Activation of potassium conductance by ophiopogonin-D in acutely dissociated rat paratracheal neurones

Abstract

1. The effect of ophiopogonin-D (OP-D), a steroidal glycoside and an active component of Bakumondo-to, a Chinese herbal antitussive, on neurones acutely dissociated from paratracheal ganglia of 2-week-old Wistar rats was investigated using the nystatin-perforated patch recording configuration. 2. Under current-clamp conditions, OP-D (10 microM) hyperpolarized the paratracheal neurones from a resting membrane potential of -65.7 to -73.5 mV. 3. At the concentration of 1 microM and above, OP-D concentration-dependently activated an outward current accompanied by an increase in the membrane conductance under voltage-clamp conditions at a holding potential of -40 mV. 4. The reversal potential of the OP-D-induced current (I(OP-D)) was -79.4 mV, which is close to the K(+) equilibrium potential of -86.4 mV. The changes in the reversal potential for a 10 fold change in extracellular K(+) concentration was 53.1 mV, indicating that the current was carried by K(+). 5. The I(OP-D) was blocked by an extracellular application of 1 mM Ba2+ by 59.0%, but other K(+) channel blockers, including 4-aminopyridine (3 mM), apamin (1 microM), charybdotoxin (0.3 microM), glibenclamide (1 microM), tolbutamide (0.3 mM) and tetraethylammonium (10 mM), did not inhibit the I(OP-D). 6. OP-D also inhibited the ACh- and bradykinin-induced depolarizing responses which were accompanied with firing of action potentials. 7. The results suggest that OP-D may be of benefit in reducing the excitability of airway parasympathetic ganglion neurones and consequently cholinergic control of airway function and further, that the hyperpolarizing effect of OP-D on paratracheal neurones via an activation of K(+) channels might explain a part of mechanisms of the antitussive action of the agent.

Figure 1

Chemical structure of ophiopogonin-D (OP-D). Fuc, Rha and Xyl stand for fucose, rhamnose and xylose, respectively.

Figure 2

The response for OP-D. (A) OP-D hyperpolarized the paratracheal neuron. A dotted line shows the level of −60 mV. The figure is representative of seven reproducible observations. (B) The outward current induced by OP-D at a holding potential (V_H) of −40 mV. A brief hyperpolarizing pulse of −20 mV with 500 ms duration was applied every 5 s. A dotted line shows the zero current level. This current response was obtained from the different neuron shown in A. The figure is representative of seven reproducible experiments.

Figure 3

Concentration-response relationship for OP-D. (A) Typical current traces elicited by various concentrations of OP-D at a V_H of −40 mV. (B) Concentration-response relationship for OP-D. Each point represents a mean outward current from 4 – 5 neurones; vertical bars show±s.e.mean.

Figure 4

Current-voltage relationships for the OP-D-induced response. (A) Currents elicited by 10 μM OP-D at various V_Hs. (B) Representative current-voltage relationships of OP-D-induced currents (I_OP-D) in external solution with two different K⁺ concentrations of 5 (open circle) and 20 mM (closed circle). The intracellular K⁺ concentration was 150 mM throughout the experiments. The data were obtained from the same neurone as shown in A. (C) The relationship between the external K⁺ concentration ([K⁺]_o) and the reversal potential of the I_OP-D (E_OP-D). The change in the E_OP-D values for a 10 fold change in [K⁺]_o was 53.1 mV. The dotted line represents the change in 58 mV for 10 fold change of [K⁺]_o predicted from the Nernst equation. Each point is the mean reversal potential of 5 – 6 neurones. No error bar was depicted, because the size of error bars was within each closed circle.

Figure 5

Effects of K⁺ channel blockers. (A) Effect of Ba²⁺ on the I_OP-D. (B) Effect of TEA on the I_OP-D. (C) Effects of various K⁺ channel blockers on the I_OP-D. Each column is the average of 4 – 7 neurones. The I_OP-D caused by OP-D alone was taken as 1.

Figure 6

Effect of OP-D on ACh- and bradykinin-induced depolarizing responses. All recordings were obtained under current clamp conditions. Dashed line represents the level of −60 mV. (A) Effect on ACh-induced depolarization with the action potential firing. This recording is representative of five reproducible experiments. (B) Effect on bradykinin-induced depolarization accompanied with the action potential firing. The recording was obtained from the different neurone shown in A. The figure is representative of four reproducible experiments.