Bincturrdca et Btoplo~ica A~a. 11360992) 155-160

155

i: 1992 Else-.ier Science Pabli:,hers B.V. All rights rescn..ed 0167-4889/92/$05.00

M e a s u r e m e n t of intraceilular Ca :+ in young and old human

erythrocytes using 19F-NMRspectroscopy N a n c i R . A i k e n a.', . l a m e s D . S a t t e r l e e

b and William R. Galey "

L'nUerstO" of New Mexico School of Medicine, Deparlment of Physiology, AIbaquerque, NM eUSA) and r, II'a.dzmgton Stare Utdl er~ty. Chemi~to" Department, Pullman, WA (USA)

(Rece~.'ed 21 November 1091} (Res'ised manu~ript received 6 April 1992)

IG.'5"v,ords: Calcium ion; Cell aging; Nuclear magnetic resonance; (Human eqahroq.le) Elevated cell calcium has been implicated in functional changes with human erythrocyte aging. However, until recently it has been difficult to measure frcc ionic intraccllular calcium in red cells. We have made use of a fluorinated calcium chelator probe (5,5'-difluoroBAPTA) and fluorine nuclear magnetic resonance (t'~F-NMR) techniques to measure changes of intracellular Ca :+ concentrations ([Ca-"" ]i) with cell aging. We have demonstrated in these studies that human crythrocytc [Ca2 +], is significantly elevated ms a funclion of in-vivo aging. Young cells, the least dense fraction of density-separated crylhrocytes, contained an average of 62 ( +_4) nM Ca: ~ ( _+S.E.), whereas the oldest, most dense cell fracti .n contained 221 nM Ca-"* ( +_25). Mechanisms by which intracellular [Ca-** ] incrca*_.eswith in-vivo aging arc currently under investigation.

Introduction The erythmcyte has been the focus of agmg studies for many years (see Ref. I for r~.wiew). The mature red ceil provides a good model for the study o i certain aspects of cell aging for several rcasous, including (1) lack of a nucleus and the capability of large scale de-novo protein synthesis necessary for cell repair; and (2) a finite lifespan, approx. 120 days for the human erythrocyte, after which time it is removed from circulation and degraded. This means that the proteins the human red cell contains can apt be replaced, and therefore are in the process o l aging from the time the nucleus is extruded from the reticulocyte. The rod cell thus provides a means of studying age-related protein changes without the complicating factors of the addition or replacement of ccl! proteins. The erythrocyte also lacks other intracellular organclles, such as mitochondria and cndoplasmic rcticulum, which serve in other cell types as stores for excess intracellular cal-

Correspondence to: W.R. Galey. Unh'ersity of New Mexico Schoc.I of Medicine. Department of Ph~,~iology,Albuquerque, NEt 87131. USA. t current address: Johns Hopkins University School of Medicine. Department of Radiology. NMR Research Laboratory. il0 MRI Bldg.. 600 N. Wolfe St.. Baltimore. MD 21205. USA.

cium. Therefore it is ideal for the study of aging effects on plasma membrane calcium ion fluxes. The identification and understanding of the mechanisms that trigger the events leading to removal of aged red cells from circulation are importam for a number of reasons. First, it is important for biood-banking purposes to understand these mechanisms in o r d e r to maintain viable, functional red cells for transfusion. Secondly, it is important to understand the aging process in general, in that knowledge gained from red-cell aging may then be applicable to aging o f other cell types. A third consideration is that understanding the means by which senescent erythro,.%'tc~ arc recognized and removed from circulation may also aid in understanding mechanisms o f recognition and destruction o f aberrant cells. Aging studies therefore have implications for understanding disease states and their consequences. Changes which havc been reported in aging cells include decreased deformability [2-4], decreased volume and surface area [5,6], increased ceil density [7-9], altered cell surface componenent [10-12], changes in enzyme activities [13-15], altered ion concentrations [13,16], and altered anion-transport kinetics [17,18]. O f particular interest are reported increases in total cell calcium wiih human red-cell aging. LaCelle et al. found an increase from 60 # M in young cells to 100 # M in old cells [2], while Shiga et al. reported an increase from 15 # M to 33 ptM Ca :+ [4]. These studies

156 wcrc performed using atomic absorption spectroscopy techniques, and thus were not direct measures of in-vivo concentrations. However, until recently, direct measurement of free ionic intracellular calcium has been difficult in the red cell. Fluorescence microscopy, using recently-developed fluorescent calcium-chelator probes such as fura-2 and the quin compounds [19,20L is difficult in erythrucytes, primarily because of the native fluorescence of hemoglobin. The development of fluorinated chelator compounds for use with SqF-NMR spectroscopy [21-23], such as 5,5'-difluoroBAPTA

column was first discarded to remove any remaining contaminating reticulocytes. The next lightest 10% of the cell column was collected as the "young' fraction, and contained less than 0.1% reticulocytes. The middle 10% and the bottom, most dense 10% were collected as the "middle" and 'old' fractions, respectively. Increased cell density has been shown to be a good correlate of increased cell age [!.8,9,13]. Lothra et al. [8] used labelled red cells to demonstrate that the dense cells had the shortest survival upon reinfusion.

(5,5'-difluoro-l,2-bis(o-amino-pheoxy)ethanc-N,N,N',

Calcium measurement

N'-tetraacetic acid), facilitates the measurement of intracellular Ca "+ in red cells. Because the red cell lacks any NMR-detectablc native XgF signal, any resonance detected in 19F-NMR experiments arises only as a result of the presence of the fluorinated calciumchelating compound. The addition of acetoxymethyl groups (AM) to the compound results in a membranepermeant form (5,5'-difluoroBAPTA-AM) which therefore equil~rates across the plasma membrane. The calcium probe is trapped within the cell due to cleavage of the AM groups by nonspecific intracellular esterases, producing a highly charged lipophob[c molecule. 5,5'-difluoroBAPTA4-, that is able to chelate free intracellular Ca 2÷. The change in the electronic environment of the fluorine atoms on the BAPTA4molecule upon binding to Ca-'+ results in a chemical shift of the 19F-NMR signal [21,23]. It is therefore possible to calculate the concentration of the free ionic intracellular calcium from the ratio of the areas under the calcium-bound BAPTA and the free BAPTA4÷ signal peaks, knowing the dissociation constant [21]. We have used this :echnique to measure differences in iutracellular free Ca-"÷ concentrations in human red blood cells of different ages. This work was been presented in abstract form [18].

Cells were allowed to equihbrate in buffer (in mM: 132 NaCI, 5A KCI, 0.8 MgSO4 -71-120, 1.25 CaCI2, 5.0 Hepes, 5.0 sodium pyruvate, 5.0 glucose; pH 7.4, 290 mOsm) for 60 rain at 3TC. They were then incubated with 50 ttM 5,5"-difiuoroBAPTA-AM (Molecular Probes, Eugene, OR) in the same buffer for 60 rain, 3TC. Following centrifugntion and resuspension in BAPTA-AM-free buffer, the cells were allowed to recover for another 60 rain (3"PC). All steps were performed at appp3x. 5% hematocrit (Hct). After washing twice in cae+-free buffer to remove any cxtracellular BAPTA4- arising from cell I~sis, cells (20% Hct, in the original buffer containing i.25 mM Ca 2÷) were placed in a 12-ram NMR sample tube. tgF-NMR experiments were performed using a GE-360 MHz instrument and a commercial ~ N / ~ H broadband probe, with the dccoupler tuned to 339.67 MHz. One pulse experiments were performed using a 40° tip angle, a sweep width of 4-10000 Hz, resulting in an acquisition time of 204.8 ms. Delay time between cycles was !.15 s, and each spectrum was an average of 4000 acquisitions, resulting in a total experimental time of approx. 90 min. All experiments were performed at 37°C under nonspinning conditions. The sample volume extended above and below the sensitive region of the coil, so that the gradual cell settling over the time course of the experiments did not cause major changes in cell density in this area. Cells were not oxygenated during the experiment (1) du¢ to the lack ol oxidative respiration in the red cells (glycolysis being the primary source of energy), and (2) the oxygen present was adequate to maintain hemoglo~i;t in t~,~ oxy state during this time. Ca 2+ concentretion wa.~ calotlated using the equation:

Materials and Methods

Cell collection and preparation All chemicals used were reagent grade or better, and were obtained from Sigma (St. Louis, MO), Aldrich (Milwaukee, WI) or Fisher Scientific (Denver, CO). Erythrocytes were obtained immediately before experiments by venipuncture from healthy adult volunteers. The plasma and burry coat were removed and the cells were then washed three times in buffer (in raM: 100 KCI, 50 NaCI, i.0 NaH2PO4, 12.5 Na2HPO4; pH 7.45; 290 mOsm). Packed cells were next separated into 'young,' "middle," and "old" fractions by means of density centrifugation. Packed cells were centrifuged for I h at 3"PC, 27000×g, in a fixed-angle rotor. This temperature was found to provide decreased viscosity while the rotor allowed maximum thermal circulation of the cells [24]. The least dense 5% of the packed cell

[Ca-'* ], = Kd([B?JrrAI/ICa-BAPTA]). where K d = 708 nM [26]. The free and bound BAPTA concentrations were the peak areas, determined from the NMR spectra. Results A typical 'OF-NMR spectrum of BAPTA-AM-Ioaded unseparated erythrocytes is shown in Fig. la. The

157

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Old

Fig. 2. Comparison of intracellular Ca2" concentrations among young, middle and old human erythrocyte fractions. The difference between old (n = 17) and both young (n = 18) and middle (n = 10) cell fractions is significant, using the SNK mullipl¢ comparison test.

2

Fig. I. Examples of VJF-NMR spectra of human eo~throcytes. (a) Unseparated ted celts,incubated ,with 5.5'-difluoroBAV['A-AM for

60 rain at 3"PC. The signal ari~ng al 2 ppm is due to free BAPTA4-. w'hile the signal at 7.5 ppm is the rcsull of a downfield shift due to the cakium-bound form of BAPTA. All NMR experiments were performed at 3TC. using a GE-360 MHz instalment eqUiplx~ with a broadband probe. T h e decoupler was tuned to IVF 039.67 M H z ) . Spectra were acquired usiag a 40 ~ tip angle and a ~04.8-ms acquisition time. Each spectrum is an average o f 4000 aoquisitions. (b)

Unseparated etylhmcytes after the addition of 0.5 mM sapouin in cells. All signal has shifted to the calcium-bound BAPTA position. Use of tbe calcium ionoph~e. A23187 (50 pM). which allows the influx of cxtracellular Ca2.. gave identical results.

o r d e r to lyse the

signal f r o m " c c a l c i u m - b o u n d B A P T A is shifted 5.5 p p m d o w r Ad f r o m t h e free B A P T A signal. U n s e p a r a t e d r e d cells w e r e f o u n d to c o n t a i n a n a v e r a g e o f 66 ( + 7 ) n M C a 2+, w h i c h is in excellent a g r e e m e n t with o t h e r published values in u n s e p a r a t e d cells, using simil a r c h e l a t o r m e t h o d s : 61 n M ( + 6 ) [26], 70 n M [25], a n d 66 n M (_+ I1) [23]. Lysis o f cells by the a d d i t i o n o f s a p o n i n (Fig. lb), o r a d d i t i o n o f t h e calcium i o n o p h o r e , A23187, in the p r e s e n c e o f b u f f e r c o n t a i n i n g 1.25 m M CaCI2, resulted in binding o f all B A P T A 4 - p r e s e n t t o C a 2 + as s h o w n by the single igF r e s o n a n c e arising a t 7.5 p l a n . O l d r e d cells, the m o s t d e n s e fraction, w e r e f o u n d to c o n t a i n 221 ( + 2 5 ) n M Ca2+ ( n = 17), while the middle fraction c o n t a i n e d 103 (+_ 11) n M ( n = 10), a n d y o u n g cells c o n t a i n e d 62 ( + 4 ) n M Ca2+ ( n = 18), as shown in Fig. 2. Initial two-way analysis o f v a r i a n c e d e m o n s t r a t e d significant d i f f e r e n c e b e t w e e n groups. T h e S t u d e n t - N e w m a n - K e u l s ( S N K ) test o f multiple c o m p a r i s o n revealed l h a t t h e r e w a s a significant differe n c e b e t w e e n old ceil fractions a n d b o t h m i d d l e a n d y o u n g fractions. However, t h e r e w a s n o statistical difference b e t w e e n m i d d l e a n d y m m g cell fractions.

T h e loading o f 5 , 5 ' - d i f l u o r o B A P T A into the cell c o u l d have p r o d u c e d s o m e effect o n cell function. In o r d e r to d e t e r m i n e w h e t h e r the loading process m i g h t have altered m e m b r a n e function, y o u n g cells w e r e l o a d e d with B A P T A , s t e a d y state intracellular C a 2+ m e a s u r e d , a n d t h e s a m p l e t h e n s t o r e d f o r 24 h a t 4°C. A f t e r allowing the cells to equilibrate at 3"PC for 60 rain, the ~gF m e a s u r e m e n t w a s r e p e a t e d . C a r e f u l w a s h ing o f t h e s a m p l e s resulted in virtually n o increase in the [Ca 2 + It- However, if s a m p l e s w e r e n o t w a s h e d p r i o r to r e m e a s u r e m e n t , [Ca2+]i w a s f o u n d to increase (Table 1). I n c u b a t i o n o f cells f o r 24 h at 37°C b e f o r e loading with the B A P T A c o m p o u n d h a d n o effect o n [Ca2+],, while t r e a t m e n t o f cells with v a n a d a t e a n d iodoacetic a d d , to inhibit C a 2 + - A T P a s e function, resulted in a n increase in intracellular Ca-'+. T h e s e d a t a are s u m m a r i z e d in T a b l e I. D u r i n g o t h e r experiments, s u p e r n a t a n t s f r o m cell s a m p l e s w e r e r e m o v e d a t t h e e n d o f the N M R e x p e r i m e n t a n d assayed by t g F - N M R

TABLE I Intracellular

Ca 2 * concentration (riM) in young human ¢r)'lhrOO'tes

measm'ed beforeand aftercarioustreatmems

Cell treatment

Pretreatment lCa2" l

Posttreatment lCa2" I

24 It. 4°C - washed 24 I1, 4°C - washed

54 74

24 h, 4~C - unwashed

55

24 h. 4°C - unwashed

48

140

vanadate + iedoacetale 24 h, 3"PC,then BAPTA loading

49 74

326 76

57 88 154

158 for the presence of BAPTA~- due to leakage of the compound from the cells. No NMR-detectahle BAPTA"z- was found in these samples. I~iscussion We have demonstrated in these studies that intracellular free ionic calcium concentration increases as a function of increao];,g ceil age. Intraeellular Ca 2~ is significantly elevated, approx. 4-fold. in old cells. This is the first time that intracellular Ca-"" has heel; measured in viable, intact populations of erythrtg'ytes of varying ages (densities). These are results consistent with earlier indirect measurements (e.g., atomic absorption of disrupted cells) that indicated an increase of total cell calcium in old cells [I,4]. This increase includes membrane bound calcium, while our studies observe free, intracellular ionic calcium. The development of fluorinated calcium chelator probes for use with '~F-NMR spectroscopy [21-23] has resulted in a method for the straightforward measurement of the free ionic form of intracellular calcium in crythrocytes. It is important to consider the difference in the K~ of 5,5'-difluoroBAPTA for calcium ad the measmed intracellular concentration of free calcium. Ideally, these values should be similar. However, the K~ used in our calculations was 708 nM [26], apparantly five timcs greater than the [CaZ'], that we measured. Other laboratories have determined a lower K,, (R.IL Gupta and B.P. Chacko, personal communications); however their experimental conditions were somewhat different from ours. The K d we used is that determined under identical experimental conditions to those used here [26]. The questions being asked in these experiments involve comparison of population groups, and therefore the comparisons of concentrations between groups remain valid. A different K,j would merely change the absolute values of the calcium concentrations, but not the relationship of the groups with each other. Longitudinal relaxation times were not measured for BAPTA~- and Ca-BAPTA in the different density-separated fractions. There may indeed be differences due to L:,e decreased cell volume in old cells, although cellula; components arc also decreased as well, and this is a question that should be addressed, if BAPTA loading resulted in preferential damage to o!d cells leading to cell lysis, the wash step preceeding the NMR cxpcrimcnt rcmovcd any such cells. We may therefore be underestimating the actual [Ca :+ j~ in old cells. Another problem with the use of this technique in red cells is the time scale of the NMR experimer.t, which precludes measurement of rapid changes of intracellular [Ca:+]. Nevertheless, within these limitations, it is possible to measure steady state values of

[Ca-" ]~. This technique allows comparison among populatious, such as the young, middle and old fractions of red cells, and hence yields useful information. Our determination of [Ca -'+ ]~ in unseparated red cells of 66 nM (_+7, S.E.) is in excellent agreement with the recent work of other laboratories, using similar techniques [23~.25,26]. We therefore were confident that application of this technique to the measurement of intracellular [Ca 2~ ] in young and old ccll fractions was valid. Increased red cell calcium has been suggested to be closely related to other age-related changes described in ei31hroc~les. This hypotbesis is supported by the observations that nearly all of the described age-related changes in old cells can also be induced by increasing intracellular [Caz+] using ionophores [2730]. Our demonstration that [Ca-'*], is significantly higher in old cclgs further supports the premise that age-related changes in erythrocytcs may be initiated or regulated by intracellular calcium concentrations. Hcm'ever, our findings do not differentiate between elevated [Ca2÷]j being the result of aging, or the cause of aging. Several mechanisms for this ch:mgc can be postulated. Since the red cell does not cot~tain cndoplasmic reticulum, mitochondria, or other p