The latency of the response of Limulus photoreceptors to inositol trisphosphate lacks the calcium-sensitivity of that to light

Summary The latent period before depolarization of Limulus ventral photoreceptors by light flashes was compared with that following brief, intracellular, pressure-injection of d-myo-inositol 1,4,5 trisphosphate. At temperatures between 18 °C and 22 °C and with an extracellular calcium concentration of 10 mM, the responses of 4 cells to light and to injections of 100 M inositol trisphosphate displayed average latencies of 71 and 56 ms, respectively. The latencies of responses to InsP₃ included an estimated 20 ms dead-time inherent in the injection method. Reducing the temperature lengthened the latency of the response to light (Q₁₀ approximately 3.2 between 7 and 22 °C) more than that to inositol trisphosphate (Q₁₀ approximately 2.3). Bathing the photoreceptors in seawater containing no added calcium and 1 mM of the calcium chelator EGTA greatly increased the latency of the light response at all temperatures, but did not increase the latency of the response to inositol trisphosphate. We conclude that the response to inositol trisphosphate lacks the calcium- and temperature-sensitive latent period which characterizes the response to light. If inositol trisphosphate acts, via the release of stored calcium, to stimulate an intermediate in the visual cascade, then that intermediate would appear to be downstream from the latency-generating mechanism.Abbreviations InsP ₃ D-myo-inositol 1,4,5 trisphosphate - ASW Artificial seawater - Ca _i Cytosolic free calcium ion concentration - Ca ₀ Extracellular calcium ion concentration     

A direct demonstration that inositol-trisphosphate induces an increase in intracellular calcium in Limulus photoreceptors

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.     

Fast desensitization of the response to InsP3 in Limulus ventral photoreceptors.

In Limulus ventral photoreceptor cells the time-course of the desensitization of InsP3 response was measured by an injection-pair paradigm. Pressure pulses of InsP3 were delivered into the cell with various interpulse intervals. The desensitization of the response to the second injection of each pair approached totality at 200 ms, which is the duration of the response to a single pressure pulse of InsP3. Lowering extracellular calcium did not affect the time-course of the desensitization. Lowering the temperature slowed down both the time-course of the response to InsP3 and the time-course of the desensitization to the same extent. These findings suggest that the desensitization is powerful enough and its onset fast enough to contribute to the transience of the InsP3 response. The time-course of the desensitization suggests it may influence light adaptation.     

Local adaptation in the ventral photoreceptors of Limulus

Local adaptation was demonstrated in the ventral photoreceptors of Lumulus using either flashes or continuous illumination. Spots of light locally desensitized the region of the photoreceptor on which they were focused. In dark-adapted photoreceptors where "quantum bumps" were clearly discernible, local adaptation of the quantum bumps was observed. Local adaptation could induce differences of threshold of 1 decade over distances of 50-80 mum. With continuous local illumination these gradients could be maintained from 2 s to 30 min. In addition, the decrease in time scale associated with light adaptation was also found to be localized to the region of illumination.     

Swim pacemakers in box jellyfish are modulated by the visual input

A major part of the cubozoan central nervous system is situated in the eye-bearing rhopalia. One of the neuronal output channels from the rhopalia carries a swim pacemaker signal, which has a one-to-one relation with the swim contractions of the bell shaped body. Given the advanced visual system of box jellyfish and that the pacemaker signal originates in the vicinity of these eyes, it seems logical to assume that the pacemakers are modified by the visual input. Here, the firing frequency and distribution of inter-signal intervals (ISIs) of single pacemakers are examined in the Caribbean box jellyfish, Tripedalia cystophora. It is shown that the absolute ambient light intensity, if kept constant, has no influence on the signal, but if the intensity changes, it has a major impact on both frequency and ISIs. If the intensity suddenly drops there is an increase in firing frequency, and the ISIs become more homogeneously distributed. A rise in intensity, on the other hand, produces a steep decline in the frequency and makes the ISIs highly variable. These electrophysiological data are correlated with behavioral observations from the natural habitat of the medusae.     

The initiation of excitation and light adaptation in Limulus ventral photoreceptors

Two types of experiments indicate that light adaptation and excitation are initiated by the same, rather than different, populations of visual pigment. (a) The criterion action spectra of light adaptation and excitation are the same. (b) Increment-threshold curves were measured with a voltage-clamp technique under conditions of high and low concentration of plasma membrane rhodopsin (Rhpm). SD, the dark-adapted sensitivity, and 1/I2, the inverse of the background irradiance that desensitized by 0.3 log units, underwent the same fractional change when the rhodopsin concentration was changed. Both quantities appear to be linearly related to Rhpm. Reversible reductions in Rhpm were achieved by orange irradiation during a brief increase of extracellular pH from 7.8 to 10. This procedure would be unlikely to produce similar concentration changes in a hypothetical intracellular pigment because the concurrent change in intracellular pH, measured using the dye, phenol red, was only 0.45 pH units. It is thus unlikely that an intracellular pigment initiates light adaptation. On the assumption that light adaptation is mediated by a light-induced release of Ca++ from an intracellular store. the results reported here imply that an intracellular transmitter is needed to couple Rhpm to the intracellular store.     

The quantal source of area supralinearity of flash responses in Limulus photoreceptors

The time integrals of the responses of dark-adapted Limulus ventral photoreceptors to flashes exhibit a supralinear dependence on intensity at intermediate intensities. By decomposing the responses into their elementary single-photon components ("bumps"), we are able to calculate the overall quantum efficiency and to display the time courses of the bump amplitude and rate of appearance. Since the time course of the flash response is not slow compared with that of the bump, it was necessary, in order to carry out the decomposition, to develop a new technique for noise analysis of dynamic signals. This new technique should have wide applications. Our main finding is that the supralinearity of the flash responses corresponds to an increase in bump amplitude, with little change in bump duration or quantum efficiency. The time courses of the bump rate and of the change in bump amplitude are peaked and have widths similar to that of the response itself. The peaks of the time courses of the bump rate and amplitude displayed against the starting times of the bumps do not coincide and occur approximately 80 and approximately 40 ms, respectively, before the peak of the response. The time from the start of a bump to its centroid is approximately 70 ms, which means that the time at which the bump centroid reaches its maximum follows the response peak by 30 ms. These results impose constraints on possible mechanisms for the amplitude enhancement.     

Spontaneous activity of the light-dependent channel irreversibly induced in excised patches from Limulus ventral photoreceptors

We have studied the properties of membrane patches excised from the transducing lobe of Limulus ventral photoreceptors. If patches are excised into an "internal" solution that resembles the ionic composition of the cytoplasm, channel activity is typically absent, but can be turned on by cyclic GMP (cGMP). In contrast, if patches are excised directly into sea water and subsequently examined in internal solution, they exhibit a high channel activity in the absence of any second messenger (spontaneous channel activity). Because these patches contained only light-dependent channels when examined before excision and because these spontaneous channels have properties in common with the light/cGMP-dependent channel, we believe that the spontaneously active channels represent light/cGMP-dependent channels that have been damaged by exposure to sea water, perhaps due to proteolysis activated by the high Ca2+ levels of the sea water. One type of the spontaneously active channel resembles the light/cGMP-dependent channel in open time, reversal potential, conductance states and voltage dependence. Application of micromolar Ca2+ to this channel produces a reversible decrease in the opening rate, indicating a high affinity binding site for Ca2+ on this channel. Another type of spontaneously active channel has a conductance state and reversal potential similar to the light/cGMP-dependent channel, but has apparently lost its dependence and sensitivity to Ca2+ and voltage.     

The initial response of Limulus ventral photoreceptors to bright flashes. Released calcium as a synergist to excitation

The leading edge of the response of Limulus ventral photoreceptors to brief flashes was investigated using a voltage clamp. The leading edge of responses increases linearly with flash intensity when dim flashes produce less than one photoisomerization per square micron of cell surface. Brighter flashes accelerate the initial portion of the response, resulting in a fourth-power relationship between the magnitude of the response at brief times after the flash and the flash intensity. The onset of this nonlinearity with increasing flash intensity is determined by the local density of photoisomerizations within the receptor. Responses to bright 10-15-mum-diam spots therefore rise faster than responses to diffuse flashes producing the same number of photoisomerizations within the receptor. Background illumination shortens the response latency and suppresses the initial nonlinearity. These phenomena can be explained by a model of transduction in which light activates two parallel cascades of reactions. Particles released by the first of these cascades open ionic channels, while the second produces an agent that accelerates the rate of production of particles by the first. Injection of the calcium buffer EGTA slows the initial portion of the response to bright flashes and suppresses its nonlinearity, which suggests that the accelerating agent released by the second cascade is calcium.     

Timing of Ca(2+) release from intracellular stores and the electrical response of Limulus ventral photoreceptors to dim flashes

Light-induced release of Ca(2+) from stores in Limulus ventral photoreceptors was studied using confocal fluorescence microscopy and the Ca(2+) indicator dyes, Oregon green-5N and fluo-4. Fluorescence was collected from a spot within 4 microm of the microvillar membrane. A dual-flash protocol was used to reconstruct transient elevations of intracellular free calcium ion concentration (Ca(i)) after flashes delivering between 10 and 5 x 10(5) effective photons. Peak Ca(i) increased with flash intensity to 138 +/- 76 microM after flashes delivering approximately 10(4) effective photons, while the latent period of the elevation of Ca(i) fell from approximately 140 to 21 ms. The onset of the light-induced elevation of Ca(i) was always highly correlated with that of the receptor potential. The time for Ca(i) to exceed 2 microM was approximately equal to that for the receptor potential to exceed 8 mV (mean difference; 2.2 +/- 6.4 ms). Ca(i) was also measured during steps of light delivering approximately 10(5) effective photons/s to photoreceptors that had been bleached with hydroxylamine so as to reduce their quantum efficiency. Elevations of Ca(i) were detected at the earliest times of the electrical response to the steps of light, when a significant receptor potential had yet to develop. Successive responses exhibited stochastic variation in their latency of up to 20 ms, but the elevation of Ca(i) and the receptor potential still rose at approximately the same time, indicating a shared process generating the latent period. Light-induced elevations of Ca(i) resulted from Ca(2+) release from intracellular stores, being abolished by cyclopiazonic acid (CPA), an inhibitor of endoplasmic reticulum Ca(2+) pumps, but not by removal of extracellular Ca(2+) ions. CPA also greatly diminished and slowed the receptor potential elicited by dim flashes. The results demonstrate a rapid release of Ca(2+) ions that appears necessary for a highly amplified electrical response to dim flashes.     

The effect of nucleotides on the rate of spontaneous quantum bumps in Limulus ventral photoreceptors

The effect of intracellular nucleotides on the rate of spontaneous quantum bumps in Limulus ventral photoreceptors has been examined. Internal dialysis of photoreceptors with solutions lacking nucleotide leads to an elevation of the quantum bump rate that can be reversed by introduction of nucleotide. Similarly, elevation occurs after treating intact cells with the metabolic inhibitor 2-deoxyglucose. This effect can be reversed by intracellular injection of ATP. The rate of spontaneous quantum bumps in unpoisoned cells can be reduced to below normal levels by injection of ATP. These results support the hypothesis that high-energy nucleotides suppress the rate of spontaneous quantum bumps.     

Dispersion of latencies in photoreceptors of Limulus and the adapting- bump model

To light stimuli of very low intensity, Limulus photoreceptors give a voltage response with a fluctuating delay. This phenomenon has been called "latency dispersion." If the generator potential is the superposition of discrete voltage events ("bumps"), and if the effect of light upon bump size is negligible, then the latency dispersion and the bump shape completely characterize the frequency response to sinusoidal flicker. For very low light intensities, the latency dispersion of the bumps, the bump shape, and the frequency response are measured. It is found that for data obtained at 20 degrees C, the frequency response can be accounted for completely by the latency dispersion and by the bump shape derived from steady-state noise characteristics. At 10 degrees C, the time scale of the response of the photoreceptor is lengthened. The dispersion of latencies and the bump shape are found not to have the same temperature dependence. However, just as those measured at 20 degrees C, the bump shape and the dispersion of latencies measured at 10 degrees C can predict the frequency response measured under the same conditions. These results strongly suggest that the major mechanisms involved in the generator potential are the latency process and the bump process. At high light intensities, the time scale of the generator potential shortens. The decrease in time scale of the generator potential can be attributed to the decreases in time scales of the bumps and of the latency dispersion process.     

Electrophysiological measurement of the number of rhodopsin molecules in single Limulus photoreceptors

Two partly independent electrophysiological methods are described for measuring the number of rhodopsin molecules (R) in single ventral photoreceptors. Method 1 is based on measurements of the relative intensity required to elicit a quantal response and the relative intensity required to half-saturate the early receptor potential (ERP). Method 2 is based on measurements of the absolute intensity required to elicit a quantal response. Both methods give values of R approximately equal to 10(9). From these and other measurements, estimates are derived for the surface density of rhodopsin (8,000/micrometer2), the charge movement during the ERP per isomerized rhodopsin (20 X 10(-21) C), and the half-time for thermal isomerization of rhodopsin (36yr).     

The photoreceptors in the high irradiance response of plants

Several studies show that the high irradiance response (HIR) of plants is probably due to two photoreceptors. One of the photoreceptors is phytochrome, and the other is an unidentified pigment provisionally named heliochrome. One of the functions of heliochrome is the synthesis of phytochrome, using far-red and blue radiations of high intensities, to replace the phytochrome destroyed by light. Another possible function could be an interaction of heliochrome with a substance produced by phytochrome. The data presented show that heliochrome is a pigment with different properties from phytochrome. It shows a far-red/green reversibility. Heliochrome has been shown to participate with phytochrome in such HIRs as leaf movement in Albizzia and flowering in a long-day plant. The first event initiated by phytochrome and by heliochrome could be the generation of a strong positive, electrostatic charge in the cell membrane.     

The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant

Background

Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1.

Results

The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs.

Conclusions

These data represent the first genome-wide analysis of translation in a eukaryote defective in the large ribosomal subunit. RPL24 and eIF3h play similar but non-identical roles in eukaryotic translation. The data also shed light on the fine structure of the regulon of ribosomal protein mRNAs.

     

Light-induced oxygen consumption in Limulus ventral photoreceptors does not result from a rise in the intracellular sodium concentration

Illumination of Limulus ventral photoreceptors leads to an increase in the intracellular concentration of sodium, [Na+]i, and to an increase in the consumption of O2 (delta QO2). After a 1-s light flash, it takes approximately 480 s for [Na+]i to return to within 10% of its preillumination level, whereas delta QO2 takes approximately 90 s. Thus, the delta QO2 is complete long before [Na+]i has returned to its resting level. Pressure injection of Na+ into the cell in order to elevate [Na+]i to the same levels as attained by illumination causes a rise in [Na+]i that returns to baseline with the same time course as the light-induced rise in [Na+]i. However, the injection of Na+ does not lead to an increase of the consumption of O2. We conclude that activation of the Na pump by a rise in [Na+]i is not a factor involved in the light-induced activation of O2 consumption in these cells.     

Characterization of an immunosuppressive factor from malignant ascites that resembles a factor induced in vitro by carcinoembryonic antigen

Oncodevelopmental antigens may cause immunologic suppression in the host through release of suppressor molecules from the host's own immunoregulatory cells. This concept has been difficult to study until recently when carcinoembryonic antigen was shown to induce the release of such molecules from normal circulating human mononuclear cells in vitro. However, the amount of the suppressor moiety generated was too small to adequately characterize, and its presence in vivo, i.e., in the cancer-bearing host, was unknown. Therefore, we sought to isolate and characterize a similar or identical macromolecule from ascites having an elevated CEA level in patients with cancer. A single malignant ascites, when precipitated at 0 to 35% ammonium sulfate saturation, was the source of suppressive factor for purposes of isolation and standardization. Suppression was quantitated by reduction of [3H]thymidine incorporation by phytohemagglutinin-stimulated normal human peripheral blood mononuclear cells. Sephadex G-200 chromatography revealed probable aggregation of the factor in isotonic buffers; aggregation was reduced in the presence of 8 M urea. Purification was achieved by precipitation with 5% trichloroacetic acid (TCA). The suppressor factor remained soluble in TCA and demonstrated a 95-fold increase in specific activity. Analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated a single protein band of 50,000 daltons. Ascites from three additional cancer patients gave identical results. Physicochemical characterization of the suppressor moiety revealed stability at 70 degrees C for 30 min and at pH 2 and pH 10 for 24 hr. Delipidation by chloroform-methanol extraction, proteolytic enzyme digestion, and protamine sulfate precipitation did not affect activity, suggesting that lipid, simple peptides, and nucleic acids were not crucial. However, periodate oxidation irreversibly destroyed suppressor activity, suggesting the importance of carbohydrate to the molecule and offering one explanation for protease resistance. Similarities in m.w. (50,000 daltons), isoelectric point (pI = 3.4), physical properties (heat and acid stability and resistance to proteases), and immunologic activity of this factor with that released from lymphocytes after in vitro exposure to carcinoembryonic antigen indicates they may be identical. Our results suggest that early aberrant events induced in the immunoregulatory network by tumor-associated antigens may be relevant and may lead to better understanding of immunosuppression in the cancer-bearing host.     

Different ionic conductances are modulated during the late receptor potential and the prolonged depolarizing afterpotential in Hermissenda type A photoreceptors

Wavelength-dependent, bistable phenomena were found in the receptor potential of Hermissenda crassicornis type A photoreceptors. Short exposure to blue light induced a prolonged depolarizing afterpotential (PDA) following the cessation of the light stimulus. Stronger adaptation to blue light, as caused by prolonged exposure and/or high intensity stimulation, effected a reduction in the early depolarizing transient of the late receptor potential (LRP) as elicited by subsequent stimuli. Vast separation of LRP emergence and PDA emergence could be obtained in photoreceptors in which a strong cancellation of the LRP was accomplished but a PDA still emerged after cessation of the light stimulus. Short exposure to yellow light cancelled the PDA, and stronger adaptation restored the LRP (opposite effect to blue light). The initial depolarizing part of the LRP had earlier been demonstrated to be mediated by the lightdependent increase of an inward conductance. In contrast, in this study the PDA was found to be accompanied by the reduction of an outward conductance, most likely a K⁺ conductance. A bistable photopigment system is thought to control the bistable receptor potential phenomenology by regulating the different membrane conductances during the LRP and the PDA.Abbreviations LRP late receptor potential - PDA prolonged depolarizing afterpotential - PHA prolonged hyperpolarizing afterpotential