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.

Purinergic inhibitory regulation of esophageal smooth muscle is mediated by P2Y receptors and ATP-dependent potassium channels in rats.

Purines such as ATP are regulatory transmitters in motility of the gastrointestinal tract. The aims of this study were to propose functional roles of purinergic regulation of esophageal motility. An isolated segment of the rat esophagus was placed in an organ bath, and mechanical responses were recorded using a force transducer. Exogenous application of ATP (10-100 µM) evoked relaxation of the esophageal smooth muscle in a longitudinal direction under the condition of carbachol (1 µM) -induced precontraction. Pretreatment with a non-selective P2 receptor antagonist, suramin (500 µM), and a P2Y receptor antagonist, cibacron blue F3GA (200 µM), inhibited the ATP (100 µM) -induced relaxation, but a P2X receptor antagonist, pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (50 µM), did not affect it. A blocker of ATP-dependent potassium channels (KATP channels), glibenclamide (200 µM), inhibited the ATP-induced relaxation and application of an opener of KATP channels, nicorandil (50 µM), produced relaxation. The findings suggest that ATP is involved in inhibitory regulation of the longitudinal smooth muscle in the muscularis mucosae of the rat esophagus via activation of P2Y receptors and then opening of KATP channels.

SHH regulates penile morphology and smooth muscle through a mechanism involving BMP4 and GREM1.

BACKGROUND: The cavernous nerve (CN) is frequently damaged in prostatectomy and diabetic patients with erectile dysfunction (ED), initiating changes in penile morphology including an acute and intense phase of apoptosis in penile smooth muscle and increased collagen, which alter penile architecture and make corpora cavernosa smooth muscle less able to relax in response to neurotransmitters, resulting in ED. AIM: Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and SHH treatment suppresses penile remodeling after CN injury through an unknown mechanism; we examine if part of the mechanism of how SHH preserves smooth muscle after CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1). METHODS: Primary cultures of smooth muscle cells were established from prostatectomy, diabetic, hypertension and Peyronie's (control) (N = 18) patients. Cultures were characterized by ACTA2, CD31, P4HB, and nNOS immunohistochemical analysis. Patient smooth muscle cell growth was quantified in response to BMP4 and GREM1 treatment. Adult Sprague Dawley rats underwent 1 of 3 surgeries: (1) uninjured or CN-injured rats were treated with BMP4, GREM1, or mouse serum albumin (control) proteins via Affi-Gel beads (N = 16) or peptide amphiphile (PA) (N = 26) for 3 and 14 days, and trichrome stain was performed; (2) rats underwent sham (N = 3), CN injury (N = 9), or CN injury and SHH PA treatment for 1, 2, and 4 days (N = 9). OUTCOMES: Western analysis for BMP4 and GREM1 was performed; (3) rats were treated with 5E1 SHH inhibitor (N = 6) or IgG (control; N = 6) for 2 and 4 days, and BMP4 and GREM1 localization was examined. Statistics were performed by analysis of variance with Scheffé's post hoc test. RESULTS: BMP4 increased patient smooth muscle cell growth, and GREM1 decreased growth. In rats, BMP4 treatment via Affi-Gel beads and PA increased smooth muscle at 3 and 14 days of treatment. GREM1 treatment caused increased collagen and smooth muscle at 3 days, which switched to primarily collagen at 14 days. CN injury increased BMP4 and GREM1, while SHH PA altered Western band size, suggesting alternative cleavage and range of BMP4 and GREM1 signaling. SHH inhibition in rats increased BMP4 and GREM1 in fibroblasts. CLINICAL IMPLICATIONS: Understanding how SHH PA preserves and regenerates penile morphology after CN injury will aid development of ED therapies. STRENGTHS AND LIMITATIONS: SHH treatment alters BMP4 and GREM1 localization and range of signaling, which can affect penile morphology. CONCLUSION: Part of the mechanism of how SHH regulates corpora cavernosa smooth muscle involves BMP4 and GREM1.

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

New phosphodiesterase-4 inhibitors present airways relaxant activity in a guinea pig acute asthma model.

Asthma is a disease characterized by chronic inflammation and hyper responsiveness of airways. We aimed to assess the relaxant potential of phosphodiesterase-4 (PDE4) inhibitors N-sulfonilhidrazonic derivatives on non-asthmatic and asthmatic guinea pig trachea. Firstly, guinea pigs were sensitized and challenged with ovalbumin, and then morphological, and contractile changes were evaluated resulting from asthma, followed by evaluation of relaxant effect of derivatives on guinea pig trachea and the cAMP levels measurement by ELISA. It has been evidenced hypertrophy of airway smooth muscle, inflammatory infiltrate, and vascular abnormalities. Moreover, only sensitized tracheal rings were responsive to OVA. Contractile response to histamine, but not to carbachol, was greater in sensitized animals, however the relaxant response to aminophylline and isoprenaline were the same in non-asthmatics and asthmatics. N-sulfonilhidrazonic derivatives presented equipotent relaxant action independent of epithelium, with exception of LASSBio-1850 that presented a low efficacy (< 50%) and LASSBio-1847 with a 4-fold higher potency on asthmatics. LASSBio-1847 relaxant curve was impaired in the presence of propranolol and potentiated by isoprenaline in both groups. Furthermore, relaxation was potentiated 54- and 4-fold by forskolin in non-asthmatics and asthmatics, respectively. Likewise, LASSBio-1847 potentiated relaxant curve of aminophylline 147- and 4-fold in both groups. The PKA inhibitor H-89 impaired the relaxant potency of the derivative. Finally, LASSBio-1847 increased tracheal intracellular cAMP levels similarly to rolipram, selective PDE4 inhibitor, in both animals. LASSBio-1847 showed to be promising to relax guinea pig trachea from non-sensitized and sensitized guinea pigs by activation of ß2-adrenergic receptors/AC/cAMP pathway.

Cellular Mechanism Underlying the Facilitation of Contractile Response Induced by Tumor Necrosis Factor-α in Mouse Tracheal Smooth Muscle.

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) augments intracellular Ca2+ signaling and contractile responses of airway smooth muscles, leading to airway hyperresponsiveness. However, the underlying mechanism has not been fully elucidated. This study aimed to investigate the cellular mechanism of the potentiated contraction of mouse tracheal smooth muscle induced by TNF-α. The results showed that TNF-α triggered facilitation of mouse tracheal smooth muscle contraction in an epithelium-independent manner. The TNF-α-induced hypercontractility could be suppressed by the protein kinase C inhibitor GF109203X, the tyrosine kinase inhibitor genistein, the Src inhibitor PP2, or the L-type voltage-dependent Ca2+ channel blocker nifedipine. Following TNF-α incubation, the α1C L-type Ca2+ channel (CaV1.2) was up-regulated in cultured primary mouse tracheal smooth muscle cells. Pronounced phosphotyrosine levels were observed in mouse tracheas. In conclusion, this study shows that TNF-α enhanced airway smooth muscle contraction via protein kinase C-Src-CaV1.2 pathways, which provides novel insights into the pathologic role of proinflammatory cytokines in mediating airway hyperresponsiveness.

Repeated Low-Dose Acrolein Triggers Irreversible Lamina Propria Edema in Urinary Bladder, Transient Voiding Behavior and Widening of Eyes to Mechanical Stimuli.

Acrolein is a metabolite of cyclophosphamide (CYP), an alkylating agent used for a wide range of benign and malignant diseases. CYP treatments are known to trigger hemorrhagic cystitis in patients and animals. Significant effort has been made to prevent CYP/acrolein-induced cystitis, while still maintaining its therapeutic benefits. As a result, supplementary therapeutic options to mediate the protective role against CYP/acrolein and lower doses of CYP are currently given to targeted patients, as compared to past treatments. There is still a need to further study the effects of the repeated low-dose CYP/acrolein on the pathophysiology of the urinary bladder. In our study, a one-time treatment of acrolein and repeated low-dose acrolein triggered the thickening of the smooth muscle and lamina propria in the urinary bladder of C57BL/6J mice, respectively. The first dose of acrolein did not trigger voiding dysfunction, but the second dose triggered high-volume low-frequency voiding. Interestingly, our new scoring criteria and concurrent behavioral assessment revealed that mice with repeated low-dose acrolein had a wider opening of eyes in response to mechanical stimuli. Our study suggests that clinical symptoms among patients undergoing prolonged low-dose CYP may differ from previously reported symptoms of CYP-induced hemorrhagic cystitis.

Identification and characterization of an atypical Gαs-biased ß2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.

Combined agonists act synergistically to increase mucociliary clearance in a cystic fibrosis airway model.

Mucus clearance, a primary innate defense mechanism of airways, is defective in patients with cystic fibrosis (CF) and CF animals. In previous work, the combination of a low dose of the cholinergic agonist, carbachol with forskolin or a ß adrenergic agonist, isoproterenol synergistically increased mucociliary clearance velocity (MCCV) in ferret tracheas. Importantly, the present study shows that synergistic MCCV can also be produced in CF ferrets, with increases ~ 55% of WT. Synergistic MCCV was also produced in pigs. The combined agonists increased MCCV by increasing surface fluid via multiple mechanisms: increased fluid secretion from submucosal glands, increased anion secretion across surface epithelia and decreased Na+ absorption. To avoid bronchoconstriction, the cAMP agonist was applied 30 min before carbachol. This approach to increasing mucus clearance warrants testing for safety and efficacy in humans as a potential therapeutic for muco-obstructive diseases.

Mechanism deconvolution of Qing Fei Pai Du decoction for treatment of Coronavirus Disease 2019 (COVID-19) by label-free integrative pharmacology assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has a long history in the prevention and treatment of pandemics. The TCM formula Lung Cleansing and Detoxifying Decoction (LCDD), also known as Qing Fei Pai Du Decoction, has been demonstrated effective against Coronavirus Disease 2019 (COVID-19). AIM OF THE STUDY: This work aimed to elucidate the active ingredients, targets and pathway mechanism of LCDD related to suppression of inflammatory, immunity regulation and relaxation of airway smooth muscle for the treatment of COVID-19. MATERIALS AND METHODS: Mining chemical ingredients reported in LCDD, 144 compounds covering all herbs were selected and screened against inflammatory-, immunity- and respiratory-related GPCRs including GPR35, H1, CB2, B2, M3 and ß2-adrenoceptor receptor using a label-free integrative pharmacology method. Further, all active compounds were detected using liquid chromatography-tandem mass spectrometry, and an herb-compound-target network based on potency and content of compounds was constructed to elucidate the multi-target and synergistic effect. RESULTS: Thirteen compounds were identified as GPR35 agonists, including licochalcone B, isoliquiritigenin, etc. Licochalcone B, isoliquiritigenin and alisol A exhibited bradykinin receptor B2 antagonism activities. Atractyline and shogaol showed as a cannabinoid receptor CB2 agonist and a histamine receptor H1 antagonist, respectively. Tectorigenin and aristofone acted as muscarinic receptor M3 antagonists, while synephrine, ephedrine and pseudoephedrine were ß2-adrenoceptor agonists. Pathway deconvolution assays suggested activation of GPR35 triggered PI3K, MEK, JNK pathways and EGFR transactivation, and the activation of ß2-adrenoceptor mediated MEK and Ca2+. The herb-compound-target network analysis found that some compounds such as licochalcone B acted on multiple targets, and multiple components interacted with the same target such as GPR35, reflecting the synergistic mechanism of Chinese medicine. At the same time, some low-abundance compounds displayed high target activity, meaning its important role in LCDD for anti-COVID-19. CONCLUSIONS: This study elucidates the active ingredients, targets and pathways of LCDD. This is useful for elucidating multitarget synergistic action for its clinical therapeutic efficacy.

TIPE2 inhibits PDGF-BB-induced phenotype switching in airway smooth muscle cells through the PI3K/Akt signaling pathway.

BACKGROUND: Childhood asthma is a common respiratory disease characterized by airway inflammation. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) has been found to be involved in the progression of asthma. This study aimed to explore the role of TIPE2 in the regulation of airway smooth muscle cells (ASMCs), which are one of the main effector cells in the development of asthma. MATERIALS AND METHODS: ASMCs were transfected with pcDNA3.0-TIPE2 or si-TIPE2 for 48 h and then treated with platelet-derived growth factor (PDGF)-BB. Cell proliferation of ASMCs was measured using the MTT assay. Cell migration of ASMCs was determined by a transwell assay. The mRNA expression levels of calponin and smooth muscle protein 22α (SM22α) were measured using qRT-PCR. The levels of TIPE2, calponin, SM22α, PI3K, p-PI3K, Akt, and p-Akt were detected by Western blotting. RESULTS: Our results showed that PDGF-BB treatment significantly reduced TIPE2 expression at both the mRNA and protein levels in ASMCs. Overexpression of TIPE2 inhibited PDGF-BB-induced ASMC proliferation and migration. In addition, overexpression of TIPE2 increased the expression of calponin and SM22α in PDGF-BB-stimulated ASMCs. However, an opposite effect was observed with TIPE2 knockdown. Furthermore, TIPE2 overexpression blocked PDGF-BB-induced phosphorylation of PI3K and Akt, whereas the expression of p-PI3K and p-Akt were aggravated by TIPE2 knockdown. Additionally, the effects of TIPE2 overexpression and TIPE2 knockdown were altered by IGF-1 and LY294002 treatments, respectively. CONCLUSIONS: Our findings demonstrate that TIPE2 inhibits PDGF-BB-induced ASMC proliferation, migration, and phenotype switching via the PI3K/Akt signaling pathway. Thus, TIPE2 may be a potential therapeutic target for the treatment of asthma.

Monomeric, Dimeric, and Trimeric Tropane Alkaloids from Pellacalyx saccardianus.

Seven new tropane alkaloids, including five monomeric (1-5), one dimeric (6), and one trimeric (7) 3α-nortropane ester, along with two known monomeric nortropane alkaloids (8 and 9), were isolated from the leaves and bark of Pellacalyx saccardianus. Their structures, including the absolute configuration of the enantiomeric pair of (±)-6, were elucidated by comprehensive spectroscopic analyses. Alkaloids 6 and 7 showed cytotoxicity toward human pancreatic cancer cell lines (AsPC-1, BxPC3, PANC-1, and SW1990). Alkaloids 1, 4, and 9 induced a smooth muscle relaxation effect comparable to that of atropine (Emax 106.1 ± 7.5%, 97.0 ± 5.2%, 100.9 ± 1.4%, 111.7 ± 1.7%, respectively) on isolated rat tracheal rings.

Cucumis sativus extract elicits chloride secretion by stimulation of the intestinal TMEM16A ion channel.

CONTEXT: Cucumber (Cucumis sativus Linn. [Cucurbitaceae]) is widely known for its purgative, antidiabetic, antioxidant, and anticancer therapeutic potential. However, its effect on gastrointestinal (GI) disease is unrecognised. OBJECTIVE: This study investigated the effect of C. sativus fruit extract (CCE) on intestinal chloride secretion, motility, and motor function, and the role of TMEM16A chloride channels. MATERIALS AND METHODS: CCE extracts were obtained from commercially available cucumber. Active fractions were then purified by HPLC and analysed by high resolution mass spectrometry. The effect of CCE on intestinal chloride secretion was investigated in human colonic T84 cells, ex vivo mouse intestinal tissue using an Ussing chamber, and the two-electrode voltage-clamp technique to record calcium sensitive TMEM16A chloride currents in Xenopus laevis oocytes. In vivo, intestinal motility was investigated using the loperamide-induced C57BL/6 constipation mouse model. Ex vivo contractility of mouse colonic smooth muscles was assessed by isometric force measurements. RESULTS: CCE increased the short-circuit current (ΔIsc 34.47 ± µA/cm2) and apical membrane chloride conductance (ΔICl 95 ± 8.1 µA/cm2) in intestinal epithelial cells. The effect was dose-dependent, with an EC50 value of 0.06 µg/mL. CCE stimulated the endogenous TMEM16A-induced Cl- current in Xenopus laevis oocytes. Moreover, CCE increased the contractility of smooth muscle in mouse colonic tissue and enhanced small bowel transit in CCE treated mice compared to loperamide controls. Mass spectrometry suggested a cucurbitacin-like analogue with a mass of 512.07 g/mol underlying the bioactivity of CCE. CONCLUSION: A cucurbitacin-like analog present in CCE activates TMEM16A channels, which may have therapeutic potential in cystic fibrosis and intestinal hypodynamic disorders.

GC/MS analysis, anti-leishmanial and relaxant activity of essential oil of Chenopodium ambrosioides (L.) from Malakand region.

Chenopodium ambrosioides is abundantly available in Malakand region. As constituents and concentrations of essential oils vary based on its geographical location, we carried our current study to extract and evaluate its possible relaxant activity in rabbits' jejunum and anti-leishmanial activity against promastigotes of Leishmania tropica. The essential oil was obtained from aerial fresh parts through steam distillation followed by GC/MS analysis. Antispasmodic activity was performed on spontaneous and KCl induced contractions. Curves for calcium concentration response (CCRCs) were prepared with and without different concentrations of essential oils and verapamil - a standard calcium channel blocker as per our reported procedures. GC/MS analysis indicated that the essential oil contains 4-carene (56.59%) and o-cymene (41.46%), the two most abundant compounds previously reported from this species. The LD50 value for acute toxicity is 279.66±2.2mg/kg. The essential oil have significant antileishmanial activity with LC50 of Log10 (1.83±0.0026) ×10-6mg/ml, potent relaxant activity on rabbits' jejunal preparations with respective EC50 = 1.46±0.15mg/ml for spontaneous activity. For KCl (80mM) induced contractions, EC50=0.26±0.02mg/ml. In CCRCs, the oil produced a right shift as exhibited by verapamil. More, its relaxant activity, which is mediated through calcium channel blocking mechanism, proves a rationale for its traditional use in gut spasm.

Quercetin affects uterine smooth muscle contractile activity in gilts.

Quercetin is a polyphenolic flavonoid occurring in leaves, stems, flowers and fruits of many plants. In traditional Chinese medicine, it is used as a natural therapeutic agent with a broad spectrum of activities (antioxidant, neuroprotective, anti-inflammatory, anticancer, antibacterial and antiviral). Moreover, quercetin affects function of the reproductive tract, however the knowledge of this activity is still fragmentary. Therefore, this study aimed to determine the influence of quercetin on the contractile activity of the porcine myometrium collected from immature (n = 6), cyclic (n = 6) and early pregnant (n = 6) gilts. Strips of the myometrium (comprising longitudinal and circular layer) were resected from the middle part of the uterine horns and the isometric contractions were recorded. After 60-90 min of preincubation, the strips were stimulated with quercetin in increasing (10-13-10-1 M) concentrations and the changes in the tension amplitude and frequency of contractions were measured. Quercetin decreased (P<0.01-0.001) the amplitude of contractions at concentrations 10-11-10-1 M and 10-10-10-1 M in cyclic and early pregnant groups, respectively. The frequency of contractions decreased in all groups but was the highest (at concentrations 10-11-10-1 M; P<0.05-0.001) in the cyclic group and the lowest (at concentrations 10-5-10-1 M; P<0.01) in the immature group. The tension decreased only in the cyclic group after quercetin administration in high concentrations (10-6-10-1 M; P<0.05-0.01). The results indicate that quercetin causes relaxation of the porcine uterine smooth muscle but this activity is strongly related to the physiological status of the gilts.

Possible Beneficial Actions of Caffeine in SARS-CoV-2.

The COVID-19 pandemic has established an unparalleled necessity to rapidly find effective treatments for the illness; unfortunately, no specific treatment has been found yet. As this is a new emerging chaotic situation, already existing drugs have been suggested to ameliorate the infection of SARS-CoV-2. The consumption of caffeine has been suggested primarily because it improves exercise performance, reduces fatigue, and increases wakefulness and awareness. Caffeine has been proven to be an effective anti-inflammatory and immunomodulator. In airway smooth muscle, it has bronchodilator effects mainly due to its activity as a phosphodiesterase inhibitor and adenosine receptor antagonist. In addition, a recent published document has suggested the potential antiviral activity of this drug using in silico molecular dynamics and molecular docking; in this regard, caffeine might block the viral entrance into host cells by inhibiting the formation of a receptor-binding domain and the angiotensin-converting enzyme complex and, additionally, might reduce viral replication by the inhibition of the activity of 3-chymotrypsin-like proteases. Here, we discuss how caffeine through certain mechanisms of action could be beneficial in SARS-CoV-2. Nevertheless, further studies are required for validation through in vitro and in vivo models.

Cooperativity between ß-agonists and c-Abl inhibitors in regulating airway smooth muscle relaxation.

Current therapeutic approaches to avoid or reverse bronchoconstriction rely primarily on ß2 adrenoceptor agonists (ß-agonists) that regulate pharmacomechanical coupling/cross bridge cycling in airway smooth muscle (ASM). Targeting actin cytoskeleton polymerization in ASM represents an alternative means to regulate ASM contraction. Herein we report the cooperative effects of targeting these distinct pathways with ß-agonists and inhibitors of the mammalian Abelson tyrosine kinase (Abl1 or c-Abl). The cooperative effect of ß-agonists (isoproterenol) and c-Abl inhibitors (GNF-5, or imatinib) on contractile agonist (methacholine, or histamine) -induced ASM contraction was assessed in cultured human ASM cells (using Fourier Transfer Traction Microscopy), in murine precision cut lung slices, and in vivo (flexiVent in mice). Regulation of intracellular signaling that regulates contraction (pMLC20, pMYPT1, pHSP20), and actin polymerization state (F:G actin ratio) were assessed in cultured primary human ASM cells. In each (cell, tissue, in vivo) model, c-Abl inhibitors and ß-agonist exhibited additive effects in either preventing or reversing ASM contraction. Treatment of contracted ASM cells with c-Abl inhibitors and ß-agonist cooperatively increased actin disassembly as evidenced by a significant reduction in the F:G actin ratio. Mechanistic studies indicated that the inhibition of pharmacomechanical coupling by ß-agonists is near optimal and is not increased by c-Abl inhibitors, and the cooperative effect on ASM relaxation resides in further relaxation of ASM tension development caused by actin cytoskeleton depolymerization, which is regulated by both ß-agonists and c-Abl inhibitors. Thus, targeting actin cytoskeleton polymerization represents an untapped therapeutic reserve for managing airway resistance.

Control of rat bladder neck relaxation with NORD-1, a red light-reactive nitric oxide releaser: In vitro study.

We aimed to control the relaxation of rat bladder neck specimens by using NORD-1, a red light-reactive nitric oxide (NO) releaser. Female and male 10-11-week-old Wistar/ST rats were divided into three groups: NORD-1, vehicle, and NORD-1+[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a soluble guanylyl cyclase inhibitor). We infused 10-4 M NORD-1 into the bladders of NORD-1 and NORD-1+ODQ group rats and the vehicle into those of vehicle group rats. Isometric tension was analyzed using circular bladder neck specimens with 10-5 M NG-nitro-l-arginine methyl ester, an NO synthase inhibitor. Moreover, 10-5 M ODQ was added into the NORD-1+ODQ group bath. After precontraction with 10-5 M carbachol, the specimens were irradiated with red light and their relaxation responses were measured. We evaluated NORD-1 tissue permeability by observing the sliced bladder neck specimens. The NORD-1 group specimens relaxed during red light irradiation; the relaxation response increased with the increase in light intensity. The vehicle and NORD-1+ODQ group specimens did not respond to irradiation. Sex-related differences in responsiveness were not noted. NORD-1 permeated into the urothelium of NORD-1 group specimens. Rat bladder neck relaxation was controlled by NORD-1 and light irradiation in vitro. NORD-1 might be a novel therapeutic agent for voiding dysfunction.

Nicotinic α7 acetylcholine receptor (α7nAChR) in human airway smooth muscle.

Diseases such as asthma are exacerbated by inflammation, cigarette smoke and even nicotine delivery devices such as e-cigarettes. However, there is currently little information on how nicotine affects airways, particularly in humans, and changes in the context of inflammation or asthma. Here, a longstanding assumption is that airway smooth muscle (ASM) that is key to bronchoconstriction has muscarinic receptors while nicotinic receptors (nAChRs) are only on airway neurons. In this study, we tested the hypothesis that human ASM expresses α7nAChR and explored its profile in inflammation and asthma using ASM of non-asthmatics vs. mild-moderate asthmatics. mRNA and western analysis showed the α7 subunit is most expressed in ASM cells and further increased in asthmatics and smokers, or by exposure to nicotine, cigarette smoke or pro-inflammatory cytokines TNFα and IL-13. In these effects, signaling pathways relevant to asthma such as NFκB, AP-1 and CREB are involved. These novel data demonstrate the expression of α7nAChR in human ASM and suggest their potential role in asthma pathophysiology in the context of nicotine exposure.

Tadalafil ameliorates bladder overactivity by restoring insulin-activated detrusor relaxation via the bladder mucosal IRS/PI3K/AKT/eNOS pathway in fructose-fed rats.

The pathophysiologies of metabolic syndrome (MS) and overactive bladder (OAB) might overlap. Using fructose-fed rats (FFRs) as a rodent model of MS we investigated the effects of tadalafil (a phosphodiesterase type 5 inhibitor) on the dysregulated insulin signalling in the bladder mucosa and bladder overactivity. Micturition behaviour was evaluated. Concentration-response curves on detrusor relaxation to insulin stimulation were examined. Expression and phosphorylation of proteins in the insulin signalling pathway were evaluated by Western blotting. Levels of detrusor cGMP and urinary nitrite and nitrate (NOx) were measured. We observed FFRs exhibited metabolic traits of MS, bladder overactivity, and impaired insulin-activated detrusor relaxation in organ bath study. A high-fructose diet also impeded insulin signalling, reflected by overexpression of IRS1/pIRS1Ser307 and pIRS2Ser731 and downregulation of PI3K/pPI3KTyr508, AKT/pAKTSer473, and eNOS/peNOSSer1177 in the bladder mucosa, alongside decreased urinary NOx and detrusor cGMP levels. Tadalafil treatment restored the reduced level of mucosal peNOS, urinary NOx, and detrusor cGMP, improved the insulin-activated detrusor relaxation, and ameliorated bladder overactivity in FFRs. These results suggest tadalafil may ameliorate MS-associated bladder overactivity by restoring insulin-activated detrusor relaxation via molecular mechanisms that are associated with preservation of IR/IRS/PI3K/AKT/eNOS pathway in the bladder mucosa and cGMP production in the bladder detrusor.