Fluorescent lipid probes 12-AS and TMA-DPH report on selective, purinergically induced fluidity changes in plasma membranes of lymphoid cells

Abstract

The effect of extracellular ATP (ATP ex) 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 ATP ex, 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°C, was observed between 20 and 60 min after ATP ex administration, and followed by a gradual decrease of fluorescence anisotropy at longer times (60–180 min). ATP ex did not change membrane fluidity of thymocytes below the phase transition temperature (at 18 °C). Oxidized ATP (oATP), a selective antagonist of P 2, purinoreceptors, blocked the ATP ex-induced decrease in membrane fluidity. Low ATP ex concentrations (100–300 μM)—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 ATP ex-treatment in the plasma membrane of the resistant human JY lymphoblast cells. No other signalling event (membrane potential change, Ca 2+ response) is elicited by ATP ex 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 Ca 2+ influx. These signals may induce changes in the conformation or lateral organization of membrane proteins, perturbing protein-lipid interactions, as well.