J Neurophysiol
87: 351–360, 2002; 10.1152/jn.00010.2001.
A2 Adenosine Receptors Inhibit Calcium Influx Through L-Type Calcium
Channels in Rod Photoreceptors of the Salamander Retina
SALVATORE L. STELLA JR., ERIC J. BRYSON, AND WALLACE B. THORESON
Department of Pharmacology and Department of Ophthalmology, University of Nebraska Medical Center,
Omaha, Nebraska 68198-5540
Received 8 January 2001; accepted in final form 23 August 2001
Adenosine is a potent neuromodulatory substance that is
widely distributed throughout the CNS. Adenosine (or P1)
receptors are divided into four known molecular and pharmacological subtypes: A1, A2A, A2B, and A3 receptors (Ralevic
and Burnstock 1998). Adenosine is well characterized as an
inhibitory transmitter in the CNS (Ribeiro 1995), but excitatory
actions of adenosine have also been observed (Sebastiao and
Ribeiro 1996). Inhibitory actions of adenosine are commonly
mediated by the A1 receptor that couples negatively to adenylyl
cyclase via a Gi protein to reduce cAMP levels in the cell
(Dolphin et al. 1986; Ralevic and Burnstock 1998; van Calker
et al. 1979). A2 receptors have been considered to mediate
excitatory effects in the brain because they couple positively to
adenylyl cyclase through a Gs protein to increase cAMP levels
(Ralevic and Burnstock 1998; Sebastiao and Ribeiro 1996; van
Calker et al. 1979). However, synaptic activation of A2 receptors is not always associated with an increase in ICa or trans-
mitter release. A2 receptor activation enhances P-type but not
other Ca2⫹ channel subtypes in neurons (Satoh et al. 1997;
Umemiya and Berger 1994). Adenosine acting at A2 receptors
has been shown to inhibit both N- and L-type Ca2⫹ channels in
PC12 cells via stimulation of cAMP and protein kinase A
(PKA) (Kobayashi et al. 1998; Park et al. 1998). A2 receptor
activation also inhibits ICa, and thus GABA release in suprachiasmatic and arcuate nuclei neurons (Chen and van den Pol
1997). It has thus been suggested that the increased excitability
associated with A2 receptor activation arises from inhibition of
ICa and the resulting decrease in release of inhibitory transmitter (reviewed in Edwards and Robertson 1999).
Adenosine is released in darkness from the retina (Blazynski
and Perez 1991; Paes de Carvalho et al. 1990; Perez et al.
1986). Adenosine is present in human photoreceptors (Braas et
al. 1987), and [3H]adenosine is taken up into photoreceptors
and horizontal cells via transporters for adenosine (Paes de
Carvalho et al. 1990; Studholme and Yazulla 1997), suggesting
that adenosine is removed from the extracellular space following release. The presence of A2 receptors in the outer retina and
particularly photoreceptors has been demonstrated by autoradiography and in situ hybridization (Blazynski 1990; Kvanta et
al. 1997). Adenosine acting on A2 receptors stimulates melatonin synthesis (Valenciano et al. 1998) and myoid elongation
in cone photoreceptors (Rey and Burnside 1999), providing
evidence for a physiological role for A2 receptors on photoreceptors.
Rod photoreceptors use dihydropyridine (DHP)-sensitive Ltype Ca2⫹ channels to control release of the neurotransmitter,
L-glutamate (Ayoub and Copenhagen 1991; Copenhagen and
Jahr 1989; Corey et al. 1984; Kourennyi and Barnes 2000;
Schmitz and Witkovsky 1997). Photoreceptor ICa can be modulated by various ions (H⫹, cations, anions) (Barnes et al.
1993; Piccolino et al. 1996; Thoreson et al. 1997, 2000) and
neurotransmitters (dopamine, somatostatin, and nitric oxide)
(Akopian et al. 2000; Kurenny et al. 1994; Stella and Thoreson
2000). Barnes and Hille (1989) found that adenosine inhibits
the Ca2⫹-activated chloride current in cones, but the actions of
adenosine on photoreceptor ICa have not previously been examined.
The purpose of the present study was to examine the effect
of adenosine on ICa and Ca2⫹ influx in rod photoreceptors.
Address for reprint requests: W. B. Thoreson, Ophthalmology Dept., University of Nebraska Medical Center, 985540 Nebraska Medical Center,
Omaha, NE 68198-5540 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
INTRODUCTION
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0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
351
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Stella, Salvatore L. Jr., Eric J. Bryson, and Wallace B. Thoreson.
A2 adenosine receptors inhibit calcium influx through L-type calcium
channels in rod photoreceptors of the salamander retina. J Neurophysiol 87: 351–360, 2002; 10.1152/jn.00010.2001. Presynaptic inhibition is a major mechanism for regulating synaptic transmission in
the CNS and adenosine inhibits Ca2⫹ currents (ICa) to reduce transmitter release at several synapses. Rod photoreceptors possess L-type
Ca2⫹ channels that regulate the release of L-glutamate. In the retina,
adenosine is released in the dark when L-glutamate release is maximal.
We tested whether adenosine inhibits ICa and intracellular Ca2⫹
increases in rod photoreceptors in retinal slice and isolated cell preparations. Adenosine inhibited both ICa and the [Ca2⫹]i increase evoked by
depolarization in a dose-dependent manner with ⬃25% inhibition at 50
␮M. An A2-selective agonist, (N6-[2-(3,5-dimethoxyphenyl)-2-(2methylphenyl)-ethyl]adenosine) (DPMA), but not the A1- or A3-selective agonists, (R)-N6-(1-methyl-2-phenylethyl)adenosine and N6-2-(4aminophenyl)ethyladenosine, also inhibited ICa and depolarizationinduced [Ca2⫹]i increases. An inhibitor of protein kinase A (PKA),
Rp-cAMPS, blocked the effects of DPMA on both ICa and the depolarization-evoked [Ca2⫹]i increase in rods. The results suggest that
activation of A2 receptors stimulates PKA to inhibit L-type Ca2⫹
channels in rods resulting in a decreased Ca2⫹ influx that should
suppress glutamate release.
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S. L. STELLA, E. J. BRYSON, AND W. B. THORESON
METHODS
Tissue preparation
Larval tiger salamanders (Ambystoma tigrinum, Kons, Germantown, 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 ⫻ 5 mm, Type GS,
0.2 ␮M pores). After the retina adhered to the filter paper, the sclera,
choroid, and retinal pigment epithelium were removed under chilled
amphibian superfusate. The isolated retina was then cut into 100- to
150-␮M 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 (⫻40, 0.7 NA or ⫻60, 1.0 NA)
and viewed on an upright fixed stage microscope (Olympus BHWI or
Nikon E600FN). All procedures were performed under dim light or
under infrared illumination using Gen III image intensifiers (Nitemate
NavIII, Litton Industries).
Solitary retinal neurons were prepared by isolating retina from the
eyecup and finely mincing it using half of a double-edged razor blade.
The minced retina was then gently triturated in amphibian superfusate
with a large-bore fire-polished Pasteur pipette. Isolated cells were
plated on slides coated with a salamander-specific antibody, Sal-1
(kindly provided by Peter MacLeish). Prior to beginning an experiment, cells were allowed to adhere to the slides for 15 min at 4°C.
Solutions and perfusion
Solutions were applied by a single-pass, gravity-fed perfusion system that delivered medium to the slice chamber (chamber volume:
⬃0.5 ml) at a rate of 1.0 ml/min. The normal amphibian superfusate
that bathed the slices contained (in mM) 111 NaCl, 2.5 KCl, 1.8
CaCl2, 0.5 MgCl2, 10 N-2-hydroxyethylpiperazine-N⬘ 2-ethanesulfonic acid (HEPES), and 5 glucose. After obtaining a photoreceptor
recording, the superfusate was switched to a Ba2⫹ solution to enhance
Ca2⫹ currents. The Ba2⫹ solution contained (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.
J Neurophysiol • VOL
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 ⬃1 ␮m OD with tip resistances of 10 –15 M⍀.
Pipettes were filled with a solution containing (in mM) 54 CsCl,
61.5 CsCH3SO4, 3.5 NaCH3SO4, and 10 HEPES. The pH was adjusted to 7.2 with CsOH 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 of ICa, we used the perforated patch
method of whole cell recording with the pore forming antibiotic,
nystatin (Rae et al. 1991). Nystatin was dissolved in dimethylsulfoxide (DMSO) at a concentration of 120 mg/ml and then diluted into the
pipette electrolyte solution to achieve a final concentration of 480
␮g/ml. The final working solution was vortexed vigorously for 20 –30
s and stored in the refrigerator. Fresh antibiotic solutions were made
every 3 h. In successful recordings, seals ⬎1 G⍀ were obtained in
ⱕ30 s and cells were usually fully perforated within 5 min of sealing.
Rods were identified by their long, rod-shaped outer segments. For
electrophysiology, electrode placement was performed under infrared
illumination using either Gen III image intensifiers mounted over the
microscope eyepieces or with an infrared-sensitive video camera
mounted on the trinocular head of the microscope.
The input resistance (Rin) of rods in the slice averaged 659.3 ⫾ 48.9
(SE) M⍀ (n ⫽ 22). Rin of isolated rods averaged 2.4 ⫾ 0.3 G⍀ (n ⫽
5). Access resistance, clamp speed, and membrane capacitance were
measured by analyzing capacitive transients evoked by hyperpolarizing steps from a holding potential of ⫺70 mV. The time constant of
the capacitative transient indicated a voltage-clamp speed of 0.97 ⫾
0.07 ms and a membrane capacitance for rods in the slice of 36.5 ⫾
3.7 pF (n ⫽ 22). For isolated rods, the clamp speed and capacitance
determined from the capacitative transient averaged 2.4 ⫾ 0.42 ms
and 13.5 ⫾ 1.8 pF (n ⫽ 5), respectively. Access resistance was
typically between 22 and 30 M⍀; recordings were considered acceptable only when the access resistance was ⬍40 M⍀. Estimates of the
voltage errors introduced by the access resistance are included in the
legends of figures showing examples of ICa.
Once whole cell access was achieved, light responses were obtained
to determine the spectral sensitivity of the cell. Light-evoked responses in rods were generated by light from a tungsten light source
that passed through a filter wheel containing interference filters of four
different wavelengths (380, 480, 580, and 680 nm). The light stimulus
was reflected into the microscope condenser using a beam splitter.
Light intensity was controlled by neutral density filters (Wratten gel).
The intensity of unattenuated light measurement with a laser power
meter (Metrologic) was ⬃1.1 ⫻ 106 photon s⫺1 ␮m⫺2 at 680 nm,
1.3 ⫻ 106 photon s⫺1 ␮m⫺2 at 580 nm, and 2.1 ⫻ 105 photon s⫺1
␮m⫺2 at 480 nm (the power meter was not accurate at 380 nm).
Generally, a flash of 1-s duration was used. The protocol for identifying the spectral sensitivity of a single photoreceptor involved successive flashes of increasing intensity (usually encompassing 2 log
units of intensity) at the four wavelengths.
After superfusion with the 10 mM BaCl2 solution was started,
voltage ramps (0.5 mV/ms) from ⫺90 to ⫹60 mV were used to assess
ICa every 30 s. Voltage ramps yield a similar current-voltage profile as
a step protocol but cause less rundown or inactivation of the current
and provide more data points for fitting for analysis (Stella and
Thoreson 2000). Drug solutions were applied after ICa appeared stable
for ⱖ90 s.
Cells were voltage clamped at ⫺70 mV using an Axopatch 200B
patch-clamp amplifier (Axon Instruments, Foster City, CA). Currents
were acquired and analyzed using Clampex 7.0 software (Axon Instruments). The leak conductance was assumed to be ohmic and equal
to the minimum conductance between ⫺75 and ⫺55 mV and then
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Driven by evidence that adenosine inhibits ICa in many other
preparations, adenosine is released from retinal neurons in the
dark, A2 receptors are located on photoreceptors, and an uptake
system for adenosine is present at the photoreceptor synapse,
we hypothesized that adenosine acting on A2 receptors may
inhibit ICa and thus decrease Ca2⫹ influx into rods, which
would in turn reduce transmitter release. Whole cell perforated
patch-clamp recordings and Ca2⫹-imaging experiments with
fura-2 were performed on rods in retinal slice and isolated cell
preparations. Our results indicate that adenosine inhibits both
ICa and the depolarization-evoked Ca2⫹ influx in a dose-dependent manner and that this inhibition is mediated by A2
receptor activation coupled to a PKA pathway. To our knowledge, this is the first report of inhibition of presynaptic ICa by
A2 receptors at a glutamatergic synapse. Abstracts describing
some of these results have previously been presented (Stella
and Thoreson 1999; Stella et al. 1999)
ADENOSINE REDUCES ROD CALCIUM INFLUX THROUGH ICa
353
digitally subtracted. Leak subtraction by blocking ICa with 0.1 mM
Cd2⫹ yields almost identical voltage profiles.
Measurement of [Ca2⫹]i transients
FIG. 1. Rod photoreceptors contain L-type ICa. A rod photoreceptor in a
retinal slice was held at ⫺70 mV and ramped from ⫺90 to ⫹60 mV at 0.5
mV/ms. The graph shows overlaid current-voltage relationships of voltage
ramps in control superfusate (thin trace) followed by perfusion with Bay K
8644 (1 ␮M, thick trace) and then nisoldipine (5 ␮M, dashed trace). Bay 8644
(1 ␮M) enhanced the peak ICa without shifting the I-V relationship along the
voltage axis. Nisoldipine (5 ␮M) suppressed ICa and shifted the I-V relationship to more positive potentials. Voltage error at the peak of the current due to
uncompensated access resistance was estimated to be ⫺5.4 mV for control, ⫺8
mV for Bay K 8644, and ⫺2.4 mV for nisoldipine.
J Neurophysiol • VOL
FIG. 2. Adenosine (50 ␮M) inhibited rod ICa. A: the graph shows I-V
relationships of voltage ramps obtained in control superfusate (thin trace) and
in the presence of adenosine (50 ␮M, thick trace). Voltage error at the peak of
the current due to uncompensated access resistance was estimated to be ⫺14
mV in control and ⫺11.5 mV with adenosine. B: time course of adenosineinduced changes in the amplitude of ICa in a rod. Voltage ramps in A were
obtained at time points 1 (control) and 2 (adenosine). The voltage was ramped
from ⫺90 to ⫹60 mV at 0.5 mV/ms.
Drug solutions
Adenosine, ATP, (R)-N6-(1-methyl-2-phenylethyl)adenosine (RPIA), N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA), N6-2-(4-aminophenyl)ethyladenosine (APNEA), and
Rp-adenosine 3⬘,5⬘-cyclic monophosphothioate triethylamine (RpcAMPS) were obtained from Sigma Chemical (St. Louis, MO). Solutions containing (⫺)Bay K 8644 (Research Biochemicals International) or nisoldipine (Zeneca Pharmaceuticals) were prepared by
diluting 10,000⫻ DMSO stock solutions into the superfusate. R-PIA,
DPMA, and APNEA stocks were dissolved in DMSO as 50 mM stock
solutions. All other stock solutions (1,000⫻) were prepared in distilled water or superfusate. Superfusion with 0.1% DMSO alone did
FIG. 3. Effect of adenosine on rod ICa as a function of concentration. Data
values were obtained from peak amplitude measurements of ICa from voltage
ramps. Amplitudes of the currents obtained in test solutions were normalized
to currents obtained in the control solution (Itest/Icontrol). Asterisks denote
significant changes with respect to control (P ⬍ 0.05). Each point represents
the mean ⫾ SE. Number of experiments is shown in parentheses. *, P ⬍ 0.05
compared with control. Adenosine concentrations: 1 ␮M: ⫺11.3 ⫾ 3.8%, n ⫽
3, P ⫽ 0.098; 10 ␮M: ⫺13.5 ⫾ 5.4%, n ⫽ 4, P ⫽ 0.089; 25 ␮M: ⫺20.6 ⫾
3.7%, n ⫽ 5, P ⫽ 0.005; 50 ␮M: ⫺22.8 ⫾ 3.3%, n ⫽ 9, P ⫽ 0.001.
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Intracellular Ca2⫹ changes were assessed using the ratiometric dye
fura-2 (Grynkiewicz et al. 1985). Retinal slices were incubated for 45
min in the dark with 0.5 ml of 10 ␮M fura-2/AM ⫹0.02% pluronic
F-127 (Molecular Probes, Eugene, OR) in a slice chamber at 4°C. This
was followed by an additional incubation in fura-2/AM alone for
1.5 h.
Digital fluorescent images were recorded with a cooled CCD camera (SensiCam, Cooke) on an upright fixed stage microscope (Nikon
E600FN) equipped with a ⫻60 (1.0 NA) water-immersion objective.
A 150-W Xe bulb (Opti-Quip) was mounted on a Sutter Lambda 10 –2
filter wheel with 340- and 380-nm interference filters and coupled to
the microscope by a liquid light guide (Sutter). The 380-nm intensity
was attenuated with a 0.5-neutral density filter to balance the intensity
of emissions evoked by 340 and 380 nm. The fluorescence emitted by
the cells on stimulation with 340- or 380-nm light was filtered through
a 510 ⫾ 20-nm band-pass emission filter. Axon Imaging Workbench
(AIW 2.2) was used to control the camera, filter wheel, and image
acquisition. Pixel binning (2 ⫻ 2) of the images was used to decrease
acquisition rate (acquisition time: 0.5–1 s). 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 to reduce any Ca2⫹dependent inactivation. Images were acquired at 5- to 10-s intervals
during elevated [K⫹]o applications. All experiments were performed
at room temperature. For analysis, a region of interest was drawn over
the rod inner segment and a change in the fluorescence ratio (340
nm/380 nm) was interpreted as reflecting changes in [Ca2⫹]i. The
ratio change produced by the elevated [K⫹]o application in the test
solution was compared with the ratio changed 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 could be analyzed in each preparation;
each experiment was performed on at least three different slice preparations.
Statistical analysis was performed using paired and unpaired Student’s t-test (GraphPad Prism 3.0). Significance was chosen as P ⬍
0.05, and variance is reported as ⫾SE.
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ADENOSINE REDUCES ROD CALCIUM INFLUX THROUGH ICa
not affect any of the properties of ICa- or Ca2⫹-imaging responses that
we studied.
RESULTS
Characterization of L-type calcium currents in rod
photoreceptors
Adenosine inhibits rod ICa
The effect of adenosine on rod ICa is shown in Fig. 2. Figure
2A illustrates current-voltage profiles of ICa in control conditions (Fig. 2B, 1) and in the presence of adenosine (Fig. 2B, 2).
Figure 2B plots the amplitude of ICa measured with voltage
ramps every 30 s. As illustrated in Fig. 2A, adenosine (50 ␮M)
inhibited ICa in rods without shifting the current-voltage relationship along the voltage axis. The mean reduction in ICa by
50 ␮M adenosine was 22.8 ⫾ 3.3% (n ⫽ 9, P ⫽ 0.0001). All
of the rods tested at this concentration displayed a similar
degree of inhibition in the presence of adenosine. Typically,
inhibition of the rod ICa began within 30 to 60 s, the time
required for complete solution exchange in the recording
chamber. Full inhibition was observed after 2 min, and currents
recovered after ⬃5 min washout of adenosine (Fig. 2B). As the
concentration of adenosine was increased from 1 to 50 ␮M, a
significant inhibition of rod ICa was seen at concentrations 10
␮M, implying that there is a concentration-dependent effect of
adenosine on rod ICa (Fig. 3).
To test whether an intermediary cell type might be involved
in adenosine modulation of rod ICa, solitary rods were mechanically isolated by gentle trituration and tested with 50 ␮M
adenosine. As in retinal slices, adenosine significantly inhibited
ICa in isolated rods by 19.0 ⫾ 5.5% (n ⫽ 5, P ⫽ 0.026),
suggesting that adenosine acts directly on rod photoreceptors to
modulate ICa.
Adenosine reduces depolarization-evoked Ca2⫹influx in rods
Ca2⫹-imaging experiments were performed on retinal slices
to test whether adenosine reduced depolarization-evoked
[Ca2⫹]i increases in rod photoreceptors. Slices were incubated
with fura-2/AM. Rod were depolarized by elevating [K⫹]o
from 2.5 to 50 mM for 1 min. Figure 4A shows a brightfield
Nomarski image of rod photoreceptors in the retinal slice.
Corresponding paired pseudocolor images of fluorescence
from the same cells are displayed in Fig. 4B (340, 380, and
340/380 nm), showing cells in both normal (2.5 mM) and
elevated (50 mM) [K⫹]o amphibian superfusate. Figure 4C
shows a plot of the [Ca2⫹]i response measured in a rod photoreceptor before, during, and after the application of adenosine (50 ␮M). Adenosine produced a reversible inhibition of
the depolarization-evoked [Ca2⫹]i increase. On average, adenosine (50 ␮M) caused a 25.9 ⫾ 3.4% (n ⫽ 14, P ⬍ 0.0001)
reduction in the K⫹-evoked 340/380 ratio change in rod photoreceptors in the slice. Significant inhibition of the K⫹-evoked
[Ca2⫹]i increase was seen at concentrations as low as 1 ␮M
(4.9 ⫾ 1.4%, n ⫽ 11, P ⫽ 0.005), and inhibition increased in a
dose-dependent fashion with concentrations ⱕ50 ␮M (Fig. 5).
A2 receptor agonists inhibit rod ICa and Ca2⫹ influx
Adenosine can interact with P2 (purinergic) receptors (Ralevic and Burnstock 1998) that have been shown to modulate
Ca2⫹ channels in various neurons (Brown et al. 2000; Dave
and Mogul 1996). To test whether a P2 receptor may be
involved in the modulation of voltage-gated Ca2⫹ channels in
rods, ATP was bath applied to rod photoreceptors in the retinal
slice preparation and examined with perforated patch recording
of rod ICa and [Ca2⫹]i imaging using fura 2. ATP did not
significantly inhibit ICa (50 ␮M ATP: ⫹7.1 ⫾ 11.3%, n ⫽ 7,
P ⫽ 0.549) or the K⫹-evoked Ca2⫹ increase in rod photoreceptors (75 ␮M ATP: ⫹11.9 ⫾ 7.3%, n ⫽ 15, P ⫽ 0.1296),
suggesting that P2 receptors do not mediate the observed
inhibition by adenosine.
The absence of an ATP effect suggests that adenosine acts at
an adenosine receptor. There are four major subtypes of adenosine receptors: A1, A2A, A2B, and A3. R-PIA is an A1-selective receptor agonist, DPMA is an A2-selective receptor agonist that interacts with both A2A and A2B receptors, and
APNEA is an A3-selective receptor agonist (Bridges et al.
1988; Ralevic and Burnstock 1998). To characterize the aden-
FIG. 4. A: a brightfield Nomarski image of rod photoreceptors in the retinal slice of a tiger salamander retinal slice.
B: corresponding paired pseudocolor images of fluorescence from rods are shown in A to illustrate the quality of fura-2 dye loading
and the resolution of images obtained in the slice preparation. Images of fura-2 fluorescence emissions are displayed in normal (2.5
mM) and elevated (50 mM) [K⫹]o amphibian superfusate for 340, 380, and the 340 nm/380 nm ratio. C: effects of adenosine on
depolarization-evoked [Ca2⫹]i increases in photoreceptor inner segments produced by a 1-min application of elevated (50 mM)
[K⫹]o.
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We will refer to the inward current evoked by a depolarizing
voltage ramp in rods in the presence of 10 mM Ba2⫹ as ICa. In
the presence of 10 mM Ba2⫹, a slowly developing U-shaped
current-voltage relationship appeared above ⫺50 mV that
peaked between ⫺25 and ⫺10 mV and then diminished at
more positive potentials. The current typically reversed between ⫹50 and ⫹60 mV. The peak amplitude of ICa in rods
averaged ⫺371.7 ⫾ 31.7 pA (n ⫽ 23) and the voltage at which
the current was half-maximal (V50) averaged ⫺31.0 ⫾ 1.6 mV
(n ⫽ 23).
Figure 1 illustrates an example of the effects of the DHP
agonist, Bay K 8644 (1 ␮M), and the DHP antagonist, nisoldipine (5 ␮M), on ICa recorded in a rod. In agreement with
other studies on photoreceptors, ICa was enhanced by Bay K
8644 and incompletely blocked by a high concentration of
nisoldipine (Kourennyi and Barnes 2000; Wilkinson and
Barnes 1996). In addition to suppressing the peak current,
nisoldipine produced a positive shift in the current/voltage
relationship for ICa similar to that shown by Wilkinson and
Barnes for cone ICa (Wilkinson and Barnes 1996). Three millivolts of the rightward shift shown in Fig. 1 can be accounted
for by a decrease in ICa flowing across the access resistance.
The remainder of this positive shift may arise from the more
potent block of L-type ICa by nisoldipine at more negative
potentials (Albillos et al. 1994). The residual ICa in photoreceptors recorded in the presence of nisoldipine has been shown
to arise largely from unblocked L-type Ca2⫹ channels and not
from a second channel type (Kourennyi and Barnes 2000;
Wilkinson and Barnes 1996).
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S. L. STELLA, E. J. BRYSON, AND W. B. THORESON
osine receptor subtype that mediates inhibition of ICa, we tested
the effects of these three agonists on the rod ICa. As shown in
Fig. 6B, only the A2-selective agonist, DPMA (2 ␮M), significantly inhibited the rod ICa. Like adenosine, DPMA did not
cause a significant shift in the current-voltage relationship
along the voltage axis (Fig. 6B, mean shift in V50 ⫽ ⫹0.8 ⫾
0.63 mV, n ⫽ 6, P ⫽ 0.1563). The A1-selective agonist, R-PIA
(2 ␮M), and A3-selective agonist, APNEA (2 ␮M), did not
significantly inhibit rod ICa (Fig. 6, A and C). Adenosine
receptor agonists were tested at 2 or 10 ␮M. Figure 6D illustrates the overall results with R-PIA (2 ␮M), DPMA (2 ␮M),
and APNEA (10 ␮M) on rod ICa. DPMA at concentrations of
2 and 10 ␮M inhibited ICa in rods by 15.1 ⫾ 4.7% (n ⫽ 11,
P ⬍ 0.0001) and 25.0 ⫾ 5.8% (n ⫽ 3, P ⫽ 0.05), respectively.
However, neither R-PIA nor APNEA had any effect on the rod
ICa at any of the concentrations tested (R-PIA, 2 ␮M: ⫺0.5 ⫾
4.7%, n ⫽ 6, P ⫽ 0.92; APNEA, 2 ␮M: 0.0 ⫾ 3.5%, n ⫽ 5,
P ⫽ 1.0; APNEA, 10 ␮M: ⫹2.4 ⫾ 5.8%, n ⫽ 7, P ⫽ 0.6982).
These results suggest that the effects of adenosine on rod ICa
are mediated by A2 receptors.
Using Ca2⫹ imaging techniques, we tested the effects of the
same three adenosine receptor agonists on depolarizationevoked [Ca2⫹]i increases. Figure 7A displays responses to a
series of K⫹-evoked depolarizations measured in the inner
segment of a rod photoreceptor in the presence of R-PIA (10
␮M), DPMA (10 ␮M), and APNEA (10 ␮M). Only the A2selective agonist, DPMA, inhibited the depolarization-evoked
[Ca2⫹]i increase in rods. Figure 7B shows the overall results of
adenosine receptor agonists on the depolarization-induced
[Ca2⫹]i increases in rods. In agreement with the effects of these
agonists on rod ICa, DPMA (10 ␮M) reduced the K⫹-evoked
[Ca2⫹]i increase in rods by 30.9 ⫾ 4.9% (n ⫽ 12, P ⬍ 0.0001),
while R-PIA (10 ␮M) and APNEA (10 ␮M) did not significantly alter the K⫹-evoked [Ca2⫹]i increase (R-PIA, ⫹3.0 ⫾
3.3%, n ⫽ 12, P ⫽ 0.355; APNEA, ⫺4.4 ⫾ 2.7%, n ⫽ 12, P ⫽
0.129).
A2 receptors inhibit rod ICa and Ca2⫹ influx through a PKAdependent pathway
The primary signaling mechanism by which A2 receptors
transduce their signals intracellularly is to couple positively to
J Neurophysiol • VOL
DISCUSSION
The present study indicates that activation of A2 receptors by
adenosine stimulates PKA activity, which in turn inhibits voltage-gated L-type Ca2⫹ channels in rod photoreceptors. Similar
concentrations of adenosine also produced a comparable inhibition of depolarization-evoked [Ca2⫹]i increases. Adenosine
released in the retina in darkness (Blazynski and Perez 1991;
Paes de Carvalho et al. 1990; Perez et al. 1986) may therefore
inhibit ICa and reduce Ca2⫹ entry into rods that would likely
inhibit their release of glutamate.
Adenosine receptor pharmacology and signaling pathways
Purine and pyrimidine receptors are divided into two large
families, P1 (or adenosine) receptors and P2 (or purinergic)
receptors (for review, Ralevic and Burnstock 1998). ATP
(50 –75 ␮M) had no effect on the ICa or the depolarizationevoked [Ca2⫹]i increase in rods, indicating that P2 receptors
are unlikely to contribute to the inhibition produced by adenosine. Adenosine receptor subtypes were classified originally
by their effects on adenylyl cyclase activity: A1 receptors
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⫹
2⫹
FIG. 5. Concentration-dependent inhibition of the K -evoked [Ca ]i increase in rods from retinal slice preparations by adenosine. Fura-2-loaded
slices were stimulated with elevated [K⫹]o in the presence of different concentrations of adenosine (1–50 ␮M). Inhibition of K⫹-evoked ratio change
produced by adenosine were normalized to the average of the initial control
and the subsequent recovery following the test solution. Each point represents
the mean ⫾ SE. Number of experiments is shown in parentheses. *, P ⬍ 0.05
compared with control. Adenosine concentrations: 1 ␮M: ⫺4.9 ⫾ 1.3%, n ⫽
11, P ⫽ 0.005; 10 ␮M: ⫺14.1 ⫾ 3.2%, n ⫽ 8, P ⫽ 0.003; 25 ␮M: ⫺12.3 ⫾
3.1%, n ⫽ 12, P ⫽ 0.002; 50 ␮M: ⫺25.9 ⫾ 3.4%, n ⫽ 14, P ⬍ 0.0001.
adenylyl cyclase and thus stimulate the production of cAMP,
which in turn activates PKA to phosphorylate various proteins
that can include Ca2⫹ channels subunits (Alexander et al.
1994; De Jongh et al. 1996; Gubitz et al. 1996; Mogul et al.
1993). Similar to the effects of the A2 receptor agonist DPMA,
stimulation of cAMP production with forskolin or activation of
PKA with Sp-cAMPS inhibits ICa in rods (Stella and Thoreson
2000). We therefore tested whether Rp-cAMPS, a cell-permeant analogue of cAMP known to inhibit PKA activity (Chik
et al. 1997; Dolphin 1995), could block the effects of DPMA.
Rp-cAMPS (10 ␮M) was applied to retinal slices prior to
application of DPMA (2 ␮M). As illustrated in Fig. 8A, DPMA
did not inhibit rod ICa while in the presence of Rp-cAMPS.
Figure 8B shows current-voltage profiles of rod ICa obtained in
control conditions (Fig. 8A, 1), in the presence of Rp-cAMPS
(Fig. 8A, 2), and in the presence of both Rp-cAMPS and
DPMA (Fig. 8A, 3). The bar graph in Fig. 8C shows that ICa
recorded in the presence of DPMA was significantly larger
when Rp-cAMPS (10 ␮M) was also present. The increase in
ICa above the baseline in the presence of Rp-cAMPS and
DPMA may reflect ability of Rp-cAMPS to enhance ICa in rods
(Stella and Thoreson 2000).
Figure 9 shows effects of Rp-cAMPS on [Ca2⫹]i increases
produced by application of elevated [K⫹]o on rod photoreceptors in the slice. Similar to rod ICa, application of Rp-cAMPS
(10 ␮M) prevented DPMA from inhibiting the K⫹-evoked
Ca2⫹ increase (Fig. 9A, ⫹3.6 ⫾ 10.8% n ⫽ 8, P ⫽ 0.7499). To
confirm the efficacy of DPMA, DPMA (2 ␮M) was reapplied
after washout of Rp-cAMPS and found to inhibit the K⫹evoked Ca2⫹ increase (Fig. 9A, ⫺36.1 ⫾ 3.9%, n ⫽ 8, P ⬍
0.0001). In agreement with the ICa results of Fig. 8, Rp-cAMPS
(10 ␮M), significantly reduced the ability of DPMA (2 ␮M) to
inhibit the depolarization-evoked [Ca2⫹]i increase (Fig. 9B,
DPMA (2 ␮M) versus DPMA (2 ␮M) ⫹ Rp-cAMPS (10 ␮M):
paired t-test, n ⫽ 8, P ⫽ 0.0212). The results of these experiments suggest that stimulation of PKA activity is primarily
responsible for A2 receptor modulation of voltage-dependent
L-type Ca2⫹ channels in rods.
ADENOSINE REDUCES ROD CALCIUM INFLUX THROUGH ICa
357
inhibit and A2 receptors stimulate adenylyl cylase (Londos et
al. 1980; van Calker et al. 1979). However, the classification
has since been broadened to include a new A3 receptor subtype
(Zhou et al. 1992) and two subtypes of A2 receptors, A2A and
A2B (Furlong et al. 1992; Maenhaut et al. 1990; Pierce et al.
1992). To discriminate among A1, A2, and A3 receptor subtypes, we used selective agonists for each receptor subtype:
R-PIA for A1 receptors, DPMA for A2 receptors, and APNEA
for A3 receptors. The ability of the A2-selective agonist,
DPMA, but not the A1 or A3 receptor agonists to inhibit both
the depolarization-evoked Ca2⫹ increase and ICa in rods (Figs.
6 and 7) is consistent with binding studies that have localized
A2 receptors to photoreceptor inner and outer segments (McIntosh and Blazynski 1994). We did not attempt to pharmacologically discriminate between A2A and A2B receptors. However, mRNA for the A2A receptor has been shown to be
expressed in the outer nuclear layer and ganglion cell layer,
whereas A2B receptor mRNA is absent from the retina (Kvanta
et al. 1997), suggesting that A2A receptors are likely to be
responsible for the effects observed in the present study. In a
FIG. 6. Effect of adenosine receptor agonists on rod photoreceptor ICa.
A: the A1-selective adenosine receptor agonist, (R)-N6-(1-methyl-2-phenylethyl)adenosine (R-PIA, 2 ␮M), did not inhibit rod ICa. Voltage error at the
peak of the current due to uncompensated access resistance was estimated to
be ⫺9 mV in both records. B: the A2-selective agonist, N6-[2-(3,5-dimethoxyphenyl)-ethyl]adenosine) (DPMA, 2 ␮M), inhibited rod ICa. Voltage error at
the peak of the current due to uncompensated access resistance was estimated
to be ⫺2.6 mV in control and ⫺2.1 mV in DPMA. C: the A3-selective
adenosine receptor agonist, N6-2-(4-aminophenyl)ethyladenosine (APNEA, 10
␮M), did not inhibit rod ICa. Voltage error at the peak of the current due to
uncompensated access resistance was estimated to be ⫺4 mV in both records.
Each graph shows I-V relationships of voltage ramps obtained in control
superfusate (—) and test solution (- - -). D: bar graph comparing effects of
R-PIA, DPMA, and APNEA on ICa in rods. Amplitude of the currents obtained
in test solutions were normalized to currents obtained in the control solution
(Itest/Icontrol). *, significant changes with respect to control (P ⬍ 0.05). R-PIA,
⫺0.5 ⫾ 4.7%, n ⫽ 6, P ⫽ 0.92; DPMA, ⫺15.1 ⫾ 4.7%, n ⫽ 11, P ⬍ 0.0001;
APNEA, ⫹2.4 ⫾ 5.8%, n ⫽ 7, P ⫽ 0.69.
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2⫹
FIG. 7. Effect of adenosine receptor agonists on [Ca ]i increases evoked
by elevated [K⫹]o in rods. A: a graphic representation of successive applications of adenosine receptor agonists. Depolarization-evoked [Ca2⫹]i changes in
photoreceptor inner segments produced by 1-min applications of elevated (50
mM) [K⫹]o were measured in the presence of the A1 receptor agonist, R-PIA
(10 ␮M), the A2 receptor agonist, DPMA (10 ␮M), and the A3 receptor
agonist, APNEA (10 ␮M). B: bar graphs comparing the effects of R-PIA,
DPMA, and APNEA on [Ca2⫹]i responses in rods. K⫹-evoked [Ca2⫹]i increases in test solutions were normalized to the average of the initial control
and subsequent recovery. Each bar graph represents the mean ⫾ SE. *, P ⬍
0.05 compared with control. R-PIA: ⫹3.0 ⫾ 3.3%, n ⫽ 12, P ⫽ 0.36; DPMA:
⫺30.9 ⫾ 4.9%, n ⫽ 12, P ⬍ 0.0001; APNEA: ⫺4.4 ⫾ 2.7%, n ⫽ 12, P ⫽
0.13.
358
S. L. STELLA, E. J. BRYSON, AND W. B. THORESON
together, these results indicate that A2 receptor-mediated
changes in Ca2⫹ influx from depolarization of ICa in rods are
likely established by stimulation of PKA.
Sources of retinal adenosine
FIG. 8. Rp-cAMPS (10 ␮M) prevented inhibition of ICa by DPMA (10 ␮M)
in rods. A: time course of changes in ICa amplitude during application of
Rp-cAMPS and Rp-cAMPS plus DPMA. B: current-voltage relationships of
voltage ramps obtained at time points 1 (control), 2 (Rp-cAMPS), and 3
(Rp-cAMPS ⫹ DPMA) from A. Control: thick solid trace. Rp-cAMPS (10
␮M): thin solid trace. DPMA (2 ␮M) ⫹ Rp-cAMPS (10 ␮M): thin trace.
Voltage error at the peak of the current due to uncompensated access resistance
was estimated to be ⫺3 mV in all 3 records. C: bar graph summarizing relative
changes in ICa amplitude induced by Rp-cAMPS or Rp-cAMPS plus DPMA.
Each bar represents the mean ⫾ SE. Asterisk, P ⬍ 0.05 compared with control.
*DPMA significantly inhibited ICa (⫺15.1 ⫾ 4.7%, n ⫽ 11, P ⬍ 0.0001) but
not in the presence of Rp-cAMPS (DPMA ⫹ Rp-cAMPS ICa: ⫹12.2 ⫾ 6.8%,
n ⫽ 4, P ⫽ 0.14). DPMA produced significantly less inhibition in the presence
of Rp-cAMPS (DPMA, n ⫽ 11 vs. DPMA ⫹ Rp-cAMP, unpaired t-test, P ⫽
0.0001).
study on cone motility in teleosts, the rank order of potency for
adenosine agonists was found to be consistent with A2-like
receptors, but antagonist potencies did not correspond precisely to either A2A or A2B receptors (Rey and Burnside 1999).
A2 receptor activation elevates cAMP levels in whole retina
(Blazynski et al. 1986; Paes de Carvalho and de Mello 1982).
A2 receptor-mediated inhibition of Ca2⫹ influx and ICa in rods
is similar to the inhibition of rod ICa produced by stimulation
of cAMP production or activation of PKA (Stella and Thoreson
2000) and inhibition of PKA with Rp-cAMPS blocked the
inhibitory effect of DPMA in rods (Figs. 8 and 9). Taken
J Neurophysiol • VOL
FIG. 9. Rp-cAMPS (10 ␮M) prevented the inhibition of depolarizationevoked [Ca2⫹]i increases by DPMA (2 ␮M) in rod photoreceptors. A: effects
of successive elevated [K⫹]o applications in control superfusate and in the
presence of Rp-cAMPS and DPMA. DPMA reversibly inhibited depolarization-evoked [Ca2⫹]i responses in the same cell following ⬃20 min washout of
Rp-cAMPS ⫹ DPMA. B: bar graph summarizing relative changes in depolarization-evoked [Ca2⫹]i responses induced by Rp-cAMPS (10 ␮M) and RpcAMPS (10 ␮M) plus DPMA (2 ␮M) in experiments similar to those illustrated in A. *DPMA (2 ␮M) significantly inhibited the K⫹-evoked Ca increase
in rods after washout of Rp-cAMPS ⫹ DPMA (⫺36.1 ⫾ 3.9%, n ⫽ 8, P ⬍
0.0001). †DPMA (2 ␮M) produced significantly less inhibition in the presence
of Rp-cAMPS (10 ␮M; paired t-test, n ⫽ 8, P ⫽ 0.02).
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Adenosine has been detected in photoreceptors of human,
monkey, guinea pig (Braas et al. 1987), fish (Ehinger and Perez
1984), and chick retinas (Paes de Carvalho et al. 1992). Adenosine has also been localized to ganglion cells (Ehinger and
Perez 1984) and cells in the inner nuclear layer (Ehinger and
Perez 1984; Paes de Carvalho et al. 1992) including rod horizontal cells (Studholme and Yazulla 1997) and amacrine cells
(Blazynski 1989). Adenosine is released tonically from retinas
in the dark (Blazynski and Perez 1991; Paes de Carvalho et al.
1990; Perez et al. 1986) and likely results from intracellular
turnover of ATP in photoreceptor inner segments. In the dark,
photoreceptors are depolarized by cations (e.g., Na⫹, Ca2⫹)
entering through cyclic nucleotide channels in the outer segment (Shimazaki and Oakley 1986; Torre 1982). Increased
intracellular adenosine levels are generated by the turnover of
ATP from a highly active Na⫹/K⫹-ATPase in the inner segment that counters the cation influx in the outer segment. The
increased ATP turnover and breakdown of adenine nucleotides
elevates intracellular adenosine levels. Increased adenosine
ADENOSINE REDUCES ROD CALCIUM INFLUX THROUGH ICa
Physiological significance
Previous studies have shown Ca2⫹ channels can be inhibited
by A2 receptors resulting in inhibition of transmitter release.
For example, activation of A2 receptors from cultured suprachiasmatic and arcuate nuclei neurons inhibit presynaptic ICa
and GABA release (Chen and van den Pol 1997). A2A activation also reduces the frequency of spontaneous and miniature
inhibitory postsynaptic currents by 30% in striatal medium
spiny neurons as a result of reduced GABA release (Mori et al.
1996). The present results show that A2 receptor activation can
also inhibit presynaptic ICa in glutamatergic neurons. Such a
mechanism might account for the decreased release of Lglutamate evoked by depolarization following activation of
A2A receptors in rat striatum and cultured chick retinal neurons
(Golembiowska and Zylewska 1997; Rego et al. 2000).
The results of the present study show that adenosine acting
on A2 receptors in rods can regulate L-type ICa and Ca2⫹ influx
in rods. Thus the changing levels of adenosine in the retina that
accompany changing levels of illumination could serve as an
autocrine or paracrine signal to regulate synaptic output from
rods.
We thank G. Rozanski, D. Monaghan, L. C. Murrin, and M. Toews for
critically reading earlier versions of the manuscript and providing helpful
discussions.
This study was supported by Nebraska Lions Clubs, National Eye Institute
Grant EY-10542, and a Research to Prevent Blindness Career Development
Award to W. B. Thoreson. S. L. Stella was supported by a Gifford Young
Investigator Award, Gifford Foundation, Omaha, NE.
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