Biomedical subjects

Peter Lipp

Publications and source records attributed to Peter Lipp.

13 recordsLinked to original sources

PubMed2006

Sustained activity of calcium release-activated calcium channels requires translocation of mitochondria to the plasma membrane.

A rise of the intracellular Ca(2+) concentration has multiple signaling functions. Sustained Ca(2+) influx across plasma membrane through calcium release-activated calcium (CRAC) channels is required for T-cell development in the thymus, gene transcription, and proliferation and differentiation of naïve T-cells into armed effectors cells. Intracellular Ca(2+) signals are shaped by mitochondria, which function as a highly dynamic Ca(2+) buffer. However, the precise role of mitochondria for Ca(2+)-dependent T-cell activation is unknown. Here we have shown that mitochondria are translocated to the plasma membrane as a consequence of Ca(2+) influx and that this directed movement is essential to sustain Ca(2+) influx through CRAC channels. The decreased distance between mitochondria and the plasma membrane enabled mitochondria to take up large amounts of inflowing Ca(2+) at the plasma membrane, thereby preventing Ca(2+)-dependent inactivation of CRAC channels and sustaining Ca(2+) signals. Inhibition of kinesin-dependent mitochondrial movement along microtubules abolished mitochondrial translocation and reduced sustained Ca(2+) signals. Our results show how a directed movement of mitochondria is used to control important cellular functions such as Ca(2+)-dependent T-cell activation.

Biological Transport, Active↗

PubMed2006

Reduced cardiac L-type Ca2+ current in Ca(V)beta2-/- embryos impairs cardiac development and contraction with secondary defects in vascular maturation.

Cardiac myocyte contraction depends on transmembrane L-type Ca2+ currents and the ensuing release of Ca2+ from the sarcoplasmic reticulum. Here we show that these L-type Ca2+ currents are essential for cardiac pump function in the mouse at developmental stages where the functional significance of the heart becomes imperative to blood flow and to the continuing growth and survival of the embryo. Disruption of the Ca(V)beta2 gene, which encodes for the predominant ancillary beta subunit of cardiac Ca2+ channels, resulted in diminished L-type Ca2+ currents in cardiomyocytes of embryonic day 9.5 (E9.5). This led to a functionally compromised heart, causing defective remodeling of intra- and extraembryonic blood vessels and embryonic death following E10.5. The defects in vascular remodeling were also observed when the Ca(V)beta2 gene was selectively targeted in cardiomyocytes, demonstrating that they are secondary to cardiac failure rather than a result of the lack of Ca(V)beta2 proteins in the vasculature. Partial rescue of the Ca2+ channel currents by a Ca2+ channel agonist significantly postponed embryonic death in Ca(V)beta2-/- mice. Taken together, these data strongly support the essential role of L-type Ca2+ channel activity in cardiomyocytes for normal heart development and function and that this is a prerequisite for proper maturation of the vasculature.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2006

PKCalpha: a versatile key for decoding the cellular calcium toolkit.

Conventional protein kinases C (cPKCs) play an essential role in signal transduction and are believed to integrate both global Ca(2+) transients and diacylglycerol signals. We provide evidence that PKCalpha is a ubiquitous readout sensor for the cellular Ca(2+) toolkit, including highly restricted elementary Ca(2+) release. Threshold stimulations of cells with Ca(2+)-mobilizing agonists resulted in PKCalpha translocation events with limited spatial spreads (<4 microm) comprising two groups of lifetimes; brief events (400-1,500 ms) exclusively mediated by Ca(2+)-C2 domain membrane interactions and long-lasting events (>4 s) resulting from longer DAG-C1a domain-mediated membrane interactions. Although upon uncaging NP-EGTA, which is a caged Ca(2+) compound, WT-PKCalpha displayed rapid membrane translocations within <250 ms, PKCalpha constructs with C2 domains mutated in their Ca(2+)-binding region lacked any Ca(2+)-dependent translocation. Flash photolysis of diazo-2, a photosensitive caged Ca(2+) buffer, revealed a biphasic membrane dissociation (slow and fast period) of WT-PKCalpha. The slow phase was absent in cells expressing PKCalpha-constructs containing mutated C1a-domains with largely reduced DAG binding. Thus, two groups of PKCalpha membrane interactions coexist; C2- and C1a-mediated interactions with different lifetimes but rapid interconversion. We conclude that PKCalpha can readout very fast and, spatially and temporally, very complex cellular Ca(2+) signals. Therefore, cPKCs are important transducers for the ubiquitous cellular Ca(2+) signaling toolkit.

Animals↗

PubMed2005

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling.

In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice. We found that the SR Ca(2+) load was reduced by 22% in cardiomyocytes from TG mice. Consistent with this, the frequency of Ca(2+) sparks was diminished by 32%. The decay of spontaneous Ca(2+) sparks was prolonged by 117% in TG. This finding was associated with a lower Na(+)-Ca(2+) exchanger (NCX) protein expression (by 67%) and a higher basal RyR phosphorylation at Ser(2809) (by 64%) in TG. The shortening- and Delta[Ca](i)-frequency relationships (0.5-4 Hz) were flat in TG compared to wild-type (WT) which exhibited a positive staircase for both parameters. Furthermore, increasing stimulation frequencies hastened the time of relaxation and the decay of [Ca](i) by a higher percentage in TG. We conclude that the impaired relaxation in TG may result from a reduced NCX expression and/or a higher SR Ca(2+) leak. The altered shortening-frequency relationship in TG seems to be a consequence of an impaired excitation-contraction coupling with depressed SR Ca(2+) release at higher rates of stimulation. Our data suggest that the more prominent frequency-dependent hastening of relaxation in TG results from a stimulation of SR Ca(2+) transport reflected by corresponding changes of [Ca](i).

Adaptation, Physiological↗

PubMed2005

Calcium imaging of individual erythrocytes: problems and approaches.

Although in erythrocytes calcium is thought to be important in homeostasis, measurements of this ion concentration are generally seen as rather problematic because of the auto-fluorescence or absorption properties of the intracellular milieu. Here, we describe experiments to assess the usability of popular calcium indicators such as Fura-2, Indo-1 and Fluo-4. In our experiments, Fluo-4 turned out to be the preferable indicator because (i) its excitation and emission properties were least influenced by haemoglobin and (ii) it was the only dye for which excitation light did not lead to significant auto-fluorescence of the erythrocytes. From these results, we conclude that the use of indicators such as Fura-2 together with red blood cells has to be revisited critically. We thus utilized Fluo-4 in erythrocytes to demonstrate a robust but heterogeneous calcium increase in these cells upon stimulation by prostaglandin E(2) and lysophosphatidic acid. For the latter stimulus, we recorded emission spectra of individual erythrocytes to confirm largely unaltered Fluo-4 emission. Our results emphasize that in erythrocytes measurements of intracellular calcium are reliably possible with Fluo-4 and that other indicators, especially those requiring UV-excitation, appear less favourable.

Aniline Compounds↗

PubMed2004

Prostaglandin E2 activates channel-mediated calcium entry in human erythrocytes: an indication for a blood clot formation supporting process.

Prostaglandin E(2) (PGE(2)) is released from platelets when they are activated. Using fluorescence imaging and the patch-clamp technique, we provide evidence that PGE(2) at physiological concentrations (10(-10) M) activates calcium rises mediated by calcium influx through a non-selective cation-channel in human red blood cells. The extent of calcium increase varied between cells with a total of 45% of the cells responding. It is well known that calcium increases elicited the calcium-activated potassium channel (Gardos channel) in the red cell membrane. Previously, it was shown that the Gardos channel activation results in potassium efflux and shrinkage of the cells. Therefore, we conclude that the PGE(2) responses of red blood cells described here reveal a direct and active participation of erythrocytes in blood clot formation.

Aniline Compounds↗

PubMed2003

Local and global spontaneous calcium events regulate neurite outgrowth and onset of GABAergic phenotype during neural precursor differentiation.

Neural stem cells can generate in vitro progenitors of the three main cell lineages found in the CNS. The signaling pathways underlying the acquisition of differentiated phenotypes in these cells are poorly understood. Here we tested the hypothesis that Ca(2+) signaling controls differentiation of neural precursors. We found low-frequency global and local Ca(2+) transients occurring predominantly during early stages of differentiation. Spontaneous Ca(2+) signals in individual precursors were not synchronized with Ca(2+) transients in surrounding cells. Experimentally induced changes in the frequency of local Ca(2+) signals and global Ca(2+) rises correlated positively with neurite outgrowth and the onset of GABAergic neurotransmitter phenotype, respectively. NMDA receptor activity was critical for alterations in neuronal morphology but not for the timing of the acquisition of the neurotransmitter phenotype. Thus, spontaneous Ca(2+) signals are an intrinsic property of differentiating neurosphere-derived precursors. Their frequency may specify neuronal morphology and acquisition of neurotransmitter phenotype.

Animals↗

PubMed2002

Spatial characteristics of sarcoplasmic reticulum Ca2+ release events triggered by L-type Ca2+ current and Na+ current in guinea-pig cardiac myocytes.

Ca2+ signals in cardiac muscle cells are composed of spatially limited elementary events termed Ca2+ sparks. Several studies have also indicated that Ca2+ signals smaller than Ca2+ sparks can be elicited. These signals have been termed Ca2+ quarks and were proposed to result from the opening of a single Ca2+ release channel of the sarcoplasmic reticulum. We used laser-scanning confocal microscopy to examine the subcellular properties of Na+ current (I(Na))- and L-type Ca2+ current (I(Ca,L))-induced Ca2+ transients in voltage-clamped ventricular myocytes isolated from guinea-pigs. Both currents, I(Na) and I(Ca,L), evoked substantial, global Ca2+ transients. To examine the spatiotemporal properties of such Ca2+ signals, we performed power spectral analysis of these Ca2+ transients and found that both lacked spatial frequency components characteristic for Ca2+ sparks. The application of 10 microM verapamil to partially block L-type Ca2+ current reduced the corresponding Ca2+ transients down to individual Ca2+ sparks. In contrast, I(Na)-induced Ca2+ responses were still spatially homogeneous and lacked Ca2+ sparks even for small current amplitudes. By using high resistance patch pipettes (> 4 MOmega) to exaggerate the loss of voltage control during I(Na), Ca2+ sparks appeared superimposed on a homogeneous Ca2+ release component and were exclusively triggered during the flow of I(Na). In the presence of 10 microM ryanodine both I(Ca,L) and I(Na) elicited small, residual Ca2+ transients that were spatially homogeneous but displayed distinctively different temporal profiles. We conclude that I(Na) is indeed able to cause Ca2+ release in guinea-pig ventricular myocytes. In contrast to I(Ca,L)-induced Ca2+ transients, which are built up from the recruitment of individual Ca2+ sparks, the I(Na)-evoked cellular responses were always homogeneous, indicating that their underlying elementary Ca2+ release event is distinct from the Ca2+ spark. Thus, I(Na)-induced Ca2+ transients are composed of smaller Ca2+ signals, most likely Ca2+ quarks.

Animals↗

PubMed2002

A therapeutic CD4 monoclonal antibody inhibits TCR-zeta chain phosphorylation, zeta-associated protein of 70-kDa Tyr319 phosphorylation, and TCR internalization in primary human T cells.

The molecular mechanisms mediating the inhibitory effects of a humanized CD4 mAb YHB.46 on primary human CD4(+) T cells were investigated. Preincubation of T cells with soluble YHB.46 caused a general inhibition of TCR-stimulated protein tyrosine phosphorylation events, including a reduction in phosphorylation of p95(vav), linker for activation of T cells, and Src homology 2 domain-containing leukocyte protein of 76-kDa signaling molecules. A marked reduction in activation of the Ras/mitogen-activated protein kinase pathway was also observed. Examination of the earliest initiation events of TCR signal transduction showed that YHB.46 inhibited TCR-zeta chain phosphorylation together with recruitment and tyrosine phosphorylation of the zeta-associated protein of 70-kDa tyrosine kinase, particularly at Tyr(319), as well as reduced recruitment of p56(lck) to the TCR-zeta and zeta-associated protein of 70-kDa complex. These inhibitory events were associated with inhibition of TCR endocytosis. Our results show that the YHB.46 mAb is a powerful inhibitor of the early initiating events of TCR signal transduction.

Antibodies, Monoclonal↗

PubMed2002

RyR1 and RyR3 isoforms provide distinct intracellular Ca2+ signals in HEK 293 cells.

Ryanodine receptors (RyRs) are expressed on the endoplasmic reticulum of many cells, where they form intracellular Ca2+-release channels that participate in the generation of intracellular Ca2+ signals. Here we report studies on the intracellular localisation and functional properties of transfected RyR1 or RyR3 channels in HEK 293 cells. Immunofluorescence studies indicated that both RyR1 and RyR3 did not form clusters but were homogeneously distributed throughout the endoplasmic reticulum. Ca2+ release experiments showed that transfected RyR1 and RyR3 channels responded to caffeine, although with different sensitivity, generating a global release of Ca2+ from the entire endoplasmic reticulum. However, video imaging and confocal microscopy analysis revealed that, in RyR3-expressing cells, local spontaneous Ca2+ release events were observed. No such spontaneous activity was observed in RyR1-expressing cells or in control cells. Interestingly, the spontaneous release events observed in RyR3-expressing cells were restricted to one or two regions of the endoplasmic reticulum, suggesting the formation of a further subcellular organisation of RyR3 in Ca2+ release units. These results demonstrate that different RyR isoforms can engage in the generation of distinct intracellular Ca2+ signals in HEK 293 cells.

Calcium↗

PubMed2002

The role of inositol 1,4,5-trisphosphate receptors in Ca(2+) signalling and the generation of arrhythmias in rat atrial myocytes.

Various cardio-active stimuli, including endothelin-1 (ET-1), exhibit potent arrhythmogenicity, but the underlying cellular mechanisms of their actions are largely unclear. We used isolated rat atrial myocytes and related changes in their subcellular Ca(2+) signalling to the ability of various stimuli to induce diastolic, premature extra Ca(2+) transients (ECTs). For this, we recorded global and spatially resolved Ca(2+) signals in indo-1- and fluo-4-loaded atrial myocytes during electrical pacing. ET-1 exhibited a higher arrhythmogenicity (arrhythmogenic index; ratio of number of ECTs over fold-increase in Ca(2+) response, 8.60; n = 8 cells) when compared with concentrations of cardiac glycosides (arrhythmogenic index, 4.10; n = 8 cells) or the beta-adrenergic agonist isoproterenol (arrhythmogenic index, 0.11; n = 6 cells) that gave similar increases in the global Ca(2+) responses. Seventy-five percent of the ET-1-induced arrhythmogenic Ca(2+) transients were accompanied by premature action potentials, while for digoxin this proportion was 25 %. The beta-adrenergic agonist failed to elicit a significant number of ECTs. Direct activation of inositol 1,4,5-trisphosphate (InsP(3)) receptors with a membrane-permeable InsP(3) ester (InsP(3) BM) mimicked the effect of ET-1 (arrhythmogenic index, 14.70; n = 6 cells). Inhibition of InsP(3) receptors using 2 microM 2-aminoethoxydiphenyl borate, which did not display any effects on Ca(2+) signalling under control conditions, specifically suppressed the arrhythmogenic action of ET-1 and InsP(3) BM. Immunocytochemistry indicated a co-localisation of peripheral, junctional ryanodine receptors with InsP(3)Rs. Thus, the pronounced arrhythmogenic potency of ET-1 is due to the spatially specific recruitment of Ca(2+) sparks by subsarcolemmal InsP(3)Rs. Summation of such sparks efficiently generates delayed after depolarisations that trigger premature action potentials. We conclude that the particular spatial profile of cellular Ca(2+) signals is a major, previously unrecognised, determinant for arrhythmogenic potency and that the InsP(3) signalling cassette might therefore be a promising new target for understanding and managing atrial arrhythmia.

Action Potentials↗

PubMed2002

Mechanisms underlying the neuronal calcium sensor-1-evoked enhancement of exocytosis in PC12 cells.

Neuronal calcium sensor-1 (NCS-1) or the originally identified homologue frequenin belongs to a superfamily of EF-hand calcium binding proteins. Although NCS-1 is thought to enhance synaptic efficacy or exocytosis mainly by activating ion channel function, the detailed molecular basis for the enhancement is still a matter of debate. Here, mechanisms underlying the NCS-1-evoked enhancement of exocytosis were investigated using PC12 cells overexpressing NCS-1. NCS-1 was found to have a broad distribution in the cells being partially distributed in the cytosol and associated to vesicles and tubular-like structures. Biochemical and immunohistochemical studies indicated that NCS-1 partially colocalized with the light synaptic vesicle marker synaptophysin. When stimulated with UTP or bradykinin, agonists to phospholipase C-linked receptors, NCS-1 enhanced the agonist-mediated elementary and global Ca2+ signaling and increased the levels of downstream signals of phosphatidylinositol 4-kinase. NCS-1 enhanced the UTP-evoked exocytosis but not the depolarization-evoked Ca2+ responses or exocytosis, suggesting that the enhancement by NCS-1 should involve phospholipase C-linked receptor-mediated signals rather than the Ca2+ channels or exocytotic machinery per se. Taken together, NCS-1 enhances phosphoinositide turnover, resulting in enhancement of Ca2+ signaling and exocytosis. This is a novel regulatory mechanism of exocytosis that might involve the activation of phosphatidylinositol 4-kinase.

Animals↗

PubMed2002

Mitochondria are morphologically and functionally heterogeneous within cells.

We investigated whether mitochondria represent morphologically continuous and functionally homogenous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or calcein. The mitochondria of HeLa, PAEC, COS-7, HUVEC, hepatocytes, cortical astrocytes and neuronal cells all displayed heterogeneous distributions and were of varying sizes. There was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could be seen clearly. Using fluorescence-recovery after photobleaching, we observed that DsRed1 and calcein were highly mobile within the matrix of individual mitochondria, and that mitochondria within a cell were not lumenally continuous. Mitochondria were not electrically coupled, since only individual mitochondria were observed to depolarize following irradiation of TMRE-loaded cells. Functional heterogeneity of mitochondria in single cells was observed with respect to membrane potential, sequestration of hormonally evoked cytosolic calcium signals and timing of permeability transition pore opening in response to tert-butyl hydroperoxide. Our data indicate that mitochondria within individual cells are morphologically heterogeneous and unconnected, allowing them to have distinct functional properties.

Animals↗