Levamisole and ryanodine receptors. I: A contraction study in Ascaris suum

Abstract

Cholinergic anthelmintics (like levamisole) are important drugs but resistance with reduced responses by the parasite to these compounds is a concern. There is a need to study and understand mechanisms that affect the amplitude of the responses of parasites to these drugs. In this paper, we study interactions of levamisole and ryanodine receptors on contractions of Ascaris suum body muscle flaps. In our second paper, we extend these observations to examine electrophysiological interactions of levamisole, ryanodine receptors (RyRs) and AF2. We report that the maximum force of contraction, g(max), was dependent on the extracellular concentration of calcium but the levamisole EC(50) (0.8 microM) was not. The relationship between maximum force of contraction and extracellular calcium was described by the Michaelis-Menten equation with a K(m) of 1.8mM. Ryanodine inhibited g(max) without effect on EC(50); ryanodine inhibited only 44% of the maximum contraction (K(i) of 40 nM), revealing a ryanodine-insensitive component in the levamisole excitation-contraction pathway. Dantrolene had the same effect as ryanodine but was less potent. The neuropeptide AF2 (1 microM) decreased the levamisole EC(50) to 0.2 microM without effect on g(max); 0.1 microM ryanodine and 100 microM dantrolene, inhibited the g(max) of the AF2-potentiated levamisole response. High concentrations of caffeine, 30 mM, produced weak contraction of the body-flap preparation. Caffeine behaved like ryanodine in that it inhibited the maximum force of contraction, g(max), without effects on the levamisole EC(50). Thus, RyRs play a modulatory role in the levamisole excitation-contraction pathway by affecting the maximum force of contraction without an effect on levamisole EC(50). The levamisole excitation-contraction coupling is graded and has at least two pathways: one sensitive to ryanodine and one not.

Fig. 1

Ascaris suum body muscle flap preparation for contraction assay. A: Flap preparation mounted between an isometric force transducer and fixed position. Maintained at 37°C in glass cylinder surrounded by a glass jacket containing heated water. B: Representative control cumulative levamisole concentration and isometric contraction relationship.

Fig. 2

Calcium sensitivity of levamisole concentration contraction plots. A: Log cumulative levamisole concentration contraction plots and fitted line to estimate g_max and EC_50. See Results for equation and estimates. B: Plot of extracellular calcium concentration vs. maximum force of contraction, g_peak and fitted line to Michaelis-Menten equation to estimate g_peakmax and Km, see Results for equation and estimates. The force of the levamisole-induced contraction was dependent on the extracellular concentration of calcium but the levamisole EC₅₀ was not. C: Log cumulative levamisole concentration contraction plots in the presence and absence of 1 μM AF2. The control levamisole g_max was 2.0 ± 0.1 g (n = 10 preparations, DF = 87); in 1 μM AF2, the levamisole g_max was not significantly different, 1.9 ± 0.1 g (n = 10 preparations, DF = 87, p >0.05). The control levamisole EC₅₀ was 1 μM (pEC₅₀ was 5.86 ± 0.13, n = 10 preparations, DF = 87) and changed in 1 μM AF2, where the levamisole EC₅₀ was 0.2 μM (pEC₅₀, 6.63 ± 0.16, n = 10 preparations, DF = 87, p < 0.05). AF2 produced a 5-fold decrease in the levamisole EC₅₀ without an effect on g_max.

Fig. 3

Ryanodine sensitivity of levamisole contractions. A: Plots of the inhibitory effect of 0.1 μM ryanodine on the cumulative-levamisole-concentration contraction response. The control levamisole g_max was 2.5 ± 0.1 g (n = 10 preparations, DF = 87); in 0.1 μM ryanodine, the levamisole g_max was significantly reduced to 1.2 ± 0.1g (n = 10 preparations, DF 87, p < 0.05). The control levamisole EC₅₀ was 0.84 μM (pEC₅₀ was 6.07 ± 0.02, n = 10 preparations, DF = 73) and not significantly changed in 0.1 μM ryanodine where the levamisole EC₅₀ was 0.79 μM (pEC₅₀ was 6.10 ± 0.03, n = 9 preparations, DF = 66 p > 0.05). B: Plot of the inhibitory effect of ryanodine concentration on the maximum force of contraction g_max. The high concentrations of ryanodine reduced the maximum levamisole contraction by 44% with an IC₅₀ of 40 μM, but did not inhibit all of the contraction. The maximum inhibitory effect of high concentrations of ryanodine suggests that RyRs modulates one but not all calcium pathways to reduce the maximum force of contraction. Because ryanodine did not inhibit all of the levamisole contractions, it suggests that levamisole also activates RyR independent calcium pathways that are able to produce contraction, see Fig. 6 C: Plots of the inhibitory effect of 0.1 μM ryanodine on 1 μM AF2 potentiated levamisole concentration contraction plots. 0.1 μM ryanodine significantly reduced g_max from 1.19 g ± 0.13 (AF2 control: n = 10 preparations, DF = 87) to 0.91 ± 0.07 g (n = 10 preparations, DF= 87 p< 0.05). Ryanodine had little effect on EC₅₀ values (1 μM AF2 EC₅₀ control: 0.16 μM ± 0.13, n = 10 preparations, DF = 87; 1 μM AF2 + 0.1 μM ryanodine EC₅₀ 0.13 μM ± 0.07, n = 10 preparations, DF = 87 p< 0.05).

Fig. 4

Inhibitory effect of dantrolene on levamisole-concentration contraction plots. A: 100 μM dantrolene reduced g_max from 3.17 ± 0.15 g (control, n = 6 preparations, DF = 51) to 2.49 ± 0.15 g (n = 6 preparation DF = 51), p < 0.05. The EC₅₀ was not affected (control EC₅₀ 0.7 μM; in 100 μM dantrolene the EC₅₀ was 0.8 μM, p > 0.05). B: Effect of 100 μM dantrolene on AF2-potentiated levamisole contractions. The control AF2 g_max was reduced from 2.6 ± 0.08 g (n= 6 preparations, DF = 51) to 1.89 ± 0.12 g (1 μM AF2 + 100 μM dantrolene, n = 5 preparations, DF = 42), p < 0.05. The AF2 control EC₅₀ was 0.20 μM and in the presence of dantrolene, the EC₅₀ was 0.35 μM, p > 0.05.

Fig. 5

Effects of caffeine. A: Caffeine, 30 mM produces a small desensitizing contraction which is much smaller than that produced by 10 μM levamisole in the same preparation. B: Effect of 30 min caffeine on levamisole cumulative concentration contraction plots. Caffeine produced a concentration dependent reduction in the maximum force, g_max, without effect on the levamisole EC₅₀. 30 mM caffeine reduced the maximum contraction from 1.86 ± 0.11 g (control, N = 8 preparations, DF = 88) to 0.77 ± 0.05 g (30 mM caffeine, n = 4 preparations, DF = 34, p < 0.05). There was no significant effect on the EC₅₀ (0.9 to 0.6 μM, p > 0.05).

Fig. 6

Summary diagram of the proposed components involved in the modulatory effects of RyRs on contraction. Levamisole activates nAChRs that are permeable to calcium and that produce depolarization and activate voltage-gated calcium channels and slow wave channels (solid red arrows) giving rise to further entry of calcium (solid blue arrows). Opening of ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR), voltage-gated calcium channels (VACCs) and sodium/calcium permeable slow wave channels (not shown for simplicity) lead to an increase in cytosolic calcium. There is a ryanodine sensitive and a ryanodine insensitive pathway that leads to an increase in cytosolic calcium and contraction.