Mehrdad Ghashghaeinia1,2, Mauro Carlos Wesseling3, Elena Ramos4,5, Polina Petkova-Kirova6, Sabrina Waibel2, Elisabeth Lang2, Rosi Bissinger2, Kossai Alzoubi2, Baerbel Edelmann7, Zohreh Hosseinzadeh8, Peter Dreischer9, Azam Shahvaroughi-Farahani10, Ulrich Mrowietz1, Martin Köberle11, Lars Kaestner6,12, Ingolf Bernhardt3, Antonio Martínez-Ruiz4,5, Thomas Wieder13, Florian Lang14. 1. Psoriasis-Center, Department of Dermatology, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Germany. 2. Department of Internal Medicine III, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany. 3. Laboratory of Biophysics, Faculty of Natural and Technical Sciences III, Saarland University, Campus A2.4, Saarbruecken, Germany. 4. Servicio de Inmunología, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Madrid, Spain. 5. Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain. 6. Institute for Molecular Cell Biology, School of Medicine, Saarland University, Homburg/Saar, Germany. 7. Otto-von-Guericke University, Department for Hematology and Oncology, Magdeburg, Germany. 8. Eye hospital, Experimental Retinal Prosthetics Group, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany. 9. Department of Internal Medicine VIII, University Hospital Tuebingen, Tuebingen, Germany. 10. Division of Neuropsychology, Center of Neurology & Hertie-Institute for Clinical Brain Research, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany. 11. Department of Dermatology and Allergology, Technische Universität Muenchen, Munich, Germany. 12. Experimental Physics, Campus E2.6, Saarland University, Saarbruecken, Germany. 13. Department of Dermatology, Eberhard Karls University, Tuebingen, Germany. 14. Department of Physiology I, Eberhard Karls University Tuebingen, Tuebingen, Germany.
Abstract
BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.
BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS:TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.