Identification of potential target genes associated with the reversion of androgen-dependent skeletal muscle atrophy.

Muscle wasting or atrophy is extensively associated with human systemic diseases including diabetes, cancer, and kidney failure. Accumulating evidence from transcriptional profiles has noted that a common set of genes, termed atrogenes, is modulated in atrophying muscles. However, the transcriptional changes that trigger the reversion or attenuation of muscle atrophy have not been characterized at the molecular level until now. Here, we applied cDNA microarrays to investigate the transcriptional response of androgen-sensitive Levator ani muscle (LA) during atrophy reversion. Most of the differentially expressed genes behaved as atrogenes and responded to castration-induced atrophy. However, seven genes (APLN, DUSP5, IGF1, PIK3IP1, KLHL38, PI15, and MKL1) did not respond to castration but instead responded exclusively to testosterone replacement. Considering that almost all proteins encoded by these genes are associated with the reversion of atrophy and may function as regulators of cell proliferation/growth, our results provide new perspectives on the existence of anti-atrogenes.

The Extracellular cAMP-Adenosine Pathway in Airway Smooth Muscle.

In the respiratory tract, intracellular cAMP has a key role in the smooth muscle relaxation induced by the ß2-adrenoceptor/Gs protein/adenylyl cyclase axis. In other tissues, cAMP also works as an extracellular messenger, after its efflux and interstitial conversion to adenosine by ectoenzymes. The aim of this study was to identify cAMP efflux and the "extracellular cAMP-adenosine pathway" in the airway smooth muscle. First, we tested the ability of ß2-adrenoceptor agonists formoterol or fenoterol to increase the extracellular cAMP in isolated tracheal rings from adult male Wistar rats. The effects of adenosine, cAMP, 8-Br-cAMP, fenoterol, or formoterol were also evaluated in the isometric contraction of control or carbachol (CCh) precontracted tracheas, normalized as the percentage of CCh-induced response. Fenoterol and formoterol induced 70%-80% relaxation and increased extracellular cAMP levels by up to 280%-450%. Although exogenous cAMP or adenosine evoked phasic contractions, the membrane-permeable cAMP analog 8-Br-cAMP induced relaxation of CCh-precontracted tracheas. The simultaneous inhibition of adenosine degradation/uptake with EHNA [erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride] plus uridine increased by 3-fold the maximum cAMP-induced contraction, whereas it was significantly reduced by AMPCP [adenosine 5'-(α,ß-methylene)diphosphate; an ecto-5'-nucleotidase inhibitor], and by adenosine receptor antagonists CGS-15943 (nonselective) or DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (A1 selective). Finally, CGS-15943 shifted to the left the concentration-relaxation curve for fenoterol. In conclusion, our results show that airway smooth muscle expresses the extracellular cAMP-adenosine pathway associated with contracting effects mediated by A1 receptors. The cAMP efflux triggered by fenoterol/formoterol indicates that the extracellular cAMP-adenosine pathway may play a role in balancing the relaxant effects of ß2-adrenoceptor agonists in airways, which may impact their bronchodilation effects.

Contraction of Rat Cauda Epididymis Smooth Muscle to α1-Adrenoceptor Activation Is Mediated by α1A-Adrenoceptors.

The cauda epididymis (CE), the site of sperm storage until the ejaculation, is densely innervated by the sympathetic nervous system. Contraction of CE smooth muscle via α1-adrenoceptors (α1-ARs) plays a key role during the seminal emission phase of ejaculation and α1-AR antagonism has been suggested as a nonhormonal and reversible male contraceptive target. Since the α1-AR subtype mediating contraction of rat CE is not known, this study investigates the expression and role of α1-AR subtypes on the proximal and distal rat CE duct contraction to norepinephrine in vitro. Alpha1a, α1b, and α1d transcripts were detected by real-time quantitative polymerase chain reaction in proximal and distal CE segments and α1a and α1d were shown to predominate over α1b The inhibition of [3H]prazosin specific binding to intact CE segments from proximal and distal CE by RS 100329 and 5-methylurapidil (α1A-selective) and BMY 7378 (α1D-selective) showed that α1A- and α1D-ARs are expressed at similar densities. Norepinephrine-induced contractions of CE were competitively antagonized with high affinity by RS 100329 (pKB ≈ 9.50) and 5-methylurapidil (pKB ≈ 9.0) and with low affinity by BMY 7378 (pKB ≈ 7.0) and the α1B-selective L-765,314 (pA2 < 7.0), suggesting contractions are mediated by α1A-ARs. The clinically used α1A/D-ARs antagonist tamsulosin potently (pA2 ≈ 10.0) inhibited the norepinephrine-induced CE contractions. Altogether, our results show that α1A- and α1D-ARs are expressed in the CE duct and α1A-AR is the main subtype mediating contraction to norepinephrine. Our results highlight the importance of α1A-AR in the peripheral control of ejaculation and strengthen the α1A-AR as a target for a nonhormonal approach to male contraception.

New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular cAMP-adenosine pathway.

G protein-coupled receptors (GPCRs) linked to stimulatory G (Gs) proteins (GsPCRs) mediate increases in intracellular cyclic AMP as consequence of activation of nine adenylyl cyclases , which differ considerably in their cellular distribution and activation mechanisms. Once produced, cyclic AMP may act via distinct intracellular signaling effectors such as protein kinase A and the exchange proteins activated by cAMP (Epacs). More recently, attention has been focused on the efflux of cAMP through a specific transport system named multidrug resistance proteins that belongs to the ATP-binding cassette transporter superfamily. Outside the cell, cAMP is metabolized into adenosine, which is able to activate four distinct subtypes of adenosine receptors, members of the GPCR family: A1, A2A, A2B, and A3. Taking into account that this phenomenon occurs in numerous cell types, as consequence of GsPCR activation and increment in intracellular cAMP levels, in this review, we will discuss the impact of cAMP efflux and the extracellular cAMP-adenosine pathway on the regulation of GsPCR-induced cell response.

Slimmer or fertile? Pharmacological mechanisms involved in reduced sperm quality and fertility in rats exposed to the anorexigen sibutramine.

Sperm acquire motility and fertility capacity during epididymal transit, under the control of androgens and sympathetic innervations. It is already known that the acceleration of epididymal sperm transit time can lead to lower sperm quality. In a previous work we showed that rats exposed to the anorexigen sibutramine, a non-selective serotonin-norepinephrine reuptake inhibitor, presented faster sperm transit time, lower epididymal sperm reserves and potentiation of the tension of epididymal duct to norepinephrine exposed acutely in vitro to sibutramine. In the present work we aimed to further investigate pharmacological mechanisms involved in these alterations and the impact on rat sperm quality. For this, adult male Wistar rats were treated with sibutramine (10 mg/kg/day) or vehicle for 30 days. Sibutramine decreased final body, seminal vesicle, ventral prostate and epididymal weights, as well as sperm transit time in the epididymal cauda. On the contrary of the in vitro pharmacological assays, in which sibutramine was added directly to the bath containing strips of distal epididymal cauda, the ductal tension was not altered after in vivo sub-chronic exposure to sibutramine. However, there is pharmacological evidence that the endogenous epididymal norepinephrine reserves were reduced in these animals. It was also shown that the decrease in prostate weight can be related to increased tension developed of the gland, due to sibutramine sympathomimetic effects. In addition, our results showed reduced sperm quality after in utero artificial insemination, a more sensitive procedure to assess fertility in rodents. The epididymal norepinephrine depletion exerted by sibutramine, associated with decreases in sperm transit time, quantity and quality, leading to reduced fertility in this experimental model, reinforces the concerns about the possible impact on fertility of man taking sibutramine as well as other non-selective serotonin-norepinephrine reuptake inhibitors, especially considering the lower reproductive efficiency of humans compared to males of other species.

Acceleration of sperm transit time and reduction of sperm reserves in the epididymis of rats exposed to sibutramine.

Sibutramine is a drug globally used for the treatment of obesity. The aim of this study was to investigate male reproductive disorders caused by sibutramine in adult rats. Wistar rats were treated for 28 consecutive days (gavage) with 10 mg/kg of sibutramine. Control animals received only vehicle (dimethylsulfoxide and saline). The rats were sacrificed for evaluation of body and reproductive organ weights, sperm parameters, hormone levels (luteinizing hormone, follicle-stimulating hormone, and testosterone), testicular and epididymal histopathology, sexual behavior, fertility and in vitro contractility of the epididymal duct. Sibutramine decreased (P < .05) weights of the epididymis and ventral prostate, but not of other reproductive organs. The sperm number and transit time in the epididymal cauda were decreased (P < .001), but the daily sperm production was not altered. Moreover, morphology and sperm motility, histopathology of the testes and epididymis, sexual behavior, fertility, and serum hormone levels were not altered by the treatment. Sibutramine increased the potency of norepinephrine and, per se, increased the mechanical activity of the epididymal duct in vitro. Thus, although sibutramine in these experimental conditions did not interfere with the reproductive process of rats, it provoked acceleration of the sperm transit time and a decrease in the sperm reserves in the epididymal cauda. This alteration is probably related to the sympathomimetic effect of this drug, as shown by the in vitro assays. In humans, use of this drug might present a threat for male fertility because sperm reserves in men are naturally lower than those in rats.