Inorg. Chem. zyxwvut 1990, Li+ T, relaxation times presumably can be used as probes of Li’ binding to the RBC membrane and for determining whether the extent and site of Li+ binding are different in RBCs from bipolar, hypertensive, and normotensive controls. Thus, the MIR approach (because of the its total noninvasiveness, easy visualization of Li+ pools, and ability to probe interactions between the Li’ ion and RBC components) will be the method of choice to investigate whether Li+ transport and distribution parameters in RBCs can be used with confidence as genetic markers of bipolar disorder^)^ and hyperten~ion.~~ 29. 3979-3985 3979 (36) Richelson, E.; Snyder, K.; Carlson, J.; Johnson, M.; Turner, S.; Lumry, A.; Boerwinkle, E.; Sing, C. F. Am. J. Psychiatry zyxwvutsrq 1986, zyxwvutsrqp 143, 457. Acknowledgment. We are grateful to Drs. Walter Dorus, Joel Stilberberg, and Vinod Bansal from the Loyola Medical Center (Maywood, IL) and Prof. Carlos Geraldes (University of Coimbra, Portugal) for helpful discussions during the course of this project. Financial support from a Grant-in-Aid from the American Heart Association of Metropolitan Chicago and USPHS Grant MH45926-01 from the National Institute of Mental Health is gratefully acknowledged by D.M.d.F. Registry No. Dy(PPP)J-, 81868-53-3; Dy(TTHA)’-, 91 264-39-0; Li, 1439-93-2; Na, 7440-23-5. (37) Wiley, J. S.; Clarke, D. A.; Bonacquisto, L. A.; Scarlett, J. D.; Harrap, S. B.; Doyle, A. E. Hypertension (Dallas) 1984, 6, 360. Contribution from the Departments of Chemistry and Biology, Loyola University of Chicago, Chicago, Illinois 60626, Department of Pediatrics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 606 12, and Department of Chemistry, University of Coimbra, 3000 Coimbra, Portugal Effects of Negatively Charged Shift Reagents on Red Blood Cell Morphology, Li+ Transport, and Membrane Potential Ravichandran Ramasamy,+ Duarte Mota de Freitas,*.+ Warren Jones,$ Frederick Wezeman,$ Richard Labotka,% and Carlos F. G. C. Geraldesll Received November 14, zyxwvutsrqp I989 Lanthanide shift reagents have been used extensively in multinuclear magnetic resonance (NMR) applications in order to obtain information regarding ion distribution and transport in cellular systems. The aqueous reagents used in this study were Dy(PPP)J-, Tm( PPP)J-, Dy(TTHA)’-, Dy(PcPcP);-, and Dy(DOTP)’-, where Dy3+ and Tm3+represent dysprosium and thulium ions and PPPs-, TTHA6-, PcPcPs-, and DOTP*- denote the triphosphate, triethylenetetraminehexaacetate, zyxw bis(dihydroxyphosphiny1- methyl)phosphinate, and I ,4,7,1 O-tetrazacyclododecane-N,N’,N”,N”’-tetrakis(methanephosphonate) ligands, respectively. The apparent size and shape of Li+-free RBCs (red blood cells), studied by both scanning electron microscopy and Coulter counter methods, were unchanged by the presence of the above shift reagents at concentrations lower than zyxwv 10 mM. However, Li+ incubation changed both the shape and size of RBCs. The rates of Na+-Li+ exchange in Li+-loadedRBCs measured by 7Li NMR spectroscopy in the presence of Dy(PPP);-, TI~(PPP),~-, or D~(PcPcP),~- were significantly higher than the rates measured in the absence of shift reagents by atomic absorption or in the presence of DY(TTHA)~- or DY(DOTP)~- by 7Li NMR spectroscopy. 31P and I9F NMR measurements of the membrane potential of Li+-free RBCs revealed that the shift reagents studied (except for Dy(TTHA)”) do change the membrane potential, with the most negatively charged reagents having the largest effect. Thus, shift reagents must be used with caution in physiological NMR studies and in particular RBC applications. Introduction Cells undergo a variety of shape changes at different stages in a cell cycle or in the process of cell maturation and differen- tiation. In the absence of hydrodynamic forces, the red blood cell (RBC) shape normally observed is that of a biconcave disc (hence the name discocyte).’,2 Several references in the literature suggest that the energy-dependent spectrin-actin network may play a role in maintaining the shape of RBCS.~” Alterations in spectrin phosphorylation by ATP depletion have been shown to be asso- ciated with crenation (shrinkage) in R B C S . ’ * ~ ~ ~ Other studies have shown that factors such as pH, ionic strength, and several drugs cause alterations of the discocyte shape.*-I2 Thus, it is apparent that RBC shape may be controlled by both energy-requiring processes and physicochemical interactions. Several paramagnetic lanthanide complexes have been applied as shift reagents for NMR-detectable alkali-metal ~ a t i o n s . l ~ - ~ ~ These reagents have become popular in recent years for distin- guishing intra- and extracellular ions, in particular Li+, Na+, and K+. In order to test the suitability of shift reagents for clinical and biological research, we have examined the effects of the negative charge on some of the most widely used shift reagents on RBC morphology, membrane potential, and Li+ transport rates. To whom correspondence should be addressed. ’ Department of Chemistry, Loyola University of Chicago. *Department of Biology, Loyola University of Chicago. 6 University of Illinois. l1 University of Coimbra. The structure of the ligands used in this study are shown in Figure I. The ligands were selected such that the shift reagents used in this study had overall charges ranging from -3 to -7. The application of the chosen shift reagents to 7Lif and 23Na+N M R (I) Bessis, M. In Red zyxwv Cell Shape; Bessis, M., Weed, R. I., Leblond, P. F., Eds.; Springer-Verlag: New York, 1973; p I. (2) Weinstein, R. S. In Red Cells; Surgenor, D. M., Ed.; Academic Press: New York, 1974; Vol. I, p 213. (3) Birchmier, W.; Singer, S. J. J. Cell Biol. 1977, 73, 647. (4) Brenner, S. L.; Korn, E. D. J. Biol. Chem. 1980, 255, 1670. (5) Cohen. C. M.; Folev; S. F. J. Cell zyxwv Biol. 1980. 86. 694. (6) Fowler, V. M.; Luna, E. J.; Hargreaves, W. R.; Taylor, D. L.; Branton, D. J. Cell Biol. 1981, 88, 388. (7) Brailsford, J. D.; Korpman, R. A,; Bull, B. S. J. Theor. Biol. 1980, 86, 513. (8) Deuticke, B. Biochim. Biophys. Acta 1968, 163, 494. (9) Glaser, R. J. Membr. Biol. 1979, 51, 217. (IO) Isomaa. B.; Paatero, G. Biochim. Biophys. Acta 1981, 647, 211. (1 1) Szaz, 1.; Hasitz, J. M.; Breuer, J. M.; Sarkadi, B.; Gardos, C. Acta Biol. Hung. 1978, 29, 1. (12) Sheetz, M. P.; Singer, S. J. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4457. (13) Chu, S. C.; Pike, M. M.; Fossel, E. T.; Smith, T. W.; Balshi, J. A.; Springer, C. S., Jr. J. Magn. Reson. 1984, 56, 33. (14) Espanol, M. T.; Mota de Freitas, D. Inorg. Chem. 1987, 26, 4356. (15) Ramasamy, R.; Espanol, M. T.; Long, K. M.; Mota de Freitas, D.; Geraldes, C. F. G. C. Inorg. Chim. Acta 1989, 163, 41. (16) Kumar, A. M.; Gupta, R. K.; Spitzer, A. Kidney Inr. 1988, 33, 792. (17) Sherry, A. D.; Malloy, C. R.; Jeffrey, F. M. H.; Cacheris, W. P.; Geraldes, C. F. G. C. J. Magn. Reson. 1988, 76, 528. 0020-1 669/90/ 1329-3979$02.50/0 0 1990 American Chemical Society Downloaded by PORTUGAL CONSORTIA MASTER on July 7, 2009 Published on May 1, 2002 on http://pubs.acs.org | doi: 10.1021/ic00345a014