Kidney International, Vol. 58 (2000), pp. 2069–2074

Autocrine effects of nitric oxide on HCO23 transport by rat

thick ascending limb

PABLO A. ORTIZ and JEFFREY L. GARVIN

Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan, USA

Autocrine effects of nitric oxide on HCO23 transport by rat thick by affecting proton and bicarbonate transport in differ-

ascending limb. ent segments of the nephron. Wang reported that NOBackground. In vivo and in vitro studies have shown that had a biphasic effect on bicarbonate transport in ratnitric oxide (NO) is an important modulator of transport pro-

proximal tubules microperfused in vivo [4]. Roczniakcesses along the nephron. The thick ascending limb (TAL)and Burns showed inhibition of Na1/H1 exchanger activ-plays a significant role in the urine-concentrating mechanism

and in the maintenance of acid/base balance. ity in the rabbit proximal tubule [5], while Tojo et alMethods. TALs from male Sprague-Dawley rats were iso- demonstrated that NO inhibited bafilomycin-sensitive

lated and perfused, and net bicarbonate flux (JHCO32) was deter-

H1/ATPase activity in the cortical collecting duct (CCD)mined.[6]. The thick ascending limb (TAL) is not only impor-Results. In perfused TALs, 0.5 mmol/L l-arginine (l-Arg),

the substrate for NO synthase, significantly lowered JHCO32 from tant for the maintenance of sodium homeostasis, but also

35.4 6 4.6 to 23.2 6 2.9 pmol · mm21 · min21, a decrease of is an important site for renal regulation of acid/base36.9 6 11.6% (P , 0.025). d-Arg (0.5 mmol/L) had no effect balance [7]. In this segment, the Na1/K1/2 Cl2 cotrans-on JHCO3

2 (N 5 7). In the presence of 5 mmol/L L-NAME, anporter can reabsorb NH1

4 [8], and the apical Na1/H1NO synthase (NOS) inhibitor, the addition of l-Arg did not

exchanger recovers the bicarbonate that escaped proxi-affect TAL JHCO32 (43.4 6 4.4 vs. 44.6 6 5.0 pmol · mm21 ·

min21). L-NAME alone (5 mmol/L) did not affect TAL JHCO32. mal tubule reabsorption [9]. Recently, our group demon-

After removing l-Arg from the bath, JHCO32 increased from strated that exogenous NO production inhibited apical

26.2 6 3.9 to 34.8 6 3.2 pmol · mm21 · min21 (P , 0.01),and basolateral Na1/H1 exchanger activity in isolatedindicating no cytotoxicity of NO. We next investigated theperfused TALs [10]. We have also reported that stimula-effect of cGMP analogues on TAL JHCO3

2. 8-Br-cGMP (50mmol/L) and db-cGMP (50 mmol/L) significantly decreased tion of NO production with l-arginine (l-Arg) inhibitsJHCO3

2 by 26.3 6 9.1% and 35.1 6 11.6%, respectively. In the chloride reabsorption in this segment [11]. However, wepresence of cGMP (50 mmol/L), the addition of l-Arg had no

have not investigated the effect of endogenously pro-effect on JHCO32. In the presence of KT-5823 (2 mmol/L), a

duced NO on bicarbonate transport. Therefore, we hy-protein kinase G inhibitor, the addition of l-Arg did not changeTAL JHCO3

2 (N 5 5). pothesized that stimulation of NO production inhibitsConclusions. We conclude that (1) endogenously produced bicarbonate transport in the TAL. Our findings indicate

NO inhibits TAL JHCO32 in an autocrine manner, (2) cGMP that (1) endogenously produced NO inhibits bicarbonatemediates all of the effects of NO, and (3) this effect is mediated

transport in the isolated perfused TAL, (2) essentiallyby protein kinase G activation.all of the effects of NO are mediated by cyclic guanosinemonophosphate (cGMP), and (3) protein kinase G medi-ates NO-induced inhibition of transport.In vivo studies have shown that nitric oxide (NO) is

important in the control of urinary sodium excretion[1–3], although its role in the regulation of acid/base METHODSbalance has not been fully studied. Recently, it has been

Preparation of isolated nephron segmentsdemonstrated that NO may regulate acid/base balanceThick ascending limbs were obtained from male

Sprague-Dawley rats weighing 120 to 150 g (CharlesKey words: acid-base balance, sodium/hydrogen exchanger, l-arginine,bicarbonate flux, protein kinase G, cGMP. River Breeding Laboratories, Wilmington, MA, USA).

Prior to use, rats were maintained on a diet containingReceived for publication December 27, 1999

0.22% sodium and 1.1% potassium (Ralston Purina, St.and in revised form May 19, 2000Accepted for publication June 5, 2000 Louis, MO, USA) for at least five days. On the day of

the experiment, rats were anesthetized with ketamine 2000 by the International Society of Nephrology

2069

Ortiz and Garvin: NO and TAL bicarbonate flux2070

(100 mg/kg body weight) and xylazine (20 mg/kg bodyweight). The abdominal cavity was opened and flushedwith ice-cold 150 mmol/L NaCl. The left kidney was re-moved and bathed in ice-cold saline. Coronal slices weretransferred to a dissection dish containing physiologicalsaline at 5 to 108C equilibrated with 95% O2/5% CO2.TALs were dissected from medullary rays under a stereo-microscope.

Thick ascending limb perfusion

Thick ascending limbs ranging from 0.48 to 0.82 mmwere transferred to a perfusion chamber, mounted onconcentric glass pipettes, and perfused at 378C as de-scribed previously [8]. In all experiments, both lumenand bath contained (in mmol/L) 114 NaCl, 25 NaHCO3,2.5 NaH2PO4, 4 KCl, 1.2 MgSO4, 6 alanine, 1 trisodiumcitrate, 5.5 glucose, and 2 calcium dilactate. All solutionswere gassed with 95% O2/5% CO2 before the experiment.The osmolality of the solution was 290 6 3 mOsm/kgH2O as measured by freezing-point depression, and thepH was 7.4. The basolateral bath was exchanged at arate of 0.5 mL/min via a continuously flowing exchange Fig. 1. Effect of 0.5 mmol/L L-arginine (L-Arg) on rat thick ascending

limb (TAL) bicarbonate flux (JHCO32). Adding l-Arg to the basolateralsystem, and tubules were perfused at 5 to 10 nL · min21 ·

bath decreased bicarbonate absorption from 35.4 6 4.6 to 23.2 6 2.9mm21. pmol · mm21 · min21. *P , 0.025, N 5 7.

Net bicarbonate flux

We tested the effects of l-Arg (0.5 mmol/L), d-Arg(0.5 mmol/L), the NO synthase (NOS) inhibitor L-NAME perfusion solution, and Cl HCO3

2 is the bicarbonate concen-(5 mmol/L), dibutyryl-cGMP (50 mmol/L; Sigma, St. tration in the collected fluid.Louis, MO, USA), 8-Br-cGMP (50 mmol/L; Bio Log,

StatisticsBremen, Germany), and KT-5823 (2 mmol/L; Biomol,Plymouth Meeting, PA, USA) on bicarbonate absorp- Results are expressed as mean 6 SE. Differences be-tion by isolated perfused TALs. This concentration of tween means were evaluated using Student’s pairedl-Arg is in the range of measured plasma levels [12, 13] t-test. P , 0.05 was considered significant.and was previously shown to inhibit chloride transportin this segment [11]. The typical experimental protocolwas as follows: tubules were equilibrated for 20 to 25 RESULTSminutes at 378C, after which at least four basal measure- Previous work from our laboratory has shown thatments corresponding to the control period were made. exogenous NO production inhibits apical Na1/H1 ex-One of the agents was then added to the bath, and after change in the TAL [10]. Therefore, we tested the effecta 20-minute re-equilibration period, four additional col- of 0.5 mmol/L l-Arg on bicarbonate flux of isolatedlections were made. In experiments designed to deter-

perfused TALs. As shown in Figure 1, bicarbonate fluxmine bicarbonate flux after stimulation of endogenous

during the control period was 35.4 6 4.6 pmol · mm21 ·NO production, l-Arg was present in the bath duringmin21. After l-Arg 0.5 mmol/L was added to the bath,the initial period and was later removed to allow thethe tubules absorbed bicarbonate at a rate of 23.2 6 2.9tubules to recover. Bicarbonate concentration in the per-pmol · mm21 · min21. Thus, the presence of 0.5 mmol/Lfusate and collected fluid was measured by microfluor-l-Arg in the bath significantly decreased TAL bicarbon-ometry [14]. All data were recorded and stored usingate transport by 36.9 6 11.6% (N 5 7, P , 0.025).data acquisition software (DATAQ Instruments, Akron,

To determine whether the inhibition of bicarbonateOH, USA). Because the TAL does not reabsorb water,transport that we observed after the addition of l-Argbicarbonate absorption (JHCO3

2) was calculated as follows:was stereo specific, we evaluated the effect of the isomer

J 2HCO3 5 CR (C 2

o HCO3 2 C 2l HCO3 ) d-Arg. During the control period, the bicarbonate reab-

sorption rate was 35.0 6 4.0 pmol · mm21 · min21. Afterwhere CR is the collection rate normalized per tubulelength; Co HCO3

2 is the bicarbonate concentration in the treatment with 0.5 mmol/L d-Arg, it was 37.4 6 4.8 pmol ·

Ortiz and Garvin: NO and TAL bicarbonate flux 2071

Fig. 3. Effect of 0.5 mmol/L L-Arg on TAL JHCO32 during NO synthesisFig. 2. Effect of D-Arg on rat TAL JHCO3

2. Incubation of perfused TALsblockade. In tubules pretreated with 5 mmol/L L-NAME, the additionwith 0.5 mmol/L d-Arg did not affect bicarbonate absorption comparedof 0.5 mmol/L l-Arg did not alter bicarbonate absorption (N 5 7).with control (35.0 6 4.0 vs. 37.4 6 4.8 pmol · mm21 · min21, N 5 7).

mm21 · min21 (Fig. 2). The lack of effect of d-Arg onTAL JHCO3

2 indicated that the inhibitory effect observedafter l-Arg treatment is specific for the l-isomer.

Next, we tested whether L-NAME is capable ofblocking the previously observed inhibitory effect ofl-Arg on TAL bicarbonate transport. During theL-NAME incubation period, bicarbonate reabsorptionrate averaged 43.4 6 4.4 pmol · mm21 · min21. Similarly,after coincubation of the tubules with 0.5 mmol/L l-Arg,net bicarbonate absorption was 44.6 6 5.0 pmol · mm21 ·min21 (Fig. 3). This indicates that the effect of l-Arg onTAL JHCO3

2 is mediated by induction of NO synthesis,ruling out the possibility that l-Arg may have an effecteither by itself or by modifying other pathways. In theabsence of l-Arg, 5 mmol/L L-NAME did not modifyTAL JHCO3

2 (N 5 7, P . 0.23), indicating that in theabsence of exogenous l-Arg, no NO is produced.

Since it has been reported that high NO concentrationsmay lead to cell damage through a variety of mecha-nisms, we tested the ability of the tubules to increasebicarbonate transport after endogenous NO productionceased. During the initial period, TALs treated with 0.5mmol/L l-Arg had an average JHCO3

2 of 26.2 6 3.9 pmol ·mm21 · min21. After l-Arg was removed from the bathand tubules were allowed to recover for at least 30 min- Fig. 4. Bicarbonate flux recovery. Removal of 0.5 mmol/L l-Arg from

the basolateral bath generated a significant increment in TAL JHCO32.utes, bicarbonate transport rates significantly increased

After the tubules recovered from l-Arg treatment, bicarbonate absorp-to 34.8 6 3.2 pmol · mm21 · min21 (P , 0.01; Fig. 4). tion rose from 26.2 6 3.9 to 34.8 6 3.2 pmol · mm21 · min21. *P , 0.01,N 5 7.These data demonstrate that the inhibition of bicarbon-

Ortiz and Garvin: NO and TAL bicarbonate flux2072

Fig. 5. Effect of cGMP analogues on TAL JHCO32. Adding the cGMP Fig. 6. Effect of 0.5 mmol/L L-Arg on TAL JHCO3

2 during protein kinaseanalogues 8-Br-cGMP 50 mmol/L (d) and dibutyryl-cGMP 50 mmol/L G blockade. In tubules pretreated with 2 mmol/L KT-5823, the addition(s) to the bath significantly decreased bicarbonate absorption from of 0.5 mmol/L l-Arg did not alter bicarbonate absorption (N 5 5).24.8 6 2.1 to 16.9 6 1.7 pmol · mm21 · min21 (N 5 10). *P , 0.05.

TAL JHCO32 (17.8 6 1.4 vs. 19.6 6 2.4 pmol · mm21 ·

ate transport observed after l-Arg treatment was not min21, N 5 5). KT-5823 alone did not change basal JHCO32.

due to cytotoxic effects of NO on TAL cells. These results indicate that PKG activation mediates thePrevious work from our laboratory has shown that effects of l-Arg on TAL JHCO3

2.NO increased the intracellular cGMP content in TAL[15] and CCD [16]. In CCDs, NO also activated cGMP-

DISCUSSIONdependent protein kinase. We hypothesized that stimula-tion of protein kinase with cell permeant cGMP ana- Our findings demonstrate that l-Arg at 0.5 mmol/Llogues would affect TAL JHCO3

2 in a manner similar to inhibits bicarbonate transport in isolated perfused ratl-Arg. Figure 5 shows that incubation of the tubules TALs whereas d-Arg has no effect. Incubating the tu-with cGMP analogues at a concentration of 50 mmol/L bules with 5 mmol/L L-NAME blocked the inhibitorydecreased TAL JHCO3

2 from 24.8 6 2.1 to 16.9 6 1.7 effect of l-Arg on TAL bicarbonate transport whereaspmol · mm21 · min21 (N 5 10, P , 0.05). Because there L-NAME alone had no effect, indicating that inhibitionwas no difference in the effects of 8-Br-cGMP and dibu- of transport was due to endogenous NO production.tyryl-cGMP, the data were pooled. These results indicate cGMP appears to mediate the effect of NO, becausethat cGMP has an inhibitory effect on TAL bicarbonate cGMP analogues inhibited bicarbonate transport, whereasabsorption that mimics treatment with 0.5 mmol/L l-Arg. in the presence of cGMP l-Arg did not have any effect.

To investigate whether all of the effects of l-Arg are Incubating the tubules with KT-5823 blocked the inhibi-mediated by cGMP, we performed a series of experi- tory effect of l-Arg on TAL bicarbonate transport whilements in which TALs were incubated with cGMP and KT-5823 alone had no effect, indicating that inhibitionthen added l-Arg. In the presence of db-cGMP (50 of transport was mediated by protein kinase G activation.mmol/L), JHCO3

2 was 22.2 6 1.8 pmol · mm21 · min21, and The TAL plays an important role in the maintenanceafter l-Arg (0.5 mmol/L) was added to the bath, JHCO3

2 of acid/base balance, since it is capable of recoveringwas 21.5 6 2.1 pmol · mm21 · min21. Thus, all of the almost all of the bicarbonate that escapes proximal tu-effects of l-Arg are mediated by cGMP. bule reabsorption [9]. Bicarbonate reabsorption occurs

Finally, we tested whether the protein kinase G (PKG) because of secretion of protons via apical Na1/H1 ex-inhibitor KT-5823 could block the effects of l-Arg. Fig- changers and subsequent dehydration of H2CO3 by lumi-ure 6 shows that in the presence of KT-5823 (2 mmol/L), nal carbonic anhydrase [9, 17]. We recently reported

that the NO donors’ spermine-nonoate and nitroglycerinadding l-Arg (0.5 mmol/L) to the bath did not change

Ortiz and Garvin: NO and TAL bicarbonate flux 2073

inhibited apical and basolateral Na1/H1 exchanger activ- and Wang, who also reported NO-dependent stimulationity in isolated perfused TALs [10]. The present data of transport, found that the effect of NO on K1 channelssuggest that endogenous NO inhibits bicarbonate reab- was dependent on the presence of superoxide anionsorption, probably by modifying Na1/H1 exchanger ac- (O2

2 ) [28]. Older animals are known to have higher levelstivity. Thus, NO not only influences sodium entry but of oxidative stress than younger ones, and it is possiblealso modifies the amount of protons and bicarbonate that the amount of free radicals being produced mayexcreted. Accordingly, Roczniak and Burns showed that influence the response to NO. Second, the diets differedextracellular NO inhibited amiloride-sensitive sodium in our study and that of Good et al. Differences in dietuptake in rabbit proximal tubules [5], and Tojo et al can modify the hormonal state of animals and thereforefound that NO may decrease H1 excretion in the CCD modify the response to different factors.by inhibiting bafilomycin-sensitive H1-ATPase [6]. The All three NOS isoforms have been reported to beproposed role for NO in regulating acid/base balance is present in the TAL. Morrissey et al reported that thein agreement with the reported inhibition of gastric H1 mTAL contains large amounts of iNOS mRNA undersecretion in rats by NO [18] and the inhibitory effects basal conditions in comparison with other nephron seg-of NO on H1-ATPases in peritoneal macrophages [19]. ments [29]. Ujiie et al found reverse transcription-poly-Despite these findings, which clearly indicate a role for merase chain reaction products corresponding to endo-NO in acid/base balance, we know of no investigations thelial NOS in rat TALs [30], and Bachmann, Bosse,of the effects of NO on blood pH or urinary excretion and Mundel reported that immunohistochemical analysisof protons, bicarbonate, and ammonia. revealed constitutive NOS in this segment [31]. Although

The inhibitory effect of NO on JHCO32 observed in our there is no doubt about the presence of NOS enzymes,

study may be due to direct inhibition of apical Na1/H1it is still not known which isoform mediates the observed

exchanger activity, but it is also possible that by decreas- effect on TAL JHCO32. Observations from our laboratory

ing basolateral Na1,K1-ATPase activity, NO diminishes demonstrated that the endothelial NOS isoform is re-the electrochemical gradient across the membrane. We sponsible for l-Arg–induced inhibition of chloride fluxhave reported that NO inhibited apical and basolateral in the mouse TAL [32]. These data strongly suggestNa1/H1 exchange to different degrees, indicating a direct that the inhibitory effect of NO reported in this work iseffect on the exchanger rather than the sodium pump mediated by the endothelial NOS isoform.[10]. In addition, our laboratory has previously reported Although there is evidence concerning the intracellu-that NO did not affect the sodium pump activity in per- lar mechanism responsible for NO actions in the CCDfused CCDs [20]. Despite these findings, some data indi- [16], little is known about how NO inhibits TAL trans-cate an effect of NO on Na1,K1-ATPase activity. Guz-

port. This study provides evidence that the inhibitoryman et al measured inhibition of sodium pump activity

actions of NO on bicarbonate reabsorption are mediatedby NO in cultured proximal tubule cells [21]. Recently,by activation of protein kinase G. The agents 8-Br-cGMPLiang and Knox reported that in opossum kidney cells,and dibutyryl-cGMP mimicked the l-Arg–induced re-NO donors inhibited Na1,K1-ATPase activity withoutduction in TAL net bicarbonate flux. Also, inhibition ofaffecting the amount of enzyme present in the membraneprotein kinase G with KT-5823 completely blocked the[22]. Thus, further studies are needed to resolve thiseffects of l-Arg on TAL bicarbonate transport. Since itmatter.is known that PKG can also be activated by increasesIn accord with the reported in vivo effects of NO,in cAMP concentration [33], our study does not rulemost studies have shown that NO inhibits transport inout the possibility that activation of the cAMP pathwaythe kidney. However, some reports suggest that NO stim-would also inhibit TAL transport as reported by Goodulates transport [23]. In contrast with our data, Good,[26]. Carefully conducted studies are needed in order toGeorge, and Wang reported a stimulatory effect ofclarify the main intracellular cascade responsible for thel-Arg and NO donors in TAL bicarbonate transportinhibitory effects of NO on TAL transport.[24]. We know of no explanation for these disparate

In summary, we found that stimulation of NO produc-results. However, the experimental designs differ in twotion in isolated perfused TALs caused a decrease inways. First, our animals were older by two to three weeks.bicarbonate reabsorption rates, that increases in cGMPNucleotide levels may vary with age in the TAL, as itcan account for all of the effects of NO, and the increasehas been reported for resting cAMP levels in the proxi-in cGMP activates PKG, which causes the decrease inmal tubule [25]. cAMP has been previously shown toTAL JHCO3

2.regulate Na1/H1 exchange in the TAL [26]. Addition-ally, age may alter the response to hormones and factors

ACKNOWLEDGMENTsuch as NO. In the proximal tubule, the response ofNa1/H1 exchanger 3 (NHE3) to dopamine is age depen- This work was supported in part by a grant from the National

Institutes of Health (HL-28982).dent [27], as is the response to angiotensin II [25]. Lu

Ortiz and Garvin: NO and TAL bicarbonate flux2074

Reprint requests to Jeffrey L. Garvin, Ph.D., Hypertension and Vas- 17. Good DW: Adaptation of HCO32 and NH1

4 transport in rat MTAL:Effects of chronic metabolic acidosis and Na1 intake. Am J Physiolcular Research Division, Henry Ford Hospital, 2799 West Grand Boule-258:F1345–F1353, 1990vard, Detroit, Michigan 48202, USA.

18. Kato S, Kitamura M, Korolkiewicz RP, Takeuchi K: Role ofE-mail: jgarvin1@hfhs.orgnitric oxide in regulation of gastric acid secretion in rats: Effectsof NO donors and NO synthase inhibitor. Br J Pharmacol 123:839–

REFERENCES 846, 199819. Swallow CJ, Grinstein S, Sudsbury RA, Rotstein OD: Nitric

1. Kone BC, Baylis C: Biosynthesis and homeostatic roles of nitric oxide derived from l-Arg impairs cytoplasmic pH regulation byoxide in the normal kidney. Am J Physiol 272:F561–F578, 1997 vacuolar-type H1 ATPases in peritoneal macrophages. J Exp Med

2. Lahera V, Salom MG, Miranda-Guardiola F, Moncada S, Ro- 174:1009–1021, 1991mero JC: Effects of NG-nitro-l-arginine methyl ester on renal func- 20. Garcia NH, Pomposiello S, Garvin JL: Nitric oxide inhibits ADH-tion and blood pressure. Am J Physiol 261:F1033–F1037, 1991 stimulated osmotic water permeability in cortical collecting ducts.

3. Majid DSA, Williams A, Navar LG: Inhibition of nitric oxide Am J Physiol 270:F206–F210, 1996synthesis attenuates pressure-induced natriuretic responses in an- 21. Guzman NJ, Fang M, Tang S, Ingelfinger JR, Garg LC: Auto-esthetized dogs. Am J Physiol 264:F79–F87, 1993 crine inhibition of Na1/K1ATPase by nitric oxide in mouse proxi-

4. Wang T: Nitric oxide regulates HCO32 and Na1 transport by mal tubule epithelial cells. J Clin Invest 95:2083–2088, 1995

cGMP-mediated mechanism in the kidney proximal tubule. Am J 22. Liang M, Knox FG: Nitric oxide reduces the molecular activityPhysiol 272:F242–F248, 1997 of Na1,K1-ATPase in opossum kidney cells. Kidney Int 56:627–634,

5. Roczniak A, Burns KD: Nitric oxide stimulates guanylate cyclase 1999and regulates sodium transport in rabbit proximal tubules. Am J 23. Lu M, Giebisch G, Wang W: Nitric oxide-induced hyperpolariza-Physiol 270:F106–F115, 1996 tion stimulates low-conductance sodium channel of rat CCD. Am

6. Tojo A, Guzman NJ, Garg LC, Tisher CC, Madsen KM: Nitric J Physiol 272:F498–F504, 1997oxide inhibits bafilomycin-sensitive H1-ATPase activity in rat cor- 24. Good DW, George T, Wang DH: Angiotensin II inhibits HCO3

2

tical collecting duct. Am J Physiol 267:F509–F515, 1994 absorption via a cytochrome P-450-dependent pathway in MTAL.7. Capasso G, Unwin R, Siani F, De Santo NG, De Tommaso G, Am J Physiol 276:F726–F736, 1999

Russo F, Giebisch G: Bicarbonate transport along the loop of 25. Garvin JL, Beierwaltes WH: Response of proximal tubules toHenle. II. Effect of acid-base dietary and neurohumoral determi- angiotensin II changes during maturation. Hypertension 31:415–nants. J Clin Invest 94:830–838, 1994 420, 1998

26. Good DW: Inhibition of bicarbonate absorption by peptide hor-8. Garvin JL, Burg MB, Knepper MA: Active NH14 absorption by

mones and cyclic adenosine monophosphate in rat medullary thickthe thick ascending limb. Am J Physiol 255:F57–F65, 1988ascending limb. J Clin Invest 85:1006–1013, 19909. Good DW: The thick ascending limb as a site of renal bicarbonate

27. Xili X, Albrecht FE, Robillard JE, Eisner GM, Jose PA: Gbreabsorption. Semin Nephrol 13:225–235, 1993regulation of Na/H exchanger-3 activity in rat renal proximal tu-10. Garvin JL, Hong NJ: Nitric oxide inhibits sodium/hydrogen ex-bules during development. Am J Physiol 278:R931–R936, 2000change activity in the thick ascending limb. Am J Physiol 277:F377–

28. Lu M, Wang WH: Reaction of nitric oxide with superoxide inhibitsF382, 1999basolateral K1 channels in the rat CCD. Am J Physiol 275:C309–11. Plato CF, Stoos BA, Wang D, Garvin JL: Endogenous nitricC316, 1998oxide inhibits chloride transport in the thick ascending limb. Am

29. Morrissey JJ, McCracken R, Kaneto H, Vehaskari M, MontaniJ Physiol 276:F159–F163, 1999D, Klahr S: Location of an inducible nitric oxide synthase mRNA12. Castillo L, Sanchez M, Vogt J, Chapman TE, Derojas-Walkerin the normal kidney. Kidney Int 45:998–1005, 1994TC, Tannenbaum SR, Ajami AM, Young VR: Plasma arginine,

30. Ujiie K, Yuen J, Hogarth L, Danziger R, Star RA: Localizationcitrulline, and ornithine kinetics in adults, with observations on and regulation of endothelial NO synthase mRNA expression innitric oxide synthesis. Am J Physiol 268:E360–E367, 1995 rat kidney. Am J Physiol 267:F296–F302, 199413. Silbernagl S, Volker K, Dantzler WH: Compartmentation of 31. Bachmann S, Bosse HM, Mundel P: Topography of nitric oxide

amino acids in the rat kidney. Am J Physiol 270:F154–F163, 1996 synthesis by localizing constitutive NO synthases in mammalian14. Star RA: Quantitation of total carbon dioxide in nanoliter samples kidney. Am J Physiol 268:F885–F898, 1995

by flow-through fluorometry. Am J Physiol 258:F429–F432, 1990 32. Plato CF, Shesely EG, Garvin JL: eNOS mediates L-arg-induced15. Garcia NH, Plato CF, Stoos BA, Garvin JL: Nitric oxide-induced inhibition of thick ascending limb chloride flux. Hypertension 35:

inhibition of transport by thick ascending limbs from Dahl salt- 319–323, 2000sensitive rats. Hypertension 34:508–513, 1999 33. Lincoln TM, Corbin JD: Characterization and biological role of

16. Garcia NH, Stoos BA, Carretero OA, Garvin JL: Mechanism the cGMP-dependent protein kinase, in Advances in Cyclic Nucleo-of the nitric oxide-induced blockade of collecting duct water per- tide Research (vol 15), edited by Greengard P, Robinson GA,meability. Hypertension 27:679–683, 1996 New York, Raven Press, 1983, pp 139–192