1. SBS 16th Annual Conference & Exhibition
11 – 15 April 2010, Phoenix, USA
Aequorin Assays in Primary Endothelial Cells Using
the Amaxa® Nucleofector® Technology
Ann Siehoff1, Corinna Thiel1, Kristin Atze1, Steffi Franke1, Alina Morsch1, Sandra Carsten1, Vincent Dupriez2, Janet Park2, Alex Batchelor1, Nicole Faust1
1
Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany, www.lonza.com
2
PerkinElmer, Inc., 940 Winter Street, Waltham, MA, USA, www.perkinelmer.com
1. Introduction 3. Results
Recombinant cell lines stably expressing a receptor of interest are still widely used in drug discovery, for which they A.) HMVEC-L B.) HUVEC
present advantaging features like their availability in large quantities, the possibility to examine a single receptor 60,000
Luminescence
90,000
Luminescence
50 µM Histamine 50 µM Histamine
subtype, and the possibility to attain the high receptor expression levels that are needed for certain assays. However, 1 mM PEA 80,000 1 mM PEA
50,000 10 µM Amthamine 10 µM Amthamine
receptor signaling and pharmacology can be modulated by several features that may not be well represented in such 100 nM Methimepip 70,000
100 nM Methimepip
100 nM 4-Methylhistamine
recombinant models, like the possibility for receptors to heterodimerize with other receptors belonging to the same 40,000 60,000 100 nM 4-Methylhistamine
family, to interact with other partner proteins, which may be absent from recombinant cells, and to trigger signal 30,000
50,000
40,000
transduction in a cell-type-specific manner. For these reasons, primary cells have increasing interest in the research
20,000 30,000
and drug discovery areas. 20,000
10,000
10,000
Signaling through various G-protein coupled receptors (GPCRs), ion channels and tyrosine kinase receptors can be 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2 0 21 2 2 23 24 2 5 2 6 27 2 8 2 9 3 0 31 3 2 3 3 3 4 3 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2 0 21 2 2 23 24 2 5 2 6 27 2 8 2 9 3 0 31 3 2 3 3 3 4 3 5
analyzed by measuring intracellular Ca2+ increase. In recent years, the Ca2+-sensitive photoprotein Aequorin has gained time (s) time (s)
more and more popularity due to its excellent signal quality and ease of assay implementation. So far, use of Aequorin
Figure 1. Histamine and the H1R agonist PEA evoke a strong Aequorin signal in HMVEC-L and HUVEC cells. Kinetics
as a Ca2+ sensor was mainly restricted to cell lines overexpressing Aequorin plus the gene(s) of interest. Transition of
of Aequorin response to various Histamine receptor agonists in HMVEC-L (A) and HUVEC (B). Cells were transiently
these assays to human primary cells enables endogenous receptors to be assayed in a relevant cell background and
transfected by Nucleofection® and treated with the agonists 24 hours post transfection.
lowers the risk of forced signaling.
We used the Amaxa® Nucleofector® Technology, which facilitates efficient transfection of primary cells, to transiently A.) HMVEC-L B.) HUVEC
transfect “Human Umbillical Vein Endothelial cells“ (HUVEC) and “Human Microvascular Endothelial cells from the lung“
12,000 20,000 Bu er/Histamine
Luminescence
Luminescence
Bu er/Histamine
(HMVEC-L) primary cells with the AequoScreen® plasmid. As histamine is known to be a vasoactive substance acting 10 µM Cimetidin/Histamine 10 µM Cimetidin/Histamine
on some endothelial cells, where it triggers intracellular calcium increase, we used it as a model agonist to activate 10,000 0.1 µM Clobenpropit/Histamine 16,000
0.1 µM Clobenpropit/Histamine
1 µM Cetirizine/Histamine 1 µM Cetirizine/Histamine
cells transiently transfected with Aequorin. The four histamine receptors described to date (H1 – H4) are members of 8000 1 µM JNJ/ Histamine 1 µM JNJ/ Histamine
0.1 µM Mepyramine/ Histamine
the GPCR family, naturally coupling to Gαq/11 (H1), Gαs (H2) and Gαi/o (H3 and H4) proteins. The main class of G proteins 0.1 µM Mepyramine/ Histamine
Bu er/Bu er
12,000
Bu er/Bu er
6000
that lead to activation of the phospholipase C (PLC)-calcium flux pathway are the Gαq/11 proteins, but calcium influx 8000
through receptors normally coupled to Gαi/o or Gαs has also been described; indicating either activation of PLC by Gαi/o 4000
proteins, or secondary couplings for the receptor of interest. We used a set of histamine receptor subtype-specific 2000
4000
agonists and antagonists to analyze which of the histamine receptors trigger calcium flux in HUVEC and HMVEC-L
cells. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2 0 21 2 2 23 24 2 5 2 6 27 2 8 2 9 3 0 31 3 2 3 3 3 4 3 5
time (s)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2 0 21 2 2 23 24 2 5 2 6 27 2 8 2 9 3 0 31 3 2 3 3 3 4 3 5
time (s)
Figure 2. The H1R antagonists Cetirizine and Mepyramine inhibit a response to Histamine in HMVEC-L and HUVEC.
2. Materials and Methods Kinetics of Aequorin response to various Histamine receptor antagonists in HMVEC-L (A) and HUVEC (B). The cells
were transiently transfected by Nucleofection® and treated with the antagonists and 10 µM histamine 24 hours post
Analysis of intracellular Calcium levels in primary cells by transient transfection of Aequorin transfection.
HUVEC and HMVEC-L were transfected with the Aequorin Plasmid pCAEQ (Perkin Elmer) using the appropriate
Optimized Nucleofection® Protocol. Briefly, 100,000 cells (HUVEC) or 50,000 cells (HMVEC-L) and 0.8 µg of DNA
were used per sample in the Amaxa® 96-well Shuttle® System. 75,000 cells (HUVEC) and 50,000 cells (HMVEC-L), A.) HMVEC-L B.) HUVEC
respectively, were transferred to each well in a 96 well plate. Approximately 20 hours post Nucleofection®, cells were
250,000 200,000
pre-treated with 10 µM coelenterazine-h for four hours at 37° C. Cells were stimulated with various concentrations
AUC (RLU)
AUC (RLU)
Histamine Histamine
PEA PEA
of histamine and different histamine receptor agonists (for details, see figures). For the antagonist experiments, 200,000
150,000
cells were pre-incubated with different antagonists for 5 – 15 minutes prior to agonist injection. Luminescence was 150,000
analyzed in a microplate reader for 30 seconds at 25°C. Dose response curves were calculated using area under 100,000
the curve (AUC) integration. 100,000
50,000
50,000 EC50 Histamine: 2 µM EC50 Histamine: 5 µM
EC50 PEA: 114 µM EC50 PEA: 64 µM
-10 -8 -6 -4 -2 0 -10 -8 -6 -4 -2 0
log [Agonists] (M) log [Agonists] (M)
H1 H2 H3 H4
non-selective Histamine Figure 3. Dose response curves to Histamine and the H1R agonist PEA in HMVEC-L (A) and HUVEC (B). Cells transiently
Histamine receptor agonist agonist (4.7 – 5.9) agonist (8.1) agonist (7.0 – 8.3) agonist (6.4 – 8.3)
selective H1 receptor
transfected by Nucleofection® were treated with different concentrations of the agonists 24 hours post transfection.
2-pyridylethylamine (PEA) agonist agonist (3.7) agonist (<4)
Agonists
high H2 receptor inactive
Amthamine selectivity (no affinity) agonist (6.2 – 8.1) agonist (4.7) inactive (<4) A.) HMVEC-L B.) HUVEC
selective H3 receptor full agonist
Methimepip agonist inactive (<5) inactive (<5) (9.0 – 9.5) agonist (5.7) 200,000 Cetirizine 300,000 Cetirizine
AUC (RLU)
AUC (RLU)
selective H4 receptor agonist 250,000
4-Methylhistamine agonist inactive (<3) inactive (5.2) inactive (5.2) (7.3 – 8.2) 150,000
200,000
selective H1 receptor inverse agonist
Mepyramine antagonist (7.9 – 9.0) 100,000 150,000
selective H1 receptor inverse agonist 100,000
Cetirizine antagonist (8.2) inactive up to 10 µM inactive up to 10 µM
Antagonists
50,000 IC50 Cetirizine: 54 nM IC50 Cetirizine: 59 nM
50,000
H2 receptor antagonist inverse agonist
Cimetidine (inverse agonist) (5.9 – 5.8) -12 -10 -8 -6 -4 -12 -10 -8 -6 -4
H3 receptor inverse inverse agonist agonist log [Cetirizine] (M) log [Cetirizine] (M)
Clobenpropit agonist antagonist (5.6) antagonist (5.2) (8.4 – 9.9) (6.4 – 8.3)
H4 receptor selective neutral antagonist Figure 4. Dose response curves to the H1R antagonist Cetirizine in HMVEC-L (A) and HUVEC (B). Cells transiently
JNJ 10191584 antagonist 4.85 (6.3 – 7.6)
transfected by Nucleofection® were treated with different concentrations of the antagonist (for 5 – 15 minutes) and
Table 1. pKi, pEC50 and pIC50 values of Histamine receptor agonists and antagonists, respectively, as stated in the 7.5 µM histamine 24 hours post transfection.
literature, or according to PerkinElmer data from functional and binding assays.
4. Conclusions
We have shown here that Calcium signalling in primary endothelial cells can be effectively monitored after transient
transfection of Aequorin. Furthermore, we found that both HUVEC and HMVEC-L respond very well to both an agonist
and antagonists of the H1 receptor suggesting that they represent an excellent system to study this receptor in a
native cellular system. EC50 and IC50 values are very similar for H1 receptor agonist and antagonists for HUVEC and
HMVEC-L, respectively. Upscaling experiments for 384 well format and the use of cryopreserved cells are currently
under way.
AequoScreen® is a trademark of PerkinElmer Inc.. All other trademarks herein are marks of the Lonza Group or its affiliates. The Nucleofector® Technology, comprising
Nucleofection® Process, Nucleofector® Device, Nucleofector® Solutions, Nucleofector® 96-well Shuttle® System and Nucleocuvette® Plates and Modules is covered by patent
and/or patent pending rights owned by Lonza Cologne AG.