(Received for publication, February 3, 1995; and in revised form, December 18, 1995)
From the
We evaluated, in renal epithelial cells with a proximal tubule
phenotype, the effect of nitric oxide (NO) on ecto-5`-nucleotidase
(5`-NU), the underlying mechanism and its functional consequence.
Sodium nitroprusside (SNP, 1-1000 µM), a NO donor,
inhibited 5`-NU activity in a time- and concentration-dependent manner.
Consequently, NO blunted the inhibition by extracellular cyclic AMP
(cAMP, 10-1000 µM) of sodium-phosphate cotransport,
a pathway which involves degradation of adenosine monophosphate (AMP)
by 5`-NU. SNP-induced inhibition of 5`-NU was not mediated by cyclic
GMP, since it was not mimicked by atrial natriuretic peptide, and was
reproduced by isosorbide dinitrate and sodium nitrate, two NO donors.
SNP and genuine NO decreased the activity of 5`-NU in renal
homogenates, and the effect of SNP was potentiated by dithiothreitol
and glutathione, but not by nicotinamide adenine dinucleotide. In
vivo in rats, kidney ischemia/reperfusion, which activates
inducible NO-synthase, inhibited renal 5`-NU. This inhibition was
prevented by N-nitro-L-arginine methyl ester, a
NO-synthase inhibitor. These results indicate that: (i) NO-related
activity inhibited the activity of an ecto-enzyme, 5`-NU, most likely
through S-nitrosylation of the enzyme; (ii) inhibition of
5`-NU activity by NO
, which can occur in vivo under pathophysiological conditions, affected the extent to which
extracellular cAMP inhibited sodium-P
cotransport.
Ecto-5`-nucleotidase (5`-ribonucleoside phosphohydrolase, 5`-NU: ()EC 3.1.3.5) is a membrane-bound glycoprotein which
hydrolyzes extracellular nucleotides into membrane-permeable
nucleosides. In the kidney, 5`-NU is expressed mainly in plasma
membranes of proximal tubular cells and, to a lesser extent, in
glomerular mesangial cells, interstitial fibroblasts and intercalated
cells of the collecting tubule (see (1) for review). Apical
brush-border membranes of proximal cells are equipped with ectoenzymes
(adenosine trisphosphatase, phosphodiesterases, and 5`-NU) which
convert adenine nucleotides, i.e. adenosine triphosphate,
adenosine diphosphate, adenosine monophosphate (AMP), and cyclic AMP
(cAMP), into adenosine(2, 3) . Released adenosine can
be taken up by proximal tubular cells through dipyridamole-sensitive
carriers (4, 5, 6, 7) and
phosphorylated into adenine nucleotides. This cascade of events was
shown to account for the protective effect of extracellular adenine
nucleotides on tubular function during and after anoxia(8) .
Our previous studies have evidenced that degradation of extracellular
cAMP in the tubular lumen followed by adenosine uptake were mandatory
steps in the well known inhibitory effect of extracellular cAMP on
renal proximal phosphate (P
) reabsorption(9) .
Through this pathway, luminal cAMP (nephrogenous cAMP), added to the
tubular fluid under the influence of parathyroid hormone, is not only a
marker of the activity of parathyroid hormone but also participates in
the overall phosphaturic effect of the hormone (9, 10, 11) . We have recently reported that
parathyroid hormone-stimulated 5`-NU activity via a mechanism which
involved protein kinase C activation and de novo protein
synthesis(12) .
Nitric oxide (NO) is a local mediator which
is synthesized from L-arginine by numerous cell types
including endothelial cells, activated macrophages, and renal tubular
cells under physiological or pathological
conditions(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) .
Cellular targets of NO and signaling pathways involved in its
pleiotropic effects have been extensively studied in the past decade.
Stimulation of soluble guanylate
cyclase(24, 25, 26) , ADP-ribosylation-like
reaction with proteins (27, 28, 29) and, more
recently, S-nitrosylation of
proteins(30, 31, 32, 33, 34, 35, 36, 37) have
been reported as biochemical events which accounted for the actions of
NO. S-Nitrosylation results from direct or indirect (via
intermediate S-nitrosothiols) transfer of NO to thiol groups of
proteins(34, 35, 37) . Such reactions were
recently shown to affect the activity of nuclear, cytosolic, and
membrane-bound proteins (34) including heterotrimeric G
proteins(33) , p21
(36) , and
glyceraldehyde-3-phosphate dehydrogenase, a key enzyme of
glycolysis(32) . However, a direct interaction between NO and
an ecto-enzyme has not been reported.
The aim of the present study
was: (i) to evaluate whether NO-related activity affected 5`-NU
activity of proximal tubular cells and the extent to which such a
modulation might influence inhibition of sodium-P cotransport by extracellular cAMP; (ii) to elucidate the
mechanism involved in the effect of NO; (iii) to identify possible
conditions in which NO inhibits 5`-NU in vivo. We show that
NO
inhibits 5`-NU activity in a cyclic GMP (cGMP)- and
protein synthesis-independent manner, most likely through S-nitrosylation of the enzyme, and that renal
ischemia/reperfusion results in NO
-dependent inhibition of
5`-NU.
On the day prior to experiments, culture medium was changed to hormone-free and serum-free medium, and, on the day of experiment, preincubations were usually performed in the same medium to which drugs were added as concentrated aliquots.
Activity of 5`-NU from renal homogenates was determined by incubating aliquots of the homogenate (2-4 µg of protein/ml) in HBS-Hepes solution in the presence of labeled and unlabeled 5`-AMP as described above during 20 min at 37 °C. The reaction was terminated as for OK cells.
At the end of the reperfusion period, both kidneys of each
animal were then removed, decapsulated, and homogenized with a Teflon
Potter-Elvehjem device in an ice-cold buffer (250 mM sucrose,
5 mM Tris-HCl, 3 mM MgCl, 1 mM EDTA, 1 mM levamisole, pH 7.4). Homogenates were
aliquoted and stored under liquid nitrogen until determination of 5`-NU
activity as described above.
Figure 1: Effect of SNP on 5`-NU activity in OK cells. Panel A, OK cells were incubated during 3 h with or without 1 mM SNP prior to determination of 5`-NU activity which was assayed over the indicated periods of time from 10 to 120 min. SNP was not present during assay. Panels B and C, OK cells were incubated during the indicated periods of time with 1 mM SNP (panel B) or during 3 h with the indicated SNP concentrations (panel C) prior to determination of 5`-NU activity which was assayed over 60 min. Results are expressed as means ± S.E. of four different experiments (n = 4) in which duplicates were obtained. *, significantly different from control values, p < 0.05. Panel D, effect of SNP on the kinetic parameters of 5`-NU in OK cells. OK cells were incubated during 3 h in the absence or presence of 1 mM SNP prior to determination of 5`-NU activity. Results are expressed as means of two different experiments in which duplicates were obtained.
We have previously reported that 5`-NU played a key role in the
inhibitory effect of extracellular cAMP on sodium-P cotransport(9, 12) . In order to evaluate the
influence of NO on this inhibitory pathway, we measured
sodium-dependent P
uptake after that OK cells had been
preincubated with increasing concentrations of cAMP in the presence or
absence of SNP or AMP-PCP. As expected, extracellular cAMP
(10-1000 µM) inhibited P
uptake in a
concentration-dependent manner (Table 1). AMP-PCP, a potent
inhibitor of 5`-NU, blunted significantly the inhibition by cAMP. The
effect of AMP-PCP was mimicked by SNP, although to a lesser extent. It
is noteworthy that neither AMP-PCP nor SNP affected P
uptake by themselves.
In order to confirm that NO was indeed responsible for the inhibitory effect of SNP on 5`-NU,
we evaluated: (i) the effect of another NO donor, isosorbide dinitrate;
(ii) the effect of the ferricyanide and ferrocyanide moieties. As shown
in Fig. 2, panel A, isosorbide dinitrate and SNP, each
of them at 1 mM, inhibited 5`-NU to a similar extent. In
contrast, neither K
Fe(CN)
nor
K
Fe(CN)
, at the same concentration, affected
significantly 5`-NU activity.
Figure 2:
Comparison of the effects of SNP, ANP,
isosorbide dinitrate, ferricyanide, and ferrocyanide on 5`-NU activity
in OK cells. Panel A, OK cells were incubated during 3 h in
the absence or presence of SNP, isosorbide dinitrate,
KFe(CN)
, or K
Fe(CN)
,
each of them at 1 mM prior to determination of 5`-NU activity.
Results are expressed as means ± S.E. of four different
experiments (n = 4) in which duplicates were obtained.
*, significantly different from the basal value, p < 0.05. Panels B and C, OK cells were incubated in the
absence or presence of 1 mM SNP or 0.1 µM ANP
during 5 min prior to determination of cGMP accumulation (panel
B) or during 3 h prior to determination of 5`-NU activity (panel C). Results are expressed as means ± S.E. of
three different experiments (n = 3) in which duplicates
were obtained. *, significantly different from the basal value, p < 0.05.
Because NO was reported in many cellular systems to act through generation of cGMP, we evaluated whether this signaling pathway was involved in inhibition of 5`-NU. For that purpose, we compared the effects of SNP and ANP on cGMP accumulation and 5`-NU activity. SNP-induced increase in intracellular cGMP content was modest and did not reach significance (Fig. 2, panel B). In contrast, ANP stimulated dramatically cGMP generation. However, ANP was without effect on 5`-NU activity (Fig. 2, panel C).
In previous studies from several
groups including ours(12) , modulation of 5`-NU activity was
reported to depend on de novo protein synthesis. Regarding the
effect of NO, this possibility was evaluated by a
pretreatment of OK cells with cycloheximide or actinomycin D at
concentrations previously reported to abolish the modulation of the
enzyme by protein kinase C activators(12) . Cycloheximide or
actinomycin did not prevent NO-induced inhibition of 5`-NU activity (Table 2).
S-Nitrosylation of proteins with NO was recently described(30, 31, 32, 33, 34, 35, 36, 37) and
was reported in some instances to account for inhibition of enzymatic
activities(32, 34) . The possibility that a similar
mechanism was involved in NO
-induced inactivation of 5`-NU
was investigated. The effect of NO donor SNP was potentiated by
addition of the reducing agent glutathione (GSH) to the incubation
medium (Fig. 3). Incubation of OK cells during 3 h with GSH
alone, 0.01 to 1 mM, had no effect on 5`-NU activity. However,
the presence of GSH together with SNP during the preincubation period
increased markedly the effect of the NO donor: SNP, at 10
µM, decreased 5`-NU activity by 7, 13, 39, and 40% in the
presence of GSH at a concentration of 0, 0.01, 0.1, and 1 mM,
respectively.
Figure 3: Effect of glutathione on SNP-induced inhibition of 5`-NU in OK cells. OK cells were incubated during 3 h in the absence or presence of SNP and GSH at the indicated concentrations prior to determination of 5`-NU activity. Results are expressed as means ± S.E. of four different experiments (n = 4) in which duplicates were obtained. *, significantly different from the homologous control value, without SNP, p < 0.05; , significantly different from the homologous value without GSH, p < 0.05.
Figure 4: Effect of renal ischemia/reperfusion on 5`-NU activity in rats. Left kidneys were subjected to 15-min ischemia followed by 60-min reperfusion while right kidneys served as controls. All along the experiments, rats were infused either with saline (panel A) or with L-NAME (50 µg/min/100 g body weight after a priming dose of 5 mg/100 g body weight) (panel B). 5`-NU activity was determined on renal homogenates. *, significantly different from the value of the right (control) kidneys, n = 4, p < 0.05.
In a last set of experiments, the in vitro effect of NO solutions and of two NO donors, SNP and SNAP, was evaluated on 5`-NU activity in homogenates prepared from control rat kidneys. 5`-NU activity (nmol/mg of protein/min, means ± S.E. n = 3 in each group) decreased from 5.4 ± 0.30 under basal conditions to 4.2 ± 0.21, 3.5 ± 0.25, 3.9 ± 0.30, and 2.8 ± 0.17 after incubation with 0.1 mM SNP, 1 mM SNAP, 10 µM NO, and 100 µM NO, respectively (basal value was significantly different from each experimental condition, p < 0.01). Furthermore, the inhibitory effect of SNP was potentiated by GSH and dithiothreitol, but not by NAD (Table 3).
The main results of the present study are that: (i) in renal
epithelial cells, NO donors inhibited 5`-NU activity in a cGMP- and
protein synthesis-independent manner; (ii) this effect resulted in
impairment of cAMP-induced inhibition of sodium-P cotransport; (iii) S-nitrosylation of the enzyme, either
direct or indirect, is likely to underlay enzymatic inactivation; and
(iv) in vivo, NO overproduction during ischemia/reperfusion
injury led to inhibition of 5`-NU activity. To our best knowledge, this
is the first demonstration of a direct interaction between a nitrogen
oxide and an ecto-enzyme.
The mechanism of 5`-NU inhibition by
NO differs from that involved in previously reported
hormonal modulation of renal
5`-NU(12, 43, 44, 45) . In cultured
glomerular mesangial cells, cAMP-protein kinase A activating
substances, such as dopamine, and tumor necrosis factor-
or
interleukin-1
were shown to stimulate 5`-NU in a
cycloheximide-dependent manner(43, 44, 45) .
In OK cells, we established that parathyroid hormone stimulation of
5`-NU through protein kinase C was dependent on cycloheximide and
actinomycin D(12) . In contrast, de novo protein
synthesis was not involved in the effect of NO as evidenced by the
short-term action of this compound (Fig. 1) and its persistence
in the presence of cycloheximide and actinomycin D (Table 2).
The inhibitory effect of NO on 5`-NU did not result from
activation of the soluble guanylate cyclase-cGMP-dependent protein
kinase pathway, a common mode of action of NO
in several
systems (24, 25, 26) which accounts for
relaxation of smooth muscle cells and for the classical vasodilatory
effect of No
(15, 16, 24) . Our
data argue against the involvement of this pathway in 5`-NU inhibition
since: (i) NO
had a modest effect on cGMP generation in OK
cells; (ii) ANP, a well known agonist of particulate guanylate cyclase,
which increased dramatically cGMP generation in OK cells ( Fig. 2and (46) ), did not affect 5`-NU activity; (iii)
the effect of NO
on the enzyme was also observed in
homogenates of renal tissue.
Nitrosylation was recently reported to
affect the activity of a large number of membrane-bound, cytosolic and
nuclear
proteins(30, 31, 32, 33, 34, 35, 36, 37) .
This expression of a wide variety of effects is achieved through
interaction of nitrogen oxides with targets via a complex redox
signaling and additive
chemistry(34, 35, 37) . As regards inhibition
of 5`-NU, it may result from interaction with NO or with congeners
NO and NO
. Indeed, SNP, which
inhibited 5`-NU in the two preparations used in the present study, is
better regarded as an NO
donor rather than an NO
donor(34, 37) . The observation that the effect of SNP
was potentiated in the presence of thiols such as dithiothreitol or
glutathione ( Fig. 3and Table 3) raises the possibility
that these compounds first interact with NO
and that
RSNO compounds then inhibit 5`-NU, probably by S-nitrosylation. It is noteworthy that neither glutathione nor
dithiothreitol alone affected 5`-NU activity, a feature which contrasts
with the reported inhibition of bull seminal plasma 5`-NU by
dithiothreitol(47) . This apparent discrepancy can be
attributed to the fact that dithiothreitol concentrations used in our
study were 2 to 3 orders of magnitude lower than those reported to
inhibit 5`-NU(47) . Alternatively, our data showing that
genuine NO solutions decreased the activity of 5`-NU from kidney
homogenates is also consistent with the possibility of a direct
interaction between NO
and the enzyme. It can be
pointed out that NO
was active within a concentration range
similar to that reported to affect the activity of heterotrimeric G
proteins and p21
(33, 36) . Along the
same line, SNAP, which can be regarded as an NO donor, inhibited renal
5`-NU as well. Finally, the possibility that decreased activity of the
enzyme resulted from an interaction between a nitrogen oxide and the
zinc moiety of 5`-NU, which is a zinc
metalloprotein(48, 49) , is unlikely since nitric
oxide does not react with zinc, an element which was shown to be
crucial for 5`-NU activity in the mammalian membrane-bound form of the
enzyme(48, 49) .
The interaction between NO and 5`-NU differs from that described between NO and
glyceraldehyde-3-phosphate dehydrogenase: in the latter case, S-nitrosylation of the protein preludes to covalent linkage of
NAD, a cofactor of the enzyme(31) . This reaction was first
interpretated as ADP-ribosylation since NO was also reported to
stimulate an
ADP-ribosyltransferase(27, 28, 29) . In our
model, the observation that NAD, alone or in combination with SNP, had
no effect on 5`-NU activity rules out such a possibility. It should be
stressed that the ectoenzymatic situation of 5`-NU made unlikely an
interaction with NAD.
In proximal tubular cells, 5`-NU was shown to
be involved in modulation of P reabsorption and in
restoration of intracellular stock of ATP following
ischemia(8, 9, 12) . (i) The inhibitory
effect of extracellular cAMP on sodium-P
cotransport was
previously shown to require extracellular degradation of the nucleotide
by phosphodiesterases and 5`-NU and subsequent uptake of
adenosine(9) ; (ii) the protective effect of extracellular
nucleotides on intracellular ATP content during ischemia also requires
degradation of extracellular nucleotides followed by adenosine
uptake(8) . Our present finding that impairment of 5`-NU
activity by NO
blunts the phosphaturic effect of
extracellular cAMP demonstrates that 5`-NU inhibition has functional
implications in terms of P
homeostasis: the relief of tonic
inhibition exerted by cAMP on sodium-P
cotransport may help
to maintain normal P
reabsorption in case of impaired ATP
content. In those situations, the fall in ATP content results in a
decrease of Na,K-ATPase activity. In turn, the impairment of this pump
may potentially increase intracellular sodium concentration and thus
affect the magnitude of membrane sodium gradient. Sodium gradient
across plasma membranes is mandatory for the efficiency of secondary
active transport systems such as sodium-P
cotransport.
Increased NO production, either by activated macrophages, or by
proximal tubular cells under the influence of cytokines or of hypoxia (15, 16, 17, 18, 19, 20) ,
was shown to occur in pathologic conditions such as ischemia or in
kidney diseases with macrophage infiltration, whatever their
cause(20, 21, 22, 23) . The present
data, however, cannot definitely discriminate between activation of
inducible NO-synthase and increased activity of constitutive
NO-synthase as the source of NO overproduction. Our
observation that transient ischemia induced inhibition of 5`-NU, and
that this effect is prevented by inhibition of NO-synthase by L-NAME, clearly demonstrates that 5`-NU inhibition can indeed
occur in vivo in relation with NO
overproduction
whatever its origin.
If indeed NO induced inhibition of
renal ecto-5`-NU, this would result in complex outcomes and three main
consequences, resulting from the distribution of 5`-NU not only in the
apical membranes of proximal tubular cells but also in glomerular
mesangial cells and in interstitial
fibroblasts(1, 43, 44, 45) , would
be: (i) at the tubular level, an impairment of ATP synthesis secondary
to a lack of precursors (8) ; (ii) at the kidney level, a
decreased production of adenosine from AMP by 5`-NU of glomerular cells
and interstitial fibroblasts which might protect against the
vasoconstrictor effects of this nucleoside (50) ; (iii)
finally, as discussed above, reduced P
excretion might help
to maintain phosphate balance and thus phosphatemia within normal
values.
In summary, we have demonstrated that NO interacts with ecto-5`-nucleotidase and that S-nitrosylation of the enzyme is likely to result in
inhibition of its activity with functional implications in renal
epithelial cells. Under in vivo conditions such as renal
ischemia, overproduction of NO may lead to impairment of 5`-NU
activity.