Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: evidence for a novel mechanism for preventing or terminating signaling by PAR1's tethered ligand

The thrombin receptor PAR1 is activated when thrombin cleaves the receptor's amino-terminal exodomain to reveal the new N-terminal sequence SFLLRN which then acts as a tethered peptide ligand. Free SFLLRN activates PAR1 independent of receptor cleavage and has been used to probe PAR1 function in various cells and tissues. PAR1-expressing cells desensitized to thrombin retain responsiveness to SFLLRN. Toward determining the mechanism of such responses, we utilized fibroblasts derived from a PAR1-deficient mouse. These cells were unresponsive to thrombin and SFLLRN and became sensitive to both ligands after transfection with human PAR1 cDNA. Moreover, PAR1-transfected cells responded to SFLLRN after thrombin-desensitization, indicating that signaling of thrombin-desensitized cells to SFLLRN was mediated by PAR1 itself. SFLLRN caused signaling in thrombin-desensitized cells when no uncleaved PAR1 was detectable on the cell surface; however, cleaved PAR1 was present. To determine whether the cleaved receptors could still signal, fibroblasts were transfected with a PAR1 mutant containing a trypsin site/SFLLRN sequence carboxyl terminal to the native thrombin site. These cells retained responsiveness to trypsin after thrombin-desensitization. Conversely, fibroblasts expressing a PAR1 mutant with the trypsin site/SFLLRN sequence amino terminal to the native thrombin site retained responsiveness to thrombin after trypsin-desensitization. This suggests that a population of thrombin-cleaved PAR1 can respond both to exogenous SFLLRN and to a second tethered ligand. In this population, the tethered ligand unmasked by thrombin cleavage must not be functional, suggesting the possibility of a novel mechanism of receptor shutoff involving sequestration or modification of the tethered ligand to prevent or terminate its function.     

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.     

Treponema denticola Major Outer Sheath Protein Impairs the Cellular Phosphoinositide Balance That Regulates Neutrophil Chemotaxis

The major outer sheath protein (Msp) of Treponema denticola inhibits neutrophil polarization and directed chemotaxis together with actin dynamics in vitro in response to the chemoattractant N-formyl-methionine-leucine-phenylanine (fMLP). Msp disorients chemotaxis through inhibition of a Rac1-dependent signaling pathway, but the upstream mechanisms are unknown. We challenged murine bone marrow neutrophils with enriched native Msp to determine the role of phospholipid modifying enzymes in chemotaxis and actin assembly downstream of fMLP-stimulation. Msp modulated cellular phosphoinositide levels through inhibition of phosphatidylinositol 3-kinase (PI3-kinase) together with activation of the lipid phosphatase, phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Impaired phosphatidylinositol[(3,4,5)]-triphosphate (PIP3) levels prevented recruitment and activation of the downstream mediator Akt. Release of the actin capping proteins gelsolin and CapZ in response to fMLP was also inhibited by Msp exposure. Chemical inhibition of PTEN restored PIP3 signaling, as measured by Akt activation, Rac1 activation, actin uncapping, neutrophil polarization and chemotaxis in response to fMLP-stimulation, even in the presence of Msp. Transduction with active Rac1 also restored fMLP-mediated actin uncapping, suggesting that Msp acts at the level of PIP3 in the hierarchical feedback loop of PIP3 and Rac1 activation. Taken together, Msp alters the phosphoinositide balance in neutrophils, impairing the cell “compass”, which leads to inhibition of downstream chemotactic events.     

An actin-nucleating activity in polymorphonuclear leukocytes is modulated by chemotactic peptides

We examined the actin-nucleating activity in polymorphonuclear leukocyte lysates prepared at various times after chemotactic peptide addition. The actin nucleation increases two- to threefold within 15 s after peptide addition, decays to basal levels within 90 s, and is largely independent of cytoplasmic calcium fluxes. The peptide-induced nucleation sites behave as free barbed ends and therefore may increase the level of polymerized actin in vivo. The new nucleation sites may also determine the cellular sites of actin polymerization. This localization of actin polymerization could be important for the directional extension of lamellipodia during chemotaxis.     

A Microfluidic Platform for Evaluating Neutrophil Chemotaxis Induced by Sputum from COPD Patients

Chronic Obstructive Pulmonary Disease (COPD) is a common lung disease characterized by breathing difficulty as a consequence of narrowed airways. Previous studies have shown that COPD is correlated with neutrophil infiltration into the airways through chemotactic migration. However, whether neutrophil chemotaxis can be used to characterize and diagnose COPD is not well established. In the present study, we developed a microfluidic platform for evaluating neutrophil chemotaxis to sputum samples from COPD patients. Our results show increased neutrophil chemotaxis to COPD sputum compared to control sputum from healthy individuals. The level of COPD sputum induced neutrophil chemotaxis was correlated with the patient’s spirometry data. The cell morphology of neutrophils in a COPD sputum gradient is similar to the morphology displayed by neutrophils exposed to an IL-8 gradient, but not a fMLP gradient. In competing gradients of COPD sputum and fMLP, neutrophils chemotaxis and cell morphology are dominated by fMLP.     

Actin polymerization in response to different chemoattractants is reduced in granulocytes from chronic myeloid leukemia patients

Chronic myeloid leukemia (CML), a hematopoietic stem cell disorder, is characterized by the presence of Philadelphia chromosome (Ph1). Earlier studies have shown that various functions, such as chemotaxis, fluid phase pinocytosis, phagocytosis, and degranulation in response to chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP), were defective in polymorphonuclear leukocytes (PMNL) from CML patients. These functions depend on actin microfilaments (MF). Further studies showed that fMLP-induced actin polymerization was lower in CML PMNL. To see if this defect is specific to stimulation by fMLP alone or is a global phenomenon involving other chemoattractant receptors, chemotaxis and actin polymerization were studied in response to fMLP, an analog of fMLP, formyl-methionine-1 aminocyclooctane 1 carboxylic acid-phenyalanine-O-methionine (FACC8), platelet-activating factor (PAF), and leukotriene B4 (LTB4). These compounds bind to different chemoattractant receptors. Chemotaxis and actin polymerization in response to all four chemoattractants were significantly lower in CML PMNL compared with PMNL from normal subjects and were differentially affected for the different chemoattractants. These results suggest a global phenomenon involving all four chemoattractant-stimulated pathways. This lower amount of F-actin may be responsible for the defective chemotaxis seen in these cells.     

Utilization of two seven-transmembrane, G protein-coupled receptors, formyl peptide receptor-like 1 and formyl peptide receptor, by the synthetic hexapeptide WKYMVm for human phagocyte activation

Trp-Lys-Tyr-Val-D-Met (WKYMVm) is a synthetic leukocyte-activating peptide postulated to use seven-transmembrane, G protein-coupled receptor(s). In the study to characterize the receptor(s) for WKYMVm, we found that this peptide induced marked chemotaxis and calcium flux in human phagocytes. The signaling induced by WKYMVm in phagocytes was attenuated by high concentrations of the bacterial chemotactic peptide fMLP, suggesting that WKYMVm might use receptor(s) for fMLP. This hypothesis was tested by using cells over expressing genes encoding two seven-transmembrane receptors, formyl peptide receptor (FPR) and formyl peptide receptor-like 1 (FPRL1), which are with high and low affinity for fMLP, respectively. Both FPR- and FPRL1-expressing cells mobilized calcium in response to picomolar concentrations of WKYMVm. While FPRL1-expressing cells migrated to picomolar concentrations of WKYMVm, nanomolar concentrations of the peptide were required to induce migration of FPR-expressing cells. In contrast, fMLP elicited both calcium flux and chemotaxis only in FPR-expressing cells with an efficacy comparable with WKYMVm. Thus, WKYMVm uses both FPR and FPRL1 to stimulate phagocytes with a markedly higher efficacy for FPRL1. Our study suggests that FPR and FPRL1 in phagocytes react to a broad spectrum of agonists and WKYMVm as a remarkably potent agonist provides a valuable tool for studying leukocyte signaling via these receptors.     

Actin polymerization in human eosinophils,unlike human neutrophils,depends on intracellular calcium mobilization

Eosinophils represent major effector cells in the allergic inflammation. In contrast to neutrophils, the mechanism of eosinophil activation during the inflammatory response is poorly understood. In this study, the relation between calcium fluxes, chemotaxis, and actin polymerization in eosinophils from healthy non-atopic donors was investigated. Pre-incubation of eosinophils with the intracellular calcium chelator BAPTA dose-dependently prevented an increase in the intracellular calcium concentration ([Ca²⁺]_i), whereas the depletion of extracellular calcium in the test medium had no effect. The chemotactic response of eosinophils, which was measured by the modified boyden chamber technique upon stimulation with RANTES, C5a and PAF, was dose-dependently inhibited by the chelation of intracellular calcium as well as inactivation of the cells in Ca²⁺-depleted medium. To evaluate whether other cell functions which are involved in the migratory response of eosinophils might be dependent on intracellular and extracellular calcium, actin polymerization was investigated. Flow-cytometric measurement of F-actin with NBD-phallacidin revealed that actin polymerization in human eosinophils in response to RANTES, C5a, and PAF was dose-dependently inhibited by the intracellular calcium chelator BAPTA. Since it is well known that actin polymerization in neutrophils is not affected by chelation of intracellular calcium, actin polymerization in these cells was investigated under the same conditions as for eosinophils. In contrast to eosinophils, BAPTA did not inhibit actin polymerization in neutrophils. In summary, these data demonstrate that intracellular calcium fluxes represent a prerequisite for eosinophil chemotaxis and actin polymerization in human eosinophils. Furthermore, regulation of actin polymerization in eosinophils differed from that of neutrophils on the level of intracellular calcium fluxes. © 1996 Wiley-Liss, Inc.     

T21/DP107, A synthetic leucine zipper-like domain of the HIV-1 envelope gp41, attracts and activates human phagocytes by using G-protein-coupled formyl peptide receptors

A leucine zipper-like domain, T21/DP107, located in the amino terminus of the ectodomain of gp41, is crucial to the formation of fusogenic configuration of the HIV-1 envelope protein gp41. We report that the synthetic T21/DP107 segment is a potent stimulant of migration and calcium mobilization in human monocytes and neutrophils. The activity of T21/DP107 on phagocytes was pertussis toxin-sensitive, suggesting this peptide uses Gi-coupled seven-transmembrane receptor(s). Since the bacterial chemotactic peptide fMLP partially desensitized the calcium-mobilizing activity of T21/DP107 in phagocytes, we postulated that T21/DP107 might preferentially use a lower affinity fMLP receptor. By using cells transfected to express cloned prototype chemotactic N-formyl peptide receptor (FPR) or its variant, FPR-like 1 (FPRL1), we demonstrate that T21/DP107 activates both receptors but has a much higher efficacy for FPRL1. In addition, T21/DP107 at nM concentrations induced migration of FPRL1-transfected human embryonic kidney 293 cells. In contrast, fMLP did not induce significant chemotaxis of the same cells at a concentration as high as 50 microM. Although a lipid metabolite, lipoxin A4, was a high-affinity ligand for FPRL1, it was not reported to induce Ca2+ mobilization or chemotaxis in FPRL1-transfected cells. Therefore, T21/DP107 is a first chemotactic peptide agonist identified thus far for FPRL1. Our results suggest that this peptide domain of the HIV-1 gp41 may have the potential to activate host innate immune response by interacting with FPR and FPRL1 on phagocytes.     

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

The effects of heparin cofactor II-derived chemotaxins on neutrophil actin conformation and cyclic AMP levels

The serine proteinase inhibitor heparin cofactor II (HC) can be cleaved by polymorphonuclear leukocyte (PMN) elastase (LE) to yield potent chemotactic activity for PMN and monocytes. In contrast to the bacterially-derived chemotaxin formyl-Met-Leu-Phe (fMLP), the HC-derived chemotaxin does not stimulate PMN degranulation or oxidative burst activity. We compared the effects of HC-derived chemotaxins to the effects of fMLP on PMN actin conformation and on the cAMP levels. Both the HC chemotaxins and fMLP rapidly induced an increase in F-actin which was similar in magnitude and time-course. However, in contrast to fMLP, HC-derived chemotaxins did not elevate cAMP levels. HC-derived chemotaxins may be useful probes of chemotactic responses, since they do not have the mixed biological activities of fMLP.     

Interaction of viper venom serine peptidases with thrombin receptors on human platelets

The serine peptidases, thrombocytin and PA-BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA-BJ, and thrombocytin on the degradation of the soluble N-terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41-Ser42. In addition, a rapid cleavage of the peptide bond at Arg46-Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA-BJ and thrombocytin both cleaved at 41-42 and 46-47 peptide bonds, and fragment 42-103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.     

Chemoattractant-induced cytoplasmic pH changes and cytoskeletal reorganization in human neutrophils. Relationship to the stimulated calcium transients and oxidative burst

The relationships between the chemotactic factor-stimulated mobilization of calcium, activation of the NADPH-oxidase, changes in cytosolic pH, and in the level of polymerized actin in human neutrophils have been examined. The approach taken was to use intracellular calcium chelators, and pharmacologic modulators (both positive and negative) of the NADPH-oxidase to measure the aforementioned responses under conditions where the calcium transients were abrogated and/or the generation of superoxide anions was either inhibited or augmented. The decrease in cytosolic pH induced by chemoattractants was inhibited by the calcium chelator BAPTA and by the diglyceride kinase inhibitor 6-[2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinylethyl ]-7-methyl-5H-thiazolo[3,2-alpha]pyriimidin-5-one (R59022) (this latter compound enhanced the oxidative response of the cells). Furthermore, a specific inhibitor of the NADPH-oxidase (diphenyleneiodonium) had no significant effect on the cytosolic acidification induced by FMLP or leukotriene B4. These results indicate that the initiation of the cytosolic acidification induced by chemotactic factors is a calcium-dependent event that is not directly linked to the activation of the NADPH-oxidase. In contrast, the stimulated polymerization of actin was insensitive to BAPTA, R59022, and diphenyleneiodonium. Thus, neither the calcium transients nor the oxidative burst play a signaling role in the initiation of actin polymerization elicited by chemoattractants. These data indicate that additional investigations are needed to uncover the biochemical basis of the signals initiated in human neutrophils by chemotactic factors that lead to the polymerization of actin and to the cytosolic acidification.     

Heat shock protein 27 regulates neutrophil chemotaxis and exocytosis through two independent mechanisms

The targets of the p38 MAPK pathway responsible for regulation of neutrophil chemotaxis and exocytosis are unknown. One target of this pathway is the actin-binding protein, heat shock protein 27 (Hsp27). Therefore, we tested the hypothesis that Hsp27 mediates p38 MAPK-dependent chemotaxis and exocytosis in human neutrophils through regulation of actin reorganization. Sequestration of Hsp27 by introduction of anti-Hsp27 Ab, but not an isotype Ab, inhibited fMLP-stimulated chemotaxis, increased cortical F-actin in the absence of fMLP stimulation, and inhibited fMLP-stimulated exocytosis. Pretreatment with latrunculin A prevented actin reorganization and the changes in fMLP-stimulated exocytosis induced by Hsp27 sequestration. To determine the role of Hsp27 phosphorylation, wild-type, phosphorylation-resistant, or phosphorylation-mimicking recombinant Hsp27 was introduced into neutrophils by electroporation. The phosphorylation-resistant mutant significantly reduced migration toward fMLP, whereas none of the Hsp27 proteins affected fMLP-stimulated or TNF-alpha-stimulated exocytosis or actin polymerization. Endogenous Hsp27 colocalized with F-actin in unstimulated and fMLP-stimulated neutrophils, whereas phosphorylated Hsp27 showed cytosolic localization in addition to colocalization with F-actin. Our results suggest that Hsp27 regulates neutrophil chemotaxis and exocytosis in an actin-dependent, phosphorylation-independent manner. Phosphorylation of Hsp27 regulates chemotaxis, but not exocytosis, independent of regulation of actin reorganization.     

Phospholipase Cγ Activation, Phosphotidylinositol Hydrolysis, and Calcium Mobilization are Not Required for FGF Receptor-Mediated Chemotaxis

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoartractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transf ected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLOγ, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLCγ following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLCγ, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.     

Thrombin downregulates muscle acetylcholine receptors via an IP3 signaling pathway by activating its G-protein-coupled protease-activated receptor-1

Regulation of thrombin activity may be required during skeletal muscle differentiation since the thrombin tissue inhibitor protease nexin-1 appears at the myotube stage before being localized at the neuromuscular synapse. Here, we have used a model of rat fetal myotube primary cultures to study the effect of thrombin on acetylcholine receptor (AChR) expression, which is enhanced at the myotube stage. Our results show that thrombin decreases both the number of surface AChRs (AChRn) and AChR alpha-subunit gene expression. Using the agonist peptide SFLLRN, we establish that the AChRn decrease is mediated by the G protein-coupled thrombin receptor "protease-activated receptor-1" (PAR-1). Moreover, the specific thrombin inhibitor hirudin increases AChRn by inhibiting the thrombin intrinsically present in the cultures. We further demonstrate that the activation of PAR-1 by thrombin induces intracellular calcium movements that are blocked by 2-APB, an inhibitor of inositol 1,4,5-triphosphate (IP3)-induced calcium release. These calcium signals are more intense in nuclei than in the cytoplasm and are consistent with the intracellular distribution of IP3 receptor that we find in the cytoplasm in a cross-striated pattern and at a high level in the nuclear envelope zone. Finally, we show that the blockade of these IP3-induced calcium signals by 2-APB prevents the AChRn decrease induced by thrombin. Our results thus demonstrate that thrombin downregulates AChR expression by activating PAR-1 and that this effect is mediated via an IP3 signaling pathway.     

Linkage between blood coagulation and inflammation: stimulation of neutrophil tissue kallikrein by thrombin

There has been major interest in the potential interaction between blood coagulation and inflammation. Most of the effort has focused on cellular interactions involving platelets and polymorphonuclear leukocytes (PMNS). The recent discovery of tissue kallikrein(TK) activity in PMNs prompted the study of the possible role of thrombin(IIa) in this process. Human PMNs were isolated by density gradient centrifugation. Human IIa was compared with fMLP with respect to chemotaxis and enzyme release. Results from the challenges by IIa and fMLP were compared to a NaCl control using Student's paired t-test. IIa was a potent chemotactic agent for PMNs (p less than or equal to 0.0121) and stimulated the release of TK (p less than or equal to 0.0001) as determined by hydrolysis of S-2266. FMLP significantly stimulated PMN chemotaxis (p less than or equal to 0.0028) but had no effect on TK release. Release of TK was confirmed by Western Blot analysis and 35S-methionine incorporation into a 35 KD protein after IIa challenge. These results demonstrate that IIa is chemotactic for PMNs and can cause release of tissue kallikrein demonstrating a direct role for blood coagulation in the regulation of the inflammatory response.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Cyclosporin A Rescues Thymocytes from Apoptosis Induced by Very Low Concentrations of Thapsigargin: Effects on Mitochondrial Function

Raising intracellular calcium levels can induce apoptosis or programmed cell death in many cells. While early rises in intracellular calcium are not universally associated with apoptotic cell death, calcium clearly plays a key role in many of the biochemical events which occur during apoptosis. In this paper we have determined intracellular calcium rises induced by 2, 10, and 100 nMthapsigargin in mouse thymocytes. These concentrations cause increases in cytosolic calcium of 100–250, 400–600, and >1000 nM,respectively. These rises are sustained for at least 85 min and the ratio between the maximum rise caused by 10 nMcompared to 2 nMthapsigargin is 2.1 ± 0.4 (n= 6). Both 2 and 10 nMthapsigargin cause apoptosis at 24 h as shown by DNA fragmentation and morphology when examined by electron microscopy. Cyclosporin A (CsA) inhibits apoptosis caused by 2 nMthapsigargin but not that caused by 10 nMthapsigargin. Electron microscopy of thymocytes treated with 2 nMthapsigargin at 24 h shows intact mitochondria although with altered morphology. There is no loss of ATP or decrease in the ATP/ADP ratio in these cells over 12 h. Mitochondria in cells treated with 10 nMthapsigargin, however, are swollen by 6 h and many are lost by 24 h. These cells show greatly diminished ATP content by 12 h and a decrease in ATP/ADP ratio. Examination of the effects of PMA, an activator of the plasma membrane calcium ATPase pump, on cells treated with 10 nMthapsigargin suggests that two pools of calcium may be responsible for the differential effects of the two calcium levels in the cells. Probing of the mitochondrial membrane potential (MMP) by rhodamine 123 staining of live cells shows that the collapse of the MMP caused by 10 nMthapsigargin is unaffected by CsA. The MMP is also reduced in cells treated with 2 nMthapsigargin but this is restored by CsA. Cells are also rescued from apoptosis caused by 2 nMthapsigargin by incubation with FK506. This immunosuppressive agent has no effect on the membrane permeability transition induced in isolated mitochondria. These results suggest that very low rises in intracellular calcium in thymocytes cause activation-induced cell death inhibited by CsA and FK506 and are without effect on ATP levels and therefore do not involve irreversible mitochondrial damage. Exceeding these calcium levels by only twofold results in apoptosis accompanied by reduced ATP levels and mitochondrial damage, although apoptotic cell death in this instance is unaffected by the classic inhibitor of mitochondrial permeability transition, CsA.