| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Pharmacology and Department of Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5540
Submitted 13 August 2002; accepted in final form 10 March 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
,
1998;
Dunwiddie et al. 1997).
Adenosine exerts its effects by activating a distinct class of four G
protein-coupled cell-surface receptors classified as A1,
A2A, A2B, and A3
(Ralevic and Burnstock 1998).
Generally it is thought that the inhibitory actions of adenosine are mediated
by A1 receptors, whereas A2 receptors and particularly
A2A receptors have been considered to mediate excitatory effects in
the CNS (Ralevic and Burnstock
1998; Sebastiao and Ribeiro
1996; van Calker et al.
1979). However, few studies demonstrate direct excitatory actions
of A2 receptors on transmission; instead, the observed excitability
produced by activation of A2 receptors appears to be mediated by
reduced release of inhibitory transmitters (e.g., GABA) produced by
presynaptic inhibition of voltage-gated Ca2+ channels (see review
by Edwards and Robertson
1999). A2 receptors have been shown to inhibit calcium
currents (ICa) in a number of preparations
(Chen and van den Pol 1997;
Kobayashi et al. 1998,
2000;
Park et al. 1998) but other
than the photoreceptor synapse considered in the present study,
A2-mediated inhibition of ICa has not been
reported at a glutamatergic synapse.
Adenosine is selectively accumulated by rod-driven horizontal cells in the
goldfish retina (Studholme and Yazulla
1997) and receptors for adenosine have been localized to the outer
nuclear layer and photoreceptors (Kvanta
et al. 1997; Paes de Carvalho et al.
1990,
1992). Adenosine release
occurs in the dark (Blazynski and Perez
1991), when photoreceptors are depolarized and
L-glutamate release from photoreceptors is at its greatest
(Schmitz and Witkovsky 1997).
We have previously shown that the L-type ICa in rod
photoreceptors that mediates synaptic release of L-glutamate is
inhibited by A2 but not A1 or A3 agonists
(Stella et al. 2002).
Therefore we hypothesized that activation of A2 receptors should
diminish transmitter release from rod photoreceptors.
The present study explores the postsynaptic consequences of activating A2 adenosine receptors on rod photoreceptors and expands the pharmacological profile of adenosine receptors to include effects of selective adenosine receptor antagonists. To accomplish this, we compared the effects of adenosine receptor antagonists and agonists on both ICa and depolarization-evoked increases of [Ca2+]i in rod photoreceptors measured using Ca2+ imaging and electrophysiological recording techniques. To assess the effects of adenosine on transmission, we examined postsynaptic currents in second-order retinal neurons and tested whether inhibition of ICa by A2 receptor activation in rod photoreceptors results in the inhibition of L-glutamate release. Our results indicate that selective A2 and A2A agonists cause a reduction in the release of L-glutamate onto second-order neurons from rods, an effect that is blocked by the selective A2A antagonist, ZM-214385. In accordance with these results, ZM-214385 alone enhanced postsynaptic light responses from horizontal and bipolar cells. ZM-214385 also prevented both adenosine and the A2A agonist, CGS-21680, from inhibiting depolarization-evoked Ca2+ increases in rod photoreceptors. This study establishes a novel A2-mediated role for adenosine at a glutamatergic synapse and suggests that as illumination levels vary they could be accompanied by changing levels of adenosine, which can modulate neurotransmission at the first synapse in vision.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Larval tiger salamanders (Ambystoma tigrinum; Kons, German-town,
WI or Charles Sullivan, TN, 7–10 in.) were cared for according to
institutional guidelines. Retinal slices were prepared according to the
methods of Werblin (1978) and
Wu (1987); exact procedures
are described in further detail by Thoreson et al.
(1997). Briefly, salamanders
were killed by decapitation and pithed; the eyes were enucleated, and the
anterior portion of an eye including the lens was removed. The resulting
eyecup was cut into sections and placed vitreal side down onto a piece of
filter paper (Millipore 2 x 5 mm, Type GS, 0.2-µm pores). After the
retina adhered to the filter paper, the retina was isolated under chilled
amphibian superfusate and cut into 100- to 150-µm slices using a razor
blade tissue chopper (Stoelting). Retinal slices were rotated 90° for
viewing of the retinal layers when placed under a water immersion objective
(x40, 0.7 NA or x60, 1.0 NA) and viewed on an upright fixed stage
microscope (Olympus BHWI or Nikon E600FN). Dissections were usually performed
under infrared light using Gen III image intensifiers (Nitemate NavIII, Litton
Industries).
Solutions and perfusion
Solutions were applied by a single-pass, gravity-feed perfusion system, which delivered medium to the slice chamber (chamber volume: approximately 0.5 ml) at a rate of 1.0 ml/min. The normal amphibian superfusate that bathed the slices contained the following (in mM): 111 NaCl, 2.5 KCl, 1.8 CaCl2, 0.5 MgCl2, 10 HEPES, and 5 glucose. When measuring light-evoked responses, the superfusate was supplemented with 100 µM picrotoxin, 1 µM strychnine, and 0.1 mM glutamine. For photoreceptor recordings of ICa, the superfusate was switched to a Ba2+ solution to enhance Ca2+ currents. The Ba2+ solution contained the following (in mM): 99 NaCl, 2.5 KCl, 10 BaCl2, 0.5 MgCl2, 10 HEPES, 5 glucose, 0.1 picrotoxin, and 0.1 niflumic acid. The pH of all solutions was adjusted to 7.8 with NaOH. The osmolarity measured with a vapor pressure osmometer (Wescor) was 242 ± 5 mOsm. Solutions were continuously bubbled with 100% O2.
Electrophysiology
Patch pipettes were pulled on a Narashige PB-7 vertical puller from
borosilicate glass pipettes (1.2 mm OD, 0.95 mm ID, omega dot) and had tips of
approximately 1 µm OD with tip resistances of 10 to 15 M
.
For bipolar and horizontal cells, pipettes were filled with a solution
containing the following (in mM): 54 KCl, 61.5 KCH3SO4,
3.5 NaCH3SO3, 10 HEPES. For ICa
recordings, pipettes were filled with a solution containing the following (in
mM): 54 CsCl, 61.5 CsCH3SO3, 3.5
NaCH3SO4, 10 HEPES. The pH of these solutions was
adjusted to 7.2 with KOH or CsOH, respectively, and the osmolarity was
adjusted, if necessary, to 242 ± 5 mOsm. To maintain endogenous second
messenger signaling pathways and avoid the rundown that accompanies
conventional whole-cell recording, we used the perforated patch method of
whole-cell recording with the pore forming antibiotic, nystatin
(Rae et al. 1991), or
gramicidin (Kyrozis and Reichling
1995). Nystatin was mixed in DMSO at a concentration of 120 mg/ml,
vortexed briefly, and then added to the pipette electrolyte solution to
achieve a final concentration of 480 µg/ml. Gramicidin was dissolved in
ethanol at a concentration of 5 mg/ml, vortexed briefly, and then added to the
pipette electrolyte solution to achieve a final concentration of 5 µg/ml.
The final working stock was vortexed vigorously for 20 to 30 s and stored in
the refrigerator. Fresh antibiotic solutions were made every 3 h. In
successful recordings, seals >1 G were obtained in 30 s or less, and
cells were usually fully perforated within 5 min of sealing. Cells were
voltage clamped using an Axopatch 200B patch-clamp amplifier (Axon
Instruments, Foster City, CA). Currents were acquired and analyzed using
Clampex 7.0 software (Axon Instruments).
From a random sample of cells, the input resistance
(RN) of rods averaged 595 ± 25.6 M
(n = 15); bipolar cell RN averaged 960 ±
138.7 M (n = 7), and horizontal cell RN
averaged 117 ± 22.7 M (n = 7). Charging curves for
bipolar cells and rods were typically well fit by a single exponential,
indicating a compact electrotonic structure (<0.1 
). Rods were
voltage clamped at –70 mV; bipolar cells were voltage clamped at
–60 mV, and horizontal cells were voltage clamped at –40 mV. Cells
in which the access resistance exceeded 40 M were excluded from
analysis.
To assess ICa, voltage ramps (0.5 mV/ms) from –90
to +60 mV were applied every 30 s. Voltage ramps yield a similar
current-voltage profile as a step protocol but cause less current rundown and
provide more data points for fitting and analysis
(Thoreson and Stella 2000).
Drug solutions were applied after ICa appeared stable for
≥90 s. The leak conductance was assumed to be ohmic and equal to the
minimum conductance between –75 and –55 mV and then digitally
subtracted. Leak subtraction by blocking ICa with 0.1 mM
Cd2+ yields almost identical voltage profiles
(Thoreson and Stella 2000).
Recordings of ICa were corrected off-line for series
resistance.
Light stimuli were generated by a tungsten light source and reflected into the microscope condenser pathway using a beam splitter. Light intensity was controlled by neutral density filters (Wratten gel) and wavelength was controlled by interference filters. The intensity of unattenuated light measured with a laser power meter (Metrologic) was 1.3 x 106 photon s–1 µm–2 at 580 nm. A flash duration of 1 s was generally used.
Pipettes were positioned under visual control using an infrared-sensitive CCD camera (Watec 902H) attached to the trinocular head of the microscope. Rods were identified by their characteristic outer segments. Horizontal cell somas are in the most distal region of the outer nuclear layer and horizontal cells exhibit large outward light-evoked currents, a low input resistance, and large inwardly rectifying currents. OFF bipolar cells also show outward light-evoked currents, but exhibit a higher input resistance and a current/voltage profile dominated by outwardly rectifying currents. ON bipolar cells show a similar outwardly rectifying current/voltage profile but exhibit inward light-evoked currents.
The intensity required to produce a half-maximal response was determined for both 580 and 680 nm light in a sample of six cells (3 horizontal cells, 2 OFF bipolar cells, and 1 ON bipolar cell). Consistent with mixed rod and cone inputs, these cells were 1.2 ± 0.36 (n = 6) log units more sensitive to 580 than 680 nm light.
Measurement of [Ca2+]i transients
Intracellular Ca2+ changes were assessed using the ratiometric
dye, fura-2, on retinal slices (Stella et
al. 2002). Briefly, retinal slices were incubated for 45 min in
the dark with 0.5 ml of 10 µM fura-2/AM + 5 µM pluronic F-127 (Molecular
Probes, Eugene, OR), followed by an additional incubation in fura-2/AM alone
for 1.5 h in a slice chamber at 4°C.
Images were acquired with a cooled CCD camera (SensiCam, Cooke) mounted on an upright fixed stage microscope (Nikon E600FN) equipped with a x60 (1.0 N.A.) water immersion objective. Epifluorescent illumination was provided by 150 W Xe lamp (Opti-Quip) attached to a Sutter Lambda 10-2 filter wheel equipped with 340 and 380 nm interference filters and coupled to the microscope by a liquid light guide (Sutter). The camera, filter wheel, and image acquisition parameters were controlled with Axon Imaging Work-bench (AIW 2.2). Binning of images (2 x 2) was used to increase rate of acquisition. Images were subtracted for background camera noise but no averaging or masking was performed.
To activate voltage-dependent Ca2+ channels, cells were
depolarized by increasing [K+]o from 2.5 to 50 mM for 1
min. Elevated KCl applications were performed at 15-min intervals. Images were
acquired at 5- to 10-s intervals during elevated [K+]o
applications. A pseudocolor fluorescence image of rods loaded with fura-2 in
the retinal slice and their responses to high K+ is illustrated in
Stella et al. (2002). For
analysis, a region of interest was drawn over the soma and the change in
fluorescence ratio (340/380 nm) was interpreted as reflecting changes in
[Ca2+]i. The ratio change produced by elevated
[K+]o applied in the presence of the test solution was
compared with the ratio change produced in control conditions. Ratio values
for control conditions were determined by averaging the prior control and
subsequent wash responses for each drug application. Multiple cells were
analyzed in each preparation; each experiment was performed on at least three
different slice preparations. In simultaneous Ca2+ imaging and
whole-cell recording experiments, it was found that the 380-nm light flashes
used for imaging experiments often evoked light responses of ≤20 pA in rods
initially placed in the recording chamber. However, these responses diminished
quickly with repeated exposure to 340/380 ratio pairs.
Statistical analysis was performed using paired and unpaired Student's t-test (GraphPad Prism 3.0); significance was chosen at P < 0.05, and variability is reported as ± SE.
Drug solutions
CGS-21680, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA), ryanodine, and 3,7-dimethyl-l-propargylxanthine (DMPX) were obtained from Sigma Chemical (St. Louis, MO). VUF-5574, 1,3-dipropyl-8-cyclopentylxathine (DPCPX), and ZM-241385 were obtained from Tocris Cookson (Ballwin, MO). DPMA and CGS-21680 were dissolved in DMSO as 50 mM stock solutions. All other solutions were prepared as 100 mM stocks with either distilled H2O or DMSO. Superfusion with 0.1% DMSO alone was not found to affect any of the properties of ICa or Ca2+ imaging responses that we studied.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Voltage-dependent Ca2+ influx in rods has previously been shown
to be mediated by dihydropyridine (DHP)-sensitive L-type calcium channels
(Kourennyi and Barnes 2000;
Stella et al. 2002; Thoreson
et al. 1997,
2000). In this study,
ICa was recorded with 10 mM Ba2+ as the charge
carrier in rods. Depolarizing voltage ramps evoked inward currents that
activated above –50 mV, peaked near –10 mV, and reversed between
+45 and +60 mV [Fig. 1, control
current-voltage (I-V) plot]. The average peak amplitude of
ICa in rods was –356.9 ± 41.6 pA (n
= 9) and the voltage at which the current was half-maximal
(V50) averaged – 30.3 ± 1.9 mV.
|
Previous work in our laboratory showed that DPMA, an A2 receptor
agonist, inhibited ICa in rods but A1,
N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]
adenosine (R-PIA) and A3,
N6-2-(4-aminophenyl)ethyladenosine (APNEA) agonists were
without effect (Stella et al.
2002). In situ hybridization has revealed the presence
A2A receptors in the outer nuclear layer
(Kvanta et al. 1997). We
therefore tested effects of an A2A selective receptor agonist,
CGS-21680, on rod ICa. Similar to effects of DPMA
(Stella et al. 2002),
application of CGS-21680 (2 µM) caused a reduction in the peak amplitude of
ICa in rods by an average of 16.8 ± 2.4%
(n = 9, P = 0.001) without any apparent shift in the
current-voltage relationship along the voltage axis
(Fig. 1A). CGS-21680
also significantly inhibited the increases in [Ca2+]i
generated by depolarizing rods with high K+ solution by an average
of 29.3 ± 3.2% (Fig.
1B, n = 33, P < 0.001).
In some cells, we found that adenosine (50 µM) produced little or no
inhibition. This may be due in part to the presence of endogenous adenosine as
suggested by results presented later in the paper. It is also possible that
release from Ca2+ stores on depolarization
(Krizaj et al. 1999) could
diminish the magnitude of inhibitory effects of adenosine on
[Ca2+]i changes in rods. We therefore examined 10 cells
in which adenosine failed to inhibit depolarization-evoked
[Ca2+]i increases (104.8 ± 2.6%, n =
10). In these cells, following block of calcium-induced calcium release (CICR)
with ryanodine (100 µM), adenosine inhibited K+-evoked
[Ca2+]i increases by 23.5 ± 2.8% (P <
0.0001), suggesting that Ca2+ release from stores can obscure the
effects of adenosine on rod voltage-dependent Ca2+ influx.
Therefore for the antagonist studies described below, we excluded cells in
which adenosine applied in control conditions inhibited depolarization-evoked
increases in [Ca2+]i by <10%.
Effects of adenosine antagonists on depolarization-evoked [Ca2+]i increase in rods
Consistent with the hypothesis that the observed effects are mediated by
adenosine activation of A2 receptors, co-application of an
A2 selective antagonist, DMPX (50 µM), with adenosine (50 µM)
prevented adenosine from inhibiting K+-evoked
[Ca2+]i increases in rod photoreceptors (paired
t-test, n = 14, P = 0.0018). After washout of both
DMPX and adenosine, adenosine (50 µM) reversibly inhibited the
K+-evoked [Ca2+]i increase (35.1 ±
5.2%, n = 14, P < 0.0001). It has been suggested that
DMPX may have actions at both A1 and A2 receptors at the
concentration tested (Seale et al.
1988). We therefore tested DPCPX, a putatively selective
antagonist for A1 receptors, and ZM-241385, a selective antagonist
for A2A receptors. Both DPCPX and ZM-241385 diminished the ability
of adenosine to inhibit K+-evoked [Ca2+]i
increases at a concentration of 1 µM but not 100 nM
(Fig. 2). The A3
antagonist, VUF-5574, failed to prevent adenosine-mediated inhibition of the
K+-evoked [Ca2+]i increase in rods at a
concentration of 1 µM (Fig.
2), which is in agreement with previous observations showing no
effect of the A3 selective agonist, APNEA, on the voltage-dependent
Ca2+ influx into rods (Stella
et al. 2002).
|
In further support of a role for A2A receptors, the A2A selective antagonist ZM-241385 (10 µM, n = 8) eliminated inhibition of the K+-evoked Ca2+ increase produced by the A2A agonist, CGS-21680 (2 µM) (Fig. 3A). ZM-241385 applied in the presence of CGS-21680 increased the K+-evoked Ca2+ increase by 41.0 ± 14.6% above control. This augmentation of the K+-evoked Ca2+ increase is consistent with results of light response experiments presented below, suggesting that there is an endogenous source of adenosine in and around the synapse that is tonically suppressing depolarization-evoked Ca2+ influx into rods.
|
8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 µM) also antagonized the
inhibitory effect of CGS-21680 (2 µM) on the K+-evoked
Ca2+ increase (Fig.
3B). Since the A1 agonist, R-PIA, did not
inhibit either the rod ICa or the K+-evoked
Ca2+ increase (Stella et al.
2002) and since DPCPX antagonized the effects of an A2A
agonist, it seems likely that the effects of DPCPX are due to its actions on
A2 receptors rather than A1 receptors.
A2 agonists inhibit light-evoked responses of second-order neurons but not photoreceptors
A hallmark of synaptic transmission from photoreceptors to second-order
neurons is the continuous release of L-glutamate, which is
regulated by the activity of DHP-sensitive calcium channels present at the
inner segment and terminals of rods
(Schmitz and Witkovsky 1997;
Thoreson et al. 1997;
Wilkinson and Barnes 1996;
reviewed by Thoreson and Witkovsky
1999). Since inhibition of photoreceptor ICa
by DHP antagonists reduces light-evoked responses in second-order neurons
(Rieke and Schwartz 1994;
Thoreson et al. 1997), we
tested whether the A2A agonist CGS-21680, which inhibits rod
ICa (Fig.
1), similarly inhibits synaptic transmission from photoreceptors
onto second-order neurons. In the tiger salamander retina, rods are
approximately 2.2 log units more sensitive to 580 nm than 680 nm light,
whereas red-sensitive cones are approximately 0.25 log units more sensitive to
680 nm light (Makino and Dodd
1996). To assess transmitter release from rods, dark-adapted
retinal slices were stimulated with 580 nm light flashes.
Figure 4 illustrates the
voltage responses of a rod and the current responses of horizontal and bipolar
cells evoked by 580-nm light flashes before, during, and after superfusion
with CGS-21680 (2 µM). In control conditions, the peak rod light-evoked
voltage responses averaged 9.6 ± 2.4 mV (n = 6). The amplitude
of rod voltage responses was not significantly reduced by CGS-21680
(–0.1 ± 0.1 mV, n = 6, P = 0.3808). In
contrast, light-evoked currents in second-order neurons were suppressed by
application of CGS-21680. In control superfusate, horizontal cell light
responses averaged 347 ± 145.4 pA (n = 4); ON
bipolar cell responses averaged 48.5 ± 7.4 pA (n = 3) and
OFF bipolar cell responses averaged 53.1 ± 11.1 pA
(n = 4). Light-evoked currents of all three types of second-order
neurons were inhibited by CGS-21680 by an average of 35.3 ± 5.1%
(n = 20, P < 0.0001). CGS-21680 inhibited horizontal
cells by 39.3 ± 10.8% (n = 9, P = 0.0067); inhibited
OFF bipolar cells by 29.4 ± 3.9 (n = 6, P
= 0.0007), and inhibited ON bipolar cells by 35.2 ± 6.1%
(n = 5, P = 0.0047). The A2 selective agonist,
DPMA (10 µM), produced a similar 43.0 ± 5.3% inhibition of
light-evoked currents of second-order neurons (n = 17, P
< 0.0001). DPMA inhibited horizontal cells by 68.0 ± 14.5%
(n = 4, P = 0.0182), inhibited OFF bipolar cells
by 34.4 ± 5.1% (n = 5, P = 0.0025), and inhibited
ON bipolar cells by 34.9 ± 4.8% (n = 8, P
= 0.0002). The ability of both A2 receptor agonists to inhibit
light-evoked currents in all three types of second-order neurons is consistent
with the hypothesis that A2-mediated inhibition of rod
ICa in turn inhibits glutamate release from rods.
|
L-glutamate release from rods is maximal in the dark. Therefore
in darkness, inhibition of photoreceptor ICa by adenosine
should suppress tonic glutamate-activated inward currents in horizontal cells
and OFF bipolar cells and glutamate-gated outward currents in
ON bipolar cells. Consistent with this prediction, the
A2 agonists, DPMA and CGS-21680, produced a large net outward
current in horizontal cells (e.g., see the baseline shift in Figs.
4,
5, and
6). These drugs produced
smaller DC current changes in ON and OFF bipolar cells,
consistent with the smaller light-evoked currents in these cells. Light-evoked
currents of horizontal cells are larger than those of bipolar cells because
they make a far greater number of synaptic contacts with rods
(Oyster 1999). The amplitude
of the dark current change induced by the A2 agonist is correlated
with the change in the amplitude of the light response (horizontal cells
n = 7, P = 0.0002, r2 = 0.95;
OFF bipolar cells: n = 7, P = 0.001,
r2 = 0.91; ON bipolar cells: n = 8,
P = 0.0127, r2 = 0.67). Moreover, the reversal
potentials of the DC currents produced by the A2 agonists were near
0 mV (CGS-21680: 1.6 ± 6.4 mV, n = 7; DPMA: –14.4
± 9.2 mV, n = 10). These results are consistent with the
conclusion that the changes in dark current were largely due to suppression of
tonic glutamate release from photoreceptors.
|
|
Inhibitory actions of CGS-21680 on Ca2+ influx were antagonized by the A2A selective antagonist, ZM-241385 (10 µM). We likewise tested whether ZM-241385 (10 µM) could antagonize the inhibitory effect of CGS-21680 on light response of second-order neurons. Figure 5 illustrates the light-evoked current responses of horizontal and bipolar cells evoked by 580-nm light flashes before, during the application of CGS-21680 (2 µM), and in the presence of CGS-21680 and ZM-241385 (10 µM). As observed previously in Fig. 4, light-evoked currents in second-order neurons were suppressed by application of CGS-21680 (2 µM). In this set of experiments, light-evoked currents of all three types of second-order neurons were inhibited by CGS-21680 by an average of 37.3 ± 7.0% (n = 7, P = 0.0018, Fig. 5B). The application of ZM-241385 reversed the inhibition of light-evoked currents produced by CGS-21680 (Fig. 5A) (n = 7, P = 0.0156, paired t-test, Fig. 5B) and in some cases caused an enhancement (e.g., the horizontal cell shown in Fig. 5A). This is similar to the effects of CGS-21680 and ZM-241385 on K+-evoked Ca2+ increases on rods (Fig. 2). The ability of ZM-241385 to reverse the inhibition of light-evoked currents by CGS-21680 in all three types of second-order neurons is consistent with the hypothesis that A2-mediated inhibition of rod ICa in turn inhibits glutamate release from rods.
An A2A antagonist enhances light-evoked currents of second-order neurons
Since application of the A2A antagonist, ZM-241385, in the presence of CGS-21680 produced an average overall increase in both the K+-evoked Ca2+ influx into rods (Fig. 2) and the amplitude of light-evoked currents in second-order neurons (Fig. 5), it appeared likely that there is endogenous adenosine present at the rod synapse. To evaluate further the impact of endogenous adenosine on transmission from photoreceptors to second-order neurons, we tested the effects of ZM-241385 alone on light responses in photoreceptors and second-order neurons. Figure 6 shows rod voltage responses recorded in current clamp and light-evoked currents recorded under voltage clamp from various second-order neurons in the outer retina before, during, and after superfusion with ZM-241385 (10 µM). In control conditions, the rod light-evoked voltage responses averaged 11.0 ± 2.7 mV (n = 4). The amplitude of the rod voltage response was unchanged by ZM-241385, indicating that the photoresponse is not altered by antagonizing the adenosine receptors (–0.13 mV, P = 0.70). However, light-evoked currents in second-order neurons were enhanced by the application of ZM-241385 (Fig. 6). In control superfusate, horizontal cell light responses averaged 77.7 ± 47.6 pA (n = 6) and ranged from 7.1 to 280.5 pA. ON bipolar cell responses averaged 33.8 ± 7.5 pA (n = 9) and ranged from 12.4 to 87.4 pA. OFF bipolar cell responses averaged 35.4 ± 1.7 pA (n = 5) and ranged from 29.6 to 38.6 pA. In ZM-241385, light-evoked currents of second-order neurons were increased to 169.7 ± 17.4% of control conditions (n = 21, P = 0.0007, Fig. 6). Similar to CGS-21680, the antagonist ZM-241385 caused a greater shift in the DC current and larger enhancement of light responses in horizontal cells compared with ON or OFF bipolar cells (Fig. 6). Horizontal cells light-evoked currents were increased to 216.6 ± 46.8% of control conditions (n = 7, P = 0.0471). ON and OFF bipolar cells showed an enhancement of light-evoked currents over control conditions of 148.4 ± 10.2% (n = 9, P = 0.0014) and 142.2 ± 14.4% (n = 5, P = 0.0428), respectively. The ability of the A2A antagonist ZM-241386 to enhance light-evoked currents of second-order neurons implies that endogenous adenosine is present at the photoreceptor synapse and provides some tonic inhibition of synaptic transmission.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
) and rat
brain stem neurons (Umemiya and Berger
1994). In addition, this study demonstrates the impact of
endogenous adenosine on glutamate release from rods. Pharmacology of adenosine receptors in rod photoreceptors
The pharmacological profile of adenosine's actions in rods is most
consistent with the presence of an A2-like receptor. CGS-21680
(Figs. 1 and
2), the A2A
selective agonist, and DPMA, the A2 receptor agonist, were
effective at inhibiting both ICa and the
K+-evoked Ca2+ increase in rods, whereas A1
(R-PIA) and A3 (APNEA) agonists were ineffective
(Stella et al. 2002). The
A2 and A2A selective agonists also inhibited light
responses of second-order neurons (Fig.
4). Inhibitory effects of the A2A agonist, CGS-21680,
on depolarization-evoked Ca2+ increases in rods and light-evoked
currents in second-order neurons were antagonized by the A2A
antagonist, ZM-241385 (Figs. 2
and 5). Similarly, antagonists
for A2 (DMPX) and A2A (ZM-241385) adenosine receptors
prevented adenosine-mediated inhibition of voltage-dependent Ca2+
influx in rods (Fig. 3). The
A1 antagonist, DPCPX, also inhibited depolarization-evoked
[Ca2+]i increases in rods. Similar to our results, 1
µM DPCPX was also found to antagonize adenosine agonist effects on cone
myoid elongation in the fish retina (Rey
and Burnside 1999). In salamander rods, the ineffectiveness of
A1 agonists and the ability of DPCPX to antagonize the
A2A-selective agonist, CGS-21680, suggest that DPCPX is acting on
A2 receptors. Thus DPCPX appears to be less selective for
A1 receptors in nonmammalian preparations than it is in mammalian
preparations.
Both A1 and A2 receptors have been detected in the
retina. In mouse and rabbit retina [3H]-NECA,
5'-(N-ethylcarboxamido)adenosine, which labels both
A1 and A2 receptors, mainly labeled photoreceptors
(Blazynski 1990). A1
receptor agonist binding sites have been localized primarily to inner retinal
layers (Blazynski 1990),
suggesting that [3H]-NECA binding in photoreceptors was mainly due
to its affinity for A2 receptors. In situ hybridization in the rat
retina localized A1 receptor mRNA entirely in the ganglion cell
layer and A2A receptor mRNA in both the inner and the outer nuclear
layers of the rat, whereas A2B receptor mRNA was absent from the
retina (Kvanta et al. 1997).
These findings are consistent with the physiological data of the present
study, suggesting the presence of an A2-like receptor mediating
adenosine's effect at the rod synapse.
Endogenous adenosine inhibits glutamate release from rods via A2-like adenosine receptor activation
The light responses of second-order neurons are generated by the
light-modulated release of L-glutamate from photoreceptors
(Thoreson and Witkovsky 1999).
Inhibition of ICa in photoreceptors by DHP antagonists
inhibits glutamate release, thereby reducing light responses in second-order
neurons (Rieke and Schwartz
1994; Schmitz and Witkvosky 1997;
Thoreson et al. 1997).
Therefore inhibition of ICa in rods by A2
receptor activation should likewise inhibit synaptic glutamate release.
Consistent with this prediction, A2 receptor agonists significantly
inhibited light-evoked currents in second-order neurons without altering the
light-evoked voltage responses of rods
(Fig. 4). The ability of
A2 adenosine receptor agonists to reduce light responses in all
three postsynaptic cell types is consistent with a common presynaptic site of
action. Although we cannot exclude the possibility that there may be
additional postsynaptic effects, the findings that CGS-21680 and DPMA produced
dark current changes that reversed near 0 mV and were correlated in amplitude
with light-evoked currents are consistent with the hypothesis that the primary
effect of these drugs on second-order neurons is to inhibit glutamate release
from rods. Furthermore, the present findings support the hypothesis that
endogenously released adenosine can inhibit L-glutamate release by
inhibiting Ca2+ influx through ICa via
activation of an A2-like adenosine receptor. This conclusion is
supported by the following: 1) the similarity between the effects of
adenosine, DPMA, and CGS-21680 on ICa and transmitter
release from rods (Figs. 1,
2, and
4;
Stella et al. 2002);
2) the finding that the antagonist ZM-241385 not only blocked the
effects of adenosine and CGS-21680 (Figs.
2,
3, and
5) but also elicited a slight
enhancement (see Figs. 2 and
5), suggesting that the synapse
contained some basal level of adenosine; and 3) the ability of
ZM-241385 alone to enhance transmitter release
(Fig. 6), presumably by
preventing adenosine binding to the A2-like receptor on rods. In
the CNS A2 receptor activation has been shown to inhibit GABA
release (Edwards and Robertson
1999), but to our knowledge this is the first example of a synapse
at which activation of A2-like adenosine receptors appears to
inhibit glutamate release.
Sources of adenosine and adenosine's role at the photoreceptor synapse
Adenosine levels reflect an equilibrium between processes that lead to the
appearance of adenosine (e.g., formation from adenine nucleotides by
nucleotidases and efflux from transporters) and mechanisms that reduce
adenosine levels (e.g., uptake of adenosine and conversion to inosine by
adenosine deaminase) (Gu and Geiger
1992; Roth et al.
1997). Adenosine formation is linked to the balance between energy
supply and demand. In darkness, ATP is metabolized by the highly active
Na+/K+ ATPase in the photoreceptor inner segment to
counter the steady influx of Na+ entering through cyclic
nucleotidegated channels in the outer segment
(Shimazaki and Oakley 1986;
Torre 1982). Increased ATP
turnover can elevate intracellular levels of adenosine
(Poulsen and Quinn 1998) and
thus adenosine is released tonically from retinas in the dark when
photoreceptors are metabolically most active
(Blazynski and Perez 1991;
Paes de Carvalho et al. 1990;
Perez et al. 1986).
Extracellular adenosine is taken up by rod horizontal cells
(Studholme and Yazulla 1997)
and ATP is released during Ca2+ waves propagating through retinal
glial cells (Newman 2001). A
discrete population of photoreceptors in the primate retina but not rodent
retina stain for adenosine immunoreactivity
(Braas et al. 1987).
Adenosine formation may also be more directly related to neurotransmission
by the release of adenosine or adenine nucleotides together with glutamate via
exocytosis (Robertson et al.
2001). Adenine nucleotides can be catabolized into adenosine by
membrane-bound ectonucleotidases (Cunha et
al. 1998; Zimmermann and Braun
1996) and by soluble nucleotidases present in vesicles
(Todorov et al. 1997).
Consistent with vesicular exocytosis of ATP in retinal neurons, the release of
ATP from cultured retinal amacrine-like neurons is Ca2+ dependent
and sensitive to botulinum neurotoxin
(Santos et al. 1999). It seems
unlikely that extracellular ATP is converted to adenosine in significant
amounts at the rod synapse since application of 50–75 µM ATP to the
retinal slice did not inhibit ICa or depolarization-evoked
Ca2+ increases in rods (Stella
et al. 2002). However, this does not eliminate the possibility
that ADP, AMP, or adenosine might be released in vesicles. Consistent with a
Ca2+-dependent release of adenosine, K+-evoked
depolarization stimulates adenosine release from isolated retinas
(Blazynski and Perez 1991).
The present results support the hypothesis that endogenously released
adenosine in the retina can regulate transmission from rods onto second-order
neurons by activating presynaptic A2-like receptors to inhibit
Ca2+ influx through L-type ICa. Increased
adenosine levels in darkness, whether arising from synaptic or metabolic
adenosine, would therefore be expected to inhibit synaptic transmission from
rods. The inhibition of glutamate release from rods may contribute to the
neuroprotective effects of adenosine in retina
(Ghiardi et al. 1999;
Roth et al. 1997) by limiting
the potentially excitotoxic actions of glutamate at second-order neurons.
Another important feature of adenosine may lie in its ability to serve as a
brake on tonic transmitter release from rods. Adenosine is likely to act in
concert with other neuromodulators such as dopamine
(Stella and Thoreson 2000),
somatostatin (Akopian et al.
2000), nitric oxide (Kurenny
et al. 1994), pH (Barnes at al.
1993), and insulin (Stella et
al. 2000). The apparent presence of adenosine in the rod synapse
raises the possibility that it may play a role in dynamic control of synaptic
transmission under constantly changing conditions of illumination at this
critical first synapse in vision.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Present address and address for reprint requests: S. L. Stella, Jr., Neurobiology Department, University of California Los Angeles, 10833 Le Conte Blvd., Box 951763 CHS, Los Angeles, California 90095-1763 (E-mail: sstella{at}mednet.ucla.edu).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Barnes S,
Merchant V, and Mahmud F. Modulation of transmission gain by protons at
the photoreceptor output synapse. Proc Natl Acad Sci
USA 90:
10081–10085, 1993.
Blazynski C. Discrete distributions of adenosine receptors in mammalian retina. J Neurochem 54: 648–655, 1990.[ISI][Medline]
Blazynski C and Perez MT. Adenosine in vertebrate retina: localization, receptor characterization, and function. Cell Mol Neurobiol 11: 463–484, 1991.[ISI][Medline]
Braas KM,
Zarbin MA, and Snyder SH. Endogenous adenosine and adenosine receptors
localized to ganglion cells of the retina. Proc Natl Acad Sci
USA 84:
3906–3910, 1987.
Chen G and van
den Pol AN. Adenosine modulation of calcium currents and presynaptic
inhibition of GABA release in suprachiasmatic and arcuate nucleus neurons.
J Neurophysiol 77:
3035–3047, 1997.
Cunha RA, Sebastiao AM, and Ribeiro JA. Ecto-5'-nucleotidase is associated with cholinergic nerve terminals in the hippocampus but not in the cerebral cortex of the rat. J Neurochem 59: 657–66, 1992.[ISI][Medline]
Cunha RA,
Sebastiao AM, and Ribeiro JA. Inhibition by ATP of hippocampal synaptic
transmission requires localized extracellular catabolism by ecto-nucleotidases
into adenosine and channeling to adenosine A1 receptors.
J Neurosci 18:
1987–1995, 1998.
Dunwiddie TV,
Diao L, and Proctor WR. Adenine nucleotides undergo rapid, quantitative
conversion to adenosine in the extracellular space in rat hippocampus.
J Neurosci 17:
7673–7682, 1997.
Edwards FA and Robertson SJ. The function of A2 adenosine receptors in the mammalian brain: evidence for inhibition vs. enhancement of voltage gated calcium channels and neurotransmitter release. Prog Brain Res 120: 265–273, 1999.[ISI][Medline]
Ghiardi GJ, Gidday JM, and Roth S. The purine nucleoside adenosine in retinal ischemia-reperfusion injury. Vision Res 39: 2519–2535, 1999.[ISI][Medline]
Gu JG and Geiger JD. Transport and metabolism of D-[3H]adenosine and L-[3H]adenosine in rat cerebral cortical synaptoneurosomes. J Neurochem 58: 1699–1705, 1992.[ISI][Medline]
Kobayashi S,
Beitner-Johnson D, Conforti L, and Millhorn DE. Chronic hypoxia reduces
adenosine A2A receptor-mediated inhibition of calcium current in
rat PC12 cells via downregulation of protein kinase A. J
Physiol 512:
351–363, 1998.
Kobayashi S, Conforti L, and Millhorn DE. Gene expression and function of adenosine A2A receptor in the rat carotid body. Am J Physiol 279: L273–L282, 2000.
Kourennyi DE and Barnes S. Depolarization-induced calcium channel facilitation in rod
photoreceptors is independent of G-proteins and phosphorylation. J
Neurophysiol 84:
133–138, 2000.
Krizaj D, Bao
JX, Schmitz Y, Witkovsky P, and Copenhagen DR. Caffeine-sensitive calcium
stores regulate synaptic transmission from retinal rod photoreceptors.
J Neurosci 19:
7249–7261, 1999.
Kurenny DE, Moroz LL, Turner RW, Sharkey KA, and Barnes S. Modulation of ion channels in rod photoreceptors by nitric oxide. Neuron 13: 315–324, 1994.[ISI][Medline]
Kvanta A, Seregard S, Sejersen S, Kull B, and Fredholm BB. Localization of adenosine receptor messenger RNAs in the rat eye. Exp Eye Res 65: 595–602, 1997.[ISI][Medline]
Kyrozis A and Reichling DB. Perforated-patch recording with gramicidin avoids artifactual changes in intracellular chloride concentration. J Neurosci Methods 57: 27–35, 1995.[ISI][Medline]
Li J and Perl ER. ATP modulation of synaptic transmission in the spinal substantia gelatinosa. J Neurosci 15: 3357–3365, 1995.[Abstract]
Londos C,
Cooper DM, and Wolff J. Subclasses of external adenosine receptors.
Proc Natl Acad Sci USA 77:
2551–2554, 1980.
Makino CL and
Dodd RL. Multiple visual pigments in a photoreceptor of the salamander
retina. J Gen Physiol 108:
27–34, 1996.
McIntosh HH and Blazynski C. Characterization and localization of adenosine A2 receptors in bovine rod outer segments. J Neurochem 62: 992–997, 1994.[ISI][Medline]
Mogul DJ, Adams ME, and Fox AP. Differential activation of adenosine receptors decreases N-type but potentiates P-type Ca2+ current in hippocampal CA3 neurons. Neuron 10: 327–334, 1993.[ISI][Medline]
Newman EA.
Propagation of intercellular calcium waves in retinal astrocytes and Muller
cells. J Neurosci 21:
2215–2223, 2001.
Oyster CW. The Human Eye: Structure and Function. Sunderland, MA: Sinauer Associates, 1999.
Paes de Carvalho R, Braas K, Adler R, and Snyder SH. Developmental regulation of adenosine A1 receptors, uptake sites and endogenous adenosine in chick retina. Dev Brain Res 70: 87–95, 1992.[Medline]
Paes de Carvalho R, Braas KM, Snyder SH, and Adler R. Analysis of adenosine immunoreactivity, uptake, and release in purified cultures of developing chick embryo retinal neurons and photoreceptors. J Neurochem 55: 1603–11, 1990.[ISI][Medline]
Park TJ, Chung S, Han MK, Kim UH, and Kim KT. Inhibition of voltage-sensitive calcium channels by the A2A adenosine receptor in PC12 cells. J Neurochem 71: 1251–1260, 1998.[ISI][Medline]
Perez MT, Ehinger BE, Lindstrom K, and Fredholm BB. Release of endogenous and radioactive purines from the rabbit retina. Brain Res 398: 106–112, 1986.[ISI][Medline]
Poulsen SA and Quinn RJ. Adenosine receptors: new opportunities for future drugs. Bioorg Med Chem 6: 619–641, 1998.[Medline]
Rae J, Cooper K, Gates P, and Watsky M. Low access resistance perforated patch recordings using amphotericin B. J Neurosci Methods 37: 15–26, 1991.[ISI][Medline]
Ralevic V and
Burnstock G. Receptors for purines and pyrimidines. Pharmacol
Rev 50:
413–492, 1998.
Rey HL and Burnside B. Adenosine stimulates cone photoreceptor myoid elongation via an adenosine A2-like receptor. J Neurochem 72: 2345–2355, 1999.[ISI][Medline]
Rieke F and Schwartz EA. A cGMP-gated current can control exocytosis at cone synapses. Neuron 13: 863–873, 1994.[ISI][Medline]
Robertson SJ, Ennion SJ, Evans RJ, and Edwards FA. Synaptic P2X receptors. Curr Opin Neurobiol 11: 378–86, 2001.[ISI][Medline]
Roth S, Rosenbaum PS, Osinski J, Park SS, Toledano AY, Li B, and Moshfeghi AA. Ischemia induces significant changes in purine nucleoside concentration in the retina-choroid in rats. Exp Eye Res 65: 771–779, 1997.[ISI][Medline]
Santos PF, Caramelo OL, Carvalho AP, and Duarte CB. Characterization of ATP release from cultures enriched in cholinergic amacrine-like neurons. J Neurobiol 41: 340–348, 1999.[ISI][Medline]
Schmitz Y and Witkovsky P. Dependence of photoreceptor glutamate release on a dihydropyridine-sensitive calcium channel. Neuroscience 78: 1209–1216, 1997.[ISI][Medline]
Seale TW, Abla KA, Shamim MT, Carney JM, and Daly JW. 3,7-Dimethyl-1-propargylxanthine: a potent and selective in vivo antagonist of adenosine analogs. Life Sci 43: 1671–1684, 1988.[ISI][Medline]
Sebastiao AM and Ribeiro JA. Adenosine A2 receptor-mediated excitatory actions on the nervous system. Prog Neurobiol 48: 167–189, 1996.[ISI][Medline]
Shimazaki H and
Oakley B. Decline of electrogenic Na+/K+ pump
activity in rod photoreceptors during maintained illumination. J
Gen Physiol 87:
633–647, 1986.
Stella SL,
Bryson EJ, and Thoreson WB. A2 adenosine receptors inhibit calcium influx
through L-type calcium channels in rod photoreceptors of the salamander
retina. J Neurophysiol 87:
351–360, 2002.
Stella SL and Thoreson WB. Differential modulation of rod and cone calcium currents in tiger salamander retina by D2 dopamine receptors and cAMP. Eur J Neurosci 12: 3537–3548, 2000.[ISI][Medline]
Stella SL Jr, Bryson EJ, and Thoreson WB. Insulin inhibits voltage-dependent calcium influx into rod photoreceptors. Neuroreport 12: 947–951, 2001.[ISI][Medline]
Studholme KM and Yazulla S. 3H-adenosine uptake selectively labels rod horizontal cells in goldfish retina. Vis Neurosci 14: 207–212, 1997.[ISI][Medline]
Thoreson WB,
Nitzan R, and Miller RF. Reducing extracellular Cl–
suppresses dihydropyridine-sensitive Ca2+ currents and synaptic
transmission in amphibian photoreceptors. J
Neurophysiol 77:
2175–2190, 1997.
Thoreson WB and Stella SL Jr. Anion modulation of calcium current voltage dependence and amplitude in salamander rods. Biochim Biophys Acta 1464: 142–150, 2000.[Medline]
Thoreson WB and Witkovsky P. Glutamate receptors and circuits in the vertebrate retina. Prog Retin Eye Res 18: 765–810, 1999.[ISI][Medline]
Thorn JA and Jarvis SM. Adenosine transporters. Gen Pharmacol 27: 613–620, 1996.[ISI][Medline]
Todorov LD, Mihaylova-Todorova S, Westfall TD, Sneddon P, Kennedy C, Bjur RA, and Westfall DP. Neuronal release of soluble nucleotidases and their role in neurotransmitter inactivation. Nature 387: 76–79, 1997.[Medline]
Torre V.
The contribution of the electrogenic sodium-potassium pump to the electrical
activity of toad rods. J Physiol
333: 315–341,
1982.
Trombley PQ and Westbrook GL. L-AP4 inhibits calcium currents and synaptic transmission via a G-protein-coupled glutamate receptor. J Neurosci 12: 2043–2050, 1992.[Abstract]
Umemiya M and Berger AJ. Activation of adenosine A1 and A2 receptors differentially modulates calcium channels and glycinergic synaptic transmission in rat brainstem. Neuron 13: 1439–1446, 1994.[ISI][Medline]
van Calker D, Muller M, and Hamprecht B. Adenosine regulates via two different types of receptors, the accumulation of cyclic AMP in cultured brain cells. J Neurochem 33: 999–1005, 1979.[ISI][Medline]
Werblin FS.
Transmission along and between rods in the tiger salamander retina.
J Physiol 280:
449–470, 1978.
Wilkinson MF and Barnes S. The dihydropyridine-sensitive calcium channel subtype in
cone photoreceptors. J Gen Physiol
107: 621–630,
1996.
Wu SM. Synaptic connections between neurons in living slices of the larval tiger salamander retina. J Neurosci Methods 20: 139–149, 1987.[ISI][Medline]
Zimmermann H and Braun N. Extracellular metabolism of nucleotides in the nervous system. J Auton Pharmacol 16: 397–400, 1996.[ISI][Medline]
This article has been cited by other articles:
