calpain and fura-2-am

Endothelial cell (EC) migration, essential for reestablishing arterial integrity after vascular injury, is inhibited by oxidized LDL (oxLDL) and lysophosphatidylcholine (lysoPC) that are present in the arterial wall. We tested the hypothesis that a mechanism responsible for lysoPC-induced inhibition is increased intracellular free calcium concentration ([Ca(2+)](i)).. LysoPC, at concentrations that inhibit in vitro EC migration to 35% of control, increased [Ca(2+)](i) levels 3-fold. These effects of lysoPC were concentration dependent and reversible. LysoPC induced Ca(2+) influx within 10 minutes, and [Ca(2+)](i) remained elevated for 2 hours. The calcium ionophore A23187 also increased [Ca(2+)](i) and inhibited EC migration. Chelators of intracellular Ca(2+) (BAPTA/AM and EGTA/AM) and nonvoltage-sensitive channel blockers (lanthanum chloride and gadolinium chloride) blunted the lysoPC-induced [Ca(2+)](i) rise and partially preserved EC migration. After lysoPC treatment, calpain, a calcium-dependent cysteine protease, was activated, and cytoskeletal changes occurred. Calpain inhibitors (calpastatin, MDL28170, and calpeptin) added before lysoPC prevented cytoskeletal protein cleavage and preserved EC migration at 60% of control levels.. LysoPC increases [Ca(2+)](i). In turn, activating calpains that can alter the cytoskeleton are activated and EC migration is inhibited.

Topics: Animals; Aorta; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calpain; Cattle; Cell Movement; Chelating Agents; Cysteine Proteinase Inhibitors; Dipeptides; Egtazic Acid; Endothelium, Vascular; Enzyme Activation; Fluorescent Dyes; Fura-2; Ionophores; Lysophosphatidylcholines

Topics: Animals; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Hypoxia; Cells, Cultured; Coumarins; Cysteine Proteinase Inhibitors; Dipeptides; Fluorescent Dyes; Fura-2; Glutathione; Hypoxia; In Situ Hybridization, Fluorescence; Liver; Oligopeptides; Rats; Substrate Specificity

The source and concentration of Ca2+ required to activate calpain I were investigated in thrombin-stimulated platelets. The concentration of cytosolic free Ca2+ ([Ca2+]i) was measured in platelets containing fura-2-AM, and exhibited a biphasic response after stimulation with 0.05, 0.1 or 0.5 NIH units of thrombin/ml. An initial transient elevation, which was predominantly dependent upon Ca2+ released from the internal stores into the cytosol, peaked at 15 s after stimulation, and a secondary sustained elevation, which was due to Ca2+ influx, was observed following the initial elevation. Calpain I was present at about 540 ng/10(8) unstimulated platelets, as measured by immunoblotting using rabbit anti-(human calpain I) IgG. Calpain I was activated 10 s after thrombin stimulation, as determined by the appearance of the 78 kDa and 76 kDa forms on immunoblots. The activation ratio of calpain I was calculated as the amount of the 78 + 76 kDa forms as a percentage of the total (80 + 78 + 76 kDa), and was influenced by the extent of the initial transient [Ca2+]i elevation after stimulation. An initial increase in [Ca2+]i of 300 nM was required to achieve the maximal activation (60%) of calpain I, and half-maximal activation occurred at 160 nM- Ca2+]i. These results suggest that the activation of calpain I in platelets is regulated by the initial elevation in Ca2+]i after thrombin stimulation, and does not necessarily require a Ca2+ influx.

Topics: Adult; Blood Platelets; Calcium; Calpain; Cytosol; Egtazic Acid; Enzyme Activation; Fluorescent Dyes; Fura-2; Humans; Immunoblotting; Immunodiffusion; In Vitro Techniques; Kinetics; Thrombin

A series of peptidyl diazomethanes and monofluoromethane with structures specific for calpain have been synthesized and tested for their ability to inhibit calpain activity in vivo, using human platelets as a model system. Calpain activity in vivo was determined by observing proteolysis of actin-binding protein and talin, two known substrates of calpain. Very potent inhibitors, which emerged from this study, were used to investigate the role of calpain in some platelet response processes. Our results show that calpain-mediated proteolysis in platelets is not an obligatory event leading to change of cell shape, adhesion to glass and spreading, aggregation and 5-hydroxytryptamine release. Two of the inhibitors were iodinated with 125I and used to radiolabel the enzyme in vivo. To our knowledge, this work also represents the first report describing the affinity labelling of calpain in human platelets using irreversible radioactive inhibitors.

Topics: Adenosine Diphosphate; Amino Acid Sequence; Blood Platelets; Calcium; Calpain; Fluorescent Dyes; Fura-2; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Oligopeptides; Platelet Activation; Platelet Adhesiveness; Platelet Aggregation; Serotonin; Spectrometry, Fluorescence