Alteration in voltage-dependent calcium channels in dog basilar artery after subarachnoid hemorrhage

Laboratory investigation

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Object

The L-type Ca++ channel antagonists like nimodipine have limited efficacy against vasospasm after subarachnoid hemorrhage (SAH). The authors tested the hypothesis that this is because SAH alters these channels, rendering them less responsible for contraction.

Methods

Basilar artery smooth muscle cells were isolated 4, 7, and 21 days after SAH in dogs, and Ca++ channel currents were recorded in 10-mmol/L barium. Proteins for α1 subunits of L-type Ca++ channels were measured by immunoblotting and isometric tension recordings done on rings of the basilar artery.

Results

High voltage–activated (HVA) Ca++ channel currents were significantly decreased and low voltage–activated (LVA) currents increased during vasospasm 4, 7, and 21 days after SAH (p < 0.05). Vasospasm was associated with a significant decrease in the number of cells with negligible LVA current while the number of cells in which the LVA current formed greater than 50% of the maximal current increased (p < 0.01). Window currents through LVA and HVA channels were significantly reduced. All changes correlated with the severity of vasospasm. There was an increase in protein for Cav3.1 and Cav3.3 α1 subunits that comprise T-type Ca++ channels, a decrease in L-type (Cav1.2 and Cav1.3) and an increase in R-type (Cav2.3) Ca++ channel α1 subunits. Functionally, however, isometric tension studies showed vasospastic arteries still relaxed with nimodipine.

Conclusions

Voltage-dependent Ca++ channels are altered in cerebral arteries after SAH. While decreased L-type channels may account for the lack of efficacy of nimodipine clinically, there may be other reasons such as inadequate dose, effect of nimodipine on other cellular targets, and mechanisms of vasospasm other than smooth muscle contraction mediated by activation of L-type Ca++ channels.

Abbreviations used in this paper: BA = basilar artery; HVA = high voltage activated; LVA = low voltage activated; PBS = phosphate-buffered saline; SAH = subarachnoid hemorrhage; VDCC = voltage-dependent Ca++ channel.

Article Information

Address correspondence to: R. Loch Macdonald, M.D., Ph.D., Division of Neurosurgery, St. Michael's Hospital, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8. email: Macdonaldlo@smh.toronto.on.ca.

Please include this information when citing this paper: published online March 12, 2010; DOI: 10.3171/2010.2.JNS091038.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Graphs showing voltage-dependent IBa in dog BA smooth muscle cells after SAH. A: Current-voltage curves evoked by voltage steps from Vh = −90 mV in cells from dogs 4 days (99 cells from 9 dogs, black squares), 7 days (168 cells from 12 dogs, black triangles), and 21 days (143 cells from 10 dogs, black triangles) after SAH. Circles on each graph are control cells (207 cells from 17 dogs). B: Current-voltage curves obtained from the same cells but from Vh = −50 mV (black symbols). C: Difference in currents obtained by subtraction of data from Vh = −50 mV and −90 mV (black symbols). The significant differences between control and SAH cells were that peak currents evoked from Vh=−90 and −50 mV were reduced at all times after SAH compared with control cells, with the most marked reduction being 7 days after SAH (p < 0.01, ANOVA). There was a significant increase in LVA current at all times after SAH (p < 0.05, ANOVA).

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    Amplitudes of components of IBa in dog BA smooth muscle cells at different times after SAH and in control cells and proportions of cells with LVA current after SAH. A: Maximum amplitude of IBa recorded at voltage step to +20 mV from Vh = −90 mV. There was a significant decrease at all times after SAH that was maximal 7 days after SAH (*p < 0.05, ANOVA). B: Maximum IBa amplitude at voltage step to +20 mV from Vh = −50 mV. There was a significant decrease at all times after SAH that was maximal 4 days after SAH (*p < 0.05, ANOVA). C: Magnitude of the difference in current at −10 mV between Vh = −50 and −90 mV, demonstrating a significant increase at all times after SAH and peaking at 4 days (*p < 0.05, ANOVA). All data are whole-cell currents normalized to cell capacitance from Fig. 1 and expressed as a percentage of the average maximal current from Vh = −90 mV in cells from control dogs. D: Number of cells with negligible LVA current (≤ 10% of total IBa) decreased from 26% of control cells to 0.6% 7 days after SAH (*p < 0.05, ANOVA). E: Number of cells in which LVA current formed more than 50% of the maximal current increased from 12% in controls to 26% 7 days after SAH (*p < 0.01, ANOVA). These changes reversed partially during recovery from vasospasm 21 days after SAH (number of cells and dogs same as Fig. 1).

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    Changes in activation-inactivation characteristics of IBa and window currents in dog BA smooth muscle cells after SAH. A: Activation (circles) and inactivation (squares) of IBa in smooth muscle cells from control dogs (43 cells from 4 dogs) and dogs 4 days (26 cells from 5 dogs), 7 days (32 cells from 7 dogs), and 21 days (30 cells from 6 dogs) after SAH. Filled squares represent total availability of Ca++ channels (test voltage step to 0 mV from Vh = ≤ 90 mV) with the curve representing data fit to double Boltzmann equation. Open squares are availability of LVA Ca++ channels (test step to −20 mV from Vh = −90 mV) fitted to a single Boltzmann equation. Voltage dependence of activation was derived from data in Fig. 1. Filled circles are for total current from Vh = −90 mV and open circles are for HVA current from Vh = −50 mV, both fitted to double Boltzmann equations. B: Current-voltage curves normalized to maximal values and expressed as percentage, showing window currents through LVA (solid line) and HVA (dashed line) Ca++ channels and the obvious reduction in LVA window current.

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    Western blots and quantification of α1 subunit expression after experimental SAH. Representative Western blots show that there was a decrease in L-type (Cav1.2 and Cav1.3) and an increase in R-type (Cav2.3) and T-type (Cav3.1 and Cav3.3) voltage-dependent Ca++ channel α1 subunits. For each blot, S is BA tissue 7 days after SAH, C is a control normal BA, and B is brain tissue. The bar graph shows densitometry results confirming the changes (4 SAH and 4 control BAs).

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    Isometric tension recordings of Ca++ channel subtypes in dog BA after experimental SAH. A: Relaxation of control BA and BA 7 days after SAH from baseline tension occurred in response to mibefradil (10 μmol/L) and nimodipine (1 μmol/L (p < 0.05 for both) but not to ω-agatoxin IVA (0.4 μmol/L), ω-conotoxin GVIA (2 μmol/L), or SNX-482 (1 μmol/L). The KCl concentration-contraction curves in absence (filled symbols) or presence (open symbols) of ω-agatoxin IVA (B, 0.4 μmol/L), mibefradil (C, 10 μmol/L), SNX-482 (D, 1 μmol/L), ω-conotoxin GVIA (E, 2 μmol/L), and nimodipine (F, 1 μmol/L) and in control BA or BA 7 days after SAH. There was significant inhibition of contraction only in the presence of mibefradil or nimodipine. Note the small increase in EC50 in response to KCl in arteries after SAH (4–8 rings from 2–4 dogs for all experiments).

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