 |
 Previous Article | Next Article 
The Journal of Neuroscience, July 15, 2000, 20(14):5254-5263
Light Transduction in Invertebrate Hyperpolarizing
Photoreceptors: Possible Involvement of a Go-Regulated
Guanylate Cyclase
Maria del Pilar
Gomez and
Enrico
Nasi
Department of Physiology, Boston University School of Medicine,
Boston, Massachusetts 02118, and Marine Biological Laboratory, Woods
Hole, Massachusetts 02543
 |
ABSTRACT |
The hyperpolarizing receptor potential of scallop ciliary
photoreceptors is attributable to light-induced opening of
K+-selective channels. Having previously
demonstrated the activation of this K+ current by
cGMP, we examined upstream events in the transduction cascade.
GTP- -S produced persistent excitation after a flash, accompanied by
decreased sensitivity and acceleration of the photocurrent, whereas
GDP- -S only inhibited responsiveness, consistent with the
involvement of a G-protein. Because Go (but not
Gt nor Gq) recently has been
detected in the ciliary retinal layer of a related species, we tested
the effects of activators of Go; mastoparan peptides
induced an outward current suppressible by blockers of the
light-sensitive conductance such as
L-cis-diltiazem. In addition, intracellular
dialysis with the A-protomer of pertussis toxin (PTX) depressed the
photocurrent. The mechanisms that couple G-protein stimulation to
changes in cGMP were investigated. Intracellular IBMX enhanced the
photoresponse with little effect on the baseline current, a result that
argues against regulation by light of phosphodiesterase activity.
LY83583, an inhibitor of guanylate cyclase (GC), exerted a reversible,
dose-dependent suppression of the photocurrent. By contrast, ODQ, an
antagonist of NO-sensitive GC, and YC-1, an activator of NO-sensitive
GC, failed to alter the light response or the holding current;
furthermore, the NO synthase inhibitor N-methyl-
L-arginine was inert, indicating that the NO signaling pathway is not implicated. Taken together, these results suggest a
novel type of phototransduction cascade in which stimulation of a
PTX-sensitive Go may activate a membrane GC to induce an increase in cGMP and the consequent opening of light-dependent channels.
Key words:
invertebrate photoreceptors; light-dependent channels; phototransduction; cGMP; guanylate cyclase; PDE; PTX; G-protein
| |
INTRODUCTION |
The retinas of the scallop and other
marine mollusks contain a layer of ciliary photoreceptors that, unlike
those of most invertebrates, hyperpolarize in response to
photostimulation (McReynolds, 1976 ), owing to a light-induced increase
in a potassium-selective conductance (Gorman and McReynolds, 1978;
Gomez and Nasi, 1994a). We previously showed that cGMP analogs activate
a K+ current with similar conduction and
pharmacology as the photocurrent. Furthermore, cGMP interacts
occlusively with light stimulation (Gomez and Nasi, 1995), suggesting
that it functions as a second messenger in phototransduction. Such a
proposition raises the question of which mechanisms link photopigment
stimulation to the changes in cytosolic cGMP concentration that are
required to regulate the light-sensitive conductance.
In rhabdomeric (depolarizing) photoreceptors rhodopsin activates a
G-protein of the Gq subtype (Lee et al., 1994;
Scott et al., 1995; Suzuki et al., 1995) and stimulates a phospholipase C (PLC) (Devary et al., 1987; Baer and Saibil, 1988) with the consequent hydrolysis of PIP2,
IP3 production (Szuts et al., 1986; Brown et al.,
1987), and Ca2+ release (Brown and Blinks,
1974; Brown and Rubin, 1984). In scallop hyperpolarizing
photoreceptors, however, neither IP3 nor
Ca2+ plays any important role in
transduction (Gomez and Nasi, 1995), making Gq a
poor candidate. The rhodopsin-stimulated G-protein of vertebrate
photoreceptors is transducin (Gt);
Gt activates a phosphodiesterase (PDE), leading
to hydrolysis of cGMP, the substance that opens the light-sensitive
channels (for review, see Yau and Baylor, 1989). Considering the
structural and functional similarities between hyperpolarizing
invertebrate photoreceptors and rods and cones (Miller, 1958; Gomez and
Nasi, 1997a), one may suggest a role for transducin; in such a case the
cascade would have to diverge downstream, because light is expected to increase cGMP. In a recent study Kojima et al. (1997) examined the
G-proteins present in the retina of a related species and demonstrated
that the only isoform that localizes to the distal layer, where ciliary
receptors are found, belongs to the
G o subtype. However, no
functional evidence was provided to confirm the involvement of a
G-protein in phototransduction nor to suggest its identity.
A further question concerns the enzymatic regulation of [cGMP]; if
the photoresponse requires an increase in cGMP, two prime light-triggered mechanisms may be a decrease in the activity of a PDE
or stimulation of a guanylate cyclase (GC). The former case is a mirror
image of the mechanism that operates in rods and cones and resembles
phototransduction in the parietal eye of the lizard, as recently
described by Xiong et al. (1998), whereas the latter has no precedent
as a mechanism to generate the receptor potential in retinal cells. In
the present report we used whole-cell patch-clamp recordings of
isolated ciliary photoreceptors to obtain evidence that a PTX-sensitive
G-protein, most likely of the Go subclass, mediates phototransduction in Pecten irradians (and probably
in other invertebrate ciliary photoreceptors). Our results do not provide support for the involvement of a light-regulated PDE as a
downstream effector; rather, the possibility of a key role of a GC
suggests itself. The departure of such a scheme from established mechanisms that generate the light response in other visual cells is
indicative of a novel type of phototransduction cascade.
| |
MATERIALS AND METHODS |
Specimens of Pecten irradians were obtained from the
Marine Resources Center at the Marine Biological Laboratory (Woods
Hole, MA) and used immediately. Isolated retinas were dispersed
enzymatically and mechanically as described (Gomez and Nasi, 1994a),
and the resulting cell suspension was plated in a recording flow
chamber pretreated with Concanavalin A (Sigma, St. Louis, MO) to
promote cell adhesion (Nasi, 1991). During the experiments the chamber was superfused continuously with artificial seawater (ASW) containing (in mM) 480 NaCl, 10 KCl, 49 MgCl2, 10 CaCl2, 10 HEPES,
and 5 glucose, pH 7.8 (adjusted with NaOH). Extracellular chemical
stimulation was accomplished by a puffer pipette (tip outer diameter of
3-4 µm) positioned ~30-50 µm from the target cell. Application
of pressurized nitrogen (1-3 psi) to the pipette via a
solenoid-operated valve permitted local solution exchange in <400 msec
(Gomez and Nasi, 1996). Patch electrodes were fabricated with thin-wall
borosilicate capillary tubing (7052, Garner Glass, Claremont, CA),
fire-polished, and filled with an "intracellular" solution
containing (in mM) 100 KCl, 200 K-aspartate or
K-glutamate, 5 Na2ATP, 12 NaCl, 6 MgCl2, 10 HEPES, 1 EGTA, 0.2 GTP and 300 sucrose,
pH 7.3. Electrode resistance, measured in ASW, ranged between 2 and 6 M . Series resistance errors were corrected via a positive feedback
circuit in the amplifier (maximal residual error typically <2 mV). For internal dialysis, test substances were added to the electrode filling
solution from stock solutions. In all experiments the holding potential
was  30 mV.
GTP--S (guanosine 5'-O-[3-thiotriphosphate]), GDP--S
(guanosine 5'-O-[2-thiodiphosphate]), 8-bromo-cyclic
guanosine monophosphate (8-Br-cGMP), EHNA
(erythro-9-[2-hydroxy-3-nonyl]adenine HCl), trequinsine, and the
A-protomer of pertussis toxin (PTX) were purchased from Sigma. PTX
(holotoxin) and mastoparan were obtained from Calbiochem (San Diego,
CA), whereas the related peptides MAS-7 and MAS 17 were
obtained from Peninsula Laboratories (San Carlos, CA). The calmodulin
antagonists W-7 hydrochloride
[N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (HCl)]
and chlorpromazine were purchased from Research Biochemicals (Natick, MA).
LY83583 (6-anilino-5,8-quinolinedione) was obtained from Calbiochem,
whereas calmidozolium chloride and YC-1
(3-[5'-hydroxymethyl-2'furyl]-1-benzyl indazole) were from Research
Biochemicals. ODQ
(1H-[1,2,4]oxadiazolol-[4,3-a]quinoxalin-1-one) and the phosphodiesterase inhibitors IBMX
(3-isobutyl-1-methylxanthine), 8-methoxymethyl-IBMX, dipyridamole,
zaprinast
[1,4-dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo-4,5-dipyrymidin-7-one], and milrinone
(1,6-dihydro-2-methyl-6-oxo-3,4-bipyridine-5-carbonitrile) were
obtained from Sigma. These water-insoluble substances were dissolved in
either EtOH or DMSO, and the stock solutions were aliquoted and stored
at 80°C. The final concentration of DMSO or EtOH present either in
the internal solution or in ASW was <0.5%. Control experiments
demonstrated that solvent concentrations up to 5% were inert on the
membrane current and on the light response of ciliary photoreceptors.
Flashes and steps of broadband light (515-650 nm) generated by a
standard optical stimulator (Gomez and Nasi, 1994a) were calibrated
in vivo, as previously described (Gomez and Nasi, 1994a,b). Light intensity is expressed either in terms of equivalent photon flux
at 500 nm or, whenever light attenuation was varied within a given
protocol, as
log10(I/Io),
where Io is the intensity of the
unattenuated light. Calibrated neutral-density filters (Melles Griot,
Irvine, CA) provided controlled attenuation. During experimental manipulations the cells were illuminated with near-IR light with a
long-pass filter ( > 780 nm; Andover, Salem, NH) and viewed with the aid of a Newvicon TV camera (model WV-1550, Panasonic, Secaucus, NJ). The infrared illuminator was turned off for several minutes before light responses were tested. All recordings were made at
room temperature (20-22°C).
| |
RESULTS |
Involvement of a G-protein
Nonhydrolyzable analogs of GTP, such as GTP--S, have been used
extensively to assess the participation of G-proteins in cell signaling
(Breitwieser and Szabo, 1988; Andrade, 1994). At low doses GTP--S is
not expected to activate the G-protein, but on stimulation of the
receptor it should induce a prolongation of the response by interfering
with a key shut-off step, namely the GTPase action of
G. Figure
1A, left,
shows the membrane current from a ciliary photoreceptor internally
perfused with 100 µM GTP--S and stimulated
repetitively with 5 sec light steps at intervals of 40 sec. A
substantial fraction of the photocurrent remained active after the
termination of the first light stimulus; subsequent lights evoked
progressively diminished responses and smaller further shifts in
baseline (n = 16). By contrast, in control cells
dialyzed with the normal intracellular solution the photocurrents shut off completely after each light (Fig. 1A,
right), and their peak amplitude remained unaltered with
successive repetitions of the stimulus (n > 20). The
progressive reduction in responsiveness in the presence of GTP--S
required photostimulation, as illustrated in Figure
1B, top; a cell internally perfused with
100 µM GTP--S for 15 min in the dark
responded to the first flash with a photocurrent of normal amplitude.
Subsequent light responses, however, were profoundly depressed
(n = 4). For comparison, the bottom trace illustrates
the outcome of a similar regime applied to a control cell.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 1.
Effects of GTP--S on the photocurrent.
A, Left, A photoreceptor was
whole-cell-clamped with an electrode containing 100 µM
GTP--S. Light-steps lasting for 5 sec were applied every 40 sec
(28 × 1014 photons × sec 1 × cm2). At the termination of the
first light stimulus, a substantial fraction of the photocurrent
remained active. A, Right, A control cell
dialyzed with the standard intracellular solution was subjected to a
similar protocol (17.6 × 1014 photons × sec1 × cm2) and displayed no sustained
activation of the photoresponses. B, The decline in
light responsiveness with GTP--S is a consequence of
photostimulation. Top, A photoreceptor internally
perfused with 100 µM GTP--S was kept in the dark for
15 min before light stimulation. The first photocurrent in response to
a flash of 7 × 1014 photons × sec1 × cm2 displayed normal amplitude,
whereas a subsequent response to the same light stimulus applied 2 min
later was severely depressed. Bottom, A cell dialyzed
with normal internal solution was stimulated immediately after
attaining the whole-cell configuration and then kept in the dark for 15 min before stimulation was resumed. The responses show no
deterioration. Light intensity, 2.3 × 1014
photons × sec1 × cm2.
|
|
When photoreceptors are stimulated with persistent background
illumination, the response to a test flash is desensitized; in
addition, it acquires a more rapid time course (Fuortes and Hodgkin,
1964; Baylor and Hodgkin, 1974). If the depression of the light
response in GTP--S-treated photoreceptors is a consequence of
sustained activation of the transduction cascade, one may expect a
similar acceleration of the photocurrent. Figure
2A, left,
shows superimposed photocurrents elicited by 100 msec flashes delivered every 2 min during intracellular dialysis with 100 µM GTP--S. In the panel on the right the
traces were normalized with respect to the peak amplitude, revealing
that the kinetics of the response became progressively faster. Similar
results were obtained in 12 cells. Figure 2B shows
superimposed light responses from a control cell, demonstrating the
lack of any acceleration of their time course (n > 50).
View larger version (25K):
[in this window]
[in a new window]
|
Figure 2.
GTP--S-induced desensitization
is accompanied by photocurrent acceleration. A,
Left, Repetitive flashes (100 msec; 4.4 × 1014 photons × sec1 × cm2) were applied every 2 min
to a photoreceptor dialyzed with 100 µM GTP--S; the
response amplitude gradually decreased. A,
Right, Normalization of the same photocurrent traces
reveals progressively faster kinetics. B, Superimposed
light responses (3.3 × 1014 photons × sec1 × cm2) in a control cell,
demonstrating the lack of any significant change in either amplitude or
time course.
|
|
Whereas the effectiveness of GTP--S at low doses requires activation
of the receptor, one would anticipate that, by the law of mass action,
higher concentrations should be capable of directly stimulating the
G-protein (Breitwieser and Szabo, 1988). This prediction is borne out
by the data in Figure 3A,
which shows membrane currents recorded from different photoreceptors in
the dark immediately after intracellular perfusion was initiated with the indicated concentrations of GTP--S. When the intracellular solution contained >100 µM GTP--S, a
conspicuous outward current gradually developed. Figure 3B
shows the mean peak amplitude of outward membrane currents recorded in
the dark, pooled from cells treated with the different concentrations
of GTP--S (total n = 14).
View larger version (14K):
[in this window]
[in a new window]
|
Figure 3.
Direct stimulatory effects of GTP--S at higher
concentrations. A, Whole-cell membrane currents measured
in the dark in four different cells immediately after the initiation of
intracellular perfusion with the concentrations of GTP--S indicated
on the right. When [GTP--S] in the pipette was
above 100 µM, a conspicuous outward current gradually
developed. B, Mean peak amplitude of outward membrane
currents recorded in the dark, pooled from cells treated with different
concentrations of GTP--S.
|
|
A complementary strategy to ascertain the involvement of a G-protein
entails the use of GDP--S. This hydrolysis-resistant GDP analog acts
as a competitive inhibitor of G-proteins by binding to the  -subunit
and preventing agonist-induced activation (Eckstein et al., 1979).
Figure 4A shows the
responses evoked by 100 msec flashes of constant intensity applied
every minute to a cell dialyzed with 200 µM
GDP--S. A marked decrease in the amplitude of the photocurrent took
place over a period of 15 min (n = 4). However, unlike
with GTP--S, the progressive decline in light responsiveness occurred irrespective of light stimulation, as illustrated in Figure
4B; a step of light was interposed between two brief
test flashes delivered 2 min apart (an interval far shorter than the time constant of the decay of the GDP--S-induced loss of
responsiveness; see Fig. 4A). The response to the
second test flash barely displayed some attenuation (top;
n = 6); by contrast, in GTP--S-treated cells light
responsiveness was reduced dramatically by the intervening conditioning
light (bottom; n = 8).
View larger version (19K):
[in this window]
[in a new window]
|
Figure 4.
GDP--S reduces light responsiveness.
A, Peak amplitude of responses elicited by 100 msec
flashes, plotted as a function of time. The photoreceptor was
voltage-clamped with an electrode containing 200 µM
GDP--S. Insets, First and last record of the series.
Calibration: 400 msec, 400 pA. B, Light stimulation does
not potentiate the effect of GDP--S. The stimulus protocol consisted
of a control flash (100 msec) followed by a conditioning 500 msec light
of the same intensity and a second test flash (identical to the
control); this regime was repeated at 2 min intervals.
Top, Responses from a cell dialyzed with GDP--S,
showing only minor changes in the photocurrent evoked by the test
flashes. Bottom, When the same protocol was applied to a
photoreceptor internally perfused with GTP--S, the conditioning
light dramatically depressed the response to the test flash. Light
intensity, 7 × 1014 photons × sec1 × cm2.
|
|
To corroborate further that the desensitizing effect of poorly
hydrolyzable guanine nucleotide analogs is not attributable to
inhibition of downstream elements in the cascade, we determined that
activation of the conductance by 8-Br-cGMP (20 µM) was
not hindered by concomitant dialysis with 100 µM
GTP--S (n = 5; data not shown).
In summary, we demonstrated the following: (1) sustained
photoexcitation in the presence of low doses of GTP--S, accompanied by response desensitization and acceleration of its kinetics; (2)
direct channel activation at high doses of GTP--S; and (3) inhibition of the photoresponse by GDP--S. Taken together, these results strongly suggest that, like in other classes of visual cells, a
G-protein mediates light transduction in the hyperpolarizing ciliary
photoreceptors of the scallop.
Identity of the G-protein
Considering that a
Go has been detected in
the ciliary retinal layer of another species of scallop (Kojima et al.,
1997), we sought physiological support for the proposition that this subclass of G-protein is involved in the phototransduction process. Among the substances that have been described as activators of G-proteins, mastoparan, a small peptide component of wasp venom, has
been shown to display a significant selectivity for
Gi and Go isoforms and to
operate by a mechanism that mimics stimulation by activated receptors
(Higashijima et al., 1988). Figure
5A shows membrane current
traces recorded in the dark from different photoreceptors immediately
after the whole-cell configuration was attained. The patch electrodes
contained 0, 20, or 50 µM mastoparan, and the holding potential was set at 30 mV. With the control internal solution no significant change in holding current was observed. With 20 µM mastoparan an outward current developed
after a latency of ~10 sec, attaining an amplitude of 226 pA (mean
222 ± 140 pA; n = 18). With 50 µM a much faster and larger response was
obtained (mean 361 ± 177 pA; n = 4). These
effects bear a striking resemblance to the direct stimulation of the
light-sensitive conductance by intracellular application of cGMP
analogs (Gomez and Nasi, 1995), although the average size of the
mastoparan-induced current tended to be smaller than that evoked by
application of 8-Br-cGMP. Extending the range of concentrations of
mastoparan proved to be problematic because at high doses this
substance also has been reported to permeabilize the plasma membrane
(Tanimura et al., 1991) by inducing the formation of nonselective
cationic membrane pores (Suh et al., 1996). In fact, a few attempts
that used larger amounts of the peptide in the pipette solution
resulted in a runaway increase in leakage. Other peptides related to
mastoparan also were tested. MAS 17, a relatively inactive analog
obtained by introducing a positively charged lysine on the hydrophobic
stretch of the molecule, proved significantly less potent than
mastoparan (20 µM; n = 5). By
contrast, MAS 7, which is produced by a lysine alanine substitution at position 12, resulting in an enhanced ability to promote GDP/GTP exchange in G-proteins (Higashijima et al., 1988), also evoked a
conspicuous outward current in the dark when dialyzed at a
concentration of 20 µM (n = 3);
in our hands, however, its effectiveness was not greater than that of
mastoparan.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 5.
Mastoparan-induced outward current.
A, Whole-cell membrane current measured in the dark
immediately after internal perfusion was initiated. The patch pipette
contained either control internal solution or mastoparan at a
concentration of 20 or 50 µM, respectively. A distinct
outward current developed in the photoreceptors treated with
mastoparan; the amplitude and speed of the response were related
directly to the peptide concentration. B, Current trace
recorded in the dark from a cell internally dialyzed with 20 µM mastoparan. When the outward current attained a steady
level, 1 mM L-cis-diltiazem was
applied repeatedly by a puffer pipette (indicated by the
bars), producing a reversible decrease in the amplitude of
the response.
|
|
To ascertain whether the mastoparan-evoked current is related to the
activation of the phototransduction cascade, we examined the effects of
blockers of light-dependent channels on the current induced by this
peptide. In Figure 5B a photoreceptor cell was held at 30
mV and dialyzed intracellularly with 20 µM
mastoparan, which induced an outward current of 284 pA. When a stable
level was attained, 1 mM
L-cis-diltiazem was applied by puffer
pipette, rapidly producing a substantial, reversible decrease in the
outward current. Similar results were obtained in six cells. No effects of the blocker were seen in the absence of mastoparan (data not shown).
The degree of blockage of the mastoparan-induced current by
L-cis-diltiazem was quantitatively
comparable to that previously reported for the light-evoked current and
for the current elicited by 8-Br-cGMP (Gomez and Nasi, 1997a).
Similarly, puffer application of 100 µM 4-AP,
which previously was shown to potently block the photocurrent in these
cells (Gomez and Nasi, 1994b), antagonized the mastoparan-elicited
outward current (n = 2; data not shown). Further clues
were provided by evidence of occlusive interactions between mastoparan
and light stimulation; after dialysis with the peptide the peak
amplitude of the current evoked by a flash of saturating intensity was
smaller than in control photoreceptors (966 ± 732 pA vs 2460 ± 907 pA; n = 9 in each condition). Moreover, in
treated cells a significant negative correlation existed between the
size of the current elicited by mastoparan and that of the residual
photocurrent (r = 0.46; p < 0.05).
Mastoparan is also known as a calmodulin (CaM) inhibitor (Barnette et
al., 1983). To rule out that the observed effects may be mediated by
CaM, we performed control experiments with chemically distinct
CaM antagonists; neither calmidazolium (100 nM;
n = 6) nor W-7 (50 µM;
n = 2) applied internally produced any change in
membrane current.
Most Go and Gi are strongly
susceptible to inhibition via ADP ribosylation by pertussis toxin
(Kaslow and Burns, 1992) (for review, see Carty, 1994). The holotoxin,
which is produced by the bacterium Bordetella pertussis, is
composed of the A-protomer, where the catalytic activity of the toxin
resides, and the B-oligomer, for which the function is to bind to the
cell surface and promote internalization (Tamura et al., 1983).
Effective PTX treatment of intact cells typically entails prolonged
incubations, sometimes in excess of 24 hr. We determined that isolated
scallop retinas can remain viable for up to 30 hr, provided they are
kept at 14-16°C. Under these conditions the addition of 1 µg/ml
PTX to the bathing medium did not lead to a significant deterioration
of the light response (data not shown). These negative results,
however, could stem from poor internalization, either because of the
lack of surface receptors for the B-oligomer (Tamura et al., 1983) or because of the rather low temperatures at which the cells had to be
incubated (Holz et al., 1986). We therefore resorted to direct
application of the toxin by intracellular dialysis via the patch
electrode. Because this approach renders the B-oligomer unnecessary, we
used the A-protomer instead of the holotoxin, because its smaller size
(28 kDa) expedites dialysis. Figure
6A, left,
shows that, when the A-protomer of PTX (3 µg/ml) was codialyzed with
500 µM nicotinamide adenosine dinucleotide
(NAD) (which serves as the donor of ADP-ribose in the
ADP-ribosyltransferase reaction) (Kaslow and Burns, 1992), the
photoresponse evoked by repetitive flashes (1/min) progressively
declined over a period of 20 min (average photocurrent decrease, 59 ±11%; n = 10). In contrast, responsiveness remained
unaltered in control photoreceptors treated with NAD alone
(right; n = 3) or with the standard internal
solution (n > 20; data not shown). In Figure
6B the peak amplitude of the light response is
plotted for the two cells to show the time course of the effect.
View larger version (26K):
[in this window]
[in a new window]
|
Figure 6.
Pertussis toxin sensitivity of the light-dependent
current. A, Left, Shown are responses to
repetitive 100 msec flashes from a cell codialyzed with the PTX
A-protomer (3 µg/ml) and NAD (500 µM). A progressive
reduction of photocurrent amplitude was observed. The pipette was
front-filled with toxin-free solution to avoid interference with seal
formation. A, Right, In a similar
experiment conducted in a photoreceptor treated with NAD alone, no sign
of deterioration of the light response was observed. B,
Shown is the normalized peak amplitude of the photoresponses for the
same two cells, plotted as a function of time. Light intensity, 9 × 1013 photons × sec1 × cm2.
|
|
Effect of PDE inhibitors
Next we explored the mechanisms by which the G-protein is coupled
to the changes in cyclic GMP necessary for opening the light-dependent conductance. Considering the well established precedent of a
light-activated PDE in vertebrate phototransduction and the
aforementioned kinship between rods and cones and invertebrate ciliary
photoreceptors, we examined the possible involvement of a PDE modulated
by light. Of course, the requirement of a photo-induced
increase in cGMP would call for a high basal level of PDE
activity in the dark, which would have to be reduced by
light stimulation, unlike in vertebrate retinas. Under this hypothesis
a pharmacological antagonist targeting PDE should be particularly
effective in the dark, when the hydrolytic activity is maximal, and
result in a build-up of cGMP levels and the consequent opening of
light-sensitive channels. In other words, an effective PDE inhibitor
should mimic photostimulation. On the other hand, the current evoked by
a flash may be expected to decrease, if anything, owing to the reduced
pool of PDE that may remain susceptible to inhibition by light and also
owing to the fact that a fraction of ion channels already would be active.
We first tested the effect of IBMX, a broad-spectrum PDE inhibitor,
applied internally via the patch pipette. As shown in Figure
7A, left, during
intracellular perfusion with 500 µM IBMX in the
dark only a small change in the holding current was observed (average,
58 ± 34 pA; n = 12); this was not significantly
different from control cells. On the other hand, IBMX induced a gradual enhancement of the size of the photocurrent (right); the
average increase was 46 ± 17% (n = 7). Although
the latter effect is consistent with the presence of PDE activity in
ciliary photoreceptors, both results argue against its involvement as a
critical light-regulated step in the generation of the photoresponse.
Attempts to narrow the identity of the PDE subtype by using more
selective pharmacological inhibitors (for review, see Beavo, 1995) were
unsuccessful. In particular, we tried to target cyclic nucleotide PDEs
that exhibit a high substrate specificity and preferentially hydrolyze
cGMP, such as types 5 and 6 (Gillespie and Beavo, 1989); however,
neither dipyridamole (100 µM; n = 7) nor zaprinast (100-500 µM;
n = 9) applied intracellularly caused any discernible
change in the holding current in the dark or in the amplitude of the
light responses (Fig. 7B,C). We also tested a variety of
compounds that are effective on other subclasses of PDEs, including
cGMP-stimulated type 2 PDE (EHNA 1-100 µM),
cGMP-inhibited type 3 PDE (trequinsin, 20-200 nM; milrinone, 5 µM), and
CaM PDE type 1 (8-methoxymethyl-IBMX, 10-30
µM; W-7, 50 µM); all
were found to be inert.
View larger version (23K):
[in this window]
[in a new window]
|
Figure 7.
Intracellular dialysis of PDE inhibitors.
A, Effect of IBMX. Left, Baseline holding
current during internal perfusion with 500 µM IBMX in the
dark. Membrane current measured at 1 min intervals starting immediately
after the whole-cell configuration was established did not change
appreciably. Each point represents the average of nine
cells; error bars indicate SD. Right, Peak light
response plotted as a function of time in a photoreceptor dialyzed with
500 µM IBMX, showing a progressive enhancement in
amplitude. The inset illustrates individual superimposed
photocurrent traces. B, C, Similar
measurements of the light-evoked current in cells dialyzed with 200 µM dipyridamole and 500 µM zaprinast,
respectively. The flash intensity was 1.1 × 1014 photons × sec1 × cm2 for all measurements shown
in the figure. Calibration in insets: 200 pA, 200 msec.
|
|
In a recent brief communication Shimatani and Katagiri (1997) reported
that bath application of IBMX and dipyridamole depressed the
photoresponse in hyperpolarizing receptors of another species of
scallop. We confirmed these effects in Pecten; as shown in Figure 8A,
extracellular local application of 1 mM IBMX
reversibly decreased the amplitude of the light response (mean
reduction, 54 ± 12%; n = 8). In the dark the
holding current was unaffected. A similar reduction of the photocurrent
also was induced by puffer administration of 200 µM dipyridamole (mean reduction, 42 ± 6%; n = 6; Fig. 8B). This inhibitory
action was not shared by other antagonists of PDE types 5 and 6, because zaprinast (200-400 µM) had no
significant effect (Fig. 8C; n = 6).
View larger version (25K):
[in this window]
[in a new window]
|
Figure 8.
Extracellular effects of PDE inhibitors on the
photoresponse. Repetitive flashes were applied in ASW, during puffer
application of PDE inhibitors, and after the compounds were washed.
A, IBMX locally superfused at a concentration of 1 mM reversibly reduced the photocurrent. Light intensity,
1.1 × 1014 photons × sec1 × cm2. B, A
similar effect was produced by 200 µM dipyridamole
(2.8 × 1014 photons × sec1 × cm2). C, Lack of
effect of local application of 200 µM zaprinast (4.4 × 1014 photons × sec1 × cm2).
|
|
The antagonistic effects of extracellular dipyridamole and IBMX on the
photoresponse contrast with the ineffectiveness of these drugs applied
directly to the cytosolic compartment (see above). Furthermore, they
are seemingly inconsistent with the notion of a light-induced increase
in cGMP. In an attempt to clarify this puzzle, we examined the
consequence of dipyridamole superfusion on the current directly evoked
by cGMP analogs. Figure 9A
shows recordings from photoreceptors voltage-clamped in the dark with electrodes containing 50 µM 8-Br-cGMP. A large
outward current developed soon after intracellular dialysis was begun;
when its amplitude reached a stable level, either 200 µM (left) or 400 µM dipyridamole (right) was applied
from a puffer pipette. A dose-dependent reversible decrease of the
nucleotide-evoked current was observed, which was quantitatively
comparable to the reduction of the light-evoked current. In Figure
9B, control experiments performed in the absence of
8-Br-cGMP showed little effect of dipyridamole, confirming that this
drug specifically acts on the cGMP-dependent
K+ current. These results, therefore,
strongly argue that the depression of the light response by
extracellular dipyridamole (and, presumably, by IBMX) stems from direct
blockage of the nucleotide-dependent channels and cannot be
attributable to any effect on PDE. The lack of effect of cytosolic
perfusion suggests that the blocking site is only accessible
extracellularly.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 9.
Photocurrent depression by dipyridamole is
attributable to channel block. A, Shown are outward
currents evoked in four cells by internal administration of 50 µM 8-Br-cGMP in the dark. At the time marked by the
thick lines, the application of
dipyridamole by puffer pipette (left, 200 µM; right, 400 µM) caused a
reversible suppression of the nucleotide-induced current.
B, Shown are dark currents in four control
photoreceptors dialyzed with standard intracellular solution and
locally perfused with the indicated concentrations of
dipyridamole.
|
|
Disposal of cGMP also could occur by extrusion via a membrane
transporter, as recently described in erythrocytes (Schultz et al.,
1998). Conceivably, modulation of efflux could control intracellular
[cGMP]. However, application of 100 µM probenecid, a
blocker of the cGMP pump, had no effect of membrane conductance in the
dark nor on the light response (n = 3).
Role of a guanylate cyclase
A plausible alternative to regulation of hydrolysis or extrusion
of cGMP as a mechanism to increase its concentration during illumination would be the modulation of a GC. The inhibitory effects of
the compound LY83583 on GC have been documented extensively in
biochemical assays and physiological measurements (Schmidt et al.,
1985). Intracellular perfusion of ciliary photoreceptors with 10-100
µM LY83583 caused a substantial, progressive decay in the
photocurrent, as shown in Figure
10A; the peak
amplitude of the response is plotted as a function of time for a
control cell (open squares) and for one voltage-clamped with
an electrode containing 100 µM LY83583
(filled squares). At this concentration of the drug
the responses decreased by (52 ± 17%; n = 6).
Taking advantage of the fact that LY83583 is significantly
membrane-permeable (Schmidt et al., 1985), we also examined the effect
of extracellular application. Figure 10B shows the
light-evoked current from a cell voltage-clamped at 30 mV;
application of 200 µM LY83583 via a puffer
pipette reversibly decreased the size of the photocurrent (mean
reduction, 53 ± 16%; n = 4). Lower doses (50 µM) were also consistently effective, although
the inhibitory effect was correspondingly less pronounced (21 ± 4%; n = 5; data not shown). Figure 10C
shows intensity series recorded from a cell in ASW and during exposure to 200 µM LY83583 (right).The
corresponding plot of the peak amplitude of the photocurrents as a
function of light intensity (left) shows that the curve
obtained in the presence of the drug is compressed considerably.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 10.
Depression of the photocurrent by inhibition of
guanylate cyclase. A, Peak amplitude of the light
response elicited by repetitive flashes, plotted as a function of time.
The pipette contained either the normal intracellular solution
(open squares) or 100 µM
LY83583 (filled squares). The
light intensity was 2.3 × 1014 photons × sec1 × cm2. B,
Reversible inhibition of the photocurrent induced by extracellular
application of 200 µM LY83583; the same light stimuli are
used as in A. C, Photocurrent amplitude
as a function of light intensity in a cell dialyzed with standard
internal solution (unattenuated light intensity, 3.4 × 1015 photons × sec1 × cm2). The intensity series was
recorded in ASW (filled squares) and during local
superfusion with 200 µM LY83583 (open
squares). The corresponding families of raw traces are shown on
the right. D, Left, Outward current
evoked by internal perfusion with 20 µM 8-Br-cGMP in the
dark. Once the cGMP-dependent current reached a steady state, LY83583
was applied with a puffer pipette but produced no measurable effect.
Subsequent application of the drug in the same cell
(right) reduced the photocurrent in a reversible manner.
Light intensity, 18 × 1014 photons × sec1 × cm2.
|
|
The depression of the photocurrent by LY83583 is compatible with an
inhibitory effect on GC activity; however, in view of a recent
observation that in olfactory neurons this substance also can block
cyclic nucleotide-gated channels (Leinders-Zufall and Zufall, 1995), it
was important to rule out any direct antagonism on the light-sensitive
conductance. To examine this possibility, we tested the effects of
LY83583 on the current activated by direct application of cGMP analogs.
Figure 10D shows a recording obtained in the dark
from a photoreceptor whole-cell-clamped with an electrode containing 20 µM 8-Br-cGMP. When the cGMP-dependent outward
current reached a stable level, LY83583 (200 µM) was applied locally (left). No
reduction in current was detected, although a subsequent test in the
same cell demonstrated that the light response exhibited a normal
susceptibility to the drug (right), confirming the
effectiveness of delivery. Similar results were obtained in 10 photoreceptors.
To ascertain the possible involvement of a nitric oxide pathway that
may target a GC, we tested the effects of ODQ, a potent and selective
inhibitor of nitric oxide-sensitive GC (Garthwaite et al., 1995).
Repetitive photostimulation during internal dialysis with 1 µM ODQ did not reveal any decrease in response amplitude; furthermore, light sensitivity, measured in a standard intensity series, was normal (n = 5). Responsiveness to light
also was maintained during local extracellular application of 1 µM ODQ, although a marginal decrease in the
peak size of the photocurrent was observed (93 ± 1.9% of
control; n = 4). In addition, we determined that the
light response was completely unaffected by intracellular administration of the NO-synthase inhibitor
N-methyl-L-arginine (NMLA) at a
concentration of 100 µM (n = 3), and neither the basal conductance not the photocurrent was altered
by YC-1 (30 µM), an activator of NO-sensitive
GC (n = 4). These results argue against the involvement
of a NO-sensitive GC in the excitatory process of ciliary photoreceptors.
| |
DISCUSSION |
In the present study we examined the intervening steps that link
photopigment stimulation to ion channel gating in ciliary invertebrate
photoreceptors. These cells share with vertebrate rods and cones the
fact that light-dependent channels are controlled via cGMP (Gomez and
Nasi, 1995), but the transduction cascade must differ at some key
enzymatic step, because the final effect of photostimulation is an
increase, rather than a decrease, in membrane conductance.
The effects of nonhydrolyzable guanine nucleotide analogs indicated
that, in common with other visual cells, activation of a G-protein is a
key early step in excitation; intracellular dialysis with low doses of
GTP--S caused part of the photocurrent to persist after termination
of a light stimulus, concomitantly with a desensitization of the
response and an acceleration of its kinetics. These effects suggest
that hindering G-protein deactivation interferes with the shut-off of
the phototransduction cascade and results in a state akin to adaptation
by background illumination. Conversely, GDP--S depressed
responsiveness in a manner that did not depend on photostimulation, as
one would expect from the fact that GDP--S targets the inactive
G-protein. The particular G-protein subtype that mediates
phototransduction must differ from those that operate in other visual
cells, because neither Gt nor
Gq is a viable candidate in this system.
Motivated by a recent report demonstrating that Go is present in the distal retina of a closely
related species (Kojima et al., 1997), we sought physiological evidence
for Go involvement in visual excitation in
Pecten. In the first place, we found that mastoparan
peptides known to activate Gi and
Go selectively, with little effect on
Gt and Gs (Higashijima et
al., 1988), consistently evoked an outward current similar to that elicited by intracellular perfusion with cGMP analogs and susceptible to the same blockers of the light/cGMP-sensitive conductance, namely
L-cis-diltiazem and 4-AP (Gomez and
Nasi, 1994b, 1997a). Light responsiveness also was depressed by
application of the A-protomer of PTX; this result dovetails with the
published amino acid sequence of the scallop retinal
Go (Kojima et al., 1997), which shows a cysteine
in the fourth position from the C terminus, the hallmark for
susceptibility to ADP ribosylation. In conjunction with the results of
Kojima et al. (1997), the present observations support the notion that
light transduction in ciliary invertebrate photoreceptors is mediated
by a G-protein of the Go subclass, in sharp
contrast with other previously studied visual receptor cells.
Interestingly, in the "on" bipolar cells from vertebrate retinae
the response triggered by stimulation of metabotropic glutamate
receptors (which also is mediated by cGMP) (Nawy and Jahr, 1990, 1991;
Shiells and Falk, 1990) appears to involve a Go
(Vardi et al., 1993).
The enzymatic target of the rhodopsin-coupled G-protein also seems to
differ from that of other photoreceptors that use cGMP as an internal
messenger, because modulation of a phosphodiesterase is an unlikely
light-regulated mechanism for controlling cGMP levels in the present
system. Such a scheme would require a high constitutive level of GC
activity; therefore, one would anticipate that inhibition of PDE in the
dark should lead to rapid cGMP accumulation and the consequent
activation of the light-sensitive conductance. Contrary to this
prediction, intracellular administration of IBMX caused only marginal
changes in the baseline holding current, although the current elicited
by light flashes was enhanced. This suggests that, although PDE
activity may be present in ciliary photoreceptors, it seems not to be
regulated by light. It is interesting to note that, in a recent study
of "on" bipolar cells, it also was found that IBMX did not affect
adversely the cGMP-mediated metabotropic response to glutamate,
indicating that a PDE may not be required for transduction in that
system either (Nawy, 1999). Surprisingly, in the presence of IBMX the
time course of the light response was not prolonged significantly. One
would have to conclude that cGMP hydrolysis is not the main
rate-limiting step in the fall of the photocurrent after a flash. The
simplest tentative explanation could be that diffusion of cGMP out of
the minute ciliary appendages (~1 µM) into the much
larger cytosolic volume of the cell body (>2000×) may account for
much of the fall in cGMP concentration in the region near the
light-dependent channels. Attempts to clarify the identity of the PDE
of Pecten photoreceptors by pharmacological means were
unsuccessful. In particular, intracellular administration of selective
inhibitors of some well characterized cGMP-specific PDEs (types 5 and
6) failed to alter membrane current, both in the dark and after
illumination. One must recognize, however, that recently discovered
PDEs (Soderling et al., 1998, 1999) bring the total number of
subclasses of cyclic nucleotide phosphodiesterases to at least 10, and
for several of them no specific antagonists have yet been found.
The depression of the light response by extracellular
dipyridamole and IBMX was unexpected, given the robust and well
documented observation that cGMP analogs activate the light-dependent
K+ conductance. Closer scrutiny of this
phenomenon led to the conclusion that it was attributable to a direct
antagonism on the light-sensitive conductance, unrelated to any effect
on PDE. Precedents for channel block by these compounds have appeared
previously in the literature; in Limulus ventral
photoreceptors IBMX blocks voltage-gated
K+ channels (Corson et al., 1979), whereas
dipyrydamole has been claimed to block
Cl channels in cultured astrocytes
(Sanchez-Olea et al., 1993). Our observations thus provide a
clarification for a recent brief communication that, on the basis of
the reduction of the photoresponse by extracellular dipyrydamole and
IBMX in hyperpolarizing photoreceptors of a related species of scallop,
suggested that a decrease in cGMP may underlie the visual
excitation process (Shimatani and Katagiri, 1997).
The results of the present study lend support to an alternative scheme,
according to which light-triggered activation of the G-protein is
coupled to stimulation of a GC to produce the increase in cGMP required
to open light-dependent channels. In agreement with this notion, the GC
inhibitor LY83583 consistently and reversibly depressed the
photoresponse in a dose-dependent manner. By contrast, no inhibition by
this compound was detected when the light-sensitive channels were
opened directly by 8-Br-cGMP, demonstrating that the site of action of
LY83583 is upstream in the cascade. GCs are broadly grouped in two
categories: soluble, which are regulated by NO (for review, see Hobbs,
1997), and particulate or membrane. LY83583 has been known to target
both soluble (Kontos and Wei, 1993) and membrane GCs (Clemo et al.,
1992). However, in Pecten ciliary photoreceptors the
manipulations designed to interfere with NO synthase produced no
effect, arguing against a NO pathway that may impinge onto cGMP
signaling. Moreover, ODQ, a selective antagonist of the NO-sensitive
GC, failed to alter the light response, and application of YC-1, a
specific activator of NO-sensitive GC, produced no effect on the basal
membrane conductance. Taken together, these results seem to rule out
the involvement of a soluble GC in phototransduction; by exclusion, a
membrane GC may be implicated. Several membrane GCs have an
extracellular receptor domain that confers them responsiveness to
ligands such as atrial natriuretic peptide or pheromones (for review,
see Wedel and Garbers, 1997). Other members of this class interact with
regulatory proteins via the membrane-proximal stretch of the cytosolic
domain and therefore can play a role in intracellular signaling. In
particular, the GCs found in the outer segment of vertebrate rods bind
GC-activating proteins (GCAPs) (for review, see Polans et al., 1996),
which can stimulate catalytic activity in a Ca-dependent manner, a
mechanism crucially implicated in light adaptation and dark current
recovery. It is clear, however, that in the case of Pecten
different functional properties would be expected, because previous
evidence argues against any critical role of calcium ions in
phototransduction (Gomez and Nasi, 1995) or its modulation (Gomez and
Nasi, 1997b). The required linkage of rhodopsin stimulation to GC
activity, whether direct or indirect, remains to be elucidated, because little is known about mechanisms that may couple G-proteins to GCs, and
clues are only beginning to emerge in other systems. For example, in
Dictyostelium the chemotactic response triggered by
extracellular cAMP has been shown to be mediated by a G-protein that
stimulates a membrane GC, causing an elevation of cGMP levels (Kuwayama
and Van Haastert, 1998). A GC/atrial natriuretic factor receptor in the
plasma membrane of murine Leydig tumor cells also appears to be
regulated by Gs/Gi (Khurana
and Pandey, 1995). Although there are no precedents for light
regulation of a GC mediating the generation of the receptor potential
in retinal receptors, a similar mechanism recently has been proposed to
operate in the extra-ocular light-sensitive neuron of a mollusk (Nishi
and Gotow, 1998); moreover, such a scheme parallels the well documented
transduction process of other sensory cells of ciliary origin, such as
the neurons of the olfactory epithelia where odorants activate an adenylate cyclase to bring about an increase in cAMP (Pace et al.,
1985). The notion that a Go mediates light
transduction in Pecten ciliary photoreceptors by stimulating
a GC would place these cells in a novel, separate subgroup among visual
receptors, which may share significant similarities with chemoreceptor cells.
| |
FOOTNOTES |
Received Jan. 31, 2000; revised April 28, 2000; accepted May 3, 2000.
This work was supported by National Institutes of Health Grant
RO1-EY07559.
Correspondence should be addressed to Dr. Maria del Pilar Gomez,
Department of Physiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. E-mail: mpgomez{at}bu.edu.
| |
REFERENCES |
-
Andrade R
(1994)
Infusion of guanine nucleotides through recording electrodes for studies on G-protein regulation of ion currents and channels.
Methods Enzymol
238:348-356[Web of Science][Medline].
-
Baer KM,
Saibil HR
(1988)
Light- and GTP-activated hydrolysis of phosphatidylinositol bisphosphate in squid photoreceptor membranes.
J Biol Chem
263:17-20[Abstract/Free Full Text].
-
Barnette MS,
Daly R,
Weiss B
(1983)
Inhibition of calmodulin activity by insect venom peptides.
Biochem Pharmacol
32:2929-2933[Web of Science][Medline].
-
Baylor DA,
Hodgkin AL
(1974)
Changes in time scale and sensitivity in turtle photoreceptors.
J Physiol (Lond)
242:729-758[Abstract/Free Full Text].
-
Beavo J
(1995)
Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms.
Physiol Rev
75:725-748[Abstract/Free Full Text].
-
Breitwieser GE,
Szabo G
(1988)
Mechanism of muscarinic receptor-induced K+ channel activation as revealed by hydrolysis-resistant GTP analogues.
J Gen Physiol
91:469-493[Abstract/Free Full Text].
-
Brown JE,
Blinks JR
(1974)
Changes in intracellular free calcium concentration during illumination of invertebrate photoreceptors.
J Gen Physiol
64:643-665[Abstract/Free Full Text].
-
Brown JE,
Rubin LJ
(1984)
A direct demonstration that inositol trisphosphate induces an increase in intracellular calcium in Limulus photoreceptors.
Biochem Biophys Res Commun
125:1137-1142[Web of Science][Medline].
-
Brown JE,
Watkins DC,
Malbon CC
(1987)
Light-induced changes in the content of inositol phosphate in squid (Loligo pealei) retina.
Biochem J
247:293-297[Web of Science][Medline].
-
Carty DJ
(1994)
Pertussis toxin-catalyzed ADP-ribosylation of G-proteins.
Methods Enzymol
237:63-71[Web of Science][Medline].
-
Clemo HF,
Feher JJ,
Baumbarten CM
(1992)
Modulation of rabbit ventricular cell volume and Na+/K+/2Cl cotransport by cGMP and atrial natriuretic factor.
J Gen Physiol
100:89-114[Abstract/Free Full Text].
-
Corson DW,
Fein A,
Schmidt J
(1979)
Two effects of phosphodiesterase inhibitors on Limulus ventral photoreceptors.
Brain Res
176:365-368[Web of Science][Medline].
-
Devary O,
Heichal O,
Blumenfeld A,
Cassel D,
Suss E,
Barash S,
Rubinstein CT,
Minke B,
Selinger Z
(1987)
Coupling of photoexcited rhodopsin to inositol phospholipid hydrolysis in fly photoreceptors.
Proc Natl Acad Sci USA
84:6939-6943[Abstract/Free Full Text].
-
Eckstein R,
Cassel D,
Levkovitz H,
Sellinger Z
(1979)
Guanosine 5'-O-(2-thiodiphosphate): an inhibitor of adenylate cyclase stimulation by guanine nucleotides and fluoride ions.
J Biol Chem
254:9829-9834[Free Full Text].
-
Fuortes MGF,
Hodgkin AL
(1964)
Changes in time scale and sensitivity in the ommatidia of Limulus.
J Physiol (Lond)
172:239-263.
-
Garthwaite J,
Southam E,
Boulton CL,
Nielsen EB,
Schmidt K,
Mayer B
(1995)
Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one.
Mol Pharmacol
48:184-188[Abstract].
-
Gillespie PG,
Beavo JA
(1989)
Inhibition and stimulation of photoreceptor phosphodiesterases by dipyridamole and M&B 22,948.
Mol Pharmacol
36:773-781[Abstract].
-
Gomez M,
Nasi E
(1994a)
The light-sensitive conductance of hyperpolarizing invertebrate photoreceptors: a patch-clamp study.
J Gen Physiol
103:939-956[Abstract/Free Full Text].
-
Gomez M,
Nasi E
(1994b)
Blockage of the light-sensitive conductance in hyperpolarizing photoreceptors of the scallop. Effects of tetraethylammonium and 4-aminopyridine.
J Gen Physiol
104:487-505[Abstract/Free Full Text].
-
Gomez M,
Nasi E
(1995)
Activation of light-dependent potassium channels in ciliary invertebrate photoreceptors involves cGMP but not the IP3/Ca cascade.
Neuron
15:607-618[Web of Science][Medline].
-
Gomez M,
Nasi E
(1996)
Ion permeation through light-activated channels in rhabdomeric photoreceptors: role of divalent cations.
J Gen Physiol
107:715-730[Abstract/Free Full Text].
-
Gomez M,
Nasi E
(1997a)
Antagonists of the cGMP-gated conductance of vertebrate rods block the photocurrent in scallop ciliary photoreceptors.
J Physiol (Lond)
500:367-378[Abstract/Free Full Text].
-
Gomez M,
Nasi E
(1997b)
Light adaptation in Pecten hyperpolarizing photoreceptors: insensitivity to Ca manipulations.
J Gen Physiol
109:371-384[Abstract/Free Full Text].
-
Gorman ALF,
McReynolds JS
(1978)
Ionic effects on the membrane potential of the hyperpolarizing photoreceptors in scallop retina.
J Physiol (Lond)
275:345-355[Abstract/Free Full Text].
-
Higashijima T,
Uzu S,
Nakajima T,
Ross EM
(1988)
Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G-proteins).
J Biol Chem
263:6491-6494[Abstract/Free Full Text].
-
Hobbs AJ
(1997)
Soluble guanylate cyclase: the forgotten sibling.
Trends Pharmacol Sci
18:484-491[Medline].
-
Holz GG,
Rane SG,
Dunlap K
(1986)
GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels.
Nature
319:670-672[Medline].
-
Kaslow HR,
Burns DL
(1992)
Pertussis toxin and target eukaryotic cells: binding, entry, and activation.
FASEB J
6:2684-2690[Abstract].
-
Khurana ML,
Pandey KN
(1995)
Catalytic activation of guanylate cyclase/atrial natriuretic factor receptor by combined effects of ANF and GTP--S in plasma membranes of Leydig tumor cells: involvement of G-proteins.
Arch Biochem Biophys
316:392-398[Web of Science][Medline].
-
Kojima D,
Terakita A,
Ishikawa T,
Tsukahara Y,
Maeda A,
Shichida Y
(1997)
A novel Go-mediated phototransduction cascade in scallop visual cells.
J Biol Chem
272:22979-22982[Abstract/Free Full Text].
-
Kontos HA,
Wei EP
(1993)
Hydroxyl radical-dependent inactivation of guanylate cyclase in cerebral arterioles by methylene blue and by LY83583.
Stroke
24:427-434[Abstract/Free Full Text].
-
Kuwayama H,
Van Haastert PJM
(1998)
Chemotactic and osmotic signals share a cGMP transduction pathway in Dictyostelium discoideum.
FEBS Lett
424:248-252[Web of Science][Medline].
-
Lee YJ,
Shah S,
Suzuki E,
Zars T,
O'Day PM,
Hyde DR
(1994)
The Drosophila dgq gene encodes a G-protein that mediates phototransduction.
Neuron
13:1143-1157[Web of Science][Medline].
-
Leinders-Zufall T,
Zufall F
(1995)
Block of cyclic nucleotide-gated channels in salamander olfactory receptor neurons by the guanylyl cyclase inhibitor LY83583.
J Neurophysiol
74:2759-2762[Abstract/Free Full Text].
-
McReynolds JS
(1976)
Hyperpolarizing photoreceptors in invertebrates.
In: Neural principles in vision (Zettler F,
Weiler R,
eds), pp 394-409. Berlin: Springer.
-
Miller WH
(1958)
Derivatives of cilia in the distal sense cells of the retina of Pecten.
J Biophys Biochem Cytol
4:227-228[Free Full Text].
-
Nasi E
(1991)
Electrophysiological properties of isolated photoreceptors from the eye of Lima scabra.
J Gen Physiol
97:17-34[Abstract/Free Full Text].
-
Nawy S
(1999)
The metabotropic receptor mGluR6 may signal through Go, but not phosphodiesterase, in retinal bipolar cells.
J Neurosci
19:2938-2944[Abstract/Free Full Text].
-
Nawy S,
Jahr CE
(1990)
Suppression by glutamate of cGMP-activated conductance in retinal bipolar cells.
Nature
346:269-271[Medline].
-
Nawy S,
Jahr CE
(1991)
CGMP-gated conductance in retinal bipolar cells is suppressed by the photoreceptor transmitter.
Neuron
7:677-683[Web of Science][Medline].
-
Nishi T,
Gotow T
(1998)
Light-increased cGMP and K+ conductance in the hyperpolarizing receptor potential of Onchidium extra-ocular photoreceptors.
Brain Res
809:325-336[Web of Science][Medline].
-
Pace U,
Hanski E,
Salomon Y,
Lancet D
(1985)
Odorant-sensitive adenylate cyclase may mediate olfactory reception.
Nature
316:255-258[Medline].
-
Polans A,
Baehr W,
Palczewski K
(1996)
Turned on by Ca2+: the physiology and pathology of Ca2+-binding proteins in the retina.
Trends Neurosci
19:547-554[Web of Science][Medline].
-
Sanchez-Olea R,
Peña C,
Moran J,
Pasantes-Morales H
(1993)
Inhibition of volume regulation and efflux of osmoregulatory amino acids by blockers of Cl transport in cultured astrocytes.
Neurosci Lett
156:141-144[Web of Science][Medline].
-
Schmidt MJ,
Sawyer BD,
Truex LL,
Marshall WS,
Fleisch JH
(1985)
LY83583: an agent that lowers intracellular levels of cyclic guanosine 3',5'-monophosphate.
J Pharmacol Exp Ther
232:764-769[Abstract/Free Full Text].
-
Schultz C,
Vaskinn S,
Kildalsen H,
Sager G
(1998)
Cyclic AMP stimulates the cyclic GMP egression pump in human erythrocytes: effects of probenecid, verapamil, progesterone, theophylline, IBMX, forskolin, and cyclic AMP on cyclic GMP uptake and association in inside-out vesicles.
Biochemistry
37:1161-1166[Medline].
-
Scott K,
Becker A,
Sun Y,
Hardy R,
Zuker C
(1995)
Gq -protein function in vivo: genetic dissection of its role in photoreceptor cell physiology.
Neuron
15:919-927[Web of Science][Medline].
-
Shiells RA,
Falk G
(1990)
Glutamate receptors of rod bipolar cells are linked to a cyclic GMP cascade via a G-protein.
Proc R Soc Lond [Biol]
242:91-94[Medline].
-
Shimatani Y,
Katagiri Y
(1997)
Phototransduction in scallop hyperpolarizing photoreceptors involves the hydrolysis of cyclic GMP.
Invest Ophthalmol Vis Sci
38:S23.
-
Soderling SH,
Bayuga SJ,
Beavo JA
(1998)
Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases.
J Biol Chem
273:15553-15558[Abstract/Free Full Text].
-
Soderling SH,
Bayuga SJ,
Beavo JA
(1999)
Isolation and characterization of a dual-substrate phosphodiesterase gene family: PDE10A.
Proc Natl Acad Sci USA
96:7071-7076[Abstract/Free Full Text].
-
Suh B-C,
Song S-K,
Kim Y-K,
Kim K-T
(1996)
Induction of cytosolic Ca2+ elevation mediated by MAS-7 occurs through membrane pore formation.
J Biol Chem
271:32753-32759[Abstract/Free Full Text].
-
Suzuki T,
Narita K,
Yoshihara K,
Nagai K,
Kito Y
(1995)
Phosphatidyl inositol-phospholipase C in squid photoreceptor membrane is activated by stable metarhodopsin via GTP-binding protein, Gq.
Vision Res
35:1011-1017[Web of Science][Medline].
-
Szuts EZ,
Woods SF,
Reid MS,
Fein A
(1986)
Light stimulates the rapid formation of inositol trisphosphate in squid retinas.
Biochem J
240:929-932[Web of Science][Medline].
-
Tamura M,
Nogimori K,
Yajima M,
Ase K,
Ui M
(1983)
A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells.
J Biol Chem
258:6756-6761[Abstract/Free Full Text].
-
Tanimura A,
Matsumoto Y,
Tojyo Y
(1991)
Mastoparan increases membrane permeability in rat parotid cells independently of action on G-proteins.
Biochem Biophys Res Commun
177:802-808[Web of Science][Medline].
-
Vardi N,
Matesic DF,
Manning DR,
Liebman PA,
Sterling P
(1993)
Identification of a G-protein in depolarizing rod bipolar cells.
Vis Neurosci
10:473-478[Web of Science][Medline].
-
Wedel BJ,
Garbers DL
(1997)
New insights on the functions of the guanylyl cyclase receptors.
FEBS Lett
410:29-33[Web of Science][Medline].
-
Xiong W-H,
Solessio EC,
Yau K-W
(1998)
An unusual cGMP pathway underlying depolarizing light responses of the vertebrate parietal-eye photoreceptor.
Nat Neurosci
1:359-365[Web of Science][Medline].
-
Yau K-W,
Baylor DA
(1989)
Cyclic GMP-activated conductance of retinal photoreceptor cells.
Annu Rev Neurosci
12:289-327[Web of Science][Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20145254-10$05.00/0
This article has been cited by other articles:
|
|
|
|
 
Y. Shichida and T. Matsuyama
Evolution of opsins and phototransduction
Phil Trans R Soc B,
October 12, 2009;
364(1531):
2881 - 2895.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. R. Ribeiro and J. C. McNamara
Cyclic Guanosine Monophosphate Signaling Cascade Mediates Pigment Aggregation in Freshwater Shrimp Chromatophores
Biol. Bull.,
April 1, 2009;
216(2):
138 - 148.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Koyanagi, K. Takano, H. Tsukamoto, K. Ohtsu, F. Tokunaga, and A. Terakita
Jellyfish vision starts with cAMP signaling mediated by opsin-Gs cascade
PNAS,
October 7, 2008;
105(40):
15576 - 15580.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D.-G. Luo, T. Xue, and K.-W. Yau
How vision begins: An odyssey
PNAS,
July 22, 2008;
105(29):
9855 - 9862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. C. Plachetzki and T. H. Oakley
Key transitions during the evolution of animal phototransduction: novelty, "tree-thinking," co-option, and co-duplication
Integr. Comp. Biol.,
November 1, 2007;
47(5):
759 - 769.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-Y. Su, D.-G. Luo, A. Terakita, Y. Shichida, H.-W. Liao, M. A. Kazmi, T. P. Sakmar, and K.-W. Yau
Parietal-eye phototransduction components and their potential evolutionary implications.
Science,
March 17, 2006;
311(5767):
1617 - 1621.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-N. Liu, S.-L. Pan, C.-Y. Peng, J.-H. Guh, D.-M. Huang, Y.-L. Chang, C.-H. Lin, H.-C. Pai, S.-C. Kuo, F.-Y. Lee, et al.
YC-1 [3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl Indazole] Inhibits Neointima Formation in Balloon-Injured Rat Carotid through Suppression of Expressions and Activities of Matrix Metalloproteinases 2 and 9
J. Pharmacol. Exp. Ther.,
January 1, 2006;
316(1):
35 - 41.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. del Pilar Gomez and E. Nasi
On the Gating Mechanisms of the Light-dependent Conductance in Pecten Hyperpolarizing Photoreceptors: Does Light Remove Inactivation in Voltage-dependent K Channels?
J. Gen. Physiol.,
April 25, 2005;
125(5):
455 - 464.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. del Pilar Gomez and E. Nasi
Calcium-Independent, cGMP-Mediated Light Adaptation in Invertebrate Ciliary Photoreceptors
J. Neurosci.,
February 23, 2005;
25(8):
2042 - 2049.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. Isoldi, M. D. Rollag, A. M. d. L. Castrucci, and I. Provencio
Rhabdomeric phototransduction initiated by the vertebrate photopigment melanopsin
PNAS,
January 25, 2005;
102(4):
1217 - 1221.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Dhingra, E. Faurobert, N. Dascal, P. Sterling, and N. Vardi
A Retinal-Specific Regulator of G-Protein Signaling Interacts with G{alpha}o and Accelerates an Expressed Metabotropic Glutamate Receptor 6 Cascade
J. Neurosci.,
June 23, 2004;
24(25):
5684 - 5693.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Gotow and T. Nishi
Light-dependent K+ Channels in the Mollusc Onchidium Simple Photoreceptors Are Opened by cGMP
J. Gen. Physiol.,
September 30, 2002;
120(4):
581 - 597.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. F. Vanhauwe, T. O. Thomas, R. D. Minshall, C. Tiruppathi, A. Li, A. Gilchrist, E.-j. Yoon, A. B. Malik, and H. E. Hamm
Thrombin Receptors Activate Go Proteins in Endothelial Cells to Regulate Intracellular Calcium and Cell Shape Changes
J. Biol. Chem.,
September 6, 2002;
277(37):
34143 - 34149.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Piccoli, M. del Pilar Gomez, and E. Nasi
Role of protein kinase C in light adaptation of molluscan microvillar photoreceptors
J. Physiol.,
September 1, 2002;
543(2):
481 - 494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
