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Neurosci Lett. Author manuscript; available in PMC Jul 25, 2009.
Published in final edited form as:
PMCID: PMC2518203

Temporal Profile of Potassium Channel Dysfunction in Cerebrovascular Smooth Muscle after Experimental Subarachnoid Haemorrhage


The pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) involves sustained contraction of arterial smooth muscle cells that is maximal 6 to 8 days after SAH. We reported that function of voltage-gated K+ (KV) channels was significantly decreased during vasospasm 7 days after SAH in dogs. Since arterial constriction is regulated by membrane potential that in turn is determined predominately by K+ conductance, the compromised K+ channel dysfunction may cause vasospasm. Additional support for this hypothesis would be demonstration that K+ channel dysfunction is temporally coincident with vasospasm. To test this hypothesis, SAH was created using the double hemorrhage model in dogs and smooth muscle cells from the basilar artery, which develops vasospasm, were isolated 4 (early vasospasm), 7 (during vasospasm) and 21 (after vasospasm) days after SAH and studied using patch-clamp electrophysiology. We investigated the 2 main K+ channels (KV and large-conductance voltage/Ca2+ activated (KCa) channels). Electrophysiologic function of KCa channels was preserved at all times after SAH. In contrast, function of KV channels was significantly decreased at all times after SAH. The decrease in cell size and degree of KV channel dysfunction was maximal 7 days after SAH. The results suggests that KV channel dysfunction either only partially contributes to vasospasm after SAH or that compensatory mechanisms develop that lead to resolution of vasospasm before KV channels recover their function.

Keywords: Cerebral vasospasm, K+ channels, subarachnoid haemorrhage


Cerebral vasospasm is transient narrowing of intradural subarachnoid arteries seen angiographically 4 to 12 days after subarachnoid hemorrhage (SAH). It causes cerebral ischemia, infarction and sometimes death in these patients. A mechanism of vasospasm may be sustained contraction of smooth muscle cells in the large conducting arteries of the circle of Willis [11].

The double hemorrhage model of SAH in dogs reproduces vasospasm with a similar temporal profile and severity to that seen in patients [14]. We reported that the function of voltage-gated K+ (KV) channels was significantly decreased during vasospasm 7 days after SAH in dogs [6]. Arterial diameter is regulated by smooth muscle membrane potential that controls influx of external Ca2+ through voltage-gated Ca2+ channels [19]. Membrane potential in turn is largely, but not solely, determined by K+ conductance. This suggests that decreased K+ channel function contributes to vasospasm. Additional evidence to support this hypothesis would be that pharmacologic or molecular manipulation of KV channels prevented vasospasm and that the time course of alteration in KV channels correlated with vasospasm. Pharmacologic blockade of K+ channels has prevented experimental vasospasm but these drugs are nonspecific [9;10;23]. Molecular manipulation of proteins in dog basilar artery is not possible at present. Therefore, this study investigated function of the 2 main K+ channels in dog basilar artery (KV and large-conductance voltage/Ca2+-activated K+ [KCa] channels) during the time course of vasospasm after SAH in dogs.


Dog Model of SAH and Isolation of Smooth Muscle Cells

Mongrel dogs weighing 15-20 kg were randomly allocated to be controls or subjected to SAH [13;21]. Dogs were placed under general anesthesia and underwent baseline cerebral angiography on day 0. After angiography, they were turned prone and the cisterna magna was punctured. 0.3 ml kg-1 cerebrospinal fluid was removed and 0.5 ml kg-1 of autologous, arterial, nonheparinized blood was injected into the cisterna magna at 5 ml min-1. The cisternal blood injection was repeated on day 2. Control animals were sacrificed after angiography on day 0, while all other animals underwent repeat angiography 4, 7 or 21 days after SAH and were then sacrificed. Animals sacrificed on day 21 had angiography on day 7. Procedures on animals were approved by the Institutional Animal Care Committee.

Smooth muscle cells were isolated from basilar arteries by enzymatic dissociation [6;15;21]. Animals were euthanized under general anesthesia by exsanguination and perfusion with ice-cold phosphate-buffered saline (PBS, pH 7.4). The basilar artery was removed and placed in dissection solution (in mmol l-1: NaCl 130, KCl 5, MgCl2 1.3, 4-[2-hydroxyethyl]piperazine-1-ethanesulfonic acid [HEPES] 10, glucose 5 with 100 units ml-1 penicillin and 0.1 mg ml-1 streptomycin). Arteries were cut into small pieces and smooth muscle cells isolated by enzymatic digestion in the dissection solution containing 500 units ml-1 collagenase type IV, 50 units ml-1 elastase, 100 units ml-1 DNase I and 1 mg ml-1 trypsin inhibitor.


Cells were plated in a chamber (180 μl, Warner Instruments, Hamden, CT) mounted on an inverted microscope (Nikon Diaphot 200, Nikon, Tokyo, Japan)[6;15;21]. Macroscopic currents were recorded using the whole-cell configuration of the patch-clamp method. Currents were amplified using an Axopatch-1D amplifier (Axon Instruments, Union City, CA) and digitized at 12-bits using a DigiData 1200 interface controlled by pClamp 6 (Axon Instruments, Union City, CA). Series resistance in whole-cell recordings was usually < 5 MΩ and was compensated by ≥ 80%. Recordings were at room temperature (22°C). Current analyses were performed using custom-written software in Igor (Wavemetrics, Lake Oswego, OR). KV currents were isolated from KCa currents by intracellular Ca2+ buffering with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA, tetrapotassium salt) and block of L-type Ca2+ channels by external Cd2+. The intracellular solution for KV currents contained (in mmol l-1): KCl 88.6, KOH 6.4, K2ATP 5, NaGTP 0.1, BAPTA 10, MgCl2 2.5, HEPES 10, pH 7.2 with KOH. The resulting currents were insensitive to paxilline (1 μmol l-1) and glyburide (10 μmol l-1), showing no contamination with KCa current and no contribution of ATP-sensitive K+ (KATP) channels. For lengthy protocols such as inactivation and recovery from inactivation, brief hyperpolarizing pulses were included at the end of each episode to facilitate calculation and subtraction of linear leak.

KCa currents recorded with standard 140 mmol l-1 K+ internal solutions usually exceeded 15-20 nA in response to the large depolarizing steps (> 120 mV), which was necessary to determine KCa voltage-dependency and kinetics. Such currents imposed significant voltage-control errors despite series resistance compensation and usually resulted in oscillations in the compensation circuitry and seal loss. To minimize this, K+ was lowered and replaced with choline, as previously reported by others [2]. The intracellular KCa solution thus consisted of (in mmol l-1): KCl 20, choline-Cl 120, MgCl2 1.5, CaCl2 2.65, ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) 5, HEPES 5, glucose 5, pH 7.2 with KOH. Under these conditions, outward K+ currents were dominated by KCa current, as demonstrated by near complete block of such currents by 1 μmol l-1 paxilline, a fungal mycotoxin that specifically blocks KCa channels with high affinity [8;18].

All chemicals were from Sigma-Aldrich (St. Louis, MO).

Data Analysis

Vasospasm was assessed by comparing diameters of basilar arteries on angiograms taken at constant exposure and magnification and measured at 5 predetermined points by 2 blinded observers using an optical micrometer. Macroscopic current analyses were performed using custom-written software in Igor. Data were compared using t-tests or analysis of variance (ANOVA, SigmaStat, SPSS, Chicago, IL). P < 0.05 was considered significant. Linear regression was performed using the least-squares method and fits to curves were performed using the Levenburg-Marquardt algorithm in Sigmaplot (Sigmaplot, SPSS) or Igor. All data are means ± standard errors of the means.


Time Course of Vasospasm

Angiography showed significant reduction in basilar artery diameter at all 3 times after SAH (4, 7 and 21 days, P < 0.001 as compared to control, Fig. 1). The reduction of basilar artery diameter was 55% 4 (0.71 ± 0.07, n = 8), 54% 7 (0.72 ± 0.34, n = 17) and 33% 21 days (1.04 ± 0.28, n = 7) after SAH compared to a control diameter of 1.55 ± 0.15 mm (n = 39). The basilar diameter was significantly larger 21 days after SAH compared to 4 or 7 days post SAH (P < 0.01) whereas there was no difference between the diameter 4 and 7 days after SAH. There were no significant differences in physiological parameters (blood pressure, endtidal PCO2, PAO2, PACO2, body temperature and heart rate) between groups at day 0 or in SAH dogs between day 0 and the day of sacrifice (data not shown).

Figure 1
Quantification of arterial diameter and myocytes properties

Temporal Changes in Size of Basilar Artery Myocytes After SAH

Cell capacitance, which is a measure of cell size, was measured during whole-cell patch clamp experiments (Fig. 1B). There was a significant decrease in cell capacitance at all 3 times after SAH as compared to controls (28.3 ± 0.6, n = 76 cells, P < 0.01). There was a 9% decrease in cell capacitance 4 (25.7 ± 0.8, n = 71), a 27% decrease 7 (20.6 ± 0.5, n = 48) and a 19% decrease 21 days after SAH (22.8 ± 0.6, n = 43, each group significantly different from any other, ANOVA, P < 0.01 - 0.001). There were no significant differences between groups in membrane and series resistance of the patch-clamped smooth muscle cells (Fig. 1C and 1D, P > 0.05)

Temporal Profile of KV Channels: Voltage-Dependence and Kinetics

We previously characterized KV current (IKV) from dog basilar artery and validated protocols to characterize these currents [6]. The same protocols were used in the present study. IKV amplitude was significantly smaller at all 3 times after SAH at voltages more positive than 0 mV (Fig. 2A to 2D, Fig. 3A, P < 0.05). Although at a holding potential of 10 to 40 mV, the absolute IKV amplitude 7 days after SAH was smaller than that 4 days after SAH (Fig. 3A), the current density was not significantly different at any holding potential between any of the 3 times after SAH (Fig. 3B, P > 0.05).

Figure 2
Current traces of KV and KCa currents from dog basilar artery smooth muscle cells
Figure 3
Changes of Kv channel functions in SAH myocytes

The voltage-dependence of activation and inactivation was similar between control and SAH myocytes (Fig. 3I and 3J) except that the conductance of myocytes from the 3 SAH groups was significantly less than control cells at holding potentials of -20 and -10 mV (Fig. 3J, P < 0.05). The mid-potentials for activation in SAH myocytes were shifted to more negative voltages at all times after SAH (-3.40 ± 0.71 mV 4 days, -3.65 ± 0.62 mV 7 days and −4.43 ± 0.61 mV 21 days after SAH, Fig. 3C, P < 0.05 - 0.001 compared to control cells [-1.33 ± 0.56 mV]). However, there were no statistically significant differences between the SAH groups (Fig. 3C, P > 0.05). There also was an identical shift of the mid-potential for inactivation at all 3 times after SAH compared to controls but no difference among the times after SAH (Fig. 3F, P < 0.01 - 0.001 as compared to controls).

In contrast, the slope factors, K, for both activation and inactivation for myocytes 4 and 21 days after SAH were not different from control (Fig. 3D and 3G, P > 0.05). However, the slope factor for activation and inactivation from cells 7 days after SAH was significantly higher than controls and cells 4 and 21 days after SAH (Fig. 3D and 3G, P < 0.05 - 0.001). Similarly, there was no difference in activation time constant (τ) for cells 4 and 21 days and controls at depolarized potentials ≥ 0 mV (0 mV to +40 mV). At these times, IKV activation was well fitted by single exponentials for both control and SAH myocytes (Fig. 3E). However, τ for myocytes 7 days after SAH at +30 and +40 mV was significantly longer than all other groups (Fig. 3E, P < 0.05 - 0.001).

Inactivation was incomplete for all groups. However, 7 days after SAH, the remainder of the inactivation-resistant fraction was significantly larger than controls as well as cells 4 and 21 days after SAH. The remainder of inactivation was 16 ± 2% for controls and 18 ± 1% 4, 22 ± 1% 7 and 17 ± 1% 21 days after SAH (Fig. 3H, P < 0.01).

Temporal Profile of KCa Channels in Vasospastic Cerebrovascular Myocytes

Whole-cell patch clamp studies of the voltage-dependence and kinetics the large-conductance KCa channel were conducted in control and SAH myocytes. Both control and vasospastic myocytes generated large outward currents upon depolarization, which could be completely blocked by the KCa-specific toxin, paxilline (data not shown)(Fig. 2E to 2H)[8]. Normalized tail current amplitudes were fit to the Boltzmann function G/ Gmax = 1 / [1 + e-(V-V1/2)/k]. There were no significant changes in half-activation potentials (V1/2, Fig. 4C), slope factors (k, Fig. 4D), activation τ (Fig. 4E), deactivation τ (Fig. 4F) and normalized tail currents (Fig. 4G). The absolute amplitudes of KCa currents at all 3 times after SAH (at 100 - 160 mV holding potential) were significantly smaller than in control cells (Fig. 4A, P < 0.05 - 0.01). However, currents adjusted for cell capacitance (current density) demonstrated only a significant difference between cells 4 and 7 days after SAH and only at more depolarized potentials (from 100 to 160 mV, Fig. 4B, P < 0.01).

Figure 4
Changes of BK channel functions in SAH myocytes


Vasospasm is a major cause of mortality and morbidity after aneurysmal SAH. It occurs in 70% of these patients and causes morbidity and mortality in up to 30% [11]. Treatment has been directed at preventing secondary injury to the brain, augmenting cerebral blood flow by hemodynamic manipulations and administering the L-type Ca2+ channel antagonist, nimodipine [11]. These measures are complicated to use and not very effective.

Vasospasm is at least initially a problem of smooth muscle contraction [4]. Contraction involves alterations in membrane potential, receptor-operated agonists, altered Ca2+ sensitivity or failure of relaxation mechanisms. Regarding the first possibility, we reported that smooth muscle cells from dogs with SAH had significantly decreased KV2.2 messenger ribonucleic acid (mRNA) and protein and mRNA but not protein of the β1 subunit of the large-conductance KCa channel [1]. There was no significant change in mRNA for L- type Ca2+ channels and the KCa α subunit. Our prior electrophysiological and functional studies of KV and KCa channels found that KV2 current density was halved in myocytes 7 days after SAH in dogs. KV2.1 and KV2.2 mRNA and protein were decreased whereas there were no changes in the KCa channel [6;7]. Vasospastic myocytes were significantly depolarized and had a smaller contribution of K+ conductance toward maintenance of their membrane potential. This depolarization was reproduced by pharmacological block of KV current in normal myocytes and the degree of membrane depolarization was compatible with the degree of vasoconstriction observed after SAH. These observations suggested KV2 dysfunction contributes to vasospasm.

The electrophysiology findings of our previous work are extended here. Vasospasm was maximal 4 and 7 days after SAH and was resolving by 21 days. This correlated with cell capacitance and probably reflects contraction of the cells during vasospasm and relaxation as vasospasm resolves. On the other hand, KV current amplitude and mid-potentials for activation and inactivation decreased with vasospasm but did not recover as vasospasm resolved. The shift in activation and inactivation mid-potentials to more negative voltage indicates these channels are more sensitive to K+, which is consistent with findings in rabbits with SAH [22]. An increased slope factor for both activation and inactivation was detected only 7 days after SAH, suggesting that KV dysfunction peaks at this time in this model. The lack of direct correlation between KV channel alteration and vasospasm raises questions about the causal effect of KV channel dysfunction in vasospasm.

One possibility is that resolution of vasospasm involves compensatory changes in other regulators of membrane potential or other pathways. For example, inwardly rectifying K+ channels were increased during vasospasm after SAH, suggesting one possible mechanism for such reversal [20]. Another is that the decrease in KV channel function does not cause vasospasm, although function studies suggested that this is not the case [6]. Also, studies reported that vasospasm in rabbits was decreased with K+ channel agonists [9;10;23]. These studies used cromakalim which is not a specific KV channel activator and they studied relatively acute, mild vasospasm. In support of a role for decreased KV channel function in vasospasm was the finding that the more specific KV channel antagonist, 4-aminopyridine, produced less depolarization and basilar artery constriction 2 days after SAH in rats compared to control arteries [16]. Furthermore, Ishiguro, et al., reported that oxyhemoglobin, a component of subarachnoid blood that may cause vasospasm [3;12], decreases 4-aminopyridine sensitive KV currents in myocytes isolated from rabbit cerebral arteries but does not directly alter activity of voltage-dependent Ca2+ or KCa channels [5]. Finally, although there was less vasospasm 21 days after SAH in this model, vasospasm did not reverse so examination of KV channel function at a longer time after SAH might show recovery. Prior studies show a 15% reduction in dog basilar artery diameter 60 days after SAH so this might be a reasonable time to examine in future studies [17].

In contrast to the decrease in KV function, we previously reported that KCa channels were not altered 7 days after SAH [7]. It is thus unlikely that a primary alteration in KCa channel function contributes to vasospasm. The present study did find significantly smaller absolute KCa current amplitude at all times after SAH although after correction for cell size, there was only a significant decrease 4 days after SAH. Whether this is important in vasospasm needs further study, although it seems unlikely given the small and transient change.

In conclusion, these data support a role for dysfunction in KV channels in vasospasm after SAH. However, recovery of vasospasm seems to occur without concomitant improvement in KV channel function, suggesting other factors contribute to resolution of vasospasm.


This work was supported by grants (to R.L.M.) from the American Heart Association, the National Institutes of Health (NS25946) and the Brain Research Foundation. B.S.J. was supported by the Canadian Institutes of Health Research and the American Association of Neurological Surgeons.


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