Departments of 1 Nephrology and 2 Endocrinology and Metabolism, Jichi Medical School, Tochigi, 329-0498 Japan
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ABSTRACT |
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The present study examined the role of protein kinase C (PKC) in the P-glycoprotein (P-gp)-induced modulation of regulatory volume increase (RVI) in the isolated nonperfused proximal tubule S2 segments from mice lacking both mdr1a and mdr1b genes (KO) and wild-type (WT) mice. The hyperosmotic solution (500 mosmol/kgH2O) involving 200 mM mannitol activated PKC and elicited RVI in the tubules from KO mice but not from WT mice. The addition of the hyperosmotic solution including the PKC activator phorbol 12-myristate 13-acetate (PMA) to the tubules of the WT mice activated PKC and elicited RVI. The hyperosmotic solution in the presence of the P-gp inhibitors (verapamil or cyclosporin A) elicited RVI in the tubules from the WT mice but not from the KO mice. The PMA- and the P-gp inhibitors-induced RVI was abolished by cotreatment with the PKC inhibitors (staurosporine or calphostin C). In the tubules of the KO mice, the PKC inhibitors abolished RVI, but PMA did not. In the tubules of the WT mice, the microtubule disruptor (colchicine), the microfilament disruptor (cytochalasin B), the phosphatidylinositol 3-kinase (PI 3-kinase) blocker (wortmannin), but not another PI 3-kinase blocker (LY-294002), inhibited the PMA-induced RVI. In the tubules of the KO mice, colchicine, cytochalsin B, and wortmannin abolished RVI, but LY-294002 did not. We conclude that 1) in the mouse proximal tubule, P-gp-induced modulation of RVI occurs via PKC; and 2) the microtubule, microfilament, and wortmannin-sensitive, LY-294002-insensitive PI 3-kinase contribute to the PKC-induced RVI.
mdr1a gene; mdr1b gene; protein kinase C; microtubule; microfilament; phosphatidylinositol 3-kinase; P-glycoprotein
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INTRODUCTION |
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P-GLYCOPROTEIN (P-GP) WAS initially identified through its ability to confer multidrug resistance (MDR) in mammalian tumor cells (reviewed in Ref. 15). P-gp is a member of the ATP-binding cassette superfamily of transporters (15) and utilizes ATP to pump hydrophobic drugs out of the cells, decreasing their intracellular concentrations and hence their toxicity. Humans have one drug-transporting P-gp (MDR1), whereas mice have two genes encoding drug-transporting P-gps, mdr1a (also called mdr3) and mdr1b (also called mdr1) (15, 19, 35). The mdr1a and mdr1b in the mouse together fulfill the same function as MDR1 in humans, and similar levels of mdr1a and mdr1b expression are observed in the kidney (5, 36). In the kidney, the apical membrane of the proximal tubule epithelium is particularly rich in P-gp (10, 39), placing this pump in the correct location to mediate the active excretion of xenobiotics. Consistent with a role for P-gp as an excretory transporter, monolayers of LLC-PK1 cells (33), opossum kidney cells, and cultured mouse proximal tubule cells (9) exhibit net secretion of several MDR substrates, including verapamil and cyclosporin A. Also, Tsuruoka et al. (42) recently demonstrated in the isolated perfused mouse proximal tubule that P-gp-mediated drug efflux capacity indeed exists in the apical membrane of proximal tubule S2 segments from wild-type (WT) mice but is lacking in that of the mice in which both the mdr1a and mdr1b genes were disrupted (KO mice).
Most animal cells regulate their volume in response to changes in the
osmolality of the environment (reviewed in Refs. 20 and
25). After cell swelling in hyposmotic medium, a regulatory volume
decrease (RVD) is generally achieved by the loss of ions and other
osmolytes and the concomitant loss of water. Of the various efflux
systems that can contribute to RVD, swelling-activated Cl
channels are present in a number of cell types (20, 25). Conversely, in most animal cells, after cell shrinkage in hyperosmotic medium, a regulatory volume increase (RVI) is generally achieved by the
gain of ions and other osmolytes (20, 25). In sharp contrast, when rabbit proximal tubule cells are exposed suddenly to
hyperosmotic mannitol, NaCl, or raffinose solutions, they rapidly shrink but remain reduced in size (12, 23). However, the
mechanisms responsible for the lack of RVI have not been fully demonstrated.
In addition to the drug-transporting activity, in cultured cells
overexpressing P-gp activity, P-gp has been suggested to be a regulator
of the swelling-activated Cl channel and RVD after the
initial cell swelling (1, 13). However, Miyata et al.
(27) very recently used isolated nonperfused proximal
tubule S2 segments from WT and KO mice to demonstrate that in intact
proximal tubule cells, P-gp is not involved in RVD under hyposmotic
stress. Instead, they have shown that the exposure of the tubules from
WT mice to the hyperosmotic mannitol solution lacked RVI, whereas the
exposure of the tubules from KO mice to the hyperosmotic mannitol
solution elicited RVI (27). They observed that in WT mice,
the peritubular addition of the P-gp inhibitors (verapamil and
cyclosporin A) resulted in RVI, whereas in KO mice it had no effect on
RVI (27). Therefore, in mouse proximal tubule, P-gp
modulates RVI during the exposure to the hyperosmotic mannitol
solution. They also observed that the basolateral
Na+/H+ exchange contributes to the P-gp-induced
modulation of RVI (27). Presently, however, it is not
known how P-gp modulates the hyperosmotic mannitol-induced RVI.
In Madin-Darby canine kidney (MDCK) cells, hyperosmolality induced by
NaCl or raffinose has been shown to enhance inositol 1,4,5-triphosphate
levels and thereby activate phorbol 12-myristate 13-acetate
(PMA)-sensitive protein kinase C (PKC) (38). In Ehrlich mouse ascites tumor cells, PKC is involved in activation of the Na+-K+-2Cl cotransporter induced
by hyperosmolality (21). Treatment of NIH/3T3 cells with
hyperosmotic NaCl solution has also been reported to trigger
phospholipase C activation and then induce an increase in
diacylglycerol levels and, as a consequence, PKC activation (46). The PKC activation induces translocation of PKC
,
PKC
, and PKC
from the cytosol to the plasma membrane after the
stimulation of NIH/3T3 cells with hyperosmotic NaCl (46).
However, it is not known whether and how, in the mouse proximal tubule,
PKC actually modulates P-gp-induced modulation of RVI after exposure to
the hyperosmotic mannitol solution.
Cytoskeletal elements may interfere in several ways with volume regulatory mechanisms (reviewed in Ref. 20). RVD is inhibited in several tissues by cytochalasin B and D, which interfere with actin assembly (20, 22). Cell swelling increases microtubule stability and stimulates the expression of tubulin (22). The addition of the microtubule inhibitor vincristine to the rabbit proximal convoluted tubule impaired RVI after the initial cell swelling (22). Colchicine, which disrupts the microtubule network, inhibits RVD in peripheral neutrophils (7). However, it is not known whether the disruptors of the microtubule and microfilament affect the hyperosmotic cell volume regulation induced by P-gp.
Phosphatidylinositol 3-kinases (PI 3-kinases) are enzymes that
phosphorylate position 3 of the head group of the membrane lipid phosphatidylinositol (41). In recent years, PI
3-kinase activity and its lipid products have been implicated in
regulation of many cellular processes, including membrane ruffling,
vesicular trafficking, and activation of membrane ion channels
(3, 4, 31, 37). In human intestine 407 cells, PI 3-kinase
has been shown to be involved in swelling-activated Cl
channels (40). In rat medullary thick ascending limb of
Henle's loop, hyposmolality, but not hyperosmolality, induces an
increase in PI 3-kinase activity, which, in turn, results in the
stimulation of HCO
Therefore, we used isolated nonperfused proximal tubule S2 segments from KO and WT mice to examine the intracellular signaling mechanisms for the P-gp-induced modulation of RVI during exposure to a hyperosmotic mannitol solution.
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METHODS |
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Animals. The KO mice used in this study were originally described by Schinkel et al. (35). Male KO and FVB (WT) mice, serving as control (body wt 25-40 g), were purchased from Taconic Engineering (Germantown, NY). The animals were maintained under a controlled environment and had free access to a standard rodent chow and tap water ad libitum until the experiments. Ages of the KO animals were matched with their WT controls.
In vitro microperfusion. Mice from both groups were anesthetized with an intraperitoneal injection of pentobarbital sodium (4 mg/100 g body wt), and both kidneys were removed. Slices of 1-2 mm were taken from the coronal section of each kidney and transferred to a dish containing a cold intracellular fluid-like solution having the following composition (in mM): 14 KH2PO4, 44 K2HPO4, 15 KCl, 9 NaHCO3, and 160 sucrose. Proximal tubule S2 segments were then dissected by fine forceps under a stereomicroscope and transferred to a bath chamber mounted on an inverted microscope (IMT-2, Olympus, Tokyo).
Both proximal and distal ends of the tubule were drawn into and crimped by glass micropipettes as described by Dellasega and Grantham (6) and Miyata et al. (27). In this preparation, both ends of the tubule are occluded so that the lumen becomes and remains collapsed. A system of the flow-through bath was utilized to permit rapid exchange of the bath fluid. The bathing solution flowed by gravity at 4.5-5.5 ml/min from the reservoirs through a water jacket to stabilize the bath temperature at 37°C.Solutions. The composition of the control isosmotic bathing solution used in the present study was as follows (in mM): 110 NaCl, 5 KCl, 25 NaHCO3, 0.8 Na2HPO4, 0.2 NaH2PO4, 10 Na-acetate, 1.8 CaCl2, 1.0 MgCl2, 8.3 glucose, and 5 alanine. The control isosmotic solution was adjusted to an osmolality of 300 mosmol/kgH2O. The hyperosmotic bathing solution was made by the addition of 200 mM mannitol, to a final osmolality of 500 mosmol/kgH2O. The osmolality of all the solutions was measured by freezing-point depression before the experiments. All the solutions were continuously gassed with a mixture of 5% CO2-95% O2 and adjusted to pH 7.4 at 37°C.
After tubules were tightly crimped between the two glass micropipettes, they were bathed in the isosmotic control solution at 37°C for 10-15 min to allow them to equilibrate. After the equilibration period, cell volume was measured as described below.Measurements of tubule cell volume.
Measurements of tubule cell volume were carried out by the methods
described by Miyata et al. (27). In brief, before, during, and after exposure of the tubules to the hyperosmotic solution, we took
serial photographs, at a magnification of ×400, focused on the center
of the tubule. The photographs were magnified, and the outer diameter
of the tubule (d) was estimated from similar photographs of
a calibrated micrometer slide as reference. Assuming the tubule is
cylindrical in shape, the apparent cell volume could then be calculated
according to the following equation
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Measurement of PKC activity.
We measured PKC activity in both groups of proximal tubules exposed to
the hyperosmotic mannitol solution, using the previously described
methods in our laboratory (8). Proximal straight tubules
from both groups of mice were dissected at 4°C in the control
isosmotic solution without enzymatic digestion of the tissue. On a
given day, they were dissected over a 4-h period, and the pooled
tubules were then divided into several groups. The groups of tubules
were then incubated in the control isosmotic solution or hyperosmotic
mannitol solution, in the absence and presence of PMA. The reaction was
then stopped by the addition of 35 µl of extraction solution (20 mM
Tris · HCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 µg/ml aprotinin, and leupeptin, pH 7.5). Cell extracts were
centrifuged at 1,500 g for 5 min. The supernatant was then
incubated with 25 µM of a synthetic peptide [4-14 amino acids
of bovine myelin basic protein
(MBP4-14)](45) and reaction mixture
containing 20 mM Tris · HCl (pH 7.5), 5 mM Mg acetate, 0.1 mM
CaCl2, 0.5 µg phosphatidyl serine, 50 ng diolein, and 50 µM [-32P]ATP (specific activity; 10 Ci/mM) for 10 min at 30°C. The reaction products were placed on P-81 paper (Whatman
International, Clifton, NJ) and were washed three times with 20 ml of
ice-cold 10% phosphoric acid. The radioactivity was counted by a
liquid scintillation counter (Aloka LSC-671, Tokyo, Japan). Specific
radioactivity was obtained by subtracting the radioactivity of the
synthetic peptide-free reaction from the synthetic peptide-directed
radioactivity. PKC activity was represented as picomoles of ATP
incorporated per milligram protein of cell extracts per minute. The
protein content of the tubules was determined by the Bio-Rad protein
assay kit (Bio-Rad Laboratories, Richmond, CA), using BSA as the standard.
Chemicals.
Verapamil, cyclosporin A, PMA, 4-phorbol, staurosporine, colchicine,
cytochalasin B, wortmannin, and MBP4-14 were purchased from Sigma (St. Louis, MO). LY-294002 was purchased from Calbiochem (La
Jolla, CA). [
-32P]ATP was purchased from New England
Nuclear (Boston, MA). Other high-grade chemicals were obtained from
Wako (Osaka, Japan).
Statistics. The data are expressed as means ± SE. Comparisons were performed by Student's t-test or one-way ANOVA in combination with Fisher's protected least significant difference or Newman-Keuls multiple-range test where appropriate. P values <0.05 were considered significant.
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RESULTS |
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Effect of hyperosmotic mannitol solution on cell volume.
At first, we examined the effect of hyperosmotic mannitol solution on
cell volume in the proximal tubules from the WT and KO mice. For this
purpose, the tubules were initially bathed in the control isosmotic
solution (300 mosmol/kgH2O) and were then perfused with the
hyperosmotic solution (500 mosmol/kgH2O) containing 200 mM
mannitol for 10 min. Figures
1 and
2 are illustrations of the cell volume in
the tubules from the WT and KO mice, respectively. When the tubules
from the WT mice were rapidly exposed to the hyperosmotic mannitol
solution, they abruptly shrank to 81.4 ± 1.4% (n = 5) of their control volume but remained in a shrunken state during
the experimental period, indicating a lack of RVI (Fig. 1A).
In sharp contrast, when the tubules from the KO mice were rapidly
treated with the hyperosmotic mannitol solution for 10 min, they
abruptly shrank to 80.0 ± 1.5% (n = 5) of their
control volume and then gradually swelled to 88.1 ± 1.3%
(n = 5) of their control volume, showing the presence
of RVI (Fig. 2).
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Role of PKC in the P-gp-induced modulation of RVI after initial
cell shrinkage.
Next, we examined whether PKC contributes to the P-gp-induced
modulation of RVI in the proximal tubule from the WT mice. For this
purpose, the tubules of the WT mice were initially exposed to the
control isosmotic solution and were then bathed in the control
isosmotic solution containing 100 nM PMA (the PKC activator) or 100 nM
4-phorbol (the inactive phorbol) for 2 min. Thereafter, they were
treated with the hyperosmotic mannitol solution in the continued
presence of PMA or 4
-phorbol for 10 min. Results are shown in Fig.
3, A (for the PMA-treated
tubules) and B (for the 4
-phorbol-treated tubules). The
peritubular addition of PMA or 4
-phorbol alone had no effect on cell
volume under isosmotic conditions. However, when the tubules of the WT
mice were rapidly exposed to the hyperosmotic mannitol solution in the
presence of PMA, they abruptly shrank to 81.4 ± 0.9%
(n = 8) of their control volume and then gradually
swelled to 88.8 ± 0.4% (n = 8) of their control
volume, indicating a presence of RVI (Fig. 3A). The
hyperosmotic mannitol-induced changes in cell volume in the presence of
PMA were markedly similar to those seen in the presence of the two P-gp
inhibitors. In sharp contrast, when the tubules were exposed to the
hyperosmotic mannitol solution containing 4
-phorbol, they did not
exhibit RVI after the initial cell shrinkage (Fig. 3B). Next, we examined the effects of the PKC inhibitors (staurosporine or
calphostin C) on the hyperosmotic mannitol-induced cell volume regulation. For this purpose, the tubules of the WT mice were initially
incubated in the control isosmotic solution, and were then bathed in
the control isosmotic solution including staurosporine (100 nM) or
calphostin C (500 nM) for 2 min. Thereafter, they were exposed to the
hyperosmotic mannitol solution in the continued presence of the PKC
inhibitors for 10 min. In the presence of either staurosporine or
calphostin C, RVI after the initial cell shrinkage was not observed at
all (data not shown). Next, we examined whether the PMA-induced RVI is
inhibited by the PKC inhibitors (staurosporine or calphostin C). For
this purpose, the tubules of the WT mice were initially incubated in
the control isosmotic solution and were then treated with the control
isosmotic solution involving PMA (100 nM) plus either of the two PKC
inhibitors for 2 min. Thereafter, they were exposed to the hyperosmotic
mannitol solution in the continued presence of PMA plus either of the
two PKC inhibitors for 10 min. Results are shown in Fig. 3,
C (for the PMA plus staurosporine-treated tubules) and
D (for the PMA plus calphostin C-treated tubules). The
peritubular addition of PMA plus staurosporine or calphostin C had no
effect on cell volume under isosmotic conditions. However, when the
tubules of the WT mice were rapidly incubated in the hyperosmotic
mannitol solution including PMA plus either staurosporine or calphostin
C, they abruptly shrank to 82.4 ± 1.0 (n = 6) or
81.8 ± 1.4% (n = 8) of their control volume,
respectively, but remained reduced in size (Fig. 3, C and
D). Therefore, the peritubular addition of PMA plus
staurosporine or calphostin C completely suppressed RVI under hyperosmotic conditions. Collectively, in the tubules of the WT mice,
PKC activation by PMA elicited RVI after the initial cell shrinkage.
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Role of cytoskeleton and PI 3-kinase in PKC-induced RVI after
initial cell shrinkage.
Next, we examined whether the cytoskeleton contributes to the
PKC-induced RVI after the initial cell shrinkage. For this purpose, the
tubules of the WT and KO mice were incubated in the control isosmotic
solution involving the microtubule disruptor (colchicine; 10 µM) or
the microfilament disruptor (cytochalasin B; 10 µM) for 2 min and
were then exposed to the hyperosmotic mannitol solution in the
continued presence of colchicine or cytochalasin B for 10 min. In the
WT mice, the addition of colchicine or cytochalasin B had no effect on
basal cell volume or the hyperosmotic mannitol-induced cell volume
(data not shown). In the KO mice, the addition of either of the
disruptors alone had no effect on cell volume under isosmotic
conditions (Fig. 7, A and
B). However, when the tubules from the KO mice were rapidly
exposed to the hyperosmotic mannitol solution including colchicine or
cytochalasin B, they abruptly shrank to 82.6 ± 1.3 (n = 7) or 81.2 ± 0.8% (n = 6)
of their control volume, respectively, but remained in a shrunken state
during the experimental period (Fig. 7, A and B).
Therefore, in sharp contrast to WT mice, in KO mice, the addition of
colchicine or cytochalasin B completely inhibited RVI induced by the
hyperosmotic mannitol solution. Next, we observed the cell volume
change, when the tubules of the WT mice were initially treated with the
control isosmotic solution including PMA (100 nM) plus colchicine (10 µM) or cytochalasin B (10 µM) for 2 min, and were then exposed to
the hyperosmotic mannitol solution in the continued presence of PMA
plus colchicine or cytochalasin B for 10 min. The coadministration with
PMA and either colchicine or cytochalasin B had no effect on cell
volume under isosmotic conditions (Fig.
8, A and B).
However, when the tubules from the WT mice were rapidly exposed to the hyperosmotic mannitol solution including PMA plus either colchicine or
cytochalasin B, they abruptly shrank to 82.7 ± 1.0%
(n = 6) or 83.0 ± 1.2% (n = 4)
of their control volume, respectively, but remained reduced in size
(Figs. 8, A and B). Therefore, the addition of
colchicine or cytochalasin B to the tubules of the WT mice completely
abolished RVI induced by the hyperosmotic mannitol solution including
PMA. Collectively, both the microtubule and microfilament are involved
in the PKC-induced RVI.
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DISCUSSION |
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We recently reported that the exposure of isolated nonperfused proximal tubules from WT mice to a hyperosmotic mannitol solution did not elicit RVI after the initial cell shrinkage (27). On the other hand, RVI was seen in the hyperosmotic mannitol solution when the P-gp activity was acutely suppressed by the P-gp inhibitors (verapamil and cyclosporin A) or when both mdr1a and mdr1b genes were genetically disrupted (27). In the present study, we extend our previous study to determine the intracellular signaling mechanisms for the P-gp-induced modulation of RVI after initial cell shrinkage.
In the present study, we observed that in the proximal tubule from the
WT mice, the peritubular addition of PMA elicited RVI after the initial
cell shrinkage, but 4-phorbol did not (Fig. 3). The treatment with
the PKC inhibitors (staurosporine and calphostin C) inhibited the
PMA-induced RVI (Fig. 3). PMA alone had no effect on cell volume under
isosmotic conditions. Either of the PKC inhibitors had no effect on
cell volume under is- or hyperosmotic conditions. These findings
indicate that, in the tubules from the WT mice, PKC activation resulted
in RVI under hyperosmotic conditions. This is supported by the fact
that in the tubules from the WT mice, the hyperosmotic mannitol
solution including PMA activated PKC, whereas the hyperosmotic mannitol
solution alone did not (see Fig. 6B). We also observed that
in the tubules of the KO mice, the two PKC inhibitors abolished RVI
under hyperosmotic conditions, although they had no effect on cell
volume under isosmotic conditions (Fig. 5). In the KO mice, PMA caused
no effect on cell volume under is- or hyperosmotic conditions (Fig. 5).
These findings indicate that in the tubules of the KO mice, PKC
inhibition with the PKC inhibitors abolished RVI under hyperosmotic
conditions. In the tubules of the KO mice, the hyperosmotic mannitol
solution in the absence of PMA increased PKC activity with a similar
magnitude to those in the presence of PMA (Fig. 6C).
Therefore, the present results are compatible with the notion that the
exposure of the proximal tubules from the KO mice to the hyperosmotic
mannitol solution elicited RVI via PKC activation. In the WT mice, the hyperosmotic mannitol-induced cell volume changes in the presence of
PMA were markedly similar to those seen in the presence of the P-gp
inhibitors. The hyperosmotic mannitol-induced cell volume changes in
the presence of PMA in the WT mice were markedly similar to those in
the KO mice during exposure to hyperosmotic mannitol. In the WT mice,
the P-gp inhibitors-induced RVI after the initial cell shrinkage was
inhibited by the two PKC inhibitors (Fig. 4). On the basis of the above
findings, we conclude that the exposure of the proximal tubule to the
hyperosmotic mannitol in the absence of P-gp activity (when the P-gp
activity is acutely suppressed by the P-gp inhibitors or when both
mdr1a and mdr1b genes are genetically disrupted)
induces PKC activation, which in turn results in RVI. On the other
hand, exposure to the hyperosmotic mannitol in the presence of P-gp
activity does not induce PKC activation and consequently abolishes RVI.
In sharp contrast, in the presence or absence of P-gp activity, PKC is
not involved in the cell volume regulation under isosmotic conditions.
Collectively, PKC contributes to the P-gp-induced modulation of RVI
after the initial cell shrinkage. This is the first demonstration of a
link between PKC and the P-gp-induced modulation of RVI. Also,
the present studies demonstrate one of the mechanisms responsible for
the lack of RVI in the mammalian proximal tubule. In the isolated
proximal tubule from frog kidney, both RVD and activation of
Cl
channels during exposure to hyposmotic stress have
also been reported to be mediated via PKC (32).
It should be noted that the exposure of the tubules from the WT mice to the hyperosmotic mannitol solution did not induce PKC activation (see Fig. 6B). In sharp contrast to the proximal tubules of the WT mice, hyperosmotic NaCl solution has been shown to activate PKC in MDCK cells (38) and NIH/3T3 cells (46). Similarly, the exposure of the tubules from the KO mice to the hyperosmotic mannitol solution resulted in PKC activation (see Fig. 6C). The mechanism for PKC activation by growth factors is well established in mammalian cells (24), and a partly common mechanism is also operating in hyperosmotic stress-induced PKC activation (38, 46). Stimulation of tyrosine kinase receptors activates phospholipase C and PI 3-kinase, leading to an increase in diacylglycerol and inositol 1,4,5-triphosphate levels, which, in turn, mediate activation of PKC (26, 28). In MDCK cells, hyperosmotic NaCl and raffinose solutions increased inositol 1,4,5-triphosphate levels and therby activated PKC (38). In NIH/3T3 cells, exposure to hyperosmotic NaCl solution triggers phospholipase C activation, then induces an increase in diacylglycerol levels, and as a consequence, PKC activation (46). However, we do not know at present how the hyperosmotic mannitol solution activates PKC in the proximal tubules of the KO mice but does not in those of the WT mice.
There are several mechanisms whereby PKC activation elicits RVI after
the initial cell shrinkage. Pedersen et al. (29) have shown that, in Ehrlich ascites tumor cells, hyperosmotic shrinkage activates Na+/H+ exchanger via PKC. Recently,
we demonstrated that in the mouse proximal tubule, the P-gp-induced
modulation of RVI partly occurs through the basolateral
Na+/H+ exchanger (27). Thus
PKC-induced RVI could be partly mediated via the basolateral
Na+/H+ exchange. Witters et al.
(44) have reported that the purified human erythrocyte
glucose transporter is phosphorylated on incubation with rat brain PKC.
Mehrens et al. (26) demonstrated that in human embryonic
epithelial cells (HEK-293 cells) transfected with the rat organic
cation transporter 1 (rOCT1) gene, PKC phosphorylates rOCT1 protein and
leads to a conformational change at the substrate binding site. Pewitt
et al. (30) have also shown that exposure of duck red
blood cells to hyperosmotic conditions stimulates phosphorylation of
the bumetanide-sensitive
Na+-K+-2Cl cotransporter itself
or a regulatory protein by activation of both cAMP-dependent and
-independent kinases, thereby activating the cotransporter. Therefore,
we propose that the PKC-induced RVI may be mediated via the direct
phosphorylation of the volume-regulated transporters like the
Na+/H+ exchanger and/or their regulatory
proteins by PKC. In support of this concept, Sardet et al.
(34) first described in hamster fibroblasts that
Na+/H+ exchanger protein is rapidly
phosphorylated in response to various mitogens, and that this
phosphorylation of the Na+/H+ exchanger protein
is temporally correlated with its activation. However, Grinstein et al.
(17) observed in rat thymic lymphocytes that
phosphorylation of the Na+/H+ exchanger protein
is not affected by hyperosmotic cell shrinkage, although
Na+/H+ exchanger is activated by
phosphorylation. They concluded that activation of the
Na+/H+ exchanger in lymphocyte RVI is due to
phosphorylation by a protein kinase other than PKC. Instead, they have
shown that direct PKC activation by PMA increases
Na+/H+ exchanger activity by causing an
alkaline shift in the cell pH dependence of the exchanger (16,
18). Therefore, it is also possible that
Na+/H+ exchanger activation may occur via a
PKC-dependent alkaline shift in the cell pH responsiveness of an
allosteric modifier site on the cytoplasmic surface of the basolateral
membrane (2, 16, 18). Further studies will be required to
determine whether and how PKC activation by PMA actually activates the
basolateral Na+/H+ exchanger in the mouse
proximal tubule.
As shown in Fig. 7, in the tubules of the KO mice both colchicine and cytochalasin B inhibited RVI under hyperosmotic conditions, although they had no effect on cell volume under isosmotic conditions. Figure 8 shows that, in the tubules of the WT mice, both colchicine and cytochalasin B inhibited PMA-induced RVI under hyperosmotic conditions. On the other hand, exposure of the tubules from WT mice to colchicine or cytochalasin B had no effect on cell volume under is- or hyperosmotic conditions. These findings indicate that both colchicine and cytochalasin B inhibit PKC-induced RVI under hyperosmotic conditions. In other words, both microtubules and microfilaments are required for the PKC-induced RVI. These findings are also the first demonstration of the involvement of both microtubules and microfilaments in the PKC-induced RVI. Because neither colchicine or cytochalasin B affected cell volume under isosmotic conditions, both colchicine- and cytochalasin B-sensitive processes are activated under hyperosmotic conditions and consequently elicit RVI. Future studies will be required to clarify the mechanisms by which PKC activation under hyperosmotic conditions elicits RVI via the microtubule- and microfilament-dependent processes.
PI 3-kinases are a family of enzymes that can be distinguished by
molecular characteristics, substrate specificity, and inhibitor sensitivities (3, 41, 43). Classic inhibitors of these enzymes are wortmannin, a fungal metabolite, which rapidly and irreversibly inhibits mammalian PI 3-kinase by alkylating the catalytic
p110 subunit and is an inhibitor effective in the nanomolar concentration range, and LY-294002, a structurally unrelated compound, which interacts with the ATP-binding site of the enzyme and is an
inhibitor effective in the micromolar concentration range
(43). In the present study, we observed that in the
tubules of the KO mice, wortmannin, but not LY-294002, inhibited RVI
under hyperosmotic conditions, although neither of the inhibitors had
an effect on cell volume under isosmotic conditions (see Fig. 7). We
also show that in the tubules of the WT mice under hyperosmotic
conditions, wortmannin abolished the PMA-induced RVI, but LY-294002 did
not (see Fig. 8). On the other hand, the exposure of the tubules from the WT mice to either wortmannin or LY-294002 had no effect on cell
volume under iso- or hyperosmotic conditions. Therefore, wortmannin,
but not LY-294002, inhibits the PKC-induced RVI under hyperosmotic
conditions, consistent with the notion that the wortmannin-sensitive, LY-294002-insensitive PI 3-kinase is required for the PKC-induced RVI
after initial cell shrinkage. Because wortmannin caused no effect on
cell volume under isosmotic conditions, the wortmannin-sensitive, LY-insensitive PI 3-kinase is activated under hyperosmotic conditions, and thereby elicits RVI, although the underlying mechanisms are unclear. This is also the first demonstration that PI 3-kinase contributes to the PKC-induced RVI after initial cell shrinkage. In
human intestine 407 cells, PI 3-kinase has been shown to be involved in
the activation of Cl channels after hyposmotic stress
(40). Feranchak et al. (11) also reported
that, in HTC hepatoma cells, both RVD and activation of the
Cl
channel under hyposmotic stress are mediated via PI
3-kinase. Good et al. (14) have very recently shown that
in the medullary thick ascending limb of Henle's loop from rat kidney,
PI 3-kinase is required for the hyposmolality-induced stimulation of
HCO
We concluded that in the mouse proximal tubule S2 segments, P-gp-induced modulation of RVI during hyperosmotic stress is mediated via PKC. On the basis of the use of pharmacological agents, we also found that the microtubule, microfilament, and wortmannin-sensitive, LY-294002-insensitive PI 3-kinase contribute to the PKC-induced RVI after initial cell shrinkage.
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ACKNOWLEDGEMENTS |
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This work was supported in part by a grant from the Japanese Kidney Foundation (Jinkenkyukai), the Salt Science Foundation, and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports (Japan).
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FOOTNOTES |
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A portion of this work was presented at the Annual Meeting of the American Society of Nephrology in Toronto, Ontario, Canada, and has been published in abstract form (J Am Soc Nephrol 11, 46A-46B, 2000).
Address for reprint requests and other correspondence: S. Muto, Dept. of Nephrology, Jichi Medical School, Minamikawachi, Tochigi 329-0498 Japan (E-mail: smuto{at}jichi.ac.jp).
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. Section 1734 solely to indicate this fact.
First published August 15, 2001; 10.1152/ajprenal. 0000036.2001
Received 6 February 2001; accepted in final form 7 August 2001.
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