点击显示 收起
【摘要】 A high-salt diet enhances nitric oxide (NO)-induced inhibition of transport in the thick ascending limb (THAL). Long exposures to NO inhibit Na-K-ATPase in cultured cells. We hypothesized that NO inhibits THAL Na-K-ATPase after long exposures and a high-salt diet would augment this effect. Rats drank either tap water or 1% NaCl for 7-10 days. Na-K-ATPase activity was assessed by measuring ouabain-sensitive ATP hydrolysis by THAL suspensions. After 2 h, spermine NONOate (SPM; 5 µM) reduced Na-K-ATPase activity from 0.44 ± 0.03 to 0.30 ± 0.04 nmol P i ·µg protein -1 ·min -1 in THALs from rats on a normal diet ( P < 0.03). Nitroglycerin also reduced Na-K-ATPase activity ( P < 0.04). After 20 min, SPM had no effect (change -0.07 ± 0.05 nmol P i ·µg protein -1 ·min -1 ). When rats were fed high salt, SPM did not inhibit Na-K-ATPase after 120 min. To investigate whether ONOO - formed by NO reacting with O 2 - was involved, we measured O 2 - production. THALs from rats on normal and high salt produced 35.8 ± 0.3 and 23.7 ± 0.8 nmol O 2 - ·min -1 ·mg protein -1, respectively ( P < 0.01). Because O 2 - production differed, we studied the effects of the O 2 - scavenger tempol. In the presence of 50 µM tempol, SPM did not inhibit Na-K-ATPase after 120 min (0.50 ± 0.05 vs. 0.52 ± 0.07 nmol P i ·µg protein -1 ·min -1 ). Propyl gallate, another O 2 - scavenger, also prevented SPM-induced inhibition of Na-K-ATPase activity. SPM inhibited pump activity in tubules from rats on high salt when O 2 - levels were increased with xanthine oxidase and hypoxanthine. We concluded that NO inhibits Na-K-ATPase after long exposures when rats are on a normal diet and this inhibition depends on O 2 -. NO donors do not inhibit Na-K-ATPase in THALs from rats on high salt due to decreased O 2 - production.
【关键词】 superoxide nitric oxide Na transport hypertension
NITRIC OXIDE (NO) is an important regulator of renal function, promoting natriuresis and diuresis ( 9 ). Intrarenal administration of NO synthesis inhibitors lowers urinary Na excretion (11-13, 17, 19, 20, 22, 38), whereas NO donors induce natriuresis ( 18, 19 ). NO-induced natriuresis and diuresis can occur in the absence of hemodynamic changes, indicating that NO directly inhibits Na and water absorption along the nephron. In vitro studies have shown that NO blunts transport in isolated proximal tubules ( 1 ), cortical collecting ducts ( 3, 39, 40 ), inner medullary collecting ducts ( 2 ), and thick ascending limbs ( 2, 34, 35 ). Additionally, NO has been reported to inhibit Na transport in several cultured renal cells ( 5, 15, 16 ).
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 O 2 - ( 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 O 2 -.
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 O 2 -; and 3 ) a high-salt diet will augment this effect.
METHODS
Animals. Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were fed chow containing 1.1% K and 0.2% Na and given either tap water or 1% NaCl in drinking water for 7-10 days. Rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip). All protocols were approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital.
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 NaHCO 3, 2.5 NaH 2 PO 4, 1.2 MgSO 4, 2.0 Ca lactate, 5.5 glucose, 6.0 alanine, and 1.0 Na 3 citrate, and it was gassed with 95% O 2 -5% CO 2. Arginine was omitted to prevent endogenous NO production. Osmolality was 290 ± 3 mosmol/kgH 2 O as measured by freezing-point depression. Solution A contained (in mM) 1 Na 2 EGTA, 5 MgCl 2, 100 imidazole, 6 Na 2 ATP, 45 NaOH, and 5 KOH, pH 7.0. Solution B contained (in mM) 1 Na 2 EGTA, 5 MgCl 2, 100 imidazole, 6 Na 2 ATP, 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 O 2 - scavenger as indicated in Protocols. Tubes were gassed with 95% air-5% CO 2 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 H 2 O 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 (P i ). 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). P i 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 O 2 - 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 NaH 2 PO 4, 1.2 MgSO 4, 2.0 Ca lactate, 5.5 glucose, 6.0 alanine, and 1.0 Na 3 citrate. pH was increased to 7.4 with NaOH, and the solution was gassed with 100% O 2. 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 O 2 - 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 O 2 -. 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 O 2 -.
Statistics. Results are expressed as means ± SE. Na-K-ATPase activity was evaluated by a Student's paired t -test. O 2 - production was evaluated by an unpaired t -test, taking P < 0.05 as significant.
RESULTS
We previously reported that NO had no effect on Na-K-ATPase activity of freshly isolated renal tubules when exposures were less than 20 min ( 29 ). In contrast, others showed that NO appeared to inhibit pump activity in cultured cells when the exposure was significantly longer ( 5, 15, 16 ). To examine whether NO-induced inhibition of Na-K-ATPase in thick ascending limbs is time dependent, we first tested the ability of 5 µM SPM, a NO donor, to reduce Na-K-ATPase activity after 20 or 120 min. In freshly isolated suspensions of thick ascending limbs from rats on a normal diet, Na-K-ATPase activity in tubules treated with vehicle for 20 min was 0.53 ± 0.05 nmol P i ·µg protein -1 ·min -1. Na-K-ATPase activity of thick ascending limbs exposed to SPM for 20 min was not significantly different (change -0.07 ± 0.05 nmol P i ·µg protein -1 ·min -1 ). In contrast, in tubules exposed to 5 µM SPM for 120 min, Na-K-ATPase activity decreased by 32% compared with vehicle (from 0.44 ± 0.03 to 0.30 ± 0.04 nmol P i ·µg protein -1 ·min -1; P < 0.03; n = 7; Fig. 1 ).
Fig. 1. Effect of vehicle or spermine NONOate (SPM) on Na-K-ATPase activity in thick ascending limbs from rats on a normal diet. Tubules were treated with either vehicle or SPM for 120 min.
To make sure the effect of SPM we observed was due to release of NO, we treated tubules with a different NO donor, nitroglycerin (10 µM), for 120 min. In vehicle-treated suspensions, Na-K-ATPase activity was 0.40 ± 0.01 nmol P i ·µg protein -1 ·min -1. In nitroglycerin-treated tubules, it was 0.31 ± 0.03 nmol P i ·µg protein -1 ·min -1 ( P < 0.04), a decrease of 23%. Taken together, these data suggest that NO-induced inhibition of Na-K-ATPase activity is time dependent.
In cultured cells, NO-induced reductions in pump activity require O 2 - ( 5 ). We therefore examined whether SPM-induced inhibition depends on O 2 - by testing the ability of O 2 - 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 P i ·µg protein -1 ·min -1 ( n = 6) after 120 min of vehicle treatment. In the presence of SPM and tempol, pump activity was 0.52 ± 0.07 nmol P i ·µg protein -1 ·min -1 ( 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 P i ·µg protein -1 ·min -1; n = 6). To make sure the tempol results were due to scavenging of O 2 -, we tested another O 2 - 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 P i ·µg protein -1 ·min -1 vs. vehicle: 0.39 ± 0.02 nmol P i ·µg protein -1 ·min -1; 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 O 2 -, probably OONO -.
Fig. 2. Effect of tempol, an O 2 - scavenger, on nitric oxide-induced inhibition of Na-K-ATPase activity in thick ascending limbs from rats on a normal diet. Tubules were treated with either tempol alone or tempol plus SPM for 120 min. N.S., not significant.
Previously, we reported that a high-salt diet enhances the ability of NO to inhibit transport in the thick ascending limb ( 28 ). Those data suggest that a high-salt diet enhances the ability of NO to inhibit Na-K-ATPase activity. Thus we tested whether the effect of SPM on pump activity would be augmented by a high-salt diet. Na-K-ATPase activity of thick ascending limbs from rats on a high-salt diet treated with vehicle for 120 min was 0.40 ± 0.03 nmol P i ·µg protein -1 ·min -1. In tubules treated with SPM for 120 min, Na-K-ATPase activity was 0.35 ± 0.04 nmol P i ·µg protein -1 ·min -1, not statistically different ( Fig. 3 ). Thus a high-salt diet reduced the ability of NO to inhibit pump activity, the opposite of what we expected.
Fig. 3. Effect of vehicle or SPM on Na-K-ATPase activity in thick ascending limbs from rats on a high-salt diet. Tubules were treated with either vehicle or SPM for 120 min.
Given that 1 ) NO-induced inhibition of Na-K-ATPase in thick ascending limbs from rats on a normal diet required O 2 - and 2 ) NO had no effect on pump activity when rats were fed a high-salt diet, we studied the effect of SPM on Na-K-ATPase activity in the presence of an exogenous source of O 2 - and measured O 2 - production by thick ascending limbs from rats on a high-salt diet. After 120 min of SPM alone, Na-K-ATPase activity was 0.64 ± 0.01 nmol P i ·µg protein -1 ·min -1. Treatment with SPM plus 0.5 mU/ml xanthine oxidase and 0.5 mM hypoxanthine reduced Na-K-ATPase activity by 24% to 0.49 ± 0.03 nmol P i ·µg protein -1 ·min -1 ( P < 0.05; Fig. 4 ). Next, we investigated whether a high-salt diet alters O 2 - production by suspensions of thick ascending limbs. In thick ascending limbs from rats on a normal diet, O 2 - production was 35.8 ± 0.3 nmol O 2 - ·min -1 ·mg protein -1; however, in thick ascending limbs from rats on a high-salt diet it was 23.7 ± 0.8 nmol O 2 - ·min -1 ·mg protein -1, a decrease of 34% ( P < 0.01; Fig. 5 ). Taken together, our data indicate that the lack of NO-induced inhibition of Na-K-ATPase in thick ascending limbs from rats on a high salt is due to reduced O 2 - levels.
Fig. 4. Effect of an exogenous O 2 - -generating system on Na-K-ATPase activity in thick ascending limbs from rats on a high-salt diet in the presence of SPM. Xanthine oxidase (XO) and hypoxanthine (HX) were used to generate O 2 -. Na-K-ATPase activity was measured after treating the tubules for 120 min.
Fig. 5. O 2 - production by thick ascending limbs from rats on a normal- or high-salt diet.
The above data indicate that a reactive nitrogen species such as OONO - is involved in the inhibition of Na-K-ATPase. Frequently, the physiological and pathophysiological effects of such radicals are mediated by formation of isoprostanes and activation of endoperoxide receptors. To investigate whether activation of endoperoxide receptors is required for inhibition of Na-K-ATPase by SPM, we examined the ability of the endoperoxide antagonist SQ-29548 to block this effect. We found no significant difference in Na-K-ATPase activity between tubules treated with SPM alone for 120 min and those treated with 1 µM SQ-29548 before the addition of SPM (0.55 ± 0.07 vs. 0.52 ± 0.08 nmol P i ·µg protein -1 ·min -1 ). Thus the inhibitory effect of SPM on Na-K-ATPase is not mediated by endoperoxide receptors.
DISCUSSION
The effects of NO on Na-K-ATPase activity in renal epithelial cells have been controversial, with reports of both inhibition ( 5, 15, 16 ) and no effect ( 29, 40 ). We set out to investigate 1 ) the time dependency of Na pump inhibition in thick ascending limbs, 2 ) the role of O 2 - in this process, and 3 ) how the effects of NO are altered by a high-salt diet. Our data show that the NO donors SPM and nitroglycerin both inhibited Na-K-ATPase activity when thick ascending limbs from rats on a normal diet were treated for 2 h but not for only 20 min. The ability of NO to reduce pump activity was prevented by treating tubules with tempol and propyl gallate, both O 2 - scavengers, but not by an endoperoxide receptor antagonist. NO did not inhibit pump activity in tubules from rats on a high-salt diet, a treatment that reduced O 2 - levels. When we artificially enhanced O 2 - in thick ascending limbs from the high-salt group, NO-induced inhibition was restored.
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 O 2 - ( 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 O 2 -. 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 O 2 - 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 O 2 - was required for NO to inhibit Na-K-ATPase in thick ascending limbs of rats on a normal diet, we measured O 2 - 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 O 2 - levels prevented NO from lowering pump activity, we applied an NO donor in the presence of an exogenous O 2 - -generating system, xanthine oxidase/hypoxanthine, and found that SPM now inhibited Na pump activity by 25%. These data indicate that reduced O 2 - 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/O 2 - reaction such as OONO -.
Our results show that inhibition of Na-K-ATPase activity by NO in freshly isolated thick ascending limbs depends on O 2 -. NO and O 2 - 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 N w -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 10 -8 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 O 2 - was involved and that NO reacted with O 2 - 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 O 2 - 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 20-30 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 O 2 - 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 O 2 - 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 O 2 -, 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 O 2 - /OONO - and the other not ( 25 ). The explanation for this discrepancy is unclear, but it may involve differences in O 2 - 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 O 2 -, because inhibition could be restored by adding an exogenous source of O 2 -. Given that NO could scavenge O 2 -, one might argue that the decrease in O 2 - 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 O 2 - 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 O 2 - in our study, NAD(P)H oxidase appears to be the most likely candidate. Several proteins produce O 2 -, 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 O 2 - 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 O 2 - production caused by high salt. However, catabolism of O 2 - may also be involved as discussed below.
The fact that O 2 - 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 O 2 -, and total medullary O 2 - 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 O 2 - 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 O 2 -, because it can be prevented by scavenging O 2 -. Although inhibition is likely mediated by OONO - formed from NO and O 2 -, 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 O 2 - levels.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute Grants HL-28982 and HL-070985 to J. L. Garvin. M. Varela was supported by a fellowship from the American Heart Association (0225424Z).
【参考文献】
Eitle E, Hiranyachattada S, Wang H, and Harris PJ. Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney. Am J Physiol Cell Physiol 274: C1075-C1080, 1998.
Garcia NH, Plato CF, Stoos BA, and Garvin JL. Nitric oxide-induced inhibition of transport by thick ascending limbs from Dahl salt-sensitive rats. Hypertension 34: 508-513, 1999.
Garcia NH, Stoos BA, Carretero OA, and Garvin JL. Mechanism of the nitric oxide-induced blockade of collecting duct water permeability. Hypertension 27: 679-683, 1996.
Garvin JL and Hong NJ. Nitric oxide inhibits sodium/hydrogen exchange activity in the thick ascending limb. Am J Physiol Renal Physiol 277: F377-F382, 1999.
Guzman NJ, Fang MZ, Tang SS, Ingelfinger JR, and Garg LC. Autocrine inhibition of Na + /K + -ATPase by nitric oxide in mouse proximal tubule epithelial cells. J Clin Invest 95: 2083-2088, 1995.
Harris RC and Breyer MD. Physiological regulation of cyclooxygenase-2 in the kidney. Am J Physiol Renal Physiol 281: F1-F11, 2001.
Huie RE and Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun 18: 195-199, 1993.
Kitiyakara CC. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. J Am Soc Nephrol 14: 2775-2782, 2003.
Kone BC and Baylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am J Physiol Renal Physiol 272: F561-F578, 1997.
Kontos HA, Wei EP, Ellis EF, Jenkins LW, Povlishock JT, Rowe GT, and Hess ML. Appearance of superoxide anion radical in cerebral extracellular space during increased prostaglandin synthesis in cats. Circ Res 57: 142-151, 1985.
Lahera V, Salom MG, Fiksen-Olsen MJ, Raij L, and Romero JC. Effects of N G -monomethyl- L -arginine and L -arginine on acetylcholine renal response. Hypertension 15: 659-663, 1990.
Lahera V, Salom MG, Fiksen-Olsen MJ, and Romero JC. Mediatory role of endothelium-derived nitric oxide in renal vasodilatory and excretory effects of bradykinin. Am J Hypertens 4: 260-262, 1991.
Lahera V, Salom MG, Miranda-Guardiola F, Moncada S, and Romero JC. Effects of N G -nitro- L -arginine methyl ester on renal function and blood pressure. Am J Physiol Renal Fluid Electrolyte Physiol 261: F1033-F1037, 1991.
Lerman LO, Nath KA, Rodriguez-Porcel M, Krier JD, Schwartz RS, Napoli C, and Romero JC. Increased oxidative stress in experimental renovascular hypertension. Hypertension 37: 541-546, 2001.
Liang M and Knox FG. Nitric oxide activates PKC and inhibits Na + -K + -ATPase in opossum kidney cells. Am J Physiol Renal Physiol 277: F859-F865, 1999.
Liang M and Knox FG. Nitric oxide reduces the molecular activity of Na +,K + -ATPase in opossum kidney cells. Kidney Int 56: 627-634, 1999.
Majid DS and Nishiyama A. Nitric oxide blockade enhances renal responses to superoxide dismutase inhibition in dogs. Hypertension 39: 293-297, 2002.
Majid DS, Omoro SA, Chin SY, and Navar LG. Intrarenal nitric oxide activity and pressure natriuresis in anesthetized dogs. Hypertension 32: 266-272, 1998.
Majid DS, Williams A, Kadowitz PJ, and Navar LG. Renal responses to intra-arterial administration of nitric oxide donor in dogs. Hypertension 22: 535-541, 1993.
Majid DS, Williams A, and Navar LG. Inhibition of nitric oxide synthesis attenuates pressure-induced natriuretic responses in anesthetized dogs. Am J Physiol Renal Fluid Electrolyte Physiol 264: F79-F87, 1993.
Mattson DL and Higgins DJ. Influence of dietary sodium intake on renal medullary nitric oxide synthase. Hypertension 27: 688-692, 1996.
Mattson DL, Lu S, Nakanishi K, Papanek PE, and Cowley AW Jr. Effect of chronic renal medullary nitric oxide inhibition on blood pressure. Am J Physiol Heart Circ Physiol 266: H1918-H1926, 1994.
Meng S, Cason GW, Gannon AW, Racusen LC, and Manning RD Jr. Oxidative stress in Dahl salt-sensitive hypertension. Hypertension 41: 1346-1352, 2003.
Meng S, Roberts LJ, Cason GW, Curry TS, and Manning RD Jr. Superoxide dismutase and oxidative stress in Dahl salt-sensitive and -resistant rats. Am J Physiol Regul Integr Comp Physiol 283: R732-R738, 2002.
Muriel P and Sandoval G. Nitric oxide and peroxynitrite anion modulate liver plasma membrane fluidity and Na + /K + -ATPase activity. Nitric Oxide 4: 333-342, 2000.
Ortiz MC, Manriquez MC, Romero JC, and Juncos LA. Antioxidants block angiotensin II-induced increases in blood pressure and endothelin. Hypertension 38: 655-659, 2001.
Ortiz MC, Sanabria E, Manriquez MC, Romero JC, and Juncos LA. Role of endothelin and isoprostanes in slow pressor responses to angiotensin II. Hypertension 37: 505-510, 2001.
Ortiz P, Stoos BA, Hong NJ, Boesch DM, Plato CF, and Garvin JL. High-salt diet increases sensitivity to NO and eNOS expression but not NO production in THALs. Hypertension 41: 682-687, 2003.
Ortiz PA, Hong NJ, and Garvin JL. NO decreases thick ascending limb chloride absorption by reducing Na + -K + -2Cl - cotransporter activity. Am J Physiol Renal Physiol 281: F819-F825, 2001.
Pagano PJ, Chanock SJ, Siwik DA, Colucci WS, and Clark JK. Angiotensin II induces p67phox mRNA expression and NADPH oxidase superoxide generation in rabbit aortic adventitial fibroblasts. Hypertension 32: 331-337, 1998.
Pagano PJ, Clark JK, Cifuentes-Pagano ME, Clark SM, Callis GM, and Quinn MT. Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II. Proc Natl Acad Sci USA 94: 14483-14488, 1997.
Pagano PJ, Ito Y, Tornheim K, Gallop PM, Tauber AI, and Cohen RA. An NADPH oxidase superoxide-generating system in the rabbit aorta. Am J Physiol Heart Circ Physiol 268: H2274-H2280, 1995.
Parks DA and Granger DN. Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiol Scand Suppl 548: 87-99, 1986.
Plato CF, Shesely EG, and Garvin JL. eNOS mediates L -arginine-induced inhibition of thick ascending limb chloride flux. Hypertension 35: 319-323, 2000.
Plato CF, Stoos BA, Wang D, and Garvin JL. Endogenous nitric oxide inhibits chloride transport in the thick ascending limb. Am J Physiol Renal Physiol 276: F159-F163, 1999.
Qayyum I, Zubrow AB, Ashraf QM, Kubin J, Delivoria-Papadopoulos M, and Mishra OP. Nitration as a mechanism of Na +,K + -ATPase modification during hypoxia in the cerebral cortex of the guinea pig fetus. Neurochem Res 26: 1163-1169, 2001.
Reckelhoff JF and Romero JC. Role of oxidative stress in angiotensin-induced hypertension. Am J Physiol Regul Integr Comp Physiol 284: R893-R912, 2003.
Salom MG, Lahera V, Miranda-Guardiola F, and Romero JC. Blockade of pressure natriuresis induced by inhibition of renal synthesis of nitric oxide in dogs. Am J Physiol Renal Fluid Electrolyte Physiol 262: F718-F722, 1992.
Stoos BA, Carretero OA, Farhy RD, Scicli G, and Garvin JL. Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct cells. J Clin Invest 89: 761-765, 1992.
Stoos BA, Carretero OA, and Garvin JL. Endothelial-derived nitric oxide inhibits sodium transport by affecting apical membrane channels in cultured collecting duct cells. J Am Soc Nephrol 4: 1855-1860, 1994.
Turkozkan N, Unlu A, Ertabak A, Cimen B, and Karabicak U. The effects of peroxynitrite on erythrocytes. Clin Chem Lab Med 39: 1263-1266, 2001.
Wang W, Wang S, Yan L, Madara P, Del Pilar CA, Wesley RA, and Danner RL. Superoxide production and reactive oxygen species signaling by endothelial nitric-oxide synthase. J Biol Chem 275: 16899-16903, 2000.
Zhang CI. Peroxynitrite and the regulation of Na +,K + -ATPase activity by angiotensin II in the rat proximal tubule. Nitric Oxide 7: 30-35, 2002.
作者单位：1 Division of Nephrology and 2 Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, Michigan 48202