JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2007, 58, 2, 321–333
www.jpp.krakow.pl
J. KARCZEWSKA1, L. MARTYNIEC3, G. DZIER¯KO3, J. STÊPIÑSKI1,2, S. ANGIELSKI1,2
THE RELATIONSHIP BETWEEN CONSTITUTIVE ATP RELEASE AND
ITS EXTRACELLULAR METABOLISM IN ISOLATED RAT KIDNEY
GLOMERULI
Laboratory of Cellular and Molecular Nephrology, Medical Research Centre of Polish Academy
of Sciences, Warsaw/Gdañsk, 2Department of Immunopathology, Medical University of Gdañsk,
3Department of Clinical Biochemistry, Medical University of Gdañsk, Poland
1
ATP and adenosine are important extracellular regulators of glomerular functions. In
this study, ATP release from glomeruli suspension and its extracellular metabolism were
investigated. Basal extraglomerular ATP concentration (1nM) increased several fold
during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate.
Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP
added to glomeruli was almost completely degraded within 20 minutes. In that time,
AMP was the main product of extracellular ATP metabolism. Significant accumulation
of AMP was observed after 5 min (194 ±16 µM) and 20 min (271 ±11 µM), whereas at
the same time concentration of adenosine was only 10 µM. A competitive inhibitor of
ecto-5-nucleotidase α-ß-methylene-ADP (AOPCP), decreased extraglomerular ATP
and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 µM)
also markedly reduced extraglomerular ATP accumulation, whereas IMP, its
deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an
inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP
from extraglomerular media caused by AMP. These findings demonstrate that the
decrease in extracellular ATP concentration observed after addition of AOPCP or AMP
is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme
catalyses reversible reaction 2ADPnATP+AMP, and a rise in the AMP concentration
can lead to fall in ATP level. The present study provides evidence the extraglomerular
accumulation of ATP reflects both release of ATP from glomeruli cells and its
metabolism by ecto-enzymes. Our data suggest that AMP, produced from ATP in the
Bowman's capsular space, might plays a dual role as a substrate for ecto-adenylate
kinase and ecto-nucleotidase reactions being responsible for the regulation of
intracapsular ATP and adenosine concentration. We conclude that AMP degrading and
converting ecto-enzymes effectively determine the balance between ATP and adenosine
concentration and thus the activation of P2 and/or adenosine receptors.
K e y w o r d s : glomerulus, ATP,
purinoreceptors
adenosine,
ecto-enzymes,
ecto-adenylate
kinase,
322
INTRODUCTION
ATP, which can be released from cytoplasm of many cells types, probably via
several efflux pathways, and from secretory granules by exocytosis, has been
shown to influence many physiological functions such as smooth, skeletal and
cardiac muscle contraction, platelet aggregation, neurotransmission and the
sensory system (1 - 3).
Biochemical and functional evidence indicates that extracellular ATP and
adenosine exerts substantial influence on kidney function via P2 and P1 receptors
(4 - 6). Growing body of evidence supports the hypothesis that ATP released from
the macula densa cells via maxi anion channels at the basolateral membrane,
directly stimulates afferent arteriolar vasoconstriction by activation of ATPsensitive P2X1 receptors (7, 8). On the other hand, adenosine hypothesis proposes
that the ATP released from the macula densa cells is hydrolysed to adenosine,
which in turn activates A1 receptors and stimulates preglomerular vasoconstriction
(9, 10). These two complementary hypotheses are being considered as an
explanation of the mechanism by which tubuloglomerular feedback signals are
transmitted from the macula densa cells to the afferent arteriole.
In a previous study we demonstrated extracellular ATP-induced relaxation of
glomerular microvasculature precontracted by Ang II, by activating P2Y
purinoreceptors, endothelial NO synthase, cytosolic guanylate cyclase pathway
(11, 12). Furthermore, using isolated glomeruli and cultured glomerular
endothelial cells we have demonstrated that nebivolol, a third-generation ßadrenoreceptor antagonist, induced relaxation of renal glomerular
microvasculature through ATP efflux with consequent stimulation of P2Y receptor
mediated NO release from cultured glomerular endothelial cells (13). The ATP
was thought to influence preferentially, if not exclusively, mesangial functions (1416). However, recent work has clearly shown that podocytes express purinergic
receptors (P2Y1, P2Y2, P2Y6) (17, 18). Extracellular ATP depolarises podocytes
in the intact glomerulus (18). ATP increases intracellular Ca2+ concentration in the
podocytes. Because podocyte foot processes possess contractile elements sensitive
to intracellular changes in Ca2+ concentration, extracellular ATP and its receptors
are good candidates for being an autocrine regulator of glomerular dynamics.
Further, because processes from the same podocyte cell extend over the surface of
two or more neighbouring capillaries, the degree of stretch capillary dilation could
be similar to that observed in mesangial cells (19).
These data clearly shows that P2 receptors may function as autocrine regulator
of glomerular dynamics. However, for purinergic expression to be relevant
physiologically, there must be adequate ATP release from the mesangial cells
and/or podocytes, or the glomerulus as a whole.
It has been shown that during adrenergic stimulation human renal cortex
releases ATP from neuronal and non-neuronal sources (20). Recently Vekaria et
al. (21), using in vitro micropuncture techniques, showed that the ATP
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concentration in the proximal tubules was more than four-times higher (32 ± 7
nmol/L) than in Bowman's space. The physiological sources of extracellular ATP
within the glomerulus are unknown. In the present work, we studied the basal
ATP release from isolated glomeruli and its extracellular metabolism with
particular emphasis on the contribution of AMP as a substrate of the ecto-5nucleotidase and ecto-adenylate kinase reactions as, the main sources of
extracellular adenosine and ATP, respectively.
MATERIAL AND METHODS
Isolation of glomeruli
Glomeruli were isolated from the renal cortex of adult male Wistar rats (200-250 g). Rats were
decapitated under Vetbutal anaesthesia and kidneys were removed and placed in ice cold phosphatebuffered saline (PBS), pH 7.4, containing (mM): 137 NaCl, 2.7 KCl, 8.1 NaHPO4, 1.5 K2HPO4, 0.9
CaCl2, 0.49 MgCl2 and 5.6 glucose. The renal capsule was removed and the cortex was minced with
a razor blade to a paste-like consistency. The minced cortex was mashed in a gradual nylon sieve (the
pore size, in sequence, 250, 120 and 70 µM). The final suspension consisted of decapsulated glomeruli
devoid of afferent and efferent arterioles. The assessed tubule contamination was less than 5% (22).
Isolation and culture of podocytes
The final suspension of glomeruli in RPMI 1640 (Sigma Chemical Co, St. Louis, Mo., USA)
supplemented with 10% FBS (Gibco-BRL, Parsley, UK) and 100U/ml penicillin with 100µg/ml
streptomycin (Gibco-BRL) was transferred to culture flasks and placed in the CO2 incubator (5%
CO2, 37°C) for 5-7 days. The outgrowing epithelial cells were trypsinized. Cells were seeded in 12well culture plates and cultivated at 37°C for 8-16 days (24).All steps of glomeruli and podocytes
isolation were under sterile condition.
Determination of glomerular inulin space
Glomerular inulin space (GIS) was measured according to previously described methods (11).
Immediately after isolation about 2000 glomeruli were suspended in 200µl PBS containing 1 %
BSA (Sigma Chemical Co, St. Louis, Mo., USA) and 0,5 µCi [H3]-inulin (Du Pont NEN Products,
Boston, MA, USA). Samples were preincubated for 20 min at 37°C in a shaking water bath.
Incubation was continued for further 5 min with 50 µM ARL (6-N,N-Diethyl-ß-γdibromomethylene-D-adenosine-5-triphosphate, Sigma Chemical Co, St. Louis, Mo., USA) or
solvent alone. Reactions were terminated by centrifugation (5 seconds at 5000g) of 200 µl of the
suspension in the microtube containing 100 µl of ice-cold silicone oil (Weeker Silicone). The tip of
the microtube with glomerular pellet was cut off and the content was resuspended in 500 µl of 0,3%
Triton X-100 (Sigma Chemical Co, St. Louis, Mo., USA). The supernatant (20 µl) was treated in
an identical manner. After solubilization the radioactivity of the samples (triplicate) was measured
in a liquid scintillation counter. The inulin space of single glomerulus was computed as follows.
GIS =
[H3] pellet
___________________________________________________________________________
[H3] supernatant x number of glomeruli in the pellet
324
ATP measurement
About 2000 of glomeruli in 200 µl of PBS were preincubated for 20 min at 37°C in water bath.
Reactions were started by adding of substrate(s), dissolved in 25 µl of PBS. Reaction was stopped
by centrifugation for 5 min at 0°C at 2000 rpm. The supernatants were boiled for 3 min and again
centrifuged. ATP concentration was measured using the luciferin/ luciferase reactions, with the
Sigma Luciferin/luciferase (LL) reagent. Standard curves of ATP at known concentrations were
constructed with 2 mg/ml LL reagent in Optimem medium or PBS solution by serial dilution from
ATP stock solution. Moreover, to each tested samples an ATP internal standard was added to
determine the possible effect of the medium on the efficiency of bioluminescence reaction. The
amount of ATP present in each tested tubes was determined from the standard calibration curve and
corrected for recovery.
A medium displacement method was used as mechanical stimulus in the case of the glomeruli.
Half of the volume of the bathing medium was gently pipetted up and down twice with a
micropipette. ATP was measured 10 min after medium displacement. Bioluminescence controls
were used with each reagent solution to eliminate reagent effect on luciferase activity as well as to
control for ATP contamination.
Adenosine and inosine measurement
The assay is based on the determination of hydrogen peroxide formed by sequential metabolism
of adenosine, inosine and hypoxantine/xantine to uric acid (26). Usually, 50µl of supernatant of the
tested medium or neutralized perchloric extracts were made up with PBS or adenosine and inosine
to yield the final volume of 100µl. Inosine and adenosine were determined sequentially. During the
first step, hypoxantine and xantine were removed by adding 0.1M Tris-Hepes buffer, pH8.2,
containing 1mM MgCl2, 25µM luminol, 1.5U/ml peroxidase (Sigma Chemical Co, St. Louis, Mo.,
USA) and 1U/ml xantine oxidase (Sigma Chemical Co, St. Louis, Mo., USA). When light emission
decayed to baseline level, the determination of inosine was initiated by the addition of a buffer
containing 0.3U/ml nucleoside phosphorylase (Sigma Chemical Co, St. Louis, Mo., USA). The
peak light emission (usually between 20-30 s) was taken as a measure of inosine content. Again,
after the light emission decayed to basal level, the determination of adenosine was initiated by
adding of 2.5U/ml adenosine deaminase (Sigma Chemical Co, St. Louis, Mo., USA). The peak light
emission was taken as measure of adenosine content. To each sample, internal standards of inosine
or adenosine were added. Each bath of peroxidase, xantine oxidase and nucleoside phosphorylase
was checked for contamination by adenosine deaminase in the case of adenosine assay, or
nucleoside phosphorylase when inosine was measured.
HPLC analysis
Analysis of metabolites was performed using a reverse-phase high performance liquid
chromatography method (HPLC) as described (39). The equipment used was a Merck-Hitachi
system connected to a Hewlett Packard 1050 diode array detector.
RESULTS
It is suggested that extracellular ATP concentration reflects the balance
between basal ATP release and hydrolysis. Extraglomerular ATP
concentrations in resting glomeruli, determined by off or on-line luminometry
325
Fig.1. Basal concentration of ATP
in extraglomerular space. ATP
concentrations were measured by
off-line measurement of samples or
real time measurement by luciferase
dissolved in incubation media
containing 2000 glomeruli in 500
µl, in the presence or absence of
ARL (50 µM) for 10 min. Data
represents means ±SE of n=24
experiments using off line
measurements and n=6 experiments
using
on-line
luminometry,
*p<0,05; **p<o,o1 vs control.
Fig.2. ATP concentrations in
extraglomerular medium containing
different amounts of mechanically
stimulated or resting glomeruli. 2000
glomeruli were suspended in 0,5 ml
of PBS in the presence of ARL (50
µM) for 10 min. Mechanical
stimulation was performed by
aspiration of glomerular suspension,
four times, and allowed to rest at
37°C for the next 10 min the medium
was removed, centrifuged and boiled
and ATP concentration was
determined by the luciferase assay as
described in Methods. The results
indicate the mean ±SE from four
experiments each involving triplicate
measurements.
were 0.5-1nM. Inhibition of ecto-ATPase resulted in ATP accumulation at a
rate of approximately 0.30 pmol/min/1000 glomeruli, reflecting the basal
release rate (Fig.1).
To examine effect of mechanical perturbation on ATP release from glomeruli,
different amounts of glomeruli were suspended in 500 µl PBS in the presence of
ARL 67156-inhibitor of ecto-ATPase. Extracellular levels of ATP were
proportional to the amounts of glomeruli in the incubation medium. ATP release
was stimulated by medium displacement and increased by about 35% in each tested
sample (Fig.2).
Figure 3A illustrates how exogenous ATP (250nM) was rapidly degraded by
the suspension of glomeruli. The ATP level in the medium decayed with t1/2
326
Fig.3. A. Time course of ATP hydrolysis by the glomeruli in the absence or presence of ARL. Following
preincubation (5 min) with vehicle of ARL (50 µM), ATP (250 nmoles) was added to suspension of
glomeruli and its hydrolysis rate was measured by off line luminometry at indicated times points.
B. Time course of ATP hydrolysis in the presence or absence of ARL by podocyte cells. Following
preincubation (5 min) with vehicle or ARL (50 µM), ATP (250 nmoles) was added to medium on resting
podocyte cells and its hydrolysis rate was measured by off line luminometry at indicated times points.
Fig.4. Metabolism of ATP by
isolated glomeruli. About 8000
glomeruli were suspended in 1 ml of
PBS. Samples were incubated with
400 µM ATP for 5 or 20 min at 37°C
in a shaking water bath. Reactions
were terminated by centrifugation.
Supernatants were boiled for 2 min
and again centrifuged.
values ~ 2min. Extraglomerular levels of ATP did not fall to zero but attained a
baseline in the low nanomolar range within 60 min (not shown). Addition of
ARL, rapidly decreased the hydrolysis of ATP. Because podocyte cells face the
urinary space and cover the capillary loops, which constitute the kidney
glomerular tuft, we measured the ecto-ATPase activity of cultured podocytes.
ATP (250nM) added to podocyte cells exhibited a half-life of ~ 2min, which was
similar to that of glomerular tufts, suggesting the presence of the activity of ectoATPase predominantly on cell surfaces of podocytes (Fig. 3B).
327
Figure 4 clearly shows that during ATP hydrolysis by the glomeruli, the
concentration of AMP was higher than that of other products of its hydrolysis.
After 5 or 20 min of incubation, the concentration of ATP decreased to 50 ± 3 and
4 ± 2, while ADP level was 106± 1 and 16±3 µM, respectively. Furthermore,
significant accumulation of AMP was observed after 5 min 194 ± 22 and 20 min
271±1 µM, whereas at the same time points the concentration of adenosine was
very low, about 10 µM. Glomeruli hydrolysed AMP to adenosine less efficiently
than ATP to ADP and AMP.
It is shown in Fig.5 that the addition of ARL 67156 (50µM) to the suspension
of glomeruli causes extracellular ATP to gradually increase to 12 nM ATP over 8
min. In the absence of ecto-ATPase, inhibition ATP remained at the 4 nM steadystate level over the same time period.
Fig.5. Effect of ARL or AOPCP on
extraglomerular ATP concentrations.
Extraglomerular ATP concentration
following addition of ARL (50µM)
or AOPCP (50µM) was measured by
on-line luminometry. Apyrase
(2U/ml) was added at the end of
incubation time. Values are means
±SE of 3 experiments.
Fig.6.
Time-dependence
of
extracellular ATP formation from
ADP in the presence or absence of
Ap5A by isolated glomeruli. About
100 glomeruli were suspended in
200 µl of PBS. Samples were
incubated with 10µM of ADP.
Reaction was terminated by
centrifugation. ATP was measured
by off-line luminometry of samples
taken at indicated time points.
Values are expressed as a mean ±SE
of 3 experiments.
328
Tab.1. Effects of various inhibitors and metabolites on extraglomerular accumulation of ATP. 2000
glomeruli were incubated with AOPCP (50µM), EHNA (5µM), AMP (100µM), IMP (100µM), and
adenosine deaminase (ADA) 0,2 U/ml for 10 min. Reaction was terminated by centrifugation.
Values are means ±SE of 4 experiments.
Added
Extraglomerular concentration (nM)
ATP
Control
+ AOPCP
+ EHNA
+ AMP
+ IMP
+ ADA
7,45
1,42
7,64
2,94
7,34
23,06
±2,15
±0,31*
±2,81
±0,72*
±1,94
±1,76**
Adenosine
35,2 ±4,1
13,6 ±2,0**
342,5 ±29**
484,1 ±16**
18,9 ±1,7**
-
*p<0,05, **p<0,005 vs Control
Fig.7. Time-dependent effect of
1µmol/L of ATP (•) or ATP in the
presence of 50µmol/L ARL. Each
point is the mean of 6 experiments.
Values are expressed as a mean ±SE,
p<0,05 ATP vs. ATP+ARL.
We have repeatedly observed that the addition of AOPCP- a strong inhibitor
of ecto-5-nucleotidase (Ki~2nM) to glomerular suspension or cultured podocytes,
decreased extracellular accumulation of ATP. Fig. 5 shows that AOPCP (50µM)
gradually decreased extraglomerular ATP concentration when added alone as
well as in the presence of ARL. In both cases, AOPCP decreased ATP below it
steady-state level (4nM). This effect was rapid, when AOPCP was added alone.
The effects of AMP, IMP and EHNA - an inhibitor of adenosine deaminase were
also tested. Table 1 shows that the AOPCP alone decreased extraglomerular ATP
concentration (7.45 ± 2.15 vs. 1.42 ± 0.31 nM). During EHNA incubation in the
absence of AOPCP, adenosine concentration increased to 342.5 ± 25 nM and
extraglomerular concentration of ATP remained unchanged. AMP alone decreased
extraglomerular ATP concentration (2.94 nM) whereas IMP was not able to reduce
it. Because AOPCP decreased both ATP and adenosine levels (35.2 ± 4.1 vs. 13.6
± 2.0 nM). The effect of adenosine deaminase on extraglomerular ATP
329
concentration was examined. Table 1 shows that in the presence of adenosine
deaminase (0.2 U/ml) extraglomerular ATP level increased 3-fold (23.06 ± 1.76 vs.
7.45 ± 2.15).
The same types of cells express ecto-adenylate kinase and ecto-nucleoside
diphosphate kinase activity at the surface (32, 33). Figure 6 illustrates that the
addition of exogenous ADP (10 µM) to glomerular suspension caused its conversion
into ATP, whereas ecto-adenylate kinase inhibitor Ap5A significantly (69%)
inhibited ATP formation. Adenylate kinase reaction catalyses transfer of phosphate
between adenine-based nucleotides by the reversible reaction 2ADP n AMP+ATP.
To assess whether glomerular ecto-ATPase activity is involved in the
glomerular dynamics the ARL -inhibitor of ecto-ATPase was used. Incubation of
the glomeruli with ATP (1 µM) induced decrease in the glomerular inulin space
(GIS). Reduction of GIS was time-dependent with maximal effect about 20 ± 2%
of basal at 1 minute (Fig. 7). ARL significantly potentiated and prolonged the
effect of ATP on glomerular inulin space.
DISCUSSION
The important finding in the present study is that ATP is constitutively
released from the glomeruli (Fig. 1) and hydrolysed by ectonucleotidases, giving
rise to extraglomerular adenosine (Fig. 4). ATP was detected in glomeruli
medium, and its concentration correlated with the number of the glomeruli (Fig.
2) Mechanical perturbation in different experimental models evoked a release of
ATP from the variety of cells (27, 28). We used a medium displacement method,
which was successfully applied to elicit ATP release from different cell types
(25). ATP release from the glomeruli stimulated by medium displacement
increased by about 40%. Mechanical stimulation did not altered lactate
dehydrogenase activity in the medium.
When ATP was added to the glomeruli or cultured podocytes it was completely
degraded within 20 minutes with half-time less than 2 min (Fig. 3A and B),
indicating that the maintenance of extracellular ATP levels by the glomeruli as a
whole represents a net balance between basal release and subsequent it hydrolysis.
It has recently been shown that ATP concentration measured in the entire medium
is unlikely to reflect its concentration at the cell surface. However, a number of
studies have confirmed that ATP concentration near cell surfaces is high enough to
activate P2 receptors (29, 30). ATP is actively released into the extracellular space
under physiological conditions through several different mechanisms (31). The
important question to be answered is the source of the glomerular ATP. It is known
that the glomerulus is composed of three different cellular components,
endothelial, mesangial and podocyte cells. The interior surface of glomerular
capillaries are lined on their by the endothelium. The glomerular endothelium is
highly fenestrated. This allows the plasma to come into direct contact with
330
glomerular basement membrane that lies between the endothelium and the
podocytes. Podocyte cells face urinary space and cover with long finger like
extensions the capillary loops, which constitute the glomerular tuft (38).
Our preparation of glomeruli consists of decapsulated glomeruli. The external
surface of the glomerular tuft is formed by the luminal cell membrane of
podocytes, and constitutes about 95% of the entire podocyte surface, which is
directly exposed to the surrounding medium. The glomerular endothelial and
mesangial cells may also contribute extraglomerular ATP. However, in the
glomerulus, the activities of ecto-nucleotidases are present on the surface of
endothelial cells as well as in the glomerular basement membrane (34, 35).
Therefore, ATP released from the endothelial cells also comes into direct contact
with the enzymes of the glomerular basement tightly covered by podocyte cells.
It is thus possible that podocyte cells significantly contribute to the basal
extraglomerular ATP concentration.
The mechanism of ATP release from the epithelial cells remains unclear. It
was suggested that maxi-anion channel might participate as an ATP-releasing
pathway in renal epithelial cells.
It has recently been shown that the maxi-anion channel serves as a basolateral
ATP-conductive pathway opening in response to changes in luminal
concentration of NaCl in the thick ascending limb, in which the macula densa
cells reside in contact with glomerular cells, expressing P2Y2 receptors (31).
A key results of this work is the demonstration that AOPCP markedly
decreased ATP accumulation in the suspension of glomeruli and in cultured
podocyte cells medium (data not shown), in either the presence or absence of
ARL. Addition of AMP, the substrate of ecto-5'-nucleotidase also resulted in
detection of low concentration of ATP, whereas IMP, the product of AMP
deaminase, did not change ATP concentration in the medium. These results
suggest that AMP per se contributed to the decrease of basal ATP concentration.
Addition of ADP to the assay medium was accompanied by the synthesis of ATP.
Because Ap5A, a specific inhibitor of adenylate kinase, markedly inhibited
conversion of ADP to ATP, the enzyme responsible for this transphosphorylation
can be an ecto-adenylate kinase which catalyses reversible reaction
AMP+ATPn2ADP. These results suggest that in the presence of AOPCP,
commonly accepted as the most powerful inhibitor of membrane -bound ecto-5nucleotidase, accumulated AMP may drive the forward of ecto-adenylate kinase
reaction, trapping ATP and converting it to ADP, and in this way decreasing basal
ATP concentration in the glomeruli medium.
Moreover, in the presence of AOPCP, adenosine, the product of ecto-5nucleotidase reaction was reduced by 62% as compared with control glomeruli,
confirming that adenosine is formed predominantly on the external surfaces of
glomerular cells by cascade of ecto-nucleotidases.
Incubations of glomeruli with an inhibitor of adenosine deaminase EHNA, did
not affect ATP accumulation but increased ~ 10-fold adenosine concentration in
331
the glomerular medium. Unexpectedly, when glomeruli were incubated in the
presence of adenosine deaminase, extraglomerular ATP concentration increased 3fold, whereas adenosine levels were below the detection limit. The basal
concentrations of ATP and adenosine measured in the entire medium are unlikely
to accurately reflect the concentrations at the cell surface (29). It is possible that
adenosine concentration in the presence of adenosine deaminase is below
threshold values for the stimulation of A2B adenosine receptors, but it might be
sufficient to stimulate high affinity adenosine receptor subtypes, such as the A1
and A2A receptors. One can assume that adenosine induces the release of ATP by
activating an Ins (1, 4, 5) P3 sensitive-Ca2+ pathway through stimulation of A1
receptors. Moreover, inhibitory effect of AOPCP on the basal concentration of
ATP in the glomerular suspension may also be due to the inhibition of adenosine
formation by ecto-5'-nucleotidase in the close vicinity of the A1 receptor.
Therefore, close association of ecto-nucleotidase with adenosine receptors may
function as an ATP-receptor system and ecto-5'-nucleotidase may be a determinant
of the cellular response to ATP. Irrespective of the source and the mechanism of
ATP release, and extracellular adenosine formation, from the functional point of
view the relevant question is what might be role of extracellular nucleotides and
adenosine in the regulation of glomerular dynamics (22, 36, 37).
Functional evidence for the presence of P2Y and P1 receptors in the intact
mammalian glomerulus has been obtained by measuring ATP-induced changes in
glomeruli size (11, 13). It has been show previously that ATP acts on isolated
glomeruli bidirectionaly, i. e. induces relaxation of the precontracted glomeruli
and contraction of the relaxed glomeruli (11).
In this study ATP in the presence of ARL significantly potentiated and
prolonged the phase of contraction of glomeruli in comparison with control
glomeruli. This may be due to a high concentration of ATP being maintained in
the vicinity of the P2Y receptors, located on podocyte or mesangial cells.
Acknowledgments: This work was supported by the Medical Research Centre of Polish
Academy of Sciences - Warsaw.
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R e c e i v e d : December 5, 2006
A c c e p t e d : April 23, 2007
Author’s address: Stefan Angielski, Laboratory of Cellular and Molecular Nephrology, Medical
Research Centre of Polish Academy of Sciences, Dêbinki 7 Street, 80-211 Gdañsk, Poland; Tel.
(+48 58) 349 27 83; e-mail: [email protected]