Angiotensin receptor subtypes in thin and muscular juxtamedullary efferent arterioles of rat kidney

Claudia M. B. Helou,1 Martine Imbert-Teboul,2 Alain Doucet,2 Rabary Rajerison,1 Catherine Chollet,1 François Alhenc-Gelas,1 and Jeannine Marchetti1

1Institut National de la Santé et de la Recherche Médicale Unité 367, Physiologie et Pathologie Expérimentale Vasculaires, Université Paris VI, 75005 Paris; and 2Laboratoire de Biologie Intégrée des Cellules Rénales, Unité de Recherche Associée 1859 au Centre National de la Recherche Scientifique, Commissariat a l'Energie Atomique-Saclay, 91191 Gif-sur-Yvette, France

Submitted 19 December 2002 ; accepted in final form 2 May 2003


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANG II controls the vascular tone of pre- and postglomerular arterioles, and thereby glomerular filtration, through binding to either AT1A, AT1B, or AT2 receptors. AT1 receptors, which are coupled to intracellular Ca2+ signaling, have vasoconstricting effects, whereas AT2 receptors, whose signaling mechanism is unknown, induce vasodilatation. The angiotensin receptors have been characterized in afferent arterioles, which express the three types of receptors, but not in efferent arterioles. Two subpopulations of juxtamedullary efferent arterioles, muscular ones which terminate as vasa rectae and thin ones which terminate as peritubular capillaries, have been described. They display functional heterogeneity with regard to the ANG II response. To evaluate whether these differences are associated with differential expression of ANG II receptors, we examined the expression pattern of AT1A, AT1B, and AT2 receptor mRNAs by RT-PCR in these arterioles and studied the effect of valsartan, a specific AT1-receptor antagonist. Results indicate that muscular arterioles express AT1A, AT1B, and AT2 receptors, whereas thin arterioles only express the AT1A and AT2 types, and at a much lower level. Valsartan fully inhibited ANG II-induced increases in intracellular Ca2+ in both arteriolar types, but with different kinetics. In muscular arterioles, inhibition was monoexponential, whereas it displayed a marked positive cooperativity in thin arterioles. Finally, the apparent affinity for valsartan was higher in muscular than in thin arterioles. In conclusion, this study further documents the differences between muscular and thin efferent arterioles with regard to ANG II signalization in the rat kidney.

angiotensin II; valsartan; calcium signaling


PRE- AND POSTGLOMERULAR ARTERIOLES are small resistance vessels that regulate renal microcirculation and glomerular filtration. ANG II is a major mediator of the regulation of glomerular filtration through its combined control of the vascular tone in afferent and efferent arterioles (AAs and EAs, respectively).

The effects of ANG II are mediated by at least two types of cell-surface receptors, namely, AT1 and AT2 receptors. AT1 receptors, which are coupled to an intracellular Ca2+ signaling pathway (33), are thought to be present in vascular smooth muscle cells and to mediate the vasoconstriction effects of ANG II in the glomerular arterioles (2, 24). In rats, two subtypes of AT1 receptors have been cloned (AT1A and AT1B receptors). These two receptors, which share 90% identity in amino acid sequence, display hardly distinguishable pharmacological properties (4, 8, 9), except in one study, which indicated that the AT1-receptor antagonist losartan was more potent on AT1B than AT1A receptors (32). AT2 receptors, which are present in endothelial cells, mediate the vasodilator effect of ANG II in AAs (3) and in EAs (12). However, the signaling pathways of AT2 receptors are not yet fully established. When AT1 and AT2 receptors are coexpressed in the same cell, AT1 receptor-mediated responses could be antagonized by AT2 receptors because of heterodimerization of the two receptors (1). Altogether, these data suggest that the vasoactive response of a given vessel to ANG II (constriction or dilatation) as well as its sensitivity to the peptide and to receptor antagonists might depend on the relative expression of the different types and subtypes of ANG II receptors.

To date, the distribution of the different types of ANG II receptors in renal vessels has been mainly investigated in AAs. Immunohistochemical (17) and radioligand binding (7) studies have shown that AT1 receptors are present in these vessels. By RT-PCR, Ruan et al. (30) found that the two subtypes of AT1 receptor mRNAs are expressed in preglomerular arterioles <50 µm in diameter, in a 4:1 AT1A/1AT1B ratio. AT2 receptor mRNAs are also expressed in AAs as well as arcuate arteries (27). In contrast, 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 the technical difficulty in isolating these different subpopulations of arterioles.

We have indeed described two subpopulations of juxtamedullary EAs that display morphological, topological, and functional differences (18). Based on morphology, one can distinguish muscular EAs (mEAs), which have a thick, regular, and muscular wall and terminate as vasa rectae, from thin EAs (tEAs), characterized by a thinner, irregular, and less muscular wall and which terminate as peritubular capillaries. Functionally, mEAs display higher increases in intracellular Ca2+ concentration ([Ca2+]i) than tEAs in response to ANG II, but they are slightly less sensitive to ANG II than are tEAs (18).

Therefore, this study aimed at determining whether these differences between mEAs and tEAs were associated with differential expression of ANG II receptors. For this purpose, we examined the expression of AT1A, AT1B, and AT2 receptor mRNAs by RT-PCR in these two subpopulations of EAs, and we compared the sensitivity of ANG II-induced increases in [Ca2+]i to the AT1-receptor antagonist valsartan.


    METHODS
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 METHODS
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Microdissection of Juxtamedullary Arterioles

Experiments were carried out in male Sprague-Dawley rats (Iffa-Credo, L'Arbresle, France) weighing 180-240 g. Juxtamedullary glomerular arterioles were isolated from collagenase-treated kidneys as previously described (18). Briefly, after anesthesia (pentobarbital sodium, 50 mg/kg ip) the left kidney was infused via the 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 containing 8 mg collagenase A (Clostridium histolyticum, 1.1 U/mg, Serva) and immediately removed, decapsulated, and longitudinally sliced. Small pyramids were cut and incubated for 8 min at 30°C in the presence of collagenase (1 mg/ml) and rinsed in cold standard solution. Juxtamedullary EAs attached to their 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 animal procedures were conducted in agreement with our institutional guidelines for the care and use of laboratory animals.

For [Ca2+]i measurements, the standard solution contained (in mM) 127 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 1 MgCl2, 4 NaHCO3, 2 CaCl2, 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 MgCl2, and 20 HEPES as well as 1 mg/ml protease-free BSA, pH 7.4, was used.

Measurements of [Ca2+]i

[Ca2+]i was evaluated with a Photoscan II microfluorimeter (Photon Technology) as previously described (25). Briefly, EAs were individually transferred on a thin glass slide within 1 µl of a standard solution containing 1% agarose (type IX, Sigma). After gelificaton of the agarose (1 min at 4°C), arterioles were loaded in the presence of 1 µl of 10 µM fura 2-AM (Molecular Probes, Leide, The Netherlands) for1hat room temperature in darkness. Then, the glass slide with the sample was mounted at the bottom of a superfusion chamber on the stage of an inverted fluorescent microscope (Nikon). The sample was continuously superfused with the standard solution (0.8 ml/min, 37°C) with or without test substances. The sample was alternatively excited at 340 and 380 nm (12 cycles/min), and the fluorescence emitted at 510 nm from a defined area (~25 x 30 µm) was measured. All values were corrected for autofluorescence determined at the two wavelengths after quenching of fura 2-AM fluorescence with 1 mM MnCl2 in the presence of 10 µM ionomycin. [Ca2+]i was calculated from the following equation (14): [Ca2+]i = Kd x {partial} (R - Rmin)/(Rmax - R), where Kd (the dissociation constant for the fura 2-Ca2+ complex) is 224 nM, R is the ratio of fluorescence emitted for each wavelength (340/380 nm), Rmax is the maximal ratio emitted in the presence of saturating Ca2+ (2 mM), Rmin is the minimal ratio measured in the absence of Ca2+ (0 mM), and {partial} is the ratio of fluorescence obtained at 380 nm in the absence and presence of 2 mM Ca2+. Values of Rmin, Rmax, and {partial} were periodically determined by external calibration using buffer that mimicked an intracellular medium (25).

The response to ANG II was evaluated either by the plateau value ({Delta}[Ca2+]i above baseline) or by the integral of the Ca2+ signal calculated as

where t0 and t1 correspond to the time for threshold of [Ca2+]i increment and for return to baseline value, respectively.

ANG II was dissolved in water, whereas the AT1-receptor antagonist 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 [Ca2+]i nor the [Ca2+]i response to ANG II.

Data are expressed as means ± SE. When each arteriole served as its own control, significance was obtained by a paired Student's t-test. Differences between the two groups of arterioles were analyzed with the use of an unpaired Student's t-test. Values were considered significantly different at P < 0.05. Commercially available Cricket Graph software was used to fit concentration-response curves and estimate IC50 and the Hill coefficient.

Expression of AT1 and AT2 Receptor mRNAs by RT-PCR

In view of the paucity and of the small size of microdissected glomerular arterioles, especially tEAs, we evaluated the expression of AT1A, AT1B, and AT2 receptor mRNAs by co-RT-PCR on the same arteriole samples in the same tube. Therefore, the number of PCR cycles was increased to allow detection of all three sequences, when coexpressed in the same 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 [GenBank] , S69961 [GenBank] , and D16840 [GenBank] for AT1A, AT1B, and AT2 receptors, respectively)] are as follows: AT1A sense, 5'-CTGGCTGATGGCTGGCTTGG-3' (bases 715-734), and antisense, 5'-TACGCTATGCAGATGGTGATGGG-3' (bases 1112-1134); AT1B sense, 5'-ATTCCCCCAACGGCCAAGTC-3' (bases 1540-1559), and antisense, 5'-GGCGGTTAACAGTGGCTTTGCTC-3' (bases 1858-1880); and AT2 sense, 5'-GAGCATGAGAGGTGGGCACTAAGG-3' (bases 1659-1682), and AT2 antisense, 5'-AAATAGCGTGCGCTCTATAACTTCAAGG-3' (bases 1881-1908). Because of the sequence homology between AT1A and AT1B receptor mRNAs (15, 20), primers for the two subtypes of AT1 receptors were checked by RT-PCR (11) on mRNAs extracted from kidney 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 the expected size (420 and 341 bp, respectively) were found in the kidney, which is known to express both AT1A and AT1B receptors, whereas a single DNA fragment (420 bp) was found in the liver, which expresses only the AT1A subtype (13, 23, 27).



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Fig. 1. Amplification of ANG II AT1A and AT1B receptor mRNAs. Because of the homology of AT1A and AT1B receptor sequences, PCR experiments were carried out in liver (250 ng total RNA/tube) and whole kidney RNA extracts (500 ng/tube) to check the specificity of oligonucleotide primers. PCR conditions were those previously described by Elalouf et al. (11), and 35 cycles were done to allow detection of putative nonspecific PCR products, if present. As shown by the representative electrophoresis gels, only 2 DNA fragments of the expected size (420 and 341 bp for AT1A and AT1B receptors, respectively) were found in the kidney, which expresses both receptor subtypes, and only 1 fragment (420 bp) was found in the liver, which does not express the AT1B subtype. No amplification product was ever found in control samples run without reverse transcriptase in the same experiments. Note that, due to the high no. of PCR cycles, all signals were saturated. Therefore, band intensity does not reflect the relative proportion of AT1A and AT1B receptor mRNAs in kidney tissue.

 

RT-PCR on arterioles. After microdissection, each single arteriolar fragment was measured with a calibrated eyepiece micrometer, washed free of contaminating cells and debris in standard solution supplemented with 5 µM DTT and 20 U RNasin (Promega, Charbonnières, France), and transferred 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-PCR was then carried out using a technique derived from that described by Lambolez et al. (22).

RT. The following compounds were added to each reaction tube: 2 µl of a 5x RT mixture containing 2.5 mM of each dNTP (Invitrogen, Paisley, UK) and 25 µM random primers [pd(N)6, Roche Diagnostics]; 1 µl of 100 mM DTT; 0.5 µl RNasin (20 U); 0.2 µl of 50 mM MgCl2;3 µl standard solution containing 3x Triton X-100; and 0.3 µl RNAse-free DNAse (3 U, Stratagene, Amsterdam, The Netherlands). Before initiation of the RT reaction (10-µl final vol), all samples were incubated at 37°C for 45-60 min to permeabilize the arteriolar fragments and digest genomic DNA and were heated at 80°C for 2 min to inactivate DNase. RT was initiated at room temperature by adding 0.5 µl (100 U) Superscript II reverse transcriptase (Invitrogen), and cDNAs were synthetized at 37°C overnight. Negative RT controls were performed by omitting reverse transcriptase. cDNAs were stored at -20°C until PCR.

PCR. Because dissection of EAs, especially tEAs, is difficult, the distribution of AT1A,AT1B, and AT2 receptor mRNAs was studied in the same samples. cDNAs were therefore coamplified in the same tube in a final volume of 100 µl. Seventy microliters of a PCR mixture containing 2.5 U Taq DNA polymerase and 10 µl of 10x PCR buffer (Qiagen, Courtaboeuf, France) were first added to the RT product (10 µl). All samples were held 1 min at 94°C and then received 20 µl of a mixture containing sense and antisense primers for AT1A, AT1B, and AT2 receptors (10 pmol each). In a first experimental series, they were then processed for 32-40 cycles at three sequential temperature steps: 95°C for 30 s; 60°C for 30 s; and 71°C for 1 min, with the exception of the last cycle, in which the elongation lasted 10 min.

An analysis of data obtained under these conditions in tEAs revealed only faint, if any, amplification products, even after 40 PCR cycles. Therefore, in a second series of experiments, two PCR steps were sequentially carried out in mEAs 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. For this purpose, 78 µl of a PCR mixture containing 10 µl 10x Taq buffer, 2.5 U Taq DNA polymerase, and 1 µl dNTPs (5 mM) were added to each tube; samples were then processed as described above and submitted to 14-25 additional PCR cycles. In all experiments, negative controls including blanks (no tissue) and arteriolar samples without reverse transcriptase were run in parallel.

PCR products were separated by electrophoresis on 2% agarose gels containing ethidium bromide and visualized by UV illumination.


    RESULTS
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 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nondesensitization of [Ca2+]i Responses of Juxtamedullary Arterioles to 1 nM ANG II

In both tEAs and mEAs, superfusion by 1 nM ANG II induced a rapid increase in [Ca2+]i followed by a sustained plateau. As previously reported (18), the magnitude of the plateau was significantly higher in mEAs than in tEAs. On ANG II removal, [Ca2+]i slowly decreased down to 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 a third application of ANG II to the same arterioles (Fig. 2 and Table 1), indicating the absence of desensitization phenomena. Thus each arteriole could be used as its own 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 application of ANG II after a 15-min washing allowed estimation of the recovery of arteriolar responsiveness from antagonist action.



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Fig. 2. Reproducibility of intracellular Ca2+ concentration ([Ca2+]i) responses to 1 nM ANG II in thin and muscular efferent arterioles [tEAs (A) and mEAs (B), respectively]. Representative tracings showing the effect of 3 successive applications of ANG II (1 nM, 7 min) on [Ca2+]i in juxtamedullary tEAs and mEAs are shown.

 

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Table 1. Absence of desensitization of Ca2+ responses to angiotensin in efferent arterioles

 

Effect of AT1-Receptor Antagonist Valsartan

Results in Table 2 and Fig. 3 indicate that changes in [Ca2+]i induced by 1 nM ANG II were inhibited by valsartan in both mEAs and tEAs, but with different sensitivities. Up to 10 nM valsartan, no significant inhibition was detected in tEAs, whereas a marked inhibition ({approx}40%) was observed in mEAs. The inhibitory effects of 30 and 50 nM valsartan were also more marked in mEAs than in tEAs (Fig. 3, A and B), whereas nearly complete inhibition (~90%) and a total suppression of [Ca2+]i responses were found with 100 and 1,000 nM valsartan, respectively, in both types of EAs.


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Table 2. Inhibition of ANG II-induced changes in [Ca2+]i by valsartan in juxtamedullary EAs

 


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Fig. 3. Effect of valsartan on ANG II-induced changes in [Ca2+]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 ANG II in a juxtamedullary mEA (left) and tEA (right), respectively. Valsartan was added 5 min before and during the 2nd application of ANG II, and each of the 3 applications of ANG II lasted 7 min. C: concentration-inhibition curve showing mean inhibition (in % ± SE) of the integral Ca2+ signal calculated from comparison of the 1st and 2nd applications of ANG II. Right: Hill's plot of the data. Values statistically different between mEAs and tEAs were determined by Student's t-test: *P < 0.05; **P < 0.01; ***P < 0.005.

 

Concentration-inhibition curves (Fig. 3C) indicate that mEAs and tEAs differ not only by their sensitivity 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 a hyperbolic curve compatible with Michaelis-Menten kinetics was observed in mEAs. Valsartan concentrations required for IC50 deduced from these curves were 14 and 57 nM for mEAs and tEAs, respectively.

Within 15 min after withdrawal of valsartan, both types of arterioles recovered partially from inhibition by concentrations of valsartan of <=100 nM, whereas no recovery from 1,000 nM valsartan was observed (data not shown).

Expression of AT1A, AT1B, and AT2 Receptor mRNAs in EAs

In a first experimental series (Fig. 4A), the distribution of AT1A, AT1B, and AT2 receptor mRNAs was studied after 32-40 PCR cycles in 11 mEAs (mean length: 200 ± 17 µm), 11 tEAs (mean length: 80 ± 6 µm), and 11 AAs (mean length: 157 ± 17 µm) used as controls. Results show that three cDNA fragments of the expected size for AT1A, AT1B, and AT2 receptors were consistently found in mEAs, as in AAs, after only 32 cycles, whereas no band (or only a faint signal for AT1A mRNA) could be observed in tEAs after 35-40 PCR cycles. Because the absence of signal in tEAs could be due to the shorter length of the samples and to the smaller number of muscular cells per unit length, the two-step PCR approach was carried out in a second experimental series on seven mEAs (146 ± 4 µm) and six tEAs (100 ± 11 µm) from four different rats. As illustrated by the example in Fig. 4B, aliquots of PCR1 products (left) were reamplified in PCR2 (right). In this experiment, as in 3 other ones (Fig. 4C), the 3 bands already observed in mEAs were not markedly enhanced after 14-25 additional cycles. Under the same conditions, after PCR2, tEA samples showed 2 bands of 420 and 250 bp corresponding to AT1A and AT2 receptors, respectively, but no signal could ever be observed at 341 bp (the size corresponding to AT1B receptors) even after 25 additional cycles.



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Fig. 4. Distribution of AT1A, AT1B, and AT2 receptor mRNAs in mEAs (m) and tEAs (t). +RTase and -RTase: arteriolar samples were treated with and without reverse transcriptase. A: arteriolar samples submitted to a single PCR step. Note that 3 DNA fragments of the expected size for AT1A, AT1B, and AT2 receptor mRNAs were detected in mEAs, as in afferent arterioles (AAs), after only 32 PCR cycles (left), whereas no or only a faint signal for AT1A mRNA (420 bp, far right lane) was observed in tEAs after 35-40 cycles (right). B: representative experiment carried out in 2 tEAs (1 +RT and 1 -RT) and 2 mEAs (1 +RT and 1 -RT) by the 2-step PCR approach (see METHODS). Left: data obtained after 40 cycles in 1st PCR (PCR1); right: data obtained after 2nd PCR (PCR2; 17 additional cycles) on the same arteriolar samples (starting from 1/50 of PCR1 product). C: results obtained after 40 PCR1 cycles (not shown) and 14-25 PCR2 cycles on 4 other samples of mEA (left) and 5 other samples of tEA (right). Note that even after 25 cycles (far right lane), AT1A and AT2, but not AT1B receptor, mRNAs were detected in tEAs. No amplification product was detected in -RT tubes (25 cycles).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Results from the present study demonstrate a qualitative and quantitative differential expression of AT receptor mRNAs in juxtamedullary EAs of the rat kidney. mEAs express the three types of AT receptor mRNAs, namely, AT1A, AT1B, and AT2, whereas tEAs lack the AT1B subtype. It is worth mentioning that mEAs express the same types of AT receptors as do AAs (Fig. 4A) (27) that display the same thick muscular wall and as do vasa rectae (27) that derive from juxtamedullary mEAs. In contrast to these data, it has been reported recently that mEAs from juxtamedullary nephrons do not exhibit a contractile response under ANG II stimulation in AT1A receptor null mice (16), an observation suggesting that mouse mEAs are devoid of AT1B receptors. Species differences might account for this apparent discrepancy.

From a quantitative point of view, expression levels of AT1A and AT2 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 is clear that detection of PCR products in tEAs required more than 10 additional PCR cycles compared with mEAs. Such a difference, which reflects a theoretical {approx}1,000-fold difference in cDNA abundance, cannot be accounted for only by the difference in sample size. From a functional point of view, quantitative differences between mEAs and tEAs were less marked. Indeed, ANG II-induced [Ca2+]i increases only differ in amplitude by a factor of <2, even though these responses were entirely abolished by the specific AT1-receptor antagonist valsartan, demonstrating that they were entirely accounted for by AT1 receptors. This apparent discrepancy between the expression levels of AT1 receptor mRNAs and the functional response to ANG II in mEAs and tEAs reflects either that there is no linear relationship between levels of mRNA and protein expression, or that the efficiency of the mechanisms between receptor occupancy and increase in [Ca2+]i is much higher in tEAs than in mEAs, or that the presence of AT1B receptors in mEAs somehow downregulates the ANG II-induced [Ca2+]i increases.

Besides AT2 receptors, which are not directly coupled to [Ca2+]i increases, tEAs only express AT1A receptors. Thus the sensitivity of the Ca2+ response to ANG II (EC50 {approx}0.12 nM) (18) and to valsartan (IC50 {approx}60 nM) observed in tEAs likely reflects the intrinsic properties of AT1A receptors. Inhibition of the ANG II response by valsartan in tEAs revealed a positive cooperativity, suggesting interaction between AT1A receptors.

In mEA, valsartan inhibited ANG II's effect according to a single exponential kinetics, a result suggesting that valsartan has the same affinity for AT1A and AT1B receptors (8, 9). Were it the case, however, the apparent IC50 for valsartan should be the same in mEAs and tEAs, which is not observed. Not only was the apparent affinity for valsartan higher in mEAs than in tEAs, which is consistent with the lower apparent affinity of mEAs for ANG II compared with tEAs (18), but also the inhibition curve in mEAs displayed no positive cooperativity. To account for this apparent discrepancy, it could be proposed that AT1A and AT1B receptors display different sensitivities to valsartan, as previously shown for losartan in adrenocortical Y-1 cells (9.7 vs. 4.7 nM, for AT1A and AT1B, respectively) (32), but that the difference in sensitivity is too modest to be detectable in a tissue coexpressing the two subtypes of receptors. If true, our findings would suggest 1) that ANG II-induced [Ca2+]i increases in mEAs are in large part accounted for by activation of AT1B receptors, which have a higher affinity for valsartan than AT1A receptors, or 2) that activation of AT1B receptors somehow downregulates the Ca2+ response triggered by AT1A receptors, as suggested above. Further studies in cells transfected with either AT1A or AT1B receptors will be necessary to definitely answer this question.

Besides AT1 receptors, both mEAs and tEAs also express AT2 receptors. At the present time, there is no published data indicating whether AT1 receptors are expressed in native endothelial cells and AT2 receptors in native muscular cells of glomerular arterioles. However, there is a large body of evidence indicating that AT2 receptors antagonizing the AT1 receptor-mediated response are localized in endothelial cells of glomerular vessels (19). For example, the vasodilation action of ANG II has been demonstrated in both glomerular AAs (3) and EAs (12). The AT2 receptor-mediated vasorelaxing effect of ANG II is a paracrine regulation thought to result from ANG II-induced release of bradykinin (6, 21, 31). In turn, bradykinin may trigger different signaling pathways, leading to the production of various relaxing factors (including nitric oxide, epoxyeicosatetranoic acids, prostacyclins, and endothelium-derived hyperpolarizing factor) by vascular endothelial cells (5, 28, 29). The nature of the relaxing factor produced by endothelial cells depends essentially on the type of vessel (26, 28). It is therefore possible that activation of AT2 receptors in endothelial cells from mEAs and tEAs releases distinct factors that would influence AT1 receptor-induced changes in [Ca2+]i differentially. Thus the distinct behaviors of calcium signals toward ANG II and valsartan in mEAs and tEAs may result from differential expression of AT1B receptors in smooth muscle cells from these two types of arterioles, but also from differential counter regulation by paracrine factors released by endothelial cells upon AT2 activation.

In summary, results from the present study further document the differences between 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 AT1 antagonist valsartan and by the molecular expression of AT1 receptor subtypes.


    DISCLOSURES
 
This study was supported in part by grants from the Bristol Myers-Squibb Institute for Medical Research (Princeton, NJ) and from Novartis Pharma. C. M. B. Helou was supported in part by a grant for foreign scientists from the Fondation pour la Recherche Médicale. She is a member of the Laboratório de Pesquisa Básica (LIM-12), Nephrology, HC-Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Marchetti, INSERM U367, 17 rue du Fer à Moulin, 75005 Paris, France (E-mail: marchett{at}ifm.inserm.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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