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Exp. Eye Res. (2001) 72, 393±401
doi:10.1006/exer.2000.0968, available online at http://www.idealibrary.com on

Voltage-dependent Calcium Channels in the Rat Retina: Involvement in


NMDA-stimulated In¯ux of Calcium
JOSE MELENA
AND
NEVILLE N. OSBORNE*
Nuf®eld Laboratory of Ophthalmology, University of Oxford, Walton Street, Oxford, OX2 6AW, U.K.
(Received Cleveland 12 June 2000, accepted in revised form 29 November 2000 and published
electronically 8 February 2001)
Rises in intracellular Ca2 induced by activation of glutamate receptors are of ultimate importance for
neuronal excitability and pathophysiological processes. In the present study, we aimed to elucidate the
types of voltage-dependent Ca2 channels involved in the NMDA-stimulated in¯ux of Ca2 into the
isolated rat retina by using selective blockers. Additionally, the number of binding sites for radioligands
labelling L- ([3H]nitrendipine), N- ([125I]o-conotoxin MVIIA) and P/Q-type ([125I]o-conotoxin MVIIC)
Ca2 channels was assessed in the rat retina and, for further comparison, in the rat cortex. Incubation of
isolated rat retinas with 100 m
M
NMDA produced a three-fold increase in the in¯ux of 45Ca2 that was
completely blunted by MK-801, a NMDA receptor antagonist, and partially attenuated (approximately
20%) by tetrodotoxin, a Na channel blocker. The L-type Ca2 channel blocker nifedipine reduced
NMDA-stimulated Ca2 in¯ux in a dose-related fashion, with a maximum reduction of approximately
50%. Similar effects were observed with verapamil and diltiazem. Blockers of N- and P/Q-type Ca2
channels had no signi®cant effect on the in¯ux of Ca2 evoked by NMDA. Co2 , a non-speci®c Ca2
channel blocker, caused an inhibition of NMDA-stimulated Ca2 in¯ux similar to that of nifedipine.
Therefore, of all voltage-dependent Ca2 channels, L-type channels appear to make the greatest
contribution (up to 50%) to the NMDA-stimulated in¯ux of Ca2 into the isolated rat retina. This ®nding
contrasts with evidence obtained in brain neurones supporting a role for L-, N- and P/Q-type channels in
NMDA-evoked Ca2 signals. A comparison of the number of radioligand binding sites associated with L-,
N- or P/Q-type Ca2 channels in the rat cortex and retina revealed that such a difference cannot be
ascribed to a distinct expression pattern of these channels in both tissues, although some variations were
found. Interestingly, a different af®nity of [3H]nitrendipine for L-type Ca2 channels in the rat retina and
cortex was observed which may re¯ect the expression of different classes of L-type channels in these
tissues. The ability of L-type Ca2 channel blockers to attenuate NMDA-stimulated Ca2 in¯ux may
underlie their neuroprotective effects in the retina. # 2001 Academic Press
Key words: voltage-dependent calcium channel; NMDA; calcium in¯ux; retina; neuroprotection;
ischaemia; glaucoma; rat.
1. Introduction
The in¯ux of Ca2 through voltage-dependent Ca2
channels (VDCCs) into cells mediates a variety of
processes which include neurotransmitter release,
excitability, excitation-contraction coupling, secretion
and gene expression. VDCCs are classi®ed into L-, N-,
P/Q-, R- and T-types based on their functional
properties, such as time- and voltage-dependent
kinetics, and sensitivity to different pharmacological
agents (Birnbaumer et al., 1994; Dunlap, Luebke and
Turner, 1995). Ca2 currents through L-type VDCCs,
exhibited both in excitable and nonexcitable cells, are
high-voltage activated, long-lasting and very sensitive
to dyhydropyridines (e.g. nifedipine), as well as
phenylalkylamines (e.g. verapamil) and benzothiaze-
pines (e.g. diltiazem). The high voltage-activated N-
and P/Q-type VDCCs are largely restricted to neurons
and selectively blocked by o-conotoxins GVIA
(o-CgTx-GVIA) and MVIIA (o-CgTx-MVIIA) and by
o-agatoxins IVA (o-AgaTx-IVA) and TK (o-AgaTx-
TK), respectively (Birnbaumer et al., 1994; Dunlap
et al., 1995). Neuronal high-voltage activated VDCCs
also include the R-type, characterized by its resistance
to both dihydropyridines and toxins and its suscepti-
bility to blockade by low concentrations of Ni2 (Avery
and Johnston, 1996; Imaizumi, Kocsis and Waxman,
1999). T-type VDCCs are low-threshold, transient
channels which are implicated in repetitive ®ring and
pacemaker activity. T-type VDCCs show resistance to
dihydropyridines and toxins, but are sensitive to
mibefradil, ¯unarizine and high concentrations of
Ni2 (Birnbaumer et al., 1994; Dunlap et al., 1995).
Rises in intracellular calcium following activation of
a-amino-3-dihydro-5-methyl-isoxazol-4-propionic
acid/kainate (AMPA/KA) and N-methyl-
D
-aspartate
(NMDA) ionotropic glutamate receptors have been
associated with critical physiological processes, such as
synaptictransmission,synapticplasticityandneuronal
differentiation (Collingridge and Singer, 1990; Ozawa,
Kamiya and Tsuzuki, 1998). Additionally, abnormally

0014-4835/01/040393 09 $35.00/0 # 2001 Academic Press

* Address correspondence to: Neville N. Osborne, Nuf®eld


Laboratory of Ophthalmology, Oxford University, Walton Street,
Oxford, OX2 6AW, U.K. E-mail: neville.osborne@eye.ox.ac.uk
high levels of glutamate released during ischaemia can
produce overstimulation of ionotropic glutamate
receptors, leading to neuronal cell death triggered by
a large in¯ux of Ca2 into cells mainly via NMDA
receptors and secondary opening of VDCCs (Lee, Zipfel
and Choi, 1999). Therefore, substances able to reduce
Ca2 overload either by interacting with NMDA
receptors and/or VDCCs should theoretically alleviate
ischaemic/excitotoxic damage to the retina. Accord-
ingly, a number of NMDA receptor antagonists have
been shown to protect retinal cells in models of
ischaemia/excitotoxicity, although their potential
therapeutic use is questioned because of their major
side effects (Osborne et al., 1999b; Ritch, 2000). An
alternative way to reduce Ca2 overload following an
ischaemic insult and hence to ameliorate neuronal cell
death would theoretically include the blockade of
VDCCs. Little is known, however, about the types of
VDCCs which are involved in the glutamate-evoked
entry of extracellular Ca2 into retinal cells. Therefore,
the aim of the present study was to determine which
kind of VDCCs are involved in the NMDA-stimulated
in¯ux of Ca2 into isolated rat retinas by applying
selective blockers. In addition, a comparative assess-
ment of the number of L-, N- and P/Q-type VDCCs in
the rat cortex and retina was performed by using
speci®c radioligands.
2. Materials and Methods
Determination of NMDA-stimulated In¯ux of Ca into
Isolated Rat Retinas
Adult Wistar rats (250±350 g) bred in our
laboratory were killed by decapitation and retinas
rapidly removed and preincubated (378C for 25 min)
in magnesium-free (138 m
M
NaCl, 5
.
6 Krebs±Ringer m
M
bicarbonate KCl, 1 m
M
CaCl
buffer
NaHCO
and 20 3
,m
1M
m
M
HEPES NaH
2
buffer, PO
4
-Na
2
pH HPO
7
4
.
, 4) 10 continuously
m
2
M
, 11 m
M
glucose
perfused then added with and 95% the O
retinas 2
/5% CO
incubated 2
. Antagonists were
for a further
10 min. Calcium in¯ux was initiated by the addition
of ®nal 1 mCi volume 45CaCl
of 2
2 and ml. 100 m
M
NMDA After 15 min or buffer in a
incubation, the
reaction 10 m
M
washed was EGTA twice halted per 0
by .
9 % the NaCl. addition Retinas of in 2 ml ice-cold 1 m
M
EGTA/0
2 ml ice-cold
were .
9% NaCl
then
and sonicated in 1 ml distilled water. Radioactivity
was determined by liquid scintillation spectrometry in
5 ml of Insta-gel Plus (Packard, Groningen, The
Netherlands) and protein concentration with a
bicinchoninic acid protein assay kit (Sigma, Poole,
U.K.) using bovine serum albumin (BSA) as standard.
Tissue Preparation for Radioligand Binding Studies
Adult Wistar rats were killed by decapitation. The
cerebral cortices and retinas were dissected over ice
and homogenized in 15 volumes of ice-cold 50 m
M

394 J. MELENA AND N. OSBORNE

.
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

CALCIUM CHANNELS IN THE RAT RETINA 395


.F
IG
1. In¯ux of 45Ca2 into isolated rat retinas. NMDA (100 m
M
(control) which was completely blunted by MK-801 and ) partially inhibited caused by a signi®cant TTX (P 5 increase
0
.
001 and in the 0
.
05, basal respectively, in¯ux of calcium
by one-
way ANOVA followed by parentheses. *Values signi®cantly Bonferroni's different post-test). (P 5 0
Data .
001) represent from control the mean+
S.E.(M.)
of group by one-way the number of experiments shown in
ANOVA followed by Bonferroni's post-
test.

.
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

45Ca2+ influx (cpm mg protein)


explore the potential role of T-type and R-type VDCCs
in the NMDA-stimulated in¯ux of Ca2 into rat
retinas. At low concentrations (530 m
M
), Ni2 is
considered to selectively block R-type VDCCs, whereas
at higher concentrations it would also inhibit T-type
channels (Avery and Johnston, 1996; Imaizumi et al.,
1999). At 10 m
M
concentration, Ni2 was found 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

396 J. MELENA AND N. OSBORNE

.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-

signi®cantly enhance the NMDA-stimulated Ca2


in¯ux into the isolated rat retina, while no signi®cant
effect was observed at 100 m
M
concentration (Fig. 3).
Finally, the non-speci®c Ca2 channel blocker Co2 ,
at a saturating concentration (500 m
M
), inhibited the
NMDA-stimulated in¯ux of Ca2 into the rat retina by
approximately 60% (Fig. 3). The in¯ux of Ca2

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

CALCIUM CHANNELS IN THE RAT RETINA 397

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.

in¯ux of Ca2 into the isolated rat retina as well to


assess the number of retinal L-, N- and P/Q-type
VDCCs by radioligand binding. Incubation of isolated
rat retinas with 100 m
M
NMDA caused a three-fold
increase in the basal total cellular Ca2 which was
blunted by MK-801, a speci®c NMDA receptor
antagonist. Such an increase is likely to re¯ect the
in¯ux of Ca2 into amacrine, ganglion and MuÈller
cells, the main types of retinal cells shown to express
functional NMDA receptors (Thoreson and Witkovsky,
1999; Fletcher et al., 2000). It has not been
demonstrated that photoreceptors, bipolar and
horizontal cells express functional NMDA receptors
(Thoreson and Witkovsky, 1999; Fletcher et al.,
2000). The speci®city of the in¯ux of Ca2 into the
isolated rat retina evoked by NMDA and the absence
of functional NMDA receptors in photoreceptors
suggest that non-speci®c uptake of Ca2 by photo-
receptor cells was negligible in our preparation. TTX,
a blocker of voltage-dependent sodium channels
(VDSCs), was found to signi®cantly reduce (approxi-
mately 20%) the NMDA-stimulated in¯ux of Ca2
into the rat retina, revealing a role for VDSCs in this
effect. It is likely that membrane depolarization
following activation of NMDA receptors leads to
opening of VDSCs, resulting in further membrane
depolarization. Blockade of VDSCs by TTX could
decrease the extent of membrane depolarization
triggered by NMDA receptor activation and hence
reduce the magnitude of other voltage-dependent
cellular processes, such as the entry of Ca2 through
VDCCs. Alternatively, TTX could reduce spontaneous
spiking activity of retinal neurones, thus reducing
membrane depolarization and increasing the prob-
ability of voltage-dependent block by Mg2 of NMDA
receptors (Mayer, Westbrook and Guthrie, 1984).
The increase in total intracellular Ca2 in the
isolated rat retina induced by NMDA was reduced by
the L-type VDCC blocker nifedipine (dihydropyridine
class) 0
.
88 in a m
M
was dose-related calculated for fashion. nifedipine An in EC
inhibiting 50
value the
of
in¯ux of Ca2 into isolated rat retinas evoked by
100 m
M
NMDA. Nifedipine caused a maximal inhibi-
tion of NMDA-stimulated in¯ux of Ca2 of approx-
imately 50 %. Similar reductions were also observed
398 J. MELENA AND N. OSBORNE

.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

CALCIUM CHANNELS IN THE RAT RETINA 399

cortex and retina was assessed by using radioligand


binding techniques.
Binding characteristics of [3H]nitrendipine and
[125I]o-CgTx-MVIIA to L- and N-type VDCCs, respect-
ively, in rat cortical membranes were in accordance
with data previously published (Ehlert et al., 1982;
Gould, Murphy and Snyder, 1984; Stoehr and Dooley,
1993; Hisamoto et al., 1998). Although no data are
available about the binding of [125I]o-CgTx-MVIIC to
rat cortical membranes, the ®nding of approximately a
two-fold higher number of binding sites for [125I]o-
CgTx-MVIIC than for [125I]o-CgTx-MVIIA in the rat
cortex is in good agreement with the results obtained
by Kristipati et al. (1994) in bovine and rat cortical
synaptosomes. Under the radioligand binding con-
ditions employed here, [125I]o-CgTx-MVIIC selectively
labels P/Q-type VDCCs (Kristipati et al., 1994). In the
rat retina, we found a reduction of the number of
radioligand binding sites associated with L- (three-
fold), N- (ten-fold) and P/Q-type (six-fold) VDCCs when
compared to the cortex. The most abundant retinal
VDCC was the P/Q-type, as in the cortex, followed by
the L-type and, ®nally, by the N-type, which is much
reduced in comparison with its levels in the cortex. A
similar distribution pattern of VDCCs in the rat retina
was reported by Kamphuis and Hendriksen (1998) by
analysing levels of mRNAs encoding the various a
1
subunits of the VDCCs. These data indicate that the
apparent lack of contribution of N- and P/Q-type
VDCCs to the NMDA-stimulated Ca2 in¯ux into the
isolated rat retina cannot be explained by an overall
reduced presence of these channels in the retina with
regard to the L-type channel. While the expression of
L-type VDCCs in the retina is relatively increased as
compared with the cortex (29% of the total of L-, N-
and P/Q-type channels in the retina vs 15 % in the
cortex), the relative levels of P/Q-type channels are the
same in both tissues (55 %), and only N-type channels
are markedly reduced in the retina (16 % in the retina
vs 30% in the cortex).
Taken together, these data suggest that L-type
VDCCs play a predominant role in the total intra-
cellular Ca2 rise evoked by NMDA in the isolated rat
retina. The possibility exists, however, that N- or P/Q-
type VDCCs may contribute to NMDA-elicited Ca2
signals in certain retinal cells, as for example the
ganglion cells, and that potential contribution could
not be detected in our experimental setting. N-type
VDCCs have been shown to signi®cantly contribute
(approximately 40 %) to the whole-cell Ca2 current
in adult rat ganglion cells (Guenther et al., 1994;
Schmid and Guenther, 1999), which express func-
tional NMDA receptors (Thoreson and Witkovsky,
1999). The limitations of the technique used, able to
detect changes in the total intracellular Ca2 but not
in free intracellular Ca2 concentration, could have
led to an underestimation of the role of N- or P/Q-type
VDCCs in the NMDA-induced Ca2 in¯ux into the
isolated rat retina. The apparent lack of effect of N- and
P/Q-type VDCC blockers on the in¯ux of Ca2 evoked
by NMDA could also be ascribed to potential
dif®culties of these high molecular weight peptides
in reaching their sites of action in the isolated rat
retina. Studies on hippocampal slices indicate, how-
ever, that these toxins do readily penetrate into whole-
tissue preparations (Fox, 1994; Keith et al., 1995).
Interestingly, [3H]nitrendipine showed a signi®-
cantly lower af®nity for binding sites in the rat retina
than in the cortex, which may result from a different
expression in these tissues. of a
1
Nitrendipine subunit isoforms of the L-type channel
the L-type VDCC and hence binds alterations to the a
in 1
subunit particular
of
amino binding acid characteristics(Striessnig sequences of the a
1
subunit et al., 1998). can modify Nitren-
its
dipine exhibits, for example, a much lower af®nity for
the L-type VDCC of skeletal muscle, which expresses
the cerebral a
1S
isoform, cortex, than for the L-type channel of the
Kamphuis and formed Hendriksen by the (1998) a
1D
(Gould reported et al., that 1984).
the
main isoform of L-type channel the rat retina isoform in the is cortex the a
1D
so , which that is a
1
also subunit the predominant
expressed in
differences in [3H]nitren-
dipine af®nity would seem unlikely. However, a new
member isoform, of has the been L-type recently VDCC a
identi®ed 1
subunit family, in the the retina
a
1F
(Bech-Hansen et al., 1998; Strom et al., 1998). This
a
demonstrated 1F
isoform, absent in the from the brain and so far only
retina, shows some amino acid
variations in the transmembrane domains critical for
conferring dihydropyridine sensitivity that may
account for the different af®nity of [3H]nitrendipine
for cortical and retinal L-type channels.
Ca2 signaling mediated by NMDA receptors
appears to be of ultimate importance for retinal
synaptic transmission and development (Thoreson
and Witkovsky, 1999; Fletcher et al., 2000; GruÈnder
et al., 2000). Additionally, overstimulation of NMDA
receptors plays a key role in ischaemic/excitotoxic
insults to the retina, as may occur in glaucoma
(Dreyer, 1998; Osborne et al., 1999a), leading to
neuronal cell death due to intracellular Ca2 overload
(Sucher, Lei and Lipton, 1991; Kitano, Morgan and
Caprioli, 1996; Zhang et al., 2000). The ability of
L-type VDCC blockers to decrease NMDA-stimulated
Ca2 in¯ux into the retina could therefore be
responsible for the neuroprotective effects of these
drugs in paradigms of ischaemia/excitotoxicity
(Crosson, Willis and Potter, 1990; Sucher et al.,
1991; Takahashi et al., 1992; Block and Schwarz,
1998; Toriu et al., 2000). To date, no evidence is
available supporting a retinal neuroprotective effect of
blockers of N- or P/Q-type VDCC in models of
ischaemia/excitotoxicity, which may be related to
the apparent inability of these drugs to inhibit NMDA-
evoked in¯ux of Ca2 into the retina reported here. It
should be noted, however, that N- or P/Q-type VDCC
blockers may theoretically exert neuroprotective
effects by reducing glutamate release during or after

400 J. MELENA AND N. OSBORNE

retinal ischaemia, as described in the brain (Kimura


et al., 1998; Kobayashi and Mori, 1998). Never-
theless, in contrast to the brain, L-type VDCCs also
appear to play a major role in controlling the release
of glutamate in the retina (Thoreson and Witkovsky,
1999), thus providing a potential additional mech-
anism for explaining the retinal neuroprotective
effects of the blockers of these channels.
In conclusion, this study demonstrates that NMDA-
stimulated in¯ux of Ca2 into the isolated rat retina
can be markedly attenuated by the blockade of L-type
VDCCs. On the contrary, other types of VDCCs do not
seem to play a major role in the in¯ux of Ca2 evoked
by NMDA. This different involvement of VDCCs in the
intracellular Ca2 rises elicited by NMDA in the retina
and in the brain does not appear to result from a
distinct expression pattern of L-, N- or P/Q-type
VDCCs in both tissues, although some variations were
found. Interestingly, a different af®nity of [3H]nitren-
dipine for L-type VDCCs in the rat retina and cortex
was observed which may re¯ect the expression of
different classes of L-type channels in these tissues.
This ability of L-type VDCC blockers to attenuate
NMDA-stimulated Ca2 in¯ux may underlie their
neuroprotective effects in the retina. Future work is
needed to elucidate the role of VDCCs in the release of
glutamate in the ischaemic retina.
Acknowledgements
J. Melena was supported by a post-doctoral Marie Curie
grant (TMR programme, European Commission).
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