Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

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.

TRPV4 Activation during Guinea Pig Airway Smooth Muscle Contraction Promotes Ca2+ and Na+ Influx.

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.

Theophylline-Induced Relaxation Is Enhanced after Testosterone Treatment via Increased KV1.2 and KV1.5 Protein Expression in Guinea Pig Tracheal Smooth Muscle.

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 ß2-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.

Important Functions and Molecular Mechanisms of Mitochondrial Redox Signaling in Pulmonary Hypertension.

Mitochondria are important organelles that act as a primary site to produce reactive oxygen species (ROS). Additionally, mitochondria play a pivotal role in the regulation of Ca2+ signaling, fatty acid oxidation, and ketone synthesis. Dysfunction of these signaling molecules leads to the development of pulmonary hypertension (PH), atherosclerosis, and other vascular diseases. Features of PH include vasoconstriction and pulmonary artery (PA) remodeling, which can result from abnormal proliferation, apoptosis, and migration of PA smooth muscle cells (PASMCs). These responses are mediated by increased Rieske iron-sulfur protein (RISP)-dependent mitochondrial ROS production and increased mitochondrial Ca2+ levels. Mitochondrial ROS and Ca2+ can both synergistically activate nuclear factor κB (NF-κB) to trigger inflammatory responses leading to PH, right ventricular failure, and death. Evidence suggests that increased mitochondrial ROS and Ca2+ signaling leads to abnormal synthesis of ketones, which play a critical role in the development of PH. In this review, we discuss some of the recent findings on the important interactive role and molecular mechanisms of mitochondrial ROS and Ca2+ in the development and progression of PH. We also address the contributions of NF-κB-dependent inflammatory responses and ketone-mediated oxidative stress due to abnormal regulation of mitochondrial ROS and Ca2+ signaling in PH.

Transient receptor potential cation channel subfamily V (TRPV) and its importance in asthma.

Transient receptor potential (TRP) ion channels play critical roles in physiological and pathological conditions. Increasing evidence has unveiled the contribution of TRP vanilloid (TRPV) family in the development of asthma. The TRPV family is a group (TRPV1-TRPV6) of polymodal channels capable of sensing thermal, acidic, mechanical stress, and osmotic stimuli. TRPVs can be activated by endogenous ligands including, arachidonic acid derivatives or endocannabinoids. While TRPV1-TRPV4 are non-selective cation channels showing a predominance for Ca2+ over Na + influx, TRPV5 and TRPV6 are only Ca2+ permeable selective channels. Asthma is a chronic inflammatory bronchopulmonary disorder involving airway hyperresponsiveness (AHR) and airway remodeling. Patients suffering from allergic asthma display an inflammatory pattern driven by cytokines produced in type-2 helper T cells (Th2) and type 2 innate lymphoid cells (ILC2s). Ion channels are essential regulators in airway smooth muscle (ASM) and immune cells physiology. In this review, we summarize the contribution of TRPV1, TRPV2, and TRPV4 to the pathogenesis of asthma. TRPV1 is associated with hypersensitivity to environmental pollutants and chronic cough, inflammation, AHR, and remodeling. TRPV2 is increased in peripheral lymphocytes of asthmatic patients. TRPV4 contributes to ASM cells proliferation, and its blockade leads to a reduced eosinophilia, neutrophilia, as well as an abolished AHR. In conclusion, TRPV2 may represent a novel biomarker for asthma in children; meanwhile, TRPV1 and TRPV4 seem to be essential contributors to the development and exacerbations of asthma. Moreover, these channels may serve as novel therapeutic targets for this ailment.

Sex Hormones and Lung Inflammation.

Inflammation is a characteristic marker in numerous lung disorders. Several immune cells, such as macrophages, dendritic cells, eosinophils, as well as T and B lymphocytes, synthetize and release cytokines involved in the inflammatory process. Gender differences in the incidence and severity of inflammatory lung ailments including asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (PF), lung cancer (LC), and infectious related illnesses have been reported. Moreover, the effects of sex hormones on both androgens and estrogens, such as testosterone (TES) and 17ß-estradiol (E2), driving characteristic inflammatory patterns in those lung inflammatory diseases have been investigated. In general, androgens seem to display anti-inflammatory actions, whereas estrogens produce pro-inflammatory effects. For instance, androgens regulate negatively inflammation in asthma by targeting type 2 innate lymphoid cells (ILC2s) and T-helper (Th)-2 cells to attenuate interleukin (IL)-17A-mediated responses and leukotriene (LT) biosynthesis pathway. Estrogens may promote neutrophilic inflammation in subjects with asthma and COPD. Moreover, the activation of estrogen receptors might induce tumorigenesis. In this chapter, we summarize the most recent advances in the functional roles and associated signaling pathways of inflammatory cellular responses in asthma, COPD, PF, LC, and newly occurring COVID-19 disease. We also meticulously deliberate the influence of sex steroids on the development and progress of these common and severe lung diseases.

Androgen Effects on the Adrenergic System of the Vascular, Airway, and Cardiac Myocytes and Their Relevance in Pathological Processes.

INTRODUCTION: Androgen signaling comprises nongenomic and genomic pathways. Nongenomic actions are not related to the binding of the androgen receptor (AR) and occur rapidly. The genomic effects implicate the binding to a cytosolic AR, leading to protein synthesis. Both events are independent of each other. Genomic effects have been associated with different pathologies such as vascular ischemia, hypertension, asthma, and cardiovascular diseases. Catecholamines play a crucial role in regulating vascular smooth muscle (VSM), airway smooth muscle (ASM), and cardiac muscle (CM) function and tone. OBJECTIVE: The aim of this review is an updated analysis of the role of androgens in the adrenergic system of vascular, airway, and cardiac myocytes. Body. Testosterone (T) favors vasoconstriction, and its concentration fluctuation during life stages can affect the vascular tone and might contribute to the development of hypertension. In the VSM, T increases α1-adrenergic receptors (α 1-ARs) and decreases adenylyl cyclase expression, favoring high blood pressure and hypertension. Androgens have also been associated with asthma. During puberty, girls are more susceptible to present asthma symptoms than boys because of the increment in the plasmatic concentrations of T in young men. In the ASM, ß 2-ARs are responsible for the bronchodilator effect, and T augments the expression of ß 2-ARs evoking an increase in the relaxing response to salbutamol. The levels of T are also associated with an increment in atherosclerosis and cardiovascular risk. In the CM, activation of α 1A-ARs and ß 2-ARs increases the ionotropic activity, leading to the development of contraction, and T upregulates the expression of both receptors and improves the myocardial performance. CONCLUSIONS: Androgens play an essential role in the adrenergic system of vascular, airway, and cardiac myocytes, favoring either a state of health or disease. While the use of androgens as a therapeutic tool for treating asthma symptoms or heart disease is proposed, the vascular system is warmly affected.

Testosterone augments ß2 adrenergic receptor genomic transcription increasing salbutamol relaxation in airway smooth muscle.

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 Ca2+ activated K+ channels were involved in the TES potentiation effect. Immunofluorescence studies showed that chronic TES augmented the ß2 adrenergic receptor (ß2-AR) expression in ASM and this finding was corroborated by q-PCR and Western blot assays. ß2-AR affinity for salbutamol after TES incubation was increased. In conclusion, chronic exposure to physiological TES concentration of the guinea pig ASM promotes ß2-AR upregulation favoring ß2 adrenergic responses and probably limiting the severity of the asthmatic exacerbations in teenage boys and men.

Maintenance of intracellular Ca2+ basal concentration in airway smooth muscle (Review).

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 L­type voltage dependent Ca2+ channel (L­VDCC), T­type voltage dependent Ca2+ channel (T­VDCC) 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 voltage­dependent Ca2+ channels (L­ and T­type) are modulated by phosphorylation processes mediated by mitogen­activated protein kinase kinase (MEK) and extracellular­signal­regulated kinase 1 and 2 (ERK1/2). The MEK/ERK signaling pathway can be activated by G­protein­coupled 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 L­VDCC on Ser496 of the ß2 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. T­VDCC 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 (PLCß or γ). Constitutive inositol 1,4,5­trisphosphate 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.

Testosterone induces hyporesponsiveness by interfering with IP3 receptors in guinea pig airway smooth muscle.

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 Ca2+ concentration ([Ca2+]i) increment. Ca2+ responses to ATP were partially modified by TES while histamine's were not. These results indicate that inositol 1,4,5-trisphosphate (IP3) signaling pathway might be involved. Photolysis of caged-IP3 increased [Ca2+]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 IP3 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 PLCß-IP3 signaling cascade. These results might be related to the low asthma prevalence in males from puberty to adulthood.

Sex steroids effects on guinea pig airway smooth muscle tone and intracellular Ca2+ basal levels.

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; 17ß-estradiol (E2) had a small effect. Blockers of L-type voltage dependent Ca2+ channel (L-VDCC, D-600) and store operated Ca2+ channel (SOC, 2-APB) also relaxed the basal tone. In tracheal myocytes, DHEA and TES diminished intracellular basal Ca2+ concentrations (b[Ca2+]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[Ca2+]i rendering this response equal to TES alone. With indomethacin, the b[Ca2+]i decrease induced by the blockade of L-VDCC and TRPC3 was not changed by the addition of TES. PGE2 or forskolin addition after D600+2-APB, decreased b[Ca2+]i resembling TES response. An adenylate cyclase inhibitor followed by D-600+2-APB lowered b[Ca2+]i, TES showed no further effect. Carbachol-induced [Ca2+]i increment was reduced by TES or DHEA. 17ß-estradiol diminished KCl-induced contraction and, in tracheal myocytes, the voltage-dependent inward Ca2+ current. CONCLUSION: DHEA and TES diminish ASM tone and b[Ca2+]i by blocking L-VDCC and probably a constitutively active TRPC3, and by PGE2 synthesis. E2 lowers ASM basal tone by blocking only L-VDCC.