Lipochondria and the light response of Aplysia giant neurons

The ultrastructure, absorbance, and elemental content of lipochondria present in the cytoplasm of Aplysia giant neurons have been investigated before and after 30–1,200 sec doses of white light at intensities which produce saturated light responses. The effects of exposure to the calcium ionophore A-23187 and to EGTA were also examined. The lipochondria of nonilluminated neurons are membrane-bound, and contain lipids, protein, Na, K, Mg, Ca, Si, Cl, Br, P, and a pigment which is probably β-carotene. The cytoplasm appeared to have little pigment. When neurons were illuminated for 20 min, 60–70% of the lipochondria showed marked ultrastructural alterations, the most notable being the appearance of membranous material. Earlier changes which occur after 30 sec of illumination include the appearance of paracrystalline arrays and mottling. Less than 10% of lipochondria in nonilluminated neurons have a similar appearance. These effects were greatly enhanced in illuminated neurons exposed to the calcium ionophore or EGTA. In nonilluminated neurons, the ionophore also produced ultrastructural changes. In frozen specimens, the calcium content of the most electron dense lipochondria of illuminated neurons was reduced. Other elements which were counted were also reduced. The lipochondria are the main intracellular site of photopigment. They may also act as an intracellular source for calcium which, as the accompanying paper indicated, may mediate phototransduction in Aplysia neurons.     

Isolation of pigmented granules involved in extra-retinal photoreception in Aplysia californica neurons

Many neurons in the ganglia of Aplysia california contain pigmented, membrane-bound granules (lipochondria), which are thought to mediate the light response of some of the neurons, including the giant cell of the abdominal ganglion. A method of isolating the lipochondria by centrifugation of ganglia homogenates has now been developed. Electron microscopy was used to demonstrate that most of the lipochondria remain morphologically intact. As shown by X-ray microanalysis, isolated lipochondria contain the same elements, including calcium, as do lipochondria in intact giant cells. The calcium can be released into the medium by treatment of the organelles with the Ca²⁺ ionophore A23187. It appears that the lipochondria of Aplysia ganglia are similar in their morphology, elemental content and susceptibility to the ionophore. Two pigments were isolated from the lipochondria, and chromatography and spectrophotometric studies indicated that they are β-carotene and a “retinol-like” compound.     

Ultrastructure of Aplysia neurons having different degrees of light sensitivity.

The relationship between ultrastructure and photosensitivity of pigmented neurons of the abdominal ganglion of Aplysia californica was investigated using electron microscopy and electrophysiological methods. Four identified neurons of similar light microscopic appearance were examined; two are photoresponsive and two are not. Illumination hyperpolarizes both responsive neurons. One of them, R2, requires roughly 100 times greater light intensities than does the other, the ventral photoresponsive neuron (VPN), for similar responses. Two neurons lying adjacent to VPN and similar in appearance to VPN do not have measurable electrophysiological responses to even the highest light intensities. All four neurons contained lipochondria, pigmented organelles associated with the light response. Therefore the presence of these organelles is not the only requirement for light sensitivity in these neurons. Illumination appeared to increase the number of membranous lipochondria in both R2 and the ventral neurons, but only in R2 was this increase significant. Factors such as the concentration of lipochondria near the plasma membrane may affect quantitative aspects of the light response, but in the insensitive cells the lipochondria are apparently uncoupled from other factors required for the light response.     

Ultrastructure of Aplysia neurons having different degrees of light sensitivity

The relationship between ultrastructure and photosensitivity of pigmented neurons of the abdominal ganglion of Aplysia californica was investigated using electron microscopy and electrophysiological methods. Four identified neurons of similar light microscopic appearance were examined; two are photoresponsive and two are not. Illumination hyperpolarizes both responsive neurons. One of them, R₂, requires roughly 100 times greater light intensities than does the other, the ventral photoresponsive neuron (VPN), for similar responses. Two neurons lying adjacent to VPN and similar in appearance to VPN do not have measurable electrophysiological responses to even the highest light intensities. All four neurons contained lipochondria, pigmented organelles associated with the light response. Therefore the presence of these organelles is not the only requirement for light sensitivity in these neurons. Illumination appeared to increase the number of membranous lipochondria in both R₂ and the ventral neurons, but only in R₂ was this increase significant. Factors such as the concentration of lipochondria near the plasma membrane may affect quantitative aspects of the light response, but in the insensitive cells the lipochondria are apparently uncoupled from other factors required for the light response.     

Spectral characterization of the neuronal pigments of Aplysia juliana.

Spectral analysis at liquid N2 temperature of the circumesophageal ganglia of Aplysia juliana showed that carotenoids and a hemoglobin-like pigment are contained in concentrations of approx. 25 and 3 micronM, respectively, in the whole ganglia. Microspectrophotometrical measurements of Aplysia neurons indicated that the carotenoids reside on lipochondria in a concentration of approx. 38 mM. In addition to lipochondria, two types of pigmented particulate having absorption maxima at about 512 and 525 nm, respectively, were found in the neurons. The neuronal carotenoids consist of violaxanthin, beta-carotene and one minor component; among them the first occupies approx. 77% of total carotenoids. Two principal absorption maxima of the carotenoids, when existing in both ganglial homogenates and Triton X-100 extracts, show a red shift of 10 nm compared with those of free pigments in hexan. The red shift may be interpreted as due to the solvation of the carotenoids by surrounding lipids.     

Spectral characterization of the neuronal pigments of Aplysia juliana

Spectral analysis at liquid N₂ temperature of the circumesophageal ganglia of Aplysia juliana showed that carotenoids and a hemoglobin-like pigment are contained in concentrations of approx. 25 and 3 μM, respectively, in the whole ganglia. Microspectrophotometrical measurements of Aplysia neurons indicated that the carotenoids reside on lipochondria in a concentration of approx. 38 mM. In addition to lipochondria, two types of pigmented particulate having absorption maxima at about 512 and 525 nm, respectively, were found in the neurons. The neuronal carotenoids consist of violaxanthin, β-carotene and one minor component; among them the first occupies approx. 77% of total carotenoids. Two principal absorption maxima of the carotenoids, when existing in both ganglial homogenates and Triton X-100 extracts, show a red shift of 10 nm compared with those of free pigments in hexan. The red shift may be interpreted as due to the solvation of the carotenoids by surrounding lipids     

Influence of calcium ion on host cell invasion and intracellular replication by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.     

Studies on the prolactin-releasing mechanism of histones H2A and H2B

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PRL)-releasing activity of histones H2A and H2B on perifused and incubated rat pituitary cells. In the perifusion experiments, freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested (stimuli) were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and PRL measured by specific RIA. In the incubation studies, freshly dispersed pituitary cells were incubated in a metabolic incubator with different stimuli at different doses and for varying times. Perifusion of cells with median eminence extract (1/30), histone H2A (30 microM) or histone H2B (30 microM), generated clear PRL release responses. Cells incubated with histone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino acid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were incubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhancers caffeine, NaF and forskolin, and the protein kinase C inhibitor, trifluoperazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were able to reduce significantly the response of lactotrophs to histones. Our results confirm previous evidence that histones may act as hypophysotropic signals. The data also suggest that calcium- and diacylglycerol-associated pathways participate in these effects.     

Intracellular calcium and extra-retinal photoreception in Aplysia giant neurons

The early or “instantaneous” current-voltage relationship for the light-activated potassium current in Aplysia giant neurons was linear during the first second of illumination. However, the light current was greatly reduced or abolished by prolonged hyperpolarization. It was also greatly reduced by the injection of calcium EGTA buffers having calcium activities of 5.6 × 10^?8 M and simulated by injecting buffers with calcium activities of 2.8–5.6 × 10^?7 M. Removal of calcium from the extracellular fluid had no effect. Both the light-and calcium-activated outward potassium currents were reduced by tetra-ethylammonium (TEA) ions. The light current was not affected by substituting rubidium for potassium nor by substituting either lithium or Tris for sodium. The calcium-activated potassium current persisted when the neuron was cooled to 5°C. However, the light response could no longer be elicited. Light hyperpolarizes Aplysia neurons probably by increasing intracellular calcium activity two-to six-fold which activates a membrane potassium conductance. Calcium levels appear to be restored within the cell and are energy dependent. The light-activated release of calcium is inhibited by cooling. The body wall of Aplysia transmits enough visible or 500-nm light to hyperpolarize some Aplysia giant neurons under ambient conditions. These neurons may be involved in the extraretinal light entrainment that occurs in Aplysia.     

Intracellular free Ca2+ concentration in fowl spermatozoa and its relationship to motility and respiration in spermatozoa.

The ability of fowl spermatozoa to accumulate and de-esterify the intracellular fluorescent Ca2+ indicator fura-2 was established. The cytosolic Ca2+ concentrations, measured by this technique, did not change after the addition of 1 mmol EGTA l-1. Subsequently, addition of the calcium ionophore A23187 caused a reduction in cytosolic Ca2+ concentrations, presumably by efflux of Ca2+ from the spermatozoa. Intracellular free Ca2+ concentrations were then significantly increased by the addition of 1 mmol CaCl2 l-1. The motility of demembranated spermatozoa gradually decreased after the addition of EGTA alone or EGTA with A23187, but was instantly restored by the addition of CaCl2 in the presence of both EGTA and A23187. Unlike demembranated spermatozoa, intact spermatozoa maintained their motility, even after the addition of EGTA, but their motility was reduced by the addition of A23187 in the presence of EGTA. The addition of A23187 also reduced the rate of oxygen consumption, but not the ATP concentrations in intact spermatozoa. These results demonstrate that the motility and respiration of fowl spermatozoa are strongly influenced by their intracellular Ca2+ concentrations.     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.     

Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels

Chemotactic factors stimulate the rate of locomotion of polymorphonuclear leukocytes (PMNs). To investigate the importance of cytoplasmic calcium we have examined the ability of the chemotactic peptide N-formylnorleucyl eucylphenalanine (FNLLP) to stimulate the locomotion of PMNs whose cytoplasmic calcium levels were reduced by incubation in EGTA or in EGTA plus the calcium ionophores, ionomycin or A23187. Locomotion was assayed by migration through micropore filters and by time-lapse videomicroscopy. Cells in EGTA exhibited similar or slightly reduced rates of locomotion compared to cells in Hanks' balanced salt solution (HBSS). The peptide dose dependence for the stimulation of locomotion was similar in medium containing calcium or EGTA. The presence of 1 microM ionophore plus EGTA had no effect on the stimulation of locomotion by peptide. The presence of ionophores (1 microM) plus external calcium inhibited locomotion.     

Modulation of nuclear rotation in neuronal interphase nuclei by nerve growth factor, by gamma-aminobutyric acid, and by changes in intracellular calcium

Nuclear rotation (NR) is typically measured as motion of nucleoli within nuclei of cells in vitro. This occurs in cycling cells. However, its observation in neurons arrested in interphase indicates that mechanisms related to mitosis are not a prerequisite. We have recently shown that NR occurs in three dimensions within the nuclear space, that it occurs within the space delineated by the outer nuclear membrane and that it includes chromatin domains in addition to nucleoli and have postulated that this motion of chromatin domains is related to changes in gene expression. We now show that exposure of dorsal root, sensory neurons in vitro to nerve growth factor (NGF) or to gamma-aminobutyric acid (GABA), agents which alter gene expression, and to agents causing redistribution of calcium, such as EGTA and the calcium ionophore A23187, significantly alters NR. The NGF increased the mean rate of NR and did so at a time after exposure when activity of RNA polymerases have been shown to rise. Exposure to GABA resulted, within minutes, in shifts of the nucleolus within the three-dimensional space of the nucleus, associated in some neurons with significant, sigmoidal increases in the rate of NR. The calcium ionophore A23187 as well as chelation of extracellular calcium with EGTA similarly increased rates. Importantly, excess calcium, with EGTA remaining present, returned NR of all nucleoli to rates not different from controls. This indicates that the increase in NR seen with EGTA is specific to the chelation of calcium and not an nonspecific response to EGTA. It is difficult to link the action of agents which alter gene expression or transmembrane ion balance with changes in NR. Nevertheless, in support of our hypothesis, the results presented here show that agents known to alter gene expression, alter NR in a temporally coincident manner and that they do so, possibly, by calcium-dependent mechanisms.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Lipopolysaccharide-mediated macrophage activation: the role of calcium in the generation of tumoricidal activity

As we have reported, calcium ionophore A23187 activates macrophages for tumor cell killing, and the activated macrophages produced a soluble cytotoxic factor (M phi-CF) that is similar, if not identical, to tumor necrosis factor. Based on these observations, we have investigated whether calcium is involved in the activation mediated by another potent macrophage activator, namely lipopolysaccharide (LPS). We first showed that A23187 caused uptake of extracellular calcium-45 by macrophage monolayers, whereas LPS did not. Because in this system rapid changes would not have been detected, several other approaches also have been used. We have examined the effect of depleting extracellular calcium by using medium containing no added calcium, supplemented with 1 mM EGTA. In no case did depletion result in decreased M phi-CF production by LPS-treated macrophages. Measurements using the fluorescent intracellular calcium indicator Quin 2 have also been performed. The calcium ionophore ionomycin caused a rapid change in the intracellular Quin 2 signal. LPS, even at a concentration in vast excess of that required to activate the macrophages, caused no change in the signal during a 2-hr period. If the macrophages were loaded with high doses of Quin 2 or another intracellular chelator, TMB-8, M phi-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M phi-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium.     

Rapid degradation of cyclooxygenase-1 and hematopoietic prostaglandin D synthase through ubiquitin-proteasome system in response to intracellular calcium level

Cyclooxygenase (COX)-1 and hematopoietic prostaglandin (PG) D synthase (H-PGDS) proteins, which are both involved in the arachidonate cascade, were stable in human megakaryocytic MEG-01 cells. In contrast, once the intracellular calcium level was increased by treatment with a calcium ionophore, both protein levels rapidly decreased with a half-life of less than 30 and 120 min for COX-1 and H-PGDS, respectively. In the presence of a proteasome inhibitor, COX-1 and H-PGDS proteins accumulated within 10 and 30 min, respectively, and concurrently appeared as the high-molecular-mass ubiquitinated proteins within 30 and 60 min, respectively, after an increase in the intracellular calcium level. The ubiquitination of these proteins was also observed when ADP, instead of a calcium ionophore, was used as an inducer to elevate the intracellular calcium level. When the entry of calcium ion into the cells was inhibited by ethylene glycol tetraacetic acid (EGTA), the ubiquitination of COX-1 and H-PGDS was clearly suppressed; and the addition of CaCl(2) to the medium cleared the EGTA-mediated suppression of the ubiquitination. These results indicate that COX-1 and H-PGDS were rapidly ubiquitinated and degraded through the ubiquitin-proteasome system in response to the elevation of the intracellular calcium level.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Calcium and Photoentrainment in Chick Pineal Cells Revisited: Effects of Caffeine, Thapsigargin, EGTA, and Light on the Melatonin Rhythm

Abstract: Chick pineal cells in dispersed cell culture display a persistent, photosensitive, circadian rhythm of melatonin production and release. Light pulses have at least two distinguishable effects on these cells, i.e., acute suppression of melatonin output and phase shifts (entrainment) of the underlying circadian pacemaker. Previous results linked calcium influx through voltage-sensitive calcium channels in the plasma membrane to acute regulation of melatonin synthesis but denied a role for such influx in entrainment. Those experiments did not, however, address the role of intracellular calcium metabolism. Here we describe the effects of pulses of caffeine, thapsigargin, and EGTA on the melatonin rhythm, and their interactions with the effects of light pulses. Caffeine had two distinguishable effects on these cells, acute enhancement of melatonin output (attributable to phosphodiesterase inhibition) and phase shifts of the circadian pacemaker with a light-like pattern (attributable to effects on intracellular calcium). Phase shifts induced by light and caffeine were not additive. Thapsigargin (which specifically blocks the pump that replenishes intracellular calcium stores, thereby increasing cytoplasmic calcium and depleting intracellular stores) had no phase-shifting effects by itself but reduced the size of the phase advances induced by caffeine or light. Low calcium solution acutely suppressed melatonin output without inducing phase shifts or affecting those induced by caffeine or light. However, addition of EGTA (which specifically chelates calcium, thereby lowering cytoplasmic calcium and depleting intracellular stores) did reduce the size of phase advances induced by caffeine or light, in normal medium or in low calcium solution, without inducing a phase shift by itself at that phase. Taken together, these results point toward a role for intracellular calcium fluxes in entrainment of the circadian pacemaker.     

Calcium ions as a mediator in GnRH action on gonadotropin release in the common carp (Cyprinus carpio L)

Collagenase-dispersed carp pituitary cells in a perifusion system were used to study the role of calcium ions in the mechanism of GnRH action on the release of maturational gonadotropin (GtH) in fish. The specific calcium chelator EGTA and the calcium antagonist manganese (Mn2+) caused a 40% inhibition in the basal GtH release and completely blocked GnRH-stimulated GtH release. Short-term application of graded doses of calcium ionophore A23187 caused a dose-dependent increase in GtH secretion. A23187 failed to stimulate GtH secretion in the presence of EGTA. Depolarization of the membrane by K+ caused a strong stimulation of GtH release similar to the action of GnRH. Stimulatory action of K+ was inhibited by EGTA. These data suggest a role for extracellular calcium as an intracellular mediator in GnRH-stimulated, as well as in basal, GtH release in carp. The stimulation of GtH release by K+ also indicates that voltage-dependent processes could be involved in this phenomenon.