The in vitro mechanism of Vaspinregulates the proliferation and steroidogenesis of rat lutein cells.

OBJECTIVE: Stable luteal cell function is an important prerequisite for reproductive ability and embryonic development. However, luteal insufficiency seriously harms couples who have the desire to have a pregnancy, and the most important thing is that there is no complete solution. In addition, Vaspin has been shown to have regulatory effects on luteal cells, but the complex mechanisms involved have not been fully elucidated. Therefore, this study aimed to explore the effect of Vaspin on rat luteal cells and its mechanism. METHODS: Granulosa lutein cells separated from the ovary of female rats were incubated for 24h with gradient concentrations of Vaspin, and granulosa lutein cells incubated with 0.5% bovine serum albumin were used as controls. The proliferation, apoptosis, angiogenesis, progesterone (P4) and estradiol (E2) were detected by CCK-8, Anneixn-FITC/PI staining, angiogenesis experiment and ELISA. Western blot was applied to observe the expression levels of proteins related to cell proliferation, apoptosis, angiogenesis and MEK/MAPK signaling pathway. RESULTS: Compared with the Control group, Vaspin could significantly up-regulate the proliferation of granulosa lutein cells and reduce the apoptosis. Moreover, Vaspin promoted the angiogenesis of granulosa lutein cells and the production of P4 and E2 in a concentration-dependent manner. Furthermore, Vaspin up-regulated the CyclinD1, CyclinB1, Bcl2, VEGFA and FGF-2 expression in granulosa lutein cells, and down-regulated the level of Bax. Also, Vaspin increased the p-MEK1 and p-p38 levels. CONCLUSION: Vaspin can up-regulate the proliferation and steroidogenesis of rat luteal cells and reduce apoptosis, which may be related to the influence of MEK/MAPK activity.

Occurrence, sources and health risk of polycyclic aromatic hydrocarbons in soils around oil wells in the border regions between oil fields and suburbs.

The Yellow River Delta (YRD) is a typical region where oil fields generally overlap cities and towns, leading to complex soil contamination from both the oil fields and human activities. To clarify the distribution, speciation, potential sources and health risk of polycyclic aromatic hydrocarbons (PAHs) in soils of border regions between oil fields and suburbs of the YRD, 138 soil samples (0-20 cm) were collected among 12 sampling sites located around oil wells with different extraction histories. The 16 priority control PAHs (16PAHs), as selected by the United States Environmental Protection Agency (USEPA), were extracted via an accelerated solvent extraction and detected by GC-MS. The results showed that soils of the study area were generally polluted by the 16PAHs. Among these pollutions, chrysene and phenanthrene were the dominant components, and 4-ring PAHs were the most abundant. A typical temporal distribution pattern of the 16PAHs was revealed in soils from different sampling sites around oil wells with different exploitation histories. The concentrations of total 16PAHs and high-ring PAHs (HPAHs) both increased with the extraction time of the nearby oil wells. Individual PAH ratios and PCA method revealed that the 16PAHs in soil with newly developed oil wells were mainly from petroleum pollutants, whereas PAHs in soils around oil wells with a long exploitation history were probably from petroleum contamination; combustion of petroleum, fuel, and biomass; and degradation and migration of PAHs from petroleum. Monte Carlo simulation was used to evaluate the health risks of the 7 carcinogenic PAHs and 9 non-carcinogenic PAHs in the study area. The results indicated that ingestion and dermal contact were the predominant pathways of exposure to PAH residues in soils. Both the carcinogenic and non-carcinogenic burden of the 16PAHs in soils of the oil field increased significantly with exploitation time of nearby oil wells.

Selective targeting of the α5-subunit of GABAA receptors relaxes airway smooth muscle and inhibits cellular calcium handling.

The clinical need for novel bronchodilators for the treatment of bronchoconstrictive diseases remains a major medical issue. Modulation of airway smooth muscle (ASM) chloride via GABAA receptor activation to achieve relaxation of precontracted ASM represents a potentially beneficial therapeutic option. Since human ASM GABAA receptors express only the α4- and α5-subunits, there is an opportunity to selectively target ASM GABAA receptors to improve drug efficacy and minimize side effects. Recently, a novel compound (R)-ethyl8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4] diazepine-3-carboxylate (SH-053-2'F-R-CH3) with allosteric selectivity for α5-subunit containing GABAA receptors has become available. We questioned whether this novel GABAA α5-selective ligand relaxes ASM and affects intracellular calcium concentration ([Ca(2+)]i) regulation. Immunohistochemical staining localized the GABAA α5-subunit to human ASM. The selective GABAA α5 ligand SH-053-2'F-R-CH3 relaxes precontracted intact ASM; increases GABA-activated chloride currents in human ASM cells in voltage-clamp electrophysiology studies; and attenuates bradykinin-induced increases in [Ca(2+)]i, store-operated Ca(2+) entry, and methacholine-induced Ca(2+) oscillations in peripheral murine lung slices. In conclusion, selective subunit targeting of endogenous α5-subunit containing GABAA receptors on ASM may represent a novel therapeutic option to treat severe bronchospasm.

Chloride channel blockade relaxes airway smooth muscle and potentiates relaxation by ß-agonists.

Severe bronchospasm refractory to ß-agonists continues to cause significant morbidity and mortality in asthmatic patients. We questioned whether chloride channels/transporters are novel targets for the relaxation of airway smooth muscle (ASM). We have screened a library of compounds, derivatives of anthranilic and indanyloxyacetic acid, that were originally developed to antagonize chloride channels in the kidney. We hypothesized that members of this library would be novel calcium-activated chloride channel blockers for the airway. The initial screen of this compound library identified 4 of 20 compounds that relaxed a tetraethylammonium chloride-induced contraction in guinea pig tracheal rings. The two most effective compounds, compounds 1 and 13, were further studied for their potential to either prevent the initiation of or relax the maintenance phase of an acetylcholine (ACh)-induced contraction or to potentiate ß-agonist-mediated relaxation. Both relaxed an established ACh-induced contraction in human and guinea pig ex vivo ASM. In contrast, the prevention of an ACh-induced contraction required copretreatment with the sodium-potassium-chloride cotransporter blocker bumetanide. The combination of compound 13 and bumetanide also potentiated relaxation by the ß-agonist isoproterenol in guinea pig tracheal rings. Compounds 1 and 13 hyperpolarized the plasma cell membrane of human ASM cells and blocked spontaneous transient inward currents, a measure of chloride currents in these cells. These functional and electrophysiological data suggest that modulating ASM chloride flux is a novel therapeutic target in asthma and other bronchoconstrictive diseases.

Calcium-activated chloride channels anoctamin 1 and 2 promote murine uterine smooth muscle contractility.

OBJECTIVE: To determine the presence of calcium activated chloride channels anoctamin 1 (ANO1) and 2 (ANO2) in human and murine uterine smooth muscle (MUSM) and evaluate the physiologic role for these ion channels in murine myometrial contractility. STUDY DESIGN: We performed reverse transcription polymerase chain reaction to determine whether ANO1 and 2 are expressed in human and murine uterine tissue to validate the study of this protein in mouse models. Immunohistochemical staining of ANO1 and 2 was then performed to determine protein expression in murine myometrial tissue. The function of ANO1 and 2 in murine uterine tissue was evaluated using electrophysiologic studies, organ bath, and calcium flux experiments. RESULTS: ANO1 and 2 are expressed in human and MUSM cells. Functional studies show that selective antagonism of these channels promotes relaxation of spontaneous MUSM contractions. Blockade of ANO1 and 2 inhibits both agonist-induced and spontaneous transient inward currents and abolishes G-protein coupled receptor (oxytocin) mediated elevations in intracellular calcium. CONCLUSION: The calcium activated chloride channels ANO1 and 2 are present in human and murine myometrial tissue and may provide novel potential therapeutic targets to achieve effective tocolysis.

Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle.

Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.

The ly-6 protein, lynx1, is an endogenous inhibitor of nicotinic signaling in airway epithelium.

Our laboratory has previously reported that bronchial epithelial cells (BEC) express a regulatory cascade of classic neurotransmitters and receptors that communicate in an almost neuronal-like manner to achieve physiological regulation. In this paper we show that the similarity between neurotransmitter signaling in neurons and BEC extends to the level of transmitter receptor allosteric modulators. Lynx1 is a member of the ly-6/three-finger superfamily of proteins, many of which modulate receptor signaling activity. Lynx1 specifically has been shown to modulate nicotinic acetylcholine receptor (nAChR) function in neurons by altering receptor sensitivity and desensitization. We now report that lynx1 forms a complex with α7 nAChR in BEC and serves to negatively regulate α7 downstream signaling events. Treatment of primary cultures of BEC with nicotine increased levels of nAChR subunits and that increase was potentiated by lynx1 knockdown. Lynx1 knockdown also potentiated the nicotine-induced increase in GABA(A) receptors (GABA(A)R) and MUC5AC mRNA expression, and that effect was blocked by α7 antagonists and α7 knockdown. In parallel with the increases in nAChR, GABA(A)R, and mucin mRNA levels, lynx1 knockdown also increased levels of p-Src. Consistent with this, inhibition of Src signaling blocked the ability of the lynx1 knockdown to increase basal and nicotine-stimulated GABA(A)R and mucin mRNA expression. Thus lynx1 appears to act as a negative modulator of α7 nAChR-induced events by inhibiting Src activation. This suggests that lynx1 agonists or mimetics are a potentially important therapeutic target to develop new therapies for smoking-related diseases characterized by increased mucin expression.

Prenatal nicotine exposure increases GABA signaling and mucin expression in airway epithelium.

Maternal smoking during pregnancy increases the risk of respiratory disease in offspring, but surprisingly little is known about the underlying mechanisms. Nicotinic acetylcholine receptors (nAChRs) expressed in bronchial epithelial cells (BECs) mediate the effects of nicotine on lung development and function. Recently, BECs were also shown to express a GABAergic paracrine loop that was implicated in mucus overproduction in asthma. We therefore investigated the interactions between cholinergic and GABAergic signaling in rhesus macaque BECs, and found that nicotine upregulated GABA signaling in BECs through the sequential activation of BEC nAChR and GABA receptors. The incubation of primary cultures of rhesus BECs increased concentrations of GAD, GABA(A) receptors, and mucin mRNA. The nicotine-induced increase in glutamatic acid decarboxylase (GAD) and GABA(A) receptor mRNA resulted in increased GABA-induced currents and increased expression of mucin. The ability of nicotine to increase mucin expression was blocked by nicotinic and GABA(A) antagonists. These results implicate GABA signaling as a middleman in nicotine's effects on mucus overproduction. Similar effects of nicotine on GABA signaling and the expression of mucin were seen in vivo after chronic exposure of rhesus monkeys to nicotine. These data provide a new mechanism linking smoking with the increased mucin seen in asthma and chronic obstructive pulmonary disorder, and suggest a new paradigm of communication between non-neuronal transmitter systems in BECs. The existence of neural-like transmitter interactions in BECs suggests that some drugs active in the central nervous system may possess previously unexpected utility in respiratory diseases.

Anoctamin 1 antagonism potentiates conventional tocolytic-mediated relaxation of pregnant human uterine smooth muscle.

BACKGROUND: Currently available tocolytic agents are not effective treatment for preterm labor beyond 48 h. A major reason is the development of maternal side effects which preclude the maintenance of an effective steady-state drug concentration. One strategy that can mitigate these side effects is utilizing synergistic drug combinations to reduce the drug concentrations necessary to elicit a clinical effect. We have previously shown that three anoctamin 1 (ANO1) antagonists mediate potent relaxation of precontracted human uterine smooth muscle (USM). In this study, we aimed to determine whether a combination of sub-relaxatory doses of tocolytic drugs in current clinical use [the L-type voltage-gated calcium channel (VGCC) blocker, nifedipine (NIF); and the ß2-adrenergic (ß2AR) agonist, terbutaline (TRB)] will potentiate USM relaxation with two ANO1 antagonists [benzbromarone (BB) and MONNA (MN)]. OBJECTIVE: This study sought to examine the synergistic potency and mechanistic basis of two ANO1 antagonists with currently available tocolytic drugs. Functional endpoints assessed included relaxation of pre-contracting pregnant human USM tissue, inhibition of intracellular calcium release, and reduction of spontaneous transient inward current (STIC) recordings in human uterine smooth muscle cells. METHODS: Human myometrial strips and primary human USM cells were used in organ bath and calcium flux experiments with different combinations of sub-threshold doses of ANO1 antagonists and terbutaline or nifedipine to determine if ANO1 antagonists potentiate tocolytic drugs. RESULTS: The combination of sub-threshold doses of two ANO1 antagonists and current tocolytic drugs demonstrate a significant degree of synergy to relax human pregnant USM compared to the effects achieved when these drugs are administered individually. CONCLUSION: A combination of sub-threshold doses of VGCC blocker and ß2AR agonist with ANO1 antagonists potentiates relaxation of oxytocin-induced contractility and calcium flux in human USM ex vivo. Our findings may serve as a foundation for novel tocolytic drug combinations.

Nicotine activates and up-regulates nicotinic acetylcholine receptors in bronchial epithelial cells.

Prenatal nicotine exposure impairs normal lung development and leads to diminished pulmonary function after birth. Previous work from our laboratory has demonstrated that nicotine alters lung development by affecting a nonneuronal cholinergic autocrine loop that is expressed in lung. Bronchial epithelial cells (BECs) express choline acetyltransferase, the choline high-affinity transporter and nicotinic acetylcholine (ACh) receptor (nAChR) subunits. We now demonstrate through a combination of morphological and electrophysiological techniques that nicotine affects this autocrine loop by up-regulating and activating cholinergic signaling. RT-PCR showed the expression of alpha 3, alpha 4, alpha 7, alpha 9, alpha 10, beta2, and beta 4 nAChR mRNAs in rhesus monkey lung and cultured BECs. The expression of alpha 7, alpha 4, and beta2 nAChR was confirmed by immunofluorescence in the cultured BECs and lung. The electrophysiological characteristics of nAChR in BECs were determined using whole-cell patch-clamp on cultured BECs. Both ACh and nicotine evoked an inward current, with a rapid desensitizing current. Nicotine induced inward currents in a concentration-dependent manner, with an EC(50) of 26.7 microM. Nicotine-induced currents were reversibly blocked by the nicotinic antagonists, mecamylamine, dihydro-beta-erythroidine, and methyllcaconitine. Incubation of BECs with 1 microM nicotine for 48 hours enhanced nicotine-induced currents by roughly 26%. The protein tyrosine phosphorylation inhibitor, genistein, increased nicotine-induced currents by 58% and enhanced methyllcaconitine-sensitive currents (alpha 7 nAChR activities) 2.3-fold, whereas the protein tyrosine phosphatase inhibitor, pervanadate, decreased the effects of nicotine. These results demonstrate that chronic nicotine exposure up-regulates nAChR activity in developing lung, and that nAChR activity can be further modified by tyrosine phosphorylation.

Functional comparison of anoctamin 1 antagonists on human uterine smooth muscle contractility and excitability.

BACKGROUND: Pre-term birth is a major health care challenge throughout the world, and preterm labor represents a potentially reversible component of this problem. Current tocolytics do not improve preterm labor beyond 48 h. We have previously shown that anoctamin 1 (ANO1) channel blockade results in relaxation of pre-contracted human uterine smooth muscle (USM). Three drug classes with reported medicinal effects in humans also have members with ANO1 antagonism. In this study, we compared the ability of representatives from these 3 classes to reduce human USM contractility and excitability. OBJECTIVE: This study sought to examine the comparative potency of 3 ANO1 antagonists on pregnant human USM relaxation, contraction frequency reduction, inhibition of intracellular calcium release and membrane hyperpolarization. METHODS: Experiments were performed using: 1) Ex vivo organ bath (human pregnant tissue), 2) Oxytocin-induced calcium flux (in vitro human USM cells) and 3) Membrane potential assay (in vitro human USM cells). RESULTS: Benzbromarone (BB) demonstrated the greatest potency among the compounds tested with respect to force, frequency inhibition, reducing calcium elevation and depolarizing membrane potential. CONCLUSION: While all 3 ANO1 antagonists attenuate pregnant human uterine tissue contractility and excitability, BB is the most potent tocolytic drug. Our findings may serve as a foundation for future structure-function analyses for novel tocolytic drug development.

Anoctamin Channels in Human Myometrium: A Novel Target for Tocolysis.

BACKGROUND: Spontaneous preterm labor leading to preterm birth is a significant obstetric problem leading to neonatal morbidity and mortality. Current tocolytics are not completely effective and novel targets may afford a therapeutic benefit. OBJECTIVE: To determine whether the anoctamin (ANO) family, including the calcium-activated chloride channel ANO1, is present in pregnant human uterine smooth muscle (USM) and whether pharmacological and genetic modulation of ANO1 modulates USM contraction. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and immunohistochemical staining were done to determine which members of the ANO family are expressed in human USM. Uterine smooth muscle strips were studied in an organ bath to determine whether ANO1 antagonists inhibit oxytocin-induced USM contractions. Anoctamin 1 small interfering RNA (siRNA) knockdown was performed to determine its effect on filamentous-/globular (F/G)-actin ratio, a measurement of actin polymerization's role in promoting smooth muscle contraction. RESULTS: Messenger RNA (mRNA) encoding all members of the ANO family (except ANO7) are expressed in pregnant USM tissue. Anoctamin 1 mRNA expression was decreased 15.2-fold in pregnant USM compared to nonpregnant. Anoctamin 1 protein is expressed in pregnant human USM tissue. Functional organ bath studies with pregnant human USM tissue demonstrated that the ANO1 antagonist benzbromarone attenuates the force and frequency of oxytocin-induced contractions. In human USM cells, siRNA knockdown of ANO1 decreases F-/G-actin ratios. CONCLUSION: Multiple members of the ANO family, including the calcium-activated chloride channel ANO1, are expressed in human USM. Antagonism of ANO1 by pharmacological inhibition and genetic knockdown leads to an attenuation of contraction in pregnant human USM. Anoctamin 1 is a potentially novel target for tocolysis.

[Studies on chemical constituents in leaves of Mallotus furetianus I].

OBJECTIVE: To investigate the chemical compounds in leaves of Mallotus furetianus. METHOD: The chemical components were isolated by solvent extraction and chromatography. The structures were identified on the basis of physico-chemical constant and spectral data. RESULT: Eight compounds were isolated and identified as 3-Hydroxy-4, 5 (R)-dimethyl-2(5H)-furanone (I), gallic acid (II), (6S, 9R)-roseoside (III), (Z)-3-Hexenyl-beta-D-glucopyranoside (IV), 3, 4, 8, 9, 10-Pentahydroxy-dibenzo [b, d] pyran-6-one (V), friedelinol (VI), beta- sitosterol (VII), friedelin (VIII). CONCLUSION: Compounds I - VIII were obtained for the first time from this plant.

Role of Lynx1 and related Ly6 proteins as modulators of cholinergic signaling in normal and neoplastic bronchial epithelium.

The ly-6 proteins are a large family of proteins that resemble the snake three finger alpha toxins such as α-bungarotoxin and are defined by their multiple cysteine residues. Multiple members of the ly-6 protein family can modulate nicotinic signaling including lynx1, lynx2, slurp-1, slurp-2 and prostate stem cell antigen (PSCA). Consistent with the expression of multiple nicotinic receptors in bronchial epithelium, multiple members of the nicotinic-modulatory ly-6 proteins are expressed in lung including lynx1 and lynx2. We studied the role of lynx1 as an exemplar of the role of ly-6 proteins in lung. Our data demonstrates that lynx1 acts as a negative modulator of nicotinic signaling in normal and neoplastic lung. In normal lung lynx1 serves to limit the ability of chronic nicotine exposure to increase levels of nicotinic receptors and also serves to limit the ability of nicotine to upregulate levels of GABAA receptors in lung. In turn this allows lynx1 to limit the ability of nicotine to upregulate levels of mucin which is mediated by GABAergic signaling. This suggests that lynx1-mimetics may have potential for treatment of asthma and COPD. In that most lung cancer cells also express nicotinic receptor and lynx1 we examined the role of lynx-1 in lung cancer. Lynx1 levels are decreased in lung cancers compared to adjacent normal lung. Knockdown of lynx1 by siRNAs increased growth of lung cancer cells while expression of lynx1 in lung cancer cell decreased cell proliferation. This suggests that lynx1 is an endogenous regulator of lung cancer growth. Given that multiple small molecule negative and positive allosteric modulators of nicotinic receptors have already been developed, this suggests that lynx1 is a highly druggable target both for development of drugs that may limit lung cancer growth as well as for drugs that may be effective for asthma or COPD treatment.

[Characteristics of dioxin-like polychlorinated biphenyls contamination in soils of Gudao Region in Dongying].

To investigate pollution characteristics of dioxin-like polychlorinated biphenyls (DL-PCBs) in Gudao Region, Dongying City, twenty surface soil samples were collected in April, 2011. The DL-PCBs in soil samples were determined by Soxhelt extraction-Florisil purification-Gas Chromatography(GC)-Electron Capture Detector (ECD). The concentration of DL-PCBs in surface soils ranged from 1.4 microg x kg(-1) to 7.4 microg x kg(-1), with the mean concentration of (3.5 +/- 1.7) microg x kg(-1). The total TEQ level was between 1.2 ng x kg(-1) and 31.8 ng x kg(-1), and the mean TEQ was 5.4 ng x kg(-1), which exceeded the Canadian soil environment quality guidelines (4.0 ng x kg(-1)). The congener profiles of DL-PCBs were similar among sites. Tetra-CB and penta-CB were the major homologues in all soil samples, together accounting for more than 80% of the total DL-PCBs. The level of DL-PCBs exhibited a decreasing trend with the increasing distance from the Gudao town to the suburb, which could be attributed to the chemical plants around the town. The correlation analysis showed that the concentration of DL-PCBs was positively correlated with the organic matter content and clay particle content (R2 were 0.732 and 0.687, separately, P < 0.01), but negatively correlated with the sand particle content (R2 = -0.438, P < 0.05).

Activated cholinergic signaling provides a target in squamous cell lung carcinoma.

The binding of exogenous nicotine to nicotinic acetylcholine (ACh) receptors (nAChR) and the binding of endogenous ACh to both nAChR and muscarinic ACh receptors (mAChR) stimulate growth of both small cell and non-small cell lung carcinomas. Understanding how cholinergic signaling is up-regulated in lung cancer may suggest new therapeutic approaches. Analysis of 28 squamous cell lung carcinomas (SCC) showed increased levels of alpha5 and beta3 nAChR mRNA and increased levels of ACh associated with increased levels of choline acetyltransferase mRNA and decreased cholinesterase mRNAs. Lynx1, an allosteric inhibitor of nAChR activity, was also decreased in SCC. Thus, cholinergic signaling is broadly increased in SCC caused by increased levels of receptors, increased levels of ligands, and decreased levels of receptor inhibitors. Partially explaining the cholinergic up-regulation seen in SCC, incubation of the H520 SCC cell line with nicotine increased levels of ACh secretion, increased expression of nAChR, and, as measured by electrophysiologic recording, increased activity of the expressed nAChR. Consistent with these effects, nicotine stimulated proliferation of H520 cells. One approach to blocking proliferative effects of nicotine and ACh on growth of lung cancers may be through M3 mAChR antagonists, which can limit the activation of mitogen-activated protein kinase that is caused by both nicotinic and muscarinic signaling. This was tested with the M3-selective muscarinic antagonist darifenacin. Darifenacin blocked nicotine-stimulated H520 growth in vitro and also blocked H520 growth in nude mice in vivo. Thus, cholinergic signaling is broadly up-regulated in SCC and blocking cholinergic signaling can limit basal and nicotine-stimulated growth of SCC.

Characterization of slowly inactivating KV{alpha} current in rabbit pulmonary neuroepithelial bodies: effects of hypoxia and nicotine.

Pulmonary neuroepithelial bodies (NEB) form innervated cell clusters that express voltage-activated currents and function as airway O(2) sensors. We investigated A-type K(+) currents in NEB cells using neonatal rabbit lung slice preparation. The whole cell K(+) current was slowly inactivating with activation threshold of approximately -30 mV. This current was blocked approximately 27% by blood-depressing substance I (BDS-I; 3 microM), a selective blocker of Kv3.4 subunit, and reduced approximately 20% by tetraethylammonium (TEA; 100 microM). The BDS-I-sensitive component had an average peak value of 189 +/- 14 pA and showed fast inactivation kinetics that could be fitted by one-component exponential function with a time constant of (tau1) 77 +/- 10 ms. This Kv slowly inactivating current was also blocked by heteropodatoxin-2 (HpTx-2; 0.2 microM), a blocker of Kv4 subunit. The HpTx-2-sensitive current had an average peak value of 234 +/- 23 pA with a time constant (tau) 82 +/- 11 ms. Hypoxia (Po(2) = 15-20 mmHg) inhibited the slowly inactivating K(+) current by approximately 47%, during voltage steps from -30 to +30 mV, and no further inhibition occurred when TEA was combined with hypoxia. Nicotine at concentrations of 50 and 100 microM suppressed the slowly inactivating K(+) current by approximately 24 and approximately 40%, respectively. This suppression was not reversed by mecamylamine suggesting a direct effect of nicotine on these K(+) channels. In situ hybridization experiments detected expression of mRNAs for Kv3.4 and Kv4.3 subunits, while double-label immunofluorescence confirmed membrane localization of respective channel proteins in NEB cells. These studies suggest that the hypoxia-sensitive current in NEB cells is carried by slowly inactivating A-type K(+) channels, which underlie their oxygen-sensitive potassium currents, and that exposure to nicotine may directly affect their function, contributing to smoking-related lung disease.

Expression of functional purinergic receptors in pulmonary neuroepithelial bodies and their role in hypoxia chemotransmission.

Adenine nucleotides act through specific cell surface receptors to invoke a variety of biological responses. Here we show that cells of neuroepithelial bodies (NEB), presumed O2 airway sensors in neonatal hamster lung, express functional P2X receptors (P2X-R). Positive immunostaining was detected in NEB cells using double-label immunohistochemistry with antibodies against P2X2 and P2X3 receptor subunits, which co-localized with serotonin (5-HT), a marker of NEB cells. For electrophysiological characterization of P2X2-R in NEB cells, fresh neonatal hamster lung slice preparation was used. Under whole-cell patch clamp, perfusion with ATP induced a concentration-dependent, non-desensitizing inward current (EC50=12 microM). Perfusion with alpha,beta-methylene ATP also induced a slow-desensitizing inward current (EC50=8.2 microM). Suramin (IC50 ca. 43 microM) and TNP-ATP (IC50 ca. 8 microM) blocked the currents evoked by both ATP and alpha,beta-methylene ATP. Using carbon fiber amperometry we observed that hypoxia and ATP induced 5-HT release from NEB cells and that this release was blocked by suramin. These data suggest that functional P2X2/3 heteromeric receptors are expressed in NEB cells. The possible function of these purinoreceptors in NEB cells could include modulation of hypoxia chemotransmission.

Expression of functional nicotinic acetylcholine receptors in neuroepithelial bodies of neonatal hamster lung.

Pulmonary neuroepithelial bodies (NEB) are presumed airway chemoreceptors involved in respiratory control, especially in the neonate. Nicotine is known to affect both lung development and control of breathing. We report expression of functional nicotinic acetylcholine receptors (nAChR) in NEB cells of neonatal hamster lung using a combination of morphological and electrophysiological techniques. Nonisotopic in situ hybridization method was used to localize mRNA for the beta 2-subunit of nAChR in NEB cells. Double-label immunofluorescence confirmed expression of alpha 4-, alpha 7-, and beta 2-subunits of nAChR in NEB cells. The electrophysiological characteristics of nAChR in NEB cells were studied using the whole cell patch-clamp technique on fresh lung slices. Application of nicotine ( approximately 0.1-100 microM) evoked inward currents that were concentration dependent (EC50 = 3.8 microM; Hill coefficient = 1.1). ACh (100 microM) and nicotine (50 microM) produced two types of currents. In most NEB cells, nicotine-induced currents had a single desensitizing component that was blocked by mecamylamine (50 microM) and dihydro-beta-erythroidine (50 microM). In some NEB cells, nicotine-induced current had two components, with fast- and slow-desensitizing kinetics. The fast component was selectively blocked by methyllcaconitine (MLA, 10 nM), whereas both components were inhibited by mecamylamine. Choline (0.5 mM) also induced an inward current that was abolished by 10 nM MLA. These studies suggest that NEB cells in neonatal hamster lung express functional heteromeric alpha 3 beta 2, alpha 4 beta 2, and alpha 7 nAChR and that cholinergic mechanisms could modulate NEB chemoreceptor function under normal and pathological conditions.