The role of intracellular Ca2+ in the degranulation of skinned mast cells

The intracellular pH of rat peritoneal mast cells was slightly acidic and compound 48/80 induced a decrease in the cytoplasmic pH of these cells. By means of chemical skinning, it was revealed that perfusion with Ca²⁺ or inositol 1,4,5-trisphosphate (IP₃) induced degranulation dose-dependently in mast cells at concentrations higher than 10 M and 0.1 M, respectively. Na⁺ was essential for the release of histamine from mast cells. An assay based on the binding of⁴⁵Ca to mast cell fragments revealed that the intracellular Ca store of the mast cell is located in the endoplasmic reticulum. IP₃ liberated Ca from the endoplasmic reticulum.     

Antiallergic Effect of Epinastine (WAL 801 CL) on Immediate Hypersensitivity Reactions: (I) Elucidation of the Mechanism for Histamine Release Inhibition

Abstract

Epinastine caused an inhibition of histamine release from rat peritoneal mast cells induced by both antigen-antibody reaction and compound 48/80. Epinastine was similarly effective in inhibiting compound 48/80-induced histamine release not only from isolated rat peritoneal mast cells but also from rat mesenterial pieces. Also, histamine release from lung pieces obtained from actively sensitized guinea pigs after exposure to antigen challenge was markedly inhibited by epinastine. The drug was effective in inhibiting not only Ca²⁺ uptake into lung mast cells in actively sensitized guinea pigs but also Ca²⁺ release from the intracellular Ca store of rat peritoneal mast cells exposed to both compound 48/80 and substance P. No significant changes were observed in phosphodiesterase activity in rat peritoneal mast cells treated with epinastine, while adenylate cyclase activity was augmented by epinastine. Epinastine has no inhibitory effect on histamine release induced by Ca²⁺ or IP3 from permeabilized mast cells. However, the drug significantly and dose-dependently suppressed calmodulin activity suggesting that histamine release inhibition due to epinastine may be partly attributable to Ca²⁺-calmodulin dependent process(es). The drug caused no visible changes in thermodynamic behavior of lipids, either in order parameter or in differential scanning calorimetry, indicating that the drug has no influence on membrane fluidity.     

The role of membrane bound sialic acid of rat mast cells in histamine release induced by compound 48/80 and derivatives as well as calcium

Histamine release induced by compound 48/80 from rat mast cells is not dependent on extracellular Ca²⁺. Preincubation of mast cells with trypsin has only little effects on histamine release induced by this polycation. This work also demonstrates that histamine release induced by compound 48/80 and its analogues in the absence of extracellular Ca²⁺ depends on membrane bound sialic acid of the mast cell. Neuraminidase treatment of the cells in the presence of extracellular Ca²⁺ leads to histamine liberation. These findings suggest that sialic acid residues of the mast cell membrane constitute the site at which polycations exert their stimulatory actions of histamine liberation.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Early kinetics of Ca2+ fluxes and histamine release in rat mast cells stimulated with compound 48/80

The kinetics of Ca²⁺ uptake and efflux have been measured in rat peritoneal mast cells stimulated with compound 48/80 using rapid mixing and a silicone oil centrifugation technique. Responses at one-second time intervals were resolved beginning as early as three seconds after initial stimulation. The results clearly demonstrate that Ca²⁺ uptake occurs after the initiation of histamine release. Ca²⁺ efflux occurs simultaneously with histamine release. The implications of these findings are discussed and the technique is described.     

Inositol 1,4,5-triphosphate receptors and NAD(P)H mediate Ca signaling required for hypoxic preconditioning of hippocampal neurons

Exposure of neurons to a non-lethal hypoxic stress greatly reduces cell death during subsequent severe ischemia (hypoxic preconditioning, HPC). In organotypic cultures of rat hippocampus, we demonstrate that HPC requires inositol triphosphate (IP₃) receptor-dependent Ca²⁺ release from the endoplasmic reticulum (ER) triggered by increased cytosolic NAD(P)H. Ca²⁺ chelation with intracellular BAPTA, ER Ca²⁺ store depletion with thapsigargin, IP₃ receptor block with xestospongin, and RNA interference against subtype 1 of the IP₃ receptor all blunted the moderate increases in [Ca²⁺]_i (50–100 nM) required for tolerance induction. Increases in [Ca²⁺]_i during HPC and neuroprotection following HPC were not prevented with NMDA receptor block or by removing Ca²⁺ from the bathing medium. Increased NAD(P)H fluorescence in CA1 neurons during hypoxia and demonstration that NADH manipulation increases [Ca²⁺]_i in an IP₃R-dependent manner revealed a primary role of cellular redox state in liberation of Ca²⁺ from the ER. Blockade of IP₃Rs and intracellular Ca²⁺ chelation prevented phosphorylation of known HPC signaling targets, including MAPK p42/44 (ERK), protein kinase B (Akt) and CREB. We conclude that the endoplasmic reticulum, acting via redox/NADH-dependent intracellular Ca²⁺ store release, is an important mediator of the neuroprotective response to hypoxic stress.     

Inhibitory effects of oxatomide on intracellular Ca mobilization, Ca uptake and histamine release, using rat peritoneal mast cells

Oxatomide at concentrations of 0.01-10 microM inhibited not only an increase in 45Ca uptake but also the intracellular Ca2+ release induced by compound 48/80 in rat peritoneal mast cells. At higher concentrations, ketotifen or other calcium antagonists caused similar inhibitory effects. However, the inhibitory effect of oxatomide on the 45Ca uptake into rat neonatal heart cells was much weaker than that of verapamil. Through image processing of quin 2-stained mast cells, it was revealed that oxatomide inhibited Ca2+ release from the intracellular store. Although oxatomide alone did not affect cAMP and cGMP contents in sensitized guinea pig lung samples, the drug effectively prevented changes in the nucleotide contents evoked by antigen challenge. These results suggest that the inhibitory effect of oxatomide on histamine release may be caused by a combination of prevention of Ca uptake, which is highly selective toward mast cells; inhibition of Ca2+ release from the intracellular Ca store, and elevation of the cAMP content in mast cells.     

Ryanodine receptors in peritoneal mast cells: possible role in the modulation of exocytotic activity

Previous studies have shown that ryanodine in low concentrations and caffeine increase intracellular [Ca²⁺] in the absence of external Ca²⁺, suggesting Ca²⁺ release from intracellular stores through ryanodine receptors (RyR). In the present study we employed amperometry to examine the effect of RyR agonists and antagonists on serotonin release elicited with compound 48/80 (10 µg/ml). Ryanodine (1 µM) or, similarly, 20 mM caffeine, in the absence of external Ca²⁺, enhanced the amperometric response to compound 48/80 and all the individual amperometric spike parameters. Ryanodine (50 µM), dantrolene (20 µM) and tetracaine (50 µM), putative antagonists of the RyR, attenuated the amperometric response significantly, decreasing the number and frequency of events as well as their amplitude. This is the first demonstration that Ca²⁺ availability from RyR-operated Ca²⁺ sources may contribute to the modulation of secretory activity in mast cells, affecting not only the cellular exocytotic response, but also the characteristics of single amperometric events. Immunocytochemical labelling, using a monoclonal RyR antibody, confirmed the presence of RyR in this preparation.     

Sequential analysis of histamine release and intracellular Ca2+ release from murine mast cells.

Stimulation of murine peritoneal mast cells with compound 48/80 at a concentration of 1 microgram/ml elicited rather slow histamine release; the onset of release was observed 5 s after stimulation, and it reached a plateau at about 60 s. Both inositol-1,4,5-trisphosphate (IP3) and inositol-1,4-bisphosphate (IP2) contents increased to their maximum 5 s after stimulation. The IP3 content decreased to the control level more rapidly than that of IP2. Changes in the intracellular Ca2+ concentration of the quin 2 loaded mast cells were determined using a video-intensified microscopy system. The fluorescence intensity due to Ca-quin 2 complex increased rapidly after 48/80 stimulation in a Ca-free medium and reached the maximum at about 6-7 s. It became clear that the increase in IP3 content and the resulting Ca2+ release from the intracellular Ca store precede histamine release from murine mast cells.     

Changes in intracellular Ca2+ distribution of rat peritoneal mast cells before and after histamine release

Rat peritoneal mast cells were stained with quin 2, a fluorescent Ca²⁺ chelator. By means of a fluorescence microscope and real time image processer, it was revealed that the fluorescence derived from the Ca-quin 2 complex was weak in the area occupied by the nucleus and distributed unevenly in the cytoplasm of the resting cells so as to encompass the individual granules. When compound 48/80 or substance P was added in a Ca-free medium, the fluorescence intensity of quin 2 increased markedly all over the cell, suggesting that a large amount of Ca²⁺ was released from intracellular Ca stores. The increase in the fluorescence intensity produced by compound 48/80 or substance P in a Ca-free medium was inhibited by pretreatment with certain drugs eliciting an increase of c-AMP levels, such as dibutyryl c-AMP and theophylline, or by some anti-allergic drugs providing a membrane stabilizing action.     

The leading role of membrane Ca(2+)-ATPase in recovery of Ca(2+) homeostasis after glutamate shock

Combined blockade of Na⁺/Ca²⁺ exchange, Ca²⁺ uptake by mitochondria and endoplasmic reticulum usually does not prevent recovery of the basal level of intracellular Ca²⁺ after 1-min action of glutamate (100 M) or K⁺ (50 mM). However, replacement of Ca²⁺ with Ba²⁺, which cannot be transported by Ca²⁺-ATPase, considerably delayed the decrease in intracellular Ba²⁺ after its rise caused by glutamate or potassium application in all examined cells, which attest to an important role of Ca²⁺-ATPase in Ca²⁺ extrusion after the action of glutamate or K⁺.     

Endoplasmic reticulum–mitochondria coupling: local Ca2+ signalling with functional consequences

Plasma membrane store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channels are a widespread and conserved Ca²⁺ influx pathway, driving activation of a range of spatially and temporally distinct cellular responses. Although CRAC channels are activated by the loss of Ca²⁺ from the endoplasmic reticulum, their gating is regulated by mitochondria. Through their ability to buffer cytoplasmic Ca²⁺, mitochondria take up Ca²⁺ released from the endoplasmic reticulum by InsP₃ receptors, leading to more extensive store depletion and stronger activation of CRAC channels. Mitochondria also buffer Ca²⁺ that enters through CRAC channels, reducing Ca²⁺-dependent slow inactivation of the channels. In addition, depolarised mitochondria impair movement of the CRAC channel activating protein STIM1 across the endoplasmic reticulum membrane. Because they regulate CRAC channel activity, particularly Ca²⁺-dependent slow inactivation, mitochondria influence CRAC channel-driven enzyme activation, secretion and gene expression. Mitochondrial regulation of CRAC channels therefore provides an important control element to the regulation of intracellular Ca²⁺ signalling.     

Cytoskeletal modulation of the response to mechanical stimulation in human vascular endothelial cells

Possible interactions of cytoskeletal elements with mechanically induced membrane currents and Ca²⁺ signals were studied in human endothelial cells by using a combined patch-clamp and Fura II technique. For mechanical stimulation, cells were exposed to hypotonic solution (HTS). The concomitant cell swelling activates a Cl^– current, releases Ca²⁺ from intracellular stores and activates Ca²⁺ influx. To interfere with the cytoskeleton, cells were loaded either with the F-actin-stabilizing agent phalloidin (10 mol/l), or the F-actin-depolymerizing substance cytochalasin B (50 mol/l). These were administered either in the bath or the pipette solutions. The tubulin structure of the endothelial cells was modulated by taxol (50 mol/l), which supports polymerization of tubulin, or by the depolymerizing agent colcemid (10 mol/l) both applied to the bath. Immunofluorescence experiments show that under the chosen experimental conditions the cytoskeletal modifiers employed disintegrate the F-actin and microtubuli cytoskeleton. Neither of these cytoskeletal modifiers influenced the HTS-induced Cl^– current. Ca²⁺ release was not affected by cytochalasin B, taxol or colcemid, but was suppressed if the cells were loaded with phalloidin. Depletion of intracellular Ca²⁺ stores by thapsigargin renders the intracellular [Ca²⁺] sensitive to the extracellular [Ca²⁺], which is indicative of a Ca²⁺ entry pathway activated by store depletion. Neither cytochalasin B nor phalloidin affected this Ca²⁺ entry. We conclude that F-actin turnover or depolymerization is necessary for Ca²⁺ release by mechanical activation. The tubulin network is not involved. The Ca²⁺ release-activated Ca²⁺ entry is not modulated by the F-actin cytoskeleton.     

Depolymerisation and rearrangement of actin filaments during exocytosis in rat peritoneal mast cells: involvement of ryanodine-sensitive calcium stores

Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca²⁺-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca²⁺ in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca²⁺. Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca²⁺ with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca²⁺ from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.     

Inhibitory Effect of MY-1250 on Histamine Release from Rat Peritoneal Mast Cells and Guinea Pig Lung Fragments: The Elucidation of the Mechanism

When the effect of MY-1250 (5, 6-dihydro-7, 8-dimcthyl-4, 5dioxo-4 H-pyrano [3, 2-c] quinolinc-2-carboxylic acid) on histamine release from rat peritoneal mast cells induced by compound 48/80 was studied, MY-1250 caused a significant inhibition of histamine release at concentrations higher than 3 _pM. Furthermore, the compound inhibited not only 45C _a uptakc into the mast cells but also Ca²⁺ release from thc intraccllular Ca store at a concentration of 10 _pM in both cases. By contrast. MY-1250 did not affect either histarnine release from permeabilized mast cells induced by TPA, IP3 and GTPyS or Ca²⁺ release from the endoplasmic reticulum induced by IP3. In the chopped lung preparations, MY-1250 at doses of 10 and 100 _pM caused a significant inhibition in histarnine release from thc pieces of actively scnsitizcd guinea pigs exposed to antigen and simultaneously prevented a decrease in intracellular CAMP contents taking place in association with antigen-antibody reaction. No significant changes were effected by MY-1250 in a-chymotrypsin activity and phospholipasc A2 activity. Also, no antagonistic effects on LTD4 and PAF were observed.     

ER-localized bestrophin 1 activates Ca2+-dependent ion channels TMEM16A and SK4 possibly by acting as a counterion channel

Bestrophins form Ca²⁺-activated Cl^? channels and regulate intracellular Ca²⁺ signaling. We demonstrate that bestrophin 1 is localized in the endoplasmic reticulum (ER), where it interacts with stromal interacting molecule 1, the ER-Ca²⁺ sensor. Intracellular Ca²⁺ transients elicited by stimulation of purinergic P2Y₂ receptors in HEK293 cells were augmented by hBest1. The p21-activated protein kinase Pak2 was found to phosphorylate hBest1, thereby enhancing Ca²⁺ signaling and activation of Ca²⁺-dependent Cl^? (TMEM16A) and K⁺ (SK4) channels. Lack of bestrophin 1 expression in respiratory epithelial cells of mBest1 knockout mice caused expansion of ER cisterns and induced Ca²⁺ deposits. hBest1 is, therefore, important for Ca²⁺ handling of the ER store and may resemble the long-suspected counterion channel to balance transient membrane potentials occurring through inositol triphosphate (IP₃)-induced Ca²⁺ release and store refill. Thus, bestrophin 1 regulates compartmentalized Ca²⁺ signaling that plays an essential role in Best macular dystrophy, inflammatory diseases such as cystic fibrosis, as well as proliferation.     

Measurement of Ca2+-release-dependent inward current reveals two distinct components of Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ current (L-type) and inward current caused by Ca²⁺ release from the sarcoplasmic reticulum and carried by electrogenic Na⁺/Ca²⁺ exchange have been measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp techniques. The pipette solution, used for internal dialysis of the cells, contained a high concentration, 60 mM or 25 mM, of citrate as a non-saturable low-affinity Ca²⁺-chelating compound. It has been shown previously that Ca²⁺-release-dependent inward current under these conditions is carried by electrogenic Na⁺/Ca²⁺ exchange. Furthermore, Ca²⁺-release-dependent inward current (the release signal) can be completely separated from triggering Ca²⁺ current if brief depolarizations for activating I _Ca are used. In the majority of cells that did not produce spontaneous Ca²⁺ release, conditions could be found that caused the release signal to be split into two components: an early component of variable amplitude and a late component of rather constant amplitude. The delay of the late component with regard to triggering I _Ca was inversely related to the amplitude of the first one. Below a certain amplitude of the first component, the second one failed to be elicited. This suggests the second component to be triggered by the first one. Weakly Ca²⁺-buffered cells produced spontaneous Ca²⁺ release, resulting in irregular transient inward currents at constant membrane-holding potential. Synchronization by trains of step depolarizations unmasked two components also in the spontaneous release signals. In none of the cells studied was any indication of more than two components of the release signal detected. The results are discussed in terms of two distinct compartments of sarcoplasmic reticulum with different properties of Ca²⁺ release.Supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

Involvement of Na/Ca Exchange and Intracellular Mobilized Ca2+ in Na,K-Pump-Mediated Control of Depression of the Cholinosensitivy of Common Snail Neorons Using a Cellular Analog of Habituation

The role of Na/Ca exchange and intracellular mobilized Ca²⁺ in modifying the depression of defensive behavior command neuron cholinosensitivity induced by the the Na,K pump inhibitor ouabain was studied in common snails using a cellular analog of habituation. Integral transmembrane acetylcholine-evoked currents (ACh currents) were recorded using a two-electrode membrane potential clamping technique. Decreases in neuron cholinosensitivity in the cellular analog of habituation were assessed in terms of the depth of depression of the amplitude of ACh currents during rhythmic local application of acetylcholine (with interstimulus intervals of 2–4 min) to the somatic membrane. The Na/Ca exchange inhibitor benzamyl (applied extracellularly, 15–35 M) and two specific endoplasmic reticulum Ca-ATPase inhibitors, cyclopiazonic acid and thapsigargin (applied intracellularly, 0.1 mM) prevented modification of depression of the ACh current by ouabain (100 M). It is concluded that Na/Ca exchange and the release of mobilized Ca²⁺ from intracellular calcium depots are involved in the mechanism by which the Na,K pump controls the depression of neuron cholinosensitivity in the cellular analog of habituation.     

Mechanism of acidic pH‐induced contraction in spontaneously hypertensive rat aorta: role of Ca2+ release from the sarcoplasmic reticulum

Aim: This study was conducted to investigate the mechanism of acidic pH‐induced contraction (APIC) with regard to Ca²⁺ handling using isometric tension recording experiments. Results: Decreasing extracellular pH from 7.4 to 6.5 produced a marked and sustained contraction of spontaneously hypertensive rat (SHR) aorta, that was 128.7 ± 2.0% of the 64.8 mm KCl‐induced contraction. Verapamil, an inhibitor of voltage‐dependent Ca²⁺ channels (VDCC) significantly inhibited the APIC. In Ca²⁺‐deficient solution, sustained contraction induced by acidic pH was abolished completely, while a transient contraction was still observed suggesting the release of Ca²⁺ from intracellular site. Ryanodine (1 μm ), a ryanodine receptor blocker, and 10 μm cyclopiazonic acid (CPA; a sarco/endoplasmic reticulum Ca²⁺ ATPase inhibitor) abolished the transient contraction induced by acidosis. In normal Ca²⁺‐containing solution, ryanodine significantly decreased the rate of rise as well as maximum level of APIC. Interestingly, ryanodine and CPA showed an additive inhibitory effect with verapamil and the combined treatment of ryanodine or CPA with verapamil nearly abolished the APIC. Conclusions: It is concluded that acidic pH induces Ca²⁺ release from ryanodine/CPA‐sensitive store of sarcoplasmic reticulum in SHR aorta. This Ca²⁺ plays an important role in the facilitation of the rate of rise of APIC, as well as contributing to the sustained contraction via a mechanism which is independent of Ca²⁺ influx through VDCC.     

Evidence for a Na+/Ca2+ exchange mechanism in rat peritoneal mast cells

 Mast cells lose their ability to secrete when incubated in nominally Ca²⁺-free medium, but the Na⁺/K⁺ pump inhibitor ouabain prevents this loss, suggesting a Na⁺ dependence of the Ca²⁺ gradient in rat mast cells. The present study includes measurements of histamine release from cell suspensions, and fura-2/AM and current-clamp experiments on single cells. KB-R7943, an inhibitor of the reverse mode of the Na⁺/Ca²⁺ exchanger, 2,4-dichlorobenzamil and La³⁺ counteracted the increase in histamine release induced by ouabain in a dose-dependent manner. The Ca²⁺ response to compound 48/80 was reduced by preincubation of the mast cells for 30 min in nominally Ca²⁺-free medium. This reduction was partly prevented by ouabain or by a low extracellular Na⁺ concentration. Superfusion of cells with a medium containing a low Na⁺concentration resulted in a hyperpolarization of the cells of 38.6±8.6 mV, n=8, followed by a repolarization after the superfusion had ceased (45.7±5.9 mV, n=4). KB-R7943 reduced the hyperpolarization and repolarization induced by a low extracellular Na⁺ concentration to 15.5±2.9 mV (n=7) and 0.2±3.4 mV (n=3), respectively. These results are consistent with the presence of a Na⁺/Ca²⁺ exchanger in rat peritoneal mast cells. Received: 3 July 1998 / Received after revision and accepted: 11 August 1998