DTIC ADA235593: Reductions in Calcium Uptake Induced in Rat Brain Synaptosomes by Ionizing Radiation

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ARMEO FORCES RAO-OiOLOGYr
RAD>ArXTON RESEARCH 125, 158-162 (1991) 1SIAPIC1 4N1STITUf
SCIErIFIC REPORT
SR91-12
Reductions in Calcium Uptake Induced in Rat Brain Synaptosomes
by Ionizing Radiation
SATHASIVA B. KANDASAMY, THOMAS C. HOWERTON, AND WALTER A. HUNT
Behaviorai So! ';,nDse partment. Armed Forces Radiobiohoqy Rewarch Institute. Bethesda, Mariand 20889-5145
Calcium channel binding sites for DHP compounds have
KANDASAMY, S. B., HOWERTON, T. C., AND HUNT. W. A. been located in the brain and heart using nimodipine and
Reductions in Calcium Uptake Induced in Rat Brain Synapto- nifedipine (I1-13). Nimodipine binding provides a molecu-
somes ly Ionizing Radiation. Padiat.R es. 125, 158-162 (1991). lar probe by which the kinetics of calcium receptors may be
Gamma irradiation (6Co) reduced KCI-stimulated voltage- evaluated (12). A slight structural modification ofDHP cal-
dlpendent 4Ca2 uptake in whole-brain, cortical, and striatal cium antagonists yields Bay K 8644 [methyl-1,4-dihydro-
synaptosomes. The time course (3, 10, 30, and 60 s) of calcium 2,6-dimethyl-3-nitro-4-2(trifluoromethylphenyl) pyridine-
uptake by irradiated (3 Gy) and nonirradiated synaptosomes, as 5-carboxylate], which confers agonist activity for calcium
well as the effect of KCI (15-65 maM), was measured in whole- influx in the brain (13-18) and smooth and cardiac muscle
brain synaptosomes. The fastest and highest rate of depolariza- (19, 20). Interestingly, a direct effect of Bay K 8644 on
tion-dependent calcium uptake occurred at 3 swith 65 mMKCI. synaptosomal calcium entry in rat brain has not been ob-
Irradiation reduced calcium uptake at all incubation times and served (21-23).
KCI concentrations. Bay K 8644 enhancement of KCI-stimu- serTe he p2re 1s-e2n3t) aexperiments indicate a reduction in calcium
lated calcium influx was also reduced by radiation exposure.
Nimodipine binding to dihydropyridine (DHP) L-type calcium uptake after irradiation in wh31e-brain, cortical, and striatal
channel receptors was not altered following radiation exposure. synaptosomes. This study characterizes the effect of ioniz-
These results demonstrate an inhibitory effect of ionizing radia- ing radiation on voltage-dependent calcium influx by mea-
tion on the voltage-sensitive calcium channels in rat brain syn- suring KCI-stimulated 4 Ca2 uptake into rat whole-brain,
aptosomes that are not mediated by DHP receptors. r i9o cortical, and striatal synaptosomes. Additionally, calcium
Aademic Pres . Inc. channel receptor binding studies were done to determine
whether the reductions were due to any alterations in DHP
L-type receptors.
INTRODUCTION
Although the central nervous system (CNS) is generally
considered to be relatively resistant to the direct effects of MATERIALS AND METHODS
ionizing radiation, exposure to ionizing radiation can have Materials. Bay K 8644 and nimodipine were gifts from Miles Labora-
a complex effect on the CNS that is dependent on the dose tories, Inc. (West Haven. CT) and were dissolved in dimethylsulfoxide
and time elapsed after radiation exposure (1-6). The bio- (DMSO). Due to the light sensitivity of these compounds, all experiments
chemical basis for radiation-induced behavioral/physiologi- were done using foil-wrapped test tubes. CaC2 and ['H]nimodipine were
cal changes mediated by the CNS is unknown. purchased from New England Nuclear (DuPont, Boston, MA). and all
other chemicals and -"agents used were of the highest analytical grade and
Calcium plays several important roles in various electro- were obtained from Sigma (St. Louis, MO).
physiological and neurochemical processes, and it is gener- Experimental animals. Male Sprague-Dawley rats weighing 200-300g
ally accepted that extracellular calcium influx can be regu- (Charles River Breeding Laboratories, Kingston, NY) were quarantined
lated by calcium channel agonists (i.e., cardiostimulatory on arrival and screened for evidence of disease by representative serology
drugs), or can be blocked by calcium channel antagonists and histopathology sampling before being released from quarantine. The
(7). These antagonists are structurally heterogenous and in- rats were housed individually in polycarbonate Micro Isolator cages (Lab
Products. Maywood, NJ) on autoclaved hardwood contact bedding (Beta
dlude verapamil, diltiazem, D-600, and the dihydropyn- Chip Northeastern Products Corp, Warrenburg, NY) and provided wAith
dine (DHP) compounds, such as nimodipine and nifedi- commerical rodent chow and acidified water (pH - 2.5) ad libitiuot, Ani-
pine (8). These drugs exhibit a common locus of action on mal holding rooms were kept at 21 t I±*C with 50 t 10% relative humidity
voltage-sensitive calcium channels (VSCC) (9, 10). Several on a 12.h light:dark cycle with no twilight,
types of VSCC have been identified, of which L, N, and T Tissue preparation and calchn uptake. Animalswerkilldbydecapi.
most i . The DHP antagonists mentioned tation, and the brain was removed from the skull and placed on ict. Whoe
are o mporant. brain, cortex, and striatum were dissected using the method ofGkiIaski
above are specific for the L-type VSCC, which is character- and Iversen (24). and the synaplosomal pellet (P2) from the vtgi ' w,,
ized by large conductances of long duration (7); or pellets were, prepared by a modification of the method of (ay v akId
t13-7597/91 $3.0
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All rights of reprdutilon in any form reerved,
91 5 07 134
IONIZING RADIATION AND CALCIU M UIPTAKF 159
.hi!taker 125i. The linal pellet was resuspended in ice-cold incubation 4 .
medium containing 136 mtNaCI, 5 ml! KCI. 1.3 mM MgCI-.0.12 mA!
CaCi- 11)m 3/lglucose. and 20 m.t Tris base, with the p11 adjusted to 7.65 0
,,it1h.0 11m aleic acid to provide a concentration range of approximately _
4-6 mp nrotcin/ml and divided into two equal portions. One portion was £ 3 '
ir-adiatcd "ith -.. photons while the other was used as a sham-irradiated TT
control. .
Aliquots (480 pl of the synaptosomal preparation were transferred into
test tutes and incubated for 14 min at 30'C in a Dubnoff metabolic shaker 0
in the presence or absence of various concentrations of Bay K 8644 as £ 2 -
detailed below. The Bay K 8644 or DMSO vehicle was added as a 20-Ul
,.iure to make a final incubation volume of 500 Al. Following the incu-
Thaion period. 500 O of depolarizing or nondepolarizing solution contain-
iHo -Ca:> (3 pCil was added. The depolarizing solution was the same 1
composition as that of the incubation medium, except that a portion of the
NaCI "as replaced by an osmotically equivalent amount of KCI to provide z I
K-1 concentrations of 15, 30, or 65 m./. Calcium uptake was termi-
addition of 5 ml of an icc-cold buffer containing 3 mM EGTA 0
c; l cu lb is,;-aminocthv ether) N, N'-tetraaceti cacid), 10 mglu-0L
>n. d 20 mOmTris base. with the pH adjusted to 7.65 with 1.0 M maleic 15mM 30mM 65mM
.Each s-ample was then immediately filtered under vacuum through a Dose of KCI
Yed Whatman GF/ filter. Excess and loosely bound 4"Ca2' was FIG. 2. Effects of 15, 30, and 65 mM KCI-stimUlated calcium uptake
,-n;natd with two 5-ml 'sashes of ice-cold incubation medium. The in nonirradiated (0) and irradiated (0) rat whole-brain synaptosomes.
%wetrre, then placed in scintillation vials and radioactivity was deter- Points and bars represent means ± SEM values from three separate experi-
in.;; quid scintillation spectrometry. ments. each using triplicate samples. A significant effect of ionizing radia-
N.ct uptake of14Ca' into synaptosomes was calculated by subtracting tion is indicated as follows: *1 < 0.05.
n:ake in the absence of depolarization (5 mM KCI) from the uptake in
"hr!presence of depolarization (15. 30. or 65 rM). This value will be
refrTed to as k (potassium-induced change) and represents net KCI-in-
duced caci um uptake (20, 27). pl/sample) for determination of nonspecific binding. A preliminary dis-
['fVii:sipic, jdnp a.. vayi,T.h e binding assay was done based on placement experiment indicated that the apparent Kd for this system was
:h. meth-ds reported by Skattebol and Triggle (28). Whole brain, cortex. apprximately I nM (data not shown).
and striatum were obtained and isolated as described above and pooled Based on preliminary experiments, maximal bindingwas obtained in 20
according to protein needs for significant binding determined in prelimi- min (data not shown). [3H]Nimodipine (NEN, 123.3 Ci/mM, 25 p1/sam-
nar.y experiments (data not shown). The P2 pellet was prepared as de- pIe) was added to the aliquoted samples (in duplicate, final volume 565 p,)
scribed aboe and resuspended in 50 mM Tris base buffer (pH 7.4) and over a concentration range of50 pM to 0.8 n.,f for I h at 4*C. The binding
divided into two equal portions. one of which was irradiated with y pho- experiment was then terminated using a 24-place Brandel Cell Harvester
tins. while the other was used as a sham-irradiated control. Synaptosomes (Gaithersburg. MD). and the membrane fragments were collected on
were irradiated to maintain procedural continuity with the calcium uptake Whatman - .- niters, followed by 3 X 3-ml washes with 50 mM Tris base
studies. Following irradiation, the svnaptosomes were homogenized for a buffir. The filters were then transferred to scintillation vials. After deter-
second time (glass/glass) in 3 ml buffer to form membrane fragments, mining radioactivity by liquid scintillation spectrometry, the data were
which were aliquoted (200 pl:1 00 pg/sample. striata: 400 pg/sample, converted to fmol and expressed as fmol/mg protein.
.whoeb rain: 400 pg/sample, cortex) into black snap-cap polypropylene Irradiationp rocedures. It has been reported that no significant oxygen
test tubes. Half of these tubes contained 5 p.M unlabeled nimodipine (25 uptake occurs in whole-brain homogenates in vitro after irradiation, which
suggests that the brain synaptosomes will not consume enough oxygen to
affect the results' (29). To maintain the maximum viability of the synapto-
somes. they were placed in glass test tubes in a Plexiglas ice bath and were
10 exposed to radiation using a 'Co source at a dose rate of I Gy/min (for the
I-Gy dose only), 10 Gy/min (for the 3-. 5-. and 10-Gy doses), or 20 Gy/
Y- Z min (for the 30-Gy dose). Dosimetry was performed using a 0.05-cm3
I i tissue-equivalent ion chamber. The ion chamber was placed in a glass test
C tube inside the Plexiglas ice bath during dosimetry measurements.
*I Miscellaneous mnethods. Protein content was determined by the
Z; -6 1 method of Lowry ci a. (30). using bovine serum albumin as the standard.
Statistical analysis was performed using Student'sI test, Multiple compari-
... I sons with a control were done by analysis of variance (RSI, BBN Softare
0 3 10 30 60 Products Corp., Cambridge. MA) and Dunnett's test (31),D ata were iden-
Time (seconds) tified as significant if P < 0.05.
FIG. I. The time course of65 m! KCI-stimulated calcium uptake in
nonirradiated (0) and irradiated (0) rat whole-brain synaptosomes. Points ' K. S. Kumar, Y. N, Vaishnav. A. M. Sancho, and J R. Weistr o,
and bars represent means ± SEM values from three separate experiments, catalyzed generation of pentanc from irradiated cr'hrocyle mcmbat-nes,
each using triplicate samples. A significant effect of ionizing radiation is In Radiation Research Societ.' ,4hsiracis, 3Sr.Imnwl ,kt' it. , L,,.!
indicated as follows: *P < 0.05. gehs. Cl p. 132, 1995, [Abstract JF-21
160 KANDSSAMY, 11OWERTON. AND IHUNT
TABLE I
Effec:t (if Radiation "n 65 mM KCI-Stimulated SCa2' Uptake by Rat Whole-Brain, Cortex, and Striatal Synaptosomes
Net uptake of "CaW' (nmol/mg protein)"
Dose k, r'ldIatI'n Dose rate
(6v (Gy/min) Whole brain Cortex Striatum
Sham Sham 3.4 ± 0.05 3.3 t 0.05 3.5 t 0.10
1 3.0 ± 0.10* 3.3 ± 0.10 3.3 ± 0.10
10 2.7 ± 0.05* 3.0 ± 0.10 3.0 ± 0.10"
10 24 ± 0.10' 2.8 ± 0.10* 2.8 t 0.10"
p0 2.0 ± 0.05* 2.5 ± 0.05* 2.5 ± 0.05"
2) 1.7 ± 0.05* 2.2 ± 0.05* 2.4 ± 0.05*
-\ ,rc mans SEM of three separate experiments. each using triplicate samples.
Sniticantt., different from sham value: P< 0.05.
RESULTS following -yp hoton exposure was reduced by irradiation (3
Gy) (Fig. 3). Because the differences in uptake for the
Tie time course (3. 10. 30. and 60 s) as well as the dose whole-brain, cortical, and striatal synaptosomes are very
effect of KCI (15-65 mll) (Figs. I and 2) of calcium uptake similar, only the whole-brain results are shown in Fig. 3.
,,asr educed following exposure of the whole brain to 3 Gy However, the same concentrations of Bay K 8644 did not
of -'p hotons. The fastest rate of depolarization-dependent enhance 30 or 65 mM KCI-stimulated calcium uptake
calcium uptake occurred at 3 s (Fig. 1). The KCI dose-re- (data not shown).
sponse curve (Fig. 2) showed that 65 mM KCI stimulated The effects described above indicate that y irradiation
calcium uptake most. In the above two studies, irradiation will reduce KCI-stimulated calcium uptake. Several hypoth-
reduced calcium uptake at all incubation times and KCI eses can be advanced to explain these data. The initial hy-
concentrations. Based on the above data. a time point of 3 s pothesis we investigated concerned calcium channel recog-
and KCI dose of 65 mM were selected for investigation of nition receptor binding. The data showing that Bay K
the radiation dose-response curve (Table I). 8644-stimulated calcium uptake is reduced by -yi rradiation
Gamma irradiation (1-30 Gy) reduced 65 mM KCI- justifies the selection of nimodipine, the antagonist for the
stimulated calcium uptake after 3 s in whole-brain, cortical, calcium L-channel, as the appropriate ligand with which to
and striatal synaptosomes. A comparison between regions perform binding studies.
showed a significant interaction term. Whole-brain synap- Data obtained from whole-brain, cortical, and striatal
tosomes were clearly more sensitive to irradiation than membranes demonstrate that the specific binding of [nH]-
those of the cortex or striatum. nimodipine is unaffected by y irradiation at 10 Gy (data
Gamma irradiation (1-30 Gy) also reduced 15 mM KCI- not shown). These data indicate that the significance of the
stimulated calcium uptake in whole-brain, cortical, and reduction in KCI and/or Bay K 8644-stimulated calcium
striatal synaptosomes after 3 s (Table II). The enhancement uptake is not due to alteration of the L-type calcium chan-
of 15 mM KCI-stimulated calcium uptake by Bay K 8644 nel recognition receptor.
TABLE 11
Effect of y Radiation on 15 nul KCI-Stimulated 'Ca2" Uptake by Rat Whole-Brain. Cortex, and Striatal Synaptosomes
Net uptake of 4'Ca2 (nmol/mg proteins)*
Dose of radiation Dose rate
(Gy) (Gy/min) Whole brain Cortex Strialum
Sham Sham 2.6 ± 0.05 2.7 ± 0.10 2.7 ± 0.10
1 1 2.4 ± 0.10 2.6 t 0.10 2,6± 0,10
3 10 2.1 ± 0.05* 2.2 t 0.05' 22 ± 0,10
10 10 1.3 ± 0.10' 1.5 ± 0.15' 1.5 ± 0.15'
30 20 1.3 ± 0.15' 1.1 ± 0.200 II t 0.20*
Values are means ± SEM of three separated experiments, each using triplicate samples.
* Significantly different from sham value: P < 0.05.
IONIZING RADIATION AND CALCIUM UPTAKE 161
34 inhibition of Bay K 8644-enhanced KCI-stimulated or
KCI-stimulated calcium uptake is not a result of an altered
32/ L-type calcium channel recognition receptor. Apparently.
radiation does not decrease calcium uptake by a DHP-sen-
3o sitive mechanism. Further work is in progress to determine
,L the role of N and T calcium channels in mediating radia-
28 * tion-induced decreased calcium uptake.
E In conclusion, the present data indicate that ionizing radi-
26., IF - /At ation tshieg npirfeicsaennttl yti mreed uwcee sh aKvCe In-sot idmautala ttoe de xcpallaciinu mth eu pdtiaffkeer-.
24 - ences in sensitivity to irradiation of whole brain, cortex,
and striatum. Additionally, iti sn ot clear how ionizing radi-
ation causes this dose-dependent decrease in calcium up-
22- take. Preliminary experiments with inositol trisphosphate,3
2prostaglandins (36), and phorbol esters (37) suggest an im-
pairment in protein kinase C activity that is linked to the
18 ,opening or closing of ion channels (38). Further work is in
0 1 10 1opnM progress to compare protein kinase C activity of irradiated
Doses Of Bay K8644 and nonirradiated synaptosomes, and we are studying G
FIG. 3. Effect of - radiation on Bay K 8644-induced 15 mMA KCI- protein receptor binding to determine whether radiation-
stimulated calcium uptake in nonirradiated (0) and irradiated (o) rat induced decreases in calcium uptake are due to impairment
whole-brain synaptosomes. Points and bars represent means ± SEM vat- of protein kinase C, or alterations in G protein receptors, or
ucs from three separate experiments, each using triplicate samples. A signif- both.
icant effect of ionizing radiation is indicated as follows: *P < 0.05.
RECEiVED: February 22, 1990, ACCEPTED: September 6. 1990
DISCUSSION
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