The phosphatidylinositol 4-kinase inhibitor phenylarsine oxide blocks evoked neurotransmitter release by reducing calcium entry through N-type calcium channels

Abstract

The effects of the phosphatidylinositol 4-kinase inhibitor, phenylarsine oxide (PAO), on acetylcholine (ACh) release and on prejunctional Ca(2+) currents were studied at the frog neuromuscular junction using electrophysiological recording techniques. Application of PAO (30 microM) increased both spontaneous ACh release reflected as miniature end-plate potential (mepp) frequencies and evoked ACh release reflected as end-plate potential (epp) amplitudes with a similar time course. Following the initial increase in epp amplitudes produced by PAO, epps slowly declined and were eventually abolished after approximately 20 min. However, mepp frequencies remained elevated over this time period. PAO (30 microM) also inhibited the perineural voltage change associated with Ca(2+) currents through N-type Ca(2+) channels (prejunctional Ca(2+) currents) at motor nerve endings. Addition of British anti-lewisite (BAL, 1 mM), an inactivator of PAO, partially reversed both the inhibition of epps and the inhibition of the prejunctional Ca(2+) current. The effects of PAO on N-type Ca(2+) channels were investigated more directly using the whole cell patch clamp technique on acutely dissociated sympathetic neurons. Application of PAO (30 - 40 microM) to these neurons decreased the voltage-activated calcium currents through N-type Ca(2+) channels, an effect that was partially reversible by BAL. In combination, these results suggest that inhibition of neurotransmitter release by PAO occurs as a consequence of the inhibition of Ca(2+) entry via N-type calcium channels. The relationship between the effects of PAO on N-type Ca(2+) channels in motor nerve endings and in neuronal soma is discussed.

Figure 1

The effect of PAO on epp amplitudes. Open circles in the graph are epps recorded in the absence of PAO and filled circles are in the presence of PAO (30 μM). Each point represents the mean±s.e.mean from four separate preparations. In each experiment the amplitudes of eight consecutive epps (0.1 Hz) corresponding to each time period were expressed as a percentage of the first eight epps. Note the initial increase in epp amplitudes following application of PAO and the subsequent inhibition of epp amplitudes. (a), (b), (c) and (d) show averages of four consecutive epps evoked at 0.1 Hz, in (a) control, (b) following 5 min application of PAO, (c) 20 min application of PAO and (d) following the addition of BAL (1 mM) in the continued presence of PAO. See text for further details.

Figure 2

The effect of PAO on the prejunctional Ca²⁺ current. Open circles in the graph were recorded in the absence of PAO and filled circles are in the presence of PAO (30 μM). Each point represents the mean amplitude of the positive component of the perineural wave form±s.e.mean from four separate preparations (see Figure 1 and Methods for further details). In each experiment the amplitudes of the eight consecutive prejunctional Ca²⁺ components corresponding to each time period (evoked at 0.05 Hz) were expressed as a percentage of the first eight perineural recordings made in the absence of PAO. Note the gradual reduction in the prejunctional Ca²⁺ current occurs during the initial increase in epp amplitude (compare with Figure 1). (a), (b), (c) and (d) are averages of eight consecutive perineural wave-forms (recorded from a separate experiment) evoked at 0.05 Hz. (a) is in control perineural recording solution (see Methods). (b) is following 5 min and (c) 20 min in the continued presence of PAO. (d) shows the perineural waveform following the addition of BAL (1 mM) in the continued presence of PAO. ^*Indicates the stimulation artefact. Note that both the abolition of the epp by PAO, shown in Figure 1, and the reduction in the prejunctional Ca²⁺ current (including a decrease in the degree of repetitive firing) by PAO occur with a similar time course. In addition both effects are partially reversed following the addition of BAL in the presence of PAO. See text for further experimental details.

Figure 3

Inhibition of Ca²⁺ currents in acutely dissociated-sympathetic neurons by the N-type Ca²⁺ channel blockers ω-CTX (a) and Cd²⁺ (b). Cells were held at −80 mV. Voltage-activated Ca²⁺ currents were triggered by voltage steps of 120 ms duration from the holding potential (−80 mV) to 0 mV. This depolarizing step was applied once every 20 s. In (a), the individual records that comprise the averaged peak current amplitudes (−1.16±0.05 nA, n=6 stimuli) ranged from −1.27 to −0.95 nA in control conditions, whilst the individual records comprising the averaged peak response made after treatment with ω-CTX ranged from −0.18 to −0.15 nA. In the Cd²⁺ experiments (b), an analysis of variance followed by multiple comparisons using Bonferroni's method revealed highly statistically-significant differences between control (−1.16±0.03 nA, n=6) and Cd²⁺ treatment (−0.18±0.02 nA, n=6) and between Cd²⁺ exposure and the wash period (−1.14±0.03 nA, n=3; P<0.05) but not between the control and wash periods. For further details of the N-type Ca²⁺ currents in acutely-dissociated autonomic neurons in guinea-pig (see Barajas-Lopez et al., 1996).

Figure 4

Effects of PAO on N-type Ca²⁺ currents and its reversal by BAL (1 mM). (a) shows individual traces depicting the inhibitory effect of a 20 min exposure to PAO (30 μM) and its partial reversal by BAL in normal Ca²⁺ solutions. (b) shows the averaged data from another experiment in which Ba²⁺ was used as the charge carrier. In this experiment, the mean peak Ba²⁺ current through N-type Ca²⁺ channels (−1.25±0.01 nA) was reduced by PAO (−0.47±0.01 nA) and reversed by coapplication of BAL (0.58±0.01 nA, n=4 for all). An analysis of variance followed by a multiple comparison using Bonferroni's method revealed highly statistically-significant differences between the three conditions (P<0.05). (c) shows that higher concentrations of PAO (40 μM), in addition to inhibiting Ca²⁺ currents, also increased the inward holding current observed before the depolarizing step. In the same experiment, BAL reversed both effects in a time dependent manner (d). Cells were held at −80 mV. Voltage-activated Ca²⁺ currents were triggered by voltage steps of 120 ms duration from the holding potential (−80 mV) to 0 mV. This depolarizing step was applied once every 20 s. For further details, see text.