Uwe Pfeil

The epithelial cholinergic system of the airways

Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types—ciliated, basal and secretory—are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs.

Coexpression of α9 and α10 nicotinic acetylcholine receptors in rat dorsal root ganglion neurons

Previous binding studies have suggested the presence of a so far unknown nicotinic acetylcholine receptor subunit in dorsal root ganglia (Pugh et al., 1995). Here, we investigated whether the most recently identified subunit, α10, and its potential interaction partner, α9 (Elgoyhen et al., 2001), are expressed in these ganglia. All neurons of rat dorsal root ganglia, but no glial cells, expressed both α9 and α10 mRNA in in situ hybridization, and exhibited α10 immunoreactivity using a newly raised antibody. These findings were confirmed by RT-PCR and western blotting. The data show that dorsal root ganglion neurons coexpress α9 and α10 nicotinic receptor subunits, thereby providing the first example of neuronal expression of this receptor subunit pair.

Hypoxic vasoconstriction of partial muscular intra-acinar pulmonary arteries in murine precision cut lung slices.

BackgroundAcute alveolar hypoxia causes pulmonary vasoconstriction (HPV) which serves to match lung perfusion to ventilation. The underlying mechanisms are not fully resolved yet. The major vascular segment contributing to HPV, the intra-acinar artery, is mostly located in that part of the lung that cannot be selectively reached by the presently available techniques, e.g. hemodynamic studies of isolated perfused lungs, recordings from dissected proximal arterial segments or analysis of subpleural vessels. The aim of the present study was to establish a model which allows the investigation of HPV and its underlying mechanisms in small intra-acinar arteries.MethodsIntra-acinar arteries of the mouse lung were studied in 200 μm thick precision-cut lung slices (PCLS). The organisation of the muscle coat of these vessels was characterized by α-smooth muscle actin immunohistochemistry. Basic features of intra-acinar HPV were characterized, and then the impact of reactive oxygen species (ROS) scavengers, inhibitors of the respiratory chain and Krebs cycle metabolites was analysed.ResultsIntra-acinar arteries are equipped with a discontinuous spiral of α-smooth muscle actin-immunoreactive cells. They exhibit a monophasic HPV (medium gassed with 1% O2) that started to fade after 40 min and was lost after 80 min. This HPV, but not vasoconstriction induced by the thromboxane analogue U46619, was effectively blocked by nitro blue tetrazolium and diphenyleniodonium, indicating the involvement of ROS and flavoproteins. Inhibition of mitochondrial complexes II (3-nitropropionic acid, thenoyltrifluoroacetone) and III (antimycin A) specifically interfered with HPV, whereas blockade of complex IV (sodium azide) unspecifically inhibited both HPV and U46619-induced constriction. Succinate blocked HPV whereas fumarate had minor effects on vasoconstriction.ConclusionThis study establishes the first model for investigation of basic characteristics of HPV directly in intra-acinar murine pulmonary vessels. The data are consistent with a critical involvement of ROS, flavoproteins, and of mitochondrial complexes II and III in intra-acinar HPV. In view of the lack of specificity of any of the classical inhibitors used in such types of experiments, validation awaits the use of appropriate knockout strains and siRNA interference, for which the present model represents a well-suited approach.

Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibro-blasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29–40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.

Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability

Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in preventing damage of the lung by stabilizing pulmonary barrier function. Intermedin (IMD), also termed adrenomedullin-2, is the most recently identified peptide targeting this receptor. Here we investigated the effect of hypoxia on the expression of IMD in the murine lung and cultured murine pulmonary microvascular endothelial cells (PMEC) as well as the role of IMD in regulating vascular permeability. Monoclonal IMD antibodies were generated, and transcript levels were assayed by quantitative RT-PCR. The promoter region of IMD gene was analyzed, and the effect of hypoxia-inducible factor (HIF)-1alpha on IMD expression was investigated in HEK293T cells. Isolated murine lungs and a human lung microvascular endothelial cell monolayer model were used to study the effect of IMD on vascular permeability. IMD was identified as a pulmonary endothelial peptide by immunohistochemistry and RT-PCR. Hypoxia caused an upregulation of IMD mRNA in the murine lung and PMEC. As shown by these results, HIF-1alpha enhances IMD promoter activity. Our functional studies showed that IMD abolished the increase in pressure-induced endothelial permeability. Moreover, IMD decreased basal and thrombin-induced hyperpermeability of an endothelial cell monolayer in a receptor-dependent manner and activated PKA in these cells. In conclusion, IMD is a novel hypoxia-induced gene and a potential interventional agent for the improvement of endothelial barrier function in systemic inflammatory responses and hypoxia-induced vascular leakage.

Localisation of the high-affinity choline transporter-1 in the rat skeletal motor unit

Abstract. The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled α-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.

Coexpression and spatial association of nicotinic acetylcholine receptor subunits α7 and α10 in rat sympathetic neurons

Fast excitatory synaptic transmission in sympathetic ganglia is mediated by nicotinic acetylcholine receptors (nAChRs). Although it is known that the nAChR alpha7-subunit occurs in sympathetic ganglia, the expression of the recently cloned subunit alpha10 (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002) has not been analyzed. Until now, functional receptors containing alpha10-subunits have been found only in combination with alpha9-subunits (Elgoyhen et al., 2001; Lustig et al., 2001; Sgard et al., 2002). The alpha9-subunit exhibits a restricted expression pattern, whereas the alpha10-subunit is expressed more widely. This broad distribution resembles more closely that known for subunit alpha7 than for subunit alpha9. On this background, we investigated the distribution of nAChR subunits alpha7, alpha9, and alpha10 in rat sympathetic ganglia and studied a possible interaction between subunit alpha7 and potential partners by double-labeling immunofluorescence and fluorescence resonance energy transfer (FRET) (Kam et al., 1995; Jares-Erijman and Jovin, 2003).

Multiple nicotinic acetylcholine receptor α-subunits are expressed in the arterial system of the rat

Abstract. Neuronal nicotinic acetylcholine receptors (nAChRs) are hetero- and homopentamers built up by nine different α-subunits and three different β-subunits. The subtype composition within the receptor determines ligand specificity, affinity and cation permeability. In this study we focused on the distribution of the ligand binding α-subunits in the rat arterial system by means of RT-PCR and immunohistochemistry. Subtypes α3, α5, α7 and α10 were found to be expressed by endothelial cells, suggesting that they are equipped both with calcium-preferring (α7 homopentamers) and monovalent cation-preferring (heteropentamers containing α3- and α5-subunits) nAChR channels. All α-subtypes except α9 were expressed by vascular smooth muscle cells with a highly specific distribution pattern along the vascular tree. While every α-subunit except α9 was detected in the thoracic aorta, intrapulmonary arterial branches contained only α7 immunoreactivity, and other vascular beds held intermediate positions with respect to the extent of α-subunit expression. Current knowledge does not allow to correlate these distribution patterns to specific functions, but it can be anticipated that at least some components of nAChR-mediated signalling in the arterial wall are highly specific for individual arteries.

Pivotal Advance: Up‐regulation of acetylcholine synthesis and paracrine cholinergic signaling in intravascular transplant leukocytes during rejection of rat renal allografts

During acute rejection, large numbers of leukocytes accumulate in the blood vessels of experimental renal allografts. About 70% of them are activated, cytotoxic monocytes that appear to be involved in allograft destruction. ACh exerts anti‐inflammatory effects upon monocytes/macrophages and has been proposed to be a key player in neuroimmunological interactions. Its short half‐life, however, makes it unlikely that neuronal ACh affects blood leukocytes. Renal transplantation was performed in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested after 4 days, and the expression of CHT1, cChAT, pChAT, and nAChR subunits was investigated by RT‐PCR, immunoblotting, and immunohistochemistry. Monocytes were identified by double‐labeling with ED1‐antibody, directed to a CD68‐like antigen. ACh content was measured by HPLC. [Ca2+]i was monitored by Fura‐2. Intravascular graft leukocytes express CHT1 and cChAT mRNA and protein and pChAT protein. Their expression is strongly up‐regulated in vivo during acute allograft rejection. Immunohistochemistry revealed CHT1, cChAT, and pChAT protein in ED1‐positive monocytes. The ACh content of allograft intravascular leukocytes was sixfold higher than that of isografts. Intravascular leukocytes express nAChR subunits, and an ATP‐induced increase in [Ca2+]i was augmented in vitro by a nAChR inhibitor in allograft but not isograft leukocytes. Intravascular graft leukocytes, among them monocytes, up‐regulate non‐neuronal ACh synthesis and develop auto‐/paracrine cholinergic attenuation of ATP signaling during acute allograft rejection.

Mechanical ventilation drives pneumococcal pneumonia into lung injury and sepsis in mice: protection by adrenomedullin

IntroductionVentilator-induced lung injury (VILI) contributes to morbidity and mortality in acute respiratory distress syndrome (ARDS). Particularly pre-injured lungs are susceptible to VILI despite protective ventilation. In a previous study, the endogenous peptide adrenomedullin (AM) protected murine lungs from VILI. We hypothesized that mechanical ventilation (MV) contributes to lung injury and sepsis in pneumonia, and that AM may reduce lung injury and multiple organ failure in ventilated mice with pneumococcal pneumonia.MethodsWe analyzed in mice the impact of MV in established pneumonia on lung injury, inflammation, bacterial burden, hemodynamics and extrapulmonary organ injury, and assessed the therapeutic potential of AM by starting treatment at intubation.ResultsIn pneumococcal pneumonia, MV increased lung permeability, and worsened lung mechanics and oxygenation failure. MV dramatically increased lung and blood cytokines but not lung leukocyte counts in pneumonia. MV induced systemic leukocytopenia and liver, gut and kidney injury in mice with pneumonia. Lung and blood bacterial burden was not affected by MV pneumonia and MV increased lung AM expression, whereas receptor activity modifying protein (RAMP) 1–3 expression was increased in pneumonia and reduced by MV. Infusion of AM protected against MV-induced lung injury (66% reduction of pulmonary permeability p < 0.01; prevention of pulmonary restriction) and against VILI-induced liver and gut injury in pneumonia (91% reduction of AST levels p < 0.05, 96% reduction of alanine aminotransaminase (ALT) levels p < 0.05, abrogation of histopathological changes and parenchymal apoptosis in liver and gut).ConclusionsMV paved the way for the progression of pneumonia towards ARDS and sepsis by aggravating lung injury and systemic hyperinflammation leading to liver, kidney and gut injury. AM may be a promising therapeutic option to protect against development of lung injury, sepsis and extrapulmonary organ injury in mechanically ventilated individuals with severe pneumonia.