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Effects of androgen manipulation on α1-adrenoceptor subtypes in the rat seminal vesicle
Life Sciences 75 (2004) 1449 – 1463 www.elsevier.com/locate/lifescie
Effects of androgen manipulation on a1-adrenoceptor subtypes in the rat seminal vesicle Fu´lvio R. Mendes 1, Margarete Hamamura 1, Daniel B.C. Queiro´z, Catarina S. Porto, Maria Christina W. Avellar * Department of Pharmacology, Section of Experimental Endocrinology, UNIFESP-Escola Paulista de Medicina, Rua 03 de maio 100, INFAR, Vila Clementino, 04044-020, Sa˜o Paulo, Brazil Received 19 September 2003; accepted 5 March 2004
Abstract This study analyses possible changes during surgical and chemical castration in the expression and pharmacological characteristics of a1-adrenoceptor in adult rat seminal vesicle. Ribonuclease protection assays indicated that a1a- was the predominant mRNA, while a1b- and a1d-adrenoceptor transcripts were detected in lower abundance in this tissue. a1a-adrenoceptor mRNA expression presented a complex dependency on androgens, while a1b- and a1d-adrenoceptor transcripts were both upregulated with surgical and chemical castration, suggesting a negative modulation by androgens. Testosterone treatment reversed the effects caused by surgical castration. Functional studies confirmed the involvement of a1A- and a1B-adrenoceptor in the seminal vesicle contractile responses, and suggested that a1B-induced contractile response was upregulated after castration. Taken together, the results suggest that a1-adrenoceptor expression in seminal vesicle is differentially regulated by the androgen status of the rat. D 2004 Elsevier Inc. All rights reserved. Keywords: Seminal vesicle; a1-adrenoceptor; Castration; Androgen
Introduction Gonadal steroids have potent effects on many aspects of neuronal function in reproductive organs during development and adulthood. These effects give rise to either permanent changes in neuronal organization or, postnatally, alterations which depend on the ongoing presence of hormones for their * Corresponding author. Tel./fax: +55-11-5576-4448. E-mail address: [email protected] (M.C.W. Avellar). 1 These two authors contributed equally to the present work. 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.011
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maintenance (see for reviews Gibson, 1981; Anderson, 1993; Keast and Saunders, 1998). For example, the interaction between androgen deprivation and adrenergic contractile response of the vas deferens and prostate has been shown (MacDonald and MacGrath, 1980; Lin et al., 1995; Lacey et al., 1996; Pupo, 1998; Akiyama et al., 1999; Homma et al., 2000; Campos et al., 2003). Three distinct a1-adrenoceptor subtypes have been cloned and characterized in tissues of various species: a1A- (Schwinn et al., 1990; Hirasawa et al., 1993; Laz et al., 1994; Perez et al., 1994; Rokosh et al., 1994), a1B- (Cotecchia et al., 1988; Voight et al., 1990; Ramarao et al., 1992) and a1D-adrenoceptors (Bruno et al., 1991; Lomasney et al., 1991; Perez et al., 1991; see for reviews Hieble et al., 1995; Langer, 1998; Docherty, 1998). A fourth a1-adrenoceptor, designated a1L-adrenoceptor, which has not been cloned yet, exhibits lower affinity to prazosin and has been proposed to mediate contraction of human, rabbit and dog lower urinary tract tissues (Muramatsu et al., 1994; Ford et al., 1996; Testa et al., 1996; Fukasawa et al., 1998). Recently, isoforms of the human and rabbit a1A-adrenoceptor generated by alternative splicing have been identified; these isoforms differ in length and sequence in the carboxylterminal region (Daniels et al., 1999; Schwinn and Price, 1999; Suzuki et al., 2000). The seminal vesicle is a contractile organ that responds to a1-adrenoceptor stimulation (Porto et al., 1988). Previous studies have shown that adrenaline is the most potent a1-adrenoceptor agonist to induce contractions of the seminal vesicle, by interacting with postsynaptic a1-adrenoceptors (Sharif et al., 1990; Soares et al., 1993; Silva et al., 1999). Presynaptic and/or postsynaptic a2-adrenoceptors are not present in this organ (Shima, 1993; Sadraei et al., 1995). Radioligand binding assays (Shima, 1993) and functional studies, using selective a1A- and a1D- adrenoceptor antagonists (Silva et al., 1999), indicated that a1A-adrenoceptor subtype predominates in the rat seminal vesicle, although a1a-, a1b- and a1dadrenoceptor subtypes have been detected in this tissue (Silva et al., 1999). The aim of the present study was to investigate whether different androgen manipulations, induced by surgical and chemical castration, have effects on the expression of a1-adrenoceptor mRNA subtypes in the rat seminal vesicle. Furthermore, the effects of castration on the contractility and pharmacological characteristics of a1adrenoceptors in the rat seminal vesicle were also explored by using functional studies.
Materials and methods Animals and treatments Male Wistar rats (120-day old, 210–220 g) were housed in the Animal Facility at Instituto Nacional de Farmacologia, Universidade Federal de Sa˜o Paulo-Escola Paulista de Medicina (UNIFESP-EPM) and maintained on a 12 h light, 12 h dark lighting schedule, at 20jC, food and water ad libitum. Animal procedures were approved by the Research Ethical Committee from UNIFESP-EPM. Animal groups included the following: NC, normal control; C, rats surgically castrated and sacrificed on days 1, 2, 7 and 15 after castration; CA, rats chemically castrated by the use of daily injections of cyproterone acetate (7.6 mg/100 g body weight, i.m.) and sacrificed on days 1, 2, 8 and 15 of treatment; C 7 days + T 3 and C 7 days + T 6, rats surgically castrated for 7 days (C 7 days) and then treated daily with testosterone propionate (1 mg/100 g body weight, s.c.) for 3 (T 3) or 6 days (T 6). Preliminary experiments with tissues from sham-operated rats or animals injected with drug vehicles were also tested as controls. Since no significant changes were observed when these two experimental groups were compared to normal control, all subsequent experiments were performed with tissues from normal rats as control.
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Testosterone propionate was dissolved in ethanol and soybean oil (1:3 v/v). Cyproterone acetate was dissolved in benzyl benzoate and soybean oil (5:1, v/v). Measurement of testosterone plasma levels Blood from the aorta artery was collected, and testosterone plasma levels were measured by RIA using Coat-A-Count Total Testosterone kit (Diagnostic Products Co., Los Angeles, CA) according to manufacturer’s instructions. The assay detection limit was 0.04 ng/ml. The intraassay and interassay coefficients of variation were 1.8% and 2.1%, respectively. RNA isolation The animals were sacrificed, seminal vesicles were immediately removed, free of connective and fat tissue, separated from coagulating gland, frozen in liquid nitrogen and stored at 75jC until use. Total RNA was extracted from frozen tissues as previously described (Chirgwin et al., 1979). RNA samples were then quantitated, using a spectrofotometer at 260/280 nm and stored at 75jC for later use. Ribonuclease protection assay Assays were performed by using MAXIscript in vitro translation and RPA IIk kits from AMBION, Inc. (Austin, TX, USA), as previously described (Queiro´z et al., 2002). The DNA templates for antisense a1adrenoceptor RNA synthesis were kindly provided by Dr. Paul C. Simpson (University of San Francisco, USA) and they are described in Rokosh et al. (1994). RNA probes were radiolabeled with [a32P]UTP in the presence of T7 RNA polymerase. Linearized plasmid containing a 400 bp glyceraldehyde phosphate dehydrogenase (GAPDH) gene fragment in the antisense orientation under the transcriptional control of T7 promoter was from AMBION. Sizes (nucleotides) of the probes and protected fragments were as follows: a1a 408/315, a1b 495/432, a1d 267/217 and GAPDH 413/316, respectively. For each a1adrenoceptor probe, the fraction of uridine residues available for radiolabeling was similar: a1a, 25%; a1b, 20% and a1d, 19%. Experiments were performed in the presence of 40 Ag of total RNA. Throughout the experiments, control lanes containing yeast RNA (40 Ag) and a1-adrenoceptor labeled probes, incubated or not with RNase A/T1 mix, were included in order to visualize full length transcripts and efficiency of RNase digestion, respectively. Total RNA from adult rat brain and heart was used as positive control for a1a-/a1d- and a1b-adrenoceptor transcritp expression, respectively (Rokosh et al., 1994). Autoradiographs were scanned using a Schimadzu densitometer (Shimadzu Scientific Instruments, Princeton, NJ, USA). The steady state level of each mRNA transcript, as estimated by the area under the curve, was normalized against that of the GAPDH mRNA in each sample, as previously described (Queiro´z et al., 2002). Since the ratio between the levels of a1-adrenoceptor subtypes and GAPDH mRNA depended on the exposure time of the autoradiogram, the normalized data of the different groups were expressed as the percentage of this ratio for normal control in each experiment. Tissue preparation and record of seminal vesicle contraction Seminal vesicles from normal control (NC) and 7 days surgically castrated rats (C 7 days) were dissected, free of connective and fat tissue, separated from coagulating gland and washed internally with
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a nutrient solution of the following composition (mM): 136.89 NaCl, 5.63 KCl, 1.80 CaCl2, 0.36 NaH2PO4, 14.88 NaHCO3 and 5.55 glucose (pH 7.6–7.8). The tissue preparation and record of seminal vesicle contraction were performed as described previously (Silva et al., 1999). Briefly, the organ was suspended, under a load of 0.5 g, into organ baths filled with nutrient solution bubbled with air, at 30jC. Propranolol (10 6M) and cocaine (10 5M) were kept in the nutrient solution throughout the experiments in order to block h-adrenoceptor and catecholamine neuronal uptake, respectively. Isometric contractions were recorded with a force-displacement transducer connected to a polygraph (Gemini 7070, Ugo Basile, Viarese, Italy). Preliminary experiments, in the absence of antagonists, were carried out and no difference in the sensitivity to phenylephrine among three consecutive concentration-effect curves were observed. BaCl2 (0.03M) was always added to the preparation, at the end of the experiment, in order to obtain the maximal contraction of the tissue (Jurkiewicz et al., 1976). After the determination of the second concentration-effect curve to phenylephrine, the agonist was washed out for at least 30 min and then an antagonist was incubated for 30 or 45 min. After this period, another concentration-effect curve to phenylephrine was obtained. The following a1-adrenoceptor antagonists were used: WB 4101, which shows higher affinity for a1A-adrenoceptors (Hieble et al., 1995; Langer, 1998; Docherty, 1998; Silva et al., 1999); BMY 7378, which recognizes preferentially a1D-adrenoceptors (Saussy et al., 1996; Deng et al., 1996; Yang et al., 1997; Xin et al., 1997); chloroethylclonidine (CEC), an irreversible a1adrenoceptor antagonist (Ford et al., 1994; Michel et al., 1995; Hieble et al., 1995; Graham et al., 1996) that inactivates 80% of both a1B- and a1D-adrenoceptors and 20% of the a1A-adrenoceptors (Laz et al., 1994; Hieble et al., 1995; Xiao and Jeffries, 1998); and (+)cyclazosin, a potent a1B-adrenoceptor antagonist, that displays a 90- to 130-fold selectivity for binding to rat a1B-adrenoceptors when compared to rat a1A- and a1D-adrenoceptor subtypes (Giardina` et al., 1995, 1996). Data analysis The data were expressed as g tension/100 mg wet tissue and analysed by the interactive nonlinear regression through the computer program GraphPad Prism (GraphPad Prism Software Inc., San Diego, CA, USA). Each tension was normalized according to the seminal vesicle weight for each treatment group. The maximal contraction was obtained from the concentration-effect curve to phenylephrine and BaCl2. The EC50 value, concentration that produces 50% of phenylephrine maximum response, was also determined. The antagonist potency was expressed as pKB values, i.e., the negative log of the dissociation constant KB, which equals to the molar concentration of the antagonist divided by the concentration-ratio minus one (Besse and Furchgott, 1976). Statistical analysis Data were expressed as mean F S.E.M. Statistical analysis was carried out using analysis of variance (ANOVA) followed by Newman-Keuls test for multiple comparisons, or by the two-tailed Student’s t-test to compare two responses (Snedecor and Cochran, 1980). P values < 0.05 were accepted as significant. Drugs and reagents The following drugs and reagents were used: phenylephrine (L-phenylephrine hydrochloride), propranolol (DL-propranolol hydrochloride), testosterone (testosterone propionate) were from Sigma
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Chemical Co. (St. Louis, MO, USA). WB 4101 (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4benzodioxane hydrochloride), BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5] decane-7,9-dione dihydrochloride), chloroethylclonidine (chloroethylclonidine dihydrochloride), (+)cyclazosin (cyclazosin hydrochloride) were from Research Biochemicals International (Natrick, MA, USA). Cocaine (cocaine hydrochloride) was from Merck (Darmstadt, FRG). Cyproterone acetate (ANDROCURR) was from Schering (Sa˜o Paulo, SP, Brazil). [a32P]UTP (800 Ci/mmol, 10 mCi/ml) was from New England Nuclear (Boston, MA, USA). MAXIscriptk transcription Kit, RPA IIIk assay Kit, RNA Century Marker Template Plus were from AMBION, Inc. (Austin, TX, USA). All other drugs and reagents were from Sigma Chemical Co. or GIBCO (Gaithersburg, MD, USA).
Results Testosterone plasma levels and seminal vesicle wet weight The rat androgen status was monitored by measuring both the testosterone plasma level and the seminal vesicle wet weight (Table 1). A reduction in circulating testosterone was already detected in the first day after surgical castration. After 7 and 15 days of the surgical procedure, hormone plasma levels were below the detection limit of the assay. Testosterone treatment for 3 or 6 days, in a group of animals that had been castrated previously for 7 days, restored hormone plasma levels above those observed in normal control rats. Chemical castration, induced by daily injections of cyproterone acetate for 1, 2 days (data not shown) or 8 days, did not affect the testosterone plasma levels when compared to normal control (Table 1). After 15 days of antiandrogen treatment, however, hormone plasma levels were significantly reduced when compared to normal control (Table 1). Surgical castration for 1 or 2 days had no effect on the wet weight of seminal vesicle (Table 1). However, a decrease of 39% and 52% on this parameter was observed after 7 and 15 days of the Table 1 Testosterone plasma level and seminal vesicle wet weight from NC, normal control; C, rats surgically castrated and sacrificed on days 1, 2, 7 and 15; C 7 days F T, rats surgically castrated for 7 days and then treated for 3 (T 3) or 6 (T 6) consecutive days with testosterone propionate; CA, rats chemically castrated by daily injections of cyproterone acetate and sacrificed on days 8 and 15 Animals
Testosterone (ng/ml)
Seminal vesicle wet weight (mg)
NC C 1 day C 2 days C 7 days C 15 days C 7 days + T 3 days C 7 days + T 6 days CA 8 days CA 15 days
1.18 F 0.18 F N.D. < 0.04a < 0.04a 16.9 F 19.0 F 1.18 F 0.12 F
169.4 147.5 140.8 103.6 81.2 190.0 264.5 120.0 83.7
0.19 0.01a
0.70a 1.00a 0.17 0.02a
Data are mean F S.E.M. of 3 – 7 different experiments. N.D. not determined. a P < 0.05 when compared with NC group. b P < 0.05 when compared with C 7 days group.
F F F F F F F F F
13.6 16.5 9.2 4.2a 2.1a 6.0b 16.5a,b 2.2a 7.4a
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surgical procedure, respectively. The reduction in the tissue wet weight, caused by 7 days of surgical castration, was reversed to levels similar or above those observed in normal control when animals were treated with testosterone for 3 or 6 days, respectively. Chemical castration, induced by
Fig. 1. Ribonuclease protection assay for a1-adrenoceptor mRNA subtypes in rat seminal vesicle. Total RNA (40 Ag) from rat seminal vesicle were hybridized to 32P-labeled cRNA of a1a-, a1b-, a1d-adrenoceptor and GAPDH transcripts. Panel (A): Representative autoradiogram of 4 – 5 different experiments. On the left, 32P-RNA molecular markers (MW). On the right, arrows indicate specific protected products. NC, normal control rats; C, rats surgically castrated and sacrificed on days 1, 2, 7 and 15; C 7 days + T, rats surgically castrated for 7 days and then treated for 3 (T 3) or 6 (T 6) consecutive days with testosterone propionate; CA, rats chemically castrated by daily injections of cyproterone acetate and sacrificed on days 1, 2, 8 and 15. Panel (B): Autoradiograms were scanned and densitometry was used to quantify the relative changes in a1a-, a1b- and a1d-adrenoceptor mRNA expression with different animal treatments. Steady state levels of each a1-adrenoceptor mRNA were normalized against that of the GAPDH mRNA and expressed as the percentage (mean F S.E.M.) of a1-adrenoceptor/GAPDH mRNA ratio for NC group in each experiment (4 – 5 different experiments). aP < 0.05 compared with NC group; bP < 0.05 compared with C 7 days group.
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daily treatment with cyproterone acetate, also induced a decrease of 29% and 50% in seminal vesicle wet weight after 8 and 15 days of treatment, respectively, when compared to normal control (Table 1). Changes in rat body weight were not observed with any of the treatments used (data not shown). Expression of a1-adrenoceptor mRNA subtypes in rat seminal vesicle Total RNA isolated from rat seminal vesicle was tested in ribonuclease protection assays to detect the steady state of a1-adrenoceptor mRNA subtypes. In normal control rats, a1a-mRNA was the predominant transcript while a1d-adrenoceptor was less abundant (Fig. 1). The expression of a1b-adrenoceptor mRNA was considerably lower when compared to the other two transcripts, being only detected when longer exposures of the gel were obtained (data not shown). Since the fraction of uridine residues available for radiolabeling of each a1-adrenoceptor cRNA probes was similar, the results show that the ratio among transcripts is a1aHHa1dHa1b. Ribonuclease protection assays were also used to investigate the androgen regulation of a1adrenoceptor transcripts in rat seminal vesicle (Fig. 1A). Densitometry was used to quantify the relative changes in mRNA expression with the different animal treatments. GAPDH mRNA, a housekeeping gene, was used as an internal control and data were expressed as the percentage of a1-adrenoceptor and GAPDH mRNA ratio for normal control in each experiment (Fig. 1B). Surgical castration induced different changes depending on the a1-adrenoceptor transcript analysed. The steadystate of a1a-adrenoceptor mRNA significantly decreased within 2 days postcastration and then increased after 7 and 15 days postcastration, when compared to normal controls. The levels of a1d-adrenoceptor mRNA, on the other hand, were already above control level in the first day postcastration. The maximum level of this mRNA specie was reached 7 days after castration. a1b-adrenoceptor mRNA transcript, although not abundant in control tissues, was significantly higher than control level after 7 and 15 days of surgical castration. Testosterone replacement for 3 days was effective to reduce the levels of a1b- and a1d-adrenoceptor transcripts observed with 7 days castration. After 6 days of testosterone treatment, on the other hand, all three mRNA subtypes were reduced to values similar or below normal control (Fig. 1B). Chemical castration, induced by cyproterone acetate, was also used to evaluate hormonal regulation of a1-adrenoceptor mRNA subtype expression. Although 1 and 2 days of treatment with this drug had no effect on the expression of a1-adrenoceptor transcripts, all three a1-adrenoceptor mRNA subtypes were up-regulated significantly after 8 and 15 days of treatment (Fig. 1B).
Table 2 Contractile response induced by BaCl2 and phenylephrine-induced contraction in seminal vesicle from normal control (NC) rats and 7 days castrated rats (C 7 days) Animals NC C 7 days
Maximal Contraction (g tension/100 mg wet tissue)
EC50 (AM)
BaCl2
Phenylephrine
Phenylephrine
1.63 F 0.12 (5) 3.36 F 0.39* (6)
1.00 F 0.06 (21) 1.95 F 0.12* (20)
17.40 F 3.98 (21) 33.50 F 8.57 (20)
Data are mean F S.E.M. of number of experiments in parenthesis. * Significantly different from control rats ( P < 0.05; Student’s t-test).
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Fig. 2. Effect of WB 4101, BMY 7378, chloroethylclonidine and (+)cyclazosin on phenylephrine-induced contractions in the rat seminal vesicle from normal control (NC, left panels) and 7 days castrated rats (C 7 days, right panels). Concentration-effect curves to phenylephrine were obtained in the absence (closed symbols) and presence of a1-adrenoceptor antagonists (open symbols). WB 4101 (10 9M, 5), BMY 7378 (10 7M, D), chloroethylclonidine (CEC) (10 5M, o) and (+)cyclazosin (10 9M, q). Each point and vertical line represents the mean F S.E.M. of n shown in Table 3.
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Table 3 pKB values for different a1-adrenoceptor antagonists tested against phenylephrine-induced contraction in seminal vesicle from normal control (NC) rats and 7 days castrated rats (C 7 days) Animal NC C 7 days
pKB WB 4101
BMY 7378
CEC
(+)Cyclazosin
8.63 F 0.10 (8) 8.58 F 0.11 (7)
5.81 F 0.05 (6) 5.70 F 0.15 (6)
4.49 F 0.01 (6) 4.49 F 0.01 (6)
9.88 F 0.04 (7) 9.93 F 0.03 (7)
Data are mean F S.E.M. of number of experiments in parenthesis. No statistical difference was observed among control and castrated rats ( P < 0.05; Student’s t-test).
Functional studies The maximal contraction induced by BaCl2 and phenylephrine, expressed as g tension/100 mg of wet tissue was approximately 2-fold higher in 7 days castrated rats than in normal control rats (Table 2). However, when the maximal contractile response to phenylephrine was normalized to the percentage of the maximal response to BaCl2, no significant difference was found between normal control and 7 days castrated rats (61% and 58%, respectively). In addition, the EC50 values for phenylephrine were similar in both groups (Table 2). WB 4101 (10 9M) and BMY 7378 (10 7M) produced rightward shifts of the concentration-effect curves to phenylephrine in seminal vesicle from normal control and 7 days castrated rats (Fig. 2). Chloroethylclonidine (CEC, 10 5M) and (+)cyclazosin (10 9M) shifted concentration-effect curves to the right and reduced the maximum contractile response induced by phenylephrine in tissue from normal control rats (40% and 67%, respectively) (Fig. 2). On the other hand, in the seminal vesicle from 7 days castrated rats, the maximum contractile response induced by phenylephrine decreased in the presence of chloroethylclonidine and (+)cyclazosin (22% and 54% respectively) (Fig. 2). The calculated pKB values for a1-adrenoceptor antagonists revealed similar values for normal control and 7 days castrated rats (Table 3). The order of potency for a1-adrenoceptor antagonists in the rat seminal vesicle from both experimental groups was (+) cyclazosin > WB 4101 { BMY 7378 > chloroethylclonidine.
Discussion In the present study, we have observed that, under normal physiological conditions, a1a-adrenoceptor mRNA was the predominant a1-adrenoceptor subtype present in this tissue, while a1d- was less abundant and a1b-adrenoceptor mRNA was only weakly expressed in seminal vesicle from adult rats. Considering that this tissue is under androgen control, we have studied the impact of changes in testosterone status caused by surgical and chemical castration on the relative expression of seminal vesicle a1-adrenoceptor mRNA subtypes. We were able to demonstrate that surgical castration differently regulates the expression of a1-adrenoceptor transcripts. a1a-adrenoceptor mRNA showed a complex dependency on androgens, since its expression was down-regulated within the first 2 days postcastration, but increased after 7 and 15 days of the surgical procedure. a1b- and a1d-transcripts, poorly expressed in seminal vesicle of control animals when compared to a1a-adrenoceptor mRNA, were both up-regulated with prolongation of surgical castration, indicating a negative dependency of these transcripts on androgens. The ability of testosterone to revert the changes in a1-adrenoceptor transcripts induced by 7
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days of surgical castration suggested that these mRNA species are physiologically dependent on androgens. Furthermore, the results show that the sustained blockade of androgen receptor with cyproterone acetate treatment for 8 and 15 days induced an up-regulation of a1a-, a1b- and a1dadrenoceptor transcripts, confirming that androgen signalling is involved on this regulation. The up-regulation in a1d-adrenoceptor mRNA level was already observed within 1 day after surgical castration. Although a1a-adrenoceptor transcript was down-regulated after 2 days, both a1a- and a1badrenoceptor were up-regulated with prolongation of castration for 7 and 15 days. Thus, the temporal changes in a1-adrenoceptor subtype expression with surgical castration suggest that the sensitivity of the a1-adrenoceptor gene subtypes to testosterone or androgen-related factors is different. A number of mRNA transcripts studied during castration-induced involution and androgen-induced regrowth of tissues in the male reproductive system, including rat seminal vesicle, present complex and negative dependency on testosterone (Collins et al., 1988; Huang et al., 1997; Nishi et al., 1996). It is difficult to correlate the a1-adrenoceptor mRNA variations in seminal vesicle during androgen manipulation of the animals to a direct hormonal effect. Although promoter regions of human a1a-adrenoceptor (Razik et al., 1997) and rat a1b-adrenoceptor gene (Gao et al., 1995; Gao and Kunos, 1998) contain multiple binding sites for sequence-specific proteins, no functional androgen response elements have been described for the three different a1-adrenoceptor genes. It is possible that the changes in a1-adrenoceptor mRNA expression after surgical and chemical castration may be due to an indirect effect of testosterone, where an androgen-sensitive factor might be involved in the expression and/or stabilization of the transcript. Furthermore, since the steady state of mRNA reflects the balance between the rate of transcription and degradation of messages, factors responsible for either mechanism could be involved. Since the changes in mRNA levels in a given tissue may not always correlate directly with changes at protein levels (Scofield et al., 1995; Docherty, 1998), functional studies were designed to observe the effects of 7 days castration on the contractile responses induced by activation of a1-adrenoceptor in rat seminal vesicle. The maximal contractile response to BaCl2 and phenylephrine, expressed as g tension/ 100 mg of tissue wet weight, was approximately 2-fold higher in 7 days castrated rat seminal vesicle. This apparent increase in phenylephrine-induced maximal response was normalized when the proportion due to BaCl2-induced contraction was taken into account. Furthermore, this increase in contractility is most likely explained by the change in tissue components of the seminal vesicle, similar to that reported for rat prostate (Lin et al., 1995; Lacey et al., 1996). Upon 30 days castration, seminal vesicle epithelium undergoes degeneration, whilst smooth muscle, responsible for the contractile property of the seminal vesicle, is preserved (unpublished data). In addition, we found that EC50 values for phenylephrine were similar in both control and 7 days castrated rats, indicating that castration caused no essential changes in the affinity of phenylephrine to a1-adrenoceptor. The pKB values calculated for all antagonists tested were similar between normal control and 7 days castrated rats. WB 4101 presented high affinity in seminal vesicle from normal control and 7 days castrated rats, while the a1D-adrenoceptor subtype selective antagonist, BMY 7378, presented a relatively low affinity, indicating that a1A-adrenoceptor is predominantly involved in the rat seminal vesicle contraction when compared to a1D-adrenoceptor subtype as previously reported in the seminal vesicle from 60-day-old rats (Silva et al., 1999). We tested the sensitivity of seminal vesicle contractions induced by phenylephrine to the alkylating agent chloroethylclonidine, an irreversible antagonist which inactivates 80% of both a1B- and a1D- and 20% of a1A-adrenoceptors (Laz et al., 1994; Hieble et al., 1995; Xiao and Jeffries, 1998). Our results showed that chloroethylclonidine shifted the phenylephrine-induced curves to the right, with 40% and
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22% reduction in the maximum contractile response induced by this agonist when seminal vesicle from normal control and 7 days castrated rats were tested, respectively. It is known that the degree to which chloroethylclonidine affects the functional response depends, on some extent, on the amount of spare receptors present in the tissue (Burt et al., 1995; Marshall et al., 1995). In the rat seminal vesicle, 0.10% of the a1-adrenoceptor pool represents a spare receptor population (Silva et al., 1999). Furthermore, the most interesting result of the present investigation was the high affinity displayed by (+)cyclazosin (pKB values = 9.88 F 0.04 and 9.93 F 0.03 in seminal vesicle from normal control and 7 days castrated rats, respectively) similar to the affinity of this antagonist for native a1B- and cloned a1b-adrenoceptors (pKi = 9.57 F 0.01 and 9.23 F 0.04, respectively) (Giardina` et al., 1995; Stam et al., 1998), indicating that functional a1B-adrenoceptor may also be involved in the seminal vesicle contractile response. The relative participation of each a1-adrenoceptor subtype (a1A- and a1B-adrenoceptors) in the seminal vesicle contraction remains to be established. The decrease of the maximum contractile response to phenylephrine was more readily detected with (+)cyclazosin in normal control rats (67%) than in 7 days castrated rats (54%). The effects of chloroethylclonidine and (+)cyclazosin on phenylephrine-induced curves obtained from normal control and 7 days castrated rats may indicate that a1B-adrenoceptor is upregulated after castration, confirming the upregulation of a1b-adrenoceptor transcripts obtained in ribonuclease protection studies. Shima (1992) observed that castration increased the number of a1-adrenoceptor in adult rat seminal vesicle, an effect reversed by androgen replacement. Lacey et al. (1996) found an apparent 2-fold increase in prostatic a1-adrenoceptor density as a consequence of androgen deprivation in rats. These authors speculated that this increase was an artifact due to a relative increase in the ratio of smooth muscle, which is mostly responsible for prostatic contraction, to epithelium in the prostate rather than from an up-regulation of a1-adrenoceptors. Lin et al. (1995) reported no changes in the in vivo measurement of dog prostatic urethral pressure by androgen deprivation. It has been shown that the androgen deprivation decreased the contraction potency induced by phenylephrine in rat prostate strips, probably associated with down-regulation of a1A-adrenoceptor spare population expressed in the intact prostate (Homma et al., 2000). Conversely, rat prostatic hypertrophy induced by testosterone resulted in a reduction of the a1-adrenoceptor density, occuring in parallel with a decrease in the amount of fibromuscular element in the entire prostate when compared to control rats (Auger-Pourmarin et al., 1998). Recently, it has been shown that the prolongation of castration for up to 30 days differently affects the potency of and maximal contractions induced by noradrenaline in the rat vas deferens (Campos et al., 2003). Castration also reduced phenylephrine-induced increase in urethral pressure in rats (Akiyama et al., 1999), while the treatment with dihydrotestosterone and prazosin increased prostate weight, its stiffness and urodynamic obstruction by prostate (Lee et al., 1998). In the present work, functional studies confirmed the involvement of a1A- and a1B-adrenoceptors in the seminal vesicle contractile response, and suggested that a1B-induced contraction is upregulated after castration. Interestingly, the expression pattern of each a1-adrenoceptor mRNA subtype (a1a, a1b and a1d) was differentially regulated by the androgen status of the rat. Taking into consideration that different cell types are present in seminal vesicle (Williams-Ashman, 1983), further studies focusing receptormediated intracellular signaling coupled to immunological techniques to localize and determine the relative levels of a1-adrenoceptor subtypes will be an important tool to understand the relative contribution of a1-adrenoceptor in the seminal vesicle.
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Acknowledgements This work was supported by Fundacßa˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, grant number 96/1777-1), Brazil and in part by T.W. Fogarty International, USA (parent grant 5H37HD0446626, subcontract UNC 5-53284). Researcher fellowships (M.C.W.A. and C.S.P.) and scholarships supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (F.R.M. and D.B.C.Q.) and by FAPESP (M.H.), Brazil. We are indebted to Dr. Paul C. Simpson (University of San Francisco, CA, USA) for provision of DNA templates for antisense a1-adrenoceptor RNA synthesis and Dr. Rodolfo P. Rothlin for kindly provided the (+)cyclazosin. We gratefully thank the technical assistance of Espedita M. Jesus and Maria D. Silva.
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Effects of androgen manipulation on a1-adrenoceptor subtypes in the rat seminal vesicle Fu´lvio R. Mendes 1, Margarete Hamamura 1, Daniel B.C. Queiro´z, Catarina S. Porto, Maria Christina W. Avellar * Department of Pharmacology, Section of Experimental Endocrinology, UNIFESP-Escola Paulista de Medicina, Rua 03 de maio 100, INFAR, Vila Clementino, 04044-020, Sa˜o Paulo, Brazil Received 19 September 2003; accepted 5 March 2004
Abstract This study analyses possible changes during surgical and chemical castration in the expression and pharmacological characteristics of a1-adrenoceptor in adult rat seminal vesicle. Ribonuclease protection assays indicated that a1a- was the predominant mRNA, while a1b- and a1d-adrenoceptor transcripts were detected in lower abundance in this tissue. a1a-adrenoceptor mRNA expression presented a complex dependency on androgens, while a1b- and a1d-adrenoceptor transcripts were both upregulated with surgical and chemical castration, suggesting a negative modulation by androgens. Testosterone treatment reversed the effects caused by surgical castration. Functional studies confirmed the involvement of a1A- and a1B-adrenoceptor in the seminal vesicle contractile responses, and suggested that a1B-induced contractile response was upregulated after castration. Taken together, the results suggest that a1-adrenoceptor expression in seminal vesicle is differentially regulated by the androgen status of the rat. D 2004 Elsevier Inc. All rights reserved. Keywords: Seminal vesicle; a1-adrenoceptor; Castration; Androgen
Introduction Gonadal steroids have potent effects on many aspects of neuronal function in reproductive organs during development and adulthood. These effects give rise to either permanent changes in neuronal organization or, postnatally, alterations which depend on the ongoing presence of hormones for their * Corresponding author. Tel./fax: +55-11-5576-4448. E-mail address: [email protected] (M.C.W. Avellar). 1 These two authors contributed equally to the present work. 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.011
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maintenance (see for reviews Gibson, 1981; Anderson, 1993; Keast and Saunders, 1998). For example, the interaction between androgen deprivation and adrenergic contractile response of the vas deferens and prostate has been shown (MacDonald and MacGrath, 1980; Lin et al., 1995; Lacey et al., 1996; Pupo, 1998; Akiyama et al., 1999; Homma et al., 2000; Campos et al., 2003). Three distinct a1-adrenoceptor subtypes have been cloned and characterized in tissues of various species: a1A- (Schwinn et al., 1990; Hirasawa et al., 1993; Laz et al., 1994; Perez et al., 1994; Rokosh et al., 1994), a1B- (Cotecchia et al., 1988; Voight et al., 1990; Ramarao et al., 1992) and a1D-adrenoceptors (Bruno et al., 1991; Lomasney et al., 1991; Perez et al., 1991; see for reviews Hieble et al., 1995; Langer, 1998; Docherty, 1998). A fourth a1-adrenoceptor, designated a1L-adrenoceptor, which has not been cloned yet, exhibits lower affinity to prazosin and has been proposed to mediate contraction of human, rabbit and dog lower urinary tract tissues (Muramatsu et al., 1994; Ford et al., 1996; Testa et al., 1996; Fukasawa et al., 1998). Recently, isoforms of the human and rabbit a1A-adrenoceptor generated by alternative splicing have been identified; these isoforms differ in length and sequence in the carboxylterminal region (Daniels et al., 1999; Schwinn and Price, 1999; Suzuki et al., 2000). The seminal vesicle is a contractile organ that responds to a1-adrenoceptor stimulation (Porto et al., 1988). Previous studies have shown that adrenaline is the most potent a1-adrenoceptor agonist to induce contractions of the seminal vesicle, by interacting with postsynaptic a1-adrenoceptors (Sharif et al., 1990; Soares et al., 1993; Silva et al., 1999). Presynaptic and/or postsynaptic a2-adrenoceptors are not present in this organ (Shima, 1993; Sadraei et al., 1995). Radioligand binding assays (Shima, 1993) and functional studies, using selective a1A- and a1D- adrenoceptor antagonists (Silva et al., 1999), indicated that a1A-adrenoceptor subtype predominates in the rat seminal vesicle, although a1a-, a1b- and a1dadrenoceptor subtypes have been detected in this tissue (Silva et al., 1999). The aim of the present study was to investigate whether different androgen manipulations, induced by surgical and chemical castration, have effects on the expression of a1-adrenoceptor mRNA subtypes in the rat seminal vesicle. Furthermore, the effects of castration on the contractility and pharmacological characteristics of a1adrenoceptors in the rat seminal vesicle were also explored by using functional studies.
Materials and methods Animals and treatments Male Wistar rats (120-day old, 210–220 g) were housed in the Animal Facility at Instituto Nacional de Farmacologia, Universidade Federal de Sa˜o Paulo-Escola Paulista de Medicina (UNIFESP-EPM) and maintained on a 12 h light, 12 h dark lighting schedule, at 20jC, food and water ad libitum. Animal procedures were approved by the Research Ethical Committee from UNIFESP-EPM. Animal groups included the following: NC, normal control; C, rats surgically castrated and sacrificed on days 1, 2, 7 and 15 after castration; CA, rats chemically castrated by the use of daily injections of cyproterone acetate (7.6 mg/100 g body weight, i.m.) and sacrificed on days 1, 2, 8 and 15 of treatment; C 7 days + T 3 and C 7 days + T 6, rats surgically castrated for 7 days (C 7 days) and then treated daily with testosterone propionate (1 mg/100 g body weight, s.c.) for 3 (T 3) or 6 days (T 6). Preliminary experiments with tissues from sham-operated rats or animals injected with drug vehicles were also tested as controls. Since no significant changes were observed when these two experimental groups were compared to normal control, all subsequent experiments were performed with tissues from normal rats as control.
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Testosterone propionate was dissolved in ethanol and soybean oil (1:3 v/v). Cyproterone acetate was dissolved in benzyl benzoate and soybean oil (5:1, v/v). Measurement of testosterone plasma levels Blood from the aorta artery was collected, and testosterone plasma levels were measured by RIA using Coat-A-Count Total Testosterone kit (Diagnostic Products Co., Los Angeles, CA) according to manufacturer’s instructions. The assay detection limit was 0.04 ng/ml. The intraassay and interassay coefficients of variation were 1.8% and 2.1%, respectively. RNA isolation The animals were sacrificed, seminal vesicles were immediately removed, free of connective and fat tissue, separated from coagulating gland, frozen in liquid nitrogen and stored at 75jC until use. Total RNA was extracted from frozen tissues as previously described (Chirgwin et al., 1979). RNA samples were then quantitated, using a spectrofotometer at 260/280 nm and stored at 75jC for later use. Ribonuclease protection assay Assays were performed by using MAXIscript in vitro translation and RPA IIk kits from AMBION, Inc. (Austin, TX, USA), as previously described (Queiro´z et al., 2002). The DNA templates for antisense a1adrenoceptor RNA synthesis were kindly provided by Dr. Paul C. Simpson (University of San Francisco, USA) and they are described in Rokosh et al. (1994). RNA probes were radiolabeled with [a32P]UTP in the presence of T7 RNA polymerase. Linearized plasmid containing a 400 bp glyceraldehyde phosphate dehydrogenase (GAPDH) gene fragment in the antisense orientation under the transcriptional control of T7 promoter was from AMBION. Sizes (nucleotides) of the probes and protected fragments were as follows: a1a 408/315, a1b 495/432, a1d 267/217 and GAPDH 413/316, respectively. For each a1adrenoceptor probe, the fraction of uridine residues available for radiolabeling was similar: a1a, 25%; a1b, 20% and a1d, 19%. Experiments were performed in the presence of 40 Ag of total RNA. Throughout the experiments, control lanes containing yeast RNA (40 Ag) and a1-adrenoceptor labeled probes, incubated or not with RNase A/T1 mix, were included in order to visualize full length transcripts and efficiency of RNase digestion, respectively. Total RNA from adult rat brain and heart was used as positive control for a1a-/a1d- and a1b-adrenoceptor transcritp expression, respectively (Rokosh et al., 1994). Autoradiographs were scanned using a Schimadzu densitometer (Shimadzu Scientific Instruments, Princeton, NJ, USA). The steady state level of each mRNA transcript, as estimated by the area under the curve, was normalized against that of the GAPDH mRNA in each sample, as previously described (Queiro´z et al., 2002). Since the ratio between the levels of a1-adrenoceptor subtypes and GAPDH mRNA depended on the exposure time of the autoradiogram, the normalized data of the different groups were expressed as the percentage of this ratio for normal control in each experiment. Tissue preparation and record of seminal vesicle contraction Seminal vesicles from normal control (NC) and 7 days surgically castrated rats (C 7 days) were dissected, free of connective and fat tissue, separated from coagulating gland and washed internally with
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a nutrient solution of the following composition (mM): 136.89 NaCl, 5.63 KCl, 1.80 CaCl2, 0.36 NaH2PO4, 14.88 NaHCO3 and 5.55 glucose (pH 7.6–7.8). The tissue preparation and record of seminal vesicle contraction were performed as described previously (Silva et al., 1999). Briefly, the organ was suspended, under a load of 0.5 g, into organ baths filled with nutrient solution bubbled with air, at 30jC. Propranolol (10 6M) and cocaine (10 5M) were kept in the nutrient solution throughout the experiments in order to block h-adrenoceptor and catecholamine neuronal uptake, respectively. Isometric contractions were recorded with a force-displacement transducer connected to a polygraph (Gemini 7070, Ugo Basile, Viarese, Italy). Preliminary experiments, in the absence of antagonists, were carried out and no difference in the sensitivity to phenylephrine among three consecutive concentration-effect curves were observed. BaCl2 (0.03M) was always added to the preparation, at the end of the experiment, in order to obtain the maximal contraction of the tissue (Jurkiewicz et al., 1976). After the determination of the second concentration-effect curve to phenylephrine, the agonist was washed out for at least 30 min and then an antagonist was incubated for 30 or 45 min. After this period, another concentration-effect curve to phenylephrine was obtained. The following a1-adrenoceptor antagonists were used: WB 4101, which shows higher affinity for a1A-adrenoceptors (Hieble et al., 1995; Langer, 1998; Docherty, 1998; Silva et al., 1999); BMY 7378, which recognizes preferentially a1D-adrenoceptors (Saussy et al., 1996; Deng et al., 1996; Yang et al., 1997; Xin et al., 1997); chloroethylclonidine (CEC), an irreversible a1adrenoceptor antagonist (Ford et al., 1994; Michel et al., 1995; Hieble et al., 1995; Graham et al., 1996) that inactivates 80% of both a1B- and a1D-adrenoceptors and 20% of the a1A-adrenoceptors (Laz et al., 1994; Hieble et al., 1995; Xiao and Jeffries, 1998); and (+)cyclazosin, a potent a1B-adrenoceptor antagonist, that displays a 90- to 130-fold selectivity for binding to rat a1B-adrenoceptors when compared to rat a1A- and a1D-adrenoceptor subtypes (Giardina` et al., 1995, 1996). Data analysis The data were expressed as g tension/100 mg wet tissue and analysed by the interactive nonlinear regression through the computer program GraphPad Prism (GraphPad Prism Software Inc., San Diego, CA, USA). Each tension was normalized according to the seminal vesicle weight for each treatment group. The maximal contraction was obtained from the concentration-effect curve to phenylephrine and BaCl2. The EC50 value, concentration that produces 50% of phenylephrine maximum response, was also determined. The antagonist potency was expressed as pKB values, i.e., the negative log of the dissociation constant KB, which equals to the molar concentration of the antagonist divided by the concentration-ratio minus one (Besse and Furchgott, 1976). Statistical analysis Data were expressed as mean F S.E.M. Statistical analysis was carried out using analysis of variance (ANOVA) followed by Newman-Keuls test for multiple comparisons, or by the two-tailed Student’s t-test to compare two responses (Snedecor and Cochran, 1980). P values < 0.05 were accepted as significant. Drugs and reagents The following drugs and reagents were used: phenylephrine (L-phenylephrine hydrochloride), propranolol (DL-propranolol hydrochloride), testosterone (testosterone propionate) were from Sigma
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Chemical Co. (St. Louis, MO, USA). WB 4101 (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4benzodioxane hydrochloride), BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5] decane-7,9-dione dihydrochloride), chloroethylclonidine (chloroethylclonidine dihydrochloride), (+)cyclazosin (cyclazosin hydrochloride) were from Research Biochemicals International (Natrick, MA, USA). Cocaine (cocaine hydrochloride) was from Merck (Darmstadt, FRG). Cyproterone acetate (ANDROCURR) was from Schering (Sa˜o Paulo, SP, Brazil). [a32P]UTP (800 Ci/mmol, 10 mCi/ml) was from New England Nuclear (Boston, MA, USA). MAXIscriptk transcription Kit, RPA IIIk assay Kit, RNA Century Marker Template Plus were from AMBION, Inc. (Austin, TX, USA). All other drugs and reagents were from Sigma Chemical Co. or GIBCO (Gaithersburg, MD, USA).
Results Testosterone plasma levels and seminal vesicle wet weight The rat androgen status was monitored by measuring both the testosterone plasma level and the seminal vesicle wet weight (Table 1). A reduction in circulating testosterone was already detected in the first day after surgical castration. After 7 and 15 days of the surgical procedure, hormone plasma levels were below the detection limit of the assay. Testosterone treatment for 3 or 6 days, in a group of animals that had been castrated previously for 7 days, restored hormone plasma levels above those observed in normal control rats. Chemical castration, induced by daily injections of cyproterone acetate for 1, 2 days (data not shown) or 8 days, did not affect the testosterone plasma levels when compared to normal control (Table 1). After 15 days of antiandrogen treatment, however, hormone plasma levels were significantly reduced when compared to normal control (Table 1). Surgical castration for 1 or 2 days had no effect on the wet weight of seminal vesicle (Table 1). However, a decrease of 39% and 52% on this parameter was observed after 7 and 15 days of the Table 1 Testosterone plasma level and seminal vesicle wet weight from NC, normal control; C, rats surgically castrated and sacrificed on days 1, 2, 7 and 15; C 7 days F T, rats surgically castrated for 7 days and then treated for 3 (T 3) or 6 (T 6) consecutive days with testosterone propionate; CA, rats chemically castrated by daily injections of cyproterone acetate and sacrificed on days 8 and 15 Animals
Testosterone (ng/ml)
Seminal vesicle wet weight (mg)
NC C 1 day C 2 days C 7 days C 15 days C 7 days + T 3 days C 7 days + T 6 days CA 8 days CA 15 days
1.18 F 0.18 F N.D. < 0.04a < 0.04a 16.9 F 19.0 F 1.18 F 0.12 F
169.4 147.5 140.8 103.6 81.2 190.0 264.5 120.0 83.7
0.19 0.01a
0.70a 1.00a 0.17 0.02a
Data are mean F S.E.M. of 3 – 7 different experiments. N.D. not determined. a P < 0.05 when compared with NC group. b P < 0.05 when compared with C 7 days group.
F F F F F F F F F
13.6 16.5 9.2 4.2a 2.1a 6.0b 16.5a,b 2.2a 7.4a
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surgical procedure, respectively. The reduction in the tissue wet weight, caused by 7 days of surgical castration, was reversed to levels similar or above those observed in normal control when animals were treated with testosterone for 3 or 6 days, respectively. Chemical castration, induced by
Fig. 1. Ribonuclease protection assay for a1-adrenoceptor mRNA subtypes in rat seminal vesicle. Total RNA (40 Ag) from rat seminal vesicle were hybridized to 32P-labeled cRNA of a1a-, a1b-, a1d-adrenoceptor and GAPDH transcripts. Panel (A): Representative autoradiogram of 4 – 5 different experiments. On the left, 32P-RNA molecular markers (MW). On the right, arrows indicate specific protected products. NC, normal control rats; C, rats surgically castrated and sacrificed on days 1, 2, 7 and 15; C 7 days + T, rats surgically castrated for 7 days and then treated for 3 (T 3) or 6 (T 6) consecutive days with testosterone propionate; CA, rats chemically castrated by daily injections of cyproterone acetate and sacrificed on days 1, 2, 8 and 15. Panel (B): Autoradiograms were scanned and densitometry was used to quantify the relative changes in a1a-, a1b- and a1d-adrenoceptor mRNA expression with different animal treatments. Steady state levels of each a1-adrenoceptor mRNA were normalized against that of the GAPDH mRNA and expressed as the percentage (mean F S.E.M.) of a1-adrenoceptor/GAPDH mRNA ratio for NC group in each experiment (4 – 5 different experiments). aP < 0.05 compared with NC group; bP < 0.05 compared with C 7 days group.
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daily treatment with cyproterone acetate, also induced a decrease of 29% and 50% in seminal vesicle wet weight after 8 and 15 days of treatment, respectively, when compared to normal control (Table 1). Changes in rat body weight were not observed with any of the treatments used (data not shown). Expression of a1-adrenoceptor mRNA subtypes in rat seminal vesicle Total RNA isolated from rat seminal vesicle was tested in ribonuclease protection assays to detect the steady state of a1-adrenoceptor mRNA subtypes. In normal control rats, a1a-mRNA was the predominant transcript while a1d-adrenoceptor was less abundant (Fig. 1). The expression of a1b-adrenoceptor mRNA was considerably lower when compared to the other two transcripts, being only detected when longer exposures of the gel were obtained (data not shown). Since the fraction of uridine residues available for radiolabeling of each a1-adrenoceptor cRNA probes was similar, the results show that the ratio among transcripts is a1aHHa1dHa1b. Ribonuclease protection assays were also used to investigate the androgen regulation of a1adrenoceptor transcripts in rat seminal vesicle (Fig. 1A). Densitometry was used to quantify the relative changes in mRNA expression with the different animal treatments. GAPDH mRNA, a housekeeping gene, was used as an internal control and data were expressed as the percentage of a1-adrenoceptor and GAPDH mRNA ratio for normal control in each experiment (Fig. 1B). Surgical castration induced different changes depending on the a1-adrenoceptor transcript analysed. The steadystate of a1a-adrenoceptor mRNA significantly decreased within 2 days postcastration and then increased after 7 and 15 days postcastration, when compared to normal controls. The levels of a1d-adrenoceptor mRNA, on the other hand, were already above control level in the first day postcastration. The maximum level of this mRNA specie was reached 7 days after castration. a1b-adrenoceptor mRNA transcript, although not abundant in control tissues, was significantly higher than control level after 7 and 15 days of surgical castration. Testosterone replacement for 3 days was effective to reduce the levels of a1b- and a1d-adrenoceptor transcripts observed with 7 days castration. After 6 days of testosterone treatment, on the other hand, all three mRNA subtypes were reduced to values similar or below normal control (Fig. 1B). Chemical castration, induced by cyproterone acetate, was also used to evaluate hormonal regulation of a1-adrenoceptor mRNA subtype expression. Although 1 and 2 days of treatment with this drug had no effect on the expression of a1-adrenoceptor transcripts, all three a1-adrenoceptor mRNA subtypes were up-regulated significantly after 8 and 15 days of treatment (Fig. 1B).
Table 2 Contractile response induced by BaCl2 and phenylephrine-induced contraction in seminal vesicle from normal control (NC) rats and 7 days castrated rats (C 7 days) Animals NC C 7 days
Maximal Contraction (g tension/100 mg wet tissue)
EC50 (AM)
BaCl2
Phenylephrine
Phenylephrine
1.63 F 0.12 (5) 3.36 F 0.39* (6)
1.00 F 0.06 (21) 1.95 F 0.12* (20)
17.40 F 3.98 (21) 33.50 F 8.57 (20)
Data are mean F S.E.M. of number of experiments in parenthesis. * Significantly different from control rats ( P < 0.05; Student’s t-test).
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Fig. 2. Effect of WB 4101, BMY 7378, chloroethylclonidine and (+)cyclazosin on phenylephrine-induced contractions in the rat seminal vesicle from normal control (NC, left panels) and 7 days castrated rats (C 7 days, right panels). Concentration-effect curves to phenylephrine were obtained in the absence (closed symbols) and presence of a1-adrenoceptor antagonists (open symbols). WB 4101 (10 9M, 5), BMY 7378 (10 7M, D), chloroethylclonidine (CEC) (10 5M, o) and (+)cyclazosin (10 9M, q). Each point and vertical line represents the mean F S.E.M. of n shown in Table 3.
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Table 3 pKB values for different a1-adrenoceptor antagonists tested against phenylephrine-induced contraction in seminal vesicle from normal control (NC) rats and 7 days castrated rats (C 7 days) Animal NC C 7 days
pKB WB 4101
BMY 7378
CEC
(+)Cyclazosin
8.63 F 0.10 (8) 8.58 F 0.11 (7)
5.81 F 0.05 (6) 5.70 F 0.15 (6)
4.49 F 0.01 (6) 4.49 F 0.01 (6)
9.88 F 0.04 (7) 9.93 F 0.03 (7)
Data are mean F S.E.M. of number of experiments in parenthesis. No statistical difference was observed among control and castrated rats ( P < 0.05; Student’s t-test).
Functional studies The maximal contraction induced by BaCl2 and phenylephrine, expressed as g tension/100 mg of wet tissue was approximately 2-fold higher in 7 days castrated rats than in normal control rats (Table 2). However, when the maximal contractile response to phenylephrine was normalized to the percentage of the maximal response to BaCl2, no significant difference was found between normal control and 7 days castrated rats (61% and 58%, respectively). In addition, the EC50 values for phenylephrine were similar in both groups (Table 2). WB 4101 (10 9M) and BMY 7378 (10 7M) produced rightward shifts of the concentration-effect curves to phenylephrine in seminal vesicle from normal control and 7 days castrated rats (Fig. 2). Chloroethylclonidine (CEC, 10 5M) and (+)cyclazosin (10 9M) shifted concentration-effect curves to the right and reduced the maximum contractile response induced by phenylephrine in tissue from normal control rats (40% and 67%, respectively) (Fig. 2). On the other hand, in the seminal vesicle from 7 days castrated rats, the maximum contractile response induced by phenylephrine decreased in the presence of chloroethylclonidine and (+)cyclazosin (22% and 54% respectively) (Fig. 2). The calculated pKB values for a1-adrenoceptor antagonists revealed similar values for normal control and 7 days castrated rats (Table 3). The order of potency for a1-adrenoceptor antagonists in the rat seminal vesicle from both experimental groups was (+) cyclazosin > WB 4101 { BMY 7378 > chloroethylclonidine.
Discussion In the present study, we have observed that, under normal physiological conditions, a1a-adrenoceptor mRNA was the predominant a1-adrenoceptor subtype present in this tissue, while a1d- was less abundant and a1b-adrenoceptor mRNA was only weakly expressed in seminal vesicle from adult rats. Considering that this tissue is under androgen control, we have studied the impact of changes in testosterone status caused by surgical and chemical castration on the relative expression of seminal vesicle a1-adrenoceptor mRNA subtypes. We were able to demonstrate that surgical castration differently regulates the expression of a1-adrenoceptor transcripts. a1a-adrenoceptor mRNA showed a complex dependency on androgens, since its expression was down-regulated within the first 2 days postcastration, but increased after 7 and 15 days of the surgical procedure. a1b- and a1d-transcripts, poorly expressed in seminal vesicle of control animals when compared to a1a-adrenoceptor mRNA, were both up-regulated with prolongation of surgical castration, indicating a negative dependency of these transcripts on androgens. The ability of testosterone to revert the changes in a1-adrenoceptor transcripts induced by 7
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days of surgical castration suggested that these mRNA species are physiologically dependent on androgens. Furthermore, the results show that the sustained blockade of androgen receptor with cyproterone acetate treatment for 8 and 15 days induced an up-regulation of a1a-, a1b- and a1dadrenoceptor transcripts, confirming that androgen signalling is involved on this regulation. The up-regulation in a1d-adrenoceptor mRNA level was already observed within 1 day after surgical castration. Although a1a-adrenoceptor transcript was down-regulated after 2 days, both a1a- and a1badrenoceptor were up-regulated with prolongation of castration for 7 and 15 days. Thus, the temporal changes in a1-adrenoceptor subtype expression with surgical castration suggest that the sensitivity of the a1-adrenoceptor gene subtypes to testosterone or androgen-related factors is different. A number of mRNA transcripts studied during castration-induced involution and androgen-induced regrowth of tissues in the male reproductive system, including rat seminal vesicle, present complex and negative dependency on testosterone (Collins et al., 1988; Huang et al., 1997; Nishi et al., 1996). It is difficult to correlate the a1-adrenoceptor mRNA variations in seminal vesicle during androgen manipulation of the animals to a direct hormonal effect. Although promoter regions of human a1a-adrenoceptor (Razik et al., 1997) and rat a1b-adrenoceptor gene (Gao et al., 1995; Gao and Kunos, 1998) contain multiple binding sites for sequence-specific proteins, no functional androgen response elements have been described for the three different a1-adrenoceptor genes. It is possible that the changes in a1-adrenoceptor mRNA expression after surgical and chemical castration may be due to an indirect effect of testosterone, where an androgen-sensitive factor might be involved in the expression and/or stabilization of the transcript. Furthermore, since the steady state of mRNA reflects the balance between the rate of transcription and degradation of messages, factors responsible for either mechanism could be involved. Since the changes in mRNA levels in a given tissue may not always correlate directly with changes at protein levels (Scofield et al., 1995; Docherty, 1998), functional studies were designed to observe the effects of 7 days castration on the contractile responses induced by activation of a1-adrenoceptor in rat seminal vesicle. The maximal contractile response to BaCl2 and phenylephrine, expressed as g tension/ 100 mg of tissue wet weight, was approximately 2-fold higher in 7 days castrated rat seminal vesicle. This apparent increase in phenylephrine-induced maximal response was normalized when the proportion due to BaCl2-induced contraction was taken into account. Furthermore, this increase in contractility is most likely explained by the change in tissue components of the seminal vesicle, similar to that reported for rat prostate (Lin et al., 1995; Lacey et al., 1996). Upon 30 days castration, seminal vesicle epithelium undergoes degeneration, whilst smooth muscle, responsible for the contractile property of the seminal vesicle, is preserved (unpublished data). In addition, we found that EC50 values for phenylephrine were similar in both control and 7 days castrated rats, indicating that castration caused no essential changes in the affinity of phenylephrine to a1-adrenoceptor. The pKB values calculated for all antagonists tested were similar between normal control and 7 days castrated rats. WB 4101 presented high affinity in seminal vesicle from normal control and 7 days castrated rats, while the a1D-adrenoceptor subtype selective antagonist, BMY 7378, presented a relatively low affinity, indicating that a1A-adrenoceptor is predominantly involved in the rat seminal vesicle contraction when compared to a1D-adrenoceptor subtype as previously reported in the seminal vesicle from 60-day-old rats (Silva et al., 1999). We tested the sensitivity of seminal vesicle contractions induced by phenylephrine to the alkylating agent chloroethylclonidine, an irreversible antagonist which inactivates 80% of both a1B- and a1D- and 20% of a1A-adrenoceptors (Laz et al., 1994; Hieble et al., 1995; Xiao and Jeffries, 1998). Our results showed that chloroethylclonidine shifted the phenylephrine-induced curves to the right, with 40% and
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22% reduction in the maximum contractile response induced by this agonist when seminal vesicle from normal control and 7 days castrated rats were tested, respectively. It is known that the degree to which chloroethylclonidine affects the functional response depends, on some extent, on the amount of spare receptors present in the tissue (Burt et al., 1995; Marshall et al., 1995). In the rat seminal vesicle, 0.10% of the a1-adrenoceptor pool represents a spare receptor population (Silva et al., 1999). Furthermore, the most interesting result of the present investigation was the high affinity displayed by (+)cyclazosin (pKB values = 9.88 F 0.04 and 9.93 F 0.03 in seminal vesicle from normal control and 7 days castrated rats, respectively) similar to the affinity of this antagonist for native a1B- and cloned a1b-adrenoceptors (pKi = 9.57 F 0.01 and 9.23 F 0.04, respectively) (Giardina` et al., 1995; Stam et al., 1998), indicating that functional a1B-adrenoceptor may also be involved in the seminal vesicle contractile response. The relative participation of each a1-adrenoceptor subtype (a1A- and a1B-adrenoceptors) in the seminal vesicle contraction remains to be established. The decrease of the maximum contractile response to phenylephrine was more readily detected with (+)cyclazosin in normal control rats (67%) than in 7 days castrated rats (54%). The effects of chloroethylclonidine and (+)cyclazosin on phenylephrine-induced curves obtained from normal control and 7 days castrated rats may indicate that a1B-adrenoceptor is upregulated after castration, confirming the upregulation of a1b-adrenoceptor transcripts obtained in ribonuclease protection studies. Shima (1992) observed that castration increased the number of a1-adrenoceptor in adult rat seminal vesicle, an effect reversed by androgen replacement. Lacey et al. (1996) found an apparent 2-fold increase in prostatic a1-adrenoceptor density as a consequence of androgen deprivation in rats. These authors speculated that this increase was an artifact due to a relative increase in the ratio of smooth muscle, which is mostly responsible for prostatic contraction, to epithelium in the prostate rather than from an up-regulation of a1-adrenoceptors. Lin et al. (1995) reported no changes in the in vivo measurement of dog prostatic urethral pressure by androgen deprivation. It has been shown that the androgen deprivation decreased the contraction potency induced by phenylephrine in rat prostate strips, probably associated with down-regulation of a1A-adrenoceptor spare population expressed in the intact prostate (Homma et al., 2000). Conversely, rat prostatic hypertrophy induced by testosterone resulted in a reduction of the a1-adrenoceptor density, occuring in parallel with a decrease in the amount of fibromuscular element in the entire prostate when compared to control rats (Auger-Pourmarin et al., 1998). Recently, it has been shown that the prolongation of castration for up to 30 days differently affects the potency of and maximal contractions induced by noradrenaline in the rat vas deferens (Campos et al., 2003). Castration also reduced phenylephrine-induced increase in urethral pressure in rats (Akiyama et al., 1999), while the treatment with dihydrotestosterone and prazosin increased prostate weight, its stiffness and urodynamic obstruction by prostate (Lee et al., 1998). In the present work, functional studies confirmed the involvement of a1A- and a1B-adrenoceptors in the seminal vesicle contractile response, and suggested that a1B-induced contraction is upregulated after castration. Interestingly, the expression pattern of each a1-adrenoceptor mRNA subtype (a1a, a1b and a1d) was differentially regulated by the androgen status of the rat. Taking into consideration that different cell types are present in seminal vesicle (Williams-Ashman, 1983), further studies focusing receptormediated intracellular signaling coupled to immunological techniques to localize and determine the relative levels of a1-adrenoceptor subtypes will be an important tool to understand the relative contribution of a1-adrenoceptor in the seminal vesicle.
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Acknowledgements This work was supported by Fundacßa˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP, grant number 96/1777-1), Brazil and in part by T.W. Fogarty International, USA (parent grant 5H37HD0446626, subcontract UNC 5-53284). Researcher fellowships (M.C.W.A. and C.S.P.) and scholarships supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (F.R.M. and D.B.C.Q.) and by FAPESP (M.H.), Brazil. We are indebted to Dr. Paul C. Simpson (University of San Francisco, CA, USA) for provision of DNA templates for antisense a1-adrenoceptor RNA synthesis and Dr. Rodolfo P. Rothlin for kindly provided the (+)cyclazosin. We gratefully thank the technical assistance of Espedita M. Jesus and Maria D. Silva.
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