Mouse Lung Slices Research Articles

Functional expression of the extracellular Ca2+‐sensing receptor in pulmonary neuroepithelial bodies

The extracellular Ca2+‐sensing receptor (CaR) is a multimodal sensor that allows cells to detect and respond to changes in the extracellular Ca2+ concentration [Ca2+]o. Besides its role in Ca2+ homeostasis, the CaR has been shown in various specialized cells, suggesting its involvement in multiple cellular functions. Prenatal CaR expression was detected in mouse airway epithelium between E11,5 and E16,5 and was shown to play a role in airway branching morphogenesis. In postnatal lung tissue, the CaR so far has not been reported. Neuroepithelial bodies (NEBs) are densely innervated groups of neuroendocrine airway epithelial cells, believed to represent complex sensory receptors that are ideally placed to detect changes in the microenvironment. Immunostaining of postnatal mouse lungs showed a selective CaR expression on the basolateral membrane of NEB cells. Confocal Ca2+ imaging of NEBs in live mouse lung slices revealed that NEBs respond with a Ca2+ rise both to changes in [Ca2+]o and to various known CaR agonists (spermine, neomycin, calcimimetic R‐568). All responses were blocked by Calhex, a calcilytic CaR antagonist. The presented expression and proven functionality of the CaR in postnatal NEBs offer interesting perspectives for further unraveling NEB cell physiology.Support: IWT fellowship SB/81162 (RL); FWO grants G.0081.08 (DA) and G.0589.11N (DA, JPT); UA grants GOA BOF 2007 (DA) and KP BOF 2006 (IB)

Intestinal, Airway, and Cardiovascular Relaxant Activities of Thymoquinone

Thymoquinone (TQ) is a bioactive component found in many medicinal herbs. In this study, we report the smooth and cardiac muscle relaxant activities of this compound. TQ concentration dependently suppressed spontaneously contracting rabbit jejunum while also relaxed high K+-(80 mM) induced contractions in jejunum and guinea-pig ileum, indicating activity at voltage-operated Ca++ channels (VOCC). Further, TQ displaced Ca++ concentration-response curves, obtained in a Ca++-free environment, to the right, showing blockade of VOCC. Similar activity was observed with verapamil, a standard VOCC blocker. TQ also exhibited nonadrenergic relaxation of agonist-induced contractions in guinea-pig trachea. When tested in fluo-4-loaded mouse lung slices, TQ inhibited ACh-induced airway narrowing and Ca++ signalling in airway smooth muscle cells. In endothelium-intact and endothelium-denuded rat aorta, TQ inhibited high K+-induced contractions at significantly lower concentrations than phenylephrine-(PE-) (1 microM) induced contractions. Relaxation of PE-induced contractions was resistant to blockade by L-NAME and atropine. In guinea-pig atria, TQ showed noncholinergic relaxation of atrial force and rate of contractions. These data suggest smooth and cardiac muscle relaxant activity of TQ possibly mediated, in part, via blockade of VOCC. The results also justify the use of TQ containing plants in related health disorders like colic, diarrhoea, cough, and asthma.

Role of CPI‐17 phosphorylation in calcium sensitization in smooth muscle of small intrapulmonary airways

Phosphorylation of protein‐kinase C (PKC)‐potentiated myosin phosphatase inhibitor (CPI‐17) plays a role in Ca2+ sensitization in different smooth muscle cell (SMC) types; however its role in small intrapulmonary airways is mostly unknown. We tested the hypothesis that Ca2+ sensitization is accompanied by CPI‐17 phosphorylation. We used phase‐contrast microscopy and Western blots to assess airway contraction, Ca2+ sensitivity and protein phosphorylation in mouse lung slices. We found that stimulation of lung slices with PKC activator phorbol‐12‐myristate‐13‐acetate (PMA, 10 μM) induced strong airway contraction in Ca2+ permeabilized airways due to Ca2+ sensitization. This contraction was reverted by addition of a selective PKC inhibitor (GF109203X, 1 μM) but not by a ROK inhibitor (Y‐27632, 10 μM). Phosphorylation of CPI‐17 increased after stimulation with PMA and CPI‐17 dephosphorylation was observed after subsequent addition of GF109203X. Furthermore, acetylcholine (ACh, 0.5 μM) and serotonin (5‐HT, 0.5 μM) induced strong airway contraction in Ca2+ permeabilized airways which was resistant to GF109203X and blocked by Y‐27632. Phosphorylation of CPI‐17 did not increase significantly after ACh or 5‐HT stimulation. Our results suggest that CPI‐17 phosphorylation is an important mechanism of Ca2+ sensitization but 5‐HT and ACh do not use this pathway in airway SMCs.

Gβγ‐activated phosphoinositide 3‐kinase γ regulates airway smooth muscle contraction by modulating calcium oscillations

Airway smooth muscle (ASM) contractile hyper‐responsiveness plays a critical role in asthma. Muscarinic receptors in ASM convey bronchoconstrictor signals by dissociating G‐proteins into active Gα and Gβγ subunits. We found that expression of a Gβγ scavenger peptide suppressed acetylcholine (ACh)‐stimulated mouse ASM cell contraction. Gβγ plays prominent roles in signal transduction through its downstream effectors including PI3Kγ. Immunohistochemistry staining and Western blot indicated expression of PI3Kγ in ASM cells of mouse trachea, lung and cultured ASM cells. Inhibitors of PI3Kγ, but not PI3Kα, β, or δ, attenuated ACh‐stimulated airway contraction of mouse lung slices. It is noteworthy that airways in lung slices pretreated with PI3Kγ inhibitors still exhibited an ACh‐induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, PI3Kγ inhibitors or silence of endogenous PI3Kγ by siRNA had a small inhibitory effect on the ACh‐stimulated initial Ca2+ transient in mouse ASM cells but significantly attenuated the sustained Ca2+ oscillations that are critical for sustained airway contraction. Thus, Gβγ/PI3Kγ signaling pathway controls contractility of airways through regulation of Ca2+ oscillations in ASM cells. Targeting this pathway may directly impact airway hyper‐responsiveness associated with asthma. Supported by Nebraska State LB595 and 692.

Phosphoinositide 3-Kinase γ Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations

Phosphoinositide 3-kinase gamma (PI3Kgamma) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kgamma can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kgamma protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kgamma inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 muM. In contrast, inhibitors of PI3Kalpha, PI3Kbeta, or PI3Kdelta, at concentrations 40-fold higher than their reported IC(50) values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kgamma inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kgamma-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca(2+) transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca(2+) oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kgamma directly controls contractility of airways through regulation of Ca(2+) oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kgamma inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.

Regulation of vascular smooth muscle contraction by Ca2+ signaling and Ca2+ sensitivity in intrapulmonary arteries

The contraction of vascular smooth muscle cells (SMCs) of small pulmonary arteries is critical for the regulation of pulmonary vascular resistance and plays an important role in the development of pulmonary hypertension. We have studied the regulation of SMC contraction by Ca2+ and Ca2+ sensitivity in intrapulmonary arteries by measuring artery contraction and Ca2+ signaling in mouse lung slices with phase‐contrast and confocal microscopy. The agonists serotonin (5‐HT) and endothelin (ET) induced artery contraction and stimulated Ca2+ oscillations in their SMCs. Conversely, nitric oxide (NO) caused relaxation and decreased the frequency of Ca2+ oscillations in arteries pre‐stimulated with agonists. Ca2+ permeabilization with caffeine and ryanodine caused artery contraction but agonists stimulate further contraction. Activation of PKC with phorbol 12‐myristate 13‐acetate (PMA, 1–10 μM) induced strong and sustained artery contraction whereas the PKC inhibitor GF‐109203X (1 μM) inhibited the agonists‐induced contraction. In conclusion, our results suggest that the contraction of intrapulmonary arteries is regulated by the frequency of Ca2+ oscillations and a PKC‐dependent increase in Ca2+ sensitivity.

Hypoxia Increases ROS Signaling and Cytosolic Ca2+ in Pulmonary Artery Smooth Muscle Cells of Mouse Lungs Slices

Precapillary arteries constrict during alveolar hypoxia in a response known as hypoxic pulmonary vasoconstriction (HPV). The mechanism by which pulmonary arterial smooth muscle cells (PASMCs) detect a decrease in Po(2) and trigger contraction is not fully understood. Previous studies in cultured PASMCs show that hypoxia induces an increase in reactive oxygen species (ROS) production, but these results may not reflect responses of PASMCs in their native tissue environment. We therefore assessed hypoxia-induced changes in cytosolic ROS in PASMCs of precision-cut mouse lung slices expressing the redox-sensitive protein, RoGFP. Superfusion of lung slices with hypoxic media (1.5% O(2)) resulted in a significant oxidation of RoGFP from normoxic baseline that was attenuated by overexpression of cytosolic catalase. Hypoxic superfusion also increased [Ca(2+)](i) above normoxic baseline; this response was significantly attenuated by cytosolic catalase overexpression or by the administration of EUK134, a synthetic SOD-catalase mimetic. The hypoxia-induced increase in [Ca(2+)](i) was abolished in the absence of extracellular Ca(2+), indicating that ROS signals trigger entry of extracellular calcium. Collectively, these results indicate that an increase in cytosolic ROS signaling is required for the increase in [Ca(2+)](i) in PASMCs in precision-cut mouse lung slices during the acute HPV response.

Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca2+ signaling, and Ca2+ sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca2+]i revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca2+ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca2+ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca2+ oscillations. NOC-5 inhibited the increase of [Ca2+]i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP3) in airway SMCs. The effect of NO on the Ca2+ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca2+ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca2+-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP–PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca2+ via the IP3 receptor to decrease the frequency of agonist-induced Ca2+ oscillations.

Effects of Formoterol on Contraction and Ca2+ Signaling of Mouse Airway Smooth Muscle Cells

Formoterol, a long-acting beta(2)-receptor agonist, is used to relieve bronchial constriction. However, formoterol is often a racemic formulation, and contains both (R,R)- and (S,S)-enantiomers. Because the activity of each isomer is poorly defined, the mechanisms by which formoterol relaxes smooth muscle cells (SMCs) of intrapulmonary airways are not well understood. Consequently, we compared the effects of (S,S)-, (R,R)-, and racemic formoterol, as well as (R)-albuterol, on the contraction and Ca(2+) signaling of airway SMCs in mouse lung slices with phase-contrast and confocal microscopy. Small airways were contracted with methacholine and the associated SMCs displayed sustained Ca(2+) oscillations and an increase in Ca(2+) sensitivity. These contracted airways displayed a substantial, concentration-dependent relaxation in response to (R,R)-formoterol. Racemic formoterol had a similar potency as (R,R)-formoterol for relaxing airways. By contrast, (S,S)-formoterol only induced a small relaxation. In conjunction with relaxation, (R,R)- and racemic formoterol stopped and decreased the methacholine-induced Ca(2+) oscillations and Ca(2+) sensitivity of the SMCs, respectively, whereas (S,S)-formoterol only decreased the Ca(2+) sensitivity. In these studies, (R,R)- and racemic formoterol had a similar, but much greater, potency than (R)-albuterol for relaxing mice airways. This action was quickly initiated at high concentrations by decreasing the frequency of Ca(2+) oscillations, but was more usually mediated at lower concentrations by decreasing the Ca(2+) sensitivity of the SMCs.

Role of Interleukin‐4 on Agonist Induced Airway Contraction in Mouse Lung Slices

Asthma is a chronic disorder of the airways characterized by inflammation and airway hyper responsiveness (AHR). Interleukin‐4 (IL‐4) is released by T‐cells and mast cells in the airway and is believe to have an important role in asthma. We hypothesize that IL‐4 has a direct effect on the airway smooth muscle cells (SMCs) by increasing their contractile response to agonist such as acetylcholine (ACh). The effect of IL‐4 on airway contraction was studied in mouse lung slices with phase‐contrast microscopy. We found that the treatment of lung slices with 100 ng/ml of IL‐4 for 12 to 24 hours resulted in an increase in contraction of the airways induced by ACh as compared with the same response measured in slices treated without IL‐4 (control). However, IL‐4 treatment did not alter the sensitivity of airways to agonists as determined by the absence of a change in the concentration of ACh that produced 50% of the maximal contraction. These results suggest that IL‐4 contributes to asthma pathology by increasing airway SMC contractility. We are investigating the mechanisms underlying the increase in airway contraction induced by IL‐4 by exploring possible changes in Ca2+ signaling and other Ca2+ independent mechanisms. This study will provide useful information to develop new specific therapeutics to treat asthma.

Nitric Oxide (NO) Induces Airway Smooth Muscle Relaxation by Inhibiting Ca2+ Oscillations

To investigate how nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, we examined airway contraction, Ca2+ signalling, and Ca2+ sensitivity in mouse lung slices in response to agonists and an NO donor, NOC‐5. In airways contracted with 5‐HT or ACh, NOC‐5 induced a concentration‐dependent relaxation consisting of an initial relaxation followed by partial re‐contraction. Inhibition of cGMP‐specific phosphodiesterases (PDE‐5) with zaprinast increased NOC‐5‐induced relaxation. 8‐Br‐cGMP, a PDE‐resistant analog of cGMP, induced sustained airway relaxation. Agonist‐induced airway contraction was mediated by SMC Ca2+ oscillations and airway relaxation correlated with a NOC‐5‐induced reduction in the frequency of these Ca2+ oscillations. NOC‐5 inhibited the Ca2+ oscillations and contraction triggered by the photolytic release of IP3 in SMCs. However, in Ca2+ permeabilized SMCs, NO only induced a small airway relaxation when the [Ca2+]i was clamped at a high level. We conclude that NO relaxes airway SMCs by decreasing the frequency of Ca2+ oscillations by blocking Ca2+ release via IP3 receptors and not by a major change in Ca2+ sensitivity.

Cardiovascular and airway relaxant activities of peony root extract

Paeonia emodi (peony) is a well known plant used medicinally to treat hypertension, palpitations, and asthma. Despite its popularity, there are few reports in the scientific literature examining its use in such conditions. We prepared a 70% ethanolic extract of peony root (Pe.Cr) and applied it to segments of guinea pig atria and trachea and rat aorta suspended separately in tissue baths. Activity against arachidonic acid (AA)-induced platelet aggregation was measured in human platelet-rich plasma. Airway relaxant effect was evaluated against acetylcholine (ACh)-induced airway contraction in mouse lung slices loaded with fluo-4. Pe.Cr (0.3-10 mg/mL) showed an atropine-resistant negative inotropic effect in atria. In rat aorta, an endothelium-independent vasodilatory effect (0.3-10 mg/mL) was seen in phenylephrine- and high-K+-induced contractions. Pe.Cr (0.01-1 mg/mL) also inhibited AA-induced platelet aggregation. In isolated trachea, Pe.Cr (0.3-10 mg/mL) relaxed carbachol- and histamine-induced contractions independently of beta-adrenergic receptors. In mouse lung slices, Pe.Cr (0.3-1 mg/mL) inhibited ACh-induced airway narrowing and oscillations of intracellular Ca2+ in airway smooth muscle cells. The results showed cardiosuppressant, vasodilatory, antiplatelet, and tracheal and airway relaxant activities of peony, providing potential justification for its medicinal use in different hyperactive cardiovascular and respiratory disorders.

Biological tissue imaging with a hybrid cluster SIMS quadrupole time-of-flight mass spectrometer

A 20 keV C 60 + ion source was mounted onto a commercial MALDI/electrospray orthogonal ToF mass spectrometer. Cross-sectional mouse brain and lung slices between 5 and 10 μm prepared by cryostat sectioning were successfully imaged using a DC C 60 + primary ion beam at a spot size of 100 μm. Analysis was performed at room temperature following vacuum drying. An abundance of ions were mapped in all samples, many whose identity can only be found using the MS/MS functionality. We have successfully identified and imaged localizations of diacylglycerol (DAG) ions – 1-palmitoyl-2-oleoyl-glycerol ( m/ z + 577.5) and 1,2-dioleoyl-glycerol ( m/ z + 603.5) – in lung tissue. The mouse brain slice revealed strong, distinct localizations of many ions revealing the potential for this technique for biological imaging. Ions throughout the mass range of m/ z + 50–800 were collected in sufficient quantities to permit unambiguous chemical mapping. Mass resolutions of 12,000 or greater were routinely obtained allowing for more accurate ion mapping than typically seen with ToF-SIMS image analysis.

Ginger attenuates acetylcholine-induced contraction and Ca2+ signalling in murine airway smooth muscle cells.

Asthma is a chronic disease characterized by inflammation and hypersensitivity of airway smooth muscle cells (ASMCs) to different spasmogens. The past decade has seen increased use of herbal treatments for many chronic illnesses. Ginger (Zingiber officinale) is a common food plant that has been used for centuries in treating respiratory illnesses. In this study, we report the effect of its 70% aqueous methanolic crude extract (Zo.Cr) on acetylcholine (ACh)-induced airway contraction and Ca(2+) signalling in ASMCs using mouse lung slices. Airway contraction and Ca(2+) signalling, recorded via confocal microscopy, were induced with ACh, either alone or after pretreatment of slices with Zo.Cr and (or) verapamil, a standard Ca(2+) channel blocker. ACh (10 micromol/L) stimulated airway contraction, seen as decreased airway diameter, and also stimulated Ca(2+) transients (sharp rise in [Ca(2+)]i) and oscillations in ASMCs, seen as increased fluo-4-induced fluorescence intensity. When Zo.Cr (0.3-1.0 mg/mL) was given 30 min before ACh administration, the ACh-induced airway contraction and Ca(2+) signalling were significantly reduced. Similarly, verapamil (1 micromol/L) also inhibited agonist-induced airway contraction and Ca(2+) signalling, indicating a similarity in the modes of action. When Zo.Cr (0.3 mg/mL) and verapamil (1 micromol/L) were given together before ACh, the degree of inhibition was the same as that observed when each of these blockers was given alone, indicating absence of any additional inhibitory mechanism in the extract. In Ca(2+) -free solution, both Zo.Cr and verapamil, when given separately, inhibited Ca(2+) (10 mmol/L)-induced increase in fluorescence and airway contraction. This shows that ginger inhibits airway contraction and associated Ca(2+) signalling, possibly via blockade of plasma membrane Ca(2+) channels, thus reiterating the effectiveness of this age-old herb in treating respiratory illnesses.

Effects of Albuterol Isomers on the Contraction and Ca2+ Signaling of Small Airways in Mouse Lung Slices

The beta(2)-adrenergic agonist, albuterol, is used as a bronchodilator by patients with asthma and consists of a racemic mixture of (R)- and (S)-albuterol. However, the action of the individual enantiomers is poorly understood. Consequently, we investigated the effects of (R)-, (S)- and racemic-albuterol on airway smooth muscle cell (SMC) contraction and Ca(2+) signaling in mouse lung slices with phase-contrast and confocal microscopy. (R)-albuterol relaxed airways contracted with methacholine (MCh) in a dose-dependent manner. By contrast, (S)-albuterol had no effect on airways. (R)-albuterol had a greater relaxant effect than a double concentration of racemic albuterol. Because MCh-induced contraction of airway SMCs is mediated by Ca(2+) oscillations and an increase in Ca(2+) sensitivity, the effects of albuterol on these responses were examined. Both (R)- and racemic albuterol decreased the frequency of the MCh-induced Ca(2+) oscillations by a similar amount. However, (R)-albuterol was more effective than racemic albuterol in decreasing the Ca(2+) sensitivity of the airway SMCs in "model" lung slices with a clamped [Ca(2+)](i). In contrast, (S)-albuterol had no effect on the Ca(2+) oscillations or the Ca(2+) sensitivity. In conclusion, (R)-albuterol consistently induced a greater airway relaxation than racemic albuterol, and (S)-albuterol appears to be responsible for this reduced efficacy.

Interactions of cholinergic agonists with pulmonary neuroepithelial bodies using an in situ mouse lung slice model

Pulmonary neuroepithelial bodies (NEBs), occurring as densely innervated groups of neuroendocrine cells in the airway epithelium, were recently suggested to represent complex sensory airway receptors. Despite the considerable amount of morphological and neurochemical data, none of the functional hypotheses have so far been fully established because reliable in vitro models are not available. Recently, our research group has developed an in situ lung model for the functional study of NEBs in a fluorescent live cell imaging set-up.

Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+oscillations and Ca2+sensitization

Endothelin-1 (ET) induces increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [Ca(2+)](i) and the Ca(2+) sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [Ca(2+)](i) and Ca(2+) sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous Ca(2+) oscillations that were propagated as Ca(2+) waves along the SMCs. These Ca(2+) oscillations were mediated by cyclic intracellular Ca(2+) release and required external Ca(2+) for their maintenance. Importantly, as the frequency of the Ca(2+) oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in Ca(2+) sensitivity. In "model" slices in which the [Ca(2+)](i) was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of Ca(2+) oscillations and by increasing the sensitivity of the contractile machinery to Ca(2+).

Functional morphology and neurochemical characterization of diverse sensory receptors in mouse lungs

Because of the importance of genetically modified mice for functional studies, and the present lack of data, aim of this study was the identification and neurochemical coding of sensory nerve terminals related to neuroepithelial bodies (NEBs) and other sensory receptors in mouse lungs. Antibodies against known selective markers for sensory nerve terminals in rat lungs were used in control and vagotomized mouse lungs. Mouse pulmonary NEBs were found to receive at least two populations of myelinated vagal afferents with intraepithelial terminals: immunoreactive (IR) for vesicular glutamate transporters (VGLUTs) and calbindin D28k; expressing P2X3 ATP receptors. CGRP IR was seen in thin vagal nerve fibers and in delicate non-vagal fibers, both in close proximity to NEBs. VGLUT 1 and P2X3 IR were further found both in receptor complexes reminiscent of the visceral pleura receptors (VPRs) and the smooth muscle-associated airway receptors (SMARs) that we characterized in rat lungs. The presented data revealed that mouse lungs harbor VPRs, SMARs and several populations of sensory nerve terminals that selectively contact NEBs. The neurochemical information on diverse sensory receptors in mouse lungs will be essential for the interpretation of functional data obtained from our recently developed in situ mouse lung slice model. Support: FWO grant G.0085.04 (D.A.); UA grants NOI-BOF 2003 (D.A.) and KP-BOF 2006 (I.B.)

Confocal fluorescent imaging of the interactions of cholinergic agonists with pulmonary neuroepithelial bodies (NEBs) using an in situ lung slice model

Evidence strongly suggests that components of tobacco smoke induce changes in the pulmonary neuroendocrine system. In this study, potential receptor mediated cholinergic interactions with NEBs are postulated based on the expression of vesicular acetylcholine transporter (VAChT) in nerve endings between the NEB cells. Aim was to explore the possible direct effects of cholinergic agonists on NEBs via Ca2+ imaging in a mouse lung slice model. NEBs were visualized by 4-Di-2-ASP and Ca2+-dependent activation by Fluo-4. Application of high extracellular K+ (5s; 50mM) was used as a positive control for NEB activation and for the identification of Clara-like cells, which cover NEBs and show a characteristic slightly delayed Ca2+ rise. Surprisingly, no rise in Fluo-4 fluorescence could be detected in NEB cells after stimulation with 10–100μM nicotine, acetylcholine or carbachol. In Clara-like cells, however, the cholinergic stimulation resulted in a clear Ca2+ rise. In conclusion, NEBs in mouse lungs did not show a Ca2+-dependent activation by externally applied cholinergic agonists. Clara-like cells that intimately interact with NEBs did reveal a cholinergic activation and, considering their possible stem cell-like properties, may be more important players in the airways in health and disease than accounted for today. Support: FWO grant G.0085.04 (D.A.); UA grants NOI-BOF 2003 (D.A.) and KP-BOF 2006 (I.B.)

Ciliary Beat Frequency Is Maintained at a Maximal Rate in the Small Airways of Mouse Lung Slices

Ciliary beat frequency (CBF) is a key factor in the defense of the airways, and ATP can stimulate CBF by increasing intracellular calcium concentration ([Ca2+]i). However, the regulatory effects of ATP have been mainly studied in cultured or isolated epithelial cells from the large cartilaginous airways. The aim of this study was to evaluate the regulation of CBF in small airways of lung slices that are representative of in vivo tissue. Mice lungs were inflated with agarose and cut into thin slices with a vibratome. CBF in the small bronchioles was observed with differential interference contrast microscopy and quantified using high-speed digital imaging (at 240 images s(-1)). We found that the in situ organization of the ciliated cells was well preserved and that their CBF was high. We verified the fidelity of our recording system by analyzing rapid changes in CBF in response to temperature. However, we found that ATP had no effect on CBF, despite the fact that the [Ca2+]i, measured with confocal fluorescence imaging, was increased. Ionomycin and purinergic or beta-adrenergic agonists also failed to increase CBF. Similar results were obtained in outgrowths of cells cultured from lung slices. By contrast, ATP increased the slower CBF of outgrowths of ciliated cells cultured from tracheal rings. Therefore, we conclude that CBF in intrapulmonary airways of mice is maintained at a maximum rate and cannot be further increased by agonist stimulation. These conditions would ensure that mucociliary clearance is constantly active to provide continuous airway protection.