Biomedical subjects

M I Glavinović

Publications and source records attributed to M I Glavinović.

At least 19 recordsLinked to original sources

PubMed2006

Glutamate, water and ion transport through a charged nanosize pore.

The transport of transmitter, ions and water through a positively-charged nanopore was investigated through computer simulations. The physics of the problem is described by a coupled set of Poisson-Nernst-Planck and Navier-Stokes equations in a computational domain consisting a cylindrical pore, whose radius ranged from 1 to 8 nm and which was flanked by two compartments representing the vesicular interior and extra-cellular space. The concentration of co-ions is suppressed and of counter-ions enhanced, especially near the pore wall owing to electrostatic interactions. Glutamate (i.e. the transmitter considered) is negatively charged and is simulated as a counter-ion. The electro-kinetically induced pressure due to the movement of ions is negative and very pronounced near the pore wall where the concentration and flux of counter-ions is very high. The water velocity peaks in the pore center, diminishes to zero at the pore wall, but is constant along the pore axis. The mean velocity of the water/fluid is proportional to the vesicular pressure and pore cross-sectional area. Interestingly it is inversely related to the vesicular glutamate concentration. The factors determining the glutamate flux are complex. The diffusive flux generally predominates for narrow pore, and convective flux may dominate for wide pore if the vesicular pressure is high. Surprisingly at low vesicular pressure the mean total glutamate flux per unit cross-sectional pore area is higher for narrow pores. Higher flux is probably due to the rise of glutamate concentration in the nanopore, which is much more pronounced for narrow nanopores, due to the maintenance of approximate neutrality of charges in the pore and on the pore wall. In conclusion intra-vesicular pressure helps 'flushing-out' the transmitter, but the induced pressure 'drags-out' the water into the extra-cellular space.

Biological Transport↗

PubMed2006

Monte Carlo simulation of release of vesicular content in neuroendocrine cells.

The release of transmitter from the vesicle, its diffusion through the fusion pore, and the cleft and its interaction with the carbon electrode were simulated using the Monte Carlo method. According to the simulation the transmitter release is largely determined by geometric factors--the ratio of the fusion pore cross-sectional and vesicular areas, if the diffusion constant is as in the aqueous solution--but the speed of transmitter dissociation from the gel matrix plays an important role during the rise phase of release. Transmitter is not depleted near the entrance to the fusion pore and there is no cleft-to-vesicle feedback, but the depletion becomes evident if the diffusion constant is reduced, especially if the pore is wide. In general, the time course of amperometric currents closely resembles the time course of the simulated transmitter concentration in the cleft and the time course of release. Surprisingly, even a tenfold change of the electrode efficiency has only a marginal effect on the amplitude or the time course of amperometric currents. Greater electrode efficiency however lowers the cleft concentration, but only if the cleft is narrow. As the cleft widens the current amplitudes diminish and rise times lengthen, but the decay times are less affected. Moreover, the amplitude dependence of the rise and decay times becomes steeper as the cleft widens and/or as the release kinetics slows. Finally, lower diffusion constant of transmitter in the narrow cleft does not further prolong the amperometric currents, whose slow time course reflects slow release kinetics.

Animals↗

PubMed2005

Rapid change of quantal size in PC-12 cells detected by neural networks.

The basic building block of synaptic transmission-the number of molecules released per vesicle (quantal size (QS)) often changes with stimulation, but there is no agreement about what factors regulate it. To throw more light on this problem spontaneous quantal release was recorded amperometrically in PC-12 cells. Amperometric current spikes, representing single vesicle release, were detected by thresholding and were separated from spurious events on the basis of their amplitude and time course using a pattern recognition system based on the principal component neural network methods. The frequency of current spikes, their amplitude, quantal size, rise time and decay time were typically non-stationary, even in the absence of stimulation. Their running values changed much more than those of memoryless stationary random data with the same probability density distribution. Irrespective of how much the quantal size, rise and decay times varied, their amplitude dependence remained constant, or changed with a very different time course. In conclusion, the quantal size is highly labile in PC-12 cells. The lability does not appear to result from the changes of fusion pore dynamics or the mechanism of release of vesicular content, but because of the preferential release of large vesicles.

Action Potentials↗

PubMed2001

Change of quantal size and parameters of release with stimulation in bovine chromaffin cells.

Changes in quantal size and in the parameters of release were examined in chromaffin cells using amperometric recordings during and following various stimuli that induce secretion. As a general rule, a greater quantal content was associated with a greater quantal size. Following a short depolarizing pulse (0.5-2 s; 100 mV from a holding potential of -80 mV), the mean value of quantal size increased by 54% over several seconds before gradually (over tens of seconds) returning toward the control value, whilst its variability rose by 62%. The changes observed following 30-s applications of high extracellular K+ (50 mM) were more modest. A rapid application of short depolarizing pulses (2 s every 10-20 s; 100 mV from a holding potential of -80 mV) also led, at least initially, to greater quantal content and quantal size. Mean quantal size rose initially by 68%, but decreased subsequently to 31% below pre-stimulation levels. In whole-clamped cells, the frequency of quantal release can rise abruptly, probably owing to a mechanical disturbance that makes the membrane leaky to Ca2+. In such cases, a marked rise in quantal content (>ten-fold) was paralleled by an almost as dramatic (up to ten-fold) rise in quantal size and an important, although less pronounced and slower, rise in its variability (up to four-fold). The return toward control values of mean quantal size occurred over several minutes, whilst its variability decayed more slowly. The release parameters were evaluated directly from the number of events to avoid a large and time-dependent contribution of the amplitude variability of spontaneous amperometric current spikes (minis). In general, the greater probability of release contributed more than the greater size of the immediately available store to the increase in quantal output. In conclusion, quantal size was found to be highly labile. Its change can alter strongly the facilitation and depression of evoked quantal output and probably occurs due to a preferential release of large vesicles that are more efficient barriers to Ca2+ diffusion when Ca2+ rises rapidly following a synchronous opening of several Ca2+ channels. When intracellular Ca2+ levels rise slowly to threshold levels for secretion, as during an asynchronous and generally spontaneous release, vesicles are less effective diffusion barriers and quantal size changes less.

Animals↗

PubMed2001

Monte Carlo evaluation of quantal analysis in the light of Ca2+ dynamics and the geometry of secretion.

Using the Monte Carlo technique, Ca2+ dynamics were simulated in the absence and presence of vesicles to gain better insight into what governs quantal release. A vesicle, represented as a flat, infinitely thin surface, was positioned parallel to the plasma membrane at a chosen distance from the locus of Ca2+ entry. Because vesicles act as important diffusion barriers after the synchronous opening of Ca2+ channels (as occurs during evoked release), [Ca2+] close to the plasma membrane reaches higher levels than it would in the absence of vesicles. The rise in [Ca2+] is greater under larger vesicles close to the plasma membrane, which thus have a higher probability of release. The power-law relationship between the [Ca2+] and the probability of release, and the cubic relationship between the vesicular diameter and its volume can make this relationship very steep. In contrast, when release occurs owing to fluctuations of [Ca2+]--as a result of Ca2+ release from an internal store or asynchronous opening of Ca2+ channels (during spontaneous release)--the effect of vesicles as diffusion barriers is less pronounced and vesicles of different sizes should have a similar probability of release. Since the preferential release of large vesicles depends on how the Ca2+ needed for secretion is raised (synchronously versus asynchronously), the quantal size of evoked and spontaneous release should differ. The main factors influencing the preferential release of large vesicles are the distance between vesicles and the plasma membrane, the concentration of Ca2+ buffers, and single-channel Ca2+ flux. Vesicles also have a pronounced effect on Ca2+ binding to buffers and on the spatio-temporal distribution of bound buffers. The greater the vesicular size and the closer their position to the plasma membrane, the more fixed buffers will be bound near the plasma membrane because of limited diffusion of Ca2+. Since bound fixed buffers act as "memory elements", such a change in their spatial distribution will further enhance the probability of release of large vesicles during stimulation.

Calcium↗

PubMed1998

Comparison of vesicular volume and quantal size in bovine chromaffin cells.

Electrochemical measurements of vesicular content released were compared with the morphometric measurements of vesicular size in bovine chromaffin cells. Cross-sectional vesicular diameters were determined from electron micrographs. Two methods were used to determine the frequency histograms of "true" vesicular diameters (i.e. diameters of the vesicles in the equatorial plane): (i) "peeling off" method [Coupland R. E. (1968), Nature 217, 384-388], and (ii) summation of individual probabilities of "true" vesicular diameters. Quantal size was estimated from the area under the spontaneous current spike detected electrochemically. The frequency histograms of "true" vesicular diameters are found to be skewed (thus not well described by a Gaussian function) irrespective of the method used to calculate them, as are the frequency histograms of the cube roots of the quantal sizes. Furthermore, we also find that the frequency histograms of electrochemical measurements (the cube roots of quantal sizes) have lower skews and coefficients of variation than those of morphometric measurements ("true" vesicular diameters), with discrepancy being especially pronounced for noradrenaline-secreting cells. Such a difference in both coefficients of variation and skews suggests that the intravesicular catecholamine concentration is not uniform, but that it is lower for vesicles of larger size. In conclusion a variety of factors--vesicular volume, vesicular surface area to volume ratio, binding capacity of chromogranin and/or ATP, likely determines the amount of catecholamine stored in the vesicle.

Adrenal Medulla↗

PubMed1996

Time course of release of content of single vesicles in bovine chromaffin cells.

The time course of the spontaneous current spikes produced by release of the catecholamine contents of individual vesicles was examined in bovine chromaffin cells using carbon filament electrodes. The rate of spontaneous release was enhanced by adding either LaCl3 (0.01-0.5 mM) or BaCl2 (2 mM) to the extracellular solution. A paucity of events of very short duration was evident from the frequency histograms of the rise and the decay times. In the scatterograms of the rise and of the decay times the regression lines are invariably positive (i.e. the longer the duration of the rise times the longer the duration of the decay times). However, the regression lines never go through the origin but intercept the ordinate (the axis of the decay times) at (+/- SD) 16.1 +/- 6.4 ms (n = 11). On the other hand, the regression lines of paired rise and decay times for the time courses of diffusion are both linear and go through the origin. This relationship holds irrespective of whether the diffusion from an instantaneous point source was assumed to occur in an infinite plane or in an infinite volume. Therefore our experimental findings are incompatible with the model(s) assuming that diffusional broadening determines entirely the time course of current spikes. However, they can be explained, although only partially, by the possible slow speed of the electrode. They thus suggest that in chromaffin cells the duration of exocytosis of individual vesicles is much longer than in synapses.

Animals↗

PubMed1996

Temperature dependence of release of vesicular content in bovine chromaffin cells.

Recent studies suggest that the time course of secretion of the vesicular content in bovine chromaffin cells is much slower than in the peripheral or the central nervous system, but the reasons for this marked difference are not known. In this study we try to assess the importance of factors that may influence the time course of release of the vesicular content of bovine chromaffin cells, namely: (1) diffusion of catecholamines in the extracellular solution, (2) dissociation of catecholamines from the matrix of chromogranin A, and (3) the kinetics of opening and closing of the fusion pore. The temperature dependence of the time course and the amplitude of the spontaneous current spikes were examined using the carbon filament recording technique in amperometric mode. The change in amplitude was not statistically significant, but both the rise and the decay times were shortened (from 29 +/- 12 to 16 +/- 5 ms, and from 87 +/- 26 to 57 +/- 11 ms respectively) as temperature was raised by 20 degrees C [from 15 to 35 degrees C; n = 6; the changes were statistically significant at the level of P = 0.05; their respective temperature coefficients (Q10) were 1.4 and 1.3]. The areas underneath the spontaneous current spikes, however, were not altered significantly. Neither the relationship between the rise and the decay times nor the frequency of occurrence of the spontaneous current spikes changed consistently as the temperature was raised. However, the frequency histograms could, in all cases, be well described by a monoexponential function. It is concluded that the release of catecholamine content from the individual vesicles in bovine chromaffin cells is probably mostly determined by the dissociation of catecholamines from the matrix of chromogranin A.

Adrenal Medulla↗

PubMed1995

Decrease of quantal size and quantal content during tetanic stimulation detected by focal recording.

End-plate potentials and miniature end-plate potentials were recorded focally (i.e., over a limited area of the end-plate with several or possibly only one active zone) in a cutaneous pectoris from neuromuscular junction during a prolonged (1-6 min) tetanic (20-100 Hz) nerve stimulation. End-plate potential amplitudes decreased and became more variable with prolonged stimulation. Synaptic depression thus occurs even when synaptic output is low, if release is evoked from only a few active zones, suggesting that there is little if any vesicular replenishment between the active zones. The probability density function of the end-plate potential amplitudes has been obtained using the Parzen estimate with a Gaussian weighting function, to reduce the number of end-plate potentials needed for the same accuracy. Quantal size of the end-plate potentials was estimated from the slope of the best fitted line to the prominent and apparently equidistant peaks of probability density functions or from the spectrogram of the probability density function of end-plate potentials. Quantal contents were initially (+/- S.D.):5.7 +/- 2.9, ranged from 2 to 12, and in all cases examined (n = 11) decreased with prolonged tetanic stimulation. The rates of the decrease of end-plate potentials amplitudes (and quantal contents) from different segments of the same nerve terminal were often different, even when they were initially comparable. This suggests that some active zones or some areas of the end-plates become depleted much faster than others. Quantal sizes of the nerve evoked and the spontaneously released quanta were generally similar at low frequencies of stimulation (0.5-2 Hz). Both decreased with high frequency stimulation, but the decrease of the quantal sizes of nerve evoked quanta was usually more pronounced. At different loci of the same end-plate the contribution of lower quantal size to the synaptic depression varied widely (from < 5% to > 80%). In conclusion lower quantal size can contribute significantly to synaptic depression. At uneven decrease of quantal sizes over the whole nerve terminal helps to explain both aspects of synaptic depression (lower synaptic efficacy and greater variability of quantal responses.

Animals↗

PubMed1992

Choline blockage of currents through Ca(2+)-activated K+ channels in bovine chromaffin cells.

The action of choline on "maxi" Ca(2+)-activated K+ channels was studied in excised patches of bovine chromaffin cell membranes. Choline (20-70 mM) applied to the internal surface of the membrane reduced the single channel current amplitudes, which can be explained by a fast channel block. The block is concentration- and voltage-dependent and is rapidly and completely reversed upon washout. The block becomes progressively greater with depolarization. The estimates of blocking parameters vary from channel to channel but appear to fall in two groups. A larger group (two-thirds of cases) with moderate affinity [KD(0) = 88.5 mM] and low voltage dependence (delta = 0.26) and a smaller group (one-third of cases) with very low affinity (KD = 306 mM) and moderate voltage dependence (delta = 0.59). The open state probability appears not to be affected at any choline concentration (up to 70 mM) or membrane potential (from -20 to +60 mV) studied, suggesting that choline does not affect the channel gating kinetics. Since the affinity of the choline block is low to moderate, the intracellular choline is not expected to alter the current flow through "maxi" Ca(2+)-activated K+ channels unless the choline concentration close to the protoplasmic membrane is much higher than the mean cellular concentration.

Adrenal Medulla↗

PubMed1992

Mastoparan blockade of currents through Ca(2+)-activated K+ channels in bovine chromaffin cells.

The action of mastoparan (a wasp venom peptide) on "maxi" Ca(2+)-activated K+ channels was studied in excised inside-out patch recordings from cultured bovine chromaffin cells, under normal conditions (160 mM K+ inside, 154 mM Na+ outside). Mastoparan, when applied on the intracellular side of the membrane reduced the open channel probability in a concentration dependent manner. Changes in the channel kinetics were complex. The histograms of the open dwell times were all described by either one or two exponentials. Mastoparan shortened the mean duration of the major (long) component and to a lesser extent the minor (short) component. Closed dwell times, were described by three exponentials. While the short (major) component was prolonged by mastoparan, and the intermediate component was unaffected, the long component was shortened. Overall mean closed times were prolonged. The changes in channel kinetics could only partly be explained by a channel-blocking mechanism, even when assuming that mastoparan acts as both an intermediate and a slow channel blocker suggesting that it affects gating mechanism. The fact that mastoparan is a calmodulin inhibitor and a G-protein activator raises the possibility that in bovine chromaffin cells, either the membrane-bound calmodulin or a G-protein, plays a role in the modulation of Ca(2+)-activated K+ channels.

Animals↗

PubMed1990

Effect of acetylcholine on single Ca2(+)-activated K+ channels in bovine chromaffin cells.

The single channel current amplitudes of "maxi" Ca2(+)-activated K+ channels from bovine chromaffin cell membranes are reduced when acetylcholine is applied to the internal surface of the membrane, which can be explained by a fast channel block. The block is concentration dependent with moderate affinity. It becomes progressively greater with depolarization although the voltage dependence is not pronounced. Acetylcholine reduces the probability of the open state in the same concentration range and in an essentially voltage independent manner. The changes in the channel kinetics are complex. Whilst the long component of the open intervals is shortened (by 48%; from 9.5 to 5.1 ms), the long component of the closed intervals is prolonged (by 96%; from 45 to 89 ms). The short components (open and closed) are essentially unaffected. Short open intervals are reduced by 4% (from 1.09 to 1.05 ms), whilst short closed intervals are reduced by 5% (from 2.3 to 2.2 ms). These changes in the channel kinetics can be explained at least partly if one assumes that acetylcholine, in addition to its fast channel blocking activity, acts also as a slow blocker. If so, both binding sites are expected to be located close to the mouth of the channel pore. Alternatively, acetylcholine may be affecting the gating mechanism, presumably by interfering with the Ca2+ binding.

Acetylcholine↗

PubMed1989

Reappearance of miniature endplate potentials in frog neuromuscular junctions "silenced" by lanthanum ions.

Miniature endplate potentials and currents were recorded from frog sartorius muscle fibres. As expected from previous work, La3+ caused a very large increase in miniature endplate potential frequency, followed by a progressive decrease, ending in the virtually complete disappearance of miniature endplate potentials. Soon after its application, La3+ caused an increase in the amplitude of miniature endplate potentials due to a lengthening of the underlying miniature endplate currents. Analysis of membrane current fluctuations produced by acetylcholine showed that La3+ caused an increase in the lifetime of the channels opened by acetylcholine. After miniature endplate potentials had vanished following La3+ treatment, the muscles were placed in organ culture conditions to see if miniature endplate potentials would reappear. After several hours miniature endplate potential activity was resumed in about 25% of the endplates, and disappeared again a few hours later, presumably because of accelerated nerve terminal degeneration. Preliminary electron microscopic observations suggest that the recovery of miniature endplate potential activity was accompanied by re-formation of synaptic vesicles in the motor endings.

Animals↗

PubMed1988

Changes in miniature end-plate currents due to high potassium and calcium at the frog neuromuscular junction.

Elevation of extracellular potassium concentration ([K+]o) in cutaneous pectoris neuromuscular junction from 2 to 20 mM slowly increased the variability of the amplitudes of miniature end-plate currents (AMEPC-s), (coefficient of variation of AMEPC-s increased by 73%). Mean AMEPC-s, however, decreased but not markedly (by 14%). Comparable MEPC changes were observed when [K+] was raised in the presence of choline chloride (50 microM), arguing that MEPC changes were not primarily due to a lower and less uniform vesicular filling. Channel kinetics were not altered by high [K+]o, since the time constant of decay of miniature end-plate currents (TMEPC-s) did not change. Acetylcholine clearance from the synaptic cleft, however, appeared to be faster in high [K+]o since with cholinesterase blocked throughout, TMEPC-s were shortened. The changes of spontaneous quantal discharge induced by high [K+]o can be almost entirely explained by altered spatial distribution of vesicular release if, as recent reports suggest, at high [K+]o, exocytosis appears randomly not only at but also in between the active zones. However, relatively greater frequency of large MEPCs suggests that in high [K+]o some, and possibly all, quanta are filled above normal levels. High [Ca2+]o appears to counteract, although not always completely, all changes in spontaneous quantal secretion induced by high [K+]o. It is possible that high [Ca2+]o reverses the changes in the spatial distribution of vesicular release induced by high [K+]o. However, high [Ca2+]o also leads to other pre- and postsynaptic changes.

Action Potentials↗

PubMed1988

Depression, recovery and facilitation of neuromuscular transmission during prolonged tetanic stimulation.

The phrenic nerve of an unparalysed "cut" rat diaphragm preparation was stimulated with a rapid sequence of short tetanic trains. The amplitudes of both the first and the last (25th) endplate potentials produced by short tetanic trains progressively decreased with repeated application, but the latter diminished faster. Both relative depression and relative recovery (defined as fractional decrease and fractional recovery of endplate potential amplitudes that occur during and after each short train) became more pronounced with time. Relative facilitation (defined as fractional increase of endplate potential amplitudes observed at the beginning of each train) when initially present, persisted or increased slightly with duration of stimulation. Present results suggest that the "classic" depletion model for depression ought to be modified. They can be explained if it is assumed that during stimulation the capacity of the immediately available store to contain transmitter increases, and/or that newly formed transmitter preferentially replenishes the store of quanta immediately available for release. (An interesting consequence of the former model is that the immediately available store is replenished even when the small store behind is relatively more depleted.

Algorithms↗

PubMed1988

Effect of blockade of choline uptake by hemicholinium-3 on synaptic output.

The decrease in amplitude of endplate potentials that occurs with high frequency nerve stimulation at the rat phrenic-diaphragm preparation is greater after blockade of choline uptake by hemicholinium-3. The effect is mainly presynaptic and occurs after presumably only a small fraction of the total number of releasable quanta has been discharged. Moreover, when the phrenic nerve is stimulated with a rapid sequence of short tetanic trains, the decrease of the amplitude of both the "first" and the "last" endplate potential of each train which is usually monoexponential becomes not only greater but also biphasic. The effect on the "first" endplate potentials is particularly large. This can be interpreted as further evidence that newly synthesized acetylcholine preferentially replenishes the immediately available store of quanta released by nerve stimulation.

Acetylcholine↗

PubMed1988

Changes in the synaptic cleft potassium concentration produced by polarizing currents.

How much the cleft K+ concentration ([K+]c) is changed by a step-like shift of an endplate membrane potential was estimated from the resulting acceleration of frequency of miniature endplate currents and the dependence of miniature endplate currents frequency on bath K+ concentration ([K+]0). The absolute change in [K+]c is generally constant, and hence relatively more pronounced the lower the [K+]0. It is estimated that in physiological solution [( K+]0 = 2 mM), [K+]c is at least tripled (approximately 6 mM) if the muscle membrane is held at the reversal potential (Vr) for the endplate currents. The same step-like shift in muscle membrane potential leads to an almost instantaneous change of endplate current amplitudes or of their time constants of decay. This indicates that cleft K+ changes have no effect on the voltage dependence of amplitudes and decay phases of endplate currents (and hence on Vr also). The lack of postsynaptic effect of change in [K+]0 from 0.1 to 10 mM and only small influence of [K+]0 changes on nerve-evoked quantal release explain these findings.

Animals↗

PubMed1988

Quinine blockade of currents through Ca2+-activated K+ channels in bovine chromaffin cells.

1. The blocking action of quinine of Ca2+-activated K+ channels was studied in excised inside-out patch recordings from cultured bovine chromaffin cells. Under normal ionic conditions (160 mM-K+ inside, 154 mM-Na+ outside), the unitary conductances measured between -20 and +40 mV were between 95 and 210 pS. 2. Quinine applied on the intracellular side of the membrane in micromolar concentrations chopped the unitary K+ currents into bursts of brief openings. These 'flickery bursts' appear to arise from discrete blocking and unblocking transitions. 3. Open and closed times within such flickery bursts were exponentially distributed suggesting a simple reaction between quinine and an open channel. 4. The blocking rate varied linearly with the concentration of quinine (rate coefficient 1.6 x 10(6) M-1 s-1). The unblocking rate was high (greater than 5000 s-1) and essentially independent of the quinine concentration. 5. The blocking rate was voltage dependent and increased with hyperpolarization which suggests that quinine acts predominantly or exclusively from the extracellular side of the membrane. The unblocking rate appeared to be independent of the membrane potential. 6. The probability of the open state was not altered by quinine indicating that quinine did not interfere directly with the gating process. 7. The total current carried by K+ ions was reduced by quinine only marginally (less than 10%) even at high quinine concentrations (1.5 mM). Moreover the reduction of the total current was also essentially voltage independent. 8. Quinine is clearly a very poor blocker. Poor block occurs, however, not only because of the brevity of the closed periods but presumably also because the channel can be closed only when it is not blocked. Only when the probability of channel opening (p) was high (greater than 0.85), i.e. when the channel's gate was predominantly open, did the flickery block appear to reduce the total current appreciably.

Adrenal Medulla↗