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M R Bennett

Publications and source records attributed to M R Bennett.

At least 109 records · Page 6Linked to original sources
PubMed1997

Autoradiography of [3H]alpha,beta-methylene-ATP binding sites in medulla oblongata and spinal cord of the rat.

Excitatory purinoceptors of P2x-type have long been known to exist on smooth muscle cells, but recently it has been shown that they are also involved in synaptic transmission in the CNS. We have used a P2x-specific agonist, alpha,beta-methylene-ATP, as a 3H-labelled radioligand, to study the distribution and characteristics of P2x receptor-binding sites in the spinal cord and medulla oblongata. Using auto-radiographic techniques, [3H]alpha,beta-methylene-ATP binding was found throughout the grey matter of the spinal cord with small areas of above-average density of binding sites in the marginal zone and substantia gelatinosa at all spinal levels and in the central grey matter of the thoracic spinal cord. In the medulla, [3H]alpha,beta-methylene-ATP binding was found to be strong in all cranial nerve nuclei, particularly those known to receive primary sensory fibres. We have found that the binding of [3H]alpha,beta-methylene-ATP in the spinal cord and medulla was inhibited by a broad-spectrum P2-receptor antagonist suramin (IC50 approximately 27 microM). This is in accordance with the data obtained previously in the forebrain and cerebellum. There was, however, no inhibition of [3H]alpha,beta-methylene-ATP binding by another close analogue of alpha,beta-methylene-ATP and P2x ligand beta,gamma-methylene-ATP (10 microM). The latter result is discussed in terms of possible involvement of Ca2+ in the binding of [3H]alpha,beta-methylene-ATP to P2x receptors in the CNS.

Adenosine Triphosphate↗
PubMed1997

Non-adrenergic non-cholinergic (NANC) transmission to smooth muscle: 35 years on.

In 1963, two substances were thought to mediate all transmission between neurons, as well as between nerve and muscle in the peripheral nervous system, namely acetylcholine and noradrenaline. This paradigm primarily was due to the research of Dale, Loewi and von Euler in the first half of the century [Dale, 1937 (Transmission of nervous effects by acetylcholine, Harvey Lect. 32, pp. 229-245)]. However, in 1963, a series of experiments were carried out using recently introduced electrophysiological techniques, which showed unequivocally for the first time that the classical paradigm was not correct. Both inhibitory and excitatory junctions between nerves and smooth muscle cells were shown to exist in which transmission was mediated by non-adrenergic, non-cholinergic (NANC) transmitters. In the succeeding 35 years, identification of these NANC transmitters has been a major task of neuropharmacology, with nitric oxide, neuropeptides, and purines being isolated. This review presents an historical account of the developments this century of the classical paradigm, of how it was displaced, and of the progress made in identifying the neuromuscular transmitters of the autonomic nervous system.

Animals↗
PubMed1997

Varicosities of single sympathetic nerve terminals possess syntaxin zones and different synaptotagmin N-terminus labelling following stimulation.

A study has been made of the probability of exocytosis of synaptic vesicles at different varicosities in single sympathetic terminal axons in the mouse was deferens. An antibody (SV2Ab) against SV2. a proteoglycan in synaptic vesicles, labelled an area of individual sympathetic varicosities that was slightly less than that occupied by dextran-rhodamine, previously orthogradely transported into the varicosities. In contrast plasma membrane bound protein syntaxin, found at active zones of motor nerve terminals, occupied an area of the varicosity that was approximately one-third that of SV2. This suggests that sympathetic varicosities possess specialized zones for exocytosis on their plasma membranes. Antibodies against the N-terminal sequence of synaptotagmin 1 (SNAb), a sequence exposed within synaptic vesicles, were used to determine the probability of exocytosis at different varicosities of single terminal branches. The area of SNAb labelling was not significantly different from that of the SV2 labelling, which implies vesicles that have undergone exocytosis may eventually return to the main pool of vesicles. Varicosities belonging to the same terminal axon, and identified with SV2Ab, showed different extents of labelling with SNAb when secretion was evoked with high potassium concentrations (80 mM) for 30 min in the presence of SNAb. There was up to an order of magnitude difference in the average intensity of SNAb labelling between different varicosities of the same terminal axon whereas there was little difference in the average intensity of SV2Ab labelling. These observations suggest that there is considerable variability in the probability of exocytosis at the specialized zones in different varicosities.

Adrenergic Fibers↗
PubMed1997

Effect of phosphorothioated oligonucleotides on neointima formation in the rat carotid artery. Dissecting the mechanism of action.

Several studies have shown that single-dose administration of agents that inhibit medial cell replication, such as antisense oligonucleotides to cell replication genes, can inhibit neointima formation after arterial injury. However, the precise mechanism of action of these agents is unknown. We analyzed the effect of phosphorothioated oligonucleotides delivered periadventitially on the response to injury in the balloon-injured rat carotid artery. Antisense oligonucleotides to c-myc suppressed medial replication 2 days after injury, but this effect was not present at 4 or 14 days. Endothelial cell proliferation was not affected by antisense oligonucleotides. There was, however, a significant suppression of intimal area and intima/media ratio at 14 days and an increase in lumen area in the antisense-treated group. Indeed, an increase in the number of medial cells at 14 days in the antisense group indicated that most of the effect of the agent was due to the suppression that most of the effect of the agent was due to the suppression of cell migration. No effect was noted on expression of two genes, osteopontin and tropoelastin, used as markers of modulation of smooth muscle cells to a "neonatal" phenotype at 4 days after injury. Because no effect on cell proliferation could be demonstrated after 2 days, our data indicate that an early effect of the antisense agent mediates its longer-term effects. We suggest that this effect may be due to the suppression of migration of medial smooth muscle cells rather than the suppression of medial or intimal cell proliferation.

Angioplasty, Balloon↗
PubMed1997

Increased sensitivity of human vascular smooth muscle cells from atherosclerotic plaques to p53-mediated apoptosis.

The recent demonstration that apoptosis of vascular smooth muscle cells (VSMCs) occurs in human atherosclerotic plaques suggests that VSMC apoptosis may promote plaque rupture and subsequent myocardial infarction. In culture, human plaque VSMCs show higher rates of apoptosis than VSMCs from normal vessels, although the mechanism of this effect is unknown. In earlier studies, we have shown that the tumor suppressor gene p53 regulates apoptosis of rat VSMCs after deregulated cell cycle control. We therefore analyzed p53 function in cultured VSMCs derived from human coronary plaques or the media of normal coronary arteries. VSMCs with reduced or increased p53 activity were created by infecting VSMCs with retroviruses containing a dominant-negative p53 minigene or a chimeric p53 protein (p53TMER), which could be activated pharmacologically. Basal p53 protein expression and transcriptional activity were similar in plaque and normal VSMCs, and suppression of p53 activity blocked growth arrest in response to DNA damage in both VSMC types. In contrast, suppression of p53 activity failed to block apoptosis of plaque or normal VSMCs in low- or high-serum conditions or after DNA damage. Furthermore, in plaque VSMCs, p53 overexpression induced apoptosis in all conditions tested and also induced growth arrest. p53-mediated apoptosis was independent of new gene transcription or protein synthesis but was suppressed by prior growth arrest of cells, indicating that growth status can regulate sensitivity to p53-mediated apoptosis. No effect of increased p53 activity was seen in normal VSMCs. We conclude that VSMCs from human plaques have an increased sensitivity to p53-mediated apoptosis compared with normal VSMCs. Our data also suggest that the mechanism of p53-mediated apoptosis of plaque VSMCs may be distinct from that inducing growth arrest.

Adult↗
PubMed1996

Vesicle-associated proteins and calcium in nerve terminals of chick ciliary ganglia during development of facilitation.

1. The developmental appearance of synaptic vesicle-associated proteins and nerve terminal calcium ([Ca2+]i) sequestering processes were determined for the chick ciliary ganglia in relation to the maturation of the different phase of increased efficacy of transmitter release following nerve impulses. The maturation phases studied were from stages 34-35, at the time of synapse formation, to stage 46 at hatching. 2. Western blots and immunohistochemical localization indicated that synaptotagmin 1 and synapsin IIa were detectable at stages 34-35 and were clearly localized at the nerve terminals by stage 37. Syntaxin was clearly localized at the nerve terminals at stage 34. 3. The relative size of the postganglionic compound action potential, used to measure the transmission efficacy through the ganglion, showed that the slope of the relationship between log efficacy and log extracellular calcium concentration ([Ca2+]o) in low [Ca2+]o was about 4 by stage 46. 4. A mature facilitatory mechanism for transmission was not present at stage 34 and did not emerge until stage 38. A mature augmentation was not present at stages 34 or 38 and was not established until stages 41-42. Post-tetanic potentiation (PTP) was not present at stage 34; it was evident at stages 37-38 and only reached maturity by stages 41-42. 5. The time course of calcium changes in the nerve terminals following trains of impulses that give rise to facilitation, augmentation and PTP was determined for different stages of development using the indicator Calcium Green-1 in the nerve terminal. The mature time course of the phases of calcium decline in the nerve terminal associated with facilitation and augmentation was observed as early as stage 38, whereas that of the PTP phase did not mature until after stage 42. 6. These results are discussed in terms of the maturation of the vesicle-associated proteins and calcium influx into the terminal following trains of impulses that give rise to the different components of increased synaptic efficacy.

Animals↗
PubMed1996

Quantal transmitter release onto different patterns of receptor distributions at somatic neuromuscular junctions.

The effect of different distributions of acetylcholine receptors at the active zone of somatic motor-nerve terminals on the amplitude-frequency histogram of spontaneous excitatory currents due to the secretion of a quantum transmitter has been analysed by numerically solving the diffusion and binding equations. The time course and amplitude of the spontaneous currents of different arrangements of receptors has been determined. Organization of receptors into stripes of different patterns beneath the active zone, which is known to occur, does not affect the amplitude or temporal characteristics of the spontaneous currents. Increases in the amount of transmitter in a quantum released onto the stripes does not introduce any subunit structure into the spontaneous currents. It is concluded that if subunits are detectable in measured amplitude-frequency histograms of spontaneous potentials they arise because of presynaptic rather than postsynaptic factors.

Action Potentials↗
PubMed1996

ATP-activated cationic and anionic conductances in cultured rat hippocampal neurons.

The effects of adenosine 5'-triphosphate (ATP) on voltage-clamped and dissociated rat hippocampal neurons was investigated. Over 30% of neurons possessed ATP-activated inward currents at a holding potential of -70 mV. The ED50 for these currents was 150 mu M. At this concentration they were blocked by suramin (1 mM), indicating the involvement of P2-purinoceptors. The P2 purinoceptor agonist potency was 2-methylthio ATP > ATP > ADP > alpha,beta-methylene ATP, thus identifying these purinoceptors as belonging to the P2x subclass. The reversal potential for the ATP-activated currents was -45 +/- 8 mV. Ion substitution experiments showed that the permeability ratio for K+/Na+/Cs+/Cl-, was 18:3:2:1, according to the Goldman-Hodgkin-Katz equation, so that ATP activates cationic and anionic conductances in hippocampal neurons.

Adenosine Triphosphate↗
PubMed1996

Quantal components of spontaneous excitatory junction potentials at visualised varicosities.

The electrical signs of spontaneous transmitter release were recorded with an extracellular electrode from single visualized sympathetic varicosities on the mouse vas deferens. Ultrastructural examination of these varicosities with the electron microscope showed that they formed close-contacts with smooth muscle cells. Amplitude-frequency histograms of the spontaneous excitatory junction potentials (SEJPs) were constructed in order to determine the statistical nature of spontaneous transmitter release from a varicosity. SEJP histograms often possessed several peaks. Statistical tests showed that these were separate modes in the histograms indicating that the SEJPs were composed of subunits. The SEJP histograms were described by a mixture of distributions in which the components were identified as quanta and there was a Poisson release of quanta. The second mode in the SEJP histograms was sometimes twice that of the first mode but generally greater, suggesting a potentiation of the effects of one quantum released nearly simultaneously with another quantum. The components in the SEJP histograms were well fitted by either a Gaussian or a gamma distribution indicating that the quantum of transmitter could be described as a Gaussian or gamma variate.

Animals↗
PubMed1996

Quantal secretion from visualized boutons on rat pelvic ganglion neurones.

Synaptic transmission from single preganglionic hypogastric nerves innervating monopolar pelvic ganglion neurones has been studied with intracellular electrodes to record transmission from all the boutons and with extracellular electrodes placed over boutons visualized with DiOC2(5) in order to record transmission from selected boutons. Intracellular electrodes revealed spontaneous excitatory postsynaptic potentials (EPSPs) with amplitude histograms that show increasing numbers of large EPSPs as the external calcium ([Ca2+]o) was increased from 0.15 to 1.0 mM. These histograms were in general well fitted by a Poisson mixture of gamma distributions. Extracellular electrodes placed over visualized boutons revealed evoked excitatory postsynaptic potentials (extracellular EPSPs) with amplitude histograms that were best described by single gamma distributions in most cases in low [Ca2+]o (less than 0.5 mM). The standard deviation of these gammas was not much larger than that of the electrical noise. In a minority of extracellular recordings the amplitude histogram of evoked extracellular EPSPs was best described by a gamma distribution in which the standard deviation was much greater than that of the noise. Confocal microscopy of boutons orthogradely labelled with dextran-rhodamine showed that about 30% of these formed closely apposing pairs on the surface of the neurones. These observations are discussed in terms of the hypothesis that multiquantal release at boutons occurs as a consequence of the coupled secretion from closely apposed boutons.

Animals↗
PubMed1996

Spatial relationships between sympathetic varicosities and smooth muscle cells in the longitudinal layer of the mouse vas deferens.

The spatial relationships between nerve varicosities and smooth muscle cells in the longitudinal muscle layer of the mouse vas deferens have been determined from serial section reconstructions of individual varicosities at the ultrastructural level. Bundles of up to five axons, together with single axons, occurred frequently at the surface of the muscle as well as at about 3-6 muscle cell diameters into the muscle. Varicosities within axon bundles at the muscle surface each became partially divested of Schwann cell processes. The smallest distance separating varicosity membrane from muscle cell membrane (apposition distance) was 100 nm (mean 170 nm) for varicosities contained in bundles. Varicosities from six single axons on the muscle surface were reconstructed and 11 of the 12 possessed a mean apposition distance of 48 nm. Varicosities in axon bundles at about 12 microns deep into the muscle came into an apposition distance of 50-90 nm (mean = 67 nm). All varicosities of single axons at this depth came into about 50 nm apposition (mean = 53 nm). These results indicate that the varicosities lie at varying distances from the muscle cells in the longitudinal muscle layer of the vas deferens.

Animals↗
PubMed1996

Neuronal cell death in the mammalian nervous system: the calmortin hypothesis.

1. This review is concerned with the calcium-dependent mechanisms involved in neuronal cell death. To this end, it provides definitions of the major types of cell death and then describes what is known of their occurrence during development and degeneration of the mammalian nervous system. 2. An analysis is presented of the different sources and compartments of calcium in neurons and of how these are related to the known calcium-dependent enzymes whose excess activation will lead to cell death. 3. The review uses the relatively large amount of pertinent information now available for other cell types, especially thymocytes, to reveal our limited knowledge of how calcium controls neuronal cell death. 4. In the final section, consideration is given to the identification of those factors that may mitigate against the calcium-dependent pathways leading to neuronal degeneration.

Animals↗
PubMed1996

Characterization of protein kinase and phosphatase systems in chick ciliary ganglion.

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary ganglion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase activities were detected in homogenates of ciliary ganglion dissected from 15-16-day-old embryos. Autophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+/calmodulin or 5 mM ZnSO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose molecular weights corresponded to the regulatory subunits of cyclic AMP-dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following incubation with [gamma-32P]ATP under most conditions had an apparent molecular weight of 65,000 which corresponds to the chicken form of myristoylated alanine-rich C kinase substrate, a known substrate of protein kinase C. Another substrate for protein kinase C was a 45,000 molecular weight protein which was tentatively identified as neuromodulin (B-50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP-dependent protein kinase were detected, protein kinase A strongly labelled a 40,000 molecular weight protein. Using 32P(i)-labelled glycogen phosphorylase, protein phosphatases 1 and 2A were identified in ciliary ganglia homogenates at levels which were indistinguishable from forebrain at the same age. The major endogenous protein substrates in ciliary ganglion homogenates from 15-16-day-old embryos were also labelled to a similar extent in homogenates of ciliary ganglia from newly hatched chickens. Intact ciliary ganglia remained viable for several hours after dissection and, after incubation with 32P(i), responded to phorbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterization of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms responsible for regulating synaptic transmission in this tissue.

Animals↗