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In invertebrate photoreceptors, light elicits the opening of cationic channels to produce a depolarizing receptor potential. One hypothesis is that cGMP is the agent that gates the channels. It has been previously proposed that the light-induced rise in intracellular Ca2+ down-regulates phosphodiesterase activity, thereby eliciting an increase in intracellular cGMP concentration. cGMP-phosphodiesterase activity from squid photoreceptors was monitored both by a continuous fluorescence assay using 2'-o-(N-methylanthraniloyl)-cGMP and by hydrolysis of [3H]cGMP. The activity of cGMP-phosphodiesterase was found to be increased by Ca2+ in the physiological range of concentrations. These findings suggest that the previously known light-induced increase in Ca2+ in invertebrate photoreceptors cannot directly account for light-elicited down-regulation of cGMP-phosphodiesterase.

Heparin is thought to inhibit InsP3 binding to receptors involved in the intracellular release of Ca2+. Injection of heparin into Limulus ventral photoreceptors to high intracellular concentrations reduces the amplitude and slows the rate of rise of voltage-clamp currents induced by brief flashes, tends to make the responses to long flashes more "square," and tends to block the light-induced rise in [Ca2+]i detected by arsenazo III. In these ways, intracellular heparin mimics the effects of high concentrations of intracellular BAPTA or EGTA. In addition, the effects of heparin are attenuated by prior injection of BAPTA to high intracellular concentrations. Neomycin and spermine are thought to inhibit phospholipase C activity. Injections of spermine or neomycin to low intracellular concentrations largely mimic the effects of intracellular heparin. These findings suggest that the predominant effect of polyamines is to inhibit light-induced production of InsP3 by phospholipase C activity and thereby reduce the light-induced increase in [Ca2+]i. Our findings suggest that excitation can proceed in the absence of InsP3-induced increases in [Ca2+]i, but (a) the gain and speed of transduction are reduced and (b) adaptation is largely blocked.

Rapid concentration jumps of Ins(1,4,5)P3 or ATP were made inside Limulus ventral photoreceptors by flash photolysis of the parent caged compounds. In intact ventral photoreceptors, the photolysis flash evokes a maximum amplitude light-activated current; therefore, a procedure was developed for uncoupling phototransduction by blocking two of the initial reactions in the cascade, rhodopsin excitation and G protein activation. Rhodopsin was inactivated by exposure to hydroxylamine and bright light. This procedure abolished the early receptor potential and reduced the quantum efficiency by 325 +/- 90-fold (mean +/- SD). G protein activation was blocked by injection of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). GDP beta S injection reduced the quantum efficiency by 1,881 +/- 1,153-fold (mean +/- SD). Together hydroxylamine exposure and GDP beta S injection reduced the quantum efficiency by 870,000 +/- 650,000-fold (mean +/- SD). After the combined treatment, photoreceptors produced quantum bumps to light that was approximately 10(6) times brighter than the intensity that produced quantum bumps before treatment. Experiments were performed with caged compounds injected into photoreceptors in which phototransduction was largely uncoupled. Photolysis of one compound, myo-inositol 1,4,5-triphosphate P4(5)-1-(2-nitrophenyl)ethyl ester (caged IP3), increased the voltage clamp current in response to the flashlamp by more than twofold without changing the latency of the response. The effect was not seen with photolysis of either adenosine-5'-triphosphate P3-1-(2-nitrophenyl)ethyl ester (caged ATP) or caged IP3 in cells preloaded with either heparin or (1,2-bis-(o-amino-phenoxy)ethane-N-N-N'-N' tetraacetic acid tetrapotassium salt (BAPTA). The results suggest that photoreleased IP3 releases calcium ions from intracellular stores and the resulting increase in [Ca2+]i enhances the amplification of the phototransduction cascade.

Na+/Ca2+exchange has been investigated in squid ( Loligo pealei) rhabdomeric membranes. Ca2+-containing vesicles have been prepared from purified rhabdomeric membranes by extrusion through polycarbonate filters of 1-μm pore size. After removal of external Ca2+, up to 90% of the entrapped Ca2+could be specifically released by the addition of Na+; this finding indicates that most of the vesicles contained Na+/Ca2+exchanger. The Na+-induced Ca2+efflux had a half-maximum value ( K1/2) of ∼44 mM and a Hill coefficient of ∼1.7. The maximal Na+-induced Ca2+efflux was ∼0.6 nmol Ca2+⋅ s−1⋅ mg protein−1. Similar Na+-induced Ca2+effluxes were measured if K+was replaced with Li+or Cs+. Vesicles loaded with Ca2+by Na+/Ca2+exchange also released this Ca2+by Na+/Ca2+exchange, suggesting that Na+/Ca2+exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate of Ca2+efflux enhanced by approximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin, Na+/Ca2+exchange was characterized by a K1/2of ∼25 mM and a Hill coefficient of 1.6. For these vesicles, the maximal Na+-induced Ca2+efflux was about twice as great as in control vesicles. We conclude that Na+/Ca2+exchange proteins localized in rhabdomeric membranes mediate Ca2+extrusion in squid photoreceptors.

Both slices of squid (Loligo) retinas and Limulus ventral photoreceptors, with or without prior stimulation by flashes of bright light, were rapidly frozen and analysed for cyclic-GMP content by radioimmunoassay. Measurement of the freezing process indicated that freezing occurred at about 100 msec after the light flash. No significant changes in cyclic-GMP content were induced by light in either tissue. These data do not confirm previous reports that cyclic-GMP content of invertebrate photoreceptors is markedly increased by illumination.

Illumination greatly increases the GTPase activity in homogenates of squid (Loligo) whole retinas or rhabdomeric membranes. Adenylylimidodiphosphate inhibits the light-insensitive (but not the light-sensitive) GTPase activity in these homogenates. Illumination also greatly increases the binding of GTP[γS] to the rhabdomeric membranes. This binding at saturating illuminations indicates that there are approximately 10–100 times more rhodopsin molecules than G-protein molecules in squid photoreceptors. Each light-activated rhodopsin molecule activates about 10 G-protein molecules which might provide amplification for the first stage of the phototransduction cascade.

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A I M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, 35SO4 was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3 M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3–7.2, to intracellular concentrations of at least 40–200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4–8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.

The sub-second time course of changes in the content of [3H]inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with [3H]inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of [3H]inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not. That is, there was rapid but transient change in the content of [3H]inositol-1,4,5-trisphosphate after the onset of stimulation.

The outer segments of solitary rods of large neotenicAmbystoma tigrinum were impaled with double-barrel micropipettes. One barrel was used to record membrane voltage and the other barrel was used both to pass voltage-clamp current and to pressure-inject cyclic GMP. Injections of cyclic GMP induced inward currents in normal cells that were bathed in Ringer's solution and studied at the dark, resting voltage. These cyclic GMP-induced currents reversed sign for voltages 25–40 mV more positive than the dark, resting voltage; however, light-induced currents did not reverse sign for voltages as depolarized as 55 mV more positive than the dark, resting voltage. The responses to light became slow and very prolonged in cells that had been injected with large amounts of cyclic GMP. In these over-injected cells, both light-induced and cyclic GMP-induced currents increased in amplitude but did not reverse sign, for voltage 35–45 mV more positive than the dark, resting voltage. We conclude that light causes the simultaneous change in concentration of an intracellular messenger for excitation in addition to cyclic GMP, or that a change in the cytosolic concentration of cyclic GMP is not a step in the excitatory process.

The spectral sensitivities of single Limulus median ocellus photoreceptors have been determined from records of receptor potentials obtained using intracellular microelectrodes. One class of receptors, called UV cells (ultraviolet cells), depolarizes to near-UV light and is maximally sensitive at 360 nm; a Dartnall template fits the spectral sensitivity curve. A second class of receptors, called visible cells, depolarizes to visible light; the spectral sensitivity curve is fit by a Dartnall template with λmax at 530 nm. Dark-adapted UV cells are about 2 log units more sensitive than dark-adapted visible cells. UV cells respond with a small hyperpolarization to visible light and the spectral sensitivity curve for this hyperpolarization peaks at 525–550 nm. Visible cells respond with a small hyperpolarization to UV light, and the spectral sensitivity curve for this response peaks at 350–375 nm. Rarely, a double-peaked (360 and 530 nm) spectral sensitivity curve is obtained; two photopigments are involved, as revealed by chromatic adaptation experiments. Thus there may be a small third class of receptor cells containing two photopigments.

A hyperpolarization can be recorded intracellularly following either a single bright light stimulus or the intracellular injection of Na(+). This after-hyperpolarization is abolished by bathing in 5 x 10(-6)M strophanthidin or removal of extracellular K(+). Both treatments also lead to a small, rapid depolarization of the dark-adapted cell. When either treatment is prolonged, light responses can still be elicited, although with repetitive stimuli the responses are slowly and progressively diminished in size. The rate of diminution is greater for higher values of [Ca(++)](out); with [Ca(++)](out) = 0.1 mM, there is almost no progressive diminution of repetitive responses produced by either K(+)-free seawater or strophanthidin. We propose that an electrogenic Na(+) pump contributes directly to dark-adapted membrane voltage and also generates the after-hyperpolarizations, but does not directly generate the receptor potential. Inhibition of this pump leads to intracellular accumulation of sodium ions, which in turn leads to an increase in intracellular Ca(++) (provided there is sufficient extracellular Ca(++)). This increase in intracellular calcium probably accounts for the progressive decrease in the size of the receptor potential seen when the pump is inhibited.

Retinal ganglion cells of the cat were stained by the techniques of Golgi (silver) and Ehrlich (in vivo methylene blue). These neurohistological techniques presumably stain a few nerve cells completely, and hence allowed the study of the form of the dendritic expansion of the cat ganglion cells. The dendritic expansions of all the cells studied ended in one plane within the inner plexiform layer; no multistratified types of cells were seen. The dendritic fields were more or less circular with the cell bodies placed centrally. The size distribution of the dendritic fields was bimodal. Sizes between 70 and 200 μ and from 400 to 700 μ were found. It is suggested that for the cat retinal ganglion cells the size of the dendritic field is related to the size of the center region of the corresponding (physiologically determined) concentric receptive field.

During intracellular iontophoretic injection of Ca(++) into Limulus ventral photoreceptor cells, there is a progressive diminution of the light response. Following Ca(++) injection, the size of the light response slowly recovers. Similarly, there is a progressive diminution of the light response during intracellular injection of Na(+) and recovery after the injection is stopped. The rate of diminution during Na(+) injection is greater for higher [Ca(++)](out). In solutions which contain 0.1 mM Ca(++), there is nearly no progressive decrease in the size of the light response during Na(+) injection. Intracellular injections of Li(+) or K(+) do not progressively decrease the size of the light response. We propose that an increase in [Na(+)](in) leads to an increase in [Ca(++)](in) and that an increase in [Ca(++)](in) by any means leads to a reduction in responsiveness to light.

The dark-adapted current-voltage (I-V) curve of a ventral photoreceptor cell of Limulus, measured by a voltage-clamp technique, has a high slope-resistance region more negative than resting voltage, a lower slope-resistance region between resting voltage and zero, and a negative slope-resistance region more positive than 0 v. With illumination, we find no unique voltage at which there is no light-induced current. At the termination of illumination, the I-V curve changes quickly, then recovers very slowly to a dark-adapted configuration. The voltage-clamp currents during and after illumination can be interpreted to arise from two separate processes. One process (fast) changes quickly with change in illumination, has a reversal potential at +20 mv, and has an I-V curve with positive slope resistance at all voltages. These properties are consistent with a light-induced change in membrane conductance to sodium ions. The other process (slow) changes slowly with changes in illumination, generates light-activated current at +20 mv, and has an I-V curve with a large region of negative slope resistance. The mechanism of this process cannot as yet be identified.

The receptor potential in Limulus photoreceptors appears to be a consequence not of permeability changes in the cell membrane but of alterations in a light-sensitive constant-current generator.

Membranes from ventral photoreceptors of Limuluswere incubated with cholera toxin and [32P]NAD+. Cholera toxin catalyzes a specific ADP-ribosylation of a 43-kDa peptide from Limulus ventral photoreceptors. Possible homologies between the 43-kDa peptide of Limulus and the α-subunits of mammalian stimulatory, guanine nucleotide-binding regulatory component of adenylate cyclase (Ns) were investigated by comparing the electrophoretic patterns of proteolytic fragments derived from each of these peptides that are radiolabeled by [32P]NAD+ and cholera toxin. Evidence is provided for structural homology between this invertebrate peptide and mammalian Ns.

Publisher Summary This chapter discusses the effects of cyclic nucleotides and calcium ions on the Bufo rods. In vertebrate rods, both calcium ions and cyclic nucleotides have been proposed to be the diffusible messengers that mediate excitation between the rhodopsin that absorbs a photon and the conductance channels of the outer segment plasma membrane. By analogy, it has been proposed that Ca 2+ and intracellular cyclic nucleotides also interact in rod outer segments (ROSs) so that a change in either produces similar changes in the electrical activity of the rods. To examine the possibility that changes in the concentration of both Ca 2+ and cyclic nucleotides produce the same changes in the electrical activity of rods, intracellularly responses from ROSs in the isolated superfused retina of Bufo marinus need to be recorded. The chapter examines the sensitivity of the rods to light, the kinetics of the receptor potential, and the membrane voltage in the dark during procedures that ought to change the intracellular concentration of either Ca 2+ or cyclic nucleotides.

Physiological studies of intracellular messengers frequently employ intracellular injections of large molecules that either monitor or modulate the metabolism of the messenger cascade. Injected molecules have unknown mobility in the cytosol and unknown accessibility to various cytosolic compartments, including those postulated to be traversed by intracellular messenger molecules. In order to determine whether injected molecules have access to the confined spaces through which messenger molecules must diffuse, we injected 5-nm colloidal gold or horseradish peroxidase, or both, into Limulus ventral photoreceptors. Injections were made by applying pressure pulses to the back of an intracellular micropipette that also monitored membrane voltage. The tissue was fixed at varying times after injection and processed for electron microscopy by conventional techniques. Cells fixed 1–3 min after injection contained HRP reaction product only in the cell body. HRP reaction product was found at varying distances down axons in direct relation to the interval between injection and fixation. Colloidal gold particles were found throughout the cell body but not in axons of tissue fixed 1–3 min after injection. Both HRP reaction product and 5-nm colloidal gold particles were observed within the microvillar projections of internal and external rhabdomere, as well as within the extracisternal spaces of endoplasmic reticulum. We conclude that large molecules injected from an intracellular micropipette into an arbitrary locus of ventral photoreceptor cells have access to all of the presumed sites of the phototransduction cascade.

Inositol-trisphosphate was pressure-injected into Limulus ventral photoreceptors; these injections induced electrical responses that mimic several aspects of the electrical responses induced by light. Single cells were also injected with aequorin. Injections of inositol-trisphosphate into such cells induced an increase in luminescence from the intracellular aequorin, even in the absence of extracellular calcium ions. These aequorin responses show directly that inositol-trisphosphate induces an increase in ionized calcium concentration within intact and functioning cells that arises from release of calcium ions from intracellular stores.

There are two classes of photoreceptor cells in the median ocellus of Limulus. One class of cells respond to long wavelength (visible) stimuli with a depolarizing receptor potential and to near ultraviolet light with a biphasic, initially hyperpolarizing, receptor potential. The other class of receptors respond with a depolarization to near ultraviolet and with a biphasic response to visible light. In the latter type of cell, visible light can counteract the depolarization elicited by near ultraviolet light. The evidence suggests that there are two photopigments in each cell and that both are involved in the generation of receptor potential.

The membranes of photoreceptor cells in Limulus have an electrogenic sodium pump which contributes directly to membrane potential and whose activity is changed by light. These light-induced changes in pump activity underlie the receptor potential.

Two types of photoreceptors are found in the median ocellus of Limulus. One type is maximally sensitive to ultraviolet (UV) light, the other to green light; they are called UV and VIS cells, respectively. Biphasic receptor potentials, consisting of a small initial hyperpolarizing phase and a later slow depolarizing phase, can be recorded from both receptor types. These biphasic responses are elicited in UV cells in response to long-wavelength light, and in VIS cells in response to ultraviolet light. Another type of hyperpolarizing response can be recorded in UV cells: after a bright ultraviolet stimulus, the cell remains depolarized; long-wavelength light rapidly returns the membrane potential to its value preceding ultraviolet illumination (this long-wavelength-induced potential change is called a "repolarizing response"). Also, a long-wavelength stimulus superimposed during a UV stimulus elicits a sustained repolarizing response. A third cell type (arhabdomeric cell) found in the median ocellus generates large action potentials and is maximally sensitive to UV light. Biphasic responses and repolarizing responses also can be recorded from arhabdomeric cells. The retina is divided into groups of cells; both UV cells and VIS cells can occur in the same group. UV cells in the same group are electrically coupled to one another and to an arhabdomeric cell.

In the UV-sensitive photoreceptors of the median ocellus (UV cells), prolonged depolarizing afterpotentials are seen following a bright UV stimulus. These afterpotentials are abolished by long-wavelength light. During a bright UV stimulus, long-wavelength light elicits a sustained negative-going response. These responses to long-wavelength light are called repolarizing responses. The spectral sensitivity curve for the repolarizing responses peaks at 480 nm; it is the only spectral sensitivity curve for a median ocellus electrical response known to peak at 480 nm. The reversal potentials of the repolarizing response and the depolarizing receptor potential are the same, and change in the same way when the external sodium ion concentration is reduced. We propose that the generation of repolarizing responses involves a thermally stable intermediate of the UV-sensitive photopigment of UV cells.

1. 1. Radioactive tracers were used to study the effect of light on the efflux of K + from Limulus polyphemus ventral eye. Eyes were labeled in artificial sea water containing 42 KCl and then perfused with non-radioactive artificial sea water. 2. 2. After the first 2 min of perfusion, 42 K in the eye decreased with time as the sum of two exponential terms of half-times 0.3 and 2 h. 3. 3. Illumination of an eye labeled with 42 K caused an abrupt increase in the rate of release of 42 K. The additional 42 K released in response to light comes from the photoreceptor cell bodies rather than from the axons. 4. 4. The proportional increase in efflux rate upon illumination did not vary with the length of time that an eye was perfused. This suggests that the two exponential terms above reflect two classes of photoreceptor cells rather than photoreceptor cells and glial cells. 5. 5. By collection of perfusate at intervals of less than one second, it was shown that the instantaneous rate of 42 K release increases up to 20-fold at the onset of illumination. With continuous illumination, the rate remains above the dark rate, but by less than a factor of 2. Thus, the response of potassium efflux to light has transient and steady phases like the receptor potential. The increased driving force on internal potassium resulting from the receptor potential is not sufficient to explain the flux changes, if one makes the constant field assumption for the photoreceptor membrane. 6. 6. A double label method with 42 K and 42 K was used to show that the average rate of influx over a 10-min period is not affected by illumination. 7. 7. In eyes cooled in the dark to 2–3 °C, light still elicited an increase in the rate of 42 K release and large, prolonged receptor potentials. Thus, the photoreception mechanism of the ventral eye is not, as has been previously suggested, inactive at low temperatures.