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Biomedical subjects

A M Vincent

Publications and source records attributed to A M Vincent.

13 recordsLinked to original sources
PubMed2000

Membrane asymmetry and DNA degradation: functionally distinct determinants of neuronal programmed cell death.

The ability to elucidate the molecular mechanisms that modulate programmed cell death (PCD) may provide the crucial clues to unravel the cellular basis of neurodegenerative disorders. Employing both a novel assay to follow serially PCD in individual living neurons and the neuroprotective agent lubeluzole as an investigative tool, we examined the development of nitric oxide (NO)-induced PCD over time through the reversible annexin V labelling of membrane phosphatidylserine (PS) exposure and the electron microscopy of genomic DNA in primary rat hippocampal neurons. Exposure to the NO generators SNP (300 microM) or NOC-9 (300 microM) alone increased annexin V-positive neurons in the population from 7% +/- 4% in untreated cultures to 13% +/- 4% at 1 hr and to 61% +/- 5% at 24 hr. Administration of a neuroprotective concentration of lubeluzole (750 nM) at the time of NO exposure initially prevented the exposure of PS residues, but consistently maintained DNA integrity over a 24 hr period. During posttreatment paradigms of lubeluzole (750 nM) at 2, 4, and 6 hr following NO exposure, progression of membrane PS inversion was reversed and subsequently suppressed over a 24 hr course. Our work illustrates that neuronal PCD is composed of at least two physiologically distinct and separate pathways that consist of the externalization of membrane PS residues and the independent maintenance of genomic DNA integrity. In addition, neuronal injury is fluid and reversible in nature, suggesting a "window of opportunity" for the repair and reversal of neurons yet to be committed to PCD.

Animals↗
PubMed2000

The metabotropic glutamate system promotes neuronal survival through distinct pathways of programmed cell death.

Activation of the metabotropic glutamate receptor (mGluR) system can prevent free radical, nitric oxide (NO)-induced programmed cell death (PCD). To investigate the mechanisms utilized by the mGluR system to regulate the induction of PCD, we examined the course of PCD in real time in individual, living, primary hippocampal neurons. We assessed both phosphatidylserine (PS) externalization, an early event in PCD, and DNA fragmentation during NO toxicity and mGluR modulation to determine the individual contributions of PS externalization and genomic DNA fragmentation during neuronal PCD. Exposure to the NO donors (300 microM SNP or 300 microM NOC-9) induced PCD in approximately 75% of neurons over a 24-h period. The externalization of PS in neurons increased to 21 +/- 2% as early as 3 h following NO exposure and then increased to 80 +/- 2% over a 24-h period. The externalization of PS was independent of the loss of membrane integrity. Agonists for individual mGluR subgroups were equally able to prevent NO-induced neuronal death and DNA degradation, yet they possessed differential abilities to regulate PS externalization. The group I agonist DHPG (750 microM) and the group III agonist L-AP4 (750 microM) both prevented and reversed NO-induced PS externalization. In contrast, activation of group II subtypes using L-CCG-I (750 microM) did not prevent PS externalization. Employing an experimental model that independently led to the externalization of PS residues, we demonstrated that PS externalization does not immediately impact on neuronal survival. Yet, subsequent neuronal survival may ultimately depend upon preventing PS externalization to avoid neuronal tagging for phagocytosis. Since group I and III mGluR subtypes possess the unique ability to maintain genomic integrity and membrane PS asymmetry, these agents may provide superior overall protection against NO-induced neuronal injury.

Animals↗
PubMed2000

Critical temporal modulation of neuronal programmed cell injury.

1. As a free radical, nitric oxide (NO) may be toxic to neurons through mechanisms that directly involve DNA damage. Lubeluzole, a novel benzothiazole compound, has recently been demonstrated to be neuroprotective through the signal transduction pathways of NO. We therefore examined whether neuroprotection by lubeluzole was dependent upon the molecular pathways of programmed cell death (PCD). 2. In primary hippocampal neurons, evidence of PCD was determined by hematoxylin and eosin (H&E) stain, transmission electron microscopy, and annexin-V binding. NO administration with the NO generators sodium nitroprusside (300 microM) or SIN-1 (300 microM) directly induced PCD. 3. Neurons positive for PCD increased from 22+/-3% (untreated) to 72+/-3% (NO) over a 24-hr period. Coadministration of NO and lubeluzole (750 nM), a neuroprotective concentration, actively decreased PCD expression on H&E stain from 72+/-3% (NO only) to 25+/-3% (NO and lubeluzole). Significant reduction in DNA fragmentation by lubeluzole also was evident on electron microscopy. Application of lubeluzole in concentrations that were not neuroprotective or administration of the biologically inactive R-isomer did not significantly alter NO-induced PCD, suggesting that neuroprotection by lubeluzole was intimately linked to the modulation of PCD. Lubeluzole also was able to prevent the initial stages of cellular membrane inversion labeled with annexin-V binding, an early and sensitive indicator of PCD. Interestingly, the critical period for lubeluzole to reverse PCD induction appeared to be within the first 4 hr following NO exposure. 4. Further investigation into the neuroprotective pathways that alter PCD may provide greater insight into the molecular mechanisms that ultimately determine neuronal injury.

Animals↗
PubMed1999

Group I metabotropic receptors down-regulate nitric oxide induced caspase-3 activity in rat hippocampal neurons.

Invoking the modulation of parallel cellular pathways, the G-protein metabotropic glutamate receptors (mGluRs) and nitric oxide (NO) have been shown to require a host of signal transduction pathways to modulate neuronal programmed cell death (PCD). Since the cysteine protease caspase-3 (CPP32) is one of the principal mediators of PCD in several nonneuronal cell systems, we investigated whether CPP32 activity was linked to both NO induced PCD and mGluR neuroprotection. We demonstrate that NO directly increases the activity of CPP32 by approximately 400% over a 6 h period that is necessary, at least in part, for the generation of neuronal PCD. Activation of only Group I mGluRs completely ameliorates the induction of CPP32 activity by NO and prevents the induction of PCD.

Animals↗
PubMed1999

Nitric oxide induction of neuronal endonuclease activity in programmed cell death.

Neuronal survival is intricately linked to the maintenance of intact DNA. In contrast, neuronal degeneration following nitric oxide (NO) exposure is dependent, in part, on the degradation of DNA through programmed cell death (PCD). We therefore investigated in primary rat hippocampal neurons the role of endogenous deoxyribonucleases, enzymes responsible for metabolically derived DNA cleavage, during NO-induced neurodegeneration. Twenty-four hours following exposure to the NO generators sodium nitroprusside (300 microM) and SIN-1 (300 microM), neuronal survival was reduced from approximately 88 to 23%. Treatment with aurintricarboxylic acid (1-100 microM), an endonuclease inhibitor, during NO exposure increased neuronal survival from 23 to 80% and decreased DNA fragmentation from 70 to 30% over a 24-h period. Enhancement of endonuclease activity alone with zinc chelation actively decreased neuronal survival from approximately 80% to approximately 34%. DNA digestion assays identified not only two constitutively active endonucleases, an acidic endonuclease (pH 4.0-7.0) and a calcium/magnesium-dependent endonuclease (pH 7.2-8.0), but also a NO-inducible magnesium-dependent endonuclease (pH 8.0). In the absence of endonuclease activity, DNA degradation did not occur during NO application, suggesting that endonuclease activity was a requisite pathway for NO-induced PCD. In addition, NO independently altered intracellular pH in ranges that were physiologically relevant for the activity of the endonucleases responsible for DNA degradation. Our identification and characterization of specific neuronal endonucleases suggest that the constitutive endonucleases may play a role in the initial stages of NO-induced PCD, but the subsequent "downstream" degradation of DNA may ultimately be dependent upon the NO-inducible endonuclease.

Animals↗
PubMed1999

Neuronal intracellular pH directly mediates nitric oxide-induced programmed cell death.

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH(i)) regulation during nitric oxide (NO)-induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 microM), 3-morpholinosydnonimine (300 microM), or 6-(2-hyrdroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex anamine (300 microM). The pH(i) was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH(i) 7.36 +/- 0.01 to approximately 7.00 (p <.0001). Within 45 min, a biphasic alkaline response was evident, with pH(i) reaching 7.40 +/- 0.02, that was persistent for a 6-h period. To mimic the effect of NO-induced acidification, neurons were acid-loaded with ammonium ions to yield a pH(i) of 7.09 +/- 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 +/- 2% (24 h) and DNA fragmentation increased to 58 +/- 8% (24 h) (p <.0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH(i) of 7.60 +/- 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO-induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration.

Animals↗
PubMed1999

Metabotropic glutamate receptors prevent programmed cell death through the modulation of neuronal endonuclease activity and intracellular pH.

Metabotropic glutamate receptor (mGluR) activation prevents neurodegeneration against nitric oxide (NO)-induced programmed cell death (PCD). We therefore investigated whether specific neuronal endogenous deoxyribonucleases, enzymes recently identified to be responsible for the maintenance of DNA integrity, mediated mGluR protection against NO. In rat primary hippocampal neurons, injury was assessed by using a 0.4% trypan blue dye exclusion method and TUNEL assay 24 h following treatment with the NO generators sodium nitroprusside (300 microM) or SIN-1 (300 microM). DNA digestion studies using neuronal cell extracts were employed to assess specific endonuclease activity. Individual application of aurintricarboxylic acid (ATA) (10 microM), an endonuclease inhibitor, or the mGluR agonists 1S,3R-ACPD (750 microM), DHPG (750 microM), L-CCG-I (750 microM), or L-AP4 (750 microM) prior to NO exposure significantly increased neuronal survival. Yet, combination therapy with ATA (10 microM) and the mGluR agonists did not synergistically improve neuronal survival, suggesting a common pathway of protection for ATA and the mGluRs that is dependent upon the modulation of neuronal endonuclease activity. In further support of this premise, protection by the mGluR agonists 1S,3R-ACPD, DHPG, L-CCG-I, and L-AP4 was significantly decreased during enhancement of endonuclease activity with the zinc chelator, N,N,N',N',-tetrakis (2-pyridylmethyl) ethylenediamine. Antagonism of the mGluR system was ineffective against endonuclease induced DNA destruction. Further assessment with DNA digestion assays identified two distinct mechanisms to maintain DNA integrity, a Ca2+/Mg2+-dependent endonuclease inhibited by L-AP4 and a magnesium dependent endonuclease inhibited by 1S,3R-ACPD. These neuroprotective mechanisms during activation of the mGluR system were also intricately linked to the active reversal of the biphasic intracellular pH changes induced by NO. Further investigation into the molecular pathways modulated by mGluRs may identify specific mechanisms that can maintain DNA integrity during adverse cellular environments.

Animals↗
PubMed1999

Direct temporal analysis of apoptosis induction in living adherent neurons.

Destruction of neurons through the genetically directed process of programmed cell death (PCD) is an area of intense interest because this is the underlying mechanism in a variety of developmental and neurodegenerative diseases. The ability to identify and track viable neurons subjected to PCD could be invaluable in development of strategies to prevent or reverse the downstream mechanisms of neuronal PCD. We have developed a novel assay for PCD in viable, adherent cells using annexin V labeling. Annexin V binds to the highly negatively charged plasma membrane phosphatidylserine residues that undergo membrane translocation during PCD. Current annexin V techniques are almost exclusively restricted to flow cytometric analysis. Our unique technique permits repeated examination of individual viable neurons without altering their survival. Correlation with electron microscopy and dye exclusion assays demonstrate both sensitivity and specificity for our method to detect PCD. To our knowledge, this is the first account of a technique that positively identifies PCD in viable, adherent cells.

Animals↗
PubMed1998

An optimized AV delay algorithm for patients with intermittent atrioventricular conduction.

Detection and promotion of an intermittent atrioventricular (AV) conduction is the objective of an AV delay hysteresis algorithm in dual chamber pacemaker (DDD) pacing. The AV delay following an atrial event is automatically extended by a programmable interval (AV hysteresis interval) if the previous cycle showed spontaneous AV conduction, i.e., a ventricular event was detected within the previous AV delay. An automatic search mode scans for spontaneous ventricular events during the hysteresis interval: a single AV delay extension (equal to the programmed AV delay hysteresis) will occur after a successive, programmable number of AV cycles with ventricular pacing. If a spontaneous AV conduction is present, the AV delay will remain extended by the hysteresis interval. Our first results in 17 patients with intermittent AV block disclosed a satisfactorily working algorithm with effective reduction of ventricular stimuli. In relation to the underlying conduction disturbance and pacemaker settings, the majority of our patients showed a reduction of ventricular pacing events up to 90% without any adverse hemodynamic or electrophysiological changes. Based on clinical (promotion of a physiological activation and contraction sequence) and technical (reduction of power consumption) advantages, the AV hysteresis principle could be of incremental value for future dual chamber pacing in patients with intermittent complete heart block.

Aged↗
PubMed1997

Metabotropic glutamate receptors prevent nitric oxide-induced programmed cell death.

Activation of metabotropic glutamate receptor (mGluR) subtypes can prevent neuronal injury through the signal transduction pathways of nitric oxide (NO). It is this link to NO free radical injury and subsequent DNA damage that is the most intriguing. We therefore examined whether neuronal protection through mGluR activation was dependent on the molecular mechanisms of programmed cell death (PCD). The NO generators sodium nitroprusside and 3-morpholino-sydnonimine were administered to induce NO toxicity in primary hippocampal neurons. PCD was documented by hematoxylin and eosin nuclear staining, DNA gel electrophoresis, transmission electron microscopy, and protein synthesis assays. Following NO exposure, PCD induction was rapid and robust in approximately 70% of the neuronal population. Activation of specific mGluR subtypes with 1S,3R-ACPD and L-AP4, agents that are neuroprotective against NO, significantly limited the progression of PCD. In contrast, antagonism of mGluRs with L-AP3 did not prevent the development of PCD. Induction of new protein synthesis, a common requisite for PCD, was evident following NO exposure, but did not appear to represent a principal pathway of modulation by the mGluR agonists. Our studies suggest that mGluR modulation of NO-induced PCD represents a primary molecular pathway responsible for neuronal survival. Further elucidation of the molecular mGluR signaling pathways may yield new insight into specific genetic regulatory mechanisms responsible for neuronal injury.

Animals↗
PubMed1996

Endothelial interactions of hairy cells: the importance of alpha 4 beta 1 in the unusual tissue distribution of the disorder.

The tissue-homing of all lymphocytes involves their interactions with endothelial cells (ECs) and with various tissue accessory cells. However, in hairy cell leukemia (HCL), these processes are particularly prominent and result in diagnostic appearances in the spleen, liver, and bone marrow. The present study explores the mechanisms that underlie these tissue reactions. Using a human umbilical vein EC (HUVEC) model, various possible receptor-ligand interactions between hairy cells (HCs) and ECs were examined and a central importance for alpha 4 beta 1/vascular cell adhesion molecule-1 (VCAM-1) was established. This receptor-ligand pair was shown to be important both for strong adhesion and for HC motility/transmigration. A similar importance for alpha 4 beta 1/VCAM-1 was established for the interaction between HCs and relevant tissue accessory cells. The in vitro relevance of these findings was confirmed by the demonstration of VCAM-1 in HCL spleen and by the fact that, in frozen sections, HCs adhered (via VCAM-1) to the red pulp, but not to other areas of normal spleen. These results indicate that alpha 4 beta 1/VCAM-1 is central to the interaction between HCs and endothelium/accessory cells. Such interactions, together with the intrinsic cell activation characteristic of HCL and the HC's consequent ability to interact with matrix, are responsible for many of the characteristic features of the disease.

Cell Movement↗
PubMed1996

Integrin function in chronic lymphocytic leukemia.

Integrins are central to many aspects of the tissue localization of normal and malignant lymphocytes. We examined how integrin function, rather than simple expression, might determine disease behavior in chronic lymphocyte leukemia (CLL). Using fluorescence-activated cell sorting (FACS) and immunoprecipitation, we first established the precise integrin heterodimer expression of a representative group of CLL patients (beta1 consistently present, with variable alpha 3, alpha 4, and alpha 5; alpha 4 beta 7 often expressed; alpha L beta 2 high; alpha V beta 3 absent). Regarding function, we initially examined the ability of CLL cells to interact with endothelium, because such interaction is the initial event determining the entry of CLL lymphocytes into tissues. The abnormal lymphocytes were shown to bind at low levels to unstimulated endothelium via beta 2/intercellular adhesion molecule (ICAM). However, when the endothelium was stimulated, markedly enhanced interaction with endothelium was observed in approximately half the cases; in these patients, the neoplastic population expressed alpha 4 beta 1, which conferred the ability to adhere strongly to stimulated endothelium via the alpha 4 beta 1 ligand, vascular cellular adhesion molecule-1 (VCAM-1). In relation to the migration of CLL cells within tissues, the abnormal lymphocytes showed differential binding to various adhesive proteins; they did not attach to basement membrane components, but displayed variable adhesion to fibronectin (FN). Finally, we examined the role of cell activation in these processes, and showed that activated CLL lymphocyte populations showed an increased capacity to adhere to both endothelium and matrix. Moreover, ex vivo CLL cells showed no capacity to migrate through endothelium/stroma, but were able to do so after cytokine stimulation. These studies show how the constitutive integrin expression/function, the intrinsic activation state of the cell, and the capacity of cytokines to modify integrin-mediated function all combine to determine the different patterns of clinical disease observed in CLL.

B-Lymphocytes↗