BI OCHEMI CAL AND BI OPHYSI CAL RESEARCH COMMUNI CATI ONS 236, 549554 (1997)

ARTI CLE NO. RC977002

Characterization of Voltage-Dependent Calcium Inux
in Human Erythrocytes by fura-2
Laura Sol dati ,
1
Renato Spaventa, Gi useppe Vezzol i , Si mona Zerbi , Donatel l a Adamo,
Andrea Caumo,* Rodol fo Ri vera, and Gi useppe Bi anchi
Chair of Nephrology, Milan University, S. RaffaeleHospital, Milan, I taly;
and *Statistics Laboratory, S. RaffaeleHospital, Milan, I taly
Recei ved June 2, 1997
K
/
efux through the acti vati on of speci c channel s
Thusfar, themethodsusedtodetermineerythrocyte
(1). The sensi ti vi ty to di hydropyri di nes, the i ncrease of
Ca
2/
inux have not allowed the assessment of the ki-
the cal ci um i nux rate by depol ari zi ng factors and the
netics of ion uptake. To overcome this drawback, we
i nux ki neti c model suggest that at l east a component
studied a newmethod, usingtheuorescent Ca
2/
-che-
of the observed cal ci um i nux coul d be acti vated by
lator fura-2, which directly quanties intracellular
membrane depol ari zati on (4,7). Recentl y erythrocyte
Ca
2/
changes in human erythrocytes. This method has
cal ci um i nux rate, esti mated as stronti um uptake i n
the advantage over previous techniques that it moni-
vanadate-treated erythrocytes, was found to be corre-
tors continuously cellular Ca
2/
levels. The Ca
2/
inux
l ated to cal ci um pump acti vi ty measured at the maxi -
is modulated by cellular membrane potential in the
mal rate (6,8).
presence of a transmembrane Ca
2/
concentration
I n the present work we propose an al ternati ve ap-
gradient andexhibitsarst slowincreaseof theintra-
proach for the measurement of Ca
2/
i nux i nto i ntact
cellular Ca
2/
concentration, followed, after the
human erythrocytes usi ng the uorescent Ca
2/
chel a-
reachment of a threshold value of 125 { 13 nM Ca
2/
,
tor fura-2, that al l ows a vi rtual l y conti nuous measure-
by a faster increase until a plateau is reached. The
ment (1 poi nt every 0.9 seconds) of the i ntracel l ul ar inux rateis inhibited by dihydropyridines in themi-
cromolar range. Thesendingssupport thehypothesis free cal ci um concentrati on [Ca
2/
]i , whi ch was not possi -
that erythrocyte Ca
2/
inux is mediated by a carrier bl e wi th the techni ques previ ousl y used. Ca
2/
i nux
similar to theslowCa
2/
channels and is dependent on
ki neti cs suggest a mechani sm of regul ati on by i ntracel -
membrane depolarization. 1997 Academic Press
l ul ar free Ca
2/
i ons never detected before i n human
erythrocytes. Moreover our resul ts conrm that the
Ca
2/
i nux occurs through vol tage-acti vated channel s.
The mechani sms underl yi ng erythrocyte Ca
2/
trans-
MATERIALS AND METHODS
port are of parti cul ar i nterest i n the study of the di s-
eases associ ated wi th an al tered cel l ul ar cal ci um han-
Drugs and chemicals. NaCl , KCl , ethyl ene gl ycol -bi s(b-ami no-
dl i ng, l i ke essenti al hypertensi on, pri mary hyperpara-
ethyl ether)-N,N,N,N-tetraaceti c aci d (EGTA), CaCl
2
, octyl phenoxy
thyroi di sm, i di opathi c hypercal ci uri a, and Al zhei mers
pol yethoxy-ethanol (Tri ton X-100), MgCl
2
, gl ucose, N-[2-hydroxy-
di sease.
ethyl ]pi perazi ne-N-[2-ethanesul foni c aci d] (HEPES), di methyl sul f-
Whi l e cal ci um pump has been extensi vel y studi ed,
oxi de (DMSO), ci tri c aci d, tri sodi um ci trate were purchased from
BDH. Carbonyl cyani de m-chl orophenyl hydrazone (CCCP), 4,4-di i - bi ochemi cal and functi onal aspects of erythrocyte cal -
sothi ocyanosti l bene-2,2-di sul foni c aci d (DI DS), fura-2-tetraki s acet-
ci um i nux remai n substanti al l y unexpl ored. Ca
2/
i n-
oxymethyl -ester (fura-2AM), i odoacetate, i nosi ne, ni cardi pi ne, ni t-
ux has been measured usi ng
45
Ca
2/
i n erythrocytes
rendi pi ne and ni fedi pi ne were purchased from Si gma Chemi cal (St.
treated wi th vanadate (1) or ATP-depl eted (2-4) i n or-
Loui s, MO). Al l sol uti ons were prepared usi ng doubl e-di sti l l ed water.
der to i nhi bi t the cal ci um pump acti vi ty. I t was found Al l reagents were of anal yti cal grade.
dependent on external cal ci um concentrati on (3), sti m-
Preparation of erythrocytes. Bl ood from normal donors was col -
ul ated by parathyroi d hormone (5,6) and promoti ng a
l ected i n tubes contai ni ng 1/10 vol ume of anti coagul ant (2.73% ci tri c
aci d, 4.48% tri sodi um ci trate, 2% gl ucose). Cel l s were preparated
i mmedi atel y after bl ood sampl i ng and stored at room temperature.
Bl ood was centri fuged at 20 C for 5 mi nutes at 750g. Pl asma and
1
To whom correspondence shoul d be addressed: Di vi si one di Nefro-
l ogi a, Ospedal e San Raffael e, Vi a Ol getti na 60, 20132 Mi l an, I tal y. buffy-coat were removed by sucti on. The hematocri t, cel l number,
mean gl obul ar vol ume, mean gl obul ar hemogl obi n concentrati on and Fax: (39) 2 26432384. E-mai l : vezzol i @rsi si .hsr.i t.
0006-291X/97 $25.00
Copyri ght 1997 by Academi c Press
Al l ri ghts of reproducti on i n any form reserved.
549
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Vol . 236, No. 3, 1997 BI OCHEMI CAL AND BI OPHYSI CAL RESEARCH COMMUNI CATI ONS
red cel l s di stri buti on wi dth were determi ned wi th a S-pl us Coul ter-
Counter.
Measurements of cytosolicfreeCa
2/
concentration in human eryth-
rocytes. Measurement of [Ca
2/
]i i n i ntact erythrocytes was carri ed
out wi th the dye fura-2. Packed cel l s were di l uted to 1% hematocri t
wi th a HEPES-buffered sal i ne (HBS) (i n mM): NaCl (123), KCl (5),
MgCl
2
(1), CaCl
2
(1), gl ucose (10), HEPES (25), pH 7.4 at 37C, 285
mOsm/l . Fura-2AM was di ssol ved i n DMSO at a concentrati on of 1
mM. Al i quots were stored at 020C and were di l uted wi th HBS
i mmedi atel y before i ncubati on to mi ni mi ze the dye degradati on.
Erythrocytes were l oaded wi th 0.5 mM fura-2AM by i ncubati on for
45 mi nutes at 37C. At the end of i ncubati on erythrocytes were di -
l uted wi th HBS (1/10 v/v) and washed by centri fugati on at 700g for
5 mi nutes at 20C to el i mi nate the extracel l ul ar fura-2AM i n excess.
Packed cel l s were resuspended i n HBS at 0.1%hematocri t and trans-
ferred i nto a quartz cuvette for the uorescence measurements, usi ng
the Perki n El mer model LS50 l umi nescence spectrometer wi th the
I ntracel l ul ar Bi ochemi stry appl i cati on software package (I CBC). Al l
experi ments were performed at 37C usi ng a magneti c sti rrer.
[Ca
2/
]i was determi ned usi ng the two wavel ength method de-
scri bed by Grynki ewi cz et al . (9), wi th exci tati on at 340 nm and 380
FIG. 1. Ca
2/
i nux i nto i ntact human erythrocytes. (A): Erythro-
nm, and emi ssi on at 510 nm wavel enghts. [Ca
2/
]i was cal cul ated
cytes (1% Ht) depl eted of ATP were tested. ATP concentrati on mea-
accordi ng to the equati on gi ven by Poeni e et al . (10):
sured before uorescence l ecture was 0, n 5. (B): Erythrocytes
(1% Ht) control sampl e. ATP concentrati on measured before uores-
[Ca
2/
]i Kd fura-2 1 [(R0Rmi n)/(Rmax 0R)] 1 (F
free380
/F
bound380
) cence l ecture was 2.73 {0.36 mmol /dl , n 5. The experi ments were
performed as descri bed under Materi al s and Methods. The phases a,
[Eq.1]
b, and c are descri bed under Resul ts. I nset shows i ntensi ty data of
340 nm (l i ne C) and 380 nm exci tati on si gnal (l i ne D) rel ated to the
Ca
2/
i nux shown i n Fi g. 1, sampl e A.
where R i s the Rati o of the uorescence i ntensi ti es measured at 340
and 380 nm, Rmi n i s the same rati o i n the absence of Ca
2/
, Rmax
i s the same rati o when saturated wi th Ca
2/
, Kd i s the di ssoci ati on
pHmeter. The pH measurements were cal i brated by means of two constant of fura-2-cal ci um compl ex, F
free380
and F
bound380
are the uo-
Radi ometers buffers (7.0 and 4.0) at 37C. rescence i ntensi ti es of Ca
2/
-free and Ca
2/
-bound fura-2 at 380 nm
Vm was cal cul ated accordi ng to (12): respecti vel y.
The parameters Rmi n, Rmax and Kd were determi ned accordi ng
to Davi d-Dul ho et al . (11).
Vm (pHi 0 pHo) 1 2.303 RT/F [Eq. 2]
Measurement of Ca
2/
inux in human erythrocytes. Erythrocytes
where Vm i s the membrane potenti al , pHi i s the measured i ntracel l u-
(1% hematocri t) were rst depl eted of ATP by i ncubati on for 60 mi n-
l ar pH, pHo i s the measured external pH, R i s the uni versal gas
utes at 37C i n a medi um contai ni ng (i n mM): NaCl (140), KCl (5),
constant, T i s absol ute temperature, and F i s Faradays constant.
i odoacetate (1), i nosi ne (10), pH 7.2 at 37C (medi um A). After 15
Vm was vari ed changi ng the Ca/EGTA rati o i n the unbuffered
mi nutes from the begi nni ng of i ncubati on 0.5 mM fura-2 AM was
medi um. The Ca-EGTA bi ndi ng caused a decrease of pHo, whi l e pHi
added to the medi um (45 mi nutes of i ncubati on). Erythrocytes were
remai ned unchanged for the hi gh bufferi ng capaci ty of haemogl obi n
then washed i n the medi um A to el i mi nate the fura-2AM i n excess
(50-60 meq/l c) (13).
as descri bed before, di l uted to 0.1% hematocri t i n medi um A pl us
EGTA-Tri s, pH 7.2 at a concentrati on rangi ng from 0.1 mM to 2 mM
Statistical analysis. Data were expressed as Mean val ues {Stan-
and transferred i nto a quartz cuvette for the uorescence measure-
dard Devi ati on. For stati sti cal eval uati on of the data Students
ment. After the stabi l i zati on of uorescence si gnal (about 1 mi nute),
pai red t-test was used, and two-tai l ed p val ues l ess than 0.05 were
CaCl
2
was added at a concentrati on rangi ng from 1 mM to 20 mM.
consi dered to be si gni cant.
[Ca
2/
]i was cal cul ated for every poi nt of uorescence l ecture (1 every
0.9 seconds) as descri bed before.
RESULTS
ATP content of erythrocytes. ATP was measured i n the superna-
tant after the removal of the preci pi tated protei ns by a coupl ed en-
Ca
2/
I nux in Human Erythrocytes
zyme assay method (Si gma), usi ng phosphogl yceri c phosphoki nase
and gl yceral dehyde phosphate dehydrogenase.
Usi ng the uorescent cal ci um chel ator fura-2 we
Membrane potential. Membrane potenti al (Vm) was measured
measured [Ca
2/
]i i n i ntact human erythrocytes. [Ca
2/
]i
accordi ng to the method of Macey et al . (12). Erythrocytes were
measured i n erythrocytes under resti ng condi ti ons
washed twi ce at 20C i n buffer-free sol uti on (145 mM NaCl , 5 mM
from normal subjects was 90 { 18.0 nM (n 14), a
KCl ) at a hematocri t of 10% i n the presence of 50 mM CCCP and
val ue qui te si mi l ar to those al ready reported (14,15).
thermostatted at 37C. pH was recorded for 1 mi nute before and 5
Ca
2/
i nux was moni tored i n human ATP-depl eted mi nutes after addi ng the erythrocytes and assumed as external pH.
To measure i nternal pH 10 ml of 20% Tri ton X-100 were added to
erythrocytes i ncubated i n a free cal ci um medi um. An
the cel l suspensi on causi ng total hemol ysi s.
exampl e of a typi cal i nward cal ci um current i s shown
pH was measured wi th a Radi ometer pHG200 gl ass mi croel ectrode
i n Fi g. 1, sampl e A: data are reported as [Ca
2/
]i vs.
wi th a REF601 Hg/Hg
2
SO
4
reference el ectrode wi th an outer sal t
ti me and as separate components of the Rati o (R) i n the
bri dge (K
2
SO
4
) to prevent KCl efux from the el ectrode to the me-
di um. The el ectrodes were connected to a Radi ometer pHM84 i nset of Fi g. 1, where R i s the rati o of the uorescence
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i ntensi ti es measured at 340 and 380 nm exci tati on
wavel engths. A few seconds after CaCl 2 was added to
the medi um, we observed rst a sl ow i ncrease of [Ca
2/
]i
(phase a i n Fi g. 1) that l asted about 500 seconds, wi th
an i nux rate of 0.099 {0.023 nM/sec (n 14), fol l owed
by a fast ri se (phase b i n Fi g. 1), wi th a rate of 0.665
{ 0.295 nM/sec l asti ng about 400 seconds. At the end
of the phase b a pl ateau was reached (phase c i n Fi g.
1), wi th val ues of [Ca
2/
]i 452 { 150 nM. The [Ca
2/
]i
remai ned stabl e for about 10 mi nutes, i n spi te of the
hi gh Ca
2/
concentrati on gradi ent. We di d not nd di f-
ferences i n the i nux rates when external Ca
2/
was
vari ed between 1 and 18 mM (data not shown).
An i nteresti ng observati on i s that the phase b, i .e.
the fast [Ca
2/
]i i ncrease, started at a threshol d val ue
of [Ca
2/
]i (124.8 { 13.3 nM versus 90 { 18.0 nM at
resti ng l evel , p 0.0001) and i t was i ndependent of
the phase a durati on and the i ncubati on temperature.
FIG. 2. Effect of membrane potenti al on Ca
2/
i nux i n i ntact
These resul ts were obtai ned i n condi ti ons of membrane
human erythrocytes. Fi gure shows the l evel s of Ca
2/
i nux rate at
depol ari zati on (Vm 0 mV), because at thi s l evel of
di fferent cel l ul ar membrane potenti al . Ca
2/
i nux i s shown as two
Vm we observed the hi gher val ues of phase b i nux
separate components: phase a and phase b. The experi ments were
rate (see Fi g. 2). performed as descri bed under Materi al s and Methods. (n 14.)
Erythrocyte [Ca
2/
]i di d not i ncrease i n control cel l s,
not submi tted to ATP depl eti on (Fi g. 1, sampl e B). I n
thi s case, the pl asma membrane Ca
2/
pump acti vi ty
was unaffected by vari ati ons of Vm between 06 and
di d not permi t to measure a cal ci um uptake, mai n-
/3 mV. Resul ts are shown i n Fi g. 2.
tai ni ng the [Ca
2/
]i at the resti ng l evel (even i n the
External potassium. Ca
2/
i nux rates were mea-
presence of hi gh external cal ci um concentrati ons).
sured i n sol uti ons wi th di fferent K
/
concentrati ons (5,
The morphol ogi cal anal ysi s of the cel l s 10 mi nutes
70 and 140 nM). The i ncreased concentrati ons of K
/
after the starti ng of the phase b by opti cal mi croscope
were compensated by decreasi ng Na
/
concentrati on, so
di d not show rel evant al terati ons. The hematocri t, cel l
that osmol ari ty di d not change. The hi gh potassi um
number, mean gl obul ar vol ume and red cel l wi dth re-
sol uti ons (70 mM and 140 mM of K
/
) caused an i n-
mai ned stabl e i n compari son to the val ues measured
crease of the cal ci um i nux rate, as seen by the el e-
i n fresh erythrocytes. We observed a cel l ul ar shri nki ng
vated tai l of the curve i n Fi g. 3 and by the i ncrease of
onl y after 1 hour from starti ng of Ca
2/
i nux experi -
the i nux rate: 0.545 nM Ca
2/
/sec at 5 mM K
/
, 1.698
ments.
nM Ca
2/
/sec at 70 mM K
/
and 1.703 nM Ca
2/
/sec at
The i nter-assay vari ati on coefci ent of the Ca
2/
i n-
140 mM K
/
.
ux phase b cal cul ated i n erythrocytes from a same
donor was 5.6 % (n 6). Dihydropyridines. To veri fy i f the observed Ca
2/
i n-
ux i s i nhi bi ted by i nhi bi tors of L-type cal ci um chan-
Factors I nuencing theCa
2/
Uptake
nel s, we studi ed the effect of the cal ci um antagoni sts
di hydropyri di nes (18). Bri ey, the desi dered antago-
Membrane potential. We measured transmem-
ni st was added to the cel l ul ar medi um duri ng the l oad-
brane potenti al i n resti ng condi ti ons and duri ng cal -
i ng wi th fura-2 and mai ntai ned unti l the end of the
ci um i nux. The Vm measured i n fresh cel l s from 9
experi ment (step experi ments). Di fferent concentra-
di fferent subjects was 06.142 {0.903 mV. Thi s val ue
ti ons were added i n si ngl e sampl e to cal cul ate the I C50
i s si mi l ar to those previ ousl y reported (16,17). We
(i .e. the drug concentrati ons requi red for a 50% i nhi bi -
found a sl i ght hyperpol ari zati on i n ATP depl eted eryth-
tory effect of antagoni st). These experi ments showed
rocytes (Vm 012 mV). Membrane potenti al of ATP
an i nhi bi tory effect i n the mi cromol ar range of the fol -
depl eted erythrocytes was vari ed as descri bed i n Mate-
l owi ng di hydropyri di nes: ni trendi pi ne (I C50: 26 mM),
ri al s and Methods, and i n the same cel l s Ca
2/
i nux
ni cardi pi ne (I C50: 20mM), ni fedi pi ne (I C50: 6mM), as
was measured. The i ncrease of the cal ci um i nux rate
shown i n Fi g. 4. I n other experi ments 10 mM ni fedi pi ne
was al ready detectabl e at Vm 09 mV, al though the
was added to the cel l ul ar medi um at the begi nni ng of
pl ateau phase was not reached i n 30 mi nutes. When
Ca
2/
i nux (Exp. A) or duri ng the phase b (Exp. B).
Vm was i ncreased at 06 mV and at 0 mV the phase b
Thi s l ast type of experi ments was carri ed out al so to
Ca i nux rate i ncreased to 0.187 nM/sec and 0.665 nM/ test i nux reversi bi l i ty, as al ready demonstrated i n
PC12 cel l s (19). The resul ts showed a smal l er i nhi bi - sec respecti vel y, whi l e the phase a of the Ca i nux rate
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FIG. 5. Effect of DI DS on Ca
2/
i nux. Sampl e (A): Ca
2/
i nux
FIG. 3. Effect of external K
/
on Ca
2/
i nux i nto i ntact human
was performed as descri bed under Materi al s and Methods. Sampl e
erythrocytes. Fi gure shows an i ndi vi dual experi ment performed wi th
(B): 2 mM DI DS were added to erythrocytes i mmedi atel y before the
l ow (5 mM) or hi gh (140 mM) potassi um sol uti on i n the external
starti ng of Ca
2/
i nux.
medi um, as descri bed under Materi al s and Methods. Resul ts compa-
rabl e to those shown were obtai ned i n 5 experi ments.
i zati on and openi ng of vol tage-operated channel s
(12,20,21). To test thi s hypothesi s, we measured the
tory acti on of the drug (about 15 %) on the i nux rate
Ca
2/
i nux i n erytrocytes exposed to DI DS, an i nhi bi tor
i n the Exp. A, whi l e i n the Exp. B the acti on was absent
of the ani on exchanger capnophori n, bl ocki ng any
and the drug was not abl e to reverse the Ca
2/
i nux.
changes i n Cl
0
cel l ul ar concentrati on. We added 2 mM
Thi s observati on, together wi th the fact that the I C50
DI DS to the medi um i mmedi atel y before the begi nni ng
was markedl y hi gher than those requi red i n exci tabl e
of the cal ci um i nux. Fi gure 5 shows an exampl e of
cel l s as al ready reported (1, 7, 14, 19), suggest ei ther
these experi ments, where the ki neti c of one Ca
2/
i nux
a di fferent afni ty of the drugs for the carri er or a
carri ed out i n the usual condi ti ons (sampl e A) i s com-
di fferent mode of acti on i n human erythrocytes.
pared wi th one carri ed out i n the presence of DI DS
DI DS. The conductance of the erythrocyte cel l
(sampl e B). Resul ts demonstrated that DI DS markedl y
membrane i s domi nated by Cl , because Cl
0
permeabi l -
i nhi bi ted the Ca
2/
entry i nto erythrocytes, conrmi ng
i ty i s much hi gher than cati on permeabi l i ty (12,20,21).
our hypothesi s.
As a consequence, Vm i s equal to the Cl equi l i bri um
Temperature. Ca
2/
i nux was i nuenced by the i n- potenti al (ECl ). The i ncrease of H
/
i ons i n the external
cubati on temperature (see Fi g. 6). When the i nux ex- unbuffered medi um predi ctabl y i nuences Cl
0
di stri -
peri ments were carri ed out at 30C i nstead of 37C, the buti on i n erythrocytes, causi ng a membrane depol ar-
durati on of the phases a and b i ncreased (from 500 to
1200 seconds and from 400 to 900 seconds respecti vel y),
the i nux rate decreased remarkabl y (from 0.09 to 0.05
FIG. 4. Effect of di hydropyri di nes cal ci um antagoni sts on Ca
2/
i nux rate. Fi gure shows the i nhi bi ti on on Ca
2/
i nux rate by di hy-
dropyri di nes i n step experi ments (see Resul ts secti on). Control exper-
i ments were performed as descri bed under Materi al s and Methods. FIG. 6. I nuence of temperature on Ca
2/
i nux i nto i ntact hu-
man erythrocytes. Ca
2/
i nux was determi ned as descri bed under 20 mM ni trendi pi ne (col umn 2), 20 mM ni cardi pi ne (col umn 3), and
10 mM ni fedi pi ne (col umn 4) were added i nto the i ncubati on medi um. Materi al s and Methods. Cel l s were i ncubated at 37 C (l i ne A) or 30
C (l i ne B). Resul ts comparabl e to those shown were obtai ned i n 5 Resul ts are expressed as Ca
2/
i nux rate (nM/mi n) of the phase b of
i nux (n 6). experi ments.
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nM/sec and from 0.66 to 0.36 nM/sec respecti vel y), i ncrease of the i nux rate when Vm was made posi ti ve,
as shown i n Fi g. 2. The i nux was i nhi bi ted by DI DS, whi l e the R val ue threshol d and the [Ca
2/
]i pl ateau
l evel were si mi l ar. that bl ocks the Cl
0
movements by an i nhi bi ti on of the
ani on exchanger capnophori n; the i nux rate was i n-
creased by depol ari zi ng sol uti ons wi th hi gh external
DISCUSSION
potassi um. These ndi ngs suggest that Ca
2/
i nux i s
a vol tage-dependent phenomenon, as al ready proposed The present i nvesti gati on shows that i t i s possi bl e to
measure a Ca
2/
i nux i n ATP depl eted human erythro- by other Authors (4,7). Thi s hypothesi s i s strengthened
by the found senti vi ty to di hydropyri di nes acti ng on cytes usi ng the uorescent Ca
2/
chel ator fura-2. Fura-2
i s a Ca
2/
-sensi ti ve uorescent dye that al l ows to detect vol tage-acti vated Ca
2/
channel s i n exci tabl e cel l s (19).
changes of [Ca
2/
]i al most conti nuousl y (1 poi nt every
0.9 seconds) i n si ngl e cel l s. I t exhi bi ts major advan-
ACKNOWLEDGMENTS
tages for measuri ng [Ca
2/
]i i n erythrocytes: i t has a
hi gh uorescence yi el d (about 30 ti mes more than qui n
The research was supported by grants from the I tal i an Mi ni stry
2), hi gh Kd and l ow sensi ti vi ty to Mg
2/
(9,22-28). Be-
of Uni versi ty and Sci enti c Research, from San Raffael e Hospi tal
and from: Programma Nazionale Ricerche sui Sistemi Neurobiolo- cause of these properti es, fura-2 can be used at l ow
gici-Tecnologiedella Trasduzionedel Segnale-Tema 2.
concentrati ons (0.5-5 mM range), resul ti ng i n mi ni mal
bufferi ng of i ntracel l ul ar Ca
2/
. Another advantage i s
that fura-2 i s a dual -wavel ength i ndi cator, permi tti ng
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