Treatment of Starfish Sperm with Egg Jelly Induces the Degradation of Histones

When spermatozoa of Asterina pectinifera are treated with a solution of homologous egg jelly, besides undergoing the acrosome reaction, they begin to degrade their histones gradually. The degradation is most prominent with histone H1, almost 75% of which is degraded within one hour at 20°C. The jelly-induced histone degradation, like the acrosome reaction, requires external Ca²⁺, prefers high pHs and is susceptible to Ca²⁺-channel antagonists such as verapamil and diltiazem. Histone degradation is also induced by nigericin as well as monensin in normal seawater, but not in Ca²⁺-free seawater. Calcium ionophore A23187, that greatly facilitates the monensin-induced histone degradation, also induces histone degradation by itself, slightly in normal seawater and markedly in Ca²⁺-enriched seawater. Concanavalin A inhibits the jelly-induced histone degradation but not the jelly-induced acrosome reaction. These results suggest that egg jelly induces the histone degradation by enhancing Ca²⁺-influx via a Ca²⁺-channel(s) and by increasing cytoplasmic pH, through a pathway which is closely related to, but not entirely the same as, the one leading to the acrosome reaction.     

The Acrosome Reaction Induced by Dimethylsulfoxide in Sea Urchin Sperm

The acrosome reaction in sea urchin sperm, as judged by disappearance of the acrosomal vesicles in Nomarski optics, was induced by dimethylsulfoxide (DMSO) at concentration above 0.1% in normal artificial sea water. The number of the acrosome-reacted spermatozoa increased in proportion to DMSO concentration. The DMSO-induced acrosome reaction, as well as the jelly water- or A23187-induced one, was inhibited by nifedipine and hardly occurred in Ca²⁺-free artificial sea water. However, the DMSO-induced acrosome reaction was found in a few number of spermatozoa in the presence of Ca²⁺at above 0.5 mM, though the jelly water- or A23187-induced acrosome reaction did not occur at external Ca²⁺levels lower than 1 mM. Dependency of the acrosome reaction by DMSO on external Ca²⁺is somewhat lower than that of the reaction by jelly water. In Ca²⁺-free artificial sea water, the acrosomal regions of DMSO-treated spermatozoa attached to their own tails. In some cases, spermatozoa thus treated with DMSO in Ca²⁺free artificial sea water caused formation of fertilization membrane in a few number of eggs kept in Ca²⁺-free artificial sea water. Even in the absence of extermal Ca²⁺, preliminary step of the acrosome reaction seems to be completed probably by DMSO-induced weak Ca²⁺-mobilization in spermatozoa.     

Activation of Ca2+ Transport System of Sea Urchin Sperm by High External pH: 220 kD Membrane Glycoprotein is Involved in the Regulation of the Ca2+ Entry

When sperm of the sea urchin, Hemicentrotus pulcherrimus , were exposed to high pH (9.0) sea water, they showed large increases in intracellular Ca²⁺ ([Ca²⁺]i) and pH (pHi) and underwent the acrosome reaction (AR) without the aid of the egg jelly. Not only [Ca²⁺]i increase but also pHi rise did not occur under Ca²⁺-free conditions. Both the increases in [Ca²⁺]i and pHi and the AR by high pH were inhibited by a Ca²⁺ channel blockers, verapamil and nisoldipine, and by a lectin, wheat germ agglutinin (WGA) which interacts with a 220 kD membrane glycoprotein of sperm. These reagents inhibited also the AR by the egg jelly. The inhibitory effects of WGA were immediately canceled by the addition of N-acetyl-D-glucosamine, a sugar which is known to remove WGA from its binding site. These results suggest that 1) the same Ca²⁺ transport system is activated by high external pH and the egg jelly, 2) increase in [Ca²⁺]i is prerequisite for the stimulation of the H⁺-efflux system(s) and 3) the 220 kD WGA-binding membrane protein functions as a regulator protein of Ca²⁺ transport system.     

Involvement of Wheat Germ Agglutinin (WGA)-Binding Protein in the Induction of the Acrosome Reaction of the Sea Urchin, Strongylocentrotus intermedius. II. Antibody against WGA-Binding Protein Induces the Acrosome Reaction

We obtained a polyclonal antibody against the WGA-binding protein (WGAbp) of Strongylocentrotus intermedius sperm, which is a membrane glycoprotein of 260 kD under non-reducing condition. Anti-WGAbp antibody induced increases in both intracellular Ca²⁺ ([Ca²⁺]i) and intracellular pH (pHi), resulting in the onset of the AR. The increases in [Ca²⁺]i and pHi required extracellular Ca²⁺ and Na⁺, respectively, and were suppressed by the pretreatment with WGA, resulting in the inhibition of the AR. Anti-WGAbp antibody-induced AR was inhibited also by lowered extracellular pH. elevated K⁺, removal of Na⁺ from seawater and the treatment with verapamil, a Ca²⁺ channel inhibitor. These inhibitory conditions are identical with those of the egg jelly-induced AR. Monovalent Fab fragments from anti-WGAbp antibody also induced the AR at relatively high concentration. These results suggest that the WGAbp on the sperm plasma membrane is involved in the regulation of Ca²⁺ influx and Na⁺/H⁺ exchange associated with the AR of S. intermedius sperm. It is a strong candidate for the receptor of the AR-inducing substance in the egg jelly.     

Effects of Cations and Other Medium Components on the Zona-induced Acrosome Reaction of Hamster Spermatozoa

Although the acrosome reaction in lively motile hamster spermatozoa can occur independently of the egg or its investments ("spontaneous" acrosome reaction), it appears to be the egg investments, particularly the zona pellucida, that induces the acrosome reaction in fertilizing spermatozoa of many mammalian species. The latter is referred to as "zona-induced" acrosome reaction. Experiments were conducted to determine if the zona-induced acrosome reaction has different ion requirements from the spontaneous reaction. Like the spontaneous acrosome reaction, the zona-induced acrosome reaction required extracellular Na⁺, K⁺ and Ca²⁺. The absence of Cl⁻ and albumin in the medium inhibited the reaction. The zona-induced acrosome reaction could occur in a HCO₃⁻-free medium, but far less efficiently than in medium containing this ion. Proteinase inhibitors, benzamidine and TLCK, inhibited the zona-induced acrosome reaction. These results suggest that the chemical reactions involved in the spontaneous and zona-induced acrosome reactions are similar although the reaction-triggering mechanism is probably different.     

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis I. Factors Participating in the Acrosome Reaction

In contrast with the case in sea urchin sperm, in starfish the acrosome reaction is not spontaneously induced by simply increasing the extracellular Ca²⁺ concentration or pH. At higher pHs, starfish sperm undergo morphological changes accompanied by exocytosis of the acrosomal vacuole, but they do not form acrosomal filaments. Nomarski-microscopic observation confirmed that spermatozoa undergo the acrosome reaction within the jelly coat. Acrosome reaction-inducing substance, a glycoprotein from the egg jelly, required a diffusible cofactor(s) present in the egg jelly for full activity. Several lines of evidence showed that this diffusible factor(s) is not merely Ca²⁺.     

Induction of the Acrosome Reaction in Starfish

In the starfish, Asterias amurensis , at least two distinct components of the egg jelly are required for inducing the acrosome reaction: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) named Co-ARIS. The following evidence suggested that ARIS and Co-ARIS cooperatively activate CA-channels of the sperm plasma membrane and eventually induce dramatic changes in sperm morphology, the acrosome reaction. 1) Pronase digest of ARIS (P-ARIS) and Co-ARIS, either as a pure or a crude preparation (Fraction M₈), were fully effective in combination for induction of the acrosome reaction in normal sea water, although they were not effective individually. P- ARIS alone induced the acrosome reaction fully in high Ca²⁺ sea water and markedly at high pHs, whereas Fraction M₈ alone did not induce the reaction even in these conditions. The reaction was not induced by increase in either the Ca²⁺ concentration or the pH of sea water, but was markedly induced in the absence of jelly components by raising both the pH and Ca²⁺ concentration together. 2) The ionophore A23187 induced the acrosome reaction appreciably when present alone and fully in the presence of monensin or Fraction M₈. Monesin alone was ineffective. 3) The jelly or a combination of ARIS and Fraction M₈ caused abrupt Ca²⁺ -uptake by the sperm. The Ca-channel blockers verapamil and diltiazem inhibited the jelly-induced acrosome reaction.     

Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca2+ and pH in Sea Urchin Spermatozoa

We examined the effects of two egg jelly components, a fucose sulfate glycoconjugate (FSG) and sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), on the intracellular pH (pH_i) and Ca²⁺ ([Ca²⁺]_i) of spermatozoa of the sea urchin Hemicentrotus pulcherrimus . FSG and/or SAP-I induced elevations of [Ca²⁺]; and pH_i in the spermatozoa at pH 8.0. At pH 8.0, a second addition of FSG did not induced further elevation of the [Ca²⁺]_i or pH_i of spermatozoa treated with FSG, but addition or FSG after SAP-I or of SAP-I after FSG induced further increases of [Ca²⁺]_i and pH_i, At pH 6.6, FSG and/or SAP-I did not induce significant elevation of the [Ca²⁺]_i, although SAP-I elevated the pH_i, its half-maximal effective concentration being 10 to 100 pM. At pH 8.0, tetraethyl-ammonium, a voltage-sensitive K⁺-channel blocker, inhibited induction of the acrosome reaction and elevations of [Ca²⁺]_i and pH_i by FSG, but did not affect those by SAP-I. These results suggest that FSG and SAP-I activate different Ca²⁺ and H⁺ transport systems.     

Mechanism Underlying the ATP-Induced Increase in the Cytosolic Ca2+ Concentration in Chick Ciliary Ganglion Neurons

Abstract: We examined the mechanism underlying the ATP-induced increase in the cytosolic Ca²⁺ concentration ([Ca]_in) in acutely isolated chick ciliary ganglion neurons, using fura-2 microfluorometry. The ATP-induced increase in [Ca]_in was dependent on external Ca²⁺, was blocked in a dose-dependent manner by reactive blue 2, and was substantially inhibited by both L- and N-type Ca²⁺ channel blockers. ATP was effective in increasing [Ca]_in in the presence of a desensitizing concentration of nicotine (100 µ M ), and simultaneous addition of maximal doses of ATP and nicotine caused an additive increase in [Ca]_in, suggesting that ATP acts on a site distinct from nicotinic acetylcholine receptors. ATP also increased the cytosolic Na⁺ concentration as determined by sodium-binding benzofuran isophthalate microfluorometry. These results suggest that ATP increases Na⁺ influx through P₂ purinoceptor-associated channels resulting in membrane depolarization, which in turn increases Ca²⁺ influx through voltage-dependent Ca²⁺ channels. However, ATP still caused a small increase in [Ca]_in under Na⁺-free conditions, and this [Ca]_in increase was little affected by Ca²⁺ channel blockers. ATP also increased Mn²⁺ influx under Na⁺-free conditions, as indicated by quenching of fura-2 fluorescence. These results suggest that nonselective cationic channels activated by ATP are permeable not only to Ca²⁺ but also to Mn²⁺, in addition to monovalent cations.     

Intracellular pH Changes of Starfish Sperm Upon the Acrosome Reaction

The acrosome reaction is accompanied by ionic changes such as increases in intracellular Ca²⁺ and intracellular pH (pH_i). Since the two jelly components essential for inducing the acrosome reaction, ARIS and Co-ARIS, were shown to activate Ca-channels (accompanying paper), we examined the jelly components to determine which was responsible for the pH_i-increase using 9-aminoacridine as a probe of pH_i. This paper presents evidence that an oligopeptide(s) is responsible for the pH_i-increase. The pH_i of swimming sperm is 7.4-7.5. Within 20 sec after the addition of jelly, their pH_i increased rapidly by 0.06 pH unit, then decreased by 0.2–0.3 pH unit, and reached a plateau level within 3 min. Similar changes in pH_i were observed on addition of a Pronase digest of ARIS (P-ARIS) and a diffusible fraction of jelly (Fraction M₈) together. Fraction M₈, but not ARIS or Co-ARIS increased the pH_i, and activated sperm respiration in sea water at pH 6.5. The two activities of Fraction M₈ depended upon Na⁺ but not Ca²⁺, and were susceptible to Pronase digestion. Fraction M₈ is also known to enhance induction of the acrosome reaction by the Ca-ionophore A23187. These results suggest that the egg jelly contains a peptide(s) that is not obligatory for the acrosome reaction but facilitates the reaction by increasing the pH_i of the sperm. The significance of the pH_i-increase upon the acrosome reaction is discussed.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Induction of the Acrosome Reaction of Hemicentrotus pulcherrimus Spermatozoa by the Egg Jelly Molecules, Fucose-Rich Glycoconjugate and Spem-Activating Peptide I

A fucose-rich glycoconjugate (FRG) was isolated from egg jelly of the sea urchin Hemicentrotus pulcherrimus by gel filtration. FRG induced the acrosome reaction in H. pulcherrimus spermatozoa in a concentration-dependent manner, although it showed about half the activity of the original unfractionated jelly. Synthetic sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) increased the rate of the acrosome reaction induced by FRG; the maximal rate of the acrosome reaction with FRG and SAP-I being that of the unfractionated jelly. The half-maximal increase in induction of the acrosome reaction by SAP-I with FRG occurred at 4 × 10⁻¹⁰ M SAP-I, which was almost the same concentration inducing half-maximal stimulation of sperm respiration. Pronase digestion of FRG resulted in an 50% decrease in induction of the acrosome reaction and also in the elevation of cAMP in sperm. Some reagents (monensin and 3-isobutyl-1-methylxanthine) which increase intracellular pH, Ca²⁺ and cyclic nucleotides also increased the rates of the acrosome reaction induced by FRG or pronase-digested FRG. However, the rates did not reach those with FRG or pronase-digested FRG with SAP-I. These results indicate that SAP-I promotes induction of the acrosome reaction by acting as a specific co-factor of FRG.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Ontogenesis of Ca++ and Mg++ Contents of Embryonic Chick Heart

The Ca⁺⁺ and Mg⁺⁺ contents of embryonic chick heart were studied by atomic absorption spectrophotometry during a period from 48 h of foetal development until 2-3 days post-hatching. The hearts were isolated and incubated for 40 min at 22°C in three different media aerated with 95% 0₂-5% C0₂. The media included: normal Ringer's; Ca⁺-free Ringer's with 3 mM EGTA; and Ca⁺⁺-free Ringer's with 3 mM EDTA. At 48 h, the tubular myocardium contained 7-3 mM Ca⁺⁺ per wet weight which decreased rapidly to 1-2 mM by 10 days of development and remained between 0-9 and 1-1 mM until hatching. The Ca⁺⁺ content paralleled the changes in Na⁺ content reported earlier. Treatment with excess chelators, EGTA or EDTA, resulted in removal of 65-75% of the Ca⁺⁺ content throughout development until the time of hatching, when 50% of the Ca⁺⁺ became firmly bound. In contrast to the results with Ca⁺⁺, myocardial Mg⁺⁺ content rose rapidly from an initial value of 3.2 mM at 48 h to 6.7 mM by the 5th day of development, and then gradually declined throughout the remaining foetal development to 4.8 mM 2-3 days post-hatching. The Mg⁺⁺ contents closely paralleled changes in K⁺ content during development, which were reported earlier. Treatment with EGTA and EDTA removed 13-22% and 19-28% of the myocardial Mg⁺⁺, respectively, during development until just prior to hatching, when only 10-12% could be removed by chelation.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Acrosome Reaction-Inducing Substance Purified from the Egg Jelly Inhibits the Jelly-Induced Acrosome Reaction in Starfish: An Apparent Contradiction

Previous studies indicated that two components of the egg jelly are required for induction of the acrosome reaction in starfish: a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) called Co-ARIS. In the present study the sites of action of ARIS and Co-ARIS and their temporal relationships were examined. When sperm had been treated for a few minutes with ARIS, or a crude preparation of Co-ARIS (Fraction M₈), or inadequate amounts of jelly, or sufficient jelly in low Ca²⁺ sea water, they did not undergo the acrosome reaction when the deficiencies were corrected. Moreover, they became nonresponsive to the jelly. Pronase digest of ARIS (P-ARIS) but not of Fraction M₈ retained this capacity. A steroidal saponin purified as Co-ARIS did not have this capacity. This suggests the presence of a third jelly component, probably an oligopeptide(s), participating in induction of the acrosome reaction. Activation of Ca²⁺ -uptake seems to be at least one, if not the only, action site of ARIS and Co-ARIS, because ARIS, P-ARIS, and Fraction M₈ inhibited jelly-induced Ca²⁺ -uptake by sperm, and because the calcium ionophore A23187 by-passed the blockage by these components of the jelly-induced acrosome reaction.     

The Role of External Potassium Ion in Activation of Sea Urchin Spermatozoa

When sperm of the sea urchin, Hemicentrotus pulcherrimus, are diluted into K⁺-free seawater, the pH of the suspension gradually decreases, whereas a rapid decline in pH is observed following dilution into regular seawater. Sperm motility and respiration are also activated after dilution into K⁺-free seawater, but levels of activity are less than those observed following dilution into regular seawater. Upon addition of 10 mM K⁺ to K⁺-free seawater, rapid acid release occurs and motility and respiratory rate in sperm are reactivated. The effect of K⁺ on respiration was competitive with respect to the external Na⁺ concentrations. Harmaline, a potent inhibitor of Na⁺/K⁺-ATPase, causes a decrease in movement and respiration of the sperm. Harmaline does not inhibit the rapid decline in pH, although it depresses the release of acid from mitochondria. These results suggest that external K⁺ plays an important role in intracellular alkalinization of sea urchin sperm.     

AMPA Receptor-Mediated Excitotoxicity in Human NT2-N Neurons Results from Loss of Intracellular Ca2+ Homeostasis Following Marked Elevation of Intracellular Na+

Abstract: Human NT2-N neurons express Ca²⁺-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors (AMPA-GluRs) and become vulnerable to excitotoxicity when AMPA-GluR desensitization is blocked with cyclothiazide. Although the initial increase in intracellular Ca²⁺ levels ([Ca²⁺]_i) was 1.9-fold greater in the presence than in the absence of cyclothiazide, Ca²⁺ entry via AMPA-GluRs in an early phase of the exposure was not necessary to elicit excitotoxicity in these neurons. Rather, subsequent necrosis was caused by a >40-fold rise in [Na⁺]_i, which induced a delayed [Ca²⁺]_i rise. Transfer of the neurons to a 5 m M Na⁺ medium after AMPA-GluR activation accelerated the delayed [Ca²⁺]_i rise and intensified excitotoxicity. Low-Na⁺ medium-enhanced excitotoxicity was partially blocked by amiloride or dizocilpine (MK-801), and completely blocked by removal of extracellular Ca²⁺, suggesting that Ca²⁺ entry by reverse operation of Na⁺/Ca²⁺ exchangers and via NMDA glutamate receptors was responsible for the neuronal death after excessive Na⁺ loading. Our results serve to emphasize the central role of neuronal Na⁺ loading in AMPA-GluR-mediated excitotoxicity in human neurons.     

Changes in the Activities of H+, K+-ATPase and Na+, K+-ATPase in Cultured Cells Derived from Micromeres of Sea Urchin Embryos with Special Reference to Their Roles in Spicule Rod Formation

In cultured cells derived from micromeres isolated at the 16-cell stage of sea urchin embryos, the activity of H⁺, K⁺-ATPase became detectable after 15 hr of culture, when the cells started to form spicules, and then increased reaching a plateau from 25 hr of culture. The Na⁺, K⁺-ATPase activity of isolated micromeres increased to a maximum at 20 hr of culture and thereafter decreased gradually. Allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, caused a decrease in intracellular pH (pHi) accompanied by blockage of ⁴⁵Ca deposition in spicule rods in spicule-forming cells at 30 hr of culture. Ouabain and amiloride had scarcely any effect on the pHi or ⁴⁵, deposition. In cultured cells exposed to nifedipine, which blocked ⁴⁵Ca deposition in spicule rods, allylisothiocyanate did not cause any decrease in pHi. These results show that H⁺, which is generated in the overall reaction to produce CaCO₃ from Ca²⁺ and HCO₃⁻, is probably released from the cells mainly in the reaction catalyzed by H⁺, K⁺-ATPase to maintain successive production of CaCO₃.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.