A novel approach for the determination of contractile and calcium responses of the basilar artery employing real-time confocal laser microscopy.
BACKGROUND
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 microM 5-hydroxytryptamine (5-HT), 10 microM alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP) and 10 nM vasopressin (VP) also induced increases in [Ca(2+)](i) and contraction dose-dependently. Additionally, 10 microM acetylcholine elicited a transient [Ca(2+)](i) decrease and sustained relaxation. In individual cells, rhythmical changes in [Ca(2+)](i) were observed after 10 microM 5-HT. VP (10 nM) evoked modest Ca(2+) oscillation in individual cells; however, Ca(2+) oscillation was not detectable with 10 microM alpha,beta-meATP.


CONCLUSIONS
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.
Hashimoto
Terumasa
T
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Japan. t-hashi@pharm.showa-u.ac.jp <t-hashi@pharm.showa-u.ac.jp>
Ohata
Hisayuki
H
Nobe
Koji
K
Sakai
Yasushi
Y
Honda
Kazuo
K
D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001488:Basilar Artery / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D010507:Periodicity; D051381:Rats; D017208:Rats, Wistar; D012701:Serotonin / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
D014667:Vasopressins; D012701:Serotonin; D000255:Adenosine Triphosphate; D000109:Acetylcholine; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium