Increase In Cytosolic Calcium Concentration Research Articles

Activation of endothelinA receptors in frog adrenocortical cells stimulates both calcium mobilization from intracellular stores and calcium influx through L-type calcium channels.

We have previously shown that endothelin (ET)-1 stimulates corticosterone and aldosterone secretion by the frog adrenal gland through activation of ETA receptors positively coupled to both the adenylyl cyclase and phospholipase C (PLC) pathways. The purpose of the present study was to investigate the involvement of calcium in ET-1-induced stimulation of corticosteroid secretion. Cytoautoradiographic labeling using [125I]ET-1 as a tracer revealed the presence of ET-1 binding sites on adrenocortical cells. Administration of graded concentrations of ET-1 in the vicinity of adrenocortical cells provoked a dose-dependent increase in cytosolic calcium concentrations ([Ca2+]i). ET-1 induced a biphasic response consisting of an immediate and transient peak of [Ca2+]i followed by a plateau phase. Preincubation of the cells with the calcium-ATPase inhibitor thapsigargin or the PLC inhibitor U-73122 reduced the amplitude of the transient phase. Administration of the calcium chelator EGTA or the protein kinase A inhibitor H-89 attenuated the plateau phase. The [Ca2+]i response to ET-1 was markedly reduced during concomitant administration of U-73122 and H-89. Preincubation of the cells with the L-type calcium channel blocker nifedipine attenuated the plateau phase. Corticosteroid secretion from perifused frog adrenal slices was almost completely suppressed by thapsigargin and reduced by nifedipine. Taken together, these data indicate that activation of ETA receptors in frog adrenocortical cells provokes immediate stimulation of PLC, which causes an early mobilization of calcium from intracellular stores, and activates adenylyl cyclase, which results in delayed calcium influx through L-type calcium channels. The resulting increase in [Ca2+]i plays a pivotal role in ET-1-induced corticosteroid secretion.

P1-156 Intraneuronal amyloid-β1-42 production triggered by sustained increase of cytosolic calcium concentration induced neuronal death

P1-156 Intraneuronal amyloid-β1-42 production triggered by sustained increase of cytosolic calcium concentration induced neuronal death

Intraneuronal amyloid-beta1-42 production triggered by sustained increase of cytosolic calcium concentration induces neuronal death.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques which contain an amyloid core made of beta-amyloid peptide (Abeta). Abeta is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signalling has been causally implicated in ageing and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurones. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the alpha-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the beta-cleavage of the protein (CTFbeta). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal Abeta1-42, which is inhibited by nimodipine, a specific antagonist of l-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal Abeta, which requires influx of extracellular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolic calcium is needed to induce the production of intraneuronal Abeta1-42 from human APP. Our results show that this accumulation of intraneuronal Abeta1-42 induces neuronal death, which is prevented by a functional gamma-secretase inhibitor.

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry.

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.

Evidence for inositol triphosphate as a second messenger for glucose-induced calcium signalling in budding yeast.

The Saccharomyces cerevisiae phospholipase C Plc1 is involved in cytosolic transient glucose-induced calcium increase, which also requires the Gpr1/Gpa2 receptor/G protein complex and glucose hexokinases. Differing from mammalian cells, this increase in cytosolic calcium concentration is mainly due to an influx from the external medium. No inositol triphosphate receptor homologue has been identified in the S. cerevisiae genome; and, therefore, the transduction mechanism from Plc1 activation to calcium flux generation still has to be identified. Inositol triphosphate (IP(3)) in yeast is rapidly transformed into IP(4) and IP(5) by a dual kinase, Arg82. Then another kinase, Ipk1, phosphorylates the IP(5) into IP(6). In mutant cells that do not express either of these kinases, the glucose-induced calcium signal was not only detectable but was even wider than in the wild-type strain. IP(3) accumulation upon glucose addition was completely absent in the plc1Delta strain and was amplified both by deletion of either ARG82 or IPK1 genes and by overexpression of PLC1. These results taken together suggest that Plc1p activation by glucose, leading to cleavage of PIP(2) and generation of IP(3), seems to be sufficient for raising the calcium level in the cytosol. This is the first indication for a physiological role of IP(3) signalling in S. cerevisiae. Many aspects about the signal transduction mechanism and the final effectors require further study.

Fetal pulmonary hypertension prolongs the endothelin-1 induced increase in pulmonary artery smooth muscle cell cytosolic calcium concentration

Introduction: At birth, pulmonary artery (PA) blood flow increases 8–10 fold and PA pressure falls by 50% within 24 hours. The mechanisms responsible for postnatal adaptation of the pulmonary circulation remain incompletely understood. High serum endothelin-1 (ET-1) levels are found in infants with persistent pulmonary hypertension of the newborn. We hypothesized that chronic intrauterine pulmonary hypertension (PHT) potentiates agonist induced increase of cytosolic calcium concentration ([Ca2+]i) in fetal PA smooth muscle cells(SMC). Methods: Chronic intrauterine pulmonary hypertension was induced by intrauterine ligation of the ductus arteriosus (DA) in late-gestation fetal lambs. 7–10 days following DA ligation, animals were euthanized. Distal PAs (>4th generation) were removed from the lung and PA SMC were isolated and maintained in normoxic (95%O2) primary culture. Age-matched non-PHT controls were sacrificed at ∼132 days gestation and cells were cultured in the same manner. Fluorescence microscopy using fura-2 was used to determine the effect of ET-1 (10−6 -10−8 M) on PA SMC [Ca2+]i from PHT and control animals. To determine the effect of PHT on endothelin A and B (ETA and ETB) receptor mRNA expression, quantitative, internally controlled RT-PCR was performed on distal PAs and SMC in culture. Results: ET-1 (10−8 M) caused a monophasic increase in [Ca2+]i (206 +/- 47%, p < 0.01 vs baseline) above baseline in controls (n = 25), while PHT SMC [Ca2+]i did not increase significantly. When PHT SMC were treated with 10−6M ET-1, in those cells that responded, [Ca2+]i increased 174 +/- 81% above baseline(p < 0.01 vs baseline). Only 3/33 (9%) of PHT SMC responded to ET-1 compared to 90% of controls. Additionally, the duration of the increase in [Ca2+]i was longer in the PHT SMC: PHT SMC [Ca2+]i fell to baseline after 328 +/- 16 sec, where control [Ca2+]i fell to baseline after 117 sec. ETA and ETB receptor mRNA expression are both increased in PHT. Conclusions: PHT decreases PA SMC sensitivity to ET-1 by ∼100 fold, however, PHT increases the duration of the ET-1 induced increase in [Ca2+]i. We conclude that PHT prolongs the response of PA SMC to ET-1. This prolongation may lead to significant increases in PA SMC [Ca2+]i that results in attenuated postnatal adaptation of the pulmonary circulation.

The metabolic inhibitor antimycin A can disrupt cell-to-cell communication by an ATP- and Ca(2+)-independent mechanism.

In cardiac myocytes of new-born rats, the degree of intercellular communication through gap junctional channels closely depends on the metabolic state of the cells. In contrast, in stably transfected HeLa cells expressing rat cardiac connexin43 (Cx43, the main channel-forming protein present in ventricular myocytes), a major part of junctional communication persisted in ATP-depleted conditions, in the presence of a metabolic inhibitor (KCN) or of a broad spectrum inhibitor of protein kinases (H7). However, another metabolic inhibitor, antimycin A, which like cyanide inhibits electron transfer in the respiratory chain, totally interrupted cell-to-cell communication between Cx43-HeLa cells, even in whole-cell conditions, when ATP (5 mM) was present. Antimycin A caused a modest increase in cytosolic calcium concentration; however, junctional uncoupling still occurred when this rise was prevented. Conditions of ischemic insult (e.g. ischemia or chemical hypoxia) frequently cause the activation of protein kinases, particularly of Src and MAP kinases, and such activations are known to markedly disrupt gap junctional communication. Antimycin-induced junctional uncoupling occurred even in the presence of inhibitors of these kinases. Antimycin A appears able to cause junctional uncoupling either through the ATP depletion it induces as a metabolic poison or via a direct action on gap junction constituents.

Calcium bursts induced by nanosecond electric pulses

We report here real-time imaging of calcium bursts in human lymphocytes exposed to nanosecond, megavolt-per-meter pulsed electric fields. Ultra-short (less than 30 ns), high-field (greater than 1 MV/m), electric pulses induce increases in cytosolic calcium concentration and translocation of phosphatidylserine (PS) to the outer layer of the plasma membrane in Jurkat T lymphoblasts. Pulse-induced calcium bursts occur within milliseconds and PS externalization within minutes. Caspase activation and other indicators of apoptosis follow these initial symptoms of nanosecond pulse exposure. Pulse-induced PS translocation is observed even in the presence of caspase inhibitors. Ultra-short, high-field, electroperturbative pulse effects differ substantially from those associated with electroporation, where pulses of a few tens of kilovolts-per-meter lasting a few tens of microseconds open pores in the cytoplasmic membrane. Nanosecond pulsed electric fields, because their duration is less than the plasma membrane charging time, develop voltages across intracellular structures without porating the cell.

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.

Role of Calcium in the Induction of TNFα Expression by Macrophages on Exposure to Ultrafine Particles

Ultrafine particles have been suggested to be one of the components responsible for the adverse health effects of environmental particulate air pollution (PM10). This study aimed to investigate the ability of fine and ultrafine carbon black (ufCB) particles to stimulate production of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα) by primary rat alveolar macrophages. We have previously demonstrated that ufCB, but not fine CB, is able to enhance calcium influx into macrophages in the presence of a secondary calcium agonist such as thapsigargin. For this reason, we also investigated the role of intracellular calcium signalling in controlling ufCB-induced TNFα production. Observation of rat alveolar macrophages stained with the calcium-sensitive dye Fura-2 and then treated with ufCB resulted in a rapid but transient increase in cytosolic calcium concentration in individual cells within 15 min of exposure. ufCB treatment of the primary macrophages also induced a significant increase in TNFα mRNA and protein production within 4 h of exposure. Inhibition of calcium influx using a calcium channel blocker, verapamil, as well as inhibition of calcium signalling events with an intracellular calcium chelator, BAPTA-AM, both prevented the ufCB-induced increase in TNFα protein production. TNFα mRNA production was inhibited by verapamil but not by BAPTA-AM. Addition of the antioxidant nacystelin, previously shown to inhibit ufCB-induced calcium influx into macrophages, also inhibited TNFα protein but not mRNA production. These data support the hypothesis that ultrafine particles, via reactive oxygen species, enhance calcium influx into macrophages and that these oxidative and calcium signalling events activate TNFα production.

Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization.

Biomineralization is a highly regulated process that plays a major role during the development of skeletal tissues. Despite its obvious importance, little is known about its regulation. Previously, it has been demonstrated that retinoic acid (RA) stimulates terminal differentiation and mineralization of growth plate chondrocytes (Iwamoto, M., I.M. Shapiro, K. Yagumi, A.L. Boskey, P.S. Leboy, S.L. Adams, and M. Pacifici. 1993. Exp. Cell Res. 207:413–420). In this study, we provide evidence that RA treatment of growth plate chondrocytes caused a series of events eventually leading to mineralization of these cultures: increase in cytosolic calcium concentration, followed by up-regulation of annexin II, V, and VI gene expression, and release of annexin II–, V–, VI– and alkaline phosphatase–containing matrix vesicles. Cotreatment of growth plate chondrocytes with RA and BAPTA-AM, a cell permeable Ca2+ chelator, inhibited the up-regulation of annexin gene expression and mineralization of these cultures. Interestingly, only matrix vesicles isolated from RA-treated cells that contained annexins, were able to take up Ca2+ and mineralize, whereas vesicles isolated from untreated or RA/BAPTA-treated cells, that contained no or only little annexins were not able to take up Ca2+ and mineralize. Cotreatment of chondrocytes with RA and EDTA revealed that increases in the cytosolic calcium concentration were due to influx of extracellular calcium. Interestingly, the novel 1,4-benzothiazepine derivative K-201, a specific annexin Ca2+ channel blocker, or antibodies specific for annexin II, V, or VI inhibited the increases in cytosolic calcium concentration in RA-treated chondrocytes. These findings indicate that annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes and mediate Ca2+ influx into these cells. The resulting increased cytosolic calcium concentration leads to a further up-regulation of annexin II, V, and VI gene expression, the release of annexin II–, V–, VI– and alkaline phosphatase–containing matrix vesicles, and the initiation of mineralization by these vesicles.

Activation of 5-HT(7) receptor in rat glomerulosa cells is associated with an increase in adenylyl cyclase activity and calcium influx through T-type calcium channels.

Serotonin (5-HT) stimulates aldosterone secretion from the rat adrenal gland through 5-HT(7) receptors. The aim of the present study was to investigate the transduction mechanisms associated with activation of 5-HT(7) receptors in rat glomerulosa cells. The stimulatory effect of 5-HT on aldosterone secretion and cAMP formation was significantly reduced by the 5-HT(7) receptor antagonist LY 215840. Pretreatment of cells with the adenylyl cyclase inhibitor SQ 22536 or the PKA inhibitor H-89 markedly attenuated the effect of 5-HT on aldosterone secretion. Conversely, type 2 and 4 phosphodiesterase inhibitors potentiated the 5-HT-induced stimulation of aldosterone secretion. Administration of 5-HT in the vicinity of cultured glomerulosa cells induced a slowly developing and robust increase in cytosolic calcium concentration ([Ca(2+)](i)). The effect of 5-HT on [Ca(2+)](i) was suppressed by mibefradil, a T-type calcium channel blocker. Patch-clamp studies confirmed that 5-HT activated a T-type calcium current. Mibefradil also induced a dose-dependent inhibition of 5-HT-induced aldosterone secretion. The sequence of events associated with activation of 5-HT(7) receptors was investigated. The PKA inhibitor H-89 markedly attenuated both the [Ca(2+)](i) response and the activation of T-type calcium current induced by 5-HT. In contrast, reduction of the calcium concentration in the incubation medium did not affect 5-HT- induced cAMP formation. Preincubation of glomerulosa cells with cholera toxin abolished the stimulatory effect of 5-HT on aldosterone secretion, but pertussis toxin had no effect. Taken together, these data demonstrate that, in rat glomerulosa cells, activation of native 5-HT(7) receptors stimulates cAMP formation through a G(salpha) protein, which in turn provokes calcium influx through T-type calcium channels. Both the adenylyl cyclase/PKA pathway and the calcium influx are involved in 5-HT-induced aldosterone secretion.

Oxidative Injury Mediated by PECAM

Reactive oxygen species, such as H 2 O 2 , can cause damage to endothelial cells. Ji et al. show that H 2 O 2 stimulated the phosphorylation of platelet endothelial cell adhesion molecule (PECAM-1) in endothelial cells (natively expressing PECAM-1) or cells transfected to express PECAM-1. Agents that block nonreceptor tyrosine kinases of the Src family inhibited phosphorylation of PECAM-1. Cells expressing PECAM-1 also responded to H 2 O 2 exposure with the activation of a nonselective cation channel, resulting in an increase in cytosolic calcium concentration. This calcium transient required the presence and phosphorylation of PECAM-1, because the current was not observed in cells lacking PECAM-1, in the presence of antibodies against the PECAM-1 intracellular domain, in the presence of agents that inhibit Src, or in cells expressing a nonphosphorylatable mutant of PECAM-1. Thus, oxidants appear to activate Src, which phosphorylates PECAM-1, which is then able to activate a nonselective cation channel that results in the increase in cytosolic calcium, which can lead to toxicity. This may represent one pathway by which oxidants can produce endothelial cell injury. G. Ji, C. D. O'Brien, M. Feldman, Y. Manevich, P. Lim, J. Sun, S. M. Albelda, M. I. Kotlikoff, PECAM-1 (CD31) regulates a hydrogen peroxide-activated nonselective cation channel in endothelial cells. J. Cell Biol. 157 , 173-184 (2002) [Abstract] [Full Text]

INVOLVEMENT OF T-TYPE CALCIUM CHANNELS IN THE MECHANISM OF ACTION OF 5-HT IN RAT GLOMERULOSA CELLS: A NOVEL SIGNALING PATHWAY FOR THE 5-HT7 RECEPTOR

We have previously demonstrated that, in rat, the stimulatory effect of 5-HT on aldosterone secretion is mediated through a 5-HT7 receptor subtype. The aim of the present study was to characterize the transduction mechanisms associated with activation of native 5-HT7 receptors. 5-HT induced a dose-dependent increase in cAMP production in rat glomerulosa cells. Pretreatment of cells with the adenylyl cyclase (AC) inhibitor SQ 22536 or the protein kinase A (PKA) inhibitor H-89 markedly attenuated the effect of 5-HT on aldosterone secretion. Administration of 5-HT in the vicinity of glomerulosa cells induced a robust increase in cytosolic calcium concentration ([Ca2+]i) and this effect was abrogated by the T-type calcium channel blocker mibefradil. Patch-clamp studies confirmed that 5-HT activated a T-type calcium current. H-89 attenuated both the [Ca2+]i response and the activation of T-type calcium current induced by 5-HT. Reduction of extracellular calcium concentration in the medium or administration of mibefradil caused a marked reduction of the maximum effect (Emax) of 5-HT on aldosterone secretion. These data demonstrate that activation of native 5-HT7 receptors stimulates cAMP formation, which in turn provokes calcium influx through T-type calcium channels. Both the activation of the AC/PKA pathway and the calcium influx are involved in 5-HT-induced aldosterone secretion.

Effects of cyclopiazonic acid on cytosolic calcium in bovine airway smooth muscle cells.

In many cells, inhibition of sarcoplasmic reticulum (SR) Ca2+-ATPase activity induces a steady-state increase in cytosolic calcium concentration ([Ca2+]i) that is sustained by calcium influx. The goal was to characterize the response to inhibition of SR Ca2+-ATPase activity in bovine airway smooth muscle cells. Cells were dispersed from bovine trachealis and loaded with fura 2-AM (0.5 microM) for imaging of single cells. Cyclopiazonic acid (CPA; 5 microM) inhibited refilling of both caffeine- and carbachol-sensitive calcium stores. In the presence of extracellular calcium, CPA caused a transient increase in [Ca2+]i from 166 +/- 11 to 671 +/- 100 nM, and then [Ca2+]i decreased to a sustained level (CPA plateau; 236 +/- 19 nM) significantly above basal. The CPA plateau spontaneously declined toward basal levels after 10 min and was attenuated by discharging intracellular calcium stores. When CPA was applied during sustained stimulation with caffeine or carbachol, decreases in [Ca2+]i were observed. We concluded that the CPA plateau depended on the presence of SR calcium and that SR Ca2+-ATPase activity contributed to sustained increases in [Ca2+]i during stimulation with caffeine and, to a lesser extent, carbachol.

An Off Switch

Sanjuán et al. investigated the role of diacylglycerol kinase α (DGKα) in the termination of signaling in T cells. The studies were performed in a T cell line that heterologously expresses a muscarinic acetylcholine receptor (which activates phospholipase C via a G protein) and measured the expression of CD69 as a marker of diacylglycerol (DAG)-mediated signaling. Stimulation of the cells with the muscarinic receptor agonist carbachol led to increased production of phosphatidic acid (the product of DGKα action on DAG), transient redistribution of DGKα-labeled with green fluorescent protein to the plasma membrane, and increased expression of CD69. Deletion of the calcium-binding EF hand domains of DGKα resulted in constitutive association of DGKα with the plasma membrane, enhanced enzymatic activity, and suppression of CD69 expression compared to that in cells expressing wild-type DGKα treated with carbachol. Activation of DGKα appeared to require two simultaneous signals: an increase in cytosolic calcium concentration and activation of tyrosine kinases.M. A. Sanjuán, D. R. Jones, M. Izquierdo, I. Mérida, Role of diacylglycerol kinase α in the attenuation of receptor signaling. J. Cell Biol. 153, 207-219 (2001). [Abstract] [Full Text]

An Off Switch

Sanjuán et al. investigated the role of diacylglycerol kinase α (DGKα) in the termination of signaling in T cells. The studies were performed in a T cell line that heterologously expresses a muscarinic acetylcholine receptor (which activates phospholipase C via a G protein) and measured the expression of CD69 as a marker of diacylglycerol (DAG)-mediated signaling. Stimulation of the cells with the muscarinic receptor agonist carbachol led to increased production of phosphatidic acid (the product of DGKα action on DAG), transient redistribution of DGKα-labeled with green fluorescent protein to the plasma membrane, and increased expression of CD69. Deletion of the calcium-binding EF hand domains of DGKα resulted in constitutive association of DGKα with the plasma membrane, enhanced enzymatic activity, and suppression of CD69 expression compared to that in cells expressing wild-type DGKα treated with carbachol. Activation of DGKα appeared to require two simultaneous signals: an increase in cytosolic calcium concentration and activation of tyrosine kinases. M. A. Sanjuán, D. R. Jones, M. Izquierdo, I. Mérida, Role of diacylglycerol kinase α in the attenuation of receptor signaling. J. Cell Biol. 153 , 207-219 (2001). [Abstract] [Full Text]

Mitochondrial uncoupling does not influence the stability of the intracellular signal activating plasma membrane calcium channels.

The effects of various concentrations of thapsigargin, a specific inhibitor of Ca2+-ATPase in the endoplasmic reticulum (ER) membrane, on calcium homeostasis in lymphoidal T cells (Jurkat) were investigated. Preincubation of these cells suspended in nominally calcium-free medium with 0.1 microM thapsigargin resulted in a complete release of Ca2+ from intracellular calcium stores. When the medium was supplemented with 3 mM CaCl2 the cells maintained constantly elevated level of cytosolic Ca2+. However, thapsigargin applied at lower concentration produced only a partial depletion of the stores. For example, in the cells pretreated with 1 nM thapsigargin and suspended in calcium-free medium approximately 75% of the calcium content was released from the intracellular stores. The addition of 3 mM CaCl2 to such cell suspension led to a transient increase in cytosolic calcium concentration, followed by a return to a lower steady-state. This phenomenon, related to the refilling of the ER by Ca2+, allowed to estimate the half-time for the process of cell recovery after activation of store-operated calcium channels. By this approach we have found that carbonyl cyanide m-chlorophenylhydrazone, which has been documented to inhibit calcium entry into Jurkat cells, does not influence the stability of the intracellular signal involved in the activation of store-operated calcium channels.

The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas

Alloxan and streptozotocin are widely used to induce experimental diabetes in animals. The mechanism of their action in B cells of the pancreas has been intensively investigated and now is quite well understood. The cytotoxic action of both these diabetogenic agents is mediated by reactive oxygen species, however, the source of their generation is different in the case of alloxan and streptozotocin. Alloxan and the product of its reduction, dialuric acid, establish a redox cycle with the formation of superoxide radicals. These radicals undergo dismutation to hydrogen peroxide. Thereafter highly reactive hydroxyl radicals are formed by the Fenton reaction. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of B cells. Streptozotocin enters the B cell via a glucose transporter (GLUT2) and causes alkylation of DNA. DNA damage induces activation of poly ADP-ribosylation, a process that is more important for the diabetogenicity of streptozotocin than DNA damage itself. Poly ADP-ribosylation leads to depletion of cellular NAD+ and ATP. Enhanced ATP dephosphorylation after streptozotocin treatment supplies a substrate for xanthine oxidase resulting in the formation of superoxide radicals. Consequently, hydrogen peroxide and hydroxyl radicals are also generated. Furthermore, streptozotocin liberates toxic amounts of nitric oxide that inhibits aconitase activity and participates in DNA damage. As a result of the streptozotocin action, B cells undergo the destruction by necrosis.

Serum amyloid A is an activator of PMN antimicrobial functions: induction of degranulation, phagocytosis, and enhancement of anti-Candida activity.

Serum amyloid A (SAA) is a 12-kDa protein secreted in large amounts by liver cells during microbial infections or inflammatory diseases. We have recently reported that SAA induces chemotaxis of polymorphonuclear cells (PMN), monocytes, and T lymphocytes and stimulates their adhesion to endothelial monolayers. In this study, we investigated whether SAA regulates PMN antimicrobial activities. We found that recombinant SAA (rSAA), at concentrations comparable to serum levels attained during an acute phase response, is a potent activator of PMN. Stimulation of PMN by rSAA results in a rapid and transient increase of cytosolic calcium concentration and up-regulation of cell-surface expression of antigens involved in adhesion and microbial recognition such as CD11c and CD16. In addition, stimulation of PMN with rSAA increases secretion of lactoferrin, an antimicrobial protein that is contained in specific granules of PMN and enhances PMN phagocytic activity against heat-killed Candida albicans. Finally, activation of PMN with rSAA enhances their anti-Candida activity within 30 min of stimulation. These results suggest that SAA is involved in up-regulating PMN antimicrobial activities and that high circulating concentrations of SAA as seen in the acute phase response may constitute a potential host defense mechanism against fungal infections.