kt-5720 and Asthma

Clinically observed differences in airway reactivity and asthma exacerbations in women at different life stages suggest a role for sex steroids in modulating airway function although their targets and mechanisms of action are still being explored. We have previously shown that clinically relevant concentrations of exogenous estrogen acutely decrease intracellular calcium ([Ca(2+)](i)) in human airway smooth muscle (ASM), thereby facilitating bronchodilation. In this study, we hypothesized that estrogens modulate cyclic nucleotide regulation, resulting in decreased [Ca(2+)](i) in human ASM. In Fura-2-loaded human ASM cells, 1 nM 17β-estradiol (E(2)) potentiated the inhibitory effect of the β-adrenoceptor (β-AR) agonist isoproterenol (ISO; 100 nM) on histamine-mediated Ca(2+) entry. Inhibition of protein kinase A (PKA) activity (KT5720; 100 nM) attenuated E(2) effects on [Ca(2+)](i). Acute treatment with E(2) increased cAMP levels in ASM cells comparable to that of ISO (100 pM). In acetylcholine-contracted airways from female guinea pigs or female humans, E(2) potentiated ISO-induced relaxation. These novel data suggest that, in human ASM, physiologically relevant concentrations of estrogens act via estrogen receptors (ERs) and the cAMP pathway to nongenomically reduce [Ca(2+)](i), thus promoting bronchodilation. Activation of ERs may be a novel adjunct therapeutic avenue in reactive airway diseases in combination with established cAMP-activating therapies such as β(2)-agonists.

Topics: Adrenergic beta-Agonists; Asthma; Bronchoconstriction; Calcium Signaling; Carbazoles; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Estradiol; Estrogens; Female; Humans; Isoproterenol; Myocytes, Smooth Muscle; Pyrroles; Receptors, Adrenergic, beta; Respiratory System

Present study was performed to examine whether ADH was implicated in psychological stress asthma and to explore the underlying molecular mechanism.. We not only examined ADH levels in the cerebrospinal fluid (CSF) via radioimmunoassay, but also measured ADH receptor (ADHR) expression in airway-related vagal preganglionic neurons (AVPNs) through real-time PCR in all experimental mice. Western blotting was performed to evaluate the relationship between ADH and PKA/PKC in psychological stress asthma. Finally, the role of PKA/PKC in psychological stress asthma was analyzed.. Marked asthma exacerbations were noted owing to significantly elevated levels of ADH and ADHR after psychological stress induction as compared to OVA alone (asthma group). ADHR antagonists (SR-49095 or SR-121463A) dramatically lowered higher protein levels of PKAα and PKCα induced by psychological stress as compared to OVA alone, suggesting the correlation between ADH and PKA/PKC in psychological stress asthma. KT-5720 (PKA inhibitor) and Go-7874 (PKC inhibitor) further directly revealed the involvement of PKA/PKC in psychological stress asthma. Some notable changes were also noted after employing PKA and PKC inhibitors in psychological stress asthma, including reduced asthmatic inflammation (lower eosinophil peroxidase (EPO) activity, myeloperoxidase (MPO) activity, immunoglobulin E (IgE) level, and histamine release), substantial decrements in inflammatory cell counts (eosinophils and lymphocytes), and decreased cytokine secretion (IL-6, IL-10, and IFN-γ), indicating the involvement of PKA/PKC in asthma exacerbations induced by psychological stress.. Our results strongly suggested that ADH participated in psychological stress-induced asthma exacerbations via PKA/PKC signal pathway in AVPNs.

Topics: Animals; Antidiuretic Hormone Receptor Antagonists; Asthma; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Cytokines; Disease Models, Animal; Eosinophils; Female; Mice; Mice, Inbred BALB C; Morpholines; Neurons; Ovalbumin; Protein Kinase C; Protein Kinase Inhibitors; Pyrroles; Receptors, Vasopressin; Signal Transduction; Spiro Compounds; Stress, Psychological; Vasopressins

To clarify heterologous regulation of a receptor is important in considering medication. Histamine constricts the airway smooth muscle through the action to the H(1) receptor (H1R), which contributes to asthma. beta(2)-Adrenergic receptor (beta2R) agonists are widely used in asthmatic therapy for their bronchodilating effects. In this study, we investigated the effect of beta2R activation on the H1R function using Chinese hamster ovary cells stably co-expressing human histamine H1R and beta2R (CHO-H1/beta2 cell). The stimulation of beta2R resulted in the decrease of H1R in the membrane. Heterologous H1R down-regulation was significantly reversed in the presence of the cyclic AMP-dependent protein kinase (PKA) inhibitor KT5720. Since phosphorylation of G protein-coupled receptor (GPCR) by second messenger-dependent kinases, is proposed to be a key step initiating heterologous receptor desensitization, we examined whether heterologous H1R down-regulation was accompanied by H1R phosphorylation. H1R was phosphorylated by beta2R stimulation; however, a PKA inhibitor did not inhibit heterologous H1R phosphorylation. Our results suggest that H1R was heterologously regulated by beta2R. Not only a direct action of beta2R agonist to beta2R causing bronchodilation but also indirect action that reduces the number of H1R responsible for bronchoconstriction might contribute to a decrease in the bronchial resistance, which proposes another possible advantage of beta2R agonists for asthmatic medication.

Topics: Adrenergic beta-Agonists; Animals; Asthma; Bronchodilator Agents; Carbazoles; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Enzyme Inhibitors; Histamine H1 Antagonists; Humans; Indoles; Isoproterenol; Phosphorylation; Pyrilamine; Pyrroles; Receptors, Adrenergic, beta-2; Receptors, Histamine H1