations of chloroquine (5 × 10-s and 2 × 10-4 g/ml) which enhanced ACh-induced contractions, decreased the potentiating effect of physostigmine. 3.1.3. On carbaehol contractions Fig. 4 shows the blocking effect of chloroquine o n carbachol-induced contractions. Chloroquine was more potent in blocking carbachol than ACh. Con-

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Fig. 3. Effect of relatively low concentrations of chloroquine (CQ in g/ml) on ACh-induced contractions of abdominis muscle in eserinized Ringer solution. Concentration of physostigmine (eserine) in the Ringer solution is 1 × l 0-4 g/ml. Control is ACh curve in the absence of CQ and physostigmine. 6 - 1 0 muscle preparations were used for the determination of each curve. The vertical bars represent

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E. A yitey-Smith, G.A. Vartanian, A ction o f chloroquine on muscle contraction

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; - Logl0Doseof Carbachol ( glml.) Fig.4. Blockingeffectof chloroquine(CQin g/ml)on carbachol-inducedcontractions.Controlis carbacholcurvein theabsence of CQ. 6-10 muscle preparations were used for the determination of each curve. *Curve obtained when carbachol was replaced by ACh; in the absence of CQ, for comparison. The vertical bars represent the standard errors. centrations of chloroquine (5 X 10 -s and 2 × 10 -4 g/ml) which enhanced contractions due to ACh, depressed those to carbachol.

3.1.4. On serum cholinesterase In fig. 5, the anticholinesterase action of chloroquine was clearly demonstrated. A, a control which contained active ACh, 5 × 10 -7 g/ml, produced contraction of the muscle, but in B, there was no response to ACh because it was hydrolysed by serum cholinesterase. In C, cholinesterase was inhibited with physostigmine, 1 × 10 -4 g/ml, therefore ACh remained active. Serum samples for D, E and F were pretreated with 5 × 10 -s, 2 × 10 -4 and 5 × 10 -4 g/ml chloroquine respectively. ACh remained active in the three samples. 3.1.5. On caffeine At concentrations of 2 X 10 -4 , 5 X 10 -4 and 2 × 10 -3 g/ml, chloroquine antagonized caffeineinduced contractions, reducing the maximal contraction by 5 0 - 7 0 % (fig. 6A). Moreover, low concentrations of chloroquine, down to 1 × 10-~ g/ml, partially blocked caffeine contractions. The blockade was non-competitive (fig. 7B) and was neither dose-dependent nor reversible after wash-out of chloroquine

from the muscle (fig. 6B). Chloroquine was 2 times more potent in blocking caffeine contractures in calcium-free Ringer solution than in normal solution (table 1).

3.1.6. On high potassium Contractions produced by high potassium were non-competitively antagonized by relatively high concentration of chloroquine (2 × 10 -3 g/ml) (fig. 8A). This blockade was also irreversible (fig. 8B).

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Fig. 5. Effect of chloroquine (CQ) on serum cholinesterase. K y m o g r a p h tracings show contractions to ACh, 5 × 10 -7 g/ml. A is a control contraction; B, absence of contraction due to hydrolysis of ACh by serum cholinesterase; C, pret r e a t m e n t of serum with physostigmine, 1 × 10 -4 g/ml, protected ACh from hydrolysis; D, E and F pretreatment of serum with various concentrations o f CQ, 5 X 10 -5, 2 x 10-4 and 5 X 10-4 g]mi respectively, protected ACh from hydrolysis. In the b a t h m e d i u m the final concentrations of physostigmine f r o m tube C and of chloroquine from D, E and F were 2 X 10 -6, 1 X 10 -6, 4 X 10 -6 and 1 X 10 -5 g/ml respectively.

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