Biomedical subjects

M R Bennett

Publications and source records attributed to M R Bennett.

At least 73 records · Page 4Linked to original sources

PubMed1999

Analysis of novel P2X subunit-specific antibodies in rat cardiac and smooth muscle.

P2X receptors are cation-selective channels gated by extracellular adenosine triphosphate (ATP). There are relatively few known types of ligand-gated receptors. In vertebrates they include acetylcholine (Ach), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA), glycine, and glutamate as well as ATP. Ach, 5-HT, GABA and glycine ligand-gated receptors are related in evolutionary terms, while glutamate and ATP receptors form separate groups. There have been seven cloned proteins identified to date as members of the P2X receptor family in a wide range of cells and species. We have carried out hydropathy investigations and sequence comparisons of each of the seven subunits in order to examine the putative transmembrane and cysteine-rich extracellular domains. Probable locations of disulphide bridges are consistent with there being two separate extracellular folding domains. Assessment of the putative surface-accessible regions was used to select small localised amino acid segments in nonglycosylated regions for raising antibodies against each of the P2X receptor subunits. To test the specificity of these novel P2X receptor antibodies and their presence in cardiac and smooth muscle, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting was undertaken in homogenised rat heart, bladder, kidney, and vas deferens.

Amino Acid Sequence↗

PubMed1999

One hundred years of adrenaline: the discovery of autoreceptors.

The active principle of suprenal extract that produces its pressor effects was isolated by the joint research of John Abel in 1899 and Jokichi Takamine in 1901. Within three years Elliott, working in Langley's laboratory, suggested that this active principle, referred to by British physiologists as "adrenaline" and named "Adrenalin" by Takamine, was released from sympathetic nerve terminals to act on smooth muscle cells. However, it was not until 1946 that von Euler showed that demythelated adrenaline (noradrenaline) rather than adrenaline is a sympathetic transmitter. The possibility that this sympathetic transmitter could also act on nerve terminals was not developed until 1971. Research on autoreceptors culminated in the identification of adrenergic receptors on nerve terminals different to those on muscle cells. This paper assesses the contributions that established the idea of the adrenergic autoreceptor, 100 years after the discovery of adrenaline.

Animals↗

PubMed1999

Apoptosis of vascular smooth muscle cells in vascular remodelling and atherosclerotic plaque rupture.

Apoptosis (programmed cell death) of vascular smooth muscle cells (VSMCs) has recently been identified as an important process in a variety of human vascular diseases, including atherosclerosis, arterial injury, and restenosis after angioplasty. VSMC apoptosis is regulated by interactions between the local cell-cell and cytokine environment within the arterial wall, and the expression of pro- and anti-apoptotic proteins by the cell, including death receptors, proto-oncogenes and tumour suppressor genes. This review summarises our current knowledge of the occurrence and mechanisms underlying VSMC apoptosis in atherosclerosis and arterial remodelling.

Animals↗

PubMed1999

Forty years of amino acid transmission in the brain.

This article is concerned with the discovery that amino acids, particularly L-glutamate and gamma-aminobutyrate (GABA), are central neurotransmitters. The crucial observations that lead to the conclusion that these two amino acids produce most of the synaptic excitation and inhibition in the central nervous system, were made in late 1950's. The combination of neurochemical knowledge and improved electrophysiological techniques was paramount in making these discoveries possible. In particular, the use of specific antagonists in microiontophoretic experiments provided the most decisive evidence. The relationship is also explored between these early findings and those of the present era characterised by extensive use of techniques of molecular biology and the development of drugs against targets identified 30 to 40 years ago.

Amino Acids↗

PubMed1999

Synapse formation molecules in muscle and autonomic ganglia: the dual constraint hypothesis.

In 1970 it was thought that if the motor-nerve supply to a muscle was interrupted and then allowed to regenerate into the muscle, motor-synaptic terminals most often formed presynaptic specializations at random positions over the surface of the constituent muscle fibres, so that the original spatial pattern of synapses was not restored. However, in the early 1970s a systematic series of experiments were carried out showing that if injury to muscles was avoided then either reinnervation or cross-reinnervation reconstituted the pattern of synapses on the muscle fibres according to an analysis using the combined techniques of electrophysiology, electronmicroscopy and histology on the muscles. It was thus shown that motor-synaptic terminals are uniquely restored to their original synaptic positions. This led to the concept of the synaptic site, defined as that region on a muscle fibre that contains molecules for triggering synaptic terminal formation. However, nerves in developing muscles were found to form connections at random positions on the surface of the very short muscle cells, indicating that these molecules are not generated by the muscle but imprinted by the nerves themselves; growth in length of the cells on either side of the imprint creates the mature synaptic site in the approximate middle of the muscle fibres. This process is accompanied at first by the differentiation of an excess number of terminals at the synaptic site, and then the elimination of all but one of the terminals. In the succeeding 25 years, identification of the synaptic site molecules has been a major task of molecular neurobiology. This review presents an historical account of the developments this century of the idea that synaptic-site formation molecules exist in muscle. The properties that these molecules must possess if they are to guide the differentiation and elimination of synaptic terminals is considered in the context of a quantitative model of this process termed the dual-constraint hypothesis. It is suggested that the molecules agrin, ARIA, MuSK and S-laminin have suitable properties according to the dual-constraint hypothesis to subserve this purpose. The extent to which there is evidence for similar molecules at neuronal synapses such as those in autonomic ganglia is also considered.

Animals↗

PubMed1999

The concept of a calcium sensor in transmitter release.

The discovery was made in the 1940s that calcium is required for transmitter release at synapses, raising the question of the identity of the sensor molecule upon which this calcium acts. Subsequently it was shown in the 1960s that this calcium acts on the inside of the nerve terminal. The channels which mediate the influx of calcium ions into the nerve terminal were identified in the 1970s. This essay is concerned with tracing the development of the concept of a calcium sensor in nerve terminals and of recent work that identifies the sensor molecule as synaptotagmin.

Action Potentials↗

PubMed1999

Phosphorylation of proteins in chick ciliary ganglion under conditions that induce long-lasting changes in synaptic transmission: phosphoprotein targets for nitric oxide action.

Production of nitric oxide and the activation of protein kinases are required for long-term potentiation of synaptic transmission at the giant synapses in chicken ciliary ganglion. In the present study, we investigated the ability of nitric oxide to regulate the phosphorylation of endogenous proteins under conditions that induced long-term potentiation in intact ciliary ganglion and the protein kinases responsible for the phosphorylation of these proteins in lysed ciliary ganglion. Using Calcium Green-1 we showed that the nitric oxide donor sodium nitroprusside did not change the intraterminal Ca2+ dynamics in ciliary ganglion. Two dimensional phosphopeptide analysis of 32Pi-labelled intact ciliary ganglion showed that the sodium nitroprusside (300 microM) increased the phosphorylation of several phosphopeptides (P50a, P50b and P41) derived from proteins at 50,000 and 41,000 mol. wts which we have called nitric oxide-responsive phosphoproteins. A similar stimulation of phosphorylation was achieved by 8-bromo-cyclic AMP (100 microM), which also induced long-term potentiation, but not by phorbol dibutyrate (2 microM) that does not induce long-term potentiation in ciliary ganglion. When subcellular fractions from lysed ciliary ganglion were labelled in vitro by [gamma-32P]ATP in the presence of purified cGMP-dependent, cAMP-dependent or Ca2+-phospholipid-dependent protein kinases, we identified cyclic GMP-dependent protein kinase substrates that gave rise to phosphopeptides co-migrating with P50a, P50b and P41 from 32Pi-labelled intact ciliary ganglion. P50a and P41 were derived from soluble proteins while P50b was derived from a membrane-associated protein. The proteins giving rise to P50a, P50b and P41 were also substrates for cyclic AMP-dependent protein kinase, but not for calcium and phospholipid-dependent protein kinase in vitro, suggesting that nitric oxide-responsive phosphoproteins are convergence points in information processing in vivo and their phosphorylation might represent an important mechanism in nitric oxide-mediated synaptic plasticity in ciliary ganglion.

Animals↗

PubMed1999

Development of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder.

Postnatal development of the distribution of different isoforms of purinergic (P2X) receptors on smooth muscle cells in relation to the development of the innervation of the cells by nerve varicosities in the rat urinary bladder has been determined with immunofluorescence and confocal microscopy. Antibodies against the extracellular domains of the P2X(1) to P2X(6) receptors were used to detect the receptors in the bladder. Several other antibodies were used to identify sympathetic varicosities and Schwann cells. At one day postnatal (D1) there were few strings of varicosities denoting isolated axons, with most axons confined to large nerve trunks. Small size clusters of P2X(1) to P2X(6) receptor subtypes (about 0.4 microm diameter) were observed in the muscle which were independent of each other, and sometimes juxtaposed to the rare isolated varicosity strings. At D4 large numbers of strings of varicosities could be discerned throughout the detrusor. Most of these clouds of small P2X(1) to P2X(6) receptor clusters in their immediate vicinity. Some of these were colocalised with the varicosities, which were of parasympathetic origin as they failed to counter-stain with antibodies to tyrosine hydroxylase. Up to D14 there was a gradual coalescence of many of the isolated P2X(1-6) small receptor clusters so that they became colocalized, often at varicosities. Most of the varicosities in isolated strings possessed receptor clusters at this time. By D21 it was rare to find varicosity strings in the detrusor that were not either in close juxtaposition with P2X small receptor clusters or possessing such clusters in colocalization. However, large numbers of small P2X receptor clusters, many of which consisted of a mixture of isoforms, could be found spatially unrelated to nerve varicosities throughout the detrusor muscle. In the adult, single axons were either coextensive with one or more isoforms of P2X receptor clusters or these were immediately juxtaposed to the axons so that is was rare to find a varicosity that did not possess a receptor cluster. However, different combinations of colocalized P2X receptor isoforms could still be discerned in small clusters unrelated to varicosities. These observations are discussed in relation to the mechanism of formation of the receptor clusters and their migration beneath parasympathetic varicosities during development.

Animals↗

PubMed1999

Vesicle-associated proteins and P2X receptor clusters at single sympathetic varicosities in mouse vas deferens.

1. Antibodies against vesicle-associated proteins of the SNARE complex (syntaxin (AbS), SNAP 25 (AbS25), synaptobrevin (VAMP; AbV) and the alpha1B subunit of calcium channels (Abalpha(1B)) were located with respect to sympathetic varicosities (labelled with the ubiquitous vesicle proteoglycan antibody AbSV2) and to clusters of P2X receptor subunits (labelled with antibodies AbP2X(1) to AbP2X(6)). In addition, these receptor clusters were located with respect to Schwann cells labelled with antibodies to S100 (AbS100).2. The spatial relation between proteins of the SNARE complex and calcium channels was determined. AbS25 patches ranged from 250-500 nm in size and were often colocalised with smaller AbS patches (250-350 nm). Abalpha(1B) patches (300-700 nm diameter) were always coincidental with AbS patches. AbV patches (400-1000 nm in diameter) also coincided with AbS patches.3. The spatial relation between different P2X subunit clusters and varicosities labelled with AbSV2 was ascertained. Large (500-700 nm diameter) AbP2X(1) receptor clusters were found colocalised with many (91%) AbSV2 labelled varicosities, although small diameter (250-350 nm) AbP2X(1) clusters occurred at random over the muscle. Small AbP2X(2) clusters were found uniquely in the vicinity of AbSV2 labelled varicosities, but were not entirely coincidental with these. Small AbP2X(3) receptor clusters were found in close association with AbSV2 labelled nerves. Small diameter AbP2X(4) clusters (250-350 nm) were found throughout the muscle with some of these coincidental with AbSV2 labelling. Small diameter AbP2X(5) (250-350 nm) cluster labelling was found in juxtaposition to strings of AbSV2 labelled varicosities but were not coincidental with these. Small (250-350 nm) diameter AbP2X(6) clusters were also found in close juxtaposition to AbSV2 labelled nerves.4. The spatial relation between different P2X subtype clusters and Schwann cells labelled with AbS100 was examined. Both AbP2X(1) and AbP2X(3) receptor clusters were found in close apposition with AbS100, with clusters of the former sometimes coincidental with patches of the latter. On the other hand AbP2X(2) was found in association with AbS100 at low levels while AbP2X(4) labelling was generally not coincidental with AbS100. AbP2X(5) and AbP2X(6) labelling was often colocalised with AbS100 labelling.5. The spatial relation between proteins of the SNARE complex and P2X(1) receptors was determined. Large AbP2X(1) clusters were often found apposed by AbS, AbV and Abalpha(1B) labelled patches.6. Destruction of the sympathetic varicosities with 6-hydroxydopamine led to the virtual disappearance of AbP2X(2) labelling, but to a large increase in the number of small AbP2X(1) receptor clusters and a reduction in the number of large AbP2X(1) clusters. AbS100 Schwann cell labelling was largely unaffected.7. These observations are interpreted as showing that most terminal sympathetic varicosities possess active zones about 250-700 nm diameter, delineated by syntaxin, SNAP 25 and N-type calcium channels and that synaptic vesicles are concentrated at these sites as indicated by the localisation of VAMP. Most of these terminal varicosities possess active zones that are precisely apposed to large clusters of P2X(1) receptors. However small clusters of P2X(2) to P2X(6) receptors can be found that are near the strings of varicosities but not usually coincidental with them except P2X(3). The functional significance of this arrangement of vesicle-associated proteins and P2X receptors for the generation of synaptic potentials at the autonomic neuromuscular junction is discussed.

Animals↗

PubMed1999

Individual sympathetic varicosities possess different sensitivities to alpha 2 and P2 receptor agonists and antagonists in mouse vas deferens.

1. The diversity of alpha(2) and purinergic autoreceptor actions on action potential evoked calcium transients in single varicosities has been investigated using the calcium indicator Oregon Green 488 BAPTA-1. 2. During long trains of impulses (10 Hz for 30 s), the change in calcium concentration in varicosities from its resting level (Delta[Ca(2+)](v)) increased in many varicosities during the first 3 s of stimulation before reaching a plateau. 3. The alpha(2) adrenoceptor agonist clonidine (1 microM) decreased Delta[Ca(2+)](v) by over 40% during short trains (five impulses at 5 Hz) in most varicosities, although some were unaffected. The alpha(2) adrenoceptor antagonist idazoxan (2 microM) increased the Delta[Ca(2+)](v) plateau following long trains in most varicosities. Hence, most varicosities possess alpha(2) adrenoceptors which are activated when noradrenaline accumulates extracellularly. 4. During long trains of impulses, the P(2y)-purinergic receptor agonist 2-methyl-thio-ATP (100 microM) decreased Delta[Ca(2+)](v) plateau by about 50% in most varicosities; alpha,beta-methylene ATP (100 microM) decreased it by about 50% in a minority of varicosities; adenosine (200 microM) had no significant effect. Suramin (100 microM) increased the Delta[Ca(2+)](v) during all stimulus protocols in most varicosities, suggesting that ambient ATP modulates Delta[Ca(2+)](v) responses. The P(2y) receptor antagonist reactive blue (100 microM) affected a minority of varicosities. Given that most varicosities respond to suramin, other P(2) receptor subtypes are probably present. 5. The ATP ectoenzyme antagonist ARL67157 (50 microM) decreased the plateau Delta[Ca(2+)](v) during long trains in complete strings of varicosities but not in others. 6. The present technique indicates that varicosities have diverse autoreceptor utilization.

Adrenergic Fibers↗

PubMed1999

Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus).

The effect of a conditioning depolarizing current pulse (80-200 micros) on quantal secretion evoked by a similar test pulse at another site was examined in visualized motor-nerve terminal branches of amphibian endplates (Bufo marinus). Tetrodotoxin (200 nM) and cadmium (50 microM) were used to block voltage-dependent sodium and calcium conductances. Quantal release at the test electrode was depressed at different distances (28-135 microm) from the conditioning electrode when the conditioning and test pulses were delivered simultaneously. This depression decreased when the interval between conditioning and test current pulses was increased, until, at an interval of approximately 0.25 ms, it was negligible. At no time during several thousand test-conditioning pairs, for electrodes at different distances apart (28-135 microm) on the same or contiguous terminal branches, did the electrotonic effects of quantal release at one electrode produce quantal release at the other. Analytic and numerical solutions were obtained for the distribution of transmembrane potential at different sites along terminal branches of different lengths for current injection at a point on a terminal branch wrapped in Schwann cell, in the absence of active membrane conductances. Solutions were also obtained for the combined effects of two sites of current injection separated by different time delays. This cable model shows that depolarizing current injections of a few hundred microseconds duration produce hyperpolarizations at approximately 30 microm beyond the site of current injection, with these becoming larger and occurring at shorter distances the shorter the terminal branch. Thus the effect of a conditioning depolarizing pulse at one site on a subsequent test pulse at another more than approximately 30 microm away is to substantially decrease the absolute depolarization produced by the latter, provided the interval between the pulses is less than a few hundred microseconds. It is concluded that the passive cable properties of motor nerve terminal branches are sufficient to explain the effects on quantal secretion by a test electrode depolarization of current injections from a spatially removed conditioning electrode.

Animals↗

PubMed1999

Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals.

A study was made to determine the disposition of vesicle-associated proteins (syntaxin, SV2, SNAP-25) and calcium channels with respect to the spatial extent of spontaneous and evoked quantal release within regions of amphibian motor-nerve terminal branches delineated by FM1-43 stained vesicle clusters (blobs). Discrete concentrations of vesicles revealed approximately 2 microm apart along the length of terminal branches through FM1-43 staining were identical in size and spacing to those identified along terminal branches with SV2 antibody (AbSV2). Fluorescent antibodies to syntaxin 1 (AbS), SNAP-25 (AbS25) and the calcium channel alpha1B subunit (Abalpha1B) were found in relatively high concentrations coincident with the AbSV2 blobs. Three extracellular recording electrodes were placed in the vicinity of individual FM1-43 blobs, and an algorithm was used to determine the spatial origin of miniature endplate potentials (MEPPs) and EPPs together with their relative amplitudes. MEPPs and EPPs originated throughout the region stained by FM1-43 but not elsewhere; amplitude-frequency distributions of MEPPs and EPPs were similar for all FM1-43 blobs with average coefficients of variation of no less than 0.28. A linear relationship existed between the size of an FM1-43 blob, measured as the integrated extent of FM1-43 staining of a blob, and the frequency of MEPPs as well as the probability of EPPs from the blob. There was a proximo-distal gradient in the size of FM1-43 blobs along the length of single terminal branches, suggesting a gradient in release probability along the branches. The frequency distribution of the distances between blobs was approximately Gaussian, whereas the frequency distribution of the size of blobs was highly skewed and was best fitted with a gamma distribution. It is concluded that there are correlations among the extent of labeling of SNAP-25, syntaxin and calcium channels at a release site, the store of vesicles to be found there, and the probability of spontaneous and evoked quantal release.

Animals↗

PubMed1999

Protection against necrosis but not apoptosis by heat-stress proteins in vascular smooth muscle cells: evidence for distinct modes of cell death.

We have reported previously that cultured vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR) show higher proliferation and cell death than normotensive controls. In addition to protecting cells against death, heat stress proteins (HSPs) appear to play a role in cell proliferation. This investigation examines the involvement of HSP72 and HSP27 in altered SHR VSMC proliferation and death. We have performed detailed discriminatory analysis to characterize which type of VSMC death is induced by heat stress (HS) and serum deprivation. Serum deprivation induced apoptosis (caspase-3 cleavage and DNA laddering) and secondary necrosis, the 2 processes being a continuum of each other. In contrast, acute HS (46 degrees C, 30 minutes), which inhibited BN. lx and SHR VSMC proliferation by 2-fold, increased necrosis (by 5-fold and 2-fold, respectively) but not apoptosis. HSP72 and HSP27 expression evoked in VSMC by mild HS (44 degrees C, 15 minutes) 6 hours before acute HS prevented the inhibition of proliferation and induction of necrosis with no effect on serum deprivation-induced or staurosporine-induced apoptosis. This induced expression of HSP72 and HSP27 did not eliminate the higher basal proliferation, apoptosis, and necrosis of SHR VSMC compared with BN.lx VSMC, suggesting that these HSPs are not involved in altered SHR VSMC proliferation and death. Also, although apoptosis and necrosis may be a continuum, in VSMC the 2 processes may be distinguished by HS, in which only necrosis is prevented by prior HSP accumulation. This observation may be of use in designing strategies for cellular protection.

Animals↗

PubMed1998

Methods for grouping shapes of synaptic currents recorded from sets of synapses.

Synaptic currents due to transmitter release at sets of synapses, such as those formed by a single nerve terminal on adjacent smooth muscle cells or a nerve terminal on a dendrite, possess different amplitudes and time courses, that is different shapes. It is possible that different shape groupings exist that may be each associated with a particular synapse in the set recorded from. Two methods, involving the distance between two synaptic currents, namely multidimensional scaling and hierarchical clustering, were used to suggest possible groupings. A test of differences between a pair of synaptic currents was developed by comparing the distances from pairs of synaptic currents with the distances from pairs of failures from the same experiment. This allowed observation of significance levels between and within the suggested groups. These methods were applied to loose-patch recordings of excitatory junctional currents (EJCs) in mouse vas deferens made with extracellular electrodes, some of which were visualised using DiOC2(5) staining to identify varicosities. Groups of EJC shapes could be distinguished supporting a hypothesis of several categories of shapes. However, each method showed that the number of shape-groupings was much larger than the number of visualised varicosities. Possible reasons for this discrepancy are analysed, including a model in which different shape groupings can be generated by a single synaptic site.

Animals↗

PubMed1998

Spontaneous calcium transients in automatic boutons and varicosities.

Spontaneous multiquantal events are recorded at many different boutons and varicosities for which there is evidence that the receptor patch at these individual synapses is saturated by the transmitter unit. In order to reconcile these observations, a model is considered in which calcium release from a ryanodine channel within a nerve terminal can reach adjacent active zones in single synapses in sufficient concentration to occasionally trigger exocytosis from adjacent zones synchronously, giving rise to multiquantal spontaneous events. It is shown that the spatial and temporal distribution of calcium concentration at the active zone after a spontaneous opening of a ryanodine channel can predict the amplitude and time course of observed calcium-activated potassium channel currents. Similar calcium transients are sufficient to give rise to multiquantal events. Such events suggest a multi hypothesis for secretion.

Animals↗

PubMed1998

Sympathetic neuromuscular transmission at a varicosity in a syncytium.

The autonomic neuromuscular junction at a varicosity in the vas deferens is defined by the localization of the vesicle-associated protein syntaxin in high concentrations in the axolemma and a high density of P2x1 receptors in a cluster beneath the varicosity. Calcium fluxes have been observed in all individual varicosities of a nerve terminal on the arrival of an impulse even though recordings made from these varicosities of the electrical signs of transmission with loose-patch electrodes over the varicosities show that they have very different probabilities for the secretion of a quantum. The fact that some varicosities seldom release a quantum on the arrival of an impulse is supported by the observation that antibodies against the N-terminus of synaptotagmin, which uniquely label the inside of synaptic vesicles when they undergo exocytosis, fail to do so in some varicosities during nerve stimulation whereas they do in others. It is suggested that the probability for secretion from a varicosity depends on the number of secretosomes that the varicosity possesses, where a secretosome is a complex of syntaxin, synaptotagmin, an N-type calcium channel, and a synaptic vesicle.

Animals↗

PubMed1998

Calcium in sympathetic boutons of rat superior cervical ganglion during facilitation, augmentation and potentiation.

The sympathetic preganglionic nerve terminals of the rat superior cervical ganglion were loaded with the calcium indicator oregon green 488 BAPTA-1 to measure the change in calcium concentration in the terminal boutons, (delta[Ca2+]b) following short (1 or 5 impulses) and long (200 impulses) trains at 30 Hz. The delta[Ca2+]b after a single action potential or a short train declined in two phases: a fast phase with a time constant of 530+/-30 ms and a moderate phase with a time constant of 4.0+/-0.2 s. The delta[Ca2+]b following a long train eventually declined with a time constant of 127+/-34 s (slow phase). The addition of either omega-agatoxin TK (100 nM), omega-conotoxin GVIA (100 nM) or nifedipine (20 microM) to block P-type, N-type or L-type calcium channels respectively showed that the rise in delta[Ca2+ ]b in boutons was predominantly mediated by an influx of calcium through P-type (53+/-7%) and N-type (46+/-4%) calcium channels. Experiments with caffeine, ryanodine and thapsigargin indicate that intracellular caffeine-sensitive calcium stores have a small but statistically significant effect on the fast and moderate phases. The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 2 microM) significantly decreased the amplitude of the slow phase of delta[Ca2+]b relaxation, and sped its time course, suggesting that mitochondria normally dump calcium during this phase. Adenosine reduced the amplitude of delta[Ca2+]b in response to single action potentials by 30+/-6%, suggesting that adenosine-mediated autoinhibition in these boutons reduces Ca2+ influx. Spontaneous increases in delta[Ca2+]b demonstrated Ca2+ coupling between adjacent boutons. The delta[Ca2+]b kinetics are compared with F2 facilitation, augmentation and post-tetanic potentiation.

Action Potentials↗

PubMed1998

Alpha and beta subunits of CaM-kinase II are localized in different neurons in chick ciliary ganglion.

The ciliary ganglion of the chicken contains only two types of neurons. Using monoclonal antibodies against the alpha and the beta subunits of Ca2+/calmodulin-stimulated protein kinase II (CaMPK-II) we found that the alpha-subunit was localized to the choroid neurons while beta subunit was associated with the ciliary neurons. As both neurons receive their inputs from the oculomotor nerve, while their postganglionic axons leave via different nerves, the ciliary ganglion of the chicken is a neuronal system in which the functional differences between alpha and beta CaMPK-II homopolymers in the regulation of synaptic transmission can be investigated.

Animals↗