No abstract

Changes in the androgen levels in asthmatic men may be associated with the severity of asthma. Androgens induce a nongenomic relaxation in airway smooth muscle, but the underlying mechanisms remain unclear. The aim of this study was to investigate the potential bronchorelaxing action of testosterone (TES) and its metabolites (5a-and 5b-dihydrotestosterone (DHT). A preventive effect on ovalbumin (OVA)-induced bronchospasm was observed in sensitized guinea pigs for each androgen. Androgens were studied in response to bronchoconstrictors: carbachol (CCh) and KCl in isolated trachea rings with and without epithelium from non-sensitized and sensitized animals as well as on OVA-induced contraction. Androgens concentration-dependently abolished the contraction in response to CCh, KCl, and OVA. There were significant differences in the sensitivity to the relaxation induced by each androgen. 5b-DHT was more potent for relaxing KCl-induced contraction, while TES and 5a-DHT were more potent for CCh-and OVA-induced contraction. No differences were found in preparations with and without epithelium or in the presence of a nitric oxide (NO) synthase inhibitor or an inhibitor of K C channels. These data indicate the absence of involvement of the epithelium-, NO-and K C channels-dependent pathway in androgen-induced relaxation. However, in dissociated tracheal myocytes loaded with the calcium-binding fluorescent dye Fura -2, physiological concentrations of androgens decreased the KCl-induced [Ca 2C ] i increment. 5b-DHT was the most potent at decreasing KCl-induced [Ca 2C ] i increment and preventing bronchospasm. We suggest that androgen-induced brochorelaxation was mediated via decreased Ca 2C influx through L-type Ca 2C channels but additional Ca 2C entry blockade may be involved. Molecular changes in androgen structure may determine its preferential site of action. Key Words" asthma " testosterone " 5a-dihydrotestosterone" 5b-dihydrotestosterone " bronchoactive steroids " airway smooth muscle

Androgen vasorelaxing action is a subject of recent interest. We investigated the involvement of l-type voltage-operated Ca(2+) channels (L-VOCCs), K(+) channels, intracellular Ca(2+) concentration ([Ca(2+)]i), and cAMP in the vasorelaxing effect of testosterone and 5beta-dihydrotestosterone (5beta-DHT) on rat thoracic aorta. Isolated aortic rings were used to study the vasorelaxing potency of testosterone and 5beta-DHT on KCl- and noradrenaline-induced contractions. Patch-clamp was used to analyze androgen effects on Ca(2+) inward and K(+) outward currents. The fluorescence technique was used to evaluate [Ca(2+)]i in single myocytes; moreover, simultaneous measurements of [Ca(2+)]i and vascular contraction were evaluated. 5beta-DHT was more potent than testosterone to relax KCl-induced contraction, but they were equipotent to relax noradrenaline contraction. l-type Ca(2+) currents were blocked by nifedipine, both androgens, and an estrogen in a concentration-dependent manner, and the order of potency was: testosterone > nifedipine > 5beta-DHT > 17beta-estradiol. We observed that testosterone has different mechanism of action by the concentration range used: at nm concentrations it was a powerful L-VOCCs antagonist, whereas at mum concentrations it was observed that: 1) its Ca(2+) antagonist property is reverted by increasing the l-type inward Ca(2+) currents (Ca(2+) agonist property); and 2) the [Ca(2+)]i and cAMP production was increased. The total K(+) currents were unaffected by testosterone or 5beta-DHT. The data show that 5beta-DHT-induced vasorelaxation is due to its selective blockade on L-VOCCs (from nm to microm concentrations), but testosterone-induced vasorelaxation involves concentration-dependent additional mechanisms: acting as an L-VOCCs antagonist at low concentrations, and increasing [Ca(2+)]i and cAMP production at high concentrations.

In airway smooth muscle, the intracellular basal Ca2+ concentration [b(Ca2+)i] must be tightly regulated by several mechanisms in order to maintain a proper airway patency. The b[Ca2+]i is efficiently regulated by sarcoplasmic reticulum Ca2+-ATPase 2b, plasma membrane Ca2+-ATPase 1 or 4 and by the Na+/Ca2+ exchanger. Membranal Ca2+ channels, including the L-type voltage dependent Ca2+ channel (L-VDCC), T-type voltage dependent Ca2+ channel (T-VDCC) and transient receptor potential canonical 3 (TRPC3), appear to be constitutively active under basal conditions via the action of different signaling pathways, and are responsible for Ca2+ influx to maintain b[Ca2+]i. The two types of voltage-dependent Ca2+ channels (L- and T-type) are modulated by phosphorylation processes mediated by mitogen-activated protein kinase kinase (MEK) and extracellular-signal-regulated kinase 1 and 2 (ERK1/2). The MEK/ERK signaling pathway can be activated by G-protein-coupled receptors through the αq subunit when the endogenous ligand (i.e., acetylcholine, histamine, leukotrienes, etc.) is present under basal conditions. It may also be stimulated when receptor tyrosine kinases are occupied by the appropriate ligand (cytokines, growth factors, etc.). ERK1/2 phosphorylates L-VDCC on Ser496 of the β2 subunit and Ser1928 of the α1 subunit, decreasing or increasing the channel activity, respectively, and enabling it to switch between an open and closed state. T-VDCC is also probably phosphorylated by ERK1/2, although further research is required to identify the phosphorylation sites. TRPC3 is directly activated by diacylglycerol produced by phospholipase C (PLCβ or γ). Constitutive inositol 1,4,5-trisphosphate production induces the release of Ca2+ from the sarcoplasmic reticulum through inositol triphosphate receptor 1. This ion induces Ca2+-induced Ca2+ release through the ryanodine receptor 2 (designated as Ca2+ ‘sparks’). Therefore, several Ca2+ handling mechanisms are finely tuned to regulate basal intracellular Ca2+ concentrations. It is conceivable that alterations in any of these processes may render airway smooth muscle susceptible to develop hyperresponsiveness that is observed in ailments such as asthma.

In vascular smooth muscle, it has been described that testosterone (TES) produces relaxation by blocking L-type Ca(2+) channels. Recently, we found that L-type Ca(2+) and store-operated Ca(2+) (SOC) channels are the main membranal structures that provide extracellular Ca(2+) for carbachol (CCh)-induced contraction in airway smooth muscle (ASM). We studied the possible interactions between L-type and SOC channels in TES-induced relaxation in guinea pig ASM. TES (10, 32, 100, and 178 μM) induced a complete relaxation of CCh-precontracted tracheal smooth muscle, and indomethacin partially inhibited this response. In single myocytes, the KCl-induced intracellular Ca(2+) increase ([Ca(2+)]i) was decreased by 32 and completely blocked by 100 nM TES. This androgen (32 and 100 μM) significantly diminished (~25 and 49 %, respectively) the capacitative Ca(2+) entry. Myocytes stimulated with CCh produced a transient Ca(2+) peak followed by a sustained plateau. D-600 was added during the plateau phase, and a partial diminution (~35 %) was observed. A greater decrease (~78 %) was seen when 2-aminoethyl diphenylborinate (2-APB, SOC antagonist) was used. The combination of both drugs completely abolished the Ca(2+) plateau induced by CCh. TES (100 μM) also completely abolished the CCh-induced Ca(2+) plateau. Indomethacin significantly diminished this effect of TES. PGE2 and butaprost proportionally decreased the Ca(2+) plateau as indomethacin blocked it. Sarcoplasmic reticulum refilling was partially, dependently, and significantly diminished by TES. We concluded that TES-induced relaxation involves blockade of L-type Ca(2+) channels at nanomolar and SOC channels at micromolar concentration and PGE2 seems to be also involved in this phenomenon.