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Measurement of changes in sarcoplasmic reticulum [Ca2+] in rat tail artery with targeted apoaequorin delivered by an adenoviral vector

Rembold, C. M., Kendall, Jonathan M. and Campbell, Anthony Keith 1997. Measurement of changes in sarcoplasmic reticulum [Ca2+] in rat tail artery with targeted apoaequorin delivered by an adenoviral vector. Cell Calcium 21 (1) , pp. 69-79. 10.1016/S0143-4160(97)90098-1

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Abstract

The physiologic relevance of Ca2+ release from the sarcoplasmicreticulum in arterial smooth muscle contraction is controversial. Therefore, we sought to measure changes in sarcoplasmicreticulum free [Ca2+] (i.e. [Ca2+]sr) in the intact rattailartery. We exploited a novel technique to measure [Ca2+]sr with genetically targetedapoaequorin acting as a pseudo-luciferase rather than as classic aequorin. Intact rattailarteries were infected with a replication deficient adenoviralvector (RAdER) containing the apoaequorin gene targeted to the sarcoplasmicreticulum. Addition of apoaequorin's substrate, coelenterazine, to the perfusate increased light production in a [Ca2+] dependent manner, consistent with apoaequorin action on coelenterazine. Within the limits of the photon counting system, imaging of infected rattailartery segments revealed light production from the whole thickness of the vascular wall. Phenylephrine stimulation decreased apoaequorin generated light and induced a contraction. Washout of phenylephrine relaxed the tissues and increased light indicating refilling of the sarcoplasmicreticulum with Ca2+. Incubation in 10 μM cyclopiazonic acid, a SERCA inhibitor, did not alter apoaequorin generated light or induce a contraction. In the presence of cyclopiazonic acid, phenylephrine contractions were enhanced and apoaequorin generated light decreased further than that observed in the absence of cyclopiazonic acid. Cyclopiazonic acid also prevented the increase in apoaequorin generated light upon washout of phenylephrine, consistent with its inhibition of sarcoplasmicreticulum refilling. These results suggest that light production from targetedapoaequorin, delivered by a replication deficient adenovirus, is a valid measure of changes in [Ca2+]sr in the intact arterial wall. There appeared to be a correlation between Ca2+ release and contraction in these lightly loaded arteries.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Pharmacy
Subjects: Q Science > QD Chemistry
Q Science > QR Microbiology
R Medicine > RM Therapeutics. Pharmacology
Publisher: Elsevier
ISSN: 0143-4160
Last Modified: 05 Jun 2017 02:58
URI: http://orca-mwe.cf.ac.uk/id/eprint/22511

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