Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes

A large conductance (～300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.     

Seasonal flooding,topography, and organic debris interact to influence the emergence and distribution of seedlings in a tropical grassland

In seasonally flooded wetlands, inundation and associated organic debris deposition followed by a drawdown period can promote plant community diversity across space and time. Post‐flood regeneration might be influenced by the direct effect of flooding on seed dispersal and seedling emergence, as well as the indirect effect of organic debris on seed trapping and germination. Our objective was to examine the influence of seasonal flooding, topography, and organic debris cover on seedling distribution in a seasonally flooded grassland. We measured species richness, seedling abundance, and organic debris cover for 3 yr in a seasonally flooded grassland in the Pantanal, Brazil, at three topographic levels at the end of the flood season and during the dry season when there was no debris deposition. A total of 43 species were recorded, with no difference in species richness detected between seasons. However, the abundance of some species was higher post‐flood than during the dry period. The greatest seedling abundance and richness were found post‐flood at intermediate elevations, followed by high and the lowest elevations. Seed germination and seedling establishment were likely suppressed at low topographic positions due to shading from organic debris and poor drainage. Therefore, areas with predictable annual floods promote diversity by creating spatial and temporal variations in environmental conditions.     

Development of an identified serotonergic neuron in the antennal lobe of the moth and effects of reduction in serotonin during construction of olfactory glomeruli

Each olfactory (antennal) lobe of the moth Manduca sexta contains a single serotonin (5-HT) immunoreactive neuron whose processes form tufted arbors in the olfactory glomeruli. To extend our present understanding of the intercellular interactions involved in glomerulus development to the level of an individual, identified antennal lobe neuron, we first studied the morphological development of the 5-HT neuron in the presence and absence of receptor axons. Development of the neuron's glomerular tufts depends, as it does in the case of other multiglomerular neurons, on the presence of receptor axons. Processes of the 5-HT neuron are excluded from the region in which the initial steps of glomerulus construction occur and thus cannot provide a physical scaffolding on which the array of glomeruli is organized. Because the neuron's processes are present in the antennal lobe neuropil throughout postembryonic development, 5-HT could provide signals that influence the pattern of development in the lobe. By surgically producing 5-HT-depleted antennal lobes, we also tested the importance of 5-HT in the construction of olfactory glomeruli. Even in the apparent absence of 5-HT, the glomerular array initiated by the receptor axons was histologically normal, glial cells migrated to form glomerular borders, and receptor axons formed terminal branches in their normal region within each glomerulus. In some cases, 5-HT-immunoreactive processes from abnormal sources entered the lobe and formed the tufted intraglomerular branches typical of most antennal lobe neurons, suggesting that local cues strongly influence the branching patterns of developing antennal lobe neurons. © 1995 John Wiley & Sons, Inc.     

High-resolution measurement and calibration of Ca(2+)-transients using Indo-1 in guinea-pig atrial myocytes under voltage clamp.

Spherical atrial myocytes obtained by enzymatic dispersion of hearts from adult guinea-pigs were loaded with the fluorescent Ca(2+)-indicator Indo-1 via patch-clamp pipettes. The dialysing solution additionally contained citrate (60 mM) as low-affinity ('linear') Ca(2+)-chelating compound in order to slow intracellular Ca(2+)-transients. Changes in Indo-1 fluorescence under voltage-clamp due to Ca(2+)-entry and/or release from the SR were calibrated using an in vivo procedure to determine the limiting fluorescence ratios. Sample recordings will be presented to demonstrate that components of a [Ca2+]i-transient due to entry via L-type Ca(2+)-channels and due to Ca(2+)-release from the SR can be directly visualized.     

Brief antecedent anoxia preserves mitochondrial function after sustained undersupply: A subcellular correlate to ischemic preconditioning?

Background: There is increasing evidence that mitochondria – owning a high degree of autonomy within the cell – might represent the target organelles of the myocardial protection afforded by ischemic preconditioning. It was the aim of the study to investigate a possible subcellular correlate to ischemic preconditioning at the mitochondrial level. In addition, we tested whether this protection depends on mitochondrial ATP-dependent potassium channels (K _ATP) and an might involve an attenuation of mitochondrial ATP hydrolysis during sustained anoxia.Methods and Results: Sustained anoxia (A, 14 min) and reoxygenation (R) completely inhibited state 3 and state 4 respiration of isolated ventricular mitochondria from Wistar rats. An antecedent brief anoxic incubation (4 min) followed by reoxygenation (2 min) prevented this loss of mitochondrial function. The protection afforded by anoxic preconditioning could be mimicked by the K _ATP opener diazoxide (30 μmol/l) and was completely inhibited by the K _ATP blocker 5-hydroxydecanoic acid (300 μmol/l). Structural mitochondrial integrity, as estimated from externalization of the mitochondrial enzymes creatine kinase and glutamateoxalacetate transaminase, remained unchanged between the groups, as did mitochondrial ATP loss during anoxia.Conclusion: For the first time, we provide direct evidence for a subcellular preconditioning-like functional mitochondrial adaptation to sustained anoxia. This effect apparently depends on opening of K_ATP but is independent of ATP preservation.     

eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca2+ in human atrial myocardium

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser¹¹⁷⁷ (Ser¹¹⁷⁷ eNOS). We have recently shown that agonist-induced Ser¹¹⁷⁷ eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca²⁺ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser¹¹⁷⁷ eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca²⁺ chelator BAPTA before and after application of BRL 37344 (BRL), a ₃-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser¹¹⁷⁷ eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca²⁺ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser¹¹⁷⁷ eNOS phosphorylation is dependent on intracellular Ca²⁺. 2) In atrial myocardium, eNOS-translocation and not Ser¹¹⁷⁷ eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca²⁺ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation. ₃-adrenoceptor; BRL 37344; cardiomyocyte; heart; Ca²⁺ regulation     

The chemical composition of throughfall beneath oak, birch and pine canopies in Northwest Germany

The chemical composition of rainwater is altered upon its passage through tree canopies. In order to investigate how rainwater chemistry is affected by canopy-dependent processes in characteristic forest types of Northwest German sandy lowland regions – oak–birch-forests, Betula pubescens Ehrh. swamp forests, and stands of Pinus sylvestris L. – comparative studies on the chemical composition of throughfall were carried out at seven forest sites, situated in close proximity within a nature reserve. Additionally, rainwater was sampled at three heathland sites for analysis of open-field precipitation and at three sites along an oak–birch-forest edge. Throughfall concentrations of most of the parameters analysed were significantly higher than open-field concentrations, especially with regard to electric conductivity, NH₄-N, K⁺, and KMnO₄-index. Ion concentrations in throughfall were the lowest in a 10-year-old stand of Betula pendula Roth. and Pinus sylvestris and in a Betula pubescens swamp forest and were highest beneath a stand of Pinus sylvestris. Except for Na⁺, Cl⁻, and NO₃⁻, ion concentrations in both throughfall and open-field precipitation increased during the growing season (May–October). In throughfall, Ca²⁺, Mg²⁺, K⁺, and Mn²⁺ were strongly correlated. Enrichment ratios between throughfall and open-field deposition varied among sites and elements and were the highest for K^‰+, Mg^2‰+, and Mn^2‰+. Estimates of canopy leaching indicated high leaching rates of K^‰+ and Mn^2‰+ and moderate leaching of Mg^2‰+. The contribution of foliar leaching to throughfall deposition was higher at the deciduous than at the coniferous stands.     

Genetic manipulation of cardiac Na+/Ca2+ exchange expression

The Na+/Ca2+ exchanger (NCX) is the primary Ca2+ extrusion mechanism in cardiomyocytes. To further investigate the role of NCX in excitation-contraction coupling and Ca2+ homeostasis, we created murine models with altered expression levels of NCX. Homozygous overexpression of NCX resulted in mild cardiac hypertrophy. Decline of the Ca2+ transient and relaxation of contraction were increased and the reverse mode of NCX was augmented. Overexpression also led to a higher susceptibility to ischemia-reperfusion injury and to a greater ability of NCX to trigger Ca2+-induced Ca2+ release. Furthermore, an increase in peak L-type Ca2+ current was observed suggesting a direct influence of NCX on L-type Ca2+ current. Whereas global knockout of NCX led to prenatal death, a recently generated cardiac-specific NCX knockout mouse was viable with surprisingly normal contractile properties. Expression levels of other Ca2+-handling proteins were not altered. Ca2+ influx in these animals is limited by a decrease of peak L-type Ca2+ current. An alternative Ca2+ efflux mechanism, presumably the plasma membrane Ca2+-ATPase, is sufficient to maintain Ca2+-homeostasis in the NCX knockout mice.     

Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective single-centre study

Psoriasis and psoriatic arthritis are inflammatory diseases that respond well to anti-tumour necrosis factor-α therapy. To evaluate the effects of anti-tumour necrosis factor-α treatment on expression of adhesion molecules and angiogenesis in psoriatic lesional skin and synovial tissue, we performed a prospective single-centre study with infliximab therapy combined with stable methotrexate therapy. Eleven patients with both active psoriasis and psoriatic arthritis received infusions of infliximab (3 mg/kg) at baseline, and at weeks 2, 6, 14 and 22 in an open-label study. In addition, patients continued to receive stable methotrexate therapy in dosages ranging from 5 to 20 mg/week. Clinical assessments, including Psoriasis Area and Severity Index (PASI) and Disease Activity Score (DAS), were performed at baseline and every 2 weeks afterward. In addition, skin biopsies from a target psoriatic plaque and synovial tissue biopsies from a target joint were taken before treatment and at week 4. Immunohistochemical analysis was performed to detect the number of blood vessels, the expression of adhesion molecules and the presence of vascular growth factors. Stained sections were evaluated by digital image analysis. At week 16, the mean PASI was reduced from 12.3 ± 2.4 at baseline to 1.8 ± 0.4 (P ≤ 0.02). The mean DAS was reduced from 6.0 ± 0.5 to 3.6 ± 0.6 (P ≤ 0.02). We found some fluctuations in DAS response as compared with the change in PASI, with the latter exhibiting a steady decrease over time. After 4 weeks the cell infiltrate was reduced in both skin and synovium. There was a significant reduction in the number of blood vessels in dermis and synovium at week 4. A significant reduction in the expression of α_vβ₃ integrin, a marker of neovascularization, was also found in both skin and synovium at week 4. In addition, a significant reduction in the expression of adhesion molecules was observed in both skin and synovium at week 4. We also observed a trend toward reduced expression of vascular endothelial growth factor in both skin and synovium. In conclusion, low-dose infliximab treatment leads to decreased neoangiogenesis and deactivation of the endothelium, resulting in decreased cell infiltration and clinical improvement in psoriasis and psoriatic arthritis.     

Middle cerebral artery blood velocity during a valsalva maneuver in the standing position.

Occasionally, lifting of a heavy weight leads to dizziness and even to fainting, suggesting that, especially in the standing position, expiratory straining compromises cerebral perfusion. In 10 subjects, the middle cerebral artery mean blood velocity (V(mean)) was evaluated during a Valsalva maneuver (mouth pressure 40 mmHg for 15 s) both in the supine and in the standing position. During standing, cardiac output decreased by 16 +/- 4 (SE) % (P < 0.05), and at the level of the brain mean arterial pressure (MAP) decreased from 89 +/- 2 to 78 +/- 3 mmHg (P < 0.05), as did V(mean) from 73 +/- 4 to 62 +/- 5 cm/s (P < 0.05). In both postures, the Valsalva maneuver increased central venous pressure by approximately 40 mmHg with a nadir in MAP and cardiac output that was most pronounced during standing (MAP: 65 +/- 6 vs. 87 +/- 3 mmHg; cardiac output: 37 +/- 3 vs. 57 +/- 4% of the resting value; P < 0.05). Also, V(mean) was lowest during the standing Valsalva maneuver (39 +/- 5 vs. 47 +/- 4 cm/s; P < 0.05). In healthy individuals, orthostasis induces an approximately 15% reduction in middle cerebral artery V(mean) that is exaggerated by a Valsalva maneuver performed with 40-mmHg mouth pressure to approximately 50% of supine rest.