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Numerous studies suggest the involvement of adenosine-5′-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5′-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males. [ABSTRACT FROM AUTHOR]

Airway smooth muscle (ASM) contraction is determined by the increase in intracellular Ca2+ concentration ([Ca2+]i) caused by its release from the sarcoplasmic reticulum (SR) or by extracellular Ca2+ influx. Major channels involved in Ca2+ influx in ASM cells are L-type voltage-dependent Ca2+ channels (L-VDCCs) and nonselective cation channels (NSCCs). Transient receptor potential vanilloid 4 (TRPV4) is an NSCC recently studied in ASM. Mechanical stimuli, such as contraction, can activate TRPV4. We investigated the possible activation of TRPV4 by histamine (His)- or carbachol (CCh)-induced contraction in guinea pig ASM. In single myocytes, the TRPV4 agonist (GSK101) evoked an increase in [Ca2+]i, characterized by a slow onset and a plateau phase. The TRPV4 antagonist (GSK219) decreased channel activity by 94%, whereas the Ca2+-free medium abolished the Ca2+ response induced by GSK101. Moreover, GSK101 caused Na+ influx in tracheal myocytes. GSK219 reduced the Ca2+ peak and the Ca2+ plateau triggered by His or CCh. TRPV4 blockade shifted the concentration–response curve relating to His and CCh to the right in tracheal rings and reduced the maximal contraction. Finally, the activation of TRPV4 in single myocytes increased the Ca2+ refilling of the SR. We conclude that contraction of ASM cells after stimulation with His or CCh promotes TRPV4 activation, the subsequent influx of Ca2+ and Na+, and the opening of L-VDCCs. The entry of Ca2+ into ASM cells via TRPV4 and L-VDCCs contributes to optimal smooth muscle contraction. [ABSTRACT FROM AUTHOR]

Theophylline is a drug commonly used to treat asthma due to its anti-inflammatory and bronchodilatory properties. Testosterone (TES) has been suggested to reduce the severity of asthma symptoms. This condition affects boys more than girls in childhood, and this ratio reverses at puberty. We reported that guinea pig tracheal tissue chronic exposure to TES increases the expression of β2-adrenoreceptors and enhances salbutamol-induced K+ currents (IK+). Herein, we investigated whether the upregulation of K+ channels can enhance the relaxation response to methylxanthines, including theophylline. Chronic incubation of guinea pig tracheas with TES (40 nM, 48 h) enhanced the relaxation induced by caffeine, isobutylmethylxanthine, and theophylline, an effect that was abolished by tetraethylammonium. In tracheal myocytes, chronic incubation with TES increased theophylline-induced IK+; flutamide reversed this effect. The increase in IK+ was blocked by 4-aminopyridine by ~82%, whereas iberiotoxin reduced IK+ by ~17%. Immunofluorescence studies showed that chronic TES exposure increased the expression of KV1.2 and KV1.5 in airway smooth muscle (ASM). In conclusion, chronic exposure to TES in guinea pig ASM promotes upregulation of KV1.2 and KV1.5 and enhances theophylline relaxation response. Therefore, gender should be considered when prescribing methylxanthines, as teenage boys and males are likely to respond better than females. [ABSTRACT FROM AUTHOR]

Testosterone (TES), other androgens and female sex steroids induce non-genomic rapid relaxing effects in airway smooth muscle (ASM). In guinea pig ASM, basal tension was relaxed by dehydroepiandrosterone (DHEA) and TES; 17β-estradiol (E2) had a small effect. Blockers of L-type voltage dependent Ca 2+ channel (L-VDCC, D-600) and store operated Ca 2+ channel (SOC, 2-APB) also relaxed the basal tone. In tracheal myocytes, DHEA and TES diminished intracellular basal Ca 2+ concentrations ( b [Ca 2+ ] i ) as D-600+2-APB but to a higher extend. TES after D-600+2APB or Pyr3, a blocker of canonical transient receptor potential 3 (TRPC3), further decreased b [Ca 2+ ] i rendering this response equal to TES alone. With indomethacin, the b [Ca 2+ ] i decrease induced by the blockade of L-VDCC and TRPC3 was not changed by the addition of TES. PGE 2 or forskolin addition after D600+2-APB, decreased b [Ca 2+ ] i resembling TES response. An adenylate cyclase inhibitor followed by D-600+2-APB lowered b [Ca 2+ ] i , TES showed no further effect. Carbachol-induced [Ca 2+ ]i increment was reduced by TES or DHEA. 17β-estradiol diminished KCl-induced contraction and, in tracheal myocytes, the voltage-dependent inward Ca 2+ current. Conclusion: DHEA and TES diminish ASM tone and b [Ca 2+ ]i by blocking L-VDCC and probably a constitutively active TRPC3, and by PGE 2 synthesis. E2 lowers ASM basal tone by blocking only L-VDCC. [ABSTRACT FROM AUTHOR]

Androgens in asthmatic men may be linked to asthma severity, acting via nongenomic and genomic effects. This ailment affects boys more than girls during infancy, and this proportion reverses in puberty. Plasmatic androgen concentration in young men increases at this age and might be related to lower asthma symptoms. Nongenomic actions occur in a brief period and are independent of the androgen receptor (AR), while genomic effects depend on AR, take hours-days and are modified by transcription or protein synthesis inhibitors. Guinea pig tracheas chronic incubation with testosterone (TES, 40 nM, 48 h) potentiates salbutamol-induced relaxation, an effect that was reversed by flutamide, not observed when tissues were pre-incubated with TES-bovine serum albumin (TES-BSA) nor when tissues were preincubated with TES for 15-60 min. In tracheal myocytes, TES chronic incubation increases salbutamol-induced K + currents (IK + ), an effect that was also reversed by flutamide, actinomycin D and cycloheximide and not seen with TES-BSA. The increment in IK + was blocked by 4-aminopyridine and iberiotoxin, indicating that delayed rectifier K + and high-conductance Ca 2+ activated K + channels were involved in the TES potentiation effect. Immunofluorescence studies showed that chronic TES augmented the β 2 adrenergic receptor (β 2 -AR) expression in ASM and this finding was corroborated by q-PCR and Western blot assays. β 2 -AR affinity for salbutamol after TES incubation was increased. In conclusion, chronic exposure to physiological TES concentration of the guinea pig ASM promotes β 2 -AR upregulation favoring β 2 adrenergic responses and probably limiting the severity of the asthmatic exacerbations in teenage boys and men., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest or financial relationships that may have infiuenced the study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Asthma symptoms have been associated with sex steroids. During childhood, this illness seems more frequent in boys than in girls and this tendency reverts in puberty when it is more severe in women. Testosterone (TES), at supraphysiological concentrations, relaxed pre-contracted airway smooth muscle, but its effects at physiological concentrations have not been thoroughly studied. We explored this possibility in guinea pig tracheal smooth muscle. In myocytes TES (10 nM) abolished carbachol (CCh)-induced intracellular Ca 2+ concentration ([Ca 2+ ]i) increment. Ca 2+ responses to ATP were partially modified by TES while histamine's were not. These results indicate that inositol 1,4,5-trisphosphate (IP 3 ) signaling pathway might be involved. Photolysis of caged-IP 3 increased [Ca 2+ ]i and TES abolished this effect. TES diminished reactivity of the smooth muscle to CCh and this effect was non-genomic since it was unchanged by flutamide. In tracheal smooth muscle, mRNA for each IP 3 receptor (ITPR) isoform was found and, by immunofluorescence, ITPR1 and ITPR3 seems to be the main isoforms observed while ITPR2 was less prominent. Comparing the amino acid sequence of ITPR1 and the sequence of the TES binding site on the androgen receptor, we found that they share a short sequence. This domain could be responsible for the TES binding to the ITPR1 and probably for its blocking effect. We conclude that TES modifies ITPR1 function in airway smooth muscle, turning this tissue less reactive to contractile agonists that act through PLCβ-IP 3 signaling cascade. These results might be related to the low asthma prevalence in males from puberty to adulthood., (Copyright © 2017 Elsevier B.V. All rights reserved.)