Reactive oxygen species and death: oxidative DNA damage in atherosclerosis.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
M R Bennett
Publications and source records attributed to M R Bennett.
Depression of transmitter release at synapses in the rat superior cervical ganglion: the role of transmitter depletion.
The characteristics of depression of the excitatory postsynaptic potential (EPSP) during a short train of impulses to the rat superior cervical ganglion (SCG) have been ascertained with the object of determining the relative contributions of transmitter depletion and autoreceptors to depression. Successive EPSPs in a short train were depressed after the first (Vo) up to about the fourth impulse when a steady-state depressed EPSP level (Vss) was reached. Vss increased with the stimulation frequency between 1 and 30 Hz. Vo recovered after a short train with a time constant of about 2.8 s in the frequency range from 5 to 30 Hz. In order to determine if depression was related to changes in calcium influx with successive impulses in the train. preganglionic boutons were loaded with the calcium indicator Oregon Green 488 BAPTA-1 and line scans taken through individual boutons with a confocal laser microscope. Successive calcium transients were of about the same amplitude in boutons during short trains of impulses at 5 Hz. The contribution of autoreceptors activated by the action of endogenously derived adenosine on the extent of depression of the EPSP during short trains was ascertained by blocking these receptors with 8-phenyltheophylline (10 microM). There was no change in the extent or time course of development of depression. Similar results were obtained with the opioid receptor antagonist naloxone (10 microM) and the adrenergic receptor antagonist yohimbine (10 microM). Factors, which increased the extent of transmitter release during a train, such as increasing the external calcium concentration from 0.8 to 2.5 mM, increased depression. Factors. which decreased the extent of transmitter release such as increasing the exogenous adenosine concentration between 1 and 200 microM decreased depression. These results are interpreted in terms of a model in which vesicles are mobilised by a calcium-dependent process from a store into an available pool of docked vesicles. Depletion of the docked vesicles during exocytosis then leads to depression of transmitter release during a train of impulses.
Animals↗
Formation and function of synapses with respect to Schwann cells at the end of motor nerve terminal branches on mature amphibian (Bufo marinus) muscle.
A study has been made of the formation and regression of synapses with respect to Schwann cells at the ends of motor nerve terminal branches in mature toad (Bufo marinus) muscle. Synapse formation and regression, as inferred from the appearance and loss of N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1-43)-stained vesicle clusters, occurred at the ends of terminal branches over a 16 hr period. Multiple microelectrodes placed in an array about FM1-43 blobs at the ends of terminal branches detected the electrical signs of neurotransmitter being released onto receptors. Injection of a calcium indicator (Oregon Green 488 BAPTA-1) into the motor nerve with subsequent imaging of the calcium transients, in response to stimulation, often showed a reduced calcium influx in the ends of terminal branches. Injection of a fluorescent dye into motor nerves revealed the full extent of their terminal branches and growing processes. Injection of the terminal Schwann cells (TSCs) often revealed pseudopodial TSC processes up to 10-microm-long. Imaging of these TSC processes over minutes or hours showed that they were highly labile and capable of extending several micrometers in a few minutes. Injection of motor nerve terminals with a different dye to that injected into their TSCs revealed that terminal processes sometimes followed the TSC processes over a few hours. It is suggested that the ends of motor nerve terminals in vivo are in a constant state of remodeling through the formation and regression of processes, that TSC processes guide the remodeling, and that it can occur over a relatively short period of time.
Animals↗
Mechanisms of depression of transmitter release at synapses.
Explore the source record for details and available documents.
Depression↗
Vesicle-associated proteins and transmitter release from sympathetic ganglionic boutons.
A method is reported for introducing peptides derived from SNARE proteins that control exocytosis of vesicles at boutons formed by sympathetic ganglion cells in tissue culture. These peptides were coupled to the DNA binding domain of the Drosophila transcription factor antennapedia, called penetratin. This facilitated the passage of peptides across the bouton membrane. FM1-43 was used to monitor the exocytosis of transmitter from depolarized boutons after their exposure to the penetratin-peptide sequences IETRHNEIIKLETSIRELHD of syntaxin and KGFLSSLFGGSSK of alpha-SNAP, both of which blocked secretion, whereas the peptide sequences SELDDRA-DALQAGASQFETSAAKLKRK of synaptobrevin did not. This report introduces a readily applicable method for determining the effect of different peptide sequences of vesicle-associated proteins on secretion at vertebrate boutons and presents an account of the effects of a selection of such peptides on exocytosis.
Amino Acid Sequence↗
Quantal and non-quantal current and potential fields around individual sympathetic varicosities on release of ATP.
The electrical phenomena that occur at sympathetic varicosities due to the release of ATP include spontaneous and evoked excitatory junction potentials (SEJPs and EJPs; recorded with an intracellular electrode) as well as fast and slow excitatory junctional currents (EJCs; recorded with a loose-patch electrode placed over varicosities). The electrical analysis of these transients is hampered by lack of a detailed theory describing how current and potential fields are generated upon the release of a quantum of ATP. Here, we supply such a theory and develop a computational model for the electrical properties of a smooth muscle syncytium placed within a volume conductor, using a distributed representation for the individual muscle cells. The amplitudes and temporal characteristics of both SEJPs and fast EJCs are predicted by the theory, but those of the slow EJCs are not. It is shown that these slow components cannot arise as a consequence of propagation of fast quantal components from their site of origin in the muscle syncytium to the point of recording. The possibility that slow components arise by a mechanism of transmitter secretion that is different from quantal release is examined. Experiments that involve inserting peptide fragments of soluble N-ethylmaleimide-sensitive fusion attachment protein (alpha-SNAP) into varicosities, a procedure that is known to block quantal release, left the slow component of release unaffected. This work provides an internally consistent description of quantal potential and current fields about the varicosities of sympathetic nerve terminals and provides evidence for a non-quantal form of transmitter release.
Adenosine Triphosphate↗
Mechanisms of angioplasty and stent restenosis: implications for design of rational therapy.
Restenosis after angioplasty or stenting remains the major limitation of both procedures. A vast array of drug therapies has been used to prevent restenosis, but they have proven to be predominantly unsuccessful. Recent trends in drug therapy have attempted to refine the molecular and biological targets of therapy, based on the assumption that a single biological process or molecule is critical to restenosis. In contrast, both stenting and brachytherapy, which are highly nonspecific, can successfully reduce restenosis after angioplasty or stenting, respectively. This review examines the biology of both angioplasty and stent stenosis, focussing on human studies. We also review the landmark human trials that have definitively proven successful therapies, such as stenting and brachytherapy. We suggest that the successful trials of stenting and brachytherapy and the failure of other treatments have highlighted the shortcomings of conventional animal models of arterial intervention, and gaps in our knowledge of human disease. In contrast to arguments advocating gene therapy, these studies suggest that the most likely successful drug therapy will have a wide therapeutic range, targeting as many of the components or biological processes contributing to restenosis as possible.
Angioplasty↗
Perception and memory in neuroscience: a conceptual analysis.
Neuroscientists, in the last half of the 20th century, provided major insights into the cellular and molecular mechanisms associated with seeing and remembering. We first identify some of the most important of these discoveries. This is done along lines familiar to neuroscientists who have read many of the recent books and reviews that provide an overview of neuroscientific discoveries. In general, these emphasize the scientific contributions this discipline has made to our understanding of the mechanisms that give rise to the psychological attributes of humans and other animals. In the next sections, we examine the claims made in these overviews; in particular, those by the standard-bearers of neuroscience, in an attempt to clarify what can and what cannot be justified in these claims. This requires a conceptual analysis of a kind that is unfamiliar to most neuroscientists. Our analysis begins with consideration of the conceptual confusions that ensue when neuroscientists attribute seeing, remembering and other psychological attributes to the brain rather than to the creature whose brain it is. Subsequently, we outline what we take to be the appropriate conceptual scheme for neuroscientists to adopt.
Animals↗
Gene therapy for coronary restenosis: is the enthusiasm justified?
Explore the source record for details and available documents.
Animals↗
New insights into atherosclerotic plaque rupture.
Explore the source record for details and available documents.
Cell Death↗
Human blood-derived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions.
Human atherosclerotic plaques that rupture are characterized by relatively low vascular smooth muscle cell (VSMC) and high inflammatory cell contents. Ruptured plaques also contain higher numbers of apoptotic VSMCs than do stable lesions, suggesting that VSMC apoptosis may promote plaque rupture. We examined the ability of human monocytes/macrophages to induce apoptosis of VSMCs derived from human carotid plaque, aortic media, and coronary media. Macrophages, but not T lymphocytes, induced a dose-dependent apoptosis of VSMCs, which required monocyte maturation to macrophages and direct cell-cell contact/proximity. VSMC apoptosis was inhibited by neutralizing antibodies to Fas-ligand (Fas-L) or an Fas-Fc fusion protein, indicating the requirement for membrane-bound Fas and Fas-L. Monocyte maturation was associated with increased surface expression of Fas-L, coincident with the onset of cytotoxicity. VSMCs expressed surface Fas, which was increased in plaque VSMCs, and plaque VSMCs also underwent Fas-induced apoptosis. We conclude that human macrophages potently induce human VSMC apoptosis, which requires direct cell-cell interactions and is in part dependent on Fas/Fas-L interactions. Macrophage-induced VSMC apoptosis may therefore directly promote plaque rupture.
Aorta↗
Localization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus. Metalloproteinase inhibition is associated with proapoptotic activity.
The tissue inhibitors of metalloproteinases (TIMPs) are a family of four secreted inhibitors of matrix metalloproteinases (MMPs). Recently, additional functions have been attributed to the TIMPs, including cell growth and inhibition of angiogenesis. In particular, we demonstrated that TIMP-3 overexpression using gene transfer induces apoptosis in a variety of cell types and can inhibit vascular neointima formation in vivo. However, little is know about the mechanisms underlying TIMP-3-mediated apoptosis. Here, using both purified recombinant proteins and novel adenoviral vectors we demonstrate that the prodeath domain of TIMP-3 is located within the N-terminal three loops of TIMP-3. Although both wild type and N-terminal TIMP-3 proteins promoted apoptosis, a T-2/T-3 chimera, in which the N-terminal three loops of TIMP-3 are replaced by those of TIMP-2, failed to induce cell death. Furthermore, a point mutation at residue 1 of TIMP-3 totally abolished MMP-inhibitory activity of TIMP-3 and also failed to promote apoptosis. This study demonstrates, using multiple apoptosis assays, that the prodeath function of TIMP-3 is located within the N-terminal three loops and the presence of functional metalloproteinase-inhibitory activity is associated with the induction of apoptosis.
Animals↗
Comparative study on the distribution patterns of P2X(1)-P2X(6) receptor immunoreactivity in the brainstem of the rat and the common marmoset (Callithrix jacchus): association with catecholamine cell groups.
The present study investigated the topographical distribution of P2X(1)-P2X(6) receptor subtypes in the rat and common marmoset hindbrain by immunohistochemistry. In addition, double-labeling immunofluorescence was used to determine the extent of colocalization between catecholamine cell groups and the various P2X receptors. The data demonstrate a widespread distribution pattern for all six P2X receptors throughout both the rat hindbrain and the marmoset hindbrain, although distinctions between species, brain nuclei, and P2X receptor subtypes exist. In rat, dense staining for the P2X receptors was found in the nucleus of the solitary tract (NTS), medial vestibular nucleus, and medial and lateral parabrachial nuclei. Moderate staining was observed in the hypoglossal nucleus, cuneate nucleus, inferior olive, prepositus hypoglossi, rostral ventrolateral medulla (RVLM), and locus coeruleus. Staining was also observed in the gracile nucleus, the mesencephalic trigeminal nucleus, and the central pontine gray. In marmoset, prominent P2X receptor-like immunoreactivity occurred in the NTS, medial cuneate nucleus, prepositus hypoglossi, and medial vestibular nucleus. Moderate staining was observed in the area postrema, dorsal motor nucleus of the vagus, lateral cuneate, lateral reticular, spinal trigeminal nucleus, RVLM, and inferior olive. Immunofluorescent double labeling of tyrosine hydroxylase (TH)-containing cells revealed that all subtypes of P2X receptors show some degree of colocalization with TH. The highest proportion of TH and P2X receptor double labeling was in the A5 region (with the P2X(2) subunit), whereas the lowest proportion of double-labeled cells occurred in the C2 region of the NTS for the P2X(5) subunit. These findings support a role for extracellular adenosine 5'-triphosphate in fast synaptic neurotransmission within the brainstem.
Adenosine Triphosphate↗
Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies.
The mechanisms involved in the initiation of vascular calcification are not known, but matrix vesicles, the nucleation sites for calcium crystal formation in bone, are likely candidates, because similar structures have been found in calcified arteries. The regulation of matrix vesicle production is poorly understood but is thought to be associated with apoptotic cell death. In the present study, we investigated the role of apoptosis in vascular calcification. We report that apoptosis occurs in a human vascular calcification model in which postconfluent vascular smooth muscle cell (VSMC) cultures form nodules spontaneously and calcify after approximately 28 days. Apoptosis occurred before the onset of calcification in VSMC nodules and was detected by several methods, including nuclear morphology, the TUNEL technique, and external display of phosphatidyl serine. Inhibition of apoptosis with the caspase inhibitor ZVAD.fmk reduced calcification in nodules by approximately 40%, as measured by the cresolphthalein method and alizarin red staining. In addition, when apoptosis was stimulated in nodular cultures with anti-Fas IgM, there was a 10-fold increase in calcification. Furthermore, incubation of VSMC-derived apoptotic bodies with (45)Ca demonstrated that, like matrix vesicles, they can concentrate calcium. These observations provide evidence that apoptosis precedes VSMC calcification and that apoptotic bodies derived from VSMCs may act as nucleating structures for calcium crystal formation.
Amino Acid Chloromethyl Ketones↗
The distribution of P2X receptor clusters on individual neurons in sympathetic ganglia and their redistribution on agonist activation.
The distribution of P2X receptors on neurons in rat superior cervical ganglia and lability of P2X receptors on exposure to agonists were determined. Antibody labeling of each P2X subtype P2X(1)-P2X(7) showed neurons isolated into culture possessed primarily P2X(2) subunits with others occurring in order P2X(7) > P2X(6) > P2X(3) > P2X(1) > P2X(5) > P2X(4). Application of ATP and alpha,beta-meATP to neurons showed they possessed a predominantly nondesensitizing P2X receptor type insensitive to alpha,beta-meATP, consistent with immunohistochemical observations. P2X(1)-green fluorescent protein (GFP) was used to study the time course of P2X(1) receptor clustering in plasma membranes of neurons and internalization of receptors following prolonged exposure to ATP. At 12-24 h after adenoviral infection, P2X(1)-GFP formed clusters about 1 microm diameter in the neuron membrane. Application of ATP and alpha,beta-meATP showed these neurons possessed a predominantly desensitizing P2X receptor type sensitive to alpha,beta-meATP. Infection converted the major functional P2X receptor type in the membrane to P2X(1). Exposure of infected neurons to alpha,beta-meATP for less than 60 s led to the disappearance of P2X(1)-GFP fluorescence from the cell surface that was blocked by monensin, indicating the chimera is normally endocytosed into these organelles on exposure to agonist.
Adenosine Triphosphate↗
P2X(1) receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras.
The P2X(1) purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different size receptor clusters, with the larger beneath autonomic nerve terminal varicosities. We have sought to determine whether these synaptic-size clusters only form in the presence of varicosities and whether they are labile when exposed to agonists. P2X(1) and a chimera of P2X(1) and green fluorescent protein (GFP) were delivered into cells using microinjection, transient transfection or infection with a replication-deficient adenovirus. The P2X(1)-GFP chimera was used to study the time course of P2X(1) receptor clustering in plasma membranes and the internalization of the receptor following prolonged exposure to ATP. Both P2X(1) and P2X(1)-GFP clustered in the plasma membranes of Xenopus oocytes, forming patches 4-6 microm in diameter. Human embryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P2X(1) antibody-labeled regions in the membrane colocalized with GFP fluorescence. The ED(50) for the binding of alpha,beta-methylene adenosine triphosphate (alpha,beta-meATP) to the P2X(1)-GFP chimera was similar to native P2X(1) receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X(1) or P2X(1)-GFP were similar. Exposure of HEK293 cells to alpha, beta-meATP for 10-20 min in the presence of 5 microM monensin led to the disappearance of P2X(1)-GFP fluorescence from the surface of the cells. These observations using the P2X(1)-GFP chimera demonstrate that P2X(1) receptors spontaneously form synaptic-size clusters in the plasma membrane that are internalized on exposure to agonists.
Adenosine Triphosphate↗
NANC transmission at a varicosity: the individuality of single synapses.
Nerve terminals consist of several hundred varicosities or synapses, each with a single active zone. The smooth muscle membrane apposing varicosities within about 50 nm is occupied by a 1-microm diameter cluster of P2X(1) receptors together with a mixture of other P2X subtypes; the rest of the membrane possesses small (0.4 microm diameter) clusters of P2X(1) to P2X(6) subunits. The small P2X clusters appear to form large clusters during development. This is supported by the observation that chimeras of P2X(1) subunits and green fluorescent protein (P2X(1)-GFP), when packaged into adenoviruses used to infect excitable cells, initially form a diffuse distribution of small clusters of P2X(1)-GFP in the membrane; these can be later observed in real time to form large clusters. Recording the electrical signs of ATP release from single adjacent varicosities, or using antibodies to label the extent of exocytosis from them, shows that they release with quite different probabilities. There are large quantitative differences in the extent of P2X autoreceptors on the membranes of individual varicosities. These will contribute to the differences in the probability of secretion from individual varicosities. The present analysis of NANC transmission at single varicosities indicates that individual synapses possess different probabilities for the secretion of transmitter as well as different complements of autoreceptors and mixtures of postjunctional receptor subunits.
Animals↗
Distribution of P2X purinoceptor clusters on individual rat dorsal root ganglion cells.
The distribution of P2X receptors on the cell bodies of acutely dissociated rat dorsal root ganglion cells has been determined using immunohistochemistry. Only medium and small ganglion cells showed P2X receptor labelling, which took the form of receptor clusters 0. 2-0.5 microm in diameter. P2X(3) was the predominant cluster type although clusters of all other subtypes, P2X(1), P2X(2), P2X(4), P2X(5) and P2X(6) were observed. There was little evidence for colocalization of the different receptor subtypes pointing to very little heteropolymerization of P2X receptors in the ganglion.
Animals↗