15
3.6 In vitro experiment 2: Acetylcholine relaxation curve
The purpose of the present protocol is to determine the sensitivity of the endothelium dependent vasodilator acetylcholine in
noradrenaline pre-contracted rat mesenteric small arteries.
3.6.1 Background
Acetylcholine causes relaxation of rat mesenteric small arteries by activating of muscarinic M3 receptors at the endothelial cell
layer leading to release of endothelium-derived relaxing factors.
Rat mesenteric arteries do not show spontaneous tone in the wire myograph, which is why it is necessary to first induce a con-
traction to be able to observe the relaxation to acetylcholine. In this protocol the contraction is induced by noradrenaline. The
required concentration of noradrenaline needs to be optimized since a too low concentration makes it impossible to evaluate the
relaxation. On the other hand it may be difficult to relax super maximally contracted arteries, which may lead to an underestima-
tion of the sensitivity to acetylcholine. Therefore it is recommended to apply a concentration of noradrenaline inducing 60-70%
of maximal contraction response. In practice this concentration is found by performing a noradrenaline concentration-response
curve as described in the previous section.
The vessel segment is exposed to the noradrenaline concentration and when the response has stabilised, increasing concentra-
tions of acetylcholine are added to relax the vessel. Each concentration is applied until a steady response has been reached and
then the next concentration is applied. When the vessel segment is either fully relaxed or does not relax more upon increasing
the acetylcholine concentration, the experiment is ended.
3.6.2 Protocol
Prepare the following stock solutions:
Acetylcholine: 10
-4
, 10
-3
, 10
-2
M
Noradrenaline: 10
-2
M
1. Mount and normalize the vessels as described in chapter 3.1 and 3.2.
2. Perform a standard start and check the vessel segment for endothelium function, as described in chapter 3.3 and 3.4.
3. Add noradrenaline to obtain a response around 60% of maximum (determined from the previous noradrenaline concen-
tration-response curve). When the contractile response is stable, add increasing concentrations of acetylcholine to the
chamber, using the table below as a guideline. Wait for a stable contractile response or a standard time such as two minutes
between each application.
[ACh] in chamber (µM)*
Volume of stock solution to add to chamber
0.1
5 μL of 10
-4
M
0.3
1 μL of 10
-3
M
0.5
1 μL of 10
-3
M
1
2.5 μL of 10
-3
M
1.3
1.5 μL of 10
-3
M
1.5
1 μL of 10
-3
M
3
7.5 μL of 10
-3
M
5
1 μL of 10
-2
M
10
2.5 μL of 10
-2
M
*In calculating the [ACh] in the Wire Myograph chamber, the applied volume of ACh is ignored.
