Tibolone Has Anti-Inflammatory Effects in Estrogen-Deficient

Regulatory Peptides 179 (2012) 55–60

Contents lists available at SciVerse ScienceDirect

Regulatory Peptides

journal homepage: www.elsevier.com/locate/regpep

Tibolone has anti-inﬂammatory effects in estrogen-deﬁcient female rats on the natriuretic peptide system and TNF-alpha

Ana Raquel Santos de Medeiros a,b, Aline Zandonadi Lamas b, Izabela Facco Caliman b, Polyana L. Meireles Dalpiaz b, Luciana Barbosa Firmes c, Gláucia Rodrigues de Abreu b, Margareth Ribeiro Moysés b, Elenice Moreira Lemos d, Adelina Martha dos Reis c, Nazaré Souza Bissoli b,⁎ a Biological and Health Sciences, Federal Institute of Espírito Santo, Vila Velha, ES, Brazil b Department of Physiological Sciences, Federal University of Espírito Santo, Vitória, ES, Brazil c Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil d Center of Infectious Diseases, Federal University of Espírito Santo, Vitória, ES, Brazil article i nfo abstract

Article history: Cardiovascular and immune system abnormalities have been reported in females with estrogen deficiency. Received 25 April 2012 To control these disorders in post-menopausal women, hormone replacement therapy (HRT) has been Received in revised form 10 August 2012 used. Tibolone has been used as a HRT, but the effects of tibolone on the natriuretic peptide system have Accepted 29 August 2012 not been determined. We investigated the effects of tibolone on the natriuretic peptide system and Available online 10 September 2012 pro-inflammatory cytokines in ovariectomized (OVX) rats. Female rats were divided into four groups: SHAM, OVX, OVX treated with 17β-estradiol (OVX+E: 14 days) and OVX treated with tibolone (OVX+T: Keywords: Ovariectomy 14 days) beginning 21 days after ovariectomy. On day 35, blood was collected to determine atrial natriuretic Tibolone peptide (ANP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels. In addition, tissues were Estrogen collected for determining ANP, natriuretic peptide receptor type-A (NPR-A), and NPR type-C (NPR-C) gene System of natriuretic peptides expression levels by RT-PCR. The cytokine levels of both IL-6 and TNF-α were increased in OVX animals. In Cytokines comparison, IL-6 and TNF-α levels were reduced in OVX+E animals. TNF-α levels were reduced similarly Hormone replacement therapy in OVX+T animals, but IL-6 levels remained elevated in this group. The concentrations of ANP in the left atrium tissue and plasma were decreased after ovariectomy, as were ANP mRNA levels in the left atrium and NPR-A mRNA levels in kidney. No variation in NPR-C gene expression in the kidney tissue was observed among the groups. Tibolone and 17β-estradiol effectively increased plasma ANP and ANP mRNA levels in the left atrium, but did not normalize renal NPR-A levels. Since HRT with tibolone normalizes plasma ANP and serum TNF-alpha levels our results suggest that treatment with tibolone has anti-inflammatory effects and could prevent cardiovascular disease in the long-term. © 2012 Elsevier B.V. Open access under the Elsevier OA license.

1. Introduction pro-inflammatory cytokines such as IL-6 and TNF-α [11–14].Thus,the impairment of the immune system and the cardiovascular system ob- Atrial natriuretic peptide (ANP) plays a vital role in regulating served with estradiol deficiency may be associated. cardiovascular function, maintaining blood pressure and body Indeed, recent studies have begun to elucidate the influence of ANP fluid homeostasis [1]. In addition, inappropriate activation of pro- on the immune system [15–17]. In mice lacking natriuretic peptide inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) receptor type A (NPR-A), enhanced activation of pro-inflammatory and interleukin-6 (IL-6) has been recognized as an important mediator cytokines is detected [18]. NPR-A has guanylate cyclase activity and me- in the development of endothelial dysfunction, cardiac hypertrophy diates the majority of the biological effects of ANP [19]. In addition, ANP and heart failure in experimental animal models and humans [2–4]. binds natriuretic peptide receptor type-C (NPR-C), which does not have Evidence from human and animal studies shows that a decrease of estro- guanylate cyclase activity, but removes natriuretic peptides from circu- gen levels is accompanied by inhibition of ANP gene expression [5–7],di- lation [20] and is abundantly expressed in the kidney [21].Further minished circulating levels of ANP [6–10], and increased production of supporting a link between ANP and the immune system, ANP and its receptors are expressed by, and regulated by, immune cells [16]. Few studies have investigated the effects of hormone replacement ⁎ Corresponding author at: Departamento de Ciências Fisiológicas, Centro de Ciências therapy (HRT) with estrogen on ANP and, to our knowledge, there are da Saúde, Universidade Federal do Espírito Santo, Av. Marechal Campos 1468, Vitória, ES, 29040‐090, Brazil. Tel.: +55 27 3335 7333; fax: +55 27 3335 7340. no data available concerning the effect of tibolone on the natriuretic E-mail address: [email protected] (N.S. Bissoli). peptide system. Tibolone, which has metabolites with estrogenic

0167-0115 © 2012 Elsevier B.V. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.regpep.2012.08.015 56 A.R.S. Medeiros et al. / Regulatory Peptides 179 (2012) 55–60 effects (3alpha-hydroxy tibolone [3α-OH] and 3beta-hydroxy tibolone and serum were stored at −80 °C. The uterus, atrium, ventricle and kid- [3β-OH]) and progestogenic and/or androgenic effects (tibolone and neys were removed, weighed, frozen in liquid nitrogen and stored at − Delta(4)tibolone), may be used as an alternative to classical HRT with 80 °C. estrogen and progesterone [22–24]. In order to further elucidate the effects of the tibolone, we examined how tibolone treatment affects the 2.4. Levels of cytokines and ANP natriuretic peptide system in rats. We hypothesized that tibolone treatment would alter ANP synthesis, secretion, and bioavailability in circu- The levels of IL-6 and TNF-α in serum were determined using lation and that a tibolone treatment that has the aforementioned effects commercial enzyme-linked immunosorbent assay (ELISA) kits on the natriuretic peptide system may also affect levels of inflammatory according to the manufacturer's instructions (Invitrogen-BioSource cytokines, such as TNF-α and IL-6, in ovariectomized rats. International). Accordingly, this study was designed to evaluate whether tibolone The level of ANP was determined by performing a double- treatment can normalize the natriuretic peptide system and promote antibody radioimmunoassay (RIA) as described by Gutkowska et al. a reduction in the levels of TNF-α and IL-6 in ovariectomized rats. [25]. The plasma was thawed and centrifuged for 5 min at 19,400×g and 4 °C, and ANP was extracted using Sep-Pak C18 columns (Waters 2. Materials and methods Associates, Milford, MA, USA). The columns were activated with 8 ml of acetonitrile and washed with 8 ml of 0.2% ammonium acetate, pH 4.0. 2.1. Animals Afterward, 1 ml of plasma was infused into the column followed by 5 ml of 0.2% ammonium acetate. Finally, the absorbed ANP was eluted Adult female Wistar rats weighing 160–180 g were obtained from with 3 ml of 60% acetonitrile in 0.2% ammonium acetate, evaporated the breeding unit at the Federal University of Espírito Santo. The rats (Speed-Vac, Eppendorf, Hamburg, Germany) and stored at −20 °C for were housed with controlled temperature (22 °C), humidity (40%) quantification by RIA. and light cycles (12-h light/dark), and they were given water and To measure the ANP concentrations in the atrial tissue, each half of standard rat chow (Purina Labina, SP-Brazil) ad libitum. The proce- the right (RA) and left atria (LA) were thawed and placed in a tube dures were carried out in compliance with the guidelines for the eth- filled with 0.1 M acetic acid and protease inhibitors (10−5 M EDTA, ical use of animals in scientific research as stated by the Federation of 10−5 M PMSF and 0.5×10−5 M pepstatin A, all purchased from the Brazilian Society of Experimental Biology and were approved by Sigma). The samples were then homogenized and centrifuged at the Ethics Committee for Animal Use at the Federal University of 20,000×g for 30 min at 4 °C, and the supernatant was diluted (final Espírito Santo (014/2008). Ketamine (70 mg/kg) and xylazine dilution: 1:2000) in phosphate buffer (0.01 mol/l sodium phosphate, (10 mg/kg) were injected intraperitoneally to anesthetize the rats 0.14 mmol/l bovine serum albumin, 0.1% Triton X-100, 0.1 mol/l NaCl prior to all surgical procedures. and 0.01% sodium azide at pH 7.4). The ANP levels were measured by Four groups of female rats were studied: sham-operated females RIA as previously described by Gutkowska et al. [25] using a specific (SHAM); ovariectomized (OVX); OVX treated with 17β-estradiol antibody that was donated by Jolanta Gutkowska. All of the samples (OVX+E: 0.5 μg/kg/day; Sigma Chemical Co., St. Louis, MO, USA) or were measured in the same assay, and the intra-assay coefficient of OVX treated with tibolone (OVX+T: 1.5 mg/kg/day). Tibolone was variation was b10%. The protein content of the tissue was determined given by gavage, and 17β-estradiol was injected subcutaneously. using the Bradford method [26]. The rats were treated for 14 days beginning 21 days after bilateral ovariectomy. 2.5. Gene expression of ANP, NPR-A and NPR-C

2.2. Measurement of arterial blood pressure (BP) and heart rate (HR) Total mRNA was extracted from the atria and kidneys by a guani- dine isothiocyanate method previously described by Chomczynski Mean arterial pressure and heart rate were determined by direct and Sacchi [27]. The mRNA expression levels of ANP and its receptors measurement in each group at the end of treatment. Forty-eight (NPR-A and NPR-C) were determined by real-time PCR. The gene ex- hours after the end of the treatment, BP and HR at rest were mea- pression of ANP was evaluated in the RA and LA, and NPR-A and sured. For this procedure, on the day before the measurement was NPR-C expression levels were determined in the right kidney. cDNA taken, a catheter filled with saline (PE-50) was inserted into the left was synthesized by the reverse transcription of mRNA. For this pro- femoral artery while the subject was under anesthesia (ketamine cess, 1 μl of mRNA from each sample was mixed in plastic tubes 70 mg/kg, xylazine 10 mg/kg). The free end of the catheter was exte- with a solution containing the following compounds: diethyl riorized at the cervical dorsal area. For the BP measurement, the arte- pyrocarbonate water (DEPC), the reverse primer of the target gene rial catheter was attached to a 40-cm polyethylene catheter during (ANP, NPR-A or NPR-C) or the reverse primer of the housekeeping the 40 min recording period. BP measurements were obtained in gene (ribosomal subunit s26), oligo dT, triphosphate deoxyribonucle- quiet, conscious rats that had complete freedom to move throughout otide (dNTP), dithiothreitol (DTT), specific buffer (10×) and a solu- the cage. The BP was recorded by a pressure transducer coupled to a tion containing the Moloney murine leukemia virus (MMLV) MP-100 System Guide (model MP100-CE; Biopac Systems, Santa reverse transcriptase enzyme, according to the manufacturer's guide- Barbara, CA, USA). The HR was calculated instantaneously from the lines (Invitrogen, CA, USA). After this process, the plastic tubes were intervals of pressure pulses. heated to 40 °C for 60 min. After reaching room temperature, the tubes were stored at −20 °C. 2.3. Collection of serum, plasma and tissues For the atria, the samples were subjected to DNAse treatment prior to reverse transcription. The DNase treatment was performed After measuring BP and HR, the females were decapitated, and 5 ml by mixing 0.5 μg of total mRNA from the atria with 4 μl of water of blood was collected in pre-chilled tubes containing heparin sulfate and 1 μl of buffer containing DNAse (1:1). This mixture was incubat- and protease inhibitors: 10−5 mol/l ethylenediaminetetraacetic acid ed for 15 min at room temperature. After the incubation period, EDTA (EDTA), 10−5 mol/l phenylmethylsulfonyl fluoride (PMSF), and was added to stop the reaction. The samples were then heated to 0.5×10−5 mol/l pepstatin A (all purchased from Sigma, St. Louis, MO, 65 °C for 10 min. USA), to obtain plasma. Blood samples were also collected in empty After synthesizing the cDNA, PCR was performed to amplify the tubes to obtain serum. The blood samples were centrifuged at 4 °C cDNA for ANP, NPR-A and NPR-C, using specific primers (ANP: and 2500 rpm (Eppendorf, Hamburg, Germany) for 15 min. Plasma 5′-GGA TTT CAA GAA CCT GCT AGA-3′ and 5′-CTT CAT CGG TCT GCT A.R.S. Medeiros et al. / Regulatory Peptides 179 (2012) 55–60 57

Table 1 Body and organ weights and organ weight/body weight ratio in the sham-operated (SHAM), ovariectomized (OVX), and OVX rats treated with 17β-estradiol (OVX+E) or tibolone (OVX+T).

Variable SHAM OVX OVX+E OVX+T ⁎⁎ †† †† Body weight, g 220±5 249±6 217±8 214±4 ⁎⁎ †† ⁎⁎†† Uterus, mg 428.3±38.9 88.4±3.6 427.5±68.2 299.8±15.9 ⁎⁎ †† ⁎†† Uterus/body weight, mg/g 1.9±0.2 0.4±0.1 2.1±0.1 1.5±0.1 Right atrium, mg 16.1±1.3 18.8±1.8 14.1±1.2 16.6±1.7 Right atrium/body weight, mg/g 0.072±0.010 0.076±0.009 0.065±0.012 0.077±0.010 Left atrium, mg 25.1±2.2 29.1±2.6 19.9±1.1 22.0±2.0 Left atrium/body weight, mg/g 0.11±0.01 0.12±0.02 0.09±0.02 0.10±0.01 Right ventricle, mg 134.8±9.9 150.8±9.4 146.0±9.8 124.6±9.9 Right ventricle/body weight, mg/g 0.61±0.03 0.61±0.03 0.67±0.04 0.58±0.03 Left ventricle, mg 490.4±33.5 495.2±26.9 430.6±33.0 455.7±26.9 Left ventricle/body weight, mg/g 2.23±0.07 2.10±0.06 2.01±0.04 2.13±0.05

Data are expressed as mean±SEM (n=8 animals per group). ⁎⁎ Pb0.01. ⁎ Pb0.05 vs. SHAM. †† Pb0.01 vs. OVX.

CGC TCA-3′; NPR-A: 5′-ATC ACA GTG AAT CAC CAG GAG TTC-3′ and 3.2. Levels of ANP and NPR in response to ovariectomy and 17β-estradiol 5′-AGA TGT AGA TAA CTC TGC CCT TTC G-3′; NPR-C: 5′-CCT ACA or tibolone treatment ATT TCG ACG AGA CCA AA-3′ and 5′-ACT CGC TCA CTG CCC TGG ATG TA-3′). In the vehicle-treated OVX rats, the plasma ANP concentrations For this procedure, 2 μl of cDNA was added to wells of real-time (92.50±19.50 pg/ml) were lower than in the SHAM group PCR plates, followed by 1.5 μl of sense primer (1 pmol/μl), 1.5 μlof (263.30±53.30 pg/ml, Pb0.05). Treatment with 17β-estradiol or anti-sense primer (1 pmol/μl), 10 μl of Power SYBR Green PCR Master tibolone increased plasma levels of ANP in OVX rats (OVX+E: Mix (Applied Biosystems, Warrington, UK) and 5 μl of DEPC water. 247.30±29.90; OVX+T: 285.70±60.40 pg/ml, Pb0.05 vs. OVX) Afterward, the plates were sealed and ampliﬁed in the thermocycler (Fig. 1). (ABI Prism 7000 SDS; Applied Biosystems, Warrington, UK) using RIA revealed that ovariectomy and estradiol treatment decreased the following cycles: [stage 1], a cycle of 52 °C/2 min; [stage 2], a and increased ANP concentrations, respectively, in the left atrium cycle of 95 °C/10 min; [stage 3], 40 cycles of 95 °C/0.15 min and but not the right atrium (Fig. 2A). The ANP concentration in the left 50 °C/1 min. atrium decreased from 5.68±0.35 μg/mg protein in the SHAM group to 3.48±0.40 μg/mg protein in the OVX group (Pb0.05). How- ever, in the OVX+E group, the ANP levels in the left atrium increased 2.6. Data analysis to 6.48±0.97 mg/mg protein (Pb0.05), which was similar to the levels observed in the SHAM group. On the other hand, the ANP levels Data are expressed as the mean±standard error of mean (SEM), in the left atrium did not return to levels similar to the SHAM group in and the statistical analysis was performed using GraphPad Prism 5. the OVX+T group (3.59±0.25 mg/mg protein, Pb0.05 vs. SHAM). As Data were compared using one-way ANOVA followed by Newman– shown in Fig. 2B, changes in the ANP mRNA levels were associated Keuls post hoc test. Data were considered statistically signiﬁcant if with increased plasma ANP levels. The OVX group had lower ANP Pb0.05. mRNA levels in the left atrium (40.00±10.10 AU) compared with the SHAM group (91.30±4.40) indicating that lower levels of ANP 3. Results synthesis were occurring in the OVX group. 17β-estradiol or tibolone treatment induced a large increase in the ANP gene expression in the 3.1. The effects of OVX and tibolone on physiological parameters, blood left atrium of ovariectomized rats (OVX+E: 122.30±12.00; OVX+T: pressure and heart rate 167.90±25.70 AU, Pb0.01 vs. OVX).

Baseline physiological parameters in SHAM and OVX animals and the effects of HRT are reported in Table 1. Ovariectomy led to increased body weight (BW); however, the treatment with 17β-estradiol (OVX+E) or tibolone (OVX+T) normalized BW (Pb0.01). The uterus weight as well as the uterus weight-to-BW ratio were largely decreased in the OVX groups (Pb0.01) indicating the efﬁciency of ovariectomy. However, in the OVX+T group, there was an increase in the uterus weight-to-BW ratio compared to the OVX group, but did not return to normal when compared to the SHAM group. This ﬁnding is different from what occur in the OVX+E group when the uterus weight-to-BW ratio returned to normal values. Ovariectomy or any of the HRT used in the present study did not alter the weight-to-BW ratio of the heart chambers (Table 1). Mean arterial pressure (MAP) and heart rate (HR) were not different in the SHAM and OVX groups (105±2 vs. 98±1 mmHg and 373±7 vs. 372±6 beats per min, respectively). In addition, administration of Fig 1. The effects of ovariectomy and 17β-estradiol or tibolone treatment on the level 17β-estradiol or tibolone, respectively, to ovariectomized rats did not of ANP in the plasma. Shown are the ANP levels from sham-operated (SHAM), ovariec- change MAP (101±2 and 103±3 mmHg) or HR (368±5 and 364± tomized (OVX), and ovariectomized rats treated with 17β-estradiol (OVX+E) or 9 beats per min). tibolone (OVX+T). n=7 animals per group. *Pb0.05 vs. SHAM; †Pb0.05 vs. OVX. 58 A.R.S. Medeiros et al. / Regulatory Peptides 179 (2012) 55–60

Changes in ANP receptor expression in the kidney tissues were compared using RT-PCR. The kidneys from the ovariectomized rats had lower NPR-A mRNA expression (0.68±0.11 AU) compared to the SHAM group (12.00±5.20 AU). On the other hand, neither 17β-estradiol nor tibolone treatment normalized the expression of NPR-A in the ovariectomized females (OVX+E: 0.92±0.28 and OVX+T: 2.04±1.04 AU) (Fig. 3A). No variation in NPR-C gene expression in the kidney tissue was observed among the groups (Fig. 3B).

3.3. Pro-inﬂammatory cytokine levels in response to ovariectomy and 17β-Estradiol or tibolone treatment

Fig. 4 shows the average level of IL-6 (A) and TNF-α (B) in the serum of rats from every group. Ovariectomy resulted in a signiﬁcant elevation of IL-6 and TNF-α levels (IL-6: 32.80±5.00 and TNF-α: 31.00±5.00 pg/ml) compared with the SHAM group (IL-6: 19.30± 6.00 and TNF-α: 20.30±2.00 pg/ml). Treatment with 17β-estradiol decreased the IL-6 and TNF-α levels in the serum of the ovariectomized rats (IL-6: 20.00±3.00 and TNF-α: 17.60±2.00 pg/ml, Pb0.01 vs. SHAM). However, tibolone treatment in the ovariectomized rats (OVX+T) decreased TNF-α levels but not IL-6 levels (34.40±2.80) in the serum compared to the OVX group (17.60± 2.00 pg/ml, Pb0.01 vs. OVX).

4. Discussion

Fig. 2. (A) Effect of 17β-estradiol (OVX+E) or tibolone (OVX+T) treatment on ANP Tibolone may be useful as an alternative to classical HRT with es- concentration in the right (RA) and left (LA) atria. (B) ANP gene expression in the atri- trogen and progesterone. However, the effects of tibolone on the na- um. The expression of ANP was examined at the mRNA level in both the right and left triuretic peptide system have not been elucidated. The present study β atria following 17 -estradiol or tibolone treatment for 14 days. n=7 animals per demonstrated that decreased ANP levels in estrogen-deﬁcient female group. **Pb0.01 and *Pb0.05 vs. SHAM; ††Pb0.01 vs. OVX.

Fig. 4. The effects of rat ovariectomy and 17β-estradiol or tibolone treatment on serum Fig. 3. Renal mRNA expression of NPR. mRNA expression of NPR-A (A) and NPR-C (B) levels of IL-6 (A) and TNF-α (B). The results are presented from samples obtained from normalized by s26 mRNA levels (arbitrary units) in the kidneys after 17β-estradiol sham-operated (SHAM), ovariectomized (OVX), and ovariectomized rats treated with (OVX+E) or tibolone (OVX+T) treatment for 14 days. n=7 animals per group. 17β-estradiol (OVX+E) or tibolone (OVX+T). n=7 animals per group. **Pb0.01 vs. **Pb0.01 vs. SHAM. SHAM; ††Pb0.01 vs. OVX. A.R.S. Medeiros et al. / Regulatory Peptides 179 (2012) 55–60 59 rats were accompanied by an increase in circulating levels of IL-6 and Delta(4)tibolone isomer, possess different affinities. The hydroxytibolone TNF-α. Plasma ANP levels were increased and serum TNF-α levels metabolites bind to estrogen receptors, triggering estrogen-like re- were decreased in tibolone treated OVX rats. sponses, whereas the Delta(4)tibolone isomer has a high affinity for pro- Although ANP is a hormone that promotes acute vasodilation, na- gesterone and androgen receptors (22). Therefore, relative to estrogen triuresis, and diuresis, with a consequent reduction in blood pressure which binds selectively to estrogen receptors, tibolone may produce [28], the reduction in plasma levels of ANP in OVX females in our more complex effects via the actions of at least one of its metabolites study did not lead to hemodynamic changes 35 d after estrogen dep- on these other receptors. rivation. Previous studies have shown that, in both women and ani- In conclusion, the present study demonstrated that estrogen defi- mals, HRT with estradiol caused plasma ANP levels to rise while BP ciency in OVX rats impaired the natriuretic peptide system, which values remained unchanged [6,9,10,29]. The role of ANP is more evi- may have contributed to the elevated concentrations of inflammatory dent when there is hypertension. HRT with 17β-estradiol has been cytokines observed in this experimental model. We showed for the reported to reduce BP in spontaneously hypertensive rats, with a con- first time the influence of tibolone on the natriuretic peptide system comitant increase in the synthesis and release of ANP, suggesting that in OVX rats. Our experiments demonstrated that tibolone possesses the natriuretic peptide system is modulated by 17β-estradiol [10]. anti-inflammatory actions in vivo, as evidenced by increased ANP In the current study, ovariectomy led to a reduction in plasma ANP levels and decreased TNF-α levels. These findings may have impor- concentrations and renal NPR-A mRNA levels, without affecting the tant implications for the treatment of post-menopausal cardiovascu- expression of NPR-C. The lower level of plasma ANP observed in lar and immunological complications. OVX rats in the absence of a change in NPR-C clearance receptors sug- gests that ANP synthesis was impaired in the OVX group. Tibolone Acknowledgments treatment induced a large increase in ANP gene expression in the left atrium as well as a large increase in plasma ANP levels in OVX This work was supported by Conselho Nacional de Desenvolvimento rats. However, the ANP levels in the left atrium were not normalized Cientifico e Tecnológico-Casadinho, Coordenação de Aperfeiçoamento by the tibolone treatment. Given that androgens have previously de Pessoal de Nível Superior, Fundação de Amparo à Pesquisa do been shown to increase ANP release [30], a likely mechanism of ac- Espírito Santo and Fundo de Apoio à Ciência e Tecnologia do Município tion of tibolone is that an androgen metabolite of tibolone is respon- de Vitória. sible for the increases in ANP levels observed in our tibolone treated rats. Our data also suggest that tibolone itself can induce ANP release, References because tibolone treatment did not normalize ANP concentration in the left atrium. The effects of tibolone on the formation of the atrial [1] de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci granules and on the secretion of ANP need to be elucidated. 1981;28:89-94. Our findings with respect to ANP receptor expression in the kidney [2] Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of demonstrated that tibolone treatment did not normalize renal NPR-A tumor necrosis factor in severe chronic heart failure. N Engl J Med 1990;323: 236-41. mRNA levels in OVX animals, while the increased plasma ANP levels [3] Hirota H, Yoshida K, Kishimoto T, Taga T. Continuous activation of gp130, a that were observed could potentially lead to an improvement in ANP bi- signal-transducing receptor component for interleukin 6-related cytokines, ological activity, since NPR-A is expressed in other tissues, such as adi- causes myocardial hypertrophy in mice. Proc Natl Acad Sci U S A 1995;92:4862-6. [4] Testa M, Yeh M, Lee P, Fanelli R, Loperfido F, Berman JW, LeJemtel TH. Circulating pose tissues [7] and immune cells [16]. Thus, we believe that ANP levels of cytokines and their endogenous modulators in patients with mild to se- synthesis was normalized in our OVX+T group due an increase ANP vere congestive heart failure due to coronary artery disease or hypertension. J Am mRNA and plasma ANP levels without a change in renal clearance re- Coll Cardiol 1996;28:964-71. [5] Hong M, Yan Q, Tao B, Boersma A, Han KK, Vantyghem MC. Estradiol, progester- ceptors. If neutral endopeptidase 24.11 (NEP) [31] is involved in ANP one and testosterone exposures affect the atrial natriuretic peptide gene expres- degradation, it could potentially play a role in the increased ANP ob- sion in vivo in rats. Biol Chem Hoppe Seyler 1992;373:213-8. served in tibolone- and 17β-estradiol-treated rats. However, this possi- [6] Jankowski M, Rachelska G, Donghao W, McCann SM, Gutkowska J. Estrogen re- bility is sustained by a prior study reporting that estrogen did not ceptors activate atrial natriuretic peptide in the rat heart. Proc Natl Acad Sci 2001;98:11765-70. significantly affect NEP activity in the renal cortex and medulla of fe- [7] Belo NO, Sairam MR, dos Reis AM. Impairment of the natriuretic peptide system in male rats [32]. follitropin receptor knockout mice and reversal by estradiol: implications for obesity-associated hypertension in menopause. Endocrinology 2008;149:1399-406. Our results also showed that OVX females had increased serum – fl α [8] de Bold MLK. Estrogen, natriuretic peptides and the renin angiotensin system. levels of pro-in ammatory cytokines (IL-6 and TNF- )andlowplasma Cardiovasc Res 1999;41:524-31. levels of ANP. Few studies have examined the actions of tibolone on [9] Maffei S, Del Ry S, Prontera C, Clerico A. Increase in circulating levels of cardiac na- the immune system, and the information we do have regarding the triuretic peptides after hormone replacement therapy in postmenopausal fl fl fl women. Clin Sci 2001;101:447-53. in uence of this drug on pro-in ammatory cytokines is con icting. [10] Belo NO, Carnio EC, Gutkowska J, Antunes-Rodrigues J, Reis AM. Involvement of One study showed that HRT with tibolone for 2 months in post- atrial natriuretic peptide in blood pressure reduction induced by estradiol in menopausal women did not decrease TNF-α, IL-4, IL-10, or IL-2 levels, spontaneously hypertensive rats. Regul Pept 2004;117:53-60. [11] Pacifici R. Estrogen, cytokines, and pathogenesis of postmenopausal osteoporosis. though it did reduce bone resorption [12]. In contrast, another study J Bone Miner Res 1996;11:1043-51. reported that tibolone had a beneficial effect on inflammation in [12] Vural P, Canbaz M, Akgul C. Effects of menopause and postmenopausal tibolone post-menopausal women, reducing TNF-α levels but not IL-6 levels treatment on plasma TNF-α, IL-4, IL-10, IL-12 cytokine pattern and some bone Here, we also found that tibolone reduced TNF-α levels without af- turnover markers. Pharmacol Res 2006;53:367-71. [33]. [13] Xing D, Nozell S, Chen YF, Hage F, Oparil S. Estrogen and mechanisms of vascular fecting serum levels of IL-6 in female rats, whereas estrogen reduced the protection. Arterioscler Thromb Vasc Biol 2009;29:289-95. levels of both cytokines. We further speculate that plasma ANP, in addi- [14] Gammier C, Romao F. Changes in the immune system during menopause and tion to its known cardiovascular effects, may also normalize levels of aging. Front Biosci (Elite Ed) 2010;2:1299-303. [15] Kiemer AK, Vollmar AM. The atrial natriuretic peptide regulates the production of pro-inflammatory cytokines. Our observations are consistent with pre- inflammatory mediators in macrophages. Ann Rheum Dis 2001;60:iii68-70. vious reports indicating that ANP-induced cyclic GMP signaling can [16] Vollmar AM. The role of atrial natriuretic peptide in the immune system. Peptides suppress the release TNF-α in vitro [16,34] and in vivo [17,35] via inhi- 2005;26:1086-94. β [17] Ladetzki-Baehs K, Keller M, Kiemer AK, Koch, Zahler S, Wendel A, Vollmar AM. bition of NF-k activity. Atrial natriuretic peptide, a regulator of nuclear factor-kappaβ activation in The differing responses induced by tibolone versus estrogen in our vivo. Endocrinology 2007;148:332-6. study may be attributable to tibolone metabolites acting on other recep- [18] Vellaichamy E, Kaur K, Pandey KN. Enhanced activation of pro-inflammatory cyto- – kines in mice lacking natriuretic peptide receptor-A. Peptides 2007;28(4):893-9. tors [22 24], in addition to the estrogen receptor. Tibolone's three [19] Levin ER, Gardner DG, Samson WK. Mechanisms of disease: natriuretic peptides. main metabolites, 3α-hydroxytibolone, 3β-hydroxytibolone, and the N Engl J Med 1998;339:321-7. 60 A.R.S. Medeiros et al. / Regulatory Peptides 179 (2012) 55–60

[20] Maack T, Suzuki M, Almeida FA, Nussenzveig D, Scarborough RM, McEnroe GA, [29] Hoeg JM, Willis LR, Weinberger MH. Estrogen attenuation of the development of Lewicki JA. Physiological role of silent receptors of atrial natriuretic factor. Science hypertension in spontaneously hypertensive rats. Am J Physiol 1977;233:369-73. 1987;238:675-8. [30] Wu S, Weng X. Regulation of atrial natriuretic peptide, thromboxane and prosta- [21] Sarzani R, Paci VM, Dessi-Fulgheri P, Espinosa E, Rappelli A. Comparative analysis glandin production by androgen in elderly men with coronary heart disease. Chin of atrial natriuretic peptide receptor expression in rat tissues. J Hypertens Suppl Med Sci J 1993;8(4):207-9. 1993;5:S214-5. [31] McGrath MF, de Bold AJ. Determinants of natriuretic peptide gene expression. [22] Kloosterboer HJ. Tissue-selectivity: the mechanism of action of tibolone. Peptides 2005;26(6):933-43. Maturitas 2004;48(Suppl. 1):S30-40. [32] Neves LA, Chappell MC, Ferrario CM, Gallagher PE, Ganten D, Brosnihan KB. Effect of [23] Escande A, Servant N, Rabenoelina F, Auzou G, Kloosterboer H, Cavaillès V, Balaguer estrogen on neprilysin expression in uterus and kidney of Sprague–Dawley normo- P, Maudelonde T. Regulation of activities of steroid hormone receptors by tibolone tensive and heterozygous (mRen2)27-transgenic hypertensive rats. Peptides and its primary metabolites. J Steroid Biochem Mol Biol 2009;116(1–2):8–14. 2006;27(11):2912-8. [24] Kloosterboer HJ. Historical milestones in the development of tibolone (Livial®). [33] Vassalle C, Cicinelli E, Lello S, Mercuri A, Battaglia D, Maffei S. Effects of meno- Climacteric 2011;14(6):609-21. pause and tibolone on different cardiovascular biomarkers in healthy women. [25] Gutkowska J, Genest J, Thibault G, Garcia R, Larochelle P, Cusson JR, Kichel O, Gynecol Endocrinol 2011;27(3):163-9. Hamet P, De Léan A, Cantin M. Circulating forms and radioimmunoassay of natri- [34] Kiemer AK, Hartung T, Vollmar AM. cGMP-mediated inhibition of TNF-alpha pro- uretic factor. Endocrinol Metab Clin North Am 1987;16:183-98. duction by the atrial natriuretic peptide in murine macrophages. J Immunol [26] Bradford M. A rapid and sensitive method for the quantiﬁcation of microgram 2000;165:175-81. quantities of protein utilizing the principle of protein–dye binding. Anal Biochem [35] Mitkiewicz M, Kuropatwa M, Kurowska E, Gorczyca WA. Different effects of solu- 1976;72:248-54. ble and particulate guanylyl cyclases on expression of inﬂammatory cytokines in [27] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium rat peripheral blood mononuclear cells. Immunobiology 2011;216:423-30. thiocynate–phenol–chloroform extraction. Anal Biochem 1987;162:155-9. [28] Potter LR, Abbey-Hosch S, Dickey DM. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 2006;27:47-72.