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. 2009 Dec 14;588(Pt 3):511–525. doi: 10.1113/jphysiol.2009.182162

Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres

M M Hamdi 1, G Mutungi 1
PMCID: PMC2825614  PMID: 20008468

Abstract

It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres.

Introduction

Testosterone, the principle sex hormone in males, is a C19 steroid synthesised from cholesterol in Leydig cells of the male testes. In healthy young adult men, its plasma concentration ranges from 300 to 1000 ng dl−1 (Basaria et al. 2001). In plasma, most of the testosterone is bound to either albumin (∼54%) or the sex hormone-binding globulin (∼44%) and only 2% (∼1.5–20 ng dl−1) is free (Pardridge, 1986; Evans, 2004). In certain target tissues such as the liver, brain, prostate and pubic skin testosterone is irreversibly converted to dihydrotestosterone (DHT) by the enzyme 5α-reductase (Bruchovsky & Wilson, 1968). Although, in healthy young men, the concentration of DHT (normal range 4–57.5 ng dl−1) in the plasma is similar to that of free testosterone, DHT is considered to be the more potent hormone because it has a higher receptor binding affinity and a lower dissociation constant than testosterone (Saartok et al. 1984). However, as skeletal muscle is thought to lack 5α-reductase (Thigpen et al. 1993), it is uncertain whether DHT has any physiological functions in this tissue.

Testosterone and DHT are lipid-soluble hormones and can cross the cell membrane freely. Once inside the cell, they bind the androgen receptor (AR) leading to a conformational change in its tertiary structure, the release of the chaperones bound to it and exposure of nuclear localisation sequences (Gelmann, 2002; Heinlein & Chang, 2002a). The hormone–receptor complex then translocates into the nucleus where it binds to the hormone-responsive elements on the promoter regions of its target genes. Depending on the co-activators/co-repressors recruited, the hormone–receptor complex leads to either the activation or repression of these genes (Rahman & Christian, 2007). This mechanism of steroid action is known as the classical or genomic pathway and because it involves gene transcription and mRNA translation, its effects usually take several hours to days to be manifested (Florini, 1970; Beato, 1989, 1996).

In addition to their genomic actions, it is now generally accepted that steroid hormones can also exert actions that are too rapid to be explained by the classical/genomic pathway (Falkenstein et al. 2000; Heinlein & Chang, 2002b; Simoncini & Genazzani, 2003). These actions occur within seconds to minutes after administration of the hormone and are generally insensitive to inhibitors of the androgen receptor (Estrada et al. 2003), suggesting that they are regulated by cellular signalling pathways involving surface membrane receptors and second messengers (Heinlein & Chang, 2002b). This mechanism of steroid action is referred to as non-genomic or non-classical (Lösel et al. 2003).

Although the non-classical actions of other steroid hormones such as oestrogen, progesterone and aldosterone have received a lot of attention and their actions in many tissues are well characterised (for details see review by Lösel et al. 2003), little is known about the non-genomic actions of steroid hormones in adult mammalian skeletal muscles. Furthermore the two studies published so far were performed on cultured muscle cells and used supra-physiological levels of testosterone (Estrada et al. 2000, 2003). Therefore, it is uncertain whether physiological levels of testosterone or its metabolite DHT have any rapid actions in isolated intact mammalian skeletal muscle fibres. It is also uncertain whether these actions have a physiological role.

Therefore, the primary aims of this study were to investigate: (1) the rapid actions of DHT in adult mammalian skeletal muscle fibres, (2) the cellular signalling pathway(s) mediating these actions, and (3) their physiological role. Our results show that the rapid actions of DHT in adult mammalian skeletal muscles include the modulation of force production. These effects are mediated through the EGF receptor (EGFR) and involve the phosphorylation of the 20 kDa regulatory myosin light chains (RMLCs) by the extracellular signal-regulated kinases (ERK) 1/2.

Methods

Preparation of intact skeletal muscle fibre bundles

All the experiments reported here were performed at 20 ± 0.1°C on small muscle fibre bundles isolated from either the extensor digitorum longus (EDL, a mainly fast twitch muscle in adult mice) or the soleus (a predominantly slow twitch muscle in adult mice) of adult female (n= 20) and male (n= 4) CD1 mice aged 49 ± 5 days (range 40–84 days). The mice were killed by cervical disarticulation as recommended in the Animals (Scientific Procedures) Act 1986 (for details of the regulations see Drummond, 2009) and all the experiments conformed to the local animal welfare committee guidelines. The EDL and soleus muscle from both hind limbs were isolated. Small muscle fibre bundles (∼10–15 fibres, mean cross-sectional diameter 230.6 ± 17.7 μm, n= 24) were dissected under dark-field illumination and care was taken to ensure that the fibres in a bundle were electrically excitable.

Determination of the effects of dihydrotestosterone (DHT) and testosterone on force

The procedure used was basically similar to that described previously by Mutungi & Ranatunga (2000). Briefly, the fibre bundles were mounted horizontally between two stainless steel hooks, one attached to a force transducer (Model 400A, Aurora Scientific Inc., Ontario, Canada) and the other to a servo-motor (Model 322C, Aurora Scientific Inc.), in a muscle chamber with a glass bottom. The bundles were then perfused, at the rate of 1 ml min−1, with the standard Ringer solution or the standard Ringer solution plus the various compounds whose effects were being tested. The standard Ringer solution contained (in mm): 109 NaCl, 5 KCl, 1 MgCl2, 4 CaCl2, 24 NaHCO3, 1 NaHPO4, 10 sodium pyruvate plus 200 mg l−1 bovine calf serum; the pH was maintained at ∼7.24 by continuously bubbling with 95% O2 and 5% CO2. The temperature of the muscle chamber was maintained using a thermoelectric controller (Model 825A, Aurora Scientific Inc.) and a Peltier device placed next to the muscle chamber.

At the beginning of each experiment, the sarcomere length of each preparation was set at 2.4 μm using a He-Ne laser (Laser Lines Ltd, Oxon, UK). The preparations were then electrically stimulated once every 90 s using a single supra-maximal stimulus to elicit twitch contractions and once every 5 min with trains of stimuli (30–60 Hz in slow twitch fibres and 90–120 Hz in the fast twitch fibres) to elicit fully fused tetani. The fibre bundles were then perfused with either the standard Ringer solution or the standard Ringer solution plus 630 ρg ml−1 androstanolone (17β-hydroxy-5α-androstan-3-one, DHT) or testosterone propionate (Sigma, Gillingham, Dorset, UK) dissolved in absolute ethanol for at least 30 min. Twitch and tetanic contractions were then recorded in the presence or absence of DHT and testosterone.

In another experiment, the fibre bundles were perfused for 15 min with Ringer solution containing the inhibitors listed in Table 1. The Ringer solution was then switched to one containing the inhibitor plus either DHT or testosterone for at least 30 min. Twitch and tetanic contractions were then recorded. Finally, the fibre bundles were switched back to the standard Ringer solution.

Table 1.

The receptors/proteins tested and the inhibitors used

Inhibitors
Receptor/protein Scientific name Common name Concentration Source
Androgen receptor (AR) 6-Chloro-1β,2β-dihydro-17-hydroxy-3′H-cyclopropa(1,2)-pregna-1,4,6-triene-3,20-dione acetate Cyproterone acetate 1 μm Sigma-Aldrich
2-Methyl-N-[4-nitro-3-(trifluoromethyl) phenyl]-propanamide Flutamide 3 μm Sigma-Aldrich
Epidermal growth factor receptor (EGFR) (N-[3-Chlorophenyl]-6,7,-dimethoxy-4-quinazolamine) Tyrphostin AG 1478 100 nm Sigma-Aldrich
Myosin light chain kinase (MLCK) 1-(5-Iodonaphthalene-1-sulfonyl) homopiperazine, HCl ML-7 hydrochloride 500 nm Sigma-Aldrich
MAP kinase kinase (MEK) 2′-Amino-3′-methoxyflavone PD98059 20 μm Alexis biochemicals
Platelet-derived growth factor receptor (PDGFR) 4-(6,7-Dimethoxy-4-quinazolinyl)-N-(4-phenoxyphenyl)-1-piperazinecarboxamide PDGFR tyrosine kinase inhibitor III 200 nm Calbiochem
Insulin-like growth factor 1 receptor (IGF-1R) α-Cyano-(3-methoxy-4-hydroxy-5-iodocinnamoyl)-(3′,4′-dihydroxyphenyl) ketone Tyrphostin AG 538 100 μm Sigma-Aldrich
Src kinase (src) 4-Amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4,d]pyrimidine PP2 10 μm Alexis biochemicals
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Most of the compounds were dissolved in either DMSO or absolute ethanol. Therefore, to ensure that the vehicle (absolute ethanol or DMSO) had no affect on maximum isometric force (Po), some of the fibre bundles were treated for 30 min with Ringer solution containing the vehicle only. Twitch and tetanic contractions were then recorded. Analysis of these data showed that, at the concentration used, the vehicle had no effect on either twitch or tetanus.

At the end of the experiments, some of the muscle fibre bundles were processed for Western blot analysis and their myosin heavy chain isoforms were analysed using monoclonal antibodies from Sigma-Aldrich (Fig. 2D).

Figure 2.

Figure 2

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Testosterone has no effect on Po in isolated intact mouse skeletal muscle fibre bundles Force records obtained from a female fast (A) and a female slow (C) twitch muscle fibre bundle before (continuous line traces), during (dotted line traces) and after (dashed line traces) treatment with 630 ρg ml−1 testosterone. Note that testosterone has no effect on Po in either fibre type. B, summary data showing the effects of DHT (hatched bars) and testosterone (filled bars) on Po in 5 fast (i) and 4 slow (ii) twitch muscle fibre bundles isolated from 6 female mice. The open bars show data recorded from the same fibre bundles in Ringer solution containing no testosterone. Note that while DHT significantly (*P= 0.02) increases Po in the fast twitch fibres and significantly (*P= 0.025) decreases it in the slow twitch fibre bundles, testosterone had no effect on Po in either fibre type. D, representative Western blots showing the myosin heavy chain (MHC) isoform composition of female fast (F) and female slow (S) twitch muscle fibre bundles probed using monoclonal antibodies against fast (i) and slow (ii) MHC. Note that each bundle consists mainly of a single MHC isoform.

Determination of the cellular signalling pathways mediating the rapid actions of DHT and testosterone

To investigate the cellular signalling pathway(s) mediating the effects of DHT and testosterone on force, small muscle fibre bundles (∼200 μm in diameter) were used. The fibre bundles were divided into two equal groups. One half was treated with the standard Ringer solution plus the vehicle (absolute ethanol/DMSO) for 1 h. Proteins from these fibres acted as controls. The other half was treated for the same period of time with Ringer solution containing 630 ρg ml−1 DHT or testosterone. In some experiments, the fibre bundles were pre-incubated for 15 min in Ringer solution containing the inhibitors listed in Table 1. They were then treated with DHT or testosterone plus the inhibitor for a further 1 h. During these experiments, the fibre bundles were subjected to twitch contractions once every 90 s.

Immunoblotting

At the end of the experiments described above, the muscle fibre bundles were snap frozen in liquid nitrogen, pulverised and proteins extracted using NP40 lysis buffer. The proteins were then immunoblotted as previously described by Mutungi (2008). Briefly, equal amounts of the proteins (10 μg per lane) were resolved in 10% SDS-polyacrylamide gels and transferred onto PVDF membranes. The membranes were blocked for non-specific antibody binding with 5% milk for at least 30 min. They were then incubated overnight with antibodies against ERK1/2 phosphorylated at threonine 202 and serine 204 (Santa Cruz Biotechnology, CA, USA), the 20 kDa regulatory myosin light chains phosphorylated at serine 20 (Abcam, Cambridge, UK), the C-terminus of the AR (Santa Cruz Biotechnology) and the N-terminus of the AR (Abcam). The following day the membranes were incubated with species-specific secondary antibodies conjugated to horseradish peroxidase. Finally, they were visualised using SuperSignal WestPico (Perbio Science UK Ltd, Cramlington, UK) chemiluminescence substrate and exposure of the membranes to film.

On the third day, the membranes were stripped and re-probed for total ERK1/2 (Santa Cruz Biotechnology) and total regulatory myosin light chains (Abcam). In some experiments, the membranes were probed for actin using a pan-actin antibody from Abcam.

Data handling and analysis

The force (produced by the muscle fibres) and the temperature (from the thermocouple) signals were collected via a CED 1401 Micro laboratory interface using Signal 2.11 software (Cambridge Electronic Design Ltd, Cambridge, UK) and stored in a computer. The amplitude of the twitch and tetanus was determined using the Signal 2.11 software. The amplitude of the tension (= force) records was normalised to the cross-sectional area of the bundles. The tension recorded from each bundle under the various experimental conditions was averaged and divided by the mean tension recorded from the same fibre bundle in the standard Ringer solution. The data are presented as a percentage ±s.e.m. of the control tension.

All the Western blots were run in triplicate and each experiment was repeated at least three times. The intensity of the protein bands from an experiment were analysed using Scion Image from NIH and are presented as mean ±s.d. Statistical analysis of the data was performed using Statistica 5.0 (StatSoft. Inc., Tulsa, USA). Comparison of the various data sets was performed using two-way ANOVA and P < 0.05 was considered statistically significant.

Results

The effects of treating small muscle fibre bundles with DHT and testosterone on maximum isometric tension (Po)

Figures 1 and 2 show the basic observations from this study. As the results show, treating small skeletal muscle fibre bundles with physiological levels of DHT for at least 30 min increased Po in the fast twitch fibre bundles (Fig. 1A) but decreased it in the slow twitch fibre bundles (Fig. 1B) irrespective of whether they were from a female (Fig. 1A and B) or male (Fig. 1C) mouse. The increase in Po observed in the fast twitch fibres was transitory and Po reverted back to its control values within 1–2 h after the fibres were transferred back into the standard Ringer solution. In contrast, the decline in Po recorded in the slow twitch fibres was considered to be permanent as none of the fibres fully recovered after they were transferred back into the standard Ringer solution.

Figure 1.

Figure 1

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The effects of DHT on maximum isometric force (Po) in isolated intact mammalian skeletal muscle fibre bundles are fibre type dependent Representative force records obtained from a female fast twitch (A), a female slow twitch (B) and a male fast twitch (C) muscle fibre bundle before (continuous line traces), during (dotted line traces) and after (dashed line traces) exposure to DHT. Note that DHT leads to a reversible increase in Po in the fast twitch fibre bundles and to an irreversible decrease in Po in the slow twitch muscle fibre bundles irrespective of whether they are from a male or female mouse. D, data summarising the effects of DHT on Po in fast (i) and slow (ii) twitch muscle fibre bundles isolated from 10 female (hatched bars) and 4 male (filled bars) mice. The data are presented as a percentage of the Po recorded from the same fibre bundles perfused with Ringer solution containing no added DHT (open bars). Note that DHT induces a statistically significant (*P= 0.02) increase in Po in the fast twitch fibre bundles but to a statistically significant (*P= 0.03) decrease in Po in the slow twitch fibre bundles.

The summary data from 10 female and 4 male mice is shown in Fig. 1D. As the data shows, DHT treatment led to a 29.6 ± 3% (n= 8 fibre bundles) and to a 24.1 ± 3% (n= 4 fibre bundles) increase in Po in the female and male fast twitch muscle fibre bundles, respectively. Conversely, it led to a 21.1 ± 3% (n= 7 fibre bundles) and to a 19.8 ± 3% (n= 4 fibre bundles) decrease in Po in the female and male slow twitch fibre bundles, respectively. Statistical analysis of the data showed that the increase or decrease in force was statistically significant. However, no statistical difference was observed between the effects of DHT in the fibres isolated from male and female mice.

On the other hand, when the muscle fibre bundles were treated with testosterone propionate, no changes in Po were observed in either fibre type (Fig. 2).

The effects of treating small muscle fibre bundles with DHT and testosterone on twitch tension

The effects of treating small muscle fibre bundles with DHT and testosterone on twitch tension are displayed in Fig. 3. As the results show, the effects of these hormones on twitch tension were similar to those on Po. Thus, treating the fibre bundles with DHT led to a 23.6 ± 1.3% (n= 6 fibre bundles) increase in peak twitch tension in fast twitch muscle fibre bundles and to a 15.3 ± 2.1% (n= 6 fibre bundles) decrease in twitch tension in slow twitch fibre bundles isolated from adult female mice (Fig. 3B). However, DHT did not alter the characteristics of the isometric twitch (Fig. 3A and C). For example, the half rise time of twitch tension in the fast fibres was 9.3 ± 0.8 ms and 9.6 ± 0.4 ms in the absence and presence of DHT, respectively. The corresponding values in the slow twitch fibres were 20.5 ± 3 ms and 21.2 ± 6 ms in the presence and absence of DHT, respectively. Treating the fibre bundles with testosterone had no effects on the amplitude of the isometric twitch tension in both fibre types (Fig. 3D). Similar effects were seen in muscle fibre bundles isolated from male mice (results not shown).

Figure 3.

Figure 3

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The effects of DHT on isometric twitch tension in isolated intact mammalian skeletal muscle fibre bundles are fibre type dependent Representative twitch myographs recorded from a female fast (A) and a female slow (C) twitch muscle fibre bundle before (continuous line traces), during (dotted line traces) and after (dashed line traces) treatment with DHT. Note that DHT reversibly increases twitch tension in the fast twitch fibre bundle and irreversibly decreases it in the slow twitch muscle fibre bundle. Summary data illustrating the effects of DHT (B, hatched bars) and testosterone (D, hatched bars) on isometric twitch tension in 6 fast (i) and 6 slow (ii) twitch muscle fibre bundles isolated from 6 adult female mice. The open and filled bars show the twitch tension recorded from the same fibre bundles before and after treatment with DHT, respectively. Note that DHT induces a statistically significant (*P= 0.021) increase in Po in the fast twitch fibre bundles and a statistically significant (*P= 0.034) decrease in Po in the slow twitch fibre bundles.

The effects of treating small muscle fibre bundles with DHT and testosterone on the activation of the mitogen-activated protein kinase (MAPK) pathway

It has been previously shown that treating cultured muscle cells with supra-physiological doses of testosterone increases the phosphorylation of ERK1/2 (Estrada et al. 2003). However, from these results it is uncertain whether similar changes occur in adult mammalian skeletal muscle fibres treated with physiological levels of either DHT or testosterone. Therefore, in another experiment we investigated the effects of treating small skeletal muscle fibre bundles isolated from adult female mice with either DHT or testosterone on the phosphorylation of ERK1/2. As the results in Fig. 4 show, treating small muscle fibre bundles with physiological concentrations of DHT for 1 h led to a 2- to 3-fold increase in the phosphorylation of ERK1/2 in both fibre types (Fig. 4A). In contrast, testosterone led to an increase in the phosphorylation of ERK1/2 in slow twitch fibres only (Fig. 4B).

Figure 4.

Figure 4

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DHT increases the phosphorylation of ERK1/2 Representative Western blots showing the concentration of phosphorylated ERK1/2 (pERK1/2; A, B and C) in female fast twitch (F) and female slow twitch (S) skeletal muscle fibre bundles treated with either the standard Ringer solution (FC and SC) or the standard Ringer solution plus 630 ρg ml−1 DHT (A, FT, ST) or testosterone (B, FT, ST). In C, the fibre bundles were pre-treated with the MEK1/2 inhibitor PD89059, followed by DHT plus the inhibitor (FMT, SMT). Note that treating the muscle fibre bundles with DHT significantly (*P= 0.02) increased the phosphorylation of ERK1/2 in both fibre types, whereas testosterone increased the phosphorylation of ERK1/2 in slow twitch fibres only. Moreover, the effects of DHT on the phosphorylation of ERK1/2 were abolished by PD89059 and fast twitch muscle fibre bundles expressed higher concentrations of total ERK1/2 (tERK1/2) than slow twitch fibres.

To determine whether there was a link between the phosphorylation of ERK1/2 and the changes in tension reported above, some of the fibre bundles were pre-treated with Ringer solution containing 20 μm of the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor PD98059. They were then treated with Ringer solution containing DHT and the inhibitor for 1 h. As the results in Fig. 4C show, pre-treating the fibre bundles with PD98059 completely abolished the effects of DHT on the phosphorylation of ERK1/2.

Androgen receptor density in fast and slow twitch skeletal muscles

Previously, it has been suggested that the diverse effects of steroid hormones on specific muscle tension observed in animal experiments arise from differences in their androgen receptor (AR) densities (Egginton, 1987; Salmons, 1992). Therefore, to test this hypothesis and to determine whether the different effects of DHT on Po in fast and slow twitch muscle fibre bundles reported above arose from differences in their AR densities, we examined the concentration of the AR in EDL (fast twitch) and soleus (slow twitch) muscles of adult male and female mice. As the results displayed in Fig. 5 show, one of the antibodies (from Santa Cruz) identified two proteins: one with a molecular mass of 110 kDa and the other ∼67 kDa in all the samples probed (Fig. 5A). On the other hand, a second antibody (from Abcam) identified only one protein with a molecular mass of ∼110 kDa (Fig. 5B). Moreover, the concentration of both proteins was similar in the fast and slow twitch muscles isolated from each animal irrespective of sex. From these results, we think that the protein with a molecular mass of ∼110 kDa corresponds to the full-length AR (also known as AR-B), whereas that with a molecular mass of ∼67 kDa represents the truncated or AR-A isoform (Wilson & McPhaul, 1996).

Figure 5.

Figure 5

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Fast twitch and slow twitch muscles express similar concentrations of the androgen receptor Western blots showing the androgen receptor expression in female fast (FF), female slow (FS), male fast (MF) and male slow (MS) twitch skeletal muscles immunoblotted with a polyclonal antibody against the C-terminus of the human AR (A) or a monoclonal antibody against the N-terminus of the human AR (B). Note that the polyclonal antibody identified two proteins, one with a molecular mass of ∼110 kDa (AR-B) and the other with a molecular mass of ∼67 kDa (AR-A), whereas the monoclonal antibody identified only a single band with a molecular mass of ∼110 kDa. C and D, summary data showing the relative concentrations of AR-B (C) and AR-A (D) in fast (F) and slow (S) twitch muscles isolated from male (M) and female (F) mice. Note that fast and slow twitch muscles from the same sex animal express similar concentrations of the AR and that no significant difference (P= 0.12) was observed when the concentration of the receptor in the male and female muscles were compared.

The effects of DHT and testosterone on the phosphorylation of the 20 kDa regulatory myosin light chains

Previously, a number of studies have shown that the motility of non-muscle cells results from an ERK1/2-dependent increase in the phosphorylation of the 20 kDa RMLCs (Klemke et al. 1997; Iwabu et al. 2004). However, it is uncertain whether the changes in the phosphorylation of ERK1/2 induced by DHT in intact mammalian skeletal muscles have similar effects on the phosphorylation of RMLCs. Therefore, in another experiment, we investigated the effects of DHT and testosterone on the phosphorylation of the 20 kDa RMLCs. As the results displayed in Fig. 6 show, treating the muscle fibre bundles with DHT led to a 20–40% increase in the phosphorylation of RMLCs in fast twitch fibre bundles only (Fig. 5A). In contrast, testosterone, which had no effect on Po led to a decrease in the phosphorylation of RMLCs in both fibre types (Fig. 6C). Furthermore, treating the fibre bundles with the myosin light chain kinase inhibitor ML-7 did not significantly affect Po (Fig. 6D) but completely reversed the effects of DHT on force (Fig. 6B).

Figure 6.

Figure 6

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DHT increases the phosphorylation of regulatory myosin light chains in fast and slow twitch muscle fibre bundles Representative Western blots showing the effects of treating female fast twitch (F), female slow twitch (S), male fast twitch (MF) and male slow twitch (MS) skeletal muscle fibre bundles treated with Ringer solution containing either DHT (A) or testosterone (C) on phosphorylation of the 20 kDa regulatory myosin light chains (pMLC; A and C). Note that treating the muscle fibre bundles with DHT significantly (*P= 0.018) increases the phosphorylation of the RMLCs in fast twitch fibre bundles (A). In contrast, treating the bundles with testosterone led to a small decrease in the phosphorylation of the RMLCs which was not statistically different from that in control fibres (P= 0.08). Furthermore, fast twitch fibres contained 15–20% more unphosphorylated myosin light chains (tMLC) than slow twitch fibres. B and D show the effects of treating small muscle fibre bundles with the standard Ringer solution (Con), the standard Ringer solution plus DHT (DHT), or the standard Ringer solution containing the myosin light chain kinase inhibitor ML-7 alone (D, ML-7) or with DHT (B, DML-7) on Po. Note that treating the fibre bundles with ML-7 alone has no effect on Po (D) but reversibly abolishes the effects of DHT on Po.

Identity of the surface membrane receptor mediating the rapid actions of DHT in mammalian skeletal muscle fibres

As stated in the Introduction, the genomic/classical actions of anabolic androgenic steroids (AAS) are mediated through the androgen receptor. Therefore, to determine whether the effects of DHT described above were exerted through this mechanism, small muscle fibre bundles were treated with Ringer solution containing the AR inhibitors cyproterone (a steroidal inhibitor) and flutamide (a non-steroidal inhibitor). As the results in Fig. 7 show, treating the fibre bundles with cyproterone had no effect on the DHT-induced changes in force (Fig. 7A), ERK1/2 phosphorylation (Fig. 7B) and the phosphorylation of the RMLCs (Fig. 7C). Similar results were obtained when the muscle fibre bundles were treated with flutamide (results not shown), the insulin-like growth factor 1 receptor (IGF-1R) inhibitor tyrphostin AG 538 (Fig. 8A and B), the Src kinase (src)-specific inhibitor PP2 (results not shown) and the platelet-derived growth factor receptor (PDGFR) inhibitor (Fig. 8C and D).

Figure 7.

Figure 7

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Inhibiting the AR does not abolish the effects of DHT in isolated intact mammalian skeletal muscle fibres A, summary data showing the effects of treating small skeletal muscle fibre bundles with the standard Ringer solution (open bars), the standard Ringer solution plus DHT (hatched bars) and the standard Ringer solution containing DHT plus the AR inhibitor cyproterone (filled bars) on Po. Note that treating the muscle fibres bundles with cyproterone does not reverse the effects of DHT on Po in either fibre type. B and C, representative Western blots and summary data showing the effects of treating fast (F) and slow (S) twitch skeletal muscle fibre bundles isolated from female mice with the standard Ringer solution (FC, SC), DHT alone (FT, ST) or DHT plus cyproterone (FCT, SCT) on the phosphorylation of ERK1/2 (B) and regulatory myosin light chains (C). Note that treating the muscle fibre bundles with DHT significantly (*P= 0.015) increases the phosphorylation of ERK1/2 in both fibre types (B) and the regulatory myosin light chains in fast twitch fibre bundles only (A) and that treating the fibre bundles with cyproterone does not reverse these effects.

Figure 8.

Figure 8

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Inhibiting the IGF-1 and the PDGF receptors does not abolish the effects of DHT in isolated intact mammalian skeletal muscle fibres Summary data showing the effects of treating small skeletal muscle fibre bundles with the standard Ringer solution (open bars), the standard Ringer solution plus DHT (hatched bars) and the standard Ringer solution containing both DHT and the IGF-1R inhibitor tyrphostin AG 538 (A) or the PDGFR inhibitor tyrosine kinase inhibitor III (C) on Po in fast (i) and slow (ii) twitch muscle fibre bundles. Note that treating the muscle fibres bundles with both inhibitors does not reverse the effects of DHT on Po in either fibre type. B and D, representative Western blots and summary data showing the effects of treating fast (F) and slow (S) twitch skeletal muscle fibre bundles isolated from female mice with the standard Ringer solution (FC, SC), DHT alone (FT, ST) or DHT plus AG 538 (FGT, SGT) or tyrosine kinase inhibitor III (FPT, SPT) on the phosphorylation of ERK1/2. Note that treating the muscle fibre bundles with DHT significantly (*P= 0.015) increases the phosphorylation of ERK1/2 in the slow twitch muscle fibre bundles and that adding the inhibitors does not abolish these effects.

A number of studies have recently suggested that the ERK1/2-dependent phosphorylation of MLCs and hence the motility of non-muscle cells is mediated through the EGFR (Klemke et al. 1997; Iwabu et al. 2004). Therefore, in another experiment we used pharmacological interventions to investigate whether the rapid actions of DHT in adult mammalian skeletal muscle fibres were mediated through the EGFR. As the results displayed in Fig. 9 show, treating the fibres with the EGFR-specific inhibitor tyrphostin AG 1473 completely abolished the effects of DHT on Po in both fibre types (Fig. 9A). It also blocked the DHT-induced increase in the phosphorylation of ERK1/2 (Fig. 9B) as well as that of RMLCs (Fig. 9C and D).

Figure 9.

Figure 9

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Inhibiting the EGF receptor abolishes the effects of DHT in isolated intact mammalian skeletal muscle fibres A, summary data showing the effects of treating small skeletal muscle fibre bundles with the standard Ringer solution (open bars), the standard Ringer solution plus DHT (hatched bars) and the standard Ringer solution containing DHT plus the EGFR inhibitor tyrphostin AG 1478 (filled bars) on Po. Note that treating the muscle fibre bundles with tyrphostin AG 1478 reverses the effects of DHT on Po in both fibre types. B, C and D, representative Western blots and summary data showing the effects of treating fast (F) and slow (S) twitch skeletal muscle fibre bundles isolated from female mice with the standard Ringer solution (FC, SC), DHT alone (FT, ST), DHT plus tyrphostin AG 1478 (FiT, SiT) or DHT plus PD89059 (FMT, SMT) on the phosphorylation of ERK1/2 (B) and regulatory myosin light chains (C and D). Note that treating the muscle fibre bundles with DHT significantly (*P= 0.01) increases the phosphorylation of ERK1/2 (B) and the regulatory myosin light chains (C and D) in fast fibres and that treating the fibre bundles with tyrphostin AG 1478 and PD89059 abolishes these effects.

Discussion

One of the key findings in the present study is the observation that treating small skeletal muscle fibre bundles with Ringer solution containing physiological levels of male sex steroids can modulate Po in a fibre type- and hormone-dependent manner. Thus, DHT increased Po in fast twitch fibres but decreased it in slow twitch fibres. In contrast, testosterone had no effect on Po in both fibre types. Although the effects of treating animals with AAS for several weeks have been the subject of a number of previous studies and some of their effects are generally accepted (Exner et al. 1973a,b; Egginton, 1987; Salmons, 1992), their effects on specific muscle force are still controversial. For example, treating female rats (Egginton, 1987), male rats (Exner et al. 1973a) and female rabbits (Salmons, 1992) with nandrolone for 4–6 weeks has been shown to have no effect on specific muscle force in the EDL and soleus muscles. On the other hand, it increased specific muscle force in the flexor hallucius muscle of female rats (Exner et al. 1973b) and the tibialis anterior of female rabbits (Salmons, 1992). Interestingly, a similar increase in specific muscle force could be induced by exercise alone or exercise plus nandrolone (Exner et al. 1973b) suggesting that the effects of the AAS may have been non-specific. It is not just in animal experiments where the effects of AAS on specific muscle force are controversial, the results from human studies are as variable (Bhasin et al. 2001).

Previously, this variability has been attributed to factors such as the generalised increase in muscle mass that accompanies chronic AAS treatment (Egginton, 1987), the sensitivity of different muscles/species to the AAS (Salmons, 1992), the androgen receptor density in the muscles (Salmons, 1992), route of administration and variability in the resorption of the drugs from muscles and fat depots (van der Vies, 1970), study design (Bhasin et al. 2001) and the effects of the AAS on the nervous system (Blanco et al. 1997; Nguyen et al. 2005). In the present study, most of these variables were eliminated by the use of small muscle fibre bundles isolated from age- and weight-matched mice kept under the same laboratory conditions. Furthermore, each fibre bundle acted as its own control and the AAS was delivered directly to the fibres. Additionally, fast and slow twitch muscles contained similar levels of AR (Fig. 5). Thus, the results we report here provide the first unequivocal evidence suggesting that AAS may have direct effects on force in isolated intact mammalian skeletal muscle fibre bundles, and that these effects may depend on both the fibre type as well as the AAS used. Therefore, it is possible that the variable effects of AAS on specific muscle force reported previously in both animal and human experiments may have arisen from the fibre type composition of the various muscles examined, the AAS used, or a combination of both factors.

In addition to the changes in force reported above, treating small muscle fibre bundles with DHT led to an increase in the phosphorylation of ERK1/2 in both fibre types and the 20 kDa RMLCs in fast twitch fibres. It is important to note that testosterone, which had no effect on maximum isometric tension in either fibre type, did not affect the phosphorylation of RMLCs in both fibre types and increased the phosphorylation of ERK1/2 in the slow twitch fibres only. In both muscle and non-muscle cells, force generation results from the interaction of myosin and actin leading to the formation of the independent force-generating units known as crossbridges (Gordon et al. 2000). Although this mechanism is basically similar in all cells, its regulation in striated muscles and other cells is different. Thus, in striated muscle, the interaction between myosin and actin is regulated mainly through the thin (actin) filament, whereas, in smooth muscles and non-muscle cells it is controlled via the thick (myosin) filament (Adelstein, 1983). The thick filament regulation of actino-myosin interaction involves the phosphorylation and dephosphorylation of the 20 kDa regulatory light chains located within the myosin molecule by myosin light chain kinase and myosin phosphatase, respectively (Adelstein, 1983; Bresnick, 1999). Thus, in smooth muscle and non-muscle cells the phosphorylation of the RMLCs is essential for actino-myosin interaction to occur, whereas in striated muscles it is not. In striated muscle, the phosphorylation of these light chains seems to have a modulatory rather than a regulatory function on force production (Manning & Stull, 1979). Therefore, from our observations we suggest that the changes in force induced by DHT in mouse skeletal fibres and the phosphorylation of the RMLCs are interlinked. This suggestion is further supported by the observations that treating fast twitch muscle fibre bundles with the myosin light chain kinase inhibitor ML-7 led to a 10% decrease in Po and completely abolished the effects of DHT on Po (Fig. 6).

Why DHT increases maximum isometric force in fast twitch fibres and decreases it in slow twitch fibres is uncertain. Previously, it has been suggested that these differences may arise from the androgen receptor (AR) density in the muscles (Egginton, 1987; Salmons, 1992). However, as our results show, fast and slow twitch muscles from the same animal contain similar concentrations of the androgen receptor (Fig. 5). Furthermore, treating muscle fibre bundles with the androgen receptor inhibitors cyproterone and flutamide does not abolish the effects of DHT on force, the phosphorylation of ERK1/2 as well as that of the 20 kDa RMLCs (Fig. 7). Therefore, it is unlikely that the opposing effects of DHT on force in fast and slow twitch muscle fibres arise from their androgen receptor densities.

In mammalian skeletal muscles, a brief period of repetitive stimulation leads to a transitory increase of the isometric twitch in fast twitch muscles and its depression in slow twitch muscles (Close & Hoh, 1968, 1969). Furthermore, this post-tetanic twitch potentiation is accompanied by an increase in the phosphorylation of the 20 kDa RMLCs (Klug et al. 1982; Moore & Stull, 1984; Houston et al. 1985). Although the phosphorylation of the RMLC does not seem to affect maximum Ca2+-activated force in chemically skinned skeletal muscle fibre bundles (Metzger et al. 1989), our results suggest that under certain circumstances it may. It is generally believed that the phosphorylation of RLMCs potentiates the isometric twitch by increasing the sensitivity of the contractile apparatus to Ca2+ and freeing crossbridges from the surface of the thick filament (Sweeny et al. 1993). Here we speculate that in addition to the phosphorylation of RMLCs, DHT treatment increases the phosphorylation of other proteins of the contractile system such as troponin I and myosin binding protein C that are involved in the regulation of actino-myosin interaction; and that this accentuates its effects on force production. However, the exact mechanism underlying the effects of DHT in isolated mammalian skeletal fibres is still uncertain and further studies to elucidate this are necessary.

In the classical or genomic pathway, testosterone exerts its effects by activating the cytosolic androgen receptor. The hormone–receptor complex then translocates to the nucleus where the hormone–receptor complex acts as a transcription factor (Simental et al. 1991). However, as this mechanism of steroid action involves gene transcription and mRNA translation, its effects usually take several hours to days to be manifested (Florini, 1970; Beato, 1989, 1996). In the present study, the effects of DHT were evident within 30 min after its application and were insensitive to both cyproterone and flutamide, both inhibitors of the intracellular androgen receptor. Therefore, from these results we suggest that the effects of DHT reported here are not exerted through the androgen receptor. They were also insensitive to inhibitors of src, IGF-1R and PDGFR; suggesting that they were not mediated via these receptors either. Instead, our results suggest that these effects are mediated through EGFR. Our hypothesis, shown in Fig. 10, is that DHT binds to the EGFR on the sarcolemma leading to the activation of the MAPK pathway. The activated MAPK pathway then drives the phosphorylation of the regulatory myosin light chains by myosin light chain kinase, culminating in the changes in force reported in this study. This mechanism of action is similar to that previously reported in Sertoli cells (Cheng et al. 2007). However, unlike that study, no evidence was observed to suggest that Src kinase is involved. In the only other study of muscles cells, it was suggested that testosterone and nandrolone exert their rapid actions through an unidentified G-protein-coupled receptor located on the cell membrane (Estrada et al. 2003). From the results presented here, we suggest that this receptor is probably the EGFR. However, whether the effects of DHT are direct or indirect remain uncertain and further studies are necessary.

Figure 10.

Figure 10

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Proposed cellular signalling pathway mediating the non-genomic actions of DHT in adult mammalian skeletal muscle fibres Schematic diagram illustrating the molecular mechanism that we suggest underlies the effects of DHT in adult mammalian skeletal muscle fibre bundles. Our hypothesis is that DHT activates the epidermal growth factor receptor (EGFR), either directly or indirectly, and this leads to an increase in the phosphorylation of ERK1/2. The activated ERK1/2 then phosphorylates MLCK which in turn phosphorylates the 20 kDa RMLCs and this increases force production in fast twitch fibres but decreases it in slow twitch fibres.

Besides the phosphorylation of myosin light chain kinase (MLCK) the activation of the MAPK pathway in most non-muscle cells leads to the transcriptional regulation of genes important for cell proliferation and differentiation (Marshall, 1995). Therefore, it is likely that in addition to its effects on force, the activation of ERK1/2 by DHT may have other functions such as the transformation of slow to fast twitch muscle fibres. Our speculation is that the irreversible decline of force induced by DHT in slow twitch fibre bundles marks the onset of this process. The atrophying slow twitch fibres are then slowly replaced by fast fibres generated from satellite cells. Indeed, in animal models, the prolonged administration of AAS is accompanied by satellite cell activation (see review by Chen et al. 2005). Thus, the rapid actions of AAS in mammalian skeletal muscles may be multifaceted and their main physiological function may be to prepare the muscles for their genomic actions. However, whether this is the case is uncertain and further studies are needed.

Acknowledgments

This work was supported by the University of East Anglia and a PhD studentship to M.M.H. from the Malaysian Government. We thank Professor Tom Wileman for discussions and useful comments on a previous version of the manuscript.

Glossary

Abbreviations

AAS

anabolic androgenic steroids

AR

androgen receptor

DHT

dihydrotestosterone

EDL

extensor digitorum longus

EGFR

epidermal growth factor receptor

ERK1/2

extracellular signal-regulated kinase1/2

IGF-1R

insulin-like growth factor 1 receptor

MAPK

mitogen-activated protein kinase

MEK1/2

MAPK/ERK kinase1/2

MLCK

myosin light chain kinase

PDGFR

platelet-derived growth factor receptor

RMLCs

regulatory myosin light chains

src

Src kinase

Author contributions

G.M. conceptualised and designed the study. He also performed the force measurement experiments. M.M.H. performed the Western blot experiments and the initial analysis of the data.

References

  1. Adelstein RS. Regulation of contractile proteins by phosphorylation. J Clin Invest. 1983;72:1863–1866. doi: 10.1172/JCI111148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Basaria S, Wahlstrom JT, Dobs AS. Anabolic-androgenic steroid therapy in the treatment of chronic diseases. J Clin Endocrinol Metab. 2001;86:5108–5117. doi: 10.1210/jcem.86.11.7983. [DOI] [PubMed] [Google Scholar]
  3. Beato M. Gene regulation by steroid hormones. Cell. 1989;56:335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  4. Beato M, Chávez S, Truss M. Transcriptional regulation by steroid hormones. Steroids. 1996;61:240–251. doi: 10.1016/0039-128x(96)00030-x. [DOI] [PubMed] [Google Scholar]
  5. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Bhasin D, Berman N, Chen X, Yarasheski KE, Magliano L, Dzekov C, Dzekov J, Bross R, Phillips J, Sinha-Hikim I, Shen R, Storer TW. Testosterone dose-response relationships in healthy young men. Am J Physiol Endocrinol Metab. 2001;281:E1172–E1181. doi: 10.1152/ajpendo.2001.281.6.E1172. [DOI] [PubMed] [Google Scholar]
  6. Blanco CE, Popper P, Micevych P. Anabolic-androgenic steroid induced alterations in choline acetyltransferase messenger RNA levels of spinal cord motoneurons in the male rat. Neuroscience. 1997;78:873–882. doi: 10.1016/s0306-4522(96)00597-0. [DOI] [PubMed] [Google Scholar]
  7. Bresnick AR. Molecular mechanisms of nonmuscle myosin-II regulation. Curr Opin Cell Biol. 1999;11:26–33. doi: 10.1016/s0955-0674(99)80004-0. [DOI] [PubMed] [Google Scholar]
  8. Bruchovsky N, Wilson JD. The conversion of testosterone to 5α-androstan-17β-ol-3-one by rat prostate in vivo and in vitro. J Biol Chem. 1968;243:2012–2021. [PubMed] [Google Scholar]
  9. Chen Y, Zajac J, MacLean HE. Androgen regulation of satellite cell function. J Endocrinol. 2005;186:21–31. doi: 10.1677/joe.1.05976. [DOI] [PubMed] [Google Scholar]
  10. Cheng J, Zhang C, Shapiro DJ. A functional serine 118 phosphorylation site in estrogen receptor-α is required for down-regulation of gene expression by 17β estradiol and 4-hydroxytamoxifen. Endocrinology. 2007;148:4634–4641. doi: 10.1210/en.2007-0148. [DOI] [PubMed] [Google Scholar]
  11. Close R, Hoh JFY. After-effects of repetitive stimulation on isometric twitch contraction of rat fast skeletal muscle. J Physiol. 1968;197:461–477. doi: 10.1113/jphysiol.1968.sp008570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Close R, Hoh JFY. Post-tetanic potentiation of twitch contractions of cross-innervated rat fast and slow muscles. Nature. 1969;221:179–181. doi: 10.1038/221179a0. [DOI] [PubMed] [Google Scholar]
  13. Drummond GB. Reporting ethical matters in The Journal of Physiology: standards and advice. J Physiol. 2009;587:713–719. doi: 10.1113/jphysiol.2008.167387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Egginton S. Effects of an anabolic hormone on striated muscle growth and performance. Pflugers Arch. 1987;410:349–355. doi: 10.1007/BF00586510. [DOI] [PubMed] [Google Scholar]
  15. Estrada M, Espinosa A, Muller M, Jaimovich E. Testosterone stimulates intracellular calcium release and mitogen-activated protein kinases via a G protein-coupled receptor in skeletal muscle cells. Endocrinology. 2003;144:3586–3597. doi: 10.1210/en.2002-0164. [DOI] [PubMed] [Google Scholar]
  16. Estrada M, Liberona JL, Miranda M, Jaimovich E. Aldosterone- and testosterone-mediated intracellular calcium response in skeletal muscle cell cultures. Am J Physiol Endocrinol Metab. 2000;279:E132–E139. doi: 10.1152/ajpendo.2000.279.1.E132. [DOI] [PubMed] [Google Scholar]
  17. Evans NA. Current concepts in anabolic-androgenic steroids. Am J Sports Med. 2004;32:534–542. doi: 10.1177/0363546503262202. [DOI] [PubMed] [Google Scholar]
  18. Exner GU, Staudte HW, Pette D. Isometric training of rats – effects upon fast and slow muscle and modification by an anabolic hormone (nandrolone decanoate). I. Female rats. Pflugers Arch. 1973a;345:1–14. doi: 10.1007/BF00587057. [DOI] [PubMed] [Google Scholar]
  19. Exner GU, Staudte HW, Pette D. Isometric training of rats – effects upon fast and slow muscle and modification by an anabolic hormone (nandrolone decanoate). II. Male rats. Pflugers Arch. 1973b;345:15–22. doi: 10.1007/BF00587058. [DOI] [PubMed] [Google Scholar]
  20. Falkenstein E, Tillmann H-C, Christ M, Feuring M, Wehling M. Multiple actions of steroid hormones – A focus on rapid, nongenomic effects. Pharmacol Rev. 2000;52:513–556. [PubMed] [Google Scholar]
  21. Florini JR. Effects of testosterone on qualitative pattern of protein synthesis in skeletal muscle. Biochemistry. 1970;9:909–912. doi: 10.1021/bi00806a027. [DOI] [PubMed] [Google Scholar]
  22. Gelmann EP. Molecular biology of the androgen receptor. J Clin Oncol. 2002;20:3001–3015. doi: 10.1200/JCO.2002.10.018. [DOI] [PubMed] [Google Scholar]
  23. Gordon AM, Homsher E, Regnier M. Regulation of contraction in striated muscle. Physiol Rev. 2000;80:853–924. doi: 10.1152/physrev.2000.80.2.853. [DOI] [PubMed] [Google Scholar]
  24. Heinlein CA, Chang C. Androgen receptor (AR) coregulators: an overview. Endocr Rev. 2002a;23:175–200. doi: 10.1210/edrv.23.2.0460. [DOI] [PubMed] [Google Scholar]
  25. Heinlein CA, Chang C. The roles of androgen receptors and androgen-binding proteins in nongenomic androgen actions. Mol Endocrinol. 2002b;16:2181–2187. doi: 10.1210/me.2002-0070. [DOI] [PubMed] [Google Scholar]
  26. Houston ME, Green HJ, Stull JT. Myosin light chain phosphorylation and isometric twitch potentiation in intact human muscle. Pflugers Arch. 1985;403:348–352. doi: 10.1007/BF00589245. [DOI] [PubMed] [Google Scholar]
  27. Iwabu A, Smith K, Allen FD, Lauffenburger DA, Wells A. Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C δ-dependent pathway. J Biol Chem. 2004;279:14551–14560. doi: 10.1074/jbc.M311981200. [DOI] [PubMed] [Google Scholar]
  28. Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, Cheresh DA. Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol. 1997;137:481–492. doi: 10.1083/jcb.137.2.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Klug GA, Botterman BR, Stull JT. The effect of low frequency stimulation on myosin light chain phosphorylation in skeletal muscle. J Biol Chem. 1982;257:4688–4690. [PubMed] [Google Scholar]
  30. Losel RM, Falkenstein E, Feuring M, Schultz A, Tillmann H-C, Rossol-Haseroth K, Wehling M. Nongenomic steroid action: controversies, questions, and answers. Physiol Rev. 2003;83:965–1016. doi: 10.1152/physrev.00003.2003. [DOI] [PubMed] [Google Scholar]
  31. Manning DR, Stull JT. Myosin light chain phosphorylation and phosphorylase-A activity in rat extensor digitorum longus muscle. Biochem Biophys Res Commun. 1979;90:164–170. doi: 10.1016/0006-291x(79)91604-8. [DOI] [PubMed] [Google Scholar]
  32. Marshall MS. Ras target proteins in eukaryotic cells. FASEB J. 1995;9:1311–1318. doi: 10.1096/fasebj.9.13.7557021. [DOI] [PubMed] [Google Scholar]
  33. Metzger JM, Greaser ML, Moss R. Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibres. Implications for twitch potentiation in intact muscle. J Gen Physiol. 1989;93:855–883. doi: 10.1085/jgp.93.5.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Moore RL, Stull JT. Myosin light chain phosphorylation in fast and slow skeletal-muscles in situ. Am J Physiol Cell Physiol. 1984;247:C462–C471. doi: 10.1152/ajpcell.1984.247.5.C462. [DOI] [PubMed] [Google Scholar]
  35. Mutungi G. The expression of NFATc1 in adult rat skeletal muscle fibres. Exp Physiol. 2008;93:399–406. doi: 10.1113/expphysiol.2007.040485. [DOI] [PubMed] [Google Scholar]
  36. Mutungi G, Ranatunga KW. Sarcomere length changes during end-held (isometric) contractions in intact mammalian (rat) fast and slow muscle fibres. J Muscle Res Cell Motil. 2000;21:565–575. doi: 10.1023/a:1026588408907. [DOI] [PubMed] [Google Scholar]
  37. Nguyen TH, Lee GS, Ji YK, Choi KC, Lee CK, Jeung EB. A calcium binding protein, calbindin-D9k, is mainly regulated by estrogen in the pituitary gland of rats during estrous cycle. Mol Brain Res. 2005;141:166–173. doi: 10.1016/j.molbrainres.2005.09.008. [DOI] [PubMed] [Google Scholar]
  38. Pardridge WM. Serum bioavailability of sex steroid hormones. Clin Endocrinol Metab. 1986;15:259–278. doi: 10.1016/s0300-595x(86)80024-x. [DOI] [PubMed] [Google Scholar]
  39. Rahman F, Christian HC. Non-classical actions of testosterone: an update. Trends Endocrinol Metab. 2007;18:371–378. doi: 10.1016/j.tem.2007.09.004. [DOI] [PubMed] [Google Scholar]
  40. Saartok T, Dahlberg E, Gustafsson JA. Relative binding affinity of anabolic-androgenic steroids: comparison of the binding to the androgen receptors in skeletal muscle and in prostate, as well as to sex hormone-binding globulin. Endocrinology. 1984;114:2100–2106. doi: 10.1210/endo-114-6-2100. [DOI] [PubMed] [Google Scholar]
  41. Salmons S. Myotrophic effects of an anabolic steroid in rabbit limb muscles. Muscle Nerve. 1992;15:806–812. doi: 10.1002/mus.880150709. [DOI] [PubMed] [Google Scholar]
  42. Simental JA, Sar M, Lane MV, French FS, Wilson EM. Transcriptional activation and nuclear targeting signals of the human androgen receptor. J Biol Chem. 1991;266:510–518. [PubMed] [Google Scholar]
  43. Simoncini T, Genazzani AR. Non-genomic actions of sex steroid hormones. Eur J Endocrinol. 2003;148:281–292. doi: 10.1530/eje.0.1480281. [DOI] [PubMed] [Google Scholar]
  44. Sweeny HL, Bowman BF, Stull JT. Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am J Physiol Cell Physiol. 1993;264:C1085–C1095. doi: 10.1152/ajpcell.1993.264.5.C1085. [DOI] [PubMed] [Google Scholar]
  45. Thigpen AE, Silver RI, Guileyardo JM, Casey ML, McConnell JD, Russell DW. Tissue distribution and ontogeny of steroid 5-α-reductase isozyme expression. J Clin Invest. 1993;92:903–910. doi: 10.1172/JCI116665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. van der Vies J. Model studies with long-acting hormonal preparations. Acta Endocrinol. 1970;64:656–669. doi: 10.1530/acta.0.0640656. [DOI] [PubMed] [Google Scholar]
  47. Wilson CM, McPhaul MJ. A and B forms of the androgen receptor are expressed in a variety of human tissues. Mol Cell Endocrinol. 1996;120:51–57. doi: 10.1016/0303-7207(96)03819-1. [DOI] [PubMed] [Google Scholar]

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