Electrophysiological effects of protopine in cardiac myocytes: inhibition of multiple cation channel currents

Abstract

Protopine (Pro) from Corydalis tubers has been shown to have multiple actions on cardiovascular system, including anti-arrhythmic, anti-hypertensive and negative inotropic effects. Although it was thought that Pro exerts its actions through blocking Ca(2+) currents, the electrophysiological profile of Pro is unclear. The aim of this study is to elucidate the ionic mechanisms of Pro effects in the heart. In single isolated ventricular myocytes from guinea-pig, extracellular application of Pro markedly and reversibly abbreviates action potential duration, and decreases the rate of upstroke (dV/dt)(max), amplitude and overshoot of action potential in a dose-dependent manner. Additionally, it produces a slight, but significant hyperpolarization of the resting membrane potential. Pro at 25, 50 and 100 microM reduces L-type Ca(2+) current (I(Ca,L)) amplitude to 89.1, 61.9 and 45.8% of control, respectively, and significantly slows the decay kinetics of I(Ca,L) at higher concentration. The steady state inactivation of I(Ca,L) is shifted negatively by 5.9 - 7.0 mV (at 50 - 100 microM Pro), whereas the voltage-dependent activation of I(Ca,L) remains unchanged. In contrast, Pro at 100 microM has no evident effects on T-type Ca(2+) current (I(Ca,T)). In the presence of Pro, both the inward rectifier (I(K1)) and delayed rectifier (I(K)) potassium currents are variably inhibited, depending on Pro concentrations. Sodium current (I(Na)), recorded in low [Na(+)](o) (40 mM) solution, is more potently suppressed by Pro. At 25 microM, Pro significantly attenuated I(Na) at most of the test voltages (-60 approximately +40 mV, with a 53% reduction at -30 mV. Thus, Pro is not a selective Ca(2+) channel antagonist. Rather, it acts as a promiscuous inhibitor of cation channel currents including I(Ca,L), I(K), I(K1) as well as I(Na). These findings may provide some mechanistic explanations for the therapeutic actions of Pro in the heart.

Figure 1

Original traces of action potentials recorded from a representative guinea-pig ventricular myocyte before (control), after exposure to Pro (25 or 50 μM) and upon washout.

Figure 2

Effects of Pro on I_Ca,L. (A) The superimposed I_Ca,L traces were from a typical myocyte. Inset: A 1-s preconditioning pulse of −40 mV from HP of −80 mV was followed by a depolarizing pulse to 0 mV for 300 ms to elicit I_Ca,L. After exposure to 25 μM Pro for 2 min (p), I_Ca,L amplitude was reduced by 18% relative to control (c). It was partially recovered subsequent to a 2-min washout (w). (B) A typical example of time course of Pro (100 μM) action on I_Ca,L and subsequent washout of the drug effect. (C) Dose-dependent inhibition of Pro on I_Ca,L amplitude. The data were calculated as the ratio to the control currents (n=5∼7 for each group). ^*P<0.05, †P<0.01 vs controls.

Figure 3

Current-voltage relationship of I_Ca,L in the absence and presence of 50 μM Pro. A 1-s preconditioning pulse of −40 mV from HP of −80 mV was followed by various depolarizing pulses (−30∼+60 mV, voltage increment 10 mV, duration 300 ms) to elicit I_Ca,L at 0.2 Hz. (A) Original recordings of I_Ca,L at different voltages under control condition (left), after exposure to 50 μM Pro, and upon 2-min washout. (B) Average data on effects of Pro on I_Ca,L over the entire voltage range. ^*P<0.05, †P<0.01 vs controls, n=7.

Figure 4

Steady-state inactivation and voltage-dependent conductance of I_Ca,L. (A) Left panel: a double-pulse protocol, 1-s conditioning pulses to various voltages (−80∼0 mV in increments of 10 mV) from HP of −80 mV followed by a fixed test pulse to 0 mV for 300 ms, which was applied to examine the steady-state inactivation of I_Ca,L. Right panel: typical traces showing I_Ca,L at different steady inactivation states. (B) Effects of Pro on I_Ca,L steady-state inactivation and conductance-voltage relationship. Smooth curves are Boltzmann fittings for control (solid line) and Pro-treated (dashed line) groups. (See methods for details on calculation of I/I_max, g/g_max and Boltzmann fitting). n=7 for each data.

Figure 5

Pro does not affect properties of I_Ca,T. (A) Pro has no effect on I_Ca,T recorded at −30 mV. The currents elicited from HP of −80 mV consist of I_Ca,T and I_Ca,L (upper traces), while only I_Ca,L is activated from a HP of −40 mV (middle traces). The subtraction of currents at a HP of −40 mV from those at −80 mV measures the I_Ca,T at −30 mV (lower traces). (B) Pro has no effect on I_Ca,T at −40 mV. I_Ca,T is the predominant component at this voltage, as the total current is completely blocked by NiCl₂ (50 μM).

Figure 6

Effects of Pro on current-voltage relationship of I_K1. (A) From left to right, representative traces of I_K1 at different voltages under control, with Pro 50 μM and after washout, respectively. (B) Continuous recordings of I_K1 during repetitive application and washout of Pro at 25, 50, and 100 μM. (C) Dose-dependent inhibition of I_K1 by Pro (25–100 μM, n=7). The inset shows the voltage protocol.

Figure 7

Effects of Pro on I-V relationship of I_K. (A) Families of I_K traces were obtained by depolarization to different voltages (−30 mV∼+50 mV) for 6 s from a HP of −40 mV in cadmium-containing bath solution (CdCl₂ 0.2 mM), prior to and after application of 50 μM Pro. (B) The averaged data on effects of Pro (50 μM) on I_K-voltage relationship. It significantly suppressed I_K at voltages higher than −10 mV. ^*P<0.05, †P<0.01 vs controls, n=8.

Figure 8

Suppression of I_Na by Pro. I_Na was recorded with large pipette (<1.25 MΩ) in low [Na⁺]_o (40 mM), cadmium-containing (CdCl₂ 0.2 mM) bath solution. Various depolarizing pulses to −60∼+40 mV from HP of −100 mV were applied to measure the whole profile of I_Na. ^*P<0.05, †P<0.01 vs controls (n=6).