Trehalose uptake through P2X7 purinergic channels provides dehydration protection.
The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.
Elliott
Gloria D
GD
Department of Mechanical Engineering and Engineering Sciences, University of North Carolina at Charlotte, 9201 University City Building, Charlotte, NC 28223-0001, USA. gdelliot@uncc.edu
Liu
Xiang-Hong
XH
Cusick
Jennifer L
JL
Menze
Michael
M
Vincent
Jill
J
Witt
Trudy
T
Hand
Steve
S
Toner
Mehmet
M
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002463:Cell Membrane Permeability; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003681:Dehydration / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D009995:Osmosis; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D013997:Time Factors; D014199:Trehalose / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014867:Water
C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D014867:Water; D000255:Adenosine Triphosphate; D014199:Trehalose