1Division of Nephrology and 2Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan 48202
Submitted 4 December 2003 ; accepted in final form 22 April 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
superoxide; nitric oxide; Na transport; hypertension
We reported that the inhibition of net Na absorption caused by NO in the thick ascending limb and cortical collecting duct is due to reduced activity of the transporters responsible for Na entry into the cell, namely, Na/H exchange (4) and Na-K-2Cl cotransport (29) in the thick ascending limb and amiloride-sensitive Na channels in the cortical collecting duct (40). We did not find inhibition of basolateral Na-K-ATPase after 20 min of exposure. However, others showed that NO inhibits Na-K-ATPase activity in cultured cells after longer exposures and that inhibition depends on O2 (5). The explanation for the disparate ability of NO to inhibit pump activity in cells of renal origin is unclear, but it could involve 1) the length of time cells are treated with NO, 2) NO concentrations, and/or 3) whether experimental conditions favor formation of other reactive nitrogen-containing compounds from NO and O2.
NO has been implicated in chronic adaptation to a high-salt diet (21). A high-salt diet increases expression of all three NO synthase (NOS) isoforms in the kidney (9, 21), and, in particular, endothelial NOS expression in the thick ascending limb (28). Additionally, a high-salt diet enhances the inhibitory effect of NO on thick ascending limb transport (28). These data suggest that NO may reduce both Na-K-ATPase activity and Na-K-2Cl cotransport in thick ascending limbs from animals on a high-salt diet. We hypothesized that 1) in contrast to short exposures to NO (<30 min), long exposures will inhibit thick ascending limb Na-K-ATPase activity; 2) this inhibition will depend on O2; and 3) a high-salt diet will augment this effect.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of thick ascending limbs. Suspensions of medullary thick ascending limbs were prepared as described previously (29). Briefly, kidneys were perfused retrograde at 6 ml/min for 6.6 min via the aorta with a physiological solution (see Solutions) containing 0.1% collagenase (Sigma, St. Louis, MO) and 2.5 U/ml heparin. The inner stripe of the outer medulla was cut from coronal slices of the kidneys, minced, and incubated at 37°C for 30 min in 0.1% collagenase. Tissue was pelleted by centrifugation at 114 g, resuspended in cold physiological solution, and stirred on ice for 30 min to release the tubules. The suspension was filtered through 250-µm nylon mesh and centrifuged at 114 g. The tubules were washed, pelleted again, and finally resuspended in cold physiological solution.
Solutions. The physiological solution contained (in mM) 114 NaCl, 4.0 KCl, 25 NaHCO3, 2.5 NaH2PO4, 1.2 MgSO4, 2.0 Ca lactate, 5.5 glucose, 6.0 alanine, and 1.0 Na3 citrate, and it was gassed with 95% O2-5% CO2. Arginine was omitted to prevent endogenous NO production. Osmolality was 290 ± 3 mosmol/kgH2O as measured by freezing-point depression. Solution A contained (in mM) 1 Na2EGTA, 5 MgCl2, 100 imidazole, 6 Na2ATP, 45 NaOH, and 5 KOH, pH 7.0. Solution B contained (in mM) 1 Na2EGTA, 5 MgCl2, 100 imidazole, 6 Na2ATP, 50 NaOH, and 2 ouabain, pH 7.0.
Na-K-ATPase assay. Thick ascending limb suspensions were resuspended in physiological solution and divided into Eppendorf tubes (see Protocols). The tubes were spun, and the pellet was resuspended in physiological saline. Thick ascending limbs were incubated for 20 min or 2 h with vehicle, a NO donor, and/or a O2 scavenger as indicated in Protocols. Tubes were gassed with 95% air-5% CO2 at 37°C. At the end of the incubation period, tubules were rinsed three times with 500 µl of cold 150 mM NaCl. Between rinses, they were spun at 148 g in a Sorvall centrifuge for 2 min at 4°C. After the last rinse, they were resuspended in 50 µl of distilled H2O and then frozen and thawed three times on dry ice to lyse the cells. After the last freeze, 6 mM ATP and 350 µl of solution A or B (prewarmed to 37°C) were added (see Solutions) and the lysates were incubated at 37°C for 10 min. To stop the reaction, a single dose of 350 µl of 5% trichloroacetic acid was added and the tubes were centrifuged at 16,000 g for 2 min. Aliquots of 100 µl were used to assay inorganic phosphate (Pi). To dissolve the protein pellet, 200 µl of 0.04% SDS and 0.05 M NaOH were added to the tubes. Protein concentration in 25-µl aliquots was measured using Coomassie protein assay reagent (Pierce, Rockford, IL). Pi was determined by a method similar to that of Fiske and Subbarrow (40). Two hundred microliters of 5% trichloroacetic acid, 300 µl of incubation buffer, and 150 µl of acid-molybdate solution were added to the 100-µl aliquots, followed by 40 µl of Fiske and Subbarrow's reducing agent (Sigma). After 10 min, absorbance was read at 660 nm.
Protocol 1. Thick ascending limb suspensions were divided into four tubes. After spinning to pellet, tubes 1 and 2 were resuspended in physiological solution plus vehicle and tubes 3 and 4 in physiological solution plus 5 µM spermine NONOate (SPM) for 120 min. Rats were placed on either a normal- or high-salt diet.
Protocol 2. Thick ascending limb suspensions were divided into four tubes. After spinning to pellet, tubes 1 and 2 were resuspended in physiological solution plus vehicle and tubes 3 and 4 in physiological solution plus 5 µM SPM for 20 min. All rats were placed on a normal diet.
Protocol 3. Thick ascending limb suspensions were divided into four tubes. After spinning to pellet, tubes 1 and 2 were resuspended in physiological solution plus vehicle and tubes 3 and 4 in physiological solution plus 10 µM nitroglycerin for 120 min. All rats were placed on a normal diet.
Protocol 4. Thick ascending limb suspensions were divided into six tubes. Tubes 1 and 2 were resuspended in physiological solution plus vehicle, tubes 3 and 4 in physiological solution plus 50 µM 4-hydroxy-2,2,6,6 tetramethylpiperidine-N-oxyl (tempol) or 10 µM n-propyl gallate, and tubes 5 and 6 in physiological solution plus 5 µM SPM and either tempol or propyl gallate. All tubes were incubated for 120 min. All rats were placed on a normal diet.
Protocol 5. Thick ascending limb suspensions were divided into four tubes. After spinning to pellet, tubes 1 and 2 were resuspended in physiological solution plus 5 µM SPM and tubes 3 and 4 in physiological solution plus 5 µM SPM, 0.5 mU xanthine oxidase, and 0.5 mM hypoxanthine (to increase O2 levels) for 120 min. All rats were placed on a high-salt diet.
Protocol 6. Thick ascending limb suspensions were divided into four tubes. After spinning to pellet, tubes 1 and 2 were resuspended in physiological solution plus 5 µM SPM and tubes 3 and 4 in physiological solution plus 5 µM SPM and 1 µM SQ-29548 (an endoperoxide receptor antagonist) for 120 min. All rats were placed on a normal diet.
Superoxide measurement. Suspensions of medullary thick ascending limbs were prepared as described previously (29). Tubules were resuspended in 1 ml HEPES-buffered perfusion solution containing (in mM) 125 NaCl, 4.0 KCl, 10 HEPES, 2.5 NaH2PO4, 1.2 MgSO4, 2.0 Ca lactate, 5.5 glucose, 6.0 alanine, and 1.0 Na3 citrate. pH was increased to 7.4 with NaOH, and the solution was gassed with 100% O2. Aliquots (100 µl) were added to 1.6-ml polypropylene tubes, diluted in HEPES-buffered perfusion solution to a final volume of 900 µl, and placed on ice. Lucigenin (100 µl; final concentration 5 µM) was added to the diluted suspensions, which were then incubated for 30 min at 37°C. Tubes were placed in a luminometer chamber (model 20e, Turner Designs, Mountain View, CA) maintained at 37°C. The average of 10 consecutive 30-s measurements was recorded for each sample. The metal-dependent O2 scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (Tiron; 10 µl) was added to the sample for a final concentration of 10 mM, and 10 consecutive 30-s measurements were made, taking the average of the last three. The difference in average luminescence between samples with and without Tiron was used to quantify O2. Measurements were normalized to protein content. The average luminescence of 10 consecutive measurements was calculated for a blank containing PBS and lucigenin. Arbitrary luminescence units were converted to nanomoles per minute per milligram of protein by means of a calibration curve (31). Tubules were resuspended in a solution lacking L-arginine, the substrate for NOS, so that NO production did not confound measurement of O2.
Statistics. Results are expressed as means ± SE. Na-K-ATPase activity was evaluated by a Student's paired t-test. O2 production was evaluated by an unpaired t-test, taking P < 0.05 as significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In cultured cells, NO-induced reductions in pump activity require O2 (5). We therefore examined whether SPM-induced inhibition depends on O2 by testing the ability of O2 scavengers to block NO-induced inhibition of thick ascending limb Na-K-ATPase activity in rats on a normal diet. In the presence of 50 µM tempol, Na-K-ATPase activity was 0.50 ± 0.05 nmol Pi·µg protein1·min1 (n = 6) after 120 min of vehicle treatment. In the presence of SPM and tempol, pump activity was 0.52 ± 0.07 nmol Pi·µg protein1·min1 (n = 6) after 120 min, not significantly different (Fig. 2). Tempol alone had no significant effect on Na-K-ATPase activity (0.44 ± 0.06 vs. 0.50 ± 0.05 nmol Pi·µg protein1·min1; n = 6). To make sure the tempol results were due to scavenging of O2, we tested another O2 scavenger, N-propyl gallate. In the presence of 10 µM propyl gallate, SPM had no significant effect on Na-K-ATPase activity after 120 min of treatment (SPM + propyl gallate: 0.44 ± 0.09 nmol Pi·µg protein1·min1 vs. vehicle: 0.39 ± 0.02 nmol Pi·µg protein1·min1; n = 5). Propyl gallate alone had no significant effect on Na-K-ATPase activity compared with vehicle. Taken together, these data indicate that NO-induced inhibition of pump activity requires the formation of a reactive nitrogen species from NO and O2, probably OONO.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Time course studies showed that the NO donor SPM reduced Na-K-ATPase activity by 30% when tubules from rats on a normal diet were treated for 120 min but not for only 20 min. This indicates that the ability of NO to inhibit Na-K-ATPase is time dependent and also confirms earlier reports showing that NO donors had no effect on Na-K-ATPase activity in freshly isolated thick ascending limbs (29) or cortical collecting ducts (40) after 20 min, whereas NO inhibits pump activity in renal epithelial cells at longer incubation times (5, 16). To make sure the inhibition caused by SPM was due to NO rather than some other degradation product, we tested nitroglycerin and found that it also inhibited pump activity. Although SPM and nitroglycerin yield chemically distinct degradation products, both produce NO; thus these data indicate that NO is responsible for the reduction in Na-K-ATPase activity.
In cultured proximal tubule cells, NO-induced inhibition of Na-K-ATPase activity has been reported to require O2 (5). For this reason, we studied the ability of tempol and propyl gallate to block SPM inhibition of Na pump activity in thick ascending limbs from rats on a normal diet. SPM by itself reduced Na-K-ATPase activity by 30%, but it had no effect on pump activity when tubules were also incubated with either tempol or propyl gallate, confirming that NO-induced reduction of Na-K-ATPase activity in thick ascending limbs from rats on a normal diet requires O2. Thus NO per se is probably not responsible for the inhibition, but rather another nitrogen-containing reactive oxygen species such as OONO. It is unlikely that the data could be explained by tempol or propyl gallate directly interacting with NO or directly affecting Na-K-ATPase activity, because the scavengers are chemically distinct and had no effect on the Na pump in the absence of NO.
Given that we previously showed that thick ascending limbs produce NO, one might reasonably ask why tempol or propyl gallate alone did not alter Na-K-ATPase activity in the absence of SPM. The answer is that these experiments were performed in the absence of L-arginine, the substrate for NOS, so that endogenously produced NO would not confound interpretation of the results. Parenthetically, the fact that neither tempol nor propyl gallate by itself altered Na-K-ATPase also indicates that endogenously produced O2 does not affect activity.
We previously observed that a high-salt diet enhances the ability of a given concentration of NO to inhibit net NaCl absorption by isolated, perfused thick ascending limbs (28). Therefore, we thought it likely that a high-salt diet would enhance the effect of NO on Na-K-ATPase. Instead, we found that NO no longer caused a significant decline in Na-K-ATPase activity after 2 h. Thus we needed to explain the varying NO-induced inhibition we observed in rats on different diets. Because O2 was required for NO to inhibit Na-K-ATPase in thick ascending limbs of rats on a normal diet, we measured O2 production with both a normal diet and a high-salt diet and found that it was significantly lower in the high-salt group. To see whether the reduced O2 levels prevented NO from lowering pump activity, we applied an NO donor in the presence of an exogenous O2-generating system, xanthine oxidase/hypoxanthine, and found that SPM now inhibited Na pump activity by 25%. These data indicate that reduced O2 production eliminates the ability of NO to inhibit Na-K-ATPase activity in thick ascending limbs from rats on a high-salt diet, thereby supporting the theory that NO per se is not responsible for inhibition, but rather a product of the NO/O2 reaction such as OONO.
Our results show that inhibition of Na-K-ATPase activity by NO in freshly isolated thick ascending limbs depends on O2. NO and O2 can react to form OONO (7). Thus it is likely that OONO mediates these effects, confirming similar reported data involving proximal tubule cells. Guzman et al. (5) reported that NO produced by inducible NOS inhibited Na-K-ATPase in cultured mouse proximal tubule cells treated with interferon- and lipopolysaccharide, although it took 4 h to do so. Inhibition was prevented by prior treatment with either Nw-nitro-L-arginine, a nonselective NOS inhibitor, or superoxide dismutase. NO donors mimicked the effects of interferon-
and lipopolysaccharide, but notably a cell-permeant analog of cGMP did not. These authors concluded that NO inhibits pump activity by generating OONO. More recently, the inhibitory phase of the biphasic dose-response relationship between angiotensin and proximal nephron transport has been shown to be due to OONO (43). In suspensions of rat proximal tubules, inhibition of Na-K-ATPase activity by angiotensin concentrations of 108 M or greater was completely blocked by L-NMMA, a nonselective NOS inhibitor, indicating that the process is dependent on NO. Inhibition was also blocked by superoxide dismutase. Thus these authors concluded that O2 was involved and that NO reacted with O2 to form OONO, which mediated the effect. Finally, there are reports that OONO inhibits Na-K-ATPase in hepatocytes (25), erythrocytes (41), and cells of the cerebral cortex (36).
Because OONO appeared to be responsible for Na pump inhibition, we tested the ability of an endoperoxide receptor antagonist, SQ-29548, to block the effects of NO on thick ascending limb Na-K-ATPase in rats on a normal diet. OONO formed from NO and O2 can cause the formation of isoprostanes through nonenzymatic oxidation of arachidonic acid. Often isoprostanes exert their effects by activating the endoperoxide receptor. However, we found that SQ-29548 did not reduce NO-induced inactivation of Na-K-ATPase. Thus either isoprostanes are not involved in such inhibition or else their effects are mediated via some other pathway.
Although we showed that OONO is responsible for the inhibition of Na-K-ATPase activity after long exposures in the thick ascending limb, it is not clear why the effect takes so long to develop. Assuming inhibition does not require de novo protein synthesis, the actions of OONO could be mediated by either a signaling pathway or a direct effect due to nitration/nitrosation/nitrosylation of one of the subunits of Na-K-ATPase. OONO probably inhibits pump activity via covalent modification by nitrogen-containing groups (25, 36). Generally, the effects of activating signaling pathways that do not involve de novo protein synthesis occur within 2030 min. Additionally, nitration/nitrosation/nitrosylation of Na-K-ATPase would essentially be expected to occur immediately after OONO was formed, due to the fact that it is highly reactive. However, it may be that a number of nitrations/nitrosations/nitrosylations must accumulate or that an intracellular buffer of the process must be overwhelmed before an effect is apparent. In either case, the rate at which these occur would depend on the rate of O2 generation. In the final analysis, however, the mechanism by which OONO reduces Na-K-ATPase activity remains undefined.
Although the inhibitory effect of NO on Na-K-ATPase depends on O2 in many cells, other mechanisms have also been implicated. Liang and Knox (15) reported that NO inhibited Na-K-ATPase activity in opossum kidney (OK) cells, a proximal nephron cell line, acting via generation of cGMP and activation of protein kinase C. They also reported that antioxidants did not alter this inhibitory effect. Thus in OK cells O2, and consequently OONO formation, is not required for NO-induced inhibition of pump activity. In hepatocytes, the reduction of Na-K-ATPase activity caused by NO is mediated by at least two different mechanisms, one involving O2/OONO and the other not (25). The explanation for this discrepancy is unclear, but it may involve differences in O2 production and NO concentrations or species differences.
Previously, we reported that a high-salt diet enhances the ability of NO to inhibit transport in the thick ascending limb (28). Thus we thought it likely that a high-salt diet would enhance the effect of NO on Na-K-ATPase. Instead, we found that NO did not reduce Na-K-ATPase activity in thick ascending limbs from rats on a high-salt diet. We concluded that this was due to a reduction in endogenously produced O2, because inhibition could be restored by adding an exogenous source of O2. Given that NO could scavenge O2, one might argue that the decrease in O2 levels was simply due to heightened NO production resulting from the increase in endothelial NOS expression caused by high salt (28). However, this cannot be the case, because all experiments presented here, including O2 measurements, were performed in the absence of L-arginine, the substrate for NO synthesis. We previously showed that in the absence of exogenously added L-arginine, thick ascending limbs produce negligible amounts of NO (35).
Although we did not identify the source of O2 in our study, NAD(P)H oxidase appears to be the most likely candidate. Several proteins produce O2, including NAD(P)H oxidase (32), xanthine oxidase (33), NO synthase (42), and cyclooxygenase (10). The thick ascending limb expresses all of these, although NAD(P)H oxidase is likely to be a major source of O2 in this segment. NAD(P)H oxidase expression is stimulated by angiotensin (30). When angiotensin concentrations fall, as they do when animals are placed on a high-salt diet, expression and assembly of NAD(P)H oxidase are also reduced. Thus changes in NAD(P)H oxidase expression are likely to explain the observed decrease in O2 production caused by high salt. However, catabolism of O2 may also be involved as discussed below.
The fact that O2 production in the thick ascending limb falls when rats are placed on a high-salt diet is consistent with the finding that oxidative stress is increased in renovascular hypertension (14) and in ANG II-induced hypertension (26, 27, 37). Given that Sprague-Dawley rats do not develop hypertension when placed on a high-salt diet, it is also consistent with findings in the Dahl salt-resistant rat. In this strain, a high-salt diet increases medullary manganese superoxide dismutase expression, which catabolizes O2, and total medullary O2 production is not increased (24). Furthermore, if one mitigates oxidative stress in the salt-sensitive strain, a high-salt diet no longer produces hypertension (23). These data appear to contrast with the reported finding that a high-salt diet increases oxidative stress in the renal cortex (8). Although the explanation for this discrepancy is unclear, it may be that isoprostane production does not precisely mimic O2 production (24), and/or superoxide dismutase mRNA levels may not correlate with protein expression. Alternatively, it may be because cortical proximal tubules respond to high salt differently from medullary thick ascending limbs. Precedence for opposite effects of high salt in cells of the cortex vs. the medulla is shown by the fact that a high-salt diet decreases cyclooxygenase-2 expression in the macula densa but increases cyclooxygenase-2 in the medullary interstitial cells (6).
In summary, we have shown that NO inhibits Na-K-ATPase in freshly isolated thick ascending limbs from rats on a normal diet after long incubations. This inhibition depends on O2, because it can be prevented by scavenging O2. Although inhibition is likely mediated by OONO formed from NO and O2, isoprostanes do not appear to be involved. The fact that NO donors do not inhibit Na-K-ATPase in thick ascending limbs from rats on a high-salt diet appears to be the result of lower O2 levels.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|