Effect of Cetiedil, an In Vitro Antisickling Agent,

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1 ffect of Cetiedil, n n Vitro Antisickling Agent, on rythrocyte Membrne Ction Permebility L R. BRKOWTZ nd UGN P. ORRNGR, Division of HemtologylOncology, Deprtments ofmedicine nd Hospitl Lbortories, University ofnorth Crolin School of Medicine, Chpel Hill, North Crolin A B S T R A C T Cetiedil hs been reported to relieve pinful crises in sickle cell nemi nd to hve ntisickling properties in vitro. The drug lters neither oxygen ffinity nor the solubility of deoxyhemoglobin S. Becuse the viscosity of the erythrocyte interior nd the kinetics of geltion re dependent on the concentrtion of hemoglobin, e postulted tht cetiedil might inhibit sickling by modifying erythrocyte sodium or potssium movements in mnner tht ould increse cell ter content nd thus dilute the cell hemoglobin. The drug hs to such effects: it inhibits the specific increse in potssium permebility tht follos rise in cytoplsmic clcium concentrtion nd it cuses rise in pssive sodium movements. These effects re further evidence tht cell ion nd ter movements my be importnt in the process of sickling nd suggest mechnism for the results reported ith cetiedil. NTRODUCTON Cetiedil, one of fmily of compounds synthesied s smooth muscle relxnts, relieves pin from cludiction nd Rynud's phenomenon (1). n ddition, cetiedil hs been reported to decrese the durtion nd intensity of sickle cell crises, lthough this study s not performed in controlled, double-blinded fshion (2). This effect my be more thn vsculr since sttisticlly significnt decrese in sickle forms hs lso been observed hen hemoglobin S-contining erythrocytes re deoxygented in the presence of cetiedil (1, 3). Concentrtions of cetiedil used in these in vitro studies ere comprble to those chieved during in vivo dministrtion (1). As the ction of the drug differs from other ntisickling gents, ffecting neither the solubility nor the oxygen ffinity of hemoglobin S (3), e postulted tht cetiedil might exert A portion of this ork hs been published in bstrct form (198. Clin. Res. 28: 823; Clin. Res. 29: 33). Address reprints requests to Dr. Lee R. Berkoit. Received for publiction 6 My 1981 nd in revised form 3 July its beneficil effect by loering the concentrtion of hemoglobin S ithin the erythrocyte. The viscosity of the erythrocyte interior is lrgely function of hemoglobin concentrtion. Thus, fll in cell ter content impirs erythrocyte deformbility (4). This effect is mgnified in the deoxygented sickle erythrocyte becuse rise in cell hemoglobin concentrtion gretly ccelertes the rte t hich hemoglobin S polymeries (5). There re lso severl lines of evidence to suggest tht the sickle erythrocyte becomes dehydrted in the process of deoxygention (6-9). Finlly, in vitro nd in vivo evidence indicte tht hypotonic conditions, by loering intrcellulr hemoglobin S concentrtion, inhibit the tendency to sickle (1, 11). For these resons drug tht incresed sodium permebility or inhibited preexistent tendency for potssium loss ould, by preventing cellulr dehydrtion, preserve the deformbility of the sickle erythrocyte. The present studies ere designed to test the in vitro effect of cetiedil on ion nd ter movements cross the membrnes of norml nd sickle erythrocytes. To effects ere found. Pssive N permebility, exmined by both net nd unidirectionl flux studies, is incresed. A preexistent tendency for K loss, studied by exposing erythrocytes to conditions knon to generte selective increse in K permebility, ATP depletion folloed by C exposure (12), is inhibited by cetiedil. These properties tht prevent cellulr dehydrtion offer potentil explntion for the reported ntisickling effects of cetiedil. MTHODS Blood from hemtologiclly norml volunteers or ptients ith sickle cell nemi ho hd not been trnsfused for minimum of 6 mo s drn into heprin-rinsed syringes nd used ithin 3 min of collection. For experiments evluting the net flux of N nd K, the plsm nd buffy cot ere discrded nd the remining cells ere shed four times t 1, g in 1 vol of N sh contining, NCl 155 mm; Hepes 5 mm; ph 7.4 t 37 C. A smll liquot of these cells s - J. Clin. nvest. The Americn Society for Clinicl nvestig tion, nc. Volume 68 November / /6 $

2 reserved for the bse-line mesurement of N, K, nd ter (the " time" smple) nd the remining cells ere suspended in buffered sline solutions, the composition of hich re detiled in the figure legends. For N, K, nd ter determintion, the shed cells ere centrifuged t 4, g in specilly constructed lucite cell ter tubes ith nrro ell t the bottom for collection of the cell button. N nd K mesurements ere performed by flme photometry, on pcked, eighed liquots of the cells. Results ere expressed s milliequivlents per kilogrm of dry cell solid. The ter content s mesured by drying knon eight of et cells to constnt eight t 9 C. Detils of these procedures hve been previously published (13). All incubtions ere crried out t 37 C in stoppered flsks under ir in ter-bth shker set t 1 oscilltions/ min over trverse of 1 in. At the timepoints indicted on the figures, n liquot of ech suspension s removed nd centrifuged t 1, g. Folloing removl of the superntnt by spirtion, the cells ere shed four times in the N sh solution nd then processed for N, K, nd ter content. The unidirectionl movement of N s evluted using 22N. 22N s obtined s 22NCl from Ne nglnd Nucler, Boston, Mss. The cells ere shed four times in 1 vol of N sh s described bove nd dded to the pproprite solutions. After 5-min equilibrtion period t 37 C, 22N s dded in concentrtion sufficient to give 5, dpm. At the indicted times, liquots ere removed nd centrifuged t 4, g in cell ter tubes. The superntnt s seprted nd the pcked cells hemolyed ith distilled ter contining 1,ul of Triton X-1. Both the superntnt nd the hemolyed cells ere counted in Pckrd gmm counter. (Pckrd nstrument Co. nc., Doners Grove, ll.). Hemoglobin in grms per deciliter s mesured from the solubilied cells s cynmethemoglobin complex t 54 OD. The influx s clculted from the rtio of cell counts to superntnt counts nd expressed s millimoles N per grm hemoglobin per hour. The trpped extrcellulr spce s estimted from the counts in the hemolyed cells of the " time" smple. To exmine the effect of cetiedil on C-dependent K permebility, cells ere shed in 1 vol of K sh contining, KC 15 mm, Hepes 5 mm; ph 7.4 t 37 C. Mesurements of N, K, nd cell ter ere performed ("fresh" smple), folloing hich ATP depletion s crried out by exposing cells for 6 min t 37 C to solution contining, KC 145 mm, Hepes 1 mm, inosine 1 mm, iodocette 1 mm; ph 7.4. The cells ere gin shed in the K sh to remove ll trces of the inhibitor, nd N, K, nd cell ter mesurements ere done (" time" smple). This repeted use of K-rich solutions prevented initition of K loss during these preprtive steps. The cells ere then plced in Nrich solutions, the composition of hich re indicted in the figure legends, nd incubted t 37 C. Aliquots of the suspension ere removed t certin timepoints, shed ith the N sh, nd processed for N, K, nd ter content. The cetiedil used in these studies s provided in poder form by Dr. Gerrd Pilley of nnother Phrmceuticls, Arcueil, Frnce. t s suspended in concentrted form in 95% ethnol nd n identicl volume of the vehicle s dded to ll control flsks. ch experiment s crried out ith both norml nd sickle erythrocytes. Figs. 1, 2, 4, nd 6 plot the men nd stndrd error of the men of four identicl experiments performed tice ith norml erythrocytes nd tice ith sickle erythrocytes. The experiments illustrted in Figs. 3 nd 5 ere lso repeted minimum offour times. As these figures sho doseresponse ptterns, men nd stndrd error ofthe men re not Cf) ' cr J l (n c7 cr y jw _' *-* CONTROL M OUABAN o--o 1-4M CTDL &-' 1-4 M OUABAN M CTDL " x TM (HOURS) FGUR 1 The effect of cetiedil (.1 mm) on ion nd ter content of erythrocytes during 24-h incubtion. rythrocytes ere divided into four flsks nd incubted in solution contining, NCl 145 mm, KC 5 mm, N2HPO4 2.5 mm, Hepes 1 mm, glucose 1 mm; ph 7.5 t 37 C. Cetiedil.1 mm nd oubin.1 mm ere dded s indicted on the figure L. R. Berkoit nd. P. Orringer

3 () -j -j C-) 19 ic cr C) V) ft... -i Y W -i u l ) C ct _ -** 5 x 1-4M CTDL x 1-4 C.) M CTDL A-- 4M CTDL i TM (HOURS) FGUR 2 The effect of cetiedil (.1,.25 nd.5 mm) on ion nd ter content of erythrocytes during 24-h incubtion. rythrocytes ere divided into three flsks nd incubted in solution contining, NCl 145 mm, KCl 5 mm, N2HPO4 2.5 mm, Hepes 1 mm, glucose 1 mm; ph 7.5 t 37 C. Cetiedil s dded to ech flsk t the indicted concentrtion. shon. ch of these to figures is representtive doseresponse curve using sickle erythrocytes. The ptterns ofdoseresponse chieved ith norml erythrocytes ere the sme. RSULTS The effect of cetiedil on the ion nd ter content of erythrocytes during 24-h net flux study is shon in Fig. 1. This experiment s performed in the presence nd bsence of oubin. During the 24-h period, exposure to cetiedil lone t concentrtion of.1 mm cused no chnge in N, K, or ter content. rythrocytes in physiologic solution plus oubin shoed the expected gin of N nd loss of K. Similr ion chnges occurred hen cetiedil s dded to oubin, but no dditionl effect s noted. At cetiedil concentrtions bove.1 mm, erythrocytes exhibited n increse in N content even in the bsence of oubin (Fig. 2). At cetiedil concentrtion of.5 mm, smples could only be obtined up to 6 h fter hich totl hemolysis occurred, mking longer incubtions invlid. To investigte further the etiology ofthe N gin seen t higher concentrtions of cetiedil, 22N influx s performed. All smples ere exposed to.1 mm oubin to eliminte ctive N efflux vi the N-K pump. Concentrtions of cetiedil identicl to those in Fig. 2 (.1,.25, nd.5 mm) ere used. At.1 mm, the N influx s identicl to control flsk here no cetiedil s present. At cetiedil concentrtions >.1 mm, N influx s incresed in dose-dependent fshion (Fig. 3). The next series of experiments s performed to evlute the effect of cetiedil on erythrocyte K permebility. rythrocytes ere exposed to in vitro conditions knon to produce selective increse in K permebility. ATP depletion s ccomplished ith inosine nd iodocette. After 1-h exposure to the inhibitor, cell ATP levels ere < 1%. The erythrocytes ere then plced into one ofthree N solutions: () solution ith GTA, C cheltor; (b) solution ith GTA plus n excess of C; or (c) solution ith GTA, n excess of C, nd cetiedil.1 mm. N, K, nd ter content ere mesured t 3, 6, nd 12 min (Fig. 4). Cells exposed to n excess of C shoed mrked K loss nd cell dehydrtion, chnges hich ere not observed if C s omitted from the incubtion vessel. This Cdependent K nd ter loss s totlly inhibited by cetiedil. The dosge dependency of cetiedil on inhibition ffect of Cetiedil on rythrocyte Membrne Ction Permebility 1217

4 ) x-o ) TM (MNUTS) FGUR 3 The effect of cetiedil on unidirectionl N influx in erythrocytes. rythrocytes ere divided into four flsks nd incubted in solution contining, NCl 145 mm, KC 5 mm, N2HPO4 2.5 mm, Hepes 1 mm, glucose 1 mm; ph 7.5 t 37 C. Oubin.1 mm s present in ll flsks. Cetiedil s dded in the concentrtions indicted. After 5 min equilibrtion period, 22N s dded nd the rte of 22N influx s mesured. of K nd ter loss is seen in Fig. 5. rythrocytes ere ATP-depleted nd exposed to grded concentrtions of cetiedil from to.1 mm. C s present -i C,) J -i J o19 cn (Resh) 2 j 18 O it 15 J 14 C. 12 TM (MNUTS) &- CoC12 + CTDL -9 GTA o--o CoC12,+\~, FGUR 4 The effect of cetiedil on C-medited K permebility in the erythrocyte. rythrocytes ere incubted for 1 h in solution contining, KCl 145 mm, Hepes 1 mm, inosine 1 mm, iodocette 1 mm; ph 7.5 t 37 C. The erythrocytes ere then plced into one of three solutions s indicted on the figure: (1) NCl 15 mm, Hepes 1 mm, GTA.25 mm (2) NCl 15 mm, Hepes 1 mm, GTA.25 mm, CCl2 2 mm; (3) NCl 15 mm, Hepes 1 mm, GTA.25 mm, CCl2 2 mm, cetiedil.1 mm. in ll solutions. A grded dose-response s seen ith mximum inhibition of K nd ter loss t.1 mm cetiedil. Little effect s seen t 1 um cetiedil. The finl experiment tested the bility of cetiedil to inhibit K nd ter loss once initited (Fig. 6). After ATP depletion, ll erythrocytes ere plced for 6 min in solution contining n excess of C. At the end of this hour, K nd ter loss hd begun. The cells ere then divided into to flsks nd cetiedil, t concentr- 7r 6 (1 5 4 Cs vr _ CTDL CONC. (M) -- o -- jo-5 *- 2.5 x1-5 D- 5 x x 1-5 *-* 1-4 l TM (MNUTS) FGUR 5 The dosge dependency of cetiedil on C-medited K permebility in the erythrocyte. rythrocytes ere incubted for 1 h in solution contining, KCL 145 mm, Hepes 1 mm, inosine 1 mm, iodocette 1 mm; ph 7.5 t 37 C. The erythrocytes ere then plced into six flsks in solution contining, NCl 15 mm, Hepes 1 mm, GTA.25 mm, CCl2 2 mm; ph 7.5 t 37 C. Cetiedil s dded to ech flsk t the concentrtion indicted on the figure L. R. Berkoit nd. P. Orringer

5 51 r TM (MNUTS) FGuR 6 The effect of cetiedil on C-medited K permebility, once initited, in the erythrocyte. rythrocytes ere incubted for 1 h in solution contining, KCl 145 mm, Hepes 1 mm, inosine 1 mm, iodocette 1 mm; ph 7.5 t 37C. rythrocytes ere next incubted for 1 h in solution contining, NCl 15 mm, Hepes 1 mm, GTA.25 mm, CCl2 2 mm. rythrocytes ere then divided into 2 flsks nd.1 mm cetiedil dded to one flsk (dshed line). tion of.1 mm, s dded to one. Those cells not exposed to cetiedil continued to lose K nd ter. Unlike the control flsk, hoever, K loss nd cell dehydrtion ere bruptly terminted s soon s the erythrocytes ere exposed to cetiedil. DSCUSSON The foregoing experiments suggest tht cetiedil might exert its ntisickling effect by preventing n increse in the concentrtion of cytoplsmic hemoglobin through n influence on erythrocyte ion nd ter content. Becuse the concentrtion of hemoglobin ithin ll erythrocytes is the mjor determinnt of cytoplsmic viscosity, rise in hemoglobin concentrtion results in fll in cell deformbility (14). An increse in hemoglobin concentrtion exerts second, prticulrly deleterious effect upon sickle erythrocytes. n these cells the rte t hich hemoglobin S ggregtes folloing deoxygention is enormously influenced by the cell hemoglobin concentrtion (5). Since hemoglobin is n imperment species, its intrcellulr concentrtion is determined by the ter content of the erythrocyte, hich in turn is function of the number of osmoticlly ctive prticles ithin the cell. Becuse K is the mjor intrcellulr ction, permebility defect specific for K nd not N ill result in the loss of cellulr ions nd ter (15). Severl lines of evidence suggest tht the sickle erythrocyte becomes dehydrted during cycles of deoxygention nd tht this loss of cell ter ith its resultnt increse in cytoplsmic viscosity nd impired cellulr deformbility is the result of K loss (6-9). The mechnism by hich such n increse in K permebility might develop is less certin. An ttrctive hypothesis ould suggest tht repeted cycles of deoxygention promote the intrcellulr ccumultion of C ions (16, 17). A rise in the C content in the erythrocyte is knon to cuse K loss by creting K-specific chnnels in the erythrocyte membrne (12, 18). Dt supporting such sequence of events in the sickle erythrocyte include the folloing: () incresed C content of the sickle erythrocyte (17, 19); (b) loss of K from sickle erythrocytes during deoxygention (6); nd (c) shrinkge of sickle erythrocytes upon deoxygention. (9). One could esily envision s the end result of this process, the severely dehydrted, energy-depleted, irreversibly sickled cell (8, 2). Unlike other ntisickling gents, cetiedil does not pper to interct ith hemoglobin S since oxygen ffect of Cetiedil on rythrocyte Membrne Ction Permebility 1219

6 ffinity nd solubility remin unchnged (3). The drug, hoever, exerts to distinct effects on monovlent ction permebility, ech of hich ould tend to prevent cell dehydrtion. ither effect of cetiedil ould inhibit the increse in cell hemoglobin concentrtion observed in sickle erythrocytes nd thus preserve their intrinsic deformbility. Such n hypothesis is consistent ith the "selling" noted by Askur et l. (3). t is resonble to speculte tht the mechnism of ction of potentil ntisickling gent involves chnge in erythrocyte membrne permebility rther thn modifiction of the hemoglobin S molecule. Before ssuming clinicl relevnce for these in vitro studies ith cetiedil, hoever, similr reduction in cell dehydrtion must be demonstrted in vivo. Controlled clinicl trils, hich include these in vivo mesurements of erythrocyte ction nd ter content, ill ttempt to correlte physiologic effects on the sickle erythrocyte ith meliortion of sickle cell crises. ACKNOWLDGMNTS We re indebted to Dr. John C. Prker for his dvice nd helpful suggestions nd to Fy Wever for expert secretril ssistnce. Supported in prt by Ntionl nstitutes of Helth grnt AM RFRNCS 1. Benjmin, L. J., G. Kokkini, nd C. M. Peterson Cetiedil: its potentil usefulness in sickle cell disese. Blood. 55: Cbnnes, R. Preliminry study on the effects of cetiedil in cute episodes of sickle cell nemi. Presented t the nterntionl Symposium on Vsculr Disese, Februry 1977, Rome, tly. 3. Askur, T., S. T. Ohnishi, K. Adchi, M. Oxcuc, K. Hshimoto, M. Singer, M.. Russell, nd. Schrt ffect of cetiedil on erythrocyte sickling. Ne type of ntisickling gent tht my ffect erythrocyte membrnes. Proc. Ntl. Acd. Sci. U. S. A. 77: Mohnds, N., W. M. Phillips, nd M. Bessis Red blood cell deformbility nd hemolytic nemis. Semin. Hemtol. 16: Hofrichter, J., P. D. Ross, nd W. A. ton Supersturtion in sickle cell hemoglobin solutions. Proc. Ntl. Acd. Sci. U. S. A. 73: Tosteson, D. C.,. She, nd R. C. Drling Potssium nd sodium of red blood cells in sickle cell nemi. J. Clin. nvest. 31: Glder, B.., nd D. G. Nthn Ction permebility ltertions during sickling: reltionship to ction composition nd cellulr hydrtion of irreversibly sickled cells. Blood. 51: Glder, B.., S.. Lux, A. Muller-Soyn,. S. Pltt, R. D. Propper, nd D. G. Nthn nergy reserve nd ction composition of irreversibly sickled cells in vivo. Brit. J. Hemtol. 4: Msys, D. R., P. A. Bromberg, nd S. P. Blcerk Red cells shrink during sickling. Blood. 44: Zrkosky, H. S., nd R. M. Hochmuth Sickling times of individul erythrocytes t ero P2. J. Clin. nvest. 56: Ros, R. M., B.. Bierer, R. Thoms, J. S. Stoff, M. Kruskll, S. Robinson, H. F. Bunn, nd F. H. pstein A study of induced hypontremi in the prevention nd tretment of sickle-cell crisis. N. ngl. J. Med. 33: Grdos, G The role of clcium in the potssium permebility of humn erythrocytes. Act Physiol. Acd. Sci. Hung. 15: Prker, J. C., H. J. Gitelmn, P. S. Glosson, nd D. L. Leonrd Role of clcium in volume regultion by dog red blood cells. J. Gen. Physiol. 65: Mohnds, N., M. R. Clrk, M. S. Jcobs, nd S. B. Shohet Anlysis of fctors regulting erythrocyte deformbility. J. Clin. nvest. 66: Orringer,. P., nd J. C. Prker on nd ter movements in red blood cells. n Progress in Hemtology.. B. Bron, editor. Grune & Strtton, nc., Ne York. 8: Bookchin, R. M., nd V. L. Le Progressive inhibition of the C pump nd C:C exchnge in sickle red cells. Nture (Lond.). 284: ton, J. W., T. D. Skelton, H. S. Sofford, C.. Kolpin, nd H. S. Jcob levted erythrocyte clcium in sickle cell disese. Nture (Lond.). 246: Plishker, G., nd H. J. Gitelmn Clcium trnsport in intct humn erythrocytes.j. Gen. Physiol. 68: Plek, J Red cell clcium content nd trnsmembrne clcium movements in sickle cell nemi. J. Lb. Clin. Med. 89: Clrk, M. R., N. Mohnds, nd S. B. Shohet Deformbility of oxygented irreversibly sickled cells. J. Clin. nvest. 65: L. R. Berkoit nd. P. Orringer