The mechanism of the anti-desensitizing action of sodium fluoride at the amphibian neuromyal junction.

Abstract Sodium fluoride was shown in these laboratories to facilite neuromyal transmission and to be a unique antagonist of desensitization. Possible mechanisms of action of sodium fluoride include a receptor effect and chelating activity. This study concerns the mechanism of the effect of sodium fluoride (0.1–2 mM) on the desensitization of the amphibian neuromyal junction and the interaction between this mechanism and calcium. Desensitization was obtained either by repetitive (10–50 Hz), brief (2 msec) iontophoretic pulses of acetylcholine (ACh), prolonged (40 sec) iontophoretic or bath application of ACh, or repetitive (10–50 Hz) presynaptic stimulation. Microelectrode and voltage-clamp methods were employed. The rate constant of desensitization induced by repetitive pulses of ACh was calculated in terms of the decay of current or potential. It depended on the frequency of pulses and on calcium ion concentration. In low calcium solution, a high frequency of pulses had to be employed to induce desensitization with a measurable rate constant. Sodium fluoride slowed down the rate of desensitization, and increased the amplitude of ACh current or potential during desensitization. The effect of sodium fluoride on the desensitization rate was pronounced at normal and low calcium ion concentrations and was slight in high calcium solutions. Desensitization did not affect reversal potential whether the latter was evaluated in terms of ACh potentials or ACh currents, and whether desensitization was induced by repeated ACh pulses or ACh perfusion. Nor did sodium fluoride (0.5–2 mM) affect the reversal potential of either a normal or desensitized endplate. Desensitization prolonged the decay time of the excitatory postsynaptic current (EPC) without affecting the slope of the decay time-membrane voltage relationship; sodium fluoride (0.1–2 mM) further prolonged decay time without affecting membrane voltage-decay time relation. The relationship between EPC or ACh current on the one hand and membrane voltage on the other was studied with respect to desensitization, sodium fluoride, and calcium concentration. Desensitization induced by repetitive (30 Hz) presynaptic stimulation did not affect the linearity of EPC-membrane voltage relationship; this remained true in different calcium solutions and when desensitization was induced in the presence of sodium fluoride. However, desensitization attenuated the slope of this relationship, the effect being similar in all three calcium solutions. This lack of effect of calcium on the slope must be considered with respect to calcium dependence of ACh release and, thus, on the EPC amplitude. The attenuation by desensitization of the slope of the EPC-voltage relationship was antagonized by sodium fluoride. Different frequencies of ACh pulses had to be employed to induce desensitization in the three calcium solutions in order to elicit desensitization with measurable rate constant. Also in this case, the ACh current-membrane voltage remained linear before and following desensitization in the three concentrations of calcium. Again, the slope of the current-voltage relationship was attenuated by desensitization at all concentrations of calcium. Sodium fluoride antagonized this effect to a significant degree at all concentrations of calcium; the antagonism was more pronounced at low than at high concentration of calcium. Desensitization induced by ACh perfusion did not affect the linearity of ACh current-membrane voltage at either of the three calcium concentrations. In this case also, desensitization attenuated the slope of the relationship in a calcium-dependent manner. Again, sodium fluoride restored significantly the amplitude of ACh current, the recovery being well-nigh complete in low, and amounting to 44% in high calcium solution. The EGTA (see Methods) did not affect the linearity of EPC-voltage or ACh current-voltage relationship, but decreased its slope; EGTA further diminished the slope of these relationships after the slope was attenuated by desensitization induced by repetitive presynaptic stimulation or ACh pulses. At that time, sodium fluoride restored to a significant degree the slope of ACh current-membrane voltage relationship; thus, sodium fluoride and EGTA exerted opposite effects on the slope of this relationship. These results support the notion that both desensitization and the effect of sodium fluoride concern the receptor rather than the channel. They also show that while calcium is involved in desensitization and the action of sodium fluoride, the anti-desensitizing effect of sodium fluoride does not depend on its chelating effect.