Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

Abstract

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 °C (18.7±1.8 kcal/mol) than below that critical temperature (7.5±1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1±1.6 kcal/mol above 15 °C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 °C, and two smaller transitions at 26 and 37 °C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 °C and smaller transitions at 26 and 35 °C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-β-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 °C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.