Redox modulation of calcium entry and release of intracellular calcium by thimerosal in GH4C1 pituitary cells

In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in GH₄C₁ pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca²⁺]_i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory.The effect of thimerosal on the IP₃-induced calcium release (IICR) was studied in permeabilized cells using the Ca²⁺ indicator Fluo-3. A low concentration of thimerosal (10 μM) stimulated IICR: the Ca²⁺ release induced by 300 nM inositol-1,4,5-trisphosphate (IP₃) was enhanced in cells treated with thimerosal for 1 or 6 min (67 ± 11 nM and 34 ± 5 nM, respectively) as compared to control cells (17 ± 2 nM). On the other hand, a high concentration of thimerosal (100 μ inhibited IICR: when IP₃ (10 μM) was added after a 5 min preincubation with thimerosal, the IP₃-induced rise in [Ca²⁺]_i (46 ± 14 nM) was 57% smaller as compared with that seen in control cells (106 ± 10 nM).The effect of thimerosal on the voltage-operated Ca ²⁺ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K⁺ to the cuvette. The depolarization-evoked increase in [Ca²⁺]_i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 μM) potently inhibited the Ba²⁺ currents through VOCCs.In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca²⁺]_i, and activated a capacitative Ca²⁺ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT).We conclude that, in GH₄C₁ pituitary cells, the mobilization of intracellular calcium and the different Ca²⁺ entry pathways are sensitive to redox modulation.     

Ca-DEPENDENT CHANGES OF ACETYLCHOLINE RELEASE AND IP3 MASS IN TORPEDO CHOLINERGIC SYNAPTOSOMES

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP₃) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP₃ mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP₃ mass levels under resting conditions. The IP₃ mass was neither modified by changes in external Ca²⁺ nor by a Ca²⁺-free medium containing EGTA. IP₃ mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K⁺ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca²⁺ emphasizing that Ca²⁺ entry, independently of the influx mechanism involved, leads to an IP₃ increase. The phospholipase C_β inhibitors U-73122 and U-73343 reduced K⁺-stimulated IP₃ levels while K⁺-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP₃ by agents that stimulate ACh release may suggest a possible link between IP₃ metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C. Copyright © 1996 Elsevier Science Ltd     

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.     

Analysis of IP3 receptors in and out of cells

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP₃R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca^2 + channels.

Scope of the review

In this brief review, we first consider a variety of complementary methods that allow the links between IP₃ binding and channel gating to be defined. How does IP₃ binding to the IP₃-binding core in each IP₃R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP₃, Ca^2 + signals and IP₃R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP₃R signalling.

Major conclusions

All IP₃R are regulated by both IP₃ and Ca^2 +. This allows them to initiate and regeneratively propagate intracellular Ca^2 + signals. The elementary Ca^2 + release events evoked by IP₃ in intact cells are mediated by very small numbers of active IP₃R and the Ca^2 +-mediated interactions between them. The spatial organization of these Ca^2 + signals and their stochastic dependence on so few IP₃Rs highlight the need for methods that allow the spatial organization of IP₃R signalling to be addressed with single-molecule resolution.

General significance

A variety of complementary methods provide insight into the structural basis of IP₃R activation and the contributions of IP₃-evoked Ca^2 + signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Endoplasmic reticulum Ca release through ryanodine and IP3 receptors contributes to neuronal excitotoxicity

Overactivation of ionotropic glutamate receptors induces a Ca²⁺ overload into the cytoplasm that leads neurons to excitotoxic death, a process that has been linked to several neurodegenerative disorders. While the role of mitochondria and its involvement in excitotoxicity have been widely studied, the contribution of endoplasmic reticulum (ER), another crucial intracellular store in maintaining Ca²⁺ homeostasis, is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine (RyR) and IP₃ (IP₃R) receptors to a neuronal in vitro model of excitotoxicity. NMDA induced a dose-dependent neuronal death, which was significantly decreased by ER-Ca²⁺ release inhibitors in cortical neurons as well as in organotypic slices. Furthermore, ryanodine and 2APB, RyR and IP₃R inhibitors respectively, attenuated NMDA-triggered intracellular Ca²⁺ increase and oxidative stress, whereas 2APB reduced mitochondrial membrane depolarization and caspase-3 cleavage. Consistent with ER-Ca²⁺ homeostasis disruption, we observed that NMDA-induced ER stress, characterized here by eIF2α phosphorylation and over-expression of GRP chaperones which were regulated by ER-Ca²⁺ release inhibitors. These results demonstrate that Ca²⁺ release from ER contributes to neuronal death by both promoting mitochondrial dysfunction and inducing specific stress and apoptosis pathways during excitotoxicity.     

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.     

The IP3 receptor-mitochondria connection in apoptosis and autophagy

The amount of Ca²⁺ taken up in the mitochondrial matrix is a crucial determinant of cell fate; it plays a decisive role in the choice of the cell between life and death. The Ca²⁺ ions mainly originate from the inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores of the endoplasmic reticulum (ER). The uptake of these Ca²⁺ ions in the mitochondria depends on the functional properties and the subcellular localization of the IP₃ receptor (IP₃R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca²⁺ ions from the ER to the mitochondria, structural interactions between IP₃Rs and mitochondria are needed. This review will focus on the key proteins involved in these interactions, how they are regulated, and what are their physiological roles in apoptosis, necrosis and autophagy. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

IP3-mediated cytosolic and nuclear calcium elevation in NRK-52E cells using ‘caged’ GPIP2

Alterations in calcium homeostasis play a pivotal role in the cellular response to injury. Increases in the concentration of cytosolic free calcium ([Ca²⁺]i) result in a variety of calcium mediated toxic responses such as cytoskeletal alterations, mitochondrial damage, and over-expression of gene products. Inositol trisphosphate is a second messenger that links external cell surface signals to [Ca²⁺]i elevation. The present study explored the use of caged glycerophosphoryl-myo-inositol-1,4,5-bisphosphate (GPIP₂) to mediate a rapid and prolonged increase in [Ca²⁺]i in a normal rat kidney epithelial cell line (NRK-52E). In intact NRK-52E cells, UV photolysis of microinjected GPIP₂ resulted in a 3–4-fold sustained increase in [Ca²⁺]_i. Graded photolytic release of GPIP2 also resulted in calcium-mediated morphologic alterations, as shown by confocal microscopy, with cellular blebs apparent within 30 min. There was no apparent increase in [Ca²⁺]i or morphologic alterations in control cells microinjected with calcium indicator and equally exposed to UV light. Subsequent application of thapsigargin or ionomycin (1.0 μM) produced a rapid and transient increase in [Ca²⁺]i. In addition, we show that activation of IN stores results in increased concentration of ionized nuclear calcium, ([Ca²⁺]n) which persists longer than the increase in [Ca²⁺]i. These findings indicate that GPIP₂ mediates a rapid and sustained elevation in [Ca²⁺]n and [Ca²⁺]i and this IP₃-mediated calcium elevation is translated to the nucleus in rat kidney epithelial cells.     

Toward a high-resolution structure of IP3R channel

The ability of cells to maintain low levels of Ca²⁺ under resting conditions and to create rapid and transient increases in Ca²⁺ upon stimulation is a fundamental property of cellular Ca²⁺ signaling mechanism. An increase of cytosolic Ca²⁺ level in response to diverse stimuli is largely accounted for by the inositol 1,4,5-trisphosphate receptor (IP₃R) present in the endoplasmic reticulum membranes of virtually all eukaryotic cells. Extensive information is currently available on the function of IP₃Rs and their interaction with modulators. Very little, however, is known about their molecular architecture and therefore most critical issues surrounding gating of IP₃R channels are still ambiguous, including the central question of how opening of the IP₃R pore is initiated by IP₃ and Ca²⁺. Membrane proteins such as IP₃R channels have proven to be exceptionally difficult targets for structural analysis due to their large size, their location in the membrane environment, and their dynamic nature. To date, a 3D structure of complete IP₃R channel is determined by single-particle cryo-EM at intermediate resolution, and the best crystal structures of IP₃R are limited to a soluble portion of the cytoplasmic region representing ∼15% of the entire channel protein. Together these efforts provide the important structural information for this class of ion channels and serve as the basis for further studies aiming at understanding of the IP₃R function.     

Selective down-regulation of IP3receptor subtypes by caspases and calpain during TNF α -induced apoptosis of human T-lymphoma cells

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP₃R) [IP₃-gated Ca²⁺channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP₃R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca²⁺channel properties in an IP₃-dependent manner. We have also demonstrated that TNFα promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca²⁺response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFα, several IP₃R subtype-related changes occur (e.g. proteolysis of IP₃R subtypes, inhibition of IP₃binding and impairment of IP₃-mediated Ca²⁺flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFα-mediated perturbation of IP₃R1 and IP₃R2 (but not IP₃R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFα on IP₃R3 function. These findings suggest that IP₃R1 and IP₃R2 serve as cellular substrates for caspases, and IP₃R3 is a substrate for calpain. We propose that the selective down-regulation of IP₃R subtype-mediated Ca²⁺function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFα in human T-cells.     

Exogenous nitric oxide-induced release of calcium from intracellular IP3 receptor-sensitive stores via S-nitrosylation in respiratory burst-dependent neutrophils

PMA-induced respiratory burst neutrophils were exposed to exogenous nitric oxide (NO) donor sodium nitroprusside (SNP) to study the effect of NO on calcium signaling. A sharp rise of cytosolic calcium concentration ([Ca²⁺]_c) was triggered by 1 mM SNP with and without external calcium. We found that GF 109203X, a specific inhibitor of protein kinase C, DPI, a putative inhibitor of the respiratory burst-generating NADPH oxidase, and 2-DG, a non-metabolizable analog of glucose, completely inhibited the SNP-induced rise of [Ca²⁺]_c in PMA-activated respiratory burst neutrophils. Meanwhile, 2-APB and TMB-8, two potent IP₃ receptor inhibitors, prevented calcium increase respectively. Furthermore, N-ethylmaleimide (NEM), a specific cysteine alkylating agent, evidently abolished the [Ca²⁺]_c elevation. In contrast, the sGC inhibitor NS2028 had little effect on the rise of [Ca²⁺]_c. Taken together, these results indicated that exogenous NO induced the release of calcium from intracellular IP₃ receptor-sensitive stores of neutrophils via S-nitrosylation in a respiratory burst-dependent manner.     

Ribosome-free Terminals of Rough ER Allow Formation of STIM1 Puncta and Segregation of STIM1 from IP3 Receptors

Store-operated Ca²⁺ entry is a ubiquitous mechanism that prevents the depletion of endoplasmic reticulum (ER) calcium [1]. A reduction of ER calcium triggers translocation of STIM proteins, which serve as calcium sensors in the ER, to subplasmalemmal puncta where they interact with and activate Orai channels ([2], [3], [4], [5], [6], [7] and [8]; reviewed in [9]). In pancreatic acinar cells, inositol 1,4,5-trisphosphate (IP₃) receptors populate the apical part of the ER. Here, however, we observe that STIM1 translocates exclusively to the lateral and basal regions following ER Ca²⁺ loss. This finding is paradoxical because the basal and lateral regions of the acinar cells contain rough ER (RER); the size of the ribosomes that decorate RER is larger than the distance that can be spanned by a STIM-Orai complex [5] and [10], and STIM1 function should therefore not be possible. We resolve this paradox and characterize ribosome-free terminals of the RER that form junctions between the reticulum and the plasma membrane in the basal and lateral regions of the acinar cells. Our findings indicate that different ER compartments specialize in different calcium-handling functions (Ca²⁺ release and Ca²⁺ reloading) and that any potential interference between Ca²⁺ release and Ca²⁺ influx is minimized by the spatial separation of the two processes.     

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca²⁺ homeostasis. We have previously shown that the IP₃ receptor (IP₃R) plays a role in elevating basal cytoplasmic Ca²⁺ and that pharmacological blockade of IP₃R restores muscle function. Moreover, we have shown that the IP₃R pathway negatively regulates autophagy by controlling mitochondrial Ca²⁺ levels. Nevertheless, it remains unclear whether IP₃R is involved in abnormal mitochondrial Ca²⁺ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP₃R was downregulated in mdx fibers. Pharmacological blockade of IP₃R in mdx fibers restored both increased mitochondrial Ca²⁺ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP₃R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP₃R1 activity with changes in autophagy, mitochondrial Ca²⁺ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP₃R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP₃R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.     

Systems Modeling of Ca Homeostasis and Mobilization in Platelets Mediated by IP3 and Store-Operated Ca Entry

Resting platelets maintain a stable level of low cytoplasmic calcium ([Ca²⁺]_cyt) and high dense tubular system calcium ([Ca²⁺]_dts). During thrombosis, activators cause a transient rise in inositol trisphosphate (IP₃) to trigger calcium mobilization from stores and elevation of [Ca²⁺]_cyt. Another major source of [Ca²⁺]_cyt elevation is store-operated calcium entry (SOCE) through plasmalemmal calcium channels that open in response to store depletion as [Ca²⁺]_dts drops. A 34-species systems model employed kinetics describing IP₃-receptor, DTS-plasmalemma puncta formation, SOCE via assembly of STIM1 and Orai1, and the plasmalemma and sarco/endoplasmic reticulum Ca²⁺-ATPases. Four constraints were imposed: calcium homeostasis before activation; stable in zero extracellular calcium; IP₃-activatable; and functional SOCE. Using a Monte Carlo method to sample three unknown parameters and nine initial concentrations in a 12-dimensional space near measured or expected values, we found that model configurations that were responsive to stimuli and demonstrated significant SOCE required high inner membrane electric potential (>−70 mV) and low resting IP₃ concentrations. The absence of puncta in resting cells was required to prevent spontaneous store depletion in calcium-free media. Ten-fold increases in IP₃ caused saturated calcium mobilization. This systems model represents a critical step in being able to predict platelets’ phenotypes during hemostasis or thrombosis.     

Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas

Summary We have previously shown that inositol-1,4,5-trisphosphate (IP₃) releases Ca²⁺ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983,Nature (London) 306:67–69). This observation suggests that IP₃ might provide the missing link between activation of the muscarinic receptor and Ca²⁺ release from intracellular stores during stimulation. In order to localize the intracellular IP₃-sensitive calcium pool, IP₃-induced Ca²⁺ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl₂. IP₃-induced Ca²⁺ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000×g, sucrose-density centrifugation, or MgCl₂ precipitation there was a close correlation of IP₃-induced Ca²⁺ release with the endoplasmic reticulum markers ribonucleic acid (r=0.96, 1.00, 0.91, respectively) and NADPH cytochromec reductase (r=0.63, 0.98, 090, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochromec oxidase (r=–0.64) and glutamate dehydrogenase (r=–0.75) and with the plasma membrane markers (Na⁺+K⁺)-ATPase (r=–0.81) and alkaline phosphatase (r=–0.77) in all fractions analyzed. IP₃-induced Ca²⁺ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca²⁺ from endoplasmic reticulum in pancreatic acinar cells.     

Vitellogenesis in Oncopeltusfasciatus: PLC/IP3, DAG/PK-C pathway triggered by CaM

In Oncopeltus fasciatus, evidence shown here indicates it is calmodulin (CaM) that activates phospholipase-C (PLC), beginning a signalling pathway necessary for endocytic uptake of yolk precursor molecules. Epithelial cell-produced CaM, transported to oocytes via gap junctions, has been shown to be required for receptor-mediated endocytic uptake of vitellogenins (Vgs, the protein precursors of yolk). To determine if CaM was directly or indirectly stimulating the phospholipase-C (PLC) signalling cascade and thus controlling Vg endocytosis we used a series of molecules known to inactivate various elements of the pathway. W-7 prevents CaM from interacting with other molecules. Neomycin isolates PIP₂ from PLC. U-73122 directly inactivates PLC. 2-APB blocks IP₃ receptors which would otherwise cause release of Ca²⁺. Verapamil and CdCl₂ block Ca²⁺ release channels. Staurosporin and calphostin are inhibitors of PK-C. 1-Hexadecyl-2-acetyl glycerol (HAG) binds to diacylglycerol (DAG). Through the use of these antagonists we show here that: (1) the activation of phospholipase-C in this system requires CaM. (2) Stimulated phospholipase-C converts PIP₂ into IP₃ and DAG. (3) IP₃ causes increase in cytosolic Ca²⁺. (4) DAG and Ca²⁺ each stimulate phosphokinase-C, resulting in endocytosis of Vgs.     

Interaction of caffeine-, IP3- and vanadate-sensitive Ca2+ pools in acinar cells of the exocrine pancreas

Summary Previous studies have shown the existence of functionally distinguishable inositol 1,4,5-trisphosphate- (IP₃) sensitive and IP₃-insensitive nonmitochondrial intracellular Ca²⁺ pools in acinar cells of the exocrine pancreas. For further characterization of Ca²⁺ pools, endoplasmic reticulum (ER) membrane vesicles were separated by Percoll gradient centrifugation which allowed us to distinguish five discrete fractions designatedP ₁ toP ₅ from the top to the bottom of the gradient. Measuring Ca²⁺ uptake and Ca²⁺ release with a Ca²⁺ electrode, we could differentiate three nonmitochondrial intracellular Ca²⁺ pools; (i) an IP₃-sensitive Ca²⁺ pool (IsCaP), vanadate- and caffeine-insensitive, (ii) a caffeine-sensitive Ca²⁺ pool (CasCaP), vanadate- and IP₃-insensitive, and (iii) a vanadate-sensitive Ca²⁺ pool (VasCaP), neither IP₃- nor caffeine-sensitive, into which Ca²⁺ uptake is mediated via a Ca²⁺ ATPase sensitive to vanadate at 10^–4 mol/liter. A fourth Ca²⁺ pool is neither IP₃- nor caffeine- or vanadate-sensitive. Percoll fractionP ₁ contained essentially the IsCaP, CasCaP and VasCaP and was mainly used for studies on Ca²⁺ uptake and Ca²⁺ release.When membrane vesicles were incubated in the presence of caffeine (2×10^–2 mol/liter), Ca²⁺ uptake up to the steady state [Ca²⁺] did not appear to be altered as compared to the control Ca²⁺ uptake. However, in control vesicles spontaneous Ca²⁺ release occurred after the steady state had been reached, whereas cfffeine-pretreated vesicles did not spontaneously release Ca²⁺. Addition of IP₃ at steady state [Ca²⁺] induced similar Ca²⁺ release followed by Ca²⁺ reuptake in both caffeine-pretreated and control vesicles. However, when caffeine was acutely added at steady state, Ca²⁺ was released from all Ca²⁺ pools including the IsCaP. Following Ca²⁺ reuptake after IP₃ had been added, a second addition of IP₃ to control vesicles induced further but smaller Ca²⁺ release, and a third addition resulted in a steady Ca²⁺ efflux by which all Ca²⁺ that had been taken up was released. This steady Ca²⁺ release started at a Ca²⁺ concentration between 5.5–8 ×10^–7 mol/liter and could also be induced by the IP₃ analogue inositol 1,4,5-trisphosphorothioate (IPS₃) or by addition of Ca²⁺ itself. Ruthenium red (10^–5 mol/liter) inhibited both caffeine-induced as well as Ca²⁺-induced but not IP₃-induced Ca²⁺ release. Heparin (100 g/m) inhibited IP₃-but not caffeine-induced Ca²⁺ release. The data indicate the presence of at least three separate Ca²⁺ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP. During Ca²⁺ uptake these Ca²⁺ pools appear to be separate. However, when steady state is reached, we assume that these Ca²⁺ pools come into contact and total Ca²⁺ release from all three pools can occur. The mechanism of this contact of Ca²⁺ pools is not clear but seems to be different from that induced by GTP in the presence of polyethylene glycol, which probably involves fusion of membranes.     

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic smooth muscle sarcoplasmic reticulum vesicles was examined using the calcium indicator antipyrylazo III. Calcium release was initiated by addition of inositol 1,4,5-trisphosphate (IP₃) to aortic vesicles 7 min after initiation of ATP-supported calcium uptake. Half-maximal calcium release occurred at 1 μM IP₃, with maximal calcium release amounting to 25±2% of the intravesicular calcium (n=12, 9 preparations). Ruthenium red (10–20 μM), which has been reported to block IP₃-induced calcium release from skeletal muscle sarcoplasmic reticulum, did not inhibit aortic IP₃-induced calcium release. Elevation of Mg²⁺ concentration from 0.06 to 7.8 mM inhibited aortic IP₃-induced calcium release 75%, which contrasts with the Mg²⁺-insensitive IP₃-induced calcium release from platelet reticular membranes. The IP₃-dependence of aortic calcium release suggested that Mg²⁺ acted as a noncompetitive inhibitor. Thus, aortic sarcoplasmic reticulum vesicles contain an IP₃-sensitive calcium pathway which is inhibited by millimolar concentrations of Mg²⁺, but which is not inhibited by Ruthenium red and so differs from the previously described IP₃-sensitive calcium pathways in skeletal muscle and platelet reticular membranes.     

Superresolution Localization of Single Functional IP3R Channels Utilizing Ca Flux as a Readout

The subcellular localization of membrane Ca²⁺ channels is crucial for their functioning, but is difficult to study because channels may be distributed more closely than the resolution of conventional microscopy is able to detect. We describe a technique, stochastic channel Ca²⁺ nanoscale resolution (SCCaNR), employing Ca²⁺-sensitive fluorescent dyes to localize stochastic openings and closings of single Ca²⁺-permeable channels within <50 nm, and apply it to examine the clustered arrangement of inositol trisphosphate receptor (IP₃R) channels underlying local Ca²⁺ puffs. Fluorescence signals (blips) arising from single functional IP₃Rs are almost immotile (diffusion coefficient <0.003 μm² s⁻¹), as are puff sites over prolonged periods, suggesting that the architecture of this signaling system is stable and not subject to rapid, dynamic rearrangement. However, rapid stepwise changes in centroid position of fluorescence are evident within the durations of individual puffs. These apparent movements likely result from asynchronous gating of IP₃Rs distributed within clusters that have an overall diameter of ∼400 nm, indicating that the nanoscale architecture of IP₃R clusters is important in shaping local Ca²⁺ signals. We anticipate that SCCaNR will complement superresolution techniques such as PALM and STORM for studies of Ca²⁺ channels as it obviates the need for photoswitchable labels and provides functional as well as spatial information.     

Comparison and characterization of retinal pericytes and retinal pigment epithelial cells on subcellular IP3-sensitive Ca2+ pools

Abstract. A comparative study of inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ mobilization in bovine retinal capillary pericytes (BRCP) and bovine retinal pigment epithelial cells (BRPE) was carried out. Both cells were permeabilized with saponin. The two cell types had similar basal levels of [Ca²⁺]i (130 nM for BRCP, 132 nM for BRPE) and responded to IP, in a dose-dependent manner. However, when stimulated by various concentrations of IP₃ (1–10 μM), the increase in [Ca²⁺]i of BRCP was always two- to threefold higher than that in BRPE. Subcellular-fractionation studies showed that a single population of IP₃ binding site with a high affinity and high specificity of IP₃ mainly localized to plasma membrane in these two cell types. Although the dissociation constant of specific [³²P]-IP₃ binding sites (Kd 1.9–2.8 nM) was similar, the profile of maximal binding capacity (Bmax) of each fraction was markedly different. In comparison, plasma membrane fractions of BRCP were with Bmax of 165 fmol/mg protein versus 90 fmol/mg protein for BRPE membranes. The ATP-dependent Ca²uptake and IP₃-dependent Ca²⁺ release were observed in the both plasma membrane fractions. With quantitative correlation, the membrane fraction (2 mg) of BRCP released 0.2 nmol Ca²⁺ whereas BRPE only released 0.07 nmol Ca²⁺ with the same dose of IP₃ (5 μM). The selectively higher density of IP, binding sites in coupling to the larger Ca²⁺-release in the membrane of BRCP suggests that the quantity of Ca²⁺ mobilized is determined by the spatially preferential distribution of membrane-associated IP₃ binding sites. These findings may provide an explanation for the differences observed between BRCP and BRPE in IP,-induced DNA replication.