Nitric oxide activates PKC
and inhibits
Na+-K+-ATPase
in opossum kidney cells
Mingyu
Liang and
Franklyn G.
Knox
Departments of Medicine and Physiology and Biophysics, Mayo
Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
Nitric oxide (NO)
reduces the molecular activity of
Na+-K+-ATPase
in opossum kidney (OK) cells, a proximal tubule cell line. In the
present study, we investigated the cellular mechanisms for the
inhibitory effect of NO on
Na+-K+-ATPase.
Sodium nitroprusside (SNP), a NO donor, inhibited
Na+-K+-ATPase
in OK cells, but not in LLC-PK1
cells, another proximal tubule cell line. Similarly, phorbol
12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited
Na+-K+-ATPase
in OK, but not in LLC-PK1, cells.
PKC inhibitors staurosporine or calphostin C, but not the protein
kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on
Na+-K+-ATPase
in OK cells. Immunoblotting demonstrated that treatment with NO donors
caused significant translocation of PKC
from cytosolic to
particulate fractions in OK, but not in
LLC-PK1, cells. Furthermore, the
translocation of PKC
in OK cells was attenuated by either the
phospholipase C inhibitor U-73122 or the soluble guanylate cyclase
inhibitor
1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on
Na+-K+-ATPase
in OK cells. The phospholipase A2
inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on
Na+-K+-ATPase
in OK cells. AACOCF3 alone, however, also decreased
Na+-K+-ATPase
activity in OK cells. In conclusion, our results demonstrate that NO
activates PKC
in OK, but not in
LLC-PK1, cells. The activation of
PKC
in OK cells by NO is associated with inhibition of
Na+-K+-ATPase.
proximal tubule; LLC-PK1
cells; phospholipase C; guanosine 3',5'-cyclic
monophosphate; phospholipase A2; protein kinase C
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INTRODUCTION |
SODIUM-POTASSIUM-ATPase located in the basolateral
membrane provides the primary driving force for solute and water
reabsorption in the proximal tubule. Alteration of the
Na+-K+-ATPase
activity, therefore, constitutes a major means for the regulation of
proximal tubular transport. A number of hormonal factors, such as
dopamine (6, 8, 34), parathyroid hormone (10, 34), glucocorticoids
(23), and angiotensin (4, 18), have been shown to affect the
Na+-K+-ATPase
activity in proximal tubule cells (21). These hormonal factors regulate
Na+-K+-ATPase
by changing the number of functional enzyme units, i.e., de novo
synthesis (23) or endocytosis (8), or modifying enzyme structure, for
example, phosphorylation (8). Several intracellular signaling pathways,
such as protein kinase A, protein kinase C (PKC), phospholipase
A2
(PLA2), arachidonic acid
metabolites, Ca2+/calmodulin-dependent protein
kinase, and
Ca2+/calmodulin-dependent protein
phosphatase, contribute to relaying messages for the regulation of
Na+-K+-ATPase
in proximal tubule cells (1, 4, 8, 10, 33, 34).
Nitric oxide (NO) is a biological molecule with a wide variety of
functions. In proximal tubule cells, NO has been shown to, among
others, cause cytotoxicity (42), decrease or increase fluid and/or
solute reabsorption (11, 40), inhibit the
Na+/H+
exchanger (36), and enhance paracellular permeability (26). With regard
to
Na+-K+-ATPase
in proximal tubule cells, in a study by Guzman et al. (17), it
was shown that induction of endogenous NO production by bacterial
lipopolysaccharide and interferon-
inhibited the catalytic activity
of
Na+-K+-ATPase
in mouse proximal tubule cells by ~30%. This inhibition of
Na+-K+-ATPase
was accompanied by a reduction of
myo-inositol uptake. However, the
mechanism for this effect of NO remains obscure. NO was also shown to
inhibit
Na+-K+-ATPase
in renal medulla (28).
Several cell lines with proximal tubule characteristics have been
established. Among the most widely used are opossum kidney (OK) cells
and LLC-PK1 cells. OK cells were
derived from kidneys of the American opossum (22), whereas
LLC-PK1 cells are of the Hampshire
pig origin (20). Interestingly, in a study by Middleton et al. (29), it
was shown that phorbol esters inhibited the transport activity of
Na+-K+-ATPase
in OK cells but not in LLC-PK1
cells, although PKC was similarly activated by phorbol esters in both
cell lines. These results suggested the presence of heterogeneity
related to the regulation of
Na+-K+-ATPase
in these two cell lines.
Previous studies in our laboratory demonstrated that NO inhibited
Na+-K+-ATPase
in OK cells by reducing the molecular activity of
Na+-K+-ATPase,
probably through mechanisms involving cGMP, without altering the number
of enzyme units (25). The present study was performed in OK and
LLC-PK1 cell lines to further
define the intracellular signaling cascade for the effect of NO on
Na+-K+-ATPase.
 |
METHODS |
Cell culture. OK and
LLC-PK1 cells were originally
obtained from the American Type Culture Collection. The cell lines were maintained in a 1:1 mixture of DMEM and Ham's F-12 nutrient mixture supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma, St. Louis, MO). Cells were incubated in 5% CO2-95% air at 37°C. Cells
were subcultured when they reached confluence by incubation with 0.25%
trypsin-0.02% EDTA.
Crude membrane fraction preparation for
Na+-K+-ATPase
assay.
After treatments, cells in six-well plastic plates were quickly washed
two times with the ice-cold scrape buffer containing (in mM) 300 D-mannitol, 10 Tris · Cl, and 1 EDTA, pH
7.4. Cells were collected in the same buffer and were counted with a
hemocytometer. Treatments in this study did not significantly change
the cell number. The cells were broken by sonication on ice and were
centrifuged at 400 g for 4 min. The
supernatant was transferred to another tube and was centrifuged at
40,000 g for 20 min (4°C). The
supernatant was decanted. The pellet was resuspended in the scrape
buffer and designated as the crude membrane fraction.
Na+-K+-ATPase
assay.
Na+-K+-ATPase
activity in the crude membrane fraction was measured as previously
described (2) with minor modification. Aliquots of the crude membrane
fraction prepared as described above were combined with an assay buffer
containing (in mM) 100 NaCl, 20 KCl, 4 MgCl2, 100 Tris · Cl, and 2 Na2 · ATP, pH
7.4, for "total" ATPase activity measurement. An assay buffer
containing (in mM) 120 NaCl, 4 MgCl2, 100 Tris · Cl, 1 ouabain, and 2 Na2 · ATP, pH
7.4, was used for ouabain-insensitive ATPase activity measurement. The
mixture was incubated at 37°C for 10 min. The reaction was stopped
by immediately returning the reaction mixture to ice water and adding
ice-cold TCA to a final concentration of 10%. The production of
Pi was measured by the Tausky and
Shorr method. Nonenzymatic degradation of ATP was
subtracted. The production of Pi
was confirmed to fall within the initial linear range in preliminary
experiments. The
Na+-K+-ATPase
activity was calculated as the difference between the total ATPase
activity and the ouabain-insensitive ATPase activity. The
Na+-K+-ATPase
activity obtained was corrected for the cell number. All experiments
were performed in parallel with controls and, when effects of various
compounds other than the NO donor were tested, treatment with NO donor
alone was also performed in parallel as "positive control." The
results were expressed as percentages of the simultaneously performed
controls. The membrane-associated Na+-K+-ATPase
activity under control conditions was ~7 nmol
Pi · min
1 · 106
cells
1 for OK cells and 11 nmol
Pi · min
1 · 106
cells
1 for
LLC-PK1 cells.
Ouabain-binding assay. The
ouabain-binding assay was performed as previously described (9) with
minor modification. After treatments, OK cells were washed three times
with a K+-free solution containing
(in mM) 140 NaCl, 1.8 CaCl2, 5 D-glucose, and 10 HEPES, pH 7.4. The
K+-free solution with 1 µM
ouabain (for total binding) or 1 mM ouabain (for nonspecific binding)
was then added together with 1 µCi/ml [3H]ouabain (50.0 Ci/mmol; Amersham Life Science, Arlington Heights, IL) and was
incubated at room temperature. After 20 min, the
K+-free solution was removed.
Cells were washed with ice-cold 100 mM
MgCl2 two times quickly and then
two times for 5 min each. Cells were then lysed in 0.5 N NaOH, and the
lysate was counted in duplicate with a
-counter (Beckman LS 6000SC).
The specific ouabain binding was calculated as the difference between
the total and the nonspecific bindings. The ouabain binding sites were
saturated within 10 min as confirmed in preliminary experiments. OK and LLC-PK1 cells under control
conditions have ~450 and 335 fmol ouabain binding
sites/cm2, respectively. The
results were expressed as percentages of the simultaneously performed controls.
Molecular activity of
Na+-K+-ATPase.
The molecular activity of membrane-associated
Na+-K+-ATPase
as previously defined (12) was calculated by dividing the
Na+-K+-ATPase
activity in the crude membrane fraction by the ouabain binding sites.
The results were expressed as percentages of the controls.
Subcellular fractionation and immunoblotting of
PKC
. After treatment, cells were quickly washed with
ice-cold PBSA (PBS without Ca2+
and Mg2+) two times and were
collected by scraping in an ice-cold homogenization buffer containing
(in mM) 20 Tris · Cl, 2 EGTA, 2 EDTA, 1 phenylmethylsulfonyl fluoride, 10
-mercaptoethanol, and 0.014 mg/ml
aprotinin, pH 7.4. The cell suspension was briefly sonicated and
centrifuged at 600 g for 5 min. The
supernatant was centrifuged at 100,000 g for 60 min at 4°C. The
supernatant of 100,000 g was collected and designated as the cytosolic fraction. The pellet was resuspended in
homogenization buffer and designated as the particulate fraction. Both
fractions were equalized for protein content and 1:1 diluted with
Laemmli buffer. Proteins were separated by SDS-PAGE using 7.5%
Tris · HCl gels and were transferred to either
nitrocellulose (Bio-Rad, Hercules, CA) or polyvinylidene difluoride
(Millipore, Bedford, MA) membranes. After blocking, membranes were
incubated with isoform-specific anti-PKC
antibodies from rabbit
(Boehringer Mannheim, Indianapolis, IN) followed by horseradish
peroxidase-conjugated anti-rabbit IgG antibodies (Boehringer Mannheim).
The detection was performed using a chemiluminescence method (Amersham
Life Science). The density of signals was quantified using a densitometer.
Statistics. Data are shown as means ± SE. The n values shown represent
the number of separate wells that were divided into several experiments
with each one done in duplicate or triplicate. Student's
t-test or one-way ANOVA followed by
Dunnett's test or Student-Newman-Keuls test were used when
appropriate. A P value of <0.05 was
considered statistically significant.
 |
RESULTS |
NO or a PKC activator inhibited the
Na+-K+-ATPase
activity in OK, but not LLC-PK1, cells.
Sodium nitroprusside (SNP) was used as a NO donor in the present study.
In previous studies, we have confirmed that SNP exerted its effect on
Na+-K+-ATPase
in OK cells via NO release (25). As shown in Fig.
1, incubation with 0.5 mM SNP for 2 h
significantly inhibited the membrane-associated
Na+-K+-ATPase
activity in OK cells, a proximal tubule cell line, to 65.5 ± 9.7%
of control (n = 6, P < 0.05 vs. control). This was due
to a reduction of the molecular activity, since the number of
Na+-K+-ATPase
enzyme units on the intact cell surface, assessed by ouabain-binding assay, was not altered. In LLC-PK1
cells, another widely used proximal tubule cell line, similar
treatments with SNP did not affect either the overall catalytic
activity of membrane-associated Na+-K+-ATPase
or the unit number of this enzyme on the intact cell surface (Fig. 1).
Similarly, incubation with 1 µM phorbol 12-myristate 13-acetate
(PMA), a PKC activator, inhibited the membrane-associated Na+-K+-ATPase
activity in OK cells to 52.7 ± 15.3% of control
(n = 9, P < 0.05 vs. control). The same
treatment was without effect in LLC-PK1 cells (92.6 ± 10.0%
of control, n = 6, P > 0.05; Fig. 2).

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Fig. 1.
Sodium nitroprusside (SNP, 0.5 mM, 2 h), a nitric oxide (NO) donor,
inhibited the molecular activity of membrane-associated
Na+-K+-ATPase
in opossum kidney (OK) cells, but not in
LLC-PK1 cells. The molecular
activity was calculated by dividing the overall catalytic activity by
the number of ouabain binding sites; n = 6. * P < 0.05 vs. control.
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Fig. 2.
Phorbol 12-myristate 13-acetate (PMA, 1 µM, 20 min), a protein kinase
C (PKC) activator, inhibited the membrane-associated
Na+-K+-ATPase
activity in OK, but not LLC-PK1,
cells; n = 6-9.
* P < 0.05 vs.
control.
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The inhibitory effect of SNP on the
Na+-K+-ATPase
activity in OK cells was abolished by PKC inhibitors.
The similar pattern of effects of SNP and PMA on the
Na+-K+-ATPase
activity in OK and LLC-PK1 cells,
as indicated by the above data, led us to suspect that PKC might be
involved in the observed effect of NO on
Na+-K+-ATPase
in OK cells. Therefore, we tested the effect of PKC inhibitors. As
shown in Fig. 3, coincubation with 20 nM
staurosporine or 0.5 µM calphostin C, both of which are PKC
inhibitors, abolished the effect of SNP (0.5 mM, 2 h) on the
membrane-associated
Na+-K+-ATPase
activity in OK cells (38.1 ± 8.4% of control for SNP alone, n = 9, P < 0.05 vs. control; 98.2 ± 29.9% of control for SNP + staurosporine,
n = 9, P > 0.05 vs. control; 129.3 ± 15.8% of control for SNP + calphostin C,
n = 9, P > 0.05 vs. control). Incubation with 20 nM staurosporine or 0.5 µM calphostin C alone for 2 h did not
have significant effects.

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Fig. 3.
Inhibitory effect of SNP (0.5 mM, 2 h), an NO donor, on the
membrane-associated
Na+-K+-ATPase
activity in OK cells was abolished by coincubation with PKC inhibitors
staurosporine (20 nM) or calphostin C (0.5 µM). Coincubation with the
cGMP-dependent protein kinase inhibitor KT-5823 (2 µM) did not
significantly affect the effect of SNP;
n = 9. * P < 0.05 vs. control.
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Because our previous studies suggested a role of cGMP in mediating the
inhibitory effect of NO on
Na+-K+-ATPase
in OK cells (25), we also tested the effect of KT-5823, a
cGMP-dependent protein kinase (PKG) inhibitor. Coincubation with 2 µM
KT-5823 did not affect the inhibitory effect of SNP on
Na+-K+-ATPase
in OK cells (53.2 ± 12.5% of control,
n = 9, P < 0.05 vs. control; Fig. 3).
KT-5823 (2 µM) alone did not have significant effects. These data
suggested that activation of PKC, but not PKG, was involved in the
inhibitory effect of NO on
Na+-K+-ATPase
in OK cells.
NO activated PKC
in OK, but not
LLC-PK1, cells.
To further confirm the role of PKC activation in the observed effect of
NO on
Na+-K+-ATPase
in OK cells, we directly assessed effects of NO on PKC in OK cells. Of
the two PKC isoforms present in OK cells, PKC
and PKC
, only
PKC
was shown to be activated by classical PKC activators (29).
Therefore, we focused on PKC
in the present study. Activation of
PKC
was measured by assessing the distribution of the enzyme between
cytosolic and particulate fractions using immunoblotting, because
translocation of the enzyme from the cytosolic fraction to the
particulate fraction correlates with activation of the enzyme. As shown
in Fig. 4, incubation with 1 µM PMA, 0.5 mM SNP, or 5 µM spermine NONOate for 2 h resulted in significant translocation of PKC
from the cytosolic fraction to the particulate fraction. These bands were abolished if purified PKC
peptide was
included during the incubation with the primary antibody, confirming
that they represented PKC
. The extent of translocation was similar
for all three treatments. A 2-h incubation with 0.5 mM SNP or 5 µM
spermine NONOate has been shown by our previous study to release a
similar amount of NO and to result in a similar extent of
Na+-K+-ATPase
inhibition in OK cells (25). Incubation with 5 µM spermine, another
substance released from spermine NONOate, for 2 h did not affect the
distribution of PKC
in OK cells. In
LLC-PK1 cells, SNP did not alter
the distribution of PKC
, although PMA caused substantial activation
of PKC
(Fig. 5). These data indicated that NO activated PKC
in OK, but not
LLC-PK1, cells.

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Fig. 4.
Effects of NO donors and PMA on the distribution of PKC in OK cells.
After incubation with various agents for 2 h, OK cells were
fractionated. Proteins of equal amounts were separated by SDS-PAGE,
transferred, incubated with anti-PKC antibodies, and detected using
a chemiluminescence method. A:
representative blotting. Lanes 1 and
2, control (2 h);
lanes 3 and
4, PMA (1 µM);
lanes 5 and
6, SNP (0.5 mM);
lanes 7 and
8, spermine NONOate (SpNONO; 5 µM);
lanes 9 and 10, control, in the presence of
purified PKC peptide. Lanes 1,
3, 5,
7, and
9, particulate fractions;
lanes 2,
4, 6,
8, and
10, cytosolic fraction.
B: amount of PKC protein estimated
by densitometry; n = 3. * P < 0.05 vs. corresponding
controls.
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Fig. 5.
Effects of SNP and PMA on the distribution of PKC in
LLC-PK1 cells. See Fig. 4 for
methods. A: representative blotting.
Lanes 1 and
2, control; lanes
3 and 4, PMA (1 µM);
lanes 5 and
6, SNP (0.5 mM).
Lanes 1,
3, and
5, cytosolic fractions;
lanes 2,
4, and
6, particulate fractions (note the
change in sequence). B: amount of
PKC proteins estimated by densitometry. Results shown are
representative of 3 independent experiments.
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Possible roles of cGMP and phospholipase
C. Because our previous study indicated that cGMP
generation was involved in the inhibitory effect of NO on
Na+-K+-ATPase
in OK cells (25), we postulated that cGMP was also involved in the
activation of PKC
in OK cells by NO. As shown in Fig. 6, coincubation with 30 µM
1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor, abolished the
translocation of PKC
induced by SNP, suggesting a role of cGMP in
the activation of PKC
in OK cells by NO. ODQ alone did not affect
the distribution of PKC
in OK cells. ODQ at the same dose has been
shown in our previous study to blunt the inhibitory effect of NO on
Na+-K+-ATPase
in OK cells (25).

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Fig. 6.
Translocation of PKC in OK cells caused by SNP was blunted by
U-73122, a phospholipase C inhibitor, or
1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one
(ODQ), a soluble guanylate cyclase inhibitor. See Fig. 4 for methods.
A: representative blotting.
Lanes 1 and
2, control; lanes
3 and 4, SNP (0.5 mM);
lanes 5 and
6, SNP (0.5 mM) + U-73122
(20 µM); lanes 7 and
8, SNP (0.5 mM) + ODQ (30 µM). Lanes 1,
3, 5,
and 7, particulate fractions;
lanes 2,
4, 6,
and 8, cytosolic fractions.
B: amount of PKC protein estimated
by densitometry. Results shown are representative of 3 independent
experiments.
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Typical pathways for PKC activation usually involve upstream activation
of phospholipase C (PLC). Indeed, 20 µM U-73122, a PLC inhibitor,
abolished the activation of PKC
in OK cells by NO (Fig. 6). U-73122
alone did not affect the distribution of PKC
in OK cells. This
effect of U-73122 was also accompanied by attenuation of the inhibitory
effect of NO on
Na+-K+-ATPase
in OK cells (63.3 ± 13.1% of control for SNP alone,
n = 9, P < 0.05 vs. control; 88.3 ± 10.6% of control for SNP + U-73122, n = 9, P > 0.05 vs. control; Fig.
7).

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Fig. 7.
Phospholipase C inhibitor U-73122 (20 µM) attenuated the inhibitory
effect of SNP (0.5 mM, 2 h) on
Na+-K+-ATPase
in OK cells, whereas the phospholipase
A2 inhibitor AACOCF3 (10 µM) did
not; n = 9. * P < 0.05 vs. control.
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It has been shown in a macrophage-like cell line (16) that NO could
activate PLA2 and release
arachidonic acid, some metabolites of which are known to activate PKC
and inhibit
Na+-K+-ATPase
in proximal tubule cells (32). In the present study, 10 µM AACOCF3, a
PLA2 inhibitor, did not affect the
inhibitory effect of SNP on
Na+-K+-ATPase
in OK cells (49.9 ± 15.2% of control,
n = 9, P < 0.05 vs. control; Fig. 7).
Unlike other inhibitors used in the present study, however, 10 µM
AACOCF3 alone also significantly inhibited the membrane-associated
Na+-K+-ATPase
activity in OK cells to 76.7 ± 5.6% of control
(n = 9, P < 0.05 vs. control), which makes
it difficult to interpret the combined effect of SNP and AACOCF3.
 |
DISCUSSION |
PKC is a family of serine/threonine-specific protein kinases with at
least 10 different isoforms (19). The interaction between NO and PKC
has been the subject of many studies, with most focused on the role
of PKC in the regulation of NO production (5, 14, 24, 38). With regard
to effects of NO on PKC, controversial results exist. Gopalakrishna et
al. (15) showed that NO inactivates purified PKC and PKC in B16
melanoma cells and in a macrophage cell line (IC-21; see Ref. 15).
This has been proposed to serve as a negative feedback mechanism for
the promoting effect of PKC on NO production. On the other hand, NO has
been shown to activate PKC in hepatocytes (7) and smooth muscle cells
(30). In rabbit hearts, NO activates specifically
and
isoforms
of PKC (35). Moreover, NO was shown to mediate the stimulation of PLC,
a typical upstream step for PKC activation, by oxidant stress (41). In the present study, SNP and spermine NONOate, two structurally unrelated
NO donors, caused significant activation of PKC
in OK cells. The
activation of PKC
was blocked by a PLC inhibitor or a soluble
guanylate cyclase inhibitor, which suggests that both PLC and cGMP play
roles in the activation of PKC by NO. Because the reversible
inactivation of PKC by NO was attributed to modification of thiol
groups in PKC by NO (15), Burgstahler and Nathanson (7) proposed
that whether NO activated or inactivated PKC might be determined by
the redox status of specific cell types. It would be interesting to
determine if this interpretation can also be applied to OK cells.
Activation of PKC is one of the major pathways leading to inhibition of
Na+-K+-ATPase
in proximal tubule cells. It contributes to the inhibition of
Na+-K+-ATPase
in proximal tubule cells by dopamine (8, 34), parathyroid hormone (34),
and 20-hydroxyeicosatetraenoic acid (32). Direct activation of PKC by
phorbol esters also causes inhibition of Na+-K+-ATPase
in proximal tubule cells mainly through phosphorylation of the
catalytic subunit of the enzyme (3, 29, 37), although this may depend
on experimental conditions (13). The results of the present study
clearly demonstrated that inhibition of
Na+-K+-ATPase
in OK proximal tubule cells by NO is also dependent on activation of
PKC because 1) the inhibitory effect
of NO was abolished by PKC inhibitors and a PLC inhibitor, but not by
PKG or PLA2 inhibitors;
2) direct activation of PKC by PMA
similarly inhibited Na+-K+-ATPase
in OK cells; and 3) NO activated
PKC
in OK cells.
The
NO-PKC-Na+-K+-ATPase
pathway appears to be cell type and/or species specific, because it was
observed in OK, but not LLC-PK1, cells. Although OK and LLC-PK1
cells both possess a variety of proximal tubule characteristics,
substantial differences exist between these two cell lines (39). For
example, parathyroid hormone inhibits phosphate transport in OK cells,
but not in LLC-PK1 cells (27).
Heterogeneity between these two cell lines also exists in the response
of
Na+-K+-ATPase
to PKC activation (29). Activation of PKC by phorbol ester
phosphorylated the
-subunit of
Na+-K+-ATPase
and reduced ouabain-sensitive 86Rb
uptake in OK cells, but not in
LLC-PK1 cells (29). The
heterogeneous response of
Na+-K+-ATPase
in OK and LLC-PK1 cells to PKC
activation was confirmed in the present study by direct measurement of
the catalytic activity of
Na+-K+-ATPase,
rather than the transport activity, as reflected by
86Rb uptake. Moreover, the results
of the present study extended the heterogeneity from the interaction
between PKC and
Na+-K+-ATPase
to their response to a physiological regulator, NO. The heterogeneity
related to the
NO-PKC-Na+-K+-ATPase
pathway between OK and
LLC-PK1 cell lines should have significant relevance in cell biology and in physiology.
The inhibitory effect of NO on the
Na+-K+-ATPase
activity in OK proximal tubule cells may have significant physiological
and/or pathophysiological implications. In in vivo microperfusion
studies, SNP at the concentrations of 0.1 or 1 mM has been shown to
inhibit fluid and/or solute reabsorption in the proximal tubule (11, 40). The inhibition of
Na+-K+-ATPase,
as shown by the present study, may serve as one of the mechanisms by
which SNP inhibits the proximal tubular reabsorption. NO donors at the
doses used in the present study resulted in the accumulation of
~3-4 µM NO
2, which was
slightly higher than that measured in normal renal cortex (43). NO
production was increased in proximal tubules exposed to hypoxia and
contributed to the hypoxia/reoxygenation injury (42). Moreover, in vivo targeting of inducible NO synthase with oligodeoxynucleotides protected
rat kidney against ischemia (31). Therefore, it appears reasonable to speculate that the inhibition of
Na+-K+-ATPase
by NO may participate in the functional alteration of the proximal
tubule under certain pathological conditions.
In summary, the present study demonstrated that NO activates PKC
and
inhibits
Na+-K+-ATPase
in OK, but not LLC-PK1 cells.
These results indicate the presence of a
NO-PKC-Na+-K+-ATPase
axis in OK proximal tubule cells.
 |
ACKNOWLEDGEMENTS |
We thank Jennifer M. Gross for technical support and Joanne
Zimmerman for secretarial assistance. We also thank Dr. Karl A. Nath
and the laboratory group for helpful discussion and critical reading of
the manuscript.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-55594.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: F. G. Knox,
Dept. of Physiology and Biophysics, Mayo Clinic and Foundation,
Rochester, MN 55905 (E-mail: knox.franklyn{at}mayo.edu).
Received 10 March 1999; accepted in final form 9 June 1999.
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