Although several second messengers are known to
be involved in invertebrate photoresponses, the mechanism underlying
invertebrate phototransduction remains unclear. In the present study,
brief injection of inositol trisphosphate into Hermissenda
photoreceptors induced a transient Na+ current followed by
burst activity, which accurately reproduced the native photoresponse.
Injection of Ca2+ did not induce a significant change in
the membrane potential but potentiated the native photoresponse.
Injection of a Ca2+ chelator decreased the response
amplitude and increased the response latency. Injection of cGMP induced
a Ca2+-dependent, transient depolarization with
a short latency. cAMP injection evoked
Na+-dependent action potentials without a rise
in membrane potential. Taken together, these results suggest that
phototransduction in Hermissenda is mediated by
Na+ channels that are directly activated by inositol
trisphosphate without mobilization of cytosolic Ca2+.
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INTRODUCTION |
There is abundant evidence that the phosphoinositide cascade is
involved in invertebrate phototransduction; however, the specific roles
of inositol trisphosphate
(IP3)1 and/or
Ca2+ in visual excitation have not yet been established
(1-3). The involvement of phosphatidylinositol 4,5-bisphosphate
(PIP2) hydrolysis by phospholipase C (PLC) in the
generation of IP3 in photoreceptors of Limulus
(4, 5), squid (6-8), Drosophila (9-12), and Hermissenda (13) is supported by several lines of
biochemical and biophysical evidence. In particular, the photoresponse
in Drosophila was found to be absolutely dependent on PLC
activity. Until recently, it was thought that IP3 is the
second messenger involved in the photoresponse in invertebrates because
a brief injection of IP3 mimicked the quantal response to
light (4, 5) and IP3-induced Ca2+ release
resulted in membrane depolarization in the Limulus ventral photoreceptor (14) by activating Ca2+/Na+
exchange (15, 16).
More recent evidence, however, indicates that the mechanism is more
complex. For example, many contradictions arise if
Ca2+/Na+ exchange is assumed to be the only
mechanism involved in phototransduction: 1) the light response precedes
the rise in Ca2+ (1), 2) excitation occurs in the absence
of an IP3-induced increase in intracellular
Ca2+ in Limulus (17), 3) abrupt Ca2+
elevation using caged Ca2+ fails to activate any channels
in Drosophila photoreceptor membrane (18), and 4) the
Drosophila mutant deficient in IP3 receptors still produces the native photoresponse (19). Furthermore, cGMP induces
an inward current in photoreceptor cells in Limulus (20) and
Drosophila (21) and activates channels directly in excised patches of molluscan microvilli membrane (1, 22). It is still uncertain, however, whether intracellular cGMP concentration is altered by light (23-25). Adding to the complexity of the system is
the possibility of second messenger cross-talk between IP3, Ca2+, and cyclic nucleotides to activate
intracellular chemical cascades (26).
Hermissenda provide an ideal animal model in which to
examine invertebrate visual transduction mechanisms. The photoreceptors are relatively large (50 µm in diameter) and are well characterized electrophysiologically (27-35). They possess two types of
photoreceptors: A and B (28, 29). Photoreceptor B responds to a bright
flash with a complex potential change involving an initial
depolarization, a hyperpolarization, and a depolarizing tail,
corresponding to a rapid and transient Na+ conductance
increase, a slower increase in K+ permeability, and a
delayed decrease in K+ permeability, respectively (32). The
initial Na+ current appears to be the primary
phototransduction event because 1) following light adaptation, light
flashes produce only the hyperpolarization component (32) and 2) the
hyperpolarization is Ca2+-dependent (33) and
Hermissenda photoreceptors respond to single photons even in
TTX containing, low Ca2+ high Mg2+ solution
(34). Our previous work also indicates that the photoresponse may be
generated through a TTX-resistant Na+ channel (36).
The present study focuses on investigating the second messengers
underlying the initial Na+ current component of the
photoresponse. Recently, channels directly gated by IP3
have been described in other cell lines (37, 38), including
Na+ channels in rat megakaryocytes (39). This raises the
possibility that the native photoresponse in Hermissenda is
generated by an IP3-gated Na+ channel. To
further characterize the role of IP3 and the possible role
of cGMP, cAMP, and Ca2+ in invertebrate phototransduction,
the effects of injection of these second messengers were examined
electrophysiologically in Type B photoreceptors of
Hermissenda.
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EXPERIMENTAL PROCEDURES |
Electrophysiological Assay--
Hermissenda
crassicornis obtained from the Sea Life Supply (Sand City, CA)
were maintained in artificial sea water (ASW) at 14 °C. Type B
photoreceptors were dissected in ASW (430 mM NaCl, 10 mM KCl, 50 mM MgCl2, 10 mM CaCl2, and 10 mM Tris-HCl, pH
7.4). Dissection and intracellular recording were performed as
described previously (13). cGMP, cAMP, and IP3 (Sigma) were
dissolved in intracellular solution (67 mM sodium acetate,
400 mM potassium gluconate, 10 mM
MgCl2, 10 mM HEPES, 1.5 mM
CaCl2, 1.5 mM EGTA, pH 7.4) at a concentration
of 1 mM. Isobutylmethylxanthine (IBMX) was dissolved in
Me2SO and diluted with intracellular solution to 1 mM. The intracellular solution alone or with
Me2SO had no effect on the photoresponse. A microelectrode
containing the drug was inserted into the photoreceptor cell, and the
drug was injected using a pressure injector (Medical System, Greenvale,
NY) at 30.0 p.s.i. for 200 ms. In some experiments the drug was
iontophoresed into the photoreceptor for at least 3 min with DC current
of 2.5 nA, after 10 min of dark adaptation. A photoresponse was
elicited every 90 s during the entire experimental period to keep
the adaptation level constant. Unattenuated light of 34.0 µW/cm2 at 550 nm delivered from a halogen lamp source was
administered to the underside of the preparation.
Assay of PIP2 Breakdown and PLC
Activity--
Isolated Hermissenda eyes were incubated in
10 µl of ASW containing 10 µCi of [3H]inositol
(Amersham International) in darkness at 23 °C for 16 h. Then
one eye of each pair was exposed to light for 30 s. The incubation
was stopped by adding chloroform/methanol/12 N HCl (200:100:0.75), and PIP2 standard was added as a carrier
and to aid in the identification of the lipids. Phospholipids were
extracted and separated by thin layer chromatography as described
previously (9). Lipids were visualized with I2 vapor, and
appropriate bands were scraped and counted. PIP2-PLC
activity was assayed using the particulate fraction obtained from the
Hermissenda circumesophageal nervous system as described
previously (13).
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RESULTS |
Comparison between the Photoresponse and the
IP3-induced Response--
The IP3-evoked
response was highly dependent on the distance between the injection
site and the rhabdomeric membrane, the light-sensitive region (Fig.
1a). Immediately after
IP3 was injected into a microvilli of the Type B
photoreceptor close to the rhabdomeric membrane, a large transient
response was observed (Fig. 1a). No response was observed
after injection approximately 10 µm away from the photosensitive
region. The latency of the photoresponse decreased with increasing
light intensity (Fig. 1b). With low light intensity, the
rate of rise and the peak amplitude of the photoresponse were low. With
increased light intensity, a more complex response was observed,
consisting of an initial depolarization, a hyperpolarization, and a
depolarizing tail, consistent with previous reports (32). The first
component was because of a rapid and transient Na+
conductance increase, the hyperpolarization was because of a slow
increase in K+ permeability, and the depolarizing tail was
because of a slow decrease in K+ permeability (33). The
Na+ current was a graded response, consistent with previous
observations in Hermissenda (36) and photoreceptors of other
species (40, 41). The spontaneous firing activity superimposed on the
photoresponse is known to arise from the distal portion of the axon and
is not involved in the visual transduction process (31). Many
characteristics of the response to IP3 injection were
similar to the response to a relatively weak light flash, including the
latency and the generation of action potentials (Fig. 1c).
Simultaneous light flash and IP3 injection were additive
(Fig. 1d). Furthermore, both responses were completely
inhibited upon removal of extracellular Na+ (Fig.
1d). Similar to the native photoresponse, the
IP3-evoked response was not dependent on the extracellular
Ca2+ concentration (data not shown). Successive injection
of IP3 resulted in a broadening and a decrease in the
amplitude of the first peak and a higher frequency of spontaneous
firing. This response mimicked the photoresponse to flickering light
stimulation (Fig. 1e). Flickering light stimulation elicited
an increase in the Na+ conductance and a decrease in the
hyperpolarization.

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Fig. 1.
Light- and IP3-induced responses
from the Type B photoreceptor of Hermissenda. a,
schematic drawing of an eye composed of five photoreceptors with the
positions of the electrodes. The rhabdomeric membranes are indicated by
the black portions. The lightly shaded portion
indicates the Type B photoreceptor. IP3 injections were
applied via electrodes placed in microvilli of the Type B photoreceptor
at position 1, close to the rhabdomeric membrane, and position 2, approximately 10 µm away. The traces are electrical
recordings of the response of the photoreceptor to IP3
injection. b, photoresponses to increasing intensities of
light (unattenuated 200-ms light flash of 34 µW/cm2 at
550 nm) from 6 log to 1 log units. c, responses of the
same cell to a light flash and an IP3 injection. The
intensity of light was adjusted to obtain a response amplitude
comparable with that of the response to the IP3 injection.
The duration of both the light flash and the IP3 injection
was 200 ms; the former was controlled by an electromagnetic shutter,
and the latter was controlled by a pressure injector with a monitor of
the pressure at the end of the electrode. d, the
IP3-induced response (dotted line) and the
response to light were additive. The additive response
(continuous line) was obtained by a simultaneous
IP3 injection and light flash, of which the intensity was
attenuated to produce a small photoresponse. Note that the
IP3-induced response was abolished in Na+-free
ASW. e, the response to repetitive injection of
IP3 mimicked the response to flickering light (bottom
panel).
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Effects of Ca2+ on the Photoresponse--
To examine
whether a transient increase in intracellular Ca2+
activated the visual transduction channel, Ca2+ (1 mM) was injected into the photosensitive region of the Type B photoreceptor. In contrast to results obtained in Limulus
(14), injection of Ca2+ alone did not induce any
significant response (Fig.
2a), but when a light flash
was applied to the photoreceptor preinjected with Ca2+, the
amplitude of the initial peak of the photoresponse and the hyperpolarization were potentiated (Fig. 2b). This response
was similar to the response to an intense light flash shown in Fig. lb.

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Fig. 2.
Ca2+ facilitated the native
photoresponse. a, Ca2+ injection alone (1 mM for 200 ms) did not produce a significant response.
b, the light-induced response amplitude increased 1.5 times
compared with control, and a significant after-hyperpolarization
(arrow) appeared after the injection of Ca2+.
The resting membrane level is indicated by a dotted line.
c, the effect of 1 mM BAPTA on the
photoresponse. Photoresponse before BAPTA injection (top
panel), 3 min later (middle panel), and 6 min later
(bottom panel). The initial depolarizing photoresponse
gradually diminished and then reversed to a hyperpolarization.
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When BAPTA (a Ca2+ chelator; 1 mM) was injected
intracellularly prior to a light flash, the latency of the
photoresponse increased, and the amplitude decreased (Fig.
2c) and was eventually completely inhibited (data not
shown). The decrease in the initial peak of the response may be because
of a decline in PLC activity dependent on the levels of cytosolic
Ca2+.
Next we examined the involvement of PLC in the photoresponse by
injecting PLC inhibitors such as aminoglycoside antibiotics and
polyamines. Our previous data indicate that neomycin completely inhibits PLC activity in Hermissenda at concentrations
higher than 0.1 mM and spermine inhibits PLC activity at
concentrations higher than 0.5 mM (13). These suppressive
effects are also carefully reconfirmed in this experiment (data not
shown). Iontophoretic injection of 1 mM neomycin into Type
B photoreceptors caused the response to light to be almost completely
abolished 90 s after the injection without a change in the resting
membrane potential (Fig. 3a).
The amplitude of the initial depolarization recovered gradually but
incompletely. The amplitude of the depolarizing tail did not recover
within 5 min after injection (data not shown) but recovered completely
within 18 min. Gentamicin and higher concentrations of neomycin
produced more prolonged inhibition of the photoresponse (data not
shown). Injection of spermine (1 mM) produced a minor
suppressive effect on the photoresponse without changing the resting
membrane potential (Fig. 3b). The amplitude of the initial
depolarization was suppressed to 50% but recovered to 90% of the
control response within 10 min after the injection (Fig.
3b). Injection of spermidine (1 mM), a weaker
inhibitor of PLC, produced a smaller (less than 40%) inhibition of the
photoresponse (data not shown). Inhibition of the hyperpolarization and
the depolarizing tail of the photoresponse by spermine and spermidine is consistent with the fact that these compounds are
Ca2+-activated K+ channel blockers (42).

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Fig. 3.
Modulation of the native photoresponse to
aminoglycosides, polyamines, and IBMX. a and
b, responses were suppressed by the injection of neomycin
(a, 1 mM) and spermine (b, 1 mM). c, IBMX reduced the amplitude of the
initial depolarization of the photoresponse. The reduced response
completely recovered 540 s after IBMX injection. d,
IBMX reduced the light-induced inward current measured using two
microelectrode voltage clamps (36) at a holding potential of 60 mV.
The numbers on the left represent the time in
seconds after the injection. The bottom panel shows the
recovery of the response at 18, 10, 9, and 9 min from a to
d, respectively.
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IBMX is a well characterized phosphodiesterase inhibitor and also a
potent blocker of PIP2 synthesis (43). Iontophoretic injection of IBMX transiently suppressed the initial depolarization and
the depolarizing tail and was completely reversible within 9 min (Fig.
3c). IBMX also reduced the light-induced Na+
current in a similar manner (Fig. 3d). These results
resemble those obtained from the photoreceptor cells of octopus (43) and Limulus (23). Other inhibitors of phosphoinositide
metabolism, such as lithium and R59022, also decreased the initial
depolarization (13). The order of potency of suppression of the initial
depolarization is as follows: gentamicin > neomycin > LiCl > spermine > R59022 > spermidine.
Effects of Cyclic Nucleotides on the Photoresponse--
Although a
significant change in the concentration of cyclic nucleotides in
invertebrate photoreceptors during light irradiation has not been
observed (25, 43), we tested the possibility that these cyclic
nucleotides are involved in visual transduction (44). Intracellular
pressure injection of 1 mM cAMP generated a train of action
potentials without a significant change in resting membrane potential
(Fig. 4a). Consistent bursts
of action potentials were observed in response to successive injections
of cAMP (Fig. 4b). Successive bursts were not significantly
different from the first burst, indicating that the effects of the
injection were transient, unlike the effects of IP3,
Ca2+, or BAPTA injection. The responses to cAMP injections
were abolished in Na+-free ASW, similar to the response to
IP3 injection (Fig. 4b). The response to cAMP in
Hermissenda also differed from that in Limulus
(44), suggesting further that the phototransduction mechanism in Hermissenda is different from the ventral eye
in Limulus.

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Fig. 4.
Responses induced by 1 mM cAMP
injection. a, comparison between the light- and
cAMP-induced response. cAMP injection evoked a train of action
potentials without a change in resting membrane potential.
b, repetitive injection of cAMP-induced responses that did
not change significantly with successive injections.
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Injection of cGMP-induced transient depolarizing responses that were
dependent on the distance between the photosensitive area and injection
site (Fig. 5a). Interestingly,
only the amplitude of the response, not the latency, was dependent on
this distance. In contrast to previous observations in
Limulus (20) and Drosophila (21), the transient
response to injection of cGMP occurred without any change in the
resting potential. The time course of the cGMP-induced response was
different from that of the native photoresponse; simultaneous
stimulation with cGMP injection and light flash resulted in a
double-peaked response (Fig. 5b).

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Fig. 5.
cGMP-induced responses. a,
cGMP injection evoked a transient depolarization without any generation
of action potentials. The numbers on the left
represent the distance in microns that the injection electrode was
advanced from the penetration site, position 2 of Fig. 1. b,
comparison of the responses to cGMP injection and light flash. The
latency of the response to cGMP was much shorter than that of the
response to light.
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We examined the effect of long term iontophoretic injection of cGMP on
the photoresponse and found that the photoresponse observed after the
injection of cGMP was dramatically reduced compared with the
photoresponse before the injection. The initial depolarization was
suppressed almost completely after a 3-min cGMP injection but then
gradually recovered (Fig. 6a).
This response is consistent with responses recorded previously in
light-adapted Type B photoreceptors (32).

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Fig. 6.
Effects of injected cGMP and extracellular
Na+ and Ca2+ concentration on Hermissenda
photoresponses. a, injection of cGMP reduced the
photoresponse in cells maintained in normal ASW containing 10 mM Ca2+. b, the inhibitory effect of
cGMP was abolished in low Ca2+ (1 mM) ASW.
c, the cGMP-evoked response was unchanged in
Na+-free ASW. d, inhibition of the cGMP-induced
response in Ca2+-free ASW (10 mM
CaCl2 was replaced with equimolar CdCl2).
e, the photoresponse was reduced in Na+-free
ASW. f, the photoresponse was unchanged in
Ca2+-free ASW.
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The effect of cGMP or phosphodiesterase inhibitors on the photoresponse
has been reported to vary with the extracellular Ca2+
concentration (45). Consistent with previous reports (43), we also
found that cumulative cGMP, by pretreatment with cGMP via iontophoretic
injection, dramatically decreased the photoresponse of light-adapted
photoreceptors maintained in normal ASW but enhanced the photoresponse
of cells maintained in low Ca2+ ASW (1 mM; Fig.
6b). The inhibitory effect of cGMP recovered after returning
to normal ASW (data not shown). The cGMP-induced response was found to
be insensitive to changes in extracellular Na+
concentration (Fig. 6c), unlike native photoresponses (Fig.
6e). Moreover, the cGMP-induced response was abolished in
Ca2+-free ASW (Fig. 6d), unlike native
photoresponses, which were independent of extracellular
Ca2+ (Fig. 6f).
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DISCUSSION |
The results of the present study demonstrate for the first time
that IP3 may mediate invertebrate visual transduction,
particularly in Hermissenda. Although cAMP, cGMP, and
Ca2+ have been proposed to activate several types of
channels, injection of these second messenger candidates did not
reproduce the native photoresponse. In contrast, injections of
IP3 produced responses that were very similar to the native
photoresponse, whereas Ca2+ acted as a modulator of the
photoresponse (46).
The hypothesis that IP3 may be the second messenger
involved in visual transduction in Hermissenda is supported
by the results of the present study and by results obtained previously:
1) light flashes result in greater increases in intracellular
IP3 than cyclic nucleotides (25) and 2) inhibition of PLC
activity and reduction of phosphoinositide turnover result in a
reduction in the amplitude of the native photoresponse (13). Although
the primary role of IP3 has been considered to be the
release of Ca2+ from intracellular stores, several
IP3-gated ion channels have been recently characterized in
olfactory cilia (37, 38) and rat megakaryocytes (39), raising the
possibility that IP3 directly activates a transduction
channel in Hermissenda.
The possibility that IP3 may be a second messenger in
Drosophila visual transduction is supported by observations
by Inoue et al. (10) that a reduction of
PIP2-PLC activity is closely correlated with
electrophysiological abnormality in the Drosophila phototransduction mutant norpA (no receptor potential A)
eye. Furthermore, the norpA gene has been shown to encode
PLC (11, 47). Yoshioka et al. (43) have also reported
previously that IBMX inhibits phosphoinositide turnover and that the
photoresponse is dependent on PIP2 hydrolysis by PLC in
squid photoreceptor. The photoresponse in Limulus has also
been shown to be diminished by the injection of the PLC inhibitor
neomycin without affecting the response to IP3 (14) and by
polyamine injection (17). Therefore, we propose that IP3
produced from PIP2 hydrolysis via PLC activation is an
essential component of invertebrate visual transduction.
The question remains about whether IP3 acts directly or via
Ca2+ in Hermissenda visual transduction. In the
present study, injection of Ca2+ into the photoreceptor in
Hermissenda induced no response in darkness, suggesting that
Ca2+ is not directly involved. Furthermore, excitation
occurs even in the absence of an IP3-induced increase in
intracellular Ca2+ in Limulus (17) and in
Drosophila mutants lacking IP3 receptors in
photoreceptor cells (19). It has also been reported that the
photoresponse precedes the rise of intracellular Ca2+ in
Limulus (48, 49). Recently, Hardie (18) reported that a
transient elevation of Ca2+ using a caged Ca2+
compound did not directly excite Drosophila photoreceptors.
These results indicate that Ca2+ is unlikely to be directly
involved in phototransduction in Drosophila, Hermissenda, and Limulus but may act as a
modulator of the photoresponse.
Several previous studies indicate that Ca2+ excites
photoreceptors (50-52) and that the injection of a Ca2+
buffer reduced the photoresponse in Limulus (50). Also, in the present study, reduction of intracellular Ca2+ by a
Ca2+ chelator resulted in a reduction of the photoresponse;
however, the mechanism underlying this response is presently unclear.
It may be because of reduced activation of PLC and a subsequent
decrease in IP3; however, a decrease in protein kinase C
activity cannot be ruled out. According to Thio and Sontheimer (53),
phorbol esters reduced the peak of the TTX-sensitive Na+
current by 25-60% and potentiated the TTX-resistant Na+
current by 60-150%. In the present study, the
Ca2+-induced increase in the photoresponse was 60-80% of
control, which is consistent with the phorbol ester-induced increase in the TTX-resistant Na+ current.
Involvement of cyclic nucleotides in visual transduction is also
unlikely. The cAMP-evoked response was very different from the
light-evoked response; injection of cAMP induced short bursts of
Na+-dependent action potentials. Repeated
injection evoked trains of similar bursts, indicating that these action
potentials may be mediated by fast-inactivating cAMP-sensitive
Na+ channels.
Interestingly, cGMP-evoked responses were similar to light-evoked
responses, but the following differences were observed: the amplitude
of the cGMP-evoked responses depended on the distance from the
rhabdomeric membrane, but the latency of the response was constant for
all trials; the rising phase of the responses to cGMP was faster than
that of responses to light; the cGMP-evoked response depended on
external Ca2+, whereas the light-evoked response depended
on external Na+; and the response induced by long term cGMP
injection and the light-evoked response from light-adapted cells were
very similar. Taken together, these findings indicate that neither cAMP
nor cGMP act as second messengers in invertebrate visual transduction. Thus the results of the present study suggest that the transduction channels of Drosophila and Hermissenda eye may be
IP3-gated Na+ channels. Cloning of
IP3-gated Na+ channels may help clarify the
mechanisms underlying invertebrate visual transduction.