Abstract
The aim of this study was to characterize the type of 5′-deiodinase activity in the prostate of pubescent rats (7–8 weeks), to establish its distribution in the lobes (ventral, dorsolateral, and anterior), and to analyze its modulation by prolactin (PRL), testosterone, dihydrotestosterone (DHT), and 17β-estradiol (E2). Our results showed that the enzymatic activity was highly susceptible to inhibition by 6-n-propyl-2-thiouracil and gold thioglucose, its preferential substrate was reverse tri-iodothyronine (rT3), it exhibited a low dithiothreitol requirement (5 mM), and the apparent Km and Vmax values for substrate (rT3) were approximately 0.25 μM and 9.0 pmol liberated/mg protein per hour, respectively. All these characteristics indicate the preferential expression of type 1 deiodinase (D1), which was corroborated by demonstrating the presence of D1 mRNA in prostate. D1 activity was detected in all lobes and was most abundant in the dorsolateral. Although we detected type 2 deiodinase (D2) mRNA expression, the D2 activity was almost undetectable. D1 activity was enhanced in animals with hyperthyroidism and hyperprolactinemia, in intact animals treated with finasteride (inhibitor of local DHT production), and in castrated animals with E2 replacement. In contrast, activity diminished in castrated animals with testosterone replacement. Our results suggest that thyroid hormones, PRL, and E2 exert a positive modulation on D1 activity, while testosterone and DHT exhibit an inhibitory effect. D1 activity may be associated with prostate maturation and/or function.
Introduction
Sex hormones, prolactin (PRL), and thyroid hormones (TH) modulate, in a synergistic or antagonistic manner, several aspects of reproductive physiology (Longcope 2000a, 2000b, Cooke et al. 2004). The prostate is a gland of the male reproductive system that differentiates and achieves functional maturity during puberty (Cunha et al. 1987). For many years, the development and functionality of the prostate was believed to be solely dependent on androgens. Although it is still widely accepted that androgens are essential, evidence exists that estrogens and PRL modulate various aspects of prostate growth, development, and metabolism (Ahonen et al. 1999, Jarred et al. 2000, Costello & Franklin 2002). With regard to TH participation, human studies show that hyperplasia and prostate cancer are associated with high serum concentrations of 3,5,3′-tri-iodothyronine (T3) (Lehrer et al. 2002). Moreover, in human cells of lymph node carcinoma prostate (LNCaP), it has been demonstrated that T3 and testosterone have synergistic effects on proliferation and cellular differentiation processes (Zhang et al. 1999). Some of these effects are explained by the well-known stimulation of androgen receptor synthesis by T3 (Esquenet et al. 1995). A few studies exist on the effects of TH on normal prostate, for example, TH modulate the activity of different enzymes involved in glycoprotein metabolism in rat prostate (Maran et al. 1998, 2000). In order for TH to exert their effects at the nuclear level, the prohormone thyroxine (T4) must be transformed intracellularly into its most active form (T3) (Bianco et al. 2002, Kohrle 2002). There is evidence showing that rat prostate produces T3 locally; however, the enzymatic mechanisms have not been characterized (Schrodervan der Elst & Van der Heide 1990). In many different tissues, T4 is converted into T3 by a deiodination catalyzed by one or both of two isoenzymes called type 1 (D1) and type 2 (D2) deiodinases. Both enzymes remove one iodine atom from position 5′ of the T4 outer ring, giving rise to T3 (Bianco et al. 2002, Kohrle 2002). These enzymes have been cloned and biochemically and molecularly characterized. D1 activity exhibits a ‘ping-pong’ kinetic pattern, its preferential substrate in vitro is 3,3′,5′-tri-iodothyronine (rT3), and it requires low co-factor concentrations (5 mM approximately). D1 exhibits a Km for T4 1000-fold higher than that of D2. In addition, its activity is highly sensitive to inhibition (IC50) by 6-n-propyl-2-thiouracil (PTU 10 μM) and gold thioglucose (GTG 0.02 μM). On the other hand, D2 activity shows a ‘sequential’ kinetic pattern, its preferential substrate is T4, and it requires higher cofactor concentrations (20 mM approximately). D2 activity is resistant to inhibition by PTU and GTG. The regulation of deiodinase activity is a complex process involving neuro-hormonal, environmental, and nutritional factors (Bianco et al. 2002, Kohrle 2002). Hepatic D1 activity is higher in male than in female rats (Harris et al. 1979); although the mechanisms have not been completely elucidated, the participation of testosterone in this sexual dimorphism has been demonstrated (Miyashita et al. 1995). The present study was designed to characterize the type of deiodinase activity present in rat prostate and to analyze its lobular distribution (ventral, dorsolateral, and anterior) and its modulation by TH, PRL, and sex hormones (androgens and estrogens). Our results show that D1 enzyme is present in rat prostate and the dorsolateral lobe exhibits the highest activity. In addition, our evidence suggests that TH, PRL, and 17β-estradiol (E2) stimulate prostatic D1 activity. In contrast, androgens appear to inhibit this activity.
Material and Methods
Reagents
Thyronines were obtained from Henning Co. (Berlin, Germany). 125I-rT3 (1174 μCi/μg) and 3H-E2 (162 μCi/nmol) were purchased from Perkin Elmer Life Sciences (Boston, MA, USA). Dithiothreitol (DTT) was obtained from Calbiochem (La Jolla, CA, USA). PTU and GTG were obtained from Sigma. Oligonucleotides were synthesized by Gibco BRL. Finasteride was obtained from Merck Co., testosterone from Organon (Mexico City, Mexico), and E2 from Schering Plough Corp. (Mexico City, Mexico). All other reagents were of the highest purity commercially available.
Animals
Male Wistar rats (7–8 weeks) were used. They weighed 230 ± 15 g. Animals were housed in stainless steel cages under controlled temperature (22 ± 1 °C) and lighting conditions (12 h light:12 h darkness cycle; lights on from 0700 to 1900 h). They had free access to rat chow (Purina, Richmond, CA, USA) and tap water. Surgical procedures were conducted under ketamine (30 mg/kg body weight) and xylazine (6 mg/kg body weight) anesthesia. Rats were killed by decapitation. Euthanasia of animals was reviewed and approved by an ad hoc ethics committee of the University of Mexico (UNAM). Whole prostates or individual lobes were removed, weighed, and analyzed for 5′-deiodinase activity. In some experiments, liver was used as a control tissue.
Experimental procedures
Experiment 1: kinetic characterization of deiodinase activity
These assays identified the type of deiodinase activity present in homogenates of prostate gland. Enzymatic activity was determined in three independent pools, each in duplicate. Characterization included the determination of the optimal conditions for the assays; protein concentration (50–500 μg), incubation time (1–4 h), and incubation temperature (4–45 °C). The affinity for substrate (rT3 vs T4) and the susceptibility to selective inhibitors for D1 activity, PTU (0.1–1.0 mM) and GTG (1–1000 nM), were also evaluated. D1 activity (PTU- and GTG-sensitive) was calculated as the difference between total activity and PTU-insensitive activity. The substrate and co-factor dependence were analyzed using the Lineweaver–Burk transformation (Copeland 1996).
Experiment 2: type and distribution of deiodinase activity in the different lobes
The enzymatic assays were performed using homogenates of the ventral, dorsolateral, and anterior lobes of the prostate, each with and without PTU (1 mM) and GTG (100 nM). The expression of mRNA for D1 and D2 in the different lobes was examined by reverse transcriptase (RT)-PCR.
Experiment 3: induction of hyperthyroidism
Hyperthyroidism was induced by adding T4 (6 μg/ml) to the drinking water for 3 weeks. The thyroid state was confirmed by circulating T3 levels.
Experiment 4: induction of hyperprolactinemia by ectopic pituitary grafts
Hyperprolactinemia was induced by implanting one pituitary under the kidney capsule of animals whose pituitary remained intact (Mena et al. 1968). The left kidney was exposed and a slit was made in the mid portion of the capsule. A small pocket was made in the kidney between the capsule and parenchyma. The pituitary was inserted into the pocket and the kidney capsule was closed using surgical catgut. Sham-operated rats were subjected to the same surgical procedure, except the pituitary was not grafted. Animals were killed 15 days after surgery. The acceptance of the heterograft was confirmed by enhanced circulating PRL. We used only those animals whose plasma PRL levels were above the control mean by at least 50%. Also we measured circulating T3 levels.
Experiment 5: finasteride treatment of intact (INT) rats
To assess whether dihydrotestosterone (DHT) participates in the regulation of prostate D1 activity, INT rats were subcutaneously injected (25 mg/kg bw) daily over the course of 10 days with finasteride. This drug is a selective inhibitor of 5-α-reductase type 2 and blocks the conversion of testosterone to DHT (Sudduth & Koronkowski 1993). The control group received vehicle (20% ethanol in oil).
Experiment 6: castration and hormonal treatments
To determine whether other sex hormones such as testosterone or E2 were responsible for the regulation of 5′-deiodinase activity, we evaluated the effects of androgen ablation and hormonal replacement in castrated rats. Rats were bilaterally castrated via the scrotal route. Hormonal treatment began 2 weeks after surgery. Testosterone (3 mg) and E2 (20 μg) were administered by i.m. injection of a slow-delivery oil solution once (testosterone) or twice (1 week apart, E2). In the sham-operated group, the testes were exposed but not removed.
Analytical procedures
Tissue homogenization
Prostatic and hepatic tissues were homogenized in 10 vol (w/v) of cold 0.01 M Hepes buffer (pH 7.6) containing sucrose (0.32 M) and EDTA (1.0 mM). Crude homogenates were centrifuged at 1500 g for 30 min at 4 °C. The supernatant was quickly frozen in dry ice and stored at −70 °C until assayed to determine deiodinase activity. In some assays, the liver was used as control tissue and was processed as above.
Enzymatic assay
Deiodinase activity was determined by a modification of the radiolabeled iodide release method (Leonard & Rosenberg 1980). The standard assay had a 100 μl final volume, 50 μl homogenate and 50 μl radiolabeled mix. Optimal assay conditions for prostate deiodinase activity were: 100 μg protein, 2 nM 125I-rT3, 0.5 μM unlabeled rT3, and 5 mM DTT, and the incubation time was 3 h at 37 °C. Liver was assessed under the same conditions except using 1–2 μg protein and a 1-h incubation. After the incubation time had elapsed, released acid-soluble radioiodide was isolated by chromatography on Dowex 50W-X2 columns (Bio-Rad). Protein was measured by the Bradford method (Bio-Rad). Results are expressed as picomole or nanomole I released/mg protein per hour.
Hormone levels
Circulating PRL and testosterone were determined with commercial enzyme immunoassay systems (rat PRL: Amersham Biosciences; testosterone: Assay Designs, Inc., MI, USA). T3 and E2 were quantified by RIA (Anguiano et al. 1991, Herrera et al. 1993).
RNA purification and RT-PCR analysis
Total RNA was isolated using Trizol reagent (Life Technologies). Analyses of D1 and D2 mRNA expression were performed by semi-quantitative RT-PCR as previously described (Aceves et al. 1999). Simultaneously, mRNA for a structural protein, cyclophilin, was amplified. Single-strand cDNA synthesis was performed with 2 μg total RNA using oligo d(T) as primer. Oligonucleotides used for RT-PCR are summarized in Table 1. The RT product was amplified in 50 μl PCR buffer containing 10 pmol of each oligonucleotide primer, 0.2 μM dNTPs, and 1 U DNA polymerase. Samples were subjected to 28 cycles consisting of 45 s at 94 °C, 45 s at 55 °C, and 45 s at 72 °C. The last extension was carried out for 10 min. As a control, a reaction mixture containing an RNA sample with the appropriate primers, but without the reverse transcriptase was included. The reaction products were analyzed by 2% agarose gel electrophoresis and the resulting bands were visualized by ethidium bromide staining. Band sizes were confirmed with a 1 Kb DNA ladder (Gibco-BRL). A Polaroid picture was taken; the pictures were digitized using a Hewlett Packard Scanner Jet 11CX and the signals were analyzed using an editing version of the NIH-image program (Bethesda, MD, USA).
Statistical analysis
All results are expressed as the mean ± s.e.m. Data were analyzed using the Student’s t-test or one-way ANOVA. Differences between means were tested by the Tuckey’s test. An asterisk or different letters identify significant differences between means (P ≤ 0.05).
Results
Assay validation and kinetic characterization
5′-Deiodinase activity was directly proportional to protein concentration (50–200 μg) and to incubation time (1–4 h). The highest activity was encountered between 20 and 37 °C (data not shown). Our data showed clear inhibition of deiodinase activity at different PTU and GTG concentrations (Fig. 1). Maximum inhibition was achieved at concentrations of 1 mM PTU and 100 nM GTG, suggesting the presence of D1 in prostate. This suggestion was tested by comparing rT3 and T4 as substrates. Using 125I-rT3 as substrate, the percentage of deiodination was suppressed more by non-radioactive rT3 than by non-radioactive T4 (Fig. 2). A slight suppression of 5 ′-deiodinase activity (10%) was observed with 125 nM unlabeled T4. A second experiment was performed using 2 nM 125T4 and different concentrations of unlabeled T4 (1–200 nM). The percentage of deiodination with 125T4 was less than 1.0%, and it did not change with increasing concentrations of unlabeled T4. Therefore, subsequent experiments were performed using only rT3 as substrate. Enzymatic activity was proportional to substrate and co-factor concentrations, and it showed a typical saturation pattern between 5 and 10 mM DTT and between 500 and 1000 nM rT3 (Figs 3 and 4). Double reciprocal plots of the data are represented in Fig. 4 (inset) and show the apparent kinetic constants for substrate of Km = 250 nM and Vmax = 9.0 pmol I liberated/mg protein per hour. Thus, the high susceptibility of 5 ′-deiodinase activity to inhibition by PTU (1 mM) and GTG (100 nM), low-cofactor requirement (5 mM), its preference for rT3 over T4, and its Km value for substrate (0.25 μM) all indicate the predominance of type D1 in rat prostate.
Distribution of deiodinases in prostate lobes
Rat prostate lobes exhibit a well-known functional heterogeneity (Cunha et al. 1987). In consequence, this experiment was designed to analyze the presence of deiodinases in the different lobes. Our results showed that D1 activity was present in all lobes with the highest activity in the dorsolateral (Fig. 5). Simultaneously, enzymatic activity was analyzed in the presence of PTU and GTG, and inhibition was found with both compounds. The presence of D1 activity in all lobes was corroborated by detection of its mRNA (Fig. 6). Contrary to expectations, amplification of D2 mRNA expression was observed (Fig. 6). Since the D2 activity was almost undetectable, the following experiments were carried out with the conditions optimal for D1 activity.
Modulation of D1 activity in prostate gland and liver
Hyperthyroidism effect
T4 treatment increased circulating levels of T3 and prostate D1 activity (Fig. 7).
PRL effect
The participation of this hormone in the modulation of prostate D1 activity was analyzed in a rat model with hyperprolactinemia. Our results showed that circulating levels of PRL significantly increased in animals with a pituitary transplant (Fig. 8). This enhancement was accompanied by a significant increase in prostate D1 activity, but liver D1 activity (Fig. 9) and circulating T3 levels (Fig. 8), as well as body and prostate weights were unmodified in animals with hyperprolactinemia.
DHTeffect
The effect of DHTon D1 activity was analyzed in a group of INT animals treated with finasteride. This treatment had no effect on the body weight of the animals, but it caused a significant decrease in prostate weight (Table 2). Figure 10 shows that the finasteride treatment selectively enhances prostate D1 activity.
Testosterone and E2 effects
In this study, we analyzed the combined effects of castration and hormonal replacement on prostate D1 activity. The results in Table 3 show that both circulating levels of testosterone and prostatic weight are reduced by castration. Testosterone replacement returned prostate weight to values similar to those in the sham group and slightly augmented circulating PRL levels. E2 replacement had no effect on prostate weight, but it did increase PRL levels. These results confirm the well-known testosterone tropic effects on the prostate gland and corroborate testosterone and E2 stimulatory effects on PRL synthesis and/or secretion in male rats (Herbert et al. 1977, De las Heras & Negro-Vilar 1979). Results of prostate D1 activity are shown in Fig. 11. Surprisingly, castration was accompanied by a significant increase in prostate D1 activity, whereas after testosterone replacement, activity values were similar to those in the sham group. In contrast, E2 administration significantly raised both circulating E2 levels and deiodinase activity, surpassing not only the values of the sham group but also those of the CX group (castrated animals injected with vehicle). However, when testosterone and E2 were simultaneously administered, the enzymatic E2 response was abolished. The effects were the opposite on hepatic D1 activity, that is, castration significantly decreased the enzymatic activity, while testosterone replacement increased basal levels (Fig. 11). Our results in liver corroborate previous studies in the literature showing that testosterone positively modulates hepatic D1 activity (Miyashita et al. 1995, Lisboa et al. 2001).
Discussion
It is now well established that during development and sexual maturation, a close functional interaction exists among TH, PRL, and sex hormones (Cooke et al. 2004, Meikle 2004). However, the effect of TH on the male reproductive system, particularly on accessory glands, has been little explored. In this study, we characterize for the first time 5′-deiodinase activity in rat prostate and analyze its possible modulation by these hormones. The high susceptibility of 5′-deiodinase activity to complete inhibition by PTU (1 mM) and GTG (100 nM), its low DTT requirement (5 mM), the preference for rT3 rather than T4 as substrate, and the identification of mRNA for D1, constitute strong evidence for the presence of the D1 enzyme in rat prostate. PTU and GTG concentrations required to inhibit more than 85% of the activity are comparable to those reported in the literature for tissues having D1 activity, such as liver and kidney (Berry et al. 1991, Sun et al. 1997). The substrate Km value (rT3) is in the same range as those found in other tissues with D1 activity (Leonard & Rosenberg 1980, St. Germain 1988, Aceves & Valverde 1989). As would be expected, the increase observed in response to hyperthyroidism is consistent with the well-documented conclusion that T3 increases D1 activity by a transcriptional mechanism (Berry et al. 1990).
The dorsolateral lobes exhibit the highest activity suggesting a differential production of T3 in prostate. Our results can only hint at potential functional implications of this lobe specificity, but the highest levels of activity could be associated with specific physiological characteristics of this lobe (i.e. the high-level expression of zinc-transporter-2, preferential secretion of specific monosaccharides, and, in some strains, the high susceptibility to develop age-dependent hyperplasia) (Maran et al. 2000, Shirai et al. 2000, Iguchi et al. 2002). An important finding in our study was the significant and positive influence of PRL on prostate D1 activity. Dorsolateral lobes possess the highest number of PRL receptors and are also the most responsive to proliferative, anti-apoptotic, and metabolic PRL effects (Thomas & Manandhar 1977, Ahonen et al. 1999). In addition, circulating levels of this hormone increase significantly during puberty (Negro-Vilar et al. 1973, Nicoll 1974). To our knowledge, the present study is the first to show a stimulatory effect of PRL on D1 activity. We have reported previously that PRL inhibits the D1 response to norepinephrine in lactating mammary gland (Aceves et al. 1999, Anguiano et al. 2004). The absence of a hepatic D1 response found here indicates that PRL modulates selectively the activity in prostate and agrees with previous work showing that deiodinase enzymes are regulated in an organ-specific manner (Bianco et al. 2002, Kohrle 2002). The mechanisms by which PRL regulates prostate D1 activity are unknown, but the absence on gene D1 of sites responsive to second messenger pathways mediated by PRL (Stat-5, for example) suggests that PRL may exert its stimulating effect indirectly by regulating sex-hormone effects. Our results showed that androgens inhibit prostate D1 activity, since the activity was significantly enhanced not only in animals treated with finasteride, but also in those that were castrated. Moreover, this activity decreased in castrated animals with testosterone replacement. These data agree with previous reports showing that hyperprolactinemia is accompanied by a reduction in circulating testosterone levels, as well as a decrease in local production of DHT in prostate (Lee et al. 1985, Sluczanowska-Glabowska et al. 2003). Therefore, it is probable that PRL effects on prostate D1 activity are secondary to its inhibitory effects on androgens.
The second interesting result in the present study was the clear-cut stimulating effect of E2 on prostate D1 activity. Indeed, our data showed that E2 causes a tenfold stimulation of the basal values of D1 activity and a twofold increase compared with the levels after castration and hyperprolactinemia. These data are consistent with previous reports showing that E2 administration significantly increases D1 activity in the pituitary gland from ovariectomized animals (Lisboa et al. 2001). Since E2 replacement was accompanied by an increase in circulating PRL levels (De las Heras & Negro-Vilar 1979, Shin 1979), our results do not distinguish whether the effects are direct or mediated by PRL. There is evidence showing that prostate expresses E2 receptors (Pelletier et al. 2000, Asano et al. 2003) and that testosterone inhibits α-estrogen receptor expression, not only in prostate but also in other tissues, such as mammary epithelium (Zhou et al. 2000, Asano et al. 2003).
Although the D2 activity in the prostate was almost undetectable, further studies will be performed to analyze the possible contribution of this type of deiodinase in the function of prostate gland. We do not know the biological significance of prostate D1 activity, but we propose that local T3 production combined with PRL and the relative amounts of androgens and estrogens may modulate the growth and/or differentiation of the gland during puberty, as well as its metabolic expenditure. This suggestion is supported by unpublished data from our laboratory showing that regular sexual activity is accompanied by a significant increase in D1 activity. Although the presence of thyroid hormone receptors has not been described in normal prostate tissue, recent reports indicate their presence in the cancer human cell line (Hsieh & Juang 2005).
On the other hand, due to the exocrine nature of the prostate, our results leave open a possible role of prostate D1 activity in seminal plasma. Moreover, we and other authors have found deiodinase activity in seminal plasma (Brzezinska et al. 2000, B Anguiano, unpublished observation) and in other types of secretions, such as milk (Slebodzinski et al. 1998).
Oligonucleotides used in the RT-PCR amplification
| Size (bp) | Sequence | Type | GenBank | |
|---|---|---|---|---|
| D1 | 561 | 66–82 CTT GGA GGT GGC TAC GG | Sense | X57999 |
| 627–610 CTG GCT GCT CTG GTT CTG | Antisense | |||
| D2 | 589 | 649–668 ACT CGG TCA TTC TGC TCA AG | Sense | U53505 |
| 1238–1219 TTC AAA GGC TAC CCC ATA AG | Antisense | |||
| Cyclophilin | 520 | 7–27 AGA CGC CGC TGT CTC TTT TCG | Sense | M19533 |
| 527–507 CCA CAC AGT CGG AGA TGG TGA TC | Antisense |
Effects of finasteride treatment
| Body weight (bw), increase (%) | Prostate weight (mg prostate/100 g bw) | |
|---|---|---|
| Data were analyzed with the Student’s t-test. *P ≤ 0.01 vs control group. n = 5 rats/group. | ||
| Groups | ||
| Control | 6.1 ± 0.8 | 110 ± 9.0 |
| Finasteride | 8.6 ± 1.0 | 70 ± 3* |
Effects of castration and hormonal replacement
| Body weight (bw), increase (%) | Prostate weight (mg/100 bw) | T (ng/ml) | E2 (pg/ml) | PRL (ng/ml) | |
|---|---|---|---|---|---|
| Body weight increases were similar in all experimental groups. Data were analyzed with one-way ANOVA, and differences between means were evaluated by the Tuckey’s test. Different letters indicate significant differences between groups (P ≤ 0.01). n = 5 rats/group. Cx, castration; T, testosterone; E2, 17β-estradiol; PRL, prolactin; ND, not detected. | |||||
| Groups | |||||
| Sham | 27.4 ± 0.9 | 191.8 ± 17a | 2.0 ± 0.3 | 107 ± 20a | 26 ± 2.6a |
| Cx | 28.5 ± 2.4 | 27 ± 5b | ND | 60 ± 11a | 21 ± 2.4a |
| Cx + T | 26.7 ± 2.7 | 188 ± 13a | 2.2 ± 0.6 | 55 ± 15a | 34 ± 8.6a |
| Cx + E2 | 21.5 ± 1.8 | 21.8 ± 10.6b | ND | 1039 ± 143b | 76 ± 20b |
| Cx + T + E2 | 24.5 ± 0.9 | 194 ± 18.5a | 1.5 ± 0.2 | 580 ± 75b | 72 ± 4.5b |
Sensitivity of prostate 5′-deiodinase activity to PTU and GTG. A 100% value corresponds to control incubations performed in the absence of inhibitors. The assay contained 150 μg protein, 2.0 nM 125I-rT3, and 20 mM DTT and was incubated for 3 h at 37 °C. n = 3 independent assays. Data were analyzed with one-way ANOVA, and differences between means were evaluated by the Tuckey’s test. Different letters indicate significant differences between groups, P ≤ 0.01.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Effect of varying concentrations of unlabeled rT3 and T4 on 5 ′-deiodination of 125I-rT3 in prostate. A 100% value corresponds to incubations performed in presence of 125I-rT3 (70 000 c.p.m.) without unlabeled iodothyronines. A wide range of iodothyronine concentrations was tested (5–3000 nM) using 20 mM DTT and 150 μg protein. Incubation conditions: 3 h at 37 °C. The figure shown is representative of n = 3 independent assays.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Effect of co-factor concentration (DTT) on 5′-deiodinase activity of prostate. The assay was performed using 2 nM 125I-rT3 as substrate and 150 μg protein. Incubation conditions: 3 h at 37 °C. n = 3 independent assays.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Effect of substrate concentration (rT3) on prostate 5′-deiodinase activity. Inset: double reciprocal plot (Lineweaver–Burk). Assays were performed using isotopic solutions of 2 nM 125I-rT3 plus unlabeled rT3 (5–1200 nM), 5 mM DTT, and 100 μg protein. Incubation conditions: 3 h at 37 °C. Figure shown is representative of n = 3 independent assays. The intercepts from these data yielded the Km and Vmax values.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Type and distribution of 5′-deiodinase activity in prostate lobes. In order to test PTU and GTG inhibition, the assay was performed using only 2 nM 125I-rT3 as substrate, 100 μg protein, and 5 mM DTT, in the presence or absence of PTU (1 mM) and GTG (100 nM). Incubation conditions: 3 h at 37 °C. n = 9 lobes for each assay. The data were analyzed with one-way ANOVA, and the differences between means were evaluated by the Tuckey’s test. Different letters indicate significant differences between groups, P ≤ 0.001. C, control group.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
RT-PCR analyses for D1 and D2 expressions in prostate gland. (A) D1 expression by prostate lobes; liver (L) was used as positive control. (B) D2 expression in total prostate; brain (B) was used as positive control. RT, RNA sample, and the appropriate oligonucleotide primers, but without RT (a representative result is shown; all tissues and lobules show negatives RT). H2O, water with all the PCR reagents, but without RT. The assays were repeated n = 3 times with independent RNA samples. V, ventral; DL, dorsolateral; A, anterior; P, total prostate.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Effect of T4 treatment on circulating T3 levels and prostate D1 activity. D1 activity was determined using optimal assay conditions (see Material and Methods). n = 5 rats/group. Data were analyzed with the Student’s t-test. *P ≤ 0.005, **P ≤ 0.0001 vs control.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Circulating levels of PRL and T3 in transplanted pituitary rats. The data were analyzed with the Student’s t-test. *P ≤ 0.05 vs sham. n = 5.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Effects of hyperprolactinemia on prostate and liver D1 activity. Liver was used as control tissue. Optimal assay conditions were used for each tissue (see Material and Methods). n = 5 rats/group. Data were analyzed with the Student’s t-test. *P ≤ 0.05 vs sham.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Effects of finasteride treatment on D1 activity of prostate and liver from intact rats. Liver was used as control tissue. Optimal assay conditions were used for each tissue (see Material and Methods). n = 5 rats/group. Data were analyzed with the Student’s t-test. *P ≤ 0.01 vs control.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
Effects of castration and hormonal replacement on prostate and liver D1 activity. Liver was used as control tissue. Optimal assay conditions were used for each tissue (see Material and Methods). n = 5 rats/group. Data were analyzed with one-way ANOVA, and differences between means were evaluated by the Tuckey’s test. Different letters indicate significant differences between groups, P ≤ 0.05. T, testosterone; E2, 17β-estradiol.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06786
We are grateful to Verónica Romero and Edith Zea for their help in some of the experiments. We also thank Felipe Ortíz-Cornejo and Martín García-Servín for animal care, Nydia Hernández and Leopoldo González-Santos for image advice, Alberto Lara and Omar González for computer assistance, Pilar Galarza for her bibliographic assistance, Leonor Casanova for academic support, and Dorothy Pless and M Sánchez-Alvarez for proofreading this manuscript. This research was supported in part by UNAM-PAPIIT (IN224602) and CONACYT (44976-M) grants. A López was supported by fellowships from CONACYT and DGEP-UNAM. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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