Kallikrei
n
Kinins
Kininas
•Bradykinin eI
1 2 3 4 5 9
6 7 8 -
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe
Arg
•DesArg9-Bradykinin
•Lys-Bradykinin (Kallidin)
Kininas
eI
1 2 3 4 5 6 10
7 8 9 -
Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-
Phe Arg
•Lys-DesArg10- Bradykinin
Kinin Receptors
B2: Bradykinin
B1: DesArg9-Bradykinin
B2/B1:Lys-Bradykinin (Kallidin)
B1:Lys-DesArg10 -Bradykinin
Kinin Receptor
Antagonists
Monomeric Antagonists
-B1 B9958
-B1/B2 B9430
Dimeric Antagonist
-B1/B2 B9870
Kinin Receptor
Antagonists
Monomeric G αq - inhibits Ca signal
Kinins
Kinins are mitogenic peptides
-regulate growth and proliferation of cancer cells
-increase vascular perm
Mesothelioma
Mesothelium: lung pleura
Grows over serosal surfaces and
inter-lobular fissures of the lung
Appearance:
-Thick, stellate shape cells with vacuoles
Aim 1
Determine expression of B1 and
B2 receptors on the human
mesothelioma cell line: NO36
Method: Dako EnVision ICC
2◦
1◦ DAB 3.3’-diaminobenzidine
R
Antibodies
Anti-tissue Polyclonal rabbit against CAP
prokallikrein (TproK) 11 (synthetic peptide) that
covers the aminoterminal
residues of TproK.
100 200
400 600
TproK
120
% cells brown
100
80
60
40
20
0
100 200 400 600
Dilutions
TK
5 100
0
200 40
0
TK
120
% cells brown
100
80
60
40
20
0
50 100 200 400
Dilutions
PPK
10 200
0
400 800
PPK
100
% cells brown
80
60
40
20
0
100 200 400 800
Dilutions
PK11
50 10
100
0
200 400
PK11
120
% cells brown
100
80
60
40
20
0
50 100 200 400
Dilutions
LK
50 100
200 40
0
LK
50
% cells brown
40
30
20
10
0
50 100 200 400
Dilutions
B1
200
100
100
100 200
400 800
400 80
0
B1
B1
120
% cells brown
100
80
60
40
20
0
100 200 400 800
Dilutions
B2
100 200
50 100
400 800
200 40
0
B2
100
% cells brown
80
60
40
20
0
50 100 200 400
Dilutions
Hypothesis 2
Yellow Insolubl
tetrazoli Reduced by e
um salt mitochondria Formaz
ancryst
al
Detergent
No.
Spectrophotom Solubilize & cell
of
eter d
viabl lysis
e Formazan
cells
Antagonist Results
NO36 24 hours NO36 48hr plate 1
1.2 ** 1.75
1.1 **
1.0 1.50
0.9
1.25
Absorbance
Absorbance
0.8
0.7
1.00
0.6
0.5 0.75
0.4
0.3 0.50
0.2
0.1 0.25
0.0
0.00
B9870 4uM
B9870 2uM
B9870 1uM
B9958 4uM
B9958 2uM
B9958 1uM
B9430 4uM
B9430 2uM
B9430 1uM
Media
B9870 4uM
B9870 2uM
B9870 1uM
B9958 2uM
B9958 1uM
B9430 2uM
B9430 1uM
Media
B9958 4uM
B9430 4uM
Treatment
Treatment
Absorbance
Absorbance
1.0 1.0
0.5 0.5
0.0 0.0
Media BK 10 BK 1 dBK 10 dBK 1 Media BK 10 BK 1 dBK 10 dBK 1
Treatment Treatment
llidi
Lys
-
Campbell 2000
Bacterial LPS, damaged basement memb
Receptors
B1 receptor: Normally absent. Upregulated
by: inflammatory mediators, ischaemia,
bacterial products,transplant rejection
Agonist: [des-Arg] kinin.
vasoactive responses
Agonist: Bradykinin
1st generation
Antibodies
PK11: monoclonal antibody to human plasma kallikrein light
chain; epitope class D; (IgG2aк), lyophilized ascites
HMW kininogen: polyclonal antibody IgG
Plasma kallikrein: antiserum to human plasma kallikrein
raised in rabbit; lyophilized antiserum, reconstituted with
0.01M PBS containing 1% IgG free BSA and 0.2% NaN3
tProk: Antibody to synthetic peptide made of the 1st 7 N-terminal
residues (Ala1-Arg7) of (TproK) followed by the 1st 3 residues (Ile8 to
Val10) of the mature enzyme, with an additional Cys residue at the
N-terminus
Cys-Ala-Pro-Pro-Ile-Gln-Ser-Arg-Ile-Val-Gly