High incorporation of [3H]inositol into phosphoinositides of human platelets during reversible electropermeabilisation.
A new method for high incorporation of [3H]inositol into human platelets is described. The method involves incorporation of [3H]inositol during reversible electropermeabilisation by high voltage discharge, followed by resealing the cells during incubation at 37 degrees C. Between 10- and 20-fold increase of isotope uptake is achieved compared to control intact cells. Permeabilised resealed platelets maintain good responses to thrombin and collagen. Analysis of the incorporation of the label amongst the phosphoinositides shows 70% to be in PI, 20% in PIP, and 10% in PIP2. Stimulation with thrombin and analysis of the formation of IP1, IP2 and IP3 shows the labelling to occur in a hormone-sensitive pool. These studies indicate that reversible electropermeabilisation can be used to achieve good uptake of non-membrane penetrating substances such as inositol.
Authi
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KS
Department of Biochemistry and Cell Biology, Hunterian Institute, Royal College of Surgeons of England, London.
Hughes
K
K
Crawford
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N
D001792:Blood Platelets / Q000378:metabolism*; D002463:Cell Membrane Permeability; D003094:Collagen / Q000493:pharmacokinetics; D004563:Electrochemistry; D006801:Humans; D007294:Inositol / Q000493:pharmacokinetics*; D010716:Phosphatidylinositols / Q000096:biosynthesis / Q000097:blood*; D013696:Temperature; D013917:Thrombin / Q000493:pharmacokinetics; D013997:Time Factors
D010716:Phosphatidylinositols; D007294:Inositol; D003094:Collagen; D013917:Thrombin