Fluorescent lipid probes 12-AS and TMA-DPH report on selective, purinergically induced fluidity changes in plasma membranes of lymphoid cells.
The effect of extracellular ATP (ATPex) on the anisotropy of 1-[4-(trimethylamino) phenyl]-6-phenyl-hexa-3,5 triene (TMA-DPH) and 12-anthroyloxi-stearic acid (12-AS) fluorescence was investigated in Balb/C mouse thymocytes and in JY human lymphoblasts. These cells have been shown recently to be sensitive and resistant to ATPex, respectively, in terms of cellular responses. Extracellular ATP (1 mM) induced a time-dependent elevation in the emission anisotropy of both probes (indicating an increased lipid packing density) in the plasma membrane of thymocytes. The maximal effect, at 37 degrees C, was observed between 20 and 60 min after ATPex administration, and followed by a gradual decrease of fluorescence anisotropy at longer times (60-180 min). ATPex did not change membrane fluidity of thymocytes below the phase transition temperature (at 18 degrees C). Oxidized ATP (oATP), a selective antagonist of P2z purinoreceptors, blocked the ATPex-induced decrease in membrane fluidity. Low ATPex concentrations (100-300 microM)--which are known to induce distinct signals (changes in membrane potential and intracellular Ph)--slightly fluidized the plasma membrane of thymocytes. This effect was partially blocked by quinine, a blocker of Ca(2+)-activated K+ channels. Neither 12-AS nor TMA-DPH showed any change in their emission anisotropy upon ATPex-treatment in the plasma membrane of the resistant human JY lymphoblast cells. No other signalling event (membrane potential change, Ca2+ response) is elicited by ATPex in this cell line. These data suggest that the changes in the membrane fluidity are likely consequences of specific, purinoreceptor-mediated signalling events, such as hyper-or depolarization of the plasma membrane or Ca2+ influx. These signals may induce changes in the conformation or lateral organization of membrane proteins, perturbing protein-lipid interactions, as well.
Matkó
J
J
Department of Biophysics, Medical University School of Debrecen, Hungary. matko@jaguar.dote.hu
Nagy
P
P
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004161:Diphenylhexatriene / Q000031:analogs & derivatives* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008560:Membrane Fluidity / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D026902:Potassium Channel Blockers; D058919:Purinergic P2 Receptor Antagonists; D011803:Quinine / Q000494:pharmacology; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction; D013229:Stearic Acids / Q000378:metabolism*; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; D026902:Potassium Channel Blockers; D058919:Purinergic P2 Receptor Antagonists; D058486:Receptors, Purinergic P2X7; D013229:Stearic Acids; C026653:12-(9-anthroyloxy)stearic acid; D004161:Diphenylhexatriene; C017199:2',3'-dialdehyde ATP; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D000255:Adenosine Triphosphate; D011803:Quinine; D002118:Calcium