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Am J Physiol Heart Circ Physiol 289: H578-H585, 2005. First published March 11, 2005; doi:10.1152/ajpheart.00958.2004
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Testosterone treatment increases thromboxane function in rat cerebral arteries

Rayna J. Gonzales, Amir A. Ghaffari, Sue P. Duckles, and Diana N. Krause

Department of Pharmacology, College of Medicine, University of California, Irvine, California

Submitted 14 September 2004 ; accepted in final form 4 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously showed that testosterone, administered in vivo, increases the tone of cerebral arteries. A possible underlying mechanism is increased vasoconstriction through the thromboxane A2 (TxA2) pathway. Therefore, we investigated the effect of chronic testosterone treatment (4 wk) on TxA2 synthase levels and the contribution of TxA2 to vascular tone in rat middle cerebral arteries (MCAs). Using immunofluorescence and confocal microscopy, we demonstrated that TxA2 synthase is present in MCA segments in both smooth muscle and endothelial layers. Using Western blot analysis, we found that TxA2 synthase protein levels are higher in cerebral vessel homogenates from testosterone-treated orchiectomized (ORX+T) rats compared with orchiectomized (ORX) control animals. Functional consequences of changes in cerebrovascular TxA2 synthase were determined using cannulated, pressurized MCA segments in vitro. Constrictor responses to the TxA2 mimetic U-46619 were not different between the ORX+T and ORX groups. However, dilator responses to either the selective TxA2 synthase inhibitor furegrelate or the TxA2-endoperoxide receptor (TP) antagonist SQ-29548 were greater in the ORX+T compared with ORX group. In endothelium-denuded arteries, the dilation to furegrelate was attenuated in both the ORX and ORX+T groups, and the difference between the groups was abolished. These data suggest that chronic testosterone treatment enhances TxA2-mediated tone in rat cerebral arteries by increasing endothelial TxA2 synthesis without altering the TP receptors mediating constriction. The effect of in vivo testosterone on cerebrovascular TxA2 synthase, observed here after chronic hormone administration, may contribute to the risk of vasospasm and thrombosis related to cerebrovascular disease.

thromboxane synthase; cerebral circulation; vascular smooth muscle; endothelium

ALTHOUGH THE RISK for cardiovascular disease and stroke is higher in males, the influence of testosterone on vascular function is not well understood (25). This lack of information also fuels concerns about the vascular impact of testosterone therapy for elderly men (33) and postmenopausal women (28) as well as the use of anabolic androgens in young athletes, both male and female (30). Androgens appear to influence a variety of cardiovascular risk factors including lipid profile, platelet aggregation, blood pressure, and vascular reactivity (1, 25, 31, 44). Data on vascular reactivity, however, are limited and often conflicting. For example, acute exposure of arteries to testosterone has been found to cause either vasodilation or vasoconstriction (6, 7, 25, 40, 43), whereas chronic testosterone exposure in vivo results in increased vascular tone (13–15, 31). A better understanding of how testosterone modulates vascular function is needed to explain these observations.

In the cerebral circulation, the critical vascular bed in stroke, arterial tone is elevated after in vivo treatment with testosterone (14, 15, 31). Using rat middle cerebral arteries (MCAs), we demonstrated that this effect is mediated, in part, by suppression of the vasodilation normally produced by EDHFs (15). There was no apparent effect of chronic testosterone treatment on the endothelium dilator nitric oxide (NO), nor was there a change in the level of the potent constrictor endothelin (15). Testosterone treatment also had no effect on cerebrovascular levels of cyclooxygenase-1 (COX-1), the rate-limiting enzyme for prostaglandin production. COX-1 converts arachidonic acid to the intermediate endoperoxide prostaglandin H2 (PGH2), which is then converted to one of a number of prostanoids, depending on the terminal enzyme present. Under normal conditions, the synthase that produces the vasodilator prostacyclin is predominant in blood vessels. Testosterone had no effect on prostacyclin production in cerebral vessels (15).

Several studies, however, suggest testosterone affects another prostanoid, thromboxane A2 (TxA2), which has potent vasoconstrictor and platelet-aggregating actions. Testosterone was shown to increase the density of TxA2-endoperoxide receptors (TP) in platelets (1, 27) and vascular smooth muscle cells cultured from the rat aorta or canine coronary artery (18, 26). Testosterone also increased constrictor responses to the TxA2 mimetic U-46619 in the isolated aorta and coronary and renal arteries (17, 21, 27, 34). Interestingly, TP receptors in vascular tissue from males were more susceptible to testosterone modulation than those in female tissue (18, 21, 26). These data suggest that the TxA2 pathway might contribute to the elevated cerebral artery tone observed after chronic testosterone treatment of orchiectomized (ORX) male rats (14, 15). To test this hypothesis, we investigated whether testosterone treatment affected cerebrovascular TxA2 synthase and/or TP-mediated constrictor responses to endogenous TxA2 and the agonist U-46619.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Procedures

Animal handling and experimental protocols were approved by the Animal Care and Use Committee of the University of California-Irvine. Male Fischer-344 rats (3 mo old; Harlan) were anesthetized with ketamine (91 mg/kg im) and xylazine (9 mg/kg im). Animals were orchiectomized under aseptic surgical conditions, and rats were divided into two groups. The first group received Silastic testosterone propionate-filled pellets (1.57 mm inner diameter, 15 mm length) implanted subcutaneously at the base of the neck (ORX+T). The second group of rats did not receive an implant (ORX; testosterone-deficient controls). Postsurgery, all animals were treated with an injection (im) of penicillin (penicillin G benzathine-penicillin G procain, 30,000 units) and, after recovery from anesthesia, were returned to housing (12:12-h light-dark cycle) with fresh water, food, and bedding.

Four weeks after the surgery/implant, animals were deeply anesthetized with pentobarbital sodium (50 mg/kg ip), and the thoracic cavity was exposed. A direct cardiac puncture was used to collect blood samples for serum testosterone measurements using a radioimmunoassay (DiSorin; Stillwater, MN). After blood samples were collected, animals were heparinized and exsanguinated. This was achieved by exposing the right and left carotid arteries via a 3-cm midline neck incision. Catheters were placed in the right and left carotid arteries using polyethylene tubing (PE-50) and secured with 3-0 surgical silk ligatures. Cannulae were connected to a small pressure transducer and pressure-servo control unit (Living Systems) to monitor infusion pressure; this did not exceed 60 Torr. Next, the right and left jugular/subclavian veins were cut, and saline containing heparin (100 U/0.1 ml) was infused until the cerebrovasculature was completely rinsed of blood and platelets. After euthanasia and exsanguination, brains were removed and placed in a Sylgard-coated dissection dish containing ice-cold PBS or rapidly frozen and stored at –80°C for future use.

Confocal Laser Scanning Microscopy

Confocal imaging was used to determine the localization of TxA2 synthase in MCA segments. The MCA was microscopically dissected from the brains of ORX+T rats, cut into small segments, fixed in 3% formaldehyde (30 min), and permeabilized with Triton X-100 (0.1%) for 5 min. After the addition of 1% BSA, arterial segments were coincubated with primary antibodies specific for TxA2 synthase or endothelial NO synthase (eNOS), each at 1:50 dilution (TxA2 synthase, Cayman Chemical; eNOS, Transduction Labs) for 3 h at room temperature. Vessels were washed (30 min, buffer change every 5 min) and incubated with a fluorescent secondary antibody at 10 µg/ml (Oregon green or Texas red, Molecular Probes) overnight at 4°C, washed again, and then arranged on microscope slides coverslipped with VectaShield mounting medium containing 4',6-diamidino-2-phenylindole dihyrochloride (DAPI; Vector Laboratories) to counterstain cellular nuclei. Images were obtained using a Bio-Rad model 1024 laser scanning confocal microscope equipped with standard and UV lasers. In control staining conditions, vessels were incubated with the secondary antibody alone but showed no fluorescence.

Western Blot Analysis

Western blot analysis was used to determine whether chronic testosterone treatment alters the protein levels of TxA2 synthase in cerebral blood vessels. Before Western blot analysis, perfused brains of ORX and ORX+T rats were thawed on ice, and cerebral vessels were isolated using a previously published procedure (29). In brief, the whole brain was gently homogenized (glass Dounce) in cold PBS (pH 7.4) and centrifuged (720 g for 10 min at 4°C). The supernatant was discarded, and pellets were resuspended in cold PBS. The mixture was layered over a 15% dextran solution (35–45 kDa) and separated by centrifugation (1,300 g for 30–40 min at 4°C), and the pellet containing blood vessels was collected and washed with ice-cold PBS over a nylon mesh (50 µm). Next, vessels were collected from the mesh using fine tip forceps and transferred to a small glass homogenizer containing lysis buffer (4°C). Light microscopy verified that the vessel fraction consisted of a mixture of arteries, arterioles, venules, veins, and capillaries.

After incubation in lysis buffer on ice (20 min), homogenates were centrifuged (4,500 g for 10 min at 4°C), the supernatant was drawn off, and an aliquot was analyzed for protein concentration using a bicinchoninic acid protein assay (Pierce; Rockford, IL). Samples were then dissolved in Tris-glycine SDS sample buffer and boiled for 6 min. Equal amounts of sample protein (40 µg/lane) were then loaded and separated in 8% polyacrylamide gels using SDS-PAGE. In addition to ORX and ORX+T samples, gels were loaded with a standard molecular weight marker (Bio-Rad), and isolated human platelet homogenate was used as a positive control. Separated proteins were transferred to polyvinylidene difluoride membranes and blocked overnight at 4°C in PBS (0.1% Tween and 6.5% nonfat dry milk). Membranes were probed with antibodies specific for TxA2 synthase (1:200, Cayman Chemical; Ann Arbor, MI) or smooth muscle {alpha}-actin (1:20,000, Sigma Chemical). Enhanced chemiluminescence development in conjunction with a horseradish peroxidase-labeled secondary antibody (1:15,000) was used to visualize the proteins of interest. Densitometry of bands was analyzed with UN-SCAN-it software (Silk Scientific; Orem, UT).

Isolated, Pressurized Cerebral Artery Preparations

Functional studies were used to assess the ability of chronic testosterone to modulate the role of the TxA2 pathway in the maintenance of vascular tone. Brains isolated from ORX and ORX+T rats were placed in ice-cold physiological saline solution (PSS) containing Ca2+ and pinned in a Sylgard-coated dissection dish aerated with an oxygen and carbon dioxide gas mixture. Small segments of second-order MCAs (200–250 µm diameter) were carefully dissected and placed in a small vessel chamber (Living Systems; Burlington, VT) with PSS. The proximal end of the vessel was mounted on a glass micropipette and secured with a nylon ligature, and the lumen was gently rinsed with PSS to ensure removal of any remaining blood elements. The proximal cannula was connected to a pressure transducer and reservoir containing PSS equilibrated with 21% O2-6% CO2-balance N2. Next, the distal end was mounted on a second cannula and tied in place. A stopcock located distal to the vessel was closed, and the vessel was gradually pressurized to 60 Torr and then maintained at constant pressure with a pressure-servo control unit (Living Systems). A constant-flow peristaltic pump continuously superfused (25 ml/min) the tissue with warmed PSS (37°C, pH 7.4) aerated with 21% O2-6% CO2-balance N2. The vessel preparation was viewed using an inverted microscope (Nikon) equipped with a charge-coupled device camera/computer interface system (IonOptix; Milton, MA). An electronic dimension analyzer (Living Systems) was used to measure intraluminal diameter. Changes in intraluminal diameter were recorded using a computer-based data-acquisition edge-detection system (IonOptix).

TxA2 receptor agonist (U-46619). After segments were equilibrated for 1 h at 60 Torr and showed the development of spontaneous tone, a concentration-response curve (10–10–10–6 mol/l) to the TxA2 mimetic (15)-hydroxy-11{alpha},9{alpha}-(epoxymethano)prosta-5,13-dienoic acid (U-46619) was generated in MCA segments isolated from ORX and ORX+T rats. At the end of the experiment, the passive intraluminal vessel diameter was measured at 60 Torr in Ca2+-free PSS containing EDTA (3 mM).

TxA2 synthase inhibition and TP receptor blockade. Arterial segments were equilibrated for 1 h at 60 Torr. After segments developed spontaneous tone, vessel diameter was recorded during increasing step pressure changes to 60, 80, and 100 Torr in the presence of PSS. Next, arteries were incubated in PSS containing the NO synthase inhibitor NG-nitro-L-arginine-methyl ester (L-NAME; 100 µM). In a separate set of experiments, artery segments were first denuded of endothelium by passing air bubbles through the lumen (2 ml total). Subsequently, L-NAME was added, and a lack of a response verified endothelial disruption. After incubation in L-NAME (30 min), intact and denuded vessels were superfused with PSS + L-NAME containing either furegrelate (TxA2 synthase inhibitor, 50 µM) or SQ-29548 (TP receptor antagonist, 1 µM) for an additional 30 min. At the end of each experiment, passive responses to step pressure changes were determined in Ca2+-free PSS containing EDTA (3 mM). Tissues were incubated in Ca2+-free PSS for 20 min, and intraluminal step pressure changes (60, 80, and 100 Torr) were then applied to obtain passive blood vessel diameters. On the basis of passive diameters, artery segments used in the isolated vessel experiments were similar in size for the ORX and ORX+T groups (Table 1).


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  		Table 1. Intraluminal diameters of MCA segments isolated from ORX and ORX+T rats in the presence of L-NAME, after endothelium removal in the presence of L-NAME, or in the presence of Ca2+-free PSS (passive diameter)

 
Solutions and Chemicals

PSS bicarbonate-phosphate buffer containing (in mM) 122 NaCl, 5.1 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25.5 NaHCO3, and 0.03 EDTA was prepared weekly as a stock solution. Before each experiment, superfusate buffer was prepared from the PSS stock solution by adding 11.5 mM glucose and 1.6 mM CaCl2. PBS was prepared from premade packets. Stock solutions for drugs in the tissue bath experiments were prepared by dissolving L-NAME and furegrelate (Cayman Chemical) in double-distilled H2O and SQ-29548 (Cayman Chemical) and U-46619 (Cayman Chemical) in ethanol. Final concentrations for all drugs were prepared in PSS. Lysis buffer for Western blots, containing {beta}-glycerophosphate (50 mmol/l), sodium orthovanadate (100 µmol/l), magnesium chloride (2 mmol/l), EGTA (1 mmol/l), and Triton X-100 (0.5%), was prepared fresh; dithiothreitol (1 mmol/l), phenylmethylsulfonyl fluoride (1 mmol/l), pepstatin (20 µmol/l), leupeptin (20 µmol/l), and aprotinin (0.1 U/ml) were then added from stock solutions (dissolved in solvent, double-distilled H2O or DMSO, and stored at –20°C). Unless noted otherwise, all drugs and chemicals were purchased from Sigma Chemical.

Data Analysis

Data are reported as means ± SE. Data were compared between ORX and ORX+T groups using Student's paired t-test or ANOVA for repeated measures as appropriate. Multiple comparisons were made with the Student-Newman-Keuls test when ANOVA indicated that differences existed. P ≤ 0.05 was considered statistically significant for all comparisons.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Serum Testosterone Levels and Body Weight

We verified our hormone replacement animal model by measuring serum levels of total circulating testosterone after 4 wk of in vivo testosterone treatment. Serum testosterone concentrations for the ORX+T group (1.29 ± 0.13 ng/ml) were in the range of those previously measured in intact male rats (14, 15). In ORX rats not receiving hormone-filled implants, serum testosterone levels were below the radioimmunoassay level of detection (0.03 ng/ml). In addition to serum levels, body weight also was used as an indicator of testosterone levels. Body weights of ORX+T (290 ± 9 g) rats were significantly greater compared with ORX controls (273 ± 4 g), as expected after chronic testosterone treatment (14, 15).

TxA2 Synthase Is Expressed in MCAs

In the present study, immunofluorescence with confocal microscopy was used to verify the presence and localization of TxA2 synthase in the MCA within the cerebral vasculature. Costaining with the nuclear specific stain DAPI was used as a focal plane marker to distinguish the endothelial and smooth muscle layers within the MCA segments. Characteristically, endothelial cell nuclei are oriented in the direction of blood flow (Fig. 1A), whereas smooth muscle nuclei are perpendicular to the direction of flow (Fig. 1B). This identification is confirmed by the presence of eNOS in the endothelial layer (Fig. 1E) and the absence of eNOS staining in the smooth muscle layer (Fig. 1F). Colabeling with an antibody selective for TxA2 synthase resulted in fluorescent staining in both the intimal (endothelial) (Fig. 1C) and medial (smooth muscle) (Fig. 1D) layers, indicating the presence of the enzyme TxA2 synthase in both cell types within the MCA.



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  		Fig. 1. Localization of thromboxane A2 (TxA2) synthase in whole mounts of cerebral arteries by immunofluorescence and confocal microscopy. A, C, and E: one confocal plane at the level of the intimal layer (endothelium). B, D, and F: second plane at the level of the medial layer (smooth muscle). A and B: nuclear staining using 4',6-diamidino-2'-phenylindole (DAPI); C and D: staining with an antibody to TxA2 synthase (green); E and F: staining with an antibody to endothelial nitric oxide synthase (eNOS; red).

 
Testosterone Increases TxA2 Synthase Levels in Cerebral Blood Vessels

Because confocal imaging verified the presence of TxA2 synthase in the cerebrovasculature, we next determined if chronic testosterone treatment affects TxA2 synthase levels in cerebral blood vessels. Using blood-free vessel homogenates, we measured the levels of TxA2 synthase with Western blot techniques. Human platelet whole cell lysate was used as a positive marker (band at 60 kDa), and levels of {alpha}-actin (40 kDa) were used to verify equal protein loading of ORX and ORX+T samples within each experiment (Fig. 2A). Interestingly, the level of TxA2 synthase was significantly greater in cerebral blood vessel homogenates from ORX+T compared with ORX rats (Fig. 2).



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  		Fig. 2. Levels of TxA2 synthase in blood-free cerebral vessel homogenates from orchiectomized rats (ORX) and ORX rats chronically treated with testosterone (ORX+T). A: representative Western blot of protein bands identified with the TxA2 antibody. A 60-kDa band was labeled in cerebral vessel homogenates from ORX and ORX+T rats that corresponded to a similar band in the positive control, human platelet lysate. Levels of {alpha}-actin are shown as loading controls (42 kDa). B: levels of TxA2 synthase expressed as the mean optical density ratio relative to ORX (control). *P ≤ 0.05 vs. ORX (n = 4).

 
Chronic Testosterone Treatment Does Not Alter Constrictor Responses to the TP Agonist U-46619 in MCAs

Possible effects of testosterone treatment on cerebrovascular TxA2 receptors (TP receptors) were assessed using a functional assay. Constrictor responses to the specific TP agonist U-46619 were measured in pressurized MCAs isolated from ORX and ORX+T rats. As shown in Fig. 3, arteries from both ORX and ORX+T rats developed significant, and similar, constriction when exposed to increasing concentrations of U-46619. The concentration-response curves of the two groups were not different, suggesting that testosterone treatment does not alter the affinity, number, or signaling of TP receptors in cerebral arteries.



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  		Fig. 3. Effect of chronic testosterone treatment on vascular contractile responses to the TxA2 receptor agonist U-46619. Diameter responses to U-46619 (10–10–10–6 mol/l) were recorded in isolated middle cerebral arteries pressurized to 60 Torr. Data are expressed as [(diameter in PSS – U-46619 diameter)/diameter in PSS] x 100 at each concentration, where PSS is physiological salt solution. There were no significant differences between groups (n = 5).

 
Testosterone Treatment Augments Dilator Responses Caused by TxA2 Synthase Inhibition or TP Receptor Blockade

Because chronic treatment with testosterone resulted in an increase in TxA2 synthase protein levels (Fig. 2), experiments were designed to investigate whether the contribution of basal TxA2 production to cerebrovascular tone is altered after chronic in vivo testosterone treatment. Two approaches were used: inhibition of TxA2 synthase with furegrelate or blockade of the TP receptor with SQ-29548. In initial experiments with pressurized MCA segments, dilatory responses to furegrelate or SQ-29548 were very weak (data not shown). However, the addition of the NO synthase inhibitor L-NAME was found to enhance tone and facilitate observation of dilation in response to TxA2 pathway inhibitors, similar to what was reported by Benyo et al. (3). Previous studies in our laboratory demonstrated that chronic testosterone treatment does not alter vasoconstrictor responses to L-NAME (15). Therefore, all inhibitor studies were conducted in the presence of L-NAME. Under this condition, diameters of both intact and endothelium-denuded vessels were not different between the ORX and ORX+T groups (Table 1). Inhibition of TxA2 synthase with furegrelate caused an increase in the diameter of arteries isolated from both ORX and ORX+T animals. As shown in Fig. 4A, this dilation was significantly greater in MCAs from ORX+T compared with ORX animals at all intraluminal pressures tested (60–100 Torr). However, in vessels denuded of endothelium, furegrelate responses were relatively small, and there were no significant differences between the ORX and ORX+T groups (Fig. 4B).



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  		Fig. 4. Vascular responses to inhibition of TxA2 synthase with furegrelate (50 µM) in pressurized middle cerebral arteries isolated from ORX and ORX+T rats. Diameter changes in response to the addition of furegrelate were determined in vessels preconstricted with NG-nitro-L-arginine methyl ester (L-NAME; 100 µM) or after endothelium removal in the presence of L-NAME. Data are calculated as the percent change of furegrelate dilation from L-NAME constriction (A) or endothelium removal constriction in the presence of L-NAME (B): [(constriction diameter – constriction diameter with furegrelate)/constriction diameter] x 100. *P ≤ 0.05 vs. ORX (n = 5 for A; n = 3 for B).

 
Inhibition of the TP receptor using the selective antagonist SQ-29548 also caused dilation of pressurized MCA segments treated with L-NAME (Fig. 5). This effect was significantly greater in MCA segments from ORX+T rats compared with the ORX rats, regardless of the intraluminal pressure.



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  		Fig. 5. Dilator responses to the TxA2-endoperoxide (TP) receptor antagonist SQ-29548 in pressurized middle cerebral arteries isolated from ORX and ORX+T rats. Diameter changes in response to SQ-29548 (1 µM) were measured in vessels constricted with L-NAME (100 µM). Data are calculated as the percent change: [(diameter in L-NAME – diameter in L-NAME with SQ-29548)/diameter in L-NAME] x 100. *P ≤ 0.05 vs. ORX (n = 5).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates functional increases in TxA2, a potent vasoconstrictor and prothrombotic factor, in cerebral blood vessels after chronic testosterone administration in vivo. Protein levels of TxA2 synthase were higher in cerebral vessels taken from ORX+T rats compared with ORX controls. Vascular TxA2 production, functionally determined in MCA segments using inhibitors of the synthase (furegrelate) and receptor (SQ-29548) for TxA2, was also increased in ORT+T arteries. TxA2 synthase was visualized by confocal microscopy to both endothelial and smooth muscle layers of the MCA, but functional experiments indicated that endothelium-derived TxA2 contributed to resting tone and was increased after chronic testosterone treatment. In contrast, no differences were found in constrictor responses to the TxA2 receptor agonist U-46619 between the ORX and ORX+T groups, implying that cerebral artery TP receptors are unaffected by in vivo testosterone treatment. Overall, these findings suggest that cerebrovascular TxA2 production may contribute to in vivo effects of testosterone treatment with possible implications for hormone therapy, androgen abuse, and gender differences in stroke.

TxA2, a prostanoid metabolite of arachidonic acid, is synthesized by a cascade of reactions terminating in the conversion of the prostaglandin endoperoxide intermediate PGH2 via the enzyme TxA2 synthase (35). Pfister et al. (32) proposed that the source of TxA2 for blood vessels is derived from an endothelium-derived precursor of the arachidonic acid pathway that is transferred to platelets for further metabolism to TxA2. In rabbit pulmonary arteries, TxA2 synthase was found in cell types, e.g., macrophages and platelets, that, when activated, adhered to the vessel wall (5). Therefore, we took special care to study cerebral blood vessels in which the lumen had been perfused, in vivo and in vitro, with heparinized PSS to remove platelets and other blood elements. A number of studies support our finding that TxA2 is produced by cells within the vascular wall (9, 12, 19). Using immunocytochemistry, Ermert et al. (12) demonstrated the presence of TxA2 synthase in the smooth muscle layer of small resistance-sized pulmonary arteries. Others have shown that endothelial cells, cultured from the porcine brain or bovine coronary microvessels, also produce measurable amounts of TxA2 (9, 19). In our study, confocal imaging confirmed the presence of TxA2 synthase within the cerebral artery wall in both the intimal and medial layers.

Our data agree with earlier reports of TxA2 activity in cerebral arteries (3, 22, 23, 36, 41). We demonstrated functional consequences of vascular TxA2 production using isolated, pressurized segments of MCAs that were pretreated with the NO synthase inhibitor L-NAME. Under these conditions, we measured small, but significant, vasodilation after inhibition of either TxA2 synthase or TP receptors, consistent with a local TxA2 constrictor influence on resting vascular tone. Although TP receptors are also activated by the prostanoid intermediate PGH2, production of this substance would not be prevented by furegrelate, a selective TxA2 synthase inhibitor. Similar to Benyo et al. (3), we used NO synthase inhibition to preconstrict the arteries and enhance the relatively weak dilator responses to furegrelate and SQ-29548. Many studies suggest that NO modulates vascular prostanoid production (3, 22, 38, 42), which may explain why block of NO synthesis allowed us to study the contribution of TxA2 to basal tone. Similarly, inhibition of NO production was found to enhance the prostacyclin prostanoid pathway in cerebral blood vessels (24).

A key finding of this study is that testosterone treatment in vivo increases TxA2 modulation of cerebral artery tone. Circulating hormone was undetectable in ORX rats, whereas plasma testosterone achieved physiological levels with the hormone implant, as we have shown previously (14, 15). This difference in testosterone exposure resulted in significant changes in the cerebrovascular TxA2 pathway. It should be noted that testosterone can be metabolized to estrogen as well as to the potent androgen dihydrotestosterone. However, our previous work indicates that estrogen has different effects on the cerebrovasculature compared with those of testosterone treatment. For example, estrogen decreases cerebral artery tone (14), whereas tone is increased after treatment with testosterone (14, 15) or dihydrotestosterone (R. J. Gonzales, D. N. Krause, and S. P. Duckles, unpublished observations).

Most notably, chronic testosterone treatment increased the level of TxA2 synthase protein in cerebral blood vessels, as demonstrated with Western blot analysis. In contrast, testosterone has no effect on levels of the rate-limiting prostanoid enzyme COX-1 or prostacyclin production in cerebral vessels (15). Together, these data indicate that testosterone shifts the cerebral vascular prostacyclin-TxA2 balance toward the constrictor prostanoid pathway. Interestingly, estrogen treatment has the opposite effect; it shifts the prostanoid balance toward vasodilation by increasing cerebrovascular levels of COX-1 and prostacyclin synthase (29). Chronic estrogen administration did not alter basal TxA2 production in cerebral blood vessels (29). Given the differences in hormonal effects, it is unlikely that testosterone conversion to estrogen underlies the changes in the TxA2 pathway observed in the present study.

We are not aware of any previous reports showing an effect of testosterone on TxA2 synthase. A number of transcription factors have been identified that regulate TxA2 synthase expression (46), but possible interactions with androgen receptors remain to be determined. Alternatively, because testosterone exposure occurred in vivo, we cannot rule out indirect effects that could modulate the levels of TxA2 synthase. For example, changes in shear stress were recently shown in human umbilical veins to affect TxA2 expression; however, unlike testosterone exposure, prostacyclin synthase was also upregulated by shear (11). While the mechanisms remain to be elucidated, an effect on TxA2 synthase is consistent with an earlier observation that monkeys treated with testosterone exhibit elevated plasma levels of TxA2 (45).

Both the selective TxA2 synthase inhibitor furegrelate and the TP receptor antagonist SQ-29548 had greater effects in arteries from ORX+T males, consistent with enhanced TxA2 synthesis. All studies were conducted in the presence of L-NAME, but this inhibitor produced similar levels of constriction in ORX and ORX+T arteries, in agreement with our previous finding that testosterone treatment does not affect the endothelial NO pathway in rat cerebral arteries (15). We also considered the possibility that in vivo testosterone treatment increased the responsiveness of TP receptors in cerebral arteries. However, concentration-response curves to the TP agonist U-46619 were identical in pressurized MCA segments from ORX and ORX+T rats. This finding implies there was no change in cerebral artery TP receptors, in contrast with studies on other vascular beds, cultured vascular smooth muscle, and platelets in which testosterone increased TP receptor density and U-46619 responses (1, 17, 21, 27, 34). Because the mechanisms underlying testosterone effects on TP receptors are unknown, it is difficult at this point to explain why cerebral arteries differ. Isoforms of the TP receptor exist, and they may be regulated differently among vascular beds (35). Alternatively, the effect of testosterone on TP receptors may require other stimuli not active in cerebral vessels under resting physiological conditions. Clearly, more investigation is needed to understand these discrepancies.

With no apparent change in TP function, our results with furegrelate and SQ-29548 point to an increase in the synthesis of TxA2 as the primary effect of testosterone on cerebrovascular prostanoid pathways. Higher production of TxA2 increases the vasoconstrictor contribution to net resting tone. This is consistent with previous findings that chronic exposure to testosterone augments the vascular tone of cerebral arteries (14, 15, 31). Interestingly, the latter effect was endothelium dependent (15). In the present study, endothelium removal also greatly reduced dilation to furegrelate, and the differences between ORX and ORX+T were abolished. Thus MCA resting tone appears to be modulated by endothelium-derived TxA2, and this pathway is influenced by chronic testosterone administration. While there was no apparent contribution of smooth muscle-derived TxA2 to smooth muscle tone under our conditions, the presence of TxA2 synthase in the medial layer no doubt has important roles, and future studies should address possible modulation by testosterone treatment.

In our earlier work, we found that testosterone treatment suppresses an endothelial vasodilator with the characteristics of EDHF (15). Therefore, the enhanced vascular tone in cerebral arteries induced by testosterone treatment involves both increases in vasoconstrictor and decresases in vasodilator factors. Interestingly, a recent study (8) in the rat mesenteric artery suggests that TxA2 suppresses EDHF-mediated hyperpolarization and dilation by reducing the activity of smooth muscle small-conductance Ca2+-activated K+ channels. This suggests a possible relationship between the increased level of TxA2 and decreased EDHF dilation observed after testosterone treatment.

In the present study, we focused on cerebral arteries under normal, physiological conditions. Under these circumstances, the contribution of TxA2 to resting tone is relatively small and is balanced by the endothelium dilators NO and prostacyclin. However, TxA2 production in cerebral arteries is stimulated by a number of vasoconstrictors including endothelin (37), serotonin (10), UTP (23), and isoprostanes (4, 19), and even relatively low levels of TxA2 can facilitate constrictor responses in cerebral resistance vessels (2, 36). Thus, by increasing levels of TxA2 synthase, testosterone may enhance cerebrovascular constriction by a number of agents. Increased levels of TxA2 in cerebral arteries also would increase the risk of thrombosis, vasospasm (39), and ischemic injury (20). The impact of cerebrovascular TxA2 is highly relevant during pathophysiological conditions such as endothelial dysfunction (3, 4, 22, 41), cerebral vasomotion (22, 39), oxidative stress (4, 19), hypoxia (38), and stroke (20). Effects of testosterone on TxA2 synthase may contribute to the detrimental influence of this hormone on ischemic brain injury (16). Future studies are needed, however, to elucidate the mechanisms by which testosterone affects cerebrovascular TxA2 synthase and directly assess the impact of this effect in pathological states.


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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-50775 and by a postdoctoral fellowship from the American Heart Association, Western States Affiliate.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. N. Krause, Dept. of Pharmacology, College of Medicine, Univ. of California, Irvine, CA 92697-4625 (E-mail: dnkrause{at}uci.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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Ajayi AA, Mathur R, and Halushka PV. Testosterone increases human platelet thromboxane A2 receptor density and aggregation responses. Circulation 91: 2694–2698, 1995.[Free Full Text]
  2. Armstead WM, Mirro R, Busija DW, and Leffler CW. Permissive role of prostanoids in acetylcholine-induced cerebral vasoconstriction. J Pharmacol Exp Ther 251: 1012–1019, 1989.[Abstract/Free Full Text]
  3. Benyo Z, Gorlach C, and Wahl M. Involvement of thromboxane A2 in the mediation of the contractile effect induced by inhibition of nitric oxide synthesis in isolated rat middle cerebral arteries. J Cereb Blood Flow Metab 18: 616–618, 1998.[CrossRef][ISI][Medline]
  4. Brault S, Martinez-Bermudez AK, Marrache AM, Gobeil F Jr, Hou X, Beauchamp M, Quiniou C, Almazan G, Lachance C, Roberts JII, and Chemtob S. Selective neuromicrovascular endothelial cell death by 8-iso-prostaglandin F2alpha: possible role in ischemic brain injury. Stroke 34: 776–782, 2003.[Abstract/Free Full Text]
  5. Buzzard CJ, Pfister SL, and Campbell WB. Endothelium-dependent contractions in rabbit pulmonary artery are mediated by thromboxane A2. Circ Res 72: 1023–1034, 1993.[Abstract/Free Full Text]
  6. Chou TM, Sudhir K, Hutchison SJ, Ko E, Amidon TM, Collins PJ, and Chatterjee K. Testosterone induces dilation of canine coronary conductance and resistance arteries in vivo. Circulation 94: 2614–2619, 1996.[Abstract/Free Full Text]
  7. Costarella CE, Stallone JN, Rutecki GW, and Whittier FC. Testosterone causes direct relaxation of rat thoracic aorta. J Pharmacol Exp Ther 277: 34–39, 1996.[Abstract/Free Full Text]
  8. Crane GJ and Garland CJ. Thromboxane receptor stimulation associated with loss of SKCa activity and reduced EDHF responses in the rat isolated mesenteric artery. Br J Pharmacol 142: 43–50, 2004.[CrossRef][ISI][Medline]
  9. Davidge ST, Baker PN, McLaughlin MK, and Roberts JM. Nitric oxide produced by endothelial cells increases production of eicosanoids through activation of prostaglandin H synthase. Circ Res 77: 274–283, 1995.[Abstract/Free Full Text]
  10. Descombes JJ, Devys M, Laubie M, and Verbeuren TJ. Endothelial thromboxane production plays a role in the contraction caused by 5-hydroxytryptamine in rat basilar arteries. Eur J Pharmacol 243: 193–199, 1993.[CrossRef][ISI][Medline]
  11. Doroudi R, Gan LM, Sjogren LS, and Jern S. Intraluminal pressure modulates eicosanoid enzyme expression in vascular endothelium of intact human conduit vessels at physiological levels of shear stress. J Hypertens 20: 63–70, 2002.[CrossRef][ISI][Medline]
  12. Ermert L, Ermert M, Duncker HR, Grimminge F, and Seeger W. In situ localization and regulation of thromboxane A2 synthase in normal and LPS primed lungs. Am J Physiol Lung Cell Mol Physiol 278: L744–L753, 2000.[Abstract/Free Full Text]
  13. Farhat MY, Wolfe R, Vargas T, Foegh ML, and Ramwell PW. Effect of testosterone treatment on vasoconstrictor response of left anterior descending coronary artery in male and female pigs. J Cardiovasc Pharmacol 25: 495–500, 1995.[ISI][Medline]
  14. Geary GG, Krause DN, and Duckles SP. Gonadal hormones affect diameter of male rat cerebral arteries through endothelium-dependent mechanisms. Am J Physiol Heart Circ Physiol 279: H610–H618, 2000.[Abstract/Free Full Text]
  15. Gonzales RJ, Krause DN, and Duckles SP. Testosterone suppresses endothelial-dependent dilation of rat middle cerebral arteries. Am J Physiol Heart Circ Physiol 286: H552–H560, 2004.[Abstract/Free Full Text]
  16. Hawk T, Zhang YQ, Rajakumar G, Day AL, and Simpkins JW. Testosterone increases and estradiol decreases middle cerebral artery occlusion lesion size in male rats. Brain Res 796: 296–298, 1998.[CrossRef][ISI][Medline]
  17. Higashiura K, Blaney B, Morgan E, Mathur RS, and Halushka PV. Inhibition of testosterone 5{alpha}-reductase: evidence for tissue-specific regulation of thromboxane A2 receptors. J Pharmacol Exp Ther 279: 1386–1391, 1996.[Abstract/Free Full Text]
  18. Higashiura K, Mathur RS, and Halushka PV. Gender-related differences in androgen regulation of thromboxane A2 receptors in rat aortic smooth-muscle cells. J Cardiovasc Pharmacol 29: 311–315, 1997.[CrossRef][ISI][Medline]
  19. Hou X, Gobeil F, Peri K Jr, Speranza G, Marrache AM, Lachapelle P, Roberts J, Varma DR, and Chemtob S. Augmented vasoconstriction and thromboxane formation by 15-F2{alpha}-isoprostane (8-iso-prostaglandin F2{alpha}) in immature pig periventricular brain microvessels. Stroke 31: 516–524, 2000.[Abstract/Free Full Text]
  20. Imamura T, Kiguchi S, Kobayashi K, Ichikawa K, Yamazaki Y, Kojima M. Effect of ozagrel, a selective thromboxane A2-synthetase inhibitor, on cerebral infarction in rats. Comparative study with norphenazone, a free-radical scavenger. Arzneimittelforschung 53: 688–694, 2003.[Medline]
  21. Karanian JW and Ramwell PW. Effect of gender and sex steroids on the contractile response of canine coronary and renal blood vessels. J Cardiovasc Pharmacol 27: 312–319, 1996.[CrossRef][ISI][Medline]
  22. Lacza Z, Herman P, Gorlach C, Hortobagyi T, Sandor P, Wahl M, and Benyo Z. NO synthase blockade induces chaotic cerebral vasomotion via activation of thromboxane receptors. Stroke 32: 2609–2614, 2001.[Abstract/Free Full Text]
  23. Lacza Z, Káldi K, Kövecs K, Görlach C, Nagy Z, Sándor P, Benyó Z, and Wahl M. Involvement of prostanoid release in the mediation of UTP-induced cerebrovascular contraction in the rat. Brain Res 896:169–174, 2001.[CrossRef][ISI][Medline]
  24. Li X, Geary G, Gonzales RJ, Krause DN, Duckles SP. Effect of estrogen on cerebrovascular prostaglandins is amplified in mice with dysfunctional NOS. Am J Physiol Heart Circ Physiol 287: H588–H594, 2004.[Abstract/Free Full Text]
  25. Liu PY, Death AK, and Handelsman DJ. Androgens and cardiovascular disease. Endocr Rev 24: 313–340, 2003.[Abstract/Free Full Text]
  26. Matsuda K, Mathur RS, Ullian ME, and Halushka PV. Sex steroide regulation of thromboxane A2 receptors in cultured rat aortic smooth muscle cells. Prostaglandins 49:183–196, 1995.[CrossRef][ISI][Medline]
  27. Matsuda K, Ruff A, Morinelli TA, Mathur RS, and Halushka PV. Testosterone increases thromboxane A2 receptor density and responsiveness in rat aortas and platelets. Am J Physiol Heart Circ Physiol 267: H887–H893, 1994.[Abstract/Free Full Text]
  28. Moynihan R. FDA panel rejects testosterone patch for women on safety grounds. Br Med J 329: 1363, 2004.[Free Full Text]
  29. Ospina JA, Krause DN, and Duckles SP. 17{beta}-Estradiol increases rat cerebrovascular prostacyclin synthesis by elevating cyclooxygenase-1 and prostacyclin synthase. Stroke 33: 600–605, 2002.[Abstract/Free Full Text]
  30. Parssinen M and Seppala T. Steroid use and long-term health risks in former athletes. Sports Med 32: 83–94, 2002.[CrossRef][ISI][Medline]
  31. Penotti M, Sironi L, Cannata L, Vigano P, Casini A, Gabrielli L, and Vignali M. Effects of androgen supplementation of hormone replacement therapy on the vascular reactivity of cerebral arteries. Fertil Steril 76: 235–240, 2001.[CrossRef][ISI][Medline]
  32. Pfister SL, Deinhart DD, and Campbell WB. Methacholine-induced contraction of rabbit pulmonary artery: role of platelet-endothelial transcellular thromboxane synthesis. Hypertension 31: 206–212, 1998.[Abstract/Free Full Text]
  33. Rhoden EL and Morgentaler A. Risks of testosterone-replacement therapy and recommendations for monitoring. N Engl J Med 350: 482–492, 2004.[Free Full Text]
  34. Schrör K, Morinelli TA, Masuda A, Matsuda K, Mathur RS, and Halushka PV. Testosterone treatment enhances thromboxane A2 mimetic induced coronary artery vasoconstriction in guinea pigs. Eur J Clin Invest 24: 50–52, 1994.[ISI][Medline]
  35. Shen RF and Tai HH. Thromboxanes: synthase and receptors. J Biomed Sci 5: 154–172, 1998.
  36. Shirahase H, Usui H, Kurahashi K, Fujiwara M, and Fukui K. Possible role of endothelial thromboxane A2 in the resting tone and contractile responses to acetylcholine and arachidonic acid in canine cerebral arteries. J Cardiovasc Pharmacol 10: 517–522, 1987.[ISI][Medline]
  37. Shirahase H, Usui H, Shimaji H, Kurahashi K, and Fujiwara M. Endothelium dependent and independent contraction induced by endothelin-1 in canine basilar arteries. Eur J Pharmacol 183: 1804–1805, 1990.[CrossRef]
  38. Sillau AH, McCullough RE, Dyckes R, White MM, and Moore LG. Chronic hypoxia increases MCA contractile response to U-46619 by reducing NO production and/or activity. J Appl Physiol 92: 1859–1864, 2002.[Abstract/Free Full Text]
  39. Takeuchi H, Tanabe M, Okamoto H, and Yamazaki M. Effects of thromboxane synthase inhibitor (RS-5186) on experimentally-induced cerebral vasospasm. Neurol Res 21: 513–516, 1999.[ISI][Medline]
  40. Teoh H, Quan A, Leung SWS, and Man RYK. Differential effects of 17{beta}-estradiol and testosterone on the contractile responses of porcine coronary arteries. Br J Pharmacol 129: 1301–1308, 2000.[CrossRef][ISI][Medline]
  41. Tsuji T and Cook DA. Origin of thromboxane mediated constriction due to neuropeptides in canine basilar artery. Eur J Pharmacol 264: 77–80, 1994.[CrossRef][ISI][Medline]
  42. Wade ML and Fitzpatrick FA. Nitric oxide modulates the activity of the hemoproteins prostaglandin I2 synthase and thromboxane A2 synthase. Arch Biochem Biophys 347: 174–180, 1997.[CrossRef][ISI][Medline]
  43. Webb CM, McNeill JG, Hayward CS, de Zeigler D, and Collins P. Effects of testosterone on coronary vasomotor regulation in men with coronary heart disease. Circulation 100: 1690–1696, 1999.[Abstract/Free Full Text]
  44. Weidemann W and Hanke H. Cardiovascular effects of androgens. Cardiovasc Drug Rev 20: 175–198, 2002.[ISI][Medline]
  45. Weyrich AS, Rejeski WJ, Brubaker PH, and Parks JS. The effects of testosterone on lipids and eicosanoids in cynomolgus monkeys. Med Sci Sports Exerc 24: 333–338, 1992.
  46. Zhang L, Xiao H, Schultz RA, and Shen RF. Genomic organization, chromosomal localization, and expression of the murine thromboxane synthase gene. Genomics 45: 519–528, 1997.[CrossRef][ISI][Medline]

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