calpain and calmidazolium

In this study we demonstrate that histone deacetylase (HDAC)-inhibitor mediated cell surface expression of the structural different NKG2D-ligands MICA/B and ULBP2 is calcium-dependent. Treatment with the calcium chelator EGTA inhibited constitutive as well as HDAC-inhibitor induced MICA/B and ULBP2 cell surface expression on melanoma cells and Jurkat T-cells. A NKG2D-dependent cytolytic assay and staining with a recombinant NKG2D-Fc fusion protein showed that calcium chelation impaired the functional ability of NKG2D-ligands induced by HDAC-inhibitor treatment. The HDAC-inhibitor induced cell surface expression of ULBP2, but not MICA/B, was sensitive to treatment calmidazolium and trifluoperazine, two agents known to block calcium signaling. siRNA-mediated knock-down of the calcium-regulated proteins calmodulin or calpain did however not affect NKG2D-ligand cell surface expression on Jurkat T-cells. We further show that secretion and cell surface binding of the calcium-regulating protein galectin-1 is enhanced upon HDAC-inhibitor treatment of melanoma cells. However, binding of galectin-1 to cell surface glycoproteins was not critical for constitutive or HDAC-inhibitor induced MICA/B and ULBP2 cell surface expression. We provide evidence that MICA/B and ULBP2 cell surface expression is controlled differently by calcium, which adds to the increasing perception that cell surface expression of MICA/B and ULBP2 is controlled by distinct signal transduction pathways.

Topics: Base Sequence; Calcium Signaling; Calmodulin; Calpain; Cell Line, Tumor; Cell Membrane; Depsipeptides; Galectin 1; Gene Knockdown Techniques; GPI-Linked Proteins; Histone Deacetylase Inhibitors; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; Jurkat Cells; Ligands; Melanoma; RNA, Small Interfering; Trifluoperazine

While the determination of postmortem interval (PMI) is a crucial and fundamental step in any death investigation, the development of appropriate biochemical methods for PMI estimation is still in its infancy. This study focused on the temperature-dependent postmortem degradation of calcineurin A (CnA), calmodulin-dependent kinase II (CaMKII), myristoylated alanine-rich C-kinase substrate (MARCKs), and protein phosphatase 2A (PP2A) in mice. The results show that MARCKS, CaMKII, and the use of lung tissue do not appear to warrant further study for the determination of PMI in humans. In skeletal muscle, CnA underwent a rapid temperature-dependent cleavage (60 --> 57 kDa) over the first 48 h of postmortem interval. At 21 degrees C, this transformation was completed within 24 h. In contrast, PP2A increased within the first 24 h after which it degraded at 21 degrees C but remained stable for up to 96 h at 5 degrees C and 10 degrees C. The 60 --> 57 kDa postmortem conversion of CnA was inhibited by addition of protease inhibitors and MDL-28170 indicating a calpain pathway mediates this breakdown. Proteasome inhibition (MG-132) and calmodulin antagonism (calmidazolium) also inhibited this conversion suggesting that other protein degradation pathways also are in play. In contrast, all of the protease inhibitors and calmidazolium but not ethylene glycol tetraacetic acid led to increased levels of PP2A. The data are discussed in terms of developing a useable field-based biochemical assay for postmortem interval determination in humans and understanding the protein degradation pathways that are initiated upon death.

Topics: Animals; Blotting, Western; Calcineurin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calpain; Centrifugation; Cysteine Proteinase Inhibitors; Egtazic Acid; Enzyme Inhibitors; Forensic Pathology; Imidazoles; Intracellular Signaling Peptides and Proteins; Leupeptins; Lung; Membrane Proteins; Mice; Muscle, Skeletal; Myristoylated Alanine-Rich C Kinase Substrate; Postmortem Changes; Proteasome Endopeptidase Complex; Protein Phosphatase 2; Temperature

Only the 80-kD catalytic subunit of smooth muscle calpain II shows a change in intrinsic fluorescence on binding calcium, but both the 80-kD and 30-kD subunits show fluorescence changes in bound toluidinyl-naphthalenesulphonate as a result of calcium binding. Both subunits also show changes in intrinsic fluorescence in the presence of calmidazolium and felodipine. These studies indicate that both subunits have binding sites for calcium and the calmodulin antagonists, which are probably located in the calmodulin-like domain of each subunit.

Topics: Animals; Calcium; Calmodulin; Calpain; Chickens; Felodipine; Fluorescence; Imidazoles; Protein Conformation

Treatment of human erythrocytes with ionophore A23187 (10 mumol.l-1) and Ca2+ (0.05-0.5 mmol.l-1) or Sr2+ (0.2-1 mmol.l-1) in results in a concentration-dependent acceleration of the transmembrane reorientation (flip) of the lipid probes lysophosphatidylcholine and palmitoylcarnitine to the inner membrane leaflet after their primary insertion into the outer leaflet. Mg2+, Mn2+, Zn2+ and La3+ do not accelerate flip. Ca2(+)-induced flip acceleration depends also on the ionophore concentration. It is reversed by removal of Ca2+ with EDTA. A causal role of Ca2(+)-induced membrane protein degradation and decrease of the polyphosphoinositide level in flip acceleration could be excluded. Likewise, calmodulin-dependent processes are probably not involved since the calmodulin antagonist calmidazolium (2-10 mumol.l-1) does not suppress but even enhances the Ca2(+)-induced flip acceleration. The same is true for the Ca2+ antagonist flunarizine. These drugs do not alter flip rate in the absence of Ca2+. At high Ca2+ (1-5 mmol.l-1) an initial flip acceleration is followed by flip normalization. High concentrations of Mn2+ and Mg2+ slow down flip rates. The selective acceleration of flip by Ca2+ and Sr2+ is discussed to be due to a local detachment of the membrane skeleton from the bilayer, whereas the unselective slow down of flip by divalent cations might be due to a stabilization of the membrane bilayer by the cations. After loading of cells with Ca2+ (but not with Mn2+) the inner membrane leaflet phospholipid phosphatidylserine becomes rapidly exposed to the outer membrane surface, as detectable by its accessibility to phospholipase A2 (5 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biological Transport; Calcimycin; Calcium; Calpain; Cations, Divalent; Erythrocyte Membrane; Flunarizine; Humans; Imidazoles; Kinetics; Lipid Bilayers; Phospholipids

1. The intrinsic fluorescence of epoxysuccinyl-inhibited calpain II undergoes a Ca2(+)-dependent decrease which contrasts with the increase observed for calmodulin. 2. Calpain II was inhibited by the calmodulin antagonist toluidinylnaphthalenesulfonate (TNS), and a Ca2(+)-dependent increase in TNS fluorescence intensity was observed for epoxysuccinyl-inhibited calpain II. 3. The calmodulin antagonists calmidazolium CDZ and felodipine both caused decreases in the intrinsic fluorescence of epoxysuccinyl-inhibited calpain II. 4. Increasing concentrations of Ca2+ caused an increase in the fluorescence intensity of the inhibited enzyme in the presence of (CDZ), and a decrease in the presence of felodipine. 5. It is concluded from these studies that Ca2+ and calmodulin antagonists induce conformational changes in calpain II, and that changes occur in regions other than the Ca2(+)-binding domains.

Topics: Binding Sites; Calcium; Calmodulin; Calpain; Felodipine; Fluorescent Dyes; Imidazoles; In Vitro Techniques; Leucine; Molecular Conformation; Naphthalenesulfonates; Spectrometry, Fluorescence

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.

Topics: Bee Venoms; Calmodulin; Calpain; Erythrocytes; Humans; Imidazoles; Kinetics; Melitten; Protein Kinase C; Sulfonamides; Trifluoperazine

Topics: Animals; Calcium-Binding Proteins; Calpain; Chickens; Cricetinae; Enzyme Inhibitors; Gizzard, Avian; Imidazoles; Kinetics; Muscle, Smooth; Muscles