A Radioimmunoassay for Ependymins β and γ: Two Goldfish Brain Proteins Involved in Behavioral Plasticity

Abstract: A radioimmunoassay (RIA) using ¹²⁵I-labeled antigen was developed for the quantitative determination of two goldfish brain proteins (ependymins β and γ). The proteins were isolated from the cerebrospinal fluid (CSF) and cells of the ependymal zone surrounding goldfish brain ventricles. The turnover rates of β and γ were previously shown to be specifically enhanced after the animals successfully acquired a new pattern of swimming behavior. Femtomole quantities of ependymin β were measurable by the RIA. In applications of the assay, β and γ ependymins were found to have common immunological properties, since ¹²⁵I-β-antigen bound to antibody could be displaced by unlabeled ependymin γ as well as ependymin β but not by a variety of other proteins including several purified glycoproteins isolated from goldfish brain. The ependymins were shown to constitute 14% of the total protein content of the brain extracellular fluid and also to be present as a minor component of the serum proteins (0.3%). Ependymins β and γ have an immunological reactivity in these fractions that can be increased by a factor of 30 on heating. The data suggest that the antigenicity of the molecules is highly masked, and that it may require some unraveling of the quaternary structure of the proteins before maximal interaction with the antisera becomes possible.     

Structural and Metabolic Relationships Between Goldfish Brain Glycoproteins Participating in Functional Plasticity of the Central Nervous System

Ependymins beta and gamma (MW 32,000 and 26,000 daltons) are two secreted goldfish brain glycoproteins that exhibit a specifically enhanced turnover rate when the animals successfully acquire a new pattern of swimming behaviour. Both proteins are bound identically to concanavalin A and can be isolated from brain extracellular fluid and from brain cytoplasm by lectin affinity chromatography. Radioimmunoassay data, using purified 125I-labeled ependymins and antisera directed against ependymin beta or ependymin gamma, show complete cross-reactivity between the two proteins. It is demonstrated by Scatchard-plot analysis that the antisera recognize identical immunological determinants in both proteins. The amino acid composition of the ependymins is similar, and several identical polypeptide fragments are obtained after limited proteolysis with Staphylococcus aureus protease. The proteins are capable of forming complexes of the compositions gamma 2, beta gamma, and beta 2. A protease present in the extracellular fluid of goldfish brain promotes proteolysis of ependymin beta to ependymin gamma. The finding that ependymin gamma is physiologically derived from ependymin beta suggests the possibility that ependymin beta might exert its biological function during consolidation of new behavioural patterns via smaller polypeptide fragments.     

Ependymins are expressed in the meninx of goldfish brain

Summary Ependymins are dimeric glycoproteins found in the extracellular fluid of goldfish brain. They were originally observed because of their enhanced turnover rates after learning. In this paper we present the first investigation concerning the expression of these secretory proteins in goldfish brain via in situ hybridization with synthetic oligonucleotides and cRNA probes. It is shown that ependymin-mRNAs are predominantly expressed in the meninx surrounding the brain and in an invaginated part of the meninx called the cavum cranii. These results have been confirmed by immunhistochemical analysis. This indicates that, in fish, the meninx synthesizes a major protein constituent of the cerebrospinal fluid; furthermore, this suggests that the functional sites of ependymins are removed from the place of their synthesis. Distribution between different compartments may be achieved via the open communication system of the perivascular spaces.     

The role of ependymin in the development of long lasting synaptic changes

1.) Three types of training experiments (a complex motor task, avoidance conditioning and classical conditioning) in the goldfish and one in the mouse (T-maze learning) indicate that the brain extracellular glycoprotein (ependymin) has a role in the consolidation process of long-term memory formation. 2.) Direct ELISA measures of the concentration of ependymin in the brain extracellular fluid (ECF) indicate that its level decreases after goldfish learn to associate a light stimulus (cs) with the subsequent arrival of a shock (US): paired CS-US gave changes whereas an unpaired presentation of CS-US gave no changes in comparison to unstimulated controls. 3.) Ependymin is released into ECF and CSF as mixtures of three types of disulfide-linked dimers of two acidic polypeptide chains (M. W. 37 kDa and 31 kDa). It contains 10% carbohydrate as an N-linked glycan. 4.) Ependymin has the capacity to polymerize in response to events that deplete Ca2+ from the brain extracellular environment. A molecular hypothesis relating polymerization properties to the process of formation of long-lasting synaptic changes is proposed. 5.) Investigations of the pattern of regeneration of goldfish optic nerve and the mechanisms of long-term potentiation (LTP) of rat brain hippocampal slices suggest that ependymin has a role in the formation of long-lasting synaptic changes. The E.M. data show that polymerized products which stain with anti-ependymin sera accumulate at synapses and in new spines after LTP.     

Ependymins and their potential role in neuroplasticity and regeneration: Calcium-binding meningeal glycoproteins of the cerebrospinal fluid and extracellular matrix

• 1.1. Ependymins are unique, highly divergent secretory proteins of the fish endomeninx. Thus far, no homologous sequences have been characterized in mammals.
• 2.2. Soluble ependymins are the predominant constituents of the cerebrospinal fluid of many teleost fish. A bound form of these glycoproteins is associated with the extracellular matrix probably with collagen fibrils. The latter may be the functional form of ependymins.
• 3.3. Ependymins bind Ca²⁺ via N-linked sialic acid residues leading to a conformational transition.
• 4.4. The molecular function of ependymins seems to be related to cell contact phenomena involving the extracellular matrix. For example, adhesive or anti-adhesive interactions may possibly influence ingrowing axons.

     

Effect of intracellular Na+ on platelet aggregation and Ca2+ mobilization

The effects of ionophores, which can carry alkali metal cations, on platelet aggregation were examined. At an alkaline extracellular pH, alkali metal cation/H+ exchanger nigericin accelerated aggregation in K+-enriched medium, whereas it rather inhibited aggregation in Na+-enriched medium, even though the intracellular pH was only slightly alkaline. The inhibitory effect of Na+ on platelet aggregation was more clearly shown with the alkali metal cation exchanger gramicidin D. The ionophore had no effect or a slightly accelerative effect on aggregation in K+-enriched medium, whereas it significantly inhibited aggregation induced by thrombin, ADP and platelet activating factor in Na+-enriched medium. Fluorescence studies on fura-2-labeled platelets revealed that in Na+-enriched medium gramicidin D inhibited agonist-induced Ca2+ mobilization both in the presence and absence of extracellular Ca2+. These results suggest that the intracellular Na+ inhibits platelet aggregation by inhibiting Ca2+ mobilization.     

Actions of two native GnRHs and protein kinase C modulators on goldfish pituitary cells. Studies on intracellular calcium levels and gonadotropin release.

Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.     

Molecular Characterization of an Ependymin Precursor from Goldfish Brain

Ependymins are thought to be implicated in fundamental processes involved in plasticity of the goldfish CNS. Gas-phase sequencing of purified ependymins beta and gamma revealed that they share the same N-terminal sequence. Each sequence displays microheterogeneities at several positions. Based on the protein sequences obtained, we constructed synthetic oligonucleotides and used them as hybridization probes for screening cDNA libraries of goldfish brain. In this article we describe the full-length sequence of a mRNA encoding a precursor of ependymins. A cleavable signal sequence characteristic of secretory proteins is located at the N-terminal end, followed directly by the ependymin sequence. Also, two potential N-glycosylation sites were detected. A computer search revealed that ependymins form a novel family of unique proteins.     

Calcium influx mediated by the inwardly rectifying K+ channel Kir4.1 (KCNJ10) at low external K+ concentration

COS-1 cells with heterologeous expression of the Kir4.1 (KCNJ10) channel subunit, possess functional Kir4.1 channels and become capable to generating cytosolic Ca2+ transients, upon lowering of the extracellular K+ concentration to 2 mM or below. These Ca2+ transients are blocked by external Ba2+ (100 microM). Acute brain stem slices from wild-type mice (second post-natal week), which were loaded with the fluorescent Ca2+ indicator Oregon Green BAPTA-1-AM, were exposed to 0.2 mM K+. Under these conditions astrocytes, but not neurons, responded with cytosolic Ca2+ elevations in wild-type mice. This astrocyte-specific response has previously been used to identify astroglial cells type [R. Dallwig, H. Vitten, J.W. Deitmer, A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28 (2000) 247-259]. In Kir4.1 knock-out (Kir4.1-/-) mice, the number of responding cells was dramatically reduced and the Ca2+ transients in responding cells were significantly smaller than in wild-type mice. Our results indicate that Kir4.1 channels are the molecular substrate for the observed Ca2+ influx in astrocytes under conditions of low external K+-concentration.     

Changes in Subcellular Distribution of Ependymins in Goldfish Brain Induced by Learning

Abstract: Goldfish were trained for 4 h to swim with an attached polystyrene foam float and tested for retention 3 days later. Intracerebroventricular injection of anti-ependymin antisera was shown to prevent long-term memory formation of this vestibulomotor learning task, as reported previously. In further experiments, fish were killed 4–14 h after the start of training. The brains were dissected, incubated in an isoosmolar solution for collection of proteins of the brain extracellular fluid (ECF), homogenized, and fractionated by differential centrifugation. The ECF, a supernatant fraction enriched in cytoplasmic constituents (S3), and various par-ticulate subcellular fractions were analyzed for their epen-dymin contents by radioimmunoassay. No statistically significant changes that might be induced by the learning were revealed in any of the participate fractions. Steady-state concentrations of ependymins in the cytoplasm, however, increased temporarily by 39% in fish that had mastered the training task as compared with nonlearning animals (passive and active controls). In the ECF, the specific concentration of ependymins first decreased to 88% of control levels (4–5 h after the start of training), but later on, it increased to 138% (8–14 h). Apparently, ependymins present in the ECF are used during biochemical reactions of memory consolidation. The resulting decrease in extracellular epen-dymin concentrations might trigger their resynthesis in the cytoplasm and lead to an increased release of these glyco-proteins into the ECF.     

High extracellular Ca2+ hyperpolarizes human parathyroid cells via Ca(2+)-activated K+ channels

Membrane potential has a major influence on stimulus-secretion coupling in various excitable cells. The role of membrane potential in the regulation of parathyroid hormone secretion is not known. High K+-induced depolarization increases secretion from parathyroid cells. The paradox is that increased extracellular Ca2+, which inhibits secretion, has also been postulated to have a depolarizing effect. In this study, human parathyroid cells from parathyroid adenomas were used in patch clamp studies of K+ channels and membrane potential. Detailed characterization revealed two K+ channels that were strictly dependent of intracellular Ca2+ concentration. At high extracellular Ca2+, a large K+ current was seen, and the cells were hyperpolarized (-50.4 +/- 13.4 mV), whereas lowering of extracellular Ca2+ resulted in a dramatic decrease in K+ current and depolarization of the cells (-0.1 +/- 8.8 mV, p < 0.001). Changes in extracellular Ca2+ did not alter K+ currents when intracellular Ca2+ was clamped, indicating that K+ channels are activated by intracellular Ca2+. The results were concordant in cell-attached, perforated patch, whole-cell and excised membrane patch configurations. These results suggest that [Ca2+]o regulates membrane potential of human parathyroid cells via Ca2+-activated K+ channels and that the membrane potential may be of greater importance for the stimulus-secretion coupling than recognized previously.     

Extracellular Fluid Proteins of Goldfish Brain: Evidence for the Presence of Proteases and Esterases

Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.     

剪切应力诱导血小板聚集

剪切应力诱导血小板聚集（shear-induced platelet aggregation, SIPA）是指在高剪切流场诱导下血小板表面的膜糖蛋白（GPⅠb/Ⅸ/Ⅴ和GPⅡb/Ⅲa）与血浆中的von Willebrand因子（vWF）相结合,介导血小板的活化、黏附和聚集,是动脉血栓的重要成因.SIPA还需要Ca2+,ADP/ATP等生化因素的参与,因而SIPA现象是生化因素和力学因素偶合作用的结果.细胞外Ca2+是高剪切应力诱导血小板发生聚集的必需条件,Ca2+的跨膜内流引起细胞骨架结构的改变和GPⅡb/Ⅲa的活化.近来对ADP/ATP位于血小板膜上的P2受体的研究表明,P2受体与细胞内Ca2+协同作用通过多种生化途径调控血小板的活化过程在SIPA的信号传导中起着关键的作用.从力学环境与生化反应的偶合关系入手研究SIPA现象的触发机制,深入研究SIPA现象中的信号转导通路是今后的研究热点之一.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

The mechanism of the effect of K+ on the steroidogenesis of rat zona glomerulosa cells of the adrenal cortex: role of cyclic AMP

The effects of various concentrations of extracellular K+ (3.6-13 mM) on the steroid (corticosterone and aldosterone) and cyclic AMP outputs of capsular cells (95% zona glomerulosa) of the rat adrenal cortex were studied at different concentrations of extracellular Ca2+. Small amounts of EGTA (50 microM) were added to reduce the free Ca2+ concentrations effectively to zero at the lowest possible total Ca2+ concentration. At a total extracellular concentration of 2.5 mM Ca2+, in 27 experiments the mean values of the steroid and cAMP outputs showed a maximum at 8.4 mM K+. The increase in steroid and cAMP outputs at 5.9, 8.4 and 13 mM K+ compared with that at 3.6 mM were highly significant (p less than 0.01). The overall correlation of either corticosterone or aldosterone with cAMP outputs was also highly significant and was even better from 3.6 to 8.4 mM K+. Lowering the effective free concentration of Ca2+ to zero decreased the steroid and cAMP outputs significantly at all K+ concentrations, and no output was then significantly higher than at 3.6 mM. With the pooled data on outputs at all total Ca2+ (2.5, 0.5, 0.25, 0.10, 0.05 and 0.0 mM) and K+ (3.6, 5.9, 8.4 and 13 mM) concentrations, the correlation of either steroid with cAMP outputs was highly significant (but again optimally from 3.6 to 8.4 mM K+). Nifedipine (10(-6) to 10(-4) M) was added to the incubations with the aim of specifically inhibiting Ca2+ influx at total extracellular Ca2+ concentrations of 2.5, 1.25 and 0.25 mM and with the usual K+ concentrations. The cAMP outputs were reduced at all K+ concentrations above 3.6 mM K+. The effect was highly significant at 10(-4) M nifedipine and a total Ca2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca2+ concentrations in vivo. These results indicate that cAMP plays a significant role in the stimulation of steroid output by K+ particularly between 3.6 and 8.4 mM K+. In this range of K+ concentrations the stimulation of cAMP seems to be controlled by increases in Ca2+ influx. The correlation of steroid and cAMP output at the higher K+ concentrations (between 8.4 and 13 mM K) and at the various total Ca2+ concentrations is less significant. Also, with all concentrations of added nifedipine there is an 'anomalous' increase in steroid output at 13 mM K+ and at total Ca2+ concentrations of 2.5 and 1.25 mM. However, at the same K+ concentrations and at 0.25 mM Ca2+, nifedipine decreases steroid outputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid induction of acetylcholine receptor aggregates by a neural factor and extracellular Ca2+

A soluble fetal brain extract (EBX) induces acetylcholine receptor (AChR) aggregation in cultured rat myotubes within 4 hr at 36 degrees C in a defined medium containing 1.8 mM (normal) extracellular Ca2+ (Olek et al., 1983). The activity of EBX was Ca2+ dependent; reducing extracellular Ca2+ significantly inhibited EBX-induced AChR aggregation and a 15-50% increase in extracellular Ca2+ synergistically enhanced the activity of EBX. Synergism was specific for Ca2+ as increases in other divalent cations (Ba2+, Co2+, Mg2+, Mn2+, Sr2+) had no effect. A large increase (300-500%) in extracellular Ca2+ alone also induced AChR aggregation within 4 hr at 36 degrees C. An equivalent increase in other cations (Ba2+, Co2+, Mg2+, Mn2+, Sr2+) did not promote AChR aggregation. An initial 15-min pulse of increased extracellular Ca2+ alone or with EBX was adequate to induce AChR aggregation. Aggregates induced by EBX, Ca2+ alone, or EBX/Ca2+ were found predominantly on the top surface of the myotube. These treatments did not detectably alter preexisting aggregates present at substrate contact sites on the bottom surface of myotubes. AChR aggregation induced by any treatment was not inhibited by cycloheximide, Ca2+ channel blockers, or protease inhibitors but was blocked by Co2+ and sodium azide.     

Store-Operated Ca2+ Influx and Voltage-Gated Ca2+ Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells

Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+ and caffeine. K+-evoked exocytosis was entirely dependent on Ca2+ influx through voltage-gated Ca2+ channels, and of the subtypes of such channels present in these cells, influx through N-type was primarily responsible for triggering exocytosis. L-type channels played a minor role in mediating K+-evoked secretion, whereas P/Q-type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+-free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+ was restored to the extracellular solution (in the absence of caffeine), store-operated Ca2+ influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L-type Ca2+ channels, but less than that caused by Ca2+ influx through N-type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+ influx through pathways other than voltage-gated Ca2+ channels.     

Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 beta-cell line

Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2',5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2',5'-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.     

Effects of Kainic Acid on Brain Calcium Fluxes Studied In Vivo and In Vitro

The effect of in vivo administration of kainic acid into the rabbit hippocampus was studied with brain dialysis and subsequent determination of the Ca2+ concentration in the dialysate. When included in the perfusing medium, kainic acid as well as veratridine induced a decrease in extracellular Ca2+. The effect of kainic acid (but not of veratridine) was insensitive to tetrodotoxin. In vitro studies revealed no effect of kainic acid on 45Ca2+ uptake by isolated astrocytes, but showed an enhancement of synaptosomal 45Ca2+ accumulation. This was, however, only 25% of the stimulatory effect of high K+ depolarization. Glutamate activated synaptosomal Ca2+ uptake, whereas dihydrokainate had no effect. The uptake evoked by kainate and glutamate was independent of the K+ level in the medium which indicates the involvement of other than voltage-sensitive Ca2+ channels. The results confirm previous finding that kainic acid promotes the uptake of Ca2+ in brain cells. Kainate affects Ca2+ fluxes pre- and postsynaptically. Presynaptic Ca2+ influx may be mediated by chemically gated mechanisms.