Wolfgang Kummer

Publications of Wolfgang Kummer

• Does bladder outlet obstruction alter the non-neuronal cholinergic system of the human urothelium?

  Authors: Thomas Bschleipfer, Wolfgang Weidner, Wolfgang Kummer, Katrin S Lips

  Life sciences. 04/2012;

  AIMS: Alterations of the bladder sensory system are considered to contribute to detrusor overactivity (DO) when patients suffer from bladder outlet obstruction (BOO). The urothelium is one part ofAIMS: Alterations of the bladder sensory system are considered to contribute to detrusor overactivity (DO) when patients suffer from bladder outlet obstruction (BOO). The urothelium is one part of this sensory system and it harbors a non-neuronal cholinergic system (NNCS). We aimed to investigate if BOO causes alterations in the NNCS. MAIN METHODS: Urothelial specimens were collected by endoscopy from six male controls and eight male patients suffering from BOO and DO. The samples were examined by immunofluorescence (IF) and real-time RT-PCR for high-affinity choline transporter-1 (CHT1), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), organic cation transporters OCT1-3, muscarinic receptor (mAChR) subtypes M1-M5 and nicotinic receptor (nAChR) subunits α7, α9 and α10. KEY FINDINGS: ChAT, VAChT and OCT2 are not present in the male urothelium. Real-time RT-PCR and IF detected all other investigated targets. Rank order of expression was M2≫M3=M5>M4=M1 for mAChR subtypes and α7≫α10>α9 for nAChR subunits. Statistical analysis of RT-PCR results did not detect significant differences between patients and controls. Only IF detected differences between both groups: α9-Immunolabeling was increased in all BOO/DO patients. SIGNIFICANCE: BOO does not induce considerable alterations of the human urothelial NNCS on mRNA level. Expression of mAChRs, CHT1, OCT1 and OCT3 is not significantly affected by BOO. Thus, transport mechanisms for choline and acetylcholine (ACh) stay unaltered. BOO increases immunolabeling of α9-nAChR but whether this sole finding contributes to the onset of DO seems questionable. Comparing the present results with our previous work, the urothelial NNCS does not differ between men and women.

• Cholinergic receptors in the murine oviduct: Inventory and coupling to intracellular calcium concentration.

  Authors: Miriam Wolff, Katharina Noreikat, Ines Ibanez-Tallon, Katrin S Lips, Sabine Kölle, Wolfgang Kummer

  Life sciences. 03/2012;

  AIMS: In the oviduct, muscarinic acetylcholine receptors (MR) are linked with motility regulation and nicotinic receptors (nAChR) with ectopic pregnancy. We here aimed to determine the repertoire ofAIMS: In the oviduct, muscarinic acetylcholine receptors (MR) are linked with motility regulation and nicotinic receptors (nAChR) with ectopic pregnancy. We here aimed to determine the repertoire of cholinergic receptor expression in the murine oviduct and their functional coupling to regulation of intracellular calcium concentration ([Ca(2+)](i)). MAIN METHODS: Cholinergic receptor transcripts were assessed by RT-PCR in oviductal segments (ampulla, isthmus, uterotubar junction) in all cyclic stages and pregnancy, and in laser-microdissected samples of epithelium and smooth muscle, nAChR subunit α3 distribution in tissue sections using an appropriate genetic reporter mouse strain. [Ca(2+)](i) responses were monitored in ciliated and non-ciliated oviductal cells isolated from wild-type and MR subtypes 1 and 3 gene deficient mice. KEY FINDINGS: Transcripts for all MR subtypes (M1-M5) are constantly expressed whereas there is some variability in nAChR expression from individual to individual. The qualitative expression pattern is independent from the hormonal status of the animal, except for nAChR α7, which is less present during pregnancy. The epithelium expresses M1, M3, nAChR α7 (data from laser-assisted microdissection) and nAChR α3 (ultrastructural investigation of reporter mice). MR dominate over nAChR in increasing [Ca(2+)](i) with being M3 the major, but not sole subtype driving this effect. The general nAChR inhibitor mecamylamine enhances muscarinic and purinergic responses. SIGNIFICANCE: In conclusion, the murine oviduct is endowed with a multiplicity of muscarinic and nicotinic receptors subtypes that, with respect to regulation of [Ca(2+)](i), are inversely linked to each other. The major, but not sole, cholinergic receptor driving increase in [Ca(2+)](i) is M3.

• Luminal cholinergic signalling in airway lining fluid: a novel mechanism for activating chloride secretion via Ca(2+) dependent Cl(-) and K(+) channels.

  Authors: Monika I Hollenhorst, Katrin S Lips, Miriam Wolff, Jürgen Wess, Stefanie Gerbig, Zoltan Takats, Wolfgang Kummer, Martin Fronius

  British journal of pharmacology. 02/2012;

  Background and purpose.  Recent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this putative non-neuronalBackground and purpose.  Recent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this putative non-neuronal cholinergic system is not known in detail unknown. Thus, this study focused on the impact of luminal acetylcholine (ACh) as a regulator of transepithelial ion transport in epithelial cells. Experimental approach.  RT-PCR experiments were performed using mouse tracheal epithelium cells for choline acetyl transferase (ChAT) and organic cation transporter (OCT) transcripts. Components of tracheal airway lining fluid were analysed with desorption electrospray ionization (DESI) mass spectrometry. Effects of nicotine on mouse tracheal epithelial ion transport were examined with Ussing-chamber experiments. Key Results.  Transcripts encoding ChAT and OCT1-3 were detected in mouse tracheal epithelial cells. The DESI experiments identified ACh in the airway lining fluid. Luminal ACh induced an immediate, dose-dependent increase of the transepithelial ion current (EC(50) : 23.3 µM), characterised by a transient peak and a sustained plateau current. This response was not affected by the Na(+) -channel inhibitor amiloride. The Cl(-) -channel inhibitor niflumic acid or the K(+) -channel blocker Ba(2+) attenuated the ACh effect. The calcium ionophore A23187 mimicked the ACh effect. Luminal nicotine or muscarine increased the ion current. Experiments with receptor gene deficient animals revealed the participation of muscarinic receptors subtypes M1 and M3. Conclusions and Implications.  The presence of luminal ACh and activation of transepithelial ion currents by luminal ACh receptors identifies a novel non-neuronal cholinergic pathway in the airway lining fluid. This pathway could represent a novel drug target in the airways. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

• Ciliary Activity in the Oviduct of Cycling, Pregnant and Muscarinic Receptor Knockout Mice.

  Authors: Katharina Noreikat, Miriam Wolff, Wolfgang Kummer, Sabine Kölle

  Biology of reproduction. 02/2012;

  The transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. As non-neuronally produced acetylcholine influences ciliary beating in the tracheaThe transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. As non-neuronally produced acetylcholine influences ciliary beating in the trachea via the muscarinic receptors M2 and M3, we supposed that components of the cholinergic system may also modulate ciliary activity in the oviduct. To address this issue, we analyzed the expression profile of muscarinic receptors (CHRM in the murine oviduct by RT-PCR and assessed ciliary beat frequency (CBF) and cilia-driven particle transport speed (PTS) on the mucosal surface of opened oviductal segments in correlation with histomorphological investigations. RT-PCR of laser-assisted microdissected epithelium revealed expression of Chrm subtypes Chrm1 and Chrm3. In opened isthmic segments, particle transport was barely seen correlating with a significantly lower number of ciliated cells compared to the ampulla. In the ampulla, basal PTS and CBF were high, 71 µm/sec and 21 Hz, respectively, both in cycling and pregnant wild-type mice and in mice with targeted deletion of the Chrm genes Chrm1, Chrm3, Chrm4, and Chrm5. In contrast to the trachea, where basal ciliary activity was low and was largely enhanced by muscarinic stimulation, muscarinic agonists and antagonists did not affect the high ampullar PTS. Our results imply that this high oviductal autonomous ciliary activity is independent from the intrinsic cholinergic system and serves to maintain optimal clearance of the tube throughout all cyclic stages and early pregnancy.

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

  Authors: Renate Paddenberg, Martina Tiefenbach, Petra Faulhammer, Anna Goldenberg, Barbara Gries, Uwe Pfeil, Katrin S Lips, José I Piruat, José López-Barneo, Ralph T Schermuly, Norbert Weissmann, Wolfgang Kummer

  Cardiovascular research. 01/2012; 93(4):702-10.

  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 molecularAlveolar 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(+/-)). 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. 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.

• Intermedin Stabilized Endothelial Barrier Function and Attenuated Ventilator-induced Lung Injury in Mice.

  Authors: Holger Christian Müller-Redetzky, Wolfgang Kummer, Uwe Pfeil, Katharina Hellwig, Daniel Will, Renate Paddenberg, Christoph Tabeling, Stefan Hippenstiel, Norbert Suttorp, Martin Witzenrath

  PloS one. 01/2012; 7(5):e35832.

  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 injuryEven 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. 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. IMD may possibly provide a new approach to attenuate VILI.

• Stereological characterization of left ventricular cardiomyocytes, capillaries, and innervation in the nondiabetic, obese mouse.

  Authors: Carina Gruber, Karin Kohlstedt, Annemarieke E Loot, Ingrid Fleming, Wolfgang Kummer, Christian Mühlfeld

  Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology. 12/2011;

  BACKGROUND: Obesity is associated with left ventricular hypertrophy and dysfunction, but little is known about the structural remodeling of cardiomyocytes, capillaries, and nerve fibers in thisBACKGROUND: Obesity is associated with left ventricular hypertrophy and dysfunction, but little is known about the structural remodeling of cardiomyocytes, capillaries, and nerve fibers in this state. We hypothesized that all three compartments should show quantitative structural alterations. METHODS: Ten C57Bl6 mice were randomly assigned to a control or obesity group. Lean mice received standard chow, whereas obese mice received a high-fat Western diet. After 28 weeks, the mice were sacrificed, and the hearts were prepared for design-based stereology using light and electron microscopy. RESULTS: Body mass and left ventricular mass were significantly elevated in obese vs. control mice. The left ventricular hypertrophy was accompanied by a significant increase in cardiomyocyte lipid droplets and total myocyte volume. The volume fractions of myofibrils, free sarcoplasm, and mitochondria did not differ between the groups. The total length of capillaries was significantly enhanced in obese vs. control mice, whereas the total length of axons ramifying between cardiomyocytes was not different. CONCLUSIONS: Obesity is associated with significant structural alterations in cardiomyocytes and capillaries, whereas no structural changes in the myocardial innervation were observed. The structural characteristics in obese mice do not provide a clear basis for functional changes observed in obesity-related cardiac hypertrophy.

• Pulmonary vascular innervation and its role in responses to hypoxia: size matters!

  Authors: Wolfgang Kummer

  Proceedings of the American Thoracic Society. 11/2011; 8(6):471-6.

  The pulmonary vasculature is innervated by specific subsets of sympathetic, parasympathetic, and sensory nerve fibers. In contrast to most other organs, innervation density is highest atThe pulmonary vasculature is innervated by specific subsets of sympathetic, parasympathetic, and sensory nerve fibers. In contrast to most other organs, innervation density is highest at large-caliber vessels and decreases toward the periphery, and reactivity to vasoactive compounds also changes along the course. In some species, such as the experimentally widely used rodents rat and mouse, autonomic efferent (sympathetic and parasympathetic) perivascular axons barely reach beyond the lung hilus, whereas in humans this innervation extends to small intrapulmonary vessels. Throughout, the most distal arterioles (i.e., intraacinar arteries equipped with an incomplete coat of intermediate cells instead of a full muscular wall) are devoid of innervation. Altogether, 10 vasoactive substances (3 small molecular transmitters and 7 neuropeptides) at minimum have been identified in various combinations (cotransmission) in pulmonary vascular axons. Analysis of this "neurochemical coding" has been provided only for the guinea pig so far, but not for humans or for animal species commonly used for pulmonary vascular research. Sympathetic pulmonary vascular neurons are reflexively activated via arterial chemoreceptors when arterial Po(2) is lowered and adapt the pulmonary vasculature to this condition of increased pulmonary blood flow by α(1)-adrenoreceptor-mediated increase in vascular impedance primarily at large vessels. In contrast, neither they nor other nerve fibers play a role in the local hypoxic vasoconstriction triggered by low alveolar Po(2), which serves to match perfusion to ventilation. The major potential role of the pulmonary vascular innervation, autonomic and sensory, lies in the pronounced trophic activities of its transmitters.

• Evidence for functional atypical nicotinic receptors that activate K+-dependent Cl- secretion in mouse tracheal epithelium.

  Authors: Monika I Hollenhorst, Katrin S Lips, Ariane Weitz, Gabriela Krasteva, Wolfgang Kummer, Martin Fronius

  American journal of respiratory cell and molecular biology. 08/2011; 46(1):106-14.

  The present study focused on the influence of nicotinic acetylcholine receptors (nAChR) on ion transport processes in mouse tracheal epithelium. RT-PCR experiments revealed expression of the α3, α4,The present study focused on the influence of nicotinic acetylcholine receptors (nAChR) on ion transport processes in mouse tracheal epithelium. RT-PCR experiments revealed expression of the α3, α4, α5, α7, α9, α10, β2, and β4 nAChR subunits in mouse tracheal epithelium. In Ussing chamber recordings of mouse tracheae, apically applied nicotine (100 μM) induced a dose-dependent increase of the transepithelial short-circuit current (EC(50): 14.6 μM). The nicotine-induced effect (I(NIC)) was attenuated by mecamylamine (25 μM, apical) and methyllycaconitine (1 μM, apical). The nAChR agonist 1.1-dimethyl-4-phenylpiperatinium iodide (DMPP) (100 μM) revealed apical and basolateral location of the receptors. I(NIC) was not affected by the sodium channel inhibitor amiloride (10 μM, apical) or the cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172 (20 μM, apical) but was reduced by the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM, apical), the Na(+)/K(+)/2Cl(-) cotransporter inhibitor bumetanide (200 μM, basolateral), the potassium channel inhibitor Ba(2+) (5 mM, basolateral), and 4.4'-diisothiocyanatostilbene-2.2'-disulfonate (100 μM, apical), indicating a contribution of Ca(2+)-activated chloride channels and potassium channels. Removal of extracellular Na(+) (apical) or Ca(2+) (apical) did not influence I(NIC) but reduced the DMPP effect. Experiments with the Ca(2+)-ionophore A23187, a mix of 3-isobutyl-1-methylxanthine and forskolin, or the inositol-1,4,5-triphospate (IP(3)) receptor inhibitor 2-aminoethyl-diphenyl-borinate (75 μM, apical) decreased I(NIC), indicating a nicotine-mediated increase of intracellular Ca(2+) and cAMP levels involving the IP(3) signaling pathway. These findings indicate the activity of Ca(2+)-permeable nAChRs and alternative metabotropic pathways by nAChR activation that mediate Cl(-) and K(+) transport in tracheal epithelium.

• Cholinergic chemosensory cells in the trachea regulate breathing.

  Authors: Gabriela Krasteva, Brendan J Canning, Petra Hartmann, Tibor Z Veres, Tamara Papadakis, Christian Mühlfeld, Kirstin Schliecker, Yvonne N Tallini, Armin Braun, Holger Hackstein, Nelli Baal, Eberhard Weihe, Burkhard Schütz, Michael Kotlikoff, Ines Ibanez-Tallon, Wolfgang Kummer

  Proceedings of the National Academy of Sciences of the United States of America. 06/2011; 108(23):9478-83.

  In the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. MorphologicallyIn the epithelium of the lower airways, a cell type of unknown function has been termed "brush cell" because of a distinctive ultrastructural feature, an apical tuft of microvilli. Morphologically similar cells in the nose have been identified as solitary chemosensory cells responding to taste stimuli and triggering trigeminal reflexes. Here we show that brush cells of the mouse trachea express the receptors (Tas2R105, Tas2R108), the downstream signaling molecules (α-gustducin, phospholipase C(β2)) of bitter taste transduction, the synthesis and packaging machinery for acetylcholine, and are addressed by vagal sensory nerve fibers carrying nicotinic acetylcholine receptors. Tracheal application of an nAChR agonist caused a reduction in breathing frequency. Similarly, cycloheximide, a Tas2R108 agonist, evoked a drop in respiratory rate, being sensitive to nicotinic receptor blockade and epithelium removal. This identifies brush cells as cholinergic sensors of the chemical composition of the lower airway luminal microenvironment that are directly linked to the regulation of respiration.

• Expression and functional characterization of P2X receptors in mouse alveolar macrophages.

  Authors: Sarah Kessler, Wolfgang G Clauss, Andreas Günther, Wolfgang Kummer, Martin Fronius

  Pflügers Archiv : European journal of physiology. 06/2011; 462(3):419-30.

  Alveolar macrophages (AM) are crucial for pulmonary host defense, and evidence emerges that ATP-gated P2X receptors are involved in inflammatory processes. This study focuses on the expression andAlveolar macrophages (AM) are crucial for pulmonary host defense, and evidence emerges that ATP-gated P2X receptors are involved in inflammatory processes. This study focuses on the expression and functional characterization of P2X receptors in AM from mouse. In RT-PCR experiments, transcripts encoding the P2X₁, P2X₃, P2X₄, P2X₅, and P2X₇ receptors were detected. In whole-cell patch-clamp recordings, ATP (1 mM) evoked an inward current (mouse and human AM) that was reversible upon washout, and the reversal potential was ~5 mV, indicating the activation of a non-selective conductance-a fingerprint of P2X receptors. Further characterization (mouse AM) revealed that the current was not desensitized by a second ATP application. The ATP-induced current was increased by the removal of extracellular Ca²⁺ (in human and mouse AM), and EC₅₀ in mouse AM were determined with ~1 mM ATP, in the presence as well as in the absence of extracellular Ca²⁺. Pharmacological characterization of mouse AM revealed that the effect was augmented by BzATP and pre-application with ivermectin, but no effect with α,β-meATP was observed. Further, the ATP effect was reduced by PPADS (300 μM), brilliant blue G (5 μM), and about A438079 (10 μM). Although different P2X receptor transcripts were detected in mouse AM, the observed functional and pharmacological characteristics indicate primarily the participation of P2X₄ and P2X₇ receptors as mediators of the ATP-induced ion current in mouse AM. These suggestions were confirmed by experiments with AM from P2X₇ -deficient animals, indicating a contribution of P2X₄ and P2X₇ receptors in pulmonary immune function.

• The cholinergic system in rat testis is of non-neuronal origin.

  Authors: S U Schirmer, I Eckhardt, H Lau, J Klein, Y C DeGraaf, K S Lips, C Pineau, I L Gibbins, W Kummer, A Meinhardt, R V Haberberger

  Reproduction (Cambridge, England). 04/2011; 142(1):157-66.

  The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known asThe cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs for Chat, Slc18a3, Slc5a7 and Slc22a2 as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three β subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.

• Acetylcholine and chronic vasculopathy in rat renal allografts.

  Authors: Joanna Wilczynska, Uwe Pfeil, Anna Zakrzewicz, Hartmut Dietrich, Christian Körner, Andreas Hecker, Ignaz Wessler, Winfried Padberg, Wolfgang Kummer, Veronika Grau

  Transplantation. 02/2011; 91(3):263-70.

  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 noChronic 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. 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. 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. We suggest that endogenous ACh contributes to the pathogenesis of CAV and may be a promising target for novel therapies preventing CAV.

• Cancer induces cardiomyocyte remodeling and hypoinnervation in the left ventricle of the mouse heart.

  Authors: Christian Mühlfeld, Suman Kumar Das, Frank R Heinzel, Albrecht Schmidt, Heiner Post, Silvia Schauer, Tamara Papadakis, Wolfgang Kummer, Gerald Hoefler

  PloS one. 01/2011; 6(5):e20424.

  Cancer is often associated with cachexia, cardiovascular symptoms and autonomic dysregulation. We tested whether extracardiac cancer directly affects the innervation of left ventricular myocardium.Cancer is often associated with cachexia, cardiovascular symptoms and autonomic dysregulation. We tested whether extracardiac cancer directly affects the innervation of left ventricular myocardium. Mice injected with Lewis lung carcinoma cells (tumor group, TG) or PBS (control group, CG) were analyzed after 21 days. Cardiac function (echocardiography), serum levels of TNF-α and Il-6 (ELISA), structural alterations of cardiomyocytes and their innervation (design-based stereology) and levels of innervation-related mRNA (quantitative RT-PCR) were analysed. The groups did not differ in various functional parameters. Serum levels of TNF-α and Il-6 were elevated in TG. The total length of axons in the left ventricle was reduced. The number of dense core vesicles per axon profile was reduced. Decreased myofibrillar volume, increased sarcoplasmic volume and increased volume of lipid droplets were indicative of metabolic alterations of TG cardiomyocytes. In the heart, the mRNA level of nerve growth factor was reduced whereas that of β1-adrenergic receptor was unchanged in TG. In the stellate ganglion of TG, mRNA levels of nerve growth factor and neuropeptide Y were decreased and that of tyrosine hydroxylase was increased. In summary, cancer induces a systemic pro-inflammatory state, a significant reduction in myocardial innervation and a catabolic phenotype of cardiomyocytes in the mouse. Reduced expression of nerve growth factor may account for the reduced myocardial innervation.

• The antimicrobial peptide cathelicidin enhances activation of lung epithelial cells by LPS.

  Authors: Renat Shaykhiev, Johannes Sierigk, Christian Herr, Gabriela Krasteva, Wolfgang Kummer, Robert Bals

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 12/2010; 24(12):4756-66.

  Epithelial cells (ECs) are usually hyporesponsive to various microbial products. Detection of lipopolysaccharide (LPS), the major component of gram-negative bacteria, is impeded, at least in part, byEpithelial cells (ECs) are usually hyporesponsive to various microbial products. Detection of lipopolysaccharide (LPS), the major component of gram-negative bacteria, is impeded, at least in part, by intracellular sequestration of its receptor, Toll-like receptor-4 (TLR4). In this study, using human bronchial ECs (hBECs) as a model of mucosal epithelium, we tested the hypothesis that the human LPS-binding, membrane-active cationic host defense peptide cathelicidin LL-37 augments epithelial response to LPS by facilitating its delivery to TLR4-containing intracellular compartments. We found that LL-37 significantly increases uptake of LPS by ECs with subsequent targeting to cholera toxin subunit B-labeled structures and lysosomes. This uptake is peptide specific, dose and time dependent, and involves the endocytotic machinery, functional lipid rafts, and epidermal growth factor receptor signaling. Cathelicidin-dependent LPS internalization resulted in significant increased release of the inflammatory cytokines IL-6 and IL-8. This indicates that, in ECs, this peptide may replace LPS-binding protein functions. In polarized ECs, the effect of LL-37 was restricted to the basolateral compartment of the epithelial membrane, suggesting that LL-37-mediated activation of ECs by LPS may be relevant to disease conditions associated with damage to the epithelial barrier. In summary, our study identified a novel role of LL-37 in host-microbe interactions as a host factor that licenses mucosal ECs to respond to LPS.

• Mitochondrial complex II participates in normoxic and hypoxic regulation of α-keto acids in the murine heart.

  Authors: Jörg Mühling, Martina Tiefenbach, José López-Barneo, José I Piruat, Paula García-Flores, Uwe Pfeil, Barbara Gries, Christian Mühlfeld, Markus A Weigand, Wolfgang Kummer, Norbert Weissmann, Renate Paddenberg

  Journal of molecular and cellular cardiology. 10/2010; 49(6):950-61.

  α-Keto acids (α-KAs) are not just metabolic intermediates but are also powerful modulators of different cellular pathways. Here, we tested the hypothesis that α-KA concentrations are regulated byα-Keto acids (α-KAs) are not just metabolic intermediates but are also powerful modulators of different cellular pathways. Here, we tested the hypothesis that α-KA concentrations are regulated by complex II (succinate dehydrogenase=SDH), which represents an intersection between the mitochondrial respiratory chain for which an important function in cardiopulmonary oxygen sensing has been demonstrated, and the Krebs cycle, a central element of α-KA metabolism. SDH subunit D heterozygous (SDHD(+/-)) and wild-type (WT) mice were housed at normoxia or hypoxia (10% O(2)) for 4 days or 3 weeks, and right ventricular pressure, right ventricle/(left ventricle+septum) ratio, cardiomyocyte ultrastructure, pulmonary vascular remodelling, ventricular complex II subunit expression, SDH activity and α-KA concentrations were analysed. In both strains, hypoxia induced increases in right ventricular pressure and enhanced muscularization of distal pulmonary arteries. Right ventricular hypertrophy was less severe in SDHD(+/-) mice although the cardiomyocyte ultrastructure and mitochondrial morphometric parameters were unchanged. Protein amounts of SDHA, SDHB and SDHC, and SDH activity were distinctly reduced in SDHD(+/-) mice. In normoxic SDHD(+/-) mice, α-ketoisocaproate concentration was lowered to 50% as compared to WT animals. Right/left ventricular concentration differences and the hypoxia-induced decline in individual α-KAs were less pronounced in SDHD(+/-) animals indicating that mitochondrial complex II participates in the adjustment of cardiac α-KA concentrations both under normoxic and hypoxic conditions. These characteristics are not related to the hemodynamic consequences of hypoxia-induced pulmonary vascular remodelling, since its extent and right ventricular pressure were not affected in SDHD(+/-) mice albeit right ventricular hypertrophy was attenuated.

• Increased expression of epidermal fatty acid-binding protein by alveolar macrophages during acute rejection of rat lungs.

  Authors: Julia Holler, Anna Zakrzewicz, Holger Garn, Markus Hirschburger, Wolfgang Kummer, Winfried Padberg, Veronika Grau

  APMIS : acta pathologica, microbiologica, et immunologica Scandinavica. 10/2010; 118(10):791-800.

  In the lung, epidermal fatty acid-binding protein (E-FABP) is expressed by alveolar macrophages (AM) and alveolar epithelial cells type II (AEII). E-FABP may regulate macrophage activation and isIn the lung, epidermal fatty acid-binding protein (E-FABP) is expressed by alveolar macrophages (AM) and alveolar epithelial cells type II (AEII). E-FABP may regulate macrophage activation and is involved in the metabolism of surfactant phospholipids. As macrophage activation and surfactant dysfunction are associated with rejection, we hypothesize that E-FABP expression is changed during acute rejection of pulmonary grafts. Orthotopic left lung transplantations were performed in the Dark Agouti to Lewis and in the isogeneic Lewis to Lewis rat strain combinations. E-FABP expression was analyzed in the lung by immunohistochemistry, immunoblotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR). Alveolar leukocytes obtained by bronchoalveolar lavage were analyzed by RT-PCR. Immunohistochemistry of isografts revealed strong E-FABP immunoreactivity in AEII and a moderate immunoreactivity in AM. In allografts undergoing acute rejection, AM exhibiting increased E-FABP immunoreactivity accumulated. Immunoblots revealed a single band at 15 kDa, which corresponds to the expected molecular mass of E-FABP. The levels of E-FABP mRNA were higher in allografts than in isografts and control lungs. Furthermore, alveolar leukocytes isolated by bronchoalveolar lavage from allografts displayed higher E-FABP mRNA expression levels than leukocytes from isografts and controls. In conclusion, we demonstrate for the first time upregulation of E-FABP expression in AM during severe inflammation.

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

  Authors: Zbigniew Mikulski, Petra Hartmann, Gitte Jositsch, Zbigniew Zasłona, Katrin S Lips, Uwe Pfeil, Hjalmar Kurzen, Jürgen Lohmeyer, Wolfgang G Clauss, Veronika Grau, Martin Fronius, Wolfgang Kummer

  Respiratory research. 09/2010; 11:133.

  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, weNicotinic 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. 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. Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. 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. 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.

• Serotonin activates murine alveolar macrophages through 5-HT2C receptors.

  Authors: Zbigniew Mikulski, Zbigniew Zaslona, Lidija Cakarova, Petra Hartmann, Jochen Wilhelm, Laurence H Tecott, Juergen Lohmeyer, Wolfgang Kummer

  American journal of physiology. Lung cellular and molecular physiology. 08/2010; 299(2):L272-80.

  Serotonin (5-HT), known as neuromodulator, regulates immune responses and inflammatory cascades. The expression and function of 5-HT receptors on alveolar macrophages (AM), which are the majorSerotonin (5-HT), known as neuromodulator, regulates immune responses and inflammatory cascades. The expression and function of 5-HT receptors on alveolar macrophages (AM), which are the major fraction of pulmonary immune cells, remain elusive. Therefore, we determined the expression of 5-HT type 2 receptors and investigated the effects evoked by stimulation with 5-HT in AM compared with alveolar epithelial cells (AEC). Quantitative PCR (qPCR) analysis revealed expression of the receptors 5-HT(2A) and 5-HT(2B) in AEC and of 5-HT(2C) in AM. In AM, 5-HT (10(-5) M) induced a rise in intracellular calcium concentration ([Ca(2+)](i)) that was initiated by release of Ca(2+) from intracellular stores and depended on extracellular Ca(2+) in a sustained phase. This 5-HT-induced increase in [Ca(2+)](i) was not observed in AM treated with the 5-HT(2C)-selective inhibitor RS-102221 and in AM derived from 5-HT(2C)-deficient mice. AM stimulated with 5-HT (10(-5) M) showed increased expression of CCL2 (MCP-1) mRNA as assayed by qPCR at 4 h and augmented production of CCL2 protein as determined by dot-blot assay and ELISA at 24 h. Notably, in 5-HT(2C)-deficient AM, CCL2 production was not induced by 5-HT treatment. Moreover, transcriptional responses to 5-HT exposure assayed by microarray experiments were only observed in AM from wild-type animals and not in AM derived from 5-HT(2C)-deficient mice. Taken together, these data demonstrate the presence of functional 5-HT(2C) receptors on AM and suggest a role of 5-HT as novel modulator of AM function. These effects are exclusively driven by the 5-HT(2C) receptor, thereby providing the potential for selective intervention.

• Is length an appropriate estimator to characterize pulmonary alveolar capillaries? A critical evaluation in the human lung.

  Authors: Christian Mühlfeld, Ewald R Weibel, Ute Hahn, Wolfgang Kummer, Jens R Nyengaard, Matthias Ochs

  Anatomical record (Hoboken, N.J. : 2007). 07/2010; 293(7):1270-5.

  Stereological estimations of total capillary length have been used to characterize changes in the alveolar capillary network (ACN) during developmental processes or pathophysiological conditions.Stereological estimations of total capillary length have been used to characterize changes in the alveolar capillary network (ACN) during developmental processes or pathophysiological conditions. Here, we analyzed whether length estimations are appropriate to describe the 3D nature of the ACN. Semi-thin sections of five human lungs, previously investigated by Gehr et al. (Respir Physiol 1978; 32:121-140), were used to estimate alveolar capillary length using a "design-based" or a "model-based" stereological approach. The design-based approach involves counting of capillary profiles related to a defined area of the reference space. The model-based approach bases on the assumption that capillaries are round tubes and length was calculated from capillary volume and surface area. The model-based approach provided a mean of 6,950 km (SD: 3,108 km) for total capillary length, the design-based approach resulted in a mean of 2,746 km (SD: 722 km). Because of the geometry of the ACN both approaches carry an unpredictable bias. The bias incurred by the design-based approach is proportional to the ratio between radius and length of the capillary segments in the ACN, the number of branching points and the winding of the capillaries. The model-based approach is biased because of the real noncylindrical shape of capillaries and the network structure. In conclusion, the estimation of the total length of capillaries in the ACN cannot be recommended as the geometry of the ACN does not fulfill the requirements for stereological length estimation. Until new methods are being developed, the unbiased estimates of capillary volume, and surface area should be preferred.