14
WIRE MYOGRAPH SYSTEM - 620M
- USER GUIDE
3.5 In vitro experiment 1: Noradrenaline contractile response
The purpose of the present protocol is to determine the sensitivity of rat mesenteric small arteries to the vasoconstrictor
noradrenaline/norepinephrine with a cumulative concentration-response curve.
3.5.1 Background
Noradrenaline (norepinephrine) causes contraction of mesenteric small arteries through activation of α-adrenoceptors whereas
noradrenaline activation of β-adrenoceptors causes vasodilatation. As the purpose is to determine the contraction sensitivity to
noradrenaline, the vasodilatory effect of noradrenaline is eliminated throughout the experiment by the constant presence of the
β-adrenoceptor antagonist, propranolol.
Rat mesenteric arteries are densely innervated by sympathetic nerves, which have a highly efficient reuptake mechanism that
removes noradrenaline from the neuromuscular junction. The reuptake mechanism will create a concentration gradient between
the solution around the vessel segment and the receptors on the smooth muscle. To correctly determine the sensitivity to
noradrenaline it is necessary to eliminate this concentration gradient by performing the experiment in the presence of cocaine
to block the noradrenaline reuptake.
To determine the sensitivity to noradrenaline the vessel segment is exposed to increasing concentrations of noradrenaline.
Each concentration is applied until a steady response has been reached and then the next concentration is applied. When the
vessel segment is fully contracted or does not response more upon increasing the noradrenaline concentration, the experiment
is ended.
3.5.2 Protocol
Prepare the following stock solutions:
Noradrenaline: 10
-4
, 10
-3
, 10
-2
M
Propranolol: 10
-3
M
Cocaine:
10
-3
M
1. Mount and normalize the vessels as described in chapter 3.1 and 3.2.
2. Perform a standard start as described in chapter 3.3.
3. Incubate the vessel segment in 1 μM propranolol (add 5 µL of 10
-3
M to 5 mL PSS in chamber) and 3 μM cocaine (add 15
µL of 10
-3
M to 5 mL PSS in chamber) for at least 10 minutes.
4. Add increasing concentrations of noradrenaline into the bath (use the table below as a guideline). Wait for a stable contractile
response or a standard time such as 2 minutes between each application.
[NA] 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 [NA] in the Wire Myograph chamber, the applied volume of noradrenaline is ignored.
CHAPTER 3
