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Oxidised α-mercaptoacrylic acid derivatives are potent conformationally restricted calpain-I inhibitors that mimic the endogenous inhibitor calpastatin., 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.

Although the mechanisms of Ca2+ signalling in neutrophils by certain chemotactic agents have been well characterized, the signalling by immune complexes is poorly understood. Here we demonstrate that immune complex stimulation, acting via Fc receptors, leads to repetitive Ca2+ spiking in neutrophils. Although the initial Ca2+ rise was the result of release of Ca2+ from intracellular stores, subsequent repetitive Ca2+ spikes resulted from transmembrane influx, as they were prevented by removal of extracellular Ca2+ and were accompanied by Mn2+ influx. The transmembrane Ca2+ spikes induced dramatic neutrophil cell shape changes. The Ca2+ spiking phase was inhibited by a phospholipase C (PLC) inhibitor, U73122, and removal of immune complex, but not by cytochalasin B. It was concluded that Ca2+ spiking was dependent upon the initial release of Ca2+ from an intracellular Ca2+ store, and driven by continued binding of immune complex, which triggered pulsatile changes in transmembrane influx.

Increases in cytoplasmic free Ca2+ concentration in rat submandibular acini were observed in response to isoprenaline (10 microM), noradrenaline (10 microM) and carbamoylcholine (10 microM). Noradrenaline and carbamoylcholine responses were decreased to 27% and 33% respectively in the absence of extracellular Ca2+, suggesting a major requirement for Ca2+ entry. beta-Adrenergic stimulation elicited a small (35-40 nM) free Ca2+ rise, approx. 75% of which was mobilized from an intracellular store. Results suggest that this Ca2+ rise is a key event in the physiological triggering of mucin secretion by exocytosis.

We demonstrate here that the recently defined cytokine hepatocyte growth factor (HGF) 'primes' human neutrophils. Recombinant human HGF over the concentration range 0.1-20 ng/ml increased the neutrophil response to f-met-leu-phe by up to 200%, and required only a short preincubation, 10 min producing the maximum effect. Priming was independent of changes in cytosolic-free calcium homeostasis. We conclude that HGF may be a physiologically important cytokine with 'priming' activity for neutrophils.

Tumour necrosis factor (TNF) is a mediator of inflammation that has been shown to enhance neutrophil responses to soluble and particulate stimuli. The release of leukotriene B4 (LTB4) by human neutrophils stimulated by unopsonized zymosan was measured in the presence or absence of recombinant human TNF-alpha (rhTNF-alpha) preincubation. There was a threefold increase in the LTB4 response at an optimal TNF concentration of 10(-9) M and an optimal preincubation time of 10-20 min. A similar time and dose dependency was observed for CR3 receptor expression and for the release of the secondary lysosomal granule marker, vitamin B12-binding protein. In contrast, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), although stimulating an increase in both CR3 receptor number and in particle phagocytosis, failed to induce an increase in LTB4 release in response to zymosan. In addition, the present study demonstrated that, unlike FMLP, the exocytotic mechanism for secondary granule release by rhTNF-alpha functioned in the absence of a rise in cytosolic free calcium. Furthermore, it was independent of changes in cyclic nucleotide concentrations and did not require an intact cytoskeleton. Thus the capacity of rhTNF-alpha to amplify the neutrophil response to zymosan through the CR3 receptor appears to be related to the amplification of post-membrane events as well as to an increase in the number of functionally active receptors.