Guinea Pig Airway Smooth Muscle Research Articles

17β-estradiol induces hyperresponsiveness in guinea pig airway smooth muscle by inhibiting the plasma membrane Ca2+-ATPase

High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17β-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases.In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases.In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17β-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.

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.

Design, Synthesis, and Biological Evaluation of Novel Spiro Imidazobenzodiazepines to Identify Improved Inhaled Bronchodilators.

Novel gamma-aminobutyric acid receptor (GABAAR) ligands structurally related to imidazobenzodiazepine MIDD0301 were synthesized using spiro-amino acid N-carboxyanhydrides (NCAs). These compounds demonstrated increased resistance to phase 2 metabolism and avoided the formation of a 6H isomer. Compound design was guided by molecular docking using the available crystal structure of the α1β3γ2 GABAAR and correlated with in vitro binding data. The carboxylic acid containing GABAAR ligands have high aqueous solubility, low permeability, and low cell toxicity. The inability of GABAAR ligands to cross the blood-brain barrier was confirmed in vivo by the absence of sensorimotor inhibition. Pharmacological activities at lung GABAARs were demonstrated by ex vivo relaxation of guinea pig airway smooth muscle and reduction of methacholine-induced airway hyperresponsiveness (AHR) in conscious mice. We identified bronchodilator 5c with an affinity of 9 nM for GABAARs that was metabolically stable in the presence of human and mouse microsomes.

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 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.

Relaxant effect of bioactive component compatibility of San-ao decoction on In vitro guinea pig airway smooth muscle: A dose-response relationship study

Background: Component compatibility is important to the modernization of traditional Chinese medicine. Studies have shown that San-ao decoction (SAD) can treat respiratory diseases by relaxing airway smooth muscle (ASM) and reducing airway hyper-responsiveness. However, whether its bioactive components and compatibility also present with similar relaxant effects remains unknown. This study aims to explore the potential relaxant property, dose-response relationship, and underlying mechanisms of the bioactive component compatibility in SAD. Methods: Network pharmacology was primarily used to identify the bioactive components of SAD and uncover its underlying mechanisms. ASM tension force measuring technique was utilized to verify the relaxant and dose-response effects on in vitro guinea pig ASM. Results: We postulated pseudoephedrine hydrochloride (PH), amygdalin (AM), and diammonium glycyrrhizate (DG) to be the bioactive components of SAD, which could effectively relax ASM in a dose-dependent manner on both acetylcholine-induced and spontaneous contraction. Both PH and AM could lead to DG dose–response curve shift. The regression equation of these three bioactive components was Y = −2.048 × X1 + 0.411 × X2 + 14.052 × X3 (X1, X2, X3 representing PH, AM, and DG, respectively). The underlying mechanisms of these components might be associated with the regulation of smooth muscle contraction. Conclusions: PH, AM, and DG are the bioactive components of SAD, which can relax ASM in a dose–response manner and exert a synergistic effect. Clinically, compatibility of these three bioactive components may serve as a new complementary and alternative treatment for respiratory diseases.

Melatonin MT2 receptor is expressed and potentiates contraction in human airway smooth muscle.

Nocturnal asthma is characterized by heightened bronchial reactivity at night, and plasma melatonin concentrations are higher in patients with nocturnal asthma symptoms. Numerous physiological effects of melatonin are mediated via its specific G protein-coupled receptors (GPCRs) named the MT1 receptor, which couples to both Gq and Gi proteins, and the MT2 receptor, which couples to Gi. We investigated whether melatonin receptors are expressed on airway smooth muscle; whether they regulate intracellular cyclic AMP (cAMP) and calcium concentrations ([Ca2+]i), which modulate airway smooth muscle tone; and whether they promote airway smooth muscle cell proliferation. We detected the mRNA and protein expression of the melatonin MT2 but not the MT1 receptor in native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by RT-PCR, immunoblotting, and immunohistochemistry. Activation of melatonin MT2 receptors with either pharmacological concentrations of melatonin (10-100 µM) or the nonselective MT1/MT2 agonist ramelteon (10 µM) significantly inhibited forskolin-stimulated cAMP accumulation in HASM cells, which was reversed by the Gαi protein inhibitor pertussis toxin or knockdown of the MT2 receptor by its specific siRNA. Although melatonin by itself did not induce an initial [Ca2+]i increase and airway contraction, melatonin significantly potentiated acetylcholine-stimulated [Ca2+]i increases, stress fiber formation through the MT2 receptor in HASM cells, and attenuated the relaxant effect of isoproterenol in guinea pig trachea. These findings suggest that the melatonin MT2 receptor is expressed in ASM, and modulates airway smooth muscle tone via reduced cAMP production and increased [Ca2+]i.

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 PLCβ-IP3 signaling cascade. These results might be related to the low asthma prevalence in males from puberty to adulthood.

Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling

Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed on airway nerve fibers that modulates afferent signals, resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca2+-ATPase-2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium channel inhibitor bupivacaine (n = 4, P < 0.05) and the neurokinin-2 receptor antagonist GR-159897 (n = 4, P < 0.05), suggesting that this contraction is neutrally mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction (n = 4, P < 0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments (n = 4-5, P < 0.05). Although capsazepine did not inhibit store-operated calcium entry in mouse ASM cells in PCLS (n = 4-7, P = nonsignificant), it did inhibit calcium oscillations (n = 3, P < 0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillations.

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; 17β-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. 17β-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.

Novel identification of the free fatty acid receptor FFAR1 that promotes contraction in airway smooth muscle.

Obesity is one of the major risk factors for asthma. Previous studies have demonstrated that free fatty acid levels are elevated in the plasma of obese individuals. Medium- and long-chain free fatty acids act as endogenous ligands for the free fatty acid receptors FFAR1/GPR40 and FFAR4/GPR120, which couple to Gq proteins. We investigated whether FFAR1 and FFAR4 are expressed on airway smooth muscle and whether they activate Gq-coupled signaling and modulate airway smooth muscle tone. We detected the protein expression of FFAR1 and FFAR4 in freshly dissected native human and guinea pig airway smooth muscle and cultured human airway smooth muscle (HASM) cells by immunoblotting and immunohistochemistry. The long-chain free fatty acids (oleic acid and linoleic acid) and GW9508 (FFAR1/FFAR4 dual agonist) dose-dependently stimulated transient intracellular Ca(2+) concentration ([Ca(2+)]i) increases and inositol phosphate synthesis in HASM cells. Downregulation of FFAR1 or FFAR4 in HASM cells by small interfering RNA led to a significant inhibition of the long-chain free fatty acids-induced transient [Ca(2+)]i increases. Oleic acid, linoleic acid, or GW9508 stimulated stress fiber formation in HASM cells, potentiated acetylcholine-contracted guinea pig tracheal rings, and attenuated the relaxant effect of isoproterenol after an acetylcholine-induced contraction. In contrast, TUG-891 (FFAR4 agonist) did not induce the stress fiber formation or potentiate acetylcholine-induced contraction. These results suggest that FFAR1 is the functionally dominant free fatty acid receptor in both human and guinea pig airway smooth muscle. The free fatty acid sensors expressed on airway smooth muscle could be an important modulator of airway smooth muscle tone.

Suppression of the increasing level of acetylcholine-stimulated intracellular Ca2+ in guinea pig airway smooth muscle cells by mabuterol.

The present study aimed to establish an effective method for the in vitro culture of guinea pig airway smooth muscle (ASM) cells, and also investigate the suppressive effect of mabuterol hydrochloride (Mab) on the increased level of intracellular Ca2+ in ASM cells induced with acetylcholine (Ach). Two different methods, i.e. with or without collagenase to pretreat tracheal tissues, were applied to the manufacture of ASM cells. Cell viability was determined with the 3-(4,5-dimethylthinazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Immunocytochemistry and immunofluorescence were used for the identification of ASM cells. Different concentration levels (10−3, 10−4, 10−5, 10−6 and 10−7 mmol/l) of Mab were administered 5 min before Ach (10−4 M) treatment, respectively. The Ca2+ fluorescent probe, Fura-2/AM or Fluo-3/AM were applied to the inspection of Ca2+ fluorescent intensity with Varioskan Flash, immunocytometry systems and an inverted system microscope, respectively. The results showed that the fresh method, in which isolated tracheal tissues were previously treated with collagenase for 20 min, was more advantageous for the preparation of guinea pig ASM cells compared to when the enzyme was not used. The time for the ASM cells to initially migrate out of the ‘tissue blocks’ and the culture having to be generated due to the thick cell density was significantly less. On identification with immunocytochemistry or immunofluorescent staining, >95% of the cells were ASM cells. Mab (10−3−10−7 mmol/l) significantly suppressed the elevation of intracellular Ca2+ induced by Ach in a concentration-dependent manner. The inhibitory rates of intracellular Ca2+ by different concentrations of Mab, from low to high, were 14.93, 24.73, 40.06, 48.54 and 57.13%, respectively, when Varioskan Flash was used for determination. In conclusion, this novel method has a shorter harvesting period for ASM cells. Mab can suppress the increasing level of intracellular Ca2+ induced by Ach in guinea pig ASM cells. Further investigation into the precise mechanisms of action is required.

17β-Aminoestrogens induce guinea pig airway smooth muscle hyperresponsiveness through L-type Ca2+ channels activation

Therapy with estrogens is frequently used in menopausal women and as hormonal contraception. Because of its thrombotic effects, long term estrogen administration used in hormonal replacement therapy (HRT) and contraception could represent a health hazard. In this regard, 17β-aminoestrogens such as aminoestrol, butolame and pentolame have shown promising HRT potential, because they have a weak agonist estrogenic action and antithrombotic activity. Additionally, estrogens play a protective role in airway smooth muscle, but the effect of 17β-aminoestrogens on the airway smooth muscle has not been tested yet. In guinea pig tracheal smooth muscle pentolame and butolame induced hyperresponsiveness to histamine (His), carbachol (Cch) and KCl. Interestingly, aminoestrol did not show this effect at the highest concentration studied, it even lowered the contraction induced by Cch. The hyperresponsiveness induced by pentolame to His was abolished by nifedipine. In single tracheal myocytes, KCl induced an increment in the intracellular Ca2+ concentration [Ca2+]i, pentolame also showed an increase in [Ca2+]i and the addition of KCl in the plateau of this rise further significantly augmented the [Ca2+]i response. Additionally, in patch clamp experiments pentolame increased the L-type Ca2+ currents. Thus, 17β-aminoestrogens such as pentolame and butolame, but not aminoestrol, activate L-type Ca2+ channel to induced hyperresponsiveness to Cch, His and KCl in guinea pig tracheal smooth muscle. Due to its lack of effect on airways and to its anticoagulant characteristics, aminoestrol seems to be the best alternative in the HRT among the 17β-aminoestrogens studied.

P6 Menthol Has Beneficial Effects In The Airways Through A Trpm8-independent Mechanism

Introduction Asthma is a debilitating disease of the airways characterised by symptoms such as bronchoconstriction, hyperresponsiveness and cough. Current therapies are associated with significant side effects and are ineffective in severe disease highlighting the need for novel treatments. Menthol, a cooling compound found in medicinal products, is commonly thought to activate the thermo-sensing cation-permeable Transient Receptor Potential Melastatin 8 (TRPM8) channel. Furthermore, menthol is known for its beneficial effects in the airways such as bronchodilation 1 and suppression of nerve activation and cough, 2 however the mechanism of action is unknown. Aim To pharmacologically characterise the role of TRPM8 and menthol in the airways. Methods TRPM8 gene expression was measured using Taqman real-time PCR. Mouse and guinea pig isolated vagal nerves were mounted in a grease-gap chamber and depolarisation (mV) recorded as an indicator of sensory nerve activity. Segments of murine and guinea pig trachea were attached to a force transducer in an organ bath and relaxation of carbachol-induced tone recorded (mg). Results TRPM8 is expressed in mouse and guinea pig vagal ganglia. The selective TRPM8 agonist, WS3, caused activation of guinea pig and mouse isolated vagal nerves, which was inhibited by the TRPM8 antagonist, JNJ41876666. Furthermore, WS3-induced depolarisation was abolished in isolated vagal nerves from Trpm8 -/- mice. (-)-menthol (active form) caused a small depolarisation of mouse and guinea pig isolated vagal nerves, which was blocked by JNJ41876666. Interestingly, pre-incubation of (-)-menthol inhibited vagal nerve activation induced by the tussive stimulus, capsaicin, an effect that was not inhibited by JNJ41876666. WS3 and (-)-menthol caused a concentration-dependent relaxation of murine and guinea pig trachea, which was not abolished by JNJ41876666 nor in the Trpm8 -/- mouse airway. No expression of TRPM8 was detected in guinea pig or mouse airway smooth muscle. Conclusions (-)-menthol caused a small TRPM8-dependent activation but a robust TRPM8-independent inhibition of vagal sensory nerve activity and relaxation of airway smooth muscle. Elucidating the mechanism behind the beneficial effects of (-)-menthol could lead to the development of promising new therapeutic targets for airway diseases such as asthma. References Ito et al. Pulm Pharmacol Ther 2008;21(5):812–817 Plekova et al. J Appl Physiol 2013;115(2):268–274

The effect of hyperpolarization-activated cyclic nucleotide-gated ion channel inhibitors on the vagal control of guinea pig airway smooth muscle tone.

Subtypes of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone. The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs(+) were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization. HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs(+) , but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres. HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes.

An improved method for guinea pig airway smooth muscle cell culture and the effect of SPFF on intracellular calcium.

The aim of the present study was to establish an improved method for in vitro guinea pig airway smooth muscle (ASM) cell culture and to evaluate the effect of 2-(4-amino-3-chloro-5-trifluomethyl-phenyl)-2-tert-butylamino-ethanol hydrochloride (SPFF), a novel β2-adrenoceptor agonist, on the release of intracellular calcium in cells. A procedure for the efficient isolation, culture, passage and characterization of the cells was described. Primary ASM cells of guinea pigs were cultured by modified tissue cultivation. The cells were identified by their morphological characteristics and immunocytochemistry. The relative inhibition of the release of intracellular calcium by drugs in the cells was measured by fluorometric quantification with fluorochrome Fura-2/AM. The results were as follows: a) The ASM cells of the guinea pigs were successfully cultured and subcultured by using our improved method and typical peak-valley characteristics were observed under the phase contrast microscope; b) data from immunocytochemical staining with specific α-smooth muscle actin (α-SMA) demonstrated that the cells were ASM cells; c) the growth characteristics and cell viability demonstrated that the cells were in good condition and were able to be applied in the follow-up studies; d) the inhibitory effect of SPFF on the release of intracellular calcium was concentration‑dependent when compared with the control and e) the potential mechanisms of SPFF on the inhibition of intracellular calcium may be independent of the ryanodine receptor, but may be closely associated with the inositol 1,4,5-trisphosphate receptor.

Testosterone-induced relaxation involves L-type and store-operated Ca2+ channels blockade, and PGE2 in guinea pig airway smooth muscle

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.

Non‐genomic relaxation effect of testosterone through L‐type and store operated Ca2+ channels blockade in guinea pig airway smooth muscle (1174.2)

In vascular smooth muscle, testosterone (TES) produces relaxation blocking L‐type Ca2+ channels. Recently, we found that L‐type and store operated (SOC) Ca2+ channels are the main membranal structures that provide extracellular Ca2+ for carbachol‐induced contraction in airway smooth muscle (ASM). Thus, we studied the possible interactions between L‐type and SOC channels in TES‐induced relaxation in guinea pig ASM. TES (10, 32 and 100 μM) induced a complete relaxation of Cch pre‐contracted tracheal smooth muscle and indomethacin partially inhibited this response. In single myocytes, the KCl induced intracellular Ca2+ increase ([Ca2+]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 Ca2+ entry. Myocytes stimulated with carbachol (Cch) produced a transient Ca2+ peak followed by a sustained plateau and 100 μM TES completely abolished the Cch induced Ca2+ plateau. Indomethacin, significantly diminished this effect of TES. When D‐600 was added during the plateau phase, a partial diminution (~35 %) was observed. A grater decrease (~78 %) was also seen when 2‐APB (SOC antagonist) was used. The combination of both drugs completely abolished the Ca2+ plateau induced by Cch. We concluded that TES blocks L‐type Ca2+ channels at nanomolar and SOC channels at micromolar concentration to induce relaxation.Grant Funding Source: PAPIIT‐DGAPA, UNAM IN200613

The In Vitro Pharmacology of GS-5759, A Novel Bifunctional Phosphodiesterase 4 Inhibitor and Long Acting β2-Adrenoceptor Agonist

Inhaled long-acting β(2)-adrenoceptor agonists (LABA) that act as bronchodilators and the oral anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor roflumilast are both approved therapies for chronic obstructive pulmonary disease (COPD). Here we describe the activity of a novel, inhaled, bifunctional, small molecule (R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl]carbamoyl}phenyl)sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), which has specific β(2) agonist and PDE4 inhibitory activity. GS-5759 demonstrated potent and full agonist activity at β(2) adrenoceptors (EC(50) = 8 ± 4 nM) and is a potent inhibitor of the PDE4 enzyme (IC(50) = 5 ± 3 nM). In cell assays, GS-5759 inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) production in human peripheral mononuclear cells (PBMC) with an IC(50) = 0.3 nM [confidence interval (CI) 0.1-0.6] and in human neutrophils formyl-methionyl-leucyl-phenylalanine (fMLP)-induced super oxide anion production with an IC(50) = 3 nM (CI 0.8-8). The addition of the β(2) antagonist ICI 118551 shifted the IC(50) in these cell assays to 4 and 38 nM, respectively, demonstrating the contribution of both β(2) agonist and PDE4 inhibitory activity to GS-5759. GS-5759 was also a potent inhibitor of profibrotic and proinflammatory mediator release from human lung fibroblasts. GS-5759 relaxed guinea pig airway smooth muscle strips precontracted with carbachol in a concentration-dependent manner with an EC(50) = 0.5 µM (CI 0.2-2) and had slow dissociation kinetics with an Off T(1/2) > 720 minutes at an EC(80) concentration of 3 µM. GS-5759 is a novel bifunctional molecule with both potent β(2) agonist and PDE4 inhibitor activity that could provide inhaled bronchodilator and anti-inflammatory therapy for COPD.

Sarcoplasmic reticulum Ca2+ refilling is determined by L-type Ca2+ and store operated Ca2+ channels in guinea pig airway smooth muscle

Sarcoplasmic reticulum Ca2+ refilling (SRREF) is crucial to sustain the agonists induced airway smooth muscle contraction. Nevertheless, its mechanisms have not been clearly described yet, although L-type voltage dependent, store operated, receptor operated channels and the Na+/Ca2+ exchanger in its reverse mode (NCXREV) have been proposed as Ca2+ handling proteins participating in this process. We found that in guinea pig and bovine tracheal smooth muscle, SRREF induced by caffeine was completely abolished by thapsigargin, even in the presence of Bay K8644, an activator of the L-type Ca2+ channel. Activation of NCXREV in guinea pig tracheal myocytes increased SRREF in ~70%, while opening of the L-type Ca2+ channels with Bay K8644 and favoring the capacitative Ca2+ entry with 2-APB (32μM) also augmented the SRREF by ~170% and ~71%, respectively. Methoxyverapamil (D-600, an L-type Ca2+ channel blocker), 2-APB (100µM, antagonist of the capacitative Ca2+ entry) and PPADS (NCXREV blocker) diminished the SRREF by ~63%, ~72% and ~31%, respectively. The simultaneous addition of D-600 and 2-APB annulled SRREF. These last results were also seen when carbachol was used instead of caffeine. In tracheal rings, 2-APB and nifedipine abolished the carbachol-induced contraction. We concluded that the sarcoplasmic reticulum Ca2+ pump is the only mechanism involved in the SRREF and that L-type Ca2+ voltage dependent and store operated Ca2+ channels are the principal membranal Ca2+ handling proteins that provide extracellular Ca2+ for SRREF and carbachol-induced contraction in the guinea pig airway smooth muscle; NCXREV seems to play a minor role.