Lanthanum in heart cell culture. Effect on calcium exchange correlated with its localization

Correlation of the localization of La⁺⁺⁺ with its effects on Ca⁺⁺ exchange in cultured rat heart cells is examined with the use of a recently developed technique. 75% of cellular Ca⁺⁺ is exchangeable and is completely accounted for by two kinetically defined phases. The rapidly exchangeable phase has a t ½ = 1.15 min and accounts for 1 1 mmoles Ca⁺⁺/kg wet cells or 43% of the exchangeable Ca⁺⁺ (cells perfused with [Ca⁺⁺]_o = 1 mM) Phase 2 has a t ½ = 19.2 min and accounts for 1.5 mmoles Ca⁺⁺/kg wet cells or 57% of the exchangeable Ca⁺⁺. 0.5 mM [La⁺⁺⁺]_o displaces 0 52 mmoles Ca⁺⁺/kg wet cells—all from phase 1—and almost completely abolishes subsequent Ca⁺⁺ influx and efflux The presence of La⁺⁺⁺ in the washout converts the washout pattern to a single phase system with a t ½ = 124 min. The effects upon Ca⁺⁺ exchange are coincident with abolition of contractile tension but regenerative depolarization of the tissue is maintained Electron microscope localization of the La⁺⁺⁺ places it exclusively in the external lamina or basement membrane of the cells. The study indicates that negatively charged sites in the basement membrane play a crucial role in the E-C coupling process in heart muscle     

Veränderung der Lichtabsorption im Facettenauge bei Belichtung

Zusammenfassung Bei Belichtung der Fliegenretinula mit Weißlicht oder mit monochromatischen Lichtern, die im Empfindlichkeitsbereich des Fliegenauges liegen, sinkt die Extinktion.Bei Erhöhung der Lichtintensität um den Faktor 10 nimmt die Extinktion jeweils um ca. 0,014 ab. Maximal wurde eine Extinktionsabnahme von 0,04 ermittelt. Wird die Lichtintensität gesenkt, nimmt die Extinktion langsam wieder zu.Die Abnahme der Extinktion, also die Anpassung an eine höhere Lichtintensität, erfolgt annähernd doppelt so schnell wie die Zunahme der Extinktion, also die Anpassung an eine niedrigere Lichtintensität. Für die Abnahme wurde ein t_50%-Wert von 29 sec und für die Zunahme ein t_50%-Wert von 60 sec bestimmt.Es läßt sich vorerst nicht entscheiden, ob diese Extinktionsänderung durch eine Änderung der Konzentration des Photopigments im Rezeptor hervorgerufen wird oder ob ihr andere, vom Sehvorgang abhängige Sekundärreaktionen (z. B. Änderung der Eintrittspupille, Verlagerung von Zellorganellen, Hydratation des Gewebes als Folge höherer Stoffwechselintensität) zugrunde liegen.

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Summary When the retinula of the fly is illuminated with white or monochromatic light which lies within the sensitivity range of the eye, the extinction drops.With each increase in light intensity by a factor of ten, the extinction sinks by about 0.014. The maximum decrease in extinction measured was 0.04. When the light intensity sinks, the extinction slowly rises.The decrease in extinction, i.e. the adaptation to a higher light intensity, takes place about twice as fast as the corresponding rise in extinction, i.e. the adaptation to lower light intensity. The t_50%-value for the decrease was found to be 29 sec. and 70 sec for the increase.As yet it could not be determined whether the change in extinction results from a change in concentration of the visual pigment in the receptor or from secondary reactions dependent on the visual process.

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Wir danken der Deutschen Forschungsgemeinschaft für die Unterstützung dieser Untersuchungen.     

Temperature Dependence of Drug Interaction with the Platelet 5-Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox

Although [3H]imipramine is a selective radioligand for the 5-hydroxytryptamine (5-HT) transporter in human platelets, its affinity for binding to the 5-HT transporter complex at 0 degrees C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5-HT uptake at the physiological temperature of 37 degrees C (Ki = 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5-HT transporter at assay temperatures between 0 degrees C and 37 degrees C, using as radioligands [3H]imipramine (0 degrees C and 20 degrees C) and [3H]paroxetine (20 degrees C and 37 degrees C), a newly available probe for the 5-HT transporter. At 20 degrees C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p less than 0.001), validating the use of these two radioligands to study the 5-HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5-HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5-HT transporter at 0 degrees C. At 37 degrees C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5-HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5-HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5-HT transporter similar to that of imipramine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Ku protein complex interacts with YY1, is up-regulated in human heart failure, and represses alpha myosin heavy-chain gene expression

Human heart failure is accompanied by repression of genes such as alpha myosin heavy chain (alphaMyHC) and SERCA2A and the induction of fetal genes such as betaMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human alphaMyHC promoter. We have now identified a region of the alphaMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human alphaMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases alphaMyHC mRNA expression and increases skeletal alpha-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the alphaMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human alphaMyHC promoter during heart failure.     

Angiotensin-converting Enzyme Inhibition Down-regulates the Pro-atherogenic Chemokine Receptor 9 (CCR9)-Chemokine Ligand 25 (CCL25) Axis

Many experimental and clinical studies suggest a relationship between enhanced angiotensin II release by the angiotensin-converting enzyme (ACE) and the pathophysiology of atherosclerosis. The atherosclerosis-enhancing effects of angiotensin II are complex and incompletely understood. To identify anti-atherogenic target genes, we performed microarray gene expression profiling of the aorta during atherosclerosis prevention with the ACE inhibitor, captopril. Atherosclerosis-prone apolipoprotein E (apoE)-deficient mice were used as a model to decipher susceptible genes regulated during atherosclerosis prevention with captopril. Microarray gene expression profiling and immunohistology revealed that captopril treatment for 7 months strongly decreased the recruitment of pro-atherogenic immune cells into the aorta. Captopril-mediated inhibition of plaque-infiltrating immune cells involved down-regulation of the C-C chemokine receptor 9 (CCR9). Reduced cell migration correlated with decreased numbers of aorta-resident cells expressing the CCR9-specific chemoattractant factor, chemokine ligand 25 (CCL25). The CCL25-CCR9 axis was pro-atherogenic, because inhibition of CCR9 by RNA interference in hematopoietic progenitors of apoE-deficient mice significantly retarded the development of atherosclerosis. Analysis of coronary artery biopsy specimens of patients with coronary artery atherosclerosis undergoing bypass surgery also showed strong infiltrates of CCR9-positive cells in atherosclerotic lesions. Thus, the C-C chemokine receptor, CCR9, exerts a significant role in atherosclerosis.     

Structural reactions to polarized light of microvilli in photoreceptor cells of the moth Spodoptera

Summary Intact armyworm moths (Spodoptera exempta, Farn. Noctuidae) were illuminated by polarized monochromatic light to induce structural changes in the rhabdomeres of the compound eyes. The degree of distortion of their microvilli depends on the light energy absorbed per time unit. Under polarized light, the number of quanta absorbed varies with the position of the plane of polarization relative to the axis of the microvilli (intrinsic dichroism). Therefore, in Spodoptera, different degrees of deformations could be demonstrated in differently oriented rhabdomeres of both types of ommatidia. Moreover, in rhabdoms of the lobed type with fan-like arranged microvilli, different reactions were regularly seen in differently oriented microvilli of one rhabdomere. This indicates that microvilli may react to light individually.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 114 (Bionach)     

Therapeutic siRNA silencing in inflammatory monocytes in mice

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.     

Differential retrotranslocation of mitochondrial Bax and Bak

The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.     

Cell-derived apolipoprotein E (ApoE) particles inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in human endothelial cells

Sub-endothelial infiltration of monocytes occurs early in atherogenesis and is facilitated by cell adhesion molecules that are up-regulated on activated endothelium. Apolipoprotein E (apoE) helps protect against atherosclerosis, in part, because apoE particles secreted by macrophages have local beneficial effects at lesion sites. Here, we hypothesize that such protection includes anti-inflammatory actions and investigate whether cell-derived apoE can inhibit tumor necrosis factor-alpha-mediated up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Two models were used to mimic endothelial exposure to macrophage-derived apoE. In the first, HUVECs were transiently transfected to secrete apoE; VCAM-1 induction inversely correlated with secretion of apoE into the media (r = -0.76, p < 0.001). In the second, incubation of HUVECs with media from recombinant Chinese hamster ovary (CHO) cells expressing apoE (CHO(apoE)) also reduced VCAM-1 in a dose-dependent manner (r = -0.70, p < 0.001). Characterization of CHO(apoE) cell-derived apoE revealed several similarities to apoE particles secreted by human blood monocyte-derived macrophages. The suppression of endothelial activation by apoE most likely occurs via stimulation of endothelial nitric oxide synthase; apoE increased levels of intracellular nitric oxide and its surrogate marker, cyclic guanosine monophosphate, while the nitric oxide synthase inhibitor, ethyl-isothiourea, blocked its effect. We propose that apoE secreted locally at lesion sites by macrophages may be anti-inflammatory by stimulating endothelium to release NO and suppress VCAM-1 expression.     

Fatty Acid Patterns in <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. as a Marker for Nutritional Regimes and Temperature under Extremely Acidic Conditions

Fatty acid profiles were used to characterize nutritional pathways in Chlamydomonas sp. isolated from an acidic mining lake (pH 2.7). Surprisingly, profiles of Chlamydomonas sp. grown in the lab under photoautotrophic, mixotrophic, and heterotrophic conditions at in situ deep strata lake water temperatures (8°C) were very similar, polyunsaturated fatty acids including -linolenic acid (18:33) and 16:43 along with palmitic acid (16:0) being most abundant. Therefore, heterotrophic growth of Chlamydomonas sp. at low temperatures can result in high concentrations of polyunsaturated fatty acids, as previously only described for some psychrophilic bacteria. By contrast, the cultivation of isolated Chlamydomonas sp. at 20°C, reflecting surface water temperatures, provided fatty acid patterns characteristic of the nutrition strategy applied: the concentration of polyunsaturated fatty acids decreased when the growth pathway changed from photoautotrophic via mixotrophic to heterotrophic. Total fatty acid concentration also diminished in this order. Principal component analysis confirmed the significance of FA profiling to mirror nutritional pathways. Lake-water analysis revealed low concentrations of dissolved organic carbon, mainly consisting of polymeric fulvic acids that are unable to support heterotrophic growth of Chlamydomonas sp. Polymeric fulvic acids present in the deeper strata of the lake turned out to be formed in situ on the basis of organic monomers including reduced sulfur-containing ones, as revealed by thermochemolysis and pyrolysis. Growth of Chlamydomonas sp. in the deep chlorophyll maximum is therefore assumed to mainly result from photosynthesis, despite very low photon densities. Phytol-including metabolites proved to be significant biomarkers to indicate the nutritional pathway of Chlamydomonas sp. , -Dicarboxylic acids—light-induced degradation products of unsaturated fatty acids—appeared to be good indicators of photooxidative alterations to the algal species under study.