Experiments on isolated vascular rings

1. Experiments on isolated vascular
rings
Ph.D. Martin Mihaly, PhD., MD.

2. PHYSIOLOGY OF BLOOD VESSELS
• Good understanding of the physiology of the vascular system is essential
for understanding the course of the experiment
• The mechanisms of vasodilation and vasoconstriction

3. Intracellular signaling
Types of receptors:
1. Receptor-dependent ion channels (ionotropic and
receptor receptors)
2. Receptors coupled with G-protein (metabotropic
receptors)
3. Kinase receptors
4. Nuclear receptors

4. R R E R
M
R
M
R
The cellular
response The cellular response
The cellular
response
The cellular
response
hyperpolarization or
depolarization
Change of
irritability
other
messengers
Discharge
Ca2+
phosphorylati
on of proteins else
phosphorylati
on of proteins
transcription of genes
protein synthesis
R
transcription of
genes
protein synthesis
First receptor-
dependent ion
channels
Second receptors coupled to G-
proteins
(Metabotropic receptors)
3rd kinase
receptors
4th Nuclear
receptor
Timeline:
msec
Example:
nicotian
ACh receptor
Timeline:
hours
Example:
cytokine
receptor
Timeline:
hours
Example:
estrogen
receptors
Timeline:
seconds
Example:
Muscarinic acetylcholine receptor

5. meta 1Target 2
α
GDP
meta 1
GTP
βγ
GDP
P
Hydrolysis
Coupled receptors G - protein

6. Intracellular signaling
Receptors coupled to G protein
meta
1
Targe
t 2 α βγ
GDP
meta
1
Targe
t 2 α βγ
GDP
GTP
meta
1
Targe
t 2 α βγ
GTP
meta
1
Targe
t 2 α βγ
GDP
+
P
receptor busy
meta activatedGTP hydrolysed
Fi
rs
t
4
t
h
T
hi
r
S
e
c
o
n
d GDP

7. Intracellular signaling

8. Adenylate cyclase
AC
α
βγ
GTP
ATP
cAMP
inactive
protein kinase A
active
protein kinase A
phosphodiesterase
ATP
cAMP
AC
Increased lipolysis
⇣ glycogen synthesis
⇡ degradation of
glycogen
...

9. Ca2+
Phospholipase C
Fos.
C
α
βγ
GTP
SEN2
DAG
IP3
IP4
activation
Protein kinase C
Liberation
Unutarst. Ca ++
The entry of Ca ++
through the
membrane
calmodulin
target enzymes
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+

11. The metabolism of arachidonic acid

12. News Physiol. Sci. 14; 238-242, 1999; Borrowed from the presentation of Prof. Ines Drenjančević - Peric
20-HETE and Eets and Control of Renal Vascular
Tone

13. α1
Fos.
C
α
βγ
GTP
SEN2
IP3
IP4
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
calmodulin
Kinase myosin light
chain
IP3
Smooth muscle cells
L Ca2+ channels
dependent on
the voltage
Ca2+ channels
dependent on
ligand
ATP
ATP
Ca2+ channels regulated
stock of
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
On+

15. L-arginine NO
eNOS
GMP
guanylate
cyclase cGMP
endothelial
station
The
smooth
muscle
cells
The basal
membrane
A.D
NO: Endothelial relaxing factor
Ca2+ - calmodulin
+ -

16. Physiological humoral regulation
vasoconstrictor
• noradrenaline
• angiotensin II.
• vasopressin (Adh)
• endothelin
Vsodilators
 bradykinin
 serotonin
 histamine
 prostaglandins
 NO (EDRF)
 C-natriuretic peptide
 Adrenomodulin
 EDHF (endothelial hyperpolarization
factor)

17. Physiological nerve regulation
Vasoconstrictor
• simpatikus: NA through α1
α2
Vasodilators
• parasympathetic: Ach
• simpatikus: For through β2

18. EFFECTS OF VASOACTIVE DRUGS ON
BLOOD VESSELS
• acetylcholine
• L-NAME
• indomethacin
• KOTRIMAZOL
• verapamil
• NO donors
• KCI
• noradrenaline

19. Noradrenaline
α1
Fos.
C
α
βγ
GTP
SEN2
DAG
IP3
activation
Protein kinase C
Liberation
Unutarst. Ca ++ Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
calmodulin
target enzymes
IP3 RIP3
Ca2+
Ca2+

20. Acetylcholine
L-arginine NO
eNOS
GMP
guanylate
cyclase cGMP
endothelial
station
The
smooth
muscle
cells
The basal
membrane
M
Phos
phor
us. C αβγ
GTP
SEN
2
IP3
Ca2+
calmodulin
Ca2+

21. L - N A M E
L-arginine NO
eNOS
GMP
guanylate
cyclase
cGMP
endothelial
station
The
smooth
muscle
cells
The basal
membrane

22. Indomethacin
arachidonic acid PGI2
COX
AMP
adenylate
cyclase
cAMP
endothelial
station
The smooth
muscle cells
- Share PGI2 (Prostacyclin) in endothelium dependent vasodilation is relatively small
The basal
membrane

23. Clotrimazole

24. On nitroprusside, SNP
L-arginine NO
eNOS
GMP
guanylate
cyclase
cGMP
endothelial
station
The
smooth
muscle
cells
The basal
membrane

25. PREPARATION FOR THE TEST
• The system (temp. 37 ⁰C, delivery ON2 etc.)
• calibration of the system
• Preparation of Krebs Henseleit solution
• Drug Therapy

26. 4-channel system vero bath for isolated organs
containing:
 Four-channel standard bath system for insulated bodies (tissue rings and straps) with all accessories
(tubes, cables, etc.)
 Four-channel amplifier of measured force
 Isometric force converter
 Software for data collection and processing
 System for Receiving and Transferring a Siganal System from Isolated Body Bath Systems to a
Computer (Quadruple with the Possibility of Expansion to the Eight Channel System)
 A 20 l water tank with heating and water circulation and a temperature adjustment range of 20 - 50
ºC and a precision of ± 0.1 ºC

28. converter forces
Well of 20 ml
Macro and micro screw
for setting the voltage

30. Ingredient Focus / mM Weight / g Weight / g
deionized water - 4 L 6L
NaCl 113 27,580 41,37
KCI 4.7 1,402 2,103
CaCl2 x 2H2ON 2.5 1,470 2,205
EDTA 0,026 .0304 0.05
MgSO4 x 6H2ON 1.2 1,185 1.78
KH2PER4 1.2 .653 0.98
NaHCO3 22 7.395 11.09
Glucose - 7,999 11,99
Krebs - Henseleit solution

31. PREPARATION OF DRUGS FOR TRIAL
• Drugs are prepared as 20 mM (2 mM and extremely 3M)
• Stored for 2 to 3 months at -20 ⁰C
• KCI

32. The drug / active matter Mr. (G / L)
Preparation
20 mM
solution
Concen-tion in the
pool
It should further dilution
20 mM solution of
Vol. which was
added to the wells
(μL)
KCI 74.56
prepared 3M
11.3 g / 50 mL
60 mM Not 400
noradrenaline 205.0 41 mg / 10 mL 10-4 M
Guinea Pig: 100 x (10-6)
Rat: 1000x (10-7)
100
acetylcholine 181.7 36,4mg / 10 mL 10-4 M
Guinea Pig: 10 x (10-5)
Rat: 100x (10-6)
100
L-NAME 268.0 53,6mg / 10 mL 3x10-4 M Not 300
indomethacin 358.0 71,6mg / 10 mL 10-4 M 10x (10-5 M) 100
clotrimazole 344.8 69mg / 10mL 10-4 M 10x (10-5 M) 100
verapamil
491.0
amp: 5 mg / 2
mL
prepared 2 mM
In 4 ml amp. + 6 mlH2ON
20 μM Not 200
* The solvent for Indometasin is 96% ethanol or DMSO / sometimes and others. Depending on the manufacturer

33. SURGERY

37. STANDARD PROTOCOL
1. Stabilization of the blood vessel
2. An initial pre-contract test
3. Endothelium intact test
4. Examination of vasoactive tissue

38. Stabilization of the first vessel (60 min)
 Blood vessels are strained to 20 mN
 Repeatedly the blood vessel tension is
corrected to 20 mN (or 0 if m N base system is
set to - 20 mN)
 During this period wells are washed 3-4puta
(Approx. Every 15 min)

39. 2. An initial pre-contract test
400μL 3M KCI
(60 mM)
Rinse
Rinse
Rinse
4 min
1 min
1 min
1 min
3 x

40. 3. Testing the endothelium intact
100μL 1000x Class. 20
mM
noradrenaline
(10-7M)
100μL 100x Class. 20 mM
acetylcholine
(10-6M)
Rinse4 min 1 min
1 min
Rinse
Rinse
4 min

41. 4. Testing vasoactive substances
100μL 1000x Class. 20 mM
noradrenaline
(10-7M)
vasodilator
in different doses
4 min

43. DATA PROCESSING
Formulas for calculating% vasodilation ...

44. X0
Xmax
X3
Vasodilation% =
Xmax - X3
Xmax - X0
% Remaining contraction =
X3 - X0
Xmax - X0

45. THE EXPERIMENT
Example Experiments: Effects Hyperbaric therapy to conductive DM rat
blood vessels

46. 24 rats
12 Ctrl.
(control)
12 DM
(diabetes,
treated with STZ)
6 Ctrl.
6 Ctrl. + Citrate
6 DM
6 DM + HOBT
L-NAME
clotrimazole
Fir
st
Se
co
nd
Ctrl. + HBOT

47. Group GUK (mmol/ L) Weight (Mr) The system on which you worked Remarks Tags rings
A - Control Citrate (Excess
10.08.2009., TH: 09.09.2009, 90 m g)
a1a 9.7 310 AND. The aorta was bent and contracted A1a 1-4
A1b 9.7 320 II. A1b 1-4
A1c 9.7 305 AND. Aorta been curled and contracted A1c 1-4
A2a 11.3 310 AND. 2a 1-4
A2b 10 290 II.
problems during experiments - tendon been longer on
dry because sust. is not was ready
2b 1-4
A2c lacking strips 300 II. A2c 1-4
B - untreated control (Excess
12.08.2009.)
B1a 9.3 360 AND. B1a 1-4
B1b 8.6 320 II. B1b 1-4
B1c 10.9 305 AND. B1b 1-4
B2a 9.5 350 AND.
Thawed serum with A 2a, A 2b and B2b - Danieli to
leave 0.5 ml of Oksi.kapac.
B2a 1-4
b2b 10.7 285 II. The serum can be diluted with thawing b2b 1-4
B2 10.3 280 AND. b2c 1-4
C - Diabetics + HBTh (Excess
25.07.2009., TH: 09.09.2009, 90 m g)
C1a > 33 250 AND. 1-4 C1a
C1b > 33 285 II. C1b 1-4
C1c 8 290 II. C1c 1-4
C2 > 33 225 AND. 1-4 C2a
C2b > 33 250 II. C2b 1-4
c2c > 33 225 II. c2c 1-4
D - Diabetics untreated (Excess
01.08.2009., TH: 09.09.2009, 90 m g)
D1a > 33 250 AND. D1a 1-4
D1b 7.8 320 II. D1b 1-4
D1c > 33 210 II. D1c 1-4
D2a > 33 200 AND. D2a 1-4
D2B > 33 170 II. D2b 1-4
d2C 10.2 300 AND. Heating the serum + white white clot d2C 1-4
They did not develop diabetes

48. An initial pre-contract test
400μL 3M KCI
(60 mM)
Rinse
Rinse
Rinse
4 min
1 min
1 min
1 min
3 x

49. A: cons.
D: diabetes.

50. A: cons.
C: diabetes. + HBTh
10 min

51. A: cons.
C: diabetes. + HBTh

52. Vasodilatation mediated by
acetylcholine

53. Ach 10 minutes. after L-NAME

54. Ach 10 minutes. after clotrimazole