Amitriptyline inhibits bronchoconstriction and directly promotes dilatation of the airways.

INTRODUCTION: The standard therapy for bronchial asthma consists of combinations of acute (short-acting ß2-sympathomimetics) and, depending on the severity of disease, additional long-term treatment (including inhaled glucocorticoids, long-acting ß2-sympathomimetics, anticholinergics, anti-IL-4R antibodies). The antidepressant amitriptyline has been identified as a relevant down-regulator of immunological TH2-phenotype in asthma, acting-at least partially-through inhibition of acid sphingomyelinase (ASM), an enzyme involved in sphingolipid metabolism. Here, we investigated the non-immunological role of amitriptyline on acute bronchoconstriction, a main feature of airway hyperresponsiveness in asthmatic disease. METHODS: After stimulation of precision cut lung slices (PCLS) from mice (wildtype and ASM-knockout), rats, guinea pigs and human lungs with mediators of bronchoconstriction (endogenous and exogenous acetylcholine, methacholine, serotonin, endothelin, histamine, thromboxane-receptor agonist U46619 and leukotriene LTD4, airway area was monitored in the absence of or with rising concentrations of amitriptyline. Airway dilatation was also investigated in rat PCLS by prior contraction induced by methacholine. As bronchodilators for maximal relaxation, we used IBMX (PDE inhibitor) and salbutamol (ß2-adrenergic agonist) and compared these effects with the impact of amitriptyline treatment. Isolated perfused lungs (IPL) of wildtype mice were treated with amitriptyline, administered via the vascular system (perfusate) or intratracheally as an inhalation. To this end, amitriptyline was nebulized via pariboy in-vivo and mice were ventilated with the flexiVent setup immediately after inhalation of amitriptyline with monitoring of lung function. RESULTS: Our results show amitriptyline to be a potential inhibitor of bronchoconstriction, induced by exogenous or endogenous (EFS) acetylcholine, serotonin and histamine, in PCLS from various species. The effects of endothelin, thromboxane and leukotrienes could not be blocked. In acute bronchoconstriction, amitriptyline seems to act ASM-independent, because ASM-deficiency (Smdp1-/-) did not change the effect of acetylcholine on airway contraction. Systemic as well as inhaled amitriptyline ameliorated the resistance of IPL after acetylcholine provocation. With the flexiVent setup, we demonstrated that the acetylcholine-induced rise in central and tissue resistance was much more marked in untreated animals than in amitriptyline-treated ones. Additionally, we provide clear evidence that amitriptyline dilatates pre-contracted airways as effectively as a combination of typical bronchodilators such as IBMX and salbutamol. CONCLUSION: Amitriptyline is a drug of high potential, which inhibits acute bronchoconstriction and induces bronchodilatation in pre-contracted airways. It could be one of the first therapeutic agents in asthmatic disease to have powerful effects on the TH2-allergic phenotype and on acute airway hyperresponsiveness with bronchoconstriction, especially when inhaled.

Platelet-derived growth factor (PDGF)-BB regulates the airway tone via activation of MAP2K, thromboxane, actin polymerisation and Ca2+-sensitisation.

BACKGROUND: PDGFR-inhibition by the tyrosine kinase inhibitor (TKI) nintedanib attenuates the progress of idiopathic pulmonary fibrosis (IPF). However, the effects of PDGF-BB on the airway tone are almost unknown. We studied this issue and the mechanisms beyond, using isolated perfused lungs (IPL) of guinea pigs (GPs) and precision-cut lung slices (PCLS) of GPs and humans. METHODS: IPL: PDGF-BB was perfused after or without pre-treatment with the TKI imatinib (perfused/nebulised) and its effects on the tidal volume (TV), the dynamic compliance (Cdyn) and the resistance were studied. PCLS (GP): The bronchoconstrictive effects of PDGF-BB and the mechanisms beyond were evaluated. PCLS (human): The bronchoconstrictive effects of PDGF-BB and the bronchorelaxant effects of imatinib were studied. All changes of the airway tone were measured by videomicroscopy and indicated as changes of the initial airway area. RESULTS: PCLS (GP/human): PDGF-BB lead to a contraction of airways. IPL: PDGF-BB decreased TV and Cdyn, whereas the resistance did not increase significantly. In both models, inhibition of PDGFR-(ß) (imatinib/SU6668) prevented the bronchoconstrictive effect of PDGF-BB. The mechanisms beyond PDGF-BB-induced bronchoconstriction include activation of MAP2K and TP-receptors, actin polymerisation and Ca2+-sensitisation, whereas the increase of Ca2+ itself and the activation of EP1-4-receptors were not of relevance. In addition, imatinib relaxed pre-constricted human airways. CONCLUSIONS: PDGFR regulates the airway tone. In PCLS from GPs, this regulatory mechanism depends on the ß-subunit. Hence, PDGFR-inhibition may not only represent a target to improve chronic airway disease such as IPF, but may also provide acute bronchodilation in asthma. Since asthma therapy uses topical application. This is even more relevant, as nebulisation of imatinib also appears to be effective.

Acid sphingomyelinase regulates TH 2 cytokine release and bronchial asthma.

BACKGROUND: Allergic diseases and especially allergic asthma are widespread diseases with high prevalence in childhood, but also in adults. Acid sphingomyelinase (ASM) is a key regulator of the sphingolipid pathway. Previous studies defined the association of ASM with the pathogenesis of TH 1-directed lung diseases like cystic fibrosis and acute lung injury. Here, we define the role of ASM in TH 2-regulated allergic bronchial asthma. METHODS: To determine the role of Asm under baseline conditions, wild-type (WT) and Asm-/- mice were ventilated with a flexiVent setup and bronchial hyperresponsiveness was determined using acetylcholine. Flow cytometry and cytokine measurements in bronchoalveolar lavage fluid and lung tissue were followed by in vitro TH 2 differentiations with cells from WT and Asm-/- mice and blockade of Asm with amitriptyline. As proof of principle, we conducted an ovalbumin-induced model of asthma in WT- and Asm-/-  mice. RESULTS: At baseline, Asm-/- mice showed better lung mechanics, but unaltered bronchial hyperresponsiveness. Higher numbers of Asm-/- T cells in bronchoalveolar lavage fluid released lower levels of IL-4 and IL-5, and these results were paralleled by decreased production of typical TH 2 cytokines in Asm-/- T lymphocytes in vitro. This phenotype could be imitated by incubation of T cells with amitriptyline. In the ovalbumin asthma model, Asm-/- animals were protected from high disease activity and showed better lung functions and lower levels of eosinophils and TH 2 cytokines. CONCLUSION: Asm deficiency could induce higher numbers of TH 2 cells in the lung, but those cells release decreased TH 2 cytokine levels. Hereby, Asm-/- animals are protected from bronchial asthma, which possibly offers novel therapeutic strategies, for example, with ASM blockade.

Tyrosine kinase inhibitors relax pulmonary arteries in human and murine precision-cut lung slices.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) inhibit the platelet derived growth factor receptor (PDGFR) and gain increasing significance in the therapy of proliferative diseases, e.g. pulmonary arterial hypertension (PAH). Moreover, TKIs relax pulmonary vessels of rats and guinea pigs. So far, it is unknown, whether TKIs exert relaxation in human and murine pulmonary vessels. Thus, we studied the effects of TKIs and the PDGFR-agonist PDGF-BB in precision-cut lung slices (PCLS) from both species. METHODS: The vascular effects of imatinib (mice/human) or nilotinib (human) were studied in Endothelin-1 (ET-1) pre-constricted pulmonary arteries (PAs) or veins (PVs) by videomicroscopy. Baseline initial vessel area (IVA) was defined as 100%. With regard to TKI-induced relaxation, K+-channel activation was studied in human PAs (PCLS) and imatinib/nilotinib-related changes of cAMP and cGMP were analysed in human PAs/PVs (ELISA). Finally, the contractile potency of PDGF-BB was explored in PCLS (mice/human). RESULTS: Murine PCLS: Imatinib (10 µM) relaxed ET-1-pre-constricted PAs to 167% of IVA. Vice versa, 100 nM PDGF-BB contracted PAs to 60% of IVA and pre-treatment with imatinib or amlodipine prevented PDGF-BB-induced contraction. Murine PVs reacted only slightly to imatinib or PDGF-BB. Human PCLS: 100 µM imatinib or nilotinib relaxed ET-1-pre-constricted PAs to 166% or 145% of IVA, respectively, due to the activation of KATP-, BKCa2+- or Kv-channels. In PVs, imatinib exerted only slight relaxation and nilotinib had no effect. Imatinib and nilotinib increased cAMP in human PAs, but not in PVs. In addition, PDGF-BB contracted human PAs/PVs, which was prevented by imatinib. CONCLUSIONS: TKIs relax pre-constricted PAs/PVs from both, mice and humans. In human PAs, the activation of K+-channels and the generation of cAMP are relevant for TKI-induced relaxation. Vice versa, PDGF-BB contracts PAs/PVs (human/mice) due to PDGFR. In murine PAs, PDGF-BB-induced contraction depends on intracellular calcium. So, PDGFR regulates the tone of PAs/PVs. Since TKIs combine relaxant and antiproliferative effects, they may be promising in therapy of PAH.

Argon reduces the pulmonary vascular tone in rats and humans by GABA-receptor activation.

Argon exerts neuroprotection. Thus, it might improve patients' neurological outcome after cerebral disorders or cardiopulmonary resuscitation. However, limited data are available concerning its effect on pulmonary vessel and airways. We used rat isolated perfused lungs (IPL) and precision-cut lung slices (PCLS) of rats and humans to assess this topic. IPL: Airway and perfusion parameters, oedema formation and the pulmonary capillary pressure (Pcap) were measured and the precapillary and postcapillary resistance (Rpost) was calculated. In IPLs and PCLS, the pulmonary vessel tone was enhanced with ET-1 or remained unchanged. IPLs were ventilated and PCLS were gassed with argon-mixture or room-air. IPL: Argon reduced the ET-1-induced increase of Pcap, Rpost and oedema formation (p < 0.05). PCLS (rat): Argon relaxed naïve pulmonary arteries (PAs) (p < 0.05). PCLS (rat/human): Argon attenuated the ET-1-induced contraction in PAs (p < 0.05). Inhibition of GABAB-receptors abolished argon-induced relaxation (p < 0.05) in naïve or ET-1-pre-contracted PAs; whereas inhibition of GABAA-receptors only affected ET-1-pre-contracted PAs (p < 0.01). GABAA/B-receptor agonists attenuated ET-1-induced contraction in PAs and baclofen (GABAB-agonist) even in pulmonary veins (p < 0.001). PLCS (rat): Argon did not affect the airways. Finally, argon decreases the pulmonary vessel tone by activation of GABA-receptors. Hence, argon might be applicable in patients with pulmonary hypertension and right ventricular failure.

Initiation of LPS-induced pulmonary dysfunction and its recovery occur independent of T cells.

BACKGROUND: The acute respiratory distress syndrome (ARDS) is a serious disease in critically ill patients that is characterized by pulmonary dysfunctions, hypoxemia and significant mortality. Patients with immunodeficiency (e.g. SCID with T and B cell deficiency) are particularly susceptible to the development of severe ARDS. However, the role of T cells on pulmonary dysfunctions in immune-competent patients with ARDS is only incompletely understood. METHODS: Wild-type (wt) and RAG2-/- mice (lymphocyte deficient) received intratracheal instillations of LPS (4 mg/kg) or saline. On day 1, 4 and 10 lung mechanics and bronchial hyperresponsiveness towards acetylcholine were measured with the flexiVent ventilation set-up. The bronchoalveolar lavage fluid (BALF) was examined for leukocytes (FACS analysis) and pro-inflammatory cytokines (ELISA). RESULTS: In wt mice, lung mechanics, body weight and body temperature deteriorated in the LPS-group during the early phase (up to d4); these alterations were accompanied by increased leukocyte numbers and inflammatory cytokine levels in the BALF. During the late phase (day 10), both lung mechanics and the cell/cytokine homeostasis recovered in LPS-treated wt mice. RAG2-/- mice experienced changes in body weight, lung mechanics, BAL neutrophil numbers, BAL inflammatory cytokines levels that were comparable to wt mice. CONCLUSION: Following LPS instillation, lung mechanics deteriorate within the first 4 days and recover towards day 10. This response is not altered by the lack of T lymphocytes suggesting that T cells play only a minor role for the initiation, propagation or recovery of LPS-induced lung dysfunctions or function of T lymphocytes can be compensated by other immune cells, such as alveolar macrophages.

PDGF-BB regulates the pulmonary vascular tone: impact of prostaglandins, calcium, MAPK- and PI3K/AKT/mTOR signalling and actin polymerisation in pulmonary veins of guinea pigs.

BACKGROUND: Platelet-derived growth factor (PDGF)-BB and its receptor PDGFR are highly expressed in pulmonary hypertension (PH) and mediate proliferation. Recently, we showed that PDGF-BB contracts pulmonary veins (PVs) and that this contraction is prevented by inhibition of PDGFR-ß (imatinib/SU6668). Here, we studied PDGF-BB-induced contraction and downstream-signalling in isolated perfused lungs (IPL) and precision-cut lung slices (PCLS) of guinea pigs (GPs). METHODS: In IPLs, PDGF-BB was perfused after or without pre-treatment with imatinib (perfused/nebulised), the effects on the pulmonary arterial pressure (PPA), the left atrial pressure (PLA) and the capillary pressure (Pcap) were studied and the precapillary (Rpre) and postcapillary resistance (Rpost) were calculated. Perfusate samples were analysed (ELISA) to detect the PDGF-BB-induced release of prostaglandin metabolites (TXA2/PGI2). In PCLS, the contractile effect of PDGF-BB was evaluated in pulmonary arteries (PAs) and PVs. In PVs, PDGF-BB-induced contraction was studied after inhibition of PDGFR-α/ß, L-Type Ca2+-channels, ROCK/PKC, prostaglandin receptors, MAP2K, p38-MAPK, PI3K-α/γ, AKT/PKB, actin polymerisation, adenyl cyclase and NO. Changes of the vascular tone were measured by videomicroscopy. In PVs, intracellular cAMP was measured by ELISA. RESULTS: In IPLs, PDGF-BB increased PPA, Pcap and Rpost. In contrast, PDGF-BB had no effect if lungs were pre-treated with imatinib (perfused/nebulised). In PCLS, PDGF-BB significantly contracted PVs/PAs which was blocked by the PDGFR-ß antagonist SU6668. In PVs, inhibition of actin polymerisation and inhibition of L-Type Ca2+-channels reduced PDGF-BB-induced contraction, whereas inhibition of ROCK/PKC had no effect. Blocking of EP1/3- and TP-receptors or inhibition of MAP2K-, p38-MAPK-, PI3K-α/γ- and AKT/PKB-signalling prevented PDGF-BB-induced contraction, whereas inhibition of EP4 only slightly reduced it. Accordingly, PDGF-BB increased TXA2 in the perfusate, whereas PGI2 was increased in all groups after 120 min and inhibition of IP-receptors did not enhance PDGF-BB-induced contraction. Moreover, PDGF-BB increased cAMP in PVs and inhibition of adenyl cyclase enhanced PDGF-BB-induced contraction, whereas inhibition of NO-formation only slightly increased it. CONCLUSIONS: PDGF-BB/PDGFR regulates the pulmonary vascular tone by the generation of prostaglandins, the increase of calcium, the activation of MAPK- or PI3K/AKT/mTOR signalling and actin remodelling. More insights in PDGF-BB downstream-signalling may contribute to develop new therapeutics for PH.

The effects of zinc- and copper-containing welding fumes on murine, rat and human precision-cut lung slices.

Recently, the pro-inflammatory effects of metal inert gas brazing welding fumes containing zinc and copper have been demonstrated in humans. Here, murine, rat and human precision cut lung slices (PCLS) were incubated in welding fume containing media with 0.1, 1, 10 and 100 µg/ml for 24 or 48 h. 24 h incubation were determined either by incubation for the total time or for only 6 h followed by a 18 h post-incubation phase. Cytotoxicity, proliferation and DNA repair rates, and cytokine levels were determined. Welding fume particle concentrations of 0.1 and 1 µg/ml showed no toxic effects on PCLS of all three species, while for 10 and 100 µg/ml a concentration-dependent toxicity occurred. Proliferation and DNA repair rates were reduced for all tested concentrations and incubation times. Additionally, the cytokine levels in the supernatants were markedly reduced, while after 6 h of exposure with 18 h of post-incubation time a trend towards increased cytokine levels occurred. PCLS are a reliable and feasible method to assess and offer a prediction of toxic effects of welding fume particles on human lungs. Rat PCLS showed similar responses compared to human PCLS and are suitable for further evaluation of toxic effects exerted by welding fume particles.

The effects of hydroxyethyl starch and gelatine on pulmonary cytokine production and oedema formation.

Recently, side effects of plasma expanders like hydroxyethyl starch and gelatine gained considerable attention. Most studies have focused on the kidneys; lungs remain unconsidered. Isolated mouse lungs were perfused for 4 hours with buffer solutions based on hydroxyethyl starch (HES) 130/0.4, HES 200/0.5 or gelatine and ventilated with low or high pressure under physiological pH and alkalosis. Outcome parameters were cytokine levels and the wet-to-dry ratio. For cytokine release, murine and human PCLS were incubated in three different buffers and time points.In lungs perfused with the gelatine based buffer IL-6, MIP-2 and KC increased when ventilated with high pressure. Wet-to-dry ratios increased stronger in lungs perfused with gelatine - compared to HES 130/0.4. Alkalotic perfusion resulted in higher cytokine levels but normal wet-to-dry ratio. Murine PCLS supernatants showed increased IL-6 and KC when incubated in gelatine based buffer, whereas in human PCLS IL-8 was elevated. In murine IPL HES 130/0.4 has lung protective effects in comparison to gelatine based infusion solutions, especially in the presence of high-pressure ventilation. Gelatine perfusion resulted in increased cytokine production. Our findings suggest that gelatine based solutions may have side effects in patients with lung injury or lung oedema.

Imatinib relaxes the pulmonary venous bed of guinea pigs.

BACKGROUND: Recently, the IMPRES study revealed that systemic imatinib improves exercise capacity in patients with advanced pulmonary arterial hypertension. Imatinib blocks the tyrosine kinase activity of the platelet-derived growth factor (PDGF)-receptor (PDGFR), acts antiproliferative and relaxes pulmonary arteries. However so far, the relaxant effects of imatinib on pulmonary veins (PVs) and on the postcapillary resistance are unknown, although pulmonary hypertension (PH) due to left heart disease (LHD) is most common and primarily affects PVs. Next, it is unknown whether activation of PDGFR alters the pulmonary venous tone. Due to the reported adverse effects of systemic imatinib, we evaluated the effects of nebulized imatinib on the postcapillary resistance. METHODS: Precision-cut lung slices (PCLS) were prepared from guinea pigs. PVs were pre-constricted with Endothelin-1 (ET-1) and the imatinib-induced relaxation was studied by videomicroscopy; PDGF-BB-related vascular properties were evaluated as well. The effects of perfused/nebulized imatinib on the postcapillary resistance were studied in cavine isolated perfused lungs (IPL). Intracellular cAMP/cGMP was measured by ELISA in PVs. RESULTS: In PCLS, imatinib (100 µM) relaxed pre-constricted PVs (126%). In PVs, imatinib increased cAMP, but not cGMP and inhibition of adenyl cyclase or protein kinase A reduced the imatinib-induced relaxation. Further, inhibition of KATP-channels, [Formula: see text]-channels or Kv-channels diminished the imatinib-induced relaxation, whereas inhibition of NO-signaling was without effect. In the IPL, perfusion or nebulization of imatinib reduced the ET-1-induced increase of the postcapillary resistance. In PCLS, PDGF-BB contracted PVs, which was blocked by imatinib and by the PDGFR-ß kinase inhibitor SU6668, whereas inhibition of PDGFR-α (ponatinib) had no significant effect. Conversely, PDGFR-ß kinase inhibitors (SU6668/DMPQ) relaxed PVs pre-constricted with ET-1 comparable to imatinib, whereas the PDGFR-α kinase inhibitor ponatinib did not. CONCLUSIONS: Imatinib-induced relaxation depends on cAMP and on the activation of K+-channels. Perfused or nebulized imatinib significantly reduces the postcapillary resistance in the pre-constricted (ET-1) pulmonary venous bed. Hence, nebulization of imatinib is feasible and might reduce systemic side effects. Conversely, PDGF-BB contracts PVs by activation of PDGFR-ß suggesting that imatinib-induced relaxation depends on PDGFR-ß-antagonism. Imatinib combines short-term relaxant and long-term antiproliferative effects. Thus, imatinib might be a promising therapy for PH due to LHD.

The cAMP response element modulator (CREM) regulates TH2 mediated inflammation.

A characteristic feature of allergic diseases is the appearance of a subset of CD4+ cells known as TH2 cells, which is controlled by transcriptional and epigenetic mechanisms. We aimed to analyze the role of CREM, a known transcriptional activator of T cells, with regard to TH2 responses and allergic diseases in men and mice. Here we demonstrate that T cells of asthmatic children and PBMCs of adults with atopy express lower mRNA levels of the transcription factor CREM compared to cells from healthy controls. CREM deficiency in murine T cells results in enhanced TH2 effector cytokines in vitro and in vivo and CREM-/- mice demonstrate stronger airway hyperresponsiveness in an OVA-induced asthma model. Mechanistically, both direct CREM binding to the IL-4 and IL-13 promoter as well as a decreased IL-2 dependent STAT5 activation suppress the TH2 response. Accordingly, mice selectively overexpressing CREMα in T cells display decreased TH2 type cytokines in vivo and in vitro, and are protected in an asthma model. Thus, we provide evidence that CREM is a negative regulator of the TH2 response and determines the outcome of allergic asthma.

Milrinone relaxes pulmonary veins in guinea pigs and humans.

INTRODUCTION: The phosphodiesterase-III inhibitor milrinone improves ventricular contractility, relaxes pulmonary arteries and reduces right ventricular afterload. Thus, it is used to treat heart failure and pulmonary hypertension (PH). However, its action on pulmonary veins (PVs) is not defined, although particularly PH due to left heart disease primarily affects the pulmonary venous bed. We examined milrinone-induced relaxation in PVs from guinea pigs (GPs) and humans. MATERIAL AND METHODS: Precision-cut lung slices (PCLS) were prepared from GPs or from patients undergoing lobectomy. Milrinone-induced relaxation was studied by videomicroscopy in naïve PVs and in PVs pre-constricted with the ETA-receptor agonist BP0104. Baseline luminal area was defined as 100%. Intracellular cAMP was measured by ELISA and milrinone-induced changes of segmental vascular resistances were studied in the GP isolated perfused lung (IPL). RESULTS: In the IPL (GP), milrinone (10 µM) lowered the postcapillary resistance of pre-constricted vessels. In PCLS (GP), milrinone relaxed naïve and pre-constricted PVs (120%) and this relaxation was attenuated by inhibition of protein kinase G (KT 5823), adenyl cyclase (SQ 22536) and protein kinase A (KT 5720), but not by inhibition of NO-synthesis (L-NAME). In addition, milrinone-induced relaxation was dependent on the activation of K ATP-, BK Ca (2+)- and Kv-channels. Human PVs also relaxed to milrinone (121%), however only if pre-constricted. DISCUSSION: Milrinone relaxes PVs from GPs and humans. In GPs, milrinone-induced relaxation is based on K ATP-, BK Ca (2+)- and Kv-channel-activation and on cAMP/PKA/PKG. The relaxant properties of milrinone on PVs lead to reduced postcapillary resistance and hydrostatic pressures. Hence they alleviate pulmonary edema and suggest beneficial effects of milrinone in PH due to left heart disease.

Levosimendan Relaxes Pulmonary Arteries and Veins in Precision-Cut Lung Slices - The Role of KATP-Channels, cAMP and cGMP.

INTRODUCTION: Levosimendan is approved for left heart failure and is also used in right heart failure to reduce right ventricular afterload. Despite the fact that pulmonary arteries (PAs) and pulmonary veins (PVs) contribute to cardiac load, their responses to levosimendan are largely unknown. MATERIALS AND METHODS: Levosimendan-induced vasorelaxation of PAs and PVs was studied in precision-cut lung slices from guinea pigs by videomicroscopy; baseline luminal area was defined as 100%. Intracellular cAMP- and cGMP-levels were measured by ELISA and NO end products were determined by the Griess reaction. RESULTS: Levosimendan relaxed control PVs (116%) and those pre-constricted with an endothelinA-receptor agonist (119%). PAs were only relaxed if pre-constricted (115%). Inhibition of KATP-channels (glibenclamide), adenyl cyclase (SQ 22536) and protein kinase G (KT 5823) largely attenuated the levosimendan-induced relaxation in control PVs, as well as in pre-constricted PAs and PVs. Inhibition of BKCa (2+)-channels (iberiotoxin) and Kv-channels (4-aminopyridine) only contributed to the relaxant effect of levosimendan in pre-constricted PAs. In both PAs and PVs, levosimendan increased intracellular cAMP- and cGMP-levels, whereas NO end products remained unchanged. Notably, basal NO-levels were higher in PVs. The KATP-channel activator levcromakalim relaxed PAs dependent on cAMP/PKA/PKG and increased cAMP-levels in PAs. DISCUSSION: Levosimendan initiates complex and divergent signaling pathways in PAs and PVs. Levosimendan relaxes PAs and PVs primarily via KATP-channels and cAMP/cGMP; in PAs, BKCa (2+)- and Kv-channels are also involved. Our findings with levcromakalim do further suggest that in PAs the activation of KATP-channels leads to the production of cAMP/PKA/PKG. In conclusion, these results suggest that levosimendan might reduce right ventricular afterload by relaxation of PAs as well as pulmonary hydrostatic pressure and pulmonary edema by relaxation of PVs.

Neurally mediated airway constriction in human and other species: a comparative study using precision-cut lung slices (PCLS).

The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS) respond to electric field stimulation (EFS) and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM) caused bronchoconstriction in human (4 from 7) and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC). However, this effect was notably smaller in human responder (30 ± 7.1%) than in guinea pig (79 ± 5.1%) PCLS. The transient receptor potential (TRP) channel blockers SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic human responses well and may thus serve as clinically relevant models to study neural airway responses.

Increasing concentrations of prothrombin complex concentrate induce disseminated intravascular coagulation in a pig model of coagulopathy with blunt liver injury.

Despite increasing use of prothrombin complex concentrate (PCC) to treat hemorrhage-associated coagulopathy, few studies have investigated PCC in trauma, and there is a particular lack of safety data. This study was performed to evaluate PCC therapy in a porcine model of coagulopathy with blunt liver injury. Coagulopathy was induced in 27 anesthetized pigs by replacing approximately 70% blood volume with hydroxyethyl starch 130/0.4 and Ringer's lactate solution; erythrocytes were collected and retransfused. Ten minutes after trauma, animals randomly received PCC (35 or 50 IU/kg) or saline. Coagulation parameters including thromboelastometry, thrombin generation, and blood loss were monitored for 2 hours. Internal organs were examined macroscopically and histologically to determine the presence of emboli and assess liver injury. Total blood loss was significantly lower and survival was higher in both PCC groups versus the control group (P < .05). These outcomes appeared to be dose-independent. Thromboembolism was found in all animals treated with 50 IU/kg PCC; 44% also showed signs of disseminated intravascular coagulation. Liver injury was similar in all animals. In conclusion, 35 IU/kg PCC safely improved coagulation and attenuated blood loss. However, the higher dose of PCC (50 IU/kg) appeared to increase the risk of thromboembolism and disseminated intravascular coagulation.

Cardiovascular agents affect the tone of pulmonary arteries and veins in precision-cut lung slices.

INTRODUCTION: Cardiovascular agents are pivotal in the therapy of heart failure. Apart from their action on ventricular contractility and systemic afterload, they affect pulmonary arteries and veins. Although these effects are crucial in heart failure with coexisting pulmonary hypertension or lung oedema, they are poorly defined, especially in pulmonary veins. Therefore, we investigated the pulmonary vascular effects of adrenoceptor agonists, vasopressin and angiotensin II in the model of precision-cut lung slices that allows simultaneous studies of pulmonary arteries and veins. MATERIALS AND METHODS: Precision-cut lung slices were prepared from guinea pigs and imaged by videomicroscopy. Concentration-response curves of cardiovascular drugs were analysed in pulmonary arteries and veins. RESULTS: Pulmonary veins responded stronger than arteries to α(1)-agonists (contraction) and ß(2)-agonists (relaxation). Notably, inhibition of ß(2)-adrenoceptors unmasked the α(1)-mimetic effect of norepinephrine and epinephrine in pulmonary veins. Vasopressin and angiotensin II contracted pulmonary veins via V(1a) and AT(1) receptors, respectively, without affecting pulmonary arteries. DISCUSSION: Vasopressin and (nor)epinephrine in combination with ß(2)-inhibition caused pulmonary venoconstriction. If applicable in humans, these treatments would enhance capillary hydrostatic pressures and lung oedema, suggesting their cautious use in left heart failure. Vice versa, the prevention of pulmonary venoconstriction by AT(1) receptor antagonists might contribute to their beneficial effects seen in left heart failure. Further, α(1)-mimetic agents might exacerbate pulmonary hypertension and right ventricular failure by contracting pulmonary arteries, whereas vasopressin might not.

Influence of temperature on the positive inotropic effect of levosimendan, dobutamine and milrinone.

BACKGROUND AND OBJECTIVE: Patients in cardiac surgery and critically ill patients often demonstrate either hypothermia or fever. In addition, owing to heart failure, they frequently require inotropic support. The relative effectiveness of modern inotropic agents at various temperatures has not yet been evaluated. Therefore, we investigated the influence of levosimendan, dobutamine and milrinone on the contractile response of myocardial trabeculae at various temperatures. METHODS: A total of 120 guinea pig ventricular trabeculae were placed in oxygenated 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) buffer, stimulated at a frequency of 1.3 Hz and randomly assigned to a temperature of 31 degrees C, 34 degrees C, 37 degrees C or 40 degrees C. Concentrations of all substances were increased stepwise from 10(-9) to 10(-5) mol l(-1) (milrinone up to 10(-4) mol l(-1)). Maximum developed force, time to peak tension, Tsystolic(50%) and Tdiastolic(50%) were continuously recorded. RESULTS: All agents showed a dose-dependent positive inotropic effect (P < 0.0001 for all). Levosimendan acted at every temperature as a positive inotrope (P = 0.0643). Dobutamine-related inotropy showed a clear trend towards temperature dependence, although statistical evaluation did not prove this (P = 0.0624). Milrinone-related inotropy was abolished at 31 degrees C and 34 degrees C, and temperature dependence was significant (P < 0.0001). Hypothermia induced a positive inotropic effect. CONCLUSION: Our results suggest no modulation of levosimendan-induced inotropy under the experimental temperatures tested. This observation is possibly due to its Ca2+-sensitizing mechanism, which might not be influenced by temperature-related changes in intracellular Ca2+ levels. In contrast, the inotropic effect of cyclic AMP-coupled dobutamine and milrinone is suppressed under hypothermia-related interaction with intracellular Ca2+ homeostasis. Hence, levosimendan might prove to be the preferred inotropic drug in hypothermic patients.