Interaction of intracellular ion buffering with transmembrane-coupled ion transport

The role of the Na/Ca exchanger in the control of cellular excitability and tension development is a subject of current interest in cardiac physiology. It has been suggested that this coupled transporter is responsible for rapid changes in intracellular calcium activity during single beats, generation of plateau currents, which control action potential duration, and control of intracellular sodium during Na/K pump suppression, which may occur during terminal states of ischemia. The actual behavior of this exchanger is likely to be complex for several reasons. First, the exchanger transports two ionic species and thus its instantaneous flux rate depends on both intracellular sodium and calcium activity. Secondly, the alteration in intracellular calcium activity, which is caused by a given transmembrane calcium flux, and which controls the subsequent exchanger rate, is a complex function of available intracellular calcium buffering. The buffers convert the ongoing transmembrane calcium fluxes into changes in activity that are a small and variable fraction of the change in total calcium concentration. Using a number of simple assumptions, we model changes in intracellular calcium and sodium concentration under the influence of Na/Ca exchange, Na/K ATPase and Ca-ATPase pumps, and passive sodium and calcium currents during periods of suppression and reactivation of the Na/K ATPase pump. The goal is to see whether and to what extent general notions of the role of the Na/Ca exchanger used in planning and interpreting experimental studies are consistent with its function as derived from current mechanistic assumptions about the exchanger. We find, for example, that based on even very high estimates of intracellular calcium buffering, it is unlikely that Na/Ca exchange alone can control intracellular sodium during prolonged Na/K pump blockade. It is also shown that Na/Ca exchange can contaminate measurements of Na/K pump currents under a variety of experimental conditions. The way in which these and other functions are affected by the dissociation constants and total capacity of the intracellular calcium buffers are also explored in detail.     

Synaptic vesicle recruitment for release explored by Monte Carlo stimulation at the crayfish neuromuscular junction

Neurotransmission at chemically transmitting synapses requires calcium-mediated fusion of synaptic vesicles with the presynaptic membrane. Utilizing ultrastructural information available for the crustacean excitatory neuromuscular junction, we developed a model that employs the Monte Carlo simulation technique to follow the entry and movement of Ca2+ ions at a presynaptic active zone, where synaptic vesicles are preferentially docked for release. The model includes interaction of Ca2+ with an intracellular buffer, and variable separation between calcium channels and vesicle-associated Ca(2+)-binding targets that react with Ca2+ to trigger vesicle fusion. The end point for vesicle recruitment for release was binding of four Ca2+ ions to the target controlling release. The results of the modeling experiments showed that intracellular structures that interfere with Ca2+ diffusion (in particular synaptic vesicles) influence recruitment or priming of vesicles for release. Vesicular recruitment is strongly influenced by the separation distance between an opened calcium channel and the target controlling release, and by the concentration and binding properties of the intracellular buffers, as in previous models. When a single opened calcium channel is very close to the target, a single synaptic vesicle can be recruited. However, many of the single-channel openings actuated by a nerve impulse are likely to be ineffective for release, although they contribute to the buildup of total intracellular Ca2+. Thus, the overall effectiveness of single calcium channels in causing vesicles to undergo exocytosis is likely quite low.     

Neuronal calcium stores

Neuronal calcium stores associated with specialized intracellular organelles, such as endoplasmic reticulum and mitochondria, dynamically participate in generation of cytoplasmic calcium signals which accompany neuronal activity. They fulfil a dual role in neuronal Ca2+ homeostasis being involved in both buffering the excess of Ca2+ entering the cytoplasm through plasmalemmal channels and providing an intracellular source for Ca2+. Increase of Ca2+ content within the stores regulates the availability and magnitude of intracellular calcium release, thereby providing a mechanism which couples the neuronal activity with functional state of intracellular Ca2+ stores. Apart of 'classical' calcium stores (endoplasmic reticulum and mitochondria) other organelles (e.g. nuclear envelope and neurotransmitter vesicles) may potentially act as a functional Ca2+ storage compartments. Calcium ions released from internal stores participate in many neuronal functions, and might be primarily involved in regulation of various aspects of neuronal plasticity.     

The effect of calcium ions on the secretion of quanta evoked by an impulse at nerve terminal release sites

A study has been made of the effects of calcium ions on the number of quanta secreted from all the release sites at an amphibian motor nerve terminal recorded with an intracellular microelectrode (m) compared with the number secreted simultaneously from a small number of release sites recorded with an extracellular microelectrode (me). If the endplate potential was made subthreshold by lowering the external calcium concentration ([Ca]o less than or equal to 0.4 mM), it was possible to find small groups of release sites for which me was comparable to m, indicating considerable nonuniformity in the probability of release of a quantum at different groups of release sites (Pe) in a given [Ca]o. Increasing [Ca]o in the range from 0.25 to 0.4 mM increased the probability of release of a quantum at groups of release sites (Pe), independent of the initial value of Pe, and the dependence of Pe on [Ca]o followed a fourth power relationship. A conditioning impulse enhanced the probability of release of a quantum by a subsequent test impulse at release sites, if Pe was less than 1.0 during the conditioning impulse. It is shown that the present observations regarding the dependence of Pe on [Ca]o and on conditioning impulses can be quantitatively predicted from previous observations regarding the dependence of the binomial parameters m, p, and n on [Ca]o and on conditioning impulses determined with intracellular electrodes, if the probability of secretion of a quantum at a release site (Pj) is different for different release sites and Pj is distributed as a beta random variable.     

Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration

Our aim was to measure the influence of sarcoplasmic reticulum (SR) calcium content ([Ca](SRT)) and free SR [Ca] ([Ca](SR)) on the fraction of SR calcium released during voltage clamp steps in isolated rabbit ventricular myocytes. [Ca](SRT), as measured by caffeine application, was progressively increased by conditioning pulses. Sodium was absent in both the intracellular and in the extracellular solutions to block sodium/calcium exchange. Total cytosolic calcium flux during the transient was inferred from I(Ca), [Ca](SRT), [Ca](i), and cellular buffering characteristics. Fluxes via the calcium current (I(Ca)), the SR calcium pump, and passive leak from the SR were evaluated to determine SR calcium release flux (J(rel)). Excitation-contraction (EC) coupling was characterized with respect to both gain (integral J(rel)/integral I(Ca)) and fractional SR calcium release. Both parameters were virtually zero for a small, but measurable [Ca](SRT). Gain and fractional SR calcium release increased steeply and nonlinearly with both [Ca](SRT) and [Ca](SR). We conclude that potentiation of EC coupling can be correlated with both [Ca](SRT) and [Ca](SR). While fractional SR calcium release was not linearly dependent upon [Ca](SR), intra-SR calcium may play a crucial role in regulating the SR calcium release process.     

Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.     

Analytical calculation of intracellular calcium wave characteristics

We present a theoretical analysis of intracellular calcium waves propagated by calcium feedback at the inositol 1,4,5-trisphosphate (IP3) receptor. The model includes essential features of calcium excitability, but is still analytically tractable. Formulas are derived for the wave speed, amplitude, and width. The calculations take into account cytoplasmic Ca buffering, the punctate nature of the Ca release channels, channel inactivation, and Ca pumping. For relatively fast buffers, the wave speed is well approximated by V(infinity) = (J(eff)D(eff)/C0)1/2, where J(eff) is an effective, buffered source strength; D(eff) is the effective, buffered diffusion constant of Ca; and C(0) is the Ca threshold for channel activation. It is found that the saturability and finite on-rate of buffers must be taken into account to accurately derive the wave speed and front width. The time scale governing Ca wave propagation is T(r), the time for Ca release to reach threshold to activate further release. Because IP3 receptor inactivation is slow on this time scale, channel inactivation does not affect the wave speed. However, inactivation competes with Ca removal to limit wave height and front length, and for biological parameter ranges, it is inactivation that determines these parameters. Channel discreteness introduces only small corrections to wave speed relative to a model in which Ca is released uniformly from the surface of the stores. These calculations successfully predict experimental results from basic channel and cell parameters and explain the slowing of waves by exogenous buffers.     

Differences in presynaptic action of 4-aminopyridine and tetraethylammonium at frog neuromuscular junction

4-Aminopyridine markedly potentiates transmitter release at the frog pectoris neuromuscular junction by increasing the quantal content even when applied at low concentrations (5-20 microM). This enhancement of transmitter release is associated with greater minimum synaptic latency, but the dispersion of the synaptic latencies does not appear much affected. This is in contrast with the action of tetraethylammonium (0.2-0.5 mM) in which case similar enhancement of transmitter release results not only in larger minimum synaptic latency but also in greater dispersion of the synaptic latencies. The time course of transmitter release associated with enhanced transmitter output is hence much more prolonged in the presence of tetraethylammonium than 4-aminopyridine, at least for low concentrations of 4-aminopyridine (5-20 microM). This indicates that their presynaptic actions differ significantly. This conclusion is further strengthened by the finding that unlike tetraethylammonium, 4-aminopyridine induces bursts of release, presumably by producing multiple action potentials in the nerve terminal. Tetraethylammonium probably acts by blocking the delayed potassium conductance, but the blockade of Ca2+-activated K+ conductance cannot be excluded. 4-Aminopyridine, however, probably blocks the fast inactivating (IA) K+ current, but it also may be acting directly on the voltage-dependent Ca2+ conductance or on the intracellular Ca2+ buffering.     

Immunoglobulin E receptor cross-linking induces oscillations in intracellular free ionized calcium in individual tumor mast cells

Fura-2 fluorescence in single rat basophilic leukemia cells was monitored to study the rise in intracellular free ionized calcium ([Ca2+]i) produced by aggregation of immunoglobulin E receptors. Repetitive transient increases in [Ca2+]i were induced by antigen stimulation and were measured using digital video imaging microscopy at high time resolution. The [Ca2+]i oscillations were not dependent upon changes in the membrane potential of the cells and were observed in cells stimulated with antigen either with or without extracellular Ca2+. Transient oscillations in [Ca2+]i were also observed when calcium influx was blocked with La3+. These results suggested that during antigen stimulation of cells under normal physiological conditions, release of Ca2+ from intracellular stores makes an important contribution to the initial increase in [Ca2+]i. Oscillations in [Ca2+]i are not induced by elevating [Ca2+]i with the calcium ionophore ionomycin. Mitochondrial calcium buffering is not required for [Ca2+]i oscillations to occur. The results show that rat basophilic leukemia cells have significant stores of calcium and that release of calcium from these stores can participate in both the initial rise and the oscillations in [Ca2+]i.     

Ways to discern the presynaptic effect of drugs on neurotransmitter release

This study is based on the authors' theory for neurotransmitter release in which explicit recognition is given to the generally accepted hypothesis that intracellular calcium ions (Ca) control the release. The new theory considers the resting level of intracellular Ca and proposes a saturating equation for the entry of Ca into the nerve terminals. The main purpose of the present paper is, by applying the new theory, to suggest experimental methods for discerning the various possible effects of drugs on release. New interpretations are given of log-log graphs of release as a function of extracellular Ca concentration. Maximal facilitation and the duration of facilitation are two other measurements that are shown to be particularly useful in determining the nature of drug action.     

Model of calcium movements in the mammalian myocardium: interval-strength relationship

A model is proposed to describe the interval-strength relationship in mammalian cardiac muscle in terms of "discrete" calcium movements associated with each cycle. The sarcoplasmic reticulum is assumed to be comprised of three functional sub-compartments: (1) The "main calcium store" which contains most of the calcium (predominantly bound) and is considered, due to its large buffering capacity, to account for the "long-term memory" lasting 7-10 beats. (2) The "releasable terminal" which contains the calcium readily available for release (all or most of it free) and accounts for the "short-term memory" which affects the subsequent beat. (3) The longitudinal network of the SR recirculating the myofibrillar calcium to the "main calcium store". The total content of calcium in the main store is determined by the transsarcolemmal influx and efflux. While influx occurs only during depolarization, efflux occurs during the whole cardiac cycle. The amount of free calcium in the main store is determined by an equilibrium equation. The release of calcium from the "releasable terminal" is governed by a "concentration-dependent" mechanism. This implies that when the concentration in the "releasable terminal" increases, the fraction released increases and the residual calcium left for the subsequent contraction decreases. The model predicts the following interval-strength relationships: steady state peak tension; changes from one steady rate to another; restitution curves; post-stimulation potentiation; paired stimulation; premature beats; post-extrasystolic potentiation following interpolated, basal or complimentary interval.     

Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibers.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.     

Theoretical analysis of Ca wave propagation along the surface of intracellular stores

A mathematical model is described that accounts propagating waves of free cytoplasmic Ca arising from the activation of single Ca release channels. The [Ca] wave moves along the surface of intracellular stores and is supported by the subsequent activation of neighbouring Ca release channels. The model considers both activation and inactivation of the channels and the buffering of excess Ca in the cytoplasm. This non-dissipating wave of Ca concentration is shown to exist only for a certain range of the single channel conductance and the rate of Ca buffering in a cytoplasm. The wave velocity depends also on the other model parameters and generally comprises the values 1-300 microns sec-1. Data obtained are used to discuss the possibility of the delivery of free Ca concentration pulse from the surface membrane to a given point of the cell interior.     

Hormone-induced calcium oscillations in liver cells can be explained by a simple one pool model

Hormone-induced oscillations of the free intracellular calcium concentration are thought to be relevant for frequency encoding of hormone signals. In liver cells, such Ca2+ oscillations occur in response to stimulation by hormones acting via phosphoinositide breakdown. This observation may be explained by cooperative, positive feedback of Ca2+ on its own release from one inositol 1,4,5-trisphosphate-sensitive pool, obviating oscillations of inositol 1,4,5-trisphosphate. The kinetic rate laws of the associated model have a mathematical structure reminiscent of the Brusselator, a hypothetical chemical model involving a rather improbable trimolecular reaction step, thus giving a realistic biological interpretation to this hallmark of dissipative structures. We propose that calmodulin is involved in mediating this cooperativity and positive feedback, as suggested by the presented experiments. For one, hormone-induced calcium oscillations can be inhibited by the (nonphenothiazine) calmodulin antagonists calmidazolium or CGS 9343 B. Alternatively, in cells overstimulated by hormone, as characterized by a non-oscillatory elevated Ca2+ concentration, these antagonists could again restore sustained calcium oscillations. The experimental observations, including modulation of the oscillations by extracellular calcium, were in qualitative agreement with the predictions of our mathematical model.     

Oscillation period of MEPP frequency at frog neuromuscular junctions is inversely correlated with release efficacy and independent of acute Ca2+ loading

Periodic oscillations in miniature endplate potential (MEPP) frequency have been described at the frog neuromuscular junction. It is assumed that the periodic oscillations in MEPP frequency reflect cytosolic oscillations in intracellular Ca2+ concentration. In the course of a study related to describing the differences between weak and strong neuromuscular junctions by using the post-tetanic potentiation of MEPP frequency, we noted periodic oscillations in MEPP frequency in the first few minutes after a tetanus. The period of this oscillation (i.e. the time interval of one complete oscillation cycle) was inversely related to synaptic release efficacy, as measured by quantal content released per 100 microns of nerve terminal length. Junctions of high release efficacy have an oscillation period of 20 s or less whereas the oscillations in weaker junctions have periods of up to 60 s or longer. This relation is very similar during post-tetanic recovery in either a calcium containing Ringer solution or in a zero calcium-EGTA Ringer solution, indicating that external calcium is not necessary to express the phenomenon. We also found that the oscillations are apparent in resting junctions preceding a tetanus and that they are similar in period and show the same inverse relation to synaptic strength.     

Calcium buffering and clearance in spider mechanosensory neurons

Spider VS-3 mechanoreceptor neurons have a low-voltage-activated Ca2+ current that raises intracellular calcium concentration [Ca2+] when they are depolarized by agonists of GABAA receptors or fire action potentials. The Ca2+ rise produces negative feedback by modulating the mechanoreceptor current and regulates Ca2+- and voltage-activated K+ currents. However, nothing is known about Ca2+ buffering in VS-3 neurons. Dynamic changes in VS-3 neuron intracellular [Ca2+] were measured using the fluorescent Ca2+ indicator Oregon Green BAPTA-1 (OG488) to understand Ca2+ buffering and clearance. Intracellular OG488 concentration increased slowly over more than 2 h as it diffused through a sharp intracellular microelectrode and spread through the cell. This slow increase was used to measure endogenous Ca2+ buffering and clearance by the added buffer technique, with OG488 acting as both added exogenous buffer and Ca2+ indicator. [Ca2+] was raised for brief periods by regular action potential firing, produced by pulsed electric current injection through the microelectrode. The resulting rise and fall of [Ca2+] were well fitted by the single compartment model of Ca2+ dynamics. With earlier ratiometric [Ca2+] estimates, these data gave an endogenous Ca2+ binding ratio of 684. Strong Ca2+ buffering may assist these neurons to deal with rapid changes in mechanical inputs.     

Investigations of calcium homeostasis mechanisms in nerve cells and their alterations during brain pathology

The paper summarises new data about the molecular mechanisms of calcium homeostasis maintenance in nerve cells and generation of intracellular calcium transients--the most general secondary messenger triggering or modulating all steps of neuronal life cycle and its main functions. It describes the low- and high-voltage activated plasmalemmal ion channels injecting Ca2+ into the cell, cytosolic buffering systems which rapidly bind the main part of injected ions, properties of intracellular stores accumulating Ca2+ ions due to the activity of CERCA-pumps and releasing them back into the cytosol via the CICR mechanism, possible participation of mitochondria in this process, extrusion of Ca2+ from the cell by PMCA-pumps. By introducing new techniques, quantitative characteristics are obtained of these mechanisms and of their participation in determining the amplitude and kinetics of calcium signals in different neurons, as well as their changes during ageing and some forms of brain pathology.     

Cooperative Ca2+ removal from presynaptic terminals of the spiny lobster neuromuscular junction

Stimulation-induced changes in presynaptic free calcium concentration ([Ca2+]i) were examined by fluorescent imaging at the spiny lobster excitor motor nerve terminals. The Ca2+ removal process in the terminal was analyzed based on a single compartment model, under the assumption that the Ca2+ removal rate from the terminal cytoplasm is proportional to nth power of [Ca2+]i. During 100 nerve stimuli at 10-100 Hz, [Ca2+]i reached a plateau that increased in a less-than-linear way with stimulation frequency, and the power index, n, was about 2. In the decay time course after stimulation, n changed with the number of stimuli from about 1.4 after 10 stimuli to about 2 after 100 stimuli. With the change of n from 1.4 to 2, the rate became larger at high [Ca2+]i (>1.5 microM), but was smaller at low [Ca2+]i (<1 microM). These results suggest that a cooperative Ca2+ removal mechanism of n = 2, such as mitochondria, may play an important role in the terminal. This view is supported by the gradual increase in the [Ca2+]i plateau during long-term stimulation at 20-50 Hz for 60 s and by the existence of a very slow [Ca2+]i recovery process after this stimulation, both of which may be due to accumulation of Ca2+ in the organelle.     

Ca2+ clearance mechanisms in neurohypophysial terminals of the rat

The importance of intracellular calcium ([Ca2+]i) in the release of vasopressin (AVP) and oxytocin from the central nervous system neurohypopyhysial nerve terminals has been well-documented. To date, there is no clear understanding of Ca2+ clearance mechanisms and their interplay with transmembrane Ca2+ entry, intracellular [Ca2+]i transients, cytoplasmic Ca2+ stores and hence the release of AVP at the level of a single nerve terminal. Here, we studied the mechanism of Ca2+ clearance in freshly isolated nerve terminals of the rat neurohypophysis using Fura-2 Ca2+ imaging and measured the release of AVP by radioimmuno assay. An increase in the K+ concentration in the perfusion solution from 5 to 50 mM caused a rapid increase in [Ca2+]i and AVP release. Returning K+ concentration to 5 mM led to rapid restoration of both responses to basal level. The K+-evoked [Ca2+]i and AVP increase was concentration-dependent, reliable, and remained of constant amplitude and time course upon successive applications. Extracellular Ca2+ removal completely abolished the K+-evoked responses. The recovery phase was not affected upon replacement of NaCl with sucrose or drugs known to act on intracellular Ca2+ stores such as thapsigargin, cyclopiazonic acid, caffeine or a combination of caffeine and ryanodine did not affect either resting or K+-evoked [Ca2+]i or AVP release. By contrast, the plasma membrane Ca2+ pump inhibitor, La3+, markedly slowed down the recovery phase. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), slightly but significantly increased the basal [Ca2+]i, and also slowed down the recovery phase of both [Ca2+]i and release responses. In conclusion, we show in nerve terminals that (i) Ca2+ extrusion through the Ca2+ pump in the plasma membrane plays a major role in the Ca2+ clearance mechanisms of (ii) Ca2+ uptake by mitochondria also contributes to the Ca2+ clearance and (iii) neither Na+/Ca2+ exchangers nor Ca2+ stores are involved in the Ca2+ clearance or in the maintenance of basal [Ca2+]i or release of AVP.