Biology Reference
In-Depth Information
chain kinase, myosin light chain phosphorylation and actin-myosin
interaction (
3
). Thus, global [Ca
2+
]
i
directly regulates the contrac-
tile state of vascular smooth muscle and therefore dictates cerebral
vascular tone and blood fl ow.
The discovery of spatially localized, transient calcium release
events (Ca
2+
sparks) has altered the view that an elevation of global
[Ca
2+
]
i
is the only effective mode of calcium signaling in smooth
muscle (
4-6
). Calcium sparks result from the opening of a cluster
of ryanodine receptors in the sarcoplasmic reticulum (SR) mem-
brane, causing a highly restricted (1% of the cell volume) and large
(
M) increase in local Ca
2+
with little direct effect on global [Ca
2+
]
i
(
5, 7
). In vascular smooth muscle, Ca
2+
sparks are functionally cou-
pled to nearby large conductance Ca
2+
-activated K
+
(BK) channels
located on the plasma membrane (
6, 8
). Elevation of intracellular
Ca
2+
causes a shift in the voltage-dependence of BK channel activa-
tion to more negative membrane potentials effectively leading to
increased channel open probability (
9
). Increased Ca
2+
spark activ-
ity therefore leads to enhanced BK channel activity, membrane
potential hyperpolarization, decreased global [Ca
2+
]
i
, and smooth
muscle relaxation. Ca
2+
spark activity is increased by vasodilators,
such as nitric oxide and forskolin, and by elevations in cytosolic
and SR calcium (
10, 11
). Thus, Ca
2+
sparks act as an important
negative feedback mechanism to oppose vasoconstriction by pro-
moting a reduction in VDCC activity and a decrease in global
[Ca
2+
]
i
.
Considering the pivotal role that vascular smooth muscle Ca
2+
plays in the regulation of cerebral blood fl ow, precise measure-
ment of local and global Ca
2+
signaling in these cells is essential for
understanding pathologies characterized by abnormal vascular
tone, such as aneurysmal subarachnoid hemorrhage (SAH). With
respect to SAH-induced “classical” or “angiographic” vasospasm,
observed in large diameter conduit arteries on the brain surface,
the role of elevated global [Ca
2+
]
i
is unclear and controversial (
12
).
However, recent evidence suggests that enhanced constriction of
much smaller “resistance” arteries and arterioles (<200
μ
m in
diameter) following SAH is associated with increased global [Ca
2+
]
i
and enhanced VDCC activity (
13
). Further, Ca
2+
spark activity is
decreased in cerebral arteries by the blood component oxyhemo-
globin (
14
) and Ca
2+
spark frequency is reduced in cerebral artery
myocytes isolated from SAH model animals (
13, 15
). In this chap-
ter, we discuss the properties of two fl uorescent Ca
2+
indicator
dyes, fura-2 and fl uo-4, that enable the measurement of global
[Ca
2+
]
i
and Ca
2+
sparks, respectively, in cerebral artery myocytes.
Further, we provide details of specifi c protocols used in our labo-
ratory to assess global and local signaling in the cerebral
vasculature.
μ
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