Inhibition of cerebral vasoconstriction by dantrolene and nimodipine

Abstract

Introduction: Cerebral vasoconstriction is associated with increased cytosolic Ca(2+) concentration in vascular smooth muscle, presumably due to Ca(2+) influx and Ca(2+) release from intracellular stores. We tested the hypothesis that dantrolene (a blocker of Ca(2+)-induced Ca(2+) release from the ryanodine receptor channel on the sarco-endoplasmic reticulum) would potentiate the action of nimodipine (a voltage-dependent L-type Ca(2+) channel blocker, considered standard therapy for SAH) in inhibiting the vasoconstriction of isolated cerebral arteries.

Method: Sprague-Dawley rat basilar and femoral arteries were analyzed for ryanodine receptor expression by immunofluorescence and PCR. Vasoconstriction of basilar artery ex vivo was measured in a wire myograph while exposed to serotonin (5-HT) or endothelin-1 (ET-1) in the presence or absence of dantrolene (10-100 muM) and/or nimodipine (30 nM). Femoral artery was examined for comparison.

Results: Basilar and femoral arteries express only the ryanodine receptor 3 (RyR3) isoform. In both basilar and femoral arteries, dantrolene significantly inhibited the constriction to 5-HT, whereas it poorly affected the constriction to ET-1. The inhibitory effect of dantrolene on 5-HT was substantially increased by nimodipine, inducing a 10-fold increase in the 50% effective concentration of 5-HT and a 46% reduction in maximum basilar constriction. In femoral artery, dantrolene modestly affected constriction to phenylephrine and there was no interaction with nimodipine.

Conclusion: Dantrolene has synergistic effects with nimodipine against 5-HT-induced vasoconstriction in isolated cerebral arteries. Dantrolene-nimodipine interaction will require testing in a pathophysiological model but might provide treatment for reducing SAH-related vasospasm or other 5-HT-related vasospastic syndromes, such as Call-Fleming syndrome.

Figure 1

Ryanodine Receptor Expression. A, cross-section of rat basilar artery immunostained for ryanodine receptor (red) and a nuclear counterstain (green). B, Agarose gel electrophoresis of PCR products from 2 samples of femoral (f) and cerebral (c) arteries. The left panel shows a 70 bp PCR fragment of the RyR3 sequence and the corresponding loading control of 18s rRNA in the right panel. Closest ladder bands to amplicon fragments are labeled 75 bp and 200bp, respectively.

Figure 2

Vehicle effect on concentration-response curves. A, Cumulative serotonin (5-hydroxytryptamine, 5-HT) concentration-response curve of basilar artery constriction normalized to 100 mM KCl induced-contraction, control arterial segments are compared to segments (n=5) incubated with 0.33% DMSO, 0.03% ethanol (ETOH) or both (n=6, for all vehicle groups), there are no statistical differences among curves. B, Cumulative endothelin-1 (ET-1) concentration-response curve of basilar artery constriction normalized to 100 mM KCl induced-contraction, control arterial segments (n=5) are compared to segments incubated with 0.33% DMSO, 0.03% ethanol (ETOH) or both (n=6, for all vehicle groups), there are no statistical differences among curves.

Figure 3

Serotonin (5-hydroxytryptamine, 5-HT) concentration-response curves in basilar artery. A, Cumulative concentration-response curve of basilar artery constriction normalized to 100 mM KCl induced-contraction, control arterial segments are compared to segments incubated with increasing concentration of dantrolene (10–100μM) (*EC₅₀ and E_max compared to control). B, The effect of dantrolene (10μM) plus nimodipine (30nM) (*EC₅₀ and E_max of nimodipine compared to control). C, The effect of dantrolene (30μM) plus nimodipine (30nM) (*EC₅₀ of dantrolene+nimodipine compared to nimodipine or dantrolene alone). D, The effect of dantrolene (100μM) plus nimodipine (30nM) (*EC₅₀ and E_max of dantrolene+nimodipine compared to nimodipine or dantrolene alone). The effective concentration of 5-HT that gave 50% contraction (EC₅₀) for each treatment is listed below the respective graph.

Figure 4

Endothelin-1 (ET-1) concentration-response curves in basilar artery. A, Cumulative concentration-response curve of basilar artery constriction normalized to 100 mM KCl induced-contraction, control arterial segments are compared to segments incubated with increasing concentration of dantrolene (10–100μM). B, The effect of dantrolene (10μM) plus nimodipine (30nM) (*EC₅₀ of nimodipine compared to control). C, The effect of dantrolene (30μM) plus nimodipine (30nM) shows no interaction. D, The effect of dantrolene (100μM) plus nimodipine (30nM) shows no interaction. The effective concentration of ET-1 that gave 50% contraction (EC₅₀) for each treatment is listed below the respective graph.

Figure 5

Serotonin (5-hydroxytryptamine, 5-HT) concentration-response curves in femoral artery. A, Cumulative concentration-response curve of basilar artery constriction normalized to 100 mM KCl induced-contraction, control arterial segments are compared to segments incubated with increasing concentration of dantrolene from 10–100μM (*EC₅₀ of 30 and 100 μM compared to control). B, The effect of dantrolene (10μM) plus nimodipine (30nM) (*EC₅₀ of nimodipine compared to control). C, The effect of dantrolene (30μM) plus nimodipine (30nM) (*EC₅₀ of dantrolene+nimodipine compared to nimodipine or dantrolene alone). D, The effect of dantrolene (100μM) plus nimodipine (30nM) (*EC₅₀ and E_max of dantrolene+nimodipine compared to nimodipine or dantrolene alone). The effective concentration of 5-HT that gave 50% contraction (EC₅₀) for each treatment is listed below the respective graph.

Figure 6

Phenylephrine (PE) concentration-response curves in femoral artery. A, Cumulative concentration-response curve of basilar artery constriction normalized to 100 mM KCl induced-contraction, control arterial segments are compared to segments incubated with increasing concentration of dantrolene from 10–100μM (*EC₅₀ of 100μM compared to control). B, The effect of dantrolene (10μM) plus nimodipine (30nM) (*EC₅₀ and E_max of nimodipine compared to control; *E_max of dantrolene+nimodipine compared to nimodipine or dantrolene alone). C, The effect of dantrolene (30μM) plus nimodipine (30nM) (*E_max of dantrolene+nimodipine compared to nimodipine or dantrolene alone). D, The effect of dantrolene (100μM) plus nimodipine (30nM) (*E_max of dantrolene+nimodipine compared to nimodipine or dantrolene alone). The effective concentration of PE that gave 50% contraction (EC₅₀) for each treatment is listed below the respective graph.