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.
Tris±HCl buffer, pH 7
4 (for [3H]nitrendipine binding),
or 50 m
M
.
4 (for [125I]o-CgTx-
MVIIA and [125I]o-CgTx-MVIIC binding). The hom-
ogenate was centrifuged at 1000 g for 10 min at 48C
and the resulting supernatant was washed by means
of two consecutive centrifugation cycles (48 000 g,
10 min, 48C) with intermittent resuspension of the
pellet in fresh buffer. The ®nal pellet was resuspended
in 50 m
M
HEPES buffer, pH 7
Tris±HCl (for [3H]nitrendipine binding) or
50 m
M
HEPES (for [125I]o-CgTx-MVIIA and [125I]o-
CgTx-MVIIC binding) buffer to yield an original tissue
concentration of 100 mg mlÀ1 and stored at À708C
until use. Protein concentration was determined as
described above.
Radioligand Binding to Voltage-dependent Ca2 Channels
[3H]Nitrendipine was used for labelling L-type
VDCCs. Saturation binding assays were performed by
incubating aliquots of cortical (100 mg protein per
tube) or retinal (250 mg protein per tube) membranes
for 90 min at 258C in 300 ml of 50 m
M
Tris±HCl
buffer containing 0
.
05±2 n
M
[3H]nitrendipine. At the
end of the incubation, samples were diluted with 4 ml
of ice-cold 50 m
M
Tris±HCl buffer (pH 7
.
4), immedi-
ately vacuum ®ltered through Whatman GF/B ®lters
and washed twice with 4 ml of ice-cold buffer.
Radioactivity on the ®lters was measured by liquid
scintillation spectrometry in 5 ml of Insta-gel Plus.
Non-speci®c binding of [3H]nitrendipine was deter-
mined in the presence of 1 m
M
nifedipine. These
experiments were carried out in subdued light to
minimize [3H]nitrendipine and nifedipine degradation.
N-type and P/Q-type VDCCs were labelled with
[125I]o-conotoxin MVIIA (o-CgTx-MVIIA) and
[125I]o-conotoxin MVIIC (o-CgTx-MVIIC), respect-
ively. Saturation binding assays were performed by
incubating aliquots of cortical (1 mg protein per tube)
or retinal (5 mg protein per tube) membranes with
1±20 p
M
[125I]o-CgTx-MVIIA or 1±20 p
M
[125I]o-
CgTx-MVIIC for 60 min at 258C in 300 ml of 20 m
M
HEPES buffer containing 75 m
M
.
1m
M
EDTA,
0
NaCl, 0
.
1m
M
EGTA and 0
.
1% BSA (pH 7
.
2). At the end of
the incubation, samples were diluted with 4 ml of ice-
cold washing buffer (20 m
M
NaCl,
and 0
HEPES, 125 m
M
.
1 % BSA, pH 7
.
2), immediately vacuum ®ltered
through Whatman GF/B ®lters and washed twice
with 4 ml of ice-cold buffer. The ®lters were presoaked
in 0.6 % polyethylenimine for 2 hr to reduce radio-
activity trapping on the ®lters. Radioactivity on the
®lters was measured using a gamma counter (NE
1600, Nuclear Enterprises Ltd, Edinburgh, U.K.). Non-
speci®c binding was determined in the presence of
20 n
M
o-CgTx-MVIIA or 100 n
M
o-CgTx-MVIIC.
Under these conditions, [125I]o-CgTx-MVIIC selec-
tively labels P/Q-type VDCCs (Kristipati et al., 1994).
Materials
[3H]Nitrendipine (72
.
4 Ci mmolÀ1), [125I]o-CgTx-
MVIIA (2200 Ci mmolÀ1) and [125I]o-CgTx-MVIIC
(2200 Ci mmolÀ1) were obtained from NEN Research
Products from Amersham (Stevenage, (Amersham, U.K.) and U.K.). 45CaCl
o-CgTx-MVIIA,
2
(2 mCi mlÀ1)
o-CgTx-MVIIC and o-AgaTx-TK were purchased from
TCS Biologicals Ltd (Botolph Claydon, U.K.) and
MK-801 and tetrodotoxin from Semat Technical Ltd
(St. Albans, U.K.). NMDA, diltiazem, nifedipine and
verapamil came from Sigma (Poole, U.K.).
Analysis of Data
Saturation binding data nonlinear regression method were (GraphPad analysed Prism using 1
.
0).
a
All experiments were performed in duplicate and data
are expressed as mean+
S.E.(M.)
. Statistical analyses
were performed by using an unpaired Student's t-test
and one-way analysis of variance Bonferroni's post-test. Values of (ANOVA) P 5 0
.
05 using were
the
considered statistically signi®cant.
3. Results
Role of Voltage-dependent Ca2 Channels in the NMDA-
stimulated In¯ux of Ca2 into Isolated Rat Retinas
As shown in Fig. 1, NMDA (100 m
M
) induced
approximately a three-fold increase in the in¯ux of
45Ca2 into isolated rat retinas from 3082+
283 cpm mgÀ1 486 cpm mgÀ1 protein protein (n 15) (n 17, P to 5 0
10329+
.
001 by
unpaired Student's t-test). Such an increase in
.
45Ca2 in¯ux was completely blunted (P 5 0
001
by one-way ANOVA followed by Bonferroni's post-test)
by the NMDA receptor antagonist MK-801 (Fig. 1).
Tetrodotoxin (TTX), at a (1 (P 5 m
M
0
), .
05 produced by saturating .
concentration
an small (19
8%), albeit signi®cant
one-way ANOVA followed by Bonferro-
ni's post-test), inhibition of NMDA-stimulated Ca2
in¯ux into the rat retina (Fig. 1), indicating a role for
voltage-sensitive Na channels in this effect.
The dihydropyridine nifedipine, a speci®c L-type
VDCC blocker, was found to decrease the NMDA-
stimulated in¯ux of Ca2 into the isolated rat retina in
a dose-related fashion, with a maximum reduction of
approximately 50% (Fig. 2). Nonlinear ®tting of the
effects of nifedipine on the in¯ux of Ca2 into the rat
retina evoked by NMDA, considering as maximum
effect that observed an and EC
a 50
slope value factor of 0
.
of 88 with 0
m
.
92. M
100 (ÀlogEC
The m
M
benzothiazepine nifedipine, 50
6
.
06+0
revealed
.
16)
and
the phenylalkylamine L-type VDCC blockers diltiazem
and verapamil, respectively, produced similar
reductions in the NMDA-stimulated in¯ux of Ca2
into the isolated rat retina (Fig. 2).
Both o-CgTx-MVIIA, a speci®c blocker of N-type
VDCCs, and o-AgaTx-TK, a P/Q-type VDCC blocker,
had no effect on the NMDA-induced in¯ux of Ca2
into isolated rat retinas at a saturating concentration
of 100 n
M
(Fig. 3). No effect was also observed with a
higher concentration (500 n
M
) of o-CgTx-MVIIA
(data not shown). Moreover, combination of 100 n
M
o-CgTx-MVIIA or 100 n
M
o-AgaTx-TK with 1 m
M
nifedipine did not result in an inhibition of Ca2 in¯ux
signi®cantly higher than that observed with 1 m
M
nifedipine alone (data not shown). Ni2 was used to
. 2. Effects of L-type Ca2 channel blockers nifedipine, verapamil and diltiazem on the NMDA-stimulated in¯ux of
45Ca2
into isolated rat retinas. Data represent the mean+
S.E.(M.)
signi®cantly different from control group by one-way ANOVA F
IG
of the followed number of by Bonferroni's experiments post-test: shown *P 5 in 0
.
01, parentheses. **P 5 0
.
001.
Values
.F
IG
3. Effects of Ca2 channel blockers on the NMDA-stimulated in¯ux of 45Ca2 into isolated rat retinas. Data
represent the
mean+
S.E.(M.)
of way ANOVA the followed number by of Bonferroni's experiments post-test: shown *P 5 in 0
.
parentheses. 01, **P 5 0
.
Values 001.
signi®cantly different from control group by one-
12000
10000
8000 6000 4000 20
100
10
10 100
Dil
apam
Ver
7) Control
l 6000
4000 -
1
8000 - 6000 4000 2000
0
Co2
measured in the presence of 500 m
M
(4276 +313, n 4) was, however, not signi®cantly
different from that observed with 10 m
M
nifedipine
(5163 +334, n 4).
Radioligand Binding to Voltage-dependent Ca2 Channels
Speci®c binding of [3H]nitrendipine to rat retinal
homogenates was saturable and consistent with the
existence of a single class of binding sites, as indicated
by values the calculated linear Scatchard by nonlinear plot [Fig. analysis 4(A)]. for K
D
the and speci®c
B
max
binding of [3H]nitrendipine to both rat cortical and
retinal homogenates are shown in Table I. [3H]Nitren-
dipine showed a signi®cantly lower af®nity (a higher
K
cortex D
value) (Table for binding I). The number sites in the of binding retina [3H]nitrendipine in the cortex was
than in the rat
approximately
sites (B
max
) for
three-fold higher than in the rat retina (Table I).
Speci®c binding of [125I]o-CgTx-MVIIA to N-type
VDCCs in rat cortical homogenates was saturable and
of high af®nity (Table I). Speci®c binding of [125I]o-
CgTx-MVIIA to rat retinal membranes showed similar
characteristics and no evidence for multiple binding
sites, given the linearity of the Scatchard plot
[Fig. 4(B)]. [125I]o-CgTx-MVIIA showed a similar
af®nity for binding sites in both the rat cortex and
retina, while the number of binding sites was
approximately 11-fold lower in the retina (Table I).
Speci®c binding of [125I]o-CgTx-MVIIC to rat retinal
membranes was saturable and consistent with the
existence of a single class of binding sites, as revealed
by the linearity and CgTx-MVIIC B
max
values to of the Scatchard for the speci®c binding plot [Fig. of 4(C)]. [125I]o-
K
D
both rat cortical and retinal hom-
ogenates are shown in Table I. No differences were
found in the af®nity of [125I]o-CgTx-MVIIC for
radioligand binding sites in the rat cortex and retina,
while the binding sites in the retina were signi®cantly
(six-fold) less abundant (Table I).
4. Discussion
In the present study we attempted to identify the
types of VDCCs involved in the NMDA-stimulated
T
ABLE
I
Af®nity (K
D
) and number of binding sites (B
max
) for radioligands labelling VDCCs in the rat cortex and retina
Radioligand
Cortex Retina
K
D
(fmol mgÀ1 protein) n K
D
(fmol mgÀ1 protein) n
[3H]Nitrendipine 216
(p
M
)B
max
(p
M
)B
max
.
0+ 20
.
5 119
.
4+ 3
.
1 3 533
.
2+ 28
.
9* 40
.
1+2
.
[125I]o-Conotoxin MVIIA [125I]o-Conotoxin MVIIC 2
5
.
.
7+0
4+0
.
.
2 4 240
448
.
.
2+ 8+ 20
10
.
.
9 0 9* 4
433
4
.
.
4+ 9+ 0
0
.
.
4 2 22
74
.
.
4+2
2+6
.
.
6* 1* 4
4
Values are mean +
S.E.(M.)
of n independent experiments performed in duplicate. *Signi®cantly different (P 5 0
.
001) from corresponding
value in cortex by unpaired Student's t-test.
.F
IG
4. Speci®c binding of [3H]nitrendipine (A), [125I]o-CgTx-MVIIA (B) and [125I]o-CgTx-MVIIC (C) to rat retinal
homogenates. Scatchard plots of the data are shown as insets. The results represent data from a single typical
experiment
performed in duplicate. Mean K
D
and B
max
values for four experiments are given in Table I.
endipine bound mg pr
otein)
A
0.10
0.00 0 IO 20 30 40
0
80 70 60 50
Free (nM) o
Bound/Free
1.5 2.0
0.3
0 0.2
Free (PM)
0.00 0 20 40 60 80
Free (PM)
after incubation with L-type VDCC blockers diltiazem
(benzothiazepine class) and verapamil (phenylalkyla-
mine class). Blockers of N-type (o-CgTx-MVIIA) and
P/Q-type (o-AgaTx-TK) VDCCs did not, however,
signi®cantly inhibit the in¯ux of Ca2 into the isolated
rat retina stimulated by NMDA, suggesting that both
N- and P/Q-type VDCCs do not play a major role in
this effect. When Ni2 was applied at a low concen-
tration (10 m
M
), at which it is considered to selectively
block R-type VDCCs (Avery and Johnston, 1996;
Imaizumi et al., 1999), a signi®cant enhancement of
the NMDA-stimulated in¯ux of Ca2 into the rat
retina was found. However, when Ni2 was applied at
a higher concentration (100 m
M
), able to block T-type
VDCCs (Avery and Johnston, 1996; Imaizumi et al.,
1999), no signi®cant effect was observed. This
biphasic effect of Ni2 on NMDA-induced in¯ux of
Ca2 could be ascribed to a direct potentiation (at low
concentrations) or inhibition (at high concentrations)
of the NMDA receptor activity, as previously reported
in cultured rat cerebellar granule cells (Eimerl and
Schramm, 1993). The unavailability of other speci®c
blockers of R- and T-type VDCCs (compounds used as
T-type antagonists such as ¯unarizine and mibefradil
can also block other VDCCs) prevented us from
exploring in more detail the possible role of these
VDCCs in the NMDA-evoked in¯ux of Ca2 into the
rat retina. Nevertheless, the fact that the non-speci®c
VDCC blocker Co2 caused an inhibition of NMDA-
stimulated in¯ux of Ca2 into the rat retina almost
identical to that elicited by nifedipine strongly
suggests that only L-type VDCCs play a major role
in the in¯ux of Ca2 into the rat retina triggered by
NMDA.
The ®nding that L-type VDCCs predominantly
contribute (up to 50 %) to the NMDA-stimulated
in¯ux of Ca2 into the rat retina contrasts with
evidence obtained in brain neurones about the
involvement of VDCCs in glutamate-induced rises in
intracellular Ca2 . In cultured rat cerebellar granule
neurones, L-, N- and P/Q-type VDCCs have been
shown to be involved in intracellular Ca2 signals
evoked by 50±200 m
M
NMDA (Qiu et al., 1998;
Netzeband et al., 1999). In rat embryonic lumbar
motoneurones in situ, blockers of L-, N- and P/Q-type
produced approximately a 25 % inhibition of gluta-
mate-induced rises in intracellular Ca2 in each case,
a 50% inhibition being observed with the joint appli-
cation of these drugs (Metzger et al., 2000). Further-
more, both L- (approximately 52 %) and N-type
(approximately 33 %) VDCCs have been reported to
mediate the in¯ux of Ca2 stimulated by kainate in
cultured rat hippocampal neurones (AmbroÂsio et al.,
1999). In order to elucidate whether differences in the
involvement of VDCCs in intracellular Ca2 responses
mediated by glutamate receptor may re¯ect a distinct
distribution of VDCCs in the brain and retina, the
number of L-, N- and P/Q-type VDCCs in both the rat