An electrogenic sodium pump in Limulus ventral photoreceptor cells

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.     

Increased intracellular sodium mimics some but not all aspects of photoreceptor adaptation in the ventral eye of Limulus

The effects of the intracellular iontophoretic injection of Na+ ions have been quantitatively compared with adaptation in ventral photoreceptors of Limulus. We find that: (a) both light adaptation and sodium injection are associated with a decrease in the variability of the threshold response amplitued; (b) both light adaptation and sodium injection are associated with a decrease in the absolute value of the temporal dispersion of the threshold response time delay; (c) the same template curve adequately fits the intensity response relationships measured under light adaptation and Na+ injection; (d) both light adaptation and Na+ injection produce a fourfold decrease in response time delay for a desensitization of 3 log units; (e) the time coures of light adaptation and dark adaptation is significantly faster than the onset of and recovery from desensitization produced by Na+ injection; (f) unlike local illumination, Na+ injection does not produce localized desensitization of the photoreceptor. These findings suggest that a rise in intracellular Na+ concentration makes at most only a minor contribution (probably less than 5%) to the total adaptation of these receptors in the intensity range we have examined (up to 3 log units above absolute threshold). However, changes in intracellular Na+ concentration may contribute to certain components of light and dark adaptation in these receptors.     

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor.

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A 1 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 3M 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.     

Lipid composition of Limulus photoreceptor membranes.

The lipid composition has been determined for rhabdomeric photoreceptor membranes of Limulus, and these data are compared with those from photoreceptor membranes of albino rats. The comparison is of interest because the membranes of these two photoreceptor cells regulate ionic transport differently during the response to illumination. 1. Phospholipid class composition of Limulus is similar, but not identical, to that of rats. The major differences are a greater percentage of sphingomyelin in Limulus and a greater percentage of phosphatidylethanolamine in the rat. 2. Ethanolamine plasmalogens, not observed in rat photoreceptor membranes, are present in Limulus photoreceptor fractions. 3. The level of cholesterol in Limulus is higher than that usually reported for vertebrate rod outer segments. 4. The predominant polyunsaturated fatty acids of Limulus photoreceptor membrane phospholipids are 20: 4(n-6) and 20: 5(n-3) with only traces of 22: 6(n-3). This is in sharp contrast with the large percentages of 22: 6(n-3) found in rat photoreceptors. 5. The fatty acid distributions of both membrane systems are highly unsaturated, but the ratio of (n-3) to (n-6) polyunsaturates is only 1.7 for Limulus as compared to 4.6 for rat.     

Protein turnover in photoreceptor cells of isolated Limulus lateral eyes

Abstract— A system was devised for the study of protein turnover in isolated lateral eyes of Limulus . In eyes incubated in a medium containing L-[³H]leucine, radioactivity of the fraction soluble in trichloroacetic acid increased steadily during 8 h. Amino-acid incorporation into proteins was investigated by scintillation counting of trichloroacetic-acid precipitates and by radioautography. At least 89% of the incorporation was inhibited by puromycin and 86% by emetine, an inhibitor of cytoplasmic protein synthesis. Label accumulated preferentially in the rhabdomes, which contain the visual pigment. Radioautography indicated that labeled protein, probably synthesized in the cytoplasm, was distributed in the microvilli of the photoreceptor cells in a diffuse pattern. In contrast to in vivo results, the photoreceptor cells in isolated eyes were labeled to a greater extent in light than in the dark. These different kinetics of labeling were both explained by the hypothesis that light increased the rate of degradation of proteins (particularly opsin) in the visual cells.     

Physiological properties of rod photoreceptor cells in green-sensitive cone pigment knock-in mice

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was approximately 11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 x 10(-7) s(-1), about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.     

Physiological roles of Na+/Ca2+ exchange in Limulus ventral photoreceptors

In previous work we have presented evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors (1989. J. Gen. Physiol. 93:473-492). This article assesses the contributions to photoreceptor physiology from Na+/Ca2+ exchange. Four separate physiological processes were considered: maintenance of resting sensitivity, light-induced excitation, light adaptation, and dark adaptation. (a) Resting sensitivity: reduction of [Na+]o caused a [Ca2+]o-dependent reduction in light sensitivity and a speeding of the time courses of the responses to individual test flashes; this effect was dependent on the final value to which [Na+]o was reduced. The desensitization caused by Na+ reduction was dependent on the initial sensitivity of the photoreceptor; in fully dark-adapted conditions no desensitization was observed; in light-adapted conditions, extensive desensitization was observed. (b) Excitation: Na+ reduction in fully dark-adapted conditions caused a Ca2+o-dependent depolarizing phase in the receptor potential that persisted beyond the stimulus duration and was evoked by a bright adapting flash. (c) Light adaptation: the degree of desensitization induced by a bright adapting flash was Na+o dependent, being larger with lower [Na+]o. Na+ reduction enhanced light adaptation only at intensities brighter than 4 x 10(-6) W/cm2. In addition to being Na+o dependent, light adaptation was Ca2+o dependent, being greater at higher [Ca2+]o. (d) Dark adaptation: the recovery of light sensitivity after adapting illumination was Na+o dependent. Dark adaptation after bright illumination in voltage-clamped and in unclamped conditions was faster in normal-Na+ saline than in reduced Na+ saline. The final sensitivity to which photoreceptors recovered was lower in reduced-Na+ saline when bright adapting illumination was used. The results suggest the involvement of Na+/Ca2+ exchange in each of these physiological processes. Na+/Ca2+ exchange may contribute to these processes by counteracting normal elevations in [Ca2+]i.     

Properties of visual cells in the lateral eye of Limulus in situ.

Excitatory properties of visual cells in the lateral eye of Limulus, investigated by optic nerve recordings in situ, differ significantly from the properties of cells in the classical, excised eye preparation. The differences suggest the possibility that two receptor mechanisms function in the eye in situ: one mechanism encodes low light intensities and the other responds to high intensities. The two mechanisms enable each ommatidium to respond over an intensity range of approximately 10 log units. This hypothesis was tested by measuring the increment threshold and the spectral sensitivity, by studying light and dark adaptation, and by analyzing the variability of the impulse discharge. Although the results do not conclusively identify two receptor mechanisms, they indicate that a process or a part of a process that functions in the eye in situ is abolished by excising the eye or cutting off its blood supply.     

Two components of the receptor current are developed from distinct elementary signals in Limulus ventral nerve photoreceptor

Transient elementary currents, bumps, stimulated by short dim light flashes were measured in ventral nerve photoreceptors of Limulus. It is demonstrated that light activates two types of bumps, which form two distinct components of the receptor current at higher light intensities. The two bump types, which are both assumed to be activated by single absorbed photons, differ in current amplitude and kinetic parameters. The current amplitude of one bump type is smaller than 0.3 nA and that of the other type is in the usual current range of up to several nanoamperes. The average latency of small bumps measured from the short stimulus flash is shorter than that of the large bumps. The small bumps have slower activation kinetics than the large bumps. It is demonstrated that with increasing flash intensity the small bumps overlap first and form a macroscopic current, on top of which the large bumps are superimposed. Results indicate that a single absorbed photon selectively activates only one kind of the enzyme cascades evoking one bump type. We conclude that the active meta conformation of a rhodopsin molecule selectively binds a specific type of G-protein, which is involved in the stimulation of one of the transduction cascades. The two bump types, which are the elements of two macroscopic current components support the previous assumption that light activates different transduction mechanisms in Limulus photoreceptors.     

Properties of barnacle photoreceptor cells in culture

1. Properties of median photoreceptor cells in cultured ocelli from the giant barnacle (Balanus nubilus) were compared in isolated ocelli, ocelli maintained with the supraesophageal ganglion, and fresh ocelli. 2. Cultured photoreceptor cells exhibited slight deterioration after 2-4 weeks. Cell bodies maintained their structure but apparently lost some dendrites. Electron micrographs revealed fewer rhabdomeres. Axons did not degenerate. 3. Intracellularly recorded responses to light in both cultured preparations were qualitatively normal with a small decrease in sensitivity and increase in input resistance. The waveforms of the light responses were normal. 4. The characteristic shadow reflex was maintained after 6 weeks.     

Dependence of bump rate and bump size in Limulus ventral nerve photoreceptor on light adaptation and calcium concentration

Bumps were recorded in Limulus ventral nerve photoreceptor as deflections in membrane voltage during 10 s illuminations by dim light which were repeated every 20 s. The bump amplitude vs frequency distribution and its dependence on the intensity of a preadapting light flash are described. Light adaptation which diminishes the average bump amplitude alters the character of the bump amplitude distribution from a curve with a convex region to a continuously falling concave curve. Weak light adaptation can increase frequency (and height) of the bumps elicited by constant stimuli. Raising the external Ca²⁺-concentration from 10 to 40 mmol/l augments the effect of a preadapting light flash in diminishing the bump amplitudes and also increases the bump frequency. The results are consistent with the assumptions

that light adaptation is based on a Ca²⁺-dependent reduction of the amplification factor which determines the bump size and

that the coupling between light induced rhodopsin reactions and bump generation is Ca²⁺-dependent.

     

Distinct lobes of Limulus ventral photoreceptors. I. Functional and anatomical properties of lobes revealed by removal of glial cells

Removing the glial cells that encase Limulus ventral photoreceptors allows direct observation of the cell surface. Light microscopy of denuded photoreceptors reveals a subdivision of the cell body into lobes. Often one lobe, but sometimes several, is relatively clear and translucent (the R lobes). The lobe adjacent to the axon (the A lobe) has a textured appearance. Scanning electron microscopy shows that microvilli cover the surface of R lobes and are absent from the surface of A lobes. When a dim spot of light is incident on the R lobe, the probability of evoking a single photon response is two to three orders of magnitude higher than when the same spot is incident on the A lobe. We conclude that the sensitivity of the cell to light is principally a function of the R lobe.     

Isolation and characterization of the inositol cyclic phosphate products of polyphosphoinositide cleavage by phospholipase C. Physiological effects in permeabilized platelets and Limulus photoreceptor cells

Cleavage of the polyphosphoinositides, catalyzed by phospholipase C purified from ram seminal vesicles, produces phosphorylated inositols containing cyclic phosphate esters (Wilson, D. B., Bross, T. E., Sherman, W. R., Berger, R. A., and Majerus, P. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4013-4017). In the present study we describe the isolation and characterization of inositol 1:2-cyclic 4-bisphosphate and inositol 1:2-cyclic 4,5-trisphosphate, the two cyclic phosphate products of phospholipase C catalyzed cleavage of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, respectively. We established the structures of these two cyclic compounds through 18O labeling of phosphate moieties, phosphomonoesterase digestion, and fast atom bombardment-mass spectrometry. We examined the physiological effects of these compounds in two systems: saponin-permeabilized platelets loaded with 45Ca2+ and intact Limulus photoreceptors. Both inositol 1:2-cyclic 4,5-trisphosphate and the noncyclic inositol 1,4,5-trisphosphate, but not inositol 1:2-cyclic 4-bisphosphate, release 45Ca2+ from permeabilized platelets in a concentration-dependent manner. Injection of inositol 1:2-cyclic 4,5-trisphosphate into Limulus ventral photoreceptor cells induces both a change in membrane conductance and a transient increase in intracellular calcium ion concentration similar to those induced by light. We injected inositol 1,4,5-trisphosphate and inositol 1:2-cyclic 4,5-trisphosphate into the same photoreceptor cell and found that the cyclic compound is approximately five times more potent than the noncyclic compound in stimulating a conductance change. We speculate that inositol 1:2-cyclic 4,5-trisphosphate may function as a second messenger in stimulated cells.     

Kinetic properties of single-ion channels activated by light in Limulus ventral nerve photoreceptors

Previous results on Limulus ventral photoreceptors have suggested that besides inositol trisphosphate, another unknown transmitter may also work in the transduction cascade. This assumption has been supported by the finding of two light-activated channel types. The present report furnishes further evidence of the dual transmitter mechanism in phototransduction by analyzing the kinetic properties and voltage dependency of these cation channels with conductances of 12 pS and 30 pS. Single-channel currents were recorded in Limulus ventral nerve photoreceptors in cell-attached configuration at 14°C. At V _m + 80 mV the open-time histograms of both channels were fit best by the sum of two exponentials; time constants (and weights) were: 0.81 ms (0.62) and 6.20 ms (0.38) for the 12 pS channels and 2.38 ms (0.43) and 19.4 ms (0.57) for the 30 pS channels. At this potential the mean open times were 2.7 ms for the 12 pS and 13.3 ms for the 30 pS channels, about two-times larger than at hyperpolarizing potentials. The deactivation kinetics were also different for the two channels. The time constants of the decay of the channel activity, after switching off the light, were 2.5 s for the 12 pS and 12.9 s for the 30 pS channels. The 12 pS channel exhibits bursting and subconductance states at positive potentials. The subconductances are about 20%, 46% and 72% of the fully open state. Results show that the two types of light-activated channels have different kinetic parameters, voltage dependence and gating mechanisms. The two channels are suggested to be gated by different transmitters or processes. It is proposed that for the 30 pS channel the transmitter could be calcium ion or a calcium-dependent transmitter.     

Inhibition in the eye of Limulus

In the compound lateral eye of Limulus each ommatidium functions as a single receptor unit in the discharge of impulses in the optic nerve. Impulses originate in the eccentric cell of each ommatidium and are conducted in its axon, which runs without interruption through an extensive plexus of nerve fibers to become a fiber of the optic nerve. The plexus makes interconnections among the ommatidia, but its exact organization is not understood. The ability of an ommatidium to discharge impulses in the axon of its eccentric cell is reduced by illumination of other ommatidia in its neighborhood: the threshold to light is raised, the number of impulses discharged in response to a suprathreshold flash of light is diminished, and the frequency with which impulses are discharged during steady illumination is decreased. Also, the activity that can be elicited under certain conditions when an ommatidium is in darkness can be inhibited similarly. There is no evidence for the spread of excitatory influences in the eye of Limulus. The inhibitory influence exerted upon an ommatidium that is discharging impulses at a steady rate begins, shortly after the onset of the illumination on neighboring ommatidia, with a sudden deep minimum in the frequency of discharge. After partial recovery, the frequency is maintained at a depressed level until the illumination on the neighboring receptors is turned off, following which there is prompt, though not instantaneous recovery to the original frequency. The inhibition is exerted directly upon the sensitive structure within the ommatidium: it has been observed when the impulses were recorded by a microelectrode thrust into an ommatidium, as well as when they were recorded more proximally in single fibers dissected from the optic nerve. Receptor units of the eye often inhibit one another mutually. This has been observed by recording the activity of two optic nerve fibers simultaneously. The mediation of the inhibitory influence appears to depend upon the integrity of nervous interconnections in the plexus: cutting the lateral connections to an ommatidium abolishes the inhibition exerted upon it. The nature of the influence that is mediated by the plexus and the mechanism whereby it exerts its inhibitory action on the receptor units are not known. The depression of the frequency of the discharge of nerve impulses from an ommatidium increases approximately linearly with the logarithm of the intensity of illumination on receptors in its vicinity. Inhibition of the discharge from an ommatidium is greater the larger the area of the eye illuminated in its vicinity. However, equal increments of area become less effective as the total area is increased. The response of an ommatidium is most effectively inhibited by the illumination of ommatidia that are close to it; the effectiveness diminishes with increasing distance, but may extend for several millimeters. Illumination of a fixed region of the eye at constant intensity produces a depression of the frequency of discharge of impulses from a nearby ommatidium that is approximately constant, irrespective of the level of excitation of the ommatidium. The inhibitory interaction in the eye of Limulus is an integrative process that is important in determining the patterns of nervous activity in the visual system. It is analogous to the inhibitory component of the interaction that takes place in the vertebrate retina. Inhibitory interaction results in the exaggeration of differences in sensory activity from different regions of the eye illuminated at different intensities, thus enhancing visual contrast.     

Mouse prolactin obtained by pituitary organ culture in a serum-free medium.

Mouse prolactin was purified by organ culture of pituitaries and electrophoresis of the medium. Mouse pituitaries were organ-cultured in 9-cm Petri dishes containing Waymouth's medium (MB 752/1) supplemented with penicillin (50 units/ml), streptomycin (100mug/ml), and bovine insulin (0.12 I.U./ml) for 8 days. Prolactin-rich culture medium was half-saturated with ammonium sulfate and centrifuged. The pellet was subjected to analytical disc electrophoresis (10% acrylamide). Gels were sectioned into 2-mm segments. Prolactin was eluted in 0.04M phosphate buffer (pH 7.2), dialyzed and lyophylized. Two hundred and forty ml medium in which 360 pituitaries were cultured yielded 29.3 mg lyophylized mouse prolactin. Although the preparation contained 2 other bands on acrylamide gel disc electrophoresis, a single precipitin line was seen in immunodiffusion and immunoelectrophoresis, showing the identity of their antigenicity. From these results, two other proteins in the preparation were suggested to be deamidated prolactin.     

Efferent control of temporal response properties of the Limulus lateral eye

The sensitivity of the Limulus lateral eye exhibits a pronounced circadian rhythm. At night a circadian oscillator in the brain activates efferent fibers in the optic nerve, inducing multiple changes in the physiological and anatomical characteristics of retinal cells. These changes increase the sensitivity of the retina by about five orders of magnitude. We investigated whether this increase in retinal sensitivity is accompanied by changes in the ability of the retina to process temporal information. We measured the frequency transfer characteristic (FTC) of single receptors (ommatidia) by recording the response of their optic nerve fibers to sinusoidally modulated light. We first measured the FTC in the less sensitive daytime state and then after converting the retina to the more sensitive nighttime state by electrical stimulation of the efferent fibers. The activation of these fibers shifted the peak of the FTC to lower frequencies and reduced the slope of the low-frequency limb. These changes reduce the eye's ability to detect rapid changes in light intensity but enhance its ability to detect dim flashes of light. Apparently Limulus sacrifices temporal resolution for increased visual sensitivity at night.