PLASMA-MEMBRANE FLUIDITY OF KERATINOCYTES OF NORMAL AND PSORIATIC SKIN - A STUDY USING FLUORESCENCE ANISOTROPY OF TRIMETHYLAMMONIUMDIPHENYLHEXATRIENE (TMA-DPH)

The aim of this study was to investigate plasma membrane fluidity in h uman keratinocytes using fluorescence anisotropy of 1,6-diphenyl-1,3,5 -hexatriene (DPH) and its cationic derivative (trimethylamino)-phenyl] -6-phenyl-1,3,5-hexatriene (TMA-DPH). Keratinocytes from normal or pso riatic skin were isolated using trypsin-EDTA or dispase. In keratinocy tes isolated from normal skin, TMA-DPH anisotropy values were higher t han those observed using DPH; the difference must be related to the di fferent localization of the two probes. In fact, DPH in whole cells lo calizes in plasma as well as intracellular membranes, yielding an aver age value of fluidity, while the cationic derivative TMA-DPH resides i n the plasma membrane of the whole cells for a sufficient time for ani sotropy measurements. Moreover, it has to be considered that plasma me mbrane is more ordered than intracellular membranes. The kinetics of i ncorporation of TMA-DPH was similar in keratinocytes isolated using tr ypsin-EDTA and those isolated using; dispase, however, the fluorescenc e anisotropy values were lower in keratinocytes isolated with dispase (0.260 +/- 0.01 vs 0.270 +/- 0.01, p = 0.029). This difference is prob ably related to modifications of lipid-protein interactions after tryp sin treatment. Since no damage to plasma membrane after incubation wit h dispase seems to have been reported, we decided to use this separati on procedure to study plasma membrane fluidity in psoriasis, a human p athological condition characterized by excessive cell proliferation an d incomplete differentiation. Lower anisotropy values (0.260 +/- 0.01 vs 0.270 +/- 0.01, p = 0.001), indicating an increase in fluidity, wer e observed in keratinocytes isolated from skin of psoriatic patients t han in epidermal cells isolated from normal human skin. We suggest tha t the measurement of fluorescence anisotropy in living cells is a conv enient and useful tool to study membrane fluidity in human keratinocyt es isolated from normal and diseased skin. Its application represents a technical advance because plasma membrane fluidity can be measured u sing very limited amounts of tissue, as obtained from biopsies.