Berkoff, H.A.

Haworth, R.A.

Hunter, D.R.

Elsevier - Publisher Connector 

Volume 110, number 2 FEBS LETTERS February 1980 
Na* RELEASES Ca2+ FROM LIVER, KIDNEY AND LUNG M~OC~ONDRIA 
R. A. HAWORTH, D. R. HUNTER and H. A. BERKOFF 
Departmenf of Surgery, 600 Highland A venue, Madison, WI 53 792, USA 
Received 5 December 1979 
1 q Introduction 
Intracelhrlar CaZ’ has assumed increasing impor- 
tance as a regulator of a variety of cellular processes, 
including muscle contraction, secretion and cell divi- 
sion [l]. A prime candidate for a controller of intra- 
cellular Cazf levels, by virtue of its high affinity Ca2* 
uptake mechanism, is the mitochondrion. Such a role 
for the mitochondrion has gamed support from the 
demonstration that mitochondri~ Ca*” release is 
under separate control from uptake [2]. Two systems 
capable of releasing mitochondria Ca”’ are the Na+- 
induced release system [3] and the Ca2+ -induced tran- 
sition in permeability of the inner membrane 243. In 
elucidating the role of these release systems in Ca’* 
homeostasis it is important o know the extent of 
their occurence in mitochondria from different issues. 
Such a study for the Na+ system has concluded that 
this system was most active in brain, heart and adrenal 
cortex, present in striated muscle, but absent in kid- 
ney, liver, lung, smooth muscie and hepatoma [5]. We 
report that the Na+ system is indeed present in mito- 
chondria from liver, kidney and lung. We have not 
studied smooth muscle or hepatoma. 
2. Materials and methods 
Sprague-Dawley female retired breeder ats were 
used. Ah types of mitochondria were prepared as in 
[6], except hat the homogenization medium (4 ml/g 
wet wt tissue) contained 250 mM sucrose, 10 mM 
Abb?MzAms: BSA, bovine serum albumin; EGTA ethylene- 
bisoxoethylenenitrolotetraacetic a id; FCCP, carbonylcyanide 
~-triflnoromethoxypheny~ydrazone; MOPS, 4-morpholine- 
propanestionic acid 
216 
Tris-HCl, 0.5 mM K+EGTA (pH 7.4). For lung and 
brain mitochondria this procedure does not give a 
completely pure preparation. However, results can be 
obtained with crude homogenates which are essentially 
identical to those obtained with pure mitochondria 
(except for kidney, where residual Na+ must be 
washed out). This indicates that mitochondrial purity 
is not a critical factor in the demonstration of Na’- 
induced Ca*’ release. 
The methodology for measuring Na+-induced fflux 
of 45Ca was as in 143. The standard buffer for Ca2* 
release studies contained 250 mM sucrose, 20 mM 
Tris-MOPS (pH 7.1) 40 mM KCl, 2 mM K’Pi, 2 mM 
MgCL, 0.8 mgjml BSA, 2 mM K’ATP and 5 mM K* 
sue&ate. Typically, to 12 ml standard buffer was 
added 12 mg mitochondria, followed immediately by 
20 nmol rotenone. CaC12 (80 nmol) conta~ing 4SCa 
(2 X lo6 dpm/&mol) was added, and the suspension 
was incubated at 30°C for 3 min in a shaking water 
bath. K’EGTA (10 pmol) was then added, and a 
0.5 ml aliquot was i~ediately centrifuged for 
I.5 mm in a Brinkmann 3200 bench centrifuge. Imme- 
diately following EGTA addition the remaining 
suspension was divided, typically into 4 and added (at 
t = 0) to separate flasks containing the agents whose 
effect on Ca2’ efflux was to be studied. Aliquots 
(0.5 ml) from each flask were centrifuged at time 
intervals. After 10 mm the contents of all flasks were 
recombined, and 10 ,ug A23 187 added. This ionophore 
releases the remaining mitochond~~ Ca” and hence 
allows the ‘zero mitochondrial Ca2+’ point to be deter- 
mined. After 10 min incubation with A23 187 the 
final 0.5 ml aliquot was centrifuged. Radioactivity in 
the supemat~ts was estimated by count~g in Aquasol 
(New England Nuclear). These counts were used to 
estimate the amount of Ca2+ accumulated by the 
mitochondria, and the time course of Ca*+ release. 
EIsevierlNorth-Holland Biomedical Press 
Volume 110, number 2 FEBS LETTERS February 1980 
3. Results and discussion 
Figure 1 shows the time course of Ca*+ release 
from mitochondria of liver, kidney and lung induced 
by the addition of 10 mM NaCi. KC1 at 10 mM caused 
no greater degree of release than no addition (fig, 1 C). 
Both the spontaneous release and the Na’-induced 
release were unaffected by the presence of ruthenium 
red (fig. 1 A). Mitochondria from kidney medulla 
released Ca2+ much faster than mitochondria from 
kidney cortex (fig. 1 B). 
We are not certain why no Na+-induced Ca*+ release 
from mitochondria from these organs was detected 
[3]. One factor could be that the Ca2’ release caused 
by the Ca2+-induced Trenton was not suf~ciently 
inhibited [4], which gave a high rate of ruthenium red- 
insensitive fflux even in the absence of Na+, at least 
A 
I LIVER 
1 
3o0 
I I I I I 2 4 6 8 
IO 
D 
TIME AFTER ADDITION OF Nat (mid 
Fig. 1. The Na+-induced release of low levels of Ca” from 
mitochondria from: (A) liver; (B) kidney; (C) lung. Ca*+ 
uptake and release was measured as in section 2. Mitochondria 
were given 6.7 nmol CaClJmg protein. After the 3 min incu- 
bation period the % uptake was: (a) liver, 79%; (b)‘kidney 
cortex, 85%; medulla, 82%; (c) lung, 87%. These % of 
6.7 nmol/mg correspond to the 100% mitochondrial Ca’+ at 
t = 0. Na+dependent Ca” release was induced by the presence 
of 10 mM NaCl. 
A 
KIDNEY 
3o0 
t I I , 
2 4 6 0 
I b 
IO 
TIME AFTER ADDITION OF Na+(min) 
I LUNG 
1 
3o0 
I I I I I 2 4 6 8 
IO 
& 
TIME AFTER ADDITION OF Na+(min) 
217 
Volume 110. number 2 FEBS LETTERS February 1980 
60- 
50- 
40 - 
TIME AFTER ADDITION OF Na+ (min) 
Fig.2. The Na+-induced release of high levels of Ca*+ from 
mitochondria of kidney and brain. The procedure was as 
before, except that mitochondria were given 150 nmol 
CaCl,/mg protein. After the 3 min incubation period the % 
uptake was: kidney cortex, 90%; kidney medulla, 93%; brain, 
54%. Na+dependent Ca’+ release was initiated by the pres- 
ence of 20 mM NaCl. 
in the case of liver and kidney [3]. In our system this 
efflux was maximally inhibited by the presence of 
ATP and Mg*+, in addition to rotenone. To obtain an 
Table 1 
Estimated V,,, for Na+-induced Ca*+ release 
V max (nmol Cal+ . min-’ . mg protein-‘) 
Liver 1.6 
Lung 2.4 
Kidney cortex 2.1 
Kidney medulla 11 
Brain 16 
Heart 10 
estimate of V,, for Na+-induced Ca*+ release, mito- 
chondria were loaded with high levels of Ca*+ 
(150 mnol/mg) and the rate of release induced by 
20 mM NaCl was measured (fig.2). This level of Na+ 
was found to maximally stimulate Ca*+ release. High 
levels of Ca*+ were used in order to promote the satu- 
ration of the Ca*+ efflux site and to overwhelm the 
endogenous (unlabelled) Ca*+ . Still higher levels of 
Ca*+ were found to affect the values of I’,, found 
by this method. The initial rate of Na+-dependent 
Ca*+ efflux gave the estimates of Vmax shown in 
table 1. The values found for brain and heart are com- 
parable to those in [3,5]. 
It is particularly important to know of the presence 
of Nat-induced Ca*+ release in mitochondria from 
these tissues, since this opens the possibility of Na+- 
induced Ca*+ release being involved in the Ca*+-mobi- 
lizing action of some hormones. It has already been 
shown that the glycogenolytic action of vasopressin, 
angiotensin II and phenylephrine on liver is accom- 
panied by a significant depletion of mitochondrial 
Ca*+ [6]. The rate of this depletion was found to be 
of the order of 0.3 mu01 Ca*+ . min-’ . mg protein-’ 
and therefore well within the Ca*+ release capability 
of the Na+-induced release system. 
Acknowledgement 
This work was supported by grant no. HL19378 
from the National Institutes of Health. 
References 
[l] Berridge, M. J. (1975) Adv. Cyclic Nucl. Res. 6, l-98. 
[2] Caroni, P., Schwerzmann, K. and Carafoli, E. (1978) 
FEBS Lett. 96, 339-342. 
[ 31 Crompton, M., Capano, M. and Carafoli, E. (1976) Eur. 
J. Biochem. 69,453-462. 
[4] Hunter, D. R. and Haworth, R. A. (1979) Arch. Biochem. 
Biophys. 195,468-477. 
[S] Crompton, M., Moser, R., Ltidi, H. and Carafoli, E. 
(1978) Eur. J. Biochem. 82,25-31. 
[6] Blackmore, P. F., Dehaye, J. P. and Exton, J. H. (1979) 
J. Biol. Chem. 254,6945-6950. 
218 


Na+ releases Ca2+ from liver, kidney and lung mitochondria 

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Na+ releases Ca2+ from liver, kidney and lung mitochondria

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