Quantal transmission at purinergic junctions: stochastic interaction between ATP and its receptors.

The time course of most quantal currents recorded with a small diameter electrode placed over visualized varicosities of sympathetic nerve terminals that secrete ATP was determined: these had a time to reach 90% of peak of 1.3-1.8 ms and a time constant of decay of 12-18 ms; they were unaffected by blocking ectoenzymes or the uptake of adenosine. Monte Carlo methods were used to analyze the stochastic interaction between ATP, released in a packet from a varicosity, and the underlying patch of purinoceptors, to reconstitute the time course of the quantal current. This leads to certain restrictions on the possible number of ATP molecules in a quantum (about 1000) and the density of purinoceptors at the junctions (about 1000 microns-1), given the known geometry of the junction and the kinetics of ATP action. The observed quantal current has a relatively small variability (coefficient of variation < 0.1), and this stochastic property is reproduced for a given quantum of ATP. Potentiation effects (of about 12%) occur if two quanta are released from the same varicosity because the receptor patch is not saturated even by the release of two quanta. The simulations show that quantal currents have a characteristically distinct shape for varicosities with different junctional cleft widths (50-200 nm). Finally, incorporation of an ectoenzyme with the known kinetics of ATPase into the junctional cleft allows for a quantal current of the observed time course, provided the number of ATP molecules in a quantum is increased over the number in the absence of the ATPase.     

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.     

Comparisons of Allergenic and Metazoan Parasite Proteins: Allergy the Price of Immunity

Allergic reactions can be considered as maladaptive IgE immune responses towards environmental antigens. Intriguingly, these mechanisms are observed to be very similar to those implicated in the acquisition of an important degree of immunity against metazoan parasites (helminths and arthropods) in mammalian hosts. Based on the hypothesis that IgE-mediated immune responses evolved in mammals to provide extra protection against metazoan parasites rather than to cause allergy, we predict that the environmental allergens will share key properties with the metazoan parasite antigens that are specifically targeted by IgE in infected human populations. We seek to test this prediction by examining if significant similarity exists between molecular features of allergens and helminth proteins that induce an IgE response in the human host. By employing various computational approaches, 2712 unique protein molecules that are known IgE antigens were searched against a dataset of proteins from helminths and parasitic arthropods, resulting in a comprehensive list of 2445 parasite proteins that show significant similarity through sequence and structure with allergenic proteins. Nearly half of these parasite proteins from 31 species fall within the 10 most abundant allergenic protein domain families (EF-hand, Tropomyosin, CAP, Profilin, Lipocalin, Trypsin-like serine protease, Cupin, BetV1, Expansin and Prolamin). We identified epitopic-like regions in 206 parasite proteins and present the first example of a plant protein (BetV1) that is the commonest allergen in pollen in a worm, and confirming it as the target of IgE in schistosomiasis infected humans. The identification of significant similarity, inclusive of the epitopic regions, between allergens and helminth proteins against which IgE is an observed marker of protective immunity explains the ‘off-target’ effects of the IgE-mediated immune system in allergy. All these findings can impact the discovery and design of molecules used in immunotherapy of allergic conditions.     

A quantitative description of the diffusion of noradrenaline in the media of blood vessels following its release from sympathetic varicosities

A quantitative model is provided which describes how noradrenaline (NAd), released from varicosities at the adventitial surface of an artery, either diffuses into the media of the vessel to reach the intimal surface, diffuses into the volume of solution surrounding the artery, or is removed by the uptake 1 process in the varicosities. These predictions are then compared with experimental evaluations of the extent of changes in NAd to be found at the adventitial and intimal surfaces of the rat-tail artery, during and after trains of impulses, as determined using amperometry. In the model of the blood vessel there is a sequential decrease in the diffusion constant of NAd from the surrounding solution, to the adventitia, to the media, to the endothelium, to rise again in the lumen of the vessel; there is also an uptake 1 NAd pump in the varicosities described by Michaelis-Menten kinetics. This model is shown to provide a quantitative account of the spatial and temporal changes in NAd observed following trains of impulses at different frequencies of stimulation (5-40 Hz) for different periods of times (10-40 s). Changes in the spatio-temporal distribution of NAd observed following block of the uptake 1 NAd pump were also successfully predicted by the model. It is concluded that, within the context of the model, there is no need to evoke special mechanisms of buffering at the sympathetic varicosities, nor distinctions on the basis that only secreting varicosities utilize the uptake 1 mechanism, in order to describe the dynamics of NAd distribution in arteries during nerve activity.     

Purinergic junctional transmission and propagation of calcium waves in spinal cord astrocyte networks

Micro-photolithographic methods have been employed to form discrete patterns of spinal cord astrocytes that allow quantitative measurements of Ca(2+) wave propagation. Astrocytes were confined to lanes 20-100 microm wide and Ca(2+) waves propagated from a point of mechanical stimulation or of application of adenosine triphosphate; all Ca(2+) wave propagation was blocked by simultaneous application of purinergic P2Y(1) and P2Y(2) antagonists. Stimulation of an astrocyte at one end of a lane, followed by further stimulation of this astrocyte, gave rise to Ca(2+) transients in the same astrocytes; however, if the second stimulation was applied to an astrocyte at the other end of the lane, then this gave rise to a different but overlapping set of astrocytes generating a Ca(2+) signal. Both the amplitude and velocity of the Ca(2+) wave decreased over 270 microm from the point of initiation, and thereafter remained, on average, constant with random variations for at least a further 350 microm. Also, the percentage of astrocytes that gave a Ca(2+) transient decreased with distance along lanes. All the above observations were quantitatively predicted by our recent theoretical model of purinergic junctional transmission, as was the Ca(2+) wave propagation along and between parallel lanes of astrocytes different distances apart. These observations show that a model in which the main determinants are the diffusion of adenosine triphosphates regeneratively released from a stimulated astrocyte, together with differences in the properties and density of the purinergic P2Y receptors on astrocytes, is adequate to predict a wide range of Ca(2+) wave transmission and propagation phenomena.     

Purinergic junctional transmission and propagation of calcium waves in cultured spinal cord microglial networks

In order to elucidate the mechanisms of purinergic transmission of calcium (Ca^2 +) waves between microglial cells, we have employed micro-photolithographic methods to form discrete patterns of microglia that allow quantitative measurements of Ca^2 + wave propagation. Microglia were confined to lanes 20–100 wide and Ca^2 + waves propagated from a point of mechanical stimulation, with a diminution in amplitude, for about 120 . The number of cells participating in propagation also decreased over this distance. Ca^2 + waves could propagate across a cell-free lane from one microglia lane to another if this distance of separation was less than about 60 , indicating that propagation involved diffusion of a chemical transmitter. This transmitter was identified as ATP since all Ca^2 + wave propagation was blocked by the purinoceptor antagonist suramin, which blocks P2Y₂ and P2Y₁₂ at relatively low concentrations. Antibodies to P2Y₁₂ showed these at very high density compared with P2Y₂, indicating a role for P2Y₁₂ receptors. These observations were quantitatively accounted for by a model in which the main determinants are the diffusion of ATP released from a stimulated microglial cell and differences in the dissociation constant of the purinoceptors on the microglial cells.     

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.     

An isotopic approach to measuring nitrogen balance in caribou

Nutritional restrictions in winter may reduce the availability of protein for reproduction and survival in northern ungulates. We refined a technique that uses recently voided excreta on snow to assess protein status in wild caribou (Rangifer tarandus) in late winter. Our study was the first application of this non-invasive, isotopic approach to assess protein status of wild caribou by determining dietary and endogenous contributions of nitrogen (N) to urinary urea. We used isotopic ratios of N (δ¹⁵N) in urine and fecal samples to estimate the proportion of urea N derived from body N (p-UN) in pregnant, adult females of the Chisana Herd, a small population that ranged across the Alaska-Yukon border. We took advantage of a predator-exclosure project to examine N status of penned caribou in April 2006. Lichens were the primary forage (>40%) consumed by caribou in the pen and δ¹⁵N of fiber tracked the major forages in their diets. The δ¹⁵N of urinary urea for females in the pen was depleted relative (−1.3 ± 1.0 parts per thousand [‰], ) to the δ¹⁵N of body N (2.7 ± 0.7‰). A similar proportion of animals in the exclosure lost core body mass (excluding estimates of fetal and uterine tissues; 55%) and body protein (estimated by isotope ratios; 54%). This non-invasive technique could be applied at various spatial and temporal scales to assess trends in protein status of free-ranging populations of northern ungulates. Intra- and inter-annual estimates of protein status could help managers monitor effects of foraging conditions on nutritional constraints in ungulates, increase the efficiency and efficacy of management actions, and help prepare stakeholders for potential changes in population trends. © 2010 The Wildlife Society.