The contribution of pigment transitions to sensitivity changes in the barnacle photoreceptor and the correlation with the prolonged depolarizing afterpotential

A conditioning light can cause a decrease (adaptation) or an increase (facilitation) in the sensitivity of barnacle photoreceptors, as measured by the amplitude of the late receptor potential (LRP). We show that a net transfer of visual pigment from the rhodopsin (R) to the metarhodopsin (M) state induces a large facilitation whereas the reverse transfer results in a much smaller facilitation or even an adaptation. These effects were not due to the response to the conditioning light but to the pigment reactions. When the conditioning light did not alter the pigment population (i.e., M M, R R) it was followed by an intermediate degree of facilitation. These conclusions are correct for cells which have relatively low sensitivity. In sensitive cells, all pigment transitions produce adaptation.LRP facilitation and the prolonged depolarizing afterpotential (PDA) show several common characteristics with respect to pigment transitions: 1.Their magnitude increases with the amount of pigment transferred from R to M. 2. Both are depressed by the M R transition. 3. Their production is impeded by the M R transition. 4. The PDA itself is facilitated by the R M transition and this facilitation decays with a time course comparable to that of LRP facilitation. These results suggest that there may be an underlying process common to LRP facilitation and PDA.     

Enhancement of sensitivity in photoreceptors of the honey bee drone by light and by Ca2+

Summary Deeply dark adapted (1 h) photoreceptor cells of the honey bee drone show a light-induced enhancement of sensitivity (facilitation) as an aftereffect of illumination or in the presence of dim backgrounds.The Ca²⁺-dependency of this effect was studied: Reduction of extracellular Ca²⁺ to 0.1 mM decreases the sensitivity of a dark adapted cell, and the light-induced increase in sensitivity due to repetitive, dim, 20 ms test flashes is slower than in normal saline. After a sensitizing conditioning light, the sensitivity drops faster in low-calcium saline. The light-induced enhancement of sensitivity is mimicked by pressure injections of low amounts of Ca²⁺ (Ca²⁺/EGTA-buffers; 0.15 M free Ca²⁺) into a dark adapted cell. Injection of EGTA alone decreases the sensitivity. Injection of a solution containing ca1 mM free Ca²⁺ sequentially decreases and later increases the sensitivity transiently.These results suggest a model in which a progressive increase in intracellular Ca²⁺ concentration by light first increases (facilitates), and, at higher concentrations, decreases (light adapts) the sensitivity of the cells. One possible site of action for this positive and negative feedback control of cell sensitivity by Ca²⁺ is the endoplasmic reticulum.     

Control of the cyclic GMP phosphodiesterase of frog photoreceptor membranes

The light-activated cyclic GMP phosphodiesterase (PDE) of frog photoreceptor membranes has been assayed in isolated outer segments suspended in a low-calcium Ringer's solution. Activation occurs over a range of light intensity that also causes a decrease in the permeability, cyclic GMP levels, and GTP levels of isolated outer segments. At intermediate intensities, PDE activity assumes constant intermediate values determined by the rate of rhodopsin bleaching. Washing causes an increase in maximal enzyme activity. Increasing light intensity from darkness to a level bleaching 5 x 10(3) rhodopsin molecules per outer segment per second shifts the apparent Michaelis constant (Km) from 100 to 900 microM. Maximum enzyme velocity increases at least 10-fold. The component that normally regulates this light- induced increase in the Km of PDE is removed by the customary sucrose flotation procedures. The presence of 10(-3) M Ca++ increases the light sensitivity of PDE, and maximal activation is caused by illumination bleaching only 5 x 10(2) rhodopsin molecules per outer segment per second. Calcium acts by increasing enzyme velocity while having little influence on Km. The effect of calcium appears to require a labile component, sensitive to aging of the outer segment preparation. The decrease in the light sensitivity of PDE that can be observed upon lowering the calcium concentration may be related to the desensitization of the permeability change mechanism that occurs during light adaptation of rod photoreceptors.     

Biochemical mechanism of light adaptation in vertebrate photoreceptors.

Vertebrate photoreceptors can adjust their sensitivity to a wide range of light intensities spanning several orders of magnitude, the phenomenon of which is called light adaptation. Electrophysiological and biochemical studies have revealed that calcium can serve as an intracellular transmitter of light adaptation under the control of cGMP metabolism. After illumination, the cytoplasmic calcium concentration of a photoreceptor decreases, which in turn strongly activates photoreceptor guanylate cyclase. This calcium-dependent effect is mediated by a novel calcium-binding protein (recoverin) and leads to the restoration of the depleted cGMP pool after illumination.     

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.     

Reduction in the calcium requirement for growth is correlated with intracellular calcium stores in normal and SV40-transformed NIH 3T3 cells.

Untransformed NIH 3T3 cells do not proliferate in media with reduced calcium, while SV40-transformed NIH 3T3 cells do. Intracellular calcium stores of untransformed cells were depleted to a higher extent than those of transformed cells under these conditions, which led to a decreased intracellular calcium transient in response to serum, compared to SV40-transformed cells. Furthermore, untransformed cells could be gradually adapted to proliferate in the low-calcium medium and, after adaptation, maintained their stores and serum response in low calcium media. Our experiments indicate that it is the ability of the cells to maintain adequate calcium stores in low calcium media that correlates with a full serum response and the ability to proliferate, rather than any differences reflected in alterations of resting calcium levels.     

Sensitivity changes of photoreceptor cells of Hirudo medicinalis caused by changes in extracellular calcium concentration

Extracellular recordings from the vacoule of photoreceptor cells of Hirudo medicinalis L. were performed using microelectrodes. The cells were adapted by white light flashes given at constant intervals (20 s). Response height versus relative intensity curves obtained from the same cell in physiological saline (PS) and in bathing solutions of either a) lowered calcium contents (2 ΜM/1 or less) or b) raised calcium contents (15 mM/1) were compared. The cells' adaptation state in PS was operationally defined by the ratio Q=h _A /h _S where h _A is the response height evoked by the adapting flashes, and h _S is the corresponding saturation response height. Sensitivity changes were measured by the half saturation intensity shift. Lowering extracellular calcium resulted in:

1. The response height increased and the shape of the response became more rounded and prolonged.
2. The total resistance between the vacuole and outside decreased from 8.2±1.4 MΩ (n=6) in PS to 4.6±0.4 MΩ (n=5). The resistance was independent of the cells' adaptation state.
3. A change of the cells' sensitivity occured either in direction to light adaptation or in direction to dark adaptation. It depended functionally on the ratio Q:

a) if Q was less or equal to about 0.6 the cells' sensitivity increased. b) if Q was greater than 0.6 the cells' sensitivity diminished. Raising extracellular calcium decreased the sensitivity of all cells tested independent of their adaptation states in PS. The results can be interpreted under the assumptions that 1. the sensitivity of leech photoreceptor cells is inversely proportional to the intracellular free calcium concentration and Z. intracellular calcium can interact with extracellular calcium in relatively dark adapted cells whereas in relatively light adapted cells the raise of intracellular free calcium is mainly effected by a release from intracellular stores. It is assumed that a Q value of about 0.6 separates relatively light adapted cells from relatively dark adapted cells.     

Sensitivity facilitation in the insect eye

Summary ERG amplitude facilitation, observed in the eye ofAtta sexdens after light adaptation, was studied as a function of duration and intensity of adaptation, of dark interval between adapting and test stimuli, and of level of steady background illumination. Results show that sensitivity facilitation in this eye cannot be regarded as a minor effect since it covers a 2 log unit range, the same as that obtained for conditions that produce sensitivity reduction. Maximum facilitation occurs with short and intense light adaptation. The time span of the effect is close to 2 min, and its maximum amplitude may be attained up to 20 s after light adaptation. Increase in background illumination gradually erases facilitation. However, the facilitated response is less sensitive to background illumination than the dark adapted response. Long durations of light adaptation cause ERG decrease, or inhibition. A comparison of these two end results of light adaptation suggests that they arise from different processes, perhaps with distinct origins.Supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo, to the senior author (Contract n 71/1141)With a Fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (N 74/388)We wish to express our appreciation to Henrique Fix for his editorial assistance, and to Celia Jablonka for laboratory help.     

Fura 2 analysis of cytosolic calcium regulation in elutriated rat gastric parietal cells

The calcium probe, Fura 2, is used to establish and partially characterize histamine-, carbachol-, and forskolin-induced calcium transients in enriched parietal cell populations prepared by centrifugal elutriation of dispersed rat fundic mucosa cell isolates. The magnitude of the maximal carbachol response, which is blocked by atropine but not cimetidine, is nearly five times that of histamine or forskolin. Time to peak responses for carbachol, forskolin, and histamine are approximately 7, 17, and 28 sec, respectively. Carbachol-, histamine-, and forskolin-induced increases in Fura 2 fluorescence appear dependent upon extracellular calcium, since these responses are attenuated in low calcium media and blocked by EGTA in low-calcium media or by lanthanum in high- or low-calcium medium. Trifluoperazine and fenoctimine, at concentrations that inhibit secretion, have no effect on either carbachol- or histamine-induced increases in cytosolic calcium. Seven major calcium/EGTA-sensitive phosphoproteins are identified by SDS-PAGE electrophoresis of ATP 32P-labeled cell sonicates. We conclude that cytosolic calcium in enriched rat gastric parietal cell populations is regulated by secretagogue receptor-controlled calcium channels. We postulate that these channels may be controlled by cyclic AMP-dependent phosphorylation, since neither changes in cyclic AMP nor calcium alone mediate the effects of secretagogues entirely, but the interplay between these two second-messenger systems potentiates the actions of these agents. The role of cytosolic calcium as a second messenger in secretagogue action appears similar to that of cyclic AMP in that a specific cellular concentration must be reached to initiate acid secretion.     

Sensitivity changes and facilitation in the photoreceptor of phalangium opilio due to light adaptation. Preliminary notes

These preliminary notes were made on sensitivity changes and facilitation in the photoreceptor of phalangium opilio, due to light adaptation. They show that facilitation is a case opposite to light adaptation. Other measurements are planned in the progress of this work.     

A reduction in the abundance of calium oxalate crystals in Oenothera improves the yield of intact chloroplasts

Abstract. Oenothera plants grown as shoot meristem cultures on media having a low calcium concentration had a reduced abundance of raphides when compared to greenhouse-grown plants or shoot meristem cultures grown on higher concentrations of calcium. Plants from the low-calcium media also yielded a higher proportion of intact chloroplasts during subcellular fractionation.     

Light Adaptation in the Ventral Photoreceptor of Limulus

Light adaptation in both the ventral photoreceptor and the lateral eye photoreceptor is a complex process consisting of at least two phases. One phase, which we call the rapid phase of adaptation, occurs whenever there is temporal overlap of the discrete waves that compose a light response. The recovery from the rapid phase of adaptation follows an exponential time-course with a time constant of approximately 75 ms at 21°C. The rapid phase of adaptation occurs at light intensities barely above discrete wave threshold as well as at substantially higher light intensities with the same recovery time-course at all intensities. It occurs in voltage-clamped and unclamped photoreceptors. The kinetics of the rapid phase of adaptation is closely correlated to the photocurrent which appears to initiate it after a short delay. The rapid phase of adaptation is probably identical to what is called the "adapting bump" process. At light intensities greater than about 10 times discrete wave threshold another phase of light adaptation occurs. It develops slowly over a period of ½ s or so, and decays even more slowly over a period of several seconds. It is graded with light intensity and occurs in both voltage-clamped and unclamped photoreceptors. We call this the slow phase of light adaptation.     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Weber and noise adaptation in the retina of the toad Bufo marinus

Responses to flashes and steps of light were recorded intracellularly from rods and horizontal cells, and extracellularly from ganglion cells, in toad eyecups which were either dark adapted or exposed to various levels of background light. The average background intensities needed to depress the dark-adapted flash sensitivity by half in the three cell types, determined under identical conditions, were 0.9 Rh*s-1 (rods), 0.8 Rh*s-1 (horizontal cells), and 0.17 Rh*s-1 (ganglion cells), where Rh* denotes one isomerization per rod. Thus, there is a range (approximately 0.7 log units) of weak backgrounds where the sensitivity (response amplitude/Rh*) of rods is not significantly affected, but where that of ganglion cells (1/threshold) is substantially reduced, which implies that the gain of the transmission from rods to the ganglion cell output is decreased. In this range, the ganglion cell threshold rises approximately as the square root of background intensity (i.e. in proportion to the quantal noise from the background), while the maintained rate of discharge stays constant. The threshold response of the cell will then signal light deviations (from a mean level) of constant statistical significance. We propose that this type of ganglion cell desensitization under dim backgrounds is due to a post-receptoral gain control driven by quantal fluctuations, and term it noise adaptation in contrast to the Weber adaptation (desensitization proportional to the mean background intensity) of rods, horizontal cells, and ganglion cells at higher background intensities.     

Long-term facilitation and long-term adaptation at synapses of a crayfish phasic motoneuron

Stimulation of the phasic (fast) motor axon of the isolated crayfish claw preparation at relatively low frequency (0.1 Hz) leads to depression of the excitatory junction potential (EJP) recorded from single muscle fibers. When the same stimulation is delivered following depression of the EJP at a higher frequency (5 Hz), a potentiated EJP appears, which is more resistant to low frequency depression. The potentiation appears to be analogous to "long-term facilitation" observed after stimulation of a tonic motor axon in crayfish and crabs. Long-term facilitation can be detected in preparations made from claws of animals in which the phasic motoneuron was stimulated at 5 Hz for 2 h in situ. This effect lasts for at least one day after one conditioning trial. Long-term facilitation is observed after stimulation of decentralized axons in situ, indicating that the change is attributable to local changes in terminal regions of the axon, and does not require the cell body. When electrodes are implanted in situ and the phasic motoneuron stimulated at 5 Hz for 2 h each day, synaptic depression becomes less pronounced and initial EJP amplitude becomes smaller over a period of several days. The latter changes, which adapt the neuron to a more tonic activity pattern, usually require several days for completion. Adaptation of fatigability occurs more rapidly than adaptation of initial EJP amplitude, and once established, remains for many days without further superimposed activity. Long-term adaptation does not occur in decentralized axons. Long-term facilitation and long-term adaptation are different responses of the neuron to enhanced activity. The former can occur in isolated or decentralized axons and leads to enhancement of EJP amplitude for a period of several hours to at least one day after a single episode of conditioning. The latter requires more time to be established, and leads to reduction of initial EJP amplitude and to lessened fatigability which persists for many days.     

Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors

The calcium sequestering agent, EGTA, was injected into Limulus ventral photoreceptors. Before injection, the inward membrane current induced by a long stimulus had a large initial transient which declined to a smaller plateau. Iontophoretic injection of EGTA tended to prevent the decline from transient to plateau. Before injection the plateau response was a nonlinear function of light intensity. After EGTA injection the response-intensity curves tended to become linear. Before injection, bright lights lowered the sensitivity as determined with subsequent test flashes. EGTA injection decreased the light-induced changes in sensitivity. Ca-EGTA buffers having different levels of free calcium were pressure-injected into ventral photoreceptors; the higher the level of free calcium, the lower the sensitivity measured after injection. The effects of inotophoretic injection of EGTA were not mimicked by injection or similar amounts of sulfate and the effects of pressure injection of EGTA buffer solutions were not mimicked by injection of similar volumes of pH buffer or mannitol. The data are consistent with the hypothesis that light adaptation is mediated by a rise of the intracellular free calcium concentration.     

The role of cytoplasmic calcium in photoreceptor light adaptation.

The process of light adaptation in vertebrate rod and cone photoreceptors is believed to involve a diffusible cytoplasmic messenger. Two lines of evidence indicate that photoreceptor light adaptation is mediated by a light-induced fall in cytoplasmic calcium concentration (Ca2+i). First, if changes in calcium concentration are slowed by the incorporation of calcium chelators into the photoreceptor cytoplasm then light adaptation is slowed also. Second, if the normal control of Ca2+i is prevented by simultaneously minimising calcium influx and efflux across the outer segment membrane by means of external solution changes, then all of the manifestations of light adaptation are abolished. Furthermore, recent results show that changes in Ca2+i imposed in the absence of light are sufficient to cause at least some of the manifestations of light adaptation. Together these results indicate that calcium acts as the messenger of light adaptation in the photoreceptors of both lower and higher vertebrates.     

Low calcium environment effects osteoprotegerin ligand/osteoclast differentiation factor

In coculture with osteoblastic cell line MC3T3-E1 (E1) and mouse bone marrow cells, we reported that numbers of osteoclasts rose significantly on exposure to a low-calcium environment. Here we examined how osteoblasts influence osteoclastogenesis under a low-calcium environment. Comparing low extracellular calcium with a regular calcium environment, osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) mRNA expression show more increase in the culture of low-calcium environment than in that of a regular calcium environment. Calcium-sensing receptor (CaSR), which was supposed as one of the mechanisms of recognizing extracellular calcium, existedon the surface of E1 cells. When E1 cells stimulated with agonists of CaSR, gadolinium, and neomycin, OPGL/ODF mRNA expression decreased. Moreover, these agonists reduced osteoclast formation in coculture. Taken together, it is possible that osteoblasts may recognize extracellular calcium via CaSR and regulate osteoclastogenesis.     

The role of steady phosphodiesterase activity in the kinetics and sensitivity of the light-adapted salamander rod photoresponse

We investigated the kinetics and sensitivity of photocurrent responses of salamander rods, both in darkness and during adaptation to steady backgrounds producing 20-3,000 photoisomerizations per second, using suction pipet recordings. The most intense backgrounds suppressed 80% of the circulating dark current and decreased the flash sensitivity approximately 30-fold. To investigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the background. The fractional responses to flashes of any fixed intensity began rising along a common trajectory, regardless of background intensity. We interpret these invariant initial trajectories to indicate that, at these background intensities, light adaptation does not alter the gain of any of the amplifying steps of phototransduction. For subsaturating flashes of fixed intensity, the fractional responses obtained on backgrounds of different intensity were found to "peel off" from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier the time of peeling off. This behavior is consistent with a background-induced reduction in the effective lifetime of at least one of the three major integrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) the inactivation of activated phosphodiesterase (E*, representing the complex G(alpha)-PDE of phosphodiesterase with the transducin alpha-subunit); or (3) the hydrolysis of cGMP, with rate constant beta. Our measurements show that, over the range of background intensities we used, beta increased on average to approximately 20 times its dark-adapted value; and our theoretical analysis indicates that this increase in beta is the primary mechanism underlying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.     

Calcium-independent growth of human ovarian carcinoma cells

Evidence in the literature suggests that cancer cell growth in vitro is generally not sensitive to external calcium. A human ovarian carcinoma cell line (SKOV3) retained 60% of its normal growth in Dulbecco modified Eagle's medium (DME) when the calcium concentration was reduced from 3 mM to 10 microM. Chinese hamster ovary cells (CHO) were growth-arrested in media containing less than 500 microM calcium. In low-calcium (10 microM) DME, 10 microM of a calmodulin antagonist W7 inhibited the growth of SKOV3 cells by more than 90%, while 100 microM of its inactive analog W5 was mildly inhibitory (20%). The growth inhibition by W7 was antagonized by increasing calcium concentrations in the culture media, while the inhibition by W5 was calcium-independent. The phorbol ester TPA was also effective in antagonizing W7's growth inhibition in low-calcium DME, suggesting that the W7 effect is mediated via protein kinase C inhibition. SKOV3 total cellular protein kinase C activity was 1.6 times higher than CHO cells when incubated in normal DME. When incubated in low-calcium DME, a large drop in protein kinase C activity in the CHO cells was observed while the enzyme activity was unchanged in the SKOV3 cells. Our data suggest that these human ovarian tumor cells have altered cellular calcium regulatory processes associated with the defective down-regulation of protein kinase C. This defect may confer these cells the ability to proliferate independently of the external calcium concentration. Targeting the cellular signal transduction components may be useful in cancer chemotherapy.