Role of Ca2+/calmodulin complex in intracellular Ca2+ mobilization in neutrophils has not been clearly elucidated. In this study, effects of chlorpromazine, trifluoperazine and imipramine on the intracellular Ca2+ mobilization, including Ca2+ influx, in C5a-activated neutrophils were investigated. Complement C5a-stimulated superoxide production and myeloperoxidase release in neutrophils were inhibited by chlorpromazine, trifluoperazine and imipramine, except no effect of imipramine on myeloperoxidase release. A C5a-elicited elevation of (Ca2+)i in neutrophils was inhibited by chlorpromazine, trifluoperazine, imipramine, staurosporine, genistein, EGTA, and verapamil but not affected by pertussin toxin. The intracellular Ca2+ release in C5a-activated neutrophils was not affected by chlorpromazine and imipramine. Chlorpromazine and imipramine inhibited Mn2+ influx by C5a-activated neutrophils. Thapsigargin-evoked Ca2+ entry was inhibited by chlorpromazine, trifluoperazine, imipramine, genistein, EGTA and verapamil, while in the activation process of neutrophils. The depressive action of calmodulin inhibitors on the elevation of cytosolic Ca2+ level in C5a-activated neutrophils appears to be accomplished by inhibition of Ca2+ influx from the extracellular medium.
Calmodulin* ; Chlorpromazine ; Complement C5a ; Complement System Proteins* ; Cytosol ; Depression* ; Egtazic Acid ; Genistein ; Imipramine ; Neutrophils* ; Peroxidase ; Staurosporine ; Superoxides ; Trifluoperazine ; Verapamil

The present study assessed the effect of calmodulin antagonists trifluoperazine and W-7 against the cytotoxicity of MPP+ and 6-hydroxydopamine (6-OHDA) in relation to the mitochondrial dysfunction and cell death in PC12 cells. Trifluoperazine (an inhibitor of the mitochondrial permeability transition and calmodulin antagonist) and W-7 (a specific calmodulin antagonist) significantly attenuated the MPP+- induced cell viability loss in PC12 cells with a maximum inhibition at 0.5~1microM; beyond these concentrations the inhibitory effect declined. Both compounds at this concentration range did not cause cell death significantly. In contrast to MPP+, the trifluoperazine and W-7 did not depress the cytotoxic effect of 6-OHDA. Addition of trifluoperazine and W-7 inhibited the cytosolic accumulation of cytochrome c and caspase-3 activation in PC12 cells treated with MPP+ and attenuated the formation of reactive oxygen species and the depletion of GSH, whereas both compounds did not reduce the effect of 6-OHDA. The results show that trifluoperazine and W-7 may attenuate the cytotoxicity of MPP+ by inhibition of the mitochondrial permeability transition and calmodulin. Meanwhile, the cytotoxic effect of 6-OHDA seems to be mediated by the actions, which are different from MPP+.
Animals ; Calmodulin ; Caspase 3 ; Cell Death ; Cell Survival* ; Cytochromes c ; Cytosol ; Oxidopamine ; PC12 Cells* ; Permeability ; Reactive Oxygen Species ; Trifluoperazine*

This paper notes the effect of vasodilators on the cerebral vasospasm induced by experimental subarachnoid hemorrhage. Artificial subarachnoid hemorrhage produced by dual injection of non-heparinized autologous blood into the cisterna magna in rabbits with 72 hours interval gave rise to considerable narrowing or spasm of the basilar artery and its branches demonstrated by cerebral angiography. SAH was induced in 12 rabbits by injecting 1.3mL/kg or autologous fresh arterial blood into the cisterna magna, followed by suspending the rabbits in a head-down position at 30 degree for 1 hour in order to accumulate blood around the basilar artery. The animals were divided into 4 groups; a group receiving nifedipine, a group receiving aminophylline, a group receiving papaverine, a group receiving trifluoperazine HCl. Angiographically vertebrobasilar arterial spasm was demonstrated 3 days after the 2nd autologous blood injection into the cisterna magna. Radiographically visible spasm was resistant to the vasodilating drugs. Rabbits were sacrificed by the overdose injection of sodium phenobarbital at the end of experiment.
Aminophylline ; Angiography ; Animals ; Basilar Artery ; Cerebral Angiography ; Cisterna Magna ; Nifedipine ; Papaverine ; Phenobarbital ; Rabbits ; Sodium ; Spasm ; Subarachnoid Hemorrhage* ; Trifluoperazine ; Vasodilator Agents* ; Vasospasm, Intracranial*

To investigate the mechanism of smooth muscle contraction induced by emptying of intracellular Ca2+ stores, we measured isometric contraction and 45Ca2+ influx. CaCl2 increased Ca2+ store emptying- induced contraction in dose-dependent manner, but phospholipase C activity was not affected by the Ca2+ store emptying-induced contraction. The contraction was inhibited by voltage-dependent Ca2+ channel antagonists dose dependently, but not by TMB-8 (intracellular Ca2+ release blocker). Both PKC inhibitors (H-7 and staurosporine) and tyrosine kinase inhibitors (genistein and methyl 2,5-dihydroxycinnamic acid) significantly inhibited the contraction, but calmodulin antagonists (W-7 and trifluoperazine) had no inhibitory effect on the contraction. The combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were greater than that of each one alone. In Ca2+ store-emptied condition, 45Ca2+ influx was significantly inhibited by verapamil, H-7 or genistein but not by trifluoperazine. However combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were not observed. Therefore, this kinase pathway may modulate the sensitivity of contractile protein. These results suggest that contraction induced by emptying of intracellular Ca2+ stores was mediated by influx of extracellular Ca2+ through voltage-dependent Ca2+ channel, also protein kinase C and/or tyrosine kinase pathway modulates the Ca2+ sensitivity of contractile protein.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Animals ; Calmodulin ; Cats* ; Genistein ; Isometric Contraction ; Muscle, Smooth* ; Phosphotransferases ; Protein Kinase C ; Protein Kinase Inhibitors ; Protein-Tyrosine Kinases ; Trifluoperazine ; Type C Phospholipases ; Verapamil

The promoting effect of hydrogen peroxide (H2O2) against the cytotoxicity of 1-methyl-4-phenylpyridinium (MPP+) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with MPP+ resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. Addition of H2O2 enhanced the MPP+-induced nuclear damage and cell death. Catalase, Carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the cytotoxic effect of MPP+ in the presence of H2O2. Addition of H2O2 promoted the change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to MPP+ in PC12 cells. The results show that the H2O2 treatment promotes the cytotoxicity of MPP+ against PC12 cells. H2O2 may enhance the MPP+-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that H2O2 as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by neurotoxins.
1-Methyl-4-phenylpyridinium ; Acetylcysteine ; Animals ; Caspase 3 ; Catalase ; Cell Death* ; Cyclosporine ; Cytochromes c ; Cytosol ; Hydrogen Peroxide* ; Hydrogen* ; Membrane Potentials ; Mitochondrial Membranes ; Neurons ; Neurotoxins ; PC12 Cells* ; Permeability ; Reactive Oxygen Species ; Trifluoperazine

The use of tumor antigen specific antibodies for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. However, several factors restrict the use of anti-PGP monoclonal antibodies(Mabs). First, Pgp is expressed in normal tissues, particularly in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroids plexus and other organs. It plays a significant role to transport drugs and toxins in these organs. Therefore, anti-PGP antibodies in combination with cytotoxic compounds or radiolabelled antibodies should neither inhibit the activity of PGP, nor harm the cells which expressed PGP normally. BiMab exploit the specificity of Mab and ensures activation of cellular cytotoxic mechanisms which kill tumor cells only, but not harm normal cells. It will provide a strategy for resistant cancer therapy using anti-PGP antibodies. Second, Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and appro ximately 90% following a second treatment. In an effort to reduce the toxicity and antigenicity, we focus to produce anti-PGP antibodies which have the binding activity only, but not inhibit the function of the "pump", and to construct a small and partially humanized recombinant molecule with dual specificity for both PGP and CD3 complex to activate the host immune response toward the tumour. PCR and overlap PCR were used to construct anti-CD3/ anti-Pgp Diabody. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by both the detection of western blot and size exclusion chromatography; its antigen-binding activity was examined by FACS, cellular RIA. Plasmid pAYZDCP which expressed the anti-CD3/anti-Pgp Diabody was constructed correctly. The diabody was recovered in high yield( up to 2mg/ L) after E-taq purification and predominantly(90%) as a dimer. The diabody can bind to Jurkat cells (CD3+) and K562/A02 cells(Pgp+). The affinities of the diabody were similar with the anti-CD3 ScFv or anti-Pgp ScFv, respectively. The anti-CD3/ anti-Pgp BsF(ab')2 was first recast into the diabody format and succeeded to obtain high level expression. The results of some biological activity experiments indicated that the diabody could bind to Jurkat cells and K562/A02 cells. Multidrug resistance can be reversed experimentally by a variety of drugs, among which the best known are verapamil and trifluoperazine, which unfortunately are of limited use in practice due to severe collateral cardiac toxicity. Anti-PGP x anti-CD3 diabody will provide another therapeutic strategy against multidrug resistance cancer.
ATP-Binding Cassette, Sub-Family B, Member 1 ; immunology ; Animals ; Antibodies, Monoclonal ; immunology ; metabolism ; Antibody Specificity ; genetics ; physiology ; Blotting, Western ; CD3 Complex ; immunology ; Chromatography, Gel ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Flow Cytometry ; Humans ; Jurkat Cells ; drug effects ; metabolism ; K562 Cells ; drug effects ; metabolism ; Mice ; Polymerase Chain Reaction ; Radioimmunoassay ; Trifluoperazine ; pharmacology ; Verapamil ; pharmacology

The early growth response gene-1 (Egr-1) is a tumor suppressor which plays an important role in cell growth, differentiation and apoptosis. Egr-1 has been shown to be down-regulated in many types of tumor tissues. Trifluoperazine (TFP), a phenothiazine class of antipsychotics, restored serum-induced Egr-1 expression in several cancer cell lines. We investigated the effect of Egr-1 expression on the TFP-induced inhibition of cell growth. Ectopic expression of Egr-1 enhanced the TFP-induced antiproliferative activity and downregulated cyclin D1 level in U87MG glioma cells. Our results suggest that antipsychotics TFP exhibits antiproliferative activity through up-regulation of Egr-1.
Cell Cycle ; Cell Proliferation/drug effects ; Cyclin D1/metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Gene Expression ; Glioma/*metabolism ; Humans ; Immediate-Early Proteins/genetics/*metabolism ; Promoter Regions (Genetics)/drug effects ; Research Support, Non-U.S. Gov't ; Transcription Factors/genetics/*metabolism ; Trifluoperazine/*pharmacology ; Tumor Cells, Cultured

Eukaryotic elongation factor eEF-2 mediates regulatory steps important for the overall regulation of mRNA translation in mammalian cells and is activated by variety of cellular conditions and factors. In this study, eEF-2 specific, Ca2+/CaM-dependent protein kinase III (CaM PK III), also called eEF-2 kinase, was examined under oxidative stress and cell proliferation state using CHO cells. The eEF-2 kinase activity was determined in the kinase buffer containing Ca2+ and CaM in the presence of eEF-2 and [gamma-32P] ATP. The eEF-2 kinase activity in cell lysates was completely dependent upon Ca2+ and CaM. Phosphorylation of eEF-2 was clearly identified in proliferating cells, but not detectable in CHO cells arrested in their growth by serum deprivation. The content of the eEF-2 protein, however, was equivalent in both cells. Using a phosphorylation state-specific antibody, we show that oxidant such as H2O2, which triggers a large influx of Ca2+, dramatically enhances the phosphorylation of eEF-2. In addition, H2O2-induced eEF-2 phosphorylation is dependent on Ca2+ and CaM, but independent of protein kinase C. In addition, okadaic acid inhibits phosphoprotein phosphatase 2A(PP2A)-mediated eEF-2 dephosphorylation. These results may provide a possible link between the elevation of intracellular Ca2+ and cell division and suggest that phosphorylation of eEF-2 is sensitive cellular reflex on stimuli that induces intracellular Ca2+ flux.
Animal ; CHO Cells ; Ca(2+)-Calmodulin Dependent Protein Kinase/*metabolism ; Cell Division ; Cells, Cultured ; Comparative Study ; Cytosol/enzymology ; Egtazic Acid/pharmacology ; Hamsters ; Human ; Hydrogen Peroxide/*pharmacology ; Mice ; Okadaic Acid/pharmacology ; Oxidants/*pharmacology ; Peptide Elongation Factors/metabolism ; Phosphoprotein Phosphatase/metabolism ; Phosphorylation ; Polyethylene Glycols/pharmacology ; Trifluoperazine/pharmacology

Adenoma ; complications ; pathology ; Adult ; Antipsychotic Agents ; adverse effects ; therapeutic use ; Aripiprazole ; Benzodiazepines ; adverse effects ; therapeutic use ; Bromocriptine ; adverse effects ; therapeutic use ; Dopamine Antagonists ; adverse effects ; therapeutic use ; Female ; Hormone Antagonists ; adverse effects ; therapeutic use ; Humans ; Hyperprolactinemia ; complications ; etiology ; Piperazines ; adverse effects ; therapeutic use ; Pituitary Neoplasms ; complications ; pathology ; Quinolones ; adverse effects ; therapeutic use ; Risperidone ; adverse effects ; therapeutic use ; Schizophrenia ; drug therapy ; etiology ; pathology ; Serotonin Antagonists ; adverse effects ; therapeutic use ; Trifluoperazine ; adverse effects ; therapeutic use