Page 1Articles of 35 in PresS. Am J Physiol Heart Circ Physiol (September 7, 2007). doi:10.1152/ajpheart.00700.2007
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Mitochondrial Arginase II Constrains Endothelial NOS-3 Activity Hyun Kyo Lim1, 4*, Hyun Kyoung Lim1*, Sungwoo Ryoo1*, Alex Benjo1, Karl Shuleri2, Victor Miriel1, Ezra Baraban1, Andre Camara1, Kevin Soucy3, Daniel Nyhan1, Artin Shoukas1, 3, Dan E. Berkowitz 1, 3
Departments of Anesthesiology and Critical Care Medicine1, Medicine2, and Biomedical Engineering3 The Johns Hopkins Medical Institutions Baltimore, Maryland Institute of Life Long Health and Department of Anesthesiology and Pain Medicine4 Yonsei University Wonju College of Medicine Wonju, Korea
Running head; Mitochondrial Arginase II and NOS-3 activity
* The following authors contributed equally to the manuscript. Address for Correspondence: Dan Berkowitz, MD Associate Professor Anesthesia; Tower 711 The Johns Hopkins Hospital 600 North Wolfe St. Baltimore, MD 21287 Phone: 410-614-1517 Fax:
410-955-0994
Email:
[email protected] Copyright Information Copyright © 2007 by the American Physiological Society.
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2 ABSTRACT Emerging evidence supports the idea that arginase expressed in the vascular endothelial cells of humans and other species, modulates endothelial nitric oxide synthase (NOS-3) activity by regulating intracellular L-arginine bioavailability. Arginase II is thought be confined to be expressed in the mitochondria of a variety of non-endothelial cells while arginase I is known to confined to the cytosol of hepatic and other cells. The isoform/s that regulate NOS-3 and their subcellular distribution however remain incompletely characterized. We therefore tested the hypothesis that arginase II, is confined to the mitochondria and that mitochondrial arginase II reciprocally regulates vascular endothelial nitric oxide (NO) production. Western blots, immunocytochemistry with MitoTracker and immuno-electron-microscopy confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly decreased, whereas NO production was significantly increased in the aorta and isolated endothelial cells from arginase II knock out (ArgII-/-) mice compared to wild type (WT) mice. The vasorelaxation response to acetylcholine (ACh) was markedly enhanced and the vasoconstrictor response to phenylephrine (PE) attenuated in ArgII-/- in pressurized mouse carotid arteries. Furthermore, inhibition of NOS-3 by NG-nitro-L-arginine methyl ester (L-NAME) impaired ACh response and restored the PE response to that observed in WT vessels. Vascular stiffness, as assessed by pulse wave velocity (PWV) was significantly decreased in ArgII-/- mice compared to WT, On the other hand 14 days of oral L-NAME treatment significantly increased PWV in both WT and ArgII-/- mice such that they were not significantly different from one another. These data suggest that arginase II is predominantly confined to the mitochondria and that this mitochondrial arginase II regulate NO production, vascular endothelial function, and vascular stiffness by modulating NOS-3 activity.
Keywords; Arginase II, Mitochondria, Nitric oxide, Vascular stiffness
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3 INTRODUCTION The endothelium plays a major role in vascular homeostasis by altering vascular tone, regulating smooth muscle cell proliferation and migration, and modulating platelet adhesion. Endothelial derived nitric oxide (NO) is a potent vasoprotective molecule and is produced from the precursor substrate L-arginine by endothelial nitric oxide synthase (NOS-3) and plays a critical role in the regulation of vascular tone and maintenance of vascular integrity. Arginase shares L-arginine as a substrate with NOS-3 and hydrolyzes L-arginine to ornithine and urea as part of the urea cycle. It is increasingly recognized that arginase modulate NOS activity, by regulating intracellular L-arginine bioavailability(2, 26). Thus, the balance between arginase and NOS-3
activities
in
part
regulates
vascular
endothelial
NO
production.
Arginase
activation/upregulation results in arginase/NOS imbalance, decreased NO production, and has been demonstrated to contribute to endothelial dysfunction, in a number of disease/pathophysiologic processes such as aging(2), diabetes(3, 6), hypertension(7, 12, 28) and atherosclerosis(18). The two isoforms of mammalian arginase, arginase I and II, encoded by different genes(25) are expressed in different tissues. Furthermore there appears to be significant species heterogeneity in isoform expression. Arginase I, located in the cytoplasm, is expressed most abundantly in the liver and is a critical enzyme in the urea cycle. Arginase II on the other hand is thought to be a mitochondrial enzyme and is expressed primarily in extrahepatic tissues such as kidney(11), brain, small intestine, mammary gland and macrophages. Accumulating evidence suggests that arginase II is the major isoenzyme in the endothelial cells of humans and other species. The role of arginase II in endothelial cells however remains incompletely understood, although there is now emerging evidence that it might be an important regulator of NO production(17, 18). In cardiac myocytes, we recently demonstrated that arginase II, confined to the mitochondria, plays and important role in regulating neuronal nitric oxide synthase (NOS-1)-dependent myocardial contractile function(21). Considering that mitochondrial arginase II in myocytes, regulates distinct and spatially confined pools of L-arginine and thus modulates NOS-1, we wished to determine if arginase
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4 II is also confined to the mitochondria in vascular endothelial cells, and whether mitochondrial arginase II regulates vascular endothelial NO production and thereby endothelial function.
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5 METHODS Animals Ten-week-old male wild-type (C57BL/6J) mice from Jackson Laboratories were used as a controls and fed a normal diet for 6 weeks. Arginase II knock out (ArgII-/-) mice were a gift from Dr. O’Brien, Baylor College of Medicine, and were bred and housed in our Institution. Male 10-week-old ArgII-/- mice were also fed a normal diet for 6 weeks. All procedures and protocols were approved by the Institutional Animal Care and Use Committee of The Johns Hopkins University School of Medicine. Aorta Preparation Heparin was administered 1 h before sacrifice. The animals were anesthetized with ketamineacepromazine intraperitoneally, and the thoracic aorta, from distal aortic arch to the diaphragmatic level, was dissected, removed, and immersed in Kreb’s solution(in mM) (NaCl 118, KCl 4.7, KH2PO4 1.2, CaCl2 2.5, MgSO4 1.2, NaHCO3 25 and glucose 11.1). The vessels were carefully cleaned of connective tissue and cut into 2-3 mm rings. Aortic rings were immediately frozen in liquid nitrogen and stored at -80°C until assayed. Isolation of mouse endothelial cells Endothelial cells (ECs) were isolated from WT and ArgII-/- mice vessel rings, as previously described(13). In brief, the dissected aorta from heparinized mice was immersed in heparin-containing 20% FBS-DMEM and washed with serum-free DMEM. After the aorta was filled with collagenase type II (2 mg/ml in serum-free DMEM, Sigma Co.) and incubated for 45 min at 37°C, ECs were removed from the aorta by flushing with 5 ml of 20% FBS-DMEM. Following centrifugation of this solution to harvest the ECs, they were cultured in a collagen Type I-coated dish. To remove smooth muscle cells, the cells were washed with warmed PBS and complete medium G (20 % FBS, 100 U/ml penicillin-G, 100 µg/ml streptomycin, 2 mM L-Glutamine, 1X non-essential amino acids, 1X sodium pyruvate, 25 mM HEPES(pH7.0-7.6), 100 µg/ml heparin, 100 µg/ml ECGS, and DMEM) was added. All experiments were performed on second passage number MAECs. Arginase Activity
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6 Arginase activity was determined by quantitating urea production using the spectrophotometric method with
-isonitrosopropiophenone as described previously(21). Briefly, cell lysates were
incubated with 75 µL manganese chloride solution at 60°C for 10 minutes and further reacted with substrate L-arginine 50 µL (0.5 mol/L, pH 9.7) at 37°C for 1 hour, and was stopped by adding 400 µL of the acid solution mixture,
-isonitrosopropiophenone (25 µL, 9% in absolute ethanol) was then
added and the mixture was heated at 100°C for 45 minutes and measured spectrophotometrically at 550 nm. NOx Measurement NO production from tissue lysates was evaluated by measuring nitrite levels using nitric oxide assay kit (Calbiochem) by Griess reaction as previously described(21). Estimation of NO generation with fluorescence probes, DAF: Endothelial cells isolated from aorta of WT and ArgII-/- mice were labeled with a fluorescentprobe to nitric oxide (DAF, 5 µmol/L, 30 min) at room temperature and protected from light. After washing with PBS, cells were then fixed with paraformaldehyde (3%) for 20 minutes. Images were acquired using a Nikon TE-200 epifluorescence microscope (with a 60 X objective, and collected using Openlab software (Improvision) and an internally cooled 12-bit CCD camera (CoolsnapHQ, Photometrics). Immunofluorescence Microscopy To stain mitochondria, cells cultured on fibronectin (Invitrogen) coated slides were incubated with 25 nM MitoTracker Red CMXRos (Molecular Probes). Cells were fixed and permeabilized with 3% paraformaldehyde and 0.5% Triton X-100 in PBS, rinsed with PBS, and incubated with polyclonal antibody against arginase II (Santa Cruz Biotechnol), and with Cy5 conjugated-anti-rabbit IgG antibody. Images were acquired using epifluorescence microscopy (Eclipse, Nikon) with a 60X objective. Epifluorescence images were collected using Openlab software (Improvision, Lexington, MA) and an internally cooled 12-bit CCD camera (CoolsnapHQ, Photometrics, Tucson, AZ). Mitochondria fractionation and Western Blot Analysis
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7 The cells were washed with twice PBS, scraped, and centrifuged at 600 g for 5 min. The pellet was resuspended in 1ml of cold mitochondria isolation buffer ( 0.3 M Sucrose, 1 mM EGTA, 5 mM MOPS, 5 mM KH2PO4 and 0.1 % BSA, pH7.4), and then homogenized. Disrupted cells were centrifuged at 2,600 g for 5 min and the supernatant was further centrifuged at 15,000 g for 10 min at 4 °C to obtain the crude mitochondrial and cytosol fraction. The resulting supernatant was centrifuged at 100,000 g for 1 h to separate the microsomal fractions. The cytosol and mitochondria fractions were mixed with 2 x SDS sample buffer (125 mM Tris, pH 6.8, 4% SDS, and 20% Glycerol) and then sonicated for 5 sec. Each sample was resolved by 10% SDS-PAGE, transferred to PVDF membrane (Bio-Rad), analyzed with antibodies according to the supplier’s protocol, and visualized with peroxidase and an enhanced-chemiluminescence system (Pierce). Normalization was performed using anti- -tubulin antibody (BD Bioscience, 1:1,000). Densitometry analysis of bands was performed with NIH ImageJ program. Immuno- Electron Microscopy Immuno-electron microscopy was performed as follows. Human aortic endothelial cells were fixed in 4% paraformaldehyde in PBS, (pH 7.4) at room temperature for one hour. The buffer was replaced with 8% paraformaldehyde PBS solution and the cells post-fixed overnight at 4°C. Cells were embedded in gelatin and ultra-thin sections were cut from the cell pellet and collected in PBS, followed by incubation in the primary Abs (rabbit anti-ArgII; 1:50 dilution) for 24h at 4°C. After washing, the secondary antibodies labeled with 6-nm gold particles were applied. The cell sections were examined with a transmission electron microscope (Hitachi 7600TEM) and images were digitally acquired. In Vitro vascular reactivity in mouse aorta Mice were terminally anesthetized using ketamine-acepromazine (100 and 10 mg/kg, respectively, ip), following which the thoracic aorta was dissected, removed, and immersed in cold oxygenated Krebs-Ringer bicarbonate solution (95% O2, 5% CO2, pH 7.4, 37°C). The vessel was carefully cleaned and cut into 1.5-mm rings, and suspended for isometric tension recording in organ chambers, as previously described(27). Protocols were performed on rings beginning at their optimum Copyright Information
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8 resting tone, previously determined to be 500 mg for mouse aorta. This resting tone was reached by stretching rings in 100 mg increments separated by 10-minute intervals. Data were collected using a MacLab system and analyzed using Dose Response Software (AD Instruments). Vessel rings were pre-constricted with PE (1µM) and their vasorelaxant dose response to ACh (1nM to 10µM) were recorded. Following washout and return to resting tension, vessels were again preconstricted with PE and their response to SNP determined (1nM to 10µM). In vitro vascular reactivity in carotid arteries Carotid arteries were dissected free from connective tissue in Kreb’s solution under a microscope and placed in a vessel chamber. Both ends of the carotid arteries were cannulated and sutured to two glass micropipettes. The chamber is filled with oxygenated Kreb’s solution (95% O2, 5% CO2, pH 7.4), which circulated from a 50-ml reservoir at a flow rate of 50 ml/min, and maintained at 37°C. Intraluminal pressure of 50 mmHg was maintained throughout the experiment. The artery was equilibrated for 30 minutes before each experiment. The external diameter of the carotid arteries was measured using video-microscopic techniques. Vasoconstrictor responses to cumulative doses of PE (PE, 1 nM to 10 µM) were measured. Vasorelaxant responses to acetylcholine (ACh, 1 nM to 100 nM) and sodium nitroprusside (SNP, 1 nM to 10 µM) were tested in PE (PE, 1 µM) pre-constricted carotid arteries. Furthermore, vasoactive response to PE and Ach were assessed after 30 min preincubation with the NOS-3 inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 µM). Vasorelaxation is expressed as percent relaxation from the PE-induced pre-constriction condition utilizing vessel external diameter. Pulse Wave Velocity (PWV) Vascular stiffness was determined pre- and post-treatment by measuring pulse wave velocity (PWV) using an EKG triggered 10 MHz Doppler probe (Indus Inst.) at thoracic and abdominal aorta locations. The animals were anesthetized and maintained with 1~1.5% isoflurane. Animals were positioned supine with legs and arms taped to electrocardiogram electrodes incorporated into a temperature controlled printed circuit board (THM100, Indus Instruments, Houston TX). Rectal temperature was monitored with a probe (Physitemp, Clifton, NJ) and maintained at 37°C throughout Copyright Information
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9 the procedure. Both thoracic and abdominal aortic flow were acquired at a depth of 2~4 and 5~6 mm, respectively, with a 2 mm diameter, 10 MHz Doppler probe (Indus Instruments). These sites of measurement were marked upon image acquisition and the separation distance between them was measured. PWV in units of meters per seconds was calculated as quotient of separation distance and time difference between pulse arrivals, as measured from EKG’s R-peaks. Data analysis of Doppler and ECG signals was performed off-line using DSPW software from Indus Instruments. Statistics All data are represented as mean ± SE. Statistical significance was determined by T-test or two-way ANOVA with Bonferroni post-tests (Graphad Prism 4 software). A values of p
