Changes In Free Ca2 Research Articles

Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca2+ -affinity targeted aequorin reporter.

SUMMARYPrecise measurements of dynamic changes in free Ca2+ concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca2+ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low‐Ca2+‐affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non‐cleavable N‐terminal ER signal peptide (fl2). The correct targeting of fl2‐AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well‐established in animal cells – consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling – was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+]ER) were recorded in response to different environmental stresses, displaying stimulus‐specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set‐up of an ER‐targeted aequorin chimera extends and complements the currently available toolkit of organelle‐targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system.

Chloroplast-localized BICAT proteins shape stromal calcium signals and are required for efficient photosynthesis.

The photosynthetic machinery of plants must be regulated to maximize the efficiency of light reactions and CO2 fixation. Changes in free Ca2+ in the stroma of chloroplasts have been observed at the transition between light and darkness, and also in response to stress stimuli. Such Ca2+ dynamics have been proposed to regulate photosynthetic capacity. However, the molecular mechanisms of Ca2+ fluxes in the chloroplasts have been unknown. By employing a Ca2+ reporter-based approach, we identified two chloroplast-localized Ca2+ transporters in Arabidopsis thaliana, BICAT1 and BICAT2, that determine the amplitude of the darkness-induced Ca2+ signal in the chloroplast stroma. BICAT2 mediated Ca2+ uptake across the chloroplast envelope, and its knockout mutation strongly dampened the dark-induced [Ca2+ ]stroma signal. Conversely, this Ca2+ transient was increased in knockout mutants of BICAT1, which transports Ca2+ into the thylakoid lumen. Knockout mutation of BICAT2 caused severe defects in chloroplast morphology, pigmentation and photosynthetic light reactions, rendering bicat2 mutants barely viable under autotrophic growth conditions, while bicat1 mutants were less affected. These results show that BICAT transporters play a role in chloroplast Ca2+ homeostasis. They are also involved in the regulation of photosynthesis and plant productivity. Further work will be required to reveal whether the effect on photosynthesis is a direct result of their role as Ca2+ transporters.

Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1

Macular dystrophy leads to progressive loss of central vision and shows symptoms similar to age-related macular degeneration. Genetic screening of patients diagnosed with macular dystrophy disclosed a novel mutation in the GUCA1A gene, namely a c.526C > T substitution leading to the amino acid substitution p.L176F in the guanylate cyclase-activating protein 1 (GCAP1). The same variant was found in three families showing an autosomal dominant mode of inheritance. For a full functional characterization of the L176F mutant we expressed and purified the mutant protein and measured key parameters of its activating properties, its Ca2+/Mg2+-binding, and its Ca2+-induced conformational changes in comparison to the wildtype protein. The mutant was less sensitive to changes in free Ca2+, resulting in a constitutively active form under physiological Ca2+-concentration, showed significantly higher activation rates than the wildtype (90-fold versus 20-fold) and interacted with an higher apparent affinity with its target guanylate cyclase. However, direct Ca2+-binding of the mutant was nearly similar to the wildtype; binding of Mg2+ occurred with higher affinity. We performed molecular dynamics simulations for comparing the Ca2+-saturated inhibiting state of GCAP1 with the Mg2+-bound activating states. The L176F mutant exhibited significantly lower flexibility, when three Ca2+ or two Mg2+ were bound forming probably the structural basis for the modified GCAP1 function.

Spatially resolved recording of transient fluorescence-lifetime effects by line-scanning TCSPC

We present a technique that records transient changes in the fluorescence lifetime of a sample with spatial resolution along a one-dimensional scan. The technique is based on scanning the sample with a high-frequency pulsed laser beam, detecting single photons of the fluorescence light, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan period. Transient lifetime effects can thus be resolved at a resolution of about one millisecond. We demonstrate the technique for recording photochemical and nonphotochemical chlorophyll transients in plants and transient changes in free Ca(2+) in cultured neurons.

Crystallization Kinetics of CaOx in Artificial Urine and in Saline System

The crystallization kinetics of calcium oxalate (CaOx) was comparatively studied by detecting the change of free Ca2+ ions concentration with the reaction time in artificial urine and in saline system. The dynamics equations of CaOx crystallization was r=kcα, and the average reaction order (α) was 3.3 regardless of the relative suprasaturation degree (RS) of CaOx in the range of RS=10.58~17.53. The average reaction rate constant (κ) was (0.97±0.1)×109 in artificial urine and κ=(3.1±1.8)×109 in saline system, due to the presence of inhibitors to CaOx crystallization in artificial urine.

Residues Contributing to the Na+-binding Pocket of the SLC24 Na+/Ca2+-K+ Exchanger NCKX2

Na(+)/Ca(2+)-K(+) exchangers (NCKX; gene family SLC24) are plasma membrane Ca(2+) transporters that mediate the extrusion of one Ca(2+) ion and one K(+) ion in exchange for four Na(+) ions. NCKX is modeled to have two sets of five transmembrane segments separated by a large cytosolic loop; within each set of transmembrane segments are regions of internal symmetry termed alpha(1) and alpha(2) repeats. The central residues that are important for Ca(2+) and K(+) liganding and transport have been identified in NCKX2, and they comprise three central acidic residues, Glu(188) in alpha(1) and Asp(548) and Asp(575) in alpha(2), as well as Ser/Thr residues one-helical turn away from these residues. In this study, we have scanned through more than 100 single-residue substitutions of NCKX2 for shifts in Na(+) affinity using a fluorescence assay to monitor changes in free Ca(2+) in HEK293 cells treated with gramicidin to control intracellular Na(+). We have identified 31 residues that, when substituted, result in shifts in Na(+) affinity, either toward higher or lower K(m) values when compared with wild type NCKX2 (K(m) for Na(+) 58 mm). These residues include the central acidic residues Glu(188), Asp(548), and Asp(575), and their neighboring residues in alpha(1) and alpha(2), in addition to a number of newly investigated residues in transmembrane segment 3. Our results relate the identification of residues important for Na(+) transport in this study to those previously identified as important in the counter-transport of Ca(2+) and K(+), lending support to the alternating access model of transmembrane transport.

The impact of paracrine signaling in brain microvascular endothelial cells on the survival of neurons

Neurons depend for survival on local neurotrophic factors which act in an autocrine/paracrine manner. However, the effect of paracrine signaling of brain microvascular endothelial cells (BMECs) under pathological conditions on neuron survival is not fully understood. In this study we cultured rat BMECs and cortical neurons. BMECs were cultured in oxygen-glucose-deprived (OGD) conditions to mimic cerebral ischemia in vitro. The conditioned media of normal BMECs or OGD-injured BMECs were used to culture normal or injured neurons. Neuron activity, free Ca 2+ concentration, NMDA receptor status, mitochondrial membrane potential and cytochrome C release level were determined. The results showed: mitochondrial activity of injured neurons was significantly increased and lactate dehydrogenase (LDH) leakage was decreased ( P < 0.05) by grown in conditioned medium of normal BMECs. Inversely, mitochondrial activity of normal or injured neurons was decreased and LDH leakage was significantly increased ( P < 0.05) by grown in conditioned medium of injured BMECs. The changes in free Ca 2+ concentration, NMDA receptor status, mitochondrial membrane potential and cytochrome C release level were consistent with the changes in neuronal activity. These findings suggest that the conditioned medium of normal BMECs has a neuroprotective effect. However, this protective effect was lost after BMECs injury; in fact, the conditioned medium became neurotoxic. Therefore, it appears that early recovery of BMECs might be helpful for neuron survival.

Transient effect of weak electromagnetic fields on calcium ion concentration in Arabidopsis thaliana

BackgroundWeak magnetic and electromagnetic fields can influence physiological processes in animals, plants and microorganisms, but the underlying way of perception is poorly understood. The ion cyclotron resonance is one of the discussed mechanisms, predicting biological effects for definite frequencies and intensities of electromagnetic fields possibly by affecting the physiological availability of small ions. Above all an influence on Calcium, which is crucial for many life processes, is in the focus of interest. We show that in Arabidopsis thaliana, changes in Ca2+-concentrations can be induced by combinations of magnetic and electromagnetic fields that match Ca2+-ion cyclotron resonance conditions.ResultsAn aequorin expressing Arabidopsis thaliana mutant (Col0-1 Aeq Cy+) was subjected to a magnetic field around 65 microtesla (0.65 Gauss) and an electromagnetic field with the corresponding Ca2+ cyclotron frequency of 50 Hz. The resulting changes in free Ca2+ were monitored by aequorin bioluminescence, using a high sensitive photomultiplier unit. The experiments were referenced by the additional use of wild type plants. Transient increases of cytosolic Ca2+ were observed both after switching the electromagnetic field on and off, with the latter effect decreasing with increasing duration of the electromagnetic impact. Compared with this the uninfluenced long-term loss of bioluminescence activity without any exogenic impact was negligible. The magnetic field effect rapidly decreased if ion cyclotron resonance conditions were mismatched by varying the magnetic fieldstrength, also a dependence on the amplitude of the electromagnetic component was seen.ConclusionConsidering the various functions of Ca2+ as a second messenger in plants, this mechanism may be relevant for perception of these combined fields. The applicability of recently hypothesized mechanisms for the ion cyclotron resonance effect in biological systems is discussed considering it's operating at magnetic field strengths weak enough, to occur occasionally in our all day environment.

Combining Voltage and Calcium Imaging from Neuronal Dendrites

The ability to monitor membrane potential (V(m)) and calcium (Ca(2+)) transients at multiple locations on the same neuron can facilitate further progress in our understanding of neuronal function. Here we describe a method to combine V(m) and Ca(2+) imaging using styryl voltage sensitive dyes and Fura type UV-excitable Ca(2+) indicators. In all cases V(m) optical signals are linear with membrane potential changes, but the calibration of optical signals on an absolute scale is presently possible only in some neurons. The interpretation of Ca(2+) optical signals depends on the indicator Ca(2+) buffering capacity relative to the cell endogenous buffering capacity. In hippocampal CA1 pyramidal neurons, loaded with JPW-3028 and 300 microM Bis-Fura-2, V(m) optical signals cannot be calibrated and the physiological Ca(2+) dynamics are compromised by the presence of the indicator. Nevertheless, at each individual site, relative changes in V (m) and Ca(2+) fluorescence signals under different conditions can provide meaningful new information on local dendritic integration. In cerebellar Purkinje neurons, loaded with JPW-1114 and 1 mM Fura-FF, V(m) optical signals can be calibrated in terms of mV and Ca(2+) optical signals quantitatively reveal the physiological changes in free Ca(2+). Using these two examples, the method is explained in detail.

Dynamic changes in free Ca-calmodulin levels in adult cardiac myocytes

Despite its multifunctional role in cardiac myocyte function, little is known about dynamic changes in activation state of calmodulin (CaM). Thus, the purpose of this study was to develop a tool to measure Ca bound CaM (Ca–CaM) levels in intact cardiac myocytes. For dynamic measurements of Ca–CaM, we generated an adenoviral vector which expresses a cyan and a yellow fluorescent protein linked by a modified version of the Ca–CaM binding domain of avian smooth muscle myosin light chain kinase. Adult rabbit cardiac myocytes were infected with the Ca–CaM sensing probe or simultaneously infected with viruses containing CaM and the Ca–CaM sensing probe for 24–48 h. Myocytes were then field stimulated (1 Hz) and excited at 440 nm with emitted fluorescence measured at 485 and 535 nm. Changes in [Ca–CaM] are expressed as the ratio of 485 nm/535 nm. Small beat-to-beat changes of [Ca–CaM] were detected, but only when CaM was co-expressed with the sensor. However, upon β-adrenergic stimulation with isoproterenol, there was an increase in the amplitude of the signals during each beat (parallel to the shortening, which is an indirect measure of [Ca] i) and also a rise in the diastolic [Ca–CaM]. Total [CaM] measured by both competitive ELISA and semi-quantitative Western blots was 5–6 μM in isolated adult ventricular myocytes. Our results indicate that there are dynamic changes in free Ca–CaM levels (a phasic component tracking [Ca] i) as well as system memory that integrates the [Ca] i signals (a tonic component).

Mode of mitochondrial Ca 2+ clearance and its influence on secretory responses in stimulated chromaffin cells

To study the role of mitochondrial Ca 2+ clearance in stimulated cells, changes in free Ca 2+ concentration in the cytosol, [Ca 2+] c and that in mitochondria, [Ca 2+] m along with secretory responses were observed using chromaffin cells co-loaded with Fura-2 and Rhod-2 in the perfused rat adrenal medulla. When the cells were stimulated with 40 mM K + in the perfusate, the duration of [Ca 2+] m response markedly increased with prolongation of the stimulation period, exhibiting a mean half-decay time of 21 min with 30 s stimulation, whereas its amplitude was not altered with stimulations of 10–30 s. A computer simulation analysis showed that such a mode of [Ca 2+] m response can be produced if excess Ca 2+ taken up by mitochondria precipitates as calcium phosphate (Pi) salt. In the presence of 5 μM rotenone plus 10 μM oligomycin, a decrease in the duration of [Ca 2+] m response and a slight but significant increase (24%) in the secretory response to 30 s stimulation with 40 mM K + were observed. Simulation analyses suggested that this effect of rotenone may be due to reduction in mitochondrial Ca 2+ uptake induced by rotenone-elicited partial depolarization of the mitochondrial membrane potential. In chromaffin cells transsynaptically stimulated through the splanchnic nerve, the intensity of NAD(P)H autofluorescence changed with time courses similar to those of [Ca 2+] m responses. The temporal profiles of those two responses were prolonged in a similar manner by application of an inhibitor of mitochondrial Na +/Ca 2+ exchanger, CGP37157. Thus, due to the unique Ca 2+ buffering mechanism, [Ca 2+] m responses associated with massive mitochondrial Ca 2+ uptake may occur within a limited concentration range in which Ca 2+-sensitive dehydrogenases are activated to control the mitochondrial redox state in stimulated chromaffin cells.

Automation highlights from literature

Automation highlights from literature

High-throughput real-time monitoring of G s-coupled receptor activation in intact cells using cyclic nucleotide-gated channels

Cyclic adenosine-monophosphate (cAMP) is one of the major second messenger molecules transmitting extracellular stimuli into short- and long-term changes of intracellular homeostasis. Measurements of cellular cAMP levels are often used to quantify and characterize signaling by G protein-coupled receptors. Current assays for cAMP determination are usually end-point assays involving cell lysis. We have developed a technology to monitor real-time changes of cAMP levels in living cells. This method uses a modified cyclic nucleotide-gated (CNG) Ca 2+ channel which is opened by intracellular cAMP. Thus, changes in cAMP levels are translated into changes in free Ca 2+ which can easily be measured using fluorimetric imaging technologies compatible with high-throughput screening formats. The new assay method was used to characterize the pharmacology of various endogenously and heterologously expressed G protein-coupled receptors and allows for the simultaneous study of G s, G i and G q-linked receptors in the same cell population.

Neuronal endoplasmic reticulum acts as a single functional Ca2+ store shared by ryanodine and inositol-1,4,5-trisphosphate receptors as revealed by intra-ER [Ca2+] recordings in single rat sensory neurones.

We addressed the fundamentally important question of functional continuity of endoplasmic reticulum (ER) Ca(2+) store in nerve cells. In cultured rat dorsal root ganglion neurones we measured dynamic changes in free Ca(2+) concentration within the ER lumen ([Ca(2+)](L)) in response to activation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs). We found that both receptors co-exist in these neurones and their activation results in Ca(2+) release from the ER as judged by a decrease in [Ca(2+)](L). Depletion of Ca(2+) stores following an inhibition of sarco(endoplasmic)reticulum Ca(2+)-ATPase by thapsigargin or cyclopiazonic acid completely eliminated Ca(2+) release via both InsP(3)Rs and RyRs. Similarly, when the store was depleted by continuous activation of InsP(3)Rs, activation of RyRs (by caffeine or 0.5 microM ryanodine) failed to produce Ca(2+) release, and vice versa, when the stores were depleted by activators of RyRs, the InsP(3)-induced Ca(2+) release disappeared. We conclude that in mammalian neurones InsP(3)Rs and RyRs share the common continuous Ca(2+) pool associated with ER.

Expression and purification of human cardiac troponin subunits and their functional incorporation into isolated cardiac mouse myofibrils

The three subunits of the human cardiac troponin complex (hcTnC, hcTnI, hcTnT) were overexpressed in E. coli, purified and reconstituted to form the hcTn complex. This complex was then incorporated into subcellular bundles of mouse cardiac myofibrils whereby the native mcTn complex was replaced. On thus exchanged myofibrils, isometric force kinetics following sudden changes in free Ca 2+ concentration were measured using atomic force cantilevers. Following the exchange, the myofibrillar force remained fully Ca 2+ regulated, i.e. myofibrils were completely relaxed at pCa 7.5 and developed the same maximum Ca 2+-activated isometric force upon increasing the pCa to 4.5 as unexchanged myofibrils. The replacement of endogenous mcTn by wild-type hcTn neither altered the kinetics of Ca 2+-induced force development of the mouse myofibrils nor the kinetics of force relaxation induced by the sudden, complete removal of Ca 2+. Preparations of functional Tn reconstituted myofibrils provide a promising model to study the role of Tn in kinetic mechanisms of cardiac myofibrillar contraction and relaxation.

A light-dependent increase in free Ca2+ concentration in the salamander rod outer segment.

1. The Ca(2+) indicator dye fluo-5F was excited by an argon ion laser to measure changes in free Ca(2+) concentration ([Ca2+]i) in the outer segments of isolated salamander rods rapidly exposed to a 0 Ca(2+), 0 Na(+) solution designed to minimise surface membrane Ca(2+) fluxes. Over 30-60 s of laser illumination, the fluorescence first increased rapidly and then declined at a rate that was much slower than in Ringer solution and consistent with previous physiological evidence that 0 Ca(2+), 0 Na(+) solution greatly retards light-induced changes in [Ca(2+)]i. 2. The initial increase in fluorescence was investigated with a sequence of 100 ms laser flashes presented at 5 s intervals. The fluorescence evoked by the second laser flash was on average 30 % larger than the first, and subsequent responses exhibited a slow decline like that measured with continuous laser exposures. The initial increase in fluorescence did not depend upon the timing of exposure to 0 Ca(2+), 0 Na(+) solution but appeared to be evoked by exposure to the laser light. 3. Both the increase and subsequent decline in fluorescence measured with brief laser flashes could be reduced by incorporation of the Ca(2+) chelator BAPTA. This and other results indicate that the fluorescence increase was unlikely to have been caused by a change in the affinity of fluo-5F for Ca(2+) or an increase in the quantity of incorporated dye available to bind Ca(2+) but reflects an actual release of intracellular Ca(2+) within the outer segment. 4. The pool of Ca(2+) available to be released could be decreased if, before the first laser flash, the rod was exposed to light bright enough to bleach a substantial fraction of the photopigment. The releasable pool could also be depleted by exposure to saturating light of much lower intensity if delivered in Ringer solution but not if delivered in 0 Ca(2+), 0 Na(+) solution. We conclude that Ca(2+) can be released within the outer segment both by the bleaching of rhodopsin and by the reduction in [Ca(2+)]i which normally accompanies illumination in Ringer solution. 5. The activation of rhodopsin appears somehow to induce the release of Ca(2+) from a binding site or store within the outer segment. Substantial release, however, required stimulating light of an intensity sufficient to bleach a considerable fraction of the visual pigment. It therefore seems unlikely that such release contributes to the normal Ca(2+)-mediated modulation of transduction during light adaptation. The mechanism and physiological function of light-induced Ca(2+) release are unknown.

Adaptation in vertebrate photoreceptors.

When light is absorbed within the outer segment of a vertebrate photoreceptor, the conformation of the photopigment rhodopsin is altered to produce an activated photoproduct called metarhodopsin II or Rh(*). Rh(*) initiates a transduction cascade similar to that for metabotropic synaptic receptors and many hormones; the Rh(*) activates a heterotrimeric G protein, which in turn stimulates an effector enzyme, a cyclic nucleotide phosphodiesterase. The phosphodiesterase then hydrolyzes cGMP, and the decrease in the concentration of free cGMP reduces the probability of opening of channels in the outer segment plasma membrane, producing the electrical response of the cell. Photoreceptor transduction can be modulated by changes in the mean light level. This process, called light adaptation (or background adaptation), maintains the working range of the transduction cascade within a physiologically useful region of light intensities. There is increasing evidence that the second messenger responsible for the modulation of the transduction cascade during background adaptation is primarily, if not exclusively, Ca(2+), whose intracellular free concentration is decreased by illumination. The change in free Ca(2+) is believed to have a variety of effects on the transduction mechanism, including modulation of the rate of the guanylyl cyclase and rhodopsin kinase, alteration of the gain of the transduction cascade, and regulation of the affinity of the outer segment channels for cGMP. The sensitivity of the photoreceptor is also reduced by previous exposure to light bright enough to bleach a substantial fraction of the photopigment in the outer segment. This form of desensitization, called bleaching adaptation (the recovery from which is known as dark adaptation), seems largely to be due to an activation of the transduction cascade by some form of bleached pigment. The bleached pigment appears to activate the G protein transducin directly, although with a gain less than Rh(*). The resulting decrease in intracellular Ca(2+) then modulates the transduction cascade, by a mechanism very similar to the one responsible for altering sensitivity during background adaptation.

Inhibition of the ecto-ATPdiphosphohydrolase of Schistosoma mansoni by thapsigargin.

ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable of hydrolyzing nucleoside di- and triphosphates. Although a number of possible physiological roles have been proposed for ATPDases, detailed studies on structure-function relationships have generally been hampered by the lack of specific inhibitors of these enzymes. We have previously characterized a Ca2+-activated ATPDase on the external surface of the tegument of Schistosoma mansoni, the etiologic agent of human schistosomiasis. In the present work, we have examined the effects of thapsigargin, a sesquiterpene lactone known as a high affinity inhibitor of sarco-endoplasmic reticulum calcium transport (SERCA) ATPase, on ATPDase activity. Whereas other lactones tested had little or no inhibitory action, thapsigargin inhibited ATP hydrolysis by the ATPDase (K(i) approximately 20 microM). Interestingly, hydrolysis of ADP was not inhibited by thapsigargin. The lack of inhibition of ATPase activity by orthovanadate, a specific inhibitor of P-type ATPases, and the inhibition of the Mg2+-stimulated ATP hydrolysis by thapsigargin ruled out the possibility that the observed inhibition of the ATPDase by thapsigargin could be due to the presence of contaminating SERCA ATPases in our preparation. Kinetic analysis indicated that a single active site in the ATPDase is responsible for hydrolysis of both ATP and ADP. Thapsigargin caused changes in both Vmax and Km for ATP, indicating a mixed type of inhibition. Inhibition by thapsigargin was little or not affected by changes in free Ca2+ or Mg2+ concentrations. These results suggest that interaction of thapsigargin with the S. mansoni ATPDase prevents binding of ATP or its hydrolysis at the active site, while ADP can still undergo catalysis.

Regulation of Intra-Golgi Membrane Transport by Calcium

Calcium cations play a critical role in regulating vesicular transport between different intracellular membrane-bound compartments. The role of calcium in transport between the Golgi cisternae, however, remains unclear. Using a well characterized cell-free intra-Golgi transport assay, we now show that changes in free Ca(2+) concentration in the physiological range regulate this transport process. The calcium-chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked transport with an IC(50) of approximately 0.8 mm. The effect of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was reversible by addition of fresh cytosol and was irreversible when performed in the presence of a Ca(2+) ionophore that depletes calcium from lumenal stores. We demonstrate here that intra-Golgi transport is stimulated by low Ca(2+) concentrations (20-100 nm) but is inhibited by higher concentrations (above 100 nm). Further, we show that calmodulin antagonists specifically block intra-Golgi transport, implying a role for calmodulin in mediating the effect of calcium. Our results suggest that Ca(2+) efflux from intracellular pools may play an essential role in regulating intra-Golgi transport.

Responses to reversible anoxia of intracellular free and bound Ca 2+ in rat cortical slices

Severe anoxia induces destabilisation of intracellular calcium homeostasis in neurones. The mechanism of this effect, and particularly the interrelationship between changes in intracellular concentration of free Ca 2+ ions and the content of the intracellular Ca 2+ stores, during and after anoxia, is not clear. We used a superfusion system of rat olfactory cortical slices for the fluorimetric estimation of changes in the intracellular concentration of free Ca 2+ ions and in the level of bound Ca 2+, utilising the fluorescent indicators Fura-2 and chlortetracycline, respectively. It was found that 10-min normoglycaemic anoxia results in simultaneous decrease in bound and increase in free Ca 2+ levels, whereas during 60-min reoxygenation, we detected an increase in both indices. The NMDA receptor antagonists MK-801 and APV attenuated changes in free Ca 2+ level during anoxia and reoxygenation and intensified anoxia-evoked decrease in bound Ca 2+ content, whereas a late post-anoxic increase in bound Ca 2+ was abolished. These data suggest that the influx of extracellular Ca 2+ to neurones via NMDA receptors, plays a critical role in the rise of intracellular free Ca 2+ concentration during and after anoxia. Biphasic changes in bound Ca 2+ content during anoxia and reoxygenation may reflect an anoxia-induced release of Ca 2+ from intracellular stores, followed later by a neuronal calcium overload and refilling of intracellular Ca 2+ binding sites.