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. Author manuscript; available in PMC: 2012 Jun 18.
Published in final edited form as: J Biol Chem. 1986 Oct 5;261(28):12965–12975.

Changes in cGMP Concentration Correlate with Some, but Not All, Aspects of the Light-regulated Conductance of Frog Rod Photoreceptors*

Richard H Cote 1,‡, Grant D Nicol 1, Sheila A Burke 1, M Deric Bownds 1
PMCID: PMC3376451  NIHMSID: NIHMS384216  PMID: 3020017

Abstract

Cyclic GMP has been implicated in controlling the light-regulated conductance of rod photoreceptors of the vertebrate retina. However, there is little direct evidence correlating changes in cGMP concentration with the light-regulated permeability mechanism in living cells. A preparation of intact frog rod outer segments suspended in a Ringer's medium containing low Ca2+ has been used to demonstrate that initial changes in total cellular cGMP concentration parallel changes in the light-regulated membrane current over a wide range of light intensities.

At light intensities bleaching from 160 to 5.6 × 106 rhodopsin molecules/rod/s, decreases in the response latency for the cGMP Kinetics parallel decreases in the latent period of the electrical response. Further, changes in the rate of the cGMP decrease parallel the rate of membrane current suppression as the light intensity is varied. Up to 105 cGMP molecules are hydrolyzed per photolyzed rhodopsin, consistent with in vitro studies showing that each bleached rhodopsin can activate over 100 phosphodiesterase molecules. Addition of the Ca2+ ionophore, A23187, does not affect the initial kinetics of the cGMP decrease or of the electrical response, excluding a direct role for Ca2+ in the initial events of phototransduction. These results are consistent with cGMP being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination.

It is unlikely, however, that light-induced changes in total cGMP concentration are the sole regulators of membrane current. This is suggested by several observations: 1) at bright light intensities, the subsecond light-induced cGMP decrease is essentially complete prior to complete suppression of membrane current; 2) maximal light-induced decreases in cGMP concentration occur at all light intensities tested, whereas the extent of membrane current suppression varies over the same range of light intensities; 3) changing the external Ca2+ concentration from 1 mM to 10 nM in the dark causes an increase in membrane current that is significantly more rapid than corresponding changes in cGMP concentration. Thus, light-induced changes in total cellular cGMP concentration correlate with some, but not all, aspects of the visual excitation process in vertebrate photoreceptors.

cGMP is a candidate for the internal messenger that mediates between the isomerization of rhodopsin by light and the subsequent electrical response of a vertebrate rod photoreceptor cell (cf. Lamb (1986) and Stryer (1986) for recent reviews). Both biochemical and electrophysiological evidence suggest that upon illumination, changes in cGMP concentration in the outer segment may regulate (either directly or indirectly) the light-sensitive ionic permeability mechanism in the plasma membrane. Injection of cGMP (Nicol and Miller, 1978; Miller, 1982; Waloga, 1983; Kawamura and Murakami, 1983; Cobbs and Pugh, 1985; Matthews et al., 1985) or slowly hydrolyzed cGMP analogs (MacLeish et al., 1984; Zimmerman et al., 1985) into intact rods causes an increase in the magnitude of the light-regulated ionic conductance. Experiments in which cGMP or cGMP analogs are perfused onto an isolated patch of outer segment membrane further suggest that cGMP can directly control an ionic conductance (Fesenko et al., 1985; Zimmerman et al., 1985; Yau et al., 1986; Stern et al., 1986). It appears that the cGMP-regulated conductance consists of an aqueous pore rather than a carrier (Haynes et al., 1986; Zimmerman and Baylor, 1986).

cGMP must satisfy several biochemical criteria to be the internal transmitter for the initial stage of phototransduction. First, its concentration must change in response to illumination. Evidence for light-induced decreases in cGMP concentration has been obtained in whole retinas, the outer segment layer of microdissected retinas, and in isolated rods (see Korenbrot, 1985, for review). Further, if the concentration of cGMP is to directly control the light-regulated permeability mechanism, there should be an exact correspondence between the concentration of cGMP in the rod and the magnitude of current flowing through the light-regulated channel.

Second, the internal transmitter must respond to the same range of light intensities required to suppress the membrane current that flows in the dark. In a 1 mM Ca2+ Ringer's solution, the total number of photolyzed rhodopsins needed to observe a light-induced cGMP decrease (Kilbride and Ebrey, 1979; Govardovskii and Berman, 1981; Woodruff and Fain, 1982; Cote et al., 1984) is much greater than the number of bleached rhodopsins (100–200) required to fully suppress the light-regulated ionic conductance of the rod (Baylor et al., 1979). However, in a low Ca2+ Ringer's Woodruff and Fain (1982) showed that light-induced changes in retinal cGMP concentration could be observed in the absence of an electrical response, suggesting that cGMP levels can be more sensitive to illumination than the ionic conductance.

A kinetic constraint for an internal transmitter is that its synthesis or degradation must precede the time course of changes in membrane permeability. Two studies of light-induced decreases in cGMP concentration in intact retinas have reported slower changes in cGMP concentration than in the expected electrical response of the photoreceptor (Kilbride and Ebrey, 1979; Govardovskii and Berman, 1981). However, Blazynski and Cohen (1985) have observed subsecond decreases in cGMP levels in the outer segment layer of microdissected retina. Also, isolated outer segments or outer segments with the ellipsoid portion of the inner segment attached (OS-IS1) undergo changes in cGMP concentration on the same time scale as changes in membrane current (woodruff and Bownds, 1979; Cote et al., 1984).

Another criterion for cGMP as an internal transmitter is that cGMP hydrolysis must be highly amplified compared to the extent of rhodopsin isomerization. Two stages of amplification are known to occur in vitro. The first occurs when a single photoexcited rhodopsin catalyzes the activation of several hundred phosphodiesterase molecules via a guanine nucleotide-binding protein (G protein or transducin; see Chabre, 1985, for review). The second stage of amplification results from each phosphodiesterase hydrolyzing many cGMP molecules (Yee and Liebman, 1978; Liebman and Pugh, 1979). Estimates of the stoichiometry of cGMP molecules hydrolyzed per rhodopsin bleached in intact rod outer segments (Woodruff and Bownds, 1979; Cote et al., 1984) confirm that this highly amplified enzyme system can operate under physiological conditions in intact outer segments.

In this study, we report that light-induced changes in total cGMP concentration meet some (but not all) of the abovementioned criteria for an internal messenger for visual excitation. We employ a purified photoreceptor preparation (OS-IS) which retains the electrophysiological properties and metabolic integrity of rods still attached to the retina (Biernbaum and Bownds, 1985a, 1985b; Cote et al., 1984). We demonstrate that OS-IS undergo light-induced decreases in cGMP concentration over a wide range of light intensities, including dim illumination that does not saturate the photocurrent (defined as the magnitude of current normally flowing in the dark which is suppressed by illumination). At all light intensities tested, the cGMP kinetics are sufficiently rapid for changes in cGMP concentration to modulate the light-regulated permeability mechanism in the plasma membrane. From the magnitude of the cGMP decrease in intact OS-IS, it is inferred that an enzymatic amplification mechanism operates to rapidly hydrolyze cGMP upon illumination. Further, neither the cGMP kinetics nor the photocurrent kinetics are altered by the presence of the calcium ionophore A23187, excluding a role for calcium concentration gradients in the initial events of phototransduction under these experimental conditions.

Nonetheless, factors other than changes in the total measured cGMP concentration must also be involved in generating the observed photoresponse. We conclude this because no exact correspondence is observed between the total measured cGMP concentration and the extent of membrane current suppression under several experimental conditions.

EXPERIMENTAL PROCEDURES

Preparation of Frog Photoreceptors

Retinas from dark-adapted Rana catesbiana or Rana grylio were removed from the eye cup and placed in a Ringer's solution containing 5% Percoll. The Ringer's solution consisted of 105 mM NaCl, 2.5 mM KCl, 2 mM MgC12, 1 mM CaC12, 5 mM NaHC03, 5 mM glucose, 10 mM HEPES, pH 7.6; solutions were oxygenated before use. Manipulations were carried out in darkness using infrared image converters. Suspensions of OS-IS were prepared by initially stretching the isolated retina, followed by gentle shaking in a solution of 5% Percoll-Ringer's. After several minutes of shaking, the retina was shredded with forceps and the retinal pieces were, gently agitated in additional 5% Percoll-Ringer's. Retinal pieces were allowed to settle and the suspension of rods layered on a discontinuous Percoll gradient (Biernbaum and Bownds, 1985a). Osmotically intact rods (i.e. rods which exclude the fluorescent dye N,N'-didansyl-L-cystine) were recovered at a density of approximately 45% Percoll-Ringer's (refractive index, 1.342–1.343).

Typically, 50–80% of the rods isolated by this technique were OS-IS, the remainder being outer segments lacking the inner segment. The metabolic integrity of the OS-IS preparation has been demonstrated with respect to electrophysiological properties, adenine and guanine nucleotide metabolism in the dark, and ability of the membrane current and nucleotide levels to regain the dark-adapted state after cessation of illumination (cf. Biernbaum and Bownds, 1985a, 1985b, for details). Outer segments lacking mitochondria might be expected to be metabolically impaired and thus adversely influence both the cGMP kinetics and the magnitude of the decrease in cGMP concentration. Although outer segments exhibit lower nucleoside triphosphate levels (Biernbaum and Bownds, 1985a) and cGMP concentration (Cote et al., 1984) than OS-IS, this degree of lowered metabolic health appears to not be critical for outer segment cGMP metabolism for several reasons. 1) The ATP, GTP, and cGMP levels in purified outer segments are stable for 1 h after Percoll gradient centrifugation (Biernbaum and Bownds, 1985a; Cote et al., 1984), suggesting maintenance of high energy phosphate metabolism and cyclic nucleotide metabolism during the time course of the experiments reported in this study. 2) Illumination results in only a slight decrease in outer segment nucleoside triphosphate levels during the time frame when cGMP measurements are carried out (Biernbaum and Bownds, 1979). 3) The light-induced decrease in outer segment cGMP concentration does not drop to zero but reaches a stable plateau with continuous illumination (Woodruff and Bownds, 1979), indicating that a new steady state condition has been attained. 4) Outer segments lacking mitochondria can restore their cGMP levels to the dark-adapted state after cessation of illumination (Woodruff and Bownds, 1979). 5) We observe no significant differences in either the cGMP kinetics or the final magnitude of the light-induced cGMP decrease as the percentage of outer segments in the rod preparation varied in individual experiments from 20 to 50%.

Purified OS-IS (in a 1 mM Ca2+ medium) were mixed with an equal volume of 4 mM EGTA (in 5% Percoll-Ringer's, pH 7.8) to give a final free Ca2+ concentration of 20 nM. The free Ca2+ concentration was calculated by the method of Caldwell (1970). The addition of EGTA to the rod suspension resulted in a pH change of less than 0.1 unit. In some experiments, the Ca2+ ionophore A23187 was also added. Within 10 s after addition of EGTA, 10 μM A23187 (1:1OO dilution in Ringer's of a 1 mM solution in 100% dimethyl sulfoxide) was added and mixed by gentle stirring. Addition of 1% dimethyl sulfoxide alone resulted in a 20–30% decrease in dark-adapted cGMP levels, but caused no detectable effect on rod morphology, osmotic intactness (assayed with the dye N,N'-didansyl-L-cystine), electrophysiological properties, or on the rate or extent of light-induced changes in cGMP concentration.

For experiments described in Fig. 10B, rods were isolated in 5% Percoll-Ringer's in which Na+ was replaced by K+. The retinas were treated as described above, and isolated rods were purified on a Percoll gradient containing K+-Ringer's. Highly enriched OS-IS preparations were not obtained under these conditions, and these experiments were performed with osmotically intact outer segments (less than 30% OS-IS).

Fig. 10. Effects of Ca2+ ionophore on cGMP concentration of dark-adapted rods in 20 nm Ca2+.

Fig. 10

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A, purified OS-IS prepared in a 1 mm Ca2+ Percoll-Ringer's (average cGMP levels, 0.011 mol of cGMP/mol of rhodopsin) were divided into two portions and incubated for 10 s in 20 nm Ca2+ medium. To one portion was added 10 μm A23187 (open circles), while to the other was added 1% dimethyl sulfoxide (filled circles). Each point represents the mean (±S.E.) from at least three experiments. B, rods were isolated from the retina in a medium in which all Na+ was replaced with K+ and purified on a K+-Ringer's-Percoll gradient. The purified intact rods (0.006 mol of cGMP/mol of rhodopsin) were mixed with EGTA (final Ca2+ concentration, 20 nm) and either 10μm A23187 (open circles) or 1% dimethyl sulfoxide (filled circles) as described in the previous figure legend. The points represent the mean (±S.E.) of three experiments.

For time course studies in the dark, 50-μl portions of OS-IS (rhodopsin concentration, 2–4 μM) were added to 100 μl of 25% perchloric acid at various times after addition of EGTA with and without A23187. The quenched samples were immediately placed on ice, and determination of cGMP concentration was carried out by radioimmunoassay (Cote et al., 1984).

Measurement of Rapid Changes in cGMP Concentration with a Rapid Quench Apparatus

A rapid quench device was utilized to measure subsecond changes in cGMP concentration in OS-IS after illumination. The technique has been previously described (Cote et al., 1984) and will be briefly summarized. Tubes containing 50 μl of OS-IS were positioned beneath a rack of syringes containing 25% perchloric acid. The ejection of the acid was timed by a signal generator that controls the flow of pressurized air. Compressed air drives a piston, forcing the acid from the syringe into a sample tube. The signal generator also controls a shutter, thus synchronizing acid ejection with illumination of a particular sample. The time resolution of the rapid quench apparatus is approximately 50 ms. The quenched samples were analyzed for cGMP concentration as described above.

The cGMP kinetics in Figs. 2–5 and Fig. 8 represent the mean of at least 3 separate experiments. Since the average cGMP concentration in OS-IS in the dark varied from experiment to experiment, cGMP concentrations were normalized to the dark average for each experiment. The data are thus expressed as a percentage of the average dark cGMP concentration.

Figs. 2–5. Rapid kinetics of light-induced cGMP decrease compared with photocurrent kinetics at 4 intensities of continuous illumination.

Figs. 2–5

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Purified OS-IS were incubated for 7–12 min in a 20 nM Ca2+ Percoll-Ringer's solution prior to illumination. Samples for cGMP determinations were automatically quenched at the indicated times using the rapid quench apparatus (see “Experimental Procedures”). The dashed line represents the best fit of the cGMP kinetics to a pseudo-first order reaction; the continuous trace is the membrane current. Note that the scales for the time and membrane current axes differ in each figure. The maximum amplitude of the membrane current was scaled to be equal to the maximum light-induced cGMP decrease permitting a direct comparison of cGMP and photocurrent kinetics.

FIG. 2 (upper left). Illumination bleaching 160 R*/rod/s. For the cGMP measurements, the actual light intensity was 180 R*/rod/s. Samples were quenched at 45-, 100-, and 250-ms intervals in different experiments, and points within a 100-ms time interval were grouped together to provide a mean (±S.D.) for 8 experiments. The average dark-adapted cGMP levels were 0.055 ± 0.020 mol of cGMP/mol of rhodopsin. For the measurement of membrane current, the actual light intensity was 140 R*/rod/s, and data were collected at 10-ms intervals. The trace represents the digitized average of 20 responses from 10 cells. The maximum photocurrent amplitude (2 PA) was attained at 9 s.

FIG. 3 (upper right). Illumination bleaching 490 R*/rod/s. For the cGMP kinetics, the actual intensity was 550 R*/rod/s. Portions of OS-IS suspensions were quenched at 45-, 100-, and 250-ms intervals as above, and the points represent the mean of 8 experiments. Dark-adapted cGMP levels in these experiments were 0.044 ± 0.006 mol of cGMP/mol of rhodopsin. The kinetics of the photocurrent were measured at a light intensity of 430 R*/rod/s, the trace representing the average of 22 responses from 11 cells. Maximum suppression of membrane current (5 PA) was reached at 5 s.

FIG. 4 (lower left). Illumination bleaching 8400 R*/rod/s. The cGMP kinetics were measured at a light intensity of 8540 R*/rod/s, and the points represent the average of 5 experiments at sampling intervals of 45 or 100 ms. Dark-adapted cGMP concentration was 0.042 ± 0.005 mol of cGMP/mol of rhodopsin. The photocurrent kinetics were sampled at 10-ms intervals (24 responses from 12 cells), and the light stimulus was 8250 R*/rod/s. The maximum suppression of the membrane current (16 PA) was reached in 1.8 s.

FIG. 5 (lower right). Illumination bleaching 5.6 × 106 R*/rod/s. For the cGMP kinetics, the light intensity was 4.8 × 106 R*/rod/s, and samples were quenched at 45-ms intervals. The data represent the mean of 3 experiments (dark cGMP concentration = 0.034 ± 0.015 mol of cGMP/mol of rhodopsin). Note that the sample which is quenched at the same time as the light is turned on has responded to the stimulus before the acid could quench the sample. For the photocurrent kinetics, the trace is the average of 12 responses from 12 cells (8-ms sampling interval). At a light intensity of 6.4 ± 106 R*/rod/s, the time to reach maximum amplitude (18 PA) is 280 ms.

Fig. 8. Ca2+ ionophore does not alter cGMP kinetics.

Fig. 8

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A, purified OS-IS were incubated in a 20 nm Ca2+ medium for 6–8 min with (open circles) or without (filled circles, taken from Fig. 4) 10 μm A23187 before illumination and quenching. The light intensity was 8540 R*/rod/s, and the rapid quench apparatus was operated at 100-ms intervals; the open circles represent the mean (±S.D.) for 3 experiments where the dark cGMP concentration was 0.027 mol of cGMP/mol of rhodopsin. In paanel B, the light intensity was increased to 4.8 × 106 R*/rod/s, and samples were quenched at 45-ms intervals. The filled circles are reproduced from Fig. 5, while the open circles represent the average of 3 experiments with 10 μm A23187 (in 1% dimethyl sulfoxide) where the dark-adapted cGMP concentration was 0.023 ± 0.003 mol of cGMP/mol of rhodopsin.

To determine the cGMP latent period (i.e. time at which the light-induced cGMP decrease was statistically significant) the data were analyzed using a cumulative sum control chart (Duncan, 1974). This method provides two estimates of the latent period of the cGMP light response: 1) the time at which there is a statistically significant difference in the mean value of the light-exposed sample compared to the mean dark value; 2) the time at which a trend leading to the significant difference between light and dark samples is initiated. These two estimates of the cGMP latent period provide the range of times over which the light-induced changes in cGMP concentration may commence. Two sources of error are associated with estimation of cGMP latent period: 1) experimental error resulting from sample-to-sample variation in the volume of acid ejected from the rapid quench apparatus; 2) limitations in the time resolution of our rapid quench apparatus resulting from the approximately 50 ms latency in the acid-quenching process.

It was not possible to determine accurately a true initial velocity of the light-induced decrease in cGMP concentration. Instead, the cGMP kinetics were modeled as a pseudo-first order reaction, and a rate constant was obtained in this way. The rate constant was used to calculate the number of cGMP molecules hydrolyzed per rod per s (Fig. 7B). The dashed lines in Figs. 2–5 represent the fit of the data points to a first-order decay of dark-adapted cGMP concentration to a new steady-state level after 15–30 s of illumination. At all light intensities the fit of the data to a first-order process was good (correlation coefficients between −0.92 and −0.98).

Fig. 7.

Fig. 7

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The latent period and rate for cGMP kinetics and photocurrent kinetics correlate over 4 decades of light intensity. A, the data of Figs. 2–5 were statistically analyzed with a cumulative sum control chart to determine the latent period for the photocurrent and the cGMP kinetics (see “Experimental Procedures”). The filled circles connected with the solid line represent the time at which the membrane current statistically differed from the current in the dark. Two estimates of the cGMP latent period are given: the filled squares represent the time at which a statistically significant decrease in cGMP concentration is observed, while the open squares represent the time at which a cGMP decrease was probably initiated. At the brightest light intensity (Fig. 5) no cGMP latent period was detected. In this case the cGMP latency was estimated to be 50 ms since there is approximately 50 ms uncertainty in the quench time. B, the rate of cGMP hydrolysis (filled squares) and the maximum rate of the photocurrent (open circles) were determined as described under “Experimental Procedures.”

Measurement of Rod Membrane Dark Current

The inner segment of an OS-IS (preincubated in Percoll-Ringer's containing 20 nM free Ca2+ for at least 5 min) was drawn into a suction electrode and the membrane current measured with a current-to-voltage converter, as described previously (Baylor et al., 1979; Biernbaum and Bownds, 1985a). Current measurements were sampled at 8- or 10-ms intervals, stored in computer memory, and reproduced by a digital plotter. We have defined the photocurrent as the magnitude of the current (normally flowing in the dark-adapted state) which is suppressed by light.

The recording chamber could be perfused with various solutions while an OS-IS was retained in the suction electrode. The time required to exchange solutions in the recording chamber was approximately 10 s. In this way, the effect of changing the external Ca2+ from 1 mM to 20 nM on the dark membrane current can be evaluated (Fig. 1). This also permits a comparison of the photocurrent kinetics for an individual rod in the absence and presence of A23187 (Fig. 9).

Fig. 1. Time course of cGMP changes upon lowering the external Ca2+ concentration to 20 nM.

Fig. 1

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Purified OS-IS were mixed with an equal volume of 4 mM EGTA in Ringer's solution to yield a final free Ca2+ concentration of 20 nM. OS-IS cGMP levels just prior to adding EGTA were 0.010 mol of cGMP/mol of rhodopsin. Samples were acid quenched at the indicated times. Points with error bars represent the mean (±S.E.) of at least 4 experiments.

Fig. 9. Ca2+ ionophore does not alter photocurrent kinetics.

Fig. 9

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Purified OS-IS were prepared as described in the previous figure, membrane current was measured with the suction electrode recording technique. In panel A, the light intensity was 790 R*/rod/s, and the photocurrent traces are averages of 18 responses from 9 cells for each condition. In panel B, the light intensity was increased to 3.7 × 105 R*/rod/s. The traces represent 14 responses from 7 cells for condition. The photocurrent of an individual OS-IS was tested under the control condition, and then the recording chamber was perfused with the solution containing 10 μm A23187, and the photoresponse of the cell was measured after 5 min in the new condition.

The latent period for the photocurrent was determined using the same statistical method as described above for changes in cGMP concentration. The statistical estimation of the latent period employed the digitized values of membrane current that had been stored in computer memory. Since the experimental error for the current recordings was about 10-fold lower and the sampling interval was 5-fold smaller than for the cGMP measurements, the photocurrent latent period could be determined much more accurately. Values for the photocurrent latent period represent the time at which the membrane current statistically differs from the dark membrane current.

The rate of change of the photocurrent was determined from the maximum negative slope of the time course of the change in membrane current. At all light intensities tested, the maximum rate of the photocurrent was linear for 40–60% of its time course.

Illumination of OS-IS

For meaningful comparison of biochemical and electrophysiological measurements, it was necessary to calibrate the two different light sources used. For illumination of OS-IS suspensions in the rapid quench apparatus, a continuous light source provided uniform illumination of all 16 samples. The light passed through a Corning CS 3–67 filter (cutoff > 600 nm) and a heat filter. The unattenuated light bleached 9.6% of the rhodopsin/min, as determined by difference spectroscopy (Bownds et al., 1971). Assuming 3 × 109 rhodopsin molecules/rod, this rate of rhodopsin bleaching is equivalent to 4.8 × 106 rhodopsins bleached R*/rod/s. Neutral density filters (calibrated in a Cary 14 spectrophotometer) were used to attenuate the light.

The light source for the suction electrode recording apparatus was calibrated in the following manner. First, a fiber optic bundle was used to position a diffuse beam of light that illuminated the entire recording chamber. The rate of rhodopsin bleaching in the chamber (measured by difference spectroscopy) was determined by illuminating a suspension of rods for various times with the diffuse spot of light. Second, the flash intensity-response amplitude relation was measured for several OS-IS using neutral density filters to attenuate the diffuse beam of light. Third, the light was then focused into a 75-μm diameter spot and the flash intensity-response relation was again determined. The amount of shift in the current-response relation that occurred when the diffuse spot was focused allowed a calculation of the light intensity of the unattenuated 75-μm diameter spot of light (1.9 × 107 R*/rod/s). In this way the light sources used for biochemical and electrophysiological measurements were calibrated to deliver nearly identical illumination to isolated photoreceptors.

Materials

Percoll was obtained from Pharmacia and HEPES from Calbiochem. All other chemicals were obtained from Sigma. Neutral density filters were from Oriel.

RESULTS

Kinetics of Light-induced cGMP Decrease Parallel Photocurrent Kinetics at Low Light Intensities

In our previous study we showed that OS-IS incubated in a physiological Percoll-Ringer's solution (1 mM Ca2+) undergo light-induced changes in cGMP concentration on the same time scale as the photocurrent (Cote et al., 1984). However, the small maximal cGMP decrease (20%) with bright illumination prevented us from studying the cGMP response at light intensities where the photocurrent was not saturated.

In this work we have increased the cGMP concentration in OS-IS by exposing purified rods to a Percoll-Ringer's solution containing 20 nM free Ca2+, enabling us to measure light-induced changes in cGMP concentration at dim intensities. Osmotically intact OS-IS were purified by discontinuous Percoll density gradient centrifugation in a 1 mM Ca2+ Percoll-Ringer's solution, and then EGTA was added to bring the final free Ca2+ concentration to 20 nM. The effect of lowering the external Ca2+ on the cGMP concentration of dark-adapted OS-IS is presented in Fig. 1. Within 4–8 min after addition of EGTA a stable 3-fold elevation of intracellular cGMP levels was attained. The average cGMP concentration for dark-adapted OS-IS incubated for 7–12 min in 20 nM Ca2+ was found to be 0.038 ± 0.011 mol of cGMP/mol of rhodopsin (n = 49), a 3.8-fold increase over the cGMP concentration of OS-IS in a 1 mM Ca2+ solution. Upon illumination, OS-IS incubated in 20 nM Ca2+ Percoll-Ringer's exhibited a 60% maximum decrease in cGMP concentration regardless of the initial dark-adapted concentration. The remaining 40% of the total cGMP content of OS-IS was not further diminished even with prolonged light exposures bleaching 10% of the rhodopsin.

Figs. 2–5 present the initial kinetics of the light-induced changes in cGMP concentration and membrane current for light intensities ranging from 160 to 5.6 × 106 R*/rod/s. For the sake of comparison, the maximum amplitudes of the cGMP decrease and the photocurrent are plotted on the same scale and superimposed. Also note that for maximum resolution of the initial kinetics, a different time axis was chosen for each light intensity.

With continuous illumination bleaching 160 R*/rod/s (Fig. 2), the kinetic profile of the light-induced cGMP decrease is indistinguishable from the photocurrent kinetics. When the light intensity is increased from 160 to 490 R*/rod/s (Fig. 3), the kinetics of the light-induced cGMP decrease and the photocurrent are both accelerated. At this 3-fold brighter light intensity there is no statistically significant difference between the two time courses, as judged by the overlap of the error bars for the cGMP data with the photocurrent trace. This similarity supports the notion that cGMP could mediate between photon absorption by rhodopsin in the disc membrane and the initial light-regulated permeability changes in the plasma membrane.

As the light intensity is increased to 8400 R*/rod/s (Fig. 4) and 5.6 × 106 R*/rod/s (Fig. 5), both the cGMP kinetics and the photocurrent kinetics are further accelerated. Once the latent period has ended, the cGMP response to light proceeds more rapidly than the photocurrent. At a bleaching rate of 8400 R*/rod/s, 40% of the light-sensitive cGMP is hydrolyzed within 300 ms, whereas the photocurrent has attained only 10% of its final amplitude. And at 5.6 × 106 R*/rod/s a 45% decrease in light-sensitive cGMP occurs within 50 ms after the onset of illumination, whereas the photocurrent latent period has just ended at 50 ms. (Note that the samples quenched at time 0 in Fig. 5 appear to have responded to the light stimulus. This is due to incomplete inactivation of light-sensitive enzymatic activities during the quenching process (see Cote et al., 1984, for discussion of this phenomenon).)

The lack of a one-to-one correspondence between light-induced cGMP decreases and changes in the photocurrent at bright light intensities is inconsistent with the idea that instantaneous changes in cGMP concentration control the light-regulated conductance. This lack of correlation cannot be due to the time required to acid quench the rods (50 ms), since given a 50-ms shift in its time axis the cGMP kinetics would still change more rapidly than the photocurrent. Therefore, processes other than changes in total cellular cGMP concentration may also be involved in regulating the waveform of the photocurrent (see “Discussion”).

This conclusion is further corroborated by the results of Fig. 6. In Fig. 6A, the cGMP kinetics from Figs. 2–5 are replotted, and measurements of the maximal light-induced cGMP decrease at each intensity are also included. At each intensity of continuous illumination, the cGMP levels fell to approximately the same final extent after 15–30 s. In contrast, the maximum amplitude of the photocurrent reached a steady state level whose magnitude varied as a function of light intensity (Fig. 6B). A graded cGMP response to continuous illumination has been reported for intact retina (Kilbride and Ebrey, 1979; Woodruff and Fain, 1982) and for isolated outer segments (Woodruff and Bownds, 1979). The different results seen in OS-IS cannot be ascribed to the low Ca2+ condition, since Woodruff and Bownds (1979) and Woodruff and Fain (1982) have observed a graded cGMP response in a low Ca2+ solution. We conclude that factors other than the measured total cGMP concentration must regulate the steady state extent of membrane current suppression by continuous light.

Fig. 6.

Fig. 6

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The amplitude of the light-induced cGMP decrease does not correlate with the photocurrent amplitude. A, the cGMP kinetics from Figs. 2–5 are replotted on the same time axis. For clarity, the data points have been omitted, and the dashed curves are the best fit lines. The maximum extent of the cGMP decrease at later times has also been included (×, 180 R*/rod/s; open circle, 550 R*/rod/s; triangle, 8540 R*/rod/s; open square, 4.8 × 106 R*/rod/s). B, the complete time courses for the photocurrent kinetics shown in Figs. 2–5, replotted on the same time scale.

The Latent Period and Rate of cGMP and Photocurrent Kinetics Co-vary over 4 Decades of Light Intensity

Two aspects of the cGMP and photocurrent kinetics presented in Figs. 2–5, namely the latent period and the initial rate, appear to be changed in a similar manner when the light stimulus is varied over 4 decades of intensity (Fig. 7). The latent period was defined as the time interval between the onset of illumination and the first detectable response to the light stimulus. A cumulative sum control chart test was employed to determine when a statistically significant change in membrane current or cGMP concentration had occurred. This method also estimates the time at which a trend leading to the statistically significant change commences (see “Experimental Procedures”). For the photocurrent, there was little (<1% ) experimental error in the measurements, and the photocurrent latent period (filled circles connected with solid line, Fig. 7A) is plotted as the time at which the membrane current statistically differed from the base-line value in the dark. The photocurrent latent period decreased as the light intensity increased, approaching a minimum value of approximately 50 ms at intensities greater than 105 R*/rod/s.

For the cGMP kinetics, two estimates of the latent period at each light intensity are prested. The filled squares in Fig. 7A represent the time at which the cGMP concentration statistically differed from the dark-adapted value. Knowing when the change in cGMP concentration becomes statistically significant also allows an extrapolation back to the time at which the trend in the cGMP data leading to the significant change commenced (Fig. 7A, open squares). These two estimates which are obtained from the cumulative sum control chart provide a range over which light-induced changes in cGMP concentration may be initiated.

The results of Fig. 7A demonstrate that increasing the light intensity caused similar reductions in the cGMP and the photocurrent latent period, except when OS-IS were illuminated with 160 R*/rod/s. The lack of covariance in latent periods at this light intensity may simply reflect experimental limitations in accurately determining the cGMP latent period. (In 4 out of 8 individual experiments a significant decrease in cGMP concentration was detected within the first second of illumination; in only one experiment did the latent period extend beyond 1.5 s.) At 490 and 8400 R*/rod/s the photocurrent latent periods (430 and 180 ms, respectively) were in good agreement with the time at which light-induced decreases in cGMP concentration were statistically significant (500 and 200 ms, respectively). The actual time at which changes in cGMP occurred may be slightly sooner, based on the time at which the trend in the cGMP decrease was first detected. At the highest light intensity, significant changes in cGMP concentration occurred in the samples that were quenched at the same time the light was turned on (Fig. 5). Because of the finite quench time (approximately 50 ms) for the rapid quench apparatus (see “Experimental Procedures”) we estimate the cGMP latent period to be no greater than 50 ms at this light intensity; at this intensity, the photocurrent latent period was 56 ms. In summary, the covariance of cGMP and photocurrent latent periods observed in Fig. 7A suggests that changes in cGMP concentration may be a necessary intermediate step in the process leading to suppression of the light-regulated conductance.

Fig. 7B demonstrates that the rate of the light-induced cGMP decrease increases over the same range of light intensities as does the rate of change of the photocurrent. It was not feasible to determine a true initial rate for the cGMP kinetics, so the entire time course was modeled as a pseudo-first order decay of cGMP concentration to the final cGMP concentration in the ligh(Figs. 2–5, dashed line). The fit of the data to this model was good at each light intensity, and pseudo-first order rate constant obtained in this way allowed calculation of the minimum number of cGMP molecules hydrolyzed per s. The maximum rate of the photocurrent was determined as the maximum slope of the photocurrent kinetics. The results in Fig. 7B indicate that changes in cGMP hydrolytic rate occur over 4 decades of light intensity and parallel changes in the photocurrent rate. Thus, the initial rate, as well as the latent period, of the photocurrent may be regulated by light-induced changes in cGMP metabolism.

The cGMP Response to Light Is Highly Amplified

The initial rate of the cGMP decrease in intact OS-IS reported in Fig. 7B represents a very large amplification of the light stimulus at low intensities of illumination (Table I). At an intensity of 180 R*/rod/s, a minimum of 1.3 × 105 cGMP molecules is hydrolyzed per R*, consistent with in vitro measurements of frog phosphodiesterase activity (Yee and Liebman, 1978). (Note that since these calculations of cGMP hydrolytic rates are determined from the change in total cGMP concentration, they reflect a minimum estimate of the phosphodiesterase activity. This is because the cGMP concentration at any time reflects the balance between cGMP hydrolysis by phosphodiesterase and cGMP synthesis by guanylate cyclase.) At a light intensity of 60 R*/rod/s a similar stoichiometry of 1 × 105 cGMP hydrolyzed per R* is obtained (data not shown). If one assumes a catalytic turnover for phosphodiesterase of 103 s−1 (Miki et al., 1975; Baehr et al., 1979), the number of phosphodiesterase molecules activated per rhodopsin bleached can be estimated (Table I). At 180 R*/rod/s, at least 130 phosphodiesterase molecules become activated for each photolyzed rhodopsin. With brighter illumination more than 1 rhodopsin is photoactivated per disc, and the efficiency of the amplification process decreases. This is because additional photolyzed rhodopsins on a disc membrane will have a greater probability of encountering a phosphodiesterase molecule which already has been activated. In contrast, the rate of cGMP hydrolysis, which depends only on the total number of activated phosphodiesterase molecules, continues to increase as the light intensity increases (cf. Liebman and Pugh, 1979, for discussion).

Table I.

Stoichiometry of cGMP hydrolysis and phosphodiesterase activation

Light intensity
 Stoichiometry
R*/rod/s R*/disc/sa cGMP hydrolyzed/R* Phosphodiesterase activated/R*b
180 0.1 1.3 × 105 130
550 0.3 6.7 × 104 67
8540 5 1.3 × 104 13
4.8 × 106 2670 5.4 × 101 0.1
Open in a new tab
a

Assuming 1800 discs/frog rod outer segment.

b

Assuming a molecular activity for phosphodiesterase of 103 s−1 (Miki et al., 1975; Baehr et al., 1979).

The question arises whether this amplification is sufficient to cause significant changes in total cGMP concentration on the time scale of membrane current suppression. From the rate data in Table I, it was calculated that at each light intensity between 25 and 40% of the total cGMP (or ⅓–⅔ of the light-sensitive cGMP) would theoretically be consumed by the time the current is half-suppressed. Thus, the light-activated phosphodiesterase of OS-IS has the hydrolytic potential to lower the intracellular cGMP concentration rapidly enough to produce changes in cGMP concentration sufficient to control membrane conductance.

Ca2+ cGMP Ionophore Does Not Alter the Initial Kinetics of the cGMP Decrease or the Photocurrent

The results presented in this section were designed to examine whether changes in intracellular Ca2+ concentration during the initial stages of phototransduction could modulate some aspects of the light-regulated permeability mechanism. Purified OS-IS were incubated in a 20 nM Ca2+ medium containing the Ca2+ ionophore A23187 which provides a shunt for Ca2+ in the rod (Schnetkamp, 1979).2 In this way, the Ca2+ concentration in the rod can be buffered at a constant value, and light-induced changes in intracellular Ca2+ concentration are expected to be minimized. The effects of 10 μM A23187 on the initial kinetics of the cGMP decrease and on the photocurrent kinetics were examined at two light intensities (Figs. 8 and 9). In both cases, little difference is seen in the cGMP kinetics or the photocurrent kinetics when A23187 is added to suspensions of OS-IS. The minor difference in the time courses at 790 R*/rod/s (Fig. 9A) was not considered significant; addition of A23187 to OS-IS at lower and higher light intensities (both continuous and flash illumination) showed no significant changes in kinetics compared to the control (data not shown).

Effects of Low External Ca2+ on the Membrane Current in the Dark

Although Figs. 8 and 9 suggest that changes in Ca2+ concentration are not required in the generation of the cGMP or photocurrent response, changes in external Ca2+ have been shown to have rapid dramatic effects on the light-regulated conductance (Yoshikami and Hagins, 1973; Woodruff and Fain, 1982; Hodgkin et al., 1984). Woodruff and Fain (1982) have also shown that the change in membrane voltage upon lowering the external Ca2+ to various final concentrations does not correlate with the final magnitude of the cGMP elevation in intact retina. In the present study, it was found that the low Ca2+-induced elevation of cGMP concentration of dark-adapted OS-IS (Fig. 1) was much slower than the change in membrane current reported by Hodgkin et al., (1984). Rapid perfusion of isolated photoreceptors with a low Ca2+ solution causes a 10-fold transient elevation of membrane current that peaks within 10 s (Hodgkin et al., 1984; Fig. 9). We have also obtained results with OS-IS wherein lowering the Ca2+ concentration elevated the membrane current prior to the increase in cGMP, although our perfusion turnover was slower (data not shown). This then suggests that the membrane current may change as rapidly as the bath concentration changes. In contrast, changes in OS-IS cGMP concentration were not statistically different (t test, p < 0.05) from the values in 1 mM Ca2+ until 12 s after lowering the Ca2+ concentration. The 20% elevation of total cGMP concentration observed at this time is probably not responsible for the 10-fold elevation of the dark current. These data are consistent with the notion that Ca2+ can act on the conductance mechanism without its effects being mediated through changes in cGMP concentration.

To determine whether the Ca2+-induced elevation in cGMP concentration was slow due to the time required for intracellular Ca2+ to reach a new equilibrium with external Ca2+, the time course of cGMP elevation in 20 nM Ca2+ was compared in the presence and absence of Ca2+ ionophore (Fig. 10A). The rate and extent of the Ca2+-induced change in cGMP concentration in OS-IS is only slightly greater in the presence of A23187. The results suggest that the slow Ca2+-induced cGMP elevation is not due to a slow equilibration of intracellular Ca2+ with the external medium.

An alternate explanation of the results of Fig. 10A is that the ionophore did not partition into the plasma and/or disc membranes under these experimental conditions. To examine this possibility, outer segments were isolated from frog retinas in a modified Percoll-Ringer's in which all Na+ was replaced by K+. In the absence of external Na+ it is thought that the sodium/calcium exchange mechanism in the plasma membrane of intact rods is inhibited (Schnetkamp, 1980; Yau and Nakatani, 1984). Under this condition, lowering external Ca2+ would be expected to not greatly affect the internal Ca2+ concentration. This expectation is confirmed in Fig. 10B by the absence of a cGMP increase during the first 4 min after the external Ca2+ concentration was lowered to 20 nM. At later times a 30% increase in cGMP concentration was observed, perhaps due to a slow leakage of Ca2+ from the rod. In contrast, the presence of A23187 provides a shunt for external and internal Ca2+ and results in a Ca2+-induced 2-fold elevation of cGMP concentration under the K+-Ringer's condition. The lowered stimulation of cGMP levels seen in Fig. 1OB compared with Fig. 10A in the presence of A23187 may result from either the metabolic stress induced by the K+-Ringer's medium or from the differences between OS-IS and OS. In any event, the results of Fig. 10 corroborate the efficacy of the Ca2+ ionophore A23187 to alter Ca2+ gradients across the plasma membrane of the photoreptor, as recently measured spectroscopically.2

DISCUSSION

The major finding of this study is that initial changes in total intracellular concentration of cGMP correlate with some, but not all, aspects of the electrical response of rod photoreceptors to illumination. These results indicate that cGMP is likely to be an internal messenger that mediates between rhodopsin activation by light in the disc membrane and the initial changes in ionic permeability of the plasma membrane. However, the lack of an exact correspondence between total cGMP concentration and the extent of membrane current suppression by light indicate that other factors (e.g. compartmentation of cGMP, metabolic flux, or other internal messengers) are also involved in regulating membrane conductance. Although it is unlikely that light-induced changes in cytoplasmic Ca2+ concentration are a required intermediate step in visual excitation, it is possible that Ca2+ can modulate the light-regulated conductance mechanism in the dark by a mechanism independent of cGMP metabolism.

Lowering the external Ca2+ concentration from 1 mM to 20 nM Ca2+ caused a 4-fold increase in total cGMP concentration in frog OS-IS. This low Ca2+-induced elevation of cGMP levels in OS-IS is smaller than the 10-fold elevation observed in intact frog retina (Kilbride, 1980) but somewhat larger than the 2-fold increase in cGMP seen in isolated frog outer segments (Polans et al., 1981). The elevation of cellular cGMP levels induced by lowering the externat Ca2+ concentration may result from stimulation of guanylate cyclase activity (Lolley and Racz, 1982) or inhibition of phosphodiesterase activity (Robinson et al., 1980), or both.

Lowering the external Ca2+ concentration also has multiple effects on rod physiology. 1) The membrane current flowing in the dark is transiently increased by a large amount when the external Ca2+ is lowered and then falls to a stable level which is somewhat greater than that observed in 1 mM Ca2+ (Hodgkin et al., 1984). 2) The light sensitivity of rod photoreceptors in low external Ca2+ is decreased (Yoshikami and Hagins, 1973; Bastian and Fain, 1982). For OS-IS incubated in 1 mM Ca2+, half-maximal suppression of OS-IS membrane current occurs with 30 R*/flash; when the external Ca2+ is lowered to 10 nM, this value is shifted to 800 R*/flash.2 3) The Ca2+ content of OS-IS is decreased to about 20% of the value (1.3 mol of Ca2+/mo1 of rhodopsin) observed in OS-IS maintained in 1 mM Ca2+.2 Since we have measured biochemical and electrophysiological responses to illumination under identical experimental conditions, these perturbations to the cell's Ca2+ metabolism do not compromise the correlations reported in this paper between cGMP and the conductance mechanism.

The relevance of the results obtained with OS-IS exposed to a low external Ca2+ solution to the case where the intracellular Ca2+ of the rod has not been perturbed remains to be established. In a Percoll-Ringer's solution containing 1 mM Ca2+, a 20% maximal light-induced decrease in cGMP concentration was observed (Cote et al., 1984), precluding further studies at subsaturating light intensities. If the correlations we observe between the cGMP and photocurrent kinetics are valid when the external Ca2+ is raised to 1 mM, it will be interesting to determine how small light-induced changes in total cellular cGMP concentration can modulate the cGMP-regulated conductance.

Limitations to the Role of Ca2+ in Visual Transduction

The results presented in this paper seriously constrain the role of Ca2+ in the initial events of visual excitation. Addition of Ca2+ ionophore does not alter either the photocurrent or the cGMP kinetics (Figs. 8 and 9). This supports the notion that light-induced changes in cytoplasmic Ca2+ concentration are not a necessary intermediate step in generating the electrical response to illumination. Nicol et al.2 demonstrate that incubation of rods in a 10 nM Ca2+ medium causes the loss of approximately 80% of total cellular Ca2+, presumably via the sodium/calcium exchange mechanism (Schnetkamp, 1980; Yau and Nakatani, 1984). Addition of the ionophore A23187 causes a negligible additional release of Ca2+ and ensures that transient Ca2+ gradients during illumination will be minimized. Both the cGMP kinetics and the photocurrent kinetics are completely unaltered at two different light intensities by the presence of ionophore (Figs. 8 and 9). The efficacy of the ionophore in releasing intracellular Ca2+ was demonstrated by preparation of rods in a solution that minimizes sodium/calcium exchange (Fig. 10B), supporting the idea that the ionophore provides a shunt for Ca2+ (cf. Nicol et al.2 for a detailed study of A23187 action on rod physiology).

Although the initial events of phototransduction do not appear to require changes in cytoplasmic Ca2+ concentration, it is also clear that changes in external Ca2+ concentration in the dark can affect the light-regulated conductance mechanism (cf. Korenbrot, 1985, for review). An effect of Ca2+ on the conductance mechanism has not been observed under physiological conditions with excised patches of rod membrane (Fesenko et al., 1985). Because there is at most a 20% change in cGMP concentration during the time when changing the external Ca2+ concentration exerts its primary effects, it is possible that the effect of changing external Ca2+ on the membrane current is mediated via a Ca2+-sensitive pathway not involving cyclic nucleotides. However, we cannot rule out the possibility that altering Ca2+ causes local changes in cGMP concentration near the conductance mechanism, thus resulting in changes in ion permeability.

Evidence Supporting cGMP as the Second Messenger for Visual Transduction

cGMP has been shown to satisfy several criteria for an internal messenger for visual excitation in rod photoreceptors. First, changes in cGMP concentration are observed at all light intensities tested, including dim illumination where the membrane current has not been fully suppressed. At an intensity where only 10% of the maximum membrane current is suppressed, the cGMP decrease is easily detected within seconds and reaches the same final concentration observed at higher light intensities (Fig. 2). A 50% maximum decrease in cGMP concentration has also been observed at a light intensity of 60 R*/rod/s, where the photocurrent is barely detectable above instrumental noise (data not shown). At 60 and 160 R*/rod/s, it appears that the response begins when approximately 100–200 R* molecules have been cumulatively bleached. In contrast, illumination of intact retina requires a cumulative bleach of several thousand rhodopsins before a significant change in cGMP levels was noted (Kilbride and Ebrey, 1979; Kilbride, 1980; Woodruff and Fain, 1982). The minimum cumulative rhodopsin bleach required to detect a cGMP response in unpurified outer segments (Woodruff and Bownds, 1979) is similar to that observed for OS-IS.

A second criterion that cGMP satisfies is that the response of the messenger concentration is highly amplified compared to the light stimulus. The results summarized in Table I are consistent with a highly amplified enzymatic cascade that acts with a high stoichiometry to lower the total cGMP concentration on the time scale required to control the observed changes in membrane permeability. With dim illumination bleaching less than 1 rhodopsin/disc/s, a minimum of 105 cGMP molecules are hydrolyzed per photolyzed rhodopsin (Table I). These results with intact photoreceptors corroborate in uitro studies of the reaction sequence leading from photolyzed rhodopsin to G protein activation and hence to the activation of phosphodiesterase (cf. Miller (1981) for reviews).

A third criterion for an internal transmitter for visual excitation is that the time course of change in the transmitter concentration must precede the time course of the photocurrent. The data presented in Figs. 2–5 indicate that the light-induced cGMP kinetics are rapid enough at all light intensities for changes in cGMP concentration to regulate changes in the light-regulated conductance mechanism. The initial onset of the decrease in cGMP concentration occurs at approximately the same time as the onset of the photocurrent, except perhaps at the lowest light intensity (Fig. 7A). A similar correlation between the rate of cGMP hydrolysis and the photocurrent rate is seen as a function of light intensity (Fig. 7B). Although these correlations between the cGMP and photocurrent kinetics do not necessarily imply a causal relationship, the evidence is consistent with light-induced changes in total cGMP both preceding and regulating the latent period and the rate of the photocurrent.

Changes in Measured cGMP Concentration Do Not Correlate with the Extent of Membrane Current Suppression

Based on recent electrophysiological results with excised patches of membrane (Fesenko et al., 1985; Zimmerman et al., 1985), truncated rod cells (Yau and Nakatani, 1985), and whole cell-attached recordings (Zimmerman et al., 1985; Matthews et al., 1985; Cobbs and Pugh, 1985), it has been proposed that binding and dissociation of cGMP to sites on the plasma membrane directly control the light-regulated ionic conductance of rods. Biochemical studies of cGMP-regulated ion fluxes in disc membranes (Caretta and Cavaggioni, 1983; Koch and Kaupp, 1985; Caretta, 1985) also suggest that the light-regulated conductance can be regulated by cGMP. One plausible hypothesis is that the light-induced decrease in cGMP concentration causes dissociation of bound cGMP from the light-regulated channel, resulting in channel closure. It is predicted that the concentration of cGMP in the rod should thus correlate with the magnitude of the membrane current.

The results of this study do not support such a simple model of equilibrium binding of cGMP to the ionic conductance mechanism. One way in which the cGMP concentration and membrane current clearly do not correlate is demonstrated in Fig. 1. When the external Ca2+ is lowered to 20 nM, the large transient increase in dark membrane current (Hodgkin et al., 1984) is accompanied by only minor changes in cGMP concentration. Second, the cGMP concentration observed after light exposure of OS-IS incubated in a 20 nM Ca2+ solution is greater than the dark-adapted levels of cGMP of OS-IS in a 1 mM Ca2+ solution. Thus, the magnitude of the membrane current cannot be solely controlled by the total intracellular cGMP concentration. Third, the lack of an exact correspondence between the cGMP kinetics and the photocurrent kinetics at two bright light intensities (Figs. 4 and 5) indicates that significant changes in cGMP concentration can precede corresponding changes in the photocurrent. Fourth, the maximum light-induced decrease in total cGMP concentration is observed at all light intensities, whereas the photocurrent reaches a steady state level whose magnitude is graded with the intensity of continuous illumination (Fig. 6). These four lines of evidence clearly demonstrate that the total intracellular cGMP concentration does not correlate with the magnitude of membrane current flowing in the dark or during continuous illumination.

Is there some way to reconcile the results of this study with the cGMP-regulated conductance that has been reported by several laboratories? Goldberg et al. (1983) have proposed that changes in cGMP metabolic flux (not changes in cGMP concentration per se) regulate the light-regulated membrane current. Observed changes in total cGMP concentration might be a secondary consequence of large changes in cGMP turnover by the coupled action of guanylate cyclase and phosphodiesterase. However, it is difficult to conceive how this metabolic flux could regulate an ion channel. Two of the chemical products of cGMP flux, namely protons and 5′-GMP, have been shown not to have a direct effect on the conductance mechanism (Brown et al., 1977; MacLeish et al., 1984; Hagins and Yoshikami, 1985), while slowly hydrolyzable cGMP analogs are effective in modulating the conductance (MacLeish et al., 1984; Zimmerman et al., 1985). Another possibility is that the cGMP metabolic flux measured by Goldberg et al. (1983) actually represents sequential activation of phosphodiesterase followed by later activation of guanylate cyclase. This would result in a transient decrease in cGMP concentration followed by a restoration of cGMP levels at a later time. This model would be consistent with the electro-physiological and biochemical experiments cited above supporting a direct interaction of cGMP with the membrane permeability mechanism.

Another intriguing possibility is that there are subcellular compartments of cGMP in the outer segment that undergo large local changes in concentration during illumination. In this case, measurements of total cellular cGMP would not necessarily reflect the relationship between changes in cGMP in a subcellular compartment and localized changes in the light-regulated conductance. There are several reasons to suspect that cGMP compartmentation may occur in rod outer segments. 1) The light-insensitive pool of cGMP in rods is often 40–75% of the total cellular cGMP concentration (Fig. 6; cf. Woodruff and Bownds, 1979; Kilbride and Ebrey, 1979; de Azeredo et al., 1981); Cote et al., 1984). 2) High-affinity cGMP-binding sites have been identified in rods that could potentially sequester a large fraction of total cGMP (Yamazaki et al., 1980, 1982; Volotovskii et al., 1984; Caretta et al., 1985). 3) It should also be noted that there are precedents for cyclic nucleotide compartmentation in other cell types (cf. Earp and Steiner, 1978; Doskeland and Oegrid, 1981; Lincoln and Corbin, 1983, for reviews). The possibility remains to be tested that the measured changes in total cGMP concentration may reflect larger changes in cytoplasmic free cGMP concentration in the region near cGMP-regulated ion channels in the plasma membrane of photoreceptors.

Acknowledgments

We thank Benjamin Kaupp for many insightful discussions and for suggesting the use of A23187 in the experiments shown in Figs. 8–10. We also thank Paul Schnetkamp, Michael Biernbaum, and Patricia Witt for helpful comments during preparation of the manuscript.

Footnotes

*

This work was supported by Research Grants EY00463 and EY05798 and Training Grants EY07049 and HD07118 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

The abbreviations used are: OS-IS, frog rod outer segments with the ellipsoid portion of the inner segment still attached; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; HEPES, N-2-hydroxyethylpiperazine-N′2-ethanesulfonacid; R*, bleached rhodop sin.

2

G. D. Nicol, U. B. Kaupp, and M. D. Bownds (1986) J. Gen. Physiol., submitted for publication.

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