In situ Ca2+ titration in the fluorometric study of intracellular Ca2+ binding

Imaging with Ca²⁺-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca²⁺ signaling. The spatiotemporal evolution of intracellular free Ca²⁺ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca²⁺ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca²⁺ imaging data, and can be exploited to investigate endogenous Ca²⁺ buffer properties. Here we extended the use of Ca²⁺ fluorometry in the characterization of Ca²⁺ binding molecules within cells, building on a method of titration of intracellular Ca²⁺ binding sites in situ with measured amounts of Ca²⁺ entering through voltage-gated Ca²⁺ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca²⁺ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca²⁺ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca²⁺ buffer and dye, this method yielded estimates of the endogenous buffer concentration and K_d, the dye K_d, and the fraction of Ca²⁺ inaccessible cellular volume. The in situ K_d of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca²⁺-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca²⁺ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca²⁺ binding species. This method offers a versatile approach to the study of endogenous Ca²⁺ binding molecules in their physiological milieu.     

Modeling of the Modulation by Buffers of Ca Release through Clusters of IP3 Receptors

Intracellular Ca²⁺ release is a versatile second messenger system. It is modeled here by reaction-diffusion equations for the free Ca²⁺ and Ca²⁺ buffers, with spatially discrete clusters of stochastic IP₃ receptor channels (IP₃Rs) controlling the release of Ca²⁺ from the endoplasmic reticulum. IP₃Rs are activated by a small rise of the cytosolic Ca²⁺ concentration and inhibited by large concentrations. Buffering of cytosolic Ca²⁺ shapes global Ca²⁺ transients. Here we use a model to investigate the effect of buffers with slow and fast reaction rates on single release spikes. We find that, depending on their diffusion coefficient, fast buffers can either decouple clusters or delay inhibition. Slow buffers have little effect on Ca²⁺ release, but affect the time course of the signals from the fluorescent Ca²⁺ indicator mainly by competing for Ca²⁺. At low [IP₃], fast buffers suppress fluorescence signals, slow buffers increase the contrast between bulk signals and signals at open clusters, and large concentrations of buffers, either fast or slow, decouple clusters.     

Calbindin-D28k and calretinin in the rat posterior pituitary; Light and electron microscopic localization and upregulation with dehydration

Ca²⁺ binding proteins (CaBPs), calbindin-D_28k (calbindin) and calretinin, are thought to contribute to the regulation of intracellular Ca²⁺ in many neuronal populations and perhaps more importantly, signal functional modulation in neuronal activity. In the present experiments, light microscopic immunohistochemistry revealed that the immunoreactivity of calbindin and calretinin was contained in varicose axons in the posterior pituitary. The dual labeling study with confocal microscopy demonstrated that calbindin immunoreactivity was present in the terminals of both oxytocin (OXT) and arginine-vasopressin (AVP) neurons. However, calretinin immunoreactivity was exclusively seen in the OXT terminals. Moreover, the dual labeling study showed that most calretinin-positive terminals contained calbindin immunoreactivity, demonstrating the colocalization of calbindin and calretinin in the same OXT nerve terminals. By electron microscopy, calbindin and calretinin immunoreactivities were seen in the neurosecretory axons and nerve terminals. These immunoreactive nerve terminals were seen to contain more clear microvesicles than dense-core neurosecretory granules. This immunoelectron microscopic observation suggests that both calbindin and calretinin localize preferentially in the active zone of the nerve terminals, which usually face the perivascular space around fenestrated capillaries. In spite of similar localization of calbindin and calretinin within the posterior pituitary, Western blot analysis showed some differences between the two CaBPs. Calbindin was present mostly in the soluble fraction with little in the insoluble fraction, but a substantial portion of calretinin was present in both the insoluble and soluble fractions. Moreover, dehydration induced by drinking 2% NaCl solution and deprivation of drinking water increased calretinin levels in the posterior pituitary as compared with control, but the calbindin level was not changed. The present findings demonstrate that calbindin and calretinin colocalize in the active zones of OXT nerve terminals, but only calretinin is upregulated with dehydration, suggesting different physiological role of calbindin and calretinin in the nerve terminals.     

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.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

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.     

Cytosolic Ca2+ and H+ buffers in green algae: A comment

Summary Recently Plieth et al. [Protoplasma (1997) 198: 107–124; 199: 223] gave a quantitative picture of the Ca²⁺ and H⁺ buffers in green algae which we would like to comment. In that paper a mechanistic model was derived which describes the relationship between cytosolic Ca²⁺ and H⁺ assuming that Ca²⁺ and H⁺ interact with the same binding site of a Ca²⁺-H⁺-exchange buffer. But the increase of the cytosolic free Ca²⁺ concentration observed upon acidification can alternatively be described by a co-operative (n=2) protonation of a Ca²⁺/H⁺-binding buffer pointing to an allosteric mechanism of Ca²⁺ liberation. Furthermore we present evidences that the cytosolic buffer capacities for H⁺ (90 mM/pH) and Ca²⁺ (20 mM/pCa) given for Eremosphaera viridis were overestimated by a factor of three and three orders of magnitude, respectively.Abbreviations [Ca²⁺]_c free cytosolic - Ca²⁺ concentration     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Calcium Domains around Single and Clustered IP3 Receptors and Their Modulation by Buffers

We study Ca²⁺ release through single and clustered IP₃ receptor channels on the ER membrane under presence of buffer proteins. Our computational scheme couples reaction-diffusion equations and a Markovian channel model and allows our investigating the effects of buffer proteins on local calcium concentrations and channel gating. We find transient and stationary elevations of calcium concentrations around active channels and show how they determine release amplitude. Transient calcium domains occur after closing of isolated channels and constitute an important part of the channel's feedback. They cause repeated openings (bursts) and mediate increased release due to Ca²⁺ buffering by immobile proteins. Stationary domains occur during prolonged activity of clustered channels, where the spatial proximity of IP₃Rs produces a distinct [Ca²⁺] scale (0.5-10 μM), which is smaller than channel pore concentrations (>100 μM) but larger than transient levels. While immobile buffer affects transient levels only, mobile buffers in general reduce both transient and stationary domains, giving rise to Ca²⁺ evacuation and biphasic modulation of release amplitude. Our findings explain recent experiments in oocytes and provide a general framework for the understanding of calcium signals.     

A calcium-induced calcium release mechanism mediated by calsequestrin

Calcium (Ca²⁺)-induced Ca²⁺ release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca²⁺ with ryanodine receptors (RyRs) and inducing Ca²⁺ release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca²⁺ may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca²⁺ buffer. We investigate how SR Ca²⁺ release via RyR is regulated by Ca²⁺ and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca²⁺ activation and inactivation, and one SR lumenal site for CSQ binding. The open probability (P_o) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca²⁺. Both peak and steady-state P_o effectively increase as SR lumenal Ca²⁺ increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca²⁺ loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca²⁺ release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca²⁺ release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca²⁺ release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes.     

Acute Restraint Stress Enhances Calcium Mobilization and Glutamate Exocytosis in Cerebrocortical Synaptosomes from Mice

Acute stress is known to enhance the memory of events that are potentially threatening to the organisms. Glutamate, the most abundant excitatory neurotransmitter in the mammalian central nervous system, plays a critical role in learning and memory formation and calcium (Ca²⁺) plays an essential role in transmitter release from nerve terminals (synaptosomes). In the present study, we investigated the effects of acute restraint stress on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in cerebrocortical synaptosomes from mice. Acute restraint stress caused a significant increase in resting [Ca²⁺]_i and significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K⁺- and 4-AP-induced Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) completely suppressed the enhancements of [Ca²⁺]_i and Ca²⁺-dependent glutamate release in acute restraint-stressed mice. These results indicate that acute restraint stress enhances K⁺- or 4-AP-induced glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through P- and N-type Ca²⁺ channels.     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

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.     

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X₇ receptor (P2X₇R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X₇R activation induced Ca²⁺-dependent vesicular glutamate release and significant Ca²⁺-independent glutamate efflux through the P2X₇R itself. In the present study, we investigated the effect of the new selective P2X₇R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca²⁺]_i signals and glutamate release, and evaluated whether P2X₇R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X₇R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X₇R-mediated [Ca²⁺]_i signals. These findings provide compelling evidence of functional presynaptic P2X₇R in cortical nerve terminals.     

Adenosine Triphosphate and the Late Steps in Calcium-dependent Exocytosis at a Ribbon Synapse

The ATP dependence of the kinetics of Ca²⁺-dependent exocytosis after flash photolysis of caged Ca²⁺ was studied by capacitance measurements with submillisecond resolution in single synaptic terminals of retinal bipolar neurons. After control experiments verified that this combination of techniques is valid for the study of exocytosis in synaptic terminals, a comparison was made between the Ca²⁺ dependence of the rate of exocytosis in synaptic terminals internally dialyzed with MgATP, MgATP-γ-S, or no added Mg²⁺ or nucleotide. The Ca²⁺ threshold for release, the maximum rate of release, and the overall relationship between the rate of synaptic vesicle fusion and [Ca²⁺]_i were found to be independent of MgATP. A decrease in the average rate at near-threshold [Ca²⁺]_i was observed in terminals with MgATP-γ-S, but due to the small sample size is of unclear significance. The Ca²⁺ dependence of the delay between the elevation of [Ca²⁺]_i and the beginning of the capacitance rise was also found to be independent of MgATP. In contrast, MgATP had a marked effect on the ability of terminals to respond to multiple stimuli. Terminals with MgATP typically exhibited a capacitance increase to a second stimulus that was >70% of the amplitude of the first response and to a third stimulus with a response amplitude that was >50% of the first, whereas terminals without MgATP responded to a second stimulus with a response <35% of the first and rarely responded to a third flash. These results suggest a major role for MgATP in preparing synaptic vesicles for fusion, but indicate that cytosolic MgATP may have little role in events downstream of calcium entry, provided that [Ca²⁺]_i near release sites is elevated above ≈30 μM.     

Pre‐synaptic GABAB receptors inhibit glutamate release through GIRK channels in rat cerebral cortex

Neuronal G protein‐gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post‐synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA_B receptors. In this study, we show for the first time that GABA_B receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA_B receptors reduces glutamate release and the Ca²⁺ influx mediated by N‐type Ca²⁺ channels in a mode insensitive to the GIRK channel blocker tertiapin‐Q and consistent with direct inhibition of this voltage‐gated Ca²⁺ channel. However, by means of weak stimulation protocols, we reveal that GABA_B receptors also reduce glutamate release mediated by P/Q‐type Ca²⁺ channels, and that these responses are reversed by the GIRK channel blocker tertiapin‐Q. Consistent with the functional interaction between GABA_B receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre‐synaptic boutons of asymmetric synapses co‐express GABA_B receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post‐synaptic level, also occurs at glutamatergic nerve terminals.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

Computational study of non-homogeneous distribution of Ca handling systems in cerebellar granule cells

The spatiotemporal distribution of cytosolic free calcium concentration ([Ca²⁺]_i) in cerebellar granule cells (GrCs) is thought to be critical in defining the occurrence and direction of long-term changes in synaptic strength at cerebellar mossy fiber-GrC synapses. Despite this, the mechanisms responsible for shaping Ca²⁺ transients in GrCs are not well understood. To investigate the interplay between Ca²⁺ entry, extrusion, buffering and dendritic morphology in shaping Ca²⁺ elevations in GrCs, we developed a model of Ca²⁺ regulation in these cells and examined the requirements for reproducing fluorescence responses to depolarization and synaptic stimulation previously described in the literature. Two conclusions can be drawn from our simulation results. First, a significant progressive decrease in the amplitudes of depolarization-evoked fluorescence transients from the dendritic endings (digits) toward the soma of GrCs, can be reproduced in the model only if the density of Ca²⁺ channels is considerably higher or the concentration of endogenous buffers is much lower in the digits than in the parent dendrites. In contrast, heterogeneities in the distribution of Ca²⁺ pumps or in cytosolic fractional volume cannot account for the formation of [Ca²⁺]_i gradients in GrCs. Second, much lower amplitudes of fluorescence transients induced by depolarization and synaptic stimulation than expected from typical measurements of Ca²⁺ and NMDA receptor-mediated currents can be reconciled with a pronounced slowing of the decay of fluorescence responses in the digits of GrCs after introducing a high-affinity Ca²⁺ indicator if a high-capacity immobile Ca²⁺ buffer (presumably plasma membrane-associated) is suggested to be present in the soma and apical part of digits. Mitochondria also are likely to modulate synaptically evoked Ca²⁺ responses in GrCs. The alternative hypotheses are thoroughly discussed and research avenues for their testing in future experiments are proposed.     

Predicting Local SR Ca Dynamics during Ca Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca²⁺) wave propagation, and 2), speed of Ca²⁺ diffusion within the SR. Ca²⁺ waves are initiated when a spontaneous local SR Ca²⁺ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca²⁺ diffusion constant in the SR (D_Ca,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca²⁺] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca²⁺] during Ca²⁺ waves. Simulations have investigated how dynamic local changes in SR [Ca²⁺] are influenced by 1), D_Ca,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca²⁺ pumps; 4), SR Ca²⁺ pump dependence on cytosolic [Ca²⁺]; and 5), the rate of transfer between network and junctional SR. Of these factors, D_Ca,SR is the primary determinant of how release from one RyR cluster alters SR [Ca²⁺] in nearby regions. Specifically, our results show that local increases in SR [Ca²⁺] ahead of the wave can potentially facilitate Ca²⁺ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca²⁺] are possible during SR Ca²⁺release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca²⁺]_SR.