Angiotensin II, reactive oxygen species, and Ca2+ signaling in afferent arterioles
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《美国生理学杂志》
Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
ABSTRACT
In afferent arteriolar vascular smooth muscle cells, ANG II induces a rise in cytosolic Ca2+ ([Ca2+]i) via inositol trisphosphate receptor (IP3R) stimulation and by activation of the adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR, which sensitizes the ryanodine receptor (RyR) to Ca2+. We hypothesize that ANG II stimulation of NAD(P)H oxidases leads to the formation of superoxide anion (O2–·), which, in turn, activates ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca2+]i. ANG II rapidly increased [Ca2+]i by 124 ± 12 nM. In the presence of apocynin, a specific inhibitor of NAD(P)H oxidase assembly, the [Ca2+]i response was reduced to 35 ± 5 nM (P < 0.01). Tempol, a superoxide dismutase mimetic, did not alter the [Ca2+]i response to ANG II at a concentration of 10–4 M (99 ± 12 nM), but 10–3 M tempol reduced the response to 32 ± 3 nM (P < 0.01). The addition of nicotinamide, an inhibitor of ADPR cyclase, to apocynin or tempol (10–3 M) resulted in no further inhibition. Measurement of superoxide production with the fluorescent probe tempo 9-AC showed that ANG II caused an increase of 48 ± 20 arbitrary units; apocynin or diphenyl iodonium (an inhibitor of flavoprotein oxidases) inhibited the response by 94%. Hydrogen peroxide (H2O2) was studied at physiological (10–7 M) and higher concentrations. In the presence of H2O2 (10–7 M), neither baseline [Ca2+]i nor the response to ANG II was altered (125 ± 15 nM), whereas H2O2 (10–6 and 10–5 M) inhibited the [Ca2+]i response to ANG II by 35 and 46%, respectively. We conclude that ANG II rapidly activates NAD(P)H oxidases of afferent arterioles, leading to the formation of O2–·, which then stimulates ADPR cyclase to form cADPR. cADPR, by sensitizing the RyR to Ca2+, augments the Ca2+ response (calcium-induced calcium release) initiated by activation of the IP3R.
cADP ribose; ryanodine; renal microcirculation; vascular smooth muscle cells; superoxide anion; tempo 9-AC
FOR MORE THAN A DECADE IT has been known that ANG II activates NAD(P)H oxidases of vascular smooth muscle cells (VSMC) to produce superoxide (O2–·) (15). Expression of NAD(P)H oxidase subunits has been demonstrated in afferent arterioles of rat kidney (2). Superoxide dismutase (SOD) subsequently converts O2–· to hydrogen peroxide (H2O2). These reactive oxygen species (ROS) were initially viewed as villains, participating in the deleterious chronic effects of ANG II on VSMC. Such conclusions were largely based on studies that examined the consequences of chronic ANG II infusion or of models of hypertension in which ANG II levels were elevated for extended periods of time (34, 40, 43). The structural alterations of resistance arteries that occur in hypertension (vascular remodeling) have been associated with upregulation of many ANG II signaling pathways. ANG II-mediated hyperactivation of vascular NAD(P)H oxidases, generation of ROS, and subsequent activation of mitogen-activated protein kinase, protein tyrosine kinases, and phosphatases and transcription factors have been detected (40). Animals chronically infused with subpressor amounts of ANG II demonstrate enhanced contractility of afferent arterioles (43). Vascular oxidative stress has been associated with a number of hypertensive animal models such as the spontaneously hypertensive rat and Dahl salt-sensitive hypertensive rat and with cyclosporine-related hypertension, human essential hypertension, and lead-induced hypertension (34). To understand the physiological role of ANG II and the renal microcirculation in pathological conditions, it is important to investigate the relationship between ANG II and ROS in normal animals. Only recently, however, has evidence begun to emerge that ROS may mediate some of the normal physiological effects of ANG II and other vasoconstrictor agonists such as endothelin-1 (ET-1) and thromboxane (TXA2) in the renal vasculature (34).
A commonly used tool to study the vascular effects of O2–· is the cell-permeable SOD mimetic 4-OH,2,2,6,6-tetramethyl piperidine-1-oxyl (tempol), which rapidly converts O2–· to H2O2, thereby immediately reducing O2–· levels. Recent studies show that the immediate vasoconstrictor effect of ANG II infused into the renal artery of normal dogs is attenuated during tempol infusion (23). In the isolated, perfused hydronephrotic rat kidney model, tempol (1–3 mM) inhibits ANG II-induced constriction of afferent arterioles by 57% (29). In microperfused normal rabbit afferent arterioles, paraquat was used to stimulate intracellular production of O2–·; tempol (1 mM) abolished the O2–·-dependent vasoconstriction (35). Studies of tubuloglomerular feedback (TGF) demonstrate that tempol diminishes the afferent arteriolar constrictive response to high salt perfusion of the macular densa (21). Catalase, which converts H2O2 to water and O2, does not alter the response to tempol, suggesting that it is O2–· rather than H2O2 that enhances the TGF response (21). To our knowledge, there have been no studies of Ca2+ signaling in afferent arterioles of normotensive rats following the acute administration of ANG II in conjunction with tempol or with the specific inhibitor of NAD(P)H oxidase, apocynin.
We recently showed that stimulation of afferent arterioles with ANG II leads to activation of the classic inositol trisphosphate (IP3) pathway and to stimulation of adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR (cADPR) (11). cADPR sensitizes the ryanodine receptor (RyR) to Ca2+, thereby amplifying calcium-induced calcium release (CICR) (13). The mechanism by which ANG II leads to activation of the ADPR cyclase in VSMC is unknown. Only one laboratory investigated the effect of ANG II on the activity of ADP-ribosyl cyclase (17). These investigators found that in membrane preparations of neonatal cardiomyocytes, ANG II increases ADPR cyclase activity in a dose-dependent fashion. Although the mechanism by which ANG II stimulates cyclase activity is unclear, this laboratory speculated that a G protein-coupled process is involved (17). Studies in VSMC from bovine coronary arteries show that generation of O2–· with xanthine/xanthine oxidase (X/XO) increases ADPR cyclase activity and that tempol inhibits this effect of X/XO (48). In fura-loaded VSMC of bovine coronary artery, X/XO increases [Ca2+]i by 200 nM; nicotinamide, an inhibitor of the ADPR cyclase, blocks the response by 36% (48). We postulate that ANG II activates NAD(P)H oxidase to increase the formation of O2–· and that O2–·, in turn, activates the membrane-bound ADPR cyclase and subsequent formation of cADPR (5, 26, 45). In the current study, we examined the effects of apocynin, tempol, and pegylated catalase (PEG catalase) on the ANG II-induced changes in [Ca2+]i in freshly isolated afferent arterioles. We also measured ANG II-stimulated production of O2–· with the fluoroprobe tempo 9-AC in the presence or absence of apocynin or the flavoprotein oxidase inhibitor diphenyl iodonium (DPI). As well, we investigated the effect of H2O2 on ANG II-mediated Ca2+ signaling.
METHODS
All studies were approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.
Preparation of fresh afferent arterioles. We used the magnetized polystyrene microsphere-sieving technique as previously described in our laboratory to isolate afferent arterioles (<20 μm in diameter) from 5-wk-old Sprague-Dawley rats maintained in the Chapel Hill Colony (11, 12). PBS, with the following composition in mM: 137 NaCl, 4.1 KCl, 0.66 KH2PO4, 3.4 Na2HPO4, 2.5 NaHCO3, 1.0 MgCl2, and 5 glucose, was adjusted daily to pH 7.4 at 4, 23, and 34°C. The vessel segments in PBS containing 0.1% BSA were treated with collagenase type IV (Worthington, 374 U/mg, 3–6 μg/ml) for 18 min at 34°C. Arterioles were loaded with fura 2-AM (2 μM) and 0.1% BSA for 45 min at 23°C in the dark. After arterioles were washed twice with PBS, the suspension was kept on ice. Ca2+ (1.1 mM) was added shortly before analysis of an arteriole.
Vessels were stimulated with ANG II (3 x 10–7 M) (9, 11). The concentrations of antagonists we chose were based either on published results: apocynin (1, 42), nicotinamide (37), PEG catalase (27), or a dose-response analysis (tempol, H2O2). Arterioles were incubated with inhibitors for 1–2 min before initiation of an experiment.
Measurement of cytosolic free calcium concentration. We measured [Ca2+]i as previously described (11, 12). Afferent arterioles were identified by their morphology and measured diameter of 15–20 μm. As well, we required visualization of microspheres in the lumen to exclude the possibility that the vessel was an efferent arteriole. The microspheres (4.0–4.5 μm) do not pass beyond the glomerular capillaries. An arteriole was centered in a small window of the optical field that was free of glomeruli or tubular fragments.
Although endothelial cells are present in these nonperfused afferent arterioles, we and others assumed that VSMC provide the major source of fura-2 fluorescence owing to the greater bulk of VSMC compared with endothelial cells (3). Studies in deendothelialized renal microvessels show that ANG II-induced Ca2+ increases are not different from those of control arterioles (32). Furthermore, all reagents were added to the bath and therefore there is not immediate intraluminal exposure of endothelial cells to agonists and antagonists. We showed previously that freshly isolated single preglomerular VSMC and afferent arterioles respond similarly, both qualitatively and quantitatively, to ET-1 (12).
The VSMC were excited alternately with light of 340- and 380-nm wavelength from a dual-excitation wavelength Delta-Scan equipped with dual monochronometers and a chopper [Photon Technology International (PTI)] as previously described (8–10). After passing signals through a barrier filter (510 nm), fluorescence was detected by a photomultiplier tube. Signal intensity was acquired, stored, and processed by an IBM-compatible Pentium computer and Felix software (PTI). Background subtraction was performed in all studies. There was no interruption in the recording during the addition of reagents to the chamber. A video camera projected images of afferent arterioles onto a video monitor permitting visualization of contraction of vessel segments.
Measurement of superoxide with tempo 9-AC. The fluoroprobe tempo 9-AC has been used to measure superoxide production in VSMC (38, 41). The fluoroprobe contains a nitroxide moiety, which quenches the fluorescence of molecule. Superoxide or hydroxyl ions result in the loss of spin trap resonance and an increase in fluorescent emission. SOD inhibited the fluorescence response (31). We loaded afferent arterioles with tempo 9-AC (20 μM) for 30 min at 23°C. After the vessels were washed three times with PBS, the fluorescence signal was measured with excitation set at 361 nm and emission at 460 nm. There was a tendency for photobleaching to occur that stabilized after 40–50 s. Background subtraction was performed for each measurement.
Reagents. We purchased ANG II, tempol, apocynin, diphenyliodonium, PEG catalase, H2O2, and nicotinamide from Sigma (St. Louis, MO), collagenase type IV from Worthington (Lakewood, NJ), fura 2-AM and tempo 9-AC from Molecular Probes (Eugene, OR), and magnetized microspheres from Spherotech (Libertyville, IL).
Statistics. The data are presented as means ± SE. Each data set was derived from afferent arterioles originating from at least three separate experiments, two rats (4 kidneys) per experiment. Individual arterioles were studied only once and then discarded. Paired data for arterioles before and after ANG II stimulation were tested with Student’s paired t-test. Unpaired t-tests were employed for comparisons of responses between two groups.
RESULTS
[Ca2+]i response to ANG II. Afferent arterioles in Ca2+-containing PBS responded to ANG II with an immediate sharp peak in [Ca2+]i. Based on the techniques employed in this study, the mean baseline [Ca2+]i was 128 ± 8, the peak 252 ± 13, and the plateau 169 ± 9 nM (n = 22, P < 0.01 for both peak and plateau values vs. baseline; Fig. 1). Thus the net peak increase in [Ca2+]i after ANG II stimulation for all control experiments is 124 ± 12 nM.
Effect of the SOD mimetic tempol on [Ca2+]i responses to ANG II. In our previous studies of the activation of the ADPR cyclase in afferent arterioles, we proposed that ANG II-mediated activation of NAD(P)H oxidase and production of O2–· importantly participate in [Ca2+]i signaling in these arterioles (11). Hence, we employed the SOD mimetic tempol to examine the role of O2–· in ANG II-mediated [Ca2+]i responses. Because there is no consistent agreement on the concentration of tempol that will block the effects of O2–· in normotensive animals, we studied concentrations of both 10–4 and 10–3 M. Neither concentration had any effect on baseline [Ca2+]i. In the presence of 10–4 M tempol, the peak [Ca2+]i response to ANG II was not statistically different from ANG II in the absence of tempol (99 ± 12 nM, n = 9, P > 0.30). In contrast, the [Ca2+]i response to ANG II in the presence of 10–3 M tempol was markedly reduced (32 ± 3 nM, n = 9, P < 0.01; Fig. 1).
Effect of the NADPH oxidase inhibitor apocynin on [Ca2+]i responses to ANG II. Apocynin, a specific inhibitor of NAD(P)H oxidase, acts by inhibiting subunit assembly of the enzyme (24). To determine whether inhibition of ANG II activation of NAD(P)H oxidase diminishes the [Ca2+]i response, we pretreated afferent arterioles with apocynin (30 μM). Apocynin did not change baseline [Ca2+]i but inhibited the ANG II peak response by 72% (35 ± 5 vs. 124 ± 12 nM, n = 11, P < 0.01; Figs. 1 and 2). The inhibitory effects of apocynin and tempol were not different from each other.
Effect of nicotinamide in addition to tempol or apocynin. We previously demonstrated that nicotinamide, a specific inhibitor of ADPR cyclase, diminishes ANG II [Ca2+]i responses by 66% (11). In the current study, similar results were obtained (67% inhibition; Fig. 2B). Superoxide is known to dimerize ADPR cyclase to enhance enzyme activity (5, 26, 45). We predicted that if ANG II-mediated generation of O2–· causes activation of the ADPR cyclase of afferent arteriolar VSMC, then inhibition of cyclase activity with nicotinamide should not be additive to the effect of tempol or apocynin. Accordingly, we pretreated afferent arterioles with nicotinamide (3 mM) and with nicotinamide plus tempol (10–3 M) or apocynin (30 μM). The peak increases in ANG II-stimulated [Ca2+]i were 28 ± 5, 29 ± 4, and 38 ± 8 nM, respectively (n = 5 for each group, P < 0.01 vs. ANG II alone; Fig. 2). Thus the inhibitory effect of tempol or apocynin was similar whether or not combined with nicotinamide, suggesting the sharing of a common signaling pathway.
H2O2 and ANG II-induced [Ca2+]i responses. A common question is whether the effects of ANG II in generating ROS are a consequence of the formation of O2–·, or of H2O2, or both. Furthermore, many studies of the effects of H2O2 in VSMC have employed concentrations that markedly exceed the presumed physiological range (vide infra). We tested the effects of three concentrations of H2O2 (10–7, 10–6, and 10 –5 M). None of the these concentrations of H2O2 had an effect on baseline [Ca2+]i (increase from baseline, 4 ± 2 nM, n = 7 in each group, P > 0.9). In the presence of 10–7 M H2O2, a concentration that likely mimics the reported physiological interstitial concentrations (4), the peak [Ca2+]i response to ANG II was 125 ± 15 nM (n = 7), a value identical to control, further suggesting that H2O2 in physiological concentrations does not alter ANG II-mediated [Ca2+]i responses. In contrast, the higher concentrations of H2O2 (10–6 and 10–5 M) diminished the ANG II-stimulated [Ca2+]i peak responses (81 ± 3 and 67 ± 8 nM, respectively, n = 7 for both, P < 0.01 vs. control, P > 0.15 for 10–6 vs. 10–5 M H2O2; Fig. 3). If H2O2 were a mediator of ANG II-induced increases in [Ca2+]i, one would expect an enhancement rather than an inhibition of the ANG II response. The inhibition suggests that high concentrations of H2O2 may exert a vasodilatory effect.
Effect of PEG catalase on ANG II-mediated increases in [Ca2+]i. To further document that O2–· but not H2O2 is involved in ANG II-induced [Ca2+]i signaling, we employed PEG catalase (250 U/ml) (27). In the presence of PEG catalase, the [Ca2+]i response to ANG II was 147 ± 26 nM, which is not different from ANG II alone (P > 0.4, n = 7).
Effect of apocynin and DPI on ANG II-mediated superoxide formation. Stimulation of afferent arterioles loaded with tempol 9-AC with ANG II produced an immediate increase in the fluorescent signal of 48 ± 20 arbitrary units (n = 10). In the presence of apocynin (50 μM) or DPI (1 mM), the signal was reduced by 94% (n = 6 for each inhibitor, P < 0.01; Fig. 4).
DISCUSSION
During the past decade, there has been an explosion in research focusing on the role of ROS in hypertension, acute renal failure, sepsis, and ischemia-reperfusion (25, 28, 34). Only recently, however, have investigators begun to examine the roles of ROS as intermediates in normal physiological processes. To understand the pathogenesis of hypertension and the important part played by the renal microcirculation, it is necessary to investigate the normal connections between ANG II and ROS in VSMC. Currently, there are no data on the acute effects of ANG II on Ca2+ signaling pathways and on the involvement of ROS in these processes in preglomerular vessels.
Previously, we showed that ANG II stimulation of afferent arterioles causes activation of the VSMC ADPR cyclase and formation of cADPR (11). We postulated that ANG II-mediated generation of IP3 and release of Ca2+ from the sarcoplasmic reticulum (SR) via the IP3R likely provides an initial and transient burst of Ca2+, which can then activate CICR via the RyR. The [Ca2+]i response to ANG II was blocked by the specific cADPR antagonist 8-Br cADPR and by a high concentration of ryanodine, which closes the RyR (11). In the current study, we provided evidence for participation of O2–· in the linkage between ANG II and cADPR in afferent arterioles.
Although a number of studies have been performed examining the effect of ROS on the ryanodine receptor of skeletal muscle (RyR1), there are fewer investigations of the cardiac ryanodine receptor (RyR2). Recent studies suggest that RyR2 is the RyR present in afferent arterioles (Wang X, Loutzenhiser K, Breaks J, and Loutzenhiser R. J Am Soc Neph 15: 211A, 2004). Investigations of cardiac RyR2 suggest that low (nanomolar) concentrations of O2–· stimulate the ADPR cyclase to enhance the Ca2+ sensitivity of the RyR, whereas high concentrations of O2–· produce a loss of function of calmodulin, thereby increasing Ca2+ release from the RyR (19, 26). Superoxide generated with hypoxanthine/xanthine oxidase stimulated Ca2+ release from cardiac myocyte vesicles and this release was abolished by the addition of SOD or high concentrations of ryanodine (19). These data not only suggest that O2–· but not H2O2 is involved, but also that the IP3R is likely not activated by O2–·. High concentrations of O2–· (produced by 400 μM X/XO) have been reported to increase the open probability of RyR channels in permeabilized cardiac myocytes (49). These data suggest that supraphysiological concentrations of O2–· may increase the activity of RyR2.
Studies of ADPR cyclase activity in cardiac myocytes show that ischemia-reperfusion and the oxidizing agent t-butyl hydroperoxide increase enzyme activity and that tempol reverses this stimulation (45). These data likewise suggest that it is O2–· and not H2O2 that is responsible for enhanced ADPR cyclase activity. As noted above, low concentrations of O2–· stimulate the synthesis of cADPR and increase Ca2+ release in cardiac myocytes (26). It has been proposed that oxidation of cysteine residues to form stable disulfide bonds and dimers of the enzyme accounts for increased enzyme activity (5, 39). The ADPR cyclase of VSMC differs from the cyclase of sea urchin eggs, Aplysia, and HL-60 cells by being inhibited rather than stimulated by nitric oxide (NO) and Zn2+ (7, 47). The activity of ADPR cyclase of VSMC derived from bovine coronary arteries nearly doubles after treatment with the O2–·-generating system X/XO. Both tempol and PEG SOD reverse the increase in ADPR cyclase enzyme activity, again suggesting that it is O2–· and not H2O2 that is responsible for enzyme activation (48).
The tempo 9-AC data in the current study provide new evidence for the premise that ANG II can stimulate the rapid generation of O2–· and that a nearly immediate increase in [Ca2+]i occurs in afferent arterioles. The time course of ANG II-induced increases in ROS has been examined in cultured aortic VSMC with DCF fluorescence (33). These investigators found that ANG II caused an abrupt increase in H2O2 synthesis is less than 5 s. Because the conversion of O2–· to H2O2 depends on SOD, it is logical to conclude that the generation of O2–· from ANG II activation of NAD(P)H oxidase is even more rapid. Another study in cultured aortic VSMC noted that after a delay of 10 s, ROS generation increased for 30 s, declined again by 60 s, followed by a plateau at 30 min (36). ANG II stimulation of cultured aortic VSMC caused an increase in O2–· production of 372% within 10 min; earlier measurements were not made (20). In homogenates of aortic VSMC, TNF- more than doubled the concentration of O2–· in less than 1 min (6).
It has been speculated that the activities of intracellular oxidases typically seen in VSMC should produce levels of O2–· in the nanomolar range (44). Basal levels of O2–· vary among different blood vessels. For example, basal levels of O2–· are significantly higher in rabbit carotid artery compared with thoracic aorta (30). To our knowledge, no measurements of O2–· levels have been made in resistance vessels.
To investigate the role of ANG II activation of NAD(P)H and the formation of O2–·, we employed the specific NAD(P)H inhibitor apocynin. In the presence of apocynin, the [Ca2+]i response to ANG II was inhibited by 72%. These results show for the first time that the ANG II-mediated formation of ROS is an important component of ANG II-induced Ca2+ signaling in afferent arterioles. If, as we hypothesize, O2–· leads to increased activity of ADPR cyclase of VSMC, increased formation of cADPR, and sensitization of the RyR to Ca2+, then the addition of an inhibitor of the cyclase should not be additive to the effect of apocynin. Therefore, we pretreated afferent arterioles with both nicotinamide and with apocynin and nicotinamide and found that nicotinamide alone causes a 68% inhibition and the combination of the two agents a 69% inhibition of the peak Ca2+ response to ANG II. This value is not different from the effect of apocynin alone, further substantiating the view that it is the formation of O2–· that activates the ADPR cyclase. Other investigators showed that generation of superoxide with X/XO causes an increase in the activity of ADPR cylcase in bovine coronary VSMC (48). In Ca2+ studies of these VSMC, nicotinamide reduced the O2–· response to X/XO by 38% (48).
The SOD mimetic tempol has been used extensively to evaluate the participation of O2–· in vascular responses to constrictor agonists. Tempol is a membrane-permeant, metal-independent compound that converts O2–· to H2O2 and oxygen. In the isolated, perfused hydronephrotic kidney model, tempol (3 x 10–4 M) had no effect on ANG II-induced vasoconstriction of the afferent arteriole, but concentrations of 1 and 3 x 10–3 M inhibited constriction in a dose-dependent manner (29). This laboratory also studied the effect of tempol on basal afferent arteriolar diameter and found that tempol (10–4 M) has no effect in Wistar-Kyoto rats but increases arteriolar diameter from 18.8 to 22.0 μm in adult spontaneously hypertensive rats (18). In isolated, microperfused rabbit afferent arterioles, tempol (10–3 M) reverses the vasoconstrictor effect of O2–· generated with the use of paraquat. A concentration of 10–4 M tempol was not tested (35). In animals chronically infused with ANG II, tempol (10–4 M) diminishes the vasoconstriction in animals infused with ANG II (60 or 200 ng·kg–1·min–1) but has no effect on ANG II-mediated constriction of afferent arterioles of sham (saline)-infused rabbits (43).
In the current study, we tested both 10–4 and 10–3 M tempol. Tempol (10–4 M) has no effect on ANG II-induced [Ca2+]i responses. In contrast, tempol (10–3 M) inhibits the ANG II response by 74%, a value identical to that produced by apocynin. Thus we conclude that in normal animals, tempol (10–4 M) may be ineffective in modifying the [Ca2+]i response to ANG II. As we did with the apocynin experiments, we combined tempol (10–3 M) with nicotinamide and found again that there is no difference in the degree of inhibition. That tempol and apocynin have identical inhibitory effects strongly supports the hypothesis that ANG II-mediated increases in O2–· are responsible for a significant portion of the total [Ca2+]i response. We also studied the [Ca2+]i response to ANG II in the presence of PEG catalase and found that responses were similar to those of ANG II alone. To evaluate ANG II-induced generation of O2–· in freshly isolated afferent arterioles, we employed the fluoroprobe tempo 9-AC (31, 38, 41). ANG II caused a rapid increase of 48 ± 20 arbitrary units in O2–· that was inhibited by 94% by the NAD(P)H oxidase inhibitors apocynin and DPI. Thus all of our experimental data support the premise that ANG II activates NAD(P)H oxidase to form O2–· in afferent arterioles.
H2O2 has been considered as both a vasodilator and a vasoconstrictor depending on the concentrations employed and the blood vessel studied (14). To interpret the significance of these findings, it is important to define physiological concentrations. Microdialysis measurements of H2O2 concentration in the interstitium of the kidney are believed to represent tissue concentrations of the freely diffusible peroxide molecule (4). Basal renal cortical interstitial H2O2 concentration is reported to be 56 nM; following infusion of tempol, the value rose to 96 nM and returned to control values with the addition of catalase (4). In mesenteric artery rings with endothelium present or removed and precontracted with phenylephrine, 10- to 100-μM concentrations of H2O2 cause only contraction. In contrast, higher concentrations of H2O2 (0.3–1 mM) cause a biphasic response characterized by contraction of 2-min duration, followed by sustained dilatation (14). In mesenteric resistance arteries preconstricted with phenylephrine and pretreated with indomethacin and NG-nitro-L-arginine methyl ester, H2O2 dose dependently relaxes the vessel; 100 per cent relaxation is achieved at a concentration of 50 μM after 4 min (22). Exposure of endothelium-denuded and endothelium-intact basilar arteries to H2O2 (2.2 x 10–5 to 4.4 x 10–3 M) causes contraction (46). Measurement of [Ca2+]i responses to H2O2 (4.4 to 440 x 10–6 M) in cultured VSMC of basilar artery shows a dose-dependent increase in [Ca2+]i that is markedly reduced in the presence of Ca2+-free medium or of verapamil (46). Studies performed in cultured aortic VSMC showed that the [Ca2+]i response to ANG II is abolished by catalase (29). Thus the majority of these studies suggest that concentrations of H2O2 of 10–6 M or greater cause vasodilatation.
We believe that the current study is the first to examine putative physiological concentrations of H2O2 as well as higher concentrations and their impact on the [Ca2+]i responses to ANG II in afferent arterioles. H2O2 (10–7 to 10–5 M) has no discernable effect on baseline [Ca2+]i. In the presence of 10–7 M H2O2, the [Ca2+]i response to ANG II was identical to control values, demonstrating that at a reported physiological concentration, H2O2 has no impact on ANG II-mediated [Ca2+]i signaling in normal afferent arterioles. These results afford further support for the hypothesis that it is O2–· and not H2O2 that is responsible for the linkage between ANG II and increases in [Ca2+]i signaling. The two higher concentration of H2O2, however, reduced the [Ca2+]i response to ANG II by 35 and 46%, respectively. The mechanism by which high concentrations of H2O2 inhibits [Ca2+]i responses to ANG II is uncertain. High concentrations of H2O2 (K0.5 = 74 ± 5 μM) may interfere with ANG II [Ca2+]i signaling by damaging the SR Ca2+ pump (16). Others have suggested that H2O2 (1 mM) activates KCa channels (14). The vasodilatory effects of 10–3 M H2O2 appear to be independent of adenylate and guanylate cyclases (29).
Although our data support the premise that ANG II-mediated generation of O2–· results in activation of ADPR cyclase of VSMC and thus enhancement of CICR, we do not suggest that such a mechanism is the exclusive pathway for the effects of O2–· in arteriolar VSMC. Among the possible important actions of O2–· on the renal vasculature are interaction of O2–· with NO to scavenge O2–· and to form peroxynitrate, stimulation of formation of 8-isoprostane PGF-2 from arachidonic acid, inhibition of prostacyclin synthesis, and increased production of adenosine (34).
In summary, we demonstrate that ANG II-mediated [Ca2+]i responses in freshly isolated afferent arterioles are closely linked to the generation of O2–·. Inhibition of NAD(P)H oxidase assembly with apocynin or treatment with the SOD mimetic tempol decreases the [Ca2+]i response to ANG II by 73%, on average. The generation of O2–· is inhibited by apocynin and DPI. H2O2 at a reported physiological concentration of 10–7 M has no effect on [Ca2+]i signaling. The ADPR cyclase inhibitor nicotinamide does not further diminish the [Ca2+]i response to ANG II, suggesting sharing of a common pathway. We propose that ANG II activates NAD(P)H oxidase to increase formation of O2–· with subsequent stimulation of ADPR cyclase activity and formation of cADPR, which sensitizes the RyR to Ca2+ and thus magnifies the overall [Ca2+]i response to ANG II.
GRANTS
This work was supported in part by an award from the Thomas H. Maren Foundation and from National Institutes of Health Research Grant HL-02334.
FOOTNOTES
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ABSTRACT
In afferent arteriolar vascular smooth muscle cells, ANG II induces a rise in cytosolic Ca2+ ([Ca2+]i) via inositol trisphosphate receptor (IP3R) stimulation and by activation of the adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR, which sensitizes the ryanodine receptor (RyR) to Ca2+. We hypothesize that ANG II stimulation of NAD(P)H oxidases leads to the formation of superoxide anion (O2–·), which, in turn, activates ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca2+]i. ANG II rapidly increased [Ca2+]i by 124 ± 12 nM. In the presence of apocynin, a specific inhibitor of NAD(P)H oxidase assembly, the [Ca2+]i response was reduced to 35 ± 5 nM (P < 0.01). Tempol, a superoxide dismutase mimetic, did not alter the [Ca2+]i response to ANG II at a concentration of 10–4 M (99 ± 12 nM), but 10–3 M tempol reduced the response to 32 ± 3 nM (P < 0.01). The addition of nicotinamide, an inhibitor of ADPR cyclase, to apocynin or tempol (10–3 M) resulted in no further inhibition. Measurement of superoxide production with the fluorescent probe tempo 9-AC showed that ANG II caused an increase of 48 ± 20 arbitrary units; apocynin or diphenyl iodonium (an inhibitor of flavoprotein oxidases) inhibited the response by 94%. Hydrogen peroxide (H2O2) was studied at physiological (10–7 M) and higher concentrations. In the presence of H2O2 (10–7 M), neither baseline [Ca2+]i nor the response to ANG II was altered (125 ± 15 nM), whereas H2O2 (10–6 and 10–5 M) inhibited the [Ca2+]i response to ANG II by 35 and 46%, respectively. We conclude that ANG II rapidly activates NAD(P)H oxidases of afferent arterioles, leading to the formation of O2–·, which then stimulates ADPR cyclase to form cADPR. cADPR, by sensitizing the RyR to Ca2+, augments the Ca2+ response (calcium-induced calcium release) initiated by activation of the IP3R.
cADP ribose; ryanodine; renal microcirculation; vascular smooth muscle cells; superoxide anion; tempo 9-AC
FOR MORE THAN A DECADE IT has been known that ANG II activates NAD(P)H oxidases of vascular smooth muscle cells (VSMC) to produce superoxide (O2–·) (15). Expression of NAD(P)H oxidase subunits has been demonstrated in afferent arterioles of rat kidney (2). Superoxide dismutase (SOD) subsequently converts O2–· to hydrogen peroxide (H2O2). These reactive oxygen species (ROS) were initially viewed as villains, participating in the deleterious chronic effects of ANG II on VSMC. Such conclusions were largely based on studies that examined the consequences of chronic ANG II infusion or of models of hypertension in which ANG II levels were elevated for extended periods of time (34, 40, 43). The structural alterations of resistance arteries that occur in hypertension (vascular remodeling) have been associated with upregulation of many ANG II signaling pathways. ANG II-mediated hyperactivation of vascular NAD(P)H oxidases, generation of ROS, and subsequent activation of mitogen-activated protein kinase, protein tyrosine kinases, and phosphatases and transcription factors have been detected (40). Animals chronically infused with subpressor amounts of ANG II demonstrate enhanced contractility of afferent arterioles (43). Vascular oxidative stress has been associated with a number of hypertensive animal models such as the spontaneously hypertensive rat and Dahl salt-sensitive hypertensive rat and with cyclosporine-related hypertension, human essential hypertension, and lead-induced hypertension (34). To understand the physiological role of ANG II and the renal microcirculation in pathological conditions, it is important to investigate the relationship between ANG II and ROS in normal animals. Only recently, however, has evidence begun to emerge that ROS may mediate some of the normal physiological effects of ANG II and other vasoconstrictor agonists such as endothelin-1 (ET-1) and thromboxane (TXA2) in the renal vasculature (34).
A commonly used tool to study the vascular effects of O2–· is the cell-permeable SOD mimetic 4-OH,2,2,6,6-tetramethyl piperidine-1-oxyl (tempol), which rapidly converts O2–· to H2O2, thereby immediately reducing O2–· levels. Recent studies show that the immediate vasoconstrictor effect of ANG II infused into the renal artery of normal dogs is attenuated during tempol infusion (23). In the isolated, perfused hydronephrotic rat kidney model, tempol (1–3 mM) inhibits ANG II-induced constriction of afferent arterioles by 57% (29). In microperfused normal rabbit afferent arterioles, paraquat was used to stimulate intracellular production of O2–·; tempol (1 mM) abolished the O2–·-dependent vasoconstriction (35). Studies of tubuloglomerular feedback (TGF) demonstrate that tempol diminishes the afferent arteriolar constrictive response to high salt perfusion of the macular densa (21). Catalase, which converts H2O2 to water and O2, does not alter the response to tempol, suggesting that it is O2–· rather than H2O2 that enhances the TGF response (21). To our knowledge, there have been no studies of Ca2+ signaling in afferent arterioles of normotensive rats following the acute administration of ANG II in conjunction with tempol or with the specific inhibitor of NAD(P)H oxidase, apocynin.
We recently showed that stimulation of afferent arterioles with ANG II leads to activation of the classic inositol trisphosphate (IP3) pathway and to stimulation of adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR (cADPR) (11). cADPR sensitizes the ryanodine receptor (RyR) to Ca2+, thereby amplifying calcium-induced calcium release (CICR) (13). The mechanism by which ANG II leads to activation of the ADPR cyclase in VSMC is unknown. Only one laboratory investigated the effect of ANG II on the activity of ADP-ribosyl cyclase (17). These investigators found that in membrane preparations of neonatal cardiomyocytes, ANG II increases ADPR cyclase activity in a dose-dependent fashion. Although the mechanism by which ANG II stimulates cyclase activity is unclear, this laboratory speculated that a G protein-coupled process is involved (17). Studies in VSMC from bovine coronary arteries show that generation of O2–· with xanthine/xanthine oxidase (X/XO) increases ADPR cyclase activity and that tempol inhibits this effect of X/XO (48). In fura-loaded VSMC of bovine coronary artery, X/XO increases [Ca2+]i by 200 nM; nicotinamide, an inhibitor of the ADPR cyclase, blocks the response by 36% (48). We postulate that ANG II activates NAD(P)H oxidase to increase the formation of O2–· and that O2–·, in turn, activates the membrane-bound ADPR cyclase and subsequent formation of cADPR (5, 26, 45). In the current study, we examined the effects of apocynin, tempol, and pegylated catalase (PEG catalase) on the ANG II-induced changes in [Ca2+]i in freshly isolated afferent arterioles. We also measured ANG II-stimulated production of O2–· with the fluoroprobe tempo 9-AC in the presence or absence of apocynin or the flavoprotein oxidase inhibitor diphenyl iodonium (DPI). As well, we investigated the effect of H2O2 on ANG II-mediated Ca2+ signaling.
METHODS
All studies were approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.
Preparation of fresh afferent arterioles. We used the magnetized polystyrene microsphere-sieving technique as previously described in our laboratory to isolate afferent arterioles (<20 μm in diameter) from 5-wk-old Sprague-Dawley rats maintained in the Chapel Hill Colony (11, 12). PBS, with the following composition in mM: 137 NaCl, 4.1 KCl, 0.66 KH2PO4, 3.4 Na2HPO4, 2.5 NaHCO3, 1.0 MgCl2, and 5 glucose, was adjusted daily to pH 7.4 at 4, 23, and 34°C. The vessel segments in PBS containing 0.1% BSA were treated with collagenase type IV (Worthington, 374 U/mg, 3–6 μg/ml) for 18 min at 34°C. Arterioles were loaded with fura 2-AM (2 μM) and 0.1% BSA for 45 min at 23°C in the dark. After arterioles were washed twice with PBS, the suspension was kept on ice. Ca2+ (1.1 mM) was added shortly before analysis of an arteriole.
Vessels were stimulated with ANG II (3 x 10–7 M) (9, 11). The concentrations of antagonists we chose were based either on published results: apocynin (1, 42), nicotinamide (37), PEG catalase (27), or a dose-response analysis (tempol, H2O2). Arterioles were incubated with inhibitors for 1–2 min before initiation of an experiment.
Measurement of cytosolic free calcium concentration. We measured [Ca2+]i as previously described (11, 12). Afferent arterioles were identified by their morphology and measured diameter of 15–20 μm. As well, we required visualization of microspheres in the lumen to exclude the possibility that the vessel was an efferent arteriole. The microspheres (4.0–4.5 μm) do not pass beyond the glomerular capillaries. An arteriole was centered in a small window of the optical field that was free of glomeruli or tubular fragments.
Although endothelial cells are present in these nonperfused afferent arterioles, we and others assumed that VSMC provide the major source of fura-2 fluorescence owing to the greater bulk of VSMC compared with endothelial cells (3). Studies in deendothelialized renal microvessels show that ANG II-induced Ca2+ increases are not different from those of control arterioles (32). Furthermore, all reagents were added to the bath and therefore there is not immediate intraluminal exposure of endothelial cells to agonists and antagonists. We showed previously that freshly isolated single preglomerular VSMC and afferent arterioles respond similarly, both qualitatively and quantitatively, to ET-1 (12).
The VSMC were excited alternately with light of 340- and 380-nm wavelength from a dual-excitation wavelength Delta-Scan equipped with dual monochronometers and a chopper [Photon Technology International (PTI)] as previously described (8–10). After passing signals through a barrier filter (510 nm), fluorescence was detected by a photomultiplier tube. Signal intensity was acquired, stored, and processed by an IBM-compatible Pentium computer and Felix software (PTI). Background subtraction was performed in all studies. There was no interruption in the recording during the addition of reagents to the chamber. A video camera projected images of afferent arterioles onto a video monitor permitting visualization of contraction of vessel segments.
Measurement of superoxide with tempo 9-AC. The fluoroprobe tempo 9-AC has been used to measure superoxide production in VSMC (38, 41). The fluoroprobe contains a nitroxide moiety, which quenches the fluorescence of molecule. Superoxide or hydroxyl ions result in the loss of spin trap resonance and an increase in fluorescent emission. SOD inhibited the fluorescence response (31). We loaded afferent arterioles with tempo 9-AC (20 μM) for 30 min at 23°C. After the vessels were washed three times with PBS, the fluorescence signal was measured with excitation set at 361 nm and emission at 460 nm. There was a tendency for photobleaching to occur that stabilized after 40–50 s. Background subtraction was performed for each measurement.
Reagents. We purchased ANG II, tempol, apocynin, diphenyliodonium, PEG catalase, H2O2, and nicotinamide from Sigma (St. Louis, MO), collagenase type IV from Worthington (Lakewood, NJ), fura 2-AM and tempo 9-AC from Molecular Probes (Eugene, OR), and magnetized microspheres from Spherotech (Libertyville, IL).
Statistics. The data are presented as means ± SE. Each data set was derived from afferent arterioles originating from at least three separate experiments, two rats (4 kidneys) per experiment. Individual arterioles were studied only once and then discarded. Paired data for arterioles before and after ANG II stimulation were tested with Student’s paired t-test. Unpaired t-tests were employed for comparisons of responses between two groups.
RESULTS
[Ca2+]i response to ANG II. Afferent arterioles in Ca2+-containing PBS responded to ANG II with an immediate sharp peak in [Ca2+]i. Based on the techniques employed in this study, the mean baseline [Ca2+]i was 128 ± 8, the peak 252 ± 13, and the plateau 169 ± 9 nM (n = 22, P < 0.01 for both peak and plateau values vs. baseline; Fig. 1). Thus the net peak increase in [Ca2+]i after ANG II stimulation for all control experiments is 124 ± 12 nM.
Effect of the SOD mimetic tempol on [Ca2+]i responses to ANG II. In our previous studies of the activation of the ADPR cyclase in afferent arterioles, we proposed that ANG II-mediated activation of NAD(P)H oxidase and production of O2–· importantly participate in [Ca2+]i signaling in these arterioles (11). Hence, we employed the SOD mimetic tempol to examine the role of O2–· in ANG II-mediated [Ca2+]i responses. Because there is no consistent agreement on the concentration of tempol that will block the effects of O2–· in normotensive animals, we studied concentrations of both 10–4 and 10–3 M. Neither concentration had any effect on baseline [Ca2+]i. In the presence of 10–4 M tempol, the peak [Ca2+]i response to ANG II was not statistically different from ANG II in the absence of tempol (99 ± 12 nM, n = 9, P > 0.30). In contrast, the [Ca2+]i response to ANG II in the presence of 10–3 M tempol was markedly reduced (32 ± 3 nM, n = 9, P < 0.01; Fig. 1).
Effect of the NADPH oxidase inhibitor apocynin on [Ca2+]i responses to ANG II. Apocynin, a specific inhibitor of NAD(P)H oxidase, acts by inhibiting subunit assembly of the enzyme (24). To determine whether inhibition of ANG II activation of NAD(P)H oxidase diminishes the [Ca2+]i response, we pretreated afferent arterioles with apocynin (30 μM). Apocynin did not change baseline [Ca2+]i but inhibited the ANG II peak response by 72% (35 ± 5 vs. 124 ± 12 nM, n = 11, P < 0.01; Figs. 1 and 2). The inhibitory effects of apocynin and tempol were not different from each other.
Effect of nicotinamide in addition to tempol or apocynin. We previously demonstrated that nicotinamide, a specific inhibitor of ADPR cyclase, diminishes ANG II [Ca2+]i responses by 66% (11). In the current study, similar results were obtained (67% inhibition; Fig. 2B). Superoxide is known to dimerize ADPR cyclase to enhance enzyme activity (5, 26, 45). We predicted that if ANG II-mediated generation of O2–· causes activation of the ADPR cyclase of afferent arteriolar VSMC, then inhibition of cyclase activity with nicotinamide should not be additive to the effect of tempol or apocynin. Accordingly, we pretreated afferent arterioles with nicotinamide (3 mM) and with nicotinamide plus tempol (10–3 M) or apocynin (30 μM). The peak increases in ANG II-stimulated [Ca2+]i were 28 ± 5, 29 ± 4, and 38 ± 8 nM, respectively (n = 5 for each group, P < 0.01 vs. ANG II alone; Fig. 2). Thus the inhibitory effect of tempol or apocynin was similar whether or not combined with nicotinamide, suggesting the sharing of a common signaling pathway.
H2O2 and ANG II-induced [Ca2+]i responses. A common question is whether the effects of ANG II in generating ROS are a consequence of the formation of O2–·, or of H2O2, or both. Furthermore, many studies of the effects of H2O2 in VSMC have employed concentrations that markedly exceed the presumed physiological range (vide infra). We tested the effects of three concentrations of H2O2 (10–7, 10–6, and 10 –5 M). None of the these concentrations of H2O2 had an effect on baseline [Ca2+]i (increase from baseline, 4 ± 2 nM, n = 7 in each group, P > 0.9). In the presence of 10–7 M H2O2, a concentration that likely mimics the reported physiological interstitial concentrations (4), the peak [Ca2+]i response to ANG II was 125 ± 15 nM (n = 7), a value identical to control, further suggesting that H2O2 in physiological concentrations does not alter ANG II-mediated [Ca2+]i responses. In contrast, the higher concentrations of H2O2 (10–6 and 10–5 M) diminished the ANG II-stimulated [Ca2+]i peak responses (81 ± 3 and 67 ± 8 nM, respectively, n = 7 for both, P < 0.01 vs. control, P > 0.15 for 10–6 vs. 10–5 M H2O2; Fig. 3). If H2O2 were a mediator of ANG II-induced increases in [Ca2+]i, one would expect an enhancement rather than an inhibition of the ANG II response. The inhibition suggests that high concentrations of H2O2 may exert a vasodilatory effect.
Effect of PEG catalase on ANG II-mediated increases in [Ca2+]i. To further document that O2–· but not H2O2 is involved in ANG II-induced [Ca2+]i signaling, we employed PEG catalase (250 U/ml) (27). In the presence of PEG catalase, the [Ca2+]i response to ANG II was 147 ± 26 nM, which is not different from ANG II alone (P > 0.4, n = 7).
Effect of apocynin and DPI on ANG II-mediated superoxide formation. Stimulation of afferent arterioles loaded with tempol 9-AC with ANG II produced an immediate increase in the fluorescent signal of 48 ± 20 arbitrary units (n = 10). In the presence of apocynin (50 μM) or DPI (1 mM), the signal was reduced by 94% (n = 6 for each inhibitor, P < 0.01; Fig. 4).
DISCUSSION
During the past decade, there has been an explosion in research focusing on the role of ROS in hypertension, acute renal failure, sepsis, and ischemia-reperfusion (25, 28, 34). Only recently, however, have investigators begun to examine the roles of ROS as intermediates in normal physiological processes. To understand the pathogenesis of hypertension and the important part played by the renal microcirculation, it is necessary to investigate the normal connections between ANG II and ROS in VSMC. Currently, there are no data on the acute effects of ANG II on Ca2+ signaling pathways and on the involvement of ROS in these processes in preglomerular vessels.
Previously, we showed that ANG II stimulation of afferent arterioles causes activation of the VSMC ADPR cyclase and formation of cADPR (11). We postulated that ANG II-mediated generation of IP3 and release of Ca2+ from the sarcoplasmic reticulum (SR) via the IP3R likely provides an initial and transient burst of Ca2+, which can then activate CICR via the RyR. The [Ca2+]i response to ANG II was blocked by the specific cADPR antagonist 8-Br cADPR and by a high concentration of ryanodine, which closes the RyR (11). In the current study, we provided evidence for participation of O2–· in the linkage between ANG II and cADPR in afferent arterioles.
Although a number of studies have been performed examining the effect of ROS on the ryanodine receptor of skeletal muscle (RyR1), there are fewer investigations of the cardiac ryanodine receptor (RyR2). Recent studies suggest that RyR2 is the RyR present in afferent arterioles (Wang X, Loutzenhiser K, Breaks J, and Loutzenhiser R. J Am Soc Neph 15: 211A, 2004). Investigations of cardiac RyR2 suggest that low (nanomolar) concentrations of O2–· stimulate the ADPR cyclase to enhance the Ca2+ sensitivity of the RyR, whereas high concentrations of O2–· produce a loss of function of calmodulin, thereby increasing Ca2+ release from the RyR (19, 26). Superoxide generated with hypoxanthine/xanthine oxidase stimulated Ca2+ release from cardiac myocyte vesicles and this release was abolished by the addition of SOD or high concentrations of ryanodine (19). These data not only suggest that O2–· but not H2O2 is involved, but also that the IP3R is likely not activated by O2–·. High concentrations of O2–· (produced by 400 μM X/XO) have been reported to increase the open probability of RyR channels in permeabilized cardiac myocytes (49). These data suggest that supraphysiological concentrations of O2–· may increase the activity of RyR2.
Studies of ADPR cyclase activity in cardiac myocytes show that ischemia-reperfusion and the oxidizing agent t-butyl hydroperoxide increase enzyme activity and that tempol reverses this stimulation (45). These data likewise suggest that it is O2–· and not H2O2 that is responsible for enhanced ADPR cyclase activity. As noted above, low concentrations of O2–· stimulate the synthesis of cADPR and increase Ca2+ release in cardiac myocytes (26). It has been proposed that oxidation of cysteine residues to form stable disulfide bonds and dimers of the enzyme accounts for increased enzyme activity (5, 39). The ADPR cyclase of VSMC differs from the cyclase of sea urchin eggs, Aplysia, and HL-60 cells by being inhibited rather than stimulated by nitric oxide (NO) and Zn2+ (7, 47). The activity of ADPR cyclase of VSMC derived from bovine coronary arteries nearly doubles after treatment with the O2–·-generating system X/XO. Both tempol and PEG SOD reverse the increase in ADPR cyclase enzyme activity, again suggesting that it is O2–· and not H2O2 that is responsible for enzyme activation (48).
The tempo 9-AC data in the current study provide new evidence for the premise that ANG II can stimulate the rapid generation of O2–· and that a nearly immediate increase in [Ca2+]i occurs in afferent arterioles. The time course of ANG II-induced increases in ROS has been examined in cultured aortic VSMC with DCF fluorescence (33). These investigators found that ANG II caused an abrupt increase in H2O2 synthesis is less than 5 s. Because the conversion of O2–· to H2O2 depends on SOD, it is logical to conclude that the generation of O2–· from ANG II activation of NAD(P)H oxidase is even more rapid. Another study in cultured aortic VSMC noted that after a delay of 10 s, ROS generation increased for 30 s, declined again by 60 s, followed by a plateau at 30 min (36). ANG II stimulation of cultured aortic VSMC caused an increase in O2–· production of 372% within 10 min; earlier measurements were not made (20). In homogenates of aortic VSMC, TNF- more than doubled the concentration of O2–· in less than 1 min (6).
It has been speculated that the activities of intracellular oxidases typically seen in VSMC should produce levels of O2–· in the nanomolar range (44). Basal levels of O2–· vary among different blood vessels. For example, basal levels of O2–· are significantly higher in rabbit carotid artery compared with thoracic aorta (30). To our knowledge, no measurements of O2–· levels have been made in resistance vessels.
To investigate the role of ANG II activation of NAD(P)H and the formation of O2–·, we employed the specific NAD(P)H inhibitor apocynin. In the presence of apocynin, the [Ca2+]i response to ANG II was inhibited by 72%. These results show for the first time that the ANG II-mediated formation of ROS is an important component of ANG II-induced Ca2+ signaling in afferent arterioles. If, as we hypothesize, O2–· leads to increased activity of ADPR cyclase of VSMC, increased formation of cADPR, and sensitization of the RyR to Ca2+, then the addition of an inhibitor of the cyclase should not be additive to the effect of apocynin. Therefore, we pretreated afferent arterioles with both nicotinamide and with apocynin and nicotinamide and found that nicotinamide alone causes a 68% inhibition and the combination of the two agents a 69% inhibition of the peak Ca2+ response to ANG II. This value is not different from the effect of apocynin alone, further substantiating the view that it is the formation of O2–· that activates the ADPR cyclase. Other investigators showed that generation of superoxide with X/XO causes an increase in the activity of ADPR cylcase in bovine coronary VSMC (48). In Ca2+ studies of these VSMC, nicotinamide reduced the O2–· response to X/XO by 38% (48).
The SOD mimetic tempol has been used extensively to evaluate the participation of O2–· in vascular responses to constrictor agonists. Tempol is a membrane-permeant, metal-independent compound that converts O2–· to H2O2 and oxygen. In the isolated, perfused hydronephrotic kidney model, tempol (3 x 10–4 M) had no effect on ANG II-induced vasoconstriction of the afferent arteriole, but concentrations of 1 and 3 x 10–3 M inhibited constriction in a dose-dependent manner (29). This laboratory also studied the effect of tempol on basal afferent arteriolar diameter and found that tempol (10–4 M) has no effect in Wistar-Kyoto rats but increases arteriolar diameter from 18.8 to 22.0 μm in adult spontaneously hypertensive rats (18). In isolated, microperfused rabbit afferent arterioles, tempol (10–3 M) reverses the vasoconstrictor effect of O2–· generated with the use of paraquat. A concentration of 10–4 M tempol was not tested (35). In animals chronically infused with ANG II, tempol (10–4 M) diminishes the vasoconstriction in animals infused with ANG II (60 or 200 ng·kg–1·min–1) but has no effect on ANG II-mediated constriction of afferent arterioles of sham (saline)-infused rabbits (43).
In the current study, we tested both 10–4 and 10–3 M tempol. Tempol (10–4 M) has no effect on ANG II-induced [Ca2+]i responses. In contrast, tempol (10–3 M) inhibits the ANG II response by 74%, a value identical to that produced by apocynin. Thus we conclude that in normal animals, tempol (10–4 M) may be ineffective in modifying the [Ca2+]i response to ANG II. As we did with the apocynin experiments, we combined tempol (10–3 M) with nicotinamide and found again that there is no difference in the degree of inhibition. That tempol and apocynin have identical inhibitory effects strongly supports the hypothesis that ANG II-mediated increases in O2–· are responsible for a significant portion of the total [Ca2+]i response. We also studied the [Ca2+]i response to ANG II in the presence of PEG catalase and found that responses were similar to those of ANG II alone. To evaluate ANG II-induced generation of O2–· in freshly isolated afferent arterioles, we employed the fluoroprobe tempo 9-AC (31, 38, 41). ANG II caused a rapid increase of 48 ± 20 arbitrary units in O2–· that was inhibited by 94% by the NAD(P)H oxidase inhibitors apocynin and DPI. Thus all of our experimental data support the premise that ANG II activates NAD(P)H oxidase to form O2–· in afferent arterioles.
H2O2 has been considered as both a vasodilator and a vasoconstrictor depending on the concentrations employed and the blood vessel studied (14). To interpret the significance of these findings, it is important to define physiological concentrations. Microdialysis measurements of H2O2 concentration in the interstitium of the kidney are believed to represent tissue concentrations of the freely diffusible peroxide molecule (4). Basal renal cortical interstitial H2O2 concentration is reported to be 56 nM; following infusion of tempol, the value rose to 96 nM and returned to control values with the addition of catalase (4). In mesenteric artery rings with endothelium present or removed and precontracted with phenylephrine, 10- to 100-μM concentrations of H2O2 cause only contraction. In contrast, higher concentrations of H2O2 (0.3–1 mM) cause a biphasic response characterized by contraction of 2-min duration, followed by sustained dilatation (14). In mesenteric resistance arteries preconstricted with phenylephrine and pretreated with indomethacin and NG-nitro-L-arginine methyl ester, H2O2 dose dependently relaxes the vessel; 100 per cent relaxation is achieved at a concentration of 50 μM after 4 min (22). Exposure of endothelium-denuded and endothelium-intact basilar arteries to H2O2 (2.2 x 10–5 to 4.4 x 10–3 M) causes contraction (46). Measurement of [Ca2+]i responses to H2O2 (4.4 to 440 x 10–6 M) in cultured VSMC of basilar artery shows a dose-dependent increase in [Ca2+]i that is markedly reduced in the presence of Ca2+-free medium or of verapamil (46). Studies performed in cultured aortic VSMC showed that the [Ca2+]i response to ANG II is abolished by catalase (29). Thus the majority of these studies suggest that concentrations of H2O2 of 10–6 M or greater cause vasodilatation.
We believe that the current study is the first to examine putative physiological concentrations of H2O2 as well as higher concentrations and their impact on the [Ca2+]i responses to ANG II in afferent arterioles. H2O2 (10–7 to 10–5 M) has no discernable effect on baseline [Ca2+]i. In the presence of 10–7 M H2O2, the [Ca2+]i response to ANG II was identical to control values, demonstrating that at a reported physiological concentration, H2O2 has no impact on ANG II-mediated [Ca2+]i signaling in normal afferent arterioles. These results afford further support for the hypothesis that it is O2–· and not H2O2 that is responsible for the linkage between ANG II and increases in [Ca2+]i signaling. The two higher concentration of H2O2, however, reduced the [Ca2+]i response to ANG II by 35 and 46%, respectively. The mechanism by which high concentrations of H2O2 inhibits [Ca2+]i responses to ANG II is uncertain. High concentrations of H2O2 (K0.5 = 74 ± 5 μM) may interfere with ANG II [Ca2+]i signaling by damaging the SR Ca2+ pump (16). Others have suggested that H2O2 (1 mM) activates KCa channels (14). The vasodilatory effects of 10–3 M H2O2 appear to be independent of adenylate and guanylate cyclases (29).
Although our data support the premise that ANG II-mediated generation of O2–· results in activation of ADPR cyclase of VSMC and thus enhancement of CICR, we do not suggest that such a mechanism is the exclusive pathway for the effects of O2–· in arteriolar VSMC. Among the possible important actions of O2–· on the renal vasculature are interaction of O2–· with NO to scavenge O2–· and to form peroxynitrate, stimulation of formation of 8-isoprostane PGF-2 from arachidonic acid, inhibition of prostacyclin synthesis, and increased production of adenosine (34).
In summary, we demonstrate that ANG II-mediated [Ca2+]i responses in freshly isolated afferent arterioles are closely linked to the generation of O2–·. Inhibition of NAD(P)H oxidase assembly with apocynin or treatment with the SOD mimetic tempol decreases the [Ca2+]i response to ANG II by 73%, on average. The generation of O2–· is inhibited by apocynin and DPI. H2O2 at a reported physiological concentration of 10–7 M has no effect on [Ca2+]i signaling. The ADPR cyclase inhibitor nicotinamide does not further diminish the [Ca2+]i response to ANG II, suggesting sharing of a common pathway. We propose that ANG II activates NAD(P)H oxidase to increase formation of O2–· with subsequent stimulation of ADPR cyclase activity and formation of cADPR, which sensitizes the RyR to Ca2+ and thus magnifies the overall [Ca2+]i response to ANG II.
GRANTS
This work was supported in part by an award from the Thomas H. Maren Foundation and from National Institutes of Health Research Grant HL-02334.
FOOTNOTES
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.
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