COVID-19 transmission in dental and oral/maxillofacial surgical practice during pandemic: questionnaire survey in 51 university hospitals in Japan.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has become a major public health problem. Dental procedures that generate aerosols are considered to impose a high risk of infection; therefore, dental professionals, such as dentists and dental hygienists, may be at high risk of viral transmission. However, few studies have reported COVID-19 clusters in dental care settings. AIM: To investigate whether dental and oral/maxillofacial procedures are associated with the occurrence of COVID-19 clusters and measures taken to prevent nosocomial infection in dental clinics. METHODS: An online questionnaire survey on clinical activities (administrative control), infection control measures (environmental/engineering control, personal protective equipment, etc.), and confirmed or probable COVID-19 cases among patients and clinical staff was administered to the faculties of the dental and oral/maxillofacial surgical departments of university hospitals. FINDINGS: Fifty-one faculty members completed the questionnaire. All members were engaged in the treatment of dental and oral surgical outpatients and actively implemented standard precautions. Fourteen faculty members treated patients with COVID-19, but no infections transmitted from the patients to the medical staff were observed. In seven facilities, patients were found to have the infection after treatment (medical staff came in close contact), but there was no transmission from patients to medical staff. Four facilities had medical staff with infections, but none of them exhibited disease transmission from staff to patients. CONCLUSION: COVID-19 clusters are unlikely to occur in dental and oral surgical care settings if appropriate protective measures are implemented.

Sequential-replenishment mechanism of exocytosis in pancreatic acini.

Here we report exocytosis of zymogen granules, as examined by multiphoton excitation imaging in intact pancreatic acini. Cholecystokinin induces Ca 2+ oscillations that trigger exocytosis when the cytosolic Ca 2+ concentration exceeds 1 microM. Zymogen granules fused with the plasma membrane maintain their Omega-shaped profile for an average of 220 s and serve as targets for sequential fusion of granules that are located within deeper layers of the cell. This secondary exocytosis occurs as rapidly as the primary exocytosis and accounts for most exocytotic events. Granule-granule fusion does not seem to precede primary exocytosis, indicating that secondary fusion events may require a plasma-membrane factor. This sequential-replenishment mechanism of exocytosis allows the cell to take advantage of a large supply of fusion-ready granules without needing to transport them to the plasma membrane.

Abnormal features in skeletal muscle from mice lacking mitsugumin29.

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.

Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns.

Inositol 1,4,5-trisphosphate (IP3) is a second messenger that elicits complex spatiotemporal patterns of calcium ion (Ca2+) mobilization and has essential roles in the regulation of many cellular functions. In Madin-Darby canine kidney epithelial cells, green fluorescent protein-tagged pleckstrin homology domain translocated from the plasma membrane to the cytoplasm in response to increased concentration of IP3. The detection of translocation enabled monitoring of IP3 concentration changes within single cells and revealed spatiotemporal dynamics in the concentration of IP3 synchronous with Ca2+ oscillations and intracellular and intercellular IP3 waves that accompanied Ca2+ waves. Such changes in IP3 concentration may be fundamental to Ca2+ signaling.

Glia-synapse interaction through Ca2+-permeable AMPA receptors in Bergmann glia.

Glial cells express a variety of neurotransmitter receptors. Notably, Bergmann glial cells in the cerebellum have Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) assembled without the GluR2 subunit. To elucidate the role of these Ca2+-permeable AMPARs, we converted them into Ca2+-impermeable receptors by adenoviral-mediated delivery of the GluR2 gene. This conversion retracted the glial processes ensheathing synapses on Purkinje cell dendritic spines and retarded the removal of synaptically released glutamate. Furthermore, it caused multiple innervation of Purkinje cells by the climbing fibers. Thus, the glial Ca2+-permeable AMPARs are indispensable for proper structural and functional relations between Bergmann glia and glutamatergic synapses.

Visualization of IP(3) dynamics reveals a novel AMPA receptor-triggered IP(3) production pathway mediated by voltage-dependent Ca(2+) influx in Purkinje cells.

IP(3) signaling in Purkinje cells is involved in the regulation of cell functions including LTD. We have used a GFP-tagged pleckstrin homology domain to visualize IP(3) dynamics in Purkinje cells. Surprisingly, IP(3) production was observed in response not only to mGluR activation, but also to AMPA receptor activation in Purkinje cells in culture. AMPA-induced IP(3) production was mediated by depolarization-induced Ca(2+) influx because it was mimicked by depolarization and was blocked by inhibition of the P-type Ca(2+) channel. Furthermore, trains of complex spikes, elicited by climbing fiber stimulation (1 Hz), induced IP(3) production in Purkinje cells in cerebellar slices. These results revealed a novel IP(3) signaling pathway in Purkinje cells that can be elicited by synaptic inputs from climbing fibers.

Subunit composition at the single-cell level explains functional properties of a glutamate-gated channel.

The diversity of known glutamate-gated channels has been markedly increased by the discovery of multiple subunits and their spliced and edited variants. These subunits can potentially form different oligomeric complexes with diverging properties. A crucial question is therefore to determine the actual subunit composition of naturally occurring glutamate receptors. We have coupled patch-clamp recordings and reverse transcription followed by PCR amplification to correlate the presence of mRNAs for each subunit and the functional properties of native glutamate receptors at the single-cell level. In a homogeneous population of functionally identified hippocampal neurons (type II) in culture bearing a glutamate receptor of the AMPA subtype with a high calcium permeability, we found that, among the multiple subunits, only two, the flop forms of GluR1 and GluR4, were expressed. In particular, GluR2 was never detected. This composition explains the uncommon properties of AMPA receptors in type II neurons.

Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons.

Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure-function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.

Efficacy and Safety of Biosimilar Filgrastim for Cord Blood Transplantation: A Single-Institution Retrospective Analysis.

BACKGROUND: Filgrastim has been widely used for hematopoietic recovery after hematopoietic stem cell transplantation. Recently, biosimilar filgrastim (BF) has been approved for the same indications as for the originator filgrastim (OF). However, evidence of the efficacy and safety of BF for unrelated cord blood transplant (CBT) is unreported. Therefore, we evaluated the efficacy and safety of BF and OF (historical control) for CBT. METHODS: Twenty-two consecutive patients with hematologic malignant tumors were assessed. Patients received BF (n = 12) or OF (n = 10) from day 1 after CBT for hematopoietic recovery. The time to hematopoietic recovery, total filgrastim dose, duration of filgrastim administration, total transfusion units, incidences of engraftment, documented infection, febrile neutropenia, acute and chronic graft-vs-host disease, incidence and severity of adverse events, hospitalization duration, and 100-day and 1-year overall survival were evaluated. RESULTS: The median total dose of BF and OF used for hematopoietic recovery were 9.68 and 10.80 mg, respectively. There were no significant between-group differences in time to hematopoietic recovery, total filgrastim dose, duration of filgrastim administration, total transfusion units, incidences of engraftment, documented infection, febrile neutropenia, acute and chronic graft-vs-host disease, incidence and severity of adverse events, hospitalization duration, and overall survival. Multivariate analysis demonstrated that filgrastim type was not a significant factor for neutrophil recovery. Median total filgrastim costs per patient were 446,405 and 910,320 yen for BF and OF, respectively. CONCLUSIONS: BF is as safe and effective as OF for hematopoietic recovery after CBT. BF is a useful option for CBT owing to its economic benefits.

A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo.

We have developed a method for visualizing phosphorylation of proteins in living cells using a novel fluorescent indicator composed of two green fluorescent protein (GFP) variants joined by the kinase-inducible domain (KID) of the transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Phosphorylation of KID by the cAMP-dependent protein kinase A (PKA) decreased the fluorescence resonance energy transfer (FRET) among the flanking GFPs. By transfecting COS-7 cells with an expression vector encoding this indicator protein (termed ART for cAMP-responsive tracer), we were able to visualize activation dynamics of PKA in living cells.

Dynamic properties of bound water studied through macroscopic water relaxations in concentrated protein solutions.

Magnetization transfer experiments using an off-resonance irradiation technique were performed on bovine serum albumin solutions by varying the irradiation frequency and concentration. Observed macroscopic magnetizations of water protons were well expressed by the rate equations of populations for spins, which gave the tumbling time of protein protons and the intrinsic relaxation rates of water and protein protons. These parameters conformed to a model of rapid-exchange water system with bound water molecules in the interior of the protein that interact with protein protons. Analysis of the data enabled the separation of relaxation rates into the respective contributions by the interior bound water and water in the hydration layer at the protein surface, and determined the amounts and the average correlation times of these water fractions. The average residence time of the interior bound water with respect to exchange with the bulk water was found to be (5 +/- 2).10(-6) s. The estimation of the hydration layer showed excellent agreement with the amount measured by a thermodynamical method.

Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci.

Ca2+ dependence of the inositol 1,4,5-trisphosphate (IP3)-induced Ca release was studied in saponin-skinned smooth muscle fiber bundles of the guinea pig taenia caeci at 20-22 degrees C. Ca release from the skinned fiber bundles was monitored by microfluorometry of fura-2. Fiber bundles were first treated with 30 microM ryanodine for 120 s in the presence of 45 mM caffeine to lock open the Ca-induced Ca release channels which are present in approximately 40% of the Ca store of the smooth muscle cells of the taenia. The Ca store with the Ca-induced Ca release mechanism was functionally removed by this treatment, but the rest of the store, which was devoid of the ryanodine-sensitive Ca release mechanism, remained intact. The Ca2+ dependence of the IP3-induced Ca release mechanism was, therefore, studied independently of the Ca-induced Ca release. The rate of IP3-induced Ca release was enhanced by Ca2+ between 0 and 300 nM, but further increase in the Ca2+ concentration also exerted an inhibitory effect. Thus, the rate of IP3-induced Ca release was about the same in the absence of Ca2+ and at 3 microM Ca2+, and was about six times faster at 300 nM Ca2+. Hydrolysis of IP3 within the skinned fiber bundles was not responsible for these effects, because essentially the same effects were observed with or without Mg2+, an absolute requirement of the IP3 phosphatase activity. Ca2+, therefore, is likely to affect the gating mechanism and/or affinity for the ligand of the IP3-induced Ca release mechanism. The biphasic effect of Ca2+ on the IP3-induced Ca release is expected to form a positive feedback loop in the IP3-induced Ca mobilization below 300 nM Ca2+, and a negative feedback loop above 300 nM Ca2+.

Effects of adenine nucleotides on inositol 1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells.

Effects of adenine nucleotides on the inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanism were studied in smooth muscle cells of the guinea pig portal vein. A microfluorometry method of fura-2 was used to measure Ca release from saponin-skinned thin muscle strips (width approximately 200 microns, thickness 50-70 microns, length 2-3 mm). About 80% of ionomycin-releasable Ca store was sensitive to IP3, of which approximately 20% was also sensitive to caffeine. The rate of Ca release by 0.1 microM IP3 depended biphasically on ATP concentration in the absence of Mg2+; it was dose-dependently enhanced by ATP up to approximately 0.5 mM, and above this concentration the enhancement became smaller. However, the decline of enhancement of the IICR at the higher ATP concentrations was absent at IP3 concentrations greater than 1 microM. This suggests competitive antagonism between IP3 and ATP. Clear effects of ATP were observed not only at pCa 7 or 8, where the Ca-induced Ca release was not activated, but after a ryanodine treatment to excise the functional compartment that possessed the Ca-induced Ca release mechanism. ATP had no effect on the rate of Ca leakage in the absence of IP3 even at pCa 5.5 after the ryanodine treatment. Therefore, ATP has direct biphasic effects on the IP3-induced Ca release mechanism. The Ca release induced by 0.1 microM IP3 at pCa 7 was potentiated not only by ATP, but by 0.5 mM ADP, AMP, or beta, gamma-methyleneadenosine 5'-triphosphate. 0.5 mM GTP had only a little effect on the IP3-induced Ca release. These results extend the functional similarities between Ca- and IP3-induced Ca release mechanisms in that adenine nucleotides enhance Ca release. Millimolar concentration of ATP, which is present physiologically, will shift the dose-response relation of IP3 toward the higher IP3 concentration and enhance the maximal effect of IP3. Thus, ATP is expected to assist the Ca release by higher concentrations of IP3 while having less effect on the Ca release by low levels of IP3. These effects of ATP may be important in the switching of Ca release from the intracellular Ca store by IP3.

Calcium-induced calcium release mechanism in guinea pig taenia caeci.

Fura-2 was used to measure the amount of Ca released from the intracellular Ca store of a saponin-skinned smooth muscle fiber bundle of the guinea pig taenia caeci (width, 150-250 microns) placed in a capillary cuvette at 20-22 degrees C. The amount of Ca actively loaded into the store was assayed when released by the application of 50 mM caffeine and/or 10 microM inositol 1,4,5-trisphosphate (IP3) in the absence of ATP, and was found to have a biphasic dependence on the loading [Ca2+] with a peak near pCa 6. After Ca loading at pCa 6, IP3 released almost all the releasable Ca, whereas caffeine discharged Ca from only approximately 40% of the store. The maximum amount of Ca in the store was some 220 mumol/liter cell water. Ca in the caffeine-releasable store was released approximately exponentially to zero with time when Ca2+ was applied in the absence of ATP, and the rate constant of the Ca-induced Ca release (CICR) increased steeply with the concentration of Ca2+ applied. Increase in [Mg2+] (0.5-5.0 mM) or decrease in pH (7.3-6.7) shifted the relation between pCa and the rate of CICR roughly in parallel toward the lower pCa. An adenine nucleotide increased the rate of the CICR, but it did not change the range of effective [Ca2+]. 5 mM caffeine greatly enhanced the CICR mechanism, making it approximately 30 times more sensitive to [Ca2+]. However the drug had no Ca-releasing action in the absence of Ca2+. Procaine in millimolar concentrations inhibited the rate of the CICR. These properties are similar to those of the skeletal muscle CICR and ryanodine receptor channels. Rates of the CICR under a physiological ionic milieu were estimated from the results, and a [Ca2+] greater than 1 microM was expected to be necessary for the activation of the Ca release. This Ca sensitivity seems too low for the CICR mechanism to play a primary physiological role in Ca mobilization, unless assisted by other mechanisms.

Blue-Light-Induced Shrinking of Protoplasts from Maize Coleoptiles and Its Relationship to Coleoptile Growth.

Protoplasts isolated from red-light-grown maize (Zea mays L.) coleoptiles shrank transiently upon brief exposure (e.g. 30 s) to blue light under background irradiation with red light. The maximal volume reduction (about 4% at a saturating fluence) occurred about 5 min after blue-light stimulation. The response was prevented by the anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Red light and far-red light did not induce any comparable response. Protoplasts of different sizes and those isolated from different coleoptile positions showed similar responses. After treatment with a saturating blue-light pulse, the protoplasts became responsive to a second pulse and gained full responsiveness within 5 min, suggesting that the photoreceptor system involves a dark-reversible component. The response to continuous blue light was also found to be transient. The protoplast volume was reduced during about 6 to 9 min of irradiation and returned within the next 30 min to the control level. The response to continuous blue light was saturated at 30 [mu]mol m-2 s-1. However, when the fluence rate was enhanced 10-fold after a period of irradiation at 30 [mu]mol m-2 s-1, the protoplasts showed another shrinking response. These and other kinetic results indicate that the photoreceptor system undergoes a photosensory adaptation. Growth in different zones of the coleoptile was inhibited by blue light transiently after pulse stimulation, as well as during continuous stimulation. It was concluded that the observed protoplast shrinking is related to the blue-light-induced inhibition of coleoptile growth.

Auxin-growth relationships in maize coleoptiles and pea internodes and control by auxin of the tissue sensitivity to auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied 3H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.

Small molecule-based laser inactivation of inositol 1,4,5-trisphosphate receptor.

BACKGROUND: Chromophore-assisted laser inactivation (CALI) is a powerful method for the study of in situ protein function in cellular processes. By using CALI, it is possible to abrogate the function of a target protein with unprecedented spatial and temporal resolution. However, CALI has some limitations, which restrict wider biological application, owing mainly to the use of antibody for target recognition. To circumvent the limitations, we have developed small molecule-based CALI (smCALI). RESULTS: The inositol 1,4,5-trisphosphate receptor (IP3R) was selected as the target protein and a malachite green-conjugated IP3 analog, MGIP3, was used as a small-molecular probe. We examined the effect of MGIP3-based CALI on Ca2+ release via IP3R using permeabilized smooth muscle cells. When the cells were treated with MGIP3 followed by laser irradiation, the IP3-induced Ca2+ release rate was decreased in a concentration- and irradiation time-dependent manner. The effect was specific for IP3R, because the Ca2+ uptake function of the co-localized sarco/endoplasmic reticulum Ca2+-ATPase was not affected. CONCLUSIONS: IP3R was specifically inactivated by smCALI using MGIP3. The efficiency of inactivation was calculated to be substantially greater than that of antibody-based CALI. The efficient and specific inactivation of IP3R would allow us to obtain an insight into spatiotemporal roles of IP3R in various cell functions. Our results may be considered to be a first step for a wider application of smCALI as a useful method to study spatiotemporal protein functions.

Influence of arthroscopically observed fibrous adhesions before and after joint irrigation on clinical outcome in patients with chronic closed lock of the temporomandibular joint.

The aim of this study was to investigate the changes in the state of arthroscopically observed fibrous adhesions (FA) after visually guided irrigation (VGIR) and the influence of FA on clinical outcome in patients with chronic closed lock of the temporomandibular joint (TMJ). Forty-eight TMJs of 48 patients with unilateral chronic closed lock were enrolled in this study. All 48 joints underwent VGIR twice. After the first VGIR (immediately before the second VGIR), clinical outcome was assessed as regards maximal interincisal opening (MIO) and self-evaluated TMJ pain (VAS). Thirty patients were symptom-free (good outcome group) and the remaining 18 patients had symptoms (poor outcome group). In each group, the changes of the MIO, VAS and severity of FA (FA score) after the first VGIR were studied. The influence of FA score in the first and second VGIR on clinical outcome was analyzed by logistic regression analysis. There was no joint with disappearance or reduction of FA after the first VGIR. In both groups, MIO and VAS were significantly improved after the first VGIR even though the state of FA became significantly worse. The multivariate logistic regression analysis showed that the risk of poor outcome for FA scores in the first and second VGIR were 0.89-times (95% CI: 0.33-2.40, P=0.82) and 1.76-times (95% CI: 0.54-5.73, P=0.35), respectively. The dose-response relationships between FA scores in the first or second VGIR were not significant. In conclusion, our results indicate that the presence of FA or a postoperative worsening of FA (including postoperative new FA formation) seems not to affect the clinical outcome as regards MIO and VAS in patients with chronic closed lock of the TMJ.

Impaired calcium regulation of smooth muscle during chronic vasospasm following subarachnoid hemorrhage.

The intracellular calcium level was determined in the canine basilar artery to investigate whether Ca2+ regulation of its smooth muscle is altered during chronic vasospasm following subarachnoid hemorrhage. A double-hemorrhage model was used. The occurrence of vasospasm was confirmed angiographically 7 days after initial hemorrhage. The intracellular calcium concentration ([Ca2+]i) of smooth muscle was measured using Fura-2. Fluorescence to excitation at 340 and 356 nm was monitored and the ration R340/356 was used as the indicator of [Ca2+]i. When the extracellular calcium concentration ([Ca2+]e) was increased from pCa 8 to 2, [Ca2+]i also increased. In the spastic arteries, the [Ca2+]e - [Ca2+]i curve was elevated as compared with the normal arteries. Treatment with ionomycin elevated the curve in the normal group, but it had little effect in the spastic arteries. Values of [Ca2+]i, calculated in multiples of Kd, were greater in the spastic arteries. Diltiazem (10(-5) mol/L) partially suppressed the augmented [Ca2+]i signal in the spastic arteries, whereas it did not affect the curve in the control group. These results indicate that the calcium regulation of smooth muscle is impaired after subarachnoid hemorrhage, which may contribute to the pathogenesis of chronic vasospasm.

Presence of functionally different compartments of the Ca2+ store in single intestinal smooth muscle cells.

Studies in smooth muscle bundles have shown the presence of functionally different compartments of Ca2+ store, one (S alpha) sensitive to both caffeine and inositol 1,4,5-trisphosphate (IP3), and the other (S beta) sensitive only to IP3. Ca2+ release in isolated single smooth muscle cells from guinea pig taenia caeci was studied to see if both compartments exist within a cell. Responses to caffeine and carbachol were consistently observed but were abolished after treatment with ryanodine, while intracellular application of IP3 induced Ca2+ release after the treatment, albeit smaller in size than control. Thus S alpha and S beta coexist in a single smooth muscle cell and agonist-induced Ca2+ release requires whole store to be loaded with Ca2+.