Angiotensin receptor subtypes in thin and muscular juxtamedullary efferent arter - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Claudia M. B. Helou, Martine Imbert-Teboul, Alain Doucet, Rabary Rajerison, Catherine Chollet, François Alhenc-Gelas,  Jeannine Marchetti作者单位：1 Institut National de la Santé et de laRecherche Médicale Unité 36 Physiologie et PathologieExpérimentale Vasculaires, Université Paris VI, 75005 Paris; and Laboratoire de Biologie Intégrée desCellules Rénales, Unité de Recherche Associ

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   ANG II controls the vascular tone of pre- and postglomerular arterioles,and thereby glomerular filtration, through binding to either AT 1A,AT 1B, or AT 2 receptors. AT 1 receptors, which are coupled to intracellular Ca 2 signaling, havevasoconstricting effects, whereas AT 2 receptors, whose signalingmechanism is unknown, induce vasodilatation. The angiotensin receptors have been characterized in afferent arterioles, which express the three types ofreceptors, but not in efferent arterioles. Two subpopulations ofjuxtamedullary efferent arterioles, muscular ones which terminate as vasarectae and thin ones which terminate as peritubular capillaries, have beendescribed. They display functional heterogeneity with regard to the ANG IIresponse. To evaluate whether these differences are associated withdifferential expression of ANG II receptors, we examined the expression pattern of AT 1A, AT 1B, and AT 2 receptor mRNAsby RT-PCR in these arterioles and studied the effect of valsartan, a specific AT 1 -receptor antagonist. Results indicate that muscular arterioles express AT 1A, AT 1B, and AT 2 receptors,whereas thin arterioles only express the AT 1A and AT 2 types, and at a much lower level. Valsartan fully inhibited ANG II-inducedincreases in intracellular Ca 2 in both arteriolartypes, but with different kinetics. In muscular arterioles, inhibition wasmonoexponential, whereas it displayed a marked positive cooperativity in thinarterioles. Finally, the apparent affinity for valsartan was higher in muscular than in thin arterioles. In conclusion, this study further documentsthe differences between muscular and thin efferent arterioles with regard toANG II signalization in the rat kidney. 1 o3 n- t+ n* L' E
      【关键词】 angiotensin II valsartan calcium signaling' g4 i8 C$ U  C6 }
   PRE - AND POSTGLOMERULAR ARTERIOLES are small resistance vessels that regulate renal microcirculation and glomerular filtration. ANG II is amajor mediator of the regulation of glomerular filtration through its combinedcontrol of the vascular tone in afferent and efferent arterioles (AAs and EAs,respectively).
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The effects of ANG II are mediated by at least two types of cell-surfacereceptors, namely, AT 1 and AT 2 receptors. AT 1 receptors, which are coupled to an intracellular Ca 2 signaling pathway ( 33 ), arethought to be present in vascular smooth muscle cells and to mediate thevasoconstriction effects of ANG II in the glomerular arterioles( 2, 24 ). In rats, two subtypes ofAT 1 receptors have been cloned (AT 1A and AT 1B receptors). These two receptors, which share 90% identity in amino acidsequence, display hardly distinguishable pharmacological properties( 4, 8, 9 ), except in one study, whichindicated that the AT 1 -receptor antagonist losartan was more potenton AT 1B than AT 1A receptors( 32 ). AT 2 receptors, which are present in endothelial cells, mediate the vasodilatoreffect of ANG II in AAs ( 3 ) andin EAs ( 12 ). However, thesignaling pathways of AT 2 receptors are not yet fully established.When AT 1 and AT 2 receptors are coexpressed in the samecell, AT 1 receptor-mediated responses could be antagonized byAT 2 receptors because of heterodimerization of the two receptors( 1 ). Altogether, these datasuggest that the vasoactive response of a given vessel to ANG II (constrictionor dilatation) as well as its sensitivity to the peptide and to receptorantagonists might depend on the relative expression of the different types andsubtypes of ANG II receptors.
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To date, the distribution of the different types of ANG II receptors inrenal vessels has been mainly investigated in AAs. Immunohistochemical ( 17 ) and radioligand binding( 7 ) studies have shown thatAT 1 receptors are present in these vessels. By RT-PCR, Ruan et al. ( 30 ) found that the twosubtypes of AT 1 receptor mRNAs are expressed in preglomerulararterioles in a 4:1 AT 1A /1AT 1B ratio. AT 2 receptor mRNAs are also expressed in AAs as well asarcuate arteries ( 27 ). Incontrast, little is known about the distribution of ANG II receptors in EAs. This may be due to the heterogeneity of the population of EAs and to thetechnical difficulty in isolating these different subpopulations ofarterioles.$ Q/ e& B0 i% A0 E- d" Y

We have indeed described two subpopulations of juxtamedullary EAs thatdisplay morphological, topological, and functional differences( 18 ). Based on morphology, onecan distinguish muscular EAs (mEAs), which have a thick, regular, and muscular wall and terminate as vasa rectae, from thin EAs (tEAs), characterized by athinner, irregular, and less muscular wall and which terminate as peritubularcapillaries. Functionally, mEAs display higher increases in intracellularCa 2 concentration([Ca 2 ] i ) than tEAs in response to ANG II,but they are slightly less sensitive to ANG II than are tEAs( 18 ).
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Therefore, this study aimed at determining whether these differences between mEAs and tEAs were associated with differential expression of ANG IIreceptors. For this purpose, we examined the expression of AT 1A,AT 1B, and AT 2 receptor mRNAs by RT-PCR in these two subpopulations of EAs, and we compared the sensitivity of ANG II-inducedincreases in [Ca 2 ] i to theAT 1 -receptor antagonist valsartan.
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METHODS
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Microdissection of Juxtamedullary Arterioles3 Q9 z/ h% p3 x5 U5 p/ q8 ~
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Experiments were carried out in male Sprague-Dawley rats (Iffa-Credo, L'Arbresle, France) weighing 180-240 g. Juxtamedullary glomerular arterioleswere isolated from collagenase-treated kidneys as previously described( 18 ). Briefly, afteranesthesia (pentobarbital sodium, 50 mg/kg ip) the left kidney was infused viathe abdominal aorta with 3-5 ml of a cold standard solution (see compositon below). The kidney was then infused with 5 ml of the same solution containing8 mg collagenase A ( Clostridium histolyticum, 1.1 U/mg, Serva) andimmediately removed, decapsulated, and longitudinally sliced. Small pyramidswere cut and incubated for 8 min at 30°C in the presence of collagenase (1mg/ml) and rinsed in cold standard solution. Juxtamedullary EAs attached totheir glomeruli were microdissected under a stereomicroscope in ice-cold standard solution. They were identified as tEAs or mEAs, respectively, according to morphological criteria( 18 ). In some experiments, juxtamedullary AAs were also isolated and served as controls. All animalprocedures were conducted in agreement with our institutional guidelines forthe care and use of laboratory animals.: M$ X9 V+ X3 Z5 U
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For [Ca 2 ] i measurements, the standardsolution contained (in mM) 127 NaCl, 5 KCl, 0.8 MgSO 4, 0.33Na 2 HPO 4, 0.44 KH 2 PO 4, 1MgCl 2, 4 NaHCO 3, 2 CaCl 2, 5 D -glucose, 10 sodium acetate, and 20 HEPES, pH 7.4, as well as 0.1%BSA. For RT-PCR experiments, Hanks' solution (Eurobio, Les Ulis, France)supplemented with (in mM) 1 sodium pyruvate, 1 glutamine, 1 sodium acetate,1.5 MgCl 2, and 20 HEPES as well as 1 mg/ml protease-free BSA, pH 7.4, was used.: I  q; z. J+ j) a$ ^
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Measurements of[Ca 2 ] i

[Ca 2 ] i was evaluated with a Photoscan IImicrofluorimeter (Photon Technology) as previously described( 25 ). Briefly, EAs wereindividually transferred on a thin glass slide within 1 µl of a standardsolution containing 1% agarose (type IX, Sigma). After gelificaton of theagarose (1 min at 4°C), arterioles were loaded in the presence of 1 µlof 10 µM fura 2-AM (Molecular Probes, Leide, The Netherlands) for1hat roomtemperature in darkness. Then, the glass slide with the sample was mounted atthe bottom of a superfusion chamber on the stage of an inverted fluorescentmicroscope (Nikon). The sample was continuously superfused with the standard solution (0.8 ml/min, 37°C) with or without test substances. The samplewas alternatively excited at 340 and 380 nm (12 cycles/min), and thefluorescence emitted at 510 nm from a defined area ( 25 x 30 µm)was measured. All values were corrected for autofluorescence determined at thetwo wavelengths after quenching of fura 2-AM fluorescence with 1 mMMnCl 2 in the presence of 10 µM ionomycin.[Ca 2 ] i was calculated from the followingequation ( 14 ):[Ca 2 ] i = K d x (R - R min )/(R max - R), where K d (the dissociation constant for the fura2-Ca 2 complex) is 224 nM, R is the ratio offluorescence emitted for each wavelength (340/380 nm), R max is themaximal ratio emitted in the presence of saturating Ca 2 (2 mM), R min is the minimal ratio measured in the absence ofCa 2 (0 mM), and is the ratio of fluorescenceobtained at 380 nm in the absence and presence of 2 mMCa 2  . Values of R min, R max, and were periodically determined by external calibration using buffer thatmimicked an intracellular medium( 25 ).9 [* r2 u8 z1 {$ _* [( F. R  k

The response to ANG II was evaluated either by the plateau value ( [Ca 2 ] i above baseline) or by theintegral of the Ca 2 signal calculated as

where t 0 and t 1 correspond to the timefor threshold of [Ca 2 ] i increment and forreturn to baseline value, respectively.

ANG II was dissolved in water, whereas the AT 1 -receptorantagonist valsartan (a gift from Novartis) was prepared from a 1 mM stock solution in ethanol. The concentration of ethanol in the superfusion solution( 0.1%) modified neither the basal [Ca 2 ] i nor the [Ca 2 ] i response to ANG II.
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Data are expressed as means ± SE. When each arteriole served as itsown control, significance was obtained by a paired Student's t -test.Differences between the two groups of arterioles were analyzed with the use ofan unpaired Student's t -test. Values were considered significantlydifferent at P Commercially available Cricket Graphsoftware was used to fit concentration-response curves and estimateIC 50 and the Hill coefficient.
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Expression of AT 1 and AT 2 Receptor mRNAs byRT-PCR( \( V5 P9 T+ y- B2 P6 H) l

In view of the paucity and of the small size of microdissected glomerulararterioles, especially tEAs, we evaluated the expression of AT 1A,AT 1B, and AT 2 receptor mRNAs by co-RT-PCR on the same arteriole samples in the same tube. Therefore, the number of PCR cycles wasincreased to allow detection of all three sequences, when coexpressed in thesame samples. This prevented the use of quantitative PCR methods.

Primers. Oligonucleotide primers specific for each type of AT receptor [chosen in divergent cDNA portions of the published rat sequences(GenBank accession nos. M74054 , S69961 , and D16840  for AT 1A,AT 1B, and AT 2 receptors, respectively)] are as follows: AT 1A sense, 5'-CTGGCTGATGGCTGGCTTGG-3' (bases 715-734),and antisense, 5'-TACGCTATGCAGATGGTGATGGG-3' (bases 1112-1134); AT 1B sense, 5'-ATTCCCCCAACGGCCAAGTC-3' (bases1540-1559), and antisense, 5'-GGCGGTTAACAGTGGCTTTGCTC-3' (bases1858-1880); and AT 2 sense,5'-GAGCATGAGAGGTGGGCACTAAGG-3' (bases 1659-1682), andAT 2 antisense, 5'-AAATAGCGTGCGCTCTATAACTTCAAGG-3'(bases 1881-1908). Because of the sequence homology between AT 1A and AT 1B receptor mRNAs( 15, 20 ), primers for the twosubtypes of AT 1 receptors were checked by RT-PCR( 11 ) on mRNAs extracted fromkidney and liver tissue according to the method of Chomczynski and Sacchi( 10 ). As shown in Fig. 1, despite the high number of PCR cycles used in this series (35 cycles), only two DNA fragments of theexpected size (420 and 341 bp, respectively) were found in the kidney, whichis known to express both AT 1A and AT 1B receptors,whereas a single DNA fragment (420 bp) was found in the liver, which expressesonly the AT 1A subtype ( 13, 23, 27 ).. I2 ]8 ^7 u# X) [- p# h2 j) w
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Fig. 1. Amplification of ANG II AT 1A and AT 1B receptor mRNAs.Because of the homology of AT 1A and AT 1B receptorsequences, PCR experiments were carried out in liver (250 ng total RNA/tube)and whole kidney RNA extracts (500 ng/tube) to check the specificity ofoligonucleotide primers. PCR conditions were those previously described byElalouf et al. ( 11 ), and 35cycles were done to allow detection of putative nonspecific PCR products, ifpresent. As shown by the representative electrophoresis gels, only 2 DNAfragments of the expected size (420 and 341 bp for AT 1A andAT 1B receptors, respectively) were found in the kidney, whichexpresses both receptor subtypes, and only 1 fragment (420 bp) was found inthe liver, which does not express the AT 1B subtype. Noamplification product was ever found in control samples run without reversetranscriptase in the same experiments. Note that, due to the high no. of PCRcycles, all signals were saturated. Therefore, band intensity does not reflectthe relative proportion of AT 1A and AT 1B receptor mRNAsin kidney tissue.

RT-PCR on arterioles. After microdissection, each singlearteriolar fragment was measured with a calibrated eyepiece micrometer, washedfree of contaminating cells and debris in standard solution supplemented with5 µM DTT and 20 U RNasin (Promega, Charbonnières, France), andtransferred with 2.5 µl of washing medium into a sterile reaction tube.Blanks were done by collecting 2.5 µl of washing medium without EAs. RT-PCRwas then carried out using a technique derived from that described by Lambolezet al. ( 22 ).

RT. The following compounds were added to each reaction tube: 2µl of a 5 x RT mixture containing 2.5 mM of each dNTP (Invitrogen,Paisley, UK) and 25 µM random primers [pd(N)6, Roche Diagnostics]; 1 µlof 100 mM DTT; 0.5 µl RNasin (20 U); 0.2 µl of 50 mM MgCl 2;3µl standard solution containing 3 x Triton X-100; and 0.3 µlRNAse-free DNAse (3 U, Stratagene, Amsterdam, The Netherlands). Before initiation of the RT reaction (10-µl final vol), all samples were incubatedat 37°C for 45-60 min to permeabilize the arteriolar fragments and digestgenomic DNA and were heated at 80°C for 2 min to inactivate DNase. RT wasinitiated at room temperature by adding 0.5 µl (100 U) Superscript IIreverse transcriptase (Invitrogen), and cDNAs were synthetized at 37°Covernight. Negative RT controls were performed by omitting reversetranscriptase. cDNAs were stored at -20°C until PCR.
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PCR. Because dissection of EAs, especially tEAs, is difficult, the distribution of AT 1A,AT 1B, and AT 2 receptor mRNAs was studied in the same samples. cDNAs were thereforecoamplified in the same tube in a final volume of 100 µl. Seventymicroliters of a PCR mixture containing 2.5 U Taq DNA polymerase and10 µl of 10 x PCR buffer (Qiagen, Courtaboeuf, France) were firstadded to the RT product (10 µl). All samples were held 1 min at 94°Cand then received 20 µl of a mixture containing sense and antisense primersfor AT 1A, AT 1B, and AT 2 receptors (10 pmoleach). In a first experimental series, they were then processed for 32-40cycles at three sequential temperature steps: 95°C for 30 s; 60°C for30 s; and 71°C for 1 min, with the exception of the last cycle, in whichthe elongation lasted 10 min.( j- Z6 ]) K9 V5 @

An analysis of data obtained under these conditions in tEAs revealed onlyfaint, if any, amplification products, even after 40 PCR cycles. Therefore, ina second series of experiments, two PCR steps were sequentially carried out inmEAs and tEAs: the first (PCR1; 40 cycles) was carried out as described above, and the second (PCR2) was performed on 2-µl aliquots of PCR1 products. Forthis purpose, 78 µl of a PCR mixture containing 10 µl 10 x Taq buffer, 2.5 U Taq DNA polymerase, and 1 µl dNTPs (5mM) were added to each tube; samples were then processed as described aboveand submitted to 14-25 additional PCR cycles. In all experiments, negativecontrols including blanks (no tissue) and arteriolar samples without reversetranscriptase were run in parallel.
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PCR products were separated by electrophoresis on 2% agarose gelscontaining ethidium bromide and visualized by UV illumination.
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RESULTS
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Nondesensitization of [Ca 2 ] i Responses of Juxtamedullary Arterioles to 1 nM ANG II! ?* ]$ ^  c! \1 O! }6 E

In both tEAs and mEAs, superfusion by 1 nM ANG II induced a rapid increasein [Ca 2 ] i followed by a sustained plateau. As previously reported ( 18 ),the magnitude of the plateau was significantly higher in mEAs than in tEAs. OnANG II removal, [Ca 2 ] i slowly decreased downto its basal level within 5-8 min ( Fig.2 ). Thereafter, identical responses (in terms of plateau values,integral signals, and duration of response) could be induced by a second and athird application of ANG II to the same arterioles( Fig. 2 and Table 1 ), indicating theabsence of desensitization phenomena. Thus each arteriole could be used as itsown control for testing the AT-receptor antagonist. In subsequent experiments,valsartan was added before and throughout the second application of ANG II,and the response obtained was compared with the first one. A third applicationof ANG II after a 15-min washing allowed estimation of the recovery ofarteriolar responsiveness from antagonist action.

Fig. 2. Reproducibility of intracellular Ca 2 concentration([Ca 2 ] i ) responses to 1 nM ANG II in thinand muscular efferent arterioles [tEAs ( A ) and mEAs ( B ),respectively]. Representative tracings showing the effect of 3 successiveapplications of ANG II (1 nM, 7 min) on[Ca 2 ] i in juxtamedullary tEAs and mEAs areshown.

Table 1. Absence of desensitization of Ca 2  responses to angiotensinin efferent arterioles+ {" p1 `$ P  c9 k6 P6 |; i
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Effect of AT 1 -Receptor Antagonist Valsartan- R& u$ h( Y  p: |# |+ O5 m) \" V

Results in Table 2 and Fig. 3 indicate that changes in[Ca 2 ] i induced by 1 nM ANG II were inhibitedby valsartan in both mEAs and tEAs, but with different sensitivities. Up to 10nM valsartan, no significant inhibition was detected in tEAs, whereas a markedinhibition ( 40%) was observed in mEAs. The inhibitory effects of 30 and 50nM valsartan were also more marked in mEAs than in tEAs( Fig. 3, A and B ), whereas nearly complete inhibition ( 90%) and atotal suppression of [Ca 2 ] i responses werefound with 100 and 1,000 nM valsartan, respectively, in both types of EAs.

Table 2. Inhibition of ANG II-induced changes in [Ca 2  ] i by valsartan in juxtamedullary EAs
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Fig. 3. Effect of valsartan on ANG II-induced changes in[Ca 2 ] i in juxtamedullary EAs. A and B: representative tracings showing the effect of 10 nM ( A )or 50 nM ( B ) valsartan (Vals) on the 2nd response induced by 1 nM ANGII in a juxtamedullary mEA ( left ) and tEA ( right ),respectively. Valsartan was added 5 min before and during the 2nd applicationof ANG II, and each of the 3 applications of ANG II lasted 7 min. C :concentration-inhibition curve showing mean inhibition (in % ± SE) ofthe integral Ca 2 signal calculated from comparison ofthe 1st and 2nd applications of ANG II. Right : Hill's plot of thedata. Values statistically different between mEAs and tEAs were determined byStudent's t -test: * P ** P *** P

Concentration-inhibition curves ( Fig.3 C ) indicate that mEAs and tEAs differ not only by theirsensitivity to valsartan but also by their inhibition kinetics. Indeed,inhibition curves in tEAs were sigmoid, with a Hill coefficient equal to 2.0, indicating positive cooperativity in the action of valsartan, whereas ahyperbolic curve compatible with Michaelis-Menten kinetics was observed inmEAs. Valsartan concentrations required for IC 50 deduced from thesecurves were 14 and 57 nM for mEAs and tEAs, respectively.
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Within 15 min after withdrawal of valsartan, both types of arterioles recovered partially from inhibition by concentrations of valsartan of 100nM, whereas no recovery from 1,000 nM valsartan was observed (data notshown).

Expression of AT 1A, AT 1B, and AT 2 Receptor mRNAs in EAs
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In a first experimental series ( Fig.4 A ), the distribution of AT 1A,AT 1B, and AT 2 receptor mRNAs was studied after 32-40 PCRcycles in 11 mEAs (mean length: 200 ± 17 µm), 11 tEAs (mean length:80 ± 6 µm), and 11 AAs (mean length: 157 ± 17 µm) used ascontrols. Results show that three cDNA fragments of the expected size forAT 1A, AT 1B, and AT 2 receptors wereconsistently found in mEAs, as in AAs, after only 32 cycles, whereas no band(or only a faint signal for AT 1A mRNA) could be observed in tEAsafter 35-40 PCR cycles. Because the absence of signal in tEAs could be due tothe shorter length of the samples and to the smaller number of muscular cellsper unit length, the two-step PCR approach was carried out in a secondexperimental series on seven mEAs (146 ± 4 µm) and six tEAs (100± 11 µm) from four different rats. As illustrated by the example in Fig. 4 B, aliquots ofPCR1 products ( left ) were reamplified in PCR2 ( right ). Inthis experiment, as in 3 other ones ( Fig. 4 C ), the 3 bands already observed in mEAs were notmarkedly enhanced after 14-25 additional cycles. Under the same conditions, after PCR2, tEA samples showed 2 bands of 420 and 250 bp corresponding toAT 1A and AT 2 receptors, respectively, but no signalcould ever be observed at 341 bp (the size corresponding to AT 1B receptors) even after 25 additional cycles.

Fig. 4. Distribution of AT 1A, AT 1B, and AT 2 receptor mRNAs in mEAs (m) and tEAs (t).  RTase and -RTase: arteriolar sampleswere treated with and without reverse transcriptase. A : arteriolarsamples submitted to a single PCR step. Note that 3 DNA fragments of theexpected size for AT 1A, AT 1B, and AT 2 receptor mRNAs were detected in mEAs, as in afferent arterioles (AAs), afteronly 32 PCR cycles ( left ), whereas no or only a faint signal forAT 1A mRNA (420 bp, far right lane) was observed in tEAsafter 35-40 cycles ( right ). B : representative experimentcarried out in 2 tEAs (1  RT and 1 -RT) and 2 mEAs (1  RT and 1 -RT) by the2-step PCR approach (see METHODS ). Left : data obtainedafter 40 cycles in 1st PCR (PCR1); right : data obtained after 2nd PCR(PCR2; 17 additional cycles) on the same arteriolar samples (starting from1/50 of PCR1 product). C : results obtained after 40 PCR1 cycles (notshown) and 14-25 PCR2 cycles on 4 other samples of mEA ( left ) and 5other samples of tEA ( right ). Note that even after 25 cycles ( farright lane), AT 1A and AT 2, but not AT 1B receptor, mRNAs were detected in tEAs. No amplification product was detectedin -RT tubes (25 cycles).

DISCUSSION: T$ ]& X9 F) \6 ~# _$ J3 h" s, {
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Results from the present study demonstrate a qualitative and quantitativedifferential expression of AT receptor mRNAs in juxtamedullary EAs of the ratkidney. mEAs express the three types of AT receptor mRNAs, namely,AT 1A, AT 1B, and AT 2, whereas tEAs lack theAT 1B subtype. It is worth mentioning that mEAs express the sametypes of AT receptors as do AAs ( Fig.4 A ) ( 27 )that display the same thick muscular wall and as do vasa rectae( 27 ) that derive fromjuxtamedullary mEAs. In contrast to these data, it has been reported recentlythat mEAs from juxtamedullary nephrons do not exhibit a contractile response under ANG II stimulation in AT 1A receptor null mice( 16 ), an observationsuggesting that mouse mEAs are devoid of AT 1B receptors. Speciesdifferences might account for this apparent discrepancy.

From a quantitative point of view, expression levels of AT 1A andAT 2 receptor mRNAs were much lower in tEAs than in mEAs. Indeed,although the RT-PCR assay used in this study was not fully quantitative, it isclear that detection of PCR products in tEAs required more than 10 additionalPCR cycles compared with mEAs. Such a difference, which reflects a theoretical 1,000-fold difference in cDNA abundance, cannot be accounted for only bythe difference in sample size. From a functional point of view, quantitativedifferences between mEAs and tEAs were less marked. Indeed, ANG II-induced[Ca 2 ] i increases only differ in amplitude bya factor of these responses were entirely abolished by thespecific AT 1 -receptor antagonist valsartan, demonstrating that theywere entirely accounted for by AT 1 receptors. This apparentdiscrepancy between the expression levels of AT 1 receptor mRNAs andthe functional response to ANG II in mEAs and tEAs reflects either that there is no linear relationship between levels of mRNA and protein expression, orthat the efficiency of the mechanisms between receptor occupancy and increasein [Ca 2 ] i is much higher in tEAs than inmEAs, or that the presence of AT 1B receptors in mEAs somehowdownregulates the ANG II-induced [Ca 2 ] i increases.

Besides AT 2 receptors, which are not directly coupled to[Ca 2 ] i increases, tEAs only expressAT 1A receptors. Thus the sensitivity of theCa 2 response to ANG II (EC 50 0.12 nM)( 18 ) and to valsartan(IC 50 60 nM) observed in tEAs likely reflects the intrinsicproperties of AT 1A receptors. Inhibition of the ANG II response byvalsartan in tEAs revealed a positive cooperativity, suggesting interactionbetween AT 1A receptors.

In mEA, valsartan inhibited ANG II's effect according to a single exponential kinetics, a result suggesting that valsartan has the same affinityfor AT 1A and AT 1B receptors( 8, 9 ). Were it the case, however,the apparent IC 50 for valsartan should be the same in mEAs andtEAs, which is not observed. Not only was the apparent affinity for valsartanhigher in mEAs than in tEAs, which is consistent with the lower apparentaffinity of mEAs for ANG II compared with tEAs( 18 ), but also the inhibition curve in mEAs displayed no positive cooperativity. To account for thisapparent discrepancy, it could be proposed that AT 1A andAT 1B receptors display different sensitivities to valsartan, aspreviously shown for losartan in adrenocortical Y-1 cells (9.7 vs. 4.7 nM, forAT 1A and AT 1B, respectively)( 32 ), but that the differencein sensitivity is too modest to be detectable in a tissue coexpressing the twosubtypes of receptors. If true, our findings would suggest 1 ) thatANG II-induced [Ca 2 ] i increases in mEAs arein large part accounted for by activation of AT 1B receptors, whichhave a higher affinity for valsartan than AT 1A receptors, or 2 ) that activation of AT 1B receptors somehow downregulatesthe Ca 2 response triggered by AT 1A receptors, as suggested above. Further studies in cells transfected witheither AT 1A or AT 1B receptors will be necessary todefinitely answer this question.! e+ @$ M& t  a. Q5 l, L" S

Besides AT 1 receptors, both mEAs and tEAs also expressAT 2 receptors. At the present time, there is no published data indicating whether AT 1 receptors are expressed in nativeendothelial cells and AT 2 receptors in native muscular cells ofglomerular arterioles. However, there is a large body of evidence indicating that AT 2 receptors antagonizing the AT 1 receptor-mediated response are localized in endothelial cells of glomerularvessels ( 19 ). For example, thevasodilation action of ANG II has been demonstrated in both glomerular AAs( 3 ) and EAs( 12 ). The AT 2 receptor-mediated vasorelaxing effect of ANG II is a paracrine regulationthought to result from ANG II-induced release of bradykinin( 6, 21, 31 ). In turn, bradykinin maytrigger different signaling pathways, leading to the production of variousrelaxing factors (including nitric oxide, epoxyeicosatetranoic acids,prostacyclins, and endothelium-derived hyperpolarizing factor) by vascularendothelial cells ( 5, 28, 29 ). The nature of therelaxing factor produced by endothelial cells depends essentially on the typeof vessel ( 26, 28 ). It is therefore possiblethat activation of AT 2 receptors in endothelial cells from mEAs andtEAs releases distinct factors that would influence AT 1 receptor-induced changes in [Ca 2 ] i differentially. Thus the distinct behaviors of calcium signals toward ANG IIand valsartan in mEAs and tEAs may result from differential expression ofAT 1B receptors in smooth muscle cells from these two types ofarterioles, but also from differential counter regulation by paracrine factors released by endothelial cells upon AT 2 activation.8 \6 B$ ?3 m4 l5 p+ A8 [, _: `

In summary, results from the present study further document the differencesbetween rat mEAs and juxtamedullary tEAs with regard to ANG II signalization.These two types of arterioles differ not only by their structure, topology,and sensitivity to ANG II but also by their sensitivity to the AT 1 antagonist valsartan and by the molecular expression of AT 1 receptor subtypes." K6 ~0 K. R& O  @
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DISCLOSURES. V$ H1 D7 `9 H2 s( H7 @

This study was supported in part by grants from the Bristol Myers-SquibbInstitute for Medical Research (Princeton, NJ) and from Novartis Pharma. C. M.B. Helou was supported in part by a grant for foreign scientists from theFondation pour la Recherche Médicale. She is a member of theLaboratório de Pesquisa Básica (LIM-12), Nephrology,HC-Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.0 [3 E9 M5 l$ o2 u0 w5 z
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