Methods for the study of ionic currents and Ca(2+)-signals in isolated colonic crypts

Isolated epithelial cells from intestinal mucosae are a suitable object for the study of the regulation of ion transport in the gut. This regulation possesses a great importance for human and veterinary medicine, as diarrheal diseases, which often are caused by an inadequate activation of intestinal anion secretion, are one of the major lethal diseases of children or young animals. The aim of this paper is to describe a method for the isolation of intact colonic crypts, e.g. for the subsequent investigation of the regulation of anion secretion by the intracellular second messenger, Ca(2+) using electrophysiological and imaging techniques

The influence of statins on the free intracellular calcium concentration in human umbilical vein endothelial cells

BACKGROUND: Statins are cholesterol-lowering drugs that are widely used to reduce the risk of cardiac infarction. Their beneficial clinical effects, however, are not restricted to their influence on cholesterol production. As several studies have shown that they have a potency of relaxing blood vessels. METHODS: We measured the effects of statins on the intracellular free calcium concentration ([Ca(2+)](i)) in human umbilical vein endothelial cells (HUVEC) after acute application and 24-h-preincubation of statins. RESULTS: Incubation of the cells for 24 h with cerivastatin or fluvastatin significantly increased the resting [Ca(2+)](i). For cerivastatin this effect manifested at a concentration of 1 μM. Increase of resting [Ca(2+)](i )in the presence of cerivastatin also occurred when the nitric oxide synthase was inhibited. Transient Ca(2+ )release induced by histamine was not affected. CONCLUSIONS: The increase of resting [Ca(2+)](i )after incubation with cerivastatin or fluvastatin may provide an explanation for the direct effects of statins on the endothelial-dependent vasodilatation and restoration of endothelial activity in vivo

The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria

Components of the death receptors-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIPL), a well- known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca2+-release as well as ER-mitochondria tethering were decreased in c-FLIP-/- mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIPL and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIPL emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4

MRI sensing based on the displacement of paramagnetic ions from chelated complexes

We introduce a mechanism for ion sensing by MRI in which analytes compete with paramagnetic ions for binding to polydentate chelating agents. Displacement of the paramagnetic ions results in alteration of solvent interaction parameters and consequent changes in relaxivity and MRI contrast. The MRI changes can be tuned by the choice of chelator. As an example, we show that calcium-dependent displacement of Mn[superscript 2+] ions bound to EGTA and BAPTA results in a T[subscript 1]-weighted MRI signal increase, whereas displacement from calmodulin results in a signal decrease. The changes are ion selective and can be explained using relaxivity theory. The ratio of T[subscript 2] to T[subscript 1] relaxivity is also calcium-dependent, indicating the feasibility of “ratiometric” analyte detection, independent of the probe concentration. Measurement of paramagnetic ion displacement effects could be used to determine analyte ion concentrations with spatial resolution in opaque specimens.National Institutes of Health (U.S.) (grant DP2-OD2441)National Institutes of Health (U.S.) (grant R01-GM65519)McGovern Institute for Brain Research at MIT. Neurotechnology (MINT) Progra

Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca2+) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca2+ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca2+-ATPase (SERCA) which is involved in the regulation of the intracellular Ca2+ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes

Development of fluorescent probes for bioimaging applications

Fluorescent probes, which allow visualization of cations such as Ca2+, Zn2+ etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems

Determination of the physical environment within the Chlamydia trachomatis inclusion using ion-selective ratiometric probes

Chlamydia trachomatis is an obligate intracellular bacterium with a biphasic life cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial inclusion is non-fusogenic with endosomal or lysosomal compartments but intersects a pathway involved in transport of sphingomyelin from the Golgi apparatus to the plasma membrane. The physical conditions within the mature chlamydial inclusion are unknown. We used ratiometric imaging with membrane-permeant, ion-selective fluorescent dyes for microanalyis of the physical environment within the inclusion. Determination of H + , Na + , K + and Ca 2 + concentrations using CFDA (carboxy fluorescein diacetate) or BCECF-AM (2 ′ ,7 ′ -bis (2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, SBFI-AM, PBFI-AM and fura-PE3-acetomethoxyester (Fura-PE3-AM), respectively, indicated that all ions assayed within the lumenal space of the inclusion approximated the concentrations within the cytoplasm. Stimulation of purinergic receptors by addition of extracellular ATP triggered a dynamic Ca 2 + response that occurred simultaneously within the cytoplasm and interior of the inclusion. The chlamydial inclusion thus appears to be freely permeable to cytoplasmic ions. These results have implications for nutrient acquisition by chlamydiae and may contribute to the non-fusogenicity of the inclusion with endocytic compartments.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72253/1/j.1462-5822.2002.00191.x.pd

Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signalling route

Astroglial cells, due to their passive electrical properties, were long considered subservient to neurons and to merely provide the framework and metabolic support of the brain. Although astrocytes do play such structural and housekeeping roles in the brain, these glial cells also contribute to the brain's computational power and behavioural output. These more active functions are endowed by the Ca2+-based excitability displayed by astrocytes. An increase in cytosolic Ca2+ levels in astrocytes can lead to the release of signalling molecules, a process termed gliotransmission, via the process of regulated exocytosis. Dynamic components of astrocytic exocytosis include the vesicular-plasma membrane secretory machinery, as well as the vesicular traffic, which is governed not only by general cytoskeletal elements but also by astrocyte-specific IFs (intermediate filaments). Gliotransmitters released into the ECS (extracellular space) can exert their actions on neighbouring neurons, to modulate synaptic transmission and plasticity, and to affect behaviour by modulating the sleep homoeostat. Besides these novel physiological roles, astrocytic Ca2+ dynamics, Ca2+-dependent gliotransmission and astrocyte–neuron signalling have been also implicated in brain disorders, such as epilepsy. The aim of this review is to highlight the newer findings concerning Ca2+ signalling in astrocytes and exocytotic gliotransmission. For this we report on Ca2+ sources and sinks that are necessary and sufficient for regulating the exocytotic release of gliotransmitters and discuss secretory machinery, secretory vesicles and vesicle mobility regulation. Finally, we consider the exocytotic gliotransmission in the modulation of synaptic transmission and plasticity, as well as the astrocytic contribution to sleep behaviour and epilepsy

Excitatory effect of ATP on rat area postrema neurons

ATP-induced inward currents and increases in the cytosolic Ca2+ concentration ([Ca]in) were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry, respectively. The ATP-induced current (IATP) and [Ca]in increases were mimicked by 2-methylthio-ATP and ATP-γS, and were inhibited by P2X receptor (P2XR) antagonists. The current–voltage relationship of the IATP exhibited a strong inward rectification, and the amplitude of the IATP was concentration-dependent. The IATP was markedly reduced in the absence of external Na+, and the addition of Ca2+ to Na+-free saline increased the IATP. ATP did not increase [Ca]in in the absence of external Ca2+, and Ca2+ channel antagonists partially inhibited the ATP-induced [Ca]in increase, indicating that ATP increases [Ca]in by Ca2+ influx through both P2XR channels and voltage-dependent Ca2+ channels. There was a negative interaction between P2XR- and nicotinic ACh receptor (nAChR)-channels, which depended on the amplitude and direction of current flow through either channel. Current occlusion was observed at Vhs between −70 and −10 mV when the IATP and ACh-induced current (IACh) were inward, but no occlusion was observed when these currents were outward at a Vh of +40 mV. The IATP was not inhibited by co-application of ACh when the IACh was markedly decreased either by removal of permeant cations, by setting Vh close to the equilibrium potential of IACh, or by the addition of d-tubocurarine or serotonin. These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR- and nAChR-channels