Biochemical Assessments of Cerebral Vasospasm: Measurement of cGMP, PKC, and PTK in Cerebral Arteries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 514

centrifuged at 2,000 × g at 4°C for 15 min. The supernatant,
containing the cytosol fraction of vascular smooth muscle, is
extracted with water-saturated dimethyl ether four times and then
lyophilized for 24 h.

Each sample is dissolved in 5.0 mL of the provided assay buffer,
and 1.0 mL of each sample is acetylated with 33.3% truethylamine.
The cGMP standard (provided in the assay kit) and collected
samples (50 mL each) are put in 96-well assay plates coated with
anti-rabbit IgG, and incubated with 100 mL of rabbit anti-cGMP
serum for 2 h at 4°C. Then, peroxidase conjugate (100 mL) is
added to each well, and the plate is incubated for 1 h at 4°C. After
washing each well with washing solution, 200 mL of tetramethyl-
benzidine is added to each well as peroxidase substrate, and incu-
bated at room temperature for 30 min. The reaction is then stopped
by addition of 1 M H 2 SO 4 (100 mL). The absorbance is read at
450 nm by a microplate spectrophotometer. The concentration of
cGMP, expressed as fmol/mg protein, is calculated using a stan-
dard curve, and normalized by protein concentration.

2.3. cGMP Assay

3. Measurement
of PKC Activity

In studies examining the pathophysiological mechanisms of cerebral
vasospasm following SAH, a great deal of effort has focused on
identifying the underlying spasmogenic substance(s). However,
many factors released from the subarachnoid blood clot have the
potential to cause sustained cerebral arterial constriction and there
is a lack of consensus among researchers as to the identity of the
primary causative agent. Thus, rather than a single compound, a
multifactorial combination of substances is likely to underlie cere-
bral vasospasm.
On the other hand, there is no doubt that (1) the contractile
proteins, myosin and actin, exist in vascular smooth muscle cells,
and (2) regardless of the specifi c identity of spasmogenic substances,
vascular smooth muscle contraction is enhanced. A number of
studies have demonstrated that PKC activation plays a pivotal role
in SAH-induced cerebral vasospasm ( 13-15 ). Regarding the role
of PKC in the development and maintenance of cerebral vasospasm
after SAH, it is important to understand when and how long PKC
is activated and the targets downstream of PKC contributing to
cerebral vasospasm.
PKC, in the inactive state, usually exists in the cytosol. However,
in response to the appropriate membrane signal, PKC translocates
to the cell membrane and is activated ( 16 ). As a result of PKC
translocation/activation, various phosphorylation cascades are ini-
tiated in vascular smooth muscle leading to enhance contraction.

3.1. Background