Alterations of voltage-dependent calcium channel currents in basilar artery smooth muscle cells at early stage of subarachnoid hemorrhage in a rabbit model

Abstract

Objective: To investigate the changes in the currents of voltage-dependent calcium channels (VDCCs) in smooth muscle cells of basilar artery in a rabbit model of subarachnoid hemorrhage (SAH).

Methods: New Zealand white rabbits were randomly divided into five groups: sham (C), normal (N), 24 hours (S1), 48 hours (S2) and 72 hours (S3) after SAH. Non-heparinized autologous arterial blood (1 ml/kg) was injected into the cisterna magna to create SAH after intravenous anesthesia, and 1 ml/kg of saline was injected into cisterna magna in the sham group. Rabbits in group N received no injections. Basilar artery in S1, S2, S3 group were isolated at 24, 48, 72 hours after SAH. Basilar artery in group C was isolated at 72 hours after physiological saline injection. Basilar artery smooth muscle cells were isolated for all groups. Whole-cell patch-clamp technique was utilized to record cell membrane capacitance and VDCCs currents. The VDCCs antagonist nifedipine was added to the bath solution to block the Ca(++) channels currents.

Results: There were no significant differences in the number of cells isolated, the cell size and membrane capacitance among all the five groups. VDCC currents in the S1-S3 groups had higher amplitudes than those in control and sham groups. The significant change of current amplitude was observed at 72 hours after SAH, which was higher than those of 24 and 48 hours. The VDCCs were shown to expression in human artery smooth muscle cells.

Conclusions: The changes of activation characteristics and voltage-current relationship at 72 hours after SAH might be an important event which leads to a series of molecular events in the microenvironment of the basilar artery smooth muscle cells. This may be the key time point for potential therapeutic intervention against subarachnoid hemorrhage.

Figure 1. Characteristics of basilar artery smooth muscle cells.

The graph A shows no significant difference for the membrane capacitance of BA smooth muscle cells in all group(P>0.05, n = 8/group).The graph B showed a representative image of smooth muscle cells isolated from the BA.

Figure 2. Activation curve of VDCCs in rabbit basilar artery smooth muscle cells after SAH.

The graph A and B showed I-V plot at HP of −90mv and −50mv respectively. The ordinate was currents density (pA/pF) and the abscissa was voltage (mV). The two I-V plots showed the current density of VDCCs in S1–S3 group, which was bigger than that in N and C group at HP of −90mV and −50mV (P<0.05, n = 8/group). The current density in S3 group was bigger than those in S1 and S2 group (P<0.05, n = 8/group).The current density was not significantly different between C and N group (P>0.05, n = 8/group).

Figure 3. Activation characteristics of VDCCs in rabbit basilar artery smooth muscle cells after SAH.

Graph A and B showed steady-state activation curves at HP of −90mV and −50mV respectively. The abscissa was voltage (mV) and the ordinate was relative conductivity (G). Compared with C and N group, steady-state activation curves of S1–S3 groups at HP of −90mV and −50mV shifted to the left (p<0.05, n = 8/group).

Figure 4. Voltage-dependent Ca+2 channel currents in rabbit basilar artery smooth muscle cells after SAH.

The graph A and B showed steady-state inactivation curves at HP of −90mV and −50mV respectively. The abscissa was voltage (mV) and the ordinate was relative conductivity (G). Compared with C group, steady-state inactivation curves of S1–S3 groups at HP of −90mV and −50mV shifted to the right, but it had only significant difference in S3 group (P<0.05, n = 8/group).

Figure 5. Analyses of current–voltage relationship for Ca+2 channel currents in rabbit basilar artery smooth muscle cells after SAH.

The graph A , B and C showed the inhibitory effect of nifedipine on the currents of VDCCs in control (C) group, sham (N) group and SAH group respectively. The results confirmed nifedipine could inhibit the currents of VDCCs in every group.

Figure 6. Expression of VDCCs in human brain vascular smooth muscle cells.

A, Expression of different types of VDCCs were detected by real-time RT-PCR. B, Wound healing assay was performed with or without nifedipine. *p<0.05 versus VEGF treated group.