Increased Levels of BAMBI Inhibit Canonical TGF-ß Signaling in Chronic Wound Tissues.

Chronic wounds affect more than 2% of the population worldwide, with a significant burden on affected individuals, healthcare systems, and societies. A key regulator of the entire wound healing cascade is transforming growth factor beta (TGF-ß), which regulates not only inflammation and extracellular matrix formation but also revascularization. This present work aimed at characterizing wound tissues obtained from acute and chronic wounds regarding angiogenesis, inflammation, as well as ECM formation and degradation, to identify common disturbances in the healing process. Serum and wound tissues from 38 patients (N = 20 acute and N = 18 chronic wounds) were analyzed. The patients' sera suggested a shift from VEGF/VEGFR to ANGPT/TIE2 signaling in the chronic wounds. However, this shift was not confirmed in the wound tissues. Instead, the chronic wound tissues showed increased levels of MMP9, a known activator of TGF-ß. However, regulation of TGF-ß target genes, such as CTGF, COL1A1, or IL-6, was absent in the chronic wounds. In wound tissues, all three TGF-ß isoforms were expressed with increased levels of TGF-ß1 and TGF-ß3 and a reporter assay confirmed that the expressed TGF-ß was activated. However, Western blots and immunostaining showed decreased canonical TGF-ß signaling in the respective chronic wound tissues, suggesting the presence of a TGF-ß inhibitor. As a potential regulatory mechanism, the TGF-ß proteome profiler array suggested elevated levels of the TGF-ß pseudo-receptor BAMBI. Also, tissue expression of BAMBI was significantly increased not only in chronic wounds (10.6-fold) but also in acute wounds that had become chronic (9.5-fold). In summary, our data indicate a possible regulatory role of BAMBI in the development of chronic wounds. The available few in vivo studies support our findings by postulating a therapeutic potential of BAMBI for controlling scar formation.

Characterization of Shear Stress Mediated Platelet Dysfunction: Data from an Ex Vivo Model for Extracorporeal Circulation and a Prospective Clinical Study.

Extracorporeal circulation (ECC) is frequently used in intensive care patients with impaired lung or cardiac function. Despite being a life-saving therapeutic option, ECC is associated with increased risk for both bleeding and thrombosis. The management of bleeding and thromboembolic events in ECC patients is still challenging partly due to the lack of information on the pathophysiological changes in hemostasis and platelet function during the procedure. Using a combination of an ex vivo model for shear stress and a sensitive and easy-to-use laboratory method, we analyzed platelet responsiveness during ECC. After shear stress simulation in an ex vivo closed-loop ECC model, we found a significantly decreased response of α-granules after activation with adenosine diphosphate and thrombin receptor activating peptide (TRAP-6) and CD63 expression after activation with TRAP-6. Mepacrine uptake was also significantly reduced in the ex vivo shear stress model.In the same line, platelets from patients under ECC with venovenous systems and venoarterial systems showed impaired CD62P degranulation after stimulation with ADP and TRAP-6 compared with healthy control on day 1, 6, and 10 after implantation of ECC. However, no correlation between platelet degranulation and the occurrence of bleeding or thromboembolic events was observed.The used whole blood flow cytometry with immediate fixation after drawing introduces a sensitive and easy-to-use method to determine platelet activation status and our data confirm that increased shear stress conditions under ECC can cause impaired degranulation of platelet.

Occurrence and Severity of Venous Air Embolism During Neurosurgical Procedures: Semisitting Versus Supine Position.

BACKGROUND: At our institution, patients undergoing neurosurgical procedures in the posterior cranial fossa are placed either in the semisitting or in the supine position. The major risk of the semisitting positioning is a venous air embolism (VAE), which may, however, also occur in the supine position. METHODS: In a prospective single-center study with 137 patients, we evaluated the occurrence of VAEs in patients in the supine and in the semisitting position during the period from January 2014 until April 2015. All patients were monitored for VAE by the use of a transesophageal echocardiography (TEE). RESULTS: In total, 50% of the patients experienced a VAE (56% of these patients underwent surgery in the semisitting and 11% in the supine position). In total, 86% of the VAEs were detected by the use of a TEE and did not lead to any changes in the end-expiratory CO2. We only observed VAEs with a decrease in end-expiratory CO2 in the semisitting position. However, none of the patients had any hemodynamic changes due to the VAE. CONCLUSIONS: The semisitting position with TEE monitoring and a standardized protocol is a safe and advantageous technique, taking account of a significant rate of VAEs. VAEs also occur in the supine position, but less frequently.

Sevoflurane Dampens Acute Pulmonary Inflammation via the Adenosine Receptor A2B and Heme Oxygenase-1.

Acute respiratory distress syndrome is a life-threatening disease associated with high mortality. The adenosine receptor A2B (Adora2b) provides anti-inflammatory effects, which are also associated with the intracellular enzyme heme oxygenase-1 (HO-1). Our study determined the mechanism of sevoflurane's protective properties and investigated the link between sevoflurane and the impact of a functional Adora2b via HO-1 modulation during lipopolysaccharide (LPS)-induced lung injury. We examined the LPS-induced infiltration of polymorphonuclear neutrophils (PMNs) into the lung tissue and protein extravasation in wild-type and Adora2b-/- animals. We generated chimeric animals, to identify the impact of sevoflurane on Adora2b of hematopoietic and non-hematopoietic cells. Sevoflurane decreased the LPS-induced PMN-infiltration and diminished the edema formation in wild-type mice. Reduced PMN counts after sevoflurane treatment were detected only in chimeric mice, which expressed Adora2b exclusively on leukocytes. The Adora2b on hematopoietic and non-hematopoietic cells was required to improve the permeability after sevoflurane inhalation. Further, sevoflurane increased the protective effects of HO-1 modulation on PMN migration and microvascular permeability. These protective effects were abrogated by specific HO-1 inhibition. In conclusion, our data revealed new insights into the protective mechanisms of sevoflurane application during acute pulmonary inflammation and the link between sevoflurane and Adora2b, and HO-1 signaling, respectively.

CXCR4 and CXCR7 Inhibition Ameliorates the Formation of Platelet-Neutrophil Complexes and Neutrophil Extracellular Traps through Adora2b Signaling.

Peritonitis and peritonitis-associated sepsis are characterized by an increased formation of platelet-neutrophil complexes (PNCs), which contribute to an excessive migration of polymorphonuclear neutrophils (PMN) into the inflamed tissue. An important neutrophilic mechanism to capture and kill invading pathogens is the formation of neutrophil extracellular traps (NETs). Formation of PNCs and NETs are essential to eliminate pathogens, but also lead to aggravated tissue damage. The chemokine receptors CXCR4 and CXCR7 on platelets and PMNs have been shown to play a pivotal role in inflammation. Thereby, CXCR4 and CXCR7 were linked with functional adenosine A2B receptor (Adora2b) signaling. We evaluated the effects of selective CXCR4 and CXCR7 inhibition on PNCs and NETs in zymosan- and fecal-induced sepsis. We determined the formation of PNCs in the blood and, in addition, their infiltration into various organs in wild-type and Adora2b-/- mice by flow cytometry and histological methods. Further, we evaluated NET formation in both mouse lines and the impact of Adora2b signaling on it. We hypothesized that the protective effects of CXCR4 and CXCR7 antagonism on PNC and NET formation are linked with Adora2b signaling. We observed an elevated CXCR4 and CXCR7 expression in circulating platelets and PMNs during acute inflammation. Specific CXCR4 and CXCR7 inhibition reduced PNC formation in the blood, respectively, in the peritoneal, lung, and liver tissue in wild-type mice, while no protective anti-inflammatory effects were observed in Adora2b-/- animals. In vitro, CXCR4 and CXCR7 antagonism dampened PNC and NET formation with human platelets and PMNs, confirming our in vivo data. In conclusion, our study reveals new protective aspects of the pharmacological modulation of CXCR4 and CXCR7 on PNC and NET formation during acute inflammation.

Sevoflurane Exerts Protective Effects in Murine Peritonitis-induced Sepsis via Hypoxia-inducible Factor 1α/Adenosine A2B Receptor Signaling.

BACKGROUND: Sepsis is one of the leading causes of mortality in intensive care units, and sedation in the intensive care unit during sepsis is usually performed intravenously. The inhalative anesthetic sevoflurane has been shown to elicit protective effects in various inflammatory studies, but its role in peritonitis-induced sepsis remains elusive. The hypothesis was that sevoflurane controls the neutrophil infiltration by stabilization of hypoxia-inducible factor 1α and elevated adenosine A2B receptor expression. METHODS: In mouse models of zymosan- and fecal-induced peritonitis, male mice were anesthetized with sevoflurane (2 volume percent, 30 min) after the onset of inflammation. Control animals received the solvent saline. The neutrophil counts and adhesion molecules on neutrophils in the peritoneal lavage of wild-type, adenosine A2B receptor -/-, and chimeric animals were determined by flow cytometry 4 h after stimulation. Cytokines and protein release were determined in the lavage. Further, the adenosine A2B receptor and its transcription factor hypoxia-inducible factor 1α were evaluated by real-time polymerase chain reaction and Western blot analysis 4 h after stimulation. RESULTS: Sevoflurane reduced the neutrophil counts in the peritoneal lavage (mean ± SD, 25 ± 17 × 105vs. 12 ± 7 × 105 neutrophils; P = 0.004; n = 19/17) by lower expression of various adhesion molecules on neutrophils of wild-type animals but not of adenosine A2B receptor -/- animals. The cytokines concentration (means ± SD, tumor necrosis factor α [pg/ml], 523 ± 227 vs. 281 ± 101; P = 0.002; n = 9/9) and protein extravasation (mean ± SD [mg/ml], 1.4 ± 0.3 vs. 0.8 ± 0.4; P = 0.002; n = 12/11) were also lower after sevoflurane only in the wild-type mice. Chimeric mice showed the required expression of the adenosine A2B receptor on the hematopoietic and nonhematopoietic compartments for the protective effects of the anesthetic. Sevoflurane induced the expression of hypoxia-inducible factor 1α and adenosine A2B receptor in the intestine, liver, and lung. CONCLUSIONS: Sevoflurane exerts various protective effects in two murine peritonitis-induced sepsis models. These protective effects were linked with a functional adenosine A2B receptor.

Inhibition of CXCR4 and CXCR7 Is Protective in Acute Peritoneal Inflammation.

Our previous studies revealed a pivotal role of the chemokine stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 on migratory behavior of polymorphonuclear granulocytes (PMNs) in pulmonary inflammation. Thereby, the SDF-1-CXCR4/CXCR7-axis was linked with adenosine signaling. However, the role of the SDF-1 receptors CXCR4 and CXCR7 in acute inflammatory peritonitis and peritonitis-related sepsis still remained unknown. The presented study provides new insight on the mechanism of a selective inhibition of CXCR4 (AMD3100) and CXCR7 (CCX771) in two models of peritonitis and peritonitis-related sepsis by injection of zymosan and fecal solution. We observed an increased expression of SDF-1, CXCR4, and CXCR7 in peritoneal tissue and various organs during acute inflammatory peritonitis. Selective inhibition of CXCR4 and CXCR7 reduced PMN accumulation in the peritoneal fluid and infiltration of neutrophils in lung and liver tissue in both models. Both inhibitors had no anti-inflammatory effects in A2B knockout animals (A2B-/-). AMD3100 and CCX771 treatment reduced capillary leakage and increased formation of tight junctions as a marker for microvascular permeability in wild type animals. In contrast, both inhibitors failed to improve capillary leakage in A2B-/- animals, highlighting the impact of the A2B-receptor in SDF-1 mediated signaling. After inflammation, the CXCR4 and CXCR7 antagonist induced an enhanced expression of the protective A2B adenosine receptor and an increased activation of cAMP (cyclic adenosine mono phosphate) response element-binding protein (CREB), as downstream signaling pathway of A2B. The CXCR4- and CXCR7-inhibitor reduced the release of cytokines in wild type animals via decreased intracellular phosphorylation of ERK and NFκB p65. In vitro, CXCR4 and CXCR7 antagonism diminished the chemokine release of human cells and increased cellular integrity by enhancing the expression of tight junctions. These protective effects were linked with functional A2B-receptor signaling, confirming our in vivo data. In conclusion, our study revealed new protective aspects of the pharmacological modulation of the SDF-1-CXCR4/CXCR7-axis during acute peritoneal inflammation in terms of the two hallmarks PMN migration and barrier integrity. Both anti-inflammatory effects were linked with functional adenosine A2B-receptor signaling.

Detrimental Effects of an Inhaled Phosphodiesterase-4 Inhibitor on Lung Inflammation in Ventilated Preterm Lambs Exposed to Chorioamnionitis Are Dose Dependent.

Background: Treatment of bronchopulmonary dysplasia in preterm infants is challenging due to its multifactorial origin. In rodent models of neonatal lung injury, selective inhibition of phosphodiesterase 4 (PDE4) has been shown to exert anti-inflammatory properties in the lung. We hypothesized that GSK256066, a highly selective, inhalable PDE4 inhibitor, would have beneficial effects on lung injury and inflammation in a triple hit lamb model of Ureaplasma parvum (UP)-induced chorioamnionitis, prematurity, and mechanical ventilation. Methods: Twenty-one preterm lambs were surgically delivered preterm at 129 days after 7 days intrauterine exposure to UP. Sixteen animals were subsequently ventilated for 24 hours and received endotracheal surfactant and intravenous caffeine citrate. Ten animals were randomized to receive twice a high (10 µg/kg) or low dose (1 µg/kg) of nebulized PDE4 inhibitor. Results: Nebulization of high, but not low, doses of PDE4 inhibitor led to a significant decrease in pulmonary PDE activity, and was associated with lung injury and vasculitis, influx of neutrophils, and increased proinflammatory cytokine messenger RNA levels. Conclusion: Contrary to our hypothesis, we found in our model a dose-dependent proinflammatory effect of an inhaled highly selective PDE4 inhibitor in the lung. Our findings indicate the narrow therapeutic range of inhaled PDE4 inhibitors in the preterm population.

The Pivotal Role of CXCR7 in Stabilization of the Pulmonary Epithelial Barrier in Acute Pulmonary Inflammation.

Acute pulmonary inflammation is still a frightening complication in intensive care units and has a high mortality. Specific treatment is not available, and many details of the pathomechanism remain unclear. The recently discovered chemokine receptor CXCR7 and its ligand stromal cell-derived factor (SDF)-1 are known to be involved in inflammation. We chose to investigate the detailed role of CXCR7 in a murine model of LPS inhalation. Inflammation increased pulmonary expression of CXCR7, and the receptor was predominantly expressed on pulmonary epithelium and on polymorphonuclear neutrophil (PMNs) after transepithelial migration into the alveolar space. Specific inhibition of CXCR7 reduced transepithelial PMN migration by affecting the expression of adhesion molecules. CXCR7 antagonism reduced the most potent PMN chemoattractants CXCL1 and CXCL2/3. After inhibiting CXCR7, NF-κB phosphorylation was reduced in lungs of mice, tight junction formation increased, and protein concentration in the bronchoalveolar lavage diminished, showing the impact of CXCR7 on stabilizing microvascular permeability. In vitro studies with human cells confirmed the pivotal role of CXCR7 in pulmonary epithelium. Immunofluorescence of human lungs confirmed our in vivo data and showed an increase of the expression of CXCR7 in pulmonary epithelium. Highlighting the clinical potential of CXCR7 antagonism, nebulization of the agent before and after the inflammation showed impressive anti-inflammatory effects. Additional CXCR7 inhibition potentiated the effect of SDF-1 antagonism, most probably by downregulating SDF-1 and the second receptor of the chemokine (CXCR4) expression. In conclusion, our data identified the pivotal role of the receptor CXCR7 in pulmonary inflammation with a predominant effect on the pulmonary epithelium and PMNs.

Anti-inflammatory Effects of Heme Oxygenase-1 Depend on Adenosine A2A- and A2B-Receptor Signaling in Acute Pulmonary Inflammation.

Acute pulmonary inflammation is still a frightening complication in intensive care units. In our previous study, we determined that heme oxygenase (HO)-1 had anti-inflammatory effects in pulmonary inflammation. Recent literature has emphasized a link between HO-1 and the nucleotide adenosine. Since adenosine A2A- and A2B-receptors play a pivotal role in pulmonary inflammation, we investigated their link to the enzyme HO-1. In a murine model of pulmonary inflammation, the activation of HO-1 by hemin significantly decreased polymorphonuclear leukocyte (PMN) migration into the lung. This anti-inflammatory reduction of PMN migration was abolished in A2A- and A2B-knockout mice. Administration of hemin significantly reduced chemokine levels in the BAL of wild-type animals but had no effects in A2A-/- and A2B-/- mice. Microvascular permeability was significantly attenuated in HO-1-stimulated wild-type mice, but not in A2A-/- and A2B-/- mice. The activity of HO-1 rose after LPS inhalation in wild-type animals and, surprisingly, also in A2A-/- and A2B-/- mice after the additional administration of hemin. Immunofluorescence images of animals revealed alveolar macrophages to be the major source of HO-1 activity in both knockout strains-in contrast to wild-type animals, where HO-1 was also significantly augmented in the lung tissue. In vitro studies on PMN migration further confirmed our in vivo findings. In conclusion, we linked the anti-inflammatory effects of HO-1 to functional A2A/A2B-receptor signaling under conditions of pulmonary inflammation. Our findings may explain why targeting HO-1 in acute pulmonary inflammation has failed to prove effective in some patients, since septic patients have altered adenosine receptor expression.

The unrecognized effects of phosphodiesterase 4 on epithelial cells in pulmonary inflammation.

Acute pulmonary inflammation is characterized by migration of polymorphonuclear neutrophils (PMNs) into the different compartments of the lung, passing an endothelial and epithelial barrier. Recent studies showed evidence that phosphodiesterase (PDE)4-inhibitors stabilized endothelial cells. PDE4B and PDE4D subtypes play a pivotal role in inflammation, whereas blocking PDE4D is suspected to cause gastrointestinal side effects. We thought to investigate the particular role of the PDE4-inhibitors roflumilast and rolipram on lung epithelium. Acute pulmonary inflammation was induced by inhalation of LPS. PDE4-inhibitors were administered i.p. or nebulized after inflammation. The impact of PDE4-inhibitors on PMN migration was evaluated in vivo and in vitro. Microvascular permeability, cytokine levels, and PDE4B and PDE4D expression were analyzed. In vivo, both PDE4-inhibitors decreased transendothelial and transepithelial migration even when administered after inflammation, whereas roflumilast showed a superior effect compared to rolipram on the epithelium. Both inhibitors decreased TNFα, IL6, and CXCL2/3. CXCL1, the strong PMN chemoattractant secreted by the epithelium, was significantly more reduced by roflumilast. In vitro assays with human epithelium also emphasized the pivotal role of roflumilast on the epithelium. Additionally, LPS-induced stress fibers, an essential requirement for a direct migration of PMNs into the alveolar space, were predominantly reduced by roflumilast. Expression of PDE4B and PDE4D were both increased in the lungs by LPS, PDE4-inhibitors decreased mainly PDE4B. The topical administration of PDE4-inhibitors was also effective in curbing down PMN migration, further highlighting the clinical potential of these compounds. In pulmonary epithelial cells, both subtypes were found coexistent around the nucleus and the cytoplasm. In these epithelial cells, LPS increased PDE4B and, to a lesser extend, PDE4D, whereas the effect of the inhibitors was prominent on the PDE4B subtype. In conclusion, we determined the pivotal role of the PDE4-inhibitor roflumilast on lung epithelium and emphasized its main effect on PDE4B in hyperinflammation.

Heme oxygenase-1 attenuates acute pulmonary inflammation by decreasing the release of segmented neutrophils from the bone marrow.

Recruiting polymorphonuclear neutrophil granulocytes (PMNs) from circulation and bone marrow to the site of inflammation is one of the pivotal mechanisms of the innate immune system. During inflammation, the enzyme heme oxygenase 1 (HO-1) has been shown to reduce PMN migration. Although these effects have been described in various models, underlying mechanisms remain elusive. Recent studies revealed an influence of HO-1 on different cells of the bone marrow. We investigated the particular role of the bone marrow in terms of HO-1-dependent pulmonary inflammation. In a murine model of LPS inhalation, stimulation of HO-1 by cobalt (III) protoporphyrin-IX-chloride (CoPP) resulted in reduced segmented PMN migration into the alveolar space. In the CoPP group, segmented PMNs were also decreased intravascularly, and concordantly, mature and immature PMN populations were higher in the bone marrow. Inhibition of the enzyme by tin protoporphyrin-IX increased segmented and banded PMN migration into the bronchoalveolar lavage fluid with enhanced PMN release from the bone marrow and aggravated parameters of tissue inflammation. Oxidative burst activity was significantly higher in immature compared with mature PMNs. The chemokine stromal-derived factor-1 (SDF-1), which mediates homing of leukocytes into the bone marrow and is decreased in inflammation, was increased by CoPP. When SDF-1 was blocked by the specific antagonist AMD3100, HO-1 activation was no longer effective in curbing PMN trafficking to the inflamed lungs. In conclusion, we show evidence that the anti-inflammatory effects of HO-1 are largely mediated by inhibiting the release of segmented PMNs from the bone marrow rather than direct effects within the lung.

Protective effects of pentoxifylline in pulmonary inflammation are adenosine receptor A2A dependent.

Pentoxifylline (PTX) has been shown to exert anti-inflammatory effects in experimental acute lung injury. However, results in humans were controversial. Recent in vitro studies suggested that the adenosine receptor A2A may be required for PTX to be effective. Therefore, we studied the association between A2A and PTX in a murine model of LPS-induced pulmonary inflammation. PTX treatment (10 mg/kg) reduced cellular influx (by 40%), microvascular permeability (30%), and the release of chemotactic cytokines into the alveolar space (TNF-α 60%, IL-6 60%, and CXCL2/3 53%, respectively). These protective effects were abolished completely in A2A(-/-) mice and in wild-type mice that had been treated with the selective A2A antagonist (1 mg/kg), but effects were not different in mice with altered adenosine levels. In vitro transmigration assays revealed a pivotal role of the endothelium in PTX-mediated PMN migration, with a reduction of 50% (2 mM PTX). This effect was also A2A dependent. Further, oxidative burst of human PMNs was A2A-dependently reduced by 53% after PTX treatment. In summary, PTX exhibits its anti-inflammatory effects in LPS-induced lung injury through an A2A-dependent pathway. These results will help to better understand previous conflicting data on PTX in inflammation and will direct further studies to consider the predominant role of A2A.

Adenosine receptor A2b on hematopoietic cells mediates LPS-induced migration of PMNs into the lung interstitium.

Uncontrolled transmigration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lungs (intravascular, interstitial, alveolar) is a critical event in the early stage of acute lung injury and acute respiratory distress syndrome. Adenosine receptor A(2b) is highly expressed in the inflamed lungs and has been suggested to mediate cell trafficking. In a murine model of LPS-induced lung inflammation, we investigated the role of A(2b) on migration of PMNs into the different compartments of the lung. In A(2b)(-/-) mice, LPS-induced accumulation of PMNs was significantly higher in the interstitium, but not in the alveolar space. In addition, pulmonary clearance of PMNs was delayed in A(2b)(-/-) mice. Using chimeric mice, we identified A(2b) on hematopoietic cells as crucial for PMN migration. A(2b) did not affect the release of relevant chemokines into the alveolar space. LPS-induced microvascular permeability was under the control of A(2b) on both hematopoietic and nonhematopoietic cells. Activation of A(2b) on endothelial cells also reduced formation of LPS-induced stress fibers, highlighting its role for endothelial integrity. A specific A(2b) agonist (BAY 60-6583) was effective in decreasing PMN migration into the lung interstitium and microvascular permeability. In addition, in vitro transmigration of human PMNs through a layer of human endothelial or epithelial cells was A(2b) dependent. Activation of A(2b) on human PMNs reduced oxidative burst activity. Together, our results demonstrate anti-inflammatory effects of A(2b) on two major characteristics of acute lung injury, with a distinct role of hematopoietic A(2b) for cell trafficking and endothelial A(2b) for microvascular permeability.

Anticholinergic antiparkinson drug orphenadrine inhibits HERG channels: block attenuation by mutations of the pore residues Y652 or F656.

The anticholinergic antiparkinson drug orphenadrine is an antagonist at central and peripheral muscarinic receptors. Orphenadrine intake has recently been linked to QT prolongation and Torsade-de-Pointes tachycardia. So far, inhibitory effects on I (Kr) or cloned HERG channels have not been examined. HERG channels were heterologously expressed in a HEK 293 cell line and in Xenopus oocytes and HERG current was measured using the whole cell patch clamp and the double electrode voltage clamp technique. Orphenadrine inhibits cloned HERG channels in a concentration dependent manner, yielding an IC(50) of 0.85 microM in HEK cells. Onset of block is fast and reversible upon washout. Orphenadrine does not alter the half-maximal activation voltage of HERG channels. There is no shift of the half-maximal steady-state-inactivation voltage. Time constants of direct channel inactivation are not altered significantly and there is no use-dependence of block. HERG blockade is attenuated significantly in mutant channels lacking either of the aromatic pore residues Y652 and F656. In conclusion, we show that the anticholinergic agent orphenadrine is an antagonist at HERG channels. These results provide a novel molecular basis for the reported proarrhythmic side effects of orphenadrine.