Diffusion rates of cell surface antigens of mouse-human heterokaryons. III. Regulation of lateral diffusion rates by calcium ions

In mouse-human heterokaryons, the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membrane is enhanced by treatment of parent cells with ouabain. Ouabain treatment is ineffective if the medium lacks calcium ion, or if Verapamil, a blocker of calcium channels, is present. The divalent ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 also enhances lateral diffusion of MHC antigens, to the same extent as ouabain, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 is effective only if calcium is present in the medium. Thus it appears that increased levels of cell calcium release constraints to lateral diffusion of MHC antigens.     

The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187

Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.     

Calcium control of ciliary reversal in ionophore-treated and ATP- reactivated comb plates of ctenophores

Previous work showed that ctenophore larvae swim backwards in high-KCl seawater, due to a 180 degrees reversal in the direction of effective stroke of their ciliary comb plates (Tamm, S. L., and S. Tamm, 1981, J. Cell Biol., 89: 495-509). Ion substitution and blocking experiments indicated that this response is Ca2+ dependent, but comb plate cells are innervated and presumably under nervous control. To determine whether Ca2+ is directly involved in activating the ciliary reversal mechanism and/or is required for synaptic triggering of the response, we (a) determined the effects of ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+ on the beat direction of isolated nerve-free comb plates dissociated from larvae by hypotonic, divalent cation-free medium, and (b) used permeabilized ATP- reactivated models of comb plates to test motile responses to known concentrations of free Ca2+. We found that 5 microM {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and 10 mM Ca2+ induced dissociated comb plate cells to beat in the reverse direction and to swim counterclockwise in circular paths instead of in the normal clockwise direction. Detergent/glycerol-extracted comb plates beat actively in the presence of ATP, and reactivation was reversibly inhibited by micromolar concentrations of vanadate. Free Ca2+ concentrations greater than 10(-6)M caused reversal in direction of the effective stroke but no significant increase in beat frequency. These results show that ciliary reversal in ctenophores, like that in protozoa, is activated by an increase in intracellular free Ca2+ ions. This allows the unique experimental advantages of ctenophore comb plate cilia to be used for future studies on the site and mechanism of action of Ca2+ in the regulation of ciliary motion.     

Immunologic mast cell-mediated responses and histamine release are attenuated by heparin.

Heparin has been shown to act as a competitive inhibitor of inositol 1,4,5-triphosphate (InsP3) receptors in various cell types. Because InsP3 is one of the second messengers involved in stimulus-secretion coupling in mast cells, it is possible that heparin may inhibit mast cell-mediated reactions. Therefore, in allergic sheep, we tested this hypothesis in two mast cell-mediated reactions induced by immunologic and nonimmunologic stimuli: immediate cutaneous reaction (ICR) and acute bronchoconstrictor response (ABR). In 12 sheep allergic to Ascaris suum antigen, the surface area of the skin wheal was determined 20 min after intradermal injection (0.05 ml) of increasing concentrations of specific antigen, compound 48/80, and histamine, without and after pretreatment with heparin (100, 300, or 1,000 U/kg i.v.). Antigen, compound 48/80, and histamine produced concentration-dependent increases in ICR. Heparin "partially" inhibited the ICR to antigen and compound 48/80 in a dose-dependent manner without modifying the ICR to histamine. The heparin preservative benzyl alcohol was ineffective. In 11 additional sheep, specific lung resistance was measured before and after inhalation challenges with antigen, compound 48/80, and histamine without and with aerosol heparin pretreatment (1,000 U/kg). Heparin blocked the antigen- and compound 48/80-induced bronchoconstriction without modifying the airway effects of histamine. In isolated human uterine mast cells, heparin inhibited the anti-immunoglobulin E- but not the calcium ionophore- (A23187) induced histamine release. These data suggest that heparin inhibits the ICR and ABR induced by stimuli that produce immunologic and nonimmunologic mast cell degranulation without attenuating the effects of histamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indoleamines and calcium channels influence morphogenesis in in vitro cultures of Mimosa pudica L.

The present article reports the interplay of indoleamine neurohormones viz. serotonin, melatonin and calcium channels on shoot organogenesis in Mimosa pudica L. In vitro grown nodal segments were cultured on MS medium with B5 vitamins containing Serotonin (SER) and Melatonin (MEL) at 100 µM and indoleamine inhibitors viz. serotonin to melatonin conversion inhibitor p-chlorophenylalanine (p-CPA) at 40 µM, serotonin reuptake inhibitor (Prozac) 20 µM. In another set of experiment, calcium at 5 mM, calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) 100 µM, and calcium channel blocker varapamil hydrochloride (1 mM) a calcium chelator EGTA (100 µM) were administered to the culture medium. The percentage of shoot multiplication, endogenous MEL and SER were monitored during shoot organogenesis. At 100 µM SER and MEL treatment 60% and 70% explants responded for shoot multiplication respectively. Medium supplemented with either SER or MEL along with calcium (5 mM) 75%–80% explants responded for organogenesis. SER or MEL along with calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) at 100 µM 70% explants responded for shoot multiplication. p-CPA, prozac, verapamil and EGTA, shoot multiplication was reduced and endogenous pools of SER, MEL decreased by 40–70%. The results clearly demonstrated that indoleamines and calcium channels positively influenced shoot organogenesis in M. pudica L.     

Ethyl Pyruvate Inhibits HMGB1 Phosphorylation and Release by Chelating Calcium

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca²⁺ and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K⁺-induced transient [Ca²⁺]_i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca²⁺ as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca²⁺ indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187-induced intracellular Ca²⁺ surge in BV2 cells and, under this condition, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187-induced activations of Ca²⁺-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca²⁺, and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.     

Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. II. Contrasting effects of local anesthetics and a calcium ionophore

In the previous study, lymphocyte surface molecules were separated into two subsets depending on whether capping was associated was associated with redistribution of cytoplasmic myosin. In the present study, the effects of the local anesthetic chlorpromazine and of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 were compared. Both drugs affected the surface redistribution of immunoglobulin (Ig), Fc receptors, and the TL antigen- -molecules that appear to cap by association with microfilaments--but had no effect on the Thy.1 (theta) and H2 antigens--molecules that cap slowly, apparently unlinked to microfilament function. The capping of Ig, Fc receptor, and TL was inhibited while that of H2 and theta was not. Both drugs reversed the Ig Fc receptor, and TL caps but not the H2 and theta caps. In the former group, the reversal of caps was accompanied by a parallel reversal of the myosin segregated to the cap area. The appearance of myosin after drug treatment varied: chlorpromazine resulted in a diffuse pattern similar to that of normal lymphocytes, whereas {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 produced an array of aggregates and coarse filaments. The results are compatible with the view that two mechanisms for capping exist in the lymphocyte. The Ca2+ ionophore may affect capping of microfilament-dependent caps by producing a systemic activation of contractile proteins while chlorpromazine may act by disrupting a Ca2+-dependent link between surface complexes and the contractile proteins.     

Modulation of the activity of cytosolic phospholipase A2�� (cPLA2��) by cellular sphingolipids and inhibition of cPLA2�� by sphingomyelin

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2α (cPLA2α) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2α inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2α. Phosphatidylcholine vesicles containing SM reduced cPLA2α activity. Furthermore, SM disturbed the binding of cPLA2α to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2α-dependent release of AA by inhibiting the binding of cPLA2α to glycerophospholipids.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Stimulation and inhibition of secretion in Paramecium: role of divalent cations

The effects of Ca2+ and Mg2+ on exocytosis in Paramecium tetraurelia cells were examined with light microscopy, freeze fracture (FEM) and transmission electron microscopy (TEM) of thin-sectioned embedded cells. Picric acid-Ca2+-induced secretion in wild type (wt) cells was captured by "quick" fixation with OsO4, and TEM demonstrated membrane fusion occurring before trichocyst matrix (tmx) expansion. Cells stimulated with picric acid in the presence of high extracellular Mg2+ showed very few sites of membrane fusion and no tmx expansion, suggesting that Ca2+ is required for both membrane fusion and tmx expansion. Further information was obtained by comparing secretory responses of wt cells with a temperature-sensitive secretory mutant, nd 9. These cells when grown at the permissive temperature (18 degrees C) possess normal rosettes at the secretory site and secrete in response to picric acid-Ca2+, but when grown at 27 degrees C they lack rosettes and do not secrete (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). Quantitation of picric acid-Ca2+-induced secretion revealed that: (a) the number of tmx secreted by wt and nd 9 cells was independent of their cultural growth phase, (b) wt cells secreted the same number of tmx when grown either at 18 or 27 degrees C, and (c) nd 9 18 degrees C cells secreted the same number of tmx as wt 18 or 27 degrees C cells. Wild type and nd 9 cells had the same frequencies of occupied and unoccupied secretory sites as determined by quantitative analysis of freeze-fracture replicas. After stimulation with divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+, wt cells showed a significant reduction in the frequency of occupied sites. FEM and TEM studies revealed that {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-Ca2+ induced tmx expansion and normal fusion of the plasma and trichocyst membranes in wt and nd 9 18 degrees C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27 degrees C cells. The lack of membrane fusion in nd 9 27 degrees C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.     

Histamine release by pharmacological agents in the absence of external free Ca2+

The involvement of extracellular free Ca2+ in histamine release was investigated in rat peritoneal mast cells. Incubation of non-antigenized cells in a media with high extracellular potassium did not increase histamine release. Secretion induced by A23187 and compound 48/80 in the presence of Ca2+ requires metabolic energy. In the absence of external free Ca2+ (2.5 microM) histamine release induced by A23187 is reduced but not abolished. Secretion induced by compound 48/80 is independent of extracellular Ca2+. These results lead us to suggest that mast cell plasma membranes probably lack voltage-gated Ca2+ channels and that external Ca2+ may not be an absolute requisite for histamine secretion.     

Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A₂α (cPLA₂α) or calcium-independent PLA₂. Surprisingly, release of arachidonic acid and LDH from cPLA₂α-deficient fibroblasts was inhibited by the cPLA₂α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca²⁺ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and H₂O₂. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.     

Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required,but mitogen-activated protein kinase activity is not sufficient for induced cell movement

We recently have demonstrated that EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation (P. Chen, K. Gupta, and A. Wells. 1994. J. Cell Biol. 124:547-555). This suggests that the immediate downstream effector molecule contains a src homology-2 domain. Phospholipase C gamma (PLC gamma) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. We utilized signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells to determine if PLC activation is necessary for EGFR-mediated cell movement. Exposure to EGF (25 nM) augmented PLC activity in all five EGFR mutant cell lines which also responded by increased cell movement. Basal phosphoinositide turnover was not affected by EGF in the lines which do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggested, but did not prove, a causal link. A specific inhibitor of PLC, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1 microM) diminished both the EGF- induced motility and PLC responses, while its inactive analogue {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 had no effect on these responses. Both the PLC and motility responses were decreased by expression of a dominant-negative PLC gamma-1 fragment in EGF-responsive infectant lines. Lastly, anti-sense oligonucleotides (20 microM) to PLC gamma-1 reduced both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC gamma as the immediate post receptor effector in this motogenic pathway. We have demonstrated previously that EGFR-mediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induced the mitogenic response while only those which are autophosphorylated induced PLC activity. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 did not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC gamma-1 fragment did not diminish EGF-induced thymidine incorporation. All kinase active EGFR stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induced cell movement; this EGF-induced MAP kinase activity was not affected by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 at concentrations that depressed the motility response. Thus, the signaling pathways which lead to motility and cell proliferation diverge at the immediate post-receptor stage, and we suggest that this is accomplished by differential activation of effector molecules.     

Human skin mast cells: their dispersion, purification, and secretory characterization

Digestion of human foreskin with collagenase and hyaluronidase disperses approximately 3.4 X 10(7) nucleated cells per gram of tissue, of which mast cells constitute 4.7%. These may be purified to 80% by use of density gradient centrifugation. The majority of mast cells (79%) measured between 9 and 13 micron in diameter, and the mean histamine content was 4.6 pg/cell. Viability was demonstrated by trypan blue exclusion by 93% of the cells and the low spontaneous histamine secretion of less than 7% in functional studies. Anti-IgE released up to 17.5% of cell-associated histamine within 5 to 7 min. Calcium ionophore-induced release was optimal with 0.3 microM A23187 when 28.6% histamine was released. Unlike human lung mast cells, skin mast cells released histamine in response to compound 48/80 and poly-L-lysine. This release, which was complete within 20 sec, was totally dependent on intact glycolysis and oxidative phosphorylation and partially dependent on extracellular calcium. The same characteristics were observed with secretion induced by substance P and morphine. The weak activity of eledoisin and physalaemin suggests that the substance P receptor, like that of the rat mast cell, is not of the classical types described for smooth muscle. Morphine-induced secretion was partially blocked by naloxone in a manner not compatible with competitive antagonism at a classical opioid receptor. The sensitivity of skin mast cells to nonimmunologic stimulation clearly distinguishes them from mast cells of the lung and lymphoid tissues and provides evidence of functional heterogeneity within human mast cells.     

The microsporidian spore invasion tube. II. Role of calcium in the activation of invasion tube discharge

A swelling response by the polaroplast organelle initiated microsporidian invasion tube extrusions by Glugea hertwigi spores. The tumescence was induced by the displacement of internal calcium. Sodium citrate, phosphate, and the calcium ionophore A23187 were effective in initiating polaroplast swelling and spore discharge; however, the addition of external CaCl2 switched the expanded polaroplasts to a contracted state and blocked spore discharge. Unlike CaCl2, equivalent concentrations of KCl, NaCl, MgCl2, and BaCl2 did not induced polaroplast contraction, and spore discharge was not blocked. 45CaCl2 readily incorporated into spores with expanded polaroplasts; however, little calcium uptake was apparent in spores with contracted polaroplasts. Metallochromic arsenazo III yielded a color spectrum characteristic of the dye-Ca++ complex in the polaroplast region; furthermore, a membrane association with calcium was indicated by strong chlorotetracycline fluorescence within the polaroplast; this fluorescence was extinguished by pretreating spores with ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. An association of the membrane with calcium was also indicated by a potassium ferrocyanide-osmium tetroxide technique. All evidence indicates that an internal calcium displacement is an important initial step in the swelling response of the polaroplast organelle.     

Role of sodium in ADP- and thrombin-induced megakaryocyte spreading

We investigated the role of sodium in megakaryocyte spreading induced by thrombin and ADP. We found that if extracellular sodium was replaced by lithium, potassium, or choline, spreading was inhibited. When extracellular sodium was present, amiloride or tetrodotoxin inhibited spreading. Using intracellular recording we found spreading to be associated with a permanent membrane depolarization. The extent and rate of thrombin-induced depolarization was reduced when lithium replaced sodium. Unspread cells had an average membrane potential of - 44.8 mV. Spread cells had an average membrane potential of -18.46 mV. When choline replaced sodium, or when in the presence of tetrodotoxin and amiloride, the spread cells repolarized, indicating that the depolarization is due to an increase in sodium permeability. Similar treatments did not change the membrane potential of unspread cells. Incubation of megakaryocytes with {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 together with monensin or methylamine induced spreading. Methylamine occasionally caused spreading by itself, but neither ionophore alone caused spreading. These results indicate that megakaryocyte spreading induced by ADP and thrombin depends on an increase in sodium conductance.     

An enhanced incorporation of fatty acid into phosphatidyl choline that parallels histamine discharge in mast cells

Summary Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24–25°C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17–37°C. Both radioactive linoleic and palmitic acids were incorporated selectively at the -position of the glycerol backbone of PC. PC labeling by [³H]choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced -acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A₂ that favors PC as a substrate.Other peritoneal cell types exhibit a very similar A23187-stimulated selective labeling of PC. Therefore, autoradiography has been used to provide a direct demonstration that in purified preparations, mast cells are the principal cell type engaged in A23187-elicited incorporation of fatty acid into PC. The efficacy of this approach has relied on special procedures devised to obtain significantly different autoradiographic grain densities between control and stimulated preparations that can be attributed to differences in the level of [³H]palmitate-labeled PC.Preliminary tests using compound 48/80 as a secretory stimulus for mast cells have identified a similar selectively enhanced PC labeling. In either case, however, consideration of possible relationships between PC metabolism and the secretory process are premature since they have not been tested directly.     

Amomum xanthiodes inhibits mast cell-mediated allergic reactions through the inhibition of histamine release and inflammatory cytokine production

In this study, we investigated the effect of Amomum xanthiodes (Zingiberaceae) extract (AXE) on the mast cell-mediated allergy model and studied the possible mechanism of action. We found that AXE inhibited compound 48/80-induced systemic reactions and plasma histamine release in mice. Additionally, AXE decreased immunoglobulin E (IgE)-mediated local allergic reactions and passive cutaneous anaphylaxis (PCA), and AXE dose-dependently attenuated the release of histamine from rat peritoneal mast cells (RPMC) activated by compound 48/80 or IgE. The amounts of AXE needed for inhibition of compound 48/80-induced plasma histamine release and PCA were similar to disodium cromoglycate, the known anti-allergic drug. We found that AXE increased the cAMP levels and decreased the compound 48/80-induced intracellular Ca2+. Furthermore, AXE attenuated the phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore (A23187)-stimulated tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 secretion in human mast cells. The inhibitory effect of AXE on the proinflammatory cytokines was nuclear factor-kappaB (NF-kappaB)-dependent. In addition, AXE decreased PMA plus A23187-induced degradation of IkappaBalphaand the nuclear translocation of NF-kappaB. Our findings provide evidence that AXE inhibits mast cell-derived immediate-type allergic reactions, and that cAMP, intracellular Ca2+, proinflammatory cytokines, and NF-kappaB are involved in these effects.     

Gap junction dynamics: reversible effects of divalent cations

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode''s solutions containing a Ca++ ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.