Lectures L10.1 Session 10. Metabolism and Signaling Role of

Lectures L10.1 Session 10. Metabolism and Signaling Role of

Session 10. Metabolism and Signaling Role of
Ectopurines and Ectopirimidines
Lectures
hibitor, may cause risk of oxidative stress resulting from
dysfunctional storage mechanism. L10.1
Antagonists of adenosine A2A receptors
as a new therapy of Parkinson’s disease
Krystyna Gołembiowska
Institute of Pharmacology, Pharmacology Department, Polish Academy
of Sciences, Poland
e-mail: Krystyna Golembiowska <[email protected]>
Parkinson’s disease (PD) is a devastating neurodegenerative disorder with not completely understood etiology. In
its pathomechanism degeneration of dopamine (DA) cell
bodies in substantia nigra pars compacta correlate with
appearance of Lewy bodies in histological investigation.
Cardinal motor symptoms of PD such as akinesia, rigidity and tremor are reduced by administration of L-DOPA
in combination with a peripheral decarboxylase inhibitor
which is “a gold standard” in treatment of PD. However,
complications such as refractory fluctuations, dyskinesias
and tolerance develop after long-term L-DOPA therapy. In
addition, L-DOPA is neurotoxic for remaining DA neurons due to free radicals generation after it is converted into
DA. In the last years adenosine A2A receptor antagonists
emerged as a new non-dopaminergic therapy of PD. Adenosine A2A antagonists were shown to alleviate symptoms
of PD in a number of behavioral studies in rodents and
primates. In rodent models of PD they increased locomotor activity in MPTP-treated or reserpinized mice, reversed
haloperidol-induced catalepsy in rats and potentiated rotational behavior produced by L-DOPA in 6-OHDA-lesioned rats. In primates treated with MPTP, adenosine A2A
antagonists increased motor activity and decreased dyskinesia induced by a prolonged administration of L-DOPA.
The mechanism of antiparkinsonian effects of A2A antagonists is based on their ability to modulate GABA release
and to decrease DA-dependent activation of the indirect
striatopallidal pathway. Presynaptically, A2A antagonists
potentiate D2 receptor control of glutamatergic transmission. Recently, a neuroprotective potential of adenosine
A2A receptor antagonists has been suggested. The protective effects of A2A receptor antagonists as well as caffeine
was observed in an animal MPTP neurotoxicity model or
in ischemia and excitotoxicity brain injury models. Oxidative stress is regarded as the main factor contributing to
the etiology o PD. Overactive glutamatergic transmission
resulting from disturbed DA-glutamate balance and a decreased vesicular monoamine transporter (VMAT2) function which leads to the disruption of DA sequestration are
important factors in generation of oxidative stress in PD.
Moreover, progressive degeneration of DA neurons is associated with chronic neuroinflammation and microglia
activation triggering free radicals production. It was found
that in 6-OHDA-model of PD, A2A adenosine receptor
antagonists administered in combination with L-DOPA
restored DA-glutamate balance and suppressed overproduction of hydroxyl radical. They also decreased hydroxyl
radical level in inflammatory model. However, under condition of disturbed VMAT2 function in an early PD, A2A
antagonists, particularly bearing properties of MAO-B in48th Congress of the Polish Biochemical Society
Session 10. Metabolism and Signaling Role of Ectopurines and Ectopirimidines155
L10.2
Oral presentations
The role of extracellular metabolism of
nucleotides in physiology, pathology
and therapy of cardiovascular diseases
O10.1
Ryszard T. Smoleński
Ecto-ATP in brain ventricular system
as a signal of brain injury in stroke
Department of Biochemistry, Medical University of Gdansk, Gdańsk,
Poland
Joanna Czarnecka1, Marek Cieślak2, Justyna
Kowal3, Michał Komoszyński1
e-mail: Ryszard Smolenski <[email protected]>
Nucleotides that are present in the extracellular space play
regulatory functions in the every organ, but these effects
are particularly important in the cardiovascular system. A
good example of practical value of studies on extracellular
nucleotides is spectacular success of drugs interacting with
purinergic receptors that are used to control platelet aggregation. Equally important from therapeutic point could
be regulatory function of nucleotides in immune system,
blood flow, heart function, angiogenesis, organogenesis
and regenerative processes. However, these mechanisms
are very complex. P2 Receptor effects of tri- and diphosphates are quite often opposite and further modulated by
P1 receptor mediated effects of adenosine. Therefore enzymes that converts extracellular triphosphates to diphosphates, monophoshates and adenosine could be as important as expression pattern of receptors for purines and
nucleotides. First two catabolic steps are catalysed by nucleoside triphosphate diphosphohydrolase (eNTPD) while
final conversion of AMP to adenosine is catalysed by ecto
5’-nucleotidase (e5N). Extracellular activity of adenosine
deaminase (eADA) that converts adenosine to inosine is
also present and can modulate adenosine receptor effects.
These enzymes are present in diverse forms (eNTPD in
particular), its expression is cell specific and could be grossly affected in different physiological states and in pathology. Effect of nucleotides may depend more on activities of
these enzymes than on amount and pattern of nucleotides
initially released. Recent clinical studies highlighted several new aspects. Loss of function genetic defect of e5N
gene was found to lead to vascular calcifications. Our own
studies demonstrated correlation of decreased e5N activity and increased eADA activity with pathologies of the
aortic valve in patients undergoing heart surgery. Our experimental studies with genetic model of atherosclerosis in
mice revealed decrease in vascular e5N activity and several
fold increase in eADA activity that progressed in parallel
with development of atherosclerosis. These effect translated into lower adenosine concentration in blood of atherosclerotic mice. We identified that drugs such as statins
can induce e5N and eNTPD. These findings demonstrate
potential to control processes regulated by purinergic and
nucleotide signaling by changes in expression of extracellular enzymes of nucleotide metabolism. This could help to
develop better therapy of cardiovascular diseases.
1Biochemistry Department, Faculty of Biology and Environment
Protection, Nicolaus Copernicus University, Toruń, Poland; 2Department
of Neurology WSZ Hospital Toruń, Poland; 3Biologisk Institut,
Department of Biology Molecular Integrative Physiology, København,
Denmark
e-mail: Joanna Czarnecka <[email protected]>
Ecto-purines may function as a switch that may bias the
physiological or the pathological outcome in the brain. In
the central nervous system extracellular ATP participates
in pain transmission and acts both as proinflammatory and
proapoptotic agent. A significant increase in ATP and other
nucleotides concentration has been observed in the brain
ventricular system of patients with neurological disease.
Based on animal model, we have identificated all three elements of purinergic signaling pathway in the brain ventricular system: 1) nucleotide receptors on the ependymocytes
surface, 2) purines and pyrimidines in the CSF 3) enzymatic
mechanisms of nucleotide signal termination. We hypothesize that qualitative and quantitative composition of purines and pyrimidines in CSF depend on the physiological
state of brain and indicate close interaction of brain and
ventricular system with CSF.
In our study we analysed concentration of nucleotides and
proinfammatory interleukins in the cerebrospinal fluid of
patients with ischemic stroke. We report that inflammatory
processes activated by ischemic stroke result in increased
concentrations of ATP, which correlates with an increased
levels of interleukin-1β in the CSF. Moreover, we indeed
found that the risk of death of patient is associated with
increasing concentrations of ATP and ADP in the cerebrospinal fluid.
Our results suggest that the ventricular system of the brain
plays a special role in the regulation of inflammatory processes in the central nervous system. This hypothesis is
confirmed by presence of all elements of purinergic signaling pathway in this region of brain and high concentration
of ATP in CSF, observed in case of brain damage. Therefore, we believe that the nucleotides in brain ventricular
system are involved in danger signaling, targeting astrocyte
activation as well as inflammatory and repair processes in
the central nervous system.
Toruń, September 2nd–5th 2013
156
Session 10. Metabolism and Signaling Role of Ectopurines and Ectopirimidines
O10.2
O10.3
Purinergic control of mesenchymal stem
cells proliferation and differentiation
Overexpression and purification
of recombinant potato (Solanum
tuberosum) apyrase 4 (StAPY4)
Katarzyna Roszek, Katarzyna Bomastek,
Agnieszka Błaszczak, Michał Komoszyński
Nicolaus Copernicus University, Faculty of Biology and Environment
Protection, Department of Biochemistry, Toruń, Poland
e-mail: Katarzyna Roszek <[email protected]>
The potential clinical applications of human mesenchymal
stem cells have increased research on correct identifying
of the high quality proliferating cells as well as their ex vivo
expansion and differentiation before therapeutic use.
Recent studies have focused on extracellular purines and
their receptors rather than the enzymes involved in purinergic signalization. Whereas, the purine nucleotides concentration outside the cell is controlled by ecto-enzymes.
Nucleotides metabolizing ecto-enzymes terminate the nucleotide signaling pathway, thus regulating the proliferation,
motility and differentiation of numerous cells, including
stem cells.
We analysed the activity of ecto-nucleotidases in mesenchymal stem cells (MSCs) as well as in MSCs-derived differentiated osteocytes and chondrocytes cultured in vitro.
We detected some substantial differences in the activity of
ecto-nucleotidases between undifferentiated stem cells and
progenitor or mature cells.
Undifferentiated MSCs have the ability to produce adenosine from AMP due to extremely high activity of ecto5’nucleotidase. Growing concentration of adenosine may
enhance the proliferation rate of mesenchymal stem cells.
We determined that these cells cultured in vitro are sensitive to adenosine added to the medium. In the opposite to
MSCs, mature cells are characterized rather by adenylate
kinase (AK) and NTPDases activity controlling the level of
ATP and ADP in the extracellular matrix.
Our results indicate that differences in the ecto-enzymes
profile correspond with the distinct proliferation capacity
of the cells. We propose that enzymes involved in ecto-nucleotides metabolism might also help to verify the effective
isolation and in vitro culture of MSCs. The ecto-nucleotidases activity determination is fast, simple and affordable.
In our opinion the results are more informative — they
indicate not only the presence of the protein on the cell
surface, but also its enzymatic activity.
Agnieszka Błaszczak1, Anna Ciarkowska2,
Michał Komoszyński3, Andrzej Wojtczak4
1Zakład Krystalochemii i Biokrystalografii/Biochemii, Polska; 2Zakład
Biochemii, Polska; 3Zakład Biochemii, Polska; 4Zakład Krystalochemii i
Biokrystalografii, Polska
e-mail: Agnieszka Błaszczak <[email protected]>
Plant NTPDases (apyrases) catalyse the removal of the
gamma phosphate from ATP and the beta phosphate from
ADP. The phosphate from AMP is not removed. As one of
the enzymes modulating the concentration of nucleotides,
apyrases are involved in many physiological processes, including the growth and development of plants or the biosynthesis of starch.
Escherichia coli (E. coli) strain BL21-CodonPlus (DE3)-RIL
was used for overexpression of the gene encoding apyrase
4 from potato Solanum tuberosum (StAPY4). We managed to
overproduce StAPY4 fused with His-tag, S-tag and Thioredoxin (Trx-tag). This fusion protein showed only traces of
enzymatic activity. Recombinant proteins produced by bacteria such as Escherichia coli, are usually accumulated in form
of inactive aggregates called inclusion bodies (IB), and that
happened in our case. In order to obtain biologically active StAPY4 we isolated IB from E. coli, partially purified
inactive StAPY4 using Immobilized Metal Ion Affinity Chromatography (IMAC) and then subjected protein to renaturation in the presence of 50 mM β-cyclodextrin (β-CD) in
suspension. Activity assays of renatured enzyme revealed
significant increase in enzymatic activity of StAPY4.
48th Congress of the Polish Biochemical Society
Session 10. Metabolism and Signaling Role of Ectopurines and Ectopirimidines157
O10.4
O10.5
The properties of adenylate kinase [EC
2.7.4.3] from Aquifex aeolicus and Bacillus
stearothermophilus and their influence of
the aggregation of human blood platelets
The role of adenylate kinase
izoenzymes 1 and 2 in regulation of
blood platelet aggregation process
initiated by ADP and collagen
Bożena Studzińska, Ewa Kubicka, Małgorzata
Kupiecka, Magdalena Wujak, Agnieszka Błaszczak,
Joanna Czarnecka, Michał Komoszyński
Magdalena Wujak, Tomasz Janczi, Michał Komoszyński
Copernicus University, Faculty of Biology and Environmental Protection,
Departament of Biochemistry, Toruń, Poland
e-mail: Michał Komoszyński <[email protected]>
e-mail: Bożena Studzińska <[email protected]>
Platelets are specialized blood elements that play a key role
in physiological and pathological processes of hemostasis, inflammation, tumor metastasis and wound healing.
Activation of platelets is crucial for blood coagulation.
Main activators of platelets: ADP, collagen thrombin and
thromboksane A2 cause the change of their shape, adhesion, secretion and lead to aggregation. Platelets hyperaggregability is associated with the risk factors for cerebral,
coronary or peripheral arterial ischemia causing stroke, cardiovascular disease and venous thrombosis. The novel therapies against platelet-dependent thrombosis are targeted at
purinergic receptors activating the platelets aggregation.
In the last few years the thienopyridyne derivatives (ticlopidine, clopidogrel, prasugrel) are mainly used. Adenylate
kinase (AK) is the enzyme involved in metabolism of ectoadenine nucleotides which can play the most important role
in hemostasis. To study the influence of AK on human
blood platelets aggregation we used enzymes obtained as a
result of over-expression in prokaryotic system. Adenylate
kinase gene derived from Aquifex aeolicus or Bacillus stearothermophilus was introduced into Escherichia coli. After purification of the recombined enzyme the influence of AK
on the platelets aggregation was examined. The platelets
aggregation was started by ADP and collagen type 1 from
calf skin. Adenylate kinase from both strains of bacteria
inhibited the platelets aggregation. In case of collagen activated aggregation, addition of AK caused the complete
desaggregation process. Anticoagulative role of AK indicates the possibility of using this enzyme in the treatment
of cardiovascular disease. Adenylate kinase may be applied
for the inhibition of uncontrolled clotting of blood and
minimalizing effects of heart attack or brain stroke.
Nicolaus Copernicus University, Faculty of Biology and Environment
Protection, Department of Biochemistry, Toruń, Poland
The blood platelets activation and aggregation is crucial
for hemostasis and thrombosis. Adenosine diphosphate
(ADP), collagen, thrombin and thromboxane A2 are main
activators of blood platelets. Meanwhile, adenylate kinases
(AK) are one of the most important enzymes involved in
metabolism of adenine nucleotides in blood.
Our previous studies revealed the significant increase of
adenylate kinase activity in the serum of patients suffering
from myocardial infarction when compared to the healthy
volunteers. Up to date, the grounds and mechanisms of
this AK activity have been not fully elucidated. However,
some other experiments indicate that bacterial adenylate kinases inhibit in vitro platelets aggregation and even dissolve
existing platelet aggregates. Whereas, the possible anti-aggregating properties of human AK izoenzymes have been
not examined so far. We hypothesize that human adenylate
kinases may be involved in regulation of platelet aggregation by removing the circulating ADP and therefore blocking activation of blood platelets.
Therefore, the aim of presented studies was to identify the
function of human blood AK izoenzymes in platelet aggregation processes.
Human adenylate kinase izoenzymes 1 and 2 were used
in our studies. The highly homogenous enzymes were obtained as result of bacterial overproduction in prokaryotic
system and purification using affinity and gel filtration
chromatography. The physicochemical and kinetic parameters of the examined enzymes were determined. We carried out series of experiments on platelet-rich plasma using
different platelet activators in order to verify the hypothesis
concerning the anti-aggregating properties of human AK
isoenzymes and define their role for hemostasis and thrombosis.
Toruń, September 2nd–5th 2013
158
Session 10. Metabolism and Signaling Role of Ectopurines and Ectopirimidines
O10.6
O10.7
Metabolism of extracellular nucleotides
in different types of cells in the aortic
valve and its control by shear stress
Purinergic-mediated increase
permeability of glomerular filtration
barrier for albumin is associated in
actin reorganization in podocytes
Ewa Kaniewska1,2, Alicja Sielicka1, Napachanok
Mongkoldhumrongkul2, Iwona Pelkant-Małecka1,
Mariola Olkowicz1, Ewa M. Słomińska1, A.
Chester2, M. Yacoub2, Ryszad T. Smoleński1
1Departments of Biochemistry1, Medical University of Gdansk, Gdańsk,
Poland; 2Heart Science Centre, Imperial College London at Harefield
Hospital, UK
e-mail: Ewa Kaniewska <[email protected]>
Background: Changes in expression of enzymes of extracellular nucleotide degradation could have major impact on
thrombosis, inflammation and immune responses. Our earlier studies demonstrated that activity of ectoenzymes such
as ecto-5’-nucleotidase and ectonucleoside triphosphate diphosphohydrolase is higher in valve endothelial cells then
in aortic valve cells. Reason for this difference is not known.
Our hypothesis is that differences in flow characteristics
such as increased shear stress on the valve surface could be
responsible for induction of ectoenzymes. We conducted
therefore baseline characteristics of extracellular nucleotide
metabolism in valve cells and tested effect of flow typical
for ventricular valve surface in endothelial cells.
Metods: Cells were isolated from healthy aortic valves by
incubation with tripsin in two steps. At first round we isolated endothelial cells in second one interstitial cells were
isolated. In both type of cells extracellular enzyme nucleotide metabolism was characterised by incubation with
adenosine, AMP and ATP. Conversion of substrates into
products was analysed by HPLC.
Effect of ventricular side valve flow was tested with
HMEC-1 cells seeded on cover slips in bioreactor mimicking ventricular flow. Incubation in static flow condition
was used as control. After that cells on cover slips were
fixed and stained with use of antibodies against e5N and
eNTPD.
Results: We examined extracellular purine metabolism in
valve cells. As a result was shown difference between levels
of nucleotide metabolites in various types of cells. In endothelial cells we can observe much higher level of AMP,
and lower level of adenosine than in interstitial cells. What
is interesting, the amount of metabolites is not influence by
passage of primary cells.
As a result of the flow we could observe decrease amount
of cells on cover slips. Despite of that fact treatment of
HMEC-1 by ventricular flow caused significant increase in
ectoenzymes expression compare to control cells in static
conditions (Table 1).
e NTPD 48h ctrl
410.5 ±68.95
Relevant fluorescence per cell [U]
eNTPD 48h
e5N 48h ctrl
1082.9± 138
10.3 x 104 ±9.4x103
e5N 48h
6.1x104 ± 5.2x103
Conclusions: Level of nucleotide metabolites in valves
cells imply that ecto and endocellular enzymes plays an important role in heart disease like aortic valve calcification
ect. We are suggest that higher level of nucleotide metabolism in valves could prevent heart from inflammation, calcification etc. Especially in ectoenzyme case. Immunohistochemical staining indicates that ventricular
flow could play an important role in ectoenzyme expression in endothelial cells what cause higher level of nucleotide metabolism. Our data from corn and plate machine experiments suggest that type of flow is important condition
which induce the proteins expression in endothelial cells. Małgorzata Kasztan1, Agnieszka Piwkowska2, Ewelina
Kreft1, Dorota Rogacka2, Robert Kowalski1, Mirosława
Szczepańska-Konkel1, Maciej Jankowski1,2
1Medical University of Gdansk, Department of Therapy Monitoring and
Pharmacogenetics, Gdańsk, Poland; 2Mossakowski Medical Research
Center Polish Academy of Sciences, Laboratory of Molecular and
Cellular Nephrology, Gdańsk, Poland
e-mail: Małgorzata Kasztan <[email protected]>
Introduction: Purinergic receptors affecting cGMPdependent protein kinase (PKG) — modulator of cytoarchitecture — are expressed on cells forming glomerular
filtration barrier — endothelial cells and podocytes. Thus,
changes of purinergic activity may affect glomerular permeability for albumin leading to albuminuria/proteinuria, a
sign of kidney disease and independent risk factor for the
progression of renal failure.
Aim: Aim of study was to investigate the effects of purinergic activation on permeability of glomerular filter for
albumin and involvement of PKG in this process.
Methods: Isolated, rat renal glomeruli were incubated with
nonselective purinergic agonists 2-methylothioATP (2-MeSATP) and ATP-γ-S (10-6M, 10 min, 37°C). Function of
glomerular barrier of single isolated glomerulus (convectional albumin permeability, Palb) was measured based on
video-microscopy (Olympus IX51) using oncopressive media (1% and 5% BSA) eliciting transglomerular fluid flux
and subsequent changes in glomerular volume (CellSens
Dimension Software). The osmotic pumps (ALZET 2001,
rate 1µl/hr) with 2-MeSATP or ATP-γ-S (both 10-6M) were
implanted subcutaneously into Wistar rats for 7 days. Afterwards pumps were removed and glomeruli were isolated
to measure Palb. Cultured rat podocytes were used for podocyte layer permeability for albumin, PKG dimerization
(Western blot) and immunofluorescence studies.
Results: 2-MeSATP and ATP-γ-S in vitro increased Palb in
time- and concentration-dependent manner with maximum
0.58±0.04 and 0.37±0.08 vs control 0.10±0.03, respectively. Effect of 2-MeSATP was abolished by L-NAME
(10–5M). Palb in glomeruli isolated from rats exposed in
vivo (ALZET) to 2-MeSATP or ATP-γ-S were significantly increased 0.35±0.03 and 0.52±0.02 vs. 0.13±0.02,
respectively. 2-MeSATP and ATP-γ-S (10-6M) increased
podocyte permeability for albumin 111.3±7.9 µg/ml and
99.7±6.2µg/ml vs. 45.3±2.1 respectively and this effect
was not dependent on PKG dimerization. 2-MeSATP and
ATP-γ-S lead to sub-cortical actin reorganization in cultured rat podocytes.
Conclusions: Our results suggest that purinergic activation leads to action reorganization in, at least, podocytes,
with subsequent increase of glomerular barrier albumin
permeability.
48th Congress of the Polish Biochemical Society