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1.
Sci Rep ; 7: 44843, 2017 03 21.
Article in English | MEDLINE | ID: mdl-28322303

ABSTRACT

Ligand gated ion channels are involved in many pathophysiological processes and represent a relevant, although challenging, target for drug discovery. We propose an innovative electro-optical approach to their analysis able to derive membrane conductance values from the local membrane potential changes imposed by test current pulses and measured by fast voltage-sensitive fluorescent dyes. We exploited the potential of this proprietary method by developing a drug testing system called "ionChannel Optical High-content Microscope" (ionChannelΩ). This automated platform was validated by testing the responses of reference drugs on cells expressing different ligand-gated ion channels. Furthermore, a double-blind comparison with FLIPR and automated patch-clamp was performed on molecules designed to act as antagonists of the P2RX7 receptor. ionChannelΩ proved highly reliable in all tests, resulting faster and more cost-effective than electrophysiological techniques. Overall, ionChannelΩ is amenable to the study of ligand gated ion channels that are receiving less attention due to limitations in current assays.


Subject(s)
Drug Discovery/methods , Ion Channel Gating/drug effects , Ligand-Gated Ion Channels/metabolism , Microscopy/methods , Optical Imaging/methods , Automation, Laboratory , Cell Membrane Permeability/drug effects , High-Throughput Screening Assays , Humans , Ligands , Membrane Potentials/drug effects , Microscopy, Fluorescence/methods , Reproducibility of Results
2.
Cell Mol Life Sci ; 63(21): 2546-53, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17058034

ABSTRACT

Astrocytes are a heterogeneous population of cells that are endowed with a great variety of receptors for neurotransmitters and neuromodulators. Recently prostaglandin E2 has attracted great interest since it is not only released by astrocytes but also activates receptors coupled to either phospholipase C or adenylyl cyclase. We report that EP2 receptor stimulation triggers cAMP production but also causes release of Ca2+ from intracellular stores. This effect is shared by other receptors similarly coupled to adenylyl cyclase and elicited by direct stimulation of the enzyme or application of cAMP analogues. However, the stimulation of the Ca2+ response by cAMP is not mediated by protein kinase A, since a specific antagonist of this kinase had no effect. Such a cross-talk between cAMP and Ca2+ was not observed in all astrocytes. It might therefore reflect a specific resource of either a subpopulation or astrocytes in a specific functional state.


Subject(s)
Adenylyl Cyclases/metabolism , Astrocytes/metabolism , Calcium Signaling , Receptors, Prostaglandin E/metabolism , Animals , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclooxygenase 2/metabolism , Dinoprostone/metabolism , Dinoprostone/pharmacology , Enzyme Activation/drug effects , Guanine Nucleotide Exchange Factors/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Prostaglandin E, EP2 Subtype
3.
Cell Mol Life Sci ; 62(1): 95-104, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15619011

ABSTRACT

Slow oscillations of cytosolic calcium ion concentration - [Ca(2+)](c) - typically originate from release by intracellular stores, but in some cell types can be triggered and sustained by Ca(2+) influx as well. In this study we simultaneously monitored changes in [Ca(2+)](c) and in the electrical activity of the cell membrane by combining indo-1 and patch-clamp measurements in single rat chromaffin cells. By this approach we observed a novel type of spontaneous [Ca(2+)](c) oscillations, much faster than those previously described in these cells. These oscillations are triggered and sustained by complex electrical activity (slow action potentials and spike bursts), require Ca(2+) influx and do not involve release from intracellular stores. The possible physiological implications of this new pathway of intracellular signalling are discussed.


Subject(s)
Action Potentials , Calcium Signaling/physiology , Calcium/metabolism , Chromaffin Cells/physiology , Animals , Calcium/analysis , Calcium Channels/physiology , Cell Membrane/physiology , Cells, Cultured , Chromaffin Cells/chemistry , Fluorometry , Permeability , Rats
4.
Diabetologia ; 45(1): 77-84, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11845226

ABSTRACT

AIMS/HYPOTHESIS: Islet transplantation is a minimally invasive approach to curing Type I (insulin-dependent) diabetes mellitus. Success has recently been reported in patients receiving solitary islet transplants but the outcome in patients receiving islets together with, or after, kidney transplants has been limited and unpredictable. METHODS: Here we report successful islet transplantation in a cohort of 15 patients with Type I diabetes who were followed for at least 1 year after islet transplantation, after having already received kidney allografts because of end-stage nephropathy. RESULTS: C-peptide after transplantation was higher than 0.17 nmol/l in all 15 recipients, reflecting the absence of primary non-function. Insulin requirement was reduced by over 50 % in all but one patient, and insulin independence was achieved in 10 (66 %) recipients, five of whom now have stable, prolonged insulin independence, well controlled fasting glycaemia, a substantial first-phase and normal second-phase response to glucose, normal insulin sensitivity (HOMA analyses) and HbA1 c of under 6.2 % (33, 26, 18, 13 and 12 months after transplantation respectively). Of importance for patient management, an assessment of fasting blood glucose and proinsulin values following overnight withdrawal of insulin administration one month after transplantation was a potent predictor of insulin independence, and could be used to decide patients who should have further islet preparations. CONCLUSION/INTERPRETATION: These findings support the use of islet transplantation as a cure for Type I diabetes in patients with severe complications.


Subject(s)
Diabetes Mellitus, Type 1/surgery , Diabetic Nephropathies/surgery , Islets of Langerhans Transplantation/physiology , Kidney Transplantation/physiology , Adult , Age of Onset , Diabetes Mellitus, Type 1/physiopathology , Female , Humans , Insulin/blood , Insulin/metabolism , Insulin Secretion , Kidney Failure, Chronic/surgery , Major Histocompatibility Complex , Male , Middle Aged , Treatment Outcome
5.
Biochem Biophys Res Commun ; 285(3): 724-7, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11453653

ABSTRACT

Fast calcium events occurring in cytoplasmic organelles after a single electrical stimulus were investigated by electron spectroscopic imaging (an electron microscope technique that reveals total calcium with high sensitivity and spatial resolution) in quick frozen presynaptic terminals of the frog neuromuscular junction. In resting preparations synaptic vesicles showed a prominent calcium signal whereas mitochondria were mostly negative and only some of the cisternae of the endoplasmic reticulum were clearly positive. In preparations quick frozen 10 ms after the application to the nerve of a single, supramaximal electric stimulus, no obvious change was observed in synaptic vesicles, while calcium levels rose to high values in the endoplasmic reticulum cisternae and in the matrix of mitochondria. Voltage-induced influx of Ca(2+) within synaptic terminals appears therefore to induce an extremely rapid uptake into selected organelles. The possible physiological role of this response is discussed.


Subject(s)
Calcium/metabolism , Neuromuscular Junction/metabolism , Presynaptic Terminals/metabolism , Animals , Cytoplasm/metabolism , Cytoplasm/ultrastructure , Electric Stimulation , Electron Probe Microanalysis , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Freeze Drying , In Vitro Techniques , Mitochondria/metabolism , Mitochondria/ultrastructure , Neuromuscular Junction/ultrastructure , Organelles/metabolism , Organelles/ultrastructure , Presynaptic Terminals/ultrastructure , Rana pipiens , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructure , Time Factors
6.
Cell Calcium ; 30(1): 1-8, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11396982

ABSTRACT

This is a review which is written on the basis of a cell calcium lecture delivered on 22 July 2000 at the European Research Meeting 'Calcium as a molecule of cellular integration'.


Subject(s)
Calcium/analysis , Cells/metabolism , Cells/ultrastructure , Animals , Calcium/metabolism , Humans , Intracellular Fluid/metabolism , Microscopy, Electron/instrumentation , Microscopy, Electron/methods , Organelles/metabolism , Organelles/ultrastructure
7.
J Physiol ; 525 Pt 2: 499-507, 2000 Jun 01.
Article in English | MEDLINE | ID: mdl-10835050

ABSTRACT

In order to further understand the role of voltage- and ligand-gated ryanodine receptors in the control of intracellular Ca2+ signalling during myogenesis, changes in cytosolic free calcium concentration ([Ca2+]i) were investigated by fura-2 videoimaging in C2C12 mouse myotubes developing in vitro. A synchronous [Ca2+]i increase was observed after depolarisation with high [K+], while the Ca2+ response propagated as a wave following caffeine administration. Application of the two stimuli to the same myotube often revealed the existence of cellular zones that were responsive to depolarisation but not to caffeine. Focal application of high [K+] promoted a [Ca2+]i response detectable only in the cellular areas close to the pipette tip, while focal application of caffeine elicited a [Ca2+]i increase which spread as a Ca2+ wave. Buffering of [Ca2+]i by BAPTA did not affect the pattern of the depolarisation-induced [Ca2+]i transient but abolished the Ca2+ waves elicited by caffeine. When high [K+] and caffeine were applied in sequence, reciprocal inhibition of the [Ca2+]i responses was observed. Our results suggest that the different spatial patterns of [Ca2+]i responses are due to uneven distribution of voltage- and ligand-gated ryanodine receptors within the myotube. These two types of receptor control two functionally distinct Ca2+ pools which are part of a common intracellular compartment. Finally, the two differently operated ryanodine receptor channels appear to be independently activated, so that a mechanism of Ca2+-induced Ca2+ release is not required to sustain the global response in C2C12 myotubes.


Subject(s)
Muscle, Skeletal/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Caffeine/pharmacology , Calcium Signaling/drug effects , Cell Line , Intracellular Fluid/metabolism , Ion Channel Gating , Ligands , Mice , Muscle Development , Muscle, Skeletal/drug effects , Muscle, Skeletal/growth & development , Potassium/pharmacology , Ryanodine Receptor Calcium Release Channel/drug effects
8.
Diabetes ; 48(10): 1971-8, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10512361

ABSTRACT

Within pancreatic islet cells, rhythmic changes in the cytosolic Ca2+ concentration have been reported to occur in response to stimulatory glucose concentrations and to be synchronous with pulsatile release of insulin. We explored the possible mechanisms responsible for Ca2+ signal propagation within islet cells, with particular regard to gap junction communication, the pathway widely credited with being responsible for coordination of the secretory activity. Using fura-2 imaging, we found that multiple mechanisms control Ca2+ signaling in pancreatic islet cells. Gap junction blockade by 18 alpha-glycyrrhetinic acid greatly restricted the propagation of Ca2+ waves induced by mechanical stimulation of cells but affected neither Ca2+ signals nor insulin secretion elicited by glucose elevation. The source of Ca2+ elevation was also different under the two experimental conditions, the first being sustained by release from inner stores and the second by nifedipine-sensitive Ca2+ influx. Furthermore, glucose-induced Ca2+ waves were able to propagate across cell-free clefts, indicating that diffusible factors can control Ca2+ signal coordination. Our results provide evidence that multiple mechanisms of Ca2+ signaling operate in beta-cells and that gap junctions are not required for intercellular Ca2+ wave propagation or insulin secretion in response to glucose.


Subject(s)
Calcium/physiology , Islets of Langerhans/physiology , Signal Transduction , Animals , Cells, Cultured , Gap Junctions/drug effects , Glucose/pharmacology , Glycyrrhetinic Acid/analogs & derivatives , Glycyrrhetinic Acid/metabolism , Islets of Langerhans/drug effects , Mice , Signal Transduction/drug effects
9.
FASEB J ; 13(11): 1395-403, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10428763

ABSTRACT

Several types of ionic channels on the outer membrane of the nuclear envelope communicate with the nuclear cisternae. These are distinct from nucleocytoplasmic pathways, the nuclear pores that span the double membrane of the envelope and are the route for RNA and protein traffic in the nucleus. Recent data indicate that the nuclear pores may also function as ion channels. The most probable candidate for nucleocytoplasmic ion flux is a 300-400 pS pathway observed in many nuclear preparations. Morphological and functional studies of nuclear envelope suggest a tight relationship between the large conductance channel and the pore complex. However, there is no direct evidence for gating of the nuclear pore or its ability to open and close as a conventional channel. This study shows that in liver nuclei isolated from newborn mouse, there is a substantial correspondence between the number of pores and the number of channels recorded during patch-clamp. This is not the case for adult nuclei. Although pore density is comparable, some nuclear cytoskeletal components, such as actin and nonmuscle myosin, show a significant increase in the adult preparation. Previous studies demonstrate the presence of these two proteins in association with the pore complex. Here we show that by using actin filament disrupter, we were able to increase the number of active channels in adult isolated nuclei. We suggest that a functional interaction between actin filaments and the nuclear pore complex could regulate nucleocytoplasmic permeability.


Subject(s)
Ion Channels/physiology , Liver/physiology , Nuclear Envelope/physiology , Animals , Cell Nucleus/physiology , Ion Channel Gating/physiology , Ion Transport , Liver/ultrastructure , Mice , Patch-Clamp Techniques
10.
Philos Trans R Soc Lond B Biol Sci ; 354(1381): 373-8, 1999 Feb 28.
Article in English | MEDLINE | ID: mdl-10212486

ABSTRACT

Until now, most ultrastructural studies on the neuromuscular junction have been carried out on samples first exposed to chemical treatments--with fixatives and/or dehydration agents--that are known to induce, or to be inadequate to prevent, artefactual changes of the native state. We report here on the potential of a physical approach to the preparation of samples that combines quick-freezing and freeze-drying (with or without exposure to OsO4 vapours) followed by direct embedding of the samples in various resins. Thin sections from physically processed frog neuromuscular junctions, when compared to their chemically fixed counterparts, exhibit an overall excellent preservation, with the organelles retaining their native density and shape. These preparations were also investigated by electron spectroscopic imaging and electron energy loss spectroscopy, obtaining high resolution maps of native total calcium distribution within the nerve terminal. Finally, thin sections from analogously processed, however unfixed, preparations embedded in Lowicryl, were immunogold labelled before exposure to OsO4. Nerve-muscle preparations treated this way exhibited adequate preservation of ultrastructure and revealed the distribution of synaptophysin with high sensitivity and resolution. In conclusion, we provide an overview of the potential of the quick-freezing-freeze-drying approach in the study of the neuromuscular junction function.


Subject(s)
Calcium/metabolism , Neuromuscular Junction/metabolism , Neuromuscular Junction/ultrastructure , Animals , Freeze Drying , Immunohistochemistry , In Vitro Techniques , Microscopy, Electron , Rana pipiens , Spectrum Analysis , Subcellular Fractions/metabolism
11.
Proc Natl Acad Sci U S A ; 96(3): 945-9, 1999 Feb 02.
Article in English | MEDLINE | ID: mdl-9927673

ABSTRACT

Regulated exocytosis triggered by the photolysis of a caged Ca2+ compound, DM-nitrophen, was investigated by patch-clamp capacitance measurements in two clones of PC12, the first wild-type and the second (PC12-27) defective of both types of classical secretory vesicles together with the neuronal-type receptors for the attachment proteins of the N-ethylmaleimide-sensitive fusion protein, the so called SNAREs. Moreover, the electrophysiological data were correlated with the ultrastructure of resting quick-frozen-freeze-dried cells of the two clones. Wild-type PC12 exhibited two-component capacitance responses, time constants of 30-100 ms and >10 s, that previous studies had suggested to reflect primarily the fusion of the small and large secretory vesicles, each contributing cell surface increases of approximately 10%. Both of these components were largely and specifically inhibited whether cells previously were microinjected with tetanus toxin light chain. In the defective clone, large responses also were recorded ( approximately 19% surface expansion; time constant, approximately 1 s) that, in contrast to those of the wild-type, were entirely resistant to the toxin. Although secretory organelles, i.e., large vesicles and also profiles of small vesicles, were abundant at the cell periphery and often docked to the plasmalemma of resting wild-type PC12, in the defective clone, no superficial accumulation of vesicles was observed. Our coordinate structural and functional results have revealed diversities between the two classical forms of regulated secretion in wild-type PC12 and have provided evidence of a toxin-insensitive form of Ca2+-induced exocytosis, prominent in the defective clone, that may play an important role(s) in cellular physiology.


Subject(s)
Cell Membrane/physiology , Exocytosis , Muscle, Skeletal/physiology , Neuromuscular Junction/physiology , PC12 Cells/physiology , Acetates/pharmacokinetics , Animals , Calcium/metabolism , Cell Membrane/ultrastructure , Chelating Agents/pharmacokinetics , Ethylenediamines/pharmacokinetics , Kinetics , Muscle, Skeletal/cytology , Neuromuscular Junction/cytology , Patch-Clamp Techniques , Photolysis , Ranidae , Rats
12.
Ann Neurol ; 43(3): 340-9, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9506551

ABSTRACT

Central nervous system (CNS) lesions typical of multiple sclerosis (MS) are characterized by demyelinating inflammatory infiltrates that contain few CNS antigen-specific autoreactive T cells and a multitude of pathogenic non-antigen-specific mononuclear cells. Here, we report that in patients with MS the combined action of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-2, and IL-6 leads to the activation of most peripheral T cells (mainly CD4 memory) by promoting a persistent intracellular calcium increase via two independent signaling pathways. The activation of these pathways, one activated by IFNgamma and the other by the combination TNFalpha/IL-2/IL-6, is independent from myelin antigens and precedes by 2 weeks phases of disease activity (eg, clinical relapses and/or appearance of gadolinium-enhancing lesions on brain magnetic resonance imaging scans during 1 year of follow-up). Our results indicate that an appropriate combination of the four cytokines, three with a proinflammatory profile and one necessary for T-cell growth and differentiation, can activate in an antigen-independent fashion most peripheral T cells from MS patients. This mechanism is likely to contribute to the recruitment of nonspecific lymphocytes into the cellular activation processes leading to CNS demyelination and may represent a major target for immune intervention in MS.


Subject(s)
Calcium/physiology , Cytokines/physiology , Inflammation Mediators/physiology , Multiple Sclerosis/physiopathology , Signal Transduction/physiology , T-Lymphocytes/physiology , Antigens/physiology , Blood Cells/metabolism , Calcium/metabolism , Cell Division/drug effects , Cell Division/physiology , Cross-Sectional Studies , Cytokines/pharmacology , Drug Synergism , Humans , Inflammation Mediators/pharmacology , Intracellular Membranes/metabolism , Lymphocyte Activation/physiology , Multiple Sclerosis/blood , Myelin Sheath/immunology , Osmolar Concentration , T-Lymphocytes/drug effects
13.
Mol Biol Cell ; 8(8): 1501-12, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9285821

ABSTRACT

The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OSO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the variations organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.


Subject(s)
Calcium/metabolism , Endoplasmic Reticulum/ultrastructure , Golgi Apparatus/ultrastructure , Animals , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Immunohistochemistry , Microscopy, Electron , PC12 Cells , Rats
14.
Proc Natl Acad Sci U S A ; 93(10): 4799-803, 1996 May 14.
Article in English | MEDLINE | ID: mdl-8643483

ABSTRACT

We report on a procedure for tissue preparation that combines thoroughly controlled physical and chemical treatments: quick-freezing and freeze-drying followed by fixation with OsO4 vapors and embedding by direct resin infiltration. Specimens of frog cutaneous pectoris muscle thus prepared were analyzed for total calcium using electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) approach. The preservation of the ultrastructure was excellent, with positive K/Na ratios revealed in the fibers by x-ray microanalysis. Clear, high-resolution EELS/ESI calcium signals were recorded from the lumen of terminal cisternae of the sarcoplasmic reticulum but not from longitudinal cisternae, as expected from previous studies carried out with different techniques. In many mitochondria, calcium was below detection whereas in others it was appreciable although at variable level. Within the motor nerve terminals, synaptic vesicles as well as some cisternae of the smooth endoplasmic reticulum yielded positive signals at variance with mitochondria, that were most often below detection. Taken as a whole, the present study reveals the potential of our experimental approach to map with high spatial resolution the total calcium within individual intracellular organelles identified by their established ultrastructure, but only where the element is present at high levels.


Subject(s)
Calcium/metabolism , Electron Probe Microanalysis/methods , Neuromuscular Junction/metabolism , Neuromuscular Junction/ultrastructure , Animals , Histocytochemistry , Histological Techniques , In Vitro Techniques , Motor Neurons/metabolism , Organelles/metabolism , Potassium/metabolism , Rana pipiens , Sarcoplasmic Reticulum/metabolism , Sodium/metabolism , Synaptic Vesicles/metabolism
15.
FEBS Lett ; 384(2): 135-7, 1996 Apr 15.
Article in English | MEDLINE | ID: mdl-8612809

ABSTRACT

Cultures of rat brain cortex astrocytes were exposed to 10(-10)-10(-9)M of the HIV-1 envelope glycoprotein, gp120. No specific binding was revealed by the iodinated protein, suggesting expression of only a few sites onto the cells. In contrast, two transduction signals were rapidly induced by gp120: increased tyrosine phosphorylation of a approximately 56 kDa protein and increased [Ca2+]i. This latter effect, present in 1/3 of the investigated astrocytes, consisted in: discrete or biphasic peaks; slowly rising plateaus; and various types of oscillations. Moreover, in apparently unresponsive cells [Ca2+]i rose slowly (45 min) to double the resting levels. Rat brain cortex astrocytes thus appear highly sensitive to gp120. The induced array of signals might contribute to neurotoxicity during HIV infection.


Subject(s)
Astrocytes/drug effects , Calcium/metabolism , Cerebral Cortex/cytology , HIV Envelope Protein gp120/pharmacology , HIV-1/physiology , Nerve Tissue Proteins/metabolism , Protein Processing, Post-Translational/drug effects , Signal Transduction/drug effects , Animals , Astrocytes/physiology , Biological Transport/drug effects , Cells, Cultured , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Phosphorylation , Rats
16.
FEBS Lett ; 379(1): 21-5, 1996 Jan 22.
Article in English | MEDLINE | ID: mdl-8566222

ABSTRACT

Insulin release was investigated in parallel with changes in cytosolic calcium concentration, [Ca2+]i, in pig islets stimulated by glucose. After two days in culture, glucose stimulation failed to induce insulin release, and caused limited [Ca2+]i changes in few cells. After ten days, insulin response was partially restored and [Ca2+]i recordings revealed a slow oscillatory activity of the whole islet. Slow oscillations appeared to be due to the average [Ca2+]i variations resulting from the spreading of waves throughout the islet. These waves demonstrate the reestablishment of functional cell coupling, which appears to play a critical role in insulin release.


Subject(s)
Calcium/metabolism , Insulin/metabolism , Islets of Langerhans/metabolism , Animals , Cytosol/metabolism , Fluorescent Dyes , Fura-2 , Glucose/pharmacology , In Vitro Techniques , Insulin Secretion , Islets of Langerhans/drug effects , Kinetics , Microscopy, Fluorescence , Microscopy, Video , Oscillometry , Swine
17.
Eur J Neurosci ; 7(6): 1333-41, 1995 Jun 01.
Article in English | MEDLINE | ID: mdl-7582107

ABSTRACT

Cultures of cerebellar cortex cells were exposed to the HIV-1 envelope glycoprotein, gp120, and investigated for cytosolic Ca2+ ion concentration ([Ca2+]i) changes by the fura-2 ratio videoimaging technique while bathed in complete, Na(+)-free or Mg(2+)-free Krebs-Ringer media. At the end of the [Ca2+]i experiments the cells were fixed and immunoidentified through the revelation of markers specific for neurons (microtubule associated protein-2), type-2 (A2B5) or all (glial fibrillary acidic protein) astrocytes, oligodendrocytes (galactocerebroside) or microglia (F4/80 antibody). In complete medium, rapid biphasic (spike-plateau) responses induced by gp120 (0.1-1 nM) were observed in a subpopulation of type-2 astrocytes. In addition, slow but progressive responses were observed in other type-2 cells and oligodendrocytes, whereas type-1 astrocytes showed small responses, if any, and granule neurons did not respond at all. Use of Na(+)-free medium (a condition that blocked another gp120-induced response, cytosolic alkalinization) resulted in an increase in [Ca2+]i response that was appreciable not only in type-2 but also in most type-1 astrocytes, possibly because of the inhibition of the Na+/Ca2+ exchanger and the ensuing decrease in Ca2+ extrusion. Granule neurons, including those in direct contact with responsive astrocytes, remained unresponsive, even when the experiments were carried out in Mg(2+)-free medium supplemented with glycine, a condition that favors activation of the glutamatergic N-methyl-D-aspartate (NMDA) receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Astrocytes/metabolism , Calcium/metabolism , Cerebellum/metabolism , HIV Envelope Protein gp120/pharmacology , HIV-1 , Oligodendroglia/metabolism , Animals , Astrocytes/classification , Cells, Cultured , Cerebellum/cytology , Cytosol/metabolism , Immunohistochemistry , Osmolar Concentration , Rats , Rats, Sprague-Dawley
18.
J Cell Biol ; 129(3): 797-804, 1995 May.
Article in English | MEDLINE | ID: mdl-7730413

ABSTRACT

Spatial and temporal aspects of Ca2+ signaling were investigated in PC12 cells differentiated with nerve growth factor, the well known nerve cell model. Activation of receptors coupled to polyphosphoinositide hydrolysis gave rise in a high proportion of the cells to Ca2+ waves propagating non decrementally and at constant speed (2-4 microns/s at 18 degrees C and approximately 10-fold faster at 37 degrees C) along the neurites. These waves relied entirely on the release of Ca2+ from intracellular stores since they could be generated even when the cells were incubated in Ca(2+)-free medium. In contrast, when the cells were depolarized with high K+ in Ca(2+)-containing medium, increases of cytosolic Ca2+ occurred in the neurites but failed to evolve into waves. Depending on the receptor agonist employed (bradykinin and carbachol versus ATP) the orientation of the waves could be opposite, from the neurite tip to the cell body or vice versa, suggesting different and specific distribution of the responsible surface receptors. Cytosolic Ca2+ imaging results, together with studies of inositol 1,4,5-trisphosphate generation in intact cells and inositol 1,4,5-trisphosphate-induced Ca2+ release from microsomes, revealed the sustaining process of the waves to be discharge of Ca2+ from the inositol 1,4,5-trisphosphate- (and not the ryanodine-) sensitive stores distributed along the neurites. The activation of the cognate receptor appears to result from the coordinate action of the second messenger and Ca2+. Because of their properties and orientation, the waves could participate in the control of not only conventional cell activities, but also excitability and differential processing of inputs, and thus of electrochemical computation in nerve cells.


Subject(s)
Calcium/physiology , Neural Conduction/physiology , Neurites/physiology , Receptors, Cell Surface/physiology , Signal Transduction/physiology , Animals , Bradykinin/pharmacology , Carbachol/pharmacology , Cell Differentiation , Cell Polarity/physiology , Enzyme Activation , Inositol 1,4,5-Trisphosphate/metabolism , Inositol Phosphates/metabolism , Microscopy, Fluorescence , Microscopy, Video , Microsomes/metabolism , Neurites/drug effects , PC12 Cells , Rats , Time Factors , Type C Phospholipases/analysis
19.
Cell Calcium ; 17(5): 367-74, 1995 May.
Article in English | MEDLINE | ID: mdl-7553789

ABSTRACT

Rat chromaffin cells in primary culture exhibit oscillations of cytosolic Ca2+ concentration, sustained by the rhythmic discharge of Ca2+ from specialized intracellular stores. Each Ca2+ spike starts from a discrete region of the cell (pacemaker), and then propagates across the entire cytosol. Spike initiation and propagation, governing the oscillation frequency and amplitude respectively, appeared to be controlled by different mechanisms. The pacemaker was found to be directly activated by increases of cytosolic Ca2+ concentration obtained by either K+ depolarization or nicotinic stimulation. On the other hand, muscarinic or B2 stimulation was required for an efficient spreading to occur, thus suggesting a key role of InsP3 in the signal propagation. The pacemaker displayed an autonomous activity, as documented by the presence of local Ca2+ discharges, which were not necessarily accompanied by spreading to the rest of the cell. This uncoupling could be stimulated by the selective increase of the pacemaker firing rate, due to the rise of the intracellular Ca2+ concentration. Modulation of Ca2+ spike amplitude by treatments affecting either the pacemaker or the spreading phase might be related to quantal Ca2+ release from functionally discrete stores.


Subject(s)
Calcium/metabolism , Chromaffin System/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Acetylcholine/pharmacology , Animals , Apamin/pharmacology , Atropine/pharmacology , Bradykinin/pharmacology , Cations , Cells, Cultured , Chromaffin System/cytology , Chromaffin System/drug effects , Muscarinic Antagonists/pharmacology , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Organelles/drug effects , Organelles/metabolism , Potassium/metabolism , Potassium Chloride/pharmacology , Rats , Signal Transduction , Tetraethylammonium , Tetraethylammonium Compounds/pharmacology
20.
J Cell Biol ; 128(5): 905-12, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7876313

ABSTRACT

Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Synapsins/metabolism , Synaptic Vesicles/metabolism , Actins/ultrastructure , Animals , Cattle , Cytoskeleton/ultrastructure , Energy Transfer , Microscopy, Fluorescence , Microscopy, Video , Phosphorylation , Prosencephalon/metabolism , Prosencephalon/ultrastructure , Protein Binding , Rabbits , Rats , Synaptic Vesicles/ultrastructure
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