UNIVERSITY OF IOANNINA / SCHOOL OF SCIENCES
DEPARTMENT OF CHEMISTRY / LABORATORY OF BIOCHEMISTRY
ΕΠΙΔΡΑΣΗ ΤΗΣ ΜΗΧΑΝΙΚΗΣ ΤΑΣΗΣ ΣΤΗ
ΒΙΟΣΥΝΘΕΣΗ ΤΗΣ ΦΩΣΦΑΤΙΔΥΛΟΧΟΛΙΝΗΣ
ΣE ΠΝΕΥΜΟΝΟΚΥΤΤΑΡΑ ΤΥΠΟΥ ΙΙ
The Effect of Mechanical Stretch on Biosynthesis of
Phosphatidylcholine (PC) on Alveolar Type II Cells (ATII)
Despoina Pantazi
Chemist, MSc, PhD
Staff of the UOI (IDAX)
http://www.chem.uoi.gr
Laboratory of Biochemistry
 Synthesized by type II alveolar cells
 Consists of surfactant specific
proteins and phospholipids
(dipalmitoylphosphatidylcholine, PC)
 Reduces surface tension (prevents
alveolar collapse during expiration)
 Exhibits host-defense properties
ALVEOLAR TYPE II CELLS
Surfactant
is
biosynthesized,
organized and stored into various
structures, such as lamellar bodies in
alveolar type II (ΑΤΙΙ) cells. Under this
form surfactant is secreted into the
alveolar space through the formation
of microvilli in the apical cell surface.
Alveolar type II cell from human lung
ATII
Golgi
LB
Υπόφαση
SER
Orgeig S. Conference proceedings, Australian Frontiers, 31 July to 1 August 2003, Canberra Australia.
EM of Lamellar Bodies and Tubular Myelin
From J. Goerke, Pulmonary surfactant: functions and molecular
composition Biochim. Biophys. Acta 1408 (1998) 79-89.
Composition of human lung surfactant
90 % Phospholipids
68.0 % PC
10.0 % PG, 5.0 % PE
90 % Lipids
4.0 % Sph
10 % Neutral Lipids
Hydrophilic proteins SP-A, SP-D (50 - 49 %)
10 % Proteins
Hydrophobic SP-B, SP-C (1 %)
Rooney S.A. 1985. Am. Rev. Respir. Dis., 131:439-460.
DPPC Structure
DPPC, the major lipid component of surfactant, is responsible for the
equilibration of pressure in the alveoli.
LUNG SURFACTANT HOMEOSTASIS
 Surfactant homeostasis is critical for lung function
and is maintained through the balanced actions of
biosynthesis,
secretion,
recycling
and
decomposition of its constituents
 Qualitative and quantitative alterations of surfactant
composition are associated to acute respiratory
distress syndrome (ARDS)
MECHANICAL VENTILATION
 Mechanical ventilation is required for critically-ill
patients
 However, it is established that certain modes of
ventilation provoque inflammation not only in
injured, but even in non-injured lung
MECHANICAL FORCES
 Lung is subjected to mechanical forces continuously during
development, normal breathing and mechanical ventilation
The main forces are
• Strain is more prominent in lung epithelium during breathing
• Shear forces acts mainly on the vascular endothelium
 Cell proliferation, differentiation, apoptosis, release of different
inflammatory cytokines and factors are some of the stretchinduced responses in AT II cells
Substantial evidence indicates that mechanical stress during
respiration is the signal for lung surfactant secretion from AT-II
cells
AIM
To investigate the effect of mechanical
stimulation on phosphatidylcholine
biosynthesis within AT-II cells
- A549-cell line was used as a model for AT-II cells
- A mode of static stretch was applied
FLOW-CHART
Force 35 g/cm2
for 1 & 4 h
Cell Count
A549 CELLS
(~ 5% Deformation)
Homogenisation
800xg, 10 min, 4 ºC
Cell Homogenate
Petriperm
Plexiglass
Pellet
Protein
Enzyme activities
Enzyme expression
Lipid analysis
RESULTS
EFFECT OF STRETCH ON PHOSPHATIDYLCHOLINE
CONTENT
(% Difference from Control)
PC
40
PC
30
*
20
10
0
-10
-20
CONTROL
STRETCH 1h
STRETCH 4h
After stretch for 1 h PC was increased by 22.9 %. At 4 h, PC
declined to the control levels
Control: unstretched cells; *: Statistical difference from the control
Enzyme activities that implicated in production of PC
CPT
DPPC
PC
LPCAT
PLA2
Lyso-- PC
ΕΝZYMIC ACTIVITIES (1)
1. Final step of the de novo formation PC
O-CO-R
CDP-choline
CMP
O-CO-R
R΄-OC-O
HO
ΟΗ
Diacylglycerol
(DAG)
R: palmitate
R’: unsaturated acyl chain
CPT
P-Cho
Phosphatidylcholine
(PC)
BIOSYNTHESIS OF PC: CPT
(% Difference from Control)
CPT Activity
100
CPTCPT
80
*
60
40
20
0
-20
CONTROL
STRETCH 1h
STRETCH 4h
-40
CPT activity was increased at 47 ± 9 % (p=0.037) after stretch for 1 h while
after stretch for 4 h the enzyme activity recurred to the control levels.
Control: unstretched cells; *: Statistical difference from the control
ΕΝZYMIC ACTIVITIES (2)
2. Remodelling of PC for DPPC formation
O-CO-R
O-CO-R
aiPLA2
R΄-OC-O
HO
P-Cho
PCho
Lyso-phosphatidylcholine
(Lyso-PC)
Phosphatidylcholine
(PC)
O-CO-R
O-CO-R
LPCAT
R-OC-O
HO
PCho
P-Cho
palmitate
(Lyso-PC)
R: palmitate
(DPPC)
TOTAL ΕΝZYMIC ACTIVITIES: variations
*
70
60
50
40
30
20
10
0
-10
-20
-30
#
‡
*
2.5
aiPLA2
(nmol C12-NBD-FA/h/mg protein)
CPT Activity
(% Difference from Control)
3.0
‡
Stretch 1 h
Stretch 4 h
- MJ33
+ MJ33
2.0
#
1.5
#
1.0
0.5
0
Stretch
1h
LPCAT activity
(% Difference from Control)
Control
45
*
40
35
Stretch
4h
D
*
30
25
20
aiPLA2 – 26 kDa
15
β-actin – 45 kDa
10
5
Hela
0
Stretch 1 h
Stretch 4 h
Control
Stretch 1h Stretch 4 h
RELATIVE GENE EXPRESSION
Relative Gene Expression
*‡
Control
Stretch 1 h
Stretch 4 h
* *
CHPT1
PRDX6
LPCAT1
IN CONCLUSION
• Static stretch of A549 cells causes a transient increase in total
phosphatidylcholine levels
• The concomitant increase in CPT justifies this alteration
• The enrichment of stretched cells in DPPC can be attributed to the
concerted actions of aiPLA2 and, in particular, of LPCAT in which the
expression is definitively increased
• Our data suggest that in A549 cells mild static mechanical stretch
promotes the biosynthesis of PC
CONCLUSIONS - CLINICAL IMPLICATIONS
Possible clinical implications of our findings
 A low level of stretch can induce surfactant production which can act in a
protective way
 This phenomenon has a limited duration and the system equilibrates after 4
h. Probably, cyclic stretch could help in the further production
 Our results are compatible with previous work where PC was reduced after
prolonged ventilation
Future Studies - Perspectives
Part I
Signaling and regulation of surfactant biosynthesis
under different stretching conditions
Relevance of our findings with mechanical ventilation
Instrumentation of Cyclic Stretcher
Future Studies
Part II
 Lipidomics is the detailed analysis and global characterization of
the structure and function of lipids (the lipidome) within a living
system
 The enrichment of the list of lipids with new structures from
biological samples, and/or after the influence of various
parameters
probably
will
help
to
the
understanding
of
mechanisms of lung diseases
 Lipidomic and further proteomic analysis of biological samples
n
(BAL, serum, etc) in the unit LC-LTQ-ORBITRAP-MS
Department of Chemistry in UOI
in
LC- LTQ – ORBITRAP – MSn INSTRUMENTATION
Solvent
Platform
Autosampler
Pump
 When the HPLC system is connected to an MS detector, the components exit the LC
column and travel through a red tubing to the MS detector. The LTQ Orbitrap features a
rapid scan rate and high mass accuracy. Enables faster, more sensitive and more reliable
detection and identification of compounds in low levels in complex mixtures. It outstands
mass accuracy, mass resolution and reliable high sensitivity MSn performance.
REFERENCES
1.
Pantazi D., Nakos G., Kitsiouli E., Trangas T., Lekka M.E. 2011. Non-Injurious mechanical stretch
induces phosphatidylcholine biosynthesis in alveolar type II cells. Under preparation.
2.
Fisher, A.B., Dodia C. 1997. Role of acidic Ca2+ -independent phospholipase A2 in synthesis of
lung dipalmitoylphosphatidylcholine. Am. J. Physiol. Lung Cell Mol. Physiol. 272:L238-L243.
3.
Edwards Y.S. 2001. Stretch stimulation: its effect on alveolar type II cell function in the lung.
Comp. Biochem. Physiol. 129:245-260.
4.
Torday J.S., Rehan V.K. 2002. Stretch-stimulated surfactant synthesis is coordinated by the
paracrine actions of PTHP and leptin. Am. J. Physiol. Lung Cell Mol. Physiol. 283:L130-L135.
5.
Fahy E., Subramaniam S., Murphy R.C., Nishijima M., Raetz C.R.H., Shimizu T., Spener F., van Meer
G., Wakelam M.J.O., Dennis E.A. 2009. Update of the LIPID MAPS comprehensive classification
system for lipids. J. Lipid Res., 50:S9-S14.
Acknowledgements
1. Professor M.E. Lekka
2. Professor G. Nakos
3. Assoc. Professor D. Galanopoulou
4. Assoc. Professor T. Trangas
5. E. Kitsiouli, PhD
6. T. Karkampounas, PhD
7. Candidate MSc & PhD students of Laboratory
of Biochemistry
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