Differential effects of uridine adenosine tetraphosphate on purinoceptors in the rat isolated perfused kidney

Article (PDF Available)inBritish Journal of Pharmacology 161(3):530-40 · October 2010with33 Reads
DOI: 10.1111/j.1476-5381.2010.00914.x · Source: PubMed
Abstract
Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine tetraphosphate (Up(4)A) as a novel and highly potent endothelial-derived contracting factor. Up(4)A induces strong vasoconstrictive effects in the renal vascular system mainly by P2X(1) receptor activation. However, other purinoceptors are also involved and were analysed here. The rat isolated perfused kidney was used to characterize vasoactive actions of Up(4)A. After desensitization of the P2X(1) receptor by α,β-methylene ATP (α,β-meATP), Up(4)A showed dose-dependent P2Y(2)-mediated vasoconstriction. Continuous perfusion with Up(4)A evoked a biphasic vasoconstrictor effect: there was a strong and rapidly desensitizing vasoconstriction, inhibited by P2X(1) receptor desensitization. In addition, there is a long-lasting P2Y(2)-mediated vasoconstriction. This vasoconstriction could be blocked by suramin, but not by PPADS or reactive blue 2. In preparations of the rat isolated perfused kidney model with an elevated vascular tone, bolus application of Up(4)A showed a dose-dependent vasoconstriction that was followed by a dose-dependent vasodilation. The vasoconstriction was in part sensitive to P2X(1) receptor desensitization by α,β-meATP, and the remaining P2Y(2)-mediated vasoconstriction was only inhibited by suramin. The Up(4)A-induced vasodilation depended on activation of nitric oxide synthases, and was mediated by P2Y(1) and P2Y(2) receptor activation. Up(4)A activated P2X(1) and P2Y(2) receptors to act as a vasoconstrictor, whereas endothelium-dependent vasodilation was induced by P2Y(1/2) receptor activation. Up(4)A might be of relevance in the physiology and pathophysiology of vascular tone regulation.
RESEARCH PAPER
Differential effects of
uridine adenosine
tetraphosphateon
purinoceptors in the rat
isolated perfused kidneybph_914 530..540
Markus Tölle, Mirjam Schuchardt, Annette Wiedon, Tao Huang,
Lars Klöckel, Joachim Jankowski, Vera Jankowski, Walter Zidek and
Markus van der Giet
Charité Universitätsmedizin Berlin, Medical. Klinik mit Schwerpunkt Nephrologie,
Hindenburgdamm 30, 12203 Berlin, Germany
Correspondence
Prof Markus van der Giet, Charite
Universitätsmedizin Berlin,
Medical. Klinik mit Schwerpunkt
Nephrologie, Hindenburgdamm
30, 12203 Berlin. E-mail:
markus.vandergiet@charite.de
----------------------------------------------------------------
Keywords
purinoceptors; uridine adenosine
tetraphosphate; P2 receptors; rat
isolated perfused kidney;
vasoconstriction; vasodilation
----------------------------------------------------------------
Received
31 July 2009
Revised
15 April 2010
Accepted
25 April 2010
----------------------------------------------------------------
This article is commented on by
Burnstock, pp. 527–529 of this
issue. To view this commentary
visit http://dx.doi.org/10.1111/
j.1476-5381.2010.00937.x
BACKGROUND AND PURPOSE
Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine
tetraphosphate (Up
4
A) as a novel and highly potent endothelial-derived contracting factor. Up
4
A induces strong
vasoconstrictive effects in the renal vascular system mainly by P2X
1
receptor activation. However, other purinoceptors are also
involved and were analysed here.
EXPERIMENTAL APPROACH
The rat isolated perfused kidney was used to characterize vasoactive actions of Up
4
A.
KEY RESULTS
After desensitization of the P2X
1
receptor by a,b-methylene ATP (a,b-meATP), Up
4
A showed dose-dependent P2Y
2
-mediated
vasoconstriction. Continuous perfusion with Up
4
A evoked a biphasic vasoconstrictor effect: there was a strong and rapidly
desensitizing vasoconstriction, inhibited by P2X
1
receptor desensitization. In addition, there is a long-lasting P2Y
2
-mediated
vasoconstriction. This vasoconstriction could be blocked by suramin, but not by PPADS or reactive blue 2. In preparations of
the rat isolated perfused kidney model with an elevated vascular tone, bolus application of Up
4
A showed a dose-dependent
vasoconstriction that was followed by a dose-dependent vasodilation. The vasoconstriction was in part sensitive to P2X
1
receptor desensitization by a,b-meATP, and the remaining P2Y
2
-mediated vasoconstriction was only inhibited by suramin. The
Up
4
A-induced vasodilation depended on activation of nitric oxide synthases, and was mediated by P2Y
1
and P2Y
2
receptor
activation.
CONCLUSIONS AND IMPLICATIONS
Up
4
A activated P2X
1
and P2Y
2
receptors to act as a vasoconstrictor, whereas endothelium-dependent vasodilation was
induced by P2Y
1/2
receptor activation. Up
4
A might be of relevance in the physiology and pathophysiology of vascular tone
regulation.
Abbreviations
a,b-meATP, a,b-methylene ATP; AngII, angiotensin II; Ap
n
A, diadenosine-n-phophate (n: number of phosphates); Ap
n
G,
adenosine-guanosine-n-phosphate (n: number of phosphates); ApoE, apolipoprotein E; CI, confidence interval; DMSO,
dimethyl sulphoxide; eNOS, endothelial NOS; Gp
n
G, diguanosine-n-phosphate (n: number of phosphates); L-NAME,
N
G
-nitro-L-arginine methyl ester; MAP, mean arterial blood pressure; MCP-1, monocyte chemoattractant protein-1;
MRS2179, 2-deoxy-N6-methyladenosine 3,5-bisphosphate; PPADS, pyridoxal-phosphate-6-azophenyl-2;4-disulphonic
acid; RB2, reactive blue 2; Up
4
A, uridine adenosine tetraphosphate
BJP
British Journal of
Pharmacology
DOI:10.1111/j.1476-5381.2010.00914.x
www.brjpharmacol.org
530 British Journal of Pharmacology (2010) 161 530–540
© 2010 The Authors
British Journal of Pharmacology © 2010 The British Pharmacological Society
Introduction
Over the past two decades, there has been an
increase in evidence that the purinoceptor system is
involved in vascular tone control (van der Giet et al.,
2002a; Buvinic et al., 2006), and also potentially
involved in the pathogenesis of hypertension (Jan-
kowski et al., 2005; Tolle et al., 2008). In the mid-
1990s, a new group of purinergic compounds, the
so-called diadenosine polyphosphates, were identi-
fied as highly potent vasoactive substances (Schluter
et al., 1994; Gabriels et al., 2002). In the following
years, it became evident that these dinucleoside
polyphosphates induce vasoconstriction in various
vascular systems, mainly via P2X
1
receptor activa-
tion. However, some of the vasoactive effects
observed with diadenosine pentaphosphate (Ap
5
A)
and the corresponding hexaphosphate (Ap
6
A) were
also mediated via activation of G-protein-coupled
P2Y receptors (Gabriels et al., 2002). In the following
years, more dinucleoside polyphosphates contain-
ing either two adenosines, one adenosine and one
guanosine, or two guanosines were identified. A
complex family of purinergic dinucleoside poly-
phosphates with a variable phosphate chain of 2–7
phosphates (Jankowski et al., 2009) has now been
described. Adenosine-containing dinucleoside poly-
phosphates act more as vasoconstrictive agents, and
guanosine-containing dinucleoside polyphosphates
act as cell-proliferating agents. One of the
guanosine-containing dinucleoside polyphosphates
is of special interest. Diguanosine pentaphosphate
(Gp
5
G) is a potent activator of Rho-kinase and
modulates the vasoactive responses of other known
vasoactive substances such as angiotensin II (AngII)
(Tolle et al., 2006). Gp
5
G activates P2Y
2
receptors
(receptor nomenclature follows Alexander et al.,
2009) to induce calcium sensitization, and such sen-
sitization is believed to be an important mechanism
in the control of vascular tone and blood pressure.
Recently, our group identified the first
pyrimidine-containing dinucleoside polyphosphate
as a highly potent, new endothelial-derived vasocon-
strictive factor. The substance was characterized as
uridine adenosine tetraphosphate (Up
4
A) (Jankowski
et al., 2005). Up
4
A shares properties of both P2X
receptor and P2Y receptor agonists. In the first study,
we demonstrated that Up
4
A acts as a vasoconstrictive
agent by P2X
1
receptor activation (Jankowski et al.,
2005). The response was only partially inhibited by
the P2X
1
and P2X
3
receptor desensitizer a,b-
methylene ATP (a,b-meATP), indicating the activa-
tion of other purinoceptors. It was proposed that P2Y
receptors are responsible for the remaining vasoac-
tive properties of Up
4
A. These Up
4
A-activated P2Y
receptors have not yet been characterized in depth.
There is some evidence that Up
4
A might have
implications in the pathogenesis of human hyper-
tension. Up
4
A plasma levels in young hypertensives
are significantly increased compared to age-matched
controls. The Up
4
A concentration is significantly
correlated with the left ventricular mass and intima
media wall thickness in these young hypertensives
(Jankowski et al., 2007).
The purpose of the current work was to identify
all purinoceptors other than P2X
1
which are acti-
vated by Up
4
A. It is necessary to know more about
the activation of the P2 receptors other than P2X
1
receptors activated by Up
4
A. We sought to under-
stand the complex vasoregulatory properties of
Up
4
A as a novel endothelium-derived vascoconstric-
tive factor. In this study, we focused on the P2Y
receptor-mediated physiological vasoactive actions
of Up
4
A. To study the vasoactive effects of Up
4
A, we
used the model of the rat isolated perfused kidney.
We demonstrated that Up
4
A exerted vasocon-
striction not only via P2X
1
receptors, but also by
activation of P2Y
2
receptors. The Up
4
A-induced
vasodilation was mediated via endothelial activa-
tion of P2Y
1
and P2Y
2
receptors.
Methods
Animal experiments
All animal care and experimental procedures were
approved by the State Ethics committee Landesamt
für Gesundheit, Ernährung und Technische Sicher-
heit Berlin.
Preparation of the rat isolated
perfused kidney
Adult male Wistar–Kyoto rats (4–6 months old)
were anaesthetized with ketamine (50 mg·kg
–1
,
intraperitoneally)/xylazine (10 mg·kg
–1
, intraperito-
neally). The abdominal cavity was opened by a mid-
ventral incision. The aorta and the left kidney were
carefully isolated from adhesive tissue by blunt dis-
section. Ligatures were placed around the left renal
artery and the infrarenal aorta. A polyethylene cath-
eter (20-gauge) was placed in the distal aorta. Imme-
diately after the insertion of the catheter, 500 U of
heparin sodium was injected. Perfusion was then
started. The catheter was gently advanced into the
left renal artery without interruption of flow. The
kidney was excised and immediately mounted in
the perfusion system (Hugo Sachs Electronic,
Freiburg, Germany).
Perfusion system
The perfusion procedure generally followed the
description given by Hofbauer et al. (1973). The
BJP
Up
4
A and purinoceptors
British Journal of Pharmacology (2010) 161 530–540 531
kidney was perfused at a constant flow rate using a
peristaltic pump equilibrated to a perfusion pressure
of about 70 mmHg Tyrode’s solution of the following
composition (in mM): NaCl, 137; KCl, 2.7; CaCl
2
, 1.8;
MgCl
2
, 1.1; NaHCO
3
, 12; NaH
2
PO
2
, 0.42; and glucose,
5.6 gassed with 95% O
2
–5% CO
2
and maintained at
37°C was used as perfusate. The pH was measured
continuously by a pH sensor included in the perfu-
sion system and was held between 7.35 and 7.45.
Responses were measured as changes in perfusion
pressure (mmHg) with a pressure transducer
(Statham Transducer P23Gb, Siemens, Erlangen,
Germany) on a side arm of the perfusion catheter,
connected to a bridge amplifier (Hugo Sachs), and
recorded digitally. Preparations were allowed to
equilibrate for 30 min prior to experimentation.
Basal tone preparations
Vasoconstrictor responses of preparations to doses
of Up
4
A, a,b-me-ATP, UTP, UDP or AngII were
assessed at basal tone. For each substance, dose–
response curves were constructed, with a minimum
of 20 min being allowed to elapse between consecu-
tive doses to avoid desensitization. This procedure
allowed dose–response curves for several agonists to
be constructed for the same preparation. A signifi-
cant degree of cross-desensitization or auto-
desensitization was not detected. The procedure has
been described previously (van der Giet et al., 1999).
The non-selective P2 receptor antagonists suramin
(100 mM), pyridoxal phosphate 6-azophenyl-2;4-
disulphonic acid (PPADS; 10 mM), reactive blue 2
(RB2, 100 mM), selective P2Y
1
receptor antagonist
MRS2179 (10 mM) or the P2X
1/3
receptor desensitiz-
ing agent a,b-meATP (10 mM) were added to the
perfusate 30 min before challenge with Up
4
A. In
some experiments, NOS was inhibited by continu-
ous perfusion with N
G
-nitro-L-arginine methyl ester
(
L-NAME) (100 mM). In some experiments, we per-
formed endothelial cell removal prior to the experi-
ments. Endothelium removal was performed with
Triton X-100. The endothelium was removed by per-
fusion of the isolated kidney for 5 s with 0.1%
Triton X-100. The lack of response to acetylcholine
was used to check endothelium removal. Unaffected
contraction to K
+
(130 mM bolus) indicated an
intact vascular smooth muscle cell function, which
was tested before and after endothelium removal.
Continuous perfusion with Up
4
A
Vasoconstrictor responses to continuous perfusion
with Up
4
A were assessed at basal tone. Dose–
response curves were constructed for each sub-
stance, with 20 min being allowed to elapse
between consecutive continuous perfusions. A
significant cross-desensitization or auto-
desensitization was not detected when substances
were being given in intervals of at least 20 min.
Desensitization was tested prior to experiments with
all substances used (data not shown), and the results
were compatible with previous observations with
purinergic substances (van der Giet et al., 1999).
Continuous perfusion with P2X
receptor antagonists
The non-selective P2 receptor antagonist suramin
(100 mM) and the P2X receptor antagonist PPADS
(10 mM) were added to the perfusate 30 min before
challenge with Up
4
A. In an additional experiment,
the P2X receptor agonist a,b-meATP (10 mM) was
also perfused before challenge with Up
4
A.
Raised tone preparations
Vasodilator responses to doses of Up
4
A and ACh
were assessed in raised-tone preparations. Perfusion
pressure was increased by continuous perfusion
with AngII (200 nM). The resistance of vasodilator
responses to desensitization and the reproducibility
of responses with time-allowed dose–response
curves for several agonists to be constructed for the
same preparation. The P2 receptor antagonist
suramin (100 mM), PPADS (10 mM, 100 mM), RB2
(100 mM), MRS 2179 (10 mM) and the antagonist of
NOS
L-NAME (100 mM) were added to the perfusate
30 min before challenge with Up
4
A.
Assessment of oedema during
perfusion experiments
To assess the development of oedema during the
perfusion experiments, rat kidneys were weighed
before and after the experiments. After perfusion,
the weight was 132 16% of the initial weight,
indicating that a slight oedema of the kidneys devel-
oped. The response to 10 nM AngII at the end of the
experiments was 106 9% of the initial response.
Data analysis
Responses were measured as changes in perfusion
pressure (mmHg), and results presented as the
means SEM and if necessary their 95% confi-
dence interval (95% CI). Statistical analysis was
performed using Friedman’s test. To compare
columns for statistical variance, we applied Dunn’s
correction where applicable. P < 0.05 were consid-
ered significant.
Materials
All vasoactive substances were applied as 100 mL
bolus into a valve proximal to the perfused kidney
preparation. Drug dilutions were performed daily
from stock solutions of 10 mM (concentrates stored
frozen) in HPLC grade water or HPLC grade
BJP
M Tölle et al.
532 British Journal of Pharmacology (2010) 161 530–540
dimethyl sulphoxide (DMSO) unless otherwise indi-
cated. Heparin (sodium salt), suramin (hexasodium
salt), a,b-meATP and ketamine/xylazine were pur-
chased from Sigma Aldrich (Schnelldorf, Germany).
Up
4
A was purchased from Jena Bioscience (Jena,
Germany). Prior to use, Up
4
A was purified according
to a procedure described by Heidenreich et al.
(1995).
Results
Vasoconstrictor responses in basal
tone preparations
The baseline perfusion pressure of the rat isolated
perfused kidneys decreased by 3.0 0.5 mmHg
during the first, and by 2.0 1.5 mmHg during the
second hour of perfusion. Vascular reactivity to
vasoactive agents did not diminish during this time.
After the equilibration period, the baseline pressure
was 68 2mmHg(n = 73). The addition of suramin
(100 mM) to the perfusate caused an increase of per-
fusion pressure of 7 5 mmHg. After addition of
L-NAME (100 mM), non-significant increases of per-
fusion pressure of 10 4 mmHg and for RB2
(100 mM) of 6 2 mmHg were observed. The addi-
tion of MRS2179 (10 mM), PPADS (10 mM) to the
perfusate did not induce any change in baseline
perfusion pressure (data not shown).
At basal tone, Up
4
A caused a dose-dependent
vasoconstriction (EC
50
[log mol] = –8.3 0.1 and
maximal change in perfusion pressure (V
max
) of 107
8 mmHg, n = 7, Figure 1A,B). In the presence of
the P2X
1
receptor desensitizer a,b-meATP (10 mM) in
the perfusate, responses to bolus application of
Up
4
A were significantly decreased, but not com-
pletely abolished (EC
50
[log mol] = –8.6 0.2, V
max
=
32 3 mmHg, n = 7, Figure 1A,B). This remaining
vasoconstriction could be almost totally blocked by
the non-selective P2 receptor antagonist suramin
(100 mM), whereas the selective P2Y
1
receptor
antagonist MRS2179 (10 mM), the non-selective P2
receptor antagonist PPADS (10 mM) or RB 2 (100 mM)
had no effect (Figure 1C). UTP induced a dose-
dependent vasoconstriction [EC
50
[log mol] = 7.9
0.2 and maximum change in perfusion pressure
(V
max
) of 39.0 1.9 mmHg, n = 7, Figure 1D]. UDP
only showed a very small change of perfusion pres-
sure at high dosages. In the presence of suramin,
there was a profound and significant (P < 0.05) inhi-
bition of UTP-induced vasoconstriction. PPADS also
showed a significant (P < 0.05) inhibitory effect.
MRS2179 showed no significant effect. (Figure 1D).
Continuous perfusion with Up
4
A
Continuous perfusion with Up
4
A led to a
concentration-dependent increase of the perfusion
pressure (Figure 2A). The perfusion pressure increase
was divided into two phases. The first phase consists
of a fast vasoconstrictive response with a rapid
desensitization (EC
50
[log mol·L
–1
] = –6.6 0.1, V
max
= 96.2 9.2 mmHg, n = 8, Figure 2B). The second
phase was characterized by a long-lasting, stable
vasoconstriction (EC
50
[log mol·L
–1
] = –6.6 0.1,
V
max
= 42 2 mmHg, n = 8, Figure 2C). Both phases
showed a concentration-dependent vasoconstric-
tion. The first, fast desensitization effect of Up
4
A
could be completely inhibited by parallel continu-
ous perfusion with a,b-meATP, suramin and PPADS.
MRS2179 and RB2 had no significant (P < 0.05)
inhibitory effect (Figure 2D).
The second, long-lasting vasoconstrictive effect
of Up
4
A was significantly (P < 0.05) blocked by
suramin, whereas PPADS, MRS2179 and RB2 showed
no significant inhibition (Figure 2E).
Vasoactive responses in raised
tone preparations
The basal pressure of the rat isolated perfused
kidney was raised by continuous perfusion with
AngII (200 nM) by 76.2 4.2 mmHg. Under condi-
tions of raised perfusion pressure, Up
4
A induced a
dose-dependent increase of the perfusion pressure
(EC
50
[log M] = –8.7 0.1, V
max
= 87.5 3.7 mmHg,
n = 7, Figure 3A,B). This vasoconstriction was fol-
lowed by a dose-dependent decline of perfusion
pressure (EC
50
[log M] = –8.0 0.1, V
max
= –65.7
9.3 mmHg, n = 7, Figure 3A,C).
As was the case under basal conditions, the initial
increase of the perfusion pressure was significantly
attenuated by a,b-meATP, suramin and PPADS,
whereas MRS2179 and RB2 had no significant effect
(Figure 4A). Under conditions of continuous perfu-
sion with AngII (200 nM) and a,b-meATP (10 mM), a
residual perfusion pressure increase by Up
4
A could
be detected. This remaining vasoconstriction was
significantly blocked by suramin. PPADS, RB2 and
MRS2179 had no significant effect on this remain-
ing perfusion pressure increase (Figure 4B).
The Up
4
A (10 pmol)-induced decrease of the per-
fusion pressure could be significantly diminished by
continuous perfusion with the non-selective NOS
antagonist
L-NAME and after chemical removal of
the endothelium with Triton X-100 (Figure 4C). The
vasodilatation could also be significantly attenuated
by the non-selective P2 receptor antagonists
suramin, PPADS and RB2, and by the selective P2Y
1
receptor antagonist MRS2179 (Figure 4D).
Discussion
Our results clearly demonstrated that Up
4
A acti-
vated at least three different purinoceptor subtypes
BJP
Up
4
A and purinoceptors
British Journal of Pharmacology (2010) 161 530–540 533
in the kidney to induce a complex vasoactive
response. Besides the P2X
1
receptor, which induces
vasoconstriction (Jankowski et al., 2005), we infer
from our data that there was also a P2Y
2
receptor
activation. The P2Y
2
receptor is responsible for addi-
tional, long-lasting vasoconstriction. Activation of
the P2Y
1
/P2Y
2
receptors results in an endothelium-
dependent, NOS-mediated vasodilation.
Studying the purinoceptor subtypes involved in
the vasoconstriction and vasodilation observed in
the current experiments is a complex undertaking.
There are no specific pharmacological active ago-
nists and antagonists available for purinoceptors.
The purinoreceptor expression profile in kidney
tissue has been extensively studied, but there are
many open questions. Turner and coworkers dem-
onstrated the expression of P2X
1
, P2X
2
and P2Y
1
receptors in rat renal vascular smooth muscle cells
(Turner et al., 2003). Using a pharmacological
approach, Inscho and colleagues identified a
receptor-mediating vasoactive response in renal
tissue that was activated by UTP (Inscho et al.,
1998). As UTP can activate P2Y
2/4
receptors, it is
tempting to speculate that these receptors can acti-
vate response in renal tissue. Potentially, these
receptor subtypes are expressed at a very low level
and therefore they cannot be detected by immuno-
histochemistry (Turner et al., 2003). In the present
work, we demonstrated that UTP showed potent
vasoactive actions, which were inhibited by
140
B
60
80
100
120
140
Up
4
A
+
α
,
β
-meATP
*
*
*
*
P (mmHg)
−11 −10 −9 −8 −7
0
20
40
*
*
*
*
*
*
*
*
log mol
40
50
C
10
20
30
+ MRS2179
+ Suramin
+ PPADS
Up
4
A
+ RB2
P (mmHg)
−11 −10
−9
−8 −7
0
log mol
200
120
140
160
180
AP (mmHg)
60
80
100
12 3 4 5 6 7 8
MA
α
,
β
-meATP
A
+
α
,
β
-meATP
40
50
UTP
UDP
10
20
30
40
UDP
UTP + Suramin
UTP + MRS2179
UTP + PPADS
P (mmHg)
−11 −10 −9
−8
−7
0
10
log mol
D
Figure 1
(A) Original tracing of a representative experiment of the rat isolated perfused kidney showing the dose-dependent increase of the perfusion
pressure (MAP) induced by Up
4
A in the absence (1–4) and the presence of a,b-meATP (1 mM; 5–8) (1:100 pmol; 2:1 nmol; 3:10 nmol;
4:100 nmol; 5:100 pmol; 6:1 nmol; 7:10 nmol; 8:100 nmol). (B) Dose–response curve of changes in perfusion pressure in the rat isolated perfused
kidney induced by Up
4
A in the absence and presence of a,b-meATP. Each point is the mean of seven determinations, and vertical lines show SEM.
For abbreviations, see text. Where error bars do not appear in figures, errors are within the symbol size. *<0.05 significant difference from baseline
perfusion pressure. (C) Dose–response curve of changes in perfusion pressure in the rat isolated perfused kidney induced by Up
4
A in the presence
of a,b-meATP (10 mM) and in the presence of the P2Y receptor antagonists suramin (50 mM), PPADS (10 mM), the specific P2Y
1
receptor antagonist
MRS2179 (10 mM), and RB2 (100 mM). Each point is the mean of seven determinations and vertical lines show SEM. Where error bars do not
appear in figures, errors are within the symbol size. *<0.05 significant difference from baseline perfusion pressure. (D) Dose–response cur ve of
changes in perfusion pressure in the rat isolated perfused kidney induced by UDP and UTP in the presence of the P2 receptor antagonists suramin
(100 mM), PPADS (10 mM) and the specific P2Y
1
receptor antagonist MRS2179 (10 mM). Each point is the mean of seven determinations, and
vertical lines show SEM. Where error bars do not appear in figures, errors are within the symbol size. *<0.05 significant difference from baseline
perfusion pressure.
BJP
M Tölle et al.
534 British Journal of Pharmacology (2010) 161 530–540
suramin, but not PPADS, indicating that the P2Y
2
receptor might be a receptor with vasoactive actions
in the kidney. UDP, which is an agonist at P2Y
6
receptors, induced only a very mild vasoconstriction
at high doses. In 2005, we identified Up
4
Aasa
potent, endothelium-derived vasoactive substance,
and we identified the P2X
1
receptor as the main
receptor mediating the observed vasoconstrictor
140
160
180
200
Hg)
A
60
80
100
120
140
P (mmH
12 3
4
56 78
100
*
*
*
B
25
50
75
*
*
P (mmHg)
−9 −8 −7 −6 −5
0
Concentration (log M)
30
40
*
*
*
C
0
10
20
30
*
*
*
P (mmHg)
−9 −8
−7
−6 −5
Concentration (log M)
100
120
D
20
40
60
80
100
*
P (mmHg)
Control
+ α,β-meATP
+ Suramin
+ PPADS
+ M
R
S2179
+ RB2
0
20
*
*
50
E
10
20
30
40
*
P (mmHg)
C
on
t
rol
+ α,β-meATP
+ Suramin
+ PPADS
+ MRS2179
+
RB2
0
*
Figure 2
(A) Original tracing of a representative experiment of the rat isolated perfused kidney showing the concentration-dependent increase of the
perfusion pressure induced by the continuous perfusion with various concentrations of Up
4
A(1= 1 nM; 2 = 10 nM; 3 = 50 nM; 4 = 100 nM, 5
= 500 nM; 6 = 1 mM; 7 = 5 mM; 8 = 10 mM). A biphasic vasoconstrictor response was observed; an initial transient effect and a subsequent
sustained phase of vasoconstriction. (B) Concentration–response curve of the first short-acting part of the perfusion pressure change induced by
continuous perfusion with Up
4
A. Each point is the mean of eight experiments, and vertical lines show SEM. Significant difference (*P < 0.05) from
baseline perfusion pressure of the Up
4
A concentration (bolus application). Where error bars do not appear in figures, errors are within the symbol
size. (C) Concentration–response curve of the sustained part of the per fusion pressure change induced by continuous perfusion with Up
4
A. Each
point is the mean of eight experiments, and vertical lines show SEM. Significant difference (*P < 0.05) from baseline perfusion pressure of the Up
4
A
concentration (bolus application). Where error bars do not appear in figures, errors are within the symbol size. (D) Influence of different P2
antagonists on the first short-acting part of the perfusion pressure increase induced by Up
4
A(5mM). The P2X
1
and P2X
3
desensitizer a,b-meATP
(10 mM), and the non-selective P2 receptor antagonist suramin (100 mM) and PPADS (10 mM) significantly inhibited (*P < 0.05) the Up
4
A-induced
perfusion pressure increase, whereas the selective P2Y
1
receptor antagonist MRS2179 (10 mM) and the non-selective P2 receptor antagonist RB2
(100 mM) had no significant influence. (E) Influence of different P2 antagonists on the second sustained part of the perfusion pressure increase
induced by Up
4
A(5mM). In the presence of the P2X
1
and P2X
3
desensitizer a,b-meATP (10 mM), there was no significant effect on the
Up
4
A-induced sustained perfusion pressure. In the presence of the non-selective P2 receptor antagonist suramin (100 mM), the sustained perfusion
pressure increase was significantly decreased (*P < 0.05), whereas the selective inhibition of the P2Y
1
receptor by MRS2179 (10 mM) and the
non-selective P2 antagonists PPADS (10 mM) and RB2 (100 mM) had no significant effect.
BJP
Up
4
A and purinoceptors
British Journal of Pharmacology (2010) 161 530–540 535
effects. There were hints that further P2 receptors
are involved in the Up
4
A-induced vasoconstriction
(Jankowski et al., 2005). Here, we could show that
the Up
4
A-induced vasoconstriction depends not
only on the activation of the P2X
1
receptor (Jan-
kowski et al., 2005), but also on the activation of the
P2Y
2
receptor. Under basal conditions, the bolus
application of Up
4
A induced a dose-dependent vaso-
constriction in the rat isolated perfused kidney. The
Up
4
A-induced vasoconstriction was blocked by the
P2X
1
receptor desensitizer a,b-meATP, the non-
selective P2 receptor antagonist suramin and PPADS.
After a,b-meATP-induced desensitization of the
P2X
1
receptor, the bolus application of Up
4
A
induced a dose-dependent vasoconstriction. This
vasoconstriction was completely blocked by the
non-selective P2 receptor antagonist suramin.
Suramin mainly inhibits activation of P2Y
1
and P2Y
2
receptors, and shows only weak low affinity at the
P2Y
6
receptor (von Kugelgen, 2006). There was no
significant inhibition of Up
4
A-induced vasoconstric-
tion by the non-selective P2 receptor antagonist
RB2. RB2 inhibits activation of the P2Y
1
, P2Y
4
and
P2Y
6
receptors. There is also a very low inhibitory
affinity at the P2Y
2
receptor (von Kugelgen, 2006).
PPADS is a potent antagonist of the P2Y
1
and P2Y
4
receptors (von Kugelgen, 2006), whereas MRS2179 is
the only tested selective antagonist at the P2Y
1
receptor (von Kugelgen, 2006). PPADS and MRS2179
showed no significant antagonistic effects on Up
4
A-
induced vasoconstriction. Taken together, these
observations suggest that the P2Y
2
receptor is
responsible for the residual vasoconstriction
induced by Up
4
A. The observations are in line with
recent findings showing that the P2Y
2
receptor
mediates the UTP-induced vasoconstriction in
porcine isolated arteries. The P2Y
4
and P2Y
6
recep-
tors were not involved (Rayment et al., 2007). In
control experiments, we can show that the UTP-
induced vasoconstriction in the rat isolated perfused
kidney was inhibited by suramin and not by PPADS.
Continuous perfusion of the rat isolated perfused
kidney with Up
4
A induced a fast, concentration-
dependent perfusion pressure increase with a sub-
sequent desensitization. In addition, continuous
perfusion with Up
4
A induced a sustained increase in
perfusion pressure without signs of desensitization.
The first part of the Up
4
A-induced vasoconstriction
60
80
100
120
140
160
180
200
220
240
1
2
3
4
5
6
8
9
7
AngII
MAP (mmHg)
−11 −10 −9 −8
−7
−20
0
20
40
60
80
100
*
*
*
*
*
log mol
Δ
P (mmHg)
−10
−9 −8
−7
−80
−60
−40
−20
0
*
*
*
*
lo
g
mol
Δ
P (mmHg)
A
CB
Figure 3
(A) Original tracing of a representative experiment performed on the rat isolated perfused kidney showing the effect of bolus application of Up
4
A
under raised tone with AngII (200 nM). The bolus application of Up
4
A induced a dose-dependent, rapid increase of the perfusion pressure with a
subsequent dose-dependent vasodilatation. 1: Up
4
A 100 pmol; 2: Up
4
A 500 pmol; 3: Up
4
A 1 nmol; 4: Up
4
A 5 nmol; 5: Up
4
A 10 nmol; 6: Up
4
A
50 nmol; 7: Up
4
A 100 nmol; 8: ACh 1 nmol; 9: wash-out. (B) Dose–response curve of the first vasoconstrictor effect of Up
4
A under raised basal tone.
Each point is the mean of seven determinations, and vertical lines show SEM. Significant difference (*P < 0.05) from increased perfusion pressure.
For abbreviations, see text. Where error bars do not appear in figures, errors are within the symbol size. (C) Dose–response curve of the second
vasodilator effect of Up
4
A under raised basal tone. Each point is the mean of six determinations, and vertical lines show SEM. Significant difference
(*P < 0.05) from baseline perfusion. For abbreviations, see text. Where error bars do not appear in figures, errors are within the symbol size.
BJP
M Tölle et al.
536 British Journal of Pharmacology (2010) 161 530–540
was attenuated by a,b-meATP, suramin and PPADS,
whereas RB2 and MRS2179 again had no significant
effect. Thus, this part of the vasoconstriction is due
to an activation of the P2X
1
receptor. The second
part of the Up
4
A-induced vasoconstriction was
significantly attenuated by suramin, whereas a,b-
meATP, PPADS, RB2 and MRS2179 had no effect.
The continuous activation of the P2Y
2
receptor was
responsible for this sustained Up
4
A-induced vaso-
constriction. Recently, it was shown in young
hypertensive patients that the Up
4
A concentration
correlates with blood pressure, left ventricular mass
and intima media thickness (Jankowski et al., 2007).
It is possible that P2Y
2
receptor activation is
involved in the physiology and pathophysiology of
blood pressure regulation. After inter-arterial appli-
cation of Up
4
A in rats, there is a potent transient
increase in the mean arterial blood pressure (MAP)
with signs of desensitization (Jankowski et al.,
2005). After the initial increase in blood pressure,
there is a sustained increase in MAP. This is further
evidence that Up
4
A is a compound that regulates
and increases blood pressure (Jankowski et al.,
2005).
In the experiments with AngII-induced raised
perfusion pressure in the rat isolated perfused
kidney, we observed a biphasic, vasoactive response
to bolus application of Up
4
A. The first response was
a dose-dependent increase in vascular tone that was
followed by a sustained decrease of perfusion pres-
sure. As was the case under basal conditions, the
perfusion pressure increase could be significantly
Up
4
A
α
,
β
-meATP
+ Suramin
+ PPADS
+ MRS2179
+ RB2
0
20
40
60
80
100
*
**
Δ
P (mmHg)
Up
4
A
+
S
ur
amin
+ RB2
+ PP
AD
S
+
MRS2179
0
10
20
30
40
*
Δ
P (mmHg)
+ E / -L-NAME
+ E / + L-NAME
- E /
-
L-NAME
−50
−40
−30
−20
−10
0
*
*
Δ
P (mmHg)
Up
4
A
+ S
u
ramin
+ M
R
S
2
179
+
P
PADS
+
R
B
2
−50
−40
−30
−20
−10
0
*
*
*
*
Δ
P (mmHg)
BA
DC
Figure 4
(A) Influence of different P2 antagonists on the first short-acting part of the perfusion pressure increase induced by Up
4
A(5mM). The P2X
1
and
P2X
3
receptor desensitizer a,b-meATP (10 mM), and the non-selective P2 receptor antagonist suramin (100 mM) and PPADS (10 mM) significantly
inhibited (*P < 0.05) the Up
4
A-induced perfusion pressure increase, whereas the selective P2Y
1
receptor antagonist MRS2179 (10 mM) and the
non-selective P2 receptor antagonist RB2 (100 mM) had no significant influence. (B) Influence of different P2 antagonists on the second sustained
part of the perfusion pressure increase induced by Up
4
A(5mM). In the presence of the P2X
1
and P2X
3
receptor desensitizer a,b-meATP (10 mM),
the Up
4
A-induced sustained perfusion pressure increase was significantly enhanced. In the presence of the non-selective P2 receptor antagonist
suramin (100 mM), the sustained perfusion pressure increase was significantly decreased (*P < 0.05), whereas the selective inhibition of the P2Y
1
receptor by MRS2179 (10 mM) and the non-selective P2 antagonists PPADS (10 mM) and RB2 (100 mM) had no significant effect. (C) In the
presence of an intact endothelium, the eNOS antagonist
L-NAME (300 mmol·L
–1
) significantly reduced (*P < 0.05) the Up
4
A- (10 nmol) induced
decrease of the perfusion pressure. After removal of the endothelium, Up
4
A (10 nmol) could not induce any significant decrease of perfusion
pressure. (D) The Up
4
A-induced perfusion pressure decrease could be significantly inhibited (*P < 0.05) by the non-selective P2 receptor antagonist
suramin (100 mM), the selective P2Y
1
receptor antagonist MRS2179 (10 mM), the non-selective P2 receptor antagonist PPADS (10 mM) and RB2
(10 mM).
BJP
Up
4
A and purinoceptors
British Journal of Pharmacology (2010) 161 530–540 537
attenuated by suramin, a,b-meATP and PPADS,
whereas MRS2179 and RB2 showed no effect. In the
presence of a,b-meATP, a perfusion pressure increase
could be seen, which was blocked by suramin,
whereas PPADS, RB2 and MRS2179 had no effect.
Therefore, this remaining perfusion pressure
increase was due to the activation of the P2Y
2
recep-
tor. The Up
4
A-induced perfusion pressure decrease
was not present after de-endothelialization or after
inhibition of NOS by
L-NAME. Up
4
A activates NOS
by stimulating P2Y
1
and P2Y
2
receptors on endothe-
lial cells. The Up
4
A-induced perfusion pressure
decrease was partially inhibited by the P2Y
1
receptor
antagonist MRS2179. In addition, the non-selective
P2Y receptor antagonist RB2, which is a potent
antagonist of the P2Y
1
and P2Y
6
receptors, and a
weak antagonist of the P2Y
2
and P2Y
4
receptors, and
the non-selective P2Y receptor antagonist PPADS, a
potent antagonist of the P2Y
1
receptor, and a weak
antagonist of the P2Y
4
and P2Y
6
receptors inhibited
the Up
4
A-induced vasodilation. A nearly complete
inhibition of the Up
4
A-induced reduction of perfu-
sion pressure could be observed using suramin, a
potent antagonist of the P2Y
1
and P2Y
2
receptors,
and a weak antagonist of the P2Y
6
receptor. Thus,
Up
4
A appears to activate NOS via stimulation of the
P2Y
1
and P2Y
2
receptors. Up
4
A-induced vasodilation
is inhibited by the P2Y
1
receptor antagonist
MRS2179 and even more pronounced inhibition by
suramin. Suramin is not an antagonist of the P2Y
4
,
but a potent antagonist of the P2Y
2
receptor. These
findings are in accordance with recent findings
showing the activation of eNOS by stimulation of
the P2Y
1
, P2Y
2
and possibly the P2Y
4
receptors (da
Silva et al., 2009). Currently, it is not possible to
study single P2Y receptor subtypes due to the lack of
selective antagonists. From our experiments, we
cannot exclude the possibility that the P2Y
4
receptor
might play a role in NOS activation, but the expres-
sion of this receptor is low in the rat kidney.
We and others were able to demonstrate previ-
ously that diadenosine polyphosphates such as
Ap
4
A activate various purinoceptor subtypes to
induce vasoconstriction and vasodilation in isolated
arterial vessels or in the rat isolated perfused kidney.
Ap
4
A mainly induces vasoconstriction in the rat iso-
lated perfused kidney and in the mesenteric bed via
activation of P2X
1
receptors (van der Giet et al.,
1998; Gabriels et al., 2002). In addition, Ap
4
A could
also induce an endothelium-dependent vasodila-
tion in isolated mesenteric arteries which was
mainly attributable to the activation of endothelial
expressed P2Y receptors (Busse et al., 1988).
However, those diadenosine polyphosphates with
more than four phosphate groups mainly activate
P2X receptors to induce vasoconstriction in most
vascular models tested (Gabriels et al., 2002). Using
the rat isolated perfused kidney model, no vasodila-
tion was observed in response to Ap
5
AorAp
6
A (van
der Giet et al., 1997). However Gabriels and cowork-
ers reported a possible vasodilation induced by Ap
5
A
or Ap
6
A in the renal microcirculation (Gabriels et al.,
2000), which might potentially be attributable to
the degradation products of Ap
5
A and Ap
6
A. Ap
4
G,
Ap
5
G and Ap
6
G are also potent vasoconstrictors acti-
vating P2X
1/3
receptors in the rat isolated perfused
kidney (Cinkilic et al., 2001; van der Giet et al.,
2001). There was no Ap
5
G- or Ap
6
G-induced vasodi-
lation observed in the rat isolated perfused kidney,
whereas in coronary arteries we were able to show
that Ap
5
A, Ap
5
G, Ap
6
A and Ap
6
G can activate P2Y
1
receptors with consequent activation of eNOS
(van der Giet et al., 2002b). The adenosine- and
guanosine-containing dinucleoside polyphosphates
mainly activate P2X
1
receptors to induce vasocon-
striction in vascular models, and P2Y receptor-
mediated vasoactive effects are rarely reported for
these substances (Gabriels et al., 2002). In the
present work, we showed that the uridine-
containing dinucleoside Up
4
A robustly activated
P2Y
1/2
receptors, as well as P2X
1
receptors. This is of
interest as there are some reports that activation
of the P2Y
2
receptor is involved in the regulation of
blood pressure. It was shown that mice lacking P2Y
2
receptor have salt-resistant hypertension and facili-
tated renal Na
+
and water reabsorption (Rieg et al.,
2007). In addition, there is paracrine regulation
of the epithelial Na
+
channel in the mammalian
collecting duct by purinergic P2Y
2
receptors
(Pochynyuk et al., 2008). All these mechanisms are
believed to be relevant in blood pressure regulation.
In addition, there are reports that activation of P2Y
2
receptor signalling might be potentially relevant in
pro-inflammatory vascular disease conditions like
atherosclerosis. Activation of P2Y
2
receptors in peri-
toneal macrophages can lead to the production of
the chemokine CCL2 (monocyte chemoattractant
protein 1; MCP-1) (Stokes and Surprenant, 2007),
and this is a key chemokine in the initiation and
progression of atherosclerotic disease (Viedt et al.,
2002). Activation of the P2Y
1
receptor also contrib-
utes to the pathogenesis of atherosclerosis, as
elegantly demonstrated with P2Y
1
/ApoE double
knock-out mice (Hechler et al., 2008). Unfortu-
nately, the precise mechanism is not fully under-
stood to date.
In summary, we were able to demonstrate that
Up
4
A induced renal vasoconstriction in perfused
kidneys via activation of the P2X
1
receptor, which
shows fast desensitization, and via activation of the
P2Y
2
receptor, which induces sustained vasocon-
striction. Up
4
A activated NOS via stimulation of the
BJP
M Tölle et al.
538 British Journal of Pharmacology (2010) 161 530–540
endothelial P2Y
1
and P2Y
2
receptors. Elevated serum
concentrations of Up
4
A can potentially induce a
significant elevation of the blood pressure. P2Y
receptor signalling initiated by Up
4
A might be rel-
evant in blood pressure control, in the pathogenesis
of human hypertension and in the progression of
vascular inflammatory disease.
Acknowledgements
The study was supported by a grant from the Deut-
sche Nierenstiftung (M.T.), Deutsche Forschungsge-
meinschaft (JA972/11-1, M.vdG. and J.J.),
Sonnenfeld-Stiftung (M.vdG. and M.T.), BMBF
(0313920D), Deutsche Hochdruckliga (M.T.), Dr
Robert-Pfleger-Stiftung (M.T. and M.vdG.),
Manchot-Stiftung (M.T.) and the Dr Werner
Jackstädt-Stiftung (M.vdG. and M.T.).
Conflict of interest
None.
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    • "Adenosine signals ATP breakdown, intra-or extracellularly, thereby providing crucial information for complex organisms and may even help regulate apoptosis (Mlejnek & Dolezel 2010). In addition to adenosine, uridine adenosine tetraphosphate (Jankowski et al. 2005, Tolle et al. 2010, Schuchardt et al. 2011 ), diadenosine tetraphosphate (Schluter et al. 1994, Hoyle & Pintor 2010) and UDP have important roles as signalling molecules (Harden et al. 2010). All these purines are important for cardiovascular physiology (Hansen et al. 2010, Andersen et al. 2011) and pathophysiology (Waldenstrom et al. 2010). "
    Article · Jan 2012
    • "Jankowski et al. [14] observed that in rat isolated perfused kidney, Up4A stimulated vasoconstriction mainly via P2X1 receptors and probably also via P2Y2 and P2Y4 receptors. Very recently, findings from this same group indicate that in the rat perfused kidney, in addition to smooth muscle P2X1 receptor-mediated vasoconstriction, Up4A showed concentration-dependent P2Y2 receptor-mediated, long-lasting vasoconstriction [37]. Moreover, they demonstrated that Up4A-induced vasoconstriction was followed by vasodilation mediated by P2Y1 and P2Y2 receptor activation on endothelial cells leading to the release of NO [37]. "
    [Show abstract] [Hide abstract] ABSTRACT: The endothelium plays a pivotal role in vascular homeostasis, and endothelial dysfunction is a major feature of cardiovascular diseases, such as arterial hypertension, atherosclerosis, and diabetes. Recently, uridine adenosine tetraphosphate (Up(4)A) has been identified as a novel and potent endothelium-derived contracting factor (EDCF). Up(4)A structurally contains both purine and pyrimidine moieties, which activate purinergic receptors. There is an accumulating body of evidence to show that Up(4)A modulates vascular function by actions on endothelial and smooth muscle cells. In this paper, we discuss the effects of Up(4)A on vascular function and a potential role for Up(4)A in cardiovascular diseases.
    Full-text · Article · Nov 2011
    • "In rat pulmonary artery, Up 4 A leads to contraction via P2Y receptors [10] . In perfused rat kidney, Up 4 Ainduced vasoconstriction depends not only on the activation of the P2X 1 receptor [7] but also on the activation of the P2Y 2 receptor [11]. Therefore, in the present study, we investigated whether DOCA-salt hypertension is associated with abnormal Up 4 A-induced vasoconstriction. "
    [Show abstract] [Hide abstract] ABSTRACT: Uridine adenosine tetraphosphate (Up(4)A) has been recently identified as a novel and potent endothelium-derived contracting factor and contains both purine and pyrimidine moieties, which activate purinergic P2X and P2Y receptors. The present study was designed to compare contractile responses to Up(4)A and other nucleotides such as ATP (P2X/P2Y agonist), UTP (P2Y(2)/P2Y(4) agonist), UDP (P2Y(6) agonist), and α,β-methylene ATP (P2X(1) agonist) in different vascular regions [thoracic aorta, basilar, small mesenteric, and femoral arteries] from deoxycorticosterone acetate-salt (DOCA-salt) and control rats. In DOCA-salt rats [vs. control uninephrectomized (Uni) rats]: (1) in thoracic aorta, Up(4)A-, ATP-, and UTP-induced contractions were unchanged; (2) in basilar artery, Up(4)A-, ATP-, UTP- and UDP-induced contractions were increased, and expression for P2X(1), but not P2Y(2) or P2Y(6) was decreased; (3) in small mesenteric artery, Up(4)A-induced contraction was decreased and UDP-induced contraction was increased; expression of P2Y(2) and P2X(1) was decreased whereas P2Y(6) expression was increased; (4) in femoral artery, Up(4)A-, UTP-, and UDP-induced contractions were increased, but expression of P2Y(2), P2Y(6) and P2X(1) was unchanged. The α,β-methylene ATP-induced contraction was bell-shaped and the maximal contraction was reached at a lower concentration in basilar and mesenteric arteries from Uni rats, compared to arteries from DOCA-salt rats. These results suggest that Up(4)A-induced contraction is heterogenously affected among various vascular beds in arterial hypertension. P2Y receptor activation may contribute to enhancement of Up(4)A-induced contraction in basilar and femoral arteries. These changes in vascular reactivity to Up(4)A may be adaptive to the vascular alterations produced by hypertension.
    Full-text · Article · Sep 2011
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