AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 287: H773-H781, 2004. First published April 8, 2004; doi:10.1152/ajpheart.01135.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/2/H773    most recent
01135.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, Z.
Right arrow Articles by Pallone, T. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, Z.
Right arrow Articles by Pallone, T. L.

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

Zhong Zhang, Kristie Rhinehart, Whaseon Kwon, Edward Weinman, and Thomas L. Pallone

Division of Nephrology, Departments of Medicine and Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1595

Submitted 1 December 2003 ; accepted in final form 5 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ANG II constricts descending vasa recta (DVR) through Ca2+ signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 µM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca2+ concentration ([Ca2+]cyt) elevation, and blocked Mn2+ influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 µM) or phorbol 12,13-dibutyrate (PDBu; 1 µM) induced both vasoconstriction and pericyte [Ca2+]cyt elevation. Diltiazem (10 µM) blocked the ability of PDBu to increase pericyte [Ca2+]cyt and enhance Mn2+ influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT2 receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 µM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca2+]cyt, divalent ion conductance into the cytoplasm, and ROS generation.

medulla; kidney; microcirculation; calcium; oxidative stress

DESCENDING VASA RECTA (DVR) supply the majority of blood flow to the renal medulla and are the site of regulation of blood flow distribution between the outer and inner medulla. DVR smooth muscle/pericytes envelop the vessel and contract to induce vasoconstriction. On the basis of these considerations, we infer that the DVR pericytes are an important regulator of regional perfusion of the kidney and potentially play roles in medullary oxygenation, salt excretion, and the urinary concentrating mechanism (30). Motivated by this, we investigated the signaling pathways and channel architecture of the DVR pericyte. ANG II depolarizes pericytes by activating a Ca2+-dependent Cl– conductance (28, 52) leading to voltage-gated Ca2+ entry into the cytoplasm and DVR contraction (26, 35, 52). Pericyte cytoplasmic Ca2+ concentration ([Ca2+]cyt) elevation and vasoconstriction can be blocked by repolarization induced by either Cl– channel blockade or K+ channel activation. Inhibition of L-type Ca2+ channels by diltiazem also reduces [Ca2+]cyt and dilates preconstricted DVR (26, 52).

In many smooth muscle cells, signaling cascades activated by contractile agonists include effects mediated by PKC. These include activation of voltage-gated Ca2+ channels and Ca2+-dependent Cl– channels (17, 25). In addition, through phosphorylation of NADPH oxidase, PKC favors generation of superoxide and other reactive oxygen species (ROS). ROS may serve to sensitize the contractile machinery to [Ca2+]cyt and modulate vasodilation by reducing bioavailable nitric oxide (NO) (10, 18, 19, 30, 37, 47). To expand our understanding of the pathways that lead to pericyte contraction, we tested the hypothesis that PKC activation is involved in ANG II constriction of DVR and ROS generation in DVR pericytes. Our results confirm that blockade of PKC prevents ANG II [Ca2+]cyt signaling and DVR contraction. Conversely, PKC activation constricts DVR and elevates [Ca2+]cyt. Both ANG II AT1 receptor stimulation and PKC activation induce ROS generation. ANG II AT2 receptor activation moderates ROS production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of DVR. The investigations involving animal use described herein were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland. Kidneys were harvested from Sprague-Dawley rats (70–150 g; Harlan Sprague Dawley, Indianapolis, IN) that were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). Tissue slices were placed in buffer and maintained at 4°C. Individual DVR was dissected from vascular bundles of the renal outer medulla and transferred to the stage of an inverted microscope where fluorescence microscopy or microperfusion studies were performed as previously described (27, 29). The solution used for dissection, microperfusion, and measurement of vasoactivity contained (in mM) 140 NaCl, 10 Na acetate, 5 KCl, 1.2 MgSO4, 1.2 Na2HPO4, 1 CaCl2, 5 HEPES, 5 L-alanine, and 5 D-glucose and 0.5 g/dl bovine albumin. The pH was adjusted to 7.55 at room temperature using NaOH to yield a pH of about 7.4 at 37°C.

Measurement of DVR diameter. Vasoactivity was monitored in DVR perfused in vitro as previously described (27, 29). Vessel images were recorded on videotape (Panasonic WV-BL90), and diameters were analyzed during playback. DVR luminal diameter was observed with a x40 objective to yield the final magnification of x1,300. Internal diameters were measured with calipers at the point of maximal constriction. Diameter changes are expressed as percent constriction given by [1 – (D/D0)] x 100, where D and D0 are the experimental and baseline diameters, respectively.

Isolation of pericytes from DVR wall. Small wedges of renal medulla were separated from kidney slices by dissection, transferred to CaCl2-free physiological saline solution (PSS) containing collagenase 1A (0.45 mg/ml; Sigma), protease XIV (0.4 mg/ml; Sigma), and albumin (1.0 mg/ml) (28, 35), incubated at 37°C for 22 min, and then transferred back to PSS containing CaCl2 (1 mM). Digested tissue was subsequently held at 4°C in a petri dish. At intervals, vessels were isolated from the digested renal tissue by microdissection and transferred to a perfusion chamber. In the chamber, DVR were captured and aspirated into a microperfusion-style collection pipette whose opening was 5–10 µm. During aspiration, pericytes were stripped from the abluminal surface of the vessel. As previously described, the DVR can be completely drawn into the pipette, leaving a preparation of isolated pericytes that adhere to the tip (34, 35, 52).

Immunolabeling of DVR pericytes and endothelia. To verify the identity of DVR pericytes and endothelium, vessels were immunolabeled with monoclonal antibody against smooth muscle actin (1:400 dilution; Sigma) and polyclonal antibody against as Na+/H+ exchanger regulating factor-2 (NHERF-2) (1:100 dilution). The NHERF-2 antibody has been described (41). DVR were fixed in 0.1 M cacodylate buffer with 3% paraformaldehyde for 5 min, washed twice with PSS, and then blocked for 30 min with PBS containing 5% bovine serum albumin and 0.1% triton, all at room temperature. Immunolabeling was performed by incubating sequentially with primary antibody and secondary antibody, 1 h each, at room temperature. Secondary antibodies were conjugated to Alexa Fluor 488 (goat anti-rabbit for NHERF-2) or Alexa Fluor 568 (Molecular Probes, Eugene, OR; goat anti-mouse for smooth muscle actin). Vessels were mounted in Antifade (Molecular Probes) and imaged with a Zeiss LSM410 laser scanning confocal microscope.

Measurement of [Ca2+]cyt. Pericytes were loaded with fura-2 by incubating them with the AM (10 µM, Molecular Probes) for 20 min at 37°C in the presence of probenecid (1 mM) (35). A photon-counting photomultiplier (PMT) assembly was employed to measure the fluorescent emission of fura-2 at 510 nm. Excitation was provided by a 75-W xenon arc lamp using a 350/380-nm wavelength combination isolated with a computer-controlled monochrometer (PTI; Lawrenceville, NJ). Fluorescent emission was isolated with a 510WB40 bandpass filter (Omega Optical; Brattleboro, VT) and collected with a Nikon CF fluor x40 oil immersion objective (1.3 numerical aperture). The background-subtracted ratio of fluorescent emission (R350/380) was converted to the [Ca2+]cyt assuming a dissociation constant of 224 nM for fura-2 at 37°C. Rmax and Rmin were measured by exposing vessels to buffer containing 5 mM CaCl2 or 0 CaCl2, 0.5 mM EGTA, respectively, and 10 µM ionomycin (29, 34, 52).

Measurement of Mn2+ influx by quench of fura-2. Mn2+ quench of fura-2 fluorescence was used to measure the effects of PKC activation and inhibition on conductance of the pericyte membrane to divalent cations (29, 34). Pericytes were loaded with fura-2 as described in Measurement of [Ca2+]cyt. To avoid changes in fura-2 fluorescence due to variations of pericyte [Ca2+]cyt, fura-2 was excited at its isosbestic (Ca2+-insensitive) wavelength, 360 nm. Fluorescent emission of fura-2 was measured at 510 nm before and after the introduction of MnCl2 (0.5 mM) into the bath. The rate of Mn2+ entry into pericytes was inferred from the rate at which fura-2 fluorescence was quenched by Mn2+ in the cytoplasm.

Measurement of ROS generation by oxidation of dihydroethidium. To measure generation of ROS, we included dihydroethidium (DHE) (10 µM) in the bath. Within cells, this probe is oxidized by superoxide (O2–·) to fluorescent products that are trapped by intercalation into DNA. This probe is generally thought to be specific for O2–· but ethidium (ETH) may not be its sole product (53). Single DVR were dissected, immobilized on pipettes in flowing bath, and excited at 535 nm. ETH emissions were monitored at 635 nm using a PMT assembly. To assess the distribution of ETH in the DVR wall, confocal images of ETH-loaded vessels were obtained with a Zeiss LSM410 laser scanning confocal microscope.

Reagents. ANG II, PD-123319, diphenylene iodonium (DPI), 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), probenecid, diltiazem, ionomycin, collagenase 1A, protease XIV, staurosporine (STP), phorbol 12,13-dibutyrate (PDBu), and 1,2-dioctanoyl-sn-glycerol (DiOG) were from Sigma (St. Louis, MO). ANG II, probenecid, diltiazem, DPI, and TEMPOL were stored in water at –20°C and diluted 1:100 or 1:1,000 on the day of the experiment. Fura-2, DHE (Molecular Probes), STP, PDBu, and DiOG were stored frozen in DMSO, thawed, and diluted for daily use. The digestion solution used to free pericytes from the endothelium was stored frozen in 2 ml aliquots and thawed once daily as needed. Excess reagents were discarded at the end of each day.

Statistics and analysis. Data in the text and figures are shown as means ± SE. Fluorescence acquisition rates were most often so rapid that graphing all error bars obscures data points. Consequently, in the figures that follow, most error bars are suppressed. Mn2+ quench experiments were analyzed by comparing the rate of decline of fluorescence, quantified as the slope of the regression line through the data points acquired during the last 2 min of the test period (after exchange of 0.5 mM Mn2+ into the bath). The oxidation of DHE to ETH was quantified by calculating the average fluorescence during the final 2 min of measurement. ETH fluorescence was normalized for display by dividing the fluorescence of individual vessels by the mean of the controls. As described in a past study of NO generation (36), that approach facilitates display without obscuring the variation or interfering with statistical analysis. The significance of differences between means was evaluated using Student's t-test (paired or unpaired, as appropriate) and ANOVA. For ANOVA, the Student-Kewman-Keuls test was used to test significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of PKC inhibition on ANG II vasconstriction and [Ca2+]cyt signaling. We tested whether the nonselective PKC inhibitor STP affects ANG II-induced vasoconstriction and [Ca2+]cyt signaling in DVR pericytes. ANG II (10 nM) constricted microperfused DVR from a mean internal diameter of 14.2 ± 0.6 to 10.5 ± 0.5 µm (time = 1 and 7 min, Fig. 1). When STP was included along with ANG II, constriction was eliminated; pre- and postexposure diameters were 13.9 ± 0.3 and 13.4 ± 0.4 µm, respectively. In the presence of STP, the response to ANG II was statistically indistinguishable from that of DMSO-exposed time controls whose pre- and posttreatment internal diameters were 13.9 ± 0.4 and 13.5 ± 0.5 µm, respectively.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1. Staurosporine (STP) inhibition of ANG II constriction. After the baseline internal diameter was recorded for 2 min, ANG II (10–8 M) was added to the bath plus either the PKC inhibitor STP (10–5 M, {blacksquare}, n = 8) or its vehicle DMSO ({bullet}, n = 9). These agents were removed from the bath after 5 min (time = 7 min). A separate group of vessels was exposed to DMSO alone ({circ}, n = 6). ANG II-induced constriction of descending vasa recta (DVR) was completely blocked by PKC inhibition. *P < 0.05 vs. ANG II + STP.

 
To test whether blockade of [Ca2+]cyt signaling might partially account for the inhibition of constriction by STP, we isolated pericytes from the underlying endothelia and examined their [Ca2+]cyt responses. We (35) previously characterized pericytes isolated in this way and showed that they respond to ANG II with a classical peak and plateau [Ca2+]cyt elevation. In contrast, the DVR endothelium responds to ANG II with [Ca2+]cyt reduction (29, 30, 34). In this study, to verify that pericyte stripping properly separates these cells from the endothelium, we performed immunochemistry (Fig. 2). Pericytes were labeled with antibody directed against smooth muscle actin and the endothelium was labeled with antibody targeted to isoform 2 of NHERF-2 (41). Stripped pericytes were positive for actin but not NHERF-2 (n = 4).



View larger version (86K):
[in this window]
[in a new window]
  		Fig. 2. Immunostaining of DVR pericytes and endothelia. A: images show an isolated DVR immunolabeled with antibody directed at smooth muscle actin (A-a, red) or Na+/H+ exchanger regulating factor-2 (NHERF-2; A-b, green). The merged images (A-c) show the abluminal (pericytes) and luminal (endothelial) location of the respective markers. B: single DVR with collapsed lumen (B-a) is aspirated into a holding pipette whose opening is ~10 µm (B-b). As the vessel is drawn into the pipette, pericytes (arrowheads) are stripped from the abluminal surface. A collection of pericytes (*) accumulates on the pipette tip. C: pericytes (*), isolated by stripping (C-a) label for smooth muscle actin (C-b) are shown but not NHERF-2 (not shown).

 
Isolated, fura-2-loaded pericytes responded to ANG II, as expected, with a peak and plateau [Ca2+]cyt response (Fig. 3). PKC inhibition with STP diminished the peak and nearly eliminated the ensuing plateau. In Fig. 3, the differences between ANG II and ANG II + STP groups were significant (P < 0.05) for times >3.3 min. Baseline pericyte [Ca2+]cyt was 50 to 100 nM, similar to that found in earlier studies (35, 52). The lack of response of [Ca2+]cyt to DMSO was shown and included in Effect of PKC activation on vasoactivity and [Ca2+]cyt.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 3. STP inhibition of ANG II pericyte cytoplasmic Ca2+ concentration ([Ca2+]cyt). [Ca2+]cyt response of pericytes exposed to ANG II (10–8 M) + vehicle (DMSO) or ANG II + STP (10–5 M) (n = 10 each group) is shown. STP markedly reduced the plateau phase of the [Ca2+]cyt response (P < 0.05 for time >3.3 min).

 
We tested whether STP can inhibit divalent cation entry into ANG II-stimulated pericytes. Fura-2 was loaded into isolated pericytes and excited at the Ca2+-insensitive wavelength, 360 nm, avoiding any fluorescence changes that might otherwise arise from variation of [Ca2+]cyt (29, 34). Mn2+ was introduced into the bath to measure the rate of its entry into the pericytes as reflected by the rate of quench of fluorescence. Results are illustrated in Fig. 4A. Even before the addition of Mn2+, fura-2 fluorescence declined slowly. That decline is due to leak of fura-2 from the cytoplasm (35). Five-minute preincubation with ANG II or STP had no effect on fura-2 leak. After Mn2+ (0.5 mM) was added to the bath, a very rapid reduction of fura-2 fluorescence occurred, related to Mn2+ entry and fluorescence quenching. In the presence of STP, the quench rate was lower (Fig. 4, A and B), verifying that PKC inhibition blocks permeation of Mn2+ into ANG II-stimulated DVR pericytes.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 4. Effect of STP on Mn2+ entry into ANG II-treated pericytes. A: protocol used to measure Mn2+ entry into ANG II-treated pericytes is shown. After a 2-min baseline, vessels were preexposed to ANG II (10 nM) with or without STP (10 µM) for 5 min. Subsequently, Mn2+ (0.5 mM) was added to the bath, quenching fura-2 in the cytoplasm. The ordinate shows fluorescence of fura-2 (F) divided by fura-2 fluorescence at time = 0 (F0). Fluorescence of fura-2 was monitored at 510 nm during isosbestic excitation at 360 nm. B: means ± SE of fluorescence quench rate in vehicle and STP-treated pericytes (n = 9, each group, *P < 0.05). Values on the ordinate are the rate of decline of fluorescence, determined by linear regression, during the final 2 min of observation.

 
Effect of PKC activation on vasoactivity and [Ca2+]cyt. We next tested whether nonspecific PKC activation mimics the ability of ANG II to increase pericyte [Ca2+]cyt and constrict DVR. Diacylglycerol (DAG) is an endogenous activator of PKC. The membrane-permeant DAG analog DiOG increased pericyte [Ca2+]cyt and induced vasoconstriction (Fig. 5). The [Ca2+]cyt response to DiOG was characterized by a slow rise toward a plateau without an early peak (contrast Figs. 3 and 5A). DiOG increased [Ca2+]cyt from a baseline of 58 ± 26 to 162 ± 37 nM (P < 0.05 vs. DMSO for time >9.1 min, Fig. 5A). Preliminary experiments showed DiOG to be very photosensitive, a property that might partially account for the slow rise in [Ca2+]cyt. In view of this, to measure vasoconstriction, the room was darkened, and the preparation was briefly illuminated with the microscope light to record pre- and posttreatment images. After 15 min of exposure to DiOG (10 µM, n = 5), DVR constricted from a baseline diameter of 14.4 ± 0.4 to 10.3 ± 0.5 µm (Fig. 5B, P < 0.05 vs. DMSO controls). For corroboration, we also tested the effect of PDBu, a less photosensitive agonist that activates PKC by a different mechanism. Like DiOG (Fig. 5A), the pericyte [Ca2+]cyt response to PDBu (Fig. 6A) was characterized by a slow increase from an initial value of 51 ± 32 nM toward a plateau of ~266 nM over an exposure time of 10 min (P < 0.05 vs. DMSO time controls for time >5.4 min, Fig. 6A). PDBu constricted DVR on a similar time scale from an internal diameter of 13.8 ± 0.4 to 11.3 ± 0.8 µm (P < 0.05 vs. DMSO time controls, Fig. 6B).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. Effect of 1,2-dioctanoyl-sn-glycerol (DiOG) on pericyte [Ca2+]cyt responses and DVR constriction. A: ability of the membrane-permeant diacylglycerol analog DiOG (10 µM) to modulate [Ca2+]cyt was tested in isolated DVR pericytes. After the baseline [Ca2+]cyt was recorded for 2 min, DiOG or DMSO was added to the bath. Compared with DMSO-treated controls (n = 11), DiOG (n = 5) increased [Ca2+]cyt (P < 0.05 vs. DMSO for time >9.1 min). B: compared with DMSO-treated controls (n = 4), exposure to DiOG (n = 7) for 15 min induced significant vasoconstriction (*P < 0.01 vs. DMSO).

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6. Effect of phorbol 12,13-dibutyrate (PDBu) on pericyte [Ca2+]cyt responses and DVR constriction. A: ability of the nonselective PKC activator PDBu (1 µM) to modulate [Ca2+]cyt was tested in isolated DVR pericytes. Compared with DMSO-treated controls (n = 11), PDBu (n = 10) increased [Ca2+]cyt (P < 0.05 vs. DMSO time controls for time >5.4 min). B: compared with DMSO-treated controls (n = 7), exposure to PDBu (n = 8) induced significant vasoconstriction (*P < 0.01 vs. DMSO).

 
Blockade of PKC [Ca2+]cyt by diltiazem. Diltiazem is a benzothiazipine L-type voltage-gated Ca2+ channel blocker with affinity variably reported in the range of 5 to 132 µM (16, 43, 46, 49). In a prior study (46), we found that diltiazem inhibits the plateau phase of the ANG II-stimulated DVR pericyte [Ca2+]cyt response. Using PDBu as a PKC agonist, we tested the hypothesis that diltiazem would also block PKC-stimulated Ca2+ responses. PDBu increased pericyte [Ca2+]cyt from a baseline of 51 ± 32 to 263 ± 64 nM at 12 min (Fig. 7). Within 2 min after the addition of diltiazem (10 µM), [Ca2+]cyt fell to 47 ± 51 nM (P < 0.05 vs. vehicle controls, time = 12–14 min, Fig. 7).



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 7. Effect of diltiazem on PDBu-stimulated pericyte [Ca2+]cyt. The ability of diltiazem to block PDBu (1 µM)-stimulated [Ca2+]cyt was tested is isolated DVR pericytes. Preexposure to PDBu elevated pericyte [Ca2+]cyt, an effect that was reversed by diltiazem (10 µM, n = 6). PDBu-stimulated [Ca2+]cyt data from Fig. 6 are reproduced for comparison.

 
The effect of diltiazem on PDBu stimulated Mn2+ entry into the pericyte cytoplasm was also tested (Fig. 8). Fura-2-loaded pericytes were exposed to PDBu (10 µM) with or without diltiazem (10 µM) for 5 min, following which Mn2+ (0.5 mM) was added to the bath (Fig. 8A). The rate of Mn2+ quench was lower with diltazem (P < 0.05 vs. control, Fig. 8B). These findings support a role for ANG II to activate Ca2+ entry pathways via PKC phosphorylation of voltage-gated channels.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 8. Effect of diltiazem on PDBu-stimulated Mn2+ entry into ANG II-treated pericytes. A: protocol used to measure Mn2+ entry into pericytes. After a 2-min baseline period, vessels were preexposed to PDBu (1 µM) with or without diltiazem (10 µM) for 5 min. Subsequently, Mn2+ (0.5 mM) was added to the bath. The ordinate shows normalized fluorescence of fura-2 monitored at 510 nm during isosbestic excitation at 360 nm. B: means ± SE of fluorescence quench rate in vehicle-exposed (n = 8) and diltiazem-treated pericytes (n = 7, each group, *P < 0.05). Values on the ordinate are the rate of decline of fluorescence, determined by linear regression, during the final 2 min of observation.

 
Activation of ROS generation. ROS can augment vasoconstriction by reacting with NO to produce peroxynitrite. They can also promote constriction through nonenzymatic generation of isoprostanes and sensitization of contractile proteins to [Ca2+]cyt (9, 12, 39). Using oxidation of DHE to ETH and other fluorescent products, we tested whether constriction of DVR by ANG II and PKC agonists leads to generation of ROS. We first determined whether ETH accumulation was greatest in DVR pericytes or endothelium. As shown in Fig. 9A, concomitant white light and confocal fluorescent images showed greater accumulation of fluorescence in pericytes. We then tested the ability of ANG II to enhance ROS generation. By itself, ANG II showed a tendency to increase oxidation of DHE, but this did not reach significance even after 40 min of observation. When ANG II AT2 receptors were blocked with PD-123319, a robust increase in ETH was readily demonstrated (Fig. 9B), suggesting a role for AT2 activation to abrogate AT1 receptor-mediated ROS generation. Statistical comparison of averaged fluorescence during the last 2 min is summarized in Fig. 9C. Identical conclusions are reached if one compares the rates of rise of fluorescence over the first 10 min rather than the total fluorescence achieved at the endpoints. The PKC agonist PDBu also enhanced ROS generation (Fig. 10A). DPI is a flavoprotein inhibitor that blocks NADPH oxidase, and TEMPOL is a membrane-permeant SOD mimetic. The effects of those two antioxidants on PDBu stimulated ROS generation as shown in Fig. 10B. Both agents blocked the rate of oxidation of DHE.



View larger version (71K):
[in this window]
[in a new window]
  		Fig. 9. Oxidation of dihydroethidium (DHE) to ethidium (ETH) by DVR pericytes. A: high- and low-power confocal images of DHE-loaded DVR (top and middle) are shown. A white light image is provided for comparison (bottom). ETH fluorescence is most prominent in pericyte cell bodies (*). B: with background subtracted, normalized ETH fluorescence is shown as a function of time in isolated DVR exposed to vehicle (n = 9), ANG II (10 nM, n = 8), or ANG II + the AT2 receptor blocker PD-123319 (10 nM, n = 9). ANG II tended to increase the rate of fluorescent labeling, but this did not achieve significance. Stimulation of AT1 receptors during concomitant AT2 receptor blockade led to a robust and significant increase in the rate of ETH labeling. C: summary of the ETH fluorescence from B shown as the mean of the final 2 min of data collection (*P < 0.05 vs. control).

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 10. Inhibition of PDBu-induced DHE oxidation by DPI and TEMPOL. A: background-subtracted fluorescence is shown as a function of time in isolated DVR exposed to DHE in the presence and absence of PDBu (1 µM, n = 8, each group). PDBu significantly enhanced the rate of labeling by ETH. B: summary of the ETH fluorescence from A is provided as the mean of the final 2 min of data collection. The effect the flavoenzyme inhibitor diphenylene iodonium (DPI; 10 µM) or the SOD mimetic TEMPOL (1 mM) on the accumulation of fluorescence is shown (n = 5, each group). Both antioxidants blocked the ability of PDBu to enhance ETH fluorescence relative to vehicle-treated controls. *P < 0.05 vs. DMSO time controls.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The DVR pericyte is an important regulator of renal medullary perfusion. Studies (28, 30, 34–36, 52) have shown that ANG II constricts DVR though mechanisms that involve pericyte plasma membrane depolarization, Ca2+-dependent Cl– channel activation, and Ca2+ influx through a diltiazem-sensitive pathway. In this work, we continued studies of DVR contraction using ANG II as a benchmark agonist by testing the hypothesis that PKC activation plays an important role in its actions. The principal findings are that ANG II-induced vasoconstriction and pericyte [Ca2+]cyt elevation are both blocked by PKC inhibition. Conversely, PKC activation induces constriction and [Ca2+]cyt elevation. Finally, PKC activation by ANG II generates ROS via AT1 receptor stimulation and AT2 modulation. These data extend our knowledge of the cellular mechanisms by which ANG II constricts DVR pericytes and reinforce a role for ROS in renal medullary physiology (6, 8, 22, 26).

STP prevented ANG II stimulated vasoconstriction (Fig. 1), pericyte [Ca2+]cyt signaling (Fig. 3), and Mn2+ influx (Fig. 4). This favors a role for PKC activation to link receptor activation and pericyte [Ca2+]cyt signaling. ANG II AT1 receptors are a member of the seven membrane-spanning, G protein-coupled, superfamily that, through activation of phospholipase C, catalyze hydrolysis of phosphoinositide bisphosphate to yield inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and DAG (1, 13). DAG activates both the Ca2+ dependent, classical, and the Ca2+-independent, novel PKC isoforms (10, 25, 37). PKC, in turn, is known to activate a variety of Ca2+ entry pathways including the voltage-gated (L-type and T-type) Ca2+ channels Cav1.x and Cav3.x, respectively (17, 25), and to modulate activity of some isoforms of transient receptor potential cation channels (50). In the DVR pericyte, PKC activation, whether resulting from ANG II AT1 receptor activation or application of agonists (DiOG, PDBu), increases divalent cation entry and elevates pericyte [Ca2+]cyt (Figs. 3, 5, and 6).

Differences were observed in the response of pericyte [Ca2+]cyt to ANG II and PKC agonists. Unlike ANG II stimulation (Fig. 3), DiOG and PDBu (Figs. 5 and 6) did not generate an early peak as part of their [Ca2+]cyt response. This is largely as expected, because PKC activation does not generate Ins(1, 4,5)P3, the signaling molecule needed to liberate Ca2+ from cellular stores via the endoplasmic/sarcoplasmic reticulum Ins(1,4,5)P3 receptor (1). In addition, the rise of [Ca2+]cyt after DiOG and PDBu was characterized by a slow increase toward a plateau (Figs. 6 and 7). In preliminary experiments, we observed that DiOG, in particular, is light sensitive to the point that strong microscope illumination could vasodilate vessels preconstricted with this agent. PDBu is less photosensitive and was therefore the PKC agonist of choice for subsequent experiments (Figs. 6– 8 and 10). The slow onset of DiOG and PDBu may also be related to their lipophilic nature and slow diffusion to cytoplasmic targets. Details of their pharmacology cannot be readily resolved in isolated perfused DVR and DVR pericytes, but consistency of the results obtained using PKC blockade (STP) and activation (DiOG, PDBu) points strongly toward the role of PKC in ANG II actions. PKC blockade with STP reduced the pericyte [Ca2+]cyt response and DVR constriction. PKC activation with DiOG and PDBu, agonists that act via different mechanisms, contracted DVR and stimulated pericyte [Ca2+]cyt.

Diltiazem is a benzothiazepine L-type Ca2+ channel blocker that binds to the channel outside the pore. Its block of Ca2+ currents in myocytes is pH and voltage dependent and exhibits an IC50 variably reported between 5 and 132 µM (16, 43, 46, 49). We (52) previously demonstrated that diltiazem (10 µM) lowers the plateau phase of the ANG II [Ca2+]cyt response. In the present work, we confirmed that PKC inhibition also blocks the plateau response, (Fig. 3). The similarity of the results is consistent with a role for PKC to activate diltiazem-sensitive channels in DVR pericytes. In support of this hypothesis, diltiazem-reduced PDBu stimulated Mn2+ entry into the pericytes (Fig. 8). To the extent that conductance of the pericyte plasma membrane to Mn2+ mirrors that of Ca2+, these data support a role for PKC to augment Ca2+ entry.

The ability of diltiazem to block ANG II- and KCl-induced vasoconstriction and pericyte [Ca2+]cyt elevation (52) is evidence for the presence of Cav1.x, low-voltage-activated L-type Ca2+ channels in DVR pericytes. Consistent with this, Hansen et al. (15) recently demonstrated expression of Cav1.2{alpha} subunits in the juxtamedullary efferent circulation, including DVR. The specificity of some L-type channel blockers has been brought into question (7, 45), and these results do not rule out the possibility that more than one pathway contributes to ANG II-stimulated Ca2+ influx. Further delineation of pericyte Ca2+ entry pathways will probably require electrophysiological study using voltage clamps above and below the threshold for activation of L- and T-type currents.

In addition to [Ca2+]cyt, the contractile state of vascular smooth muscle is regulated through several intracellular signaling cascades that control the phosphorylation of myosin light chains (MLC). The balance of phosphorylation is determined by the activities of Ca2+/calmodulin-dependent MLC kinase (MLCK) and MLC phosphatase (31, 33). The latter two enzymes are targets of signaling cascades that control sensitivity to [Ca2+]cyt. Such sensitization can result in smooth muscle contraction even in the absence of overt [Ca2+]cyt elevation (5, 37). The ability of STP to nearly eliminate DVR vasoconstriction (Fig. 1) can be taken as evidence for PKC-mediated sensitization of pericytes to [Ca2+]cyt, but this is not conclusive. PKC blockers are not entirely specific and might block other kinases including MLCK (32, 38). The elevation of [Ca2+]cyt by PKC stimulation (Figs. 5A and 6A) reinforces the possibility that blockade of ANG II-stimulated pericyte [Ca2+]cyt signaling by STP (Fig. 2) involves PKC inhibition.

We (36) previously observed that the SOD mimetic TEMPOL blocks DVR constriction by ANG II, implying a role for O2–· and/or its reaction products in acute signaling. The present data (Figs. 9 and 10) suggest a role for generation of O2–· in ANG II constriction. We employed oxidation of DHE to measure ANG II- and PKC-stimulated ROS generation. Although many ROS probes are nonspecific, DHE is generally felt to react with O2–·, although ETH may not be its sole reaction product (53). Many enzymes, including NO synthase, cyclooxygenases, lipoxygenases, epoxygenases, xanthine oxidase, and NADPH oxidase can generate ROS (9, 12, 39). It is increasingly accepted, however, that the dominant source of ROS for bacterocidal activity in leukocytes and signaling in other cells is NADPH oxidase (14, 19, 44, 47). This enzyme consists of cytosolic components (p47phox, p67phox), a G protein (Rac1 or Rac2), and a membrane-associated cytochrome composed of two other subunits (p22phox, gp91phox). The latter provide the final transfer of electrons from NADPH to O2 in leukocytes. In nonphagocytic cells, including vascular smooth muscle, the gp91phox subunit may be substituted by another isoform such as NOX1 or NOX4, and several NOX isoforms are expressed in individual cell types (4, 19, 20). A role for elevation of [Ca2+]cyt and PKC phosphorylation of p47phox to activate NADPH oxidase is established, and PKC inhibitors reduce vascular production of ROS after stimulation with ANG II (13, 14, 40) and other agonists (23). Activation of ANG II AT1 receptors also enhances vascular smooth muscle production of O2–· through stimulation of phospholipase D, formation of phosphatidic acid, and activation of NADPH oxidases (14, 20, 48, 51).

DVR are contractile vessels that supply blood flow to the renal medulla. The importance of ROS generation to the tonic maintenance of renal medullary blood flow has been well documented (6, 8, 54). SOD inhibition and infusion of the SOD mimetic TEMPOL reduce and enhance medullary blood flow, respectively (54), and chronic renal medullary SOD inhibition induces hypertension (22). The thick ascending limb of Henle's loop has been identified as an important source of both O2–· and NO production (6, 8, 21, 26). We confirmed that ANG II tends to increase ROS formation in DVR pericytes, but the effect did not achieve significance unless AT2 receptors were concomitantly blocked with PD-123319 (Fig. 9C). These results agree with the recent report of Mori and Cowley (24) showing that ANG II fails to increase ETH fluorescence in DVR pericytes during 250 s of measurement. The scale of the abscissa in Fig. 9C shows that a significant effect of ANG II on DHE oxidation could not be demonstrated, even with 10-fold longer observations. The trend, however, does favor ANG II enhancement of ROS production. As a consequence of intravessel variation and the requirement for across-group comparison, the DHE oxidation method is probably too insensitive to demonstrate ROS generation unless AT2 blockade is employed to unmask AT1 receptor-mediated effects.

Our prior finding (36) that TEMPOL abrogates ANG II constriction favors a role for it to stimulate significant O2–· production in the DVR wall. We (34) recently described the effect of AT2 receptor blockade (PD-123319) and activation (CGP-42112A) on DVR endothelial Ca2+ signaling and vasoconstriction. In that study, AT2 blockade enhanced the ability of ANG II to inhibit the endothelial [Ca2+]cyt response to vasodilators, but it did not affect the magnitude of the ANG II-stimulated pericyte [Ca2+]cyt response. The effect of ANG II to inhibit endothelial [Ca2+]cyt responses persisted after pericytes were removed from the vessel (35). The ability of PD-123319 to enhance ROS formation in this study and the dominance of the ETH signal in pericytes (Fig. 9, A and C) suggests that the AT2 receptors might exert their influences through actions in both endothelia and pericytes.

The ability of ANG II to inhibit DVR endothelial [Ca2+]cyt signaling by vasodilators implies that the increase in tissue NO that buffers vasoconstrictor effects to preserve medullary blood flow (6) originate from sources other than DVR endothelia. Increasing attention has been focused on the possibility that signaling between the medullary thick ascending limb of Henle and outer medullary vascular bundles through diffusible paracrine agents might mediate local feedback to preserve oxygenation of the outer medullary interbundle region where vectoral NaCl transport occurs (6, 8, 29). The effect of AT2 stimulation to reduce ROS formation (Fig. 9) and facilitate endothelial [Ca2+]cyt signaling (34) agrees with the general observation that AT2 receptor actions favor intrarenal NO formation (2, 42), because endothelial NO synthase is a Ca2+-dependent enzyme (11) and O2–· inactivates NO by reacting with it to form peroxynitrite (9). The recent demonstration by Chabrashvili et al. (3) shows that AT2 inhibition during chronic ANG II infusion increases urinary markers of oxidative stress and that NADPH oxidase subunit expression supports the role of AT2 receptors in the abrogation of intrarenal ROS generation. Thus it is reasonable to postulate that physiological modulators of AT2 activity or receptor expression might be an important link to the control of renal medullary perfusion and renal salt and water excretion.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Studies in our laboratory have been supported by National Institutes of Health Grants DK-42495, HL-62220, and HL-68686.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. L. Pallone, Div. of Nephrology, N3W143, Univ. of Maryland at Baltimore, Baltimore, MD 21201-1595 (E-mail: tpallone{at}medicine.umaryland.edu).

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Berridge MJ, Lipp P, and Bootman MD. The versatility and universality of calcium signaling. Nat Rev 1: 11–21, 2000.
  2. Carey RM, Jin X, Wang Z, and Siragy HM. Nitric oxide: a physiological mediator of the type 2 (AT2) angiotensin receptor. Acta Physiol Scand 168: 65–71, 2000.[CrossRef][ISI][Medline]
  3. Chabrashvili T, Kitiyakara C, Blau J, Karber A, Aslam S, Welch WJ, and Wilcox CS. Effects of ANG II type 1 and 2 receptors on oxidative stress, renal NADPH oxidase, and SOD expression. Am J Physiol Regul Integr Comp Physiol 285: R117–R124, 2003.[Abstract/Free Full Text]
  4. Cheng G, Cao Z, Xu X, van Meir EG, and Lambeth JD. Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5. Gene 269: 131–140, 2001.[CrossRef][ISI][Medline]
  5. Chitaley K, Weber D, and Webb RC. RhoA/Rho-kinase, vascular changes, and hypertension. Curr Hypertens Rep 3: 139–144, 2001.[Medline]
  6. Cowley AW Jr, Mori T, Mattson D, and Zou AP. Role of renal NO production in the regulation of medullary blood flow. Am J Physiol Regul Integr Comp Physiol 284: R1355–R1369, 2003.[Abstract/Free Full Text]
  7. Curtis TM and Scholfield CN. Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle. J Physiol 532: 609–623, 2001.[Abstract/Free Full Text]
  8. Dickhout JG, Mori T, and Cowley AW Jr. Tubulovascular nitric oxide crosstalk: buffering of angiotensin II-induced medullary vasoconstriction. Circ Res 91: 487–493, 2002.[Abstract/Free Full Text]
  9. Droge W. Free radicals in the physiological control of cell function. Physiol Rev 82: 47–95, 2002.[Abstract/Free Full Text]
  10. Gokina NI, Knot HJ, Nelson MT, and Osol G. Increased Ca2+ sensitivity as a key mechanism of PKC-induced constriction in pressurized cerebral arteries. Am J Physiol Heart Circ Physiol 277: H1178–H1188, 1999.[Abstract/Free Full Text]
  11. Goligorsky MS, Li H, Brodsky S, and Chen J. Relationships between caveolae and eNOS: everything in proximity and the proximity of everything. Am J Physiol Renal Physiol 283: F1–F10, 2002.[Abstract/Free Full Text]
  12. Griendling KK and FitzGerald GA. Oxidative stress and cardiovascular injury: Part I: basic mechanisms and in vivo monitoring of ROS. Circulation 108: 1912–1916, 2003.[Free Full Text]
  13. Griendling KK, Lassegue B, and Alexander RW. Angiotensin receptors and their therapeutic implications. Annu Rev Pharmacol Toxicol 36: 281–306, 1996.[Medline]
  14. 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/Free Full Text]
  15. Hansen PB, Jensen BL, Andreasen D, and Skott O. Differential expression of T- and L-type voltage-dependent calcium channels in renal resistance vessels. Circ Res 89: 630–638, 2001.[Abstract/Free Full Text]
  16. Hart J, Wilkinson MF, Kelly ME, and Barnes S. Inhibitory action of diltiazem on voltage-gated calcium channels in cone photoreceptors. Exp Eye Res 76: 597–604, 2003.[CrossRef][ISI][Medline]
  17. Keef KD, Hume JR, and Zhong J. Regulation of cardiac and smooth muscle Ca2+ channels (Ca(V)1.2a,b) by protein kinases. Am J Physiol Cell Physiol 281: C1743–C1756, 2001.[Abstract/Free Full Text]
  18. Kitazawa T, Takizawa N, Ikebe M, and Eto M. Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in triton X-100-demembranated rabbit arterial smooth muscle. J Physiol 520: 139–152, 1999.[Abstract/Free Full Text]
  19. Lassegue B and Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol 285: R277–R297, 2003.[Abstract/Free Full Text]
  20. Lassegue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD, and Griendling KK. Novel gp91phox homologues in vascular smooth muscle cells: nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ Res 88: 888–894, 2001.[Abstract/Free Full Text]
  21. Li N, Yi FX, Spurrier JL, Bobrowitz CA, and Zou AP. Production of superoxide through NADH oxidase in thick ascending limb of Henle's loop in rat kidney. Am J Physiol Renal Physiol 282: F1111–F1119, 2002.[Abstract/Free Full Text]
  22. Makino A, Skelton MM, Zou AP, Roman RJ, and Cowley AW Jr. Increased renal medullary oxidative stress produces hypertension. Hypertension 39: 667–672, 2002.[Abstract/Free Full Text]
  23. Marumo T, Schini-Kerth VB, Fisslthaler B, and Busse R. Platelet-derived growth factor-stimulated superoxide anion production modulates activation of transcription factor NF-{kappa}B and expression of monocyte chemoattractant protein 1 in human aortic smooth muscle cells. Circulation 96: 2361–2367, 1997.[Abstract/Free Full Text]
  24. Mori T and Cowley AW Jr. Angiotensin II-NAD(P)H oxidase-stimulated superoxide modifies tubulovascular nitric oxide cross-talk in renal outer medulla. Hypertension 42: 588–593, 2003.[Abstract/Free Full Text]
  25. Obejero-Paz CA, Auslender M, and Scarpa A. PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells. Am J Physiol Cell Physiol 275: C535–C543, 1998.[Abstract/Free Full Text]
  26. Ortiz PA and Garvin JL. Superoxide stimulates NaCl absorption by the thick ascending limb. Am J Physiol Renal Physiol 283: F957–F962, 2002.[Abstract/Free Full Text]
  27. Pallone TL. Microdissected perfused vessels. Methods Mol Med 86: 443–456, 2003.[Medline]
  28. Pallone TL and Huang JM. Control of descending vasa recta pericyte membrane potential by angiotensin II. Am J Physiol Renal Physiol 282: F1064–F1074, 2002.[Abstract/Free Full Text]
  29. Pallone TL, Silldorff EP, and Zhang Z. Inhibition of calcium signaling in descending vasa recta endothelia by ANG II. Am J Physiol Heart Circ Physiol 278: H1248–H1255, 2000.[Abstract/Free Full Text]
  30. Pallone TL, Zhang Z, and Rhinehart K. Physiology of the renal medullary microcirculation. Am J Physiol Renal Physiol 284: F253–F266, 2003.[Abstract/Free Full Text]
  31. Pfitzer G. Invited review: regulation of myosin phosphorylation in smooth muscle. J Appl Physiol 91: 497–503, 2001.[Abstract/Free Full Text]
  32. Pfitzer G, Sonntag-Bensch D, and Brkic-Koric D. Thiophosphorylation-induced Ca2+ sensitization of guinea-pig ileum contractility is not mediated by Rho-associated kinase. J Physiol 533: 651–664, 2001.[Abstract/Free Full Text]
  33. Rembold CM. Modulation of the [Ca2+] sensitivity of myosin phosphorylation in intact swine arterial smooth muscle. J Physiol 429: 77–94, 1990.[Abstract/Free Full Text]
  34. Rhinehart K, Handelsman CA, Silldorff EP, and Pallone TL. ANG II AT2 receptor modulates AT1 receptor-mediated descending vasa recta endothelial Ca2+ signaling. Am J Physiol Heart Circ Physiol 284: H779–H789, 2003.[Abstract/Free Full Text]
  35. Rhinehart K, Zhang Z, and Pallone TL. Ca2+ signaling and membrane potential in descending vasa recta pericytes and endothelia. Am J Physiol Renal Physiol 283: F852–F860, 2002.[Abstract/Free Full Text]
  36. Rhinehart KL and Pallone TL. Nitric oxide generation by isolated descending vasa recta. Am J Physiol Heart Circ Physiol 281: H316–H324, 2001.[Abstract/Free Full Text]
  37. Ruegg JC. Smooth muscle: PKC-induced Ca2+ sensitisation by myosin phosphatase inhibition. J Physiol 520: 3, 1999.
  38. Ruegg UT and Burgess GM. Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases. Trends Pharmacol Sci 10: 218–220, 1989.[CrossRef][Medline]
  39. Schnackenberg CG. Physiological and pathophysiological roles of oxygen radicals in the renal microvasculature. Am J Physiol Regul Integr Comp Physiol 282: R335–R342, 2002.[Abstract/Free Full Text]
  40. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, and Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res 91: 406–413, 2002.[Abstract/Free Full Text]
  41. Shenolikar S, Voltz J, Minkoff CM, Wade J, and Weinman EJ. Targeted disruption of the mouse gene encoding a PDZ domain-containing protein adaptor, NHERF-1, promotes Npt2 internalization and renal phosphate wasting. Proc Natl Acad Sci USA 99: 11470–11475, 2002.[Abstract/Free Full Text]
  42. Siragy HM, Inagami T, Ichiki T, and Carey RM. Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor. Proc Natl Acad Sci USA 96: 6506–6510, 1999.[Abstract/Free Full Text]
  43. Smirnov SV and Aaronson PI. pH dependent block of the L-type Ca2+ channel current by diltiazem in human mesenteric arterial myocytes. Eur J Pharmacol 360: 81–90, 1998.[CrossRef][ISI][Medline]
  44. Sorescu D, Somers MJ, Lassegue B, Grant S, Harrison DG, and Griendling KK. Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells. Free Radic Biol Med 30: 603–612, 2001.[CrossRef][ISI][Medline]
  45. Stepien O and Marche P. Amlodipine inhibits thapsigargin-sensitive Ca2+ stores in thrombin-stimulated vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 279: H1220–H1227, 2000.[Abstract/Free Full Text]
  46. Striessnig J, Grabner M, Mitterdorfer J, Hering S, Sinnegger MJ, and Glossmann H. Structural basis of drug binding to L Ca2+ channels. Trends Pharmacol Sci 19: 108–115, 1998.[CrossRef][Medline]
  47. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 279: L1005–L1028, 2000.[Abstract/Free Full Text]
  48. Touyz RM and Schiffrin EL. Ang II-stimulated superoxide production is mediated via phospholipase D in human vascular smooth muscle cells. Hypertension 34: 976–982, 1999.[Abstract/Free Full Text]
  49. Triggle DJ. The pharmacology of ion channels: with particular reference to voltage-gated Ca2+ channels. Eur J Pharmacol 375: 311–325, 1999.[CrossRef][ISI][Medline]
  50. Venkatachalam K, Zheng F, and Gill DL. Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C. J Biol Chem 278: 29031–29040, 2003.[Abstract/Free Full Text]
  51. Wang JP, Tsao LT, Raung SL, Hsu MF, and Kuo SC. Inhibition by HAJ11 of respiratory burst in neutrophils and the involvement of protein tyrosine phosphorylation and phospholipase D activation. Br J Pharmacol 120: 79–87, 1997.[CrossRef][ISI][Medline]
  52. Zhang Z, Rhinehart K, and Pallone TL. Membrane potential controls calcium entry into descending vasa recta pericytes. Am J Physiol Regul Integr Comp Physiol 283: R949–R957, 2002.[Abstract/Free Full Text]
  53. Zhao H, Kalivendi S, Zhang H, Joseph J, Nithipatikom K, Vasquez-Vivar J, and Kalyanaraman B. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic Biol Med 34: 1359–1368, 2003.[CrossRef][ISI][Medline]
  54. Zou AP, Li N, and Cowley AW Jr. Production and actions of superoxide in the renal medulla. Hypertension 37: 547–53, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Just, A. J. M. Olson, C. L. Whitten, and W. J. Arendshorst
Superoxide mediates acute renal vasoconstriction produced by angiotensin II and catecholamines by a mechanism independent of nitric oxide
Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H83 - H92.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
Q. Zhang, C. Cao, M. Mangano, Z. Zhang, E. P. Silldorff, W. Lee-Kwon, K. Payne, and T. L. Pallone
Descending vasa recta endothelium is an electrical syncytium
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2006; 291(6): R1688 - R1699.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
D. Wang, P. Jose, and C. S. Wilcox
beta1 Receptors Protect the Renal Afferent Arteriole of Angiotensin-Infused Rabbits from Norepinephrine-Induced Oxidative Stress
J. Am. Soc. Nephrol., December 1, 2006; 17(12): 3347 - 3354.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
W. Neuhofer and F.-X. Beck
Survival in Hostile Environments: Strategies of Renal Medullary Cells
Physiology, June 1, 2006; 21(3): 171 - 180.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
C. S. Wilcox
Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension?
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2005; 289(4): R913 - R935.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
T. L. Pallone
Microvascular Effects of Aldosterone and Angiotensin Type 2 Receptors
Hypertension, May 1, 2005; 45(5): 845 - 846.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
W. Lee-Kwon, J. B. Wade, Z. Zhang, T. L. Pallone, and E. J. Weinman
Expression of TRPC4 channel protein that interacts with NHERF-2 in rat descending vasa recta
Am J Physiol Cell Physiol, April 1, 2005; 288(4): C942 - C949.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Z. Zhang, K. Rhinehart, G. Solis, J. Pittner, W. Lee-Kwon, W. J. Welch, C. S. Wilcox, and T. L. Pallone
Chronic ANG II infusion increases NO generation by rat descending vasa recta
Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H29 - H36.
[Abstract] [Full Text] [PDF]