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 513

2. Measurement
of cGMP

In the fi eld of the vascular research, the role of the endothelium was
in the mist for many decades. In the 1980s, it was fi rst reported that
vascular endothelium could produce and release vasodilatory
substances to regulate arterial diameter. Physical removal of the
endothelial layer ablated vasodilations caused by agonists, such as
acetylcholine ( 8, 9 ). This unidentifi ed vasodilator substance was
named endothelium-derived relaxing factor (EDRF) ( 8, 9 ). However,
most researchers remained skeptical regarding the existence EDRF
until it was fi nally identifi ed as nitric oxide (NO) ( 2, 3 ). Since this
ground-breaking discovery, the role of vascular endothelium in
health and disease has been the subject of intense study.
It is now known that NO is released from endothelial cells, and
activates guanylate cyclase in vascular smooth muscle leading to
increased cGMP levels and arterial relaxation ( 10 ). Due to its short
half life (approximately 5 s), the direct measurement of NO poses
a signifi cant technical challenge. Alternatively, measurements of
more stable metabolites of NO or downstream NO-mediated sig-
nals (e.g., cGMP) have routinely been used by researchers to
examine the actions of NO.
In vasospastic arteries following SAH, damage to both vascular
smooth muscle and endothelial cells can occur. Endothelial detach-
ment from the internal elastic lamina leading to a corrugated
appearance of the blood vessel, when viewed in cross-section, has
been reported in vasospastic conduit arteries. Endothelial dysfunc-
tion after SAH may contribute to abnormal contraction of arterial
smooth muscle cells leading to cerebral vasospasm. Thus, it is
important to investigate the function and the role of endothelium
in the pathophysiological mechanisms of cerebral vasospasm.
One common approach to examine the impact of SAH on
endothelial function is to measure cGMP levels in vascular smooth
muscle cells ( 11 ). Here, we outline a procedure for the measure-
ment of basal cGMP levels in vascular smooth muscle cells using an
enzyme immunoassay system for cGMP (Amersham, GE healthcare,
Piscataway, NJ).

2.1. Background

2.2. Sample
Preparation

After dissection, canine basilar arteries are incubated for 30 min in
modifi ed Krebs-Henseleit solution (in mM: 118.1 NaCl, 4.1 KCl,
1.19 MgSO 4 , 2.49 CaCl 2 , 1.53 NaH 2 PO 4 , 24.90 NaHCO 3 , 5.0
glucose; aerated with 20% O 2 , 5% CO 2 , and 75% N 2 ; pH 7.38 ± 2.0)
containing 100 mM of isobuthyllxanthine (IBMX), a phosphodi-
esterase inhibitor ( 12 ). For this procedure, IBMX is also added
into dissection buffer. Tissue is then minced and sonicated (20 s,
six times) in 6.0% trichloroacetic acid, and the homogenate is