Inhibition of ezrin phosphorylation by NSC305787 attenuates procaterol-stimulated ciliary beating in airway cilia.

Ciliary beating in the airway epithelium plays an important role in preventing infection by eliminating small particles and pathogens. Stimulation of ß2 adrenergic receptor (ß2AR) increases [cAMP]i levels and strongly activates this ciliary beating. ß2AR is localized to the apical membrane of the airways by indirectly binding to ezrin, an actin-binding protein. Ezrin takes active phosphorylated and inactive dephosphorylated states at Thr-567. Previously we showed that procaterol-stimulated ciliary beating was impaired in the ezrin-knockdown mice. In this study, we examined the roles of ezrin and its phosphorylation in regulating ciliary beating by using NSC305787, an ezrin inhibitor, in normal human airway epithelial cells (NHBE). We found that NSC305787 inhibits the phosphorylation of ezrin with an IC50 of 50 µM in NHBE. Treatment with NSC305787 for 4 h or more decreased the expression of ß2AR in the cell membrane and induced vesicle- or dot-like expression of ezrin and ß2AR inside the cell. As a result, inhibition of ezrin phosphorylation by NSC305787 attenuated the effect of procaterol-induced activation of ciliary beating in both frequency and distance indices.

Ezrin knockdown reduces procaterol-stimulated ciliary beating without morphological changes in mouse airway cilia.

Mucociliary clearance, which is conducted by beating cilia cooperating with the surface mucous layer, is a major host defense mechanism of the airway epithelium. Ezrin, a crosslinker between membrane proteins and the actin cytoskeleton, is located in microvilli and around the basal bodies in airway ciliary cells. It is also likely that ezrin plays an important role in apical localization of ß2 adrenergic receptor (ß2AR) in airway ciliary cells. Here, we studied the physiological roles of ezrin by using trachea and airway epithelial cells prepared from ezrin-knockdown (Vil2kd/kd) mice. The trachea and airway ciliary cells of Vil2kd/kd mice presented a normal morphology and basal body orientation, suggesting that ezrin is not directly involved in development and planar cell polarity of cilia. Procaterol stimulates ciliary beating (frequency and amplitude) via ß2AR in the airway ciliary cells. In the Vil2kd/kd mice, airway ciliary beating stimulated with procaterol was partly inhibited due to the impairment of cell surface expression of ß2AR. These results suggest that ezrin regulates the beating of airway ciliary cells by promoting the apical surface localization of ß2AR. This article has an associated First Person interview with the first author of the paper.

CDK12 and PAK2 as novel therapeutic targets for human gastric cancer.

Background: Gastric cancer remains the second leading cause of cancer-related death, and the third in mortality due to lack of effective therapeutic targets for late stage cancer patients. This study aims to identify potential druggable target biomarkers as potential therapeutic options for patients with gastric cancer. Methods: Immunohistochemistry of human gastric tumor tissues was conducted to determine the expression level of cyclin-dependent kinase 12 (CDK12). Multiple in vitro and in vivo assays such as RNAi, mass spectrometry, computer docking models, kinase assays, cell xenograft NU/NU mouse models (CDXs) and patient-derived xenograft NOD/SCID mouse models (PDXs) were conducted to study the function and molecular interaction of CDK12 with p21 activated kinase 2 (PAK2), as well as to find CDK12 inhibitors as potential treatment options for human gastric cancer. Results: Here we identified that CDK12 is a driver gene in human gastric cancer growth. Mechanistically, CDK12 directly binds to and phosphorylates PAK2 at T134/T169 to activate MAPK signaling pathway. We further identified FDA approved clinical drug procaterol can serve as an effective CDK12 inhibitor, leading to dramatic restriction of cancer cell proliferation and tumor growth in human gastric cancer cells and PDXs. Conclusions: Our data highlight the potential of CDK12/PAK2 as therapeutic targets for patients with gastric cancer, and we propose procaterol treatment as a novel therapeutic strategy for human gastric cancer.

Seihai-to (TJ-90)-Induced Activation of Airway Ciliary Beatings of Mice: Ca2+ Modulation of cAMP-Stimulated Ciliary Beatings via PDE1.

Sei-hai-to (TJ-90, Qing Fei Tang), a Chinese traditional medicine, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP (3',5'-cyclic adenosine monophosphate) accumulation modulated by Ca2+-activated phosphodiesterase 1 (PDE1A). A high concentration of TJ-90 (≥40 µg/mL) induced two types of CBF increases, a transient increase (an initial increase, followed by a decrease) and a sustained increase without any decline, while it only sustained the CBA increase. Upon inhibiting increases in intracellular Ca2+ concentration ([Ca2+]i) by 10 µM BAPTA-AM (Ca2+-chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or Ca2+/calmodulin-dependent PDE1 by 8MmIBMX (a selective PDE1 inhibitor), TJ-90 (400 µg/mL) induced only the sustained CBF increase without any transient CBF increase. The two types of the CBF increase (the transient increase and the sustained increase) induced by TJ-90 (≥40 µg/mL) were mimicked by the stimulation with both procaterol (100 pM) and ionomycin (500 nM). Thus, TJ-90 stimulates small increases in the intracellular cAMP concentration ([cAMP]i) and [Ca2+]i in airway ciliary cells of mice. These small increases in [cAMP]i and [Ca2+]i cause inducing a transient CBF increase or a sustained CBF increase in an airway ciliary cells, depending on the dominant signal, Ca2+-signal, or cAMP-signal.

ADRB2 gene polymorphism and emphysema heterogeneity can modulate bronchodilator response in patients with emphysema.

BACKGROUND: Genetic variation in the ß2-adrenergic receptor (ADRB2) gene has been thought to have an important role in the differential response to ß2-agonist therapy for asthma. However, previous studies have shown little evidence for an association between these ADRB2 variants and the bronchial dilator response (BDR) in chronic obstructive pulmonary disease (COPD) patients. This discrepancy could be explained by differences in the distribution and heterogeneity of pulmonary emphysema in COPD patients, since emphysema distribution and heterogeneity are thought to have a role in pulmonary function in COPD patients. We hypothesized that differences in emphysema distribution and heterogeneity may have masked significant alterations of the bronchodilator response among ADRB2 genotypes in COPD patients in previous studies. METHODS: The BDR (induced by 20 µg of procaterol) was measured in 211 patients who had a smoking history of more than 10 pack/years and had undergone chest high resolution computed tomography examination. A low attenuations area (<960 Hounsfield Units) was identified and the emphysema heterogeneity index (EHI%) was calculated with a range in value from -100% to 100%. ADRB2 Arg16Gly genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The BDR was augmented in patients with homogenous emphysema compared with those with upper-dominant emphysema. In patients carrying the AA genotype of ADRB2, the BDR was significantly increased in patients with upper-dominant emphysema, but not in patients with lower-dominant emphysema. CONCLUSION: Combination analysis of ADRB2 Arg16Gly polymorphism and EHI% may predict the effectiveness of ß2-adrenergic receptor agonist treatment in patients with COPD and emphysema.

A low [Ca2+]i-induced enhancement of cAMP-activated ciliary beating by PDE1A inhibition in mouse airway cilia.

This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an ß2-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca2+ concentration ([Ca2+]i) caused by switching to an EGTA-containing Ca2+-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca2+-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca2+ chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia.

Responsiveness to bronchodilator procaterol in COPD as assessed by forced oscillation technique.

The aim of this retrospective study was to assess responses to a bronchodilator by forced oscillation technique (FOT) and to relate the results of respiratory impedance (Zrs) to spirometric parameters in patients with chronic obstructive pulmonary disease (COPD). Zrs was measured as a function of frequency from 4 to 36Hz before and after inhalation of procaterol, a short-acting ß2-agonist (n=60). Respiratory resistance (Rrs) and reactance (Xrs) were significantly frequency-dependent, and inspiratory and expiratory phases were different both before and after procaterol inhalation. The Rrs at 4Hz and Xrs at 4-20Hz during a whole breath were significantly improved after procaterol inhalation. The response to procaterol inhalation varied among patients, and changes in Xrs at 4Hz significantly correlated with% change in forced expiratory volume in one second and changes in forced vital capacity. Taken together, Zrs, and specifically Xrs parameters, are sensitive to acute physiological responses to a bronchodilator in COPD.

Bronchial biopsy and reactivity in patients with chest tightness relieved with bronchodilator.

OBJECTIVE: It has been hypothesized that some patients with chest tightness of unknown origin can be successfully treated with a bronchodilator and that they should be diagnosed with chest pain variant asthma. We conducted a prospective study to characterize newly diagnosed patients with chest tightness relieved with bronchodilator use and without characteristic bronchial asthma attacks. METHODS: Eleven patients were registered following recurrent positive responses of chest tightness to inhalation of a ß2-agonist. These patients underwent assessments of airway responsiveness to methacholine, bronchial biopsy and bronchial lavage under fiber-optic bronchoscopy before receiving treatment. RESULTS: For the patients with chest tightness relieved with bronchodilator use, the bronchial biopsy specimens exhibited significant increases in lymphocyte and macrophage infiltration (p < 0.05) and no significant increase in eosinophils (p = 0.2918) compared with the control subjects. The bronchial responsiveness to methacholine was increased in two of the patients with chest tightness, and it was not increased in seven; in addition, increased percentages of eosinophils were detected in bronchial lavage fluid (5% or more) from two patients, but no increase was detected in eight patients. CONCLUSIONS: We suspect that the chest tightness was induced by airway constriction in these patients, but further study is necessary to validate this hypothesis. We propose that the chest tightness relieved with bronchodilator use was attributed to airway constriction resulting from inflammation with lymphocytes and macrophages and/or that the chest tightness was directly attributed to airway inflammation. This clinical trial is registered at www.umin.ac.jp (UMIN13994 and UMIN 16741).

Pulmonary liposomal formulations encapsulated procaterol hydrochloride by a remote loading method achieve sustained release and extended pharmacological effects.

Drug inhalation provides localized drug therapy for respiratory diseases. However, the therapeutic efficacy of inhaled drugs is limited by rapid clearance from the lungs. Small hydrophilic compounds have short half-lives to systemic absorption. We developed a liposomal formulation as a sustained-release strategy for pulmonary delivery of procaterol hydrochloride (PRO), a short-acting pulmonary ß2-agonist for asthma treatment. After PRO-loaded liposomes were prepared using a pH gradient (remote loading) method, 100-nm liposomes improved residence times of PRO in the lungs. PRO encapsulation efficiency and release profiles were examined by screening several liposomal formulations of lipid, cholesterol, and inner phase. Although PRO loading was not achieved using the conventional hydration method, PRO encapsulation efficiency was >60% using the pH gradient method. PRO release from liposomes was sustained for several hours depending on liposomal composition. The liposomal formulation effects on the PRO behavior in rat lungs were evaluated following pulmonary administration in vivo. Sustained PRO release was achieved using simplified egg phosphatidylcholine (EPC)/cholesterol (8/1) liposome in vitro, and greater PRO remnants were observed in rat lungs following pulmonary administration. Extended pharmacological PRO effects were observed for 120min in a histamine-induced bronchoconstriction guinea pig model. We indicated the simplified EPC/cholesterol liposome potential as a controlled-release PRO carrier for pulmonary administration.

Cyclic Adenosine Monophosphate-Mediated Enhancement of Vascular Endothelial Growth Factor Released by Differentiated Human Monocytic Cells: The Role of Protein Kinase A.

OBJECTIVE: Our investigation was designed to examine the signaling pathway involved in the enhancement of vascular endothelial growth factor (VEGF) release by ß-adrenoceptor agonists. MATERIALS AND METHODS: Human U937 cells differentiated into macrophages were primed with lipopolysaccharide (LPS) in the absence or presence of ß-adrenoceptor agonists and antagonists. The VEGF released and the intracellular cyclic adenosine monophosphate (cAMP) generated were assayed by ELISA. Where necessary, differences between mean values were tested for significance using Student's t test. RESULTS: Isoprenaline, procaterol and salbutamol concentration-dependently enhanced the release of VEGF induced by LPS in U937 cells. R*,R*-(±)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344), a selective ß3-adrenoceptor agonist, did not enhance VEGF release. Using isoprenaline as an agonist, propranolol, ICI 118551 and atenolol produced a parallel rightward shift of the concentration-response curve with no reduction in the maximum response. The -logKB values were 8.12 ± 0.17, 8.03 ± 0.05 and 7.23 ± 0.05 for propranolol, ICI 118551 and atenolol, respectively, indicating the possible involvement of both ß1- and ß2-adrenoceptor subtypes. Isoprenaline and prostaglandin E2 concentration-dependently increased cAMP generation in U937 cells. Isoprenaline, db-cAMP and 6-Bnz-cAMP, a protein kinase A (PKA) activator, all enhanced VEGF release induced by LPS, and this effect was abolished by KT 5720 and Rp-cAMPS, which are both selective PKA inhibitors, suggesting that PKA is the downstream effector of cAMP activity. 8-CPT-cAMP, a selective activator of the Epac system, had no effect on VEGF release induced by LPS, indicating that the Epac pathway played no role in the release process. CONCLUSION: In this study, we established that ß1- and ß2- but not ß3-adrenoceptors mediated cAMP-dependent enhancement of VEGF release induced by LPS in differentiated U937 cells, and that PKA was the downstream effector of cAMP activity.

Design of highly sensitive phosphorescence sensor for determination of procaterol hydrochloride based on inhibition of KClO3 oxidation fluorescein isothiocyanate.

Procaterol hydrochloride (Prh) can inhibit KClO3 oxidation of fluorescein isothiocyanate (FITC) to form a non-phosphorescent compound, which causes room temperature phosphorescence (RTP) of FITC in the system to enhance sharply the linear relationship between ∆Ip and the Prh content. Thus, a rapid response and highly sensitive phosphorescence sensor for the determination of Prh has been developed based on the inhibiting effect of Prh on KClO3 oxidation of FITC. This simple, high sensitivity (detection limit (LD) calculated by 3Sb /k was 0.019 fg/spot, sample volume 0.40 µl, corresponding concentration 4.8 × 10(-14) g ml(-1) ) and selective sensor with a wide linear range (0.080-11.20 g/spot) has been applied to detect Prh in blood samples, and the results were consistent with those obtained by high-performance liquid chromatography (HPLC). Simultaneously, the mechanism of the phosphorescence sensor for the detection of Prh was also investigated using infrared spectroscopy.

UGT1A1*28 is associated with greater decrease in serum K⁺ levels following oral intake of procaterol.

BACKGROUND AND OBJECTIVE: Procaterol is a potent ß2-agonist frequently used for the management of asthma and chronic obstructive pulmonary disease. The efficacy and adverse effects of ß2-agonists are heterogeneous in individual patients, which may be partly caused by genetic variations in metabolizing enzymes and receptor molecules. The present study was designed to analyze the relationship between gene polymorphisms and physiological effects of procaterol in healthy subjects. METHODS: Ninety-two non-smoking healthy volunteers were given 1 µg/kg body weight (max 50 µg) of procaterol as a dry syrup preparation, and the serum concentrations of procaterol, serum K(+), and the physical responses were monitored for 240 min. We genotyped ß2-adrenergic receptor (ADRB2) (Arg16Gly and Gln27Glu), cytochrome P450 3A4 (rs2246709, rs4646437), and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) (rs4148323 [allele A, *6], rs12479045, rs4148328, rs4663971, rs12052787, rs4148329, A (TA)6/7 TAA [seven-repeat allele, *28]). Procaterol concentrations in serum were measured by liquid chromatography-tandem mass spectrometry. RESULTS: No gene polymorphisms affected serum procaterol concentrations. Meanwhile, overall serum K(+) level changes were significantly lower in carriers of UGT1A1*28 than in non-carriers after correcting for strong effects of serum procaterol concentrations and baseline K(+) levels. No other polymorphisms were associated with serum K(+) levels. None of polymorphisms of ADRB2 were associated with any physical responses. CONCLUSION: The present study indicates that significant hypokalemia may occur in carriers of UGT1A1*28 by systemic administration of procaterol and potentially by other ß2-agonists metabolized in the liver.

Procaterol but not dexamethasone protects 16HBE cells from H2O2-induced oxidative stress.

Oxidative stress is an important pathophysiological factor of asthma and chronic obstructive pulmonary disease (COPD). We hypothesized that procaterol and dexamethasone might treat inflammation through inhibiting oxidative stress in vitro. This study evaluated procaterol and dexamethasone in the hydrogen peroxide (H2O2)-induced immortal human bronchial epithelial cell model of oxidative stress and investigated the underlying mechanisms. Results showed that exposure to 125 µM H2O2 for 2 h led to a 50% reduction in the cell viability, significantly increased the percentage of apoptosis, and elevated levels of malondialdehyde and reactive oxygen species. Pretreatment with procaterol (25 - 200 nM) could reduce these effects in a dose-dependent manner. In contrast, pretreatment with dexamethasone (100 nM, 1000 nM) was inefficient. Pretreatment with procaterol plus dexamethasone (100 nM procaterol + 1000 nM dexamethasone) was effective, but the combined effect was not more effective than the sole pretreatment with 100 nM procaterol. The nuclear factor kappa-B (NF-κB) pathway was involved in the pathogenic mechanisms of H2O2. Procaterol may indirectly inhibit H2O2-induced activation of the NF-κB pathway due to its capability of antioxidation. Glucocorticoids may be not recommended to treat asthma or COPD complicated with severe oxidative stress.

Aging causes a slowing in ciliary beat frequency, mediated by PKCε.

The elderly are at much higher risk for developing pneumonia than younger individuals. Pneumonia is a leading cause of death and is the third most common reason for hospitalization in the elderly. One reason that elderly people may be more susceptible to pneumonia is a breakdown in the lung's first line of defense, mucociliary clearance. Cilia beat in a coordinated manner to propel out invading microorganisms and particles. Ciliary beat frequency (CBF) is known to slow with aging, however, little is known about the mechanism(s) involved. We compared the CBF in BALB/c and C57BL/6 mice aged 2, 12, and 24 mo and found that CBF diminishes with age. Cilia in the mice at age 12 and 24 mo retained their ability to be stimulated by the ß2 agonist procaterol. To help determine the mechanism of ciliary slowing, we measured protein kinase C alpha and epsilon (PKCα and PKCε) activity. There were no activity differences in PKCα between the mice aged 2, 12, or 24 mo. However, we demonstrated a significantly higher PKCε activity in the mice at 12 and 24 mo than the in the mice 2 mo of age. The increase in activity is likely due to a nearly threefold increase in PKCε protein in the lung during aging. To strengthen the connection between activation of PKCε and ciliary slowing, we treated tracheas of mice at 2 mo with the PKCε agonist 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). We noted a similar decrease in baseline CBF, and the cilia remained sensitive to stimulation with ß2 agonists. The mechanisms for the slowing of baseline CBF have not been previously determined. In this mouse model of aging we were able to show that decreases in CBF are related to an increase in PKCε activity.

M1-muscarinic receptors promote fear memory consolidation via phospholipase C and the M-current.

Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with ß2-adrenergic (ß2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating ß2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.

Efficacy of procaterol combined with inhaled budesonide for treatment of cough-variant asthma.

BACKGROUND AND OBJECTIVE: Procaterol, a selective, short-acting beta-2 adrenoceptor agonist, is effective in treating 'classical' asthma, but its efficacy for cough-variant asthma (CVA) is unknown. We evaluated the efficacy and safety of procaterol combined with budesonide for CVA. METHODS: A prospective, randomized, double-blind, placebo-controlled, multicenter trial in China was conducted. One hundred and fifty-nine patients diagnosed with CVA (aged 18-75 years) were randomly divided into two groups to receive twice daily for 8 weeks, inhaled budesonide 100 µg plus either oral procaterol 25 µg or placebo. Primary and secondary efficacy variables were cough symptom severity scores and Leicester Cough Questionnaire (LCQ) life quality scores. Adverse events were also assessed. RESULTS: The budesonide/placebo and budesonide/procaterol groups contained 80 and 78 participants (one excluded for later diagnosis of eosinophilic bronchitis), respectively, with similar baseline characteristics. Daily cough score declined during treatment in both groups and was lower in the budesonide/procaterol group at 8 (0.44 vs 0.73) and 10 (0.36 vs 0.69) weeks (P < 0.05). Compared with the budesonide/placebo group, the proportion of patients with a reduction of 3 points or greater (66% vs 42%) and that of patients scoring 0 points (63% vs 51%) was higher in the budesonide/procaterol group for daily cough scores (P < 0.05). At 8 weeks, LCQ score improvement was superior in the budesonide/procaterol group (38.94 ± 19.24 vs 32.71 ± 18.92; P < 0.05). CONCLUSION: Procaterol combined with budesonide was well tolerated and effective at improving cough symptoms and quality of life in patients with CVA.

Dietary antioxidants prevent alcohol-induced ciliary dysfunction.

Previously we have shown that chronic alcohol intake causes alcohol-induced ciliary dysfunction (AICD), leading to non-responsive airway cilia. AICD likely occurs through the downregulation of nitric oxide (NO) and cyclic nucleotide-dependent kinases, protein kinase G (PKG) and protein kinase A (PKA). Studies by others have shown that dietary supplementation with the antioxidants N-acetylcysteine (NAC) and procysteine prevent other alcohol-induced lung complications. This led us to hypothesize that dietary supplementation with NAC or procysteine prevents AICD. To test this hypothesis, C57BL/6 mice drank an alcohol/water solution (20% w/v) ad libitum for 6 weeks and were concurrently fed dietary supplements of either NAC or procysteine. Ciliary beat frequency (CBF) was measured in mice tracheas, and PKG/PKA responsiveness to ß-agonists and NOx levels were measured from bronchoalveolar lavage (BAL) fluid. Long-term alcohol drinking reduced CBF, PKG and PKA responsiveness to ß-agonists, and lung NOx levels in BAL fluid. In contrast, alcohol-drinking mice fed NAC or procysteine sustained ciliary function and PKG and PKA responsiveness to ß-agonists. However, BAL NO levels remained low despite antioxidant supplementation. We also determined that removal of alcohol from the drinking water for as little as 1 week restored ciliary function, but not PKG and PKA responsiveness to ß-agonists. We conclude that dietary supplementation with NAC or procysteine protects against AICD. In addition, alcohol removal for 1 week restores cilia function independent of PKG and PKA activity. Our findings provide a rationale for the use of antioxidants to prevent damage to airway mucociliary functions in chronic alcohol-drinking individuals.

Treatment responses of procaterol and CD38 inhibitors in an ozone-induced airway hyperresponsiveness mice model.

Airway hyperresponsiveness (AHR) and airway inflammation are key pathophysiological features of many respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). To evaluate the treatment responses of procaterol and CD38 inhibitors in an ozone-induced AHR mice model, we hypothesized that procaterol and two synthetic CD38 inhibitors (Compounds T and H) might have therapeutic effects on the ozone-induced AHR mice model, and the nuclear factor-kappaB (NF-κB) pathway and the CD38 enzymatic activity might be involved in the mechanisms. With the exception of the Control group, ozone exposure was used to establish an AHR model. Male Kunming mice in the Procaterol and CD38 inhibitors groups were treated with an emulsifier of procaterol hydrochloride, Compound T or H. Results indicated that (1) no drug showed severe toxicity in this study; (2) ozone exposure induced airway inflammation and AHR; (3) intragastric treatment with procaterol and Compound T achieved potent therapeutic effects, but Compound H did not show any therapeutic effect; (4) the NF-κB pathway was involved in both the pathogenic mechanisms of ozone and therapeutic mechanisms of procaterol and Compound T; (5) however, the in vivo effect of Compound T was not caused by its inhibitory activity on CD38. Taken together, procaterol and Compound T are potentially good drugs to treat asthma and COPD complicated with ozone exposure.

Expression and functional role of ß3 -adrenoceptors in the human ureter.

OBJECTIVES: To investigate the presence of ß-adrenoceptor subtypes in the human ureter, and to examine whether ß(3) -adrenoceptors modulate relaxation of the human ureter. METHODS: Expression of messenger ribonucleic acid of ß-adrenoceptors in the human ureter was determined by reverse transcription polymerase chain reaction, and distribution of ß-adrenoceptors was examined by immunohistochemistry. In functional studies, the relaxant effects of isoproterenol, procaterol, TRK-380, salbutamol and BRL 37344 on KCl-induced contraction of the human ureter were evaluated, and the inhibitory effects of isoproterenol, procaterol and TRK-380 on electrical field stimulation-induced contractions were determined. RESULTS: Expression of ß(1) -, ß(2) - and ß(3) -adrenoceptor messenger ribonucleic acid in the human ureter was confirmed by reverse transcription polymerase chain reaction. Positive staining for ß(1) -, ß(2) - and ß(3) -adrenoceptor was identified not only in smooth muscle, but also in the urothelium of the human ureter. All ß-adrenoceptor agonists decreased the tone of KCl-induced contractions of the human ureter with a rank order of relaxant effects of isoproterenol > procaterol > TRK-380 > salbutamol > BRL 37344. Furthermore, isoproterenol, procaterol and TRK-380 significantly decreased the amplitude of electrical field stimulation-induced contractions with a rank order of inhibitory effects of isoproterenol > procaterol > TRK-380. CONCLUSIONS: Human ureteral relaxation is mediated by both ß(2) - and ß(3) -adrenoceptor stimulation. ß(3) -Adrenoceptor agonists have the potential to relax the human ureter, and their clinical application in the treatment of ureteral stones is expected.

Procaterol-stimulated increases in ciliary bend amplitude and ciliary beat frequency in mouse bronchioles.

The beating cilia play a key role in lung mucociliary transport. The ciliary beating frequency (CBF) and ciliary bend amplitude (CBA) of isolated mouse bronchiolar ciliary cells were measured using a light microscope equipped with a high-speed camera (500 Hz). Procaterol (aß(2)-agonist) increased CBA and CBF in a dose dependent manner via cAMP. The time course of CBA increase is distinct from that of CBF increase: procaterol at 10 nM first increased CBA and then CBF. Moreover, 10 pM procaterol increased CBA, not CBF, whereas 10 nM procaterol increased both CBA and CBF. Concentration-response studies of procaterol demonstrated that the CBA curve was shifted to a lower concentration than the CBF curve, which suggests that CBA regulation is different from CBF regulation. Measurements of microbead movements on the bronchiole of lung slices revealed that 10 pM procaterol increased the rate of ciliary transport by 37% and 10 nM procaterol increased it by 70%. In conclusion, we have shown that increased CBA is of particular importance for increasing the bronchiolar ciliary transport rate, although CBF also plays a role in increasing it.