A novel approach for the determination of contractile and calcium responses of the basilar artery employing real-time confocal laser microscopy

Abstract

Introduction Intracellular calcium concentration ([Ca 2+] i) modifications in endothelial and smooth muscle cells represent a key element in the pathogenesis of cerebral artery vasospasm. Therefore, the present study documented potential application of confocal laser microscopy in the determination of contractile and [Ca 2+] i responses in basilar artery. Methods Experiments were performed on the rat isolated basilar artery. Changes in [Ca 2+] i were determined by ratiometry involving Fluo-4/AM and Fura Red/AM. Contractile function was calculated from the change in fluorescent area by Fluo-4/AM. Results KCl (50 mM) elicited an increase in [Ca 2+] i and contraction in basilar artery; moreover, nearly well maintained responses were evident for at least 120 min following the first application. 10 μM 5-hydroxytryptamine (5-HT), 10 μM α,β-methyleneadenosine 5′-triphosphate (α,β-meATP) and 10 nM vasopressin (VP) also induced increases in [Ca 2+] i and contraction dose-dependently. Additionally, 10 μM acetylcholine elicited a transient [Ca 2+] i decrease and sustained relaxation. In individual cells, rhythmical changes in [Ca 2+] i were observed after 10 μM 5-HT. VP (10 nM) evoked modest Ca 2+ oscillation in individual cells; however, Ca 2+ oscillation was not detectable with 10 μM α,β-meATP. Discussion These results indicate that this method offers reproducibility and quantifiable effects. Imaging technology may therefore be applied to the estimation of [Ca 2+] i responses at the tissue level as well as at the level of the individual cell. Thus, confocal laser microscopy is a suitable tool for estimation of small artery function.