Involvement of phospholipase A2 and arachidonic acid in the depolarization-evoked accumulation of Ca2+ in hippocampal mossy fiber nerve endings

Depolarization-evoked increases in intraterminal free Ca²⁺ are required for the induction of neurotransmitter release from nerve terminals. Although the mechanisms that regulate the voltage-induced accumulation of presynaptic Ca²⁺ remain obscure, there is evidence that the phospholipase-dependent accumulation of arachidonic acid, or its metabolites, may be involved. Therefore, fura-2 loaded hippocampal mossy fiber nerve endings were used to investigate the relationships between membrane depolarization, lipid metabolism and presynaptic Ca²⁺ availability. It was observed that depolarization of the nerve terminals with KCl induced an increase in intraterminal free calcium that was inhibited more than 90% by a combination of voltage-sensitive Ca²⁺ channel blockers. In addition, the K⁺-dependent effects on Ca²⁺ concentrations were attenuated in the presence of phospholipase A₂ inhibitors, but were mimicked by the phospholipase A₂ activator melittin and exogenous arachidonic acid. Both the melittin- and arachidonic acid-induced increases in presynaptic Ca²⁺ were reduced by voltage-sensitive Ca²⁺ channel blockers. The stimulatory effects of arachidonic acid appeared to be independent of its further metabolism to prostaglandins. In fact, inhibition of either cyclooxygenase or lipoxygenase pathways resulted in a potentiation of the depolarization-evoked increase in intraterminal free Ca²⁺. From these results, we propose that some portion of the depolarization-evoked increase in intraterminal free calcium depends on the activation of phospholipase A₂ and the subsequent accumulation of unesterified arachidonic acid.     

A Ca2+-induced Ca2+ Release Mechanism Involved in Asynchronous Exocytosis at Frog Motor Nerve Terminals

The extent to which Ca²⁺-induced Ca²⁺ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20–50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca²⁺ ([Ca²⁺]_i) in presynaptic terminals (Ca²⁺-hump) in frog skeletal muscles over a period of minutes in a low Ca²⁺, high Mg²⁺ solution. Mn²⁺ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca²⁺ channels. MEPP-hump depended on extracellular Ca²⁺ (0.05–0.2 mM) and stimulation frequency. Both the Ca²⁺- and MEPP-humps were blocked by 8-(N,N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN⁻. Thus, Ca²⁺-hump is generated by the activation of CICR via ryanodine receptors by Ca²⁺ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca²⁺ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca²⁺-hump affected little MEPP frequency or [Ca²⁺]_i, but caused a quick increase (faster than MEPP- or Ca²⁺-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca²⁺ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca²⁺ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca²⁺ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.     

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca²⁺ release through ryanodine receptors. In the present study we aimed at further understanding how Ca²⁺ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca²⁺ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca²⁺ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca²⁺ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca²⁺ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca²⁺ sparks while the rest take either the form of lower amplitude, longer duration Ca²⁺ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca²⁺ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca²⁺-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.     

Lipid-Dependent Modulation of Ca2+ Availability in Isolated Mossy Fiber Nerve Endings

An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca²⁺, since exocytosis requires Ca²⁺ and a potentiation of Ca²⁺ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca²⁺ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca²⁺ accumulation was reduced by inhibitors of phospholipase A₂ and voltage-sensitive Ca²⁺ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A₂ was also indicated by the ability of staurosporine to reduce the Ca²⁺ accumulation induced by arachidonic acid or the phospholipase A₂ activator melittin. Similarly, the synergistic facilitation of evoked Ca²⁺ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca²⁺ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes.     

Suppression of Ca2+ syntillas increases spontaneous exocytosis in mouse adrenal chromaffin cells

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca²⁺] in the vicinity of plasmalemmal Ca²⁺ channels due to Ca²⁺ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca²⁺] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca²⁺ syntillas. Ca²⁺ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca²⁺ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca²⁺ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca²⁺: one due to influx from the cell exterior through voltage-gated Ca²⁺ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca²⁺ influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca²⁺ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.     

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca²⁺ is a central step in the process of Ca²⁺-induced Ca²⁺ release, but the molecular basis of RyR2 activation by cytosolic Ca²⁺ is poorly defined. It has been proposed recently that the putative Ca²⁺ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca²⁺ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca²⁺ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca²⁺-dependent activation of [³H]ryanodine binding or the cytosolic Ca²⁺ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca²⁺ activation of RyR2 and spontaneous Ca²⁺ release in HEK293 cells during store Ca²⁺ overload or store overload-induced Ca²⁺ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca²⁺, it plays an important role in luminal Ca²⁺ activation and SOICR.     

Ca2+-induced Ca2+ Release Phenomena in Mammalian Sympathetic Neurons Are Critically Dependent on the Rate of Rise of Trigger Ca2+

The role of ryanodine-sensitive intracellular Ca²⁺ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced Ca²⁺ release in individual fura-2–loaded sympathetic neurons. Two ryanodine-sensitive release components were distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR component shows refractoriness, depends on the filling status of the store, and requires caffeine concentrations ≥10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca²⁺-mediated feedback loops, suggesting that it constitutes a Ca²⁺-induced Ca²⁺-release phenomenon. The dynamics of release is markedly affected when Sr²⁺ substitutes for Ca²⁺, indicating that Sr²⁺ release may operate with lower feedback gain than Ca²⁺ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca²⁺ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca²⁺ and Ca²⁺-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses: When the Ca²⁺ reservoir is full, the rapid initial Ca²⁺ rise determines a faster occupation of the ryanodine receptor Ca²⁺ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial Ca²⁺ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating site, thereby suppressing the initial fast release. The PR component is less dependent on the store''s Ca²⁺ content. This study suggests that transmembrane Ca²⁺ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca²⁺] near the Ca²⁺ release channels sufficiently fast to overcome their Ca²⁺-dependent inactivation. Conversely, caffeine-induced Ca²⁺ release can undergo considerable amplification especially when Ca²⁺ stores are full. We propose that the primary function of ryanodine-sensitive stores in neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca²⁺ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on the filling status of intracellular Ca²⁺ stores.     

The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle

In vascular smooth muscle cells, Ca²⁺ release via IP₃ receptors (IP₃R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca²⁺ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca²⁺ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca²⁺ store is a single continuous interconnected network or multiple separate Ca²⁺ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca²⁺ available throughout the store was examined using localized photolysis of caged-IP₃ and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP₃R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP₃R and RyR was regulated by the Ca²⁺ concentration within the SR (luminal [Ca²⁺]). As the luminal [Ca²⁺] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP₃R. The SR Ca²⁺ store is a single luminally continuous entity which contains both IP₃R and RyR and within which Ca²⁺ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP₃R and RyR by luminal [Ca²⁺] explains the appearance of multiple stores in some functional studies.     

Miro1‐dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca²⁺ indicator, SyGCaMP5, to address whether presynaptic Ca²⁺ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca²⁺ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca²⁺‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca²⁺ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.     

Parallel computation enables precise description of Ca2+ distribution in nerve terminals

Parallel computation employing a domain decomposition method was used to calculate precisely without approximations the spatio-temporal distribution of Ca²⁺ in nerve terminals. The results showed, contrary to expectations, that for equal admitted Ca²⁺ currents at low (one channel open) and high (four channels open) depolarization, the average Ca²⁺ concentration at the release area is higher at the low depolarization. These calculations provide additional support for the Ca²⁺-voltage hypothesis for neurotransmitter release.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

Implications of G-Protein-Mediated Ca2+ Channel Inhibition for Neurotransmitter Release and Facilitation

G-protein-mediated inhibition of Ca²⁺ current is ubiquitous in neurons, and in synaptic terminals it can lead to a reduction in transmitter release (presynaptic inhibition). This type of Ca²⁺ current inhibition can often be relieved by prepulse depolarization, so the disinhibition of Ca²⁺ current can combine with Ca²⁺-dependent mechanisms for activity-induced synaptic facilitation to amplify this form of short-term plasticity. We combine a mathematical model of a G-protein-regulated Ca²⁺ channel with a model of transmitter secretion to study the potential effects of G-protein-mediated Ca²⁺ channel inhibition and disinhibition on transmitter release and facilitation. We investigate several scenarios, with the goal of observing a range of behaviors that may occur in different synapses. We find that the effects of Ca²⁺ channel disinhibition depend greatly on the location and distribution of inhibited channels. Facilitation can be greatly enhanced if all channels are subject to inhibition or if the subpopulation of channels subject to inhibition are located closer to release sites than those insensitive to inhibition, an arrangement that has been suggested by recent experiments (Stanley and Mirotznik, 1997). We also find that the effect of disinhibition on facilitation is greater for longer action potentials. Finally, in the case of homosynaptic inhibition, where Ca²⁺ channel inhibition occurs through the binding of transmitter molecules to presynaptic autoreceptors, there will be little reduction in transmitter release during the first of two successive bursts of impulses. The reduction of release during the second burst will be significantly greater, and if the unbinding rate of autoreceptors is relatively low, then the effects of G-protein-mediated channel inhibition become more pronounced as the duration of the interburst interval is increased up to a critical point, beyond which the inhibitory effects become less pronounced. This is in contrast to presynaptic depression due to the depletion of the releasable vesicle pool, where longer interburst intervals allow for a more complete replenishment of the pool. Thus, G-protein-mediated Ca²⁺ current inhibition leads to a reduction in transmitter release, while having a highly variable amplifying effect on synaptic facilitation. The dynamic properties of this form of presynaptic inhibition are very different from those of vesicle depletion.     

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca²⁺ release channel (ryanodine receptor) in excitable tissues. Using [³H]ryanodine, a 30 S protein complex comprised of four polypeptides of M_r 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [³H]ryanodine binding and Ca²⁺ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca2+-induced Ca2+ Release

The relationship between relative cell volume and time-dependent changes in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ _vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca²⁺] _i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca²⁺] _i transients were inhibited by either: (a) Ca²⁺-free medium; (b) 5 mm Ni²⁺ (inhibitor of plasmalemma Ca²⁺ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm ryanodine (inhibitor of Ca²⁺ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca²⁺ influx and the emptying of intracellular Ca²⁺ stores during the [Ca²⁺] _i transients, Mn²⁺ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn²⁺ permeability 6-fold. However, Mn²⁺ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca²⁺] _i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca²⁺ release from an intracellular store leads to a subsequent increase in plasmalemma Ca²⁺ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997     

Mechanisms of Ca2+ liberation at fertilization

The mechanisms underlying the Ca²⁺ release at fertilization of several animal organisms are reported. Four main classical theories are described, i.e., that of Ca²⁺ release following simple sperm contact and a G protein stimulation; that of simple sperm contact followed by a tyrosine kinase receptor activation; that of the necessity of introduction by sperm into the egg of molecules for Ca²⁺ release; and that the molecule introduced into the marine eggs for Ca²⁺ release is the same Ca²⁺. Two other mechanisms for Ca²⁺ release are also illustrated: that of ryanodine receptor stimulation and that of NAADP formation.     

The luminal Ca2+ chelator,TPEN, inhibits NAADP-induced Ca2+ release

The regulation of Ca²⁺ release by luminal Ca²⁺ has been well studied for the ryanodine and IP₃ receptors but has been less clear for the NAADP-regulated channel. In view of conflicting reports, we have re-examined the issue by manipulating luminal Ca²⁺ with the membrane-permeant, low affinity Ca²⁺ buffer, TPEN, and monitoring NAADP-induced Ca²⁺ release in sea urchin egg homogenate. NAADP-induced Ca²⁺ release was almost entirely blocked by TPEN (IC₅₀ 17–25 μM) which suppressed the maximal extent of Ca²⁺ release without altering NAADP sensitivity. In contrast, Ca²⁺ release via IP₃ receptors was 3- to 30-fold less sensitive to TPEN whereas that evoked by ionomycin was essentially unaffected. The effect of TPEN on NAADP-induced Ca²⁺ release was not due to an increase in the luminal pH or chelation of trace metals since it could not be mimicked by NH₄Cl or phenanthroline. The fact that TPEN had no effect upon ionophore-induced Ca²⁺ release also argued against a substantial reduction in the driving force for Ca²⁺ efflux. We propose that, in the sea urchin egg, luminal Ca²⁺ is important for gating native NAADP-regulated two-pore channels.     

Ca2+ homeostasis,Ca2+ signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca²⁺ homeostasis and provide for Ca²⁺ signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca²⁺ clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca²⁺ homeostatic molecules on cytosolic Ca²⁺ ([Ca²⁺]_i) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca²⁺]_i dynamics. When bathing the cells in a Na⁺-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca²⁺-ATPase (PMCA), La³⁺, all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca²⁺]_i transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca²⁺ transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca²⁺ homeostatic pathways, the Na⁺/Ca²⁺ exchanger, the endoplasmic reticulum Ca²⁺ pump, the plasmalemmal Ca²⁺ pump and mitochondria, are complementary in actively clearing Ca²⁺ from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca²⁺]_i dynamics; (iii) there is (are) Ca²⁺ clearance mechanism(s) distinct from the four outlined above; and (iv) Ca²⁺ homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.