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M R Bennett

Publications and source records attributed to M R Bennett.

At least 55 records · Page 3Linked to original sources
PubMed2000

Sensitivity to Fas-mediated apoptosis is determined below receptor level in human vascular smooth muscle cells.

Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1+/-2.3% viable, n = 4, P = NS) and Fas-sensitive (31.3+/-2.6% viable, n = 3, P<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The concept of transmitter receptors: 100 years on.

It is nearly one hundred years since John Langley of Cambridge developed the idea of the 'receptive substance' or 'receptors' as we now call them. This historical review traces the background to his introduction of this concept of the transmitter receptor and of how succeeding generations built on his ideas to generalise the applicability of this concept to synapses in general. It starts with a consideration of the discovery by Bernard (1844) that curare could paralyse rabbits without affecting their hearts because, as Vulpian (1866) suggested, curare acts on some intermediate zone between nerve and muscle. No further progress could be made without establishing the idea of chemical transmission, which Elliott (1904) then achieved, building on observations concerning sympathetic transmission to smooth muscle made previously by his mentor Langley (1901). Then between 1905 and 1907 Langley, in a wonderful act of creative ability, carried out a series of experiments on the somatic neuromuscular junction which established the idea of transmitter receptors. This review gives details of the experiments which persuaded both Langley and a recalcitrant Ehrlich that pharmacological substances could possess the necessary structure for them to combine with appropriate molecules on cells. The subsequent identification by Dale and his colleagues (1936) of acetylcholine as the transmitter acting on the receptors first discovered by Langley at the somatic neuromuscular junction as well as of acetylcholine on receptors in the heart by Loewi (1921) is then detailed. The review concludes with the triumph of the first recordings of the electrical signs of single channel openings by Neher and Sakmann (1976) at the receptors which Langley had first described.

Animals↗
PubMed2000

Quantal potential fields around individual active zones of amphibian motor-nerve terminals.

The release of a quantum from a nerve terminal is accompanied by the flow of extracellular current, which creates a field around the site of transmitter action. We provide a solution for the extent of this field for the case of a quantum released from a site on an amphibian motor-nerve terminal branch onto the receptor patch of a muscle fiber and compare this with measurements of the field using three extracellular electrodes. Numerical solution of the equations for the quantal potential field in cylindrical coordinates show that the density of the field at the peak of the quantal current gives rise to a peak extracellular potential, which declines approximately as the inverse of the distance from the source at distances greater than about 4 microm from the source along the length of the fiber. The peak extracellular potential declines to 20% of its initial value in a distance of about 6 microm, both along the length of the fiber and in the circumferential direction around the fiber. Simultaneous recordings of quantal potential fields, made with three electrodes placed in a line at right angles to an FM1-43 visualized branch, gave determinations of the field strengths in accord with the numerical solutions. In addition, the three electrodes were placed so as to straddle the visualized release sites of a branch. The positions of these sites were correctly predicted on the basis of the theory and independently ascertained by FM1-43 staining of the sites. It is concluded that quantal potential fields at the neuromuscular junction that can be measured with available recording techniques are restricted to regions within about 10 microm of the release site.

Algorithms↗
PubMed2000

The probability of quantal secretion near a single calcium channel of an active zone.

A Monte Carlo analysis has been made of calcium dynamics and quantal secretion at microdomains in which the calcium reaches very high concentrations over distances of <50 nm from a channel and for which calcium dynamics are dominated by diffusion. The kinetics of calcium ions in microdomains due to either the spontaneous or evoked opening of a calcium channel, both of which are stochastic events, are described in the presence of endogenous fixed and mobile buffers. Fluctuations in the number of calcium ions within 50 nm of a channel are considerable, with the standard deviation about half the mean. Within 10 nm of a channel these numbers of ions can give rise to calcium concentrations of the order of 100 microM. The temporal changes in free calcium and calcium bound to different affinity indicators in the volume of an entire varicosity or bouton following the opening of a single channel are also determined. A Monte Carlo analysis is also presented of how the dynamics of calcium ions at active zones, after the arrival of an action potential and the stochastic opening of a calcium channel, determine the probability of exocytosis from docked vesicles near the channel. The synaptic vesicles in active zones are found docked in a complex with their calcium-sensor associated proteins and a voltage-sensitive calcium channel, forming a secretory unit. The probability of quantal secretion from an isolated secretory unit has been determined for different distances of an open calcium channel from the calcium sensor within an individual unit: a threefold decrease in the probability of secretion of a quantum occurs with a doubling of the distance from 25 to 50 nm. The Monte Carlo analysis also shows that the probability of secretion of a quantum is most sensitive to the size of the single-channel current compared with its sensitivity to either the binding rates of the sites on the calcium-sensor protein or to the number of these sites that must bind a calcium ion to trigger exocytosis of a vesicle.

Action Potentials↗
PubMed2000

The probability of quantal secretion within an array of calcium channels of an active zone.

A Monte Carlo analysis has been made of calcium dynamics in submembranous domains of active zones in which the calcium contributed by the opening of many channels is pooled. The kinetics of calcium ions in these domains has been determined using simulations for channels arranged in different geometries, according to the active zone under consideration: rectangular grids for varicosities and boutons and lines for motor-nerve terminals. The effects of endogenous fixed and mobile buffers on the two-dimensional distribution of free calcium ions at these active zones are then given, together with the extent to which these are perturbed and can be detected with different affinity calcium indicators when the calcium channels open stochastically under an action potential. A Monte Carlo analysis of how the dynamics of calcium ions in the submembranous domains determines the probability of exocytosis from docked vesicles is also presented. The spatial distribution of exocytosis from rectangular arrays of secretory units is such that exocytosis is largely excluded from the edges of the array, due to the effects of endogenous buffers. There is a steeper than linear increase in quantal release with an increase in the number of secretory units in the array, indicating that there is not just a local interaction between secretory units. Conditioning action potentials promote an increase in quantal release by a subsequent action potential primarily by depleting the fixed and mobile buffers in the center of the array. In the case of two parallel lines of secretory units exocytosis is random, and diffusion, together with the endogenous calcium buffers, ensures that the secretory units only interact over relatively short distances. As a consequence of this and in contrast to the case of the rectangular array, there is a linear relationship between the extent of quantal secretion from these zones and their length, for lengths greater than a critical value. This Monte Carlo analysis successfully predicts the relationship between the size and geometry of active zones and the probability of quantal secretion at these, the existence of quantal versus multiquantal release at different active zones, and the origins of the F1 phase of facilitation in synapses possessing different active zone geometries.

Action Potentials↗
PubMed2000

The regulation of vascular smooth muscle cell apoptosis.

Apoptosis describes the morphological changes that identify a specific form of regulated cell death. Over recent years, the importance of either aberrant onset or suppression of apoptosis within tissues has become apparent and is associated with the development of several terminal diseases. Here we describe the relevance of apoptosis to the maintenance of vascular homeostasis. Specifically, we address the role of vascular smooth muscle cell death, how this may be regulated at the molecular level and whether any of these molecular mediators will provide targets for intervention in diseases such as atherosclerosis.

Animals↗
PubMed2000

The concept of long term potentiation of transmission at synapses.

The phenomenon of long term potentiation (LTP) of synaptic transmission, whereby a series of conditioning trains of impulses potentiate the size of synaptic potentials for periods in excess of hours, was discovered in the mammalian hippocampus by Lomo [1966, Acta Physiol. Scand. 68(Suppl. 277), 128] and subsequently characterized by Bliss and Lomo (1970, J. Physiol. 207, 61P). The search for the underlying mechanisms of LTP makes for fascinating reading. The induction of associative LTP was shown by Collingridge et al. (1982, J. Physiol. 334, 33-46) to be dependent on the presence of N-methyl-D-aspartate receptors, following the discovery of these receptors by Watkins and Evans (1981, A. Rev. Pharmac. Toxic. 21, 165-204). There has, however, been continuing controversy as to whether the maintenance phase of LTP over periods of hours may be attributed to an increase in the amount of transmitter released or to an increase in the number of glutamate receptors or both. There is more agreement on the important role or protein kinases in the maintenance phase of LTP. The role of LTP in memory is just now being elucidated.

Animals↗
PubMed2000

Statistics of transmitter release at nerve terminals.

This review presents an historical account of the developments of the statistical analysis of quantal transmission over the past half century and of the progress made in using this approach to reveal new properties of nerve terminals. In the early 1950s, Katz and his colleagues showed that evoked transmitter release occurred in quanta at the neuromuscular junction, opening up the study of transmitter release at nerve terminals to statistical analysis. In the subsequent two decades attempts were made to see if evoked quantal release could be described by binomial or compound binomial statistics, as originally suggested by Katz, and to relate the parameters of the statistic to various structures of the nerve terminal. During this period two hypotheses were enunciated, namely the 'vesicle hypothesis', which states that quanta arise as a consequence of the packaging of transmitter in vesicles; and the 'active zone hypothesis', which states that vesicles undergo exocytosis at discrete sites on the nerve terminal. Unsuccessful attempts were made to relate the binomial parameter n to the elements in these hypotheses, that is to the number of active zones possessed by the terminal or the number of vesicles available for release at these zones. This difficulty was part resolved in the late 1970s with the application of non-uniform binomial statistics to transmitter release from nerve terminals, in which n is the number of active zones each with their individual probabilities, p(j). Autocorrelation functions were subsequently introduced to detect if transmitter release is quantised at a particular nerve terminal. Statistical methods which would allow discrimination between different models of transmitter release over the active zones of a terminal were then developed. The introduction of maximum likelihood estimation procedures then allowed estimates to be made of the parameters in the statistical models of quantal release. The application of these procedures to experimental data from a variety of nerve terminals provided evidence for the concept that each synapse, taken as possessing a single active zone, possesses its own individual probability of secretion of a quantum by the exocytosis of a vesicle. In the late 1960s Stevens introduced the first stochastic approach to the analysis of the kinetics of the release of a quantum of transmitter at the neuromuscular junction following an impulse. In the subsequent decades this was developed into an explicit theory for the interaction of proteins involved in regulated exocytosis of a vesicle at an active zone. The parameters were the number of transition steps in the release process (k), each occurring at the same rate (alpha), with the possibility of each of these steps becoming blocked at the same rate (gamma). Maximum likelihood estimation procedures could then be used to obtain these parameter values. The discovery was made in the 1990s of the core proteins of the SNARE complex that govern regulated exocytosis. This offers the possibility in the near future of identifying the kinetic interaction of these proteins with the parameters of the stochastic process of exocytosis which confer a particular probability on individual synapses.

Animals↗
PubMed2000

Changes in the distribution of different subtypes of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the pregnant rat urinary bladder.

Clusters of purinergic receptor subunits, about 1 microm diameter, are found on the smooth muscle cell membrane beneath junctional varicosities in the detrusor muscle of the rat urinary bladder. We have examined the extent of redistribution of the six different subunit clusters, P2X(1) to P2X(6), with respect to junctional varicosities during pregnancy, as it is known that the detrusor muscle undergoes changes in purinergic innervation during this period. Before pregnancy, clusters at junctional varicosities are principally composed of the subtypes P2X(1), P2X(2), P2X(3) and P2X(5). However this subtype distribution changes dramatically during pregnancy, such that by day 14 of pregnancy, the extent of P2X(1), P2X(2), P2X(3) and P2X(5) junctional clusters has decreased by more than 80% whereas the extent of P2X(4) and P2X(6) junctional clusters has increased by more than 80%. These changes were confirmed with Western blots for different subtypes. It is suggested that the changes in the purinergic innervation of the detrusor muscle during pregnancy reflect changes in the P2X subtypes found on the smooth muscle membrane beneath junctional varicosities.

Animals↗
PubMed2000

P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding.

The redistribution of purinergic P2X receptor subunits (P2X(1) to P2X(7)) within the rabbit aorta wall three weeks after endothelial balloon injury/cholesterol feeding was examined. P2X(1) receptor cluster density was elevated in the media following balloon injury/cholesterol feeding by about 30% and these clusters appeared on smooth muscle cells throughout the greatly expanded neointima but they did not change significantly on the endothelial cells following balloon injury. P2X(4) clusters were found in high density throughout the media and in very high density in the enlarged neointima following balloon injury, particularly on the endothelial cells where the density increased about 10-fold after balloon injury. P2X(5) clusters were found in high density in the media of normal aorta but with little change following balloon injury. P2X(3), P2X(6) and P2X(7) cluster density was low in normal aorta and remained unchanged following balloon injury. All receptor subunits were found on endothelial cells. It is suggested that the release of ATP from damaged endothelial cells and from smooth muscle cells sufficient to activate P2X(4) receptors may contribute to neointimal proliferation.

Animals↗
PubMed1999

Distribution of purinergic P2X receptors in the rat heart.

The distribution of P2X purinergic receptor subtypes has been determined in relation to nerve varicosities in the rat heart with immunohistochemistry. Large clusters (about 1 microm diameter) of co-localised and sometimes co-extensive P2X1 and P2X3 receptors were found at sites of tyrosine hydroxylase (TH) positive axon varicosities in the atrium and the ventricle. Varicosities that were labelled with antibodies to the synaptic vesicle epitope SV2 were frequently labelled also with antibodies to P2X3, P2X5 and P2X6 but not always with antibodies to P2X1. Especially prominent were large numbers of small clusters (about 400 nm diameter) of co-localised P2X2 and P2X5 receptors on the sarcolemma unrelated to nerves at all. During development the 1 day-old heart possessed an abundance of co-localised P2X2 and P2X5 small receptor clusters on the sarcolemma. These observations are discussed in relation to the role of purinergic receptors in the mammalian heart.

Adenosine Triphosphate↗
PubMed1999

Mechanisms of p53-induced apoptosis.

The p53 tumour suppressor gene functions in both cell cycle arrest and apoptosis. Despite considerable advances in understanding as to how p53 regulates growth arrest, the mechanisms by which p53 regulates apoptosis are only just emerging. In particular, there appears to be a structural and functional separation between the ability of p53 to induce growth arrest and apoptosis. This review examines the interactions between p53-induced growth arrest and apoptosis, and the mechanisms of p53-induced apoptosis, both via induction of p53 transcriptional targets and via nontranscriptional mechanisms.

Animals↗
PubMed1999

The early history of the synapse: from Plato to Sherrington.

One hundred years ago, in 1897, Sherrington adopted the name synapse. However, the concept of the synapse emerged from considerations of how muscles are contracted and so locomotion affected over a period of 2400 years, from the time of Plato and Aristotle in the 4th century BC to the early part of the 20th century. This early history is considered in the present review. In terms of duration of influence, the early history was dominated by Aristotle's concept of vital pneuma. This was derived from the ether which filled all space, taken in by the lungs, transformed to vital pneuma in the heart, and then conducted in the blood stream to be transmitted to muscles. The vital pneuma then initiated the final phase of the muscle's psyche, that is, its contraction leading to locomotion. Aristotle's ideas had to be modified with the discovery by Galen and his students in the 2nd and 3rd centuries AD that nerves arising from the brain and spinal cord are necessary for the initiation of muscle contraction. They modified the Aristotlean account so that the vital pneuma delivered by blood vessels to the brain was converted there to psychic pneuma, from whence it was conducted along nerves to be transmitted to muscle, so allowing the muscle to contract. There matters rested for about 1300 years until Descartes. Descartes rejected the idea of organs and muscles possessing a psyche with a final cause that was released by the conduction and transmission of psychic pneuma in nerves, emphasising that mechanical explanations must be sought when determining the function of an organ or muscle. He argued in his corpuscular theory that fine particles derived from the blood in the brain, which he gave the unfortunate name of animal spirits, were conducted and transmitted along nerves to enter muscle during transmission, so leading to the increase in width of the muscle fibres, their shortening and contraction. This description was elaborated on in great detail by Descartes, and by his contemporary Borelli, in the 17th century. In the 18th century, Swammerdam carried out a series of brilliant experiments that showed that the Descartes/Borelli theory could not be correct, muscles did not change their volume during contraction, and so could not be contracted by being swollen due to an influx of the corpuscles that made up the animal spirits. These results were published at about the time of the birth of Galvani (1737), whose work was to show that animal spirits were not corpuscular but electrical. The triumph of 19th century physiology, primarily due to Matteucci, du Bois-Reymond and Helmholtz, was to take Galvani's discoveries and show that nerves possessed a potential across their walls that could give rise to a propagating transient potential change which was transmitted to muscles with a finite velocity. Although Sherrington refined the concept and adopted the word "synapse" at the end of that century, it was not until the early part of the 20th century that a conceptual scheme for the synapse involving transmitters and receptors was developed. This clearly delineated a new period following the early history of synaptic transmission.

Animals↗
PubMed1999

cdc25A is necessary but not sufficient for optimal c-myc-induced apoptosis and cell proliferation of vascular smooth muscle cells.

Increasing evidence indicates that the control of cell proliferation and apoptosis are linked. The c-myc proto-oncogene is induced early after cell-cycle entry in vascular smooth muscle cells (VSMCs) in vitro and after arterial injury and regulates both cell proliferation and apoptosis. Although both proliferation and apoptosis are likely to be mediated via transcriptional activation of target genes, few c-myc targets have been identified. Therefore, the recent identification that cdc25A, a cell-cycle phosphatase involved in G1 progression, is transcriptionally activated by c-myc and regulates c-myc-induced apoptosis has suggested that cdc25A may be the principal mediator of c-myc in VSMCs. We examined cdc25A regulation of c-myc-induced proliferation and apoptosis by expressing cdc25A or antisense cdc25A in primary rat VSMCs or in VSMCs expressing deregulated c-myc or adenovirus E1A. Ectopic c-myc increased cdc25A expression, but cdc25A was still responsive to serum components, which indicated that c-myc alone is not the main determinant of cdc25A expression. Antisense cdc25A inhibited c-myc-induced proliferation and apoptosis; however, drug and metabolic blocks indicated that this effect was limited to G1. Ectopic cdc25A augmented the proproliferative and proapoptotic action of c-myc but did not increase cell proliferation or apoptosis in the absence of ectopic c-myc. In contrast, E1A/E2F-induced apoptosis was independent of cdc25A. We conclude that cdc25A expression modulates the ability of c-myc to induce apoptosis in G1. However, cdc25A alone does not induce apoptosis and cannot substitute for c-myc in VSMCs. Additional targets of c-myc are therefore involved in apoptosis of both G1 and post-G1 VSMCs.

Animals↗
PubMed1999

P2X (purinergic) receptor distributions in rat blood vessels.

The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2X1 receptor clusters were observed on the smooth muscle cells of mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32+/-0.21 microm long and 0.96+/-0.10 microm in diameter; and a smaller spherical cluster with a diameter of 0.32+/-0.05 microm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.

Animals↗
PubMed1999

Altered circadian rhythmicity is an early sign of murine dietary thiamine deficiency.

To determine if circadian clock function is affected by thiamine deficiency, the rhythm of locomotor (wheel-running) activity was measured in a murine model of dietary thiamine deficiency. About 1 month before the expected onset of overt neurological illness, locomotor rhythmicity in deficient animals exhibited a shortened free-running period without a change in amplitude. This effect was fully reversible by thiamine administration. Disordered circadian timekeeping may contribute to altered physiological responses in Wernicke's encephalopathy.

Animals↗
PubMed1999

Localisation of P2X receptors in human salivary gland epithelial cells and human embryonic kidney cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting and immunofluorescence.

Human salivary gland epithelial cells, a continuous cell line derived from an irradiated human salivary gland and human embryonic kidney cell line human embryonic kidney (HEK)293 were examined for the purpose of establishing whether they expressed endogenous P2X ionotropic receptors at any stage in their cycles. HSG cells were found to express P2X1-6 subtypes using both Western blotting and immunofluorescence labeling. HEK293 cells had no detectable levels of P2X1-3 and P2X6 under normal circumstances along with very low levels of P2X4 and P2X5 but when the cells were grown past confluence then all subtypes were expressed on the surface membrane with the exception of P2X2. The results are discussed in terms of the likely influence of ATP acting as an intercellular signaling molecule.

Antibody Specificity↗