§
From the * Howard Hughes Medical Institute, Department of Neuroscience, and § Department of Ophthalmology, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205;
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki
305, Japan
Biochemical experiments by others have indicated that protein kinase C activity is present in the rod
outer segment, with potential or demonstrated targets including rhodopsin, transducin, cGMP-phosphodiesterase
(PDE), guanylate cyclase, and arrestin, all of which are components of the phototransduction cascade. In particular, PKC phosphorylations of rhodopsin and the inhibitory subunit of PDE (PDE ) have been studied in some detail, and suggested to have roles in downregulating the sensitivity of rod photoreceptors to light during illumination.
We have examined this question under physiological conditions by recording from a single, dissociated salamander rod with a suction pipette while exposing its outer segment to the PKC activators phorbol-12-myristate,13-acetate (PMA) or phorbol-12,13-dibutyrate (PDBu), or to the PKC-inhibitor GF109203X. No significant effect of
any of these agents on rod sensitivity was detected, whether in the absence or presence of a background light, or
after a low bleach. These results suggest that PKC probably does not produce any acute downregulation of rod
sensitivity as a mechanism of light adaptation, at least for isolated amphibian rods.
Phototransduction in retinal rods involves a cGMP signaling cascade (for recent reviews, see Lagnado and
Baylor, 1992; Kawamura, 1993
; Pugh and Lamb, 1993
;
Yarfitz and Hurley, 1994
; Yau, 1994
; Fain et al., 1996
;
Koutalos and Yau, 1996
; Palczewski and Saari, 1997
). In
this process, light isomerizes the visual pigment rhodopsin into an active form, which, via the G protein
transducin, stimulates a cGMP-phosphodiesterase to
lead to an increase in cGMP hydrolysis. In darkness, cytoplasmic cGMP binds to and opens cGMP-activated cation channels on the plasma membrane of the rod's
outer segment (see Yau and Baylor, 1989
; Finn et al.,
1996
). These open channels sustain an inward dark
current to keep the cell partially depolarized and maintain a steady release of glutamate from the rod's synaptic terminal. In the light, the hydrolysis of cGMP leads
to the closure of these channels, producing a membrane hyperpolarization as the light response and reducing the glutamate release from the cell. The turn-off of phototransduction involves several mechanisms, an important one being the phosphorylation of the
photoactivated rhodopsin by a rhodopsin kinase, which
reduces its ability to activate transducin; this is followed
by the binding of a protein called arrestin to the phosphorylated rhodopsin to cap its activity.
For many years, there have also been reports of protein kinase C activity in the rod outer segment (Kapoor
and Chader, 1984; Kelleher and Johnson, 1985
; Hayashi et al., 1987
; Kapoor et al., 1987
; Binder et al.,
1989
; Wolbring and Cook, 1991
), with potential or
demonstrated substrates including rhodopsin (Kelleher and Johnson, 1986
; Newton and Williams, 1991
,
1993a
; Greene et al., 1995
, 1997
; Udovichenko et al.,
1997
), the
-subunit of transducin (Zick et al., 1986
),
the cGMP-phosphodiesterase (PDE)1 (Hayashi et al.,
1991
; Udovichenko et al., 1993
, 1994
, 1996
), guanylate
cyclase (Wolbring and Schnetkamp, 1995
) and arrestin (Weyand and Kühn, 1990
). In particular, the PKC
phosphorylations of rhodopsin and the inhibitory subunit of PDE (PDE
) have been studied in some detail.
The phosphorylation of rhodopsin by PKC is reported
to be triggered or enhanced by light, and involves both
photoactivated rhodopsin and rhodopsin that has not
absorbed any light (Newton and Williams, 1991
, 1993a
,
1993b
; Kelleher and Johnson, 1986
). Because PKC
phosphorylation of rhodopsin also weakens the latter's
ability to activate transducin (Kelleher and Johnson,
1986
), the action of PKC on both photoactivated and nonphotoactivated rhodopsin has been proposed to be
a highly effective mechanism for reducing the light sensitivity of rods, much analogous to the heterologous desensitization of the
-adrenergic receptor through
phosphorylation by protein kinase A and possibly protein kinase C (Newton and Williams, 1993b
). As for the
PDE
, PKC phosphorylation is reported to have several
consequences, including an increased ability of PDE
to inhibit the catalytic subunits PDE
, a decreased ability of transducin to activate the PDE
holoenzyme,
and an accelerated GTPase activity of the transducin
bound to PDE
, all of which would also reduce the sensitivity of rods to light (Udovichenko et al., 1994
, 1996
;
see also Tsuboi et al., 1994a
, 1994b
).
In view of the above biochemical findings, we have undertaken experiments to look for a physiological role of PKC in phototransduction, by recording from single, mechanically dissociated salamander rods and testing the effects of agents that either activate or inhibit PKC. However, we have not found any significant effect of these chemicals on the sensitivity of rods to light under various illumination conditions.
Preparation
Single dissociated rods from the retina of the dark-adapted, larval
tiger salamander (Ambystoma tigrinum) were used. The animals were purchased from Kons Scientific Co., Inc. (Germantown,
WI). The procedure for isolating the cells was as described elsewhere (Nakatani and Yau, 1988a). Briefly, the eyes were removed
from the pithed animal and coronally hemisected. The retina was
peeled from the back half of the eye, divided into several pieces,
and stored under normal Ringer at room temperature for up to
several hours until use. When needed, a piece of retina was finely
chopped with a razor blade under Ringer solution and on a bed
of cured Sylgard elastomer (Dow Corning Corp., Midland, MI),
yielding many dissociated rod photoreceptors with intact outer
and inner segments. These intact cells were selected for recording. The enucleation of the eyes was done in deep red light, and
all other procedures were carried out in infrared light with the
help of an infrared viewing scope or TV system.
Recordings and Optics
The suction-pipette recording technique (Baylor et al., 1979a)
was used to record membrane current from a rod. In all experiments, carried out at room temperature (19-24°C), the inner
segment of the cell was drawn into the pipette for recording
membrane current, and the outer segment was subjected to bath
perfusion (see, for example, Nakatani and Yau, 1989
). The position of the cell was adjusted so that the cilium between the outer
and inner segments was situated near the point of narrowest constriction at the tip lumen. With the electrode resistance typically
around 1-2 Mohm when empty and 5-10 Mohm with a rod in
place, the fraction of membrane current recorded should be
>80% (assuming that the resistance of the empty electrode was
equally distributed between its very tip and the shank). No corrections were made for this imperfect current collection. However, the time course of the light response, as well as the light
sensitivity when expressed as a fractional suppression of the dark
current, should not be affected. All records were stored on an FM
tape recorder and simultaneously digitized on-line on a PC computer. Data acquisition and analysis were carried out using the
pCLAMP software. Low-pass filtering was set at 100 Hz. In figures
where flash-response families are shown, the traces were generally derived from averages of multiple flash trials.
The optical bench design was as previously described (Baylor
et al., 1979a). Diffuse, unpolarized light at 520 nm (peak absorption wavelength for the "red" rods studied here; see Harosi,
1975
) was used in all experiments, incident approximately perpendicular to the longitudinal axis of the recorded cell. Illumination consisted of 8-ms test flashes, 1- or 5-s bleaching light, or
long steps of background light. For sensitivity calculations, we obtained the number of photoisomerized rhodopsin molecules
from light calibrations and an individual cell's effective collecting
area derived from video images. The effective collecting area, A
(µm2), of an outer segment for incident light normal to the longitudinal axis of the outer segment is given by A =
r2l × Q × 2.303
f (Baylor et al., 1979b
), where r and l are the radius and
length of the rod outer segment (in microns), respectively, Q is
the quantum efficiency,
is the transverse specific optical density
of the outer segment, and f is a correction factor depending on
the polarization of the illuminating light. This expression for A is
a linear approximation of an exponential function for absorption, and holds when 2.303
f × 2r << 1, roughly true for tiger-salamander rods (r
4 µm). For unpolarized light used in our
experiments, f is 0.5 (Baylor et al., 1979b
). We have also adopted an
of 0.012 µm
1 at the peak absorption wavelength and a Q of 0.5 (see Harosi, 1975
; Baylor et al., 1979b
; Nakatani and Yau, 1989
).
In the bleaching experiments, the fractional bleach can be calculated as follows. For a bleaching light of intensity I (photons µm2 s
1 at 520 nm) and duration T (s), the number of photoisomerizations (Rh*) is then I × T × A, or I × T ×
r2lQ × 2.303
f = I × T ×
r2l × 6.9 × 10
3, from above. With a rhodopsin concentration in the outer segment of 3.5 mM (Harosi,
1975
), the total number of rhodopsin molecules is
r2l × 2.1 × 106. Thus the fractional bleach is simply I × T × 3.3 × 10
9.
Again, this expression only holds when the percentage of pigment bleach is low, which applies to our experiments.
Perfusion and Solutions
The perfusion system and the recording chamber were as described previously (Nakatani and Yau, 1988a, 1989
). The tip of
the pipette was bent at right angles to the main shank so that during the experiment the long axis of the recorded cell was oriented parallel to the solution flow (Nakatani and Yau, 1988a
,
1989
).
Normal Ringer solution was at pH 7.6 and contained (mM):
110 NaCl, 2.5 KCl, 1.6 MgCl2, 1.0 CaCl2, 5.0 NaHEPES, 5.0 dextrose. The phorbol esters PMA and PDBu (phorbol-12,13-dibutyrate) were purchased from Research Biochemicals International
(Natick, MA). The PKC inhibitor GF109203X {2-[1-(3-dimethyl-aminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide} was purchased
from Tocris Cookson (St. Louis, MO). Stock solutions of the
chemicals were prepared by dissolving in DMSO at 16.2 mM for
PMA, 19.8 mM for PDBu, and 24.2 mM for GF109203X, and
stored at 20°C. Immediately before the experiments, the stock
solutions were diluted to the final concentrations (1 µM for
PMA, 20 µM for PDBu, and 1 or 5 µM for GF109203X) with
Ringer solution. The 0.1% or less DMSO by itself had no effects
in control experiments. The outer segment of a recorded rod was
usually perfused with a chemical-containing solution for >10
min before any effect on light sensitivity was examined. In the literature, this interval is generally more than sufficient for an effect to take place when these chemicals are applied extracellularly at comparable or much lower concentrations to other cell
types (see, for example, Stea et al., 1995
; Gillette and Dacheux,
1996
; Toullec et al., 1991
; Li and Cathcart, 1994
; Qiu and Leslie,
1994
; Ikeuchi et al., 1996
; Poulin et al., 1996
). During the entire
period of sensitivity testing, which could take many minutes, perfusion with the chemical-containing solution was continued. In
experiments where sensitivity was retested after washout of the
chemical, again at least 10 min was allowed after chemical removal before testing of sensitivity began.
PKC Activators
The phorbol esters PMA and PDBu were chosen as
PKC activators because of their potency and general effectiveness in other cell types (see, for example, Stratton et al., 1989; Stea et al., 1995
; Gillette and Dacheux,
1996
). PMA was also reported to be effective in promoting PKC phosphorylation of rhodopsin in biochemical experiments with intact retinae (Newton and Williams,
1991
,1993a).
We first
examined any effect of the two phorbol esters on rod
sensitivity in the absence of background light. Fig. 1 A
shows a family of responses of a salamander rod to
flashes of increasing intensity in normal Ringer, and
Fig. 1 B shows the responses of the same cell to identical flashes after being exposed to 1 µM PMA in the perfusing Ringer. The two families of responses are extremely similar, both in amplitudes (absolute and relative) and kinetics. Results from six cells are averaged
and plotted in the form of peak response-intensity relations in Fig. 1 C, in the presence (filled symbols) and
absence (open symbols) of 1 µM PMA. It is clear that 1 µM PMA had practically no effect on rod sensitivity.
The smooth curve is the Michaelis equation with a half-saturating flash intensity at ~8 Rh*/µm2, which is
broadly similar to previous measurements (Nakatani and Yau, 1989).
Identical experiments were carried out with 20 µM
PDBu. During PDBu application, the dark current generally decreased by ~10%. The reason for this current
decrease is unknown, but the change is opposite to
what would be expected for an increase in desensitization due to activation of PKC. Despite this small reduction in dark current, there was no change in flash sensitivity, based on averaged results from six experiments
(Fig. 2). The dark current reduction was reversible
upon PDBu removal. Previously, a similar inhibitory effect on the dark current by oleoylacetylglycerol, another PKC activator, was reported (Binder et al., 1989).
Occasional effects of PKC activators unconnected with
PKC activity have also been described for other retinal
neurons (Gillette and Dacheux, 1996
).
Flash responses in the presence of background light.
Next we
examined rod sensitivity in the presence of background
light. Three background intensities were chosen, producing steady responses of ~30, 50, and 80% to saturation,
respectively, in order to cover a fairly broad range. Superposed on these backgrounds were incremental flashes
to test for sensitivity. Fig. 3 A shows selected records
from one such experiment in which the effect of PMA
was examined. Each panel in Fig. 3 A corresponds to a
different background light, with the traces showing the
averaged responses to an incremental dim and bright
flash, respectively; the DC level of the traces corresponds to the steady response of the cell to a particular
background light, and the labels a and b indicate control condition and the presence of 1 µM PMA, respectively. Again, no effect of the chemical is evident for either the steady response to background light or the incremental flash response. Fig. 3 B shows averaged data
from five complete experiments, plotted in the form of
incremental flash response-intensity relations under the
three different background lights, as indicated by square, circle, and diamond symbols, respectively. As expected,
the response-intensity relation shifted progressively to
the right with increasing background light. However, at
each background intensity there was no apparent difference in rod behavior whether PMA was absent (Fig. 3 B,
open symbols) or present (Fig. 3 B, filled symbols).
Four experiments with 20 µM PDBu led to the same
conclusion (Fig. 4).
After a bleach
Finally we examined rod sensitivity after
a low bleach because Newton and Williams (1991, 1993a
)
have reported that the phosphorylation of rhodopsin
in the presence of phorbol ester is strongest in intact
rat retina after a
10% bleach. Fig. 5 A shows such an experiment, carried out initially in control condition
(upper trace), and then in the presence of 1 µM PMA
(lower trace). In each condition, a series of flashes at different intensities were delivered to a rod before bleaching, after which an ~2% bleach was affected by a 1-s intense flash at time zero. As a result, the dark current
stayed completely suppressed for seconds before slowly
recovering. During this recovery, dim and bright flashes were again delivered to produce small and saturating
responses for comparison with those elicited before the
bleach. The time course of recovery of the light sensitivity paralleled that of the dark current. After the run
in the presence of PMA, the chemical was removed
from the bath solution and a second control run was repeated (not shown in Fig. 5 A, but see data analysis in
B-D). Fig. 5, B-D show analyzed results averaged from
six cells. Three parameters were measured as a function of time after the bleach: (a) the amplitude of the
dark current, (b) the sensitivity to a dim flash, expressed
as a fractional suppression of the dark current per Rh*,
and (c) the half-recovery time of a (saturated) response
to a bright flash of fixed intensity. For the dark current,
there was a slight indication that the final phase of recovery was more complete when PMA was present (Fig.
5,
) than absent (at least the first run; Fig. 5,
),
which would be consistent with a desensitization effect
induced by hyperactivated PKC. On the other hand,
this was not corroborated by the recoveries of the dim-flash sensitivity and of the half-recovery time of the responses to bright flashes, which appeared similar with
and without PMA.
Four experiments were also carried out with a more
intense bleach, namely ~10%. After such a bleach, the
cells took much longer to recover, but again PMA had
no obvious effect (Fig. 6). We did not carry out still
more intense bleaches because the extremely slow recoveries in these cases would preclude any comparison
between absence and presence of the chemical from
the same cell.
Finally, we performed identical bleach experiments
with 20 µM PDBu, and obtained the same results (Fig. 7).
PKC Inhibitor
We have also tested for possible constitutive PKC activity with an inhibitor of the enzyme. The recently developed PKC inhibitor GF109203X was chosen because of
its high specificity (Toullec et al., 1991).
Fig. 8, A and B shows an experiment with GF109203X
on a dark-adapted rod. Again, the flash response-intensity families in the absence and presence of the drug
were very similar, a conclusion borne out by averaged
results from six cells (Fig. 8 C). Experiments with different background light intensities (Fig. 9) likewise gave
negative results. With 2 and 10% bleaches, the rod dark
current and flash responses recovered perhaps a little
more slowly in the presence of GF109203X (Fig. 10),
which would be consistent with a role of PKC in desensitization under these conditions. However, the difference between absence and presence of the PKC inhibitor was very minor and probably not statistically significant.
The existence of a light-activated, phosphoinositide signaling pathway in the rod outer segment has been a
topic of interest for many years (Ghalayini and Anderson, 1984; Hayashi and Amakawa, 1985
; Brown et al.,
1987
; Millar et al., 1988
). The hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) by PLC produces two second messengers: inositol-1,4,5-trisphosphate (IP3)
and diacylglycerol, with the former leading to intracellular Ca2+ release and the latter activating PKC. The
role of IP3 in the rod outer segment remains elusive because so far there is no evidence of an IP3 receptor in
this location (Peng et al., 1991
; Day et al., 1993
). The
evidence of a diacylglycerol-PKC pathway in the rod
outer segment, on the other hand, is more substantial
(INTRODUCTION). The concentration of PKC in the rod
outer segment is reported to be at least as high as that
of rhodopsin kinase, ~1:1,000 in mole ratio with respect to rhodopsin (Kelleher and Johnson, 1986
; Wolbring and Cook, 1991
). The PKC isozymes identified so
far are grouped into three classes: conventional (
,
I,
II, and
), novel (
,
,
, and
), and atypical (
,
,
,
and µ) isoforms (see, for example, Asaoka et al., 1992
;
Nishizuka, 1992
). Biochemical studies suggest that the
PKC in the rod outer segment is of the conventional
class (i.e., Ca2+ dependent and phorbol ester activatable [see, for example, Kelleher and Johnson, 1986
;
Newton and Williams, 1991
, 1993a
; Greene et al., 1995
;
but see Wolbring and Schnetkamp, 1995
]), and is perhaps the
-isoform (Wolbring and Cook, 1991
; Udovichenko et al., 1993
). This isoform identification in the
rod outer segment, nonetheless, is still tentative and
has not been confirmed immunocytochemically, despite numerous studies employing a variety of isoform-specific antibodies (Wood et al., 1988
; Negishi et al.,
1988
; Suzuki and Kaneko, 1990
; Ghalayini et al., 1991
,
1994
; Usuda et al., 1991
; Zhang et al., 1992
; Kolb et al.,
1993
; Osborne et al., 1992
, 1994
; Koistinaho and Sagar,
1994
; Ohki et al., 1994
). Some of these studies could
have produced negative results because of the failure of
a given antibody to recognize the same protein isoform
across different vertebrate species.
As pointed out in INTRODUCTION, several rod proteins involved in phototransduction have been found
to be substrates of PKC. Many of these studies were
carried out in vitro, so it is difficult to be certain that
the findings are really relevant to the intact tissue. On
the other hand, the light-triggered phosphorylation of
bleached and unbleached rhodopsin by PKC has been
reported in intact retinae of both mammalian and amphibian species (Newton and Williams, 1991, 1993a
,
1993b
; Green et al., 1995, 1997; Udovichenko et al.,
1997
). Furthermore, this phosphorylation by PKC has
been found to reduce the ability of rhodopsin to activate transducin (Kelleher and Johnson, 1986
). Because
the action of PKC is less specific than rhodopsin kinase,
which phosphorylates only photoactivated rhodopsin
(but see below), it has been proposed to provide an effective mechanism for light adaptation, by analogy to
the heterologous desensitization of the
-adrenergic
receptor through PKA phosphorylation (Newton and
Williams, 1991
, 1993a
, 1993b
). To test this idea, we have
examined the effect of hyperactivation of PKC by phorbol esters on the sensitivity of single, intact salamander
rods under three conditions: in the absence and presence of background light, and after a low bleach. The
choice of one of the phorbol esters (PMA) and its concentration (1 µM) was based on the biochemical experiments in intact rat retina (Newton and Williams, 1991
,
1993a
). For the bleaching experiments, the degrees of
bleach (~2 and 10%) we used also broadly matched
those (
10%) reported in the same biochemical experiments to be effective for enhancing the phosphorylation of rhodopsin with PMA. However, in none of
these conditions were we able to detect any significant
effect of PMA on rod sensitivity. The second phorbol
ester we used, PDBu, is an equally potent PKC activator,
and the concentration adopted (20 µM) is up to 100-fold higher than that found effective in other cell types
when applied extracellularly (Stratton et al., 1989
; Stea
et al., 1995
; Gillette and Dacheux, 1996
). However, we
also did not find any effect of PDBu on rod sensitivity.
Because our experiments generally lasted 30 min or
less (longer for some of the bleaching experiments), we could have missed any effects of PKC that developed
beyond these time durations upon illumination. However, this possibility seems unlikely because, most recently, Udovichenko et al. (1997)
have reported that,
in intact frog retina, the phosphorylation of rhodopsin
due to hyperactivation of PKC by phorbol ester is quite
fast and transient, peaking at 10-15 min after light onset, and declining shortly thereafter. This time window
should be well within detection in our experiments.
We have also tested for any possible constitutive PKC
activity on rod sensitivity using a PKC inhibitor. Ideally,
the inhibitor of choice for these experiments would be
a chemical, such as calphostin C, that acts on the regulatory domain of PKC and is highly potent and specific
(Kobayashi et al., 1989). However, the action of calphostin C is light dependent (Bruns et al., 1991
), making it incompatible with the nature of our experiments.
Most other PKC inhibitors act on the catalytic domain
of the enzyme, which shares sequence homology with
other protein kinases, and are thus less specific. Indeed, we have tried several PKC inhibitors of this class
before GF109203X, including H-7 (250 µM), staurosporine (1-10 µM), and chelerythrine (10 µM), and
found these to decrease the dark current substantially
(~30-50%), as well as slow down the kinetics of the
light response (data not shown). Because these inhibitors may also inhibit rhodopsin kinase, we found it difficult to interpret the results. The recently developed
chemical GF109203X is one of the most selective
among PKC inhibitors that act on the kinase's catalytic
site (Toullec et al., 1991
). At the concentration of 1 (and occasionally 5) µM used here, this chemical is
found to be effective in inhibiting PKC activity in other
cell types when applied extracellularly (Toullec et al.,
1991
; Li and Cathcart, 1994
; Qiu and Leslie, 1994
; Ikeuchi et al., 1996
; Poulin et al., 1996
). However, it did not
affect rod sensitivity under the different light conditions described above. Thus, there is no obvious indication of any constitutive PKC activity, in either darkness
or light, that affects phototransduction, at least for isolated rod photoreceptors.
While our experiments strongly suggest that PKC is
unlikely to have a significant role in light adaptation,
they cannot be viewed as absolutely conclusive because
of the negative findings, and because of an inevitable
element of uncertainty associated with pharmacological experiments. However, judging from the degree of
rhodopsin phosphorylation by PKC reported so far, it
would be very surprising if the conclusion should eventually turn out to be any different. This point can be appreciated from the following order-of-magnitude calculations. Using calphostin C as a specific PKC inhibitor,
Udovichenko et al. (1997) most recently estimated that, in intact frog retina, PKC accounted for ~50% of
the overall phosphorylation of rhodopsin induced by illumination. Furthermore, they found that PKC-phosphorylated rhodopsin was dephosphorylated more rapidly than rhodopsin kinase-phosphorylated rhodopsin.
For argument sake, let us assume that rhodopsin kinase
can phosphorylate photoactivated rhodopsin at multiple
residues (though, in reality, probably just one residue is
phosphorylated; see Ohguro et al., 1995
), whereas PKC
phosphorylates rhodopsin only at one residue (Newton
and Williams, 1993a
, 1993b
). Thus, even most optimistically, the maximum stoichiometry of unbleached to
bleached pigment that becomes phosphorylated in the
light is still less than a factor of 10 (but see below). Furthermore, let us assume that the unbleached, but phosphorylated, rhodopsin has negligible transducin-activating ability upon photoisomerization (though, in reality, this ability is only reduced by <50%; see Kelleher
and Johnson, 1986
). Thus, even in the extreme case,
there cannot be more than 10 "inactivated," unbleached
rhodopsin molecules associated with each Rh*. A salamander rod has ~3 × 109 rhodopsin molecules, but its
response already saturates when a steady light effectively produces 105 Rh* at a given instant (see Fig. 2 a in
Nakatani and Yau, 1988b
; and by adopting a typical effective collecting area of 10 µm2 for salamander rods illuminated by unpolarized light, together with an integration time of 1 s for their single-photon response). With this level of Rh*, at most 106 unbleached pigment
molecules would be rendered "inactive" by PKC phosphorylation, leading to a reduction in the photon-capturing ability (or sensitivity) of the cell by no more than
a factor of 10
3, or 0.1%; more realistically, this percentage could be as low as 0.01%. In either case, the
change is well below detectability. With higher steady
light intensities, the fraction of unbleached pigment
that is "inactivated" will increase, but this is immaterial because rods cannot signal after response saturation.
Similar arguments apply to mammalian rods. A complexity not mentioned so far in this paper and not included in the above calculations is the possible phenomenon of so-called "high gain" phosphorylation, in
which rhodopsin kinase supposedly phosphorylates
both bleached and unbleached rhodopsin in the light,
much like the reported action of PKC being evaluated in
this paper, and thought to lead to light adaptation as well
(see, for example, Aton, 1986
; Binder et al., 1990
, 1996
;
Chen et al., 1995
; Dean and Akhtar, 1993
, 1996
; Palczweski, 1997). The existence of this phenomenon has been controversial and, most recently, not confirmed
(Rim et al., 1997
); even if present, however, its contribution to light adaptation also appears insignificant, based
on the most recent experiments (Binder et al., 1996
).
If included in our calculations above, this high gain
phosphorylation will only make the phosphorylation of
rhodopsin by PKC even less significant compared with
that by rhodopsin kinase. The situation could be a little
different for cones, if the same PKC biochemistry exists,
because these cells continue to function at very high
steady light intensities (see, for example, Nakatani and
Yau, 1988b
).
Finally, it might be added that, while several groups
have reported light-activated phosphoinositide hydrolysis in the rod outer segment (see above), this observation is by no means universal (see Gehm and McConnell, 1990; Panfoli et al., 1990
for negative results). The
phosphorylation of rhodopsin by PKC is likewise not an
unequivocal finding (compare Binder et al., 1989
;
Ohguro et al., 1996
). Thus, it seems that even the basic
questions of which in vivo stimuli trigger PKC and
which substrates this enzyme acts on in the rod outer
segment are still unsettled. Because the activation of
PKC can also be enhanced or sustained by phospholipid signaling pathways involving phospholipase A2 or
phospholipase D (Asaoka et al., 1992
; Nishizuka, 1992
),
these alternative pathways perhaps need to be considered and examined. On the other hand, based on our recent immunocytochemical and immunoblotting studies, it appears that G
11 and PLC
4 (or isoforms immunologically identical to them) are present in the rod outer segment (Peng et al., 1997
; but see Ferreira and Pak, 1994
).
These proteins are specific for the phosphatidylinositol-4,5-bisphosphate pathway, suggesting that this pathway is
truly present in this location. The PKC in the rod outer
segment may have nothing to do with the modulation of
phototransduction but affects other cellular functions instead. In this respect, rod photoreceptors differ from
invertebrate rhabdomeric photoreceptors, such as those
in the fly, where PKC has indeed been demonstrated to
be involved in light adaptation (Hardie et al., 1993
).
This difference may lie in the fact that a phosphoinosi-tide pathway is central for phototransduction in fly photoreceptors (see, for example, Hardie and Minke, 1995
; Ranganathan et al., 1995
), whereas a cGMP phototransduction pathway is used in rods.
Address correspondence to Dr. King-Wai Yau, Room 907, Preclinical Teaching Building, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205. Fax: 410-614-3579; E-mail: kwyau{at}welchlink.welch.jhu.edu
Received for publication 30 May 1997 and accepted in revised form 23 July 1997.
1 Abbreviations used in this paper: PDBu, phorbol-12,13-dibutyrate; PDE, phosphodiesterase; Rh*, photoisomerization.We thank Drs. James B. Hurley, George I. King, David J. Linden, Alexandra C. Newton, Krzysztof Palczewski, and David S. Williams for discussions, and Drs. James B. Hurley, Alexandra C. Newton, and Krzysztof Palczewski for sending us preprints.
This work was supported by National Institutes of Health grant EY-06837.
1. | Asaoka, Y., S.-I. Nakamura, K. Yoshida, and Y. Nishizuka. 1992. Protein kinase C, calcium and phospholipid degradation. Trends Biochem. Sci. 17: 414-417 [Medline]. |
2. | Aton, B.R.. 1986. Illumination of bovine photoreceptor membranes causes phosphorylation of both bleached and unbleached rhodopsin molecules. Biochemistry 25: 677-680 [Medline]. |
3. | Baylor, D.A., T.D. Lamb, and K.-W. Yau. 1979a. The membrane current of single rod outer segments. J. Physiol. 288: 589-611 [Abstract]. |
4. | Baylor, D.A., T.D. Lamb, and K.-W. Yau. 1979b. Responses of retinal rods to single photons. J. Physiol. 288: 613-634 [Abstract]. |
5. |
Binder, R.M.,
M.S. Biernbaum, and
M.D. Bownds.
1990.
Light activation of one rhodopsin molecule causes the phosphorylation of
hundreds of others.
J. Biol. Chem.
265:
15333-15340
|
6. |
Binder, B.M.,
E. Brewer, and
M.D. Bownds.
1989.
Stimulation of
protein phosphorylations in frog rod outer segments by protein
kinase activators.
J. Biol. Chem.
264:
8857-8864
|
7. |
Binder, B.M.,
T.M. O'Connor,
M.D. Bownds, and
V.Y. Arshavsky.
1996.
Phosphorylation of non-bleached rhodopsin in intact retinas and living frogs.
J. Biol. Chem.
271:
19826-19830
|
8. | Brown, J.E., C. Blazynski, and A.I. Cohen. 1987. Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments. Biochem. Biophys. Res. Commun. 146: 1392-1396 [Medline]. |
9. | Bruns, R.F., F.D. Miller, R.L. Merriman, J.J. Howbert, W.F. Heath, E. Kobayashi, I. Takahashi, T. Tamaoki, and H. Nakano. 1991. Inhibition of protein kinase C by calphostin C is light-dependent. Biochem. Biophys. Res. Commun. 176: 288-293 [Medline]. |
10. |
Chen, C.-K.,
J. Inglese,
R.J. Lefkowitz, and
J.B. Hurley.
1995.
Ca2+-dependent interaction of recoverin with rhodopsin kinase.
J.
Biol. Chem.
270:
18060-18066
|
11. | Day, N.S., C.A. Koutz, and R.E. Anderson. 1993. Inositol-1,4,5-trisphosphate receptors in the vertebrate retina. Curr. Eye Res. 12: 981-991 [Medline]. |
12. | Dean, K.R., and M. Akhtar. 1993. Phosphorylation of solubilized dark-adapted rhodopsin: insights into the activation of rhodopsin kinase. Eur. J. Biochem 213: 881-890 [Abstract]. |
13. | Dean, K.R., and M. Akhtar. 1996. Novel mechanism for the activation of rhodopsin kinase: implications for other G protein-coupled receptor kinases (GRKs). Biochemistry. 35: 6164-6170 [Medline]. |
14. | Fain, G.L., H.R. Matthews, and M.C. Cornwall. 1996. Dark adaptation in vertebrate photoreceptors. Trends Neurosci. 19: 502-507 [Medline]. |
15. |
Ferreira, P.A., and
W.L. Pak.
1994.
Bovine phospholipase C highly
homologous to the NorpA protein of Drosophila is expressed specifically in cones.
J. Biol. Chem.
269:
3129-3131
|
16. | Finn, J.T., M.E. Grunwald, and K.-W. Yau. 1996. Cyclic nucleotide-gated ion channels: an extended family with diverse functions. Annu. Rev. Physiol. 58: 395-426 [Medline]. |
17. | Gehm, B.D., and D.G. McConnell. 1990. Phosphatidylinositol-4,5-bisphosphate phospholipase C in bovine rod outer segments. Biochemistry. 29: 5447-5452 [Medline]. |
18. | Ghalayini, A.J., and R.E. Anderson. 1984. Phosphatidylinositol 4,5-bisphosphate: light-mediated breakdown in the vertebrate retina. Biochem. Biophys. Res. Commun. 124: 503-506 [Medline]. |
19. |
Ghalayini, A.J.,
C.A. Koutz,
W.C. Wetsel,
Y.A. Hannun, and
R.E. Anderson.
1994.
Immunolocalization of PKC![]() |
20. | Ghalayini, A.J., A.P. Tarver, W.M. Mackin, C.A. Koutz, and R.E. Anderson. 1991. Identification and immunolocalization of phospholipase C in bovine rod outer segments. J. Neurochem. 57: 1405-1412 [Medline]. |
21. |
Gillette, M.A., and
R.F. Dacheux.
1996.
Protein kinase modulation
of GABAA currents in rabbit retinal rod bipolar cells.
J. Neurophysiol.
76:
3070-3086
|
22. |
Greene, N.M.,
D.S. Williams, and
A.C. Newton.
1995.
Kinetics and
localization of the phosphorylation of rhodopsin by protein kinase C.
J. Biol. Chem.
270:
6710-6717
|
23. |
Greene, N.M.,
D.S. Williams, and
A.C. Newton.
1997.
Identification
of protein kinase C phosphorylation sites on bovine rhodopsin.
J.
Biol. Chem.
272:
10341-10344
|
24. | Hardie, R.C., and B. Minke. 1995. Phosphoinositide-mediated phototransduction in Drosophila photoreceptors: the role of Ca2+ and trp. Cell Calcium. 18: 256-274 [Medline]. |
25. | Hardie, R.C., A. Peretz, E. Suss-Toby, A. Rom-Glas, S.A. Bishop, Z. Selinger, and B. Minke. 1993. Protein kinase C is required for light adaptation in Drosophila photoreceptors. Nature (Lond.). 363: 634-637 [Medline]. |
26. |
Harosi, F.I..
1975.
Absorption spectra and linear dichroism of cone
amphibian photoreceptors.
J. Gen. Physiol.
66:
357-382
|
27. | Hayashi, F., and T. Amakawa. 1985. Light-mediated breakdown of phosphatidylinositol-4,5-bisphosphate in isolated rod outer segments of frog photoreceptor. Biochem. Biophys. Res. Commun. 128: 954-959 [Medline]. |
28. | Hayashi, F., G.Y. Lin, H. Matsumoto, and A. Yamazaki. 1991. Phosphatidylinositol-stimulated phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in vertebrate rod photoreceptors. Proc. Natl. Acad. Sci. USA. 88: 4333-4337 [Abstract]. |
29. | Hayashi, F., M. Sumi, and T. Amakawa. 1987. Phosphatidylinositol stimulates phosphorylation of protein components I and II in rod outer segments of frog photoreceptors. Biochem. Biophys. Res. Commun. 148: 54-60 [Medline]. |
30. | Ikeuchi, Y., T. Nishizaki, M. Mori, and Y. Okada. 1996. Adenosine activates K+ channel and enhances cytosolic Ca2+ release via a P2Y purinoceptor in hippocampal neurons. Eur. J. Pharmacol. 304: 191-199 [Medline]. |
31. | Kapoor, C.L., and G.J. Chader. 1984. Endogenous phosphorylation of retinal photoreceptor outer segment proteins by calcium phospholipid-dependent protein kinase. Biochem. Biophys. Res. Commun. 122: 1397-1403 [Medline]. |
32. | Kapoor, C.L., P.J. O'Brien, and G.J. Chader. 1987. Phorbol ester- and light-induced endogenous phosphorylation of rat rod outer segment proteins. Exp. Eye Res. 45: 545-556 [Medline]. |
33. | Kawamura, S.. 1993. Molecular aspects of photoreceptor adaptation in vertebrate retina. Int. Rev. Neurobiol. 35: 43-86 [Medline]. |
34. | Kelleher, D.J., and G.L. Johnson. 1985. Purification of protein kinase C from bovine rod outer segments. J. Cyclic Nucleotide Protein Phosphorylation Res. 10: 579-591 [Medline]. |
35. |
Kelleher, D.J., and
G.L. Johnson.
1986.
Phosphorylation of rhodopsin by protein kinase C in vitro.
J. Biol. Chem.
261:
4749-4757
|
36. | Kobayashi, E., H. Nakano, M. Morimoto, and T. Tamaoki. 1989. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 159: 548-553 [Medline]. |
37. | Koistinaho, J., and S.M. Sagar. 1994. Localization of protein kinase C subspecies in the rabbit retina. Neurosci. Lett. 177: 15-18 [Medline]. |
38. | Kolb, H., L. Zhang, and L. Dekorver. 1993. Differential staining of neurons in the human retina with antibodies to protein kinase C isozymes. Vis. Neurosci. 10: 341-351 [Medline]. |
39. | Koutalos, Y., and K.-W. Yau. 1996. Regulation of sensitivity in vertebrate rod photoreceptors by calcium. Trends Neurosci. 19: 73-81 [Medline]. |
40. | Lagnado, L., and D.A. Baylor. 1992. Signal flow in visual transduction. Neuron. 8: 995-1002 [Medline]. |
41. |
Li, Q., and
M.K. Cathcart.
1994.
Protein kinase C activity is required for lipid oxidation of low density lipoprotein by activated
human monocytes.
J. Biol. Chem.
269:
17508-17515
|
42. | Millar, F.A., S.C. Fisher, C.A. Muir, E. Edwards, and J.N. Hawthorne. 1988. Polyphosphoinositide hydrolysis in response to light stimulation of rat and chick retina and retinal rod outer segments. Biochim. Biophys. Acta. 970: 205-211 [Medline]. |
43. | Nakatani, K., and K.W. Yau. 1988a. Calcium and magnesium fluxes across the plasma membrane of the toad rod outer segment. J. Physiol. 395: 695-729 [Abstract]. |
44. | Nakatani, K., and K.-W. Yau. 1988b. Calcium and light adaptation in retinal rods and cones. Nature (Lond.). 334: 69-71 [Medline]. |
45. | Nakatani, K., and K.W. Yau. 1989. Sodium-dependent calcium extrusion and sensitivity regulation in retinal cones of the salamander. J. Physiol. 409: 525-548 [Abstract]. |
46. | Negishi, K., S. Kato, and T. Teranishi. 1988. Dopamine cells and rod bipolar cells contain protein kinase C-like immunoreactivity in some vertebrate retinas. Neurosci. Lett. 94: 247-252 [Medline]. |
47. |
Newton, A.C., and
D.S. Williams.
1991.
Involvement of protein kinase C in the phosphorylation of rhodopsin.
J. Biol. Chem.
266:
17725-17728
|
48. |
Newton, A.C., and
D.S. Williams.
1993a.
Rhodopsin is the major in
situ substrate of protein kinase C in rod outer segments of photoreceptors.
J. Biol. Chem.
268:
18181-18186
|
49. | Newton, A.C., and D.S. Williams. 1993b. Does protein kinase C play a role in rhodopsin desensitization? Trends Biochem. Sci. 18: 275-277 [Medline]. |
50. | Nishizuka, Y.. 1992. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science (Wash. DC). 258: 607-614 [Medline]. |
51. |
Ohguro, H.,
M. Rudnicka-Nawrot,
J. Buczylko,
X. Zhao,
J.A. Taylor,
K.A. Walsh, and
K. Palczewski.
1996.
Structural and enzymatic aspects of rhodopsin phosphorylation.
J. Biol. Chem.
271:
5215-5224
|
52. |
Ohguro, H.,
J.P. Van Hooser,
A.H. Milam, and
K. Palczewski.
1995.
Rhodopsin phosphorylation and dephosphorylation in vivo.
J.
Biol. Chem.
270:
14259-14262
|
53. | Ohki, K., K. Yoshida, J. Imaki, T. Harada, and H. Matsuda. 1994. The existence of protein kinase C in cone photoreceptors in the rat retina. Curr. Eye Res. 13: 547-550 [Medline]. |
54. |
Osborne, N.N.,
N.L. Barnett,
N.J. Morris, and
F.L. Huang.
1992.
The occurrence of three isoenzymes of protein kinase C (![]() ![]() ![]() |
55. |
Osborne, N.N.,
J. Wood, and
N. Groome.
1994.
The occurrence of
three calcium-independent protein kinase C subspecies (![]() ![]() ![]() |
56. | Palczewski, K.. 1997. GTP-binding-protein-coupled receptor kinases: two mechanistic models. Eur. J. Biochem 248: 261-269 [Abstract]. |
57. | Palczewski, K., and J.C. Saari. 1997. Activation and inactivation steps in the visual transduction pathway. Curr. Opin. Neurobiol. 7: 500-504 [Medline]. |
58. | Panfoli, I., A. Morelli, and I. Pepe. 1990. Calcium ion-regulated phospholipase C activity in bovine rod outer segments. Biochem. Biophys. Res. Commun. 173: 283-288 [Medline]. |
59. |
Peng, Y.-W.,
S.G. Rhee,
W.-P. Yu,
Y.K. Ho,
T. Schoen,
G.J. Chader, and
K.-W. Yau.
1997.
Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments.
Proc.
Natl. Acad. Sci. USA.
94:
1995-2000
|
60. | Peng, Y.-W., A.H. Sharp, S.H. Snyder, and K.-W. Yau. 1991. Localization of the inositol 1,4,5-trisphosphate receptor in synaptic terminals in the vertebrate retina. Neuron. 6: 525-531 [Medline]. |
61. |
Poulin, B.,
N. Rich,
Y. Mitev,
J.-P. Gautron,
C. Kordon,
A. Enjalbert, and
S.V. Drouva.
1996.
Differential involvement of calcium channels and protein kinase-C activity in GnRH-induced phospholipase-C, -A2, and -D activation in a gonadotrope cell line (![]() |
62. | Pugh, E.N. Jr, and T.D. Lamb. 1993. Amplification and kinetics of the activation steps in phototransduction. Biochim. Biophys. Acta. 1141: 111-149 [Medline]. |
63. |
Qiu, Z.-H., and
C.C. Leslie.
1994.
Protein kinase C-dependent and
-independent pathways of mitogen-activated protein kinase activation in macrophages by stimuli that activate phospholipase A2.
J. Biol. Chem.
269:
19480-19487
|
64. | Ranganathan, R., D.M. Malicki, and C.S. Zuker. 1995. Signal transduction in Drosophila photoreceptors. Annu. Rev. Neurosci. 18: 283-317 [Medline]. |
65. | Rim, J., E. Faurobert, J.B. Hurley, and D.D. Oprian. 1997. In vitro assay for trans-phosphorylation of rhodopsin by rhodopsin kinase. Biochemistry 36: 7064-7070 [Medline]. |
66. | Stea, A., T.W. Soong, and T.P. Snutch. 1995. Determinants of PKC-dependent modulation of a family of neuronal calcium channels. Neuron. 15: 929-940 [Medline]. |
67. | Stratton, K.R., P.F. Worley, R.L. Huganir, and J.M. Baraban. 1989. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices. Proc. Natl. Acad. Sci. USA. 86: 2498-2501 [Abstract]. |
68. | Suzuki, S., and A. Kaneko. 1990. Identification of bipolar cell subtypes by protein kinase C-like immunoreactivity in the goldfish retina. Vis. Neurosci. 5: 223-230 [Medline]. |
69. |
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakane,
V. Baudet,
P. Boissin,
E. Boursier,
F. Loriolle, et al
.
1991.
The bisindolymaleimide GF109203X is a potent and selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781
|
70. |
Tsuboi, S.,
H. Matsumoto,
K.W. Jackson,
K. Tsujimoto,
T. Williams, and
A. Yamazaki.
1994a.
Phosphorylation of an inhibitory subunit of cGMP phosphorylation in Rana catesbiana rod photoreceptors. I. Characterization of the phosphorylation.
J. Biol. Chem.
269:
15016-15023
|
71. |
Tsuboi, S.,
H. Matsumoto, and
A. Yamazaki.
1994b.
Phosphorylation of an inhibitory subunit of cGMP phosphorylation in Rana
catesbiana rod photoreceptors. II. A possible mechanism for the
turnoff of cGMP phosphorylation without GTP hydrolysis.
J. Biol.
Chem.
269:
15024-15029
|
72. | Udovichenko, I.P., J. Cunnick, K. Gonzalez, and D.J. Takemoto. 1993. Phosphorylation of bovine rod photoreceptor cyclic GMP phosphodiesterase. Biochem. J. 295: 49-55 [Medline]. |
73. |
Udovichenko, I.P.,
J. Cunnick,
K. Gonzalez, and
D.J. Takemoto.
1994.
Functional effect of phosphorylation of the photoreceptor
phosphodiesterase inhibitory subunit by protein kinase C.
J. Biol.
Chem.
269:
9850-9856
|
74. | Udovichenko, I.P., J. Cunnick, K. Gonzalez, A. Yakhnin, and D.J. Takemoto. 1996. Protein kinase C in rod outer segments: effects of phosphorylation of the phosphodiesterase inhibitory subunit. Biochem. J. 317: 291-295 [Medline]. |
75. |
Udovichenko, I.P.,
A.C. Newton, and
D.S. Williams.
1997.
Contribution of protein kinase C to the phosphorylation of rhodopsin
in intact retinae.
J. Biol. Chem.
272:
7952-7959
|
76. | Usuda, N., Y. Kong, M. Hagiwara, C. Uchida, M. Terasawa, T. Nagata, and H. Hidaka. 1991. Differential localization of protein kinase C isozymes in retinal neurons. J. Cell Biol. 112: 1241-1247 [Abstract]. |
77. | Weyand, I., and H. Kühn. 1990. Subspecies of arrestin from bovine retina. FEBS Lett. 193: 459-467 . |
78. | Wolbring, G., and N.J. Cook. 1991. Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments. FEBS Lett. 201: 601-606 . |
79. | Wolbring, G., and P.P.M. Schnetkamp. 1995. Activation by PKC of the Ca2+-sensitive guanylyl cyclase in bovine retinal rod outer segments measured with an optical assay. Biochemistry. 34: 4689-4695 [Medline]. |
80. | Wood, J.G., C.E. Hart, G.J. Mazzei, P.R. Girard, and J.F. Kuo. 1988. Distribution of protein kinase C immunoreactivity in rat retina. Histochem. J. 20: 63-68 [Medline]. |
81. |
Yarfitz, S., and
J.B. Hurley.
1994.
Transduction mechanisms of vertebrate and invertebrate photoreceptors.
J. Biol. Chem.
269:
14329-14332
|
82. | Yau, K.-W.. 1994. Phototransduction mechanism in retinal rods and cones. The Friedenwald Lecture. Invest. Ophthalmol. Vis. Sci. 35: 9-32 [Medline]. |
83. | Yau, K.-W., and D.A. Baylor. 1989. Cyclic GMP-activated conductance of retinal photoreceptor cells. Annu. Rev. Neurosci. 12: 289-327 [Medline]. |
84. | Zhang, L., L. Dekorver, and H. Kolb. 1992. Immunocytochemical staining with antibodies against protein kinase C and its isozymes in the turtle retina. J. Neurocytol. 21: 833-847 [Medline]. |
85. |
Zick, Y.,
R. Sagi-Eisenberg,
M. Pines,
P. Gierschik, and
A.M. Spiegel.
1986.
Multisite phosphorylation of the ![]() |