Presence of Ca2+ is obligatory for the cytotoxic activity of dengue virus-induced cytotoxic factor.

The present study was undertaken to investigate the role of calcium ions (Ca2+) in the cytotoxic activity of the cytotoxic factor (CF) produced by T lymphocytes of the dengue type 2 virus (DV)-infected mouse spleen. It was observed that CF prepared in Ca2(+)-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca2(+)-free medium but had activity on cells suspended in medium having Ca2+. The cytotoxic activity of CF was restored by substitution with calcium chloride, the optimal dose being 10(-7) M. CF induced influx of Ca2+ as measured by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, macrophages (M phi) and T lymphocytes, but had no effect on CF-resistant B lymphocytes. Calcium channel blocking drugs, like verapamil, nifedipine and diltiazem, inhibited the cytotoxic activity of CF and also the CF-induced influx of 45Ca in M phi and T cells. Thus, presence of Ca2+ is obligatory for the cytotoxic activity of CF and the cell death is associated with increased intracellular Ca2+.     

Extracellular calcium is not an absolute requirement for tumoricidal activation of RAW-264 macrophage-like cell line

The purpose of these studies was to establish whether extracellular calcium (Cao2+) plays a role in the process of activation of RAW-264 macrophages for tumor cell killing. We found that these cells were capable of developing a significant level of cytolytic activity under treatment with lymphokine (LK) and lipopolysaccharide (LPS), in the absence of Cao2+ and that responses developed in Ca2+-free media were only 6-18% lower in comparison with the responses developed in the presence of Cao2+. The determination of 45calcium uptake in RAW-264 cells treated with LK and LPS showed that the rate of 45calcium uptake has displayed no increase during either the course of activation or in activated, highly cytolytic cells. Finally, three calcium channel blockers examined here: verapamil, diltiazem and flunarizine, with concentrations ranging from 1 X 10(-7) M - 2.5 X 10(-5) M, showed no inhibitory effect on the process of activation. Nifedipine, another calcium channel blocker, inhibited the development of cytolytic activity with concentrations ranging from 1 X 10(-6) M - 2.5 X 10(-5) M. It could be argued, however, that this inhibition was nonspecific, since this agent was 13 times more potent with regard to the calcium ionophore A23187-induced release of beta-glucuronidase, the function which is entirely dependent on Cao2+. Taken together, these results suggest that Cao2+ is not an absolute requirement for the process of tumoricidal activation of RAW-264 macrophages but it may play some supportive role in this process.     

两型TNF-α的细胞毒效应与靶细胞内Ca2+浓度变化的关系

目的 :比较分泌型TNF α(S TNF α)和跨膜型TNF α(TM TNF α)发挥细胞毒效应时 ,引起靶细胞内Ca2 浓度的变化。方法 :采用生物学活性检测法 ,观察两型TNF α对不同靶细胞的杀伤效应 ;用Fura 2检测细胞内Ca2 浓度的变化。结果 :TM TNF α可杀伤实验所用 6株靶细胞 ;而S TNF α则仅对其中两株有细胞毒效应。两型TNF α杀伤靶细胞时 ,均伴有明显的Ca2 浓度升高。用钙螯合剂EGTA(10mmol/L)预先处理靶细胞30min ,只能降低S TNF α作用的靶细胞内的Ca2 浓度 ,并使其细胞毒效应明显减弱 (P <0 .0 1) ;对TM TNF α无影响。结论 :两型TNF α发挥细胞毒效应时 ,均可引起靶细胞内钙离子的重分布 ,导致靶细胞内Ca2 浓度的升高 ,但S TNF α的作用还可能与促进胞外Ca2 内流有关     

Variable effects of dengue virus-induced cytotoxic factors on different subpopulations of macrophages.

Dengue virus (DV) induces T lymphocytes of the spleen to produce a cytotoxic factor (CF) that induces a subpopulation of macrophages (M phi) to produce a soluble cytotoxin (CF2). Both these factors kill normal lymphoid cells and M phi. The present study was undertaken to investigate the effects of these factors on the I-A-positive and I-A-negative subpopulations of mouse peritoneal M phi. It was observed that CF kills I-A-negative M phi and induces I-A-positive M phi to produce the CF2 that kills both types of cells. However, even when combined together, CF and CF2 do not kill 100% of the M phi. The two-step mechanism involving co-operation between T cells and M phi appears to be biologically economical for maintaining the cytotoxic pathway.     

Production of dengue virus-induced macrophage cytotoxin in vivo.

We have observed that dengue virus-induced cytotoxic factor (CF) induces peritoneal and splenic macrophages in vitro to produce a cytotoxin (CF2). This study demonstrates also production of CF2 in vivo in DV-infected mice and following inoculation with CF. The cell-type responsible for CF2 production in vivo is the macrophage (M phi) as M phi-depleted mice failed to produce CF2. CF2 activity could not be observed in the serum or peritoneal fluid though it is produced in peritoneal M phi. Once stimulated, CF2 is present for 4 h in M phi. M phi can be restimulated to produce CF2 only after a refractory period of 48 h.     

Production of dengue virus-induced macrophage cytotoxin in vivo

We have observed that dengue virus-induced cytotoxic factor (CF) induces peritoneal and splenic macrophages in vitro to produce a cytotoxin (CF2). This study demonstrates also production of CF2 in vivo in DV-infected mice and following inoculation with CF. The cell-type responsible for CF2 production in vivo is the macrophage (M phi) as M phi-depleted mice failed to produce CF2. CF2 activity could not be observed in the serum or peritoneal fluid though it is produced in peritoneal M phi. Once stimulated, CF2 is present for 4 h in M phi. M phi can be restimulated to produce CF2 only after a refractory period of 48 h.     

Potency of depolarization-induced transmitter release is determined by divalent cation influx in PC 12 cells.

Evoked release of [3H]dopamine ([3H]DA) from pheochromocytoma cells (PC 12) is dependent on extracellular calcium ([Ca2+]ex), but it can take place if calcium ions (Ca2+) are substituted by other divalent ions such as strontium (Sr2+) and barium (Ba2+). The potency of the divalent cations at supporting release varies with the cell type; in PC 12 cells the order of potency is Ba2+ > Sr2+ > Ca2+. The close correlation between depolarization-evoked Ca2+ entry and depolarization-evoked transmitter release prompted us to examine whether the higher evoked transmitter release in the presence of Sr2+ correlates with an increased evoked Sr2+ influx. Influx studies were conducted on PC12 cells using a radioactive tracer (45Ca2+ or 85Sr2+, < 1 microM) in the presence of either Sr2+ (0.5 mM) or Ca2+ (0.5 mM). Depolarization with K Cl (60 mM) increased evoked 45Ca2+ influx 2-fold when Ca2+ was substituted with Sr2+. Similarly, evoked 85Sr2+ influx increased 1.87-fold by substituting Ca2+ for Sr2+. Thus the amount of evoked cation influx is determined by the type of divalent ion which is accessible in the extracellular medium, independently of the radioactive tracer used. Increased evoked transmitter release in the presence of Sr2+ was associated with increased evoked Sr2+ influx. This suggests that the potency of evoked transmitter release is determined predominantly by the influx of divalent cations. Furthermore, the steps subsequent to cation influx in the release process are equally efficient for both cations.     

Bursting activity in leech Retzius neurons induced by low external chloride

We have investigated the bursting activity of Retzius neurons in the central nervous system of the leech Hirudo medicinalis as induced in Cl(-)-free saline by measuring membrane potential, membrane current and the intracellular calcium concentration ([Ca2+]i), using fura-2 or Oregon-Green488-Bapta-1. The Retzius neurons changed their low tonic firing to rhythmical bursting activity when the extracellular Cl- concentration ([Cl-]o) was lowered to 1 mM or less. In Cl(-)-free saline (Cl- exchanged by gluconate), bursting was accompanied by a rise in intracellular Ca2+ in both cell body and axon, which oscillated in synchrony with the bursts. The Ca2+ transients depended on the amplitude and duration of the depolarization underlying the burst, and were presumably due to Ca2+ influx through voltage-dependent Ca2+ channels. In Ca(2+)-free, EGTA-buffered saline or in the presence of Ca2+ channel blockers verapamil (1 mM) or diltiazem (500 microM) the depolarizations underlying the bursts in Cl(-)-free saline were enhanced in amplitude and duration. Bursting was not affected by depleting the intracellular Ca2+ stores with cyclopiazonic acid. The depolarization in Cl(-)- and Ca(2+)-free saline did not evoke intracellular Ca2+ changes. The burst-underlying membrane depolarization induced by Cl- removal was found to be due to a Na(+)-dependent persistent inward current and could be inhibited by saxitoxin (25-50 microM). The results suggest that a persistent Na+ current is generated in Cl(-)-free saline and induces the depolarization underlying rhythmic activity, and that presumably Ca(2+)-induced K+ currents modulate the bursting behaviour.     

Mechanism of action of magnesium on acetylcholine-evoked secretory responses in isolated rat pancreas.

This study investigates the effects of magnesium (Mg2+) on acetylcholine (ACh)-evoked secretory responses and calcium (Ca2+) mobilization in the isolated rat pancreas. ACh induced marked dose-dependent increases in total protein output and amylase release from superfused pancreatic segments in zero, normal (1 x 1 mM) and elevated (10 mM) extracellular Mg2+. Elevated Mg2+ attenuated the ACh-evoked secretory responses compared to zero and normal Mg2+. In the absence of extracellular Ca2+, but presence of 1 mM-EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid), ACh elicited a small transient release of protein from pancreatic segments compared to a larger and more sustained secretion in the absence of both Ca2+ and Mg2+. Incubation of pancreatic segments with 45Ca2+ resulted in time-dependent uptake with maximum influx of 45Ca2+ occurring after 20 min of incubation period. ACh stimulated markedly the 45Ca2+ uptake compared to control tissues. In elevated extracellular Mg2+ the ACh-induced 45Ca2+ influx was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. ACh also evoked dose-dependent increases in cytosolic free Ca2+ concentrations ([Ca2+]i) in pancreatic acinar cells loaded with the fluorescent dye Fura-2 AM. In elevated Mg2+ the ACh-induced cytosolic [Ca2+]i was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. These results indicate that Mg2+ can influence ACh-evoked secretory responses possibly by controlling both Ca2+ influx and release in pancreatic acinar cells.     

Profiles of cell-to-cell interaction of Mycobacterium intracellulare-induced immunosuppressive macrophages with target T cells in terms of suppressor signal transmission

Previously, we have found that immunosuppressive macrophages (M(phi)s) induced by Mycobacterium intracellulare-infection (MI-M(phi)s) required cell contact with target T cells to express their suppressor activity against concanavalin A (Con A)-induced T cell mitogenesis. In this study, we examined the profiles of cell-to-cell interaction of MI-M(phi)s with target T cells. First, MI-M(phi)s displayed suppressor activity in an H-2 allele-unrestricted manner, indicating that MHC molecules are not required for cell contact. The suppressor activity of MI-M(phi)s was reduced markedly by paraformaldehyde fixation or treatment with cytochalasin B or colchicine, indicating that vital membrane functions are required for their suppressor activity. Secondly, the suppressor activity of MI-M(phi)s was independent of cell-to-cell interaction via CD40 ligand/CD40 and M(phi)-derived indoleamine 2,3-dioxygenase, which causes rapid degradation of tryptophan in T cells. Thirdly, precultivation of splenocytes with MI-M(phi)s, allowing cell-to-cell contact, reduced Con A- or anti-CD3 antibody-induced mitogenesis but not phorbol myristate acetate/calcium ionophore A23187-elicited proliferation of T cells. In addition, co-cultivation of T cells with MI-M(phi)s caused marked changes in profiles of the tyrosine phosphorylation of 33 kDa, 34 kDa and 35-kDa proteins and, moreover, the activation of protein kinase C and its translocation to the cell membrane. It thus appears that suppressor signals of MI-M(phi)s, which are transmitted to the target T cells via cell contact, principally cross-talk with the early signalling events before the activation of PKC and/or intracellular calcium mobilization.     

Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis.

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.     

Evidence that TRH stimulates secretion of TSH by two calcium-mediated mechanisms

Thyrotropin-released hormone (TRH) stimulation of thyrotropin (TSH) release from mouse thyrotropic tumor (TtT) cells is dependent on Ca2+. We demonstrate that TRH action in TtT cells does not require extracellular Ca2+ but that Ca2+ influx induced by TRH can augment TSH secretion. TRH caused a 46% increase in 45Ca2+ uptake by TtT cells in medium with 100 micro M Ca2+. The increment in 45Ca2+ uptake caused by TRH was dependent on the concentration of Ca2+ in the medium. In contrast to the effect of 50 mM K+, which also causes Ca2+ influx, TRH caused 45Ca2+ efflux and TSH release from TtT cells even when the concentration of Ca2+ in the medium was lowered below 100 micro M. TRH stimulated TSH release during perifusion in medium in which the free Ca2+ concentration was lowered to approximately 0.02 micro M, and reintroduction of Ca2+ into the medium simultaneously with TRH markedly increased TSH release. We suggest that TRH may affect Ca2+ metabolism in TtT cells by both extracellular Ca2+-independent and -dependent mechanisms and that this dual mechanism of action serves to augment further TSH secretion induced by TRH.     

NPC-14686, a novel anti-inflammatory agent, increased intracellular Ca(2+) concentrations in MDCK renal tubular cells

The effect of NPC-14686 (Fmoc-L-homophenylalanine), a novel anti-inflammatory agent on intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) renal tubular cells, was investigated, using fura-2 as a Ca(2+) dye. At concentrations between 10 and 200 microM NPC-14686 increased [Ca(2+)](i) concentration dependently. The [Ca(2+)](i) signal comprised an initial rise and a sustained phase. Ca(2+) removal inhibited the Ca(2+) signals by 90%. In Ca(2+)-free medium, pretreatment with 100 microM NPC-14686 nearly abolished the [Ca(2+)](i) increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) and abolished the [Ca(2+)](i) increase induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) (a mitochondrial uncoupler). NPC-14686 (100 microM) induced a slight [Ca(2+)](i) increase after pretreatment with 2 microM CCCP and 1 microM thapsigargin. Addition of 3 mM Ca(2+) elicited a [Ca(2+)](i) increase in cells pretreated with 100 microM NPC-14686 in Ca(2+)-free medium. Inhibition of inositol-1,4,5-trisphosphate (IP(3)) production by suppressing phospholipase C with 2 microM U73122 did not alter NPC-14686-induced Ca(2+) release. Trypan blue exclusion revealed that incubation with 10 or 200 microM NPC-14686 for 1-30 min decreased cell viability by 10-20% concentration dependently. Collectively, the results demonstrate that, in MDCK tubular cells, NPC-14686 induced Ca(2+) release followed by Ca(2+) entry, with the latter playing a major role. NPC-14686 appears to release intracellular Ca(2+) in an IP(3)-uncoupled manner. NPC-14686 may be of mild cytotoxicity.     

Antigen-induced and non-antigen-induced histamine release from rat mast cells sensitized with mouse antiserum

Marked IgE-mediated histamine release from rat mast cells sensitized in vitro with mouse antiserum occurs in the presence of added Ca++ and phosphatidylserine (PS), although a considerable degree of antigen-induced histamine release which may utilize intracellular or cell-bound calcium is also observed. The decay in the responsiveness to Ca++ of the sensitized cells stimulated by antigen in Ca++-free medium in the presence of PS is relatively slow, and maximum release is produced by Ca++ added 1 min after antigen. Histamine release also occurs when Ca++ is added after PS in the absence of antigen to the sensitized cells suspended in Ca++-free medium. Unlike the antigen-induced release, the intensity of this non-antigen-induced release varies depending on both mast-cell and antiserum pools. A heat-labile factor(s), which is different from antigen-specific IgE antibody and is also contained in normal mouse serum, is involved in this reaction. In the antigen-nondependent (PS + Ca++)-induced release, no decay in the responsiveness to Ca++ is observed after PS addition. Both the antigen-induced and non-antigen-induced release are completed fairly rapidly and are dependent of temperature, pH and energy.     

Trifluoperazine, a calmodulin inhibitor, blocks secretion in cultured chromaffin cells at a step distal from calcium entry

Trifluoperazine, a calmodulin antagonist, inhibited the secretory response of cultured bovine adrenal medullary chromaffin cells to acetylcholine (10(-4) M) or a depolarizing concentration of [K+] (56 mM KCl) in a dose-related fashion. The ID50s of this effect were 2 x 10(-7) M and 2.2 x 10(-6) M for acetylcholine and high [K+], respectively. A decrease in external [Ca2+] concentration of the incubation medium from 4.4 to 0.275 mM resulted in an increase in the percentage of inhibition produced by trifluoperazine on the acetylcholine-evoked secretory response from 20.7 to 96.5%, respectively. However, trifluoperazine inhibited the acetylcholine-evoked catecholamine output by a similar absolute magnitude for all [Ca2+] concentrations tested with the exception of 4.4 mM [Ca2+]. Trifluoperazine, unlike the [Ca2+] channel blocker Ni2+, in concentrations (10(-6)-10(-5) M) that were found to inhibit significantly [K+]-induced amine output did not modify [K+]-induced 45Ca uptake or 45Ca efflux. However, trifluoperazine at a concentration of 2.5 x 10(-5) M was found to produce a small decrease in the 45Ca efflux curve and a decrease in the [K+]-evoked 45Ca uptake of 30 +/- 14% (n = 6). In addition, 2.5 x 10(-6) M trifluoperazine, a concentration which was found to suppress high [K+]-induced amine release by 64 +/- 5%, did not inhibit the 45Ca2+-Ca2+ exchange mechanism. These results demonstrate that trifluoperazine, an antipsychotic agent with anticalmodulin activity, blocks catecholamine release from cultured chromaffin cells at a step distal from calcium entry and, consequently, suggests a role for calmodulin in the secretory process of these cells.     

Bone-like tissue formation by three-dimensional culture of MG63 osteosarcoma cells in gelatin hydrogels using calcium-enriched medium

The aim of this study was to investigate the effect of Ca(2+) concentration in culture medium on the promotion of osteogenesis by MG63 osteoblast-like cells and to prepare bone-like tissues by supplying Ca(2+)-enriched medium to MG63 cells immobilized in three-dimensional gelatin hydrogels. Human osteosarcoma MG63 cells were cultured on tissue culture dish under various Ca(2+) concentrations to evaluate the effect of Ca(2+) concentration on calcium deposition. When Ca(2+) concentration was 8 mM, the maximum calcium deposition was obtained at day 28. Then MG63 cells were entrapped in gelatin hydrogels cross-linked by transglutaminase and cultured for 28 days, either in a standard culture medium or in medium containing 8 mM Ca(2+). Effects of Ca(2+)-enriched medium on osteoblastic phenotype of MG63 cells in gelatin hydrogels were analyzed in terms of cell number, calcium deposition content, and alkaline phosphatase (ALP) activity. The characteristics of calcified gelatin hydrogels were evaluated by x-ray diffraction (XRD), histological analysis, and scanning electron microscopy (SEM). After 28 days of culture, no significant difference in cell numbers was found between the different culture conditions. However, calcium content of gelatin hydrogels with cells cultured in Ca(2+)-enriched media was significantly higher than that of hydrogels with cells cultured in standard Ca(2+) concentration medium. After 14 days of culture, ALP activity of cells cultured in Ca(2+)-enriched media was down-regulated compared with that of cells cultured in standard Ca(2+) concentration media. XRD analysis indicated the formation of hydroxyapatite in gelatin hydrogels cultured in the Ca(2+)-enriched media at day 14, and the XRD pattern of the composite at day 21 was almost similar to that of mouse tibia. Moreover, histological analysis and SEM analysis revealed that cross-sections of hydrogels cultured in Ca(2+)-enriched media had an organic/mineral layer structure analogous to that of mouse tibia.     

On the functional relationship between 45Ca2+ release and prolactin secretion in cultured rat pituitary tumour (GH3) cells

We have evaluated the role of cellular Ca2+ transport associated with stimulus-secretion coupling in prolactin (PRL) producing rat pituitary adenoma cells (GH3 cells). The action of different substances, known to modify PRL secretion, on release of 45Ca2+ from preloaded cells were examined. Surface-bound 45Ca2+ was removed by pretreatment with trypsin in EDTA buffer. During the first 6 min, basal efflux of 45Ca2+ occurred at a constant rate (0.24 min-1) at 37 degrees C. Addition of TRH (5 X 10(-7) M) resulted in an immediate enhancement of 45Ca2+ release representing about 20% of the remaining cellular 45Ca2+. In the same experiments PRL secretion increased by 45%. The EDTA in the external medium reduced the basal rate of 45Ca2+ release by 60%, but did not apparently affect the TRH-stimulated release. Somatostatin (10(-6) M) and verapamil (5 X 10(-5) M) inhibited both basal and TRH-stimulated PRL secretion, whereas high extracellular concentration of K+ (5 X 10(-2) M) had a stimulatory effect. However, neither of these treatments changed cellular 45Ca2+ release. Interference with energy-dependent Ca2+ transport by using metabolic inhibitors (iodoacetate, 6 X 10(-3) M; and antimycin, 2 X 10(-6) M) or by replacing Na+ in the medium by choline or by lowering the incubation temperature from 37 to 25 degrees C, had no effect on TRH-stimulated 45Ca2+ release although basal and TRH-stimulated PRL secretion were reduced. Thus, TRH apparently releases 45Ca2+ from calcium binding sites in the cell membrane.     

In situ imaging of intracellular calcium with ischemia in lung subpleural microvascular endothelial cells

We propose that generation of reactive oxygen species (ROS) during ischemia is associated with an increase in intracellular calcium ([Ca2+]i) in pulmonary capillary endothelial cells. We used an isolated rat lung model and epifluorescence microscopy to evaluate [Ca2+]i in subpleural microvascular endothelial cells in situ by ratio imaging of the fluorophores, Calcium Green and Fura Red (CG/FR). Lungs were ventilated continuously under control (continuously perfused) or global ischemia (no perfusion) and thus remained adequately oxygenated even with ischemia. Ischemia for 5 min led to increase in CG/FR, indicating increase in [Ca2+]i in endothelial cells in situ; CG/FR remained elevated during a subsequent 10 min of ischemia. Ca(2+)-free perfusion and gadolinium (100 microM) inhibited the increase in [Ca2+]i, while thapsigargin (250 nM) had no effect. These results indicate that increase in endothelial cell [Ca2+]i with ischemia was due to influx from the extracellular medium. Perfusion with N-acetyl-L-cysteine (20 mM) or diphenyleneiodonium chloride (10 microM) prevented the ischemia-mediated [Ca2+]i increase, suggesting a role for ROS in the Ca2+ changes with ischemia. Membrane depolarization by perfusion with high potassium (K+) or glyburide also resulted in increased [Ca2+]i whereas the K(+)-channel agonist cromakalim, inhibited ischemia-mediated Ca2+ influx. We conclude that increased ROS generation with 'oxygenated' lung ischemia is associated with influx of Ca2+ and an increase in endothelial cell cytosolic calcium concentration.     

Effects of ionic calcium on the responses of canine testicular polymodal receptors to algesic substances

1. To explore possible mechanisms of the responses to algesic substances (bradykinin, hypertonic saline, and high K+ solution) of polymodal receptors in the canine testis, the Ca2+ concentration was varied in vitro. 2. After 1 min in Ca2+-free media, the responses to both high K+ solution (60 mM K+) and hypertonic saline (0.6 M Na+) were significantly augmented and tended to increase further with time; return to normal Ca2+ concentration quickly reversed these changes. These augmenting effects were blocked by the substitution of Mg2+ for Ca2+. The excitation produced by 60 mM K+ was decreased by increasing Ca2+ in a concentration-dependent fashion. 3. Reducing the bath concentration of K+ decreased responses evoked by 9 X 10(-8) M bradykinin (BK), whereas increased K+ concentration had the opposite effect. 4. The excitatory effects of BK were significantly suppressed in extracellular Ca2+-free condition. The suppression was not affected by the addition of Mg2+. Prostaglandin E2, which has been known to be released by BK and to augment the BK response, failed to restore the suppressed response by either preapplication (2.8 X 10(-7) M) or simultaneous application in high concentration (1.4 X 10(-5) M). 5. On the basis of these observations, it was postulated that Ca2+ concentration-dependent changes of the responses to 60 mM K+ and 0.6 M Na+ results from Ca2+-dependent "membrane surface potential" changes. The suppressed response to BK by Ca2+ depletion may be explained by the intervention of Ca2+-dependent processes other than PG production.     

Acute application of interleukin-1beta induces Ca(2+) responses in human microglia

The effects of the pro-inflammatory cytokine interleukin-1-beta (IL-1beta) on levels of intracellular calcium [Ca(2+)](i) in cultured human microglia have been studied using the fluorescent Ca(2+) indicator fura-2. IL-1beta (2 ng/ml) caused a slow, progressive increase in [Ca(2+)](i) in standard Ca(2+)-containing physiological solution (PSS). A similar effect was observed in separate studies using Ca(2+)-free PSS, however, the mean rate of increase was significantly lower than that measured with PSS. Similar results were obtained in a separate protocol, where cells were exposed to both IL-1beta in Ca(2+)-free PSS and PSS. The slope of the IL-1beta induced increase of [Ca(2+)](i) in Ca(2+)-free PSS was not altered when adenosine triphosphate was added prior to application of the cytokine. These results suggest that IL-1beta-induced responses in human microglia involve both a Ca(2+) entry pathway and a mechanism of intracellular increase other than from IP(3)-sensitive stores.