ORIGINAL ARTICLE © Copyright 2006 by Humana Press Inc. All rights of any nature whatsoever reserved. 1085-9195/(Online)1559-0283/06/46:265–276/$30.00 Cell Biochemistry and Biophysics 265 Volume 46, 2006 INTRODUCTION It is widely accepted that human red blood cells (RBCs) become selectively sequestered at spleen level after nearly 120 d of uninterrupted circulation (1). Little is known about the physiological mechanisms involved in the recognition and removal of aged cells. It has been suggested earlier that the internal Ca increase that occurs on cell aging might trigger removal of senescent RBCs (2,3). Such a Ca increase apparently results from unbalance between a steadily raising influx and an increasingly deficient pump activity as the cell ages (4,5). Opening of mechanosensitive Ca channels appears the main mechanism leading to a raised influx during RBC passage through restricted areas of microcircula- tion (5–8). However, other Ca-selective channels may also be involved. In this context, presence of putative, Voltage-Dependent Calcium Channels in Young and Old Human Red Cells Pedro J. Romero, 1, * Eneida A. Romero, 1 Dania Mateu, 1 Concepción Hernández, 1 and Irina Fernández 1 1 Laboratory of Membrane Physiology, Institute of Experimental Biology, Faculty of Sciences, Central University of Venezuela. Aptdo. 48088, Caracas 1041-A (Venezuela) Abstract Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of α 1 subunit of high-voltage activated Ca channels (pan α 1 ) and against conserved residues of α 1C and α 1E subunits. No specific reaction was detected with anti- bodies against conserved residues of α 1A , α 1B , or α 1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan α 1 or anti-α 1E blots, whereas two bands (200 and 230 kDa) were detected by anti-α 1C exposure. Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl + 5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15–50% reduction of 45 Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Q- and N-type blockers also reduced Ca influx to various extents (25–60%). Old cells were generally insensitive to L-type but not to non–L-type blockers. Raising external K to about 70–80 mM reduced by 50–100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non–L-type. The results clearly demonstrate pres- ence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non–L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed. Index Entries: Erythrocyte Ca channels; human erythrocytes; voltage-dependent Ca channels; Ca channel immunoblotting; young and old red cells; L- and R-type channels *Author to whom all correspondence and reprint requests should be addressed. E-mail: romepe@yahoo.com.