In Vivo Evidence for Microvascular Oxidative Stress in Spontaneously Hypertensive Rats
Abstract The factors that predispose to the accelerated organ injury that accompanies the hypertensive syndrome have remained speculative and without a firm experimental basis. Indirect evidence has suggested that a key feature may be related to an enhanced oxygen radical production. The purpose of this study was to refine and use a technique to visualize evidence of spontaneous microvascular oxidative stress in vivo in the spontaneously hypertensive rat (SHR) compared with its normotensive control, the Wistar-Kyoto rat (WKY). We investigated the effects of adrenal glucocorticoids on the microvascular oxidative stress sequence. The mesentery was superfused with hydroethidine, a reduced, nonfluorescent precursor of ethidium bromide. In the presence of oxidative challenge, hydroethidine is transformed intracellularly into the fluorescent compound ethidium bromide, which binds to DNA and can be detected by virtue of its red fluorescence. The fluorescent light emission from freshly exteriorized and otherwise unstimulated mesentery microvessels was recorded by digital microscopy. The number of ethidium bromide–positive nuclei along the arteriolar and venular walls in SHR was found to be significantly increased above the level exhibited by WKY. The elevation in ethidium bromide fluorescence in SHR arterioles could be attenuated by a synthetic glucocorticoid inhibitor and in rats subjected to adrenalectomy. The administration of glucocorticoids after adrenalectomy by injection of dexamethasone restored the oxidative reaction in SHR arterioles. Treatment with dimethylthiourea and with a xanthine oxidase inhibitor attenuated the superoxide formation. Although a nitric oxide synthase inhibitor (NG-nitro-l-arginine methyl ester) enhanced the ethidium bromide staining in WKY, it did not affect that in SHR. Our findings suggest an enhancement of spontaneous oxidative stress in the microvascular wall of SHR that appears to be associated with glucocorticoid synthesis.
Recently, we obtained direct evidence that oxygen radicals are produced spontaneously from activated circulating leukocytes of spontaneously hypertensive rats (SHR) but not in their normotensive control, the Wistar-Kyoto rat (WKY).1 Our analysis of the SHR model of hypertension has shown that key aspects of the syndrome might be associated with glucocorticoid pathways.2 It was demonstrated that the increase in the number of activated circulating leukocytes in SHR could be overridden by treatment with a synthetic glucocorticoid inhibitor (RU486)3 as well as by bilateral adrenalectomy.4 These observations led us to entertain the question of whether other cells in the cardiovascular system, such as endothelium, might also be involved in spontaneous oxygen radical production and whether the overproduction of oxygen radicals could be related to the action of steroid hormones.
Hydroethidine, the sodium borohydride–reduced derivative of ethidium bromide (EB),5 6 which permeates the cell membranes easily, is an oxygen radical–sensitive fluorescent probe. The intracellular hydroethidine can be directly oxidized to form red fluorescent EB, which in turn is trapped in the nucleus by intercalation into DNA.7 Hydroethidine is especially sensitive to superoxide anion and to a lesser degree to hydrogen peroxide. The end product EB emits light at a longer wavelength (590 nm), with comparatively little light contamination of the signal from the background fluorescence in the tissue.
We designed the present series of studies to investigate the occurrence of spontaneous mesenteric microvascular oxidative stress in SHR compared with WKY with the use of digital microfluorography to determine the presence of EB formation. The involvement of adrenal glucocorticoid in spontaneous oxidative stress and its correlation to arteriolar tone were explored.
All animal procedures were previously reviewed and approved by the University of California at San Diego Animal Subject Committee. SHR (n=33) and WKY (n=32) (Charles River Breeding Laboratories, Wilmington, Mass) were used at 13 to 14 weeks of age. A cohort of SHR (n=10) and WKY (n=9) was subjected to bilateral adrenalectomy at 12 weeks.4 These bilaterally adrenalectomized SHR and WKY were fed standard rat chow with 0.9% (wt/vol) NaCl in their drinking water ad libitum and were maintained in a light-controlled holding facility for 1 week.4 Control SHR and WKY were fed standard rat chow and water ad libitum. The rats appeared alert and without overt signs of infection or weight loss. Completeness of the surgical adrenalectomy was ascertained by postmortem examination of the suprarenal region.
After general anesthesia with pentobarbital sodium (40 mg/kg IM), a femoral catheter (PE-50, Clay-Adams) was inserted and mean arterial pressure measured. Rats were placed on a heating pad and covered with a blanket maintained at 37°C. The abdomen was opened by a small midline incision. The ileocecal portion of the mesentery was gently exteriorized and draped over a plastic support for intravital microscopy, as described previously.8 The preparation was kept at 37°C and continuously superfused (1.0 mL/min) with a Krebs-Henseleit bicarbonate-buffered solution saturated with 95% N2/5% CO2. Special precautions were taken to avoid interruption of the suffusion solution on the tissue because even superficial drying causes rapid cell injury.
To provide further support that the difference in EB stain between SHR and WKY was not related to differences in strain, we investigated the microvascular oxidation of hydroethidine in normotensive Sprague-Dawley rats.
Intravital Fluorescence Microscopy
The mesenteric microcirculation was visualized through an intravital microscope (×55 water immersion objective lens, Leitz) with the use of a digital color charge coupled device (CCD) camera (DEI-470, Optronics Engineering). Single unbranched arterioles in the field with a diameter between 20 and 30 μm and approximately 150 μm in length and venules with a diameter between 30 and 40 μm and approximately 150 μm in length were selected for study. Arterioles are easily identified by their position in the microvascular network (at the inflows), the existence of vascular smooth muscle, and divergent bifurcations. At the level at which arterioles were tested in our experiments, they exhibited very little leukocyte margination. Vessels were classified as venules on the basis of their position in the convergent part of the network with at least two to three convergent capillary channels. A digital gain-control mode in the color CCD camera allowed suitable transmission images to be obtained. The camera sensitivity and shutter speed were set at constant values (contrast=0, brightness=0, manual integration=) so that the camera served as a light intensity indicator. To elicit fluorescent images, we illuminated the preparation with a 200-W mercury lamp. The light was passed through a quartz collector, heat filter (model KG-2, Zeiss), and excitation filter (490 nm, Leitz) for epi-illumination. Fluorescence emission from the specimen was passed through a band-pass filter (590 nm) and onto the CCD camera.
Photobleaching of the fluorescent images was avoided by keeping the light exposure time of the tissue limited to less than 1 second by means of a shutter between the light source and a filter cube. Transillumination images were also recorded immediately after the fluorescence images. During the intervening periods, the shutter for the excitation light was kept closed. The images were recorded with a videocassette recorder (model AG-127OP, Panasonic) for playback analysis. Fluorescence images of the microvessels were transferred into an image digitizer (512×512, 8-bit deep, Image 1.53 with a Macintosh IIci laboratory computer) and stored on a retractable hard disk for subsequent analysis at a fixed camera control setting on the digital CCD camera controller. The number of EB-positive nuclei along arterioles or venules (NEB) was counted every 15 minutes for 120 minutes. At the end of the experiments, the tissue was superfused with absolute ethanol for 10 minutes followed by EB superfusion to specify the total number of the nuclei lining the vessel wall (NT). The EB-positive number of nuclei was computed as (NEB/NT)×100 (%).
Vessel diameters were measured off-line with a videoimage-shearing monitor (model 907, IPM). The diameters reported in the study refer to inner lumen measurements; no corrections were made for noncircular cross sections.9 In addition to steady-state values, each microvessel was studied after local application of a vasodilator (1.0 mg/mL papaverine in normal saline). This dose was sufficient to eliminate active tone in the arterioles, because only vessel dilation, not narrowing at constant pressure, was observed in the presence of papaverine. Measurements before and after application of papaverine provided steady-state lumen diameters (dss) and maximal diameters (dmax), respectively. All diameter measurements were made at constant central blood pressures native to each animal. The tone (T) was computed as T=(dmax−dss)/dmax and is a nondimensional parameter that specified the degree of active smooth muscle constriction, such that T=0% in dilated vessels and T=100% in fully constricted vessels and vessels with an occluded lumen.10
After an initial 20-minute stabilization period, the mesenteric preparation was superfused with the perfusate, and a background autofluorescence image in the selected tissue area was recorded and stored in the memory of a laboratory computer (Macintosh Computer IIci, Apple Computer Co, assisted by Image 1.53 software, National Institute of Health public domain software). The preparation was then superfused with a buffer solution containing hydroethidine (5.0 μmol/L, Polyscience, Inc) for 120 minutes. The number of EB-stained nuclei was counted per unit length of microvessel. The selection of microvessels was limited to arterioles and venules.
To modify the extent of glucocorticoid involvement, we administered RU486 (33.3 mg/kg body wt IM mifepristone, Roussel-Uclaf), a synthetic glucocorticoid inhibitor, 6 hours before the microvascular experiments. Intramuscular injection of RU486 has a significant effect on the systemic leukocyte count in SHR.3 To explore the effect of glucocorticoid restoration in adrenalectomized rats on EB fluorescence, we injected dexamethasone 21-acetate (Sigma Chemical Co) at a dose of 0.5 mg/kg body wt per day IM for 5 days in a separate set of rats.
In additional experiments, to verify the role of oxygen radical formation in the mesenteric vascular wall, we pretreated the mesentery with dimethylthiourea (2 mmol/L in superfusate, Aldrich Chemical Co), which decomposes the hydroxyl radical and hydrogen peroxide.11 To explore the value of xanthine oxidase–mediated superoxide formation, we also pretreated the mesentery with (−)-8-(3-methoxy-4-phenylsulfinyl-phenyl)pyrazolo(1,5-α)-1,3,5-triazine-4-olate monohydrate [(−)BOF 4272, Otsuka Pharmaceutical Co, 10 nmol/L in superfusate], which is a synthetic xanthine oxidase inhibitor, as well as its negative control reagent, (+)-8-(3-methoxy-4-phenylsulfinyl-phenyl)pyrazolo(1,5-α)-1,3,5-triazine-4-olate monohydrate [(+)BOF 4272)]. (−)BOF 4272 at 10 nmol/L attenuated more than 60% of superoxide production in an in vitro cell-free reaction mixture containing hypoxanthine and xanthine oxidase but did not attenuate phorbol ester–induced superoxide release by isolated neutrophils, suggesting that this reagent does not have a direct scavenging effect on superoxide anions.12
To investigate the role of nitric oxide (NO) for the oxidation of hydroethidine, an NO synthase inhibitor, we superfused NG-nitro-l-arginine methyl ester (L-NAME, 50 μmol/L) for 20 minutes before the onset of hydroethidine superfusion and continued it for the entire experimental period.
Statistical comparison of EB-stained nuclear number (percent) among groups was determined by one-way layout ANOVA and Scheffé-type multiple comparison test. All values are reported as mean±SD. A value of P<.05 was considered statistically significant.
During continuous superfusion of hydroethidine, no significant changes in microvascular velocity as well as in leukocyte–endothelial cell adhesive interaction were observed.
EB fluorescence was strikingly enhanced in the wall of SHR arterioles compared with WKY arterioles without application of a stimulator (Fig 1a⇓). The elevated level of EB fluorescence in SHR arterioles (Fig 1b⇓) was attenuated by surgical adrenalectomy (Fig 1c⇓).
A similar enhancement of EB fluorescence was seen in mesenteric venules of SHR compared with those of WKY. Again, no additional stimulation of the microcirculation was required (Fig 2⇓). In contrast to the arterioles, the elevated EB fluorescence in SHR venules (Fig 2b⇓) was not significantly attenuated after adrenalectomy (Fig 2c⇓).
The majority of EB-stained nuclei in arterioles and venules had a longitudinally oriented spindle shape and were positioned on the inner lining of the vascular wall, suggesting that they were endothelial cells.
Fig 3a⇓ depicts the time course for the relative number of EB-positive nuclei (percent) along the mesenteric arteriolar wall. The number was significantly greater in SHR mesentery compared with WKY mesentery. The number of EB-positive nuclei along mesenteric venules was also significantly higher in SHR (Fig 3b⇓). In both strains, the increase in the number of EB-positive nuclei was significantly greater in arteriolar walls compared with venular walls.
The enhanced number of EB-positive nuclei (percent) along mesenteric arterioles 60 minutes after the onset of hydroethidine superfusion in SHR was blunted by adrenalectomy or by treatment with a glucocorticoid inhibitor (RU486) but was restored by glucocorticoid supplementation with dexamethasone (Fig 4a⇓). In contrast, in WKY the number was lower and there was no significant difference among groups.
The number of EB-positive nuclei (percent) along mesenteric venules 60 minutes after the onset of hydroethidine superfusion was significantly increased in SHR compared with WKY. All groups had low values of EB-positive nuclei, and there were no significant differences between groups (Fig 4b⇑).
The enhanced oxidation of hydroethidine along mesenteric arterioles at 60 minutes was significantly attenuated by dimethylthiourea or (−)BOF 4272 treatment (Fig 5a⇓). L-NAME superfusion did not elicit a significant increase in EB-positive nuclei in SHR over the number seen in untreated, control SHR (Fig 5a⇓). L-NAME superfusion induced a significant increase in EB-positive nuclei in WKY to the level observed in untreated, control SHR (Fig 5a⇓).
The enhanced number of EB-positive nuclei (percent) along mesenteric venules in SHR 60 minutes after the onset of hydroethidine superfusion could be attenuated by treatment with dimethylthiourea or (−)BOF 4272 (Fig 5b⇑). In WKY venules, all groups except the L-NAME group had low values of EB-positive nuclei, and there were no significant differences between groups (Fig 5b⇑). L-NAME superfusion induced a significant increase in EB-positive nuclei in WKY to the level observed in untreated, control SHR (Fig 5b⇑).
There was a positive correlation between arteriolar tone and the local EB-positive nuclei (percent) for the different groups (Fig 6⇓). Arteriolar tone in SHR was significantly higher than in WKY (P<.05). There was a significant linear correlation between the relative EB-positive nuclei and arteriolar tone at 60 minutes after the onset of hydroethidine superfusion in SHR, WKY, and adrenalectomized WKY.
In mesenteric arterioles or venules of normotensive Sprague-Dawley rats, no statistically significant differences compared with WKY could be demonstrated in the number of EB-positive nuclei 60 minutes after the onset of hydroethidine superfusion (arteriole, 32.7±12.0%; venule, 18.8±3.8%).
The present microfluorographic studies provide the first in vivo demonstration of oxidative stress in a hypertensive animal model without any additional stimulation. Quantitative estimates of the extent of EB staining as a consequence of oxidative stress were obtained by recording the number of EB-positive nuclei per unit length of a vessel. On this basis, the staining in the arterioles was greater than in venules and greater in SHR for both types of microvessels.
The agent hydroethidine may serve as a tool for detecting spontaneous oxidative changes in a microcirculation under several other in vivo conditions. Hydroethidine has been used in the past as a measure of the oxidative burst in neutrophils7 that results from activation of the membrane-bound NADPH oxidase via an electron transfer reaction. The superoxide anion produced is then dismutated to H2O2 either spontaneously or via superoxide dismutase. The hydroethidine moiety, which is the sodium borohydride–reduced product of EB, was initially developed as a vital dye that could cross cell membranes and label DNA.5 13 The resulting visual assessment of the EB staining in a cell after oxidation of hydroethidine is thus primarily a measure of nuclear fluorescence.14 Earlier studies on phagocytic cells have demonstrated that EB fluorescence reflected the generation of superoxide anion O2− and that O2− was acting to oxidize the hydroethidine to EB.7 Carter et al14 reported that superoxide anion resulted in a 10-fold increase in fluorescence intensity of EB compared with other oxidizing agents. According to their cell-free assay,14 when the relative intensity of EB fluorescence with potassium superoxide (KO2, equivalent to 200 μmol/L O2−) was defined as 100%, the intensities with 200 μmol/L H2O2 and with 200 μmol/L H2O2 plus 200 U/mL horseradish peroxidase were 7.8% and 10.43%, respectively.
We have previously used a method for demonstrating in vivo H2O2 levels directly with a fluorescence precursor, 2′,7′-dichlorofluorescein (DCFH) diacetate8 or 5-(6)-carboxy-2′,7′-dichlorofluorescin (CDCFH) diacetate.15 In the present study we elected to rely on hydroethidine microfluorography as a measure of the oxidative stress in microvessels for the following reason: hydroethidine is more superoxide sensitive. DCFH and CDCFH methods depend on intracellular esterase activity; therefore, it has been difficult to compare groups of cells that have different levels of esterase activity. Furthermore, inasmuch as DCFH and CDCFH fluorescence is emitted by the same range of wavelengths as the background autofluorescence characteristic of collagen fibers, it was difficult to make a comparison in a situation that may also be accompanied by a change in tissue structure involving collagen fibers. Another problem with esterase-dependent fluorescent probes was their leakage after damage of the cell membrane. Although DCFH and CDCFH allow quantification of the oxidative stress in tissue, all-or-none determination of oxidative stress with hydroethidine is well suited for the purpose of the present study.
The demonstration that the arteriolar wall in the SHR microcirculation is a site of oxygen free radical production may be relevant to a number of the different abnormalities observed in the hypertensive strain. The release of H2O2 was found to activate intercellular adhesion molecule-116 and major histocompatibility complex-1 expression in human umbilical vein endothelial cells.17 It has also been proposed that such high levels of oxygen radicals may also underlie the elevated arteriolar tone that has been documented in the SHR.10
Dimethylthiourea is a small, permeable, and relatively nontoxic scavenger of H2O2 and the H2O2-derived product hydroxyl radical.11 In the present study, dimethylthiourea significantly attenuated the microvascular EB staining in SHR, even in the arterioles of WKY, suggesting that oxygen radicals could be spontaneously produced along the arteriolar wall and that their extent was enhanced in SHR. In view of the fact that a xanthine oxidase inhibitor, (−)BOF 4272, significantly attenuated the elevated oxidative changes in SHR mesentery, the possibility exists that an overproduction of oxygen radicals might be generated at least in part by way of the xanthine–xanthine oxidase system. The fact that significant attenuation of hydroethidine oxidation occurred by specific inhibition of a xanthine oxidase, which is predominantly located in endothelial cells,18 and the shape and position of EB-stained nuclei along the innermost cell layer in microvessels suggest that hydroethidine oxidation occurred mainly in endothelial cells.
Glucocorticoid blockade by RU486, as well as by bilateral adrenalectomy, serves to attenuate EB staining in arterioles and also modify vascular responses under identical conditions. In a recent report, we were able to show that the dilator response of the mesenteric arterioles (the maximal change in tone) after histamine superfusion was blunted in SHR and that such a blunted response could be circumvented by bilateral adrenalectomy (H.S. et al, unpublished data, 1994). Pretreatment with dexamethasone significantly counteracted this mesenteric arteriolar dilator response in the adrenalectomized SHR (H.S. et al, unpublished data, 1994). The present study directly supports a strong correlation between local EB fluorescence and arteriolar tone (Fig 6⇑).
Our data demonstrate that the degree of EB staining is considerably higher along the arteriolar wall than along the venular wall. Such a distinctive difference could possibly be ascribed to the heterogeneity of the mechanisms leading to the production of oxygen radical species. Since the transformation of hydroethidine is in large part a result of O2− and H2O2 formation, the arteriolar wall, which is exposed to higher levels of oxygen than the venular walls, would be more avidly stained by EB. However, the fact that venular levels were also elevated in SHR indicates that some mechanism other than high oxygen content and the high blood pressure seen in arterioles may determine the levels of superoxide formation in SHR.
The evolution of the hypertension syndrome has been assumed to involve a glucocorticoid pathway in an important way.19 The present evidence demonstrates that a synthetic glucocorticoid inhibitor, as well as bilateral adrenalectomy, can significantly diminish the elevated levels of EB staining in the arteriolar wall of SHR mesentery. The experimental elevation of glucocorticoid levels by injection after adrenalectomy restored this visual index of oxidative stress, suggesting a possible involvement of adrenal glucocorticoids in the observed oxidative change along the microvascular wall of SHR. Glucocorticoids are potent inhibitors of the induction of a calcium-independent (inducible) NO synthase.20 21 A number of studies indicate that the flow-induced dilation of arterioles mediated by NO is impaired in SHR.22 Thus, there may be in SHR an impairment of the shear stress stimulation of NO synthesis in response to a shift in shear stress22 through activation of a potassium channel that has been shown to be coupled to a pertussis toxin–sensitive G protein.23 Our current data support the concept that endogenous NO release from microvascular endothelium may minimize spontaneous oxidative stress by inactivating O2−.15 The present data on L-NAME–treated WKY are consistent with the previous data in Wistar rats,15 which showed that L-NAME superfusion induced leukocyte-independent and leukocyte-dependent oxidative stress in arterioles and venules. The fact that L-NAME effects on EB staining in SHR arterioles and venules were not significantly different from those in the untreated groups would suggest that NO activity is suppressed in SHR. One of the possibilities in this respect is that elevated levels of endogenous glucocorticoids in SHR evoke an overexpression of oxygen radicals in microvascular endothelium by suppressing inducible NO synthase. It has also been reported that glucocorticoids decrease prostaglandin formation in endothelial cells24 by way of an induced biosynthesis of lipocortin25 so as to inhibit phospholipase A2 activity. Superoxide might also be generated from prostaglandin H synthase during the conversion from prostaglandin G2 to prostaglandin H2 in the presence of NADH or NADPH.26 27
Glucocorticoids are known to enhance catecholamine-stimulated inotropism,28 modulate β-adrenergic receptor density,29 and regulate the ability of β-adrenergic receptors to form a high-affinity state.30 In addition, glucocorticoids enhance vascular reactivity to norepinephrine.31 Adrenergic modification along these various lines by a glucocorticoid may also serve as one of the modulating factors leading to a higher level of EB staining in SHR. Such reciprocal effects of glucocorticoids and adrenergic components can contribute to a significant suppression of oxidative stress in SHR after adrenalectomy.
In conclusion, microfluorography with the use of hydroethidine suggests an enhanced formation of oxygen radicals in the wall of the SHR microvasculature. Ancillary experiments further suggest that such oxidative changes in hypertensives may be related to the elevated levels of adrenal glucocorticoids in SHR.
This work was completed during the tenure of a research fellowship for Dr H. Suzuki from the American Heart Association, California Affiliate. This work was supported by National Institutes of Health grant HL-10881. We thank Prof D.T. O’Connor, Veterans Administration Medical Center, La Jolla, Calif, for providing RU486 and Otsuka Pharmaceutical Factory, Inc, Naruto, Japan, for providing (−)BOF 4272 and (+)BOF 4272.
- Received October 28, 1994.
- Revision received November 21, 1994.
- Accepted January 20, 1995.
Schmid-Schönbein GW, Seiffge D, DeLano FA, Shen K, Zweifach BW. Leukocyte counts and activation in spontaneously hypertensive and normotensive rats. Hypertension. 1991;17:323-330.
Ruch W, Baumann JB, Häusler A, Otten UH, Siegl H, Girard J. Importance of the adrenal cortex for the development and maintenance of hypertension in spontaneously hypertensive rats. Acta Endocrinol. 1984;105:417-424.
Suzuki H, Schmid-Schönbein GW, Suematsu M, DeLano FA, Forrest MJ, Miyasaka M, Zweifach BW. Impaired leukocyte-endothelial cell interaction in spontaneously hypertensive rats. Hypertension. 1994;24:719-727.
Suzuki H, Zweifach BW, Forrest MJ, Schmid-Schönbein GW. Modification of leukocyte adhesion in spontaneously hypertensive rats by adrenal corticosteroids. J Leukoc Biol. 1995;57:20-26.
Bucana C, Saiki I, Nayer R. Uptake and accumulation of the vital dye hydroethidine in neoplastic cells. J Histochem Cytochem. 1986;34:1109-1115.
Rothe G, Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′-dichlorofluorescin. J Leukoc Biol. 1990;47:440-448.
Suematsu M, Schmid-Schönbein GW, Chavez-Chavez RH, Yee TT, Tamatani T, Miyasaka M, DeLano FA, Zweifach BW. In vivo visualization of oxidative changes in microvessels during neutrophil activation. Am J Physiol. 1993;264:H881-H891.
Schmid-Schönbein GW, Zweifach BW, DeLano FA, Chen P. Microvascular tone in a skeletal muscle of spontaneously hypertensive rats. Hypertension. 1987;9:164-171.
Jackson JH, White CW, Parker NB, Ryan JW, Repine JE. Dimethylthiourea consumption reflects H2O2 concentrations and severity of acute lung injury. J Appl Physiol. 1985;59:1995-1998.
Suzuki H, Suematsu M, Ishii H, Kato S, Miki H, Mori M, Ishimura Y, Nishino T, Tsuchiya M. Prostaglandin E1 abrogates early reductive stress and zone-specific paradoxical oxidative injury in hypoperfused rat liver. J Clin Invest. 1994;93:155-164.
Gallop PM, Paz MA, Henson E, Latt SA. Dynamic approaches to the delivery of receptor reagents into living cells. Biotechniques. 1984;2:32-36.
Carter WO, Narayanan PK, Robinson JP. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J Leukoc Biol. 1994;55:253-258.
Suematsu M, Tamatani T, DeLano FA, Miyasaka M, Forrest MJ, Suzuki H, Schmid-Schönbein GW. Microvascular oxidative stress preceding leukocyte activation elicited by in vivo nitric oxide suppression. Am J Physiol. 1994;266:H2410-H2415.
Siren AL, McCarron RM, Liu Y, Spatz M, Feuerstein G, Hallenbeck JM. Adhesion receptor expression and perivascular monocyte accumulation in carotid arteries and brains of hypertensive rats. In: Tomita M, Mohedlishville G, Rosenblum WI, Heiss W-D, Fukuuchi Y, eds. Microcirculatory Stasis in the Brain. Amsterdam, Netherlands: Elsevier; 1993:169-175.
Jarasch ED, Bruder G, Heid HW. Significance of xanthine oxidase in capillary endothelial cells. Acta Physiol Scand Suppl. 1986;548:39-46.
Radomski MW, Palmer RMJ, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci U S A. 1990;87:10043-10047.
Koller A, Huang A. Impaired nitric oxide-mediated flow-induced dilation in arterioles of spontaneously hypertensive rats. Circ Res. 1994;74:416-421.
Ohno M, Gibbons GH, Dzau VJ, Cooke JP. Shear stress elevates endothelial cGMP: role of a potassium channel and G protein coupling. Circulation. 1993;88:193-197.
Hirata F, Schiffmann E, Venkatasubramanian K, Solomon D, Axelrod J. A phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids. Proc Natl Acad Sci U S A. 1980;77:2533-2536.
Kukreja RC, Kontos HA, Hess ML, Ellis EF. PGH synthase and lipoxygenase generate superoxide in the presence of NADH or NADPH. Circ Res. 1986;59:612-619.
Wolfe BB, Harden TK, Molinoff PB. Beta-adrenergic receptors in rat liver: effect of adrenalectomy. Proc Natl Acad Sci U S A. 1976;73:1343-1347.
Schmid PG, Eckstein JW, Abboud FM. Comparison of effects of deoxycorticosterone and dexamethasone on cardiovascular responses to norepinephrine. J Clin Invest. 1967;46:590-598.