Increased cation conductance in human erythrocytes artificially aged by glycation.
Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which may result from activation of Ca(2+) permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca(2+)](i) from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca(2+) channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30-48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37 degrees C) significantly increased glycation of membrane proteins, hemoglobin (HbA(1c)), TRPC3/6/7, and L-type Ca(2+) channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca(2+)](i), and phosphatidylserine exposure, and led to significant cell shrinkage. Ca(2+) removal and addition of Ca(2+) chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca(2+) entry from extracellular space.
Kucherenko
Yuliya V
YV
Department of Physiology, Institute I, Eberhard-Karls-Universität Tübingen, Gmelinstr. 5, 72076, Tübingen, Germany.
Bhavsar
Shefalee K
SK
Grischenko
Valentin I
VI
Fischer
Uwe R
UR
Huber
Stephan M
SM
Lang
Florian
F
D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D004553:Electric Conductivity; D004905:Erythrocyte Aging / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D006442:Glycated Hemoglobin / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D018408:Patch-Clamp Techniques; D010718:Phosphatidylserines / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000187:drug effects / Q000378:metabolism*
D002118:Calcium; D020746:Calcium Channels, L-Type; D005947:Glucose; D006442:Glycated Hemoglobin; D010718:Phosphatidylserines; D050052:TRPC Cation Channels; C517652:hemoglobin A1c protein, human