Nitric oxide activates PKCalpha 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 PKCalpha from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCalpha 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 PKCalpha in OK, but not in LLC-PK1, cells. The activation of PKCalpha 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 PKCalpha . 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 beta -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-PKCalpha 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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 PKCalpha 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, PKCalpha and PKCzeta , only PKCalpha was shown to be activated by classical PKC activators (29). Therefore, we focused on PKCalpha in the present study. Activation of PKCalpha 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 PKCalpha from the cytosolic fraction to the particulate fraction. These bands were abolished if purified PKCalpha peptide was included during the incubation with the primary antibody, confirming that they represented PKCalpha . 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 PKCalpha in OK cells. In LLC-PK1 cells, SNP did not alter the distribution of PKCalpha , although PMA caused substantial activation of PKCalpha (Fig. 5). These data indicated that NO activated PKCalpha in OK, but not LLC-PK1, cells.


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Fig. 4.   Effects of NO donors and PMA on the distribution of PKCalpha 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-PKCalpha 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 PKCalpha peptide. Lanes 1, 3, 5, 7, and 9, particulate fractions; lanes 2, 4, 6, 8, and 10, cytosolic fraction. B: amount of PKCalpha 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 PKCalpha 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 PKCalpha proteins estimated by densitometry. Results shown are representative of 3 independent experiments.

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 PKCalpha 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 PKCalpha induced by SNP, suggesting a role of cGMP in the activation of PKCalpha in OK cells by NO. ODQ alone did not affect the distribution of PKCalpha 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 PKCalpha 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 PKCalpha protein estimated by densitometry. Results shown are representative of 3 independent experiments.

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 PKCalpha in OK cells by NO (Fig. 6). U-73122 alone did not affect the distribution of PKCalpha 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.

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 epsilon  and iota  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 PKCalpha in OK cells. The activation of PKCalpha 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 PKCalpha 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 alpha -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 PKCalpha 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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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