TP receptor-mediated vasoconstriction in microperfused afferent arterioles: roles of O2minus and NO

Christine G. Schnackenberg, William J. Welch, and Christopher S. Wilcox

Division of Nephrology and Hypertension, Georgetown University Medical Center, Washington, District of Columbia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Thromboxane A2 (TxA2) preferentially constricts the renal afferent arteriole. Nitric oxide (NO) modulates vasoconstriction and is rapidly degraded by superoxide radical (O2-). We investigated the roles of NO and O2- in rabbit isolated, perfused renal afferent arteriole responses to the TxA2/prostaglandin H2 (TP) receptor agonist U-46,619. U-46,619 (10-10-10-6 M) dose-dependently reduced afferent arteriolar luminal diameter (ED50 = 7.5 ± 5.0 nM), which was blocked by the TP receptor antagonist ifetroban (10-6 M). Tempol (10-3 M) pretreatment, which prevented paraquat-induced vasoconstriction in afferent arterioles, blocked the vasoconstrictor responses to U-46,619. To test whether U-46,619 stimulates NO and whether tempol prevents U-46,619-induced vasoconstriction by enhancing the biological activity of NO, we examined the luminal diameter response to U-46,619 in arterioles pretreated with Nw-nitro-L-arginine methyl ester (L-NAME, 10-4 M) or L-NAME + tempol. During L-NAME, the sensitivity and maximal responses of the afferent arteriole to U-46,619 were significantly (P < 0.05) enhanced. Moreover, L-NAME restored a vasoconstrictor response to U-46,619 in vessels pretreated with tempol. In conclusion, in isolated perfused renal afferent arterioles TP receptor activation stimulates NO production, which buffers the vasoconstriction, and stimulates O2- production, which mediates the vasoconstriction, in part, through interaction with NO.

thromboxane A2; nitric oxide; superoxide; afferent arteriole; tempol


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THROMBOXANE A2 (TxA2) produced in the endothelium constricts vascular smooth muscle cells through activation of thromboxane A2/prostaglandin H2 (TP) receptors. In the kidney, TxA2 reduces renal blood flow and glomerular filtration rate through preferential action on the afferent arteriole and glomerulus (3, 15, 25). Some of these effects are mediated via the tubuloglomerular feedback (TGF) response (41), whereas others are apparent in hydronephrotic kidneys that lack a TGF response and therefore must be due to direct action on afferent arterioles (15,25). Several studies implicate TxA2 to be an important mediator of the renal hemodynamic and blood pressure effects of angiotensin II (ANG II) under normal conditions (42, 43) and in ANG II-dependent forms of hypertension (28). However, the mechanisms by which TP receptor activation causes vasoconstriction remain to be fully elucidated.

Nitric oxide (NO) produced by the endothelium is a ubiquitous vasodilator and modulator of vascular tone. NO buffers the vasoconstrictor action of TxA2 in the isolated aorta (5, 7), coronary artery (37), and pulmonary artery (40). NO also modulates vasoconstriction induced by ANG II and endothelin-1 but not by norepinephrine in the renal afferent arteriole (18, 19). Whether NO plays an important role in modulating vasoconstriction induced by TxA2 in the renal afferent arteriole remains unknown.

Several studies suggest that the oxygen radical superoxide (O2-) interacts with NO and thus limits its bioavailability. The affinity of NO for O2- is so high that its rate of reaction is limited only by diffusion (31). Since O2- effectively degrades NO, the biological activity of NO may be determined by the availability of O2- (13, 31). NO-mediated vasodilation is impaired in aorta with enhanced generation of O2- (12) and can be restored by blockade of TP receptors (2, 33, 38). This led us to the hypothesis that TP receptor activation may be a potent source for the generation of O2- and hence for degrading NO in resistance vessels. The objectives of this study were to 1) determine the role of O2- in TP receptor activation, 2) to examine the role of NO in TP receptor activation, and 3) to investigate the interaction between NO and O2- in TP receptor activation in renal afferent arterioles. The response to the stable TP receptor agonist U-46,619 was studied in rabbit isolated, perfused renal afferent arterioles. The role of NO was assessed from the responses to inhibition of NO synthase with Nw-nitro-L-arginine methyl ester (L-NAME). The stable nitroxide 4-hydroxy-[2,2,6,6]-tetramethylpiperidine-1-oxyl (tempol) was used to scavenge O2-. Tempol is a metal-independent, membrane-permeable superoxide dismutase mimetic that scavenges O2- to hydrogen peroxide (H2O2) and oxygen. Tempol has been validated as an electron paramagnetic resonance spin-label molecule specifically for O2- (16, 29) and does not donate NO or scavenge H2O2 (16, 29). In vivo studies have shown that tempol reduces damage caused by oxygen radicals in ischemia/reperfusion injury (8), inflammation (21), and radiation (27).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and Microperfusion of Afferent Arterioles

Male New Zealand White rabbits (1.4-1.8 kg) were maintained on tap water and standard chow. Protocols were approved by the Institutional Animal Care and Use Committee of Georgetown University Medical Center and were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health as well as the guidelines of the Animal Welfare Act. Rabbits were anesthetized with xylazine (9 mg/kg im), ketamine (47 mg/kg im), and pentobarbital sodium (11 mg/kg iv) followed by heparin (1,000 USP iv) for anticoagulation. Microdissection and microperfusion of the afferent arteriole were performed as previously described (18, 19). Briefly, the right kidney was extracted via an abdominal incision and immediately placed in ice-cold preservation solution (24). Slices of the kidney were made along the corticomedullary axis and replaced in the preservation solution. A single superficial afferent arteriole with glomerulus attached was microdissected under a stereomicroscope (model SZ40, Olympus) on a temperature-controlled stage maintained at 4°C. The arteriole was transferred to a temperature-regulated chamber mounted on the stage of an inverted microscope (model IX70, Olympus) modified with micromanipulators. The afferent arteriole was cannulated with a series of concentric glass pipettes including holding, perfusion, and exchange pipettes and perfused with alpha modification of minimum essential media (MEMalpha ) at 60 mmHg. The arteriole was superfused at ~1 ml/min with MEMalpha bubbled with 95% O2-5% CO2. The microperfused arteriole was displayed at ×400 magnification (Nomarski optics, Olympus) on a video monitor via a black-and-white camera (model NC 70, Dage-MTI) attached to the inverted microscope and recorded on VHS tape. The area of the afferent arteriole with the consistently most constricted point during an experiment was selected for measurement. Measurements were made with standard vernier calipers (Mitutoyo, Japan).

Protocol

Rabbit microperfused afferent arterioles were gradually warmed to 37°C and allowed to equilibrate for 30 min. Drugs were added to the superfusion solution, and measurements of luminal diameter were made after 10-15 min. To test the viability of the tissue at the completion of the studies, norepinephrine (10-7 M) was administered. Only those vessels showing a >25% contraction were selected. Figure 1 illustrates an example of an isolated, perfused rabbit afferent arteriole during basal conditions and after norepinephrine (10-7 M).


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Fig. 1.   Example of an isolated, perfused rabbit afferent arteriole (perfusion pressure = 60 mmHg) during basal conditions (A) and after norepinephrine (10-7 M, B).

Experimental Design

Series 1. The aim of this series was to assess the specificity of tempol as a superoxide dismutase mimetic in the isolated, perfused afferent arteriole. We used the classic quinoline agent paraquat to stimulate intracellular production of O2- in afferent arterioles. Paraquat redox cycles with cellular diaphorases and molecular oxygen to generate superoxide (30). This occurs at doses that do not affect cell viability (9). Luminal diameter was measured during graded doses of paraquat (10-7-10-3 M) in normal vessels (n = 4) and in vessels pretreated with tempol (10-3 M, n = 4). To assess the stability of the response to tempol, the luminal diameter response to tempol (10-3 M) alone was examined in microperfused afferent arterioles after 15 and 60 min of superfusion (n = 4).

Series 2. The aim of this series was to determine the afferent arteriolar response to TP receptor activation. Luminal diameter was measured during basal conditions and after increasing doses of U-46,619 (10-10-10-6 M, n = 6). To determine whether the arteriolar response to U-46,619 was mediated through activation of TP receptors, the dose response was repeated in separate vessels pretreated with the TP receptor antagonist ifetroban (10-6 M, n = 4).

Series 3. The aim of this series was to investigate the role of O2- in TP receptor activation. The luminal diameter response to U-46,619 (10-10-10-6 M) was determined in afferent arterioles pretreated with tempol (10-3 M, n = 6).

Series 4. The objective of this series was to assess the role of NO on the afferent arteriolar response to TP receptor activation. The NO synthase inhibitor L-NAME was used at a dose which has previously been shown to block acetylcholine-induced, endothelium-dependent vasodilation of the rabbit afferent arteriole (19). The luminal diameter response to U-46,619 (10-10-10-6 M) was determined in afferent arterioles pretreated with L-NAME (10-4 M, n = 6).

Series 5. The objective of this series, was to assess whether the effect of tempol on the response to TP receptor activation could be ascribed to potentiation of the effects of NO. The arteriole response to U-46,619 was measured during blockade of NO synthesis and scavenging of O2-. The luminal diameter response to graded concentrations of U-46,619 (10-10-10-6 M) was measured in vessels pretreated with tempol (10-3 M) + L-NAME (10-4 M, n = 6).

Drugs and Solutions

U-46,619 (Cayman Chemical) was evaporated under N2 and reconstituted using 97% ethanol and 55 mM Tris. After further evaporation with nitrogen, aliquots of U-46,619 (10-3 M) were made in tissue culture grade H2O and stored at -20°C until use. All other solutions were prepared fresh daily. Heparin was dissolved in 0.9% NaCl at 1,000 USP/ml. Ifetroban (BMS-180291) was prepared in tissue culture grade H2O and diluted in superfusion solution before use daily. All other agents including norepinephrine, tempol (4-hydroxy TEMPO), and L-NAME were purchased from Sigma Chemical and prepared similarly. Preservation solution consisted of 150 mM sucrose, 52 mM NaHPO4 (anhydrous), 16 mM NaH2PO4, and 5% BSA which was filtered (0.8 µm), saturated with 95% O2-5% CO2 (pH 7.40-7.45), and prepared fresh daily. MEMalpha solution containing 126.40 mg/l L-arginine and an additional 26 mM NaHCO3 and 5% BSA for perfusion and 26 mM NaHCO3 and 0.15% BSA for superfusion were filtered (0.2 µm), saturated with 95% O2-5% CO2, and buffered to pH 7.40-7.45 before use daily.

Statistics

All values are reported as means ± SE. Overall significance between dose responses was determined from repeated measures analysis of variance and the Scheffé post hoc test where appropriate. A Student's t-test was used to determine significance between groups. P < 0.05 was determined to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Series 1

Figure 2 shows the luminal diameter response to paraquat (10-7-10-3 M) in vehicle pretreated vessels and in vessels pretreated with tempol (10-3 M). From a baseline of 18.41 ± 1.37 µm, paraquat caused dose-dependent reductions in luminal diameter (ED50 = 8.6 ± 3.1 µM) of microperfused afferent arterioles that were abolished by tempol (P < 0.05). Superfusion of tempol alone (10-3 M) had no effect on basal luminal diameter (15.42 ± 1.98 µm) after 15 (15.50 ± 1.62 µm) or 60 (15.74 ± 1.43 µm) min.


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Fig. 2.   Series 1. Line graph displaying the change in luminal diameter during the superoxide stimulating agent paraquat in microperfused afferent arterioles in the presence (tempol, n = 4) or absence (vehicle, n = 4) of scavenging superoxide with tempol (10-3 M). *P < 0.05 vs. vehicle.

Series 2

Figure 3 illustrates the luminal diameter response to TP receptor activation with U-46,619 in microperfused afferent arterioles in the presence and absence of ifetroban. From a baseline of 15.63 ± 0.89 µm, U-46,619 (10-10-10-6 M) caused graded reductions in luminal diameter (ED50 = 7.5 ± 5.0 nM). Ifetroban (10-6 M) pretreatment had no significant effect on basal luminal diameter (17.83 ± 1.18 to 19.26 ± 1.59 µm) of microperfused afferent arterioles but abolished the response to U-46,619 (P < 0.001). An additional series was undertaken to contrast the luminal diameter response to U-46,619 when given in the bath + lumen compared with in the bath alone. U-46,619 (10-10-10-6 M) given in the bath + lumen had no significant effect on the luminal diameter (18.23 ± 1.98 to 19.86 ± 1.56 µm). Furthermore, U-46,619 (10-10-10-6 M) given in the bath during sham exchange through the lumen also had no significant effect on luminal diameter (17.77 ± 1.01 to 20.48 ± 3.15 µm). Therefore, all further series were conducted using U-46,619 given in the bath only.


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Fig. 3.   Series 2. Line graph displaying the change in luminal diameter during U-46,619 in microperfused afferent arterioles in the presence (ifetroban, n = 4) or absence (vehicle, n = 6) of blockade of thromboxane A2/prostaglandin H2 (TP) receptors with ifetroban (10-6 M). Comparing the two groups, *P < 0.05 and **P < 0.01.

Series 3

Figure 4 illustrates the luminal diameter response to U-46,619 (10-10-10-6 M) in microperfused afferent arterioles in the presence and absence of the membrane-permeable superoxide dismutase mimetic tempol. Tempol (10-3 M) abolished the contractile response to U-46,619 across the dose range studied (P < 0.001). Basal luminal diameter was 16.57 ± 0.77 µm and remained unchanged in response to tempol + U-46,619 (17.25 ± 0.38 µm). At the end of the experiments, luminal diameter responses to norepinephrine (10-7 M) were not significantly different in vessels treated with U-46,619 alone or with U-46,619 + tempol (-54 ± 7% vs. -36 ± 16%).


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Fig. 4.   Series 3. Line graph showing the change in luminal diameter during U-46,619 in microperfused afferent arterioles in the presence (tempol, n = 6) or absence (vehicle, n = 6) of scavenging superoxide with tempol (10-3 M). *P < 0.05 vs. vehicle. **P < 0.01 vs. vehicle.

Series 4

Figure 5 illustrates the luminal diameter response to low (10-10 M) and high (10-6 M) doses of U-46,619 in the presence or absence of NO synthesis blockade with L-NAME. Pretreatment with L-NAME (10-4 M) significantly (P < 0.001) reduced basal afferent arteriolar luminal diameter by 20 ± 1% (from 15.30 ± 1.22 to 12.23 ± 0.50 µm) and significantly (P < 0.05) enhanced the vasoconstrictor response to U-46,619 (10-10-10-6 M). L-NAME significantly (P < 0.05) increased the sensitivity and maximal responses of afferent arterioles to U-46,619. Additional studies were conducted to examine the luminal diameter response to L-NAME (10-4 M) given in the bath + lumen. This reduced the luminal diameter by 22 ± 2%, which is not significantly different compared with the bath alone as indicated above or compared with Ito et al. (19).


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Fig. 5.   Series 4. Bar graph displaying the change in luminal diameter during two concentrations of U-46,619 (10-10 M, 10-6 M) in microperfused afferent arterioles in the presence (L-NAME, n = 6) or absence (vehicle, n = 6) of blockade of nitric oxide synthesis with L-NAME (10-4 M). *P < 0.05 vs. vehicle. L-NAME, Nw-nitro-L-arginine methyl ester.

Series 5

Figure 6 shows the luminal diameter response to U-46,619 (10-10-10-6 M) in microperfused afferent arterioles pretreated with tempol (10-3 M) in the presence or absence of NO synthesis blockade with L-NAME (10-4 M). L-NAME significantly (P < 0.05) restored a vasoconstrictor response to U-46,619 in tempol pretreated arterioles. Whereas U-46,619 had no effect in vessels pretreated with tempol (16.57 ± 0.77 to 17.25 ± 0.38 µm), U-46,619 significantly decreased luminal diameter by 32% in vessels pretreated with tempol + L-NAME (12.23 ± 0.51 to 8.27 ± 1.04 µm). Clearly, the inhibitory action of tempol during U-46,619-induced vasoconstriction was blunted by L-NAME.


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Fig. 6.   Series 5. Line graph showing the change in luminal diameter during U-46,619 in microperfused afferent arterioles during scavenging O2- with tempol (10-3 M) in the presence (tempol + L-NAME, n = 6) or absence (tempol, n = 6) of blockade of NO synthesis with L-NAME (10-4 M). Comparing the two groups, *P < 0.05 and **P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The stable TP receptor agonist U-46,619 vasoconstricts isolated rabbit microperfused afferent arterioles. This response can be attributed to activation of TP receptors because it is blocked by ifetroban. These results are similar to those previously reported for TP receptor-mediated vasoconstriction of isolated aorta (7), pulmonary arteries (40), and afferent arterioles of the hydronephrotic rat kidney preparation (25). The new findings in the present study are that the vasoconstrictor response to U-46,619 is abolished by a membrane-permeable superoxide dismutase mimetic and is enhanced by NO synthesis blockade. We conclude that TP receptor activation leads to generation of O2-, which is permissive in the vasoconstriction, and to NO, which buffers the vasoconstriction. L-NAME restored a vasoconstrictor response to U-46,619 in vessels pretreated with tempol. This suggests an important role for the interaction between NO and O2- in TP receptor-mediated vasoconstriction in the afferent arteriole.

NO is a powerful endothelium-derived vasodilator that maintains basal vascular tone and modulates the vasoconstrictor actions of several agonists within the kidney. Ito et al. demonstrated that NO blunts the vasoconstriction caused by ANG II (19) and endothelin-1 (18) of isolated, microperfused afferent arterioles. The present study confirms that blockade of NO synthesis significantly reduces basal luminal diameter, indicating that NO is produced tonically in rabbit isolated, perfused afferent arterioles (19). The data show, for the first time, that TP receptor activation stimulates NO, which buffers the vasoconstrictor action of U-46,619 in afferent arterioles.

The mechanism by which U-46,619 stimulates NO in afferent arterioles remains unknown. One possible mechanism may involve a TP receptor located on the endothelium. Kent et al. (22) identified a TP receptor on isolated human endothelial cells that stimulates increases in intracellular calcium, which can activate endothelial-derived NO synthase. The importance of the endothelium in modulating the vasoconstrictor actions of TP receptor activation depends on the vascular site. For example, removal of the endothelium or blockade of NO synthesis in the aorta (7) and coronary artery (37) enhances U-46,619-induced vasoconstriction. However, blockade of NO synthase in the pulmonary vasculature either enhances (40) or has no effect (20) on U-46,619-induced vasoconstriction. Endothelial NO synthase may also be activated indirectly by a rise in intracellular calcium generated in response to a primary action of TP receptors on vascular smooth muscle cells where TP receptors are readily expressed. Dora et al. (6) have shown such electromechanical coupling between vascular smooth muscle and endothelial cells of the hamster cheek pouch. Activation of TP receptors in afferent arterioles of the hydronephrotic kidney increases intracellular calcium (25). However, the role of electromechanical coupling or endothelial TP receptors in mediating the stimulation of NO in the afferent arteriole remains to be elucidated.

O2- is scavenged extracellularly by copper-zinc superoxide dismutase (CuZnSOD) and intracellularly by CuZnSOD and manganese SOD. Mehta et al. (26) showed that inhibition of TxA2 synthesis decreases the production of O2- in activated human neutrophils. They suggested that TxA2 stimulates the production of O2-. Griendling et al. (10) provided direct evidence that ANG II stimulates O2- production in vascular smooth muscle cells via activation of NADPH oxidase. We selected the nitroxide tempol in our studies. It is a stable, membrane-permeable, metal-independent SOD mimetic (16, 29). U-46,619 caused a dose-dependent vasoconstriction of afferent arterioles. This response was completely prevented in vessels pretreated with tempol. We conclude that U-46,619 stimulates the production of O2-, which is permissive for TP receptor-induced vasoconstriction of the afferent arteriole.

The source of O2- production in the renal afferent arteriole remains unknown. O2- is produced by cellular electron transport chains such as those in mitochondria and endoplasmic reticulum (14), NO synthase (4, 32), cyclooxygenase (23), lipoxygenase (23), xanthine oxidase (12), and NADPH oxidase (10). All of these enzymes are expressed in the kidney. NO synthase and cyclooxygenase are known to be expressed in the afferent arteriole. The importance of these enzymes in mediating U-46,619-induced stimulation of O2- in the afferent arteriole remains to be determined.

Our data suggest that one mechanism whereby O2- mediates U-46,619-induced vasoconstriction is through interaction with NO. Gryglewski et al. (13) first showed in vascular endothelial cells that O2- is involved in the inactivation of NO. Since then, several studies have demonstrated that scavenging of O2- increases the release of bioactive NO in the vasculature in situ (39) and in cultured vascular endothelial cells (12). In the present study, we show that blockade of the U-46,619-induced vasoconstriction by tempol is largely prevented by inhibition of NO synthesis with L-NAME. This data suggests that production of O2- after U-46,619 decreases the bioactivity of stimulated NO and that this promotes the vasoconstriction of afferent arterioles. However, additional mechanisms must be involved since L-NAME augmented the response to U-46,619 in the absence of tempol. Superoxide can also stimulate inositol 1,4,5 trisphosphate (IP3) formation and thus increase intracellular calcium in vascular smooth muscle cells (44). Therefore, tempol may reduce superoxide-mediated increases in intracellular calcium and thus block the vasoconstrictor response to U-46,619.

Tempol has been evaluated extensively as a scavenger of O2- in vitro and in vivo. We evaluated the specificity of tempol's actions in the rabbit isolated perfused afferent arteriole. Tempol given alone for 60 min did not alter the diameter of the vessel. This indicates that tempol does not have nonspecific effects on afferent arteriole tone. Vessels treated with paraquat, which is a O2--generating quinoline, showed graded but reversible vasoconstriction consistent with the contractile effects of O2- on blood vessels (34). Vessels pretreated with tempol were protected fully from paraquat-induced contractions, consistent with tempol's proposed mechanism of actions as a superoxide dismutase mimetic. Furthermore, we have previously reported (35) that tempol given in vivo reduces a marker for oxygen radical production. This confirms its ability to scavenge superoxide radical. The afferent arteriole responses to norepinephrine were unaffected by tempol (-69 ± 17% vs. -67 ± 13%, unpublished observations), indicating that G protein coupled signal transduction was intact in vessels treated with tempol. However, we have not performed direct studies to exclude the possibility that tempol impaired the vasoconstrictor response to U-46,619 by uncoupling G proteins. We have recently reported that the elevated blood pressure and renal vasoconstriction in the spontaneously hypertensive rat (SHR) are normalized by tempol (35, 36); however, TP receptor antagonism is not effective in this model (11). This suggests that tempol is not a TP receptor antagonist. We found that the antihypertensive and renal vasodilatory effect of tempol in the SHR is blocked when NO synthesis is reduced (36). This suggests an important role for the interaction between NO and O2- in genetic hypertension, similar to what we have found for the isolated afferent arteriole stimulated by U-46,619 in the present studies.

Metabolism of O2- by superoxide dismutase or tempol yields oxygen and H2O2. H2O2 can be either a vasodilator or a vasoconstrictor in the aorta (17, 45) and pulmonary artery (1). After blockade of NO generation with L-NAME and scavenging of O2- with tempol, the response of the afferent arteriole to U-46,619 was not fully restored to the level seen with U-46,619 alone. One possible explanation for this finding is that H2O2 generated in tempol-treated arterioles in response to U-46,619 may contribute to vasodilation. However, the action of H2O2 in the afferent arteriole remains to be investigated.

In conclusion, this study provides evidence that generation of O2- contributes to the contractile response to activation of TP receptors in afferent arterioles. TP receptor activation also stimulates NO, which buffers the contraction. The balance between O2- and NO is important in the vasoconstrictor response. These results suggest a role for oxygen radical therapy in conditions of endothelial dysfunction associated with oxidative stress and increased TxA2 production in the kidney such as hypertension, diabetes, and renal failure.


    ACKNOWLEDGEMENTS

We sincerely appreciate the assistance of Drs. Tom Pallone and Erik Silldorff from the University of Maryland Medical Center and of Drs. Oscar Carretero and YiLin Ren from Henry Ford Hospital for their technical advice on microdissection and microperfusion of the afferent arteriole.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-36079 and DK-49870 and by the George E. Schreiner Chair of Nephrology. Dr. Schnackenberg is a recipient of an American Heart Association Scientist Development Grant.

Present address of C. G. Schnackenberg and address for reprint requests and other correspondence: Renal Pharmacology, UW2521, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, PO Box 1539, King of Prussia, PA 19406-0939 (Email: Christine_G_Schnackenberg{at}sbphrol.com).

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. §1734 solely to indicate this fact.

Received 16 August 1999; accepted in final form 22 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Asano, K, Yamaguchi K, Kawai A, Mori M, Takasugi T, Umeda A, and Kawashiro T. Mechanism of constriction and dilatation of pulmonary artery induced by hydrogen peroxide. Nippon Kyobu Shikkan Gakkai Zasshi 31: 795-901, 1993[Medline].

2.   Auch-Schwelk, W, Katusic ZS, and Vanhoutte PM. Thromboxane A2 receptor antagonists inhibit endothelium-dependent contractions. Hypertension 15: 699-703, 1990[Abstract].

3.   Baylis, C. Effects of administered thromboxanes on the intact, normal rat kidney. Renal Physiol 10: 110-121, 1987[ISI][Medline].

4.   Cosentino, F, Patton S, D'Uscio LV, Werner ER, Werner-Felmayer G, Moreau P, Malinski T, and Luscher TF. Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats. J Clin Invest 101: 1530-1537, 1998[Abstract/Free Full Text].

5.   DelliPizzi, A, and Nasjletti A. Involvement of nitric oxide and potassium channels in the reduction of basal tone produced by blockade of thromboxane A2/prostaglandin H2 receptors in aortic rings of hypertensive rats. Clin Exp Hypertens 20: 903-916, 1998[ISI][Medline].

6.   Dora, KA, Doyle MP, and Duling BR. Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles. Proc Natl Acad Sci USA 94: 6529-6534, 1997[Abstract/Free Full Text].

7.   Folger, WH, Lawson D, Wilcox CS, and Mehta JL. Response of rat thoracic aortic rings to thromboxane mimetic U-46,619: roles of endothelium-derived relaxing factor and thromboxane A2 release. J Pharmacol Exp Ther 258: 669-675, 1991[Abstract].

8.   Gelvan, D, Saltman P, and Powell SR. Cardiac reperfusion damage prevented by a nitroxide free radical. Proc Natl Acad Sci USA 88: 4680-4684, 1991[Abstract].

9.   Gobbel, TG, Chan TYY, and Chan PH. Nitric oxide- and superoxide-mediated toxicity in cerebral endothelial cells. J Pharmacol Exp Ther 282: 1600-1607, 1997[Abstract/Free Full Text].

10.   Griendling, KK, Minieri CA, Ollerenshaw JD, and Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141-1148, 1994[Abstract].

11.   Grone, HJ, Grippo RS, Arendshorst WJ, and Dunn MJ. Role of thromboxane in control of arterial pressure and renal function in young spontaneously hypertensive rats. Am J Physiol Renal Fluid Electrolyte Physiol 250: F488-F496, 1986[ISI][Medline].

12.   Grunfeld, S, Hamilton CA, Mesaros S, McClain SW, Dominiczak AF, Bohr DF, and Malinski T. Role of superoxide in the depressed nitric oxide production by the endothelium of genetically hypertensive rats. Hypertension 26: 854-857, 1995[Abstract/Free Full Text].

13.   Gryglewski, RJ, Palmer RMJ, and Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived relaxing factor. Nature 320: 454-456, 1986[ISI][Medline].

14.   Halliwell, B, and Gutteridge JMC Role of free radicals and catalytic metal ions in human disease: an overview. Methods in Enzymology San Diego, CA: Academic, 1990, vol. 186 Chapt. 1.

15.   Hayashi, K, Loutzenhiser R, and Epstein M. Direct evidence that thromboxane mimetic U44069 preferentially constricts the afferent arteriole. J Am Soc Nephrol 8: 25-31, 1997[Abstract].

16.   Iannone, A, Bini A, Swartz HM, Tomasi A, and Vannini V. Metabolism in rat liver microsomes of the nitroxide spin probe tempol. Biochem Pharmacol 38: 2581-2586, 1989[ISI][Medline].

17.   Iesaki, T, Okada T, Shimada I, Yamaguchi H, and Ochi R. Decrease in Ca2+ sensitivity as a mechanism of hydrogen peroxide-induced relaxation of rabbit aorta. Cardiovasc Res 31: 820-825, 1996[ISI][Medline].

18.   Ito, S, Juncos LA, Nushiro N, Johnson CS, and Carretero OA. Endothelium-derived relaxing factor modulates endothelin action in afferent arterioles. Hypertension 17: 1052-1056, 1991[Abstract].

19.   Ito, S, Arima S, Ren YL, Juncos LA, and Carretero OA. Endothelium-derived relaxing factor/nitric oxide modulates angiotensin II action in the isolated perfused rabbit afferent but not efferent arteriole. J Clin Invest 91: 2012-2019, 1993[ISI][Medline].

20.   Jourdan, KB, Evans TW, Curzen NP, and Mitchel JA. Evidence for a dilator function of 8-iso prostaglandin F2 alpha in rat pulmonary artery. Br J Pharmacol 120: 1280-1285, 1997[Abstract].

21.   Karmeli, F, Eliakim R, Okon E, Samuni A, and Rachmilewitz D. A stable nitroxide radical effectively decreases mucosal damage in experimental colitis. Gut 37: 386-393, 1995[Abstract].

22.   Kent, KC, Collins LJ, Schwerin FT, Raychowdhury MK, and Ware JA. Identification of functional PGH2/TxA2 receptors on human endothelial cells. Circ Res 72: 958-965, 1993[Abstract].

23.   Kukreja, RC, Kontos HA, Hess ML, and Ellis EF. PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH. Circ Res 59: 612-619, 1986[Abstract].

24.   Lam, FT, Mavor AID, Potts DJ, and Giles GR. Improved 72-hour renal preservation with phosphate-buffered sucrose. Transplantation 47: 767-771, 1989[ISI][Medline].

25.   Loutzenhiser, R, Epstein M, Horton C, and Sonke P. Reversal of renal and smooth muscle actions of thromboxane mimetic U-44069 by diltiazem. Am J Physiol Renal Fluid Electrolyte Physiol 250: F619-F626, 1986[Abstract/Free Full Text].

26.   Mehta, JL, Lawson D, and Mehta P. Modulation of human neutrophil superoxide production by selective thromboxane synthetase inhibitor U63,557A. Life Sci 43: 923-928, 1988[ISI][Medline].

27.   Mitchell, JB, DeGraff W, Kaufman D, Krishna MC, Samuni A, Finkelstein E, Ahn MS, Hahn SM, Gamson J, and Russo A. Inhibition of oxygen-dependent radiation-induced damage by the nitroxide superoxide dismutase mimic tempol. Arch Biochem Biophys 289: 62-70, 1991[ISI][Medline].

28.   Nasjletti, A. The role of eicosanoids in angiotensin-dependent hypertension. Hypertension 31: 194-200, 1998[Abstract/Free Full Text].

29.   Nilsson, UA, Olsson LI, Carlin G, and Bylund-Fellenius AC. Inhibition of lipid peroxidation by spin labels: relationships between structure and function. J Biol Chem 19: 11131-11135, 1989.

30.   Ody, C, and Junod AF. Direct toxic effects of paraquat and oxygen on cultured endothelial cells. Lab Invest 52: 77-84, 1985[ISI][Medline].

31.   Oury, TD, Day BJ, and Crapo JD. Extracellular superoxide dismutase: a regulator of nitric oxide bioavailability. Lab Invest 75: 617-636, 1996[ISI][Medline].

32.   Pou, S, Pou WS, Bredt DS, Snyder SH, and Rosen GM. Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem 267: 24173-24176, 1992[Abstract/Free Full Text].

33.   Rapoport, RM, and Williams SP. Role of prostaglandins in acetylcholine-induced contraction of aorta from spontaneously hypertensive and Wistar-Kyoto rats. Hypertension 28: 64-75, 1996[Abstract/Free Full Text].

34.   Rubanyi, GM. Vascular effects of oxygen-derived free radicals. Free Radic Biol Med 4: 107-120, 1988[ISI][Medline].

35.   Schnackenberg, CG, and Wilcox CS. Two-week administration of tempol attenuates both hypertension and renal excretion of 8-iso prostaglandin F2alpha . Hypertension 33: 424-428, 1999[Abstract/Free Full Text].

36.   Schnackenberg, CG, Welch WJ, and Wilcox CS. Normalization of blood pressure and renal vascular resistance in SHR with a membrane-permeable superoxide dismutase mimetic: role of nitric oxide. Hypertension 32: 59-64, 1998[Abstract/Free Full Text].

37.   Szwahkun, KK, Lamping G, and Dole WP. Role of endothelium-derived relaxing factor and prostaglandins in responses of coronary arteries to thromboxane in vivo. Circ Res 66: 1729-1737, 1990[Abstract].

38.   Tesfamariam, B. Selective impairment of endothelium-dependent relaxations by prostaglandin endoperoxide. J Hypertens 12: 41-47, 1994[ISI][Medline].

39.   Tschudi, MR, Mesaros S, Luscher TF, and Malinski T. Direct in situ measurement of nitric oxide in mesenteric resistance arteries: increased decomposition by superoxide in hypertension. Hypertension 27: 32-35, 1996[Abstract/Free Full Text].

40.   Valentin, JP, Bessac AM, Maffre M, and John GW. Nitric oxide regulation of TP-receptor-mediated pulmonary vasoconstriction in the anesthetized, open-chest rat. Eur J Pharmacol 317: 335-342, 1996[ISI][Medline].

41.   Welch, WJ, and Wilcox CS. Potentiation of tubuloglomerular feedback in the rat by thromboxane mimetic: role of macula densa. J Clin Invest 89: 1857-1865, 1992[ISI][Medline].

42.   Wilcox, CS, and Welch WJ. Thromboxane mediation of the pressor response to infused angiotensin II. Am J Hypertens 3: 242-249, 1990[ISI][Medline].

43.   Wilcox, CS, Welch WJ, and Snellen H. Thromboxane mediates renal hemodynamic response to infused angiotensin II. Kidney Int 40: 1090-1097, 1991[ISI][Medline].

44.   Wu, L, and de Champlain J. Effects of superoxide on signaling pathways in smooth muscle cells in rats. Hypertension 34: 1247-1253, 1999[Abstract/Free Full Text].

45.   Yand, ZW, Zheng T, Zhang A, Altura BT, and Altura BM. Mechanism of hydrogen peroxide-induced contraction of rat aorta. Eur J Pharmacol 344: 169-181, 1998[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 279(2):F302-F308
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