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Androgens Differentially Potentiate Mouse Intestinal Smooth Muscle by Nongenomic Activation of Polyamine Synthesis and Rho Kinase Activation

Laboratorio de Fisiología Animal (M.C.G.-M., T.G., M.D.), Departamento de Biología Animal, Facultad de Biología; Departamento de Fisiología (R.M.), Facultad de Medicina; and Institute of Biomedical Technologies (R.M., T.G., M.D.), Universidad de La Laguna, 38206 Tenerife, Spain

Address all correspondence and requests for reprints to Dr. Mario Díaz, Laboratorio de Fisiología Animal, Departamento de Biología Animal, Facultad de Biología, Universidad de La Laguna, 38206 Tenerife, Spain. E-mail: madiaz{at}ull.es.

	Abstract

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We demonstrate that testosterone and its active metabolite 5-dihydrotestosterone acutely (30 min) potentiate mouse ileal, but not duodenal, muscle activity. Androgens augment the amplitude of spontaneous peak-to-peak oscillations, alter the spontaneous activity frequency spectrum, and increase the amplitude of calcium-induced and carbachol-induced contractions. Concentration-dependence analyses revealed that maximal potentiation (449–910%) occurred at physiological concentrations of androgens (100 pM to 10 nM) with EC₅₀ values in the picomolar range (8–20 pM). Western blot analyses using an antiandrogen receptor (anti-AR) antibody revealed the presence of two different AR proteins migrating at 87 and 110 kDa in ileal, but not duodenal, extracts. Androgen-induced potentiation was prevented by preincubation with AR antagonists flutamide or cyproterone acetate but was unaffected by pretreatment with cycloheximide plus actinomycin D, indicating that potentiation was mediated by ARs via a novel nongenomic mechanism. Androgen effects were mimicked by polyamines putrescine and spermine and were blocked by the ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase inhibitors -difluoromethylornithine and berenil, respectively. Accordingly, androgens increase -difluoromethylornithine-sensitive ornithine-decarboxylase- mediated L-ornithine decarboxylation in ileal tissues within the same time course as isometric potentiation. Both putrescine and dihydrotestosterone induced Ca²⁺ sensitization of ionomycin-permeabilized ileal smooth muscle. Finally, inhibition of the Rho kinase (ROK) pathway with the specific inhibitor Y27632 completely prevented androgen-induced potentiation. In agreement, androgens elicited ROK-induced Ser¹⁹ phosphorylation of myosin light chain 2 in ileal muscle. These data indicate that androgens potentiate ileal contractile activity by an AR-dependent nongenomic mechanism involving intracellular polyamine signaling and Ca²⁺ sensitization via ROK activation.

	Introduction

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ANDROGENS HAVE BEEN traditionally linked to the normal regulation of spermatogenesis, skeletal development, maintenance of bone metabolism, and development of secondary sexual characteristics in males and females. These actions are considered to be genotropic, and the major mechanisms accounting for these actions are through activation of high-affinity receptors that modify DNA transcription upon binding to specific response elements in target gene promoters (reviewed in Ref. 1). However, in recent years, a variety of evidence has demonstrated that androgens and other steroid hormones can also exert many actions by transcription-independent (nongenomic or alternative) mechanisms (2). Often these effects are initiated by interaction of androgen molecules with membrane and/or cytoplasmic target proteins, followed either by modulation of intracellular signaling pathways, including cAMP, phospholipase C-inositol triphosphatase, ERK1/2, p21-activated kinase, phosphoinositide 3-kinase/Akt, and polyamines (3, 4, 5, 6, 7, 8), or by altering the activity of calcium channels and/or different types of potassium channels (9, 10, 11, 12). A demonstrated paradigm of acute androgen effects is vascular smooth muscle, where pharmacological doses of androgens reduce vascular tone and induce vasorelaxation both in vivo and in vitro by directly modulating ion channel activity rather than by activating NO generation at the endothelial lining (reviewed in Ref. 10). The mechanisms of vascular relaxation appear to be independent of androgen receptor (AR) activation and aromatase-mediated conversion of testosterone into estradiol (10). Likewise, few recent in vitro studies have demonstrated acute relaxing effects of androgens and 4- and 5-reduced androgens on myometrium and vaginal smooth muscles (13, 14, 15). However, although androgens appear to acutely modify smooth muscle activity, it is noteworthy that similar effects have not been observed to date for intestinal muscle cells, especially considering that gastrointestinal smooth muscle contractile mechanisms are regulated by complex pathways involving most molecular targets reported to be rapidly modulated by androgens (16, 17). Therefore, the aim of the present work was to assess the hypothesis that intestinal contractile activity might be acutely modified by androgens. In addition, we have explored the putative mechanisms responsible for such rapid effects. Our results show, for the first time, that intestinal smooth muscle differentially responds to physiologically relevant concentrations of androgens. In addition, we demonstrate that androgens potentiate force development by calcium sensitization of contractile machinery via stimulation of myosin light chain (MLC) phosphorylation.

	Materials and Methods

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Animals and tissue preparation
Intestinal segments were dissected from male mice weighing 20–30 g after diethyl ether anesthesia. Animal procedures complied with and were approved by the Animal Care and Use Committee of Universidad de La Laguna. Longitudinal strips of smooth muscle (1.5 cm long) were immediately placed in cold physiological salt solution (PSS), containing (in mM) 126 NaCl, 4.5 KCl, 1.0 MgSO₄, 2.0 CaCl₂, 0.56 NaH₂PO₄, 1.44 Na₂HPO₄ (adjusted to pH 7.4), and 15.0 glucose. Ca²⁺-free solutions were made by replacing all CaCl₂ from the standard saline solution and supplementing with 25 µM EGTA. Tissues were incubated in aerated PSS at 37 C in a water-jacketed organ bath and left for 30 min before starting the experiments. Bath solutions were replaced every 15 min. Tissues were equilibrated at a resting tension value of 0.5 g. The maximal contraction produced by 50 mM KCl was recorded for each muscle strip at the beginning of the experiment to determine the contractile range.

Recording system
The isometric tension of isolated intestinal strips was measured using an isometric force transducer (TRI110, Letica, Barcelona, Spain) connected to a DC amplifier (Letica). Voltage signals were digitized at a sampling rate of 20 Hz using an A/D card (LabPC+; National Instruments, Austin, TX) and stored onto the computer using a data acquisition and analysis program (PHYSCAN) written by one of the authors (M.D.). Data were low-pass filtered at 5 Hz and analyzed using computer routines included in the acquisition software.

Effects of testosterone on spontaneous activity
Intestinal tissues were incubated in PSS, and spontaneous activity was recorded for 10 min after the equilibration period (control activity). Testosterone or 5-dihydrotestosterone (DHT) dissolved in ethanol or dimethylsulfoxide (DMSO) was then added in small volumes (10 µl) directly to the bath solution at time 0, and the time course of muscle activity was recorded throughout the experiments (up to 90 min). Comparison of spontaneous activity was assessed by frequency analysis on activity data from control and androgen-treated tissues using Fourier methods (see below).

CaCl₂-induced and carbachol (CCH)-induced contractions
Intestinal preparations were incubated in Ca⁺²-free solutions for 5 min and then exposed to either 2.0 mM CaCl₂ or 1.0 µM CCH. The resulting peak contraction (T_ctrl) was used as a control value for subsequent effects. Afterward, the solution was replaced with fresh PSS and left for 5 min to stabilize. Tissues were then exposed to vehicle (ethanol or DMSO) or androgens (testosterone or DHT) at the desired concentration for different times (45, 90, and, occasionally, 180 min). At the end of these periods, bath solutions were replaced with Ca²⁺-free solutions for 5 min and the peak contraction (T_test) elicited by a second application of 2.0 mM CaCl₂ or 1.0 µM CCH was measured. Bath solutions were then replaced and washed three times with fresh PSS and left in the presence of vehicle or androgen until next measuring time. Antagonists [flutamide (FLU), cyproterone acetate (CPA), finasteride, chelerythrine, Y27632, -difluoromethylornithine (DFMO), berenil, and cycloheximide plus actinomycin D] were allowed to preincubate for 30–60 min (depending on the chemical) before the initial application of androgens and were maintained thereafter at the same concentrations in the bath.

Calcium permeabilization
Experiments in calcium-permeabilized smooth muscles were performed by incubation of ileal strips in permeabilization solution (PS) containing 25 µM ionomycin, 1.6 mM EGTA, 126 mM NaCl, 4.5 mM KCl, 1.2 mM MgCl₂, 20 mM Tris-HCl (pH 7.4), and 15 mM glucose. To obtain simultaneous recordings of control and experimental conditions within the same animal, ileal tissues were cut into two halves (0.4 cm long) and incubated for 30 min in aerated PSS. Afterward, tissues were exposed to appropriate concentrations of either vehicles or chemicals (androgens or polyamines) for 90 min, washed three times with Ca²⁺-free solution, and finally incubated in PS. Intestinal preparations were incubated in PS for 5 min and supplemented afterward with nifedipine (5 µM) for an additional 2 min before the application of the calcium pulse (free [Ca²⁺], 50 µM). In some experiments, intracellular calcium stores were depleted by preexposure of ileal strips to CCH (10 µM) under nominal zero calcium conditions.

Ornithine decarboxylase (ODC) activity assays
ODC activity was determined following the procedure described by Bordallo et al. (18). Briefly, intestinal muscles were dissected off the epithelial lining in cold aerated PSS and cut into two longitudinal halves. After incubation for 90 min in either vehicle (DMSO) or DHT, tissues were homogenized in a Polytron in 1 ml ice-cold buffer containing (in mM) 10.0 Tris-HCl, 0.05 pyridoxal-5-phosphate, 2.0 dithiothreitol (pH 7.2). The homogenate was centrifuged for 15 min at 26,000 x g at 4 C. Then, 300 µl of the supernatant and 0.250 µCi of L-[1-¹⁴C]ornithine (final concentration, 100 µM) were incubated for 60 min at 37 C in a closed tube equipped with a filter paper wetted in 50 µl of 10% KOH to trap released ¹⁴CO₂. Nonspecific ¹⁴CO₂ released was determined in test tubes containing DFMO (10 mM) added before the supernatant. The incubation was terminated by addition of 150 µl trichloroacetic acid (10%) and incubated for an additional 45 min at 37 C to release ¹⁴CO₂ from the incubation buffer. The filter papers were removed, and the trapped ¹⁴CO₂ was measured by liquid scintillation. The protein content was determined according to the Bradford procedure, and specific ODC activity was expressed as pmol of ¹⁴CO₂ per hour per milligram protein.

Western blot analyses
For total protein extraction, male murine tissues were dissected out, homogenized in SDS lysis buffer (62.5 mM Tris-HCl, 2.3% SDS, 10% glycerol, pH 6.8) using a tissue homogenizer, and tissue homogenates were kept on ice for 15 min. After centrifugation at 13,000 x g for 15 min at 4 C, supernatants were recovered for gel analysis. An aliquot of each protein extract was preserved for protein quantification using a commercial DC protein assay, and 5% ß-mercaptoethanol and 0.001% bromophenol blue were then added and samples boiled at 95 C for 5 min. Equal amounts of extracted protein (50–60 µg) were loaded, electrophoresed on 12% SDS-PAGE, and transferred to Hybond-P membranes. The immunodetection on the Western blots was carried out by, first, membrane preincubation in 5% blotting-grade blocker nonfat dry milk (Bio-Rad, Madrid, Spain) in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T) at room temperature for 1 h. Membranes were then incubated with specific polyclonal anti-AR antibody (diluted 1:150 in nonfat dry milk blotting solution) overnight at 4 C with gentle agitation. For Western blot assays using rabbit polyclonal anti-phospho-MLC2 (Ser¹⁹) (Cell Signaling Technology, Barcelona, Spain) and goat polyclonal anti-regulatory MLC2 (LC₂₀), nonfat dry milk was replaced by 5% BSA diluted in TBS-T throughout all the experiments. These antibodies were used at, respectively, 1:750 and 1:200 diluted in 5% BSA. Membranes were washed several times (10 min each) in TBT-T (TBS plus 0.05% Tween 20) and incubated for 2 h at room temperature with specific goat antirabbit (1:5000 in nonfat dry milk solution or in BSA) or with rabbit antigoat (1:1000 in BSA) horseradish peroxidase (HRP)-linked secondary antibodies. After washing three times for 10 min each in TBS-T, specific bands corresponding to proteins recognized by the different antibodies were visualized with the Amersham Biosciences (Arlington Heights, IL) enhanced chemiluminescence kit. As a control of experiments performed with anti-AR antibody, membranes were reprobed with polyclonal rabbit anti-estrogen receptor- (anti-ER) antibody (1:200) overnight at 4 C with gentle agitation, followed by incubation with a HRP-conjugated goat antirabbit secondary antibody (1:5000), and processed for specific band visualization by chemiluminescence enhancement.

The immunoreactive bands were analyzed using a GS-670 imaging densitometer (Bio-Rad) by quantifying relative bands intensity after subtraction of local background at the vicinity of the bands. Each band density was evaluated with the Molecular Analyst software. Values from experimental groups are expressed as a percentage of densitometric units of phospho-LC₂₀ relative to total LC₂₀. Values were normalized to the average of immunosignals obtained with control tissues treated with vehicle only.

Statistics and mathematical analyses
Differences between sample means were assessed by one-way ANOVA or Kruskal-Wallis test followed by either Student-Newman-Keuls t test, post hoc Tukey, or Mann-Whitney U test where appropriate. Results are expressed as mean ± SEM. Dose-response curves were fitted to a logistic equation using nonlinear regression analysis tools provided in SigmaPlot software (Jandel Scientific, San Rafael, CA). Free calcium concentrations were computed using EQCAL, specific software for multiple equilibrium calculation (Biosoft, Cambridge, UK). Frequency analyses were assessed using the fast Fourier transform (FFT) and short-time Fourier transform (STFT or Gabor transform) algorithms implemented in the acquisition and analysis software (PHYSCAN). Analyses were performed following the procedures described in Refs. 9 and 19 .

Materials
Testosterone, DHT, FLU, CPA, CCH, pyridoxal-5-phosphate, dithiothreitol, finasteride, putrescine, spermidine, spermine, actinomycin D, and cycloheximide were obtained from Sigma-Aldrich (Biosigma, Madrid, Spain). DFMO was purchased from Bachem (Cymit Química, Spain). Y27632 was obtained from Tocris Cookson Ltd. (Bristol, UK). L-[1-¹⁴C]Ornithine was obtained from PerkinElmer España (Madrid, Spain). The rabbit polyclonal anti-AR antibody (N-20), raised against a peptide mapping at the N terminus of AR of human origin, the rabbit polyclonal anti-ER antibody (MC-20) raised against a region at the vicinity of the ligand-binding domain, and goat polyclonal antibody recognizing LC₂₀ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal antibody recognizing endogenous levels of Ser¹⁹ phosphorylated LC₂₀ was from Cell Signaling Technology (Barcelona, Spain). The antirabbit and antigoat HRP-linked whole antibodies, the Hybond-P polyvinylidene difluoride membranes, and the ECL Western blotting kit were from Amersham Biosciences (Arlington Heights, IL).

	Results

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Effects of testosterone and DHT on intestinal contractile activity
Results in Fig. 1 show that under control conditions, duodenal and ileal tissues respond to application of calcium chloride (2.0 mM) and the muscarinic agonist CCH (1 µM). Responses were consistent with a rapid transient increase of isometric tension followed by a sustained phase that, in the presence of extracellular calcium, featured a peristaltic pattern. Preincubation of ileal, but not duodenal, tissues with a physiological concentration of testosterone (10 nM) for 45 or 90 min brought about a considerable increase of contractile responses to both calcium and CCH (Fig. 1A). The stimulatory effect of testosterone on ileum was reproduced by the active metabolite DHT in the same range of concentrations (Fig. 1B). Unlike ileal tissues, both vehicle-treated and androgen-treated duodenal tissues displayed a tendency to reduce amplitude of spontaneous activity as well as contractile responses to CaCl₂ and CCH, a finding that may be attributable to the exhaustion of some preparations in long-lasting in vitro experiments.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Effects of androgens on intestinal duodenal and ileal longitudinal smooth muscles. A, Representative recordings of contractile responses elicited by CaCl2 and CCH. Tissues were exposed to testosterone (10 nM) for 90 min. The contractile responses to CaCl2 and CCH were recorded at the beginning of the experiment (control) and 90 min after exposure to the hormone. Stimulants were applied at the instant indicated by the arrows after a 5-min period in which tissues were maintained under calcium-free conditions. B, Effects of preincubation of ileal smooth muscle with androgens testosterone (T, 10 nM) and DHT (10 nM), on CaCl2-induced and CCH-induced contractions. Results represent the mean ± SEM of 15 different experiments. Statistical significance was assessed by one-way ANOVA, followed by Student’s t test. ***, P < 0.005; *, P < 0.05 as compared with vehicle (V).

View larger version (36K): [in this window] [in a new window]   FIG. 2. Androgens alter the frequency spectrum of ileal peristaltic activity. A and B, Recordings of spontaneous activity in the presence of vehicle DMSO (0.1%) (A) and after application of testosterone (10 nM) (B). Frequency analyses using the FFT at time 0 (dotted line) and after 90 min (solid line) in the presence of vehicle (A) or testosterone (B) are shown in lower panels. C, Application of the STFT to continuous mechanical signals from the same tissue initially maintained under control conditions (0.1% DMSO) for 90 min and exposed afterward to testosterone (10 nM) for an additional 90 min. Exposure to testosterone gives rise to the generation of high-energy low-frequency components (<0.05 Hz) after approximately 30 min that are nonexistent in DMSO-treated periods. Results are representative of another five different experiments. Color scale encodes for power spectral densities. For details see Materials and Methods and Results.

View larger version (31K): [in this window] [in a new window]   FIG. 3. Dose-response curves for the effects of androgens on CaCl2- and CCH-induced contractions. Curves were fitted to logistic four-parameter models using nonlinear regression analyses. Each point represents the mean ± SEM of at least six different experiments. Results were submitted to one-way ANOVA, and statistical significance of fitted parameters was considered valid with P < 0.05 and convergence satisfied.

Effect of 5-reductase inhibitor on the stimulating effect of androgens

AR expression in intestinal tissues
The fact that mouse ileal muscle contractile activity was potentiated by testosterone and DHT treatments led us to wonder whether AR might be involved. Therefore, we tested for the presence of possible ARs in duodenum and ileum by Western blotting, using a specific polyclonal antibody directed to AR (Fig. 4). Results showed that the antibody recognized a strong band at the expected molecular mass of the receptor (110 kDa) in mouse ileum. An additional band was observed at about 87 kDa, probably corresponding to an N-terminally truncated AR form of this protein migrating at a lower molecular mass (21). In contrast, no AR expression was observed in duodenal protein extracts. Membranes were then reprobed with a specific polyclonal rabbit antibody directed to ER. This antibody recognized a specific ER in both tissues, therefore confirming our previous data in duodenum (9). Thus, AR seems to be highly expressed in ileal tissues, whereas it may not be present in duodenum.

View larger version (31K): [in this window] [in a new window]   FIG. 4. Analysis of AR expression in ileum and duodenum. Total protein extracts from ileal (I) and duodenal (D) smooth muscle were electrophoresed on SDS-PAGE and processed for Western blotting, using a specific polyclonal antibody to AR. As a control of protein load membranes were reblotted with an antibody directed to ER. Molecular weights of the different specific bands recognized by the antibodies are indicated. Results are representative of five experiments.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Effects of AR inhibitors on testosterone-induced potentiation of ileal contractile activity. A, Illustrative recordings showing the effect of 30 min preincubation with the nonsteroidal antiandrogen FLU (10 µM) or the steroidal antagonist CPA (10 µM) on testosterone-induced potentiation of spontaneous peristaltic activity. B and C, Effects of preincubation with antiandrogens FLU (10 µM) and CPA (10 µM) on CaCl2-induced (B) and CCH-induced (C) contractions in ileal preparations exposed to vehicle (V, DMSO) or testosterone (T, 10 nM in B and 1 nM in C). Results are expressed as mean ± SEM of six different experiments under each condition. a, P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.005 as compared with vehicle (V). b, P < 0.05; #, P < 0.01; ##, P < 0.005 as compared with testosterone (T).

View larger version (22K): [in this window] [in a new window]   FIG. 6. Effects of de novo protein synthesis inhibitors on testosterone-induced potentiation of CaCl2-induced (A) and CCH-induced (B) contractions. Cycloheximide (CHX) and actinomycin D (ACTD) were applied together at a concentration of 10 µM each 30 min before exposure to vehicle (0.1% DMSO) or testosterone (T, 10 nM in A and 1 nM in B). Representative recordings, taken at time 0 and 90 min after exposure to CHX plus ACTD, are shown on the left panels of A and B. CaCl2 and CCH were applied at the time indicated by the arrows after a brief (5-min) incubation of tissues under calcium-free conditions. Results in vertical bar charts are expressed as mean ± SEM of six experiments for each treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.005 as compared with vehicle (V). ##, P < 0.05 compared with testosterone (T).

View larger version (24K): [in this window] [in a new window]   FIG. 7. Polyamine synthesis inhibitors prevent testosterone-induced potentiation of ileal contractile activity. A, Left, Illustrative recordings showing the effect of preincubation with the irreversible ODC inhibitors berenil (20 µM) and DFMO (5 mM) on ileal spontaneous peristaltic activity; right, illustrative recordings showing the effect of 30 min preincubation with berenil on testosterone-induced (10 nM) potentiation of spontaneous peristaltic activity taken at time 0 and 90 min after addition of testosterone. B and C, Effects of preincubation with berenil (BER, 20 µM) on CaCl2-induced (B) and CCH-induced (C) contractions in ileal preparations exposed to vehicle (V, DMSO) or testosterone (T, 10 nM in B and 1 nM in C). Results are expressed as mean ± SEM of six different experiments under each condition. *, P < 0.05; ***, P < 0.005 as compared with vehicle (V). #, P < 0.05; ##, P < 0.01 compared with testosterone (T).

View larger version (18K): [in this window] [in a new window]   FIG. 8. Androgens stimulate ODC activity in ileal smooth muscle. A, Effect of DFMO (10 mM) on basal (unstimulated ileal tissues) ODC activity; B, DHT induces a dose-dependent increase on L-ornithine decarboxylation by ODC; C, linear relationships between basal and DHT-stimulated ODC activities. ODC activities in ileal preparations obtained from the same animal were measured simultaneously in response to DMSO and DHT (100 nM) and in the presence or absence of DFMO (10 mM). Regression equation is indicated for the DHT vs. control plot. In all cases, tissues were preincubated with vehicle (0.1% DMSO) or DHT at the indicated concentrations for 90 min before ODC activity measurements. Results are expressed as mean ± SEM of at least six different assays under each experimental condition. ***, P < 0.001; *, P < 0.05 as compared with vehicle (V). #, P < 0.05 vs. DHT (10 pM).

View larger version (19K): [in this window] [in a new window]   FIG. 9. Androgens induce Ca2+ sensitization in ileal smooth muscle. A, Calcium-dependence curves for control and DHT (10 nM) measured as peak responses to CaCl2 in longitudinal ileal preparations. Tissues were allowed to preincubate with control or DHT for 90 min before addition of calcium to the bath. Traces on the left panelillustrate typical responses to calcium pulses (100 µM). B and C, Responses of ionomycin-permeabilized ileal longitudinal strips to calcium (50 µM and 1.6 mM). After preincubation with DHT (10 nM, in B) or putrescine (500 µM, in C) for 90 min, tissues were sequentially exposed to ionomycin (5 min) and the L-type calcium channel blocker nifedipine (2 min) before the calcium challenge. Recordings (control and experimental) were obtained in smooth muscle preparations from the same animal. Similar results were obtained in another seven experiments.

Mechanism of calcium sensitization
A number of studies linking intracellular signaling to Ca²⁺ sensitization in smooth muscles have demonstrated the existence of at least two mechanisms mediated by protein kinase C (PKC) and/or Rho kinase (ROK) activation (28, 29, 30, 31). Therefore, we have assessed the participation of PKC and/or ROK Ca²⁺-sensitizing pathways in the stimulation of contractile activity after androgen administration. First, we have explored the effects of PKC activation with phorbol 12-myristate 13-acetate (PMA; 0.3–3 µM) on ileal mechanical activity. Our results showed that PMA failed to induce any appreciable effect on either calcium-dependent spontaneous activity or CCH-induced contractions (Fig. 10A), indicating that PKC was unlikely to be mediating the androgen-induced potentiation in ileal tissues. We also tested the effect of preincubation with the general PKC inhibitor chelerythrine (1–10 µM) on DHT-induced potentiation. However, even the lowest dose of this inhibitor induced significant changes on intestinal mechanical activity in control tissues (not shown), and therefore this strategy was not further explored.

View larger version (27K): [in this window] [in a new window]   FIG. 10. Mechanism of Ca2+ sensitization of ileal muscle by androgens. A, Effects of phorbol ester PMA on CCH-induced contractions. Tissues were incubated under calcium-free conditions for 5 min before initial application of CCH (1 µM) and maintained in zero calcium thereafter. B, Effects of preteatment with the ROK inhibitor Y27632 (10 µM) on CaCl2- and CCH-induced contractions. Tissues were preincubated for 45 min with Y27632 before exposure to either vehicle (0.1% DMSO) or DHT (10 nM) for an additional 90 min. The left panel illustrates representative traces taken at 0 and 90 min in the presence of DHT (10 nM) and Y27632. C, Western blot analyses for total and phosphorylated (Ser19) MLC2 (LC20) in ileal and duodenal muscle extracts. In each experiment, equivalent segments from ileum and duodenum were obtained from the same animal and exposed to the different treatments (0.1% DMSO vehicle, 10 nM DHT, and 10 nM DHT plus 10 µM Y27632). Total lysates from each segment were collected and analyzed for changes in LC20 phosphorylation using anti-phospho(Ser19)-LC20 antibody, followed by reblotting with an antibody that recognizes both phosphorylated and unphosphorylated LC20. Top, A representative immunoblot assay after immunoblotting with either anti-phospho-LC20 or total LC20 antibody; bottom, densitometric values of phospho-LC20 relative to total LC20. Values were normalized to the average of immunosignals obtained with control tissues (V). *, P < 0.05 vs. vehicle-treated tissues; #, P < 0.05 vs. DHT. Four assays were performed per treatment.

	Discussion

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The results reported in the present article demonstrate for the first time that androgens are potent modulators of intestinal contractile activity. At physiological concentrations, both testosterone and its active metabolite DHT increase the contractile response of intestinal tissues to external stimuli, i.e. CaCl₂ and CCH. These effects are tissue specific and restricted to ileum, whereas duodenal tissues were completely irresponsive. In ileal tissues, androgens also stimulate endogenous spontaneous activity by increasing not only the amplitude of contractile signals but also the energy of high-frequency components, often accompanied by induction of low-frequency high-energy components in the frequency domain of mechanical signals. Time-course analyses of these changes are consistent with a significant stimulation after 30 min exposure to the hormones. To the best of our knowledge, the present data provide the first demonstration that androgens might be physiological modulators of intestinal muscle activity.

Acute effects of androgens have been demonstrated for different in vitro smooth muscle preparations, including coronary arteries (11, 34), cerebral arteries (35), mesenteric beds (36), thoracic aorta (37, 38), and uterus (13). More relevant is the fact that most reports dealing with the acute effects of androgens on smooth muscles (mainly vascular) have led to the general concept that androgens exert a rapid relaxing effect on this type of muscle. Indeed, in rat and rabbit agonist-precontracted aortic rings, testosterone elicits endothelium-independent relaxations within minutes after exposure to the androgen (34, 39). However, a fact worth noting is that the relaxing effects of androgens in most in vitro observations require supraphysiological concentrations (in the micromolar range) of testosterone. In contrast, we have found that low physiological concentrations of testosterone or DHT are effective in stimulating intestinal contractile activity. Indeed, in view of results on the dose-response analyses performed here, we report EC₅₀ values for testosterone and DHT that lay in the low picomolar range with maximal stimulations around 1 nM, indicating the exquisite sensitivity of intestinal-responsive tissues to androgens.

The fact that ileal muscle activity was potentiated in response to physiological doses of testosterone and DHT strongly suggested the involvement of AR underlying the stimulatory effect of androgens. Using a specific polyclonal antibody directed to AR, we demonstrated in the mouse ileum the presence of two different immunoreactive bands, one migrating at the expected molecular mass of canonical AR (about 110 kDa) and another band at about 87 kDa, likely corresponding to the N-terminal truncated form of AR (AR-A) reported several times in the literature (3, 21). Paralleling our observations on duodenal tissues, which exhibit no potentiation by androgens, we observed no AR expression in this muscle, which indicates that both AR expression and androgen-induced potentiation of contractile activity are tissue-specific. In addition, these results represent, to the best of our knowledge, the first demonstration of the existence of AR protein expression in male mouse ileum. Furthermore, our experiments using two well-known inhibitors of ARs, namely CPA and FLU, showed that both compounds were able to significantly prevent the increase in the responses to CaCl₂ and CCH in intestinal-responsive tissues exposed to testosterone. These results provide strong evidence that ARs are involved in the stimulation of intestinal contractility by androgens and are well in line with previous reports in pig coronary arteries (40), where modulation of smooth muscle activity by physiological doses of androgens involve activation of ARs that are (hydroxy-)flutamine and/or CPA sensitive. In contrast, most in vitro studies aimed at assessing the effects of androgens on vascular and uterine tissue contractility have provided evidence that acute relaxation induced by androgens is completely insensitive to (or even stimulated by) AR antagonists and therefore not mediated by ARs (reviewed in Ref. 10), although the physiological significance of these findings is doubtful given the high supraphysiological (micromolar) doses of androgens required to induce smooth muscle relaxation.

The time course of androgen-induced potentiation of intestinal contractility (evident after 30 min incubation with androgens as assessed by frequency analyses), together with the lack of sensitivity to inhibitors of protein and RNA synthesis demonstrate that the modulatory mechanisms triggered by androgens are irrefutably nongenomic. In fact, acute nongenomic modulation of smooth muscles by androgens appear to be a generalized feature of arteries from systemic, coronary, and pulmonary beds as well as uterine muscles (see above). Remarkably, recent reports have also demonstrated the existence of acute nongenomic androgen effects on cultured skeletal (41) and cardiac myocytes (42) as well as in nonmuscle cell types like rat Sertoli cells (43), rat lymphocytes (44), C6 glioma cells (3), and human prostatic cells (43) among others.

One potential mechanism by which testosterone might stimulate intestinal contractility is through its conversion to estrogens. It is well known that testosterone is readily converted into 17ß-estradiol via the enzyme aromatase in target and nontarget tissues, including smooth muscle (reviewed in Ref. 45). However, different evidence ruled out such an action: first, estrogens induce a fast relaxation of mouse intestinal tissues (9); second, the nonaromatizable physiological metabolite DHT mimicked the stimulatory effect of testosterone on intestinal tissues in the same time course and range of concentrations (see Results); and third, the effects were partially prevented by the steroid 5-reductase inhibitor finasteride (observed here for testosterone-induced potentiation of CCH-induced contractions), indicating that conversion of testosterone into DHT might be a requisite for the potentiation mechanism.

We have attempted to delineate the putative signaling pathway(s) involved in androgen-induced potentiation of ileal contractile activity. As such, we explored different cellular modulators previously associated with acute androgen effects in different muscle cells. In cultured skeletal muscle cells, testosterone (and testosterone conjugated to BSA) produced rapid intracellular calcium release from inositol 1,4,5-triphosphate-sensitive stores (41). This doesn’t seem to be the case of mouse ileal muscle because incubation with testosterone or DHT either in the presence of verapamil or under calcium-free conditions failed to produce any appreciable increase for basal isometric tension over the time course of experiments. On the other hand, there is evidence that activation of signaling pathways involving stimulation of protein kinase A and/or PKC accounts for AR-regulated mechanisms (trans-activation) through interaction of components of these pathways with the AR protein (46, 47). For instance, it has been demonstrated that the rapid increase in intracellular cAMP in response to androgens accounts for the positive inotropism in rat left atrium (4). However, our previous experiments using the permeable analog dibutyryl cAMP or the adenylate cyclase activator forskolin completely relaxed mouse small intestinal activity (9). Similarly, exposure to the PKC activator PMA stimulated neither calcium-dependent spontaneous activity nor CCH-induced contractions in intestinal tissues (shown in Fig. 10A for CCH-induced contractions).

Several studies have shown that androgens can affect calcium signaling not only in a variety of nonmuscle cell types, including Sertoli cells (48), human prostate cells (43), and activated T cells (44), but also in rat heart myocytes (5, 42) and skeletal muscle (41). Depending on the system studied, androgen-induced elevation of cytosolic calcium is secondary to rapid stimulation of Ca²⁺ influx from extracellular space via voltage-gated Ca²⁺ channels, non-voltage-gated Ni²⁺-sensitive Ca²⁺ channels, or mobilization from intracellular stores (41, 42, 44, 49). However, two pieces of evidence suggest that these are not the mechanisms underlying the potentiation of mechanical activity observed in the present study. First, incubation of androgen-responsive tissues with testosterone or DHT either in the presence of voltage-gated channel blockers verapamil or nifedipine or under calcium-free conditions failed to produce any appreciable increase in basal tone over the time course of the experiments (>90 min). Second, permeabilization of intestinal tissues to calcium with ionomycin in the presence of the specific L-type calcium-channel blocker nifedipine (Fig. 9B), does not prevent the DHT-induced potentiation of the response to clamped low external calcium concentration induced by androgens. Therefore, androgen stimulation of mechanical activity of intestinal tissues by acute changes of cytosolic calcium availability can be ruled out.

In a completely different way, several studies performed in nonmuscle and muscle preparations have shown that androgens affect polyamine metabolism at different levels, from genomic activation of ODC gene expression (which is the first and rate-limiting enzyme in the biosynthetic pathway in polyamine metabolism) (22) to direct stimulation of ODC enzymatic activity (6, 18). In the polyamine biosynthetic pathway, ODC converts the amino acid ornithine to the diamine putrescine, which is the obligatory precursor of the subsequent synthesis of polyamines spermidine and spermine. To convert putrescine into polyamines spermidine and spermine, an aminopropyl donor, the decarboxylated S-adenosylmethionine, is required. In mammalian cells this aminopropyl transfer is catalyzed by SAMDC, which not only is essential for polyamine synthesis but also is regulated by androgens (50).

Polyamines have been shown to inhibit contractile and electrical activities in uterine muscle (51) and portal vein (52) from rat and in guinea pig taenia coli smooth muscle (53) by mechanisms associated with decreased intracellular calcium. These effects are generally observed after extracellular exposure of polyamines (mainly spermine and spermidine) in the millimolar range and are caused by interaction of these polycations with voltage-activated calcium channels responsible for action potentials in smooth muscle cells (54). In agreement with these findings, we have also observed that when used at high (millimolar) concentrations, the three polyamines used in this study rapidly inhibit spontaneous ileal and duodenal activities (our manuscript in preparation). However, studies using intracellular loading of guinea pig intestinal muscle (27) and rat portal veins (52) with polyamines by reversible permeabilization with ß-escin, increased the Ca²⁺-force relationship of depolarized smooth muscle cells by increasing calcium sensitivity of contractile proteins.

In consonance with these observations, we have demonstrated that exposure of intestinal tissues to low (micromolar) concentrations of putrescine, spermidine, and spermine increased mechanical response to CaCl₂ and CCH, therefore mimicking the effect of androgens. On the basis of the results discussed up to now, it is reasonable to think that polyamines could enter the intestinal smooth muscle cells to mimic the stimulatory effect of androgens. In smooth muscle cells, polyamines can be transported by at least two different saturable carriers exhibiting differential kinetics and selectivity toward polycations (55), and we hypothesized that these might be the transmembrane route for intracellular polyamines in the present paradigm. The most convincing evidence for the involvement of polyamines in the stimulatory effect of androgens comes from the experiments using two sets of inhibitors of polyamine synthesis, namely DFMO and berenil. We have observed that preincubation with berenil effectively prevented the stimulatory effect of androgens on both calcium-induced and CCH-induced contractions. In agreement with this, we have observed that berenil blockade of testosterone-induced potentiation of CCH-induced contraction in ileal tissues could be reversed by application of putrescine (500 µM, not shown). Berenil was found to be a better blocker of androgen effects because DFMO, used at millimolar concentrations (those conventionally used to block ODC activity), readily inhibited spontaneous activity and relaxed intestinal tissues, thereby resembling the effect of high polyamine concentrations (not shown). Furthermore, we have demonstrated that acute exposure to DHT, in a dose-dependent manner, increases DFMO-sensitive ODC activity and L-[1-¹⁴C]ornithine decarboxylation independently of tissue basal ODC activity. Similarly, acute stimulation of ODC enzyme by androgens, independently of ODC gene transcription, has been postulated to underlie the positive inotropic effect elicited by DHT in the rat left atrium (18). Altogether, our observations demonstrate that androgens effectively stimulate polyamine synthesis and increase intracellular polyamine concentration in intestinal smooth muscle cells. In addition, in view of the effects of ODC and SAMDC inhibitors on androgen-induced potentiation, it can be concluded that polyamines are modulators downstream of AR activation, leading to sensitization of contractile machinery to calcium in intestinal smooth muscle. A key proof for this hypothesis was the observation that preincubation of ileal muscle with putrescine induces a considerable increase of contractile force in ionomycin-permeabilized tissues exposed to nifedipine. Under these conditions L-type calcium channels are blocked by the presence of the dihidropyridine nifedipine, and therefore, changes in tension are entirely a result of calcium influx through ionomycin pores. These results strongly indicate that putrescine (and perhaps also other polyamines) induced Ca²⁺ sensitization of ileal smooth muscle. Indeed, these results, together with the facts that 1) polyamine synthesis inhibitors prevent androgen-induced potentiation of mechanical signals, 2) DHT stimulates ODC activity, and 3) exogenous polyamines mimicked the effect of androgens, lead us to conclude that polyamines are the signaling transducers of androgen-induced Ca²⁺ sensitization in ileal smooth muscle.

The mechanism of calcium sensitization of intestinal smooth muscle machinery by polyamines appear to involve a positive modulation of MLC₂₀ phosphorylation as a result of an inhibitory effect on MLCP activity (26, 56). Intracellular mechanisms linking neurotransmitter receptor activation to Ca²⁺ sensitization in intestinal smooth muscle have been investigated in several laboratories. Two main pathways have been characterized. On one hand is the Rho pathway, which involves activation of Rho protein and Rho-activated kinase, which, in turn, phosphorylates both LC₂₀ and the myosin-binding subunit of MLCP thereby increasing the phosphorylation state of the 20-kDa regulatory MLC (16, 32, 33). On the other hand is the second Ca²⁺-sensitization mechanism, the PKC pathway, which activates CPI-17, a smooth muscle cell inhibitor of MLCP (28). In the rat colon, PKC and Rho pathways in parallel mediate the Ca²⁺ sensitization coupled to activation of muscarinic receptors in the distal colon, whereas the Rho pathway alone mediates this action in the proximal colon (31). Our experiments conducted to establish the participation of either pathway in the Ca²⁺ sensitization induced by androgens in ileal tissues showed that stimulation of the PKC pathway with phorbol ester PMA changed neither the basal tone nor the response to CCH. On the contrary, we observed that the specific Rho-associated protein kinase inhibitor Y27632 completely abolished the potentiation of mouse ileal tissues to DHT without affecting basal, unstimulated responses. Paralleling the observed potentiation of ileal mechanical activity, androgens also induced a significant increase of LC₂₀ phospho-Ser¹⁹ within the same time course specifically in ileal, but not in duodenal, tissues. Furthermore, androgen-induced augmentation of LC₂₀ phospho-Ser¹⁹ in ileal smooth muscle was completely abolished by Y27632. Taken together, these novel observations demonstrate that the Rho protein pathway, but not PKC pathway, is activated by androgens to elicit Ca²⁺ sensitization of intestinal tissues. In agreement with our observations, only the Rho pathway is involved in the calcium sensitization to agonists in guinea pig and rat ileum (29, 30). Interestingly, recent reports have demonstrated that polyamines regulate ROK activity and myosin phosphorylation in intestinal cells and that depletion of intracellular polyamines using DFMO reduces Rho activation and Rho-associated ROK (57).

From our present experiments, we cannot establish the precise mechanism by which intracellular polyamines activate ROK to induce calcium sensitization. Considering that Rho proteins are GTP-binding proteins usually associated with G protein-coupled receptors (GPCR), the question arises that in our present paradigm a molecular link, other than GPCR activation, should enable (either directly or indirectly) interaction of polyamines with Rho protein. In this respect, Masuda and coworkers (58) have shown that RhoA becomes polyaminated in the presence of spermidine at a concentration as low as 100 µM (in the range of those used in this study) by Bordetella dermonecrotizing toxin in a GPCR-independent pathway. Furthermore, polyamination endows Rho with the ability to interact with its downstream effector, ROK, in a GTP-independent manner (58, 59). In addition, it has been shown that polyamine-linked Rho, even in its GDP-bound form, associates more effectively with ROK (58). Taking these observations into consideration, it is conceivable in the mouse ileum that androgen-induced polyamine synthesis may lead to interaction of these polycations with Rho protein in the cytosol, causing the release of its GDP dissociation inhibitor and the exchange of GDP by GTP on RhoA. According to the well-established mechanism of RhoA activation in smooth muscle cells, RhoA·GTP may translocate to the membrane where it may interact with ROK to initiate MLCP and MLCK phosphorylation upon release toward the smooth muscle cell cytoplasm (16).

In summary, our data demonstrate for the first time that androgens potentiate ileal contractile activity in an androgen-receptor-dependent manner that does not involve transcriptional activation or de novo protein synthesis but rather transduce through polyamine signaling secondary to stimulation of ODC (and perhaps SAMDC) activities. The potentiation mechanism triggered during androgen-induced stimulation of intracellular polyamines commits calcium sensitization of smooth muscle cells by activation of the ROK pathway and, eventually, inhibition of MLCP activity. A cellular model depicting the hypothetical mechanism of ileal smooth muscle potentiation by androgens is shown in Fig. 11. Future experiments will help to unravel the precise molecular interactions involved in acute androgen-induced signaling pathways leading to mechanical stimulation of ileal contractile machinery.

View larger version (62K): [in this window] [in a new window]   FIG. 11. Hypothetical cellular model depicting the mechanisms of Ca2+ sensitization of mouse ileal muscle induced by androgens. For details see Discussion. CaM, Calmodulin; 5R, steroid 5-reductase; IP3, inositol 1,4,5-triphosphate.

	Acknowledgments

 
This article is dedicated to the memory of Dr. Javier Corzo, an excellent biochemist and better friend who made some of us believe that the good ideas are the most precious treasure in science. We thank Dr. Guadalberto Hernández (Department of Physiology, Universidad de La Laguna) for helpful comments on androgen metabolism in target tissues and Jorge Marrero-Alonso for his collaboration in some experiments and critical discussions on this study. We also recognize Lupe Acosta for her technical contribution. We are grateful to Francisco Lopez (Ligand Pharmaceuticals Inc., San Diego, CA) for kindly providing the polyclonal anti-AR antibody.

	Footnotes

 
This work was supported by research Grants PI042640 (FISS, Ministerio de Sanidad y Consumo, Spain) and PI84/04 (Gobierno de Canarias, Spain) and the Spanish Network for Neurological Disorders (CIEN). R.M. is a fellow of the "Ramon y Cajal" Programe (Spain).

Disclosure of potential conflict of interest: M.C.G.-M., R.M., T.G., and M.D. have nothing to disclose.

First Published Online August 31, 2006

Abbreviations: AR, Androgen receptor; CPA, cyproterone acetate; DFMO, -difluoromethylornithine; DHT, 5-dihydrotestosterone; DMSO, dimethylsulfoxide; ER, estrogen receptor-; FFT, fast Fourier transform; FLU, flutamide; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; ODC, ornithine decarboxylase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PS, permeabilization solution; PSS, physiological salt solution; ROK, Rho kinase; SAMDC, S-adenosylmethionine decarboxylase; STFT, short-time Fourier transform; TBS-T, Tris-buffered saline with 0.1% Tween 20.

Received June 9, 2006.

Accepted for publication August 24, 2006.

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