Biology Reference
In-Depth Information
3 KCl, 18 NaHCO
3
, 1.25 NaH
2
PO
4
, 1 MgCl
2
, 2 CaCl
2
, 5 glucose
aerated with 5% CO
2
, 20% O
2
, 75% N
2
(pH, 7.30-7.35). Freshly
isolated cerebral artery segments are cannulated on glass micropi-
pettes mounted in a 5 ml myograph chamber (Fig.
1
) (
18
).
Following cannulation, blood vessels are incubated with previously
aerated aCSF containing fura-2AM (acetoxymethyl ester mem-
brane-permeable form; 5
M; Invitrogen, Carlsbad, CA) and
pluronic acid (0.1%; Invitrogen) at room temperature (~22°C) for
45 min in the dark. The myograph chamber is then mounted on an
inverted fl uorescence microscope and arteries are pressurized and
continuously superfused with aCSF (37°C, 30 min) to allow equil-
ibration and deesterifi cation of fura-2.
It has previously been demonstrated that Ca
2+
can be selec-
tively measured in vascular smooth muscle or endothelium by
loading fura-2 from the extraluminal or luminal side of the vessel,
respectively (
19
). However, the specifi c loading time and dye con-
centration described above may need to be slightly adjusted based
on the specifi c tissue studied to enable suffi cient permeation of dye
into smooth muscle while avoiding loading of the dye into the
endothelium. To examine whether dye loading is smooth muscle-
specifi c, studies can be performed on arteries in which the endothe-
lium is physically removed by luminal passage of an air bubble (
2
).
Endothelial removal can be confi rmed by lack of response to
endothelium-specifi c pharmacological agents (e.g., acetylcholine,
bradykinin, NS309) (
20, 21
).
μ
First, an aperture located adjacent to the dichroic mirror is adjusted
so that the blood vessel completely fi lls the rectangular fi eld of view
in the data acquisition software. This minimizes the contribution
of extraneous light to the fl uorescence signal. The dichroic mirror
refl ects fl uorescence emission to the PMT while the use of a red
fi lter in the microscope condenser allows the vessel image to be
transmitted to the CCD camera. Thus, the image collected by the
CCD camera for diameter measurement represents the same fi eld
from which fl uorescence is detected by the PMT.
Following equilibration, fl uorescence ratio (
F
340
:
F
380
) is
obtained from background-corrected 510 nm emission from arter-
ies alternately excited at 340 and 380 nm (Fig.
2
) using software
developed by IonOptix (Milton, MA). Arterial wall [Ca
2+
] is
estimated
2.3.2. Data Acquisition
and Estimation of
Intracellular Ca
2+
using
the
following
equation:
(
17
)
⎡
2
+
⎤
=
K R R R R
R
min
and
R
max
values represent the ratios of emission signal
under Ca
2+
-free and Ca
2+
-saturated conditions, respectively. To
obtain these values, arteries are fi rst loaded with fura-2 and equili-
brated as described above. Arteries are then perfused (10 min,
37°C) with a baseline solution containing the following (in mM):
5 NaCl, 140 KCl, 5 HEPES, and 1 MgCl
2
(pH 7.4). Next, to
obtain
R
min
, arteries are perfused (30 min) with baseline solution
Ca
×−
(
) / (
−
).
⎣
⎦
d
min
max
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