Inhibition of mouse neuromuscular transmission and contractile function by okadaic acid and cantharidin

Abstract

1. Phosphorylations of cellular proteins modulate biological activities. The effects of okadaic acid (0.1 - 10 microM) and cantharidin (1 - 100 microM), inhibitors of protein phosphatases, on the synaptic transmission at the mouse neuromuscular junction were explored. 2. Both inhibitors almost completely depressed twitch forces elicited by electrical stimulation of diaphragm muscles (the IC(50)s for okadaic acid and cantharidin were 1.1+/-0.2 and 13+/-1 microM, n=5, respectively) and suppressed contractures evoked by high K(+) and ryanodine more than 70%. Contractures caused by cardiotoxin, which destroys the integrity of sarcolemma, were not depressed. 3. Both okadaic acid (10 microM) and cantharidin (100 microM) depolarized muscle membranes from approximately -80 to approximately -60 mV in a partially reversible and tetrodotoxin-sensitive manner. The initial short-term enhancement of twitch responses (up to approximately 40%) was correlated with the inhibitors-induced repetitive firings of muscle action potential. 4. Treatment with either agent resulted in nearly complete inhibitions of endplate potential (epp). The IC(50)s were 0.8+/-0.2 and 9+/-2 microM (n=5), respectively, for okadaic acid and cantharidin. On high frequency stimulation, the coefficient of epps was increased more than 10 fold and the extent of epp run-down during stimulations intensified from approximately 25 to approximately 75%. Analyses of presynaptic quantal releases revealed decreases in epp quantal content and the immediately available vesicle pool. 5. The frequency of miniature epp was initially elevated up to 2 fold then suppressed down to approximately 30%. The small reduction in the amplitude was antagonized when the membrane of endplate area was repolarized. 6. The data suggest that okadaic acid and cantharidin inhibit mobilizations of synaptic vesicles and depress Ca(2+) release from sarcoplasmic reticulum and that protein phosphatases participate in the modulation of motor function.

Figure 1

Effects of okadaic acid (OA) and cantharidin on the contractility of mouse diaphragm. (A) Biphasic effects of OA on twitch forces. Left panel: tetanic contraction evoked by direct stimulation at 100 Hz for 5 s. Middle: twitches were elicited by alternate direct and indirect stimulations at 0.1 Hz. The larger responses in each couple are twitches on direct stimulation. Right panel: tetanic contraction 120 min after treatment with OA. The breaks between panels indicate 10 fold changes in the gain of contractility. (B) Concentration-dependent inhibitions of twitch responses in diaphragms treated with OA or cantharidin for 90 min. Data (mean±s.e.mean) pooled from five different preparations.

Figure 2

Biphasic effects on twitch force and inhibition of K⁺-contracture by cantharidin. (A) Twitches and K⁺-contracture in control preparation. In this and the following figures, direct and indirect stimulations were terminated 5 min before the challenge of contracture inducer. (B) Another preparation, high KCl Tyrode solution was applied after twitches were depressed to <10% by cantharidin (pretreated for 60 min).

Figure 3

Inhibition of ryanodine-contracture by OA and cantharidin. Ryanodine-contracture was induced in control (A), in preparations pretreated with OA (B), or with cantharidin (C) for 60 min. The ordinate in (C) denotes per cent contracture-time integral (each data point pooled from five different experiments), and the integral produced by 30 min treatment with ryanodine 100 μM was arbitrarily set 100%.

Figure 4

OA did not depress contracture evoked by cardiotoxin. (A) Control twitch responses and contractures elicited by cardiotoxin, a protein that disrupt the integrity of sarcolemma. (B) Another preparation. Cardiotoxin was applied after twitches were depressed to <10% by OA (pretreated for 60 min).

Figure 5

OA and cantharidin depolarized muscle membrane. Preparations were treated either with OA or cantharidin for 120 min. Thereafter, at the arrow mark, OA was washed out or tetrodotoxin was added to the preparations already incubated with cantharidin. Data pooled from five experiments. Note the partial restorations of membrane potential after washout or treatment with tetrodotoxin.

Figure 6

Effects of OA on miniature endplate potential (mepp). (A) In preparations bathed in normal Tyrode solution (2.8 mM KCl), OA was added at t=5 min, causing an initial facilitation, then suppression, of spontaneous quantal release. Data pooled from five preparations. (B) Elevating KCl concentration to 17 mM (at t=5 min) increased mepp frequency. OA, applied at t=25 min, progressively reversed this increase. Data pooled from five experiments.

Figure 7

Effects of OA and cantharidin on endplate potential (epp). Nerve-muscle preparations were immobilized with μ-conotoxin. Epps were evoked with low frequency pulses (at 0.3 Hz, A) or with trains of high frequency stimulation (50 Hz for 5 s, B and C). OA or cantharidin was added at t=5 min and washed out at t=135 min (A, data pooled from five preparations). Trains of epps were obtained before (B) or after treatment with OA for 120 min (C). The resting membrane potential of the endplate area were repolarized to −78–−82 mV during samplings of epps and mepps. The randomly-occurred ticks (B, C) with amplitudes delimited between baseline and horizontal arrows were mepps. Note that OA increased the extent of epp run-down but left mepp amplitude undepressed.

Figure 8

Inhibitions of evoked quantal releases by ω-agatoxin IVA, OA or cantharidin and the accompanied changes in epp profile. Quantal releases were evoked with trains of high frequency pulses (50–100 Hz for 5 s every 3 min). Abscissa is the absolute amplitude of the first epp (Epp₁) on a train of stimulation. The amplitudes are assorted in six categories and the group associated with the epp amplitude >38 mV depicts the control data (before any treatment). All the three agents depressed epp amplitude progressively down to 3–7 mV during 120 min experimental period. Ordinate is the per cent amplitude deviations of the at steady-state, the mean amplitude of the 20–100th epps (Epp_(20–100)) during a train of stimulation, from the respective Epp₁. The parameter signifies epp profiles (minus sign for run-down of train of epps, positive sign for epp run-up) and reflects overall facilitation (treated with ω-agatoxin IVA) or depression (with OA or cantharidin) of the mobilization of releasable transmitter quanta during intense stimulations. Data pooled from 5–6 preparations.