TP receptors regulate renal hemodynamics during ...

Am J Physiol Renal Physiol 287: F753–F759, 2004. First published June 22, 2004; 10.1152/ajprenal.00423.2003.

TP receptors regulate renal hemodynamics during angiotensin II slow pressor response Noritaka Kawada,1 Kathryn Dennehy,1 Glenn Solis,1 Paul Modlinger,1 Rebecca Hamel,1 Julie T. Kawada,1 Shakil Aslam,1 Toshiki Moriyama,2 Enyu Imai,2 William J. Welch,1 and Christopher S. Wilcox1 1

Cardiovascular Kidney Institute and Division of Nephrology and Hypertension, Georgetown University, Washington, DC 20007; and 2Division of Nephrology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan Submitted 3 December 2003; accepted in final form 15 June 2004

thromboxane A2-prostaglandin H2 receptor; isoprostane; hypertension; renal vascular resistance

infusion at 400 ng䡠 kg⫺1 䡠min⫺1 causes a slow pressor response accompanied by oxygen free radical (O⫺ 2 䡠) formation and increased filtration fraction (FF) and renal vascular resistance (RVR). The important role of increased O⫺ 2䡠 formation from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the vasoconstrictor response to ANG II was first showed by Harrison and colleagues (15). We showed later that ANG II increased NADPH oxidase activity in the kidney and the expression of the p22phox component in the renal afferent arteriole (6, 35). We and others also proposed a significant role for thromboxane A2 (TxA2) and other ligands for the TxA2-PGH2 receptor (TP-R) in the pressor and renal SUBCUTANEOUS ANG II

Address for reprint requests and other correspondence: C. S. Wilcox, Division of Nephrology and Hypertension, Georgetown Univ. Medical Center, 3800 Reservoir Rd. NE, PHCF6003, Washington, DC 20007-2197 (E-mail: [email protected]). http://www.ajprenal.org

hemodynamic response to ANG II (17, 18, 20 –22, 40, 42, 43). Under normal conditions, blood pressure (BP) is unaffected by a TxA2 synthase inhibitor, TP-R blocker, or by TP-R gene knockout (13, 28, 40). However, during ANG II infusion, there is increased prostaglandin (PG) and TxA2 generation (19), and blockade of TP-Rs blunts or prevents the pressor response and the increase in RVR (20 –22, 40, 43). However, the conclusion that TP-Rs have a critical role in the response to ANG II depends on the specificity of the drugs used. Therefore, this study was conducted in mice lacking functional TP-Rs by gene deletion. TP-Rs are expressed on systemic blood vessels and renal microvessels, glomeruli, mesangial cells, thick ascending limbs (TALs) of the loops of Henle, and collecting ducts (2, 10, 11, 32). This receptor is activated not only by TxA2, but also by PGH2, 8-isoprostanes, and a cyclooxygenase (COX) metabolite of 20-HETE, presumably 20-hydroxy-PGH2 (9, 14, 29). As 8-isoprostanes are mainly generated through the nonenzymatic oxidation of arachidonate, TP-Rs can potentially be activated both by prostanoids and by products of O⫺ 2 䡠 formation. The aim of this study is to determine the role of the TP-Rs in the ANG II slow pressor response. TP-R-deficient mice were used to investigate the requirement for TP-Rs in BP and the renal hemodynamic responses. The primary outcome is mean arterial pressure (MAP) at 12–14 days. The entire experiment error rate (␣ ⫽ 0.05) is allocated to this endpoint. The plasma ANG I, and the renal excretion of aldosterone, nitrate plus nitrite (NOx), 8-isoprostane PGF2␣ (8-iso), thiobarbituric acidreactive substances (TBARS), and PGs or their metabolites, TxB2 or the prostacyclin (PGI2) metabolite, 6-keto-PGF1␣ were measured to assess their potential roles in the functional changes observed. METHODS

Animals. Pathogen-free male and female TP-R heterozygous mice (TP ⫹/⫺; C57/black6J background, Jackson Lab) were generously provided by Dr. T. Coffman (Division of Nephrology, Duke Univ., Durham, NC) (33). These had been backcrossed for more than nine generations. They were inbred and genotyped in our laboratory. Male homozygous (TP ⫺/⫺) and wild-type (TP ⫹/⫹) mice were housed in a quiet room at 25°C with a 12:12-h light-dark cycle and free access to food and water. This study was approved by the Georgetown University Animal Care and Use Committee.

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Kawada, Noritaka, Kathryn Dennehy, Glenn Solis, Paul Modlinger, Rebecca Hamel, Julie T. Kawada, Shakil Aslam, Toshiki Moriyama, Enyu Imai, William J. Welch, and Christopher S. Wilcox. TP receptors regulate renal hemodynamics during angiotensin II slow pressor response. Am J Physiol Renal Physiol 287: F753–F759, 2004. First published June 22, 2004; 10.1152/ajprenal. 00423.2003.—We investigated the hypothesis that thromboxane A2 (TxA2)-prostaglandin H2 receptors (TP-Rs) mediate the hemodynamic responses and increase in reactive oxygen species (ROS) to ANG II (400 ng 䡠 kg⫺1 䡠 min⫺1 sc for 14 days) using TP-R knockout (TP ⫺/⫺) and wild-type (⫹/⫹) mice. TP ⫺/⫺ had normal basal mean arterial blood pressure (MAP) and glomerular filtration rate but reduced renal blood flow and increased filtration fraction (FF) and renal vascular resistance (RVR) and markers of ROS (thiobarbituric acid-reactive substances and 8-isoprostane PGF2␣) and nitric oxide (NOx). Infusion of ANG II into TP ⫹/⫹ increased ROS and thromboxane B2 (TxB2) and increased RVR and FF. ANG II infusion into TP ⫺/⫺ mice reduced ANG I and increased aldosterone but caused a blunted increase in MAP (TP ⫺/⫺: ⫹6 ⫾ 2 vs. TP ⫹/⫹: ⫹15 ⫾ 3 mmHg) and failed to increase FF, ROS, or TxB2 but increased NOx and paradoxically decreased RVR (⫺2.1 ⫾ 1.7 vs. ⫹2.6 ⫾ 0.8 mmHg 䡠 ml⫺1 䡠 min⫺1 䡠 g⫺1). Blockade of AT1 receptor of TP ⫺/⫺ mice infused with ANG II reduced MAP (⫺8 mmHg) and aldosterone but did not change the RVR or ROS. In conclusion, during an ANG II slow pressor response, AT1 receptors activate TP-Rs that generate ROS and prostaglandins but inhibit NO. TP-Rs mediate all of the increase in RVR and FF, part of the increase in MAP, but are not implicated in the suppression of ANG I or increase in aldosterone. TP ⫺/⫺ mice have a basal increase in RVR and FF associated with ROS.

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TXA2 AND TP-RS WITH ANG II RESULTS

Hematocrit, total body and kidney weights, heart rate, plasma electrolytes, and urine volume. Table 1 shows the data after 12–14 days of vehicle (Veh) or ANG II 400 (ANG II) infusion. There were no differences in total body weight, heart rate, or plasma electrolytes. ANG II increased urine volume (P ⬍ 0.05), body weight gain (⌬BW), and Hct (P ⬍ 0.05), whereas TP-R deletion increased kidney weight (P ⬍ 0.01) and prevented the ANG II-induced increase in body weight and hematocrit. MAP with infusion of Veh or ANG II and the effect of AT1 receptor blockade of TP ⫺/⫺ mice infused with ANG II. Figure 1 shows the MAP measured by direct intra-arterial catheterization under anesthesia. The baseline BP was identical in TP ⫹/⫹ and TP ⫺/⫺ mice. There was a significant interaction between TP-Rs and ANG II with MAP (P ⬍ 0.05). ANG II increased the MAP by an average of 15 mmHg in TP ⫹/⫹ (P ⬍ 0.005) but by a significantly smaller degree of 6 mmHg in TP ⫺/⫺ mice (P ⬍ 0.05). AT1 receptor blockade of TP ⫺/⫺ mice infused with ANG II reduced the MAP (P ⬍ 0.05). Renal function with ANG II and the effect of AT1 receptor blockade of TP ⫺/⫺ mice infused with ANG II. Figure 2 shows the glomerular filtration rate (GFR), renal blood flow (RBF), FF, and RVR in TP ⫹/⫹ and TP ⫺/⫺ mice after 12–14 days of Veh or ANG II infusion. TP ⫺/⫺ mice had lower baseline RBF (P ⬍ 0.001) and higher FF and RVR (FF: P ⬍ 0.01, RVR: P ⬍ 0.001) than TP ⫹/⫹ mice. There was a significant interaction between TP-Rs and ANG II for RBF (P ⬍ 0.05), FF (P ⬍ 0.01), and RVR (P ⬍ 0.005), but not GFR. ANG II infusion in TP ⫹/⫹ mice increased the GFR (P ⬍ 0.05), FF, and RVR (FF: P ⬍ 0.01, RVR: P ⬍ 0.05), whereas a similar infusion into TP ⫺/⫺ mice did not change the FF and actually increased the RBF (P ⬍ 0.005) and reduced the RVR (P ⬍ 0.05). This implies that TP-Rs mediate ANG II-induced reduction in RBF and increase in FF and RVR. AT1 receptor blockade of TP ⫺/⫺ mice infused with ANG II reduced the GFR (P ⬍ 0.05) but did not significantly change the RBF, FF, or RVR. Plasma ANG I and renal excretion of aldosterone. Figure 3 shows the plasma ANG I and renal aldosterone excretion in TP ⫺/⫺ and ⫹/⫹ mice after 12–14 days of Veh or ANG II infusion. As anticipated, ANG II infusion decreased the plasma

Table 1. Total body and kidney weight, heart rate, hematocrit, plasma electrolytes, and urine volume of TP-R-deficient mice infused with ANG II

n Total body weight, g ⌬BW, % Kidney weight, g %Kidney weight, g BW Heart rate, min Hematocrit, % Plasma Na⫹, meq/l Plasma K⫹, meq/l Plasma Cl⫺, meq/l n 24-H urine volume, ml

TP-R (⫹/⫹) Vehicle Day 14

TP-R (⫺/⫺) Vehicle Day 14

TP-R (⫹/⫹) ANG II Day 14

TP-R (⫺/⫺) ANG II Day 14

7 25.2⫾1.3 3.4⫾0.7 0.29⫾0.01 1.21⫾0.04 444⫾10 38⫾1 146⫾1 3.8⫾0.2 113⫾1 6 1.2⫾0.2

7 25.9⫾1.1 3.0⫾1.3 0.35⫾0.02 1.27⫾0.03 419⫾20 38⫾1 148⫾1 3.9⫾0.2 114⫾1 6 1.5⫾0.2

7 24.1⫾1.0 8.9⫾0.9 0.32⫾0.02 1.16⫾0.04 428⫾16 41⫾1 146⫾1 3.6⫾0.3 112⫾3 6 2.0⫾0.3

9 25.0⫾1.2 2.9⫾1.3 0.35⫾0.02 1.25⫾0.03 431⫾13 37⫾1 149⫾1 3.6⫾0.2 117⫾1 6 1.8⫾0.2

Statistics for Two-Way ANOVA ANG II

TP-R

Interaction

n.s. N/A n.s. n.s. n.s. N/A n.s. n.s. n.s.

n.s. N/A P ⬍ 0.01 n.s. n.s. N/A n.s. n.s. n.s.

n.s. P ⬍ 0.05 n.s. n.s. n.s. P ⬍ 0.01 n.s. n.s. n.s.

P ⬍ 0.05

n.s.

n.s.

Values are means ⫾ SE. n, No. of mice; BW, body weight; TP-R, thromboxane A2-prostaglandin H2 receptor; n.s., not significant; N/A, not available. AJP-Renal Physiol • VOL

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Delivery of ANG II and AT1A receptor antagonist. The details of the mouse slow pressor response model have been published (16). ANG II (Peninsula Laboratory, San Carlos, CA) was infused at 0 [vehicle (V)] and 400 ng 䡠 kg⫺1 䡠 min⫺1 (ANG II 400) through subcutaneous (sc) osmotic minipumps (model 1002; Alza, Palo Alto, CA). One group of TP ⫺/⫺ mice infused with ANG II received the selective AT1A receptor antagonist candesartan (Cand; Astra Zeneca). This was dissolved in the drinking water at a dose of 25 mg/l. This is a fully effective dose in a rat model of renovascular hypertension (36). Renal function studies. Twelve to 14 days after implantation of the minipumps, mice were prepared for clearance experiments, as described previously (16). The following groups were studied: TP ⫹/⫹ Veh (n ⫽ 7), TP ⫺/⫺ Veh (n ⫽ 7), TP ⫹/⫹ ANG II (n ⫽ 7), TP ⫺/⫺ ANG II (n ⫽ 9), and TP ⫺/⫺ ANG II with Cand (n ⫽ 6). Renal excretion of aldosterone, 8-isoprostane, TxB2, 6-ketoPGF1␣, TBARS, and NOx. On days 12 and 13, mice were housed in mouse metabolic cages (Nalgene Nunc International, Rochester, NY) and were fed a NOx-free synthetic diet. Urine was collected for 24 h into antibiotics (penicillin G: 0.8 mg, streptomycin: 2.6 mg, and amphotericin B: 5.0 mg). Aldosterone was measured by a RIA kit (Diagnostic Systems Lab). Total 8-isoprostane (8-iso-PGF2␣), TxB2, and 6-keto-PGF1␣ in urine were purified, extracted, diluted, and assayed with an enzyme immunoassay (EIA) procedure (Cayman Chemical, Ann Arbor, MI) using a method that we validated (27). TBARS were measured by an OXItec TBARS assay kit (ZeptoMetric). Nitrate plus nitrite (NOx) was measured in a NO Chemiluminescence Analyzer (model 270B, Sievers Instrument). These values are factored by creatinine, which was measured in a Creatinine Analyzer2 (Beckman Instruments). Plasma ANG I and TBARS. Blood was collected from the femoral artery into EDTA-containing tubes under anesthesia with 1% isoflurane and was centrifuged to obtain plasma. Plasma ANG I was measured by an RIA kit (Peninsula Laboratories). TBARS were measured by an OXItec TBARS assay kit (ZeptoMetric). Statistics. Results are expressed as means ⫾ SE. Statistical analyses of four groups were performed with two-way factorial ANOVA to determine the interaction of two factors. If there was a positive interaction (shown in the figures as Interaction ANG II ⫻ TP-R), a post hoc t-test was performed to assess the difference between groups (shown in the figures by bars). If there was no interaction, the individual effects of ANG II and TP-Rs were assessed by two-way factorial ANOVA (shown in the figures as ANG II or TP-R). Statistical analyses of two groups were performed with a Student’s t-test, and the differences are reported with a nominal P value. Statistical significance was defined as P ⬍ 0.05. Professional statistical advice was kindly provided by Franca Benedicty Barton M.S. (Dept. of Biostatistics, Georgetown University).

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excretion of NOx (P ⬍ 0.01). This implies that TP-Rs counteract NO production during ANG II infusion. Renal excretion of TxB2 and 6-keto-PGF1␣. Figure 6 shows the renal excretion of TxB2 and 6-keto-PGF1␣ in mice after 12 days of Veh or ANG II infusion. TP-R deletion increased basal TxB2 excretion (P ⬍ 0.01). There was a significant interaction between TP-Rs and ANG II for TxB2 (P ⬍ 0.05) and 6keto-PGF1␣ (P ⬍ 0.05). ANG II infusion in TP ⫹/⫹ mice increased the excretion of TxB2 (P ⬍ 0.01) and 6-keto-PGF1␣ (P ⬍ 0.05). In contrast, a similar infusion into TP ⫺/⫺ mice did not change excretion of TxB2 or 6-keto-PGF1␣. This implies that TP-Rs mediate ANG II-induced increases in TxB2 and 6-keto-PGF1␣.

Fig. 1. The pressor response to ANG II is attenuated in TP ⫺/⫺ mice, and AT1 receptor blockade blunted the pressor response in TP ⫺/⫺ mice infused with ANG II. Mean arterial pressure (MAP) is shown in anesthetized thromboxane A2-prostaglandin H2 receptor (TP-R) wild-type (TP ⫹/⫹) and TP knockout (TP ⫺/⫺) mice infused subcutaneously with vehicle (Veh) or ANG II (400 ng 䡠 kg⫺1 䡠 min⫺1) ⫾ candesartan (Cand; 25 mg/l in drinking water) at day 14. n.s., Not significant; N/A, not available.

ANG I and increased the aldosterone (TP-R: P ⬍ 0.0001). This effect was not affected by TP-R deletion. AT1 receptor blockade of TP ⫺/⫺ mice infused with ANG II reduced the renal excretion of aldosterone (P ⬍ 0.001). Plasma TBARS and renal excretion of TBARS and 8-isoPGF2␣. Figure 4 shows the plasma TBARS and the renal excretion of TBARS and 8-iso-PGF2␣ in TP ⫺/⫺ and ⫹/⫹ mice after 12–14 days of Veh or ANG II infusion. TP-R deletion increased basal TBARS and 8-iso-PGF2␣ excretion (P ⬍ 0.05). There was a significant interaction between TP-Rs and ANG II for plasma TBARS (P ⬍ 0.05) and the excretion of TBARS and 8-iso-PGF2␣ (TBARS: P ⬍ 0.05, 8-iso-PGF2␣: P ⬍ 0.05). ANG II infusion in TP ⫹/⫹ mice increased plasma TBARS (P ⬍ 0.05) and excretion of TBARS (P ⬍ 0.05) and 8-iso-PGF2␣ (P ⬍ 0.01). In contrast, a similar infusion into TP ⫺/⫺ mice did not change excretion of TBARS and 8-isoPGF2␣ and reduced plasma TBARS (P ⬍ 0.05). This implies that TP-Rs mediate ANG II-induced change in TBARS and 8-iso-PGF2␣. AT1 receptor blockade of TP ⫺/⫺ mice infused with ANG II did not significantly change the renal excretion of TBARS or 8-iso-PGF2␣. Renal excretion of NOx. Figure 5 shows the renal excretions of NOx in TP ⫺/⫺ and ⫹/⫹ mice after 12 days of Veh or ANG II infusion. TP-R deletion increased basal NOx excretion (P ⬍ 0.05). There was a significant interaction between TP-Rs and ANG II for NOx (P ⬍ 0.05). As anticipated, infusion of ANG II into TP ⫹/⫹ mice did not change the excretion of NOx (8). In contrast, a similar infusion into TP ⫺/⫺ mice increased AJP-Renal Physiol • VOL

We confirmed that ANG II increases MAP, FF, and RVR and increases parameters of oxidative stress (16) in wild-type mice and that the pressor response to ANG II is diminished in the TP ⫺/⫺ mouse (12). The main new findings are that TP ⫺/⫺ mice infused with ANG II fail to increase oxidative stress or PGs but have an enhanced NO production with a paradoxical reduction in RVR. The RVR and oxidative stress in TP ⫺/⫺ mice infused with ANG II are not responsive to AT1 receptor blockade. As in the study by Coffman et al. (12), using a higher dose of ANG II, TP-R deletion blunted the rise in MAP with ANG II. This cannot be ascribed to a failure to stimulate aldosterone (Fig. 3). RVR normally accounts for 20% of the total peripheral vascular resistance. Whereas ANG II increased RVR by ⫹19% in TP ⫹/⫹ mice, it reduced the RVR by ⫺12% in TP ⫺/⫺ mice. Therefore, this paradoxical reduction in RVR may account for a part of the blunted rise in MAP with ANG II in the TP ⫺/⫺ mice. The blunted pressor response and the paradoxical reduction in renal vasoconstriction to ANG II in TP ⫺/⫺ mice may relate to an absence of vasoconstrictive TxA2 and oxidative stress (as indicated by reduced plasma TBARS and unchanged excretion of 8-iso and TBARS) and an increase in the NO (as indicated by increased excretion of NOx). Thus mice infused with the permeant nitroxide superoxide dismutase mimetic tempol at a dose that prevents oxidative stress do not have a rise in MAP and have blunted renal vasoconstriction with ANG II, as in the TP ⫺/⫺ mice in this study (16). We concluded that the principal site of vasoconstriction of O⫺ 2 䡠 generated during ANG II was on the preglomerular arterioles (6, 16, 31). The present finding that ANG II failed to increase RVR or FF in TP ⫺/⫺ mice suggests that activation of TP-Rs by ANG II enhances the pre- and postglomerular vascular resistance in the mouse kidney. Afferent arterioles from ANG II-infused rabbit have an enhanced vasoconstriction to ANG II that depends on O⫺ 2 䡠 and TP-Rs (26, 34, 35). Activation of TP-Rs can also cause renal vasoconstriction by promotion of tubuloglomerular feedback (TGF) (37). O⫺ 2䡠 and TxA2 generated in response to prolonged ANG II potentiate vasoconstriction (38). Thus the effect of TP-Rs to mediate renal vasoconstriction during oxidative stress accompanying prolonged ANG II infusion likely entails direct effects in the renal microvessels and enhanced TGF responses. The renal circulation is uniquely sensitive to TP-R activation during infusion of a TP-R agonist, U-46,619, perhaps reflecting activation of TGF (37). Indeed, Yamaguchi et al. (44) showed

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DISCUSSION

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Fig. 2. TP-Rs mediate ANG II-induced increases in filtration fraction (FF) and renal vascular resistance (RVR), and AT1 receptor blockade has no effect on FF and RVR in TP ⫺/⫺ mice infused with ANG II. See Fig. 1 legend. RBF, renal blood flow; GFR, glomerular filtration rate.

that renal vasoconstriction during TP-R activation is prevented by blockade of TGF by a loop diuretic. TGF causes vasoconstriction of the afferent arteriole that is enhanced during ANG II infusion (3, 23) or TP-R activation (37). Therefore, the failure of TP ⫺/⫺ mice to decrease RVR during ANG II infusion could be a consequence of a failure to enhance TGF-induced preglomerular vasoconstriction. Schnermann et al. (28) reported that whereas TP ⫺/⫺ mice reduce glomerular capillary pressure with increased delivery to NaCl to the macula densa, they have a blunted macula densa regulation of single-nephron GFR. This implies a blunted role for TGF in the regulation of glomerular hemodynamics. ANG II increases the FF in normal mice. This effect is not prevented by tempol (16), but, in the present study, was prevented by TP-R deletion. This suggests that ANG II induces AJP-Renal Physiol • VOL

vasoconstriction of the postglomerular vessels through activation of TP-Rs and that, in contrast to the preglomerular effect, this is likely independent of O⫺ 2 䡠 formation. Arima et al. (4) and Ren et al. (25) proposed that prostanoids and 20-HETE released from the glomeruli by ANG II regulate postglomerular vascular resistance. Because metabolites of 20-HETE and PGH2 both provide ligands for the TP-R (9, 14, 29), those findings are consistent with our present study. Our result with an AT1 receptor antagonist confirms the conclusion of Coffman et al. (5, 7) that AT1 receptors mediate the increase in aldosterone with ANG II but shows further that this is independent of TP-Rs. In contrast, an AT1 receptor antagonist did not change renal hemodynamics or excretion of oxidative stress markers in TP ⫺/⫺ mice infused with ANG II. This indicates that, during the ANG II slow pressor response,

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Fig. 3. Neither ANG I nor aldosterone was not affected by TP-R deletion. See Fig. 1 legend.

response in TP ⫺/⫺ mice during ANG II infusion could be an unopposed vasodilatator effect mediated via AT2 receptors (1). Activation of TP-R in the aorta or kidneys releases endogenous TxA2 (11, 41). The present study demonstrates that ANG II fails to increase excretion of TxB2 or 6-keto-PGF1␣ in TP ⫺/⫺ mice. This indicates that TP-Rs mediate the effect of ANG II to activate phospholipase or COXs. The present study confirms that prolonged ANG II infusion does not increase NOx excretion (8), despite an increase in NO synthase isoform expression in the kidney (24). The present study implicates TP-Rs in preventing NO generation, because NOx excretion increased with ANG II only in TP ⫺/⫺ mice. The failure of NOx to increase normally with ANG II may be

⫺ Fig. 4. TP-Rs mediate ANG II-induced O⫺ 2 䡠 formation and AT1 receptor blockade has no effect on O2 䡠 formation in TP ⫺/⫺ mice infused with ANG II. See Fig. 1 legend. TBARS, thiobarbituric acid-reactive substances.

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AT1 receptors mediate the vasoconstriction and O⫺ 2 䡠 formation indirectly through activation of TP-Rs. Because NADPH oxidase is the major source for O⫺ 2 䡠 generation in the blood vessels and kidneys and is activated by AT1 receptors (6, 15), induction of NADPH oxidase during ANG II infusion apparently may require TP-Rs. AT1 receptors activate TP-Rs, but the precise mechanisms are not explored in this study. Whether this relates to an increase in the amount or the sensitivity of TP-Rs or simply to an increase in the production of eicosanoid agonists, which activate TP-Rs, requires further study. Mice with AT2 receptor gene deletion have an enhanced pressor response to ANG II (30). Thus the renal vasodilatator

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a consequence of increased O⫺ 2 䡠 generation. Accordingly, it may be a failure to generate O⫺ 2 䡠 with ANG II in TP ⫺/⫺ mice that promotes increased NOx excretion. Whether the paradoxical reduction in RVR with ANG II in TP ⫺/⫺ mice relates to

ACKNOWLEDGMENTS We are grateful to Dr. T. Coffman (Division of Nephrology, Duke University, Raleigh-Durham, NC) for the TP-R (⫹/⫺) mice and to F. B. Barton (Dept. of Biostatistics, Georgetown University) for expert statistical advice.

Fig. 6. TP-Rs mediate ANG II-induced increase in thromboxane B2 (TxB2) and 6-keto-PGF1␣. See Fig. 1 legend.


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Fig. 5. TP-Rs prevent an increase in nitric oxide (NOx) excretion during ANG II. See Fig. 1 legend.

a failure to enhance O⫺ 2 䡠 generation or to an accompanying increase in NO generation requires further study. The higher baseline FF and RVR in TP ⫺/⫺ mice in the present study are not consistent with TP-Rs being a vasoconstrictor signaling pathway. This could indicate compensatory activation of other vasoconstrictive signals in TP ⫺/⫺ mice. The FF and RVR commonly increase during activation of the renin-angiotensin-aldosterone system, and we reported that inhibition of TP-Rs or TxA2 synthase increases plasma renin activity (39). However, TP ⫺/⫺ mice had a normal level of ANG I and aldosterone and a normal regulation during ANG II infusion. Moreover, TP ⫺/⫺ mice had no change in FF or RVR during AT1 receptor blockade with candesartan. It is possible that an increased oxidative stress may account for the renal vasoconstriction, but the cause for the oxidative stress is obscure. Rats infused with TP-R antagonists do not have an increase in RVR or FF (37, 39, 41). Therefore, the increase in RVR and FF in TP ⫺/⫺ mice may be a consequence of a developmental defect in these mice. TP ⫺/⫺ mice in this study had consistently enlarged kidneys. In conclusion, a slow pressor dose of ANG II increases generation of PGs including TxA2 and causes oxidative stress. AT1 receptors activate TP-Rs to mediate the increase in RVR, FF, O⫺ 2 䡠 formation, and a part of the pressor response, but suppression of ANG I and stimulation of aldosterone secretion are independent of TP-Rs. The paradoxical reduction in RVR with ANG II in the absence of TP-Rs may relate to the absence of an increase in oxidative stress or to a rise in NO generation.

TXA2 AND TP-RS WITH ANG II GRANTS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-36,079 and DK-49,870, National Heart, Lung, and Blood Institute Grant HL-68686, the American Heart Association Beginning Grant-in-Aid (PI: N. Kawada; 0465440U), and by funds from the George E. Schreiner Chair of Nephrology.

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