Conformationally restricted calpain inhibitors.

The cysteine protease calpain-I is linked to several diseases and is therefore a valuable target for inhibition. Selective inhibition of calpain-I has proved difficult as most compounds target the active site and inhibit a broad spectrum of cysteine proteases as well as other calpain isoforms. Selective inhibitors might not only be potential drugs but should act as tools to explore the physiological and pathophysiological roles of calpain-I. α-Mercaptoacrylic acid based calpain inhibitors are potent, cell permeable and selective inhibitors of calpain-I and calpain-II. These inhibitors target the calcium binding domain PEF(S) of calpain-I and -II. Here X-ray diffraction analysis of co-crystals of PEF(S) revealed that the disulfide form of an α-mercaptoacrylic acid bound within a hydrophobic groove that is also targeted by a calpastatin inhibitory region and made a greater number of favourable interactions with the protein than the reduced sulfhydryl form. Measurement of the inhibitory potency of the α-mercaptoacrylic acids and X-ray crystallography revealed that the IC50 values decreased significantly on oxidation as a consequence of the stereo-electronic properties of disulfide bonds that restrict rotation around the S-S bond. Consequently, thioether analogues inhibited calpain-I with potencies similar to those of the free sulfhydryl forms of α-mercaptoacrylic acids.

Ca2+ activation of cytosolic calpain induces the transition from apoptosis to necrosis in neutrophils with externalized phosphatidylserine.

Physiologically, apoptotic neutrophils are ingested before they undergo necrosis. However, failure of ingestion will lead to necrosis of neutrophils and the unregulated release of neutrophil-derived pathogenic molecules, such as protease and hydrolases. Understanding the mechanism of postapoptotic necrosis is thus clearly important. Here, we monitored the apoptotic-to-necrotic transition in individual-aged human neutrophils in vitro by imaging fluorescent probes for externalized PS, cytosolic Ca(2+), and membrane integrity. This showed that prenecrotic-aged neutrophils with externalized PS had a significantly elevated cytosolic-free Ca(2+) level. A further unregulated Ca(2+) influx into PS-externalized neutrophils always preceded the necrotic transition. Ca(2+) elevation was not simply a consequence of aging, as PS externalization was not uniform in similarly aged neutrophil populations. PS-externalized neutrophils could be induced to undergo necrosis experimentally by simply elevating cytosolic Ca(2+) further with ionomycin. This effect was observed only in neutrophils that had externalized PS, and was independent of the time after their isolation from blood (i.e., in vitro age). As pharmacological inhibition of calpain-1 inhibition significantly reduced this CAIN, it was concluded that the apoptotic-to-necrotic transition was a consequence of uncontrolled calpain activation that resulted from Ca(2+) overload in PS-externalized neutrophils.

Optical complexities of living cytoplasm--implications for live cell imaging and photo-micromanipulation techniques.

The ability to manipulate the intracellular environment within living cells and to monitor the cytosolic chemical changes which occur during cell stimulation has lead to major advances in our understanding of how cells read and respond to their environment. Perhaps the most powerful suite of techniques for achieving these dual objectives is based on the use of light (photons). Because cells are 'transparent', light has been used to both interrogate and manipulate the chemistry inside living cells, exploiting technical advances in both the physical and biochemical sciences. However, cells are neither transparent nor homogeneous with respect to their optical properties. The interface between light and the living cell cytoplasm thus represent an important, yet largely ignored, interface. There has been no review of the optical properties of cytoplasm and little discussion about how the optical properties of living cytoplasm influence the outcome of such measurements and manipulations. In this short review, we discuss the importance of understanding the optical properties of cytoplasm for such techniques and how imperfections in experimental interpretation can arise.

Reduced iC3b-mediated phagocytotic capacity of pulmonary neutrophils in cystic fibrosis.

Cystic fibrosis (CF) is characterized by a neutrophil-dominated chronic inflammation of the airways with persistent infections. In order to investigate whether neutrophils contribute to an inadequacy in the pulmonary defence mechanism, the phagocytic activity of pulmonary and peripheral blood neutrophils from CF and non-CF respiratory patients were compared. Neutrophils were isolated from both the blood and bronchoalveolar lavage fluid of 21 patients with CF (12 male, 9 female; mean age 7.5 years, range 0.25-16.4 years) and 17 non-CF subjects (9 male, 8 female; mean age 5.4 years, range 0.2-13.1 years). The ex vivo phagocytic rate of normal pulmonary neutrophils to internalize zymosan particles opsonized with iC3b was faster than that of circulating neutrophils (P < 0.05), but the maximum capacity (9 particles/cell) was similar. In contrast, pulmonary neutrophils from patients with CF had a lower phagocytic capacity than circulating neutrophils either from the same patients or from normal subjects. This deficiency could not be attributed to (i) the cell surface density of CR3 (CD18/CD11b) receptors, which were not significantly different between the other groups (ii) the signalling ability of the CR3 receptors, using cytosolic free Ca(2+) signalling as the receptor activity read-out or (iii) a decrease in cellular ATP concentration. As CFTR was not detectable on neutrophils from any source by either histochemistry or Western blotting, it was concluded that the reduced phagocytic capacity was not the direct result of a CFTR mutation, but was attributed to a failure of neutrophil phagocytic priming during translocation into the CF lung.

Requirement for CD28 co-stimulation is lower in SHP-1-deficient T cells.

This study provides biochemical and functional evidence pertaining to the role of the intracellular protein tyrosine phosphatase, SHP-1, in influencing thresholds for TCR activation. Although the loss of SHP-1 in thymocytes from motheaten mice had minimal effects on the initial rise of cytosolic Ca(2+) concentration following TCR triggering, the post-stimulation equilibrium levels of Ca(2+) were consistently elevated. In keeping with a SHP-1 effect on PLCgamma function, IP3 generation was increased in SHP-1 deficient thymocytes. Importantly, we demonstrate that loss of SHP-1 results in a relaxation of the normally stringent co-stimulatory requirements for IL-2 production. SHP-1 deficient single-positive CD4(+) thymocytes revealed a significantly enhanced capacity to produce IL-2 in response to anti-CD3 stimulation alone. In contrast, the simultaneous triggering of CD3 and CD28 was required for equivalent IL-2 production in control single-positive CD4(+) thymocytes. Furthermore, SHP-1 deficient thymocytes generated an increased and prolonged proliferative response to anti-CD3 stimulation alone. In addition, the simultaneous triggering of CD28 and CD3 resulted in equivalent proliferative responses in SHP-1-deficient and control thymocytes, suggesting that a strong co-stimulatory signal is able to override the effect of SHP-1 loss on TCR hyperresponsiveness. Collectively, these results suggest that SHP-1, rather than acting directly on TCR signaling, may indirectly raise thresholds for TCR triggering by modulating co-stimulatory signals.

Osmotically induced cytosolic free Ca(2+) changes in human neutrophils.

Cytosolic free Ca(2+) concentration in neutrophils was measured by ratiometric fluorometry of intracellular fura2. Increasing the extracellular osmolarity, by either NaCl (300-600 mM) or sucrose (600-1200 mM), caused a rise in cytosolic free Ca(2+) (Delta(max) approximately equal to 600 nM). This was not due to cell lysis as the cytosolic free Ca(2+) concentration was reversed by restoration of isotonicity and a second rise in cytosolic free Ca(2+) could be provoked by repeating the change in extracellular osmolarity. Furthermore, the rise in cytosolic free Ca(2+) concentration occurred in the absence of extracellular Ca(2+), demonstrating that release of intracellular fura2 into the external medium did not occur. The osmotically-induced rise in cytosolic free Ca(2+) was not inhibited by either the phospholipase C-inhibitor U73122, or the microfilament inhibitor cytochalasin B, suggesting that neither signalling via inositol tris-phosphate or the cytoskeletal system were involved. However, the rise in cytosolic free Ca(2+) may have resulted from a reduction in neutrophil water volume in hyperosmotic conditions. As these rises in cytosolic Ca(2+) (Delta(max) approximately equal to 600 nM) were large enough to provoke changes in neutrophil activity, we propose that conditions which removes cell water may similarly elevate cytosolic free Ca(2+) to physiologically important levels.

Control of Ca2+ influx in human neutrophils by inositol 1,4,5-trisphosphate (IP3) binding: differential effects of micro-injected IP3 receptor antagonists.

Neutrophils signal Ca2+ changes in response to occupancy of G-protein-linked receptors such as the formylated peptide receptor. This Ca2+ signal is composed of two parts, inositol 1,4,5-trisphosphate (IP3)-triggered release of Ca2+ from an intracellular store and Ca2+ influx. In order to probe the relationship between these events, cytosolic free Ca2+ changes in neutrophils were monitored after micro-injection of agents which inhibit IP3 binding. Micro-injection of heparin into neutrophils totally inhibited both formylmethionyl-leucylphenylalanine-induced Ca2+ release and the subsequent Ca2+ influx. This effect was not due to prior depletion of Ca2+ stores. Furthermore, micro-injection with anti-IP3-receptor antibody also inhibited Ca2+ release. However, anti-IP3-receptor antibody and another high-molecular-mass IP3-binding antagonist, heparin-albumin conjugate, failed to inhibit the accompanying Ca2+ influx. It was concluded that two IP3-binding sites exist in neutrophils: one accessible by both heparin and the high-molecular-mass inhibitors of IP3 binding and responsible for Ca2+ release, and another inaccessible to high-molecular-mass molecules and responsible for Ca2+ influx.

Nonuniform distribution of Ca(2+) uptake sites within human neutrophils.

The rate at which Ca(2+) returns towards the basal concentration is controlled by the action of Ca(2+) pumps, both on the plasma membrane and on organelles within the cytosol. The distribution of Ca(2+) uptake sites within the cytosol was investigated using rapid confocal imaging (55 ms/frame) of fluo3-loaded human neutrophils. In some zones within the cell, the uptake of Ca(2+) from the cytosol followed a single exponential time course, whereas in others, there was accelerated kinetics after about 3 s. Using the full array of data, to produce a cell-map of Ca(2+) uptake rates a clear nonuniformity of Ca(2+) uptake sites throughout the neutrophil cytosol was observed. The location of the Ca(2+) uptake sites did not correlate with the granules or the main body of the nucleus, but Ca(2+) uptake was highest near the vestigial Golgi/ER, the edges of the nuclear lobes and at the leading cell edge. The possibility exists that the nonuniform distribution of Ca(2+) uptake sites plays a role in restricting Ca(2+) signals with the neutrophil cytosol.

Effect of prostanoids and their precursors on the aggregation of rainbow trout thrombocytes.

The role of prostanoids and their precursor fatty acids in the aggregatory response of thrombocytes (platelet equivalents of fish) from the rainbow trout, Oncorhynchus mykiss, was studied. Aggregation of these cells was induced by the thromboxane mimetic U-46619 or arachidonic acid (AA) in the presence of human or trout fibrinogen. The production of TXB2/3 by thrombocytes in response to stimulation with AA was inhibited by aspirin, ibuprofen, and indomethacin. However, thrombocyte aggregation in response to AA stimulation was not significantly altered by these agents at the concentrations tested (10-100 microM), with the exception of indomethacin at 20 and 40 microM. Effects on cytosolic calcium concentration have been suggested as an alternative mechanism for the inhibitory action of indomethacin on human platelet aggregation. The present study, however, failed to identify this as a mechanism for the inhibition of U-46619-induced trout thrombocyte aggregation by indomethacin. The polyunsaturated fatty acids docosahexaenoic acid and eicosapentaenoic acid both exhibited an inhibitory effect on U-46619-induced thrombocyte aggregation similar to that observed with mammalian platelets. Unlike the case in mammalian hemostasis, prostacyclin inhibited thrombocyte aggregation only at high concentrations (>5 microM). Prostaglandin E2, however, inhibited thrombocyte aggregation at much lower concentrations (>0.01 microM), suggesting that it may be the major inhibitory eicosanoid in trout.

Techniques for measuring and manipulating free Ca2+ in the cytosol and organelles of neutrophils.

Ca(2+) signalling in neutrophils is important for triggering and coordinating the behaviour of neutrophils. Fluorescent probes for cytosolic free Ca(2+) concentration, e.g., fura2 and fluo3, have been widely used in neutrophils. These probes can be used to monitor Ca(2+) in the cytosol, the nucleus, near the plasma membrane and theoretically within Ca(2+) storage organelles. The longer wavelength indicators, e.g., fluo3 and calcium green, can be used confocally to monitor subcellular Ca(2+) changes in the cytosol of neutrophils and in the nucleus. Confocal techniques also permit "impossible views" imaging of Ca(2+) and newer scanning techniques promise very fast temporal resolution. Techniques using chlortetracycline (CTC) and DiOC(6)(3) are also described for monitoring the position of Ca(2+) storage sites in neutrophils and for manipulating their activity. Thus, in this review, a spectrum of new (and older) optical techniques are presented which are useful for measuring, monitoring and manipulating cytosolic free Ca(2+) concentration and Ca(2+) storage in neutrophils. With these techniques, it is hoped that more insight will be gained into both the mechanism of and the consequences of Ca(2+) signalling in neutrophils.

Cytosolic Ca2+ signalling in inflammatory neutrophils: implications for rheumatoid arthritis (Review).

Recognition of the ways in which neutrophil behaviour is regulated may be crucial for a full understanding of their role in inflammation and in rheumatoid arthritis. Although it is well established that changes in cytosolic free Ca2+ play a central role in triggering neutrophil responses, only recently has evidence accumulated which points strongly to the existence of two distinct Ca2+ pathways in neutrophils. One pathway is mediated by conventional agonists, such as formylated peptides, IL-8, C5a and PAF, and the other by cross-linking and immobilisation of surface receptors, such as integrins, and the Fc receptors, CD32 and CD16. In this review, we give evidence for these two signalling pathways in neutrophils, highlighting the roles of two Ca2+ storage and release organelles, one centrally located and stationary, and the other peripheral and mobile. We point out the significance of these two routes of Ca2+ signalling for the correct sequence of neutrophil responses, and suggest that aberration of this sequence could result in pathogenic neutrophil activation.

Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells.

The microinjection of synthetic molecules, proteins, and nucleic acids into the cytosol of living cells is a powerful technique in cell biology. However, the insertion of a glass micropipette into the cell is a potentially damaging event, which presents significant problems, especially for small mammalian cells (spherical diameter = 2-15 micron), especially if they are only loosely adherent. The current technique is therefore limited to cells that are both sufficiently large or robust and firmly attached to a substrate. We describe here a modification of the standard technique that overcomes some of the problems associated with conventional microinjection but that does not involve the insertion of a micropipette deep into the cell cytoplasm. Instead, this method depends on lipid fusion at the micropipette tip to form a continuous but temporary conductance pathway between the interiors of the micropipette and cell. This technique thus also provides a novel method of transferring lipids and lipid-associated molecules to the plasma membrane of cells.

High micromolar Ca2+ beneath the plasma membrane in stimulated neutrophils.

Ca2+ near the inner face of the plasma membrane, as reported by the membrane associated fluorescent Ca2+ probe FFP-18, was higher than the bulk cytosolic free Ca2+ concentration both in resting neutrophils and in response to f-met-leu-phe. Influx caused Ca2+ close to the plasma membrane to rise more rapidly than the bulk cytosolic free Ca2+ and to reach a peak concentration of at least 30 microM. This zone of high Ca2+ was localised to just beneath the plasma membrane and did not extend more than 0.1 micron into the cell, as it was undetected by the bulk cytosolic free Ca2+ probes magfura2 and fura2. From these data, reconstruction of the distribution of Ca2+ within the neutrophil showed that the high Ca2+ signal at the cell cortex rapidly subsided to give a uniform free Ca2+ across the cell.

Release of 'caged' cytosolic Ca2+ triggers rapid spreading of human neutrophils adherent via integrin engagement.

The role of the transient rise in cytosolic free Ca2+ which occurs during neutrophil adhesion and cell spreading is unclear. In order to establish whether such a Ca2+ signal triggers neutrophil shape change, neutrophils co-loaded with fluo3 and Nitr5 ('caged' Ca2+) were used with rapid-time confocal laser scanning microscopy. Here we show that the photolytic generation of a Ca2+ rise in neutrophils which were adherent to an integrin-engaging surface, triggered a rapid change in cell morphology, with increases in cell diameter of approximately 175% occurring within 90 seconds of the Ca2+ signal. In non-adhered neutrophils or neutrophils on plain glass, no acceleration of the rate of spreading occurring in response to the release of 'caged Ca2+' could be demonstrated. It was concluded that although a rise in cytosolic free Ca2+ was not the sole trigger for neutrophil shape change, with other signals generated by integrin engagement, a rise in cytosolic free Ca2+ accelerated the rate of neutrophil spreading.

Ca2+ signalling delays in neutrophils: effects of prior exposure to platelet activating factor or formyl-met-leu-phe.

Rapid-time confocal scanning of fluo3-loaded neutrophils revealed that in individual cells there were grossly heterogeneous time intervals between stimulation with either f-met-leu-phe or platelet activating factor (PAF) and the initiation of Ca2+ influx, ranging from 75 msec to several seconds. The distribution of lag times after stimulation with f-met-leu-phe (100 nM) was influenced by prior stimulation with either f-met-leu-phe or PAF. However, whereas prior stimulation with f-met-leu-phe (50 nM) caused the subsequent cytosolic free Ca2+ response to second challenge with f-met-leu-phe to be delayed, prior stimulation with PAF (100 nM) caused an increase in the rapidity of the onset of the second response to f-met-leu-phe. With both stimuli, the cytosolic free Ca2+ in some neutrophils (non-Ca2+ responders) in the population did not increase significantly. However, some of these cells responded to the subsequent challenge. However, with both pre-treatment stimuli, those cells in which a significant Ca2+ response was provoked by the first stimulus, responded significantly faster than the initial 'non-Ca2+ responders.' However, the reduced lag time provoked by pre-stimulation was not inhibited in neutrophils in which cytosolic free Ca2+ changes were dampened by intracellular BAPTA. These data point to post-receptor events, other than prior cytosolic free Ca2+ elevation, being important in determining the response Ca2+ delay to subsequent stimulation.

Two distinct Ca2+ storage and release sites in human neutrophils.

It is well established that changes in cytosolic free Ca2+ play a role in a number of neutrophil responses such as cell shape change and oxidase activation. The release of intracellular stored Ca2+ occurs with stimuli that either act by occupancy of seven membrane-spanning domain receptors or those which act by receptor cross-linking. Here, two distinct Ca2+ storage and release sites have been identified in neutrophils. Using chlortetracycline fluorescence as an indicator of Ca2+ storage, two separate Ca2+ storage sites have been identified. One site was located peripherally under the plasma membrane and the other was in the juxtanuclear space. Confocal imaging of Fluo3-loaded neutrophils demonstrated that the central Ca2+ storage site released Ca2+ in response to N-formyl-methionyl-leucyl-phenylalanine, whereas engagement and clustering of CD11b/CD18 integrins causes Ca2+ release from the peripheral stores. The release sites also correlated with organelles that stained with DiOC6(3). Localized phototoxicity generated by DiOC6(3) excitation resulted in inhibition of the release of stored Ca2+, which was selective for the stimulus used. The presence of two distinct cellular locations for these Ca2+ stores and their independent release raises the possibility that separate intracellular messengers for their release are generated.

Spatial and temporal separation of calcium signals in myeloid cells stimulated by immune complexes.

Stimulation of large (100 microns) human myeloid cells with immune complexes resulted in Ca2+ spiking. Both global and regional changes in the intracellular cytosolic free Ca2+ concentration were detected in response to immune complex stimulation. The regional changes were mediated by release of Ca2+ from stores, whereas global changes were mediated by Ca2+ influx. They occurred independently of each other, with release of Ca2+ from intracellular Ca2+ stores being separated from transmembrane influx of Ca2+. Bromophenacyl bromide, an 1-plastin binding agent, inhibited store release without preventing transmembrane influx of Ca2+. The large size of the myeloid cells used here allowed the visualisation of the spatial and temporal separation of store release from transmembrane influx of Ca2+, providing further evidence for the existence of independent Ca2+ store release and Ca2+ influx mechanisms in these cells.