Fluorescence anisotropy of diphenylhexatriene and its cationic Trimethylamino derivative in liquid dipalmitoylphosphatidylcholine liposomes: opposing responses to isoflurane.

BACKGROUND: The mechanism of action of volatile general anesthetics has not yet been resolved. In order to identify the effects of isoflurane on the membrane, we measured the steady-state anisotropy of two fluorescent probes that reside at different depths. Incorporation of anesthetic was confirmed by shifting of the main phase transition temperature. RESULTS: In liquid crystalline dipalmitoylphosphatidylcholine liposomes, isoflurane (7-25 mM in the bath) increases trimethylammonium-diphenylhexatriene fluorescence anisotropy by ~0.02 units and decreases diphenylhexatriene anisotropy by the same amount. CONCLUSIONS: The anisotropy data suggest that isoflurane decreases non-axial dye mobility in the headgroup region, while increasing it in the tail region. We propose that these results reflect changes in the lateral pressure profile of the membrane.

Combined use of steady-state fluorescence emission and anisotropy of merocyanine 540 to distinguish crystalline, gel, ripple, and liquid crystalline phases in dipalmitoylphosphatidylcholine bilayers.

The various lamellar phases of dipalmitoylphosphadtidylcholine bilayers with and without cholesterol were used to assess the versatility of the fluorescent probe merocyanine 540 through simultaneous measurements of emission intensity, spectral shape, and steady-state anisotropy. Induction of the crystalline phase (Lc') by pre-incubation at 4°C produced a wavelength dependence of anisotropy which was strong at 15 and 25°C, weak at 38°C, and minimal above the main transition (>~41.5°C) or after returning the temperature from 46 to 25°C. The profile of anisotropy values across this temperature range revealed the ability of the probe to detect crystalline, gel (Lß'), and liquid crystalline (Lα) phases. The temperature dependence of fluorescence intensity was additionally able to distinguish between the ripple (Pß') and gel phases. In contrast, the shape of the emission spectrum, quantified as the ratio of merocyanine monomer and dimer peaks (585 and 621 nm), was primarily sensitive to the crystalline and gel phases because dimer fluorescence requires a highly-ordered environment. This requirement also explained the diminution of anisotropy wavelength dependence above 25°C. Repetition of experiments with vesicles containing cholesterol allowed creation of a phase map. Superimposition of data from the three simultaneous measurements provided details about the various phase regions in the map not discernible from any one of the three alone. The results were applied to assessment of calcium-induced membrane changes in living cells.PACS Codes: 87.16.dt.