Dihydropyridine inhibition of neuronal calcium current and substance P release

Abstract

Dihydropyridine (DHP) calcium channel antagonists, which inhibit the slowly inactivating or L-type cardiac calcium (Ca) current, have been shown to be ineffective in blocking 45Ca influx and Ca-dependent secretion in a number of neuronal preparations. In the studies reported here, however, the antagonist DHP nifedipine inhibited both the L-type Ca current and potassium-evoked substance P (SP) release from embryonic chick dorsal root ganglion (DRG) neurons. These results suggest that, in DRG neurons, Ca entry through L-type channels is critical to the control of secretion. The inhibition of Ca current by nifedipine was both voltage and time-dependent, significant effects being observed only on currents evoked from relatively positive holding potentials maintained for several seconds. As expected from these results, nifedipine failed to inhibit L-type Ca current underlying the brief plateau phase of the action potential generated from the cell's normal resting potential; likewise, no significant effect of the drug was observed on action potential-stimulated SP release evoked by electrical field stimulation. The results of this work are discussed in terms of an assessment of the role of L-type Ca channels in neurosecretion.

Fig. 1

Nifedipine inhibition of neuronal calcium current is voltage-dependent. Amplitudes of dorsal root ganglion cell calcium currents were measured within the last 5 ms of a 40 ms voltage step to 0 mV from one of three holding potentials, −90, −60, or −30 mV. Voltage commands were given every 10 s and nifedipine was applied continuously for 30 s. Since complete reversibility was not achieved following drug application, each cell was tested at only a single holding potential. Representative control and nifedipine reduced current records are shown with the control records normalized to peak current measured in the cell held at −60 mV. For the three holding potentials, −90, −60, −30 mV (left to right), the actual control current amplitudes were −2.7, −3.4, and −1.5 nA respectively. The calibration bar represents 10 ms

Fig. 2

A, B. Nifedipine affects steady-state inactivation of neuronal calcium current. (A) Nifedipine causes progressively greater attenuation of DRG cell calcium current as membrane holding potential is made more positive. This effect can be seen as a 12 mV hyperpolarizing shift in the steady-state inactivation curve. The durations of the prepotentials and the test pulse were 20 s and 40 ms respectively. Data points are averages (± SEM) of normalized current amplitudes either in the presence (●) or absence (■) of 100 nM nifedipine. The maximal current amplitude was defined as the current produced from a holding potential of −80 mV either with or without nifedipine. The maximal current during nifedipine application was 86±4% of the control current. Control results were obtained from 8 cells and nifedipine was applied to 4 of these cells. (B) Representative voltage clamp records show, for the same cell, the currents recorded at each of the 5 different holding potentials before (left) and after nifedipine application. Maximal current measured in the presence of nifedipine was normalized to control current amplitude to aid visual comparisons. The calibration bars represent 2 nA for control, 1.5 nA for drug, 10 ms for both. Linear washout of calcium current (less than 1%/min), which occurs under the whole cell dialysis conditions (Forscher and Oxford 1985 and unpublished data), makes some contribution to decreases in currents seen over the course of this and other long duration experiments

Fig. 3

A, B. Nifedipine inhibition of neuronal calcium current is time-dependent. (A) Data points are averages (±SEM) of normalized current amplitudes either with (●) or without (■) 100 nM nifedipine. The duration of the test pulse was 40 ms. The maximal current amplitude was defined as the current measured with a nominal prepulse duration (10 or 100 ms), with or without nifedipine. The maximal current during nifedipine application was 83 ± 3% of control. Results are averages from 4 cells. A one-tailed t-test was used to compare the average responses of nifedipine-treated and control cells (**, p < 0.05). (B) Superimposed representative currents following prepulses of 0.01, 1, 5, 10, 15, and 20 s before (left) and after application of 100 nM nifedipine. Scale bars indicate 3 nA for the control records and 2 nA for the nifedipine records, 10 ms for both

Fig. 4

A, B. Nifedipine inhibits the 60 mM KCl-induced release of substance P from DRG neurons, but does not affect release induced by electrical field stimulation. (A) Nifedipine reduced the amount of substance P released in response to electrical field stimulation to 85% of the vehicle-treated control, but this inhibitory action was not statistically significant (n.s., p > 0.10; paired Student’s t-test). (B) Nifedipine significantly reduced the amount of SP released in response to 60 mM KCl to 40% of control (**, p < 0.001). For both (A) and (B) the concentration of nifedipine was 5 μM. Substance P release is expressed as a percentage of total cellular content prior to stimulation, and baseline release was subtraced from stimulated release to calculate percentage inhibition by the DHP