Lysophosphatidylcholine Increases Ca Current via Activation of Protein Kinase C in Rabbit Portal Vein Smooth Muscle Cells

Abstract

Lysophosphatidylcholine (LPC), a metabolite of membrane phospholipids by phospholipase A(2), has been considered responsible for the development of abnormal vascular reactivity during atherosclerosis. Ca(2+) influx was shown to be augmented in atherosclerotic artery which might be responsible for abnormal vascular reactivity. However, the mechanism underlying Ca(2+) influx change in atherosclerotic artery remains undetermined. The purpose of the present study was to examine the effects of LPC on L-type Ca(2+) current (I(Ca(L))) activity and to elucidate the mechanism of LPC-induced change of I(Ca(L)) in rabbit portal vein smooth muscle cells using whole cell patch clamp. Extracellular application of LPC increased I(Ca(L)) through whole test potentials, and this effect was readily reversed by washout. Steady state voltage dependency of activation or inactivation properties of I(Ca(L)) was not significantly changed by LPC. Staurosporine (100 nM) or chelerythrine (3 microM), which is a potent inhibitor of PKC, significantly decreased basal I(Ca(L)), and LPC-induced increase of I(Ca(L)) was significantly suppressed in the presence of PKC inhibitors. On the other hand, application of PMA, an activator of PKC, increased basal I(Ca(L)) significantly, and LPC-induced enhancement of I(Ca(L)) was abolished by pretreatment of the cells with PMA. These findings suggest that LPC increased I(Ca(L)) in vascular smooth muscle cells by a pathway that involves PKC, and that LPC-induced increase of I(Ca(L)) might be, at least in part, responsible for increased Ca(2+) influx in atherosclerotic artery.

Keywords: Ca2+ current; Lysophosphatidylcholine; Protein kinase C; Vascular smooth muscle.

Fig. 1

(A) Plot of Ca²⁺ currents evoked by repetitive step depolarization from -80 mV to 0 mV versus time. Horizontal bar indicates the time of LPC (1 µM), and nifedipine (1 µM) application in the bath. (B) Summary of the LPC-induced change of I_Ca(L). The value of LPC-induced change was expressed as % of control. Bath application of LPC increased the current amplitude to 142.6±8.4% (n=19). (C) Effect of LPC on current-voltage relations of I_Ca(L). I_Ca(L) was recorded at 10 mV increments between -50 and +60 mV from a holding potential of -80 mV. (D) Summary of voltage dependence of I_Ca(L) for control (○), 1 µM LPC (●), and 1 µM nifedipine. At each voltage steps, peak values of current were determined, and averaged (n=8).

Fig. 2

Steady state activation and inactivation curves of I_Ca(L) obtained before and after LPC application. (A) Steady state activation curves of I_Ca(L) in control and LPC. Chord conductance (G) was measured at different membrane potentials and normalized to the maximal chord conductance (G_max). Each data points were the means of 8 experiments and fitted using a following form of Boltzman equation; Y={1+exp[(V_1/2 - V)/k]}^-1, where V_1/2 represents half maximal activation potential and k is slope factor. Measured V_1/2 was -5.8±0.2 mV and -3.1±0.1 mV in control and LPC, respectively. (B) Steady state inactivation curves of I_Ca(L) in control and LPC. Current amplitudes (I) of test potential to 0 mV from different pretest potentials were normalized to the maximal current (I_max). Each data points were the means of 6 experiments. Curves were obtained from following form of Boltzman equation; Y={1+exp[(V - V_1/2)/k]}^-1, where V_1/2 represents half maximal inactivation potential and k is slope factor. V_1/2 was -31.4±1.2 mV and -30.2±1.6 mV in control and LPC, respectively.

Fig. 3

Effect of PKC on LPC-induced change of I_Ca(L). Plot of Ca²⁺ currents evoked by repetitive step depolarization from -80 mV to 0 mV versus time. Temporal course of change of I_Ca(L) during treatment of LPC (1 µM) alone and LPC with 3 µM chelerythrine (A), or with 100 nM PMA (B). Horizontal bars indicate the time of drugs application. (C) Summary of the effect of pretreatment of staurosporine (100 nM), chelerythrine (3 µM), and PMA (100 nM) on LPC-induced change of I_Ca(L). Data are expressed as the % of control. Number of cells in each experimental condition is indicated in parenthesis.