Macula densa nitric oxide synthase: Expression, regulation, and function
Authors: Christopher S. Wilcox, William J. Welch
Journal: Kidney International (1998)

Macula densa nitric oxide synthase: Expression, regulation, and function. The type 1 brain nitric oxide synthase (bNOS) isoform occurs in macula densa (MD) cells where it functions to vasodilate the afferent arteriole and blunt expression of tubuloglomerular feedback (TGF). Dietary salt restriction enhances bNOS expression, yet microperfusion studies with NOS inhibitors imply that it is functionally inactive. We thus assessed the hypothesis that reduced l-arginine (L-Arg) availability during low salt (LS) intake limits MD NO generation. Maximal TGF responses were recorded during Henle's loop perfusion with artificial tubular fluid (ATF). Microperfusion of L-Arg into the MD of LS, but not normal or high-salt (HS) rats blunted maximal TGF responses (8.0 ± 0.4 to 6.0 ± 0.5 mm Hg; N = 23; P < 0.01). Response to L-Arg was stereospecific, inhibited by coperfusion with monomethyl-l-arginine (L-NMA), and dependent on system y+ transport, because it was blocked by coperfusion with the competitors l-lysine or l-homoarginine. Absorption of [3H]-L-Arg from the perfused loop, via an L-Arg— or l-homoarginine—inhibitable process, was enhanced during HS. Salt restriction thus diminishes TGF attenuation by NO in the MD despite enhanced bNOS expression because of limited delivery and/or uptake of L-Arg via system y+. This defines a novel mechanism of renal microcirculatory adaptation to salt restriction via L-Arg—dependent changes in TGF. Macula densa nitric oxide synthase: Expression, regulation, and function. The type 1 brain nitric oxide synthase (bNOS) isoform occurs in macula densa (MD) cells where it functions to vasodilate the afferent arteriole and blunt expression of tubuloglomerular feedback (TGF). Dietary salt restriction enhances bNOS expression, yet microperfusion studies with NOS inhibitors imply that it is functionally inactive. We thus assessed the hypothesis that reduced l-arginine (L-Arg) availability during low salt (LS) intake limits MD NO generation. Maximal TGF responses were recorded during Henle's loop perfusion with artificial tubular fluid (ATF). Microperfusion of L-Arg into the MD of LS, but not normal or high-salt (HS) rats blunted maximal TGF responses (8.0 ± 0.4 to 6.0 ± 0.5 mm Hg; N = 23; P < 0.01). Response to L-Arg was stereospecific, inhibited by coperfusion with monomethyl-l-arginine (L-NMA), and dependent on system y+ transport, because it was blocked by coperfusion with the competitors l-lysine or l-homoarginine. Absorption of [3H]-L-Arg from the perfused loop, via an L-Arg— or l-homoarginine—inhibitable process, was enhanced during HS. Salt restriction thus diminishes TGF attenuation by NO in the MD despite enhanced bNOS expression because of limited delivery and/or uptake of L-Arg via system y+. This defines a novel mechanism of renal microcirculatory adaptation to salt restriction via L-Arg—dependent changes in TGF. artificial tubular fluid brain nitric oxide synthase high salt juxtaglomerular apparatus loop of Henle l-arginine monomethyl-l-arginine nitro-L-Arg methyl ester low salt macula densa nitric oxide nitric oxide synthase glomerular capillary hydraulic pressure proximal stop-flow pressure renin-angiotensin system single-nephron glomerular filtration rate thick ascending limb tubuloglomerular feedback In pioneering experiments, Thurau and Schnermann showed that increasing Na reabsorption at the macula densa (MD) led to a fall in single-nephron glomerular filtration rate (SNGFR)1.Thurau K. Schnermann J. Die Natriumkonzentration an den Macula densa Zellen als regulierender Faktor für das Glomerulumfiltrat (Mikropunktionsversuche).Klin Wochenschr. 1965; 43: 410-413Crossref PubMed Scopus (183) Google Scholar that they termed tubuloglomerular feedback (TGF). This response entails vasoconstriction of the afferent arteriole, but the constrictor mediators remain to be defined precisely2.Briggs J.P. Schnermann J. Control of renin release and glomerular vascular tone by the juxtaglomerular apparatus,.in: Laragh J.H. Brenner B.M. Hypertension: Pathophysiology, diagnosis, and management. Raven, New York1995: 1359Google Scholar. Wilcox et al have proposed recently that nitric oxide (NO) generated in the MD by l-arginine (L-Arg) metabolism via NO synthase (NOS) is a negative mediator blunting expression of TGF responses3.Wilcox C.S. Welch W.J. Murad F. Gross S.S. Taylor G. Levi R. Schmidt H.H. Nitric oxide synthase in macula densa regulates glomerular capillary pressure.Proc Natl Acad Sci USA. 1992; 89: 11993-11997Crossref PubMed Scopus (464) Google Scholar. Three or more NOS isoforms are expressed in the normal rat kidney4.Mohaupt M.G. Madsen K.M. Wilcox C.S. Kone B.C. Molecular characterization and localization of three nitric oxide synthase isoforms in the rat kidney.Endothelium-J Endothel Cell Res: Third International Meeting Biology of Nitric Oxide. 1993: S25Google Scholar. Brain-type NOS (bNOS) is expressed densely in MD cells3.Wilcox C.S. Welch W.J. Murad F. Gross S.S. Taylor G. Levi R. Schmidt H.H. Nitric oxide synthase in macula densa regulates glomerular capillary pressure.Proc Natl Acad Sci USA. 1992; 89: 11993-11997Crossref PubMed Scopus (464) Google Scholar, 5.Tojo A. Zhang L. Wilcox C.S. Welch W.J. Tisher C.C. Madsen K.M. Gross S.S. Schmidt H.H.H.W. Differential localization and regulation of nitric oxide synthase isoforms in the juxtaglomerular apparatus.J Am Soc Nephrol. 1992; 3 (abstract): 554Google Scholar, 6.Bachmann S. Bosse H.M. Mundel P. Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney.Am J Physiol. 1995; 268: F885-F898PubMed Google Scholar. bNOS is expressed additionally in some adjacent thick ascending limb (TAL) cells, cells of Bowman's capsule, some intrarenal nerves, and tubular structures5.Tojo A. Zhang L. Wilcox C.S. Welch W.J. Tisher C.C. Madsen K.M. Gross S.S. Schmidt H.H.H.W. Differential localization and regulation of nitric oxide synthase isoforms in the juxtaglomerular apparatus.J Am Soc Nephrol. 1992; 3 (abstract): 554Google Scholar,6.Bachmann S. Bosse H.M. Mundel P. Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney.Am J Physiol. 1995; 268: F885-F898PubMed Google Scholar. Both bNOS mRNA4.Mohaupt M.G. Madsen K.M. Wilcox C.S. Kone B.C. Molecular characterization and localization of three nitric oxide synthase isoforms in the rat kidney.Endothelium-J Endothel Cell Res: Third International Meeting Biology of Nitric Oxide. 1993: S25Google Scholar, 6.Bachmann S. Bosse H.M. Mundel P. Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney.Am J Physiol. 1995; 268: F885-F898PubMed Google Scholar, 7.Bosse H.M. Bohm R. Resch S. Bachmann S. Parallel regulation of constitutive NO synthase and renin at JGA of rat kidney under various stimuli.Am J Physiol. 1995; 269: F793-F805PubMed Google Scholar, 8.Singh I. Grams M. Wang W. Yang T. Killen P. Smart A. Schnermann J. Briggs J.P. Coordinate regulation of renal expression of nitric oxide synthase, renin, and angiotensinogen mRNA by dietary salt.Am J Physiol. 1996; 270: F1027-F1037PubMed Google Scholar, 9.Terada Y. Tomita K. Nonoguchi H. Marumo F. Polymerase chain reaction localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger RNAs in microdissected rat nephron segments.J Clin Invest. 1992; 90: 659-665Crossref PubMed Scopus (241) Google Scholar and nicotinamide adenine dinucleotide phosphate diaphorase3.Wilcox C.S. Welch W.J. Murad F. Gross S.S. Taylor G. Levi R. Schmidt H.H. Nitric oxide synthase in macula densa regulates glomerular capillary pressure.Proc Natl Acad Sci USA. 1992; 89: 11993-11997Crossref PubMed Scopus (464) Google Scholar are also expressed in the MD. MD solute absorption appears to activate NOS and generate NO that blunts the expression of TGF-induced reductions in glomerular capillary hydraulic pressure (PGC). Thus, microperfusion of NG-methyl-L-Arg (L-NMA) into the lumen of the MD or adjacent interstitium via the peritubular capillaries enhances the TGF response in normal rats3.Wilcox C.S. Welch W.J. Murad F. Gross S.S. Taylor G. Levi R. Schmidt H.H. Nitric oxide synthase in macula densa regulates glomerular capillary pressure.Proc Natl Acad Sci USA. 1992; 89: 11993-11997Crossref PubMed Scopus (464) Google Scholar. This effect of L-NMA is stereospecific and is prevented by coperfusion with excess L-Arg (demonstrating dependency on NOS) and by microperfusion of furosemide and thus depends on MD solute reabsorption. Studies with L-NMA or the relatively bNOS-specific inhibitor 7-nitroindazole have confirmed that blockade of MD NOS enhances rat TGF responses10.Welch W.J. Wilcox C.S. Role of nitric oxide in tubuloglomerular feedback: Effects of salt intake.Clin Exp Physiol Pharmacol. 1997; 24: 582-586Crossref PubMed Scopus (37) Google Scholar. In a perfused rat juxtaglomerular apparatus (JGA) preparation, microperfusion of the MD by nitro-L-Arg methyl ester (L-NAME) leads to graded constriction of the adjacent afferent arteriole11.Ito S. Ren Y. Evidence for the role of nitric oxide in macula densa control of glomerular hemodynamics.J Clin Invest. 1993; 92: 1093-1098Crossref PubMed Scopus (168) Google Scholar. This response is dependent on MD solute absorption, as it is prevented by MD perfusion with a low NaCl solution. These studies directly implicate MD NOS in this response because during MD microperfusion of L-NAME, the vasodilator response to intraarterial infusion of acetylcholine, which activates endothelial NO generation, is preserved. MD microperfusion with L-Arg in normal rats blunts TGF12.Thorup C. Persson A.E.G. Macula densa-derived nitric oxide in regulation of glomerular capillary pressure.Kidney Int. 1996; 49: 430-436Abstract Full Text PDF PubMed Scopus (115) Google Scholar. Collectively, these studies demonstrate a powerful MD-afferent arteriolar signaling pathway dependent on NO, which vasodilates the afferent arteriole and blunts the expression of TGF. MD-derived NO might also blunt the generation of the signal initiating TGF, as NO, or its second messenger cGMP, can inhibit TAL solute reabsorption13.Bailly C. Neant F. Amiel C. Cyclic GMP mediates the inhibition by platelet-activating factor of Cl reabsorption in the cTAL.J Am Soc Nephrol. 1992; 4 (abstract): 450Google Scholar. TGF responses are enhanced by dietary salt restriction or activation of the renin-angiotensin system (RAS)2.Briggs J.P. Schnermann J. Control of renin release and glomerular vascular tone by the juxtaglomerular apparatus,.in: Laragh J.H. Brenner B.M. Hypertension: Pathophysiology, diagnosis, and management. Raven, New York1995: 1359Google Scholar,14.Thurau K. Renal hemodynamics.Am J Med. 1964; 36: 698-719Abstract Full Text PDF PubMed Scopus (250) Google Scholar. The immunocytochemical expression of bNOS in the MD is enhanced by dietary salt restriction15.Tojo A. Madsen K.M. Wilcox C.S. Expression of immunoreactive nitric oxide synthase isoforms in rat kidney: Effects of dietary salt and losartan.Jpn Heart J. 1995; 36: 389-398Crossref PubMed Scopus (66) Google Scholar. This effect is dependent partly on enhanced RAS activity, because it could be blunted by the angiotensin II type 1 (AT1) receptor antagonist, losartan, in low-salt rats15.Tojo A. Madsen K.M. Wilcox C.S. Expression of immunoreactive nitric oxide synthase isoforms in rat kidney: Effects of dietary salt and losartan.Jpn Heart J. 1995; 36: 389-398Crossref PubMed Scopus (66) Google Scholar. Studies of microdissected vascular poles containing intact MD segments have shown enhanced mRNA expression for bNOS in rats adapted to low salt intake that parallels increases in afferent arteriolar renin gene expression7.Bosse H.M. Bohm R. Resch S. Bachmann S. Parallel regulation of constitutive NO synthase and renin at JGA of rat kidney under various stimuli.Am J Physiol. 1995; 269: F793-F805PubMed Google Scholar,8.Singh I. Grams M. Wang W. Yang T. Killen P. Smart A. Schnermann J. Briggs J.P. Coordinate regulation of renal expression of nitric oxide synthase, renin, and angiotensinogen mRNA by dietary salt.Am J Physiol. 1996; 270: F1027-F1037PubMed Google Scholar. Despite this clear evidence for enhanced expression of bNOS mRNA and protein in the MD during salt restriction, results from functional studies suggest that the MD-afferent arteriolar NO signaling pathway is inactive in salt-restricted rats. Thus, microperfusion of L-NMA into the MD of salt-restricted rats fails to modify TGF, whereas similar microperfusions in rats adapted to normal or high salt intake regularly enhance the response10.Welch W.J. Wilcox C.S. Role of nitric oxide in tubuloglomerular feedback: Effects of salt intake.Clin Exp Physiol Pharmacol. 1997; 24: 582-586Crossref PubMed Scopus (37) Google Scholar,16.Wilcox C.S. Welch W.J. TGF and nitric oxide: Effects of salt intake and salt-sensitive hypertension.Kidney Int. 1996; 49: S9-S13PubMed Google Scholar. These studies were designed to examine the hypothesis that delivery and/or uptake of L-Arg at the MD limits the blunting of TGF by NO generation during salt restriction. L-Arg is transported across cell membranes by Na-dependent and -independent pathways, the latter including system y+, for which L-Arg competes with l-ornithine, l-lysine, and l-homoarginine17.Schmidt H.H.H.W. Baeblich S.E. Zernikow B.C. Klein M.M. Bohme E. L-arginine and arginine analogues: Effects on isolated blood vessels and cultured endothelial cells.Br J Pharmacol. 1990; 101: 145-151Crossref PubMed Scopus (30) Google Scholar, 18.Schmidt K. Klatt P. Mayer B. Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors.Mol Pharmacol. 1993; 44: 615-621PubMed Google Scholar, 19.Schmidt K. List B.M. Klatt P. Mayer B. Characterization of neuronal amino acid transporters: Uptake of nitric oxide synthase inhibitors and implication for their biological effects.J Neurochem. 1995; 64: 1469-1475Crossref PubMed Scopus (44) Google Scholar. L-Arg is transported both into and out of the TAL by a saturable, carrier-mediated process20.Dantzler W.H. Silbernagl S. Amino acid transport by juxtamedullary nephrons: Distal reabsorption and recycling.Am J Physiol. 1988; 255: F397-F407PubMed Google Scholar,21.Silbernagl S. Volker K. Dantzler W.H. Cationic amino acid fluxes beyond the proximal convoluted tubule of rat kidney.Pflügers Arch. 1994; 429: 210-215Crossref PubMed Scopus (11) Google Scholar. Male Sprague-Dawley rats (220 to 300 g) were fed identical diets (Teklad, Madison, WI, USA) with a low salt (LS; Na content 0.03 g/100 g) or high salt (HS; Na content 2.3 g/100 g) content for 8 to 12 days prior to testing. The LS diet is sufficient for normal growth over this period. Rats were prepared for micropuncture, microperfusion, and TGF studies, as described in detail previously22.Welch W.J. Wilcox C.S. Potentiation of tubuloglomerular feedback in the rat by thromboxane mimetic: Role of macula densa.J Clin Invest. 1992; 89: 1857-1865Crossref PubMed Scopus (71) Google Scholar. In brief, the TGF response was assessed from changes in proximal stop-flow pressure (PSF) recorded via an ultramicropipette inserted in a proximal tubule upstream from a wax block and connected to a servo-null micropressure recorder (IPS, La Jolla, CA, USA). TGF was activated maximally by microperfusion of artificial tubular fluid (ATF) containing vehicle or drugs from the end proximal tubule into the loop segment at 40 nl/min. Responses of HS and LS rats were compared in the following protocols: TGF responses were contrasted in nephrons of LS rats (N = 23) or HS rats (N = 19) during microperfusion of ATF plus vehicle or ATF plus L-Arg (10-3m) perfused alone. Additional studies were undertaken in nephrons perfused with l-lysine (2 × 10-3m) or l-homoarginine (2 × 10-3m) perfused alone or with L-Arg as competitive inhibitors of system y+ transport. ATF containing [3H]-L-Arg (15 cpm/nl; Amersham, Arlington Heights, IL, USA) and [14C]-inulin (15 cpm/nl; ICN, Costa Mesa, CA, USA) as a recollection marker was microperfused from a precisely calibrated microperfusion pump into the late proximal tubule and collected upstream from an oil block at the early distal tubule. Perfusions were delivered at 20 nl/min, and collections were made over 2-minute periods. To the perfusates were added vehicle, L-Arg (1 mm), or l-homoarginine (1 mm). Aliquots of the perfusate and the collectate were counted in scintillation vials. Data were analyzed by analysis of variance, and results were considered statistically significant at P < 0.05. Data are presented as mean ± sem. There were no significant differences in body wt, kidney weight, or blood pressure in HS and LS rats. Moreover, PSF during zero loop perfusion was similar (LS, 36.1 ± 0.6 vs. HS, 37.2 ± 0.8 mm Hg; N = 24; NS). Therefore, TGF responses are presented as changes in PSF during LH perfusion at 40 nl/min compared with zero perfusions. During LH perfusion with ATF + vehicle, maximal TGF responses, assessed from changes in PSF, were enhanced in nephrons of salt-restricted rats (LS, 8.1 ± 0.4 vs. HS, 6.6 ± 0.4 mm Hg; P < 0.01). For series 1, the addition of L-Arg to ATF perfusates blunted the maximal TGF responses in nephrons of LS rats Figure 1a. Microperfusion of l-lysine or l-homoarginine did not alter TGF responses when given alone but, when coperfused with L-Arg, blocked the effects of L-Arg in blunting TGF responses. In contrast, L-Arg did not significantly change TGF responses in nephrons of HS rats Figure 1b. l-Lysine or l-homoarginine had no effects whether microperfused alone or with L-Arg. We conclude that L-Arg delivery and uptake from the tubular lumen via system y+ blunts TGF specifically in nephrons of salt-restricted rats. For series 2, 90% to 110% of the [14C]-inulin microperfused from the late proximal tubule was collected at the early distal tubule in each nephron. Absorption of [3H]-L-Arg from the perfused LH averaged 92% ± 3% and did not differ between LS and HS rats. However, the mechanism of absorption was strongly salt dependent. Addition of excess L-Arg significantly (P < 0.01) blunted [3H]-L-Arg absorption, but this effect was significantly (P < 0.01) greater in HS than in LS rats. Likewise, addition of l-homoarginine significantly (P < 0.01) blunted [3H]-L-Arg absorption. This effect also was significantly (P < 0.05) greater in HS than in LS rats. We conclude that dietary salt restriction diminishes the saturable y+ system reabsorptive flux of L-Arg from the perfused LH. The finding that bNOS is heavily expressed in the MD during dietary salt restriction15.Tojo A. Madsen K.M. Wilcox C.S. Expression of immunoreactive nitric oxide synthase isoforms in rat kidney: Effects of dietary salt and losartan.Jpn Heart J. 1995; 36: 389-398Crossref PubMed Scopus (66) Google Scholar, but apparently does not function to produce NO, because TGF is unaltered during NOS blockade10.Welch W.J. Wilcox C.S. Role of nitric oxide in tubuloglomerular feedback: Effects of salt intake.Clin Exp Physiol Pharmacol. 1997; 24: 582-586Crossref PubMed Scopus (37) Google Scholar,16.Wilcox C.S. Welch W.J. TGF and nitric oxide: Effects of salt intake and salt-sensitive hypertension.Kidney Int. 1996; 49: S9-S13PubMed Google Scholar implies that the NOS expressed might not be functional. However, these studies demonstrate a robust blunting of TGF by microperfusion of L-Arg into the MD of LS rats. This response to MD perfusion with L-Arg apparently entails NO generation, as it is stereospecific and blocked by coperfusion of L-NMA23.Welch W.J. Wilcox C.S. Salt-sensitive regulation of PGC by macula densa l arginine delivery.J Am Soc Nephrol. 1994; 5 (abstract): 615Google Scholar. Therefore, the NOS expressed during salt restriction is functional but apparently is limited by L-Arg availability. These single-nephron studies have parallels with whole-kidney results in which dietary salt restriction reduces plasma levels and rates of excretion of the NO metabolites, NO2 + NO3 and cGMP, enhances the renal vasoconstrictor response to NOS blockade with L-NAME24.Deng X. Welch W.J. Wilcox C.S. Renal vasoconstriction during inhibition of NO synthase: Effects of dietary salt.Kidney Int. 1994; 46: 639-646Abstract Full Text PDF PubMed Scopus (73) Google Scholar, and increases the NOS-dependent renal vasodilator response to L-Arg25.Deng X. Welch W.J. Wilcox C.S. Renal vasodilation with l-arginine: Effects of dietary salt.Hypertension. 1995; 26: 256-262Crossref PubMed Scopus (38) Google Scholar. These whole-kidney data complement the more specific single-nephron studies and indicate that dietary salt restriction diminishes NO generation via an L-Arg—dependent mechanism. Rat plasma [L-Arg] averages 80 μm during salt restriction but increases significantly to 160 μm during salt loading26.Deng X. Welch W.J. Wilcox C.S. Plasma arginine concentration and renal nitric oxide generation during changes in dietary salt and angiotensin II.J Am Soc Nephrol. 1995; 6 (abstract): 658Google Scholar, demonstrating a systemic effect of salt intake on arginine metabolism. However, the contrast between this evidence of L-Arg—stimulated NO generation and the extracellular concentrations that are one or two orders of magnitude above the Km for NOS measured in purified systems has been considered paradoxical27.Arnal J.F. Munzel T. Venema R.C. James N.L. Bai C. Mitch W.E. Interactions between l-arginine and l-glutamine change endothelial NO production: An effect independent of NO synthase substrate availability.J Clin Invest. 1995; 95: 2565-2572Crossref PubMed Scopus (165) Google Scholar. Nevertheless, the kidney generates methylarginine derivatives that both inhibit arginine uptake from the LH via system y+ and inhibit NOS activity in the MD28.Tojo A. Wilcox C.S. Bremer V. Kimoto M. Welch W.J. Kimura K. Omata M. Ogawa T. Vallance P. Methyl arginines in rat kidney: Comparison of immunohistochemical localization of demethylating enzyme NG, NG-dimethylarginine dimethylaminohydrolase (DDAH) with nitric oxide synthase, and effects on arginine transport.J Am Soc Nephrol. 1996; 7 (abstract): 1574Google Scholar. Immunocytochemical studies have located the MD as a site of expression of the enzyme NG-NG-dimethylarginine dimethylaminohydrolase that recycles dimethylarginines to citrulline28.Tojo A. Wilcox C.S. Bremer V. Kimoto M. Welch W.J. Kimura K. Omata M. Ogawa T. Vallance P. Methyl arginines in rat kidney: Comparison of immunohistochemical localization of demethylating enzyme NG, NG-dimethylarginine dimethylaminohydrolase (DDAH) with nitric oxide synthase, and effects on arginine transport.J Am Soc Nephrol. 1996; 7 (abstract): 1574Google Scholar. Therefore, cellular levels of L-Arg in the MD may be limited because of competition with dimethylarginine for uptake, and the intracellular NOS activity in vivo may be inhibited by the presence of these methylarginines. This could account for a higher apparent Km for L-Arg in vivo. This finding of a saturable, carrier-mediated L-Arg reabsorptive pathway in the LH, for which l-homoarginine competes, confirms previous studies21.Silbernagl S. Volker K. Dantzler W.H. Cationic amino acid fluxes beyond the proximal convoluted tubule of rat kidney.Pflügers Arch. 1994; 429: 210-215Crossref PubMed Scopus (11) Google Scholar. Our data are consistent with system y+ transport, in which L-Arg competes with other cationic amino acids17.Schmidt H.H.H.W. Baeblich S.E. Zernikow B.C. Klein M.M. Bohme E. L-arginine and arginine analogues: Effects on isolated blood vessels and cultured endothelial cells.Br J Pharmacol. 1990; 101: 145-151Crossref PubMed Scopus (30) Google Scholar, 18.Schmidt K. Klatt P. Mayer B. Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors.Mol Pharmacol. 1993; 44: 615-621PubMed Google Scholar, 19.Schmidt K. List B.M. Klatt P. Mayer B. Characterization of neuronal amino acid transporters: Uptake of nitric oxide synthase inhibitors and implication for their biological effects.J Neurochem. 1995; 64: 1469-1475Crossref PubMed Scopus (44) Google Scholar. Tracer quantities of L-Arg were almost completely absorbed from the LH, regardless of salt intake. However, the pathways of absorption differed significantly. During dietary salt loading, absorption through the system y+ transport pathway was increased substantially. Because luminal L-Arg uptake into MD cells for NO generation also depends on system y+ transport, the results suggest that, during HS, the robust L-Arg uptake into the MD via system y+ accounts for the lack of response to provision of further L-Arg via the tubular lumen. In contrast, during salt restriction, system y+ uptake of L-Arg is greatly diminished. This is compounded by reduced plasma L-Arg concentration26.Deng X. Welch W.J. Wilcox C.S. Plasma arginine concentration and renal nitric oxide generation during changes in dietary salt and angiotensin II.J Am Soc Nephrol. 1995; 6 (abstract): 658Google Scholar and, probably, enhanced reabsorption of L-Arg proximally via Na+-linked absorption29.Silbernagl S. Deetjen P. Molecular specificity of the l-arginine reabsorption mechanism: Microperfusion studies in the proximal tubule of rat kidney.Pflügers Arch. 1973; 340: 325-334Crossref PubMed Scopus (21) Google Scholar, which would limit arginine delivery to the MD segment and uptake into cells. This could account for the requirement of extra L-Arg during LS to generate NO via MD bNOS. In conclusion, these studies demonstrate that MD NO generation is limited during salt restriction. This is important physiologically because a limited NO generation could contribute to TGF enhancement and thereby to renal vasoconstriction. The failure of NO generation cannot be ascribed to decreased NOS expression but rather to decreased L-Arg availability. Reduced availability can be ascribed to a reduced plasma level, enhanced proximal reabsorption, and diminished uptake in the LH and MD cells via sodium-independent system y+. These findings that L-Arg levels and transport are regulated and that arginine can act systemically and within the kidney to determine NOS activity, elevate it to a level of a hormone involved in body fluid volume homeostasis. These hypotheses are summarized in Figure 2. These studies were supported by grants from the NIDDK to CSW (DK 49870 and DK 36079) and by funds from the George E. Schreiner Chair of Nephrology. We thank Marly Davidson for preparing the manuscript.