I. INTRODUCTION

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Sensory receptors respond to specific stimuli with an electrical response and are vital to our perception of the external world. Most sensory receptors give graded responses, whose amplitude increases proportionally with the intensity of the stimulus. The relationship between stimulus intensity and response is, however, not fixed for most sensory receptors but is altered as the mean level of stimulation is changed. This process, called adaptation, is a nearly universal feature of sensory transduction, responsible for example for the fading of intense odors and our accommodation to the sound of city traffic (see for example Ref. 65). It is also responsible for adjustments in the sensitivity of photoreceptors and the visual system, which make it possible for us to detect objects in our environment at nearly constant contrast despite large changes in the level of ambient illumination.

For rod photoreceptors, which are specialized for the detection of dim illumination and can respond to single photons of visible light (13), the linearity of signal detection and very high absolute sensitivity has the necessary consequence that response amplitude reaches a maximum value for flashes of relatively dim intensity (see for example Refs. 14, 60, 64, 132). The process of adaptation acts to decrease the sensitivity of the transduction cascade, reducing the amplitude of the single-photon response. In steady light bright enough initially to produce a response of maximum amplitude in a dark-adapted receptor, the sensitivity of transduction is rapidly reduced so that saturation does not occur and the receptor can respond to increments or decrements of illumination in the presence of the background illumination.

The electrical responses in Figure 1 illustrate the phenomenon of photoreceptor adaptation. These recordings were all made from the same rod, isolated from the retina of the salamander Ambystoma tigrinum. The responses in Figure 1A were recorded from the dark-adapted photoreceptor. In darkness there was a steady current of ~40 pA entering the outer segment of the cell through the cGMP-gated channels. This is usually referred to as the circulating current or dark current (see Ref. 220). Brief flashes of illumination reduce this current by closing the cGMP-gated channels, as we describe in detail in section II. This produces a change in circulating current whose peak amplitude, for dim flash intensities, varies approximately linearly with the intensity of the stimulus. Bright flashes close all of the cGMP channels and reduce the dark current to zero, effectively saturating the rod (see Ref. 184).

View larger version (22K): [in this window] [in a new window]   Fig. 1. Light adaptation of responses from a salamander rod recorded with a suction pipette. Timing of flashes and steps of light are indicated by upward deflections in top trace. Intensities of flashes (IF) are given for the responses in A-C by the numbers above each of the flash markers, in units of log10 photons ()·µm2. Flashes increase progressively in intensity with each flash about 4 times as bright as its predecessor, in darkness (A) or in the presence of steady backgrounds of intensities 0.24 (B) and 1.57 (C) log10 photons·µm2·s1. Note decrease in amplitude and speeding of kinetics of the response to any given flash intensity for backgrounds of increasing brightness. [From Matthews (171). Reprinted with the permission of Cambridge University Press.]

When the rod in Figure 1 was stimulated with steady light (Fig. 1, B and C), there was an initial relaxation in the response waveform to the steady illumination, indicating that the sensitivity of the rod was decreasing. The sensitivity to flashes also decreased so that a brighter flash intensity was now required to produce a response of the same amplitude. This had the effect of shifting the entire operating range of the receptor to brighter intensities. Careful examination of the responses in Figure 1, B and C, shows that the sensitivity to flashes is approximately inversely proportional to the intensity of the background.

This relationship of inverse proportionality between sensitivity and background intensity is known as Weber's law and was first proposed not only for the visual system but for all sensory systems by Weber in the 1800s (278). Careful psychophysical investigation (summarized in Refs. 8, 23, 243) shows that Weber's law is valid for visual perception particularly for large visual fields and for flashes of relatively long duration. For single photoreceptors, Weber's law seems to be followed quite closely for background lights over at least two to three orders of magnitude. This is true for both rods (14, 64) and cones (12) in lower vertebrates, and also for both rods (139, 172, 196, 267, 268) and cones (254) in mammals.

For photoreceptors, Weber's law can be expressed in the following way (see Refs. 12, 14, 64). We define the flash sensitivity (S) as the amplitude in millivolts or picoamps of the response to a flash (evoking only a small response) divided by the intensity of the flash, in units of photons per square centimeters or rhodopsin molecules bleached per photoreceptor. If S_F is the flash sensitivity of the receptor in the presence of a background and S_F^D the flash sensitivity in darkness, then

(1)

where I_B is the intensity of the backgound. The constant I₀, sometimes called the dark light (8), is equal to the background intensity required to reduce sensitivity by one-half. This equation says that for background lights sufficiently greater than I₀ (i.e., for I_B I₀), S_F is nearly equal to (S_F^D · I₀)/I_B. Since S_F^D and I₀ are both constants, sensitivity is inversely proportional to background intensity, as Weber's law predicts. Equation 1 can also be expressed in the following form (see Ref. 12)

(2)

This relationship has been plotted in Figure 2.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Flash sensitivity (SF) of salamander rods as a function of background intensity (IB). Flash sensitivity has been linearized with Equation 2. Each symbol represents data for a different cell. The results have been normalized for each cell to Io, the background intensity required to decrease the flash sensitivity by a factor of 2. [From Jones et al. (121), by copyright permission of The Rockefeller University Press.]

The decrease in sensitivity in the presence of steady background light is often referred to as light adaptation or background adaptation. The sensitivity of photoreceptors is also decreased by previous exposure to bright light that bleaches a significant fraction of the visual pigment. It is a common experience that bright light produces a long-lasting desensitization, called bleaching adaptation, that recovers slowly with a time course of many minutes during dark adaptation. This process has also been studied in considerable detail by visual psychophysicists (summarized in Refs. 7, 8, 23, 153, 243).

Stiles and Crawford (265) first suggested that light bright enough to produce significant bleaching of the photopigment desensitizes by producing an "equivalent" background light. This equivalent background was later proposed to be produced in some way by bleached pigment and to persist until the pigment was regenerated (see Refs. 7, 8). Although the equivalent background hypothesis was originally proposed without reference to a molecular mechanism, we now know that in single photoreceptors, bleached photopigment can in fact stimulate the visual cascade and produce a steady excitation much like the one produced by steady background light.

In this review, we summarize what is presently known about the molecular mechanisms of both background and bleaching adaptation. We begin by describing visual transduction, since a detailed knowledge of this process is fundamental to our understanding of adaptation. The regulation of key components of the transduction cascade is almost certainly responsible for the changes in sensitivity produced by background light and bleaches. We then describe what we know about adaptation itself, beginning first with the role of Ca²⁺ as a second messenger controlling the sensitivity of the photoreceptor and then presenting recent evidence for possible pathways regulated by Ca²⁺. Finally, in section IV, we present our current understanding of the mechanism of bleaching adaptation. We describe the evidence for excitation of the cascade by bleached pigment and then explain how this excitation is believed to occur at a molecular level and how it may be responsible for the changes in photoreceptor sensitivity after exposure to bright light. Some of this material has been the subject of previous articles, which may also be usefully consulted (11, 50, 66, 69, 102, 138, 162, 191, 215, 225, 229-231).

	II. VISUAL TRANSDUCTION

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Visual detection begins with the absorption of a photon by the photopigment rhodopsin (summarized in Refs. 101, 102, 215, 233), a member of the G protein-coupled receptor family also including many hormone receptors, odorant receptors, and metabotropic synaptic receptors. These proteins are all integral membrane proteins having seven, mostly -helical, transmembrane domains (253, 261, 273). Rhodopsin is unusual among the G protein-coupled receptors in that it is bound in darkness (in its inactive conformation) to a small-molecular-weight chromophore, 11-cis-retinal, that regulates its activity. The 11-cis-retinal is covalently bound via a Schiff base linkage to the terminal () amino group of a lysine (Lys-296 in bovine rhodopsin), and this Schiff base linkage is protonated (see Refs. 32, 233). The absorption of a photon by the 11-cis-retinal chromophore produces a photoisomerization to all-trans-retinal, resulting in a subtle change in the conformation of rhodopsin (73, 75, 261).

The active form of the photopigment is a spectrally distinct intermediate called metarhodopsin II or Rh^* (61), and it is this form of the pigment that triggers the transduction cascade (see Fig. 3). Rh^* is inactivated by two sequential processes: first the phosphorylation of rhodopsin (21, 146) and then the binding of arrestin to the phosphorylated photopigment. The COOH terminus of rhodopsin contains seven serines and threonines that are capable of being phosphorylated, and in rod outer segments in vitro, an average of seven phosphorylations per rhodopsin has been observed (281). Nevertheless, it is likely in vivo that the phosphorylation of fewer amino acids, and possibly only of one, at (in the bovine pigment) Ser-334 or Ser-338, normally contributes to rhodopsin turnoff (207). In mouse rods containing COOH-terminal-truncated rhodopsin and lacking Ser-334 and Ser-338, the termination of transduction is greatly retarded (31).

View larger version (62K): [in this window] [in a new window]   Fig. 3. Schematic diagram illustrating mechanisms of activation and inactivation in vertebrate photoreceptors. [From Fain (65). Copyright © 1999 by The President and Fellows of Harvard College. Reprinted by permission of Harvard University Press.]

Phosphorylation of rhodopsin can, of itself, reduce the ability of Rh^* to activate the transduction cascade (see Refs. 158, 284). A complete inactivation of Rh^*, however, requires in addition the binding of either arrestin (143) or perhaps its splice variant p44 (263). Both p44 and arrestin have been shown to bind preferentially to phosphorylated rhodopsin, although p44 also will bind to nonphosphorylated rhodopsin. The binding to rhodopsin turns off the transduction cascade, probably by sterically hindering the binding of the G protein transducin (141, 147, 232). In mouse rods lacking the arrestin gene (and therefore both arrestin and p44), the decay of the photoresponse is greatly prolonged (284). Furthermore, phytic acid, which inhibits the interaction of arrestin and phosphorylated Rh^*, also greatly prolongs the light response (214, 177).

The time course of phosphorylation and arrestin binding determines the lifetime of Rh^*, which probably varies considerably for rods from different species and between rods and cones. Pepperberg et al. (222) have argued from an analysis of the time course of decay of the photoresponse that the lifetime of Rh^* in tiger salamander rods is on the order of 1-2 s and is rate limiting for the recovery of the photoresponse. A similar value was obtained by Rieke and Baylor (236) from dialyzed rod outer segments; they used the clever technique of abruptly increasing the gain of transduction by presenting elevated GTP concentrations at fixed times after the presentation of a light flash. Other experiments, however, suggest that the lifetime of Rh^* may be much shorter than this and not rate limiting (176, 249). We shall return to this subject in section IIID, since regulation of the lifetime of Rh^* has been proposed to be an important mechanism of sensitivity regulation.

After the inactivation of Rh^* by phosphorylation and arrestin binding, the photopigment must be regenerated before a new photon can be absorbed. This is a multistep process; the protein moiety (opsin) must be dephosphorylated, and the chromophore must be converted from all-trans-retinal back to 11-cis-retinal. The retinal in the outer segment is first reduced to all-trans-retinol by a membrane-associated all-trans-retinol dehydrogenase within the outer segment (99). The chromophore then leaves the photoreceptor and is transported to an adjacent layer of epithelium, called the retinal pigment epithelium (or RPE; see Ref. 215). There it is isomerized to the 11-cis-isomer and then retransported back to the photoreceptor (see Ref. 44), where it recombines with dephosphorylated opsin. This process of pigment regeneration is often referred to as the visual cycle, and we shall describe some of its features in more detail in section IVD.

The change in the conformation of rhodopsin exposes groups of amino acids on the cytoplasmic side of the protein that form a binding site for the heterotrimeric G protein transducin. The binding of transducin facilitates an exchange of GTP for GDP on the guanosine nucleotide binding site of the transducin -subunit, producing the active form T·GTP, which binds to and activates a membrane-bound cyclic nucleotide phosphodiesterase (PDE). This enzyme hydrolyzes cGMP and effectively reduces the concentration of this nucleotide in the cytoplasm of the outer segment, decreasing the probability of the opening of cyclic nucleotide-gated channels. The closing of these channels generates the electrical response of the photoreceptor (see Fig. 3).

Transducin resembles other heterotrimeric G proteins in structure and function (see Refs. 17, 20, 96, 100, 202). There are three subunits, called T, T, and T (or just , , and ), with a stoichiometry of 1:1:1. The rod and cone T subunits are distinct molecules but share 80% sequence identity; both are members of the _i/_o subfamily of G protein -subunits. Rods and cones also contain different -subunits. Inactive T is normally membrane bound; after the exchange of GTP for GDP on the -subunit, the T remains membrane bound, but the T·GTP appears to be less tightly associated with the membrane and may very well be released into the space between adjacent disks (6, 144, 275).

The T·GTP then diffuses and binds to a membrane-associated photoreceptor-specific PDE. This protein consists of four subunits (see Refs. 15, 74, 102), two catalytic and two inhibitory. In rods the two catalytic subunits of the PDE are different and are called  and ; in cones, they are apparently identical and called '. Each catalytic subunit is associated with an inhibitory subunit called , but there appear to be different  in rods and cones (210). In addition, a subunit called  found in rod but not cone outer segments can bind to the rod ₂ complex and solubilize it, disrupting its normally close association with the disk membrane (78). It is possible that  somehow regulates PDE activity by removing it from its normal site of interaction with T·GTP.

Because the PDE has two inhibitory subunits each of which can bind T·GTP (49), and because the catalytic activities of the PDE - and -subunits are approximately the same, the gain of the activation of the PDE by T·GTP is 0.5. The PDE then hydrolyzes cGMP with high velocity so that the total gain from Rh^* to cGMP in a dark-adapted rod is of the order of 10⁵-10⁶ molecules hydrolyzed during the lifetime of a single activated pigment molecule (294). This reaction is thought to be terminated as for other heterotrimeric G proteins, when the GTP of T·GTP is hydrolyzed to GDP. Alternative mechanisms of termination have also been suggested, including inactivation of PDE by excess PDE (63) and phosphorylation of PDE by a specific kinase, leading to inactivation of PDE even without GTP hydrolysis (271, 272). The significance of these mechanisms is not yet clear.

The rate of hydrolysis of GTP to GDP may be critically important in determining the time course of the photoresponse and may be the rate-limiting reaction that determines the decline of the photocurrent back to the dark-adapted level (249). The rate of hydrolysis of bound GTP is quite slow in vitro, taking many seconds (see, for example, Ref. 3). This is too slow to account for the turn off of transduction, and it is likely that hydrolysis is much more rapid in the rod outer segment (274). Recent experiments indicate that hydrolysis is accelerated in vivo by a membrane-bound factor (1) that acts as a GTPase accelerating protein, or GAP. The most important GAP in photoreceptors seems to be the membrane-associated protein RGS9 (104), which is expressed in rods and at even higher levels in cones (42). RGS9 can by itself produce a large acceleration of the rate of the GTPase hydrolysis, although the rate of hydrolysis is further accelerated by the addition of PDE, which is also known to function as a photoreceptor GAP (4, 270). Other RGS proteins called RGS4 (203) and RGS-r or RGS16 (30, 280) have also been reported to be found in retina, but their role in transducin GTPase modulation is less clear.

It is possible that the rate of GTP hydrolysis by T·GTP is also regulated by noncatalytic binding sites for cGMP on the PDE molecule (286). Both the - and -subunits have noncatalytic sites for cGMP binding, and the affinity of these sites for cGMP decreases after PDE activation (41, 285). Because the rate of GTP hydrolysis by T·GTP is accelerated when cGMP dissociates from these noncatalytic sites, it would be possible to imagine that activation of the PDE by the binding of T·GTP to PDE would decrease the concentration of cGMP in the outer segment, leading to a decrease in the binding of cGMP to noncatalytic sites on the PDE - and -subunits and an increase in the rate of hydrolysis of GTP bound to T. This would in effect modulate the duration of the light response. Recent evidence suggests, however, that the affinity of the noncatalytic sites for cGMP is so great (the off rate constant is so slow) that release of cGMP from the noncatalytic binding sites would be unlikely to participate either in the photoresponse or in short-term exposure to background light (26). On the other hand, a role for this mechanism during longer exposures to backgrounds or after bleaches remains a possible mechanism of long-term modulation of sensitivity.

The total concentration of cGMP in an amphibian rod is of the order of 30-60 µM (see, for example, Ref. 282), but the free concentration is much smaller than this, of the order of 6 µM (199). Some of the cGMP in the outer segment is bound to noncatalytic sites on the PDE, as we have seen, and some may be bound to other proteins, including the cyclic nucleotide-gated channels. Activation of the PDE hydrolyzes free cGMP, and the free concentration of cGMP declines rapidly. If the rod is to generate a maintained electrical response to steady illumination (see Fig. 1), then the cGMP must be resynthesized as it is hydrolyzed by the PDE so that the free cGMP concentration can reach a steady-state concentration to produce a steady-state probability of opening of the cGMP-gated channels.

The synthesis of cGMP in the rod is largely produced by specialized photoreceptor guanylyl cyclases, present in the rod and cone outer segments. These enzymes are members of a family of membrane-bound cyclases that includes proteins that act as receptors for hormones and peptides, for example, atrial natriuretic peptide (see Refs. 85, 297). Two cyclases have been cloned and identified in photoreceptors: GC-E (retGC-1 or GC-1) and GC-F (retGC-2 or GC-2; see Refs. 166, 262, 287). The two forms of cyclase appear to be present in both rods and cones, but GC-E is apparently more abundant in cones (52, 124); both forms of cyclase can be found in the same cell (288).

One of the most interesting features of the photoreceptor guanylyl cyclase is its dependence on Ca²⁺ concentration. First discovered by Lolley and Racz (165), this dependence was subsequently shown to be a quite steep function of cytoplasmic Ca²⁺, probably as a result of a cooperative interaction of Ca²⁺ with the enzyme (134). The cyclase activity increases as the Ca²⁺ concentration declines, with a half-maximal activity at ~100-200 nM free Ca²⁺ (52, 82, 86, 134, 136).

There is now excellent evidence that the Ca²⁺ regulation of photoreceptor guanylyl cyclase is produced by novel, small-molecular-weight Ca²⁺-binding proteins (see Ref. 228). At least two such proteins, called guanylyl cyclase activating proteins or GCAPs, are present in photoreceptor outer segments (see, for example, Refs. 46, 124) and have three Ca²⁺-binding EF-hand regions (see, for example, Ref. 216), which appear all to be necessary for the proper functioning of the protein (51). The number of Ca²⁺ binding sites of the GCAPs is thought to be responsible at least in part for the high cooperativity of Ca²⁺ regulation of the cyclase. In low free Ca²⁺, these sites are unoccupied, and GCAP in this form can stimulate the cyclase. The Ca²⁺-loaded form, on the other hand, inhibits the cyclase. As we shall see shortly, the Ca²⁺ concentration of the photoreceptor is relatively high in darkness and decreases in the light. Thus the rate of synthesis of cGMP would be expected to be low in darkness and to increase in light, in the same direction as the change in the rate of PDE activity produced by light stimulation.

In addition to GCAPs, a newly discovered protein in amphibians, called guanylyl cyclase inhibitory protein (GCIP), may also participate in cyclase regulation (163). This protein shares conserved sequences with the GCAPs and is of a similar molecular weight, but GCIP does not stimulate guanylate cyclase in low Ca²⁺ as the GCAPs do, although it does inhibit the cyclase when the Ca²⁺ increases. The role of this protein in cyclase regulation is presently unclear. We shall return to the subject of the Ca²⁺ regulation of the cyclase in section IIID.

Since the discovery of cyclic nucleotide-gated channels (76), many studies have carefully investigated their molecular biology and physiology in photoreceptors and in other cell types (notably olfactory receptor neurons). We give only a brief summary of the most recent experiments, focusing on the modulation of the channels. Excellent summaries of earlier literature are given in Yau and Baylor (289) and McNaughton (184). Comprehensive treatments of the molecular biology and structure/function of cyclic nucleotide-gated channels can be found in Finn et al. (77), Zagotta and Siegelbaum (298), Frings (81), and Molday (192).

Cyclic nucleotide-gated channels in rods may be tetramers, perhaps consisting both of -subunits and of -subunits, having different structures. The -subunit can be expressed by itself to form homomeric channels, whose physiological properties are somewhat different from native channels. Coexpression of both  and  produces channels more closely resembling those in rod outer segments. Cones contain different -channel proteins (19), and some as yet indirect evidence suggests that they may also have -subunits different from the ones in rods (97, 235).

Both the - and -subunits have binding sites for cyclic nucleotides, and binding is cooperative with a mean affinity constant (K_0.5) variously reported but of the order of 50 µM and a Hill coefficient also variously reported to be of the order of 2-3 (see, for example, Ref. 199). Recent evidence indicates that the cooperativity suggested by the Hill coefficient is the result of changes in the probability of channel opening as successive cGMPs bind to the channel (239). In homomeric rod -channels expressed in Xenopus oocytes, photoaffinity analogs of cGMP have been used to produce channels with persistently bound nucleotide. These experiments (239) show that channel opening probability increases as successive nucleotide binding sites are filled, but in a way not previously predicted. Each liganded state can produce channel openings to more than one conductance level; however, for each channel open state, the binding equilibrium constant does not increase by a constant factor as each nucleotide binds. This has the consequence that the subunits interact in some way not presently understood to favor openings to smaller conductance levels for partially liganded channels, but to favor openings to the highest conductance state for the fully liganded channel.

The cyclic nucleotide-gated channels of both rods and cones are permeable to a variety of monovalent cations, including Na⁺, K⁺, and Li⁺. In addition, the channels in both rods and cones are permeable to divalent cations, including Ca²⁺. This was first shown for rods (107, 291), for which the influx of Ca²⁺ through the channels is estimated to be of the order of 10⁷ ions per outer segment per second in toad, or ~10-15% of the circulating current entering the outer segment in darkness (198, 293). The channels in cones are also permeable to Ca²⁺ (226, 190) and contribute an even larger fraction of the dark current entering the outer segment (226). Measurements from membrane patches indicate that the ratio of permeabilities for Ca²⁺ and Na⁺ (P_Ca/P_Na) is considerably larger for cone than for rod channels (227) and may vary with the gating of the channel (98).

There is considerable evidence that the channels can be modulated, although the details are still controversial (299). Hsu and Molday (111) first reported that the apparent affinity of the channels for cGMP in isolated rod outer segments was decreased upon addition of exogenous Ca²⁺/calmodulin. Although this report has been confirmed in several subsequent studies (10, 89, 112), there is no agreement about whether calmodulin is actually the molecule responsible for the modulation in rods, or what role this modulation plays in functioning photoreceptors. Bauer (10) has argued that the affinity of the channels for Ca²⁺/calmodulin is so high that calmodulin, or a similar small-molecular-weight protein, is likely to remain bound to the channels in the rods except at very low intracellular Ca²⁺ concentration. Changes in Ca²⁺ concentration have been shown to produce changes in the apparent affinity of the channels for cGMP in permeabilized rods, with affinity increasing for decreased Ca²⁺, but the EC₅₀ for Ca²⁺ is ~50 nM for bullfrog (195) and salamander (248). As we shall see in section IIE, this is very near the limit of the range of Ca²⁺ concentration in the rod cytoplasm and is only reached in saturating bright light.

Quite different results have recently been obtained for cones. Exogenous calmodulin seems to have little or no effect on the cone channels in isolated membrane patches (97, 103), suggesting that different proteins may mediate modulation in the two kinds of photoreceptors. Furthermore, the EC₅₀ for Ca²⁺ modulation in permeabilized cones has been reported to be quite different from that in rods (235); it is apparently of the order of 250-300 nM, within the physiological range of outer segment Ca²⁺ in cones (see sect. IIE and Ref. 250). This would suggest that channel modulation produced by a change in outer segment Ca²⁺ may have greater importance for the normal physiology of cones than of rods.

In addition to modulation by Ca²⁺, channels in isolated patches have been reported to be affected by diacylglycerol (DAG) analogs (88) and by phosphorylation (87). This could in principle be quite important, but at present, there is no other evidence in support of either DAG or channel phosphorylation as a mechanism in the response or adaptation of photoreceptors (see for example Ref. 283).

Because the channels in the outer segments of both rods and cones are permeable to Ca²⁺, and because these channels are partially open in darkness, there is a steady influx of Ca²⁺ into the photoreceptor that is reduced by illumination. Ca²⁺ is transported out of the outer segment by a Na⁺/Ca²⁺-K⁺ exchange protein, which is located almost exclusively in the membrane of the outer segment (131, 140). The physiology of this exchanger has been studied in some detail (see Refs. 184, 185, 255). The stoichiometry of exchange is four Na⁺ (normally moving inward) for one Ca²⁺ and one K⁺ (both normally moving outward) (28). This has the consequence that the exchange is electrogenic, and the extrusion of Ca²⁺ produces an inward current that can be easily recorded from both rods (108, 292) and cones (200, 226).

When the probability of opening of the cGMP-gated channels decreases in the light, the influx of Ca²⁺ into the outer segment also decreases. Na⁺/Ca²⁺-K⁺ exchange does not appear to be affected directly by illumination (137, 198) so that Ca²⁺ continues to be transported out of the cell, resulting in a decrease in the intracellular Ca²⁺ (293). The Michaelis constant (K_m) for the exchanger for Ca²⁺ in salamander is of the order of 1-2 µM (150), severalfold higher than the dark resting Ca²⁺ concentration in a rod or cone (see below). This has the consequence that the rate of Ca²⁺ extrusion is nearly proportional to the Ca²⁺ concentration over most of the physiological range, and Ca²⁺ efflux continues in the presence of a steady light until influx and efflux are equal and the Ca²⁺ concentration reaches a new steady level, lower than its value in darkness.

The light-induced decrease in Ca²⁺ has been demonstrated directly, first in isolated salamander rods with aequorin (150, 186) and then in populations of photoreceptors with fluorometric dyes in the isolated retina of toad (135) and bullfrog (182, 183, 234, 296). Subsequently, outer segment Ca²⁺ was measured from isolated outer segments during simultaneous current recording with indo 1 dextran from Gekko rods (93) and from isolated intact salamander rods with fluo 3 (251). These experiments indicate that Ca²⁺ declines from ~500-700 nM in darkness to ~30-50 nM in saturating light, corresponding to a change in the free Ca²⁺ concentration of ~10- to 20-fold.

Upon the presentation of saturating light and sudden closure of all of the cGMP-gated channels, the Ca²⁺ concentration in the outer segment declines with two exponential components, which in both Gekko and salamander have time constants of several tenths of a second and several seconds. The monotonic decline of Ca²⁺ in the light, together with electrical measurements of channel and transport activity, suggest that the major mechanisms responsible for determining the Ca²⁺ concentration in the rod are influx through the cGMP-gated channels and efflux from Na⁺/Ca²⁺-K⁺ exchange.

There is, however, some reason to suppose that there may be other mechanisms of Ca²⁺ homeostasis in photoreceptors, although their relationship to transduction remains unclear. Rod outer segments have been reported to contain large amounts of Ca²⁺ (70, 257, 260), although this claim has been disputed (264). This Ca²⁺ has been shown to behave as if it were bound very tightly within the cytosol or held within a sequestered pool (70, 71, 256, 257), from which it can be released by light (72, 260). This light-induced release is controversial, and its mechanism is unclear. Although rod outer segments appear to contain one of the isoforms of PLC- (PLC-4) (see Ref. 219), the enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) to inositol 1,4,5-trisphosphate (IP₃) and DAG and thus might evoke Ca²⁺ release, there are few if any IP₃ receptors in rods (48), and the knocking out of PLC-4 has been reported to have little effect on the receptor light response (117). On the other hand, a light-induced release of Ca²⁺ would be consistent with Ca²⁺ electrode measurements of the Ca²⁺ concentration in the extracellular spaces around the rods in isolated rat retina (133). These measurements show that light bright enough to close all the channels in the rods produces a steady increase in the extracellular Ca²⁺ that persists even 5 min after the beginning of a saturating stimulus. This increase disappears after the light is turned off and Ca²⁺ reenters the rod, but less Ca²⁺ reenters than was extruded, resulting in a decrease in total rod outer segment Ca²⁺ (as in Ref. 260). Finally, recent experiments indicate that bright bleaching lights can produce a release of Ca²⁺ within the photoreceptor outer segment (68). The function of this Ca²⁺ release is not yet clear.

In rods in bright light, Ca²⁺ declines to about 30-50 nM (93, 182, 251). This in itself is surprising, since the exchanger could theoretically reduce Ca²⁺ below 1 nM (28). One explanation for this difference is that the exchanger is inhibited at low Ca²⁺ concentrations (259), but it is also possible that there is a steady release of Ca²⁺ from some internal compartment in the presence of bright light (258, 260).

Although Na⁺/Ca²⁺-K⁺ exchange is apparently the dominant mechanism of Ca²⁺ transport in the rod outer segment plasma membrane, the inner segment plasma membrane appears not to contain the Na⁺/Ca²⁺-K⁺ exchange protein (131) and instead extrudes Ca²⁺ with a Ca²⁺-ATPase (140, 193). A Ca²⁺-ATPase protein has been localized by immunohistochemistry to the inner segment and synaptic terminal but is apparently absent from the plasma membrane of the rod outer segment (140, 193). It would appear therefore that the inner and outer segments of rods regulate Ca²⁺ through different mechanisms and represent functionally distinct pools of Ca²⁺, since there may be little movement of Ca²⁺ between these two parts of the cell (140).

Much less is known about Ca²⁺ in cones, in part because their much smaller outer segments make optical measurements with fluorometric dyes more difficult. The first direct measurements of Ca²⁺ in cones have recently been made from salamander by Sampath et al. (250). Some interesting differences between rods and cones have emerged from this work. 1) The Ca²⁺ concentration is less than in rods (mean values were 410 nM in the dark, 5.5 nM in saturating illumination). Because the Ca²⁺ concentration falls to a lower level in the cone than in the rod, either the exchange proteins in the two cells are different, and/or there is some difference in the release of Ca²⁺ from buffers or internal stores in the two cell types. 2) The Ca²⁺ concentration in salamander cones changes by nearly 100-fold from darkness to saturating light, a greater range than for rods from the same species. This means that any transduction reactions regulated by Ca²⁺ could in theory be modulated to a greater extent in cones than in rods. 3) The time constants of Ca²⁺ decay are biexponential in cones as in rods but are much faster in cones, averaging 43 and 640 ms for red-sensitive cone photoreceptors. The amplitude of the fast time constant (the coefficient multiplying the exponential term) is significantly larger than that of the slow one (the amplitudes of these two time constants are nearly equal for rods). Blue-sensitive cones have somewhat slower time constants of decline, corresponding to the slower kinetics of their photoresponses (226), but the time constants of decay of Ca²⁺ in blue-sensitive cones were still a factor of two faster than in rods. The reason for the faster decay of Ca²⁺ in cones is unknown but may be the result of the greater surface-to-volume ratio of cone outer segments, or some difference in the properties of the exchange proteins or Ca²⁺ buffering or sequestration in the two kinds of photoreceptors (see Ref. 190).

One difficulty of making Ca²⁺ measurements from photoreceptors with fluorometric dyes is that light bright enough to elicit dye fluorescence will also produce considerable bleaching of the photopigment. This makes repeated measurements from the same rod almost impossible with present methods. For cones, however, the photopigment can be regenerated with exogenous 11-cis-retinal rapidly enough to measure the Ca²⁺ concentration from the same cell repeatedly, for example, in the presence of steady backgrounds over a range of light intensities. This has made it possible to make a direct correlation between circulating current and Ca²⁺ concentration in cones (250). Within the limits of experimental measurement, these two appear to be linearly related, with the free Ca²⁺ concentration declining in parallel with the circulating current. There seems therefore to be no nonlinearity in the relationship between photocurrent and Ca²⁺ in salamander cones like the one reported by Gray-Keller and Detwiler for Gekko rods (93), and it is possible that the homeostasis of Ca²⁺ in cones is simpler than that in rods, for reasons that are still unclear.

	III. LIGHT ADAPTATION

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Background adaptation in rods is mediated by a cytoplasmic messenger. Although it would be possible to imagine a mechanism for desensitization that was very local and restricted to single disks or even to small subregions of the disk membrane, several observations indicate that this does not occur. In the first place, rods are desensitized by very dim steady illumination; in amphibians the intensity of background light necessary to reduce the sensitivity of the receptor by one-half (I₀; see Eq. 1) is of the order of only 5-10 rhodopsins bleached per second (9, 14, 57, 64, 156). Because sensitivity reaches steady state after a few seconds of steady light, only a few tens of pigment molecules need be excited to reduce sensitivity by a factor of two, and because amphibian rods have of the order of 1,000-2,000 disks, some molecule must diffuse between the disks so that a pigment molecule bleached in one disk can affect the response produced by subsequent pigment molecules bleached in other disks (57, 9). For mammalian rods, I₀ is larger: 35 Rh^* s¹ in cats (267), 20-50 Rh^* s¹ in guinea pigs (172), and 30-50 Rh^* s¹ in primates (268). However, because measurements from mammalian rods are made at a higher temperature, the integration time of the receptor is considerably less, and steady state is reached more quickly. The same arguments used for the spread of desensitization in amphibian rods would therefore also apply to mammalian photoreceptors.

The spread of light adaptation has been measured directly by illuminating the outer segment either at the wavelength of peak sensitivity with narrow slits of light (156, 36) or at a much longer wavelength using two-photon excitation (92). These measurements give a space constant for the spatial spread of desensitization of ~5-10 µm in amphibian rods, larger than can be accounted for by light scatter from the slit. Using a different technique, Hemila and Reuter (106) illuminated rods obliquely to produce a preferential absorption of light by the distal end of the outer segments. They then compared adaptation produced by this stimulus with adaptation produced by homogeneous illumination and estimated that desensitization spreads 5-20 µm from the site of photon absorption. These experiments all support the requirement for an internal messenger in light adaptation, at least in rods. Comparable measurements have not been attempted in cones, but the similarity of the effects of background illumination on the two kinds of photoreceptors suggests that desensitization in cones may occur by a similar mechanism.

Bownds (22) first proposed that steady light might produce a maintained decrease in the intracellular Ca²⁺ concentration that in some way regulates the transduction cascade to produce desensitization of the photoreceptor response. A role for Ca²⁺ in light adaptation was subsequently indicated by the experiments of Torre et al. (269), who used whole cell patch recording to incorporate the Ca²⁺ buffer BAPTA into salamander photoreceptors. BAPTA incorporation produces an increase in the maximum amplitude of the light response and a marked retardation of its decay without affecting the initial time course of the response. These changes in amplitude and kinetics are effectively the inverse of the effects of background light on the response waveform (12, 14). It is therefore plausible that, by slowing the decrease in internal Ca²⁺ concentration, BAPTA retards the changes in sensitivity and response waveform that are normally responsible for desensitization. Similar experiments on amphibian cones (180) and mammalian rods (172) gave comparable results, thus indicating that Ca²⁺ is also likely to play a role in the control of response sensitivity and kinetics in these photoreceptors.

Clear evidence for Ca²⁺ as an internal messenger for light adaptation emerged from experiments which attempted to prevent or greatly reduce both the influx and efflux of Ca²⁺ from the photoreceptor outer segment and thereby minimize light-induced changes in intracellular free Ca²⁺ concentration. The outer segment was exposed to a solution that contained a lowered concentration of Ca²⁺ (to reduce Ca²⁺ entry) and no Na⁺ (181, 197). Because Na⁺/Ca²⁺-K⁺ transport requires external Na⁺, the absence of Na⁺ should prevent or greatly reduce Ca²⁺ efflux. Control experiments showed that when rods (67) and cones (180) are exposed to such a low-Ca²⁺/zero-Na⁺ solution with either Li⁺ or guanidinium⁺ as a Na⁺ substitute, a circulating current can be recorded that is reasonably stable over a 5- to 10-s interval. This contrasts with the rapid increase in circulating current that ensues when the outer segment is exposed to zero-Ca²⁺ solution in the presence of Na⁺ , the majority of which originates from a reduction in cytoplasmic Ca²⁺ concentration via Na⁺/Ca²⁺-K⁺ transport (154). Thus the relative stability of circulating current in low-Ca²⁺/zero-Na⁺ solution is a good indication that Ca²⁺ is remaining nearly constant with time, since stability of photocurrent implies stability of cGMP concentration, which in turn is an excellent indication of stability of Ca²⁺ because of the strong dependence of the cyclase rate on intracellular Ca²⁺ (see sect. IIC).

Even greater stability of photocurrent and Ca²⁺ can be achieved by using solutions containing the impermeant ion choline⁺ instead of Li⁺ or guanidinium⁺, and by removing Mg²⁺ from the bathing solution (168, 173-175). Under these conditions, there appears again to be little influx or efflux of Ca²⁺ and also Mg²⁺, which may, like Ca²⁺, affect the enzymes of the transduction cascade (see, for example, Ref. 136). With the impermeant ion choline⁺ substituted for Na⁺, the current through the cGMP-gated channels is outward instead of inward, presumably carried by K⁺ (290). Cells seem to survive longer in this solution than in Li⁺- or guanidinium⁺-substituted solution, perhaps because Li⁺ or guanidinium⁺ enters the outer segments and accumulates to toxic levels, since the photoreceptors may lack a mechanism for specifically removing them.

In low-Ca²⁺/zero-Na⁺ solution, the adaptation of both rods and cones to background light is eliminated (67, 180, 181, 197, 268). One way of demonstrating this is shown in Figure 4 (67). Figure 4A compares the responses of a rod to a brief flash in Ringer solution (labeled R) and in low-Ca²⁺/zero-Na⁺ solution (larger response). Exposure to the low-Ca²⁺/zero-Na⁺ solution produced an increase in the peak amplitude of the light response and a marked prolongation of the time to peak and slowing of the decay phase, much as after BAPTA incorporation (172, 180, 269). A similar prolongation of the light response is also observed in mammalian rods exposed to low-Ca²⁺/zero-Na⁺ solution (268). If the alteration in the waveform reflects the removal of a component that desensitizes the transduction cascade, then responses in low-Ca²⁺/zero-Na⁺ solution should sum linearly with no evidence of gain adjustment other than through response compression. Response compression is the decrease in response amplitude and sensitivity, produced simply by the instantaneous reduction in the probability of channel closing, as the response-intensity curve saturates and progressively fewer channels remain open and available to close.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Prediction of step responses from flash responses for a salamander rod. A: flash responses in Ringer solution (labeled R) and in low-Ca2+/zero-Na+ solution. Flash intensity for both responses was 2.8 photons·µm2. B and C: responses to steps (noisy traces) and predictions (smooth traces) in Ringer solution (B) and in low-Ca2+/zero-Na+ solution (C). Predictions were calculated from the flash responses in A by integration (after scaling according to the measured step intensity) and compression (according to the measured response-intensity relation) (see text and Ref. 67). Intensities of steps for traces D and 1-4 were 0 (dark), 2.4, 9.1, 37, and 140 photons·µm2·s1. Traces have been normalized according to the circulating current in darkness. [From Fain et al. (67).]

The notion that exposure to low-Ca²⁺/zero-Na⁺ solution eliminates adaptation is tested in Figure 4, B and C, in the following way. The responses to flashes in Figure 4A were corrected for response compression, scaled according to the flash and step intensities, integrated with respect to time, and then compressed according to the exponential saturation relation. These predicted step responses in the absence of adaptation (smooth traces) were then compared with responses to steady light (noisy traces). In normal Ringer solution (Fig. 4B), the recorded responses initially rose according to the predicted curves but then sagged below them at later times, corresponding to the onset of light adaptation (12). In contrast, when exposed to low-Ca²⁺/zero-Na⁺ solution (Fig. 4C), the compressed integral of the flash response came close to predicting the responses to steps of light, as if the adaptation to the steady light produced in normal Ringer solution was essentially eliminated. Similar results have also been obtained for cones (180).

The relaxation in the response to steps in normal Ringer solution is a well-established manifestation of light adaptation (12) that results in the characteristically shallow response-intensity relation for steady light, illustrated for salamander rods in Figure 5A by the solid symbols. In contrast, when steady light is presented in low-Ca²⁺/zero-Na⁺ solution, and this relaxation is abolished, the response rises more steeply as a function of intensity (Fig. 5A, open symbols) and can be fitted by the exponential saturation relation (181), which in Ringer solution is appropriate only for responses to flashes or to steps during the early rising phase before the onset of adaptation (157).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Normalized response-intensity curves for steady responses (A) and incremental sensitivity (B) as functions of background intensity (photons·µm2·s1) for salamander rods. Solid circles are from cells in Ringer solution, and open circles are from cells superfused with low-Ca2+/zero-Na+ solution before presentation of the background light. Half-filled circles (in B) are control experiments, for which rods were exposed to the background light before superfusion with low-Ca2+/zero-Na+ solution. In A, steady photocurrent response (i) has been normalized to the maximal current response (imax) obtained with a saturating bright steady light, and in B, flash sensitivity (SF) has been normalized to the sensitivity in the appropriate solution in darkness (SFD). The curve in A for the open circles is 1  exp(kIB), with k1 equal to 0.38 photons·µm2·s1. The curves in B are as follows: for the open circles, exp(kIB) with the same value of k as for the open circles in A; and for the half-filled and closed circles, I0/(IB + I0) with I0 equal to 0.8 photons·µm2·s1. Error bars give ±SD, and downward arrow indicates upper limit for responses too small to be measured above the noise of the recording. [From Matthews et al. (181). Copyright (1988) Macmillan Magazines Limited.]

Another way to investigate the effect of superfusion with low-Ca²⁺/zero-Na⁺ solution is to measure changes in sensitivity from flashes delivered in darkness and during exposure to backgrounds of increasing intensity (181). The curves in Figure 5B compare the normalized flash sensitivity (S_F/S_F^D) as a function of steady intensity for rods in normal Ringer solution (solid symbols) and in low-Ca²⁺/zero-Na⁺ solution (open symbols). In normal bathing solution, the sensitivity decreases with increasing steady intensity according to Weber's law (Eq. 1 in sect. I). In low-Ca²⁺/zero-Na⁺ solution, on the other hand, sensitivity declines much more steeply. The change in sensitivity can be adequately fit by the derivative of the response-intensity curve in low-Ca²⁺/zero-Na⁺ solution from Figure 5A. Thus the precipitous decline in sensitivity that is seen under these conditions is not the result of any feedback modulation of the transduction cascade but instead is caused simply by response compression. Background light closes a fraction of the channels in the outer segment, and this fraction increases as the background intensity increases. This leaves fewer channels available to close. It is of some interest that a relatively dim light in low-Ca²⁺/zero-Na⁺ solution is sufficient to close all of the channels and completely saturate the rod, whereas in normal bathing solution the dynamic range of the photoreceptor is greatly extended by mechanisms that the results in Figure 5 show to be dependent on a change in the Ca²⁺ concentration in the outer segment. Similar results have also been obtained for cones (180, 197), indicating that Ca²⁺ plays a broadly similar role in the adaptation of both types of photoreceptor.

If exposure to low-Ca²⁺/zero-Na⁺ solution can be assumed greatly to retard changes in outer segment Ca²⁺ concentration, as is argued above, then the results in Figures 4 and 5 show that a change in Ca²⁺ is necessary for light adaptation to occur. In the absence of a change in Ca²⁺, the photoreceptors simply sum single photon responses with no modulation of the transduction mechanism until all of the cGMP-gated channels are closed and the photoreceptor is saturated. The only change in sensitivity that the photoreceptors show under these conditions is due to response compression, that is, to the nonlinearity of the response-intensity curve. Thus Ca²⁺ appears to function as an internal messenger that regulates the sensitivity of the transduction mechanism.

Is Ca²⁺ the only messenger responsible for adaptation? One way of asking this question is to ask whether changes in Ca²⁺ are sufficient to produce light adaptation in a photoreceptor. This question would seem fairly easy to answer but is in fact more complicated than it initially appears. The principal reason for this difficulty is that the transduction mechanism is influenced by Ca²⁺ via a number of powerful feedback mechanisms that influence cGMP homeostasis and that are described in detail in section IIID. Any reduction in Ca²⁺ imposed in darkness leads to an acceleration in the rate of cGMP synthesis by guanylyl cyclase (see sect. IIC), and this produces an increase in cGMP concentration and a corresponding elevation in the circulating current. If the extracellular (9, 164) or cytoplasmic (67, 174, 205) Ca²⁺ concentration is maintained in darkness at a substantially reduced level corresponding to exposure to steady light of moderate to high intensity, then a considerable increase in circulating current results (67), which is accompanied by supralinearity of the response to light (9, 174). These changes, which are presumably due to electrical and biochemical nonlinearities, alter the waveform and sensitivity of the flash response in ways that are difficult to interpret. These difficulties can be avoided in part by recording under voltage clamp and by making only a modest reduction in external (and hence internal) Ca²⁺ concentration (94). Nevertheless, any direct comparison between the effects of Ca²⁺ and steady light must inevitably be restricted to a fairly small reduction in Ca²⁺ concentration, corresponding to relatively dim intensities of steady light.

An approach that avoids this complication and that allows the entire range of adapting intensities to be addressed is to use the time spent in saturation by the response to a bright flash of constant intensity to probe the sensitivity of the transduction mechanism and to examine its dependence on Ca²⁺ and light. The way in which this can be done is illustrated by the recordings in Figure 6A, made in normal Ringer solution. First, the rod is allowed to adapt to steady light, then a bright flash is delivered, sufficient to hold the response in saturation for several seconds. As the intensity of the background is increased, the time spent in saturation by the bright flash response progressively decreases as the cell adapts (67, 174, 223). However, if the rod is maintained in low-Ca²⁺/zero-Na⁺ solution throughout the time that the steady light is present, then this shortening of the time spent in saturation is abolished (67). This observation demonstrates that a change in Ca²⁺ concentration is necessary for this particular manifestation of adaptation over the full range of background intensities, instead of only the rather modest intensity that is able to saturate the electrical response when the outer segment is stepped to low-Ca²⁺/zero-Na⁺ solution in darkness (see Fig. 4C).

View larger version (26K): [in this window] [in a new window]   Fig. 6. Response recovery for salamander rods in Ringer solution and in low-Ca2+/zero-Na+ solution with increasing steady background intensity. Each trace is the average of two responses obtained at the start and two at the end of each experiment. Light monitors above traces indicate timing of 20-ms saturating flashes, of intensity 1,600 photons·µm2, and of steady backgrounds. For traces 1-4, background intensities were 4.36, 20.6, 93.7, and 473 photons·µm2·s1; for trace D, there was no background exposure. A: responses recorded in Ringer solution. B: recordings begun in Ringer solution; at the time indicated, the rod was perfused with a zero-Ca2+/zero-Mg2+/zero-Na+ solution in which choline was substituted for Na+. This would have the effect of holding the intracellular free Ca2+ near to its level in darkness (trace D) or its presumably lower level in each of the backgrounds for the duration of exposure to the solution. Traces have been corrected by subtraction of junction currents. [From Matthews (174).]

The progressively earlier onset of the recovery from saturation of the response in Ringer solution during such a step-flash protocol seems likely to represent actions of Ca²⁺ principally on early stages in the transduction cascade which sense the Ca²⁺ concentration at or near the time of the flash (174-176). The reason for this is that the kinetics of Ca²⁺ extrusion are sufficiently rapid in normal Ringer solution that by the time the response to the flash begins to recover from saturation, the cytoplasmic Ca²⁺ concentration will have fallen greatly from its value in darkness or moderate background illumination to levels approaching those attained during steady saturating light (93, 108, 251, 293). Hence, any process (such as modulation of the rate of guanylyl cyclase) that can also be influenced by Ca²⁺ at later times during the response will be maximally affected by the time the response begins to recover from saturation, and thus would have essentially the same effect on response recovery regardless of the intensity of the preexisting background. Consequently, only events occurring near the time of the flash would be likely to affect the time at which recovery from saturation commences.

To assess whether a reduction in Ca²⁺ concentration alone is sufficient to evoke this change in the time course of recovery to a saturating flash, the procedure illustrated in Figure 6B was adopted (174). First the rod was allowed to adapt to the steady light and then the outer segment was exposed to zero-Ca²⁺/zero-Na⁺ solution, thereby holding the Ca²⁺ concentration near the appropriate light-induced level for that particular intensity. The steady light was then extinguished and the rod remained in darkness for a period that would have been sufficient for substantial recovery from even the brightest background (interrupted curve, Fig. 6A). Finally, a bright flash was delivered and the outer segment was returned to normal Ringer solution before the response began to recover. Comparison of the two panels in Figure 6 reveals the striking observation that the onset of response recovery was speeded to an equivalent degree by each steady intensity irrespective of whether the background was actually present at the time of the bright flash. In other words, the decrease in Ca²⁺ concentration induced by the background is sufficient to evoke this particular manifestation of adaptation without a requirement for light itself (174).

Another manifestation of light adaptation is the alteration of the time course of responses to dim flashes (12). When dim flashes are presented during steady background light, the response kinetics are considerably accelerated, corresponding to both a shortening of the time to peak of the response and a speeding of the subsequent recovery (12, 14, 64). Is this characteristic change in the dim flash response solely due to the change in Ca²⁺ concentration and the cyclic nucleotide economy of the photoreceptor, or might some further feedback messenger also be involved?

One way to address this question is to illuminate a photoreceptor with background light while preventing changes in Ca²⁺ concentration and in cyclic nucleotide economy, and to examine the consequences for the dim flash response. Changes in Ca²⁺ can be minimized by superfusing the outer segment with zero-Ca²⁺/zero-Na⁺ solution. The light-induced changes in cyclic nucleotide economy can be counteracted by reducing the activity of the PDE (which is accelerated by the background light) to close to the initial dark-adapted level with the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX).

An example of such an experiment is shown in Figure 7. In Figure 7, trace DI, both Ca²⁺ concentration and PDE activity were maintained near their original dark-adapted levels by first stepping the outer segment to zero-Ca²⁺/zero-Na⁺ solution and then presenting bright steady light. The outer segment was then exposed to zero-Ca²⁺/zero-Na⁺ solution containing IBMX at a concentration sufficient to return the circulating current, and hence, the cGMP concentration, to approximately its original level in darkness. During the exposure to this solution, only relatively modest and gradual changes in circulating current were observed (174), indicating that Ca²⁺ concentration and the cyclic nucleotide economy remained relatively stable during this period. Because the Ca²⁺ concentration was maintained near its dark-adapted level, the rate of cGMP synthesis by guanylyl cyclase will also have remained unchanged. Consequently, the restoration of the dark current implies that PDE activity was reduced by IBMX to around its dark-adapted level. Under these conditions, the waveform of the dim flash response was similar to trace DG in Figure 7, which was obtained in zero-Ca²⁺/zero-Na⁺ solution in darkness, even though for trace DI the rod was continuously exposed to light. This is despite the fact that in Figure 7, trace DI, a considerably brighter flash was required to evoke a criterion response than in trace DG, presumably because of the inhibition by IBMX of the increase in PDE activity evoked by the flash. This constancy of response kinetics contrasts with the acceleration of both time to peak and recovery seen in Figure 7, trace LG, in which the outer segment was exposed to zero-Ca²⁺/zero-Na⁺ solution after adaptation to steady light so that the free Ca²⁺ concentration was permitted to decline before it was maintained at a reduced level. The preservation of dark-adapted response kinetics even in the presence of bright background illumination implies that steady light per se is unable to alter the kinetics of the dim flash response, provided there is no change in Ca²⁺ concentration or cGMP economy (174).

View larger version (20K): [in this window] [in a new window]   Fig. 7. Comparison of flash response waveform in darkness and in steady background light for a salamander rod. For trace DG, response was recorded in darkness after exposure to zero-Ca2+/zero-Mg2+/zero-Na+ solution, to hold the intracellular free Ca2+ concentration near its dark-adapted level. For trace DI, the rod was also first exposed to zero-Ca2+/zero-Mg2+/zero-Na+ solution to hold the intracellular free Ca2+ concentration near its dark-adapted level; the rod was then exposed to background light in the presence of 500 µM 3-isobutyl-1-methylxanthine (IBMX; trace DI) to inhibit the phosphodiesterase (which was stimulated by the steady light) to bring its velocity near to that in darkness. For trace LG, the perfusion with the zero-Ca2+/zero-Mg2+/zero-Na+solution was begun after turning on the background light so that the intracellular free Ca2+ concentration would have been held at a lower level than for traces DG and DI. Steady light delivered 70.7 photons·µm2·s1 for trace DI and 33.2 photons·µm2·s1 for trace LG. Flashes suppressed around one-third of the circulating current in each case and delivered 1.42 photons·µm2 for trace DG, 121 photons·µm2 for trace DI, and 31.5 photons·µm2 for trace LG. In each case, a record obtained under the same conditions in the absence of the flash has been subtracted from the flash response to correct for slow changes in circulating current. Responses have been normalized to the same peak amplitude. [From Matthews et al. (174).]

An alternative approach to investigate this same question was adopted by Gray-Keller and Detwiler (94). They compared the dim flash response in isolated voltage-clamped outer segments after adaptation to steady light with that recorded from outer segments in which the cytoplasmic Ca²⁺ concentration had been reduced artificially in darkness. In each case, the mean Ca²⁺ concentration was carefully measured with the fluorescent dye indo 1 dextran, so as to allow the responses to dim flashes to be compared for an equivalent reduction in Ca²⁺ concentration in the presence and absence of steady light. They found that while steady light both decreas