Reference Gene U2 Enables Direct Comparison between Relative Gene Expression Levels of Vascular Smooth Muscle Cells in Tissue and Culture Using Real-Time Quantitative PCR.

In nearly every lab, real-time quantitative polymerase chain reaction (qPCR) is used to quantify gene expression. However, a comparison of different samples requires the careful selection of suitable reference genes (RGs), sometimes referred to as housekeeping genes. In the case of vascular smooth muscle cells (vSMCs), it is important to know under which conditions gene expression in isolated and cultured vSMCs can be compared with vSMCs in a healthy blood vessel. We isolated the vSMC-containing layer of the rat aorta (tunica media) and used one (longitudinal) half for direct RNA extraction, while the other half served to isolate and culture vSMCs prior to RNA extraction. First, the expression of the routinely used RGs beta-actin (Actb) and Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is investigated in intact media and corresponding cultured vSMCs. Significant differences in their Ct values show that these RGs could not be used for such direct comparisons; therefore, we select 15 different RGs. Only the gene expression of the small ribonuclear protein (snRNP) U2 shows no significant differences between the absolute Ct values of cultured vSMCs and the intact media; moreover, no differences were found between male and female rats in our experimental setup. In conclusion, U2 was shown to be an appropriate (sex-independent) RG to compare relative expression levels of vSMCs in culture to those vSMCs within their physiological tissue environment.

A succinate/SUCNR1-brush cell defense program in the tracheal epithelium.

Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cß2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.

Cysteinyl leukotrienes and acetylcholine are biliary tuft cell cotransmitters.

The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.

CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung.

The conducting airways are lined by distinct cell types, comprising basal, secretory, ciliated, and rare cells, including ionocytes, solitary cholinergic chemosensory cells, and solitary and clustered (neuroepithelial bodies) neuroendocrine cells. Airway neuroendocrine cells are in clinical focus since they can give rise to small cell lung cancer. They have been implicated in diverse functions including mechanosensation, chemosensation, and regeneration, and were recently identified as regulators of type 2 immune responses via the release of the neuropeptide calcitonin gene-related peptide (CGRP). We here assessed the expression of the chemokine CXCL13 (B cell attracting chemokine) by these cells by RT-PCR, in silico analysis of publicly available sequencing data sets, immunohistochemistry, and immuno-electron microscopy. We identify a phenotype of neuroendocrine cells in the naïve mouse, producing the chemokine CXCL13 predominantly in solitary neuroendocrine cells of the tracheal epithelium (approx. 70% CXCL13+) and, to a lesser extent, in the solitary neuroendocrine cells and neuroepithelial bodies of the intrapulmonary bronchial epithelium (< 10% CXCL13+). In silico analysis of published sequencing data of murine tracheal epithelial cells was consistent with the results obtained by immunohistochemistry as it revealed that neuroendocrine cells are the major source of Cxcl13-mRNA, which was expressed by 68-79% of neuroendocrine cells. An unbiased scRNA-seq data analysis of overall gene expression did not yield subclusters of neuroendocrine cells. Our observation demonstrates phenotypic heterogeneity of airway neuroendocrine cells and points towards a putative immunoregulatory role of these cells in bronchial-associated lymphoid tissue formation and B cell homeostasis.

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

Hypoxia-induced pulmonary vasoconstriction of intra-acinar arteries is impaired in NADPH oxidase 4 gene-deficient mice.

We show that genetic deficiency of the reactive oxygen species generating enzyme NADPH oxidase 4 (NOX4) impairs hypoxic pulmonary vasoconstriction in small (25-40 µm) intra-acinar, but not pre-acinar, arteries in murine precision cut lung slices. These data suggest an involvement of NOX4 in ventilation-perfusion matching at the acinar level.

Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice.

Several species of the Gram-negative genus Bordetella are the cause of respiratory infections in mammals and birds, including whooping cough (pertussis) in humans. Very recently, a novel atypical species, Bordetella pseudohinzii, was isolated from laboratory mice. These mice presented no obvious clinical symptoms but elevated numbers of neutrophils in bronchoalveolar lavage fluid and inflammatory signs in histopathology. We noted that this species can occur at high prevalence in a mouse facility despite regular pathogen testing according to the FELASA-recommendations. Affected C57BL/6 J mice had, in addition to the reported pulmonary alterations, tracheal inflammation with reduced numbers of ciliated cells, slower ciliary beat frequency, and largely (>50%) compromised cilia-driven particle transport speed on the mucosal surface, a primary innate defence mechanism. In an in vitro-model, Bordetella pseudohinzii attached to respiratory kinocilia, impaired ciliary function within 4 h and caused epithelial damage within 24 h. Regular testing for this ciliotropic Bordetella species and excluding it from colonies that provide mice for lung research shall be recommended. On the other hand, controlled colonization and infection with Bordetella pseudohinzii may serve as an experimental model to investigate mechanisms of mucociliary clearance and microbial strategies to escape from this primary innate defence response.

TASK-1 potassium channel is not critically involved in mediating hypoxic pulmonary vasoconstriction of murine intra-pulmonary arteries.

The two-pore domain potassium channel KCNK3 (TASK-1) is expressed in rat and human pulmonary artery smooth muscle cells. There, it is associated with hypoxia-induced signalling, and its dysfunction is linked to pathogenesis of human pulmonary hypertension. We here aimed to determine its role in hypoxic pulmonary vasoconstriction (HPV) in the mouse, and hence the suitability of this model for further mechanistic investigations, using appropriate inhibitors and TASK-1 knockout (KO) mice. RT-PCR revealed expression of TASK-1 mRNA in murine lungs and pre-acinar pulmonary arteries. Protein localization by immunohistochemistry and western blot was unreliable since all antibodies produced labelling also in TASK-1 KO organs/tissues. HPV was investigated by videomorphometric analysis of intra- (inner diameter: 25-40 µm) and pre-acinar pulmonary arteries (inner diameter: 41-60 µm). HPV persisted in TASK-1 KO intra-acinar arteries. Pre-acinar arteries developed initial HPV, but the response faded earlier (after 30 min) in KO vessels. This HPV pattern was grossly mimicked by the TASK-1 inhibitor anandamide in wild-type vessels. Hypoxia-provoked rise in pulmonary arterial pressure (PAP) in isolated ventilated lungs was affected neither by TASK-1 gene deficiency nor by the TASK-1 inhibitor A293. TASK-1 is dispensable for initiating HPV of murine intra-pulmonary arteries, but participates in sustained HPV specifically in pre-acinar arteries. This does not translate into abnormal rise in PAP. While there is compelling evidence that TASK-1 is involved in the pathogenesis of pulmonary arterial hypertension in humans, the mouse does not appear to serve as a suitable model to study the underlying molecular mechanisms.

Avertin®, but Not Volatile Anesthetics Addressing the Two-Pore Domain K+ Channel, TASK-1, Slows Down Cilia-Driven Particle Transport in the Mouse Trachea.

RATIONALE: Volatile anesthetics inhibit mucociliary clearance in the airways. The two-pore domain K+ channel, TASK-1, represents one of their molecular targets in that they increase its open probability. Here, we determine whether particle transport speed (PTS) at the mucosal surface of the mouse trachea, an important factor of the cilia-driven mechanism in mucociliary clearance, is regulated by TASK-1. METHODOLOGY/RESULTS: RT-PCR analysis revealed expression of TASK-1 mRNA in the manually dissected and laser-assisted microdissected tracheal epithelium of the mouse. Effects of anesthetics (isoflurane and Avertin®) and TASK-1 inhibitors (anandamide and A293) on ciliary activity were investigated by assessment of PTS at the mucosal surface of the explanted and opened murine trachea. Neither TASK-1 inhibitors nor isoflurane had any impact on basal and ATP-stimulated PTS. Avertin® reduced basal PTS, and ATP-stimulated PTS decreased in its presence in wild-type (WT) mice. Avertin®-induced decrease in basal PTS persisted in WT mice in the presence of TASK-1 inhibitors, and in two different strains of TASK-1 knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that TASK-1 is expressed by the tracheal epithelium but is not critically involved in the regulation of tracheal PTS in mice. Avertin® reduces PTS independent of TASK-1.

Calcitonin Peptide Family Members Are Differentially Regulated by LPS and Inhibit Functions of Rat Alveolar NR8383 Macrophages.

Members of the calcitonin peptide family-calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin2/intermedin (IMD)-exert modulatory effects upon monocytes and macrophages of various extrapulmonary origins. Utilizing the rat alveolar macrophage (AMφ) cell line NR8383, we here set out to determine to which extent these three peptides and their receptors are differentially regulated in AMφ and what specific effects they have on AMφ key functions. LPS treatment differentially up-regulated expression of the peptides and receptors. Among the three peptides, IMD mRNA content was lowest both in primary rat AMφ and NR8383 cells, whereas IMD peptide dominated in basal and LPS-stimulated secretion from NR8383 cells. Fcγ receptor-mediated phagocytosis and TNF-α production were inhibited by AM, IMD, and CGRP, whereas pro-IL-1ß mRNA was slightly down-regulated exclusively by CGRP. Neither of these peptides affected IL-6 or IL-10 production. None increased intracellular calcium concentration, but AM significantly inhibited store-operated calcium entry. In conclusion, the rat AMφ cell line NR8383 is both a source and a target of the calcitonin peptide family members AM, IMD, and CGRP. Despite sharing proteins of the receptor complexes, AM, IMD, and CGRP each showed a characteristic pattern of effects and regulation, suggesting that these closely related peptides are not just redundant members of one common signaling pathway but act in concert by addressing parallel signaling cascades. Since peptide and receptor expression are up-regulated by LPS, these signaling pathways might act as inhibitory feedback mechanisms in pulmonary bacterial infection.

Spatial expression of components of a calcitonin receptor-like receptor (CRL) signalling system (CRL, calcitonin gene-related peptide, adrenomedullin, adrenomedullin-2/intermedin) in mouse and human heart valves.

Heart valves are highly organized structures determining the direction of blood flow through the heart. Smooth muscle cells within the valve are thought to play an active role during the heart cycle, rather than being just passive flaps. The mature heart valve is composed of extracellular matrix (ECM), various differentiations of valvular interstitial cells (VIC), smooth muscle cells and overlying endothelium. VIC are important for maintaining the structural integrity of the valve, thereby affecting valve function and ECM remodelling. Accumulating evidence suggests an important role of calcitonin receptor-like receptor (CRL) signalling in preventing heart damage under several pathological conditions. Thus we investigate the existence of a putative CRL signalling system in mouse and human heart valves by real-time RT-PCR, laser-assisted microdissection, immunofluorescence and NADPH-diaphorase histochemistry. Mouse and human heart valves expressed mRNAs for the CRL ligands adrenomedullin (AM), adrenomedullin-2 (AM-2) and calcitonin gene-related peptide (CGRP) and for their receptor components, i.e., CRL and receptor-activity-modifying proteins 1-3. Immunofluorescence analysis revealed AM-, AM-2- and CRL-immunolabelling in endothelial cells and VIC, whereas CGRP immunoreactivity was restricted to nerve fibres and some endothelial cells. Nitric oxide synthase activity, as demonstrated by NADPH-diaphorase histochemistry, was shown mainly in valvular endothelial cells in mice, whereas in human aortic valves, VIC and smooth muscle cells were positive. Our results showed the presence of an intrinsic AM/AM-2/CGRP signalling system in murine and human heart valves with distinct cellular localization, suggesting its involvement in the regulation of valve stiffness and ECM production and turnover.

Expression and localization of GPR91 and GPR99 in murine organs.

Energy substrates and metabolic intermediates are proven ligands of a growing number of G-protein coupled receptors. In 2004, GPR91 and GPR99 were identified as receptors for the citric acid cycle intermediates, succinate and α-ketoglutarate, respectively. GPR91 seems to act as a first responder to local stress and GPR99 participates in the regulation of the acid-base balance through an intrarenal paracrine mechanism. However, a systematic analysis of the distribution of both receptors in mouse organs is still missing. The aim of this study was to examine the expression of GPR91 and GPR99 in a large number of different murine organs both at mRNA and protein level. Whereas GPR91 mRNA was detectable in almost all organs, GPR99 mRNA was mainly expressed in neuronal tissues. Widespread expression of GPR91 was also detected at the protein level by western blotting and immunohistochemistry. In addition to neuronal cells, GPR99 protein was found in renal intercalated cells and epididymal narrow cells. Double-labeling immunohistochemistry demonstrated the colocalization of GPR99 with the B1 subunit isoform of vacuolar H(+)-ATPases which is expressed only by a very limited number of cell types. In summary, our detailed expression analysis of GPR91 and GPR99 in murine tissues will allow a more directed search for additional functions of both receptors.

Low-dose adrenomedullin-2/intermedin(8-47) reduces pulmonary ischemia/reperfusion injury.

Adrenomedullin-2/intermedin stabilizes the pulmonary microvascular barrier challenged by application of thrombin ex vivo and by experimental ventilation in vivo. Here, we test the hypothesis that adrenomedullin-2/intermedin(8-47) protects mouse lungs from ischemia/reperfusion injury in vivo. C57BL/6 mice were anesthetized, intubated, ventilated, and heparinized. Blood vessels and the main bronchus of the left lung were clamped for 90min. Thereafter, lungs were reperfused for 120min. Five min before clamping and before reperfusion, mice obtained intravenous injections of adrenomedullin-2/intermedin(8-47). After reperfusion, mice were sacrificed and bronchoalveolar lavage of the left and the right lung was performed separately. The integrity of the blood-air barrier was investigated by electron microscopy using stereological methods. In response to ischemia/reperfusion injury, intraalveolar leukocytes accumulated in the ischemic lung. Two applications of 10ng/kg body weight adrenomedullin-2/intermedin(8-47) dramatically reduced leukocyte infiltration to about 15% (p≤0.001). Also the proportion of the subpopulation of neutrophil granulocytes decreased (12% vs 5%, p=0.013). Electron microscopy revealed a protection of the blood-air barrier by adrenomedullin-2/intermedin(8-47). Adrenomedullin-2/intermedin(8-47) ameliorates early ischemia/reperfusion injury in mouse lungs by protecting the integrity of the blood-air barrier and by potently reducing leukocyte influx into the alveolar space. Adrenomedullin-2/intermedin(8-47) might be of therapeutic interest in lung transplantation and cardiopulmonary bypass.

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

INTRODUCTION: Ventilator-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. METHODS: We 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. RESULTS: In 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). CONCLUSIONS: MV 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.

Intrinsic vascular dopamine - a key modulator of hypoxia-induced vasodilatation in splanchnic vessels.

Dopamine not only is a precursor of the catecholamines noradrenaline and adrenaline but also serves as an independent neurotransmitter and paracrine hormone. It plays an important role in the pathogenesis of hypertension and is a potent vasodilator in many mammalian systemic arteries, strongly suggesting an endogenous source of dopamine in the vascular wall. Here we demonstrated dopamine, noradrenaline and adrenaline in rat aorta and superior mesenteric arteries (SMA) by radioimmunoassay. Chemical sympathectomy with 6-hydroxydopamine showed a significant reduction of noradrenaline and adrenaline, while dopamine levels remained unaffected. Isolated endothelial cells were able to synthesize and release dopamine upon cAMP stimulation. Consistent with these data, mRNAs coding for catecholamine synthesizing enzymes, i.e. tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase, and dopamine-ß-hydroxylase were detected by RT-PCR in cultured endothelial cells from SMA. TH protein was detected by immunohistochemisty and Western blot. Exposure of endothelial cells to hypoxia (1% O2) increased TH mRNA. Vascular smooth muscle cells partially expressed catecholaminergic traits. A physiological role of endogenous vascular dopamine was shown in SMA, where D1 dopamine receptor blockade abrogated hypoxic vasodilatation. Experiments on SMA with endothelial denudation revealed a significant contribution of the endothelium, although subendothelial dopamine release dominated. From these results we conclude that endothelial cells and cells of the underlying vascular wall synthesize and release dopamine in an oxygen-regulated manner. In the splanchnic vasculature, this intrinsic non-neuronal dopamine is the dominating vasodilator released upon lowering of oxygen tension.

Adrenomedullin and the calcitonin receptor-like receptor system mRNA expressions in the rat heart and sensory ganglia in experimentally-induced long-term diabetes.

Both adrenomedullin and calcitonin gene-related peptide (CGRP) regulate vascular tone in the heart, being cardioprotective in hypoxia. Additionally, adrenomedullin exhibits antiproliferative and antiapoptotic functions in the myocardium, while CGRP exerts positive chronotropic effect. Their actions are mediated through the specific G protein-coupled receptor, CRLR, whose ligand affinity is determined by receptor activity modifying proteins RAMP1-3. CGRP binds to the complex formed by CRLR/RAMP1, whereas CRLR/RAMP2 and CRLR/RAMP3 serve as receptors for adrenomedullin. Here, we quantified expression of this signaling system in the rat heart and supplying sensory ganglia (dorsal root ganglia T1-T4 and vagal nodose ganglia) in streptozotocin-induced diabetes. In the course of diabetes, an increase of CRLR mRNA was noticed in the right ventricle 8 weeks and of RAMP3 mRNA in the left ventricle and right atrium 26 weeks after induction of diabetes. Relative expressions of other tested genes were not significantly altered. In the nodose vagal supplying specific cardiac afferents, but not in dorsal root ganglia which provide cardiac pain fibres, a small upregulation of CGRP expression was detected. In summary, the shifts observed in diabetes may favour a trend of a pronounced adrenomedullin signaling. These observations may provide a new possible therapeutic strategy for diabetic cardiomyopathy.

Intermedin stabilized endothelial barrier function and attenuated ventilator-induced lung injury in mice.

BACKGROUND: Even protective ventilation may aggravate or induce lung failure, particularly in preinjured lungs. Thus, new adjuvant pharmacologic strategies are needed to minimize ventilator-induced lung injury (VILI). Intermedin/Adrenomedullin-2 (IMD) stabilized pulmonary endothelial barrier function in vitro. We hypothesized that IMD may attenuate VILI-associated lung permeability in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Human pulmonary microvascular endothelial cell (HPMVEC) monolayers were incubated with IMD, and transcellular electrical resistance was measured to quantify endothelial barrier function. Expression and localization of endogenous pulmonary IMD, and its receptor complexes composed of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) 1-3 were analyzed by qRT-PCR and immunofluorescence in non ventilated mouse lungs and in lungs ventilated for 6 h. In untreated and IMD treated mice, lung permeability, pulmonary leukocyte recruitment and cytokine levels were assessed after mechanical ventilation. Further, the impact of IMD on pulmonary vasoconstriction was investigated in precision cut lung slices (PCLS) and in isolated perfused and ventilated mouse lungs. IMD stabilized endothelial barrier function in HPMVECs. Mechanical ventilation reduced the expression of RAMP3, but not of IMD, CRLR, and RAMP1 and 2. Mechanical ventilation induced lung hyperpermeability, which was ameliorated by IMD treatment. Oxygenation was not improved by IMD, which may be attributed to impaired hypoxic vasoconstriction due to IMD treatment. IMD had minor impact on pulmonary leukocyte recruitment and did not reduce cytokine levels in VILI. CONCLUSIONS/SIGNIFICANCE: IMD may possibly provide a new approach to attenuate VILI.

Mitochondrial complex II is essential for hypoxia-induced pulmonary vasoconstriction of intra- but not of pre-acinar arteries.

AIMS: Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II (SDHD(+/-)). METHODS AND RESULTS: Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD(+/-) mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20-50 µm) intra-acinar and larger (51-100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD(+/-) mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD(+/-) than in wild-type mice. CONCLUSION: SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.

Acetylcholine and chronic vasculopathy in rat renal allografts.

BACKGROUND: Chronic allograft vasculopathy (CAV) is an important aspect of chronic allograft injury, which limits the long-term success of renal transplantation. The pathogenesis of CAV is ill defined, and no effective therapies exist. Acute rejection episodes are a major risk factor for CAV. Recently, we demonstrated that leukocytes, which strongly accumulate in allograft blood vessels during fatal acute rejection, produce acetylcholine (ACh), which has the potential to provoke CAV. Herein, we test the hypothesis that ACh is also produced by leukocytes during the development of CAV. METHODS: Kidneys were transplanted in the Fischer 344 to Lewis rat strain combination, an established experimental model for CAV. Isografts were performed in Lewis rats. The capacity of intravascular graft leukocytes to synthesize ACh was investigated during reversible acute rejection on day 9 posttransplantation and during the process of vascular remodeling on day 42. Furthermore, allograft recipients were treated with rivastigmine, which blocks enzymatic degradation of ACh. RESULTS: The protein expression of the high-affinity choline transporter-1 and choline acetyltransferase was increased in leukocytes from allografts on day 9 and 42 posttransplantation. In addition, leukocytes accumulating in the lumina of allograft blood vessels were by far more numerous compared with isografts. In line with our hypothesis, ACh itself was detected by high-pressure liquid chromatography in graft leukocytes but not in leukocytes from untreated kidneys. Treatment with rivastigmine drastically exacerbated CAV compared with placebo. CONCLUSION: We suggest that endogenous ACh contributes to the pathogenesis of CAV and may be a promising target for novel therapies preventing CAV.

Nicotinic receptors on rat alveolar macrophages dampen ATP-induced increase in cytosolic calcium concentration.

BACKGROUND: Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. METHODS: Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. RESULTS: Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, ß1, and ß2, with most stable expression being noted for subunits α9, α10, ß1, and ß2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 µM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. CONCLUSIONS: Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.