RAPSE (ANOTHER TRIAL) OCT 18th 2014

Noradrenaline stimulates the release of

panglossin, an amine from a granulated cell in
glandular tissues. Given below are data from a
study exploring the effects of a series of
synthetic organic compounds on the responses
to noradrenaline in glandular pieces incubated in
physiological solutions. These are multicellular
preparations. The compounds tested have not
yet been patented and are given code numbers.
They are all however, closely related.
They glands are pretreated with the compounds
(1mM) for fifteen minutes prior to addition of
noradrenaline and the responses measured ten
minutes later with a very sensitive and reliable
assay. Complete concentration response curves
to are constructed and EC50s to noradrenaline
estimated.
The results are shown below:
Compound

EC50

None (CONTROL)

1M

KR 236

0.1M

KR564

0.05 M

KR973

0.0075M

What is happening: compounds are changing

the EC50 of the noradrenaline, shifts the dose
response curve to the left with KR
Hypothesis: KR compounds block re-uptake
of norepinephrine with varying affinities for
the presynaptic heteroreceptor
Experiment: test whether KR compounds bind
with varying affinity for the presynaptic
heteroreceptor test for 2 things:
All kr compounds bind to presynaptic
heteroreceptor
2. kr compounds bind with varying affinities
1. look at microscope to see whether compound
actually binds
- way to small, cant look at ligand binding to a
receptor, also light vs electron microscopes
specify
- instead use binding assay, get colours,
fluorescent assay
- you need to show that it actually happening
here, and not somewhere else

- if experiment allows other hypothesis to occur,

then not a good experiment
- positive control: you know it works, negative
control: you know it doesnt work
- good to physically see that it binds
2. reproduce test mentioned and create dose
response curve EC50 will tell affinities

Experiment 2
Set up 3 organ baths, each with some glandular
tissue
- Positive control: inhibit function of PSR with
known inhibitor
- Negative: inhibit function of PSR with
molecule not found in noradrenergic
neurons, such as histamine
Negative control, poorly worded and cant use
histamine for tissue
Use saline in cases where theres injection
- We get functional data, get results but we
dont know how
Add NE to each organ bath, measure amount of
panglossin released with western blot
Next add each KR236, KR 564, KR 973
sequentially and detect level of activity of PSR
using detection technique
Maximal blockage of receptor seen upon with
addition of KR 973