Dihydropyridine-insensitive calcium currents contribute to function of small cerebral arteries

Abstract

Although dihydropyridines are widely used for the treatment of vasospasm, their effectiveness is questionable, suggesting that other voltage-dependent calcium channels (VDCCs) contribute to control of cerebrovascular tone. This study therefore investigated the role of dihydropyridine-insensitive VDCCs in cerebrovascular function. Using quantitative PCR and immunohistochemistry, we found mRNA and protein for L-type (Ca(V)1.2) and T-type (Ca(V)3.1 and Ca(V)3.2) channels in adult rat basilar and middle cerebral arteries and their branches. Immunoelectron microscopy revealed both L- and T-type channels in smooth muscle cell (SMC) membranes. Using patch clamp electrophysiology, we found that a high-voltage-activated calcium current, showing T-type channel kinetics and insensitivity to nifedipine and nimodipine, comprised approximately 20% of current in SMCs of the main arteries and approximately 45% of current in SMCs from branches. Both components were abolished by the T-type antagonists mibefradil, NNC 55-0396, and efonidipine. Although nifedipine completely blocked vasoconstriction in pressurized basilar arteries, a nifedipine-insensitive constriction was found in branches and this increased in magnitude as vessel size decreased. We conclude that a heterogeneous population of VDCCs contributes to cerebrovascular function, with dihydropyridine-insensitive channels having a larger role in smaller vessels. Sensitivity of these currents to nonselective T-type channel antagonists suggests that these drugs may provide a more effective treatment for therapy-refractory cerebrovascular constriction.

Figure 1

Distribution of L- and T-type channels in basilar and middle cerebral arteries. (A–F) L-type channels (Ca_V1.2) are expressed in membranes of SMCs (A–C, arrow) but not endothelial cells (ECs; D, E). Staining of Ca_V1.2 is blocked with the immunogenic peptide (F). (G–L) T-type channels (Ca_V3.1) are expressed in cytoplasm and membranes (arrows) of SMCs (G–I, arrow) and ECs (J, K). Staining is abolished after incubation with immunogenic peptide (L). (M–R) Ca_V3.2 channels are expressed in SMCs (M–O) and ECs (P, Q) similar to Ca_V3.1. Staining is abolished after incubation with immunogenic peptide (R). Nuclei (Nu) are stained with propidium iodide (red). Images in C, I, and O are higher power magnifications of MCA VDCC staining. The longitudinal vessel axis is left to right in all panels. Staining is indicative of at least four preparations.

Figure 2

Location of L- and T-type channels at the ultrastructural level. (A) Tissue prepared for immunoelectron microscopy shows normal vessel morphology with smooth muscle cells (SMCs) and endothelial cells (ECs) separated by the internal elastic lamina (IEL); with myosin filaments oriented to the longitudinal cell axis and accumulations of mitochondria within the cells. (B–G) Gold labelled Ca_V1.2 (10 nm) and Ca_V3.1 (5 nm) were found in the SMC cytoplasm and at cell membranes (B, C, E–G). Ca_V3.1 was also found in ECs (D). Boxes in C–E shown at higher magnification in panels a–e. Gold particles were rarely found over nuclei (nu: C, D, panel c), adventitia (adv: C, panel d), IEL (B, E, panels a, b, and e, F, G), or lumen (panel c), and staining was abolished after incubation with appropriate corresponding immunogenic peptide (insets in B, E).

Figure 3

Voltage-dependent calcium currents in cerebrovascular SMCs. (A) Isolated SMCs were spindle-shaped and expressed L- and T-type channels (Ca_V1.2 and 3.1) with reduced Ca_V3.2 expression. Immunohistochemistry images were converted to black on white. (B) Ba²⁺ currents evoked from a holding potential of −70 mV with incremental 10 mV depolarizing steps between −50 and +50 mV. (C, D) Group data for currents evoked from −50, −70, and −100 mV, normalized to cell capacitance (C) or peak current (D). (E) Activation and inactivation curves from −70 and −100 mV. n=number of cells/animals.

Figure 4

Effect of nifedipine on calcium currents. (A) Calcium currents evoked from holding potential of −70 mV were reduced by nifedipine (1 μmol/L) and restored on wash out. (B) Group data of nifedipine-sensitive and insensitive currents, n=8 cells. (C) Effects of nifedipine did not increase with time of application to current evoked by repetitive depolarizing steps from −70 to +10 mV, n=4 cells. (D–F) Nifedipine-insensitive currents had significantly faster activation (D, E) and inactivation time constants (D, F). Traces in D have been normalized to maximum current evoked with a depolarizing step to 0 mV from a holding potential of −70 mV. (G, H) Nifedipine-insensitive tail currents were fitted with two time constants that were both significantly slower than control, n=9 cells. ^*P<0.05 significantly different from paired control.

Figure 5

Effect of L-type blockers on calcium currents. (A) Calcium currents evoked from holding potential of −70 mV were reduced by nimodipine (10 μmol/L) and abolished by addition of mibefradil (1 μmol/L). (B) Group data n=10 cells. (C, D) After inhibition of L-type currents with nifedipine (C) or nimodipine (D), window currents were left shifted due to a significant change in the activation and inactivation curves of the dihydropyridine-resistant current (see Supplementary Table 3 for details). (E) Calcium currents evoked from holding potential of −70 mV were reduced by nifedipine (1 μmol/L), but not affected by addition of diltiazem (10 μmol/L). (F) Group data n=4 cells.

Figure 6

Effect of L- and T-type blockers on calcium currents. (A) Calcium currents in SMCs from the main basilar artery, evoked from holding potential of −70 mV, were reduced by nifedipine (1 μmol/L) and abolished by addition of mibefradil (1 μmol/L; n=4 cells). (B) In SMCs from basilar side branches, there was a larger nifedipine-insensitive component (1 μmol/L) that was abolished with mibefradil (1, 3 μmol/L; n=9 cells). (C–F) T-type blockers, mibefradil (1 μmol/L), and NNC 55-0396 (1 μmol/L) blocked all evoked current in SMCs from the main basilar artery. (D, F) Group data n=4 cells.

Figure 7

Effect of L- and T-type blockers on myogenic tone of basilar artery (A) and branches (B–F). (A–C) Nifedipine-resistant tone increased with decreasing vessel size. In the main basilar artery (vessel diameter 319 μm), nifedipine (1 μmol/L) completely abolished vascular tone, and mibefradil (1 μmol/L) had no additional effect (A). In the larger branches (vessel diameter 172 μm), addition of mibefradil (1 μmol/L) after nifedipine (1 μmol/L) caused a further significant relaxation (B). In the smaller side branches (vessel diameter 101 μm), nifedipine (1 μmol/L) only partially reduced tone at pressures above 60 mm Hg and addition of mibefradil (3 μmol/L) significantly reduced tone at all pressures (C). (D–F) Effects of L- and T-type blockers in the larger branches of the basilar artery. Nifedipine (0.1, 1 μmol/L) caused a dose-dependent relaxation, but did not completely abolish tone (D). Mibefradil alone inhibited constriction at 1 μmol/L, but induced constriction at 10 μmol/L (E). The T-type blocker, NNC 55-0396, caused a dose-dependent inhibition of constriction that was further inhibited by the addition of nifedipine (1 μmol/L) (F). ^*P<0.05, two-way analysis of variance, n=4 to 6 vessels.