Endothelin resets renal blood flow autoregulatory efficiency during acute blockade of NO in the rat

R. Kramp, P. Fourmanoir, and N. Caron

Service de Physiologie et Pharmacologie, Faculté de Médecine et de Pharmacie, Université de Mons-Hainaut, 7000 Mons, Belgium


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First published August 15, 2001; 10.1152/ajprenal.00078.2001.---Renal blood flow (RBF) autoregulatory efficiency may be enhanced during NO inhibition in the rat, as recently reported. Under these conditions, endothelin (ET) synthesis and release may be increased. Our purpose was therefore to determine the role of ET in RBF autoregulatory changes induced by NO inhibition. To address this point, ETA/B receptors were blocked in anesthetized rats with bosentan, or selectively with BQ-610 or BQ-788. NO synthesis was inhibited with NG-nitro-L-arginine methyl ester (L-NAME). Mean arterial pressure (MAP) was decreased after bosentan (-10 mmHg; P < 0.01) or increased after L-NAME (25 mmHg; P < 0.001). RBF measured with an electromagnetic flow probe was reduced by L-NAME (-50%) and by BQ-788 (-24%). The pressure limits of the autoregulatory plateau (PA ~100 mmHg) and of no RBF autoregulation (Po ~80 mmHg) were significantly lowered by 15 mmHg after L-NAME but were unchanged after bosentan, BQ-610, or BQ-788. During NO inhibition, autoregulatory resetting was completely hindered by bosentan (PA ~100 mmHg) and by ETB receptor blockade with BQ-788 (PA ~106 mmHg), but not by ETA receptor blockade with BQ-610 (PA ~85 mmHg). These results suggest that the involvement of ET in the RBF autoregulatory resetting occurs during NO inhibition, possibly by preferential activation of the ETB receptor. However, the relative contribution of ET receptor subtypes remains to be further specified.

renal vascular resistance; renal hemodynamics; autoregulation; angiotensin; nitric oxide


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INTRODUCTION
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ENDOTHELIAL CELLS PRODUCE several vasoconstrictor and vasodilator factors that contribute to the control of hemodynamics. Among these factors, nitric oxide (NO), a potent vasodilator, has been postulated to set the vasomotor tone and to be implicated in autoregulation of blood flow (21). In this regard, it has been shown that renal blood flow (RBF) autoregulation in the dog and rat was maintained during an acute and systemic inhibition of NO synthesis, despite a marked increase in renal vascular resistance (RVR) (2, 5, 19). Recently, the efficiency of RBF autoregulation was shown to be enhanced during an acute inhibition of NO synthesis in anesthetized euvolemic rats (16, 29). Under these conditions, the autoregulatory plateau was significantly extended to a lower mean arterial pressure (MAP) (16). This autoregulatory resetting occurring during inhibition of NO synthesis was due, at least in part, to activation of voltage-dependent Ca2+ channels in smooth muscle cells of the preglomerular vasculature (16).

Besides NO, other paracrine factors synthesized in the endothelium, such as endothelin (ET), may also intervene in the setting of vasomotor tone through modulation of different Ca2+ transport systems in vascular smooth muscle cells, including voltage-dependent Ca2+ channels (22). Interactions between NO and ET could thus occur at these levels and influence renal hemodynamics. This likelihood is further emphasized by the demonstration that the synthesis as well as the release of ET in cultured human endothelial cells and in porcine aortas were enhanced after an acute inhibition of NO synthesis (6, 14). It has also been reported that NO can displace ET from its receptors located on vascular smooth muscle (10). Therefore, hemodynamic effects of ET could only become conspicuous in vivo when the activity of NO synthase is reduced or blocked. In view of these considerations, the purpose of our study was to delineate the involvement of ET in the autoregulatory resetting of RBF that occurs during an acute inhibition of NO synthesis. To do so, experiments in renal hemodynamics were undertaken in anesthetized euvolemic rats that were submitted to an acute blockade of ETA and ETB receptors and of NO synthesis with the use of bosentan and L-NAME, respectively. Furthermore, ETA or ETB receptors were selectively blocked with BQ-610 and BQ-788, respectively, to unravel the type of ET receptor involved in the autoregulatory resetting (12, 20). For comparative purposes, interactions between NO and ANG II were also investigated with the use of losartan to block angiotensin AT1 receptors.


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Animal preparation. Male Wistar rats were maintained on a standard diet as previously reported (15, 16). Before experimentation, the rats, weighing ~300 g, were deprived of food overnight but had free access to tap water. They were anesthetized with Inactin (10 mg/100 g body wt ip; Byk-Gulden or Research Biochemicals International, Natick, MA) and placed on a heated table to maintain a rectal temperature between 37 and 38°C. The left femoral artery was first catheterized to determine the initial hematocrit (Hct) and to measure blood pressure, using a Statham P23 ID pressure transducer connected to a pressure monitor (Hugo Sachs, Elektronik, March-Freiburg, Germany) and a recorder. The right femoral artery, for subsequent blood sampling, and vein were then rapidly catheterized. To avoid fluid shifts during further surgery, a 0.85% saline solution containing 2.5% albumin was infused immediately into the right femoral vein as previously specified (15, 16). After a tracheostomy was performed, the right jugular vein was catheterized for subsequent infusions. The left kidney was exposed through a midline and subcostal abdominal incision as previously described (15). The segment of the aorta located between or above the two renal arteries as well as the left renal artery were then carefully dissected from surrounding tissues, avoiding interference with nerve bundles as much as possible (15). Finally, each ureter was cannulated for urine collection. After completion of surgery, a 0.85% NaCl solution was infused at a rate of 50 µl/min. The kidneys were removed, decapsulated, blotted dry, and weighed at the end of the experiment.

Renal hemodynamics. An adjustable constriction clamp was placed around the aorta above or beneath the right renal artery to reduce renal perfusion pressure (RPP) in a stepwise manner. A small-diameter, noncannulating, and factory-precalibrated electromagnetic flow transducer, connected to a square-wave electromagnetic flowmeter (MDL 1401 compact; Skalar Medical, Delft, The Netherlands) and a recorder, was vertically fitted around the left renal artery to continuously measure RBF. Calibration and use of these flow sensors as well as the final tests to check the accuracy of RBF measurements at the end of the experiment have been previously described in detail (15). No marked changes in RBF measurements before or after these final tests were found.

RBF autoregulatory efficiency was investigated by stepwise aortic constrictions inducing 5-mmHg decrements in RPP from spontaneous mean arterial pressure (MAP) down to 60 mmHg and by measurement of RBF during at least 30-s periods. No measurements were undertaken during increased MAP.

Experimental protocol. After a 60- to 90-min equilibration, baseline measurements of hemodynamics were carried out during three control periods of 20-min duration each. Control autoregulatory maneuvers (A1) were undertaken during the second control period. After the control periods, vehicle (200 µl/kg body wt of 0.85% NaCl in doubly distilled water), bosentan (10 mg/kg body wt, Hoffman-La Roche, Basel, Switzerland) (7, 24); NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg body wt; Sigma, St Louis, MO) (4, 5, 15); BQ-610 or BQ-788 (8 µg · kg body wt-1 · min-1 after a priming injection of 100 µg/kg body wt, American Peptide, Sunnyvale, CA); or losartan (3 mg/kg body wt unless stated otherwise, DuPont-Merck, Wilmington, DE) was administered intravenously. Preliminary experiments demonstrated that this dose of losartan rapidly and fully blocked the effects of repeated injections of 10 ng of ANG II on MAP and RBF until the end of the experiment (data not shown). After a brief equilibration period, six experimental periods of 20-min duration each were then undertaken. Autoregulatory maneuvers were carried out during the second (A2) and fifth (A3) experimental periods to allow time for reequilibration. After the first three experimental periods, L-NAME was injected intravenously in some of the rats treated with bosentan, BQ-610, BQ-788, or losartan for 1 h, while bosentan or losartan was administered intravenously in some of the rats treated with L-NAME for 1 h. Bosentan and L-NAME, or losartan, bosentan and L-NAME, were simultaneously coadministered in other rats after the last control period.

Three main study groups, consisting of the following 12 experiments, were considered.

Group 1 included, for control purposes, six rats treated with vehicle, seven rats injected with bosentan, and seven rats injected with L-NAME (Vehicle, Bosentan, and NAME experiments, respectively).

Group 2 included, for assessment of ET and NO interactions, six rats injected with bosentan and, after 1 h, with L-NAME; seven rats injected with L-NAME and, after 1 h, with bosentan; seven rats treated concomitantly with bosentan and L-NAME; six rats continuously infused with BQ-610 and, after 1 h, injected with L-NAME; and five rats continuously infused with BQ-788 and, after 1 h, injected with L-NAME. These are hereinafter referred to as Bosentan/NAME, NAME/Bosentan, Bosentan+NAME, BQ-610/NAME, and BQ-788/NAME experiments, respectively.

Group 3 included, for assessment of specificity of ET and NO interactions, six rats injected with losartan; six rats injected with losartan and, after 1 h, with L-NAME; six rats injected with L-NAME and, after 1 h, with losartan (at a dose of 10 mg/kg body wt in 4 rats); and four rats treated concomitantly with losartan (at a dose of 10 mg/kg body wt iv), bosentan, and L-NAME. These are hereinafter referred to as Losartan, Losartan/NAME, NAME/Losartan, and Losartan+Bosentan+ NAME experiments, respectively.

Calculations and statistics. Renal hemodynamic data were obtained from the left kidney, henceforth referred to as the experimental kidney. Mean values of MAP and RBF were obtained by averaging measurements carried out on the recordings every 5 min, except during autoregulatory maneuvers. RVR was calculated as MAP/RBF, where MAP refers to the pressure in the femoral artery. RPP was equated with femoral MAP. The relationship between RBF and MAP was assessed independently by three investigators for each experiment. To do so, progressive linear regressions were applied using the least squares method. They were determined on segments composed of three RBF values corresponding to three pressure levels, starting from the initial MAP and progressing down to lower pressures by successive substitutions in steps of 5 mmHg. The magnitude of the correlation coefficient, the slope, and the intercept of the best-fit regression lines, as well as the amplitude of the change in RBF, were taken as criteria to attribute a data point with a precision of 5 mmHg to one of the following components of the RBF-MAP relationship: 1) the autoregulatory plateau, which corresponds to the pressure range in which autoregulatory efficiency was maximal; 2) a subautoregulatory or transition zone, which corresponds to the pressure range in which RBF autoregulation became less efficient; and 3) an absence of autoregulation, which corresponds to the pressure range in which RBF changes were fully pressure dependent. The perfusion pressure, corresponding to the lower limit of the autoregulatory plateau, was defined as the pressure limit of efficient RBF autoregulation (PA). The perfusion pressure, corresponding to disappearance of RBF autoregulation, was defined as the pressure limit of no RBF autoregulation (Po).

One-way ANOVA was applied for multiple intergroup comparisons, and one-way ANOVA for repeated measurements was applied for multiple intragroup comparisons, followed by a post hoc t-test (30). The paired t-test was used for a single ecomparison within a group. The unpaired t-test was used for a single comparison between two groups. All results are presented as means ± SE. P < 0.05 was considered to be statistically significant.


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Experiments were undertaken in 73 rats. Their body weight and left and right kidney weights averaged 292 ± 1, 1.167 ± 0.014, and 1.179 ± 0.015 g, respectively.

Temporal evolution of MAP and RBF during ET and NO blockade. As shown in Table 1, mean baseline values for Hct, MAP, RBF,and RVR did not differ significantly between experiments in groups 1 and 2. Figure 1, A and B, illustrates, respectively, the temporal variations in MAP and RBF, from before each treatment until the end of the experiment. These variables did not change after injection of vehicle, nor did they with time. In the Bosentan experiment, MAP decreased progressively by 10 mmHg, whereas RBF remained stable. Conversely, in the NAME experiment, MAP increased rapidly by 20-25 mmHg and remained stable thereafter, whereas RBF decreased rapidly by 50%, averaging 3.96 ± 0.25 ml/min at the end of the experiment. In the Bosentan/NAME experiment, MAP decreased to 104 ± 2 mmHg 1 h after injection of bosentan but increased rapidly to 125 ± 3 mmHg after the superimposed injection of L-NAME. In the NAME/Bosentan experiment, MAP increased again markedly after injection of L-NAME but decreased progressively after the superimposed injection of bosentan, averaging 121 ± 7 mmHg at the end of the experiment. In the Bosentan+NAME experiment, MAP increased to a similar extent as with L-NAME alone and was almost constant until the end of the experiment. In these three experiments, RBF was similarly decreased at the end of the experiment. In the BQ-610/NAME experiment, MAP decreased by 8 mmHg and RBF increased by 6% after 1 h infusion of BQ-610. They increased and decreased, respectively, as usual after the superimposed injection of L-NAME. In the BQ-788/NAME experiment, MAP did not change whereas RBF decreased rapidly by 24% during the infusion of BQ-788. These variables increased and further decreased, respectively, by 21 mmHg and by 57% after the superimposed injection of L-NAME. At the end of the experiment, RVR did not differ from baseline in the Vehicle experiment but was reduced or elevated, respectively, to 12.7 ± 0.5 (P < 0.05, from baseline) and 34.1 ± 2.0 (P < 0.0001) mmHg · ml-1 · min in the Bosentan and NAME experiments. RVR was also markedly enhanced by L-NAME, averaging, respectively, 31.1 ± 3.9 (P < 0.0032), 39.9 ± 3.4 (P < 0.0002), 37.8 ± 3.2 (P < 0.0001), 34.5 ± 2.2 (P < 0.0001), and 52.7 ± 4.5 mmHg · ml-1 · min (P < 0.0001), whether bosentan was given before, after, or coadministered with L-NAME, as well as after pretreatment with BQ-610 or BQ-788.

                              
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Table 1.   Baseline values for hematocrit, mean arterial pressure, renal blood flow, and renal vascular resistance in experiments undertaken to determine ET and NO interactions in study groups 1 and 2 



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Fig. 1.   Temporal variations of mean arterial pressure (MAP; A) and renal blood flow (RBF; B) in Vehicle (; n = 6), Bosentan (open circle ; n = 7), NG-nitro-L-arginine methyl ester (L-NAME; NAME; black-triangle; n = 7), Bosentan/NAME (Delta ; n = 6), NAME/Bosentan (; n = 7), Bosentan+NAME (; n = 7), BQ-610/NAME (black-lozenge ; n = 6), or BQ-788/NAME (diamond ; n = 5) experiments. Values are means ± SE illustrated for the last control and the 6 experimental periods and set in the middle of each 20-min period; some SE are too small to be shown. Differences in temporal variations of MAP and RBF were evaluated by ANOVA for repeated measurements followed by a Newman-Keuls test between control and experimental values (*P < 0.05) and between the experimental values at the end of the first hour of treatment and the last 3 experimental values (+P < 0.05).

RBF autoregulation during ET and NO blockade. Figure 2 illustrates the RBF-MAP relationship during control (A1) and experimental (A2 and A3) periods in the Vehicle (A), Bosentan (B), and NAME (C) experiments. The three autoregulatory curves were superimposed and were essentially similar in the Vehicle and Bosentan experiments (Fig. 2, A and B). In contrast, the RBF autoregulatory plateau was markedly extended into the subautoregulatory zone (A2 and A3) in the NAME experiment (Fig. 2C). Figure 3, A, B, and C, illustrates autoregulatory curves determined 90 min after different treatments were started (A3). At each level of MAP, RBF is now expressed as a percentage of RBF at a reference MAP of 100 mmHg to emphasize changes in RBF autoregulation, especially so when RBF was reduced. For comparative purposes, RBF autoregulatory curves in the Vehicle, Bosentan, and NAME experiments are presented in Fig. 3A. In the NAME experiment, the autoregulatory plateau was markedly extended to a lower MAP, whereas RBF at a pressure of 60 mmHg was less decreased than in the Vehicle or Bosentan experiments. In contrast, the RBF autoregulatory plateau was not extended to a lower MAP in the Bosentan/NAME or NAME/Bosentan experiments (Fig. 3B). As illustrated in Fig. 3C, in the Bosentan+NAME experiment, the RBF-MAP relationship in four rats was similar to the profiles shown in Fig. 3B and to the profile of the NAME experiment presented in Fig. 3A in three other rats. Note that the autoregulatory plateau was not extended to a lower MAP in these seven rats 30 min after coadministration was started (data not illustrated).


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Fig. 2.   RBF-MAP relationships, with RBF expressed in ml/min during stepwise decrements by 5 mmHg in MAP, in Vehicle (n = 6; A), Bosentan (n = 7; B), or NAME (n = 7; C) experiments. Autoregulatory curves were determined 30 min before (A1; ) and 30 (A2; open circle ) and 90 min (A3; triangle ) after treatment was started. Values are means ± SE.



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Fig. 3.   RBF-MAP relationships with RBF expressed at each pressure decrement as a percentage of RBF at a reference MAP of 100 mmHg, determined 90 min (A3) after treatment was started, in Vehicle (nabla ; n = 6), Bosentan (Delta ; n = 7), or NAME (black-triangle; n = 7; A) experiments; in Bosentan/NAME (Delta ; n = 6), or NAME/Bosentan (nabla ; n = 7; B) experiments; or in Bosentan+NAME experiment (n = 7; C). In the latter experiments, 2 distinct autoregulatory profiles were observed during the second experimental hour (Delta ; n = 4; black-triangle; n = 3). In these different experiments, RBF at a MAP of 100 mmHg averaged, respectively, 8.30 ± 0.56, 9.38 ± 0.41, and 3.96 ± 0.21 ml/min (A); 4.19 ± 0.56 and 3.25 ± 0.19 ml/min (B); and 3.67 ± 0.59 and 3.21 ± 0.37 ml/min (C). Values are means ± SE. Bos, bosentan.

As shown in Fig. 4, after the superimposed injection of L-NAME, the autoregulatory plateau in the BQ-610/NAME experiment was almost similar to the profile in the NAME experiment but was disrupted in the BQ-788/NAME experiment. RBF tended then not to stabilize at each pressure decrement. Autoregulatory profiles during infusion of BQ-610 and BQ-788 alone were similar and comparable to Vehicle (not illustrated).


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Fig. 4.   RBF-MAP relationships with RBF expressed at each pressure decrement as a percentage of RBF at a reference MAP of 100 mmHg. Autoregulatory profiles were determined 90 min (A3) after treatment was started in BQ-610/NAME (black-triangle; n = 6) and BQ-788/NAME (Delta ; n = 5) experiments. RBF at a MAP of 100 mmHg averaged, respectively, 3.54 ± 0.23 and 2.54 ± 0.39 ml/min. Values are means ± SE.

Table 2 presents the autoregulatory pressure limits, PA and Po, during the control (A1) and experimental (A2 and A3) periods for the control experiments and those undertaken to study ET and NO interactions. PA and Po did not change in the Vehicle or Bosentan experiments but were significantly lowered by 15 mmHg in the NAME experiment. Resetting of PA and Po by L-NAME was, however, completely hindered by bosentan, whether administered before, after, or concomitantly with L-NAME, except during the second hour of coadministration (A3) in three rats. In the BQ-610/NAME and BQ-788/NAME experiments, PA after the superimposed injection of L-NAME was lowered or enhanced by ~10 mmHg, respectively. Po tended then to be lowered in the two groups.

                              
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Table 2.   Pressure limits of efficient and no RBF autoregulation in experiments undertaken to determine ET and NO interactions in study groups 1 and 2 

Hemodynamic effects of AT1 and NO or AT1, ETA/B, and NO blockade. To assess the specificity of the ET and NO interactions, autoregulatory experiments were also carried out during blockade of AT1 receptors and NO synthesis. As shown in Table 3, baseline values of systemic and renal hemodynamic variables, as well as PA and Po did not differ significantly between the different experiments. Note that baseline values of Hct (data not shown) were in the similar range as those presented in Table 1. MAP and RBF markedly decreased or increased, respectively, in the Losartan experiment, so that RVR was reduced by ~20%. In contrast, MAP and RBF were again markedly increased or decreased, respectively, by L-NAME in the three other groups. RVR was then significantly enhanced. As illustrated in Fig. 5A, the autoregulatory profile in the Losartan experiment (A3) was almost similar to the profile in the Vehicle or Bosentan experiment (see Fig. 3A). In the Losartan/NAME and NAME/Losartan experiments, the autoregulatory profiles were comparable to the profile in the NAME experiment, especially so in the NAME/Losartan experiment (see Fig. 3A). In this group, PA and Po were significantly decreased by 15 and 10 mmHg, respectively (Table 3). As shown in Fig. 5B, the autoregulatory profile in the Losartan+ Bosentan+NAME experiment was nearly identical to the profile in the Vehicle or Bosentan experiment shown in Fig. 3A. PA and Po were then similar to baseline (Table 3).

                              
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Table 3.   Baseline and experimental values for MAP, RBF, renal vascular resistance, and pressure limits of efficient and no RBF autoregulation in experiments undertaken to determine ANG II, ET, and NO interactions in group 3 



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Fig. 5.   RBF-MAP relationships with RBF expressed at each pressure decrement as a percentage of RBF at a reference MAP of 100 mmHg. Autoregulatory profiles were determined 90 min (A3) after treatment was started in Losartan (nabla ; n = 6), Losartan/NAME (Delta ; n = 6), and NAME/Losartan (black-triangle; n = 6) experiments (A) or in Losartan+Bosentan+NAME (Delta ; n = 4) experiment (B). In these experiments, RBF at a MAP of 100 mmHg averaged, respectively, 9.24 ± 0.52, 4.35 ± 0.39, 4.00 ± 0.19, and 4.79 ± 0.47 ml/min. Values are means ± SE. Los, losartan.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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Pharmacological manipulations revealed a role for ET in the resetting of RBF autoregulation when NO synthesis was acutely inhibited in our rats. Under these conditions, systemic pressor as well as renal vasoconstrictor effects were marked, as usually described (4, 15, 16). Interestingly, pretreatment with bosentan did not prevent the hemodynamic changes induced by the superimposed treatment with L-NAME, but the pressor effect of L-NAME was then less marked. Similarly, the pressor effect induced by prior treatment with L-NAME was only partially reversed by bosentan, whereas RBF was unaffected. The hemodynamic effects of L-NAME were even more predominant during the simultaneous coadministration of bosentan and L-NAME because enhancement of MAP was then not influenced by bosentan. Combined treatments with bosentan and L-NAME thus revealed some systemic effect of the blockade of ETA/B receptors on the acute hypertension induced by L-NAME but only when this blockade occurred before or after inhibition of NO synthesis. These findings do correspond, in general, to those obtained in conscious and anesthetized rats (24, 25). Selective blockade of ETA receptors with BQ-610, which induced a slight renal vasodilator effect, did not hinder the pressor and vasoconstrictor effects of L-NAME. On the other hand, when the ETB receptor was selectively blocked with BQ-788 infused at a dose that did not change MAP, RBF decreased by ~20%, as previously reported by Matsuura et al. (20). Subsequent blockade of NO synthesis induced the usual pressure increase but a greater RBF decrease. Overall, the effects of L-NAME on systemic pressure and on RBF were thus predominant.

During inhibition of NO synthesis, the RBF-MAP relationship was characterized by a significant extension of the autoregulatory plateau to a lower MAP despite the enhanced RVR, as previously reported (15). Blockade of ETA and ETB receptors with bosentan, whether undertaken before or after the acute inhibition of NO synthesis, hindered this change in RBF autoregulation. These observations strongly suggest that ET is implicated in the autoregulatory resetting, the more so that blockade of ETA and ETB receptors per se did not modify RBF autoregulation. Similarly, prior treatment with phosphoramidon, a nonspecific inhibitor of the enzyme converting Big ET to ET, prevented the autoregulatory resetting induced by L-NAME (9). All of these observations were further substantiated during coadministration of bosentan and L-NAME. Curiously, resetting of RBF autoregulatory pressure limits was not maintained in a few rats during the second experimental hour. This unexpected observation was unpredictable because no differences in any of the tested physiological or environmental variables were noticed among our rats. The transient autoregulatory effect presumably resulted from a temporal limitation in the activation of ET synthesis and release in these animals.

During the combined blockade of ETA receptors and NO synthesis, the RBF-MAP relationship was similar to the autoregulatory profile induced by L-NAME alone, and the autoregulatory pressure limits were again lowered. These results thus differ from those observed during the combined blockade of ETA and ETB receptors and NO synthesis. In contrast, during the combined blockade of ETB receptors and NO synthesis, the RBF-MAP relationship was markedly modified because the steadiness of the autoregulatory plateau usually observed after inhibition of NO synthesis was now not achieved. RBF tended even to become pressure-dependent at each pressure decrement. Also, the pressure limit of efficient RBF autoregulation was reset to higher values whereas the subautoregulatory zone was extended to the right. Although its physiological significance remains to be specified, changes in the extent of the subautoregulatory zone may represent differences in autoregulatory adjustments, as suggested by Aukland (1), and/or in the responsiveness to vasoactive factors of the different preglomerular vascular segments. Overall, the combined blockade of the ETB receptor and NO synthesis induced RBF autoregulatory changes of a magnitude greater than the simultaneous blockade of ETA and ETB receptors and NO synthesis. These effects seemed to offset the apparent lack of effect of ETA receptor blockade on the autoregulatory resetting during inhibition of NO synthesis.

The effects of AT1 receptor blockade with losartan and of NO synthesis on RBF autoregulation differed, at least to some extent, from those occurring during blockade of ET receptors. Indeed, although AT1 receptor blockade before the inhibition of NO synthesis prevented, at least partially, the autoregulatory resetting, this change could not be reversed, even when the dose of losartan was increased. The reasons for the differences between ANG II and ET blockade remain unknown at present. However, it should be noted that Baylis et al. (3) suggested that renal hemodynamic effects of "EDRF" blockade were not mediated by endogenous ANG II, at least in conscious rats. Nevertheless, the simultaneous blockade of AT1, ETA, and ETB receptors and of NO synthesis did not further modify the autoregulatory resetting induced by a blockade limited to ET receptors and NO synthesis. These results suggest, therefore, that ET may be specifically implicated in the RBF autoregulatory changes induced by an acute inhibition of NO synthesis. They are consistent with recent results of Zhang and Baylis (32) suggesting that ET mediates renal vascular memory of a transient rise in perfusion pressure due to NO synthase (NOS) inhibition in anesthetized rats.

The mechanisms underlying the differential effects of separate pharmacological inactivation of the two ET receptor subtypes on the autoregulatory resetting remain elusive at present. They could, however, be related to differences in the renal localization of the receptors and/or in cellular signaling, as well as to interactions between ET and other paracrine factors. For example, although both ETA and ETB receptors are localized on vascular smooth muscle cells of the renal microvessels, activation of the ETA receptor induced preglomerular vasoconstriction, whereas activation of the ETB receptor induced preglomerular, as well as efferent, arteriolar vasoconstriction in the split hydronephrotic rat kidney (8). The ETB receptor is further present on the vascular endothelium, where its autocrine activation may result in vasodilation through release of NO and PGI2 (22). However, this vasodilatory effect will vanish, at least to a great extent, during inhibition of NO synthesis. ETB receptors are also expressed in different tubular segments of the nephron, including the thick ascending limb of the loop of Henle, where chloride absorption is inhibited by ET via an ETB receptor-mediated release of NO (23). This interaction between ET and NO affecting salt transport along the thick ascending limb is of interest concerning our findings because it may interfere with the sensitivity and reactivity of the tubuloglomerular feedback component of RBF autoregulation, which is enhanced during intrarenal inhibition of NO synthesis in the rat (28). In contrast, ET per se does not seem to be involved in tubuloglomerular feedback in the rat (26). However, at present it is not known to what extent this ET-NO tubular interaction could eventually influence the autoregulatory resetting occurring during inhibition of NO synthesis in our rats. On the other hand, it should be emphasized that ET is chiefly released toward the vascular smooth muscle cells, because inhibition of NO synthesis in the rat only moderately increased plasma levels of ET (25). Therefore, effects of ET on RBF autoregulation, made conspicuous by inhibition of NO synthesis, may essentially result from direct myogenic adjustments. These occurred by inhibition of NOS in dynamic RBF experiments and in the perfused hydronephrotic kidney of the rat (13, 31).

During inhibition of NO synthesis, which induces the release of ET, activation of the ETB receptor, when the ETA receptor was blocked, maintained the autoregulatory resetting to a lower pressure limit, whereas activation of the ETA receptor, when the ETB receptor was blocked, markedly impaired RBF autoregulation. These pharmacological manipulations suggest an implication of the ETB receptor in the autoregulatory resetting during NOS inhibition. Nevertheless, the relative contribution of each ET receptor subtype may be somewhat contingent to be specified under the present experimental conditions. Indeed, they do not precisely reflect the impact of the simultaneous activation of the two ET receptor subtypes, which will be balanced by differences in their cellular localization and affinity for ET. In this regard, it is worthy to underline that low doses of ET preferentially activate ETA receptors, whereas high doses of ET preferentially activate ETB receptors in the rat (22). At present, it is not known to what extent this preferential ET receptor activation may occur with endogenous ET released in greater amounts when the synthesis of NO is suppressed (6, 14).

Interactions between ET and other paracrine factors, such as ANG II, may also interfere with the autoregulatory resetting induced by inhibition of NO synthesis, because they share some transport systems that adjust intracellular Ca2+ levels in vascular smooth muscle. However, the effect of ET, but not of ANG II, is likely to be predominant because its vasoconstrictor effect is long-lasting and because ET can inhibit renin synthesis (17, 18). Finally, recent findings by Hercule and Oyekan (11), suggesting that both ETA and ETB receptor-mediated vasoconstrictor effects of ET implicate 20-hydroxyeicosatetraenoic acid in the preglomerular arteriole of the rat, represent an interesting perspective for future investigation of RBF autoregulatory mechanisms.

In summary, blockade of ETA and ETB receptors with bosentan hinders autoregulatory resetting of RBF, which occurs during an acute inhibition of NO synthesis in the rat. Under the latter conditions, selective blockade of the ETA receptor did not alter autoregulatory resetting, in contrast to selective blockade of the ETB receptor, which markedly impaired RBF autoregulation. Taken together, these findings suggest that ET is involved in RBF autoregulatory changes induced by inhibition of NOS, possibly by a preferential activation of ETB receptors.


    ACKNOWLEDGEMENTS

Present address of P. Fourmanoir: Baxter R&D Europe, 7 rue du Progrès, 1400 Nivelles, Belgium.


    FOOTNOTES

We appreciate the dedicated technical assistance of B. Blairon, F. Coulon, and V. Jenart and the secretarial assistance of M. Fontaine. We gratefully acknowledge the collaboration of A. El Hajjam and C. Matteoti in some of the experiments. We thank Hoffman-La Roche, Belgium, for the generous gift of bosentan and Dr. R. D. Smith, DuPont-Merck, for providing losartan.

This study was presented, in part, at the meeting of the Société Belge de Physiologie et de Pharmacologie, in Brussels, June 13, 1998, the 68th Congrès de la Société de Physiologie in Liège, September 18-22, 2000, and at the 33rd Annual Meeting of the American Society of Nephrology in Toronto, Canada, on October 13-16, 2000. It appeared in abstract form in Fund Clin Pharmacol 13: 516, 1999; Eur J Physiol 440: R228, 2000, and J Am Soc Nephrol 11: 361A, 2000.

Address for reprint requests and other correspondence: R. A. Kramp, Service de Physiologie et Pharmacologie, Faculté de Médecine et de Pharmacie, Université de Mons-Hainaut, 7000 Mons, Belgium (E-mail: ronald.kramp{at}umh.ac.be).

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.

First published August 15, 2001;10.1152/ajprenal.00078.2001

Received 8 March 2001; accepted in final form 30 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 281(6):F1132-F1140
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