Androgen deprivation increases neuronal nitric oxide metabolism and its vasodilator effect in rat mesenteric arteries

NITRIC OXIDE

Biology and Chemistry

Nitric Oxide 12 (2005) 163–176 www.elsevier.com/locate/yniox

Androgen deprivation increases neuronal nitric oxide metabolism and its vasodilator effect in rat mesenteric arteries Ma del Carmen Martı´n, Gloria Balfago´n, Nuria Minoves, Javier Blanco-Rivero, Mercedes Ferrer * Departamento de Fisiologı´a, Facultad de Medicina, Universidad Auto´noma de Madrid, Spain Received 14 October 2004; revised 25 January 2005

Abstract This study examines the effects of male sex hormones on the vasoconstrictor response to electrical field stimulation (EFS), as well as neuronal NO modulation of this response. For this purpose, denuded superior mesenteric artery from orchidectomized and control male Sprague–Dawley rats was used. EFS induced similar frequency-dependent contractions in segments from both groups. The NO synthase (NOS) inhibitor Nx-nitro-L-arginine methyl ester strengthened EFS-elicited contractions more in arteries from orchidectomized than from control male rats. The expression of nNOS was more pronounced in segments from control than from orchidectomized animals. Basal and EFS-induced NO release was similar in segments from both groups. In noradrenaline (NA)-precontracted segments, sodium nitroprusside (SNP) induced a concentration-dependent relaxation, that was greater in segments from orchidectomized than control male rats. 8-Bromo-cGMP induced a similar concentration-dependent relaxation in NA-precontracted segments from either group, and the cGMP levels induced by SNP were also similar in the two groups. Superoxide dismutase (SOD), a superoxide anion scavenger, did not modify the relaxation in segments from control male rats. In contrast, SOD enhanced the relaxation induced by SNP in segments from orchidectomized rats, and the effect was reversed by preincubation with SOD plus catalase. The generation of superoxide anion and of peroxynitrite was greater in segments from orchidectomized than control rats. In NA-precontracted segments from control or orchidectomized rats, exogenous peroxynitrite and H2O2 induced a concentration-dependent relaxation. These results suggest that EFS induces a similar nNOS-derived NO release in segments from orchidectomized and control male rats, despite the decrease in nNOS expression in orchidectomized rats. The NO metabolism is higher in segments from orchidectomized male rats due to the increases in anion superoxide generation and peroxynitrite formation. The vasodilator effects of the peroxynitrite and H2O2 generated from the NO metabolism are what enhance the functional role of the nNOS-derived NO release in the orchidectomized rats.
2005 Elsevier Inc. All rights reserved. Keywords: Sex hormones; Nitric oxide; Vasodilation; Oxygen radicals; Mesenteric artery

The incidence of cardiovascular diseases is lower in premenopausal women than in men of similar age [1]. While the beneficial effects of estrogens on arterial function are well established [2,3], studies concerning the vascular role of androgens have produced conflicting

*

Corresponding author. Fax: +34 91 497 5478. E-mail address: [email protected] (M. Ferrer).

1089-8603/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.02.003

results. In general, androgens have been associated with impaired vascular reactivity [4], although recent experimental studies report a preventive effect with regard to cardiovascular diseases [5,6] similar to that shown by estrogens in women. Nitric oxide (NO) is known to play an important role in vasomotor tone regulation [7,8]. NO is formed through several NO synthases (NOS), i.e., endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal

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NOS (nNOS). Most of the investigations on the effects of gender on NO release/function have been focused on the actions of estrogens. In addition, particular attention has been paid recently to the effects of androgens on NO function. Thus, it has been reported that testosterone increases the activity of eNOS [9] and nNOS [10], while it inhibits that of iNOS [11]. Increasing evidence suggests that superoxide anion is an important contributor to reduced NO bioactivity, which has been associated with cardiovascular disorders [12,13]. On the other hand, it has been reported that the generation of superoxide anion also depends on gender [14], and again, most of the studies have focused on the effects of estrogen [15,16]. However, several recent studies have also emerged showing antioxidant properties of androgens [17,18]. Mesenteric artery has perivascular nitrergic innervation that releases NO when electrically stimulated [19,20], and that participates in the regulation of vascular tone. Recently, we have studied how physiological levels of female sex hormones affect the release and metabolism of neuronal NO in rat mesenteric artery [21] and, since there are no studies analysing the effect of male sex hormones on neuronal NO release in vascular tissues, we hypothesize that neuronal NO release as well as its bioavailability and vasomotor response may be affected by the presence or absence of male sex hormones. Therefore, the aim of the present study was to investigate a possible role for male sex hormones in the release, response, and/or metabolism of neuronal NO in mesenteric arteries from male rats.

Materials and methods Animal housing and protocols Male Sprague–Dawley rats (6 months old) were used. They were divided into two groups: control and orchidectomized males. All animals were housed in accordance with the institutional guidelines (constant temperature; 12 h dark/light cycle; and standard feeding with rat chow and water ad libitum). Deprivation of male sex hormones was surgically induced at 7 weeks of age, and 4 months later the animals were sacrificed. The observation of atrophy of the seminal vesicles confirmed successful gonadectomy. Rats were sacrificed by CO2 inhalation; the first branch of the mesenteric artery was carefully dissected out, cleaned of connective tissue, and placed in Krebs–Henseleit solution (KHS, in mM: NaCl 115, CaCl2 2.5, KCl 4.6, KH2PO4 1.2, MgSO4Æ7H2O 1.2, NaHCO3 25, glucose 11.1, and Na2-EDTA 0.03) at 4 C. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the USA National Institutes of Health (NIH publication No. 85.23 revised 1985).

Vascular reactivity The method used for isometric tension recording has been described in full elsewhere [22]. Briefly, two parallel stainless steel pins were introduced through the lumen of the vascular segment: one was fixed to the bath wall, and the other connected to a force transducer (Grass FTO3C; Quincy, MA, USA); this was connected in turn to a model 7D Grass polygraph. For electrical field stimulation (EFS) experiments, segments were mounted between two platinum electrodes 0.5 cm apart and connected to a stimulator (Grass, model S44) modified to supply adequate current strength. Segments were suspended in an organ bath containing 5 ml KHS at 37 C continuously bubbled with a 95% O2–5%CO2 mixture (pH of 7.4). Experiments were performed in endothelium-denuded segments to eliminate the main source of vasoactive substances, including NO. This avoided possible actions by different drugs on endothelial cells that could lead to misinterpretation of results. Endothelium was removed by gently rubbing the luminal surface of the segments with a wooden stick. The segments were subjected to a tension of 0.5 g which was readjusted every 15 min during a 90 min equilibration period before drug administration. After this, the vessels were exposed to 75 mM KCl to check their functional integrity. Endothelium removal did not alter the contractions elicited by 75 mM KCl. After a washout period, the absence of vascular endothelium was tested by the inability of 10 lM acetylcholine (ACh) to relax segments precontracted with 1 lM noradrenaline (NA). Frequency–response curves to EFS (1, 2, 4, 8, and 16 Hz) or concentration–response curves to NA (10 nM–10 lM) were performed. The parameters used for EFS were: 200 mA, 0.3 ms, 1–16 Hz, for 30 s with an interval of 1 min between each stimulus, the time required to recover basal tone. A washout period of at least 1 h was necessary to avoid desensitization between consecutive curves. Two successive frequency–response curves separated by 1 h intervals produced similar contractile response. When assessing the effect of tetrodotoxin, a blocker for nerve impulse (TTX, 0.1 lM) propagation, and the effect of phentolamine, the antagonist of a-adrenoceptors (1 lM) on the contraction elicited by EFS, the substance was added to the bath 30 min in advance. To determine the participation of NO on EFS- and/ or NA-induced responses in segments from both groups of rats, 10 lM Nx-nitro-L-arginine methyl ester (LNAME) or 5 nM 2-amino-5,6-dihydro-6-methyl-4H1,3-thiazine (AMT), respective inhibitors of constitutive and inducible NOS, was added to the bath 30 min before performing the second frequency–response curve or concentration–response curve to NA. To analyse the possible participation of sensitive innervation on EFS-induced response in control and

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orchidectomized male rats, 0.5 lM capsaicin, a sensitive neurotoxin, was added to the bath 60 min before performing the second frequency–response curve or concentration–response curve to NA. To analyse the possible differences in the NO pathway, the ability of sodium nitroprusside (SNP, 10 nM–10 lM) and 8-bromo-cyclicGMP (8Br-cGMP, 0.1 lM–0.1 mM) to induce relaxation was analysed in segments from both rat groups precontracted with 1 lM NA. To analyse the effects of superoxide anions and H2O2 on the response elicited by SNP, response curves to this agent were performed in segments from both groups of rats, in the presence of either superoxide dismutase (SOD, 13 U/ml; a scavenger of superoxide anions) or catalase (1000 U/ml; an inactivator of H2O2). The response to SNP in the presence of SOD plus catalase was also performed only in segments from orchidectomized animals. In NA-precontracted arteries from control and orchidectomized animals, the vasodilator effects of exogenous peroxynitrite (1 lM–0.3 mM) and H2O2 (10 lM–1 mM) were tested. Nitric oxide release Segments of rat mesenteric arteries (10.8 ± 0.6) were immersed for 30 min in 10 ml KHS at 37 C, continuously gassed with a 95% O2–5% CO2 mixture (stabilization period). Afterwards, the arteries were transferred to a 0.5 ml chamber containing two parallel platinum electrodes, 0.5 cm apart, connected to a stimulator (Grass model S44) for EFS. After two washout periods of 6.5 min, the medium was collected to measure the basal NO release. Once the chambers had been refilled, EFS periods of 30 s at 1, 2, 4, 8, and 16 Hz at 1 min intervals were applied. Aliquots (500 ll) of the KHS were taken from the bath to measure the concentration of NO2 , according to the colorimetric method based on the Griess reaction [23]. The interference of TTX in NO release was studied by incubating the arteries with this substance 30 min before being placed in the EFS chamber. The stimulation-induced NO release was calculated by subtracting basal NO release from that evoked by EFS. The amount of NO released was expressed in nmol/mg tissue. To avoid possible interferences of products derived from the NO metabolism, the fluorescent probe 4,5-diaminofluorescein (DAF-2) was also used to specifically quantify the NO release induced by EFS. Briefly, mesenteric arteries were subjected to a resting tension of 0.5 g, as in the reactivity experiments. After an equilibration period of 60 min, arteries were incubated with DAF-2 (2 lM) for 45 min. The medium was collected to measure the basal NO. Once the organ bath was refilled, EFS periods of 30 s at 1, 2, 4, 8, and 16 Hz at 1 min

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intervals were applied. The fluorescence of the medium was measured at room temperature using a luminescence spectrometer LS50 (Perkin-Elmer Instruments) with the excitation wavelength set at 495 nm and emission wavelength at 515 nm. The stimulation-induced NO release was calculated by subtracting basal NO release from that evoked by EFS. Also, blank measures were collected in the same way from medium without mesenteric segments to subtract background emission. The amount of NO released was expressed as arbitrary unit per milligram tissue. Western blot analysis of neuronal NOS expression For Western blot analysis of nNOS protein expression, superior mesenteric arteries were homogenized in a boiling buffer composed of 1 mM sodium vanadate (a protease inhibitor), 1% SDS, and 0.01 M, pH 7.4 Tris–HCl. Homogenates containing 16.5 lg protein were electrophoretically separated on a 7.5% SDS–polyacrylamide gel (SDS–PAGE) and then transferred to polyvinyl difluoride membranes (Bio-Rad Immun-Blot overnight at 4 C, 230 mA, using a Bio-Rad Mini Protean III system (Bio-Rad Laboratories, Hercules, CA, USA) containing 25 mM Tris, 190 mM glycine, 20% methanol, and 0.05% SDS. Prestained SDS–PAGE broad range standards (Bio-Rad Laboratories) were used as molecular mass markers. The membrane was blocked for 3 h at room temperature in Tris-buffered-saline solution 100 mM, 0.9%w/v NaCl, 0.1% SDS) with 5% non-fat powdered milk before being incubated overnight at 4 C with mouse monoclonal antibody for nNOS (1:750 dilution), purchased from Transduction Laboratories (Lexington, UK). After washing, the membrane was incubated with a 1:5000 dilution of antimouse Immunoglobulin G antibody conjugated to horseradish peroxidase (Sigma–Aldrich). The membrane was thoroughly washed and the immunocomplexes were detected using an enhanced horseradish peroxidase/luminol chemiluminescence system (ECL Plus, Amersham International, Little Chalfont, UK) and subjected to autoradiography (Hyperfilm ECL, Amersham International). Signals on the immunoblot were quantified using a computer program (NIH Image V1.56). The same membrane was used to determine a-actin expression, and the content of the latter was used to correct n-NOS expression in each sample, by means of a monoclonal antibody anti a-actin (1:2000 dilution, Sigma). Determination of cGMP Denuded rat mesenteric arteries were subjected to a resting tension of 0.5 g, as indicated above in the reactivity experiments. After an equilibration period of 60 min, segments were contracted with NA during 3 min (considered the basal level), and then some segments were

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incubated with 0.1 lM SNP for 10 s. Segments were immediately frozen in liquid nitrogen and stored at 70 C. Levels of cGMP were determined using the Cyclic GMP Enzyme Immunoassay Kit from Assay Designs. For this assay, the frozen arteries were homogenized in 0.1 M HCl and centrifuged at 600g for 10 min at 4 C. Non-soluble fraction was used to measure protein content with a DC protein assay kit (Bio-Rad). The supernatant was then collected and used for the assay. cGMP levels were measured following the manufacturer
s protocol. Briefly, samples were acetylated with one volume of acetic anhydride and two volumes of triethylamine mix, then transferred to a 96-well plate coated with goat antibody specific for rabbit IgG, and were incubated overnight with a cGMP antibody at 4 C. After washing, a solution of p-nitrophenyl phosphate was added for 1 h at room temperature to reveal the reaction. The reaction was stopped and the plate was read at 405 nm using a microplate reader. Results were expressed as pmol cGMP/mg protein. Detection of superoxide anions Superoxide anion levels were measured using lucigenin chemiluminescence, as previously described [24,25]. Briefly, mesenteric segments were rinsed in KHS for 30 min, equilibrated for 30 min in Hepes buffer at 37 C, transferred to test tubes that contained 1 ml Hepes buffer (pH 7.4) containing lucigenin (250 lM) and then kept at 37 C. The luminometer was set to report arbitrary units of emitted light; repeated measurements were collected during 5 min at 10 s intervals and averaged. 4,5-Dihydroxy-1,3-benzene-disulphonic acid ‘‘Tiron’’ (10 mM), a cell permeant, non-enzymatic scavenger of the superoxide anion, was added to quench the superoxide anion-dependent chemiluminescence. Also, blank measures were collected in the same way without mesenteric segments to subtract background emission. Some assays were performed by adding SOD 13 U/ml to ensure the specificity of the method. Oxidative fluorescent microphotography Hydroethidine (HE), an oxidative fluorescent dye, was used to evaluate superoxide anion levels in situ, as described [26,27]. Briefly, mesenteric arteries from control and orchidectomized male rats were cryoprotected with 30%w/v sucrose in PBS, embedded in optimum cutting temperature compound, OCT Tissue Tek, and 20 lm cryostat sections were placed on a glass slide. HE (5 lM) was topically applied to each tissue section and coverslipped. To ensure the specificity of the method, vessels from orchidectomized rats were incubated with tempol (4-hydroxy-2,2,6,6,-tetramethyl piperidinoxyl, 1 mM), a membrane-permeable mimetic of SOD, during 40 min prior to cryoprotection. Once the cryostat

sections were obtained, they were also treated with tempol during sample processing and HE incubation. Slides were incubated in a light-protected, humidified chamber at 37 C for 30 min. Images were obtained with a LEICA (TCS ST2 DM IRE2) laser scanning confocal microscope (excitation 488 nm, emission 610 nm). Laser and image settings were fixed for the acquisition of images from both control and orchidectomized rats. The photomicrographs show the intensity and location of the oxidized HE, which reflects superoxide production, so that comparison of these groups could be made. Peroxynitrite detection The oxidation of dihydrorhodamine-123 was determined and used as an indirect measure of peroxynitrite formation [28]. The arteries were washed three times with PBS. After that, the arteries were incubated for 45 min in PBS containing 0.1 mM of the metal chelator diethylenetriamine penta-acetic acid and 0.1 mM dihydrorhodamine-123 at 37 C; in another set of experiments, 0.1 lM SNP was also added. The medium was collected and absorbance was determined at 500 nm. Blank samples were collected by incubating the assay mixture without tissue, and in the presence of 0.1 lM SNP to subtract the absorbance induced by SNP alone. Dihydrorhodamine-123 oxidation was calculated using an extinction coefficient of 78,800 M 1 cm 1 at 500 nm. Immunohistochemistry for peroxynitrite formation Mesenteric arteries were fixed in 4% formaldehyde, 0.5% acetic acid, 0.1 M phosphate buffer, then cryoprotected with 30%w/v sucrose in PBS, and cut in OCT Tissue Tek embedded cross-sections. Sections were incubated with a rabbit antinitrotyrosine polyclonal antibody (1:125, Upstate Biotechnology), washed, and then incubated for an additional 1 h 30 min with a biotinylated secondary antirabbit IgG (1:250, Vector) antibody. Detection was enhanced by means of a streptavidin–HRP complex (1:250, Vector). A 0.02%w/v diaminobenzidyne solution 0.003%v/v hydrogen peroxide in 0.2 M Tris–HCl was used as chromogenic substrate, and the reaction was stopped after 5 min by dilution with 0.05 M Tris–HCl. Sections were mounted, dehydrated, and coverslipped for light microscopy photography. Drugs used L-NA hydrochloride, ACh chloride, TTX, AMT, capsaicin, catalase, phentolamine, SNP, SOD, tempol, L-NAME hydrochloride, tiron, lucigenin, dihydrorhodamine-123, diethylenetriamine penta-acetic acid, and hydroethidine (Sigma, St. Louis, MO, USA).

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Statistical analysis The responses elicited by EFS, KCl, or NA were expressed in milligram for comparison between control and orchidectomized male rats, and also as a percentage of the contraction induced by 75 mM KCl. Results are given as means ± SEM. Statistical analysis was done by comparing the curve obtained in the presence of the different substances with the previous or control curve by means of repeated-measures analysis of variance (ANOVA). For the experiments of NO release, cGMP formation, superoxide anion, and peroxynitrite quantification, the statistical analysis was done by means of Student
s t test for unpaired experiments. A p value of less than 0.05 was considered significant.

Results Vascular reactivity to electrical field stimulation and exogenous noradrenaline The response induced by 75 mM KCl was decreased in segments from orchidectomized rats (control, 1530 ± 93, n = 30, and orchidectomized rats, 1161 ± 73 mg, n = 42, p = 0.002). The contractions induced by EFS were similar in arteries from both groups of rats when the results were expressed in milligram (Fig. 1A), and were increased in segments from orchidectomized male rats when the results were expressed as a percentage of the contraction induced by 75 mM KCl (Fig. 1B). EFS-induced contractions were practically abolished by the blocker for nerve impulse propagation, tetrodotoxin (0.1 lM), and markedly reduced by the a-adrenoceptor antagonist, phentolamine (1 lM), in segments from both groups (Table 1). The contractile response induced by exogenous NA (10 nM–10 lM) was greater in segments from control than orchidectomized rats when the results were ex-

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pressed in milligram (Fig. 2A); however, when the results were referred to the contraction induced by 75 mM KCl, NA induced similar contractions in both groups of rats (Fig. 2B). These results indicate that the contraction induced by EFS evokes NA release from adrenergic nerve endings thereby activating a-adrenoceptors in both experimental groups. The contraction induced by EFS was significantly increased by preincubation with the NOS inhibitor LNAME (10 lM) in segments from both groups of rats, but it was greater in segments from orchidectomized than from control male rats (Figs. 3A and B). Neither the blocker of iNOS, 5 nM AMT, nor the sensitive neurotoxin, 0.5 lM capsaicin, modified the contractile response induced by EFS in segments from either group (Figs. 4 and 5). These results indicate the involvement of neuronal NO in the response induced by EFS, and that the vascular effect is more pronounced in mesenteric segments from orchidectomized than control animals. The sensory innervation does not have a functional role in the response induced by EFS in the control or orchidectomized rats. Expression of neuronal NOS The expression of nNOS was detected in homogenates from fresh rat superior mesenteric arteries and was more evident in homogenates from control than from orchidectomized male rats (Fig. 6). The doublet band observed in homogenates from control animals, but not the orchidectomized animals, could be due to an alternative spliced nNOS isoform. Vascular relaxation to sodium nitroprusside and 8Br-cGMP In segments precontracted with NA (1 lM), SNP (0.1 nM–10 lM) induced a concentration-dependent

Fig. 1. Isometric tension recording of the frequency-dependent contractions in denuded mesenteric artery segments from control and orchidectomized male rats. Results (means ± SEM) are expressed in milligram (A) and as a percentage of tone induced by 75 mM KCl (control, 1453 ± 124; orchidectomized, 997 ± 75 mg) (B). n, Number of animals. *p < 0.05 compared with control rats.

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Table 1 Effects of tetrodotoxin (TTX, 0.1 lM) and phentolamine (Phent., 1lM) on the frequency–contraction curves performed in mesenteric artery segments from control and orchidectomized male rats Frequency (Hz)

Control TTX Phent. Orchidectomized TTX Phent.

Contraction (%) 1

2

4

8

16

7.4 ± 1.2 0 0 12 ± 0.5 0 0

16.7 ± 2.1 0 0 25.2 ± 1.8 0 0

30.7 ± 3 0 0 38.9 ± 2 0 0

45 ± 3 2.8 ± 0.5 3.5 ± 0.6 56 ± 3.5 1.5 ± 0.5 3.8 ± 0.6

68 ± 4.8 3.0 ± 1.5 6.2 ± 0.7 78.9 ± 4.1 2.6 ± 1.5 7.1 ± 1.3

Results (means ± SEM) are expressed as percentages of the response elicited by 75 mM KCl; zeros are used when contraction was not detected. n = 5–7 animals.

Fig. 2. Contractile response to NA in mesenteric artery segments from control and orchidectomized male rats. Cumulative concentration–response curves to NA were carried out. Results (means ± SEM) are expressed in milligram (A) and as a percentage of tone induced by 75 mM KCl (control, 1527 ± 93; orchidectomized, 1152 ± 72 mg) (B). n, Number of animals. *p < 0.01 compared with control rats.

Fig. 3. Effect of L-NAME on the frequency–response curves performed in mesenteric artery segments from control and orchidectomized male rats. Results (means ± SEM) are expressed as a percentage of a previous tone with 75 mM KCl (control, 1344 ± 98; orchidectomized, 1049 ± 130 mg). n, Number of animals. *p < 0.05; **p < 0.01 compared with control rats.

relaxation, that was greater in segments from orchidectomized than from control male rats (Fig. 7A). However, the vasodilator response to the non-hydrolyzable analog of cGMP, 8Br-cGMP (0.1 lM–0.1 mM), was

not significantly different in segments from either control or orchidectomized animals (Fig. 7B). In NA-precontracted segments from control rats, preincubation with 13 U/ml SOD, a superoxide anion

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Fig. 4. Effect of AMT on the frequency–response curves performed in mesenteric artery segments from control and orchidectomized male rats. Results (means ± SEM) are expressed as a percentage of a previous tone with 75 mM KCl (control, 1555 ± 119; orchidectomized, 1244 ± 125 mg ). n, Number of animals.

Fig. 5. Effect of capsaicin on the frequency–response curves performed in mesenteric artery segments from control and orchidectomized male rats. Results (means ± SEM) are expressed as a percentage of a previous tone with 75 mM KCl (control, 1579 ± 224; orchidectomized, 1234 ± 159 mg ). n, Number of animals.

scavenger, did not modify the relaxation induced by SNP (Fig. 8A). In contrast, in segments from orchidectomized male rats, SOD increased the vasorelaxation

Fig. 6. Western blot for nNOS expression in mesenteric artery segments from control and orchidectomized male rats. Arterial homogenates were subjected to SDS–PAGE followed by immunoblot analysis using anti-nNOS antibody (see Materials and methods for more details). Figure is representative of preparations from four rats.

induced by SNP (Fig. 8B); this effect was reversed by the simultaneous presence of SOD and catalase (Fig. 8B). Preincubation with catalase alone (1000 U/ml) did not modify the relaxation induced by SNP in either group (data not shown). In NA-precontracted segments from control and orchidectomized rats, exogenous peroxynitrite (1 lM– 0.3 mM) and H2O2 (10 lM–1 mM) induced a concentration-dependent relaxation (Figs. 9A and B). All these data indicate a more pronounced NO metabolism through superoxide anion formation in the mesenteric segments from orchidectomized than control rats, and suggest the formation of vasodilator products from the NO metabolism in the former.

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Fig. 7. Vasorelaxant response to SNP (A) and 8Br-cGMP (B) in mesenteric artery segments from control and orchidectomized male rats. Cumulative concentration–response curves to SNP and 8Br-cGMP were carried out. Results (means ± SEM) are expressed as a percentage of the inhibition of contraction induced by 1 lM NA (control, 1344 ± 98; orchidectomized, 1049 ± 130 mg). Number of animals is indicated in parentheses. *p < 0.05; **p < 0.01 compared with control rats.

Fig. 8. Effect of superoxide dismutase (SOD) on the concentration–response curves to SNP in precontracted segments from control male rats, and effect of SOD and SOD plus catalase on the concentration–response curves to SNP in NA-precontracted segments from orchidectomized male rats. Results (means ± SEM) are expressed as a percentage of the inhibition of contraction induced by 1 lM NA (control, 1475 ± 195; orchidectomized, 1083 ± 104 mg). The number of animals is indicated in parentheses. *p < 0.01 compared with control condition.

Fig. 9. Concentration–response curves to peroxynitrite and H2O2 in NA-precontracted segments from control and orchidectomized male rats. Results (means ± SEM) are expressed as a percentage of the inhibition of contraction induced by 1 lM NA (control, 1360 ± 215; orchidectomized, 1079 ± 94 mg). The number of animals is indicated in parentheses.

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Nitric oxide release Basal levels of NO2 were similar in segments from both control and orchidectomized rats. EFS induced a similar level of NO2 formation in segments from both groups of rats. The NO2 formation induced by EFS was strongly decreased by pretreatment with TTX (Table 2). The incubation medium containing DAF-2 and artery from control or orchidectomized rat, in basal conditions, emitted similar fluorescent levels (control, 1.20 ± 0.1; orchidectomized, 1.22 ± 0.2 arbitrary unit per milligram tissue; n = 4; p > 0.05). EFS induced a similar intensity of fluorescence in the incubation medium with artery from control or orchidectomized rats Table 2 EFS induces nitrite release in mesenteric artery segments from control and orchidectomized male rats: effects of preincubation with 0.1 lM tetrodotoxin (TTX) Nitrite release (pmol/mg) Basal

Control (8) Orchidectomized (10)

EFS

Control

TTX

Control

TTX

6.5 ± 2.2 8.1 ± 1.9

5.9 ± 2.5 7.5 ± 3

18.2 ± 1.7* 15.9 ± 2.8*

7.5 ± 3** 6.7 ± 2**

Results (means ± SEM) are expressed in pmol/mg tissue. The number of animals is indicated in parenthesis. * p < 0.05 compared with the respective basal nitrite release. ** p < 0.05 compared with the respective EFS-induced nitrite release.

Table 3 Basal and stimulated with sodium nitroprusside (SNP) cGMP levels in NA precontracted mesenteric segments from control and orchidectomized male rats

Control (4) Orchidectomized (4)

Basal (pmol/mg)

SNP (pmol/mg)

1.9 ± 0.5 2.0 ± 1

21.8 ± 3* 15.9 ± 3.7*

Results (means ± SEM) are expressed in pmol/mg protein. The number of animals is indicated in parenthesis. * p < 0.01 compared with the respective basal cGMP levels.

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(control, 2.2 ± 0.3; orchidectomized, 2.1 ± 0.2 arbitrary unit per milligram tissue; n = 4; p < 0.05 vs basal conditions). These results indicate that orchidectomy does not modify the nNOS-derived NO release induced by EFS. Determination of cGMP Basal cGMP levels were similar in segments from both groups. Incubation with 0.1 lM SNP for 10 s similarly increased cGMP levels in segments from control and orchidectomized animals (Table 3). These data suggest that differences in cGMP level formation are not responsible for the more pronounced vasodilator effect of SNP and nNOS-derived NO release. Detection and confocal microscopic examination of superoxide anion After subtracting the lucigenin chemiluminescence obtained in the presence of tiron from that obtained in its absence, the calculated tiron-quenchable chemiluminescence was significantly higher in segments from orchidectomized rats (546 ± 58 U/mg/min n = 5) than in segments from control rats (117 ± 43 U/mg/min; n = 5; p < 0.001). Preincubation of segments with SOD strongly decreased the tiron-quenchable chemiluminescence (data not shown). Cross-sections of mesenteric arteries from control and orchidectomized male rats were evaluated for the presence of superoxide anion, and the results are summarized in Fig. 10. After incubation with hydroethidine, the arteries from orchidectomized male rats showed a markedly higher level of expression of EtBr fluorescence than the arteries from control rats, in which fluorescence had been inhibited by pretreating the vessel with the membrane-permeable mimetic of SOD, tempol 1 mM. These results indicate higher concentrations of superoxide anion in the arteries from orchidectomized male rats,

Fig. 10. Fluorescent confocal micrographs showing in situ detection of superoxide anion in denuded mesenteric arteries from control (A) orchidectomized (B) and orchidectomized rats pretreated with tempol (C). Arterial sections were labelled with the oxidative dihydroethidium, which fluoresces red when oxidized to EtBr by superoxide (see Materials and methods). The sections shown are typical of preparations from four rats. Magnification: 400·.

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Fig. 11. Immunohistochemical localization of nitrotyrosine in denuded mesenteric arteries from control (A) and orchidectomized (B) male rats. Appropriate negative control, treated only with immunoglobulin G, is shown in C. Figure is representative of preparations from four rats. Magnification: 400·.

and reinforce the functional involvement of superoxide anion in the vasodilator effect of SNP. Detection and immunohistochemical localization of peroxynitrite Basal dihydrorhodamine-123 oxidation was not detected in segments from either group, control or orchidectomized animals. The dihydrorhodamine-123 oxidation induced by sodium nitroprusside (0.1 lM) was greater in segments from orchidectomized than from control animals (control, 265.3 ± 63, orchidectomized 488.6 ± 76 pmol/mg; n = 5; p < 0.05). To measure the expression of nitrotyrosine, a specific marker of peroxynitrite formation, immunohistochemical localization of nitrotyrosine was performed in arteries from control and orchidectomized male rats in basal conditions. Immunohistochemical staining of nitrotyrosine was markedly stronger in arteries from orchidectomized male rats than in arteries from control male rats (Fig. 11). These data support the earlier suggestion that the higher peroxynitrite formation in basal and SNP-stimulated arteries from orchidectomized animals than in the controls.

Discussion The results of the present study demonstrate that the absence of male sex hormones decreases nNOS expression and does not modify the nNOS-derived NO release induced by EFS, although it does raise the NO metabolism through superoxide anion and peroxynitrite generation. Nevertheless, the functional role of the nNOS-derived NO release in segments from orchidectomized male rats is increased, probably due to products generated by the NO metabolism such as peroxynitrite and H2O2, which can act as alternative vasodilator mechanisms to compensate for the loss of NO bioavailability. Rat mesenteric arteries possess rich sympathetic [29], sensory [30], and nitrergic innervations [20,31], which

participate in vasomotor tone modulation. The present results show that EFS induced contractile responses in endothelium-denuded mesenteric segments from control and orchidectomized male rats; these responses were practically abolished by tetrodotoxin and markedly reduced by phentolamine, the respective blockers for nerve impulse propagation and a-adrenoceptors. Therefore, these responses appear to be mediated by NA release from adrenergic nerve terminals and the subsequent activation of a-adrenoceptors in both experimental hormonal conditions, as has been described in other rat strains [32,33]. We found that the vasoconstrictor responses to KCl and exogenous NA are diminished in segments from orchidectomized male rats, which agrees with most studies demonstrating that testosterone treatment enhances the action of several contractile agents [34–36]. The fact that the absolute values of the responses-one of the most informative measurements to determine real differences between groups-induced by EFS were similar in both groups of rats, while the absolute values of the response induced by exogenous NA were diminished in orchidectomized animals, suggests that EFS increased the NA release from adrenergic endings in segments from orchidectomized animals. However, studies on this question, mostly in the central nervous system, have produced contradictory results: a decrease [37,38], an increase [39,40], and no modification [41,42] in NA release have been reported in orchidectomized male animals. Nevertheless, and even NA release was increased in orchidectomized rats, the involvement of vasodilator neurotransmitters other than NA cannot be ruled out. As mentioned above, an involvement of the vasodilator neurotransmitters, NO [20,30] and calcitonin gene related peptide (CGRP) [31] in the response to EFS has been demonstrated. Since male sex hormone have been shown to modulate CGRP release [43], we first investigated the possibility of CGRP participation in the EFS-induced responses in controls and orchidectomized animals. Capsaicin, which selectively depletes CGRP in the sensory nerves [29], did not significantly affect the vasomotor response to either EFS or exoge-

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nous NA in mesenteric arteries from control and orchidectomized rats, indicating that sensory innervation does not modulate the vasomotor response to EFS in our experimental conditions, as is also true in male Wistar [20] or Sprague–Dawley [32] rat mesenteric arteries. Using the same methodology, we demonstrated a functional role for sensitive innervation in hypertension [44]. The results obtained here indicate that endogenous male sex hormones do not modify the functionality of sensory innervation. The involvement of NO in the EFS-induced response is demonstrated by the increased vasoconstrictor response to EFS in segments from both control and orchidectomized rats, preincubated with the NOS inhibitor L-NAME. The fact that the endothelium was removed and that AMT, an inhibitor of iNOS [32,45,46], did not modify the response induced by EFS in segments from both rat groups, suggests a neuronal origin for the NO release. The greater response to EFS in the presence of L-NAME in segments from orchidectomized animals compared with the response obtained in segments from control rats indicates that male sex hormone levels induce differences in neuronal synthesis, release at this point, and/or steps downstream from NO release. The reported effects of androgens on NO release are contradictory. Testosterone impairs relaxation and worsens endothelial dysfunction in male rabbits [4], although it has also been reported that testosterone or its derivatives increase eNOS [9,47] and nNOS activity in the central nervous system of guinea pig [47] and mouse [10]. Nevertheless, to our knowledge, there is no experimental evidence on the effect of male sex hormones on the nNOS expression and nNOS-derived NO release in vascular tissues. Therefore, first we analysed the expression of nNOS, and it was higher in segments from control rats than from orchidectomized rats. We also quantified the NO release induced by EFS in segments from both groups of rats by measuring nitrite production and the fluorescence emitted by DAF-2, and the results showed that the EFS-induced NO release was similar in segments from both control and orchidectomized rats. In addition, the released NO seems to come from nerve endings, since preincubation with tetrodotoxin abolished the nitrite release in segments from both groups of rats. This result indicates that male sex hormones apparently do not modulate nNOS-derived NO release. Since the response to EFS in the presence of LNAME was greater in segments from orchidectomized rats than in the controls, although nNOS-derived NO release was the same as in the control rats, the next step was to study the possible changes in the sensitivity of the smooth muscle cells to the released NO. Therefore, we analysed the vasodilator response induced by the NO donor, SNP. The response to SNP was more pronounced in segments from orchidectomized than from

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control rats, suggesting a possible different sensitivity to NO in orchidectomized and control animals. This result is in line with other reports in which testosterone impaired the relaxation induced by SNP in porcine coronary artery [48,49] and contrasts with reports in rat aorta [50], where castration did not modify the relaxation induced by SNP. Our findings indicate that male sex hormones seem to influence downstream factors in the NO signalling pathway, possibly cGMP formation and/or its vasodilator effect. The fact that the vasodilator response to the cGMP analogue 8Br-cGMP was similar in segments from both of our rat groups initially suggested that the cGMP formation in response to SNP might be increased in segments from orchidectomized rats. Therefore, we analysed the activation of guanylate cyclase by measuring the basal and SNP-stimulated cGMP formation in control and orchidectomized animals. Our results show that the basal and stimulated cGMP levels induced by SNP were unaffected by orchidectomy, as was also reported [51,52] although other reports differ [53,54]. This apparent discrepancy could be because the tissue and/or animal species used in the latter studies were different from those used in the present work, and it is also possible that the products of the NO can also activate, entire or partially, guanylate cyclase [55,56]. Therefore, our results suggest that factor(s) with a vasodilator effect, other than the NO, seem to be produced in arteries from orchidectomized male rats, and this would explain the greater effect of SNP and nNOS-derived NO release in these animals. Since the antioxidant role of androgen has been recently reported [17,18], the involvement of reactive oxygen species in vascular responses cannot be ruled out; these would alter the neuronal NO metabolism, and consequently affect vascular response to EFS and SNP. Among all the reactive oxygen species, superoxide anion plays a critical role since it is a source of many other reactive oxygen intermediates. Therefore, we first studied the possible functional role of superoxide anion in vessels from control and orchidectomized rats, by analysing the effect of the superoxide anion scavenger, SOD, on the relaxation induced by SNP. We observed that the presence of SOD did not modify the response to SNP in segments from control male rats, but the response was increased in segments from orchidectomized rats. This result seems to indicate that loss of male sex hormones leads to an increase in superoxide anion generation, and this induces a vascular effect. It is known that the interaction between NO and superoxide anion results in the formation of peroxynitrite, which has a wide range of biological activities, including biomolecule oxidation and protein tyrosine nitration [57,58], which reduce NO function. Additionally, a vasodilator action by peroxynitrite has been also reported [56,57], and we have confirmed that, in NA-precontracted arteries from control and orchidectomized animals, peroxynitrite in-

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duced a concentration-dependent relaxation. Therefore, we first focused on analysing the possible differences in superoxide anion and peroxynityrite generation in the two groups of rats. The lucigenin chemiluminescence measurement showed that the formation of superoxide anion was greater in segments from orchidectomized rats than in segments from control rats. This finding was further reinforced by the in situ detection of superoxide anion using hydroethidine fluorescence. It is important to note that in our experimental conditions-denuded arteries-, the source of superoxide anion is the smooth muscle layer and the adventitia, mostly the former, as can be observed in the confocal microscope images. This result agrees with those of previous studies showing that the NADPH (oxidase) expressed in the smooth muscle cells is a major source of superoxide anion [59,60]. Therefore, the next step was to investigate the possible differences in peroxynitrite formation in arteries from control or orchidectomized animals. Immunohistochemical localization of nitrotyrosine, a marker of peroxynitrite formation [57], showed a marked increase in arteries from orchidectomized rats compared to results in segments from control rats. The immunostaining pattern of nitrotyrosine is similar to that of superoxide anion in confocal microscopy, and superoxide anion or peroxynitrite formation predominantly takes place in the smooth muscle layer. The increased basal production of peroxynitrite in arteries from orchidectomized male rats, as a consequence of the increased superoxide anion generation, could modulate cell signalling pathways [61], and thereby might induce adaptative changes in the vasomotor response of the vessel. We also tried to quantify the production of peroxynitrite through dihydrorhodamine-123 oxidation, and we observed that, in basal conditions, peroxynitrite formation could not be detected. Since the functional studies on NO metabolism were performed analysing the vasodilator action of SNP, we quantified the formation of peroxynitrite induced by the NO donor sodium nitroprusside, and we observed that peroxynitrite formation was more pronounced in arteries from orchidectomized than from control rats, which would explain the increased relaxation to SNP in the former. It is important to note that the presence of exogenous SOD diminishes peroxynitrite formation by removing superoxide anion at the same time that it increases the formation of H2O2, and, since a vasodilator effect for H2O2 has been described in several vascular beds [62,63], it is reasonable to speculate that the increased relaxation to SNP observed in the presence of SOD could be due to H2O2 generation. We first confirmed that H2O2 induced a concentrationdependent relaxation in NA-precontracted segments from orchidectomized animals. We also found that simultaneous incubation of SOD and catalase reversed the vasodilator response to SNP, indicating that the

H2O2, synthesized in the presence of SOD, would participate in that vasodilator effect. These results seem to indicate that products derived from the NO metabolism, mainly peroxynitrite and H2O2, contribute to the relaxation induced by SNP. In addition, the fact that the sensitivity to L-NAME was increased in arteries from orchidectomized animals demonstrated the involvement of products derived from the NO metabolism in nNOSderived NO release in these animals, since inhibiting NO synthesis also prevents the formation of products generated from the NO metabolism, which mediate the vasodilator effect. In conclusion, the present study demonstrated that the loss of male sex hormones did not modify the nNOS-derived NO release induced by EFS although it did increase the NO metabolism through superoxide anion and peroxynitrite generation; however, the functional role of the nNOS-derived NO release was more pronounced in arteries from orchidectomized animals, due to products generated from the NO metabolism, such as peroxynitrite and H2O2, that seem to be able to compensate for the loss of NO bioavailability, probably through their direct vasodilator effect.

Acknowledgments This research was supported by the Fondo de Investigaciones Sanitarias (FIS, PI020335) and the Direccio´n General de Ciencia y Tecnologı´a (DGCYT, BFI20011324). We thank the veterinarian Dr. Ma del Carmen Ferna´ndez Criado for the care of animals, Dr. Ana Guadan˜o for assistance in processing the immunohistochemistry samples, Ms Rocio Baena for her technical help, and Ms Esther Martı´nez for typing the manuscript.

References [1] C. Isles, Blood pressure in males and females, J. Hypertens 13 (1995) 285–290. [2] J.K. Williams, M.R. Adams, H.S. Klopfenstein, Estrogen modulates responses of atherosclerotic coronary arteries, Circulation 81 (1990) 1680–1687. [3] T.B. Clarkson, Effects of estrogens, progestins, and androgens on coronary vasomotion and atherosclerosis, J. Reprod. Med. 43 (1998) 741–745. [4] S.J. Hutchison, K. Sudhir, T.M. Chou, et al., Testosterone worsens endothelial dysfunction associated with hypercholesterolemia and environmental tobacco smoke exposure in male rabbit aorta, J. Am. Coll. Cardiol. 29 (1997) 800–807. [5] P. Alexandersen, J. Haarbo, I. Byrjalsen, H. Lawaetz, C. Christiansen, Natural androgens inhibit male atherosclerosis a study in castrated, cholesterol-fed rabbits, Circ. Res. 84 (1999) 813–819. [6] W. Weidemann, H. Hanke, Cardiovascular effects of androgens, Cardiovasc. Drug Rev. 20 (2002) 175–198. [7] J. Marı´n, M.A. Rodrı´guez-Martı´nez, Role of vascular nitric oxide in physiological and pathological conditions, Pharmacol. Ther. 75 (1997) 111–114.

M.C. Martı´n et al. / Nitric Oxide 12 (2005) 163–176 [8] S. Moncada, R.M.J. Palmer, E.A. Higgs, Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev. 43 (1997) 109–142. [9] D. Liu, J.S. Dillon, Dehydroepiandrosterone activates endothelial cell nitric oxide synthase by a specific plasma membrane receptor coupled to G alpha (i2,3), J. Biol. Chem. 277 (2002) 21379–21388. [10] E.M. Scordalakes, D.B. Imwalle, E.F. Rissman, Oestrogen
s masculine side: mediation of mating in male mice, Reproduction 124 (2002) 331–338. [11] S.W. Barger, J.A. Chavis, P.D. Drew, Dehydroepiandrosterone inhibits microglial nitric oxide production in a stimulus-specific manner, J. Neurosci. Res. 62 (2000) 503–509. [12] D.G. Harrison, Endothelial dysfunction in atherosclerosis, Basic Res. Cardiol. 89 (1994) 87–102. [13] T. Munzel, T. Heitzer, D.G. Harrison, The physiology and pathophysiology of the nitric oxide/superoxide system, Herz 22 (1997) 22–158. [14] R.F. Brandes, A. Mu¨gge, Gender differences in the generation of superoxide anions in the rat aorta, Life Sci. 60 (1997) 391–396. [15] S. Ayres, W. Abplanalp, J.H. Liu, M.T.R. Subbiah, Mechanisms involved in the protective effect of estradiol-17b on lipid peroxidation and DNA damage, Am. J. Physiol. 274 (1998) E1002–E1008. [16] K. Ghanam, J. Javellaud, L. Ea-Kim, N. Oudar, The protective effect of 17 b-estradiol on vasomotor responses of aorta from cholesterol-fed rabbit is reduced by inhibitors of superoxide dismutase and catalase, Biochem. Biophys. Res. Commun. 249 (1998) 858–864. [17] G. Be´ke´si, R. Kakucs, S. Va´rbı´ro´, et al., In vitro effects of different steroid hormones on superoxide anion production of human neutrophil granulocytes, Steroids 65 (2000) 889–894. [18] M.A. Yorek, L.J. Coppey, J.S. Gellett, et al., Effect of treatment of diabetic rats with dehydroepiandrosterone on vascular and neural function, Am. J. Physiol. Endocrinol. Metab. 283 (2002) E1067–E1075. [19] X.J. Yu, Y.J. Li, H.W. Deng, The regulatory effect of bradykinin on the actions of sensory nerves in the perfused rat mesentery is mediated by nitric oxide, Eur. J. Pharmacol. 241 (1993) 35–40. [20] J. Marı´n, G. Balfago´n, Effect of clenbuterol on non-endothelial nitric oxide release in rat mesenteric arteries and the involvement of b-adrenoceptors, Br. J. Pharmacol. 124 (1998) 124–473. [21] N. Minoves, G. Balfago´n, M. Ferrer, Role of female sex hormones in neuronal nitric oxide release and metabolism in rat mesenteric arteries, Clin. Sci. 103 (2002) 239–247. [22] K.C. Nielsen, C. Owman, Contractile response and amine receptor mechanism in isolated middle cerebral artery of the cat, Brain Res. 27 (1971) 25–32. [23] J.P. Griess, Bemerjungen zu der abhandlung der HH. Weselsky und Benedikt ‘‘Ueber einigre Azoverdindungen’’, Ber. Dtsch. Chem. Ges. 12 (1979) 426–428. [24] P.J. Pagano, Y. Ito, K. Tornheim, et al., An NADPH oxidase superoxide-generating system in the rabbit aorta, Am. J. Physiol. 268 (1995) H2274–H2280. [25] H.D. Wang, S. Hope, Y. Du, et al., Paracrine role of adventitial superoxide anion in mediating spontaneous tone of the isolated rat aorta in angiotensin II-induced hypertension, Hypertension 33 (1999) 1225–1232. [26] L. Li, E. Crockett, D.H. Wang, et al., Gene transfer of endothelial NO synthase and manganese superoxide dismutase on arterial vascular cell adhesion molecule-1 expression and superoxide production in deoxycorticosterone acetate-salt hypertension, Arterioscler. Thromb. Vasc. Biol. 22 (2002) 249–255. [27] T.J. Bivalacqua, J.S. Armstrong, J. Biggerstaff, et al., Gene transfer of extracellular SOD to the penis reduced O2  and improves erectile function in aged rats, Am. J. Physiol. Heart Circ. Physiol. 284 (2003) H1408–H1421. [28] S. Pfeiffer, A. Lass, K. Schmidt, B. Mayer, Protein tyrosine in mouse peritoneal macrophages activated in vitro and in vivo:

175

evidence against an essential role of peroxynitrite, FASEB J. 15 (2001) 2355–2364. [29] Y.J. Li, S.P. Duckles, Effect of endothelium on the actions of sympathetic and sensory nerves in the perfused rat mesentery, Eur. J. Pharmacol. 210 (1992) 23–40. [30] H. Kawasaki, K. Takasaki, A. Saito, K. Goto, Calcitonin generelated peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat, Nature 335 (1988) 164–167. [31] G.E. Boeckxstaens, P.A. Pelckmans, Nitric oxide and the nonadrenergic non-cholinergic neurotransmission, Comp. Biochem. Physiol. 118A (1997) 925–937. [32] M. Ferrer, J. Marı´n, G. Balfago´n, Diabetes alters neuronal nitric oxide release from rat mesenteric arteries. Role of protein kinase C, Life Sci. 66 (2000) 337–345. [33] M. Ferrer, M.J. Alonso, M. Salaices, J. Marı´n, G. Balfago´n, Angiotensin II increases neurogenic nitric oxide metabolism in mesenteric arteries from hypertensive rats, Life Sci. 68 (2001) 1169–1179. [34] S. Greenberg, W.R. George, P.J. Kadowitz, W.R. Wilson, Androgen-induced enhancement of vascular reactivity, Can J. Physiol. Pharmacol. 52 (1974) 14–22. [35] P.J. Baker, E.R. Ramey, P.W. Ramwell, Androgen-mediated sex differences of cardiovascular responses in rats, Am. J. Physiol. 235 (1978) H242–H246. [36] V. Calderone, B. Baragatti, M.C. Breschi, P. Nieri, E. Martinotti, Hormonal influence on the release of endothelial nitric oxide: gender related dimorphic sensitivity of rat aorta for noradrenaline, J. Pharm. Pharmacol. 54 (2002) 523–528. [37] X.B. Guan, D. Dluzen, Castration reduces potassium-stimulated norepinephrine release from superfused olfactory bulbs of male rats, Brain Res. 568 (1991) 147–151. [38] F. Holmquist, K. Persson, A. Bodker, K.E. Anderson, Some pre- and postjunctional effects of castration in rabbit isolated corpus cavernosum and urethra, J. Urol. 152 (1994) 1011– 1016. [39] A. Siddiqui, B.H. Shah, Neonatal androgen manipulation differentially affects the development of monoamine systems in rat cerebral cortex, amygdale and hypothalamus, Dev. Brain Res. 98 (1997) 247–252. [40] Y. Shang, D.E. Dluzen, Castration increases nisoxetine-evoked norepinephrine levels in vivo within the olfactory bulb of male rats, Neurosci. Lett. 328 (2002) 81–84. [41] M.C. Agostini, E.S. Borda, M.F. Gimeno, A.L. Gimeno, Differences in the effects of acetylcholine on the vas deferens from normal and castrated rats. A participation of adrenergic mechanisms, Arch. Int. Pharmacodyn. Ther. 250 (1981) 212– 220. [42] D.C. Chen, S.P. Duckles, D.N. Krause, Postjunctional a2adrenoceptors in the rat tail artery: effect of sex and castration, Eur. J. Pharmacol. 372 (1999) 247–252. [43] C. Sun, M. Chen, J. Mao, X. Wang, Biphasic effects of orchidectomy on calcitonin gene-related peptide synthesis and release, Neuroreport 12 (2001) 3497–3502. [44] J. Marı´n, M. Ferrer, G. Balfago´n, Role of protein kinase C in electrical-stimulation-induced neuronal nitric oxide release in mesenteric arteries from hypertensive rats, Clin. Sci. 99 (2000) 277–283. [45] W.R. Tracey, M. Nakane, F. Basha, G. Carter, In vivo pharmacological evaluation of two novel type II (inducible) nitric oxide synthase inhibitors, Can. J. Physiol. Pharmacol. 73 (1995) 665– 669. [46] M. Ishikawa, R.M. Quock, Role of nitric oxide synthase isoforms in nitrous oxide antinociception in mice, J. Pharmacol. Exp. Ther. 306 (2003) 484–489. [47] C.P. Weiner, I. Lizasoain, S.A. Baylis, R.G. Knowles, I.G. Charles, Induction of calcium-dependent nitric oxide synthases by sex hormones, Proc. Natl. Acad. Sci. USA 91 (1994) 5212– 5216.

176

M.C. Martı´n et al. / Nitric Oxide 12 (2005) 163–176

[48] A. Quan, H. Teoh, R.Y. Man, Acute exposure to a low level of testosterone impairs relaxation in porcine coronary arteries, Clin. Exp. Pharmacol. Physiol. 26 (1999) 830–832. [49] H. Teoh, A. Quan, R.Y. Man, Acute impairment of relaxation by low levels of testosterone in porcine coronary arteries, Cardiovasc. Res. 45 (2000) 1010–1018. [50] M. Ferrer, N. Tejera, J. Marı´n, G. Balfago´n, Androgen deprivation facilitares acetylcholine-induced relaxation by superoxide anion generation, Clin. Sci. 97 (1999) 625–631. [51] K. Kanda, K. Ogawa, N. Miyamoto, T. Hatano, H. Seo, N. Matsui, Potentiation of atrial natriuretic peptide-stimulated cyclic guanosine monophosphate formation by glucocorticoids in cultured rat renal cells, Br. J. Pharmacol. 96 (1989) 795–800. [52] A.M. Traish, K. Park, V. Dhir, N.N. Kim, R.B. Moreland, I. Goldstein, Effects of castration and androgen replacement on erectile function in a rabbit model, Endocrinology 140 (1999) 1861–1868. [53] O.R. Hayek, A. Shabsigh, S.A. Kaplan, A.J. Kiss, M.W. Chen, T. Burchardt, M. Burchadt, C.A. Olsson, R. Buttyan, Castration induces acute vasoconstriction of blood vessels in the rat prostate concomitant with a reduction of prostatic nitric oxide synthase activity, J. Urol. 162 (1999) 1527–1531. [54] T. Hayashi, T. Esaki, E. Muto, H. Kano, Y. Asai, N.K. Thakur, D. Sumi, M. Jayachandran, A. Iguchi, Dehydroepiandrosterone retards atherosclerosis formation trough its conversion to estrogen: the possible role of nitric oxide, Arterioscler. Thromb. Vasc. Biol. 20 (2000) 782–792.

[55] A. Sato, I. Sakuma, D.D. Gutterman, Mechanism of dilation to reactive oxygen species in human coronary arterioles, Am. J. Physiol. 285 (2003) H2345–H2354. [56] J. Li, W. Li, B.T. Altura, B.M. Altura, Peroxynitrite-induced relaxation in isolated canine cerebral arteries and mechanisms of action, Toxicol. Appl. Pharmacol. 196 (2004) 176–182. [57] J.S. Beckman, W.H. Koppenol, Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly, Am. J. Physiol. 271 (1996) C1424–C1437. [58] U. Hink, M. Oelze, P. Kolb, et al., Role for peroxynitrite in the inhibition of prostacyclin synthase in nitrate tolerance, J. Am. Coll. Cardiol. 42 (2003) 1826–1834. [59] B. Lassegue, D. Sorescu, K. Szocs, et al., Novel gp91(phox) homologues in vascular smooth muscle cells: nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signalling pathways, Circ. Res. 88 (2001) 888–894. [60] D. Sorescu, M.J. Somers, B. Lassegue, et al., Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells, Free Radic. Biol. Med. 30 (2001) 603–612. [61] R.A. Oeckler, M.S. Wolin, New concepts in vascular nitric oxide signalling, Curr. Atheroscler. Rep. 2 (2000) 437–444. [62] G.M. Rubanyi, P.M. Vanhoutte, Oxygen-derived free radicals, endothelium, and responsiveness of vascular smooth muscle, Am. J. Physiol. 250 (1986) H815–H821. [63] E.P. Wei, H.A. Kontos, J.S. Beckman, Mechanisms of cerebral vasodilation by superoxide, hydrogen peroxide, and peroxynitrite, Am. J. Physiol. 271 (1996) H1262–H1266.