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发表于 2009-4-21 13:50 |只看该作者 |倒序浏览 |打印

作者：DoronSchwartz, Idit F.Schwartz, EhudGnessin, YoramWollman, TamaraChernichovsky, MiriamBlum,  AdrianIaina作者单位：Nephrology Department, The Tel Aviv Sourasky MedicalCenter, Tel Aviv 6423 Israel , z0 m; @; I+ s, S


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      【摘要】
   The decrease inglomerular filtration rate (GFR) that is characteristic of sepsis hasbeen shown to result from inhibition of glomerular endothelial nitricoxide synthase (eNOS) by nitric oxide (NO) generated from the inducibleisoform of NOS (iNOS). Although L -arginine is the soleprecursor for NO biosynthesis, its intracellular availability inglomeruli from septic animals has never been investigated. Arginineuptake was measured in freshly harvested glomeruli from the followingexperimental groups: 1 ) untreated rats; 2 ) ratspretreated with LPS (4 mg/kg body wt, 4 h before experiments); 3 ) rats treated with LPS as above with either L - N 6 -(1-iminoethyl)lysinehydrochloride ( L -NIL), a selective iNOS antagonist, or7-nitroindazole, a selective neuronal NOS antagonist; and 4 )rats treated with L -NIL only. Both glomeular and mesangial arginine transport characteristics were found compatible with ay system. Arginine uptake was augmented in glomeruli fromLPS-treated rats. Treatment with L -NIL completely abolishedthis effect whereas L -NIL alone had no effect. Similarresults were obtained when primary cultures of rat mesangial cells werepreincubated with LPS (10 µg/ml for 24 h) with or without L -NIL. Using RT-PCR, we found that in vivo administrationof LPS resulted in a significant increase in glomerular cationic aminoacid transporter-2 (CAT-2) mRNA expression whereas CAT-1 mRNA wasundetected. Northern blotting further confirmed a significant increasein glomerular CAT-2 by LPS. In mesangial cells, the expression of bothCAT-1 and CAT-2 mRNA was augmented after incubation with LPS. Inconclusion, in vivo administration of LPS augments glomerular argininetransport through upregulation of steady-state CAT-2 mRNA whiledownregulating CAT-1 mRNA. These results may correspond to the changesin glomerular iNOS and eNOS activity in sepsis.
      【关键词】 cationic amino acid transporter sepsis nitric oxide argininetransport
   INTRODUCTION1 L) A9 n8 t3 E2 r, T
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THE PRODUCTION AND RELEASE of nitric oxide (NO) have been claimed to play animportant role in diverse renal pathophysiological conditions ( 2, 8 ). Considerable evidence indicates that LPS, via induction ofinducible nitric oxide synthase (iNOS) and generation of copiousquantities of NO, mediates the protracted hypotension and renal failureof septic shock. ( 4, 30 ).# r* I' N& y9 V# Z

We have recently suggested that the renal vasoconstriction observed insepsis is due to local inhibition of the glomerular endothelial NOS(eNOS) by NO, generated from cytokine-induced iNOS. High basal NOactivity after induction of iNOS by LPS exerts a capacity toautoinhibit the constitutive eNOS. Suppression of glomerular eNOSresults in renal vasoconstriction and decrease in glomerular filtrationrate (GFR) ( 26 ).* O! ?: |) C# s: l: }
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Enhanced NO production depends on accelerated uptake of circulating L -arginine, the sole precursor for NO biosynthesis.Although most cells can synthesize some arginine, the cellular uptakeroute is believed to be of primary importance ( 3, 11, 15 ).
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Among several transport system that mediate L -arginineuptake (y  , b 0, , B 0, , andy L), system y is widely expressed andconsidered to be a major arginine transporter in most tissues andcells. Encoded by cationic amino acid transporters (CAT-1, CAT-2, andCAT-3), system y is characterized by high affinity forcationic amino acids, sodium independence, and stimulation of transportby substrate on the opposite ( trans ) side of the membrane( 1, 18, 20, 31-33 ). CAT-1 is widely expressed inmurine tissues and appears to have a greater capacity than CAT-2 for trans -stimulation ( 6 ). CAT-2 has been detectedin activated murine macrophages and lymphocytes. An alternately splicedtranscript of CAT-2, cloned from murine hepatocytes, encodes akinetically distinct low-affinity form of CAT-2A, which is highlyexpressed in murine liver ( 6, 7 ). CAT-3 expression wasfound to be present in adult brain and only recently in rat renal innermedullary collecting duct. ( 13, 14, 34 ). Little is knownabout factors that regulate arginine transport across the cellmembrane. In fact, the presence of arginine transporters in glomerulartissue has never been confirmed.

The present experiments, in a rat model of sepsis, were designed tostudy the glomerular presence of CAT-1 and CAT-2 and to examine theexpression and activity of these transporters after in vivo and ex vivoexposure to LPS. We have also attempted to define a link betweendirectional changes in CAT-1 and CAT-2 mRNA expression and the activityof eNOS and iNOS during sepsis.& J. S/ J* n" M! m3 q; z

METHODS
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All animal experiments described in this study were conducted inaccord with the protocol approved by the institutional committee onethics in animal experiments. Studies were performed using male Wistarrats weighing 200-250 g.  T5 L" v2 X. v: c+ X: R" J
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Four hours after a single intraperitoneal injection of LPS (4 mg/kgbody wt, serotype 0111:B4, Sigma) or saline, animals were allocated todifferent experimental groups. The above time course was chosen basedon the observation demonstrating that one dose of LPS exerts itsmaximal effect on glomerular arginine uptake after 4-6 h (see Fig. 3 ).$ A, `3 W9 i9 W7 u+ Z4 J( V

Experimental groups. The experimental groups were as follows. In group 1, controlrats were untreated ( n = 6). Group 2 ratswere treated with LPS only ( n = 5). Group 3 rats were treated LPS  L - N 6 -(1-iminoethyl)lysinehydrochloride (L -NIL), a selective inhibitor ofiNOS ( 26 ) [ 5 injections at a dose of 3 mg/kgip 36, 24, and 12 h before LPS and simultaneously with and 2 h after the administration of LPS ( n = 5); Sigma]. Group 4 rats were treated with L -NIL only ( n = 5) as in group 3 without LPS. Group 5 rats received LPS 7-nitroindazole (7-NI), selectiveneuronal (brain) NOS (bNOS) inhibitor ( n = 5; agenerous gift from R. C. Blantz). 7-NI was dissolved in DMSO andpeanut oil (25/75%) and was administered intraperitonally (25 mg/kgbody wt) 30 min before the experiments. This dose has been previouslyshown to exert antinociceptive activity and affect tubuloglomerularfeedback response, both effects related to bNOS activity. However, itdid not affect systemic blood pressure, suggesting selectivity to bNOS( 22 ).

Isolation of glomeruli. Kidneys from all experimental groups were decapsulated and bisected,and the cortex was carefully dissected free. Glomeruli were preparedusing a sieving technique. Cortices were minced into a fine paste witha razor blade and gently pressed through a 106-µm stainless steelsieve. The resulting material was suspended in HEPES buffer (in mM: 5 KCl, 0.9 CaCl 2, 1 MgCl 2, 5.6 D -glucose, 25 HEPES, and 140 NaCl, pH 7.4) at 4°C. Thesuspension was forced through a 20-gauge needle to decapsulate theglomeruli and then passed through a 75-µm sieve. The glomeruli thatwere trapped on the sieve were washed and pelleted by centrifugation at1,000 rpm for 1 min. This was repeated three times. This fractionconsisted 95% glomeruli, the majority of which were decapsulated.The isolated glomeruli were used for RNA extraction and for arginine uptake assessments.
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Mesangial cell isolation and culture. Wistar rats 3-4 wk old were killed. The kidneys were surgicallyremoved and kept on ice in sterile DMEM with 1% FCS. The kidneys wereminced, and glomeruli were separated from the remaining renal tissue bysequential mechanical sieving as described above. Glomeruli were platedonto culture flasks using a selective RPMI medium (in which D -valine was substituted with L -valine toinhibit fibroblast growth) containing 15% FCS, 10 mg/ml streptomycin,10,000 U/ml penicillin, and 2 mM L -glutamine at 37°Cunder 5% CO 2. Media were replaced every 48 h, whereasadherent cells were retained. After cells reached confluence (in ~4wk) they were passaged using trypsin/EDTA. The cells utilized in theseexperiments exhibited typical morphological characteristics ofmesangial cells and stained uniformly positive for -smooth muscleactin. Cells between passages 2 and 3 were usedfor subsequent experimental procedures.

L -Arginine uptake in freshly harvested glomeruli. Arginine uptake was determined essentially as described by Gazzola etal. ( 10 ). Glomerular suspensions from the various experimental groups were incubated and shaken for 10 min in HEPES buffer, pH 7.4, at 37°C. L -[H 3 ]arginine and L -arginine, in a final concentration of 1 mM, were added toa total volume of 1 ml for an additional 4 min. The duration of 4 minwas chosen because it was within the linear portion of uptake curves(data not shown). Transport activity was terminated by rapidly washingthe cells with ice-cold PBS buffer (4 times, 2 ml/tube). The glomeruliwhere than dried and solubilized in 1 ml of 0.5% SDS in 0.5 N NaOH. Tomonitor radioactivity by liquid scintillation spectrometry (Betamatic,Kontron),700 µl of the extract were used. The remaining 300 µl wereused for protein content determination by using the Lowry method. Tocorrect for nonspecific uptake or cell membrane binding, glomeruli wereincubated with 10 mM unlabeled arginine in HEPES buffer, and theassociated radioactivity was subtracted from each data point. Theresults are expressed as means ± SE of at least 5 different experiments.
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L -Arginine uptake in mesangial cells. Cells were seeded onto six-well plates (Corning) at a density of10 6 cells/well. When confluent, cells were washed with 2 mlHEPES buffer, pH 7.4. L -[H 3 ]arginine and L -arginine, in a final concentration of 1 mM, were added toa total volume of 1 ml for 1 min (within the linear portion of theuptake curve). Transport assays were performed as described above inthe following conditions: 1 ) control (untreated cells); 2 ) LPS (mesangial cells preincubated with 10 µg/ml LPS for24 h); 3 ) LPS  L -NIL (LPS as in the previousgroup 50 µM L -NIL); and 4 ) L -NIL only." `8 m3 b& i' O4 O% ]: F

Analysis of mRNA levels for CAT-1 and CAT-2 by RT-PCR. Total cellular RNA was extracted from glomeruli or mesangial cellsfollowing the method described by Chomeczynski and Sacchi ( 5 ). RT was carried out for 1.5 h at 42°C, and PCRin 1× Jeffrrey's buffer ( 16 ), for 35 cycles each of94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and 7 min (finalcycle). The first pair of primers was designed to bind to a portion ofthe rat CAT-1 gene: 5'-GCCATCGTCATCTCCTTCCTG-3' (forward 21-mer), and5'- CCCTCCCTCACCGTATTTCAC-3' (reverse 21-mer) ( 17 ). Asecond pair of primers, which hybridizes to a sequence common to bothCAT-2 and CAT-2A, comprised 5'-AACGTGCTTTTATGCCTTTGT-3' (forward24-mer) and 5'-GGTGACCTGGGACTCGCTCTT-3' (reverse 23-mer) ( 9 ). To differentiate between the two variants of CAT-2(CAT-2 and CAT-2A), a third pair of primers, which hybridizes to asequence specific to CAT-2A, was used: 5'-CCTTACCCCGCATTCTGTTTG-3'(forward 21-mer) and 5'-AAATGACCCCTGCAGTCATCG-3' (reverse 21-mer)( 12 ). To exclude the possibility of contamination bygenomic DNA amplification and to assess the adequacy of cDNA,experiments in the absence of RT were carried out and amplification ofGAPDH was performed, respectively. PCR products were electrophoresed ona 1.6% agarose gel and visualized by UV-induced fluorescence. All PCRreactions resulted in the amplification of a single product of thepredicted size for CAT-1, CAT-2, CAT-2A, and GAPDH.

Northern blot analysis. CAT-2 mRNA level was determined by Northern hybridization. Fifteenmicrograms of total RNA were denatured and fractionated by size on1.3% formaldehyde-agarose gels. RNA was transferred overnight, bycapillary action, to a nylon membrane (Hybond-N, Amersham) andcross-linked by short-wave UV illumination. Purified end products ofCAT-2 and GAPDH cDNA (5 ng/ml) were used directly for radiolabelingafter electrophoresis in 1.5% (wt/vol) low-melting-point agarose gels.The probes used were labeled to a specific activity 10 9 cpm/µg with [ - 32 P]dCTP by a randomprimer labeling method (GIBCO BRL), where cpm is counts/min.After hybridization with the 32 P-labeled cDNA overnight at50°C, the membranes were sequentially washed twice in 1× SSC, 0.1%SDS for 15 min at room temperature, once in 1× SSC, 0.1% SDS for 15 min at 50°C, followed by 0.5× SSC, 0.1% SDS at 55°C for 30 min,and then washed at high stringency in 0.1× SSC, 0.1% SDS at 57°Cfor 15 min. Autoradiography was carried out with Kodak XAR film for24-48 h at 70°C. Relative mRNA abundance was quantified bymeasuring the density of the exposed film with a densitometer (B.I.S202D). CAT-2 mRNA level was normalized to GAPDH mRNA and is expressedin arbitrary units as the ratio of CAT-2 to GAPDH expression of threedifferent experiments.
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Statistical analysis. One-way ANOVA for comparison between groups (means ± SE) andStudent's t -test between two groups were computed and usedto assess statistical significance. P values to be statistically significant.; l' N0 L3 ^; M* V+ t
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RESULTS

While the membrane transport of arginine has been found to occurlargely via a Na -independent pathway, we wished to examinethe possibility of a Na -dependent transport component.Accordingly, initial experiments were designed to examine the possiblecontribution of Na -dependent glomerular argininetransport. Figure 1, A and B, demonstrates that the presence of sodium had no effect oneither glomerular or mesangial arginine transport. This indicates thatarginine transport in glomeruli and mesangial cells occurs exclusively via Na - independent pathways. In addition, an excessconcentration of lysine strongly inhibited L -arginineuptake in both glomeruli and mesangial cells. In contrast, the neutralamino acid methionine was found to be a poor inhibitor (Fig. 1, A and B ). These data establish that systemy is the predominant arginine transport system inglomeruli and mesangial cells.8 F5 m) U/ g1 m' a

Fig. 1. Sodium independence and cis inhibition bylysine of glomerular and mesangial arginine uptake. Uptake ofradiolabeled arginine ( L -[ 3 H]arginine) byfreshly harvested glomeruli ( A ) and mesangial cells( B ) in the presence of either 100 mM sodium or 100 mMcholine and sodium lysine or 10 mM methionine. Values are means ± SE of 4 different experiments. * P

To characterize the kinetics of L -arginine transport inglomeruli and mesangial cells, the saturable uptake of L -arginine (0-1 mM) was measured. The plot of L -arginine uptake as a function of extracellular L -arginine concentration is shown in Fig. 2, A and B. A high-affinitytransporter was present in both glomeruli and mesangial cells(glomeruli: K m 110 µM; V max 4.3 nmolarginine · µgprotein 1 · 4 min 1;mesangial cells: K m 120 µM, V max : 540 fmol arginine/µg protein). On thebasis of previous reports, the kinetic properties of the argininetransport system in both glomeruli and mesangial cells resemble thoseof system y  , CAT-1, and CAT-2.- G) e) \, K6 G) T5 @- Y1 b/ ?
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Fig. 2. Concentration dependence of L -arginine uptakeby glomeruli ( A ) and mesangial cells ( B ). Uptakeof L -[ 3 H]arginine was measured for 4 min infreshly harvested glomeruli from normal rats and for 1 min in primarycultures of mesangial cells over a range of concentrations (0-1mM). Values are means ± SE of 4 different experiments.

Effects of LPS on arginine transport in glomeruli and mesangialcells. In rats, a significant increase in glomerular arginine uptake wasobserved 2 h after LPS administration, peaking at 4-6 h anddecreasing to control levels at 16 h (Fig. 3 ). These results correspond to the timecourse of glomerular iNOS activation after LPS ( 25 ). Invivo administration of LPS resulted in a significant increase inglomerular arginine uptake compared with untreated rats. The effect wascompletely abolished by the coadministration of L -NIL, aselective iNOS inhibitor. L -NIL alone had no effect onglomerular arginine transport. Because L -NIL couldpotentially block the effect of bNOS as well, 7-NI, a selective bNOSinhibitor, was also administered to LPS-treated rats. Incontrast to L -NIL, 7-NI did not affect arginine uptake byglomeruli harvested from these animals (Fig. 4 ). We repeated the critical experimentsusing primary cultures of renal mesangial cells and found thatmesangial cells exposed to LPS (10 µg/ ml for 24 h) exhibitedmaximal nitrite production (data not shown). Therefore, we chose to usethe above concentration and time course in all of the followingexperiments. Similar to our findings in glomeruli, incubating mesangialcells with LPS induced a significant increase in uptake of 1 mMextracellular arginine, an effect that was prevented by coincubatingthe cells with L -NIL. L -NIL alone had no effecton mesangial arginine uptake (Fig. 5 ).After the administration of LPS to both glomeruli and mesangial cells,arginine uptake remained sodium independent. Lysine, but notmethionine, exerted an inhibitory effect on arginine uptake, implyingthat the y system remains the predominant argininetransport system in tissues exposed to LPS (Fig. 6, A and B ).

Fig. 3. Time course of L -[ 3 H]arginineuptake by freshly harvested glomeruli from rats pretreated with LPS (0, 2, 4, 6, and 16 h after an ip injection of LPS, 4 mg /kg body wt).Values are means ± SE of 4 different experiments.* P

Fig. 4. Uptake of L -[ 3 H]arginine byfreshly harvested glomeruli from the various experimental groups.Values are means ± SE of at least 5 different experiments. Seetext for description of groups. CTL, control; L -NIL, L - N 6 -(1-iminoethyl)lysinehydrochloride; 7-NI, 7- nitroindazole. * P
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Fig. 5. Uptake of L -[ 3 H]arginine byprimary cultures of mesangial cells preincubated with LPS (10 µg/ml)for 24 h. For comparison with nontreated cells, cells werecoincubated with LPS plus L -NIL (a selective iNOSantagonist, 50 µM) and treated with L -NIL only. Valuesare means ± SE of 4 different experiments. * P

Fig. 6. Characterization of the arginine transporters inglomeruli and mesangial cells after the administration of LPS. Uptakeof L -[ 3 H]arginine by freshly harvestedglomeruli from LPS-treated rats (4 mg/kg body wt ip 4 h beforeexperiments; A ) and mesangial cells preincubated with LPS(10 µg/ml for 24 h; B ) in the presence of either 100 mM sodium or 100 mM choline and sodium lysine or 10 mM methionine.Values are means ± SE of 4 different experiments.* P# X% Y# n: X, r5 K5 }" {2 T
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Differential regulation of CAT-1 and CAT-2 gene expression by LPS. To determine whether the observed LPS-induced changes in arginineuptake are associated with similar directional changes in levels formRNA for the CAT family of transporters, total glomerular RNA wasanalyzed by RT-PCR to amplify portions of CAT-1 and CAT-2. RT-PCRidentified cDNA encoding both CAT-1 and CAT-2 in glomeruli harvestedfrom control rats. Surprisingly, in vivo administration ofLPS abolished the expression of CAT-1 mRNA. In contrast, LPS significantly augmented the expression of mCAT-2 mRNA. Thecoadministration of L -NIL (a selective iNOS inhibitor) toLPS-treated rats reversed the effects of LPS on both transporters.Because a low-affinity component of the glomerular arginine transportercould not be excluded, specific primers to CAT-2A were used. The mRNAexpression of CAT-2A was constitutively present but did not vary amongthe experimental groups (Fig. 7, A and B ). Because CAT-2A wasminimally expressed in glomeruli, positive controls from rat livercells were performed (Fig. 7 C ). No RT-PCR products wereobtained with RNA samples in the absence of RT or when cDNA was omittedfrom PCR.) p4 I! M4 g0 v  z5 F5 q
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Fig. 7. A : ethidium-stained agarose electrophoresisgel showing PCR-amplified cationic amino acid (CAT)-1, CAT-2, CAT-2A,and GAPDH cDNA of glomeruli harvested from the various experimentalgroups. The blots are representative of 3 different experiments. Seetext for description of groups. B : relative amount of CAT-1,CAT-2, and CAT-2A mRNA quantitated by densitometry and expressed asCAT/GAPDH ratio of the same experiments shown in Fig. 6 A.Values are means ± SE of 3 different experiments.* P C : ethidium-stainedagarose electrophoresis gel showing PCR-amplified CAT-2A cDNA of ratliver cells (in triplicate).
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To further confirm this observation, Northern blotting was performed.CAT-1 and CAT-2 mRNA were not detectable in glomeruli taken fromuntreated animals. Therefore, we were unable to confirm our PCR resultson CAT-1. Administration of LPS resulted in a significantincrease in steady-state glomerular CAT-2 mRNA, an increasethat was abolished by coadministration of L -NIL (Fig. 8, A and B ).
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Fig. 8. Effect of LPS on steady-state CAT-2 mRNA in ratglomeruli. A : Northern blot analysis of glomerular CAT-2 andGAPDH mRNA from the various experimental groups. The blots arerepresentative of 3 different experiments. B : graphicrepresentation of relative changes in mRNA levels. The ratio of thesignal of the 7.9-kb CAT-2 mRNA to the signal of GAPDH mRNA was plottedagainst the appropriate experimental group. Values are means ± SEof 3 different experiments. * P# x# x5 O5 y' s$ `6 `6 {5 e

RT-PCR for the two arginine transporters was also performed inmesangial cells treated with LPS. In contrast to our findings inglomeruli from LPS-treated rats, incubating mesangial cells with LPSfor 24 h significantly augmented the expression of both CAT-1 andCAT-2 mRNA, when normalized to GAPDH (Fig. 9, A and B ).

Fig. 9. A : ethidium-stained agarose electrophoresisgel showing PCR-amplified CAT-1, CAT-2, and GAPDH cDNA of rat mesangialcells preincubated with LPS (10 µg/ml) for 24 h. A comparisonwith nontreated cells is shown. B : relative amount of CAT-1and CAT-2 mRNA quantitated by densitometry and expressed as CAT/GAPDHratio of the same experiments shown in Fig. 6 A. Values aremeans ± SE of 3 different experiments. * P. M5 W2 p; l9 f" v/ f
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DISCUSSION

Despite the enormous interest being focused on the various renaleffects of arginine metabolites, most importantly NO, the present studyis the first to investigate the regulation of arginine uptake byglomeruli. We demonstrate that both glomerular and mesangial argininetransport systems are sodium independent, subject to cis inhibition by lysine, but not methionine, and constitutively expressthe two major arginine transporters, namely, CAT-1 and CAT-2. Ourresults imply that arginine uptake in those tissues occurs largely viaa y system and the above transporters play a crucial rolein glomerular arginine traffic. Therefore, changes in their levels oractivity could potentially alter NO production under certainpathophysiological conditions, including sepsis.

We have also demonstrated that LPS-treated rats exhibit an augmentedglomerular arginine uptake through modulation of CAT-2 mRNA. Theobservations above suggest that increased glomerular arginine transportin sepsis is related to the induction of iNOS by LPS and results fromupregulation of CAT-2 mRNA. The fact that coadministration of L -NIL (a selective iNOS antagonist) but not 7-NI (aselective bNOS inhibitor) completely abrogated the increase inglomerular arginine uptake and CAT-2 mRNA expression after LPSadministration further supports the above hypothesis. The associationbetween activation of iNOS and CAT-2 has been previously described innonrenal tissues. The first indication that CAT-2 may provide iNOS withits substrate came from the observation that CAT-2 and iNOS transcriptswere coinduced in concert with increased system y activityafter appropriate cytokine stimulation. Simmons et al. ( 29 ) have shown in cardiac myocytes that IL-1 andinterferon simultaneously increase mRNA expression of both CAT-1 andCAT-2, thereby enhancing arginine transport into the cells. Nicholson et al. ( 23 ) have reported that iNOS activity was reducedin macrophages from CAT-2 knockout mice. We have recently demonstrated that tetrahydrobiopterin (BH 4 ) acts as a cofactor thatsimultaneously upregulates both iNOS and CAT-2 mRNA ( 27 ).Together, these data support the view that on induction of iNOS, theexcess arginine required is delivered by CAT-2. Whether the associationbetween activation of iNOS and CAT-2 stems from dependency on the same regulatory cofactors, such as BH 4, or relies on an adjacentintracellular localization remains elusive.* S: p' E5 o! e- ?, X0 b: w2 D
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In contrast to the effect of LPS on glomerular CAT-2, the expression ofCAT-1 was completely abolished. Although a direct comparison of mRNAlevels does not necessarily reflect the amount or activity of thetransport proteins, the fact that CAT-1 mRNA was undetectable makes theassumption of decreased activity conceivable. The mechanism responsiblefor decreased CAT-1 mRNA level is not clear. Could regulation of onetransporter be dictated by changes in expression of another CATprotein? Similar findings were reported by us in a study of the effectof BH 4 on CAT-1 and CAT-2 mRNA expression in rat cardiacmyocytes ( 27 ). Nicholson et al. ( 24 ) demonstrated that CAT-3 can compensate for the loss of functional CAT-1in cells from CAT-1 knockout mice. It was hypothesized that upregulation of an individual CAT decreases the steady-state mRNA ofthe other transporters via a direct effect or, alternatively, anincrease in an individual CAT could reflect a compensatory response toa decrease in the expression of a different CAT isoform. It seemslogical that NO or one of its metabolites, most importantly peroxynitrite, plays a role in the attenuation of CAT-1 expression. This led us to conduct a series of in vitro experiments in which weexposed mesangial cells to LPS in an attempt to explore a possible explanation for the decrease in CAT-1 mRNA. Surprisingly, in contrast to our findings in glomeruli from LPS-treated rats, when primary cultures of mesangial cells were treated with LPS, mRNA expression ofboth transporters was increased, contradicting the above assumption. Onthe basis of the discrepancy between the results in freshly harvestedglomeruli and mesangial cell cultures, one could speculate that certainevents occurring in the kidney after systemic administration of LPS areresponsible for the downregulation of CAT-1 mRNA. Such a critical eventafter LPS administration is renal vasoconstriction andischemia. Preliminary data from our laboratory suggest that peroxynitrite, a toxic oxidant formed from the reaction of NO andsuperoxide during ischemia-reperfusion, increased rather than decreased the expression of both CAT-1 and CAT-2 when administered tomesangial cells (Schwartz and Iaina, unpublished observations). Therefore, its production could not serve as a possible explanation ofthe above-mentioned phenomenon. At this juncture, the mechanism fordownregulation of glomerular CAT-1 mRNA by LPS remains enigmatic.
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A major question posed by this observation is whether selectiveinhibition of CAT-1, while total glomerular uptake of arginine issignificantly increased, has any pathophysiological meaning. Inendothelial cells, extracellular arginine seems to drive NO productioneven when excess intracellular levels are available. This phenomenonhas been termed "the arginine paradox." One paradigm that couldexplain this observation is that intracellular arginine is sequesteredin one or more pools that are poorly accessible to eNOS, whereasextracellular arginine transported into the cell is preferentiallydelivered to eNOS. It has recently been shown that CAT-1 and eNOS arecolocalized in a caveolar complex ( 21 ). Such a complex hasbeen suggested to serve as a mechanism for channeling newly acquiredextracellular arginine to eNOS for NO synthesis. Thus selectivedelivery of transported arginine to membrane-bound eNOS could explainthe arginine paradox discussed above. We have previously demonstratedthat induction of iNOS by LPS induces selective eNOS inhibition( 26 ). One can speculate that inhibition of CAT-1 after invivo administration of LPS, as shown in the present experiments, mayprovide a possible mechanism for selective eNOS inhibition in thepresence of increased arginine uptake and iNOS activity. In theaggregate, the effects of LPS on the expression of the two transportersdescribed here parallel the changes in their corresponding NOSactivity, as shown previously ( 26 ). Namely, increasedactivity/expression of CAT-2 and iNOS, while activity/expression ofCAT-1 and eNOS is diminished, emphasizes the dependency of these twopairs of proteins.
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Using glomeruli as a source of renal tissue in our experiments revealsa significant limitation because the specific cells in which the aboveevents occur cannot be defined. One can argue that the induction ofiNOS by LPS, which involves mainly macrophages and mesangial cells,would not influence NO metabolism in endothelial cells. Nevertheless,Shultz and Raij ( 28 ) demonstrated in LPS-treated rats thatinhibition of NO generation induces glomerular capillary thrombosis, apurely endothelial event. We have found that induction of iNOS inhibitseNOS within the glomerulus ( 26 ). These publications suggest that the glomerulus exhibits a microenvironment, in which events in one cell can affect NO metabolism in neighboring cells. Moreover, the importance of using freshly harvested glomeruli ratherthan cell cultures is greatly supported by the above findings, whichdemonstrated an opposite effect of LPS on CAT-1 in glomeruli vs.mesangial cells.

Arginine transport velocities in our experiments were found to besignificantly higher than the average values published by others. Thesedifferences can be partly explained by higher extracellular arginineconcentrations and longer incubation periods used in the presentstudies, as well as improved accessibility of glomeruli in suspensionto extracellular arginine compared with that of adherent cells. Withregard to the high transport rates reported in our studies, equilibriumshould have been reached earlier. Glomeruli tend to stick to tubewalls, and therefore the participation of cells in a transport assay isprobably unsynchronized. This phenomenon could have caused a deviationof the equilibrium point in our studies.
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In summary, our findings suggest that administration of LPS to rats, asan experimental model of sepsis, augments glomerular arginine uptakethrough upregulation of CAT-2 mRNA while decreasing CAT-1 mRNA levels.The complex regulation of genes encoding proteins required for L -arginine transport by LPS could potentially play a rolein altered NOS isoform activity in sepsis./ j, K) x$ n+ k" K2 j0 W) W+ k
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ACKNOWLEDGEMENTS8 B% a% j  _* |/ t' [& \. F8 r
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This study was supported by grants from the Chief Scientist of theIsraeli Minister of Health.9 q5 v+ }5 @0 n: x1 A* i7 E  b7 l
      【参考文献】) R5 \% A4 T7 |; w# k' H0 r
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