Materials and Methods

Results

SAH

After SAH, basilar artery diameter was constricted by 23.0±3.3% (mean±SE; n=8; Fig 1⇓), from a resting control diameter of 838±11 μm (mean±SE; n=50). Ca²⁺-free solution relaxed the spasm by 60.3±7.1% (mean±SE; n=5; Figs 2⇓ and 3). Readdition of Ca²⁺ to spastic vessels exposed to Ca²⁺-free solution restored the magnitude of spasm to the original level (data not shown). Exposure of Ca²⁺-free solution−treated spastic vessels to 1 μmol/L verapamil did not cause further relaxation (Fig 2⇓).

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Figure 1.

Effects of SAH and agonists on basilar artery tone in situ. Rabbits were subjected to SAH, and control rabbit basilar artery was challenged with 40 to 50 mmol/L KCl, 3 nmol/L ET-1, and 2 to 8 μmol/L serotonin (5-HT). Additional control vessels were exposed to 1 μmol/L verapamil for 30 minutes before KCl and ET-1. The magnitudes of constriction were calculated as described in “Materials and Methods.” Values shown are mean±SE. n, shown in parentheses, represents the number of rabbits. Statistical significance was tested between values of agonists not exposed to verapamil and between values of the same agonist in the presence and absence of verapamil. *Significantly less than KCl.

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Figure 2.

Effects of Ca²⁺-free solution, verapamil, and Ni²⁺ on SAH-induced spasm of rabbit basilar artery in situ. SAH vessels were exposed to Ca²⁺-free solution, 1 μmol/L verapamil, Ca²⁺-free solution plus verapamil, or verapamil plus 0.1 or 0.3 mmol/L Ni²⁺. The magnitudes of relaxation were calculated as described in “Materials and Methods.” Values shown are mean±SE. n, shown in parentheses, represents the number of rabbits. *Significantly less than other values. ^†Significantly greater than other values.

Verapamil (1 μmol/L) decreased the spasm by only 16.5±4.2% (mean±SE; n=5; Figs 2 and 3⇑⇓). Ni²⁺ further relaxed verapamil-treated spastic vessels (66.7±8.9%, n=4, and 100.7±7.4%, n=5, relaxation due to verapamil plus 0.1 or 0.3 mmol/L Ni²⁺, respectively; mean±SE; Fig 2⇑).

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Figure 3.

Effects of Ca²⁺-free solution and verapamil on SAH-induced spasm and KCl-, ET-1−, and serotonin-induced constriction of control rabbit basilar artery in situ. Rabbits were subjected to SAH, and control rabbit basilar artery was challenged with 40 to 50 mmol/L KCl, 3 nmol/L ET-1, and 2 to 8 μmol/L serotonin (5-HT). Vessels were then exposed to Ca²⁺-free solution (A) or to 1 μmol/L verapamil (B) in the continued presence of agonist. The magnitudes of relaxation were calculated as described in “Materials and Methods.” The SAH, KCl, and ET-1 data are also shown in Figs 2, 4, and 5⇑⇓⇓, respectively. Values shown are mean±SE. n, shown in parentheses, represents the number of rabbits. *Significantly less than KCl and 5-HT.

Basal tone of control vessels was decreased by Ca²⁺-free solution, by verapamil, and by verapamil in the presence of 0.1 or 0.3 mmol/L Ni²⁺ by 6.3±0.2%, 0.2±0.1%, 0.4±0.4%, and 0.9±0.4%, respectively (mean±SE; n=3 in each case).

KCl

To test whether Ca²⁺-free solution and verapamil were able to completely reverse contractions entirely dependent on Ca²⁺ influx through L-type Ca²⁺ channels, control vessels were challenged with KCl followed by these agents. KCl (40 to 50 mmol/L) constricted the basilar artery by 34.0±2.7% (mean±SE; n=15; Fig 1⇑). Both Ca²⁺-free solution and 1 μmol/L verapamil essentially completely relaxed 40 to 50 mmol/L KCl−constricted vessels (Figs 3 and 4⇑⇓). Exposure to verapamil before KCl also greatly inhibited the KCl constriction (3.4±1.1%; mean±SE; n=3; Fig 1⇑).

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Figure 4.

Effects of Ca²⁺-free solution, verapamil, and Ni²⁺ on KCl-induced constriction of rabbit basilar artery in situ. Control rabbit basilar artery was challenged with 40 to 50 mmol/L KCl followed by Ca²⁺-free solution, 1 μmol/L verapamil, or 0.1 or 0.3 mmol/L Ni²⁺ in the continued presence of KCl. The magnitudes of relaxation were calculated as described in “Materials and Methods.” Values shown are mean±SE. n, shown in parentheses, represents the number of rabbits. *Significantly less than other values.

To test whether Ni²⁺ also inhibited L-type Ca²⁺-channel−dependent contraction, KCl-constricted vessels were challenged with Ni²⁺. In contrast to the complete inhibition of the KCl contraction by verapamil, 0.1 mmol/L Ni²⁺ relaxed the KCl constriction by only 39.8±4.7% (mean±SE; n=3), although 0.3 mmol/L Ni²⁺ induced 90.7±6.4% relaxation (mean±SE; n=3; Fig 4⇑).

ET-1

To test whether the relaxant effects of Ca²⁺-free solution, verapamil, and Ni²⁺ in SAH vessels were mimicked in ET-1−constricted vessels, control vessels were challenged with ET-1 followed by these agents. ET-1 (3 nmol/L) constricted the basilar artery by 29.0±2.3% (mean±SE; n=18; Fig 1⇑). Ca²⁺-free solution relaxed the ET-1 constriction by 69.2±3.9% (mean±SE; n=5; Fig 5⇓), which was not significantly different from the magnitude of Ca²⁺-free solution−induced relaxation of SAH vessels (Fig 3⇑). Exposure of Ca²⁺-free solution−treated ET-1−constricted vessels to 1 μmol/L verapamil did not cause further relaxation (Fig 5⇓).

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Figure 5.

Effects of Ca²⁺-free solution, verapamil, and Ni²⁺ on ET-1−induced constriction of rabbit basilar artery in situ. Control rabbit basilar artery was challenged with 3 nmol/L ET-1 followed by Ca²⁺-free solution, 1 μmol/L verapamil, Ca²⁺-free solution plus verapamil, 0.1 or 0.3 mmol/L Ni²⁺, or verapamil plus 0.1 or 0.3 mmol/L Ni²⁺ in the continued presence of ET-1. Additional vessels were exposed to 1 μmol/L verapamil for 30 minutes before exposure to 3 nmol/L ET-1, followed by 0.1 or 0.3 mmol/L Ni²⁺ in the continued presence of verapamil and ET-1. The magnitudes of relaxation were calculated as described in “Materials and Methods.” Values shown are mean±SE. n, shown in parentheses, represents the number of rabbits. *Significantly less than all post−ET-1 treatment values except Ni²⁺ 0.1 mmol/L. †Significantly less than all post−ET-1 treatment values except Verapamil 1 μmol/L. ‡Significantly greater than post−ET-1 treatment values of Ni²⁺ 0.1 mmol/L in the absence and presence of verapamil.

Verapamil (1 μmol/L) reversed the ET-1 constriction by only 20.4±4.7% (mean±SE; n=10; Fig 5⇑), which was not significantly different from the magnitude of verapamil-induced relaxation of SAH vessels (Fig 3⇑). Ni²⁺ (0.1 and 0.3 mmol/L) further relaxed these verapamil-treated ET-1−constricted vessels (50.1±8.5% and 90.8±7.6% relaxation due to verapamil plus 0.1 or 0.3 mmol/L Ni²⁺, respectively; mean±SE; n=8 in each case), and the magnitudes of relaxation were not significantly different from those observed in SAH vessels (compare Figs 2 and 5⇑⇑). The magnitude of relaxation due to 0.1 mmol/L Ni²⁺ alone in these verapamil-treated ET-1−constricted vessels, 30.5±5.2% (mean±SE; n=8; column 2 subtracted from column 4 in Fig 5⇑), was not significantly different from that due to 0.1 mmol/L Ni²⁺ in ET-1−constricted vessels not exposed to verapamil (28.9±5.4%; mean±SE; n=3; Fig 5⇑).

Pretreatment of vessels with verapamil did not inhibit the ET-1 constriction (30.8±3.5%; mean±SE; n=7; Fig 1⇑), although the time to achieve a plateau response was approximately double the 15 to 20 minutes required in the absence of verapamil (data not shown). Ni²⁺ (0.1 mmol/L) relaxed these verapamil-pretreated vessels by 80.8±6.4% (mean±SE; n=6), which was greater than relaxations to 0.1 mmol/L Ni²⁺ in vessels exposed to verapamil after ET-1 and in ET-1−constricted vessels not exposed to verapamil (Fig 5⇑).

Discussion

The present study demonstrates that extracellular Ca²⁺-dependent and -independent contractile mechanisms are responsible for the maintenance of SAH-induced chronic vasospasm in the two-hemorrhage rabbit model. This conclusion is supported by the observation that Ca²⁺-free solution, which completely reversed the KCl-induced constriction, only partially reversed (60%) the constriction due to SAH.

The present results further demonstrate that the influx of extracellular Ca²⁺ required to maintain SAH-induced spasm is only in part due to the opening of L-type Ca²⁺ channels. This conclusion is supported by the observation that verapamil, which completely reversed the KCl-induced constriction, reversed the constriction due to SAH by only 17%. The limited ability of verapamil to reverse the spasm in situ is consistent with the inability of L-type Ca²⁺-channel antagonists to significantly reverse the spasm in vivo in animal models^{2 3 4 5 6 7 8 9 10 11 12 13 14} and in the human.¹⁵

The question then arises as to the identity of the non−L-type Ca²⁺ channel or channels (verapamil-insensitive) responsible for a significant component of the spasm. We tested whether this component of the spasm may be mediated through Ca²⁺ influx through SOCs (SOC indicates store-operated influx channels, generally described as depletion-activated channels, I_CRAC, and capacitative Ca²⁺ entry; International Conference on Receptor-Regulated Calcium Influx, Asilomar, Calif, May, 1995). The possibility that SOCs may be responsible, at least in part, for the spasm was based on the considerable evidence in support of the mediation of the spasm by ET-1 (see References 18 and 19 and references therein), and that ET-1−induced contraction of rabbit aorta and porcine coronary artery was mediated through Ca²⁺ influx via a Ni²⁺-sensitive, non−L-type Ca²⁺ channel.^{21 22} Moreover, ET-1 has been shown to open a nonselective cation channel in A10 vascular smooth muscle cells,²³ primary cultures of rat aorta and mesenteric artery smooth muscle cells,²⁴ and freshly dissociated smooth cells from rabbit aorta,²⁵ and the channel was blocked by Ni²⁺.^{23 24} Also of relevance is the recent demonstration that ATP and hemolysate open SOCs in freshly dissociated smooth muscle cells from rat basilar artery and that Ni²⁺, albeit at a high concentration, blocked the SOCs.²⁶

The present results suggest that the non−L-type Ca²⁺-channel−dependent component of the spasm dependent on the influx of extracellular Ca²⁺ is due to the opening of SOCs, since 0.1 mmol/L Ni²⁺ in the presence of verapamil relaxed the spasm to a magnitude not significantly different from Ca²⁺-free solution (67% and 60%, respectively). However, this conclusion must be viewed with caution, since 0.3 mmol/L Ni²⁺ in the presence of verapamil completely relaxed the spasm. Thus, higher concentrations of Ni²⁺ may also induce relaxation through a mechanism or mechanisms independent of blockade of Ca²⁺ influx, and the following possibilities were considered. First, since considerable evidence suggests that the spasm results from continuous ET-1 release (see References 18 and 19 and references therein), Ni²⁺ may inhibit ET-1 release and/or receptor binding. However, neither of these possibilities appear likely since the ET-1 constriction, similar to the spasm after SAH, was partially relaxed by Ca²⁺-free solution and completely relaxed by verapamil plus 0.3 mmol/L Ni²⁺. In addition, 10 mmol/L Ni²⁺ did not inhibit the binding of ET-1 to the solubilized ET receptor from human placenta.²⁷ Alternatively, it may be speculated that Ni²⁺ blocks the internalization of the ET-1−ET receptor complex, which has been proposed to account for the prolonged ET-1−induced contraction.²⁸

Secondly, Ni²⁺ may induce relaxation through an intracellular site. In possible support of this suggestion is the observation that Ni²⁺ relaxed SAH- and ET-1−constricted vessels exposed to Ca²⁺-free solution and verapamil (authors' unpublished data, 1996). However, it should be considered that the ability of Ni²⁺ to induce relaxation in the absence of extracellular Ca²⁺ may result from increased cellular permeability to Ni²⁺. In support of this possibility is the increased skeletal muscle Ca²⁺-channel permeability observed on removal of extracellular Ca²⁺.²⁹ Furthermore, 20 and 5 mmol/L Ni²⁺ did not enter A10 vascular smooth muscle cells and neutrophils, respectively.^{23 30} Determining the effects of Ni²⁺ on intracellular Ca²⁺ levels in basilar artery after SAH, as well as in ET-1−constricted vessels, with fura-2 would further test this possibility.

Also of some concern regarding the selectivity of Ni²⁺ as a blocker of SOCs is the present observation that the KCl-induced constriction, which was almost entirely inhibited by verapamil, was relaxed by 0.1 and 0.3 mmol/L Ni²⁺ by 40% and 91%, respectively. Thus, the action of Ni²⁺ in rabbit basilar artery may also include blockade of L-type Ca²⁺ channels. This apparent relative lack of selectivity is in contrast to demonstrations that Ni²⁺ did not inhibit the KCl-induced constriction of rabbit aorta and porcine coronary artery in vitro.^{21 22} The relative selectivity of Ni²⁺ for SOCs as compared with L-type Ca²⁺ channels may depend on the blood vessel and/or preparation (eg, in vitro versus in situ). It is important to note, however, that in the present study the relaxant effects of Ni²⁺ on the spasm and on the ET-1−induced constriction were determined in the continued presence of verapamil. Thus, the Ni²⁺-induced relaxation was not due to blockade of L-type Ca²⁺ channels.

The present study also suggests that the profile of extracellular Ca²⁺ dependency and Ca²⁺ independency of the SAH-induced constriction results from the underlying ET-1 mediation of the spasm. In support of this suggestion is the observation that the relaxation profile due to Ca²⁺-free solution, verapamil, and verapamil plus 0.1 mmol/L Ni²⁺ was similar in SAH- and ET-1−constricted vessels, in contrast to serotonin- and KCl-constricted vessels.

The present results can be placed in the context of the mechanism underlying SAH-induced cerebral vasospasm as illustrated in the working model of Fig 6⇓. Although several factors contribute to the initial spasm, chronic SAH-induced cerebral vasospasm, as defined in the present rabbit model (6 days after the initial SAH), is completely dependent on ET-1 release, because the combination of an ET_A and ET_B receptor antagonist or an ET_A/B receptor antagonist completely reverses the spasm.³¹ Endothelin-1 then activates ET_A and possibly ET_B receptors that, among other actions, results in the opening of L-type Ca²⁺ channels (verapamil-sensitive) and releases intracellular Ca²⁺. The depletion of the intracellular Ca²⁺ store also results in the opening of SOCs (Ni²⁺-sensitive).

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Figure 6.

Working model of the role of Ca²⁺ in SAH-induced cerebral vasospasm. This model is based on the two-hemorrhage rabbit model of SAH-induced cerebral vasospasm. SAH induces ET-1 release from the vascular endothelium. ET-1 acts at ET_A, and possibly at ET_B, receptors³¹ to cause inositol triphosphate (IP₃) formation, and IP₃ releases intracellular Ca²⁺. Ca²⁺ is also depleted from a store that regulates SOCs. The IP₃-sensitive and SOC-regulatory Ca²⁺ pools may be distinct. Contraction is elicited by Ca²⁺ influx through L-type Ca²⁺ channels (voltage-operated channels; VOCs) and SOCs and by mechanisms independent of Ca²⁺ influx, including IP₃-induced Ca²⁺ release. The Ca²⁺ filling of the store that regulates SOCs occurs via Ca²⁺ influx through an L-type Ca²⁺ channel coupled to the store. Opening of SOCs by the Ca²⁺ store is indicated by the dashed line. The model of SOC regulation is based on models presented in References 33 and 37. Additional signal transduction pathways that may underlie ET-1 constriction and not discussed in this article (eg, protein kinase C−dependent and Ca²⁺-independent pathways) are not presented in the model. See text for further details.

Also illustrated in Fig 6⇑ is the mechanism that may underlie the present observation that verapamil addition during the ET-1 constriction induced partial relaxation, whereas verapamil pretreatment did not inhibit the ET-1 constriction. Since Ca²⁺ influx through L-type Ca²⁺ channels is thought to replenish the Ca²⁺ store that regulates SOCs in smooth muscle,^{32 33 34} verapamil pretreatment of basilar artery presumably prevented refilling of the regulatory Ca²⁺ store, resulting in increased opening of SOCs. Greater Ca²⁺ influx through SOCs induced further SOC-dependent ET-1−induced constriction, as shown by the almost threefold increase in 0.1 mmol/L Ni²⁺−induced relaxation of vessels pretreated with verapamil. Thus, the lack of inhibition of the ET-1 constriction in verapamil-pretreated vessels was likely due to the increased ET-1−induced constriction mediated through SOCs. It should also be noted that these observations represent the first demonstration, to the best of our knowledge, of a change in the dependency of a contractile response on Ca²⁺ influx through L-type Ca²⁺ channels and SOCs to one dependent on Ca²⁺ influx through SOCs.

Studies have demonstrated that L-type Ca²⁺-channel blocker administration before SAH prevents the development of vasospasm.^{2 3 4 5 6 7 8 9 10 11 12 13 14} Although the mechanism underlying this inhibitory effect is not known, the following mechanism is speculated. On the basis of the present demonstration that verapamil pretreatment did not inhibit ET-1−induced constriction (although nicardipine pretreatment inhibited ET-1–induced constriction of the canine basilar artery in vivo³⁵ ), and the considerable evidence in support of mediation of the spasm by ET-1 (see References 18 and 19 and references therein), the ability of L-type Ca²⁺-channel blockers to prevent the spasm may be due to inhibition of ET-1 release through a process dependent on Ca²⁺ influx through L-type Ca²⁺ channels. One might further speculate that the inhibition of ET-1 release by L-type Ca²⁺-channel blockers is not due to their action at the endothelium, the source of ET-1,¹⁸ since endothelial cells are not thought to express L-type Ca²⁺ channels (see Reference 36 and references therein). Thus, the Ca²⁺-channel blockers may prevent the release of factors and the development of conditions responsible for ET-1 release (Fig 6⇑).

It should be noted that the present rabbit model of SAH-induced vasospasm, as well as other animal models of SAH-induced vasospasm, may not reflect the entirety of the human pathophysiology. Differences between the present rabbit model of SAH-induced vasospasm and the human pathophysiology include the apparent lack of neurological deficit (authors' unpublished data, 1996) and of functional damage to the smooth muscle and endothelium (as found in the present study and References 38 and 39). However, these differences may be related to the difficulty in the assessment of neurological damage in nonprimate models of SAH-induced vasospasm and to the severity and duration of the spasm.

In summary, SAH-induced vasospasm in the two-hemorrhage rabbit model is maintained by contractile mechanisms independent of extracellular Ca²⁺ influx and by Ca²⁺ influx through L-type Ca²⁺ channels and an additional channel or channels, possibly SOCs. The possible involvement of SOCs in the spasm in the two hemorrhage rabbit model clearly awaits more direct investigation. The extracellular Ca²⁺-dependent and -independent mechanisms are consistent with the mediation of the spasm by ET-1. The possible involvement of SOCs in the maintenance of the spasm suggests that selective SOC blockers, and inhibitors of the signal transduction pathway responsible for SOC opening, represent potential therapeutic modalities for pathophysiologies involving agonists that open SOCs, including SAH-induced vasospasm.

Selected Abbreviations and Acronyms