Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons

Abstract

Aim: To investigate the effect of ginsenoside Rb1 on voltage-gated calcium currents in cultured rat hippocampal neurons and the modulatory mechanism.

Methods: Cultured hippocampal neurons were prepared from Sprague Dawley rat embryos. Whole-cell configuration of the patch-clamp technique was used to record the voltage-gated calcium currents (VGCCs) from the hippocampal neurons,and the effect of Rb1 was examined.

Results: Rb1 (2-100 μmol/L) inhibited VGCCs in a concentration-dependent manner, and the current was mostly recovered upon wash-out. The specific L-type Ca(2+) channel inhibitor nifedipine (10 μmol/L) occluded Rb1-induced inhibition on VGCCs. Neither the selective N-type Ca(2+) channel blocker ω-conotoxin-GVIA (1 μmol/L), nor the selective P/Q-type Ca(2+) channel blocker ω-agatoxin IVA (30 nmol/L) diminished Rb1-sensitive VGCCs. Rb1 induced a leftward shift of the steady-state inactivation curve of I(Ca) to a negative potential without affecting its activation kinetics or reversal potential in the I-V curve. The inhibitory effect of Rb1 was neither abolished by the adenylyl cyclase activator forskolin (10 μmol/L), nor by the PKA inhibitor H-89 (10 μmol/L).

Conclusion: Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels, without affecting the N-type or P/Q-type Ca(2+) channels in hippocampal neurons. cAMP-PKA signaling pathway is not involved in this effect.

Figure 1

(A) Phase-contrast image showing a single patch recording from 7-d cultured hippocampal neurons for the recording of the VGCCs. Scale bar, 10 μm. (B) Pharmacological separation of the VGCC subtypes in hippocampal neurons. Upper panel, inward Ca²⁺ channel Ba²⁺ currents evoked by pulses from −60 mV to 0 mV at the times indicated in the lower panel. Lower panel, time course of effects of ω-conotoxin GVIA (1 μmol/L), ω-agatoxin IVA (30 nmol/L) and nifedipine (10 μmol/L) on the Ba²⁺ current amplitude.

Figure 2

Effect of the extracellular solution, Rb1 (1 μmol/L) and Rb1 (10 μmol/L), on the I_Ba (A, B, and C, respectively). (1) Before drug application; (2) drug application; (3) washout. (D) represents the time course of the experiment corresponding to (C).

Figure 3

Rb1 inhibited the I_Ba in hippocampal neurons, and this inhibitory effect was eliminated after the application of nifedipine (A). Neither ω-conotoxin-GVIA nor ω-agatoxin IVA diminished the Rb1-sensitive I_Ba (B and C, respectively). Upper panel, pairs of the inward currents evoked by pulses from −60 to +0 mV (0–+20 mV) at the times indicated in the lower panel. Lower panel, time course of the effects of 10 μmol/L Rb1 on the I_Ba amplitude before and after application of the Ca²⁺ channel antagonists (10 μmol/L nifedipine, 1 μmol/L ω-conotoxin GVIA and 30 nmol/L ω-agatoxin IVA). The bar graphs for Rb1 inhibition (mean±SEM, n=5 for Rb1) on the I_Ba in cells untreated or treated with Ca²⁺ channel antagonists. ^cP<0.01 compared with Rb1 treatment alone.

Figure 4

The I-V relationships of the I_Ba showed the inhibitory effects of 10 μmol/L Rb1 on the VGCCs (n=6). The holding potential was −60 mV, and the test potentials ranged from −70 mV to +70 mV in 10 mV increments.

Figure 5

The effects of 10 μmol/L Rb1 on the voltage-dependence of the steady-state of I_Ba inactivation (n=6). The data were fitted to the Boltzmann equation.

Figure 6

(A) The percentage of inhibitory action by 10 μmol/L Rb1 and Rb1 co-administered with forskolin. (B) The percentage of inhibitory action by 10 μmol/L Rb1 in the presence and absence of H-89.