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Exposure to aerosolized citric acid (CA, 150 mM) and prostaglandin E

The interaction between Chronic lymphocytic leukemia (CLL) cells and monocyte-derived nurse-like cells (NLCs) is fundamentally important to CLL biology. However, studies of how CLL cells and NLCs interact have been hampered by the need for freshly obtained CLL blood samples, coupled with wide variation in the number of monocytes present in the blood of individual patients. Here, we report the development and validation of a cell-line model of NLCs which overcomes these difficulties. Co-culture of primary CLL cells with THP-1 cells induced to differentiate into macrophages by phorbol 12-myristate 13-acetate (PMA) significantly reduced both spontaneous and fludarabine-induced cell death of leukemic cells. Furthermore, compared with their M1-polarized counterparts, M2-polarized macrophages derived from PMA-differentiated THP-1 cells conferred to CLL cells greater protection from spontaneous and fludarabine-induced apoptosis. Since NLCs resemble M2 tumor-associated macrophages, this cell-line model could be useful for investigating the mechanisms through which NLCs protect CLL cells from spontaneous and drug-induced apoptosis.

Liquiritin apioside (LA), a main flavonoid component of licorice, reportedly suppresses cough responses to inhalation of aerosolized capsaicin [CAP; a stimulant to transient receptor potential vanilloid 1 (TRPV1)] in conscious guinea pigs via acting on peripheral nerves. However, the evidence of LA having a direct effect on airway sensory fibers is lacking. Considering the important role laryngeal chemoreceptors and mechanoreceptors play in triggering apnea and cough, we studied whether LA suppressed the apneic responses to stimulation of these receptors via directly acting on the superior laryngeal nerve (SLN). Intralaryngeal delivery of chemical [CAP, HCl, and distilled water (DW)] and mechanical [an air-pulse (AP)] stimulations was applied in anesthetized rat pups to evoke the apnea. These stimuli were repeated after intralaryngeal LA treatment or peri-SLN LA treatment to determine the direct effect of LA on the SLN. Our results showed that all stimuli triggered an immediate apnea. Intralaryngeal LA treatment significantly attenuated the apneic response to chemical but not mechanical stimulations. The same attenuation was observed after peri-SLN LA treatment. Owing that TRPV1 receptors of laryngeal C fibers are responsible for the CAP-triggered apneas, the LA impact on the activity of laryngeal C neurons retrogradely traced by DiI was subsequently studied using a patch-clamp approach. LA pretreatment significantly altered the electrophysiological kinetics of CAP-induced currents in laryngeal C neurons by reducing their amplitudes, increasing the rise times, and prolonging the decay times. In conclusion, our results, for the first time, reveal that LA suppresses the laryngeal chemoreceptor-mediated apnea by directly acting on the SLN (TRPV1 receptors of laryngeal C fibers).

Systemic 8-OH-DPAT (a 5-HT

Aerosolized adenosine 5′-triphosphate (ATP) induces cough and bronchoconstriction by activating vagal sensory fibers’ P2X3 and P2X2/3 receptors (P2X3R and P2X2/3R). The goal of this study is to determine the effect of these receptors on the superior laryngeal nerve (SLN)-mediated cardiorespiratory responses to ATP challenge. We compared the cardiorespiratory responses to intralaryngeal perfusion of either ATP or α,β-methylene ATP in rat pups before and after 1) intralaryngeal perfusion of A-317491 (a P2X3R and P2X2/3R antagonist); 2) bilateral section of the SLN; and 3) peri-SLN treatment with capsaicin (to block conduction in superior laryngeal C-fibers, SLCFs) or A-317491. The immunoreactivity (IR) of P2X3R and P2X2R was determined in laryngeal sensory neurons of the nodose/jugular ganglia. Lastly, a whole cell patch clamp recording was used to determine ATP- or α,β-methylene ATP (α,β-mATP)-induced currents without and with A-317491 treatment. It was found that intralaryngeal perfusion of both ATP and α,β-mATP induced immediate apnea, hypertension, and bradycardia. The apnea was eliminated and the hypertension and bradycardia were blunted by intralaryngeal perfusion of A-317491 and peri-SLN treatment with either A-317491 or capsaicin, although all of the cardiorespiratory responses were abolished by bilateral section of the SLN. P2X3R- and P2X2R-IR were observed in nodose and jugular ganglionic neurons labeled by fluoro-gold (FG). ATP- and α,β-mATP-induced currents recorded in laryngeal C-neurons were reduced by 75% and 95%, respectively, by the application of A-317491. It is concluded that in anesthetized rat pups, the cardiorespiratory responses to intralaryngeal perfusion of either ATP or α,β-mATP are largely mediated by the activation of SLCFs’ P2X3R-P2X2/3R. NEW & NOTEWORTHY Aerosolized ATP induces cough and bronchoconstriction via activating P2X3 and P2X2/3 receptors (P2X3R and P2X2/3R) localized on vagal pulmonary sensory fibers. The superior laryngeal nerve (SLN), particularly SLN C-fibers (SLCFs), is involved in generating apnea, hypertension, and bradycardia. This study demonstrates for the first time that either ATP or α,β-mATP applied onto the laryngeal mucosa elicit these cardiorespiratory responses predominately through the activation of P2X3R-P2X2/3R localized on SLCFs.

Prenatal nicotinic exposure (PNE) reportedly sensitizes bronchopulmonary C-fibers (PCFs) and prolongs PCF-mediated apnea in rat pups, contributing to the pathogenesis of sudden infant death syndrome. Serotonin, or 5-hydroxytryptamine (5-HT), induces apnea via acting on 5-HT receptor 3 (5-HT3R) in PCFs, and among the 5-HT3R subunits, 5-HT3B is responsible for shortening the decay time of 5-HT3R-mediated currents. We examined whether PNE would promote pulmonary 5-HT secretion and prolong the apnea mediated by 5-HT3Rs in PCFs via affecting the 5-HT3B subunit. To this end, the following variables were compared between the control and PNE rat pups: 1) the 5-HT content in bronchoalveolar lavage fluid, 2) the apneic response to the right atrial bolus injection of phenylbiguanide (a 5-HT3R agonist) before and after PCF inactivation, 3) 5-HT3R currents and the stimulus threshold of the action currents of vagal pulmonary C-neurons, and 4) the immunoreactivity (IR) and mRNA expression of 5-HT3A and 5-HT3B in these neurons. Our results showed that PNE up-regulated the pulmonary 5-HT concentration and strengthened the PCF 5-HT3R-mediated apnea. PNE significantly facilitated neural excitability by shortening the decay time of 5-HT3R currents, lowering the stimulus threshold, and increasing 5-HT3B IR. In summary, PNE prolongs the apnea mediated by 5-HT3Rs in PCFs, likely by increasing 5-HT3B subunits to enhance the excitability of 5-HT3 channels.-Zhao, L., Gao, X., Zhuang, J., Wallen, M., Leng, S., Xu, F. Prolongation of bronchopulmonary C-fiber-mediated apnea by prenatal nicotinic exposure in rat pups: role of 5-HT3 receptors.

Chronic lymphocytic leukaemia (CLL) exhibits variable clinical course and response to therapy, but the molecular basis of this variability remains incompletely understood. Data independent acquisition (DIA)-MS technologies, such as SWATH (Sequential Windowed Acquisition of all THeoretical fragments), provide an opportunity to study the pathophysiology of CLL at the proteome level. Here, a CLL-specific spectral library (7736 proteins) is described alongside an analysis of sample replication and data handling requirements for quantitative SWATH-MS analysis of clinical samples. The analysis was performed on 6 CLL samples, incorporating biological (IGHV mutational status), sample preparation and MS technical replicates. Quantitative information was obtained for 5169 proteins across 54 SWATH-MS acquisitions: the sources of variation and different computational approaches for batch correction were assessed. Functional enrichment analysis of proteins associated with IGHV mutational status showed significant overlap with previous studies based on gene expression profiling. Finally, an approach to perform statistical power analysis in proteomics studies was implemented. This study provides a valuable resource for researchers working on the proteomics of CLL. It also establishes a sound framework for the design of sufficiently powered clinical proteomics studies. Indeed, this study shows that it is possible to derive biologically plausible hypotheses from a relatively small dataset.

Prostaglandin E2(PGE2)-induced coughsin vivoand vagal nerve depolarizationin vitroare inhibited by systemic and local administration of prostaglandin EP3 receptor (L-798106) and TRPV1 antagonists (JNJ 17203212). These results indicate a modulating effect of TRPV1 on the EP3 receptor-mediated cough responses to PGE2likely through the vagal sensory nerve. This study aimed to determine whether 1) inhalation of aerosolized JNJ 17203212 and L-798106 affected cough responses to citric acid (CA, mainly stimulating TRPV1) and PGE2; 2) TRPV1 and EP3 receptor morphologically are co-expressed and electrophysiologically functioned in the individual of vagal pulmonary C-neurons (cell bodies of bronchopulmonary C-fibers in the nodose/jugular ganglia); and 3) there was a cross-effect of TRPV1 and EP3 receptor on these neural excitations. To this end, aerosolized CA or PGE2was inhaled by unanesthetized guinea pigs pretreated without or with each antagonist given in aerosol form. Immunofluorescence was applied to identify the co-expression of TRPV1 and EP3 receptor in vagal pulmonary C-neurons (retrogradely traced by DiI). Whole-cell voltage patch clamp approach was used to detect capsaicin (CAP)- and PGE2-induced currents in individual vagal pulmonary C-neurons and determine the effects of the TRPV1 and EP3 receptor antagonists on the evoked currents. We found that PGE2-induced cough was attenuated by JNJ 17203212 or L-798106 and CA-evoked cough greatly suppressed only by JNJ 17203212. Approximately 1/4 of vagal pulmonary C-neurons co-expressed EP3 with a cell size < 20 μm. Both CAP- and PGE2-induced currents could be recorded in the individuals of some vagal pulmonary C-neurons. The former was largely inhibited only by JNJ 17203212, while the latter was suppressed by JNJ 17203212 or L-798106. The similarity of the cross-effect of both antagonists on cough and vagal pulmonary C-neural activity suggests that a subgroup of vagal pulmonary C-neurons co-expressing TRPV1 and EP3 receptor is, at least in part, responsible for the cough response to PGE2.

Systemic 8-OH-DPAT (a 5-HT1A receptor agonist) challenge evokes hyperventilation independent of peripheral 5-HT1A receptors. Though the pre-Botzinger Complex (PBC) is critical in generating respiratory rhythm and activation of local 5-HT1A receptors induces tachypnea via disinhibition of local GABAA neurons, its role in the respiratory response to systemic 8-OH-DPAT challenge is still unclear. In anesthetized rats, 8-OH-DPAT (100 μg/kg, iv) was injected twice to confirm the reproducibility of the evoked responses. The same challenges were performed after bilateral microinjections of (S)-WAY-100135 (a 5-HT1A receptor antagonist) or gabazine (a GABAA receptor antagonist) into the PBC. Our results showed that: 1) 8-OH-DPAT caused reproducible hyperventilation associated with hypotension and bradycardia; 2) microinjections of (S)-WAY-100135 into the PBC attenuated the hyperventilation by ˜60 % without effect on the evoked hypotension and bradycardia; and 3) the same hyperventilatory attenuation was also observed after microinjections of gabazine into the PBC. Our data suggest that PBC 5-HT1A receptors play a key role in the respiratory response to systemic 8-OH-DPAT challenge likely via disinhibiting local GABAergic neurons.

Prostaglandin E2 (PGE2)-induced coughs in vivo and vagal nerve depolarization in vitro are inhibited by systemic and local administration of TRPV1 (JNJ 17203212) and prostaglandin EP3 receptor antagonists (L-798106) respectively. These results indicate an interaction of TRPV1 and EP3 receptor in cough responses to PGE2 likely through the vagal sensory nerve. This study aimed to determine whether 1) inhalation of aerosolized JNJ 17203212 and L-798106 affected cough responses to citric acid (CA to mainly stimulate TRPV1) and PGE2; 2) TRPV1 and EP3 receptor morphologically co-expressed and electrophysiologically functioned in the individual of vagal pulmonary C-neurons (cell bodies of bronchopulmonary C-fibers in the nodose/jugular ganglia); and 3) the interaction of the two receptors occurred in these neurons. To this end, aerosolized CA or PGE2 was inhaled by unanesthetized guinea pigs pretreated without or with JNJ 17203212 or L-798106 given in aerosol. Immunofluorescence was applied to identify the expression of the two receptors in vagal pulmonary C-neurons (retrogradely traced by DiI). Whole-cell patch clamp approach was used to detect capsaicin (CAP)- and PGE2-induced currents in the same neurons and determine the effects of the TRPV1 and EP3 receptor antagonists on the currents. We found that PGE2-evoked cough was attenuated by JNJ 17203212 or L-798106 and CA-evoked cough greatly suppressed only by JNJ 17203212. EP3-labeled neurons always co-expressed TRPV1 with a cell size 2-induced currents could be recorded in the individuals of some vagal pulmonary C-neurons. The former was largely inhibited only by JNJ 17203212, while the latter was suppressed by JNJ 17203212 or L-798106. The similarity of the interaction in cough and vagal pulmonary C-neural activity suggests that a subgroup of vagal pulmonary C-neurons co-expressing TRPV1 and EP3 receptor is, at least in part, responsible for the interaction of the two receptors in the cough response to PGE2.

The functional significance of AKT in chronic lymphocytic leukemia (CLL) remains unclear. Given the importance of non-malignant T cells in regulating clonal expansion in CLL, we investigated the role of AKT in T cell-mediated cytoprotection and proliferation using an established co-culture system in which primary CLL cells were incubated on a monolayer of transfected mouse fibroblasts expressing human CD40L (CD154). Stimulation of CLL cells via CD40 induced activation of AKT, which was closely associated with downregulation of its negative regulator PTEN, and protected CLL cells from killing by bendamustine. This cytoprotective effect of CD40 stimulation was prevented by a selective inhibitor of AKT. Stimulation of CLL cells with CD154 + IL-4 or IL-21 induced proliferation detected as reduced fluorescence of cells pre-stained with CFSE. AKT inhibition produced a significant, consistent reduction in proliferation induced by CD154 + IL-4 and a reduction in proliferation induced by CD154 + IL-21 in most but not all cases. In contrast, AKT inhibition had no effect on the proliferation of normal B cells induced by CD154 + IL-4 or IL-21. These findings indicate that AKT contributes in a significant way to T-cell mediated survival and proliferation signalling in CLL and support the clinical evaluation of AKT inhibitors in this disease.

Maternal cigarette smoke, including prenatal nicotinic exposure (PNE), is responsible for sudden infant death syndrome (SIDS). The fatal events of SIDS are characterized by severe bradycardia and life-threatening apneas. Although activation of transient receptor potential vanilloid 1 (TRPV1) of superior laryngeal C fibers (SLCFs) could induce bradycardia and apnea and has been implicated in SIDS pathogenesis, how PNE affects the SLCF-mediated cardiorespiratory responses remains unexplored. Here, we tested the hypothesis that PNE would aggravate the SLCF-mediated apnea and bradycardia via up-regulating TRPV1 expression and excitation of laryngeal C neurons in the nodose/jugular (N/J) ganglia. To this end, we compared the following outcomes between control and PNE rat pups at postnatal days 11-14: 1) the cardiorespiratory responses to intralaryngeal application of capsaicin (10 µg/ml, 50 µl), a selective stimulant for TRPV1 receptors, in anesthetized preparation; 2) immunoreactivity and mRNA of TRPV1 receptors of laryngeal sensory C neurons in the N/J ganglia retrogradely traced by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; and 3) TRPV1 currents and electrophysiological characteristics of these neurons by using whole-cell patch-clamp technique in vitro Our results showed that PNE markedly prolonged the apneic response and exacerbated the bradycardic response to intralaryngeal perfusion of capsaicin, which was associated with up-regulation of TRPV1 expression in laryngeal C neurons. In addition, PNE increased the TRPV1 currents, depressed the slow delayed rectifier potassium currents, and increased the resting membrane potential of these neurons. Our results suggest that PNE is capable of aggravating the SLCF-mediated apnea and bradycardia through TRPV1 sensitization and neuronal excitation, which may contribute to the pathogenesis of SIDS.-Gao, X., Zhao, L., Zhuang, J., Zang, N., Xu, F. Prenatal nicotinic exposure prolongs superior laryngeal C-fiber-mediated apnea and bradycardia through enhancing neuronal TRPV1 expression and excitation.

This article describes the preclinical pharmacology and pharmacokinetics (PK) of hexadecyl-treprostinil (C16TR), a prodrug of treprostinil (TRE), formulated in a lipid nanoparticle (LNP) for inhalation as a pulmonary vasodilator. C16TR showed no activity (>10 µM) in receptor binding and enzyme inhibition assays, including binding to prostaglandin E2 receptor 2, prostaglandin D2 receptor 1, prostaglandin I2 receptor, and prostaglandin E2 receptor 4; TRE potently bound to each of these prostanoid receptors. C16TR had no effect (up to 200 nM) on platelet aggregation induced by ADP in rat blood. In hypoxia-challenged rats, inhaled C16TR-LNP produced dose-dependent (0.06-6 µg/kg), sustained pulmonary vasodilation over 3 hours; inhaled TRE (6 µg/kg) was active at earlier times but lost its effect by 3 hours. Single- and multiple-dose PK studies of inhaled C16TR-LNP in rats showed proportionate dose-dependent increases in TRE Cmax and area under the curve (AUC) for both plasma and lung; similar results were observed for dog plasma levels in single-dose PK studies. In both species, inhaled C16TR-LNP yielded prolonged plasma TRE levels and a lower plasma TRE Cmax compared with inhaled TRE. Inhaled C16TR-LNP was well tolerated in rats and dogs; TRE-related side effects included cough, respiratory tract irritation, and emesis and were seen only after high inhaled doses of C16TR-LNP in dogs. In guinea pigs, inhaled TRE (30 µg/ml) consistently produced cough, but C16TR-LNP (30 µg/ml) elicited no effect. These results demonstrate that C16TR-LNP provides long-acting pulmonary vasodilation, is well tolerated in animal studies, and may necessitate less frequent dosing than inhaled TRE with possibly fewer side effects.

We tested the hypothesis that mu-opioid receptors (MORs) in the caudomedial nucleus tractus solitarius (cmNTS) are important for the ventilatory responses to stimulation of bronchopulmonary C-fibers (PCFs), the carotid body-mediated hypoxia, and hypercapnia independent of the carotid body. First, we used immunohistochemistry to map MORs distribution in the caudal medulla. Then we compared the effects of intra-cmNTS microinjection of DAMGO (a MOR agonist) with or without a combination of CTAP (a MOR antagonist) on the ventilatory responses to: 1) right atrial injection of capsaicin (to stimulation of PCFs) and 2) acute hypoxia (HVR, to stimulate the carotid body) in awake intact rats; and 3) hypercapnia (HCVR) in the carotid body ablated rats. The cmNTS presented the highest MORs in the caudal medulla. Microinjection of DAMGO blocked the PCF-mediated apnea, attenuated HVR (70%) and HCVR (21%), while microinjection of CTAP+DAMGO failed to affect these chemoreflexes. Our data demonstrate a critical role of activation of cmNTS MORs in regulating these chemoreflexes and imply a presence of MORs in the synapse of the 2nd-order neurons receiving inputs from PCFs and the carotid body, and NTS chemosensitive neurons.

Prenatal nicotinic exposure (PNE) reportedly sensitizes bronchopulmonary C-fibers (PCFs) and prolongs PCF-mediated apnea in rat pups, contributing to the pathogenesis of sudden infant death syndrome. Serotonin, or 5-hydroxytryptamine (5-HT), induces apnea via acting on 5-HT receptor 3 (5-HT(3)R) in PCFs, and among the 5-HT(3)R subunits, 5-HT(3B) is responsible for shortening the decay time of 5-HT(3)R–mediated currents. We examined whether PNE would promote pulmonary 5-HT secretion and prolong the apnea mediated by 5-HT(3)Rs in PCFs via affecting the 5-HT(3B) subunit. To this end, the following variables were compared between the control and PNE rat pups: 1) the 5-HT content in bronchoalveolar lavage fluid, 2) the apneic response to the right atrial bolus injection of phenylbiguanide (a 5-HT(3)R agonist) before and after PCF inactivation, 3) 5-HT(3)R currents and the stimulus threshold of the action currents of vagal pulmonary C-neurons, and 4) the immunoreactivity (IR) and mRNA expression of 5-HT(3A) and 5-HT(3B) in these neurons. Our results showed that PNE up-regulated the pulmonary 5-HT concentration and strengthened the PCF 5-HT(3)R–mediated apnea. PNE significantly facilitated neural excitability by shortening the decay time of 5-HT(3)R currents, lowering the stimulus threshold, and increasing 5-HT(3B) IR. In summary, PNE prolongs the apnea mediated by 5-HT(3)Rs in PCFs, likely by increasing 5-HT(3B) subunits to enhance the excitability of 5-HT(3) channels.—Zhao, L., Gao, X., Zhuang, J., Wallen, M., Leng, S., Xu, F. Prolongation of bronchopulmonary C-fiber–mediated apnea by prenatal nicotinic exposure in rat pups: role of 5-HT(3) receptors.

The highly pathogenic H5N1 (HK483) viral infection causes a depressed hypercapnic ventilatory response (dHCVR, 20%↓) at 2 days postinfection (dpi) and death at 7 dpi in mice, but the relevant mechanisms are not fully understood. Glomus cells in the carotid body and catecholaminergic neurons in locus coeruleus (LC), neurokinin 1 receptor (NK1R)-expressing neurons in the retrotrapezoid nucleus (RTN), and serotonergic neurons in the raphe are chemosensitive and responsible for HCVR. We asked whether the dHCVR became worse over the infection period with viral replication in these cells/neurons. Mice intranasally inoculated with saline or the HK483 virus were exposed to hypercapnia for 5 min at 0, 2, 4, or 6 dpi, followed by immunohistochemistry to determine the expression of nucleoprotein of H5N1 influenza A (NP) alone and coupled with 1) tyrosine hydroxylase (TH) in the carotid body and LC, 2) NK1R in the RTN, and 3) tryptophan hydroxylase (TPH) in the raphe. HK483 viral infection blunted HCVR by ∼20, 50, and 65% at 2, 4, and 6 dpi. The NP was observed in the pontomedullary respiratory-related nuclei (but not in the carotid body) at 4 and 6 dpi, especially in 20% of RTN NK1R, 35% of LC TH, and ∼10% raphe TPH neurons. The infection significantly reduced the local NK1R or TPH immunoreactivity and population of neurons expressing NK1R or TPH. We conclude that the HK483 virus infects the pontomedullary respiratory nuclei, particularly chemosensitive neurons in the RTN, LC, and raphe, contributing to the severe depression of HCVR and respiratory failure at 6 dpi.

Supplemental material, DT_paper_suppl_2 for DT-0111: a novel drug-candidate for the treatment of COPD and chronic cough by Amir Pelleg, Fadi Xu, Jianguo Zhuang, Bradley Undem and Geoffrey Burnstock in Therapeutic Advances in Respiratory Disease

Supplemental material, Reviewer_2_v.1 for DT-0111: a novel drug-candidate for the treatment of COPD and chronic cough by Amir Pelleg, Fadi Xu, Jianguo Zhuang, Bradley Undem and Geoffrey Burnstock in Therapeutic Advances in Respiratory Disease

Prenatal nicotinic exposure (PNE) as a SIDS model reportedly sensitizes bronchopulmonary C-fibers (PCFs), contributing to the prolonged PCF-mediated apnea in rat pups, but the relevant mechanisms are not fully understood. Pulmonary IL-1β upregulated by cigarette smoke is known to stimulate or sensitize PCFs acting via IL-1 type I receptor (IL1RI) and inhibit inspiration frequency. Because of its upregulation observed in SIDS victims, we hypothesized that PNE increased pulmonary IL-1β release and IL1RI expression in pulmonary C-neurons via action on α7 nicotinic acetylcholine receptors (α7nAChR) to induce the prolonged PCF-mediated apnea. IL-1β in BALF and IL1RI in the nodose/jugular (N/J) ganglion and vagal pulmonary C-neurons retrogradely-traced were compared between Ctrl (saline) and PNE pups and among the vehicle-treated Ctrl and PNE and methyllycaconitine (a selective α7nAChR antagonist)-treated PNE pups. The effect of IL-1RI blockade (IL-1Ra) on the PCF-mediated apnea was also compared between Ctrl and PNE pups. PNE significantly elevated IL-1β in BALF and upregulated IL1RI gene and protein expression in N/J ganglia and gene in vagal pulmonary C-neurons. All of these responses were eliminated by pretreatment with blockade of α7nAChR. In addition, the prolonged PCF-mediated apnea in PNE pups was significantly shortened by right atrial bolus injection of IL-1Ra. We conclude that PNE enhances pulmonary IL-1β release and PCF IL1RI expression acting via α7nAChR in contributing to sensitization of PCFs and prolongation of the PCF-mediated apneic response.

Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA1 receptor, ADA1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA1R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to change BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChRs or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin.