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 endothelin_A-receptor agonist (119%). PAs were only relaxed if pre-constricted (115%). Inhibition of K_ATP-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 BK_Ca ²⁺-channels (iberiotoxin) and K_v-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 K_ATP-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 K_ATP-channels and cAMP/cGMP; in PAs, BK_Ca ²⁺- and K_v-channels are also involved. Our findings with levcromakalim do further suggest that in PAs the activation of K_ATP-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.     

Electric field stimulation of precision-cut lung slices

The precision-cut lung slice (PCLS) technique is widely used to examine airway responses in different species. We developed a method to study nerve-dependent bronchoconstriction by the application of electric field stimulation (EFS) to PCLS. PCLS prepared from Wistar rats were placed between two platinum electrodes to apply serial rectangular impulses (5-100 Hz), and bronchoconstriction was studied by videomicroscopy. The extent of airway contractions increased with higher frequencies. Stable repeated airway contractions were obtained at a frequency of 50 Hz, a width of 1 ms, and an output of 200 mA for 2.5 s each minute. Larger airways showed stronger responses. The EFS-triggered contractions were increased by the acetylcholine esterase inhibitor neostigmine (10 μM) and reversed by the muscarinic antagonist atropine (10 μM), whereas the thromboxane protanoid receptor antagonist SQ29548 (10 μM) had no effect. Magnesium ions (10 mM) antagonized airway contractions induced by EFS, but not by methacholine, indicating that nerve endings remain intact in PCLS. Our data further show that the electrically evoked airway contractions in PCLS are mediated by cholinergic nerves, independent of thromboxane and more prominent in larger airways. Taken together these findings show that nerve endings remain intact in PCLS, and they suggest that the present method is useful to study neurogenic responses in airways of different size.     

Intrazelluläre Zellzotten in Nahrungsvakuolen von Ciliaten

Zusammenfassung 1. Die Nahrungsvakuolen vonMetafolliculina andrewsi entwickeln mit zunehmendem Alter einen Faltensaum, der seine höchste Differenzierung in dem Altersstadium erreicht, wo die stärkste Resorption der Nährstoffe zu vermuten ist.2. Dieser Faltensaum besteht aus lamellenartigen Cytoplasmaprotuberanzen und bewirkt — ähnlich wie der Bürstensaum an der freien Zelloberfläche — innerhalb der Zelle eine erhebliche Oberflächenvergrößerung der Nahrungsvakuole zugunsten der Permeation der Nährstoffe.3. Bei alten Nahrungsvakuolen werden die Falten wieder zurückgebildet, eine Tatsache, die die funktionelle Bedeutung dieser Struktur weiterhin unterstreicht.4. An Hand des Verdauungsgrades des Vakuoleninhalts und der morphologischen Veränderungen der Vakuolenwand lassen sich bei den Nahrungsvakuolen vonMetafolliculina deutlich vier Alters- beziehungsweise Funktionsstadien unterscheiden: 1. Ingestionsvakuolen, 2. Digestionsvakuolen, 3. Resorptionsvakuolen und 4. Egestionsvakuolen.5. Die morphologischen Befunde an der Resorptionsvakuole, die strukturelle und funktionelle Parallelen mit den resorbierenden Darmepithelien von Metazoen aufweist, werden im Rahmen der heutigen Vorstellungen über die zelluläre Stoffaufnahme diskutiert.

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Lamellar microvilli in food vacuoles of ciliates

With progressing age, the food vacuoles ofMetafolliculina adrewsi develop a border of folds, which attains its most pronounced differentiation at a stage where maximum resorption of nutrient substances presumably takes place. This border of folds, similar to the well known brush border at the free cell surface, consists of lamellar cytoplasm protuberances and causes a considerable enlargement of the membrane area of the food vacuole within the cell, thus favoring the permeation of nutrient substances. In old food vacuoles the border of folds undergoes involution, an observation that also points to the functional significance of this structure. InMetafolliculina the degree of digestion of the vacuole contents and the morphological changes of the vacuole wall enable a distinction to be made between 4 different stages of age and function of the food vacuoles respectively: (1) ingestion vacuoles, (2) digestion vacuoles, (3) resorption vacuoles and (4) egestion vacuoles. The resorption vacuole evidently shows structural and functional analogies to the resorbing intestinal epithelia of Metazoa. The morphological findings are discussed in the light of current concepts on substance uptake by the cell.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Positive anti-citrullinated protein antibody status and small joint arthritis are consistent predictors of chronic disease in patients with very early arthritis: results from the NOR-VEAC cohort

Introduction  

The current 1987 American College of Rheumatology (ACR) classification criteria for rheumatoid arthritis (RA) have proven less useful in early arthritis. The objective of this study was to identify and compare predictors of three relevant outcomes of chronic arthritis in a cohort of very early arthritis patients.     

Unilateral nephrectomy leads to up-regulation of syndecan-2- and TGF-beta-mediated glomerulosclerosis in syndecan-4 deficient male mice.

Syndecan-4 is an ubiquitous, plasma membrane-spanning heparan sulfate proteoglycan involved in proliferation, differentiation, adhesion and migration of cells in vitro. Syndecan-4 knockout (KO) mice show no obvious defects but respond abnormally to experimental stress conditions. In the adult, syndecan-4 is the most abundant syndecan of renal tissue. We therefore investigated the consequences of syndecan-4 deficiency during progression of kidney disease using unilaterally nephrectomized mice, a model of glomerular hyperfiltration and renal hypertrophy. 60 days after unilateral nephrectomy (UNX), mesangial expansion, enhanced matrix production (collagens I and IV, fibronectin) and focal segmental glomerulosclerosis, resembling early stages of diabetic nephropathy, was apparent in male but not female syndecan-4 KO mice. No defect was detected in wild type UNX males. Syndecan-2 mRNA and protein were not detectable in renal glomeruli of wild type mice, but were induced specifically in the glomeruli of the syndecan-4 deficient kidneys after unilateral nephrectomy. Due to the structural similarities of syndecans-2 and -4 we hypothesize that de novo-production of syndecan-2 in kidneys after unilateral nephrectomy reflects a compensatory response. However, this response is counterproductive since syndecan-2 supports the pro-sclerotic activity of TGF-beta1 which is increased in parallel with syndecan-2 synthesis. By contrast, signaling through syndecan-4 negatively controls the production of pro-sclerotic TGF-beta1.     

Changes in airway resistance by simultaneous exposure to TNF-alpha and IL-1beta in perfused rat lungs

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are formed simultaneously under inflammatory conditions such as asthma and acute respiratory distress syndrome. Here we investigated the effects of TNF-alpha (10 ng/ml) and/or IL-1beta (10 ng/ml) in isolated blood-free perfused rat lungs. In lungs precontracted with methacholine, IL-1beta alone and IL-1beta/TNF-alpha decreased airway resistance 10 min after administration, whereas TNF-alpha alone had no effect. In untreated lungs, airway resistance was unaltered by either cytokine alone but started to increase 40 min after treatment with both cytokines together, indicating bronchoconstriction. The bronchoconstriction was accompanied by a steroid-sensitive increase in cyclooxygenase (COX)-2 mRNA expression and thromboxane formation. The cytokine-induced bronchoconstriction was blocked by the thromboxane receptor antagonist SQ-29548, indomethacin, the selective COX-2 inhibitor NS-398, and the steroid dexamethasone. We conclude that IL-1beta has an early bronchodilatory effect (after 10 min) that is unchanged by TNF-alpha. However, at later time points (after 40 min), IL-1beta and TNF-alpha in concert cause a COX-2- and thromboxane-dependent bronchoconstriction. Our findings show that TNF-alpha and IL-1beta exert complex and time-dependent effects on lung functions that cannot be predicted by studying each cytokine alone.     

Intercellular adhesion molecule-1 mediates cellular cross-talk between parenchymal and immune cells after lipopolysaccharide neutralization

The mechanisms by which parenchymal cells interact with immune cells, particularly after removal of LPS, remain unknown. Lung explants from rats, mice deficient in the TNF gene, or human lung epithelial A549 cells were treated with LPS and washed, before naive alveolar macrophages, bone marrow monocytes, or PBMC, respectively, were added to the cultures. When the immune cells were cocultured with LPS-challenged explants or A549 cells, TNF production was greatly enhanced. This was not affected by neutralization of LPS with polymyxin B. The LPS-induced increase in the expression of ICAM-1 on A549 cells correlated with TNF production by PBMC. The cellular cross talk leading to the TNF response was blunted by an anti-ICAM-1 Ab and an ICAM-1 antisense oligonucleotide. In A549 cells, a persistent decrease in the concentration of intracellular cAMP was associated with colocalization of LPS into Toll-like receptor 4 and the Golgi apparatus, resulting in increased ICAM-1 expression. Inhibition of LPS internalization by cytochalasin D or treatment with dibutyryl cAMP attenuated ICAM-1 expression and TNF production by PBMC. In conclusion, lung epithelial cells are not bystanders, but possess memory of LPS through the expression of ICAM-1 that interacts with and activates leukocytes. This may provide an explanation for the failure of anti-LPS therapies in sepsis trials.