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M R Bennett

Publications and source records attributed to M R Bennett.

At least 91 records · Page 5Linked to original sources
PubMed1998

Ligand recognition sites on P2X receptors studied by quantitative autoradiography of [3H]alpha,beta-methylene-ATP binding in rat brain.

The specificity of alpha, beta-methylene-ATP for P2X receptor binding sites in the CNS has been examined by testing the effects of several ATP analogues and other ATP-related substances on the binding of 10 nM [3H]alpha,beta-methylene-ATP to 20 microns thick sections of fresh-frozen rat brain. The labelling of the putative P2X receptor binding sites by [3H]alpha,beta-methylene-ATP was evaluated by quantitative densitometry. [3H]alpha,beta-methylene-ATP binding was strongly inhibited by two close ATP analogues, 3'-O-(trinitrophenyl)-adenosine-5'-triphosphate and beta,gamma-imido-ATP (IC50 2.5 microM). beta,gamma-Methylene-ATP was, however, less potent (< 50% inhibition at 25 microM). Inosine-5'-triphosphate, guanosine-5'-triphosphate, uridine-5'-triphosphate, and cytidine-5'-triphosphate were practically inactive up to concentrations of 100 microM. Periodate oxidised ATP and 1, N6-etheno-ATP produced < 50% inhibition at 100 and 500 microM concentrations, respectively. Cations (K+, Rb+, Cs+, and Mg2+ at 5 mM and Na+ at 150 mM) reduced [3H]alpha,beta-methylene-ATP binding by no more than 50%. Several agents known to interact with Ca2+- and/or ATP-related cationic channels (Cd2+, glibenclamide, dantrolene, nifedipine, and thapsigargin) had no effect. We conclude that [3H]alpha,beta-methylene-ATP at low nanomolar concentrations binds to a site that has very strict structural requirements and is pharmacologically similar to ATP P2X receptors.

Adenosine Triphosphate↗
PubMed1998

Calcium transients evoked by action potentials in the somata of chick ciliary neurons.

The effect of action potentials on the calcium concentration in the somata of chick ciliary neurons ([Ca2+]s) was determined by loading these with the calcium indicator calcium green-1. Following trains of 1-10 impulses (30 Hz) to the postganglionic nerve, the [Ca2+]s increased rapidly and then declined along a single exponential with a time constant of 0.70 +/- 0.04 s (fast phase). After trains of 20 or 50 impulses, the elevated [Ca2+]s declined as the sum of two exponentials, with time constants of 0.78 +/- 0.12 s (fast phase) and 4.0 +/- 0.4 s (moderate phase). After a 600-impulse postganglionic train of impulses, the elevated [Ca2+]s declined quickly over about 1 s, and then as the sum of two exponentials: that of the moderate phase and a slower component with a time constant of 109 +/- 16 s (slow phase). Similar time courses were observed following stimuli to the preganglionic nerve. Caffeine (3 mM) and ryanodine (20 microM) both sped the fast phase and slowed the moderate phase of [Ca2+]s decline. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 2 microM) slowed the slow phase, without affecting the other phases of decline. These results are discussed in relation to identifying the mechanisms responsible for these different phases of Ca2+ removal.

Action Potentials↗
PubMed1998

Probability of quantal secretion and the mobilization of vesicles at the active zones of endplates.

A vesicle mobilization model is developed for active zones of motor-nerve terminals. In the model, vesicles from a storage pool are recruited to an available pool where they are docked at the active zone, the latter pool being replenished by endocytosis. The predictions of this model are then compared with experimental results on these active zones. If a styryl dye (FM1-43) is used to stain all of the vesicles at motor-nerve terminals then the stores of vesicles associated with different active zones are shown to vary considerably in size. Under the assumption that the available pool size is proportional to the storage pool size, the mobilization model shows that the extent of destaining of these stores during a particular period of stimulation is proportional to the size of the storage pool, this size being indicated by the initial staining. This is not so under the alternative assumption that the available pool size does not vary proportionally with store size. If the two pools are proportional then the number of quanta released from the available pool is proportional to the storage pool size. Experimental results indicate that there is a linear relationship between destaining and staining, as well as between quantal release and staining at different sites along motor-nerve terminals, thus favouring the hypothesis that available pool size (or the size of the active zone) and store size are proportional. Given that there are very different store sizes along the length of motor-nerve terminals, this analysis provides further evidence for the non-uniform probability of quantal secretion at different active zones within a single motor-nerve terminal.

Amphibians↗
PubMed1998

Cooperative interactions between RB and p53 regulate cell proliferation, cell senescence, and apoptosis in human vascular smooth muscle cells from atherosclerotic plaques.

Compared with vascular smooth muscle cells (VSMCs) from normal vessels, VSMCs from human atherosclerotic plaques proliferate more slowly, undergo earlier senescence, and demonstrate higher levels of apoptosis in culture. The tumor suppressor genes p105RB (retinoblastoma, acting through the E2F transcription factor family) and p53 regulate cell proliferation, cell senescence, and apoptosis in many cell types. We have therefore determined whether these stable growth properties of plaque VSMCs reflect altered activity of RB and/or p53. VSMCs were derived from coronary atherectomies or from normal coronary arteries from transplant recipients. Compared with normal VSMCs, plaque VSMCs showed a higher ratio of the active (hypophosphorylated) to the inactive (phosphorylated) form of RB and a lower level of E2F transcriptional activity. Cells were stably transfected with retrovirus constructs that inhibited RB or p53 alone or in combination. Suppression of RB alone increased rates of cell proliferation and apoptosis and inhibited cell senescence in normal VSMCs. Suppression of p53 and RB together had similar effects but, additionally, resulted in immortalization of normal VSMC cultures. In contrast, inhibition of RB binding to E2F or ectopic expression of E2F-1 in plaque VSMCs induced massive apoptosis, which required suppression of p53 to rescue cells. Suppression of RB and p53 together increased cell proliferation and delayed senescence but failed to immortalize plaque VSMCs. Inhibition of p53 alone had minimal effects on plaque VSMCs but increased the lifespan of normal VSMCs. We conclude that human plaque VSMCs have slower rates of cell proliferation and earlier senescence than do cells from normal vessels because of a defect in phosphorylation of RB. Furthermore, both disruption of RB/E2F and inhibition of p53 are required for plaque VSMCs to proliferate without apoptosis. This observation may explain the relatively low level of cell proliferation and high level of apoptosis seen in VSMCs in human atherosclerotic plaques.

Apoptosis↗
PubMed1998

On the origin of skewed distributions of spontaneous synaptic potentials in autonomic ganglia.

The histograms of spontaneous synaptic potentials at synapses in autonomic ganglia are described by distributions consisting of mixtures of Gaussians, rather than by single Gaussian distributions. The possible origin of these mixed distributions is investigated, using Monte-Carlo simulations of the action of spontaneously released units of transmitter. A single unit of acetylcholine of fixed size, released from an active zone with receptor patches both beneath and adjacent to the zone, does not give rise to the observed histograms. But if the unit is of variable size, consisting of integer multiples of smaller units, and release is from an active zone onto either the receptor patch beneath, or in addition onto adjacent patches, then the histogram is well described by a mixture of Gaussians. However, this explanation is unlikely to be correct as present evidence suggests that in most cases the released unit of transmitter saturates the postsynaptic receptor patch beneath the active zone. The final case considered is where a unit of transmitter is spontaneously released from an active zone, simultaneously with a unit in an adjacent zone less than one micron away. The histogram of potentials then conforms to those observed even when there are differences in the sizes of the receptor patches. It is suggested that this kind of release could provide an explanation for distributions of spontaneous potentials that are mixtures of Gaussians.

Animals↗
PubMed1998

Alterations in the expression of P2X1 receptors in failing and nondiseased human atria.

This is the first report of the analysis of the ATP-specific P2X1 receptor subunit in human hearts. We have examined homogenate samples of human left atria for the presence of P2X1 receptors using Western blots. Anti-P2X1 immunoreactivity was detected in populations of nondiseased atria as well as in atria from explanted hearts from patients with terminally failing heart conditions such as dilated cardiomyopathy. At least three groups of P2X1 immunoreactive proteins were detected in the Western blots with approximate molecular mass values of 50, 70, and 160 kDa. We report changes in expression of their 50 and 70 kDa components. These changes may be related to the type of deficit in these hearts since the changes have been observed in hearts with decreased ejection fractions characteristic of dilated cardiomyopathy.

Animals↗
PubMed1998

Apoptosis of vascular smooth muscle cells in atherosclerosis.

Apoptosis (programmed cell death) of vascular smooth muscle cells and macrophages has recently been demonstrated in the following: (a) human atherosclerotic plaques; (b) physiological remodelling of the vessel; and (c) a variety of disease states. Apoptosis is a highly regulated mechanism of death, controlled by the interactions between the following: (i) cellular receptors; (ii) cytoplasmic and nuclear gene products; and (iii) the local cytokine and cellular environment within the plaque. The knowledge of the key regulators of apoptosis does, however, offer novel therapeutic targets in both the prevention and treatment of atherosclerosis.

Apoptosis↗
PubMed1998

Neurotransmitter release at individual sympathetic varicosities, boutons.

The secretosome hypothesis has been studied at autonomic nerve terminals. Evidence is presented that there is a nonuniform secretion probability at different release sites of these terminals. This can be correlated with their influx of calcium ions following an impulse, as expected according to the secretosome hypothesis.

Adrenergic Fibers↗
PubMed1998

The distribution of single P2x1-receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder.

The distribution of purinergic (P2x1) receptors on smooth muscle cells in relation to autonomic nerve varicosities in the rat urinary bladder has been determined using immunofluorescence and confocal microscopy. P2x1 receptors were visualized using rabbit polyclonal antibodies against the extracellular domain of the P2x1 receptor, and varicosities were visualized 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 the detrusor, namely, a large ellipse of mean long axis 1.23+/-0.21 microm and short axis 0.92+/-0.17 microm and a smaller spherical cluster with a mean diameter of 0.40+/-0.04 microm. The latter occured in much greater numbers than the former in selected areas, with a density as high as 0.8 per microm2 or two orders of magnitude more than the larger-sized clusters. The large clusters are in general located beneath varicosities, with only 4.5% of P2x1 clusters not possessing an overlying varicosity. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2x1 and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2x1 receptor clusters. On occasions, two or more small SV2-labelled varicosities about 0.7 microm in diameter each with a receptor patch were found juxtaposed to each other; these might represent the splitting up of a single large varicosity. These observations are discussed in relation to the identity of the autonomic neuromuscular junction.

Animals↗
PubMed1998

Monoaminergic synapses and schizophrenia: 45 years of neuroleptics.

In 1952 Delay and Deniker introduced the first antipsychotic, chlorpromazine, into the treatment of mental patients. They subsequently defined the word 'neuroleptic' to describe drugs as different as reserpine and chlorpromazine which seemed to have similar effects on the mental life of patients. In the 1960s the hypothesis was developed, mainly due to Carlsson, that the principal mode of action of neuroleptics was to interfere with synaptic transmission mediated by dopamine (DA) in the brain. This concept was given substantial credence with the discovery by Seeman and Snyder in the 1970s that many of the neuroleptics acted as DA receptor blockers. Subsequently two different classes of DA receptor were defined on the basis of their coupling to adenylate cyclase by Kebabian. In the 1980s molecular biology led to the cloning of five different DA receptors, and at the end of this period vanTol and his colleagues cloned the D4 DA receptor, which has been of considerable interest in the 1990s as it is greatly elevated in the brains of schizophrenics. This historical review ends with a consideration of the possibility that in addition to DA receptors, serotonin and perhaps other transmitter receptors are involved in the aetiology of schizophrenia.

Animals↗
PubMed1997

P2 purinoceptor blocker suramin antagonises NMDA receptors and protects against excitatory behaviour caused by NMDA receptor agonist (RS)-(tetrazol-5-yl)-glycine in rats.

It has been reported that suramin, an anthelminthic, trypanocidal agent and an inhibitor of P2 receptors, may antagonise N-methyl-D-aspartate (NMDA) subtype of the excitatory amino acid receptors. Both NMDA receptors and P2X subclass of P2 receptors are ligand-gated Ca2+-selective channels and, since the increased influx of Ca2+ into neurons has been linked to neurotoxicity, simultaneous inhibition of P2X and NMDA receptors in vivo by suramin could represent an effective neuroprotective treatment. We have found that suramin inhibited the binding of [3H]CGP 39653 to NMDA receptor binding sites in vitro and reduced the frequency of NMDA channel openings in patch-clamp studies. Suramin (1 mM) had no effect on [3H]kainate binding in vitro. In vivo, intracerebroventricular (I.C.V.) injections of suramin (70 nmol/brain) antagonised convulsive effects of the NMDA agonist (RS)-(tetrazol-5-yl)-glycine (TZG, LY 285265). Suramin, however, did not prevent neurotoxic lesions in the hippocampus caused by I.C.V. administration of TZG. Increasing the dose of suramin resulted in death from severe respiratory depression.

Animals↗
PubMed1997

Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition.

1. The sympathetic nerve terminals of the mouse vas deferens were loaded with the calcium indicator Oregon Green 488 BAPTA-1 by orthograde transport along the postganglionic nerves. Changes in the calcium concentration in the varicosity (delta [Ca2+]v) were determined following single impulses, and short (5-impulse) and long (200-impulse) trains at 5 Hz. 2. All varicosities showed a significant delta [Ca2+]v in response to every single impulse. The elevated delta [Ca2+]v declined in two phases with similar kinetics for all varicosities: a fast phase (time constant, 0.42 +/- 0.05 s) and a moderate phase (3.6 +/- 0.4 s). 3. Line scanning confocal microscopy revealed that the delta [Ca2+] of a single terminal following single impulses was smaller for the intervaricose regions than for the varicosities. 4. Blockade of the voltage-sensitive calcium channels with Cd2+ (in calcium-free solution) completely blocked the delta [Ca2+]v on stimulation. The addition of either nifedipine (10 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin TK (100 nM) showed that 47 +/- 6% of the evoked response was mediated by N-type calcium channels. 5. Ryanodine (10 microM) did not significantly change the amplitude of delta [Ca2+]v in response to short trains. 6. Spontaneous increases in delta [Ca2+]v were observed in individual varicosities, with coupling in the increase of delta [Ca2+]v between varicosities. 7. The presynaptic alpha 2-receptor antagonist yohimbine (10 microM) increased the amplitude of delta [Ca2+]v in response to five impulses (5 Hz) by 54 +/- 14%, while the alpha 2-receptor agonist clonidine (1 microM) decreased the delta [Ca2+]v by 55 +/- 4%. 8. These results are discussed in terms of the hypotheses that the increased probability for secretion at sympathetic nerve terminals which accompanies facilitation and augmentation is due to the residual delta [Ca2+]v remaining after the calcium influx following impulses and that noradrenaline acts presynaptically to decrease the probability of secretion by modifying calcium influx.

Adenosine Triphosphate↗
PubMed1997

Structural motif and characteristics of the extracellular domain of P2X receptors.

Seven sequences of the proteins recently cloned from rat tissues and identified as P2X receptors are examined. Their putative transmembrane and extracellular domains and the most probable locations of disulphide bridges are discussed. Some surface accessible regions are identified and the structure of the putative ATP binding domain is discussed in the light of known selectivities and physiological characteristics of individual P2X subunits. The summarised information and the theoretical considerations regarding the protein structure of P2X receptors have been used to select amino acid sequences for raising antibodies against the P2X1 receptor subunit. Initial results suggest that, in rat brainstem, P2X1 receptor subunits are found on a distinct subpopulation of neuronal perikarya in distributions consistent with distributions of known alpha,beta-methylene-ATP autoradiography and physiological effects of purinoceptor activation.

Adenosine Triphosphate↗
PubMed1997

Thrombin generation by apoptotic vascular smooth muscle cells.

Thrombin activation requires assembly of a prothrombinase complex of activated coagulation factors on an anionic phospholipid surface, classically provided by activated platelets. We have previously shown that anionic phosphatidylserine is exposed by rat vascular smooth muscle cells (VSMCs) undergoing apoptosis after serum withdrawal. In this study, using a chromogenic assay, we have shown thrombin generation by apoptotic VSMCs expressing c-myc (VSMC-myc) with an area under the thrombin-generation curve (AUC) of 305 +/- 17 nmol x min/L and a peak thrombin (PT) of 154 +/- 9 nmol/L. The thrombin-generating potential of the apoptotic VSMC-myc cells was greater than that of unactivated platelets (P = .003 for AUC; P = .0002 for PT) and similar to calcium-ionophore activated platelets (AUC of 332 +/- 15 nmol x min/L, P = .3; PT of 172 +/- 8 nmol/L, P = .2). Thrombin activation was also seen with apoptotic human VSMCs (AUC of 211 +/- 8 nmol x min/L; PT of 103 +/- 4 nmol/L) and was inhibited by annexin V (P < .0001 for AUC and PT). VSMC-myc cells maintained in serum generated less thrombin than after serum withdrawal (P = .0002 for AUC and PT). VSMCs derived from human coronary atherosclerotic plaques that apoptose even in serum also generated thrombin (AUC of 260 +/- 2 nmol x min/L; PT of 128 +/- 4 nmol/L). We conclude that apoptotic VSMCs possess a significant thrombin-generating capacity secondary to phosphatidylserine exposure. Apoptotic cells within atherosclerotic plaques may allow local thrombin activation, thereby contributing to disease progression.

Adenovirus E1A Proteins↗
PubMed1997

Quantal currents and potential in the three-dimensional anisotropic bidomain model of smooth muscle.

The potential generated in the smooth muscle of the vas deferens on release of a quantum of transmitter from a varicosity was analyzed using a three-dimensional bidomain continuum model. Current was injected at the origin of the bidomain; this current had the temporal characteristics of the junctional current. The membrane potential, intracellular potential, and extracellular potential, as well as the extracellular current, were then calculated throughout the bidomain at different times. Calculations were performed to show the effect of changing the anisotropy ratios of the intracellular and extracellular conductivities on the spread of current and potential in each of the three dimensions. These results provide a theoretical framework for ascertaining the time course of transmitter interaction at a varicosity following the secretion of a quantum of transmitter.

Animals↗
PubMed1997

Probabilistic secretion of quanta and the synaptosecretosome hypothesis: evoked release at active zones of varicosities, boutons, and endplates.

A quantum of transmitter may be released upon the arrival of a nerve impulse if the influx of calcium ions through a nearby voltage-dependent calcium channel is sufficient to activate the vesicle-associated calcium sensor protein that triggers exocytosis. A synaptic vesicle, together with its calcium sensor protein, is often found complexed with the calcium channel in active zones to form what will be called a "synaptosecretosome." In the present work, a stochastic analysis is given of the conditions under which a quantum is released from the synaptosecretosome by a nerve impulse. The theoretical treatment considers the rise of calcium at the synaptosecretosome after the stochastic opening of a calcium channel at some time during the impulse, followed by the stochastic binding of calcium to the vesicle-associated protein and the probability of this leading to exocytosis. This allows determination of the probabilities that an impulse will release 0, 1, 2,... quanta from an active zone, whether this is in a varicosity, a bouton, or a motor endplate. A number of experimental observations of the release of transmitter at the active zones of sympathetic varicosities and boutons as well as somatic motor endplates are described by this analysis. These include the likelihood of the secretion of only one quantum at an active zone of endplates and of more than one quantum at an active zone of a sympathetic varicosity. The fourth-power relationship between the probability of transmitter release at the active zones of sympathetic varicosities and motor endplates and the external calcium concentration is also explained by this approach. So, too, is the fact that the time course of the increased rate of quantal secretion from a somatic active zone after an impulse is invariant with changes in the amount of calcium that enters through its calcium channel, whether due to changes consequent on the actions of autoreceptor agents such as adenosine or to facilitation. The increased probability of quantal release that occurs during F1 facilitation at the active zones of motor endplates and sympathetic boutons is predicted by the residual binding of calcium to a high-affinity site on the vesicle-associated protein. The concept of the stochastic operation of a synaptosecretosome can accommodate most phenomena involving the release of transmitter quanta at these synapses.

Action Potentials↗
PubMed1997

Synaptic transmission at visualized sympathetic boutons: stochastic interaction between acetylcholine and its receptors.

Excitatory postsynaptic currents (EPSCs) were recorded with loose patch electrodes placed over visualized boutons on the surface of rat pelvic ganglion cells. At 34 degrees C the time to peak of the EPSC was about 0.7 ms, and a single exponential described the declining phase with a time constant of about 4.0 ms; these times were not correlated with changes in the amplitude of the EPSC. The amplitude-frequency histogram of the EPSC at individual boutons was well described by a single Gaussian-distribution that possessed a variance similar to that of the electrical noise. Nonstationary fluctuation analysis of the EPSCs at a bouton indicated that about 120 ACh receptor channels were available beneath boutons for interaction with a quantum of ACh. The characteristics of these EPSCs were compared with the results of Monte Carlo simulations of the quantal release of 9000 acetylcholine (ACh) molecules onto receptor patches of density 1400 microns-2 and 0.41 micron diameter, using a kinetic scheme of interaction between ACh and the receptors similar to that observed at the neuromuscular junction. The simulated EPSC generated in this way had temporal characteristics similar to those of the experimental EPSC when either the diffusion of the ACh is slowed or allowance is made for a finite period of transmitter release from the bouton. The amplitude of the simulated EPSC then exhibited stochastic fluctuations similar to those of the experimental EPSC.

Acetylcholine↗