Behavior of Na+/K+-ATPase α-subunit alleles in sea urchin eggs upon fertilization

The allelic division of the Na⁺/K⁺-ATPase α-subunit gene was found in eggs of the sea urchin Hemicentrotus pulcherrimus by polymerase chain reaction (PCR). Two PCR products of different lengths were detected from a genome in one embryo derived from a fertilized egg, although only one product in one embryo derived from an artificially activated egg by parthenogenesis was detected, indicating one copy of the gene in a haploid genome. One of the two PCR products from each fertilized egg was identical in size to the product from an artificially activated egg in the same batch. The other PCR product was the same in length as one of the products from the sperm with which the eggs were fertilized. These results indicate that recombination of the heteromeric alleles of the Na⁺/K⁺-ATPase α-subunit gene occurs in the sea urchin egg due to meiosis and fertilization. The sequencing of these products demonstrated that their exon sequences were identical and that the intron, inserted in the PCR products, generated polymorphism in length due to the frequency of the repeating 53 bp sequence and insertion/deletion of other two segments.     

Effect of Cytochalasin B on the Development of Membrane Transports in Sea Urchin Eggs after Fertilization

Investigations were made on the role of the cytoskeleton in the onset of ionic events following fertilization of sea urchin eggs. Events which depend upon phosphoinositide metabolism, such as the cortical reantion and acid release are affected by cytochalasin B (CB) after fertilization but not after activation of eggs with the ionophore A23187. These findings suggest that the sequence of events following sperm-egg attachment depends on the cytoskeleton. CB also inhibits the Na⁺ pump and alanine uptake when added before insemination and during the following 30 min. These results argue for a role of the egg cortex cytoskeleton in activation of the Na⁺ pump by fertilization. We propose that the inhibitory effect of CB on the development of amino-acid uptake after fertilization may result from an increase in the Na⁺ content of the egg resulting from Na⁺ pump suppression rather than from direct blockage of the carrier.     

Ca2+-induced change of dorsal-ventral polarity in frog (Rana temporaria) eggs

Abstract. The effect of local Ca²⁺ administration 10–20 min after fertilization and during artificial activation was examined in Rana temporaria eggs. Ca²⁺ was injected into the pigmented region near the boundary between pigmented and unpigmented domains. The locations of egg gray crescent (GC) and dorsal lip of blastopore (DLB), as predictors of the dorsal region in embryos, as well as the measurements of angles between GC middle and sperm entry site were observed. In more than 70% of the cases, microinjection of Ca^{2 +} into subcortical cytoplasm and egg pricking in high-Ca^{2 +} solutions induced GC and DLB formation near the injection site. The formation of Ca²⁺-induced GC occurred mostly as in control eggs. In addition premature displacement of the egg surface was observed near the prick site in high-Ca^{2 +} solutions. GC formation occurred by displacement of the pigmented surface in the same direction as earlier wound translocations. These results show that Ca²⁺ injection determines the direction of the surface movement.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.     

Acid Release during Activation of Barnea candida (Mollusca, Pelecypoda) Oocytes

Barnea caridida oocytes release acid (1.35 pmole H⁺/oocyte) upon fertilization. After artificial activation by an excess of KCl, germinal vesicle breakdown (GVBD) occurs normally and a quite similar, but not identical, acid release is recorded (1.10 pmole H⁺/oocyte). KCl activation of Barnea oocytes is completely inhibited in 100 mM sodium-acetate sea water at pH 6.5 and fertilization does not result in activation when the oocytes are transferred after one minute into 100 mM sodium-acetate sea water at pH 6.3. When D–600, a calcium transmembrane fluxes inhibitor, is added 20 seconds after fertilization, GVBD is inhibited but a normal acid release is recorded. The presence of at least 10 mM sodium ions in the external medium is required for 100% activation of these oocytes by an excess of KCl. These results suggest that while an intracellular pH increase may be a requisite for GVBD, this can not be a sufficient condition to trigger it unless a calcium influx is allowed to occur. Moreover, the acid release does not result from a Ca⁺⁺-H⁺ exchange transport but appears more likely to be due to a Na⁺-H^* exchange as it has been demontrated in sea urchin eggs.     

The Na+ and K+ Content of Isolated Torpedo Synaptosomes and Its Effect on Choline Uptake

Abstract: The Na⁺ and K⁺ concentrations in isolated Torpedo marmorata synaptosomes were determined. Synaptosomes made according to the method of Israël et al. have high internal Na⁺ (290 MM) and low internal K⁺ (30 mM) concentrations. Modification of the homogenisation media permitted the isolation of synaptosomes which could maintain transmembrane ion gradients (internal Na⁺, 96 mM; K⁺, 81 mM); 0.1 mM-ouabain abolished these gradients. The trans-membrane Na⁺ gradient started to dissipate after 15 min at 20°C. Inclusion of ATP in the homogenisation medium enabled the synaptosomes to maintain the Na⁺ gradient for about 90 min. The presence of these transmembrane ion gradients stimulated choline uptake sevenfold. It is concluded that (a) by selecting the isolation media, Torpedo synaptosomes can be prepared with transmembrane ion gradients; (b) these gradients are ouabain-sensitive and stimulate choline uptake: (c) the synaptosomes require additional ATP to maintain the ion gradients.     

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

Regulation of haemolymph sodium and potassium during dehydration in the cricket Teleogryllus oceanicus

ABSTRACT. Male crickets ( Teleogryllus oceanicus ) when dehydrated for 3 days lost 51% of their body water, and 65% of their haemolymph volume. Haemolymph osmolality rose from 391 to 572mOs/kg; [Na⁺] from 149 to 289 HIM; and [K⁺] from 13.0 to 26.3 mM. During dehydration 385 μig Na (expressed as NaCl) and 41 μug K (expressed as KCI) were removed from the haemolymph. Rehydration of the dehydrated insects failed to restore the Na⁺ and K⁺ concentrations to near their original levels. Approximately 62% of the missing Na⁺ was excreted, whilst five times the amount of K⁺ removed from the haemolymph was excreted. It is presumed that the excess represents K⁺ removed from intracellular fluid.     

Fertilization Reaction without Changes in Intracellular Ca2+ in Medaka Eggs—An Experiment with Acetone-Treated Eggs

To investigate whether or not causal relationship exists between the increase in intracellular Ca²⁺ and other cortical reactions at fertilization in the medaka, Oryzias latipes , intracellular Ca²⁺ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca²⁺. Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone-treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca²⁺ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca²⁺ and exocytosis.     

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca²⁺ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca²⁺ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca²⁺ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca²⁺ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca²⁺ into the cortex also induced a Ca²⁺ wave. When the egg was stimulated by microinjection of Ca²⁺ at the equatorial region, the Ca²⁺ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca²⁺ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca²⁺ wave preceded the wave of cortical change.
A Ca²⁺ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.     

Effects of Sperm-Activating Peptide I on Hemicentrotus pulcherrimus Spermatozoa in High Potassium Sea Water

The effects of sperm-activating peptide I (SAP-I: Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) on Hemicentrotus pulcherrimus spermatozoa in high [K⁺] sea water were examined. In high [K⁺] sea water, the respiration rates and motility of H. pulcherrimus spermatozoa were lower than those in normal sea water. SAP-I did not stimulate the lowered respiration rate or motility, although the peptide bound to the spermatozoa as it does in normal sea water. SAP-I elevated the sperm cGMP level in 100 mM K⁺ sea water (from 0.37 to 4.81 pmol/mg wet weight spermatozoa) more than those in normal sea water (from 0.21 to 0.93 pmol/mg wet weight). A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and SAP-I synergistically elevated the cGMP level from 0.35 to 33.08 pmol/mg wet weight in 100 mM K⁺ sea water. However, in high [K⁺] sea water, SAP-I did not increase the cAMP level even in the presence of IBMX. SAP-I caused rapid, transient elevation of the intracellular pH and Ca²⁺ concentration of spermatozoa in normal sea water but not in 100mM K⁺ sea water. SAP-I did not decrease the apparent molecular weight of sperm guanylate cyclase from 131,000 to 128,000 in high [K⁺] sea water. These results suggest that the SAP-I-induced elevation of the cGMP level in sea urchin spermatozoa occurs before or independently of membrane hyperpolarization induced by the opening of K⁺ channels.     

Paradoxical effect of ethanol on potassium channel currents and cell survival in cerebellar granule neurons

Transient exposure to ethanol (EtOH) results in a massive neurodegeneration in the developing brain leading to behavioral and cognitive deficits observed in fetal alcohol syndrome. There is now compelling evidence that K⁺ channels play an important role in the control of programmed cell death. The aim of the present work was to investigate the involvement of K⁺ channels in the EtOH-induced cerebellar granule cell death and/or survival. At low and high concentrations, EtOH evoked membrane depolarization and hyperpolarization, respectively. Bath perfusion of EtOH (10 mM) depressed the I _A (transient K⁺ current) potassium current whereas EtOH (400 mM) provoked a marked potentiation of the specific I _K (delayed rectifier K⁺ current) current. Pipette dialysis with GTPγS or GDPβS did not modify the effects of EtOH (400 mM) on both membrane potential and I _K current. In contrast, the reversible depolarization and slowly recovering inhibition of I _A induced by EtOH (10 mM) became irreversible in the presence of GTPγS. EtOH (400 mM) induced prodeath responses whereas EtOH (10 mM) and K⁺ channel blockers promoted cell survival. Altogether, these results indicate that in cerebellar granule cells, EtOH mediates a dual effect on K⁺ currents partly involved in the control of granule cell death.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

Long distance transport of potassium in cereals during grain filling in intact plants

Translocation of labeled potassium (K⁺) from the root to the ear and its distribution within the culm during 4, 8 and 12 h of uptake was studied in intact wheat plants ( Triticum aestivum L. cv. Kolibri) 3 and 5 weeks after anthesis at 0.5 and 5.0 m M K⁺ concentration in the uptake solution. Uptake of labeled K⁺ into the shoot was proportional to the K⁺ concentration applied. After 4 h of uptake about 2% and after 12 h about 7% of labeled K⁺ applied to the roots were taken up into the shoot at both K⁺ concentrations. After 12 h of uptake only 6% of the total label in the culm had reached the ear, while about 40% of the label was found in the upper three internodes. In spite of an increasing concentration of labeled K⁺ during 12 h in the uppermost internode (peduncle), translocation of K⁺ into the rachis was low. The low and uniform K⁺ content found generally in grain dry weight seems therefore to be due to a controlled K⁺ supply to the ear.     

Roles for Potassium and Calcium Channels in the Initiation of Sperm Motility in Rainbow Trout.

It is well known that the motility of spermatozoa in rainbow trout is suppressed by K⁺. We showed here that although trout sperm are completely immotile in medium containing 5 mM K⁺, motility was initiated by the subsequent addition of several mM Ca²⁺, suggesting that both K⁺and Ca²⁺are related to the process of the initiation of sperm motility. It was further found that K⁺channel blockers tetraethylammonium, nonyltriethylammonium, Ba²⁺and Cs⁺, as well as the Ca²⁺channel blocker verapamil, inhibited the initiation of sperm motility at doses at which these reagents inhibit chnnel-related functions in other cells. However, Na⁺channel blocker, tetrodotoxin and anion channel blocker 4, 4-diisothiocyatatostilbene-2, 2'-disulfonic acid inhibited the motility only at extremely high doses. These results suggest that transport of K⁺and Ca²⁺through ion channels at the plasma membrane of spermatozoa is the first event that triggers the initiation of sperm motility in rainbow trout.     

Passive uptake of K+(86Rb+) in sunflower roots at low external K+ concentrations

Passive fluxes of K⁺ (⁸⁶Rb) into roots of sunflower ( Helianthus annuus L. cv. Uniflorus) were determined at low K⁺ concentration (0.1 and 1.0 mM K⁺) in the ambient solution. Metabolic uptake of K⁺ was inhibited by 10⁻⁴M 2,4-dinitrophenol (DNP). K⁺ (⁸⁶Rb) fluxes were studied both continuously and by time differentiation of uptake. In high K⁺ roots passive uptake was directly proportional to the K⁺ concentration of the uptake solution, indicating free diffusion. This assumption was supported by the fact that passive Rb⁺ uptake was not affected by high K⁺ concentrations. In low K⁺ roots the passive uptake of K⁺ was higher than in high K⁺ roots. The increase was possibly due to carrier-mediated K⁺ transport. As K⁺ effluxes were quantitatively similar to influxes, it is suggested that passive K⁺ fluxes represent exchange diffusion without relation to net K⁺ transport.     

MAGNESIUM REQUIREMENT IN FERTILIZATION PROCESS OF SEA URCHINS

The Mg²⁺ requirement in fertilization was investigated in sea urchins. It was found that when sea urchin eggs were inseminated in sea water free of Mg²⁺, little fertilization took place. Even when spermatozoa pre-treated with dissolved egg-jelly to induce the acrosome reaction, which needs Ca²⁺, were used, the fertilization rate remained quite low in the absence of Mg²⁺. In Strongylo-centrotus intermedius , the lowest concentration of Mg²⁺ required for 50% fertilization was 0.05 mM in the presence of 10 mM Ca²⁺, whereas that of calcium was 3 mM in the presence of 49 mM Mg²⁺. These critical concentrations increased when the concentration of the other ion decreased. Removal of Mg²⁺ or Ca²⁺ or both from the suspending medium had little adverse effect on sperm motility. The elevation of the fertilization membrane was also induced by butyric acid independent of the presence or absence of Mg²⁺ and/or Ca²⁺. These results indicate that Mg²⁺ are required at least in some process(es) between acrosome reaction and fertilization membrane elevation, such as sperm penetration or membrane fusion.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca²⁺-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN^— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN^— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN^— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN^— for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN^— on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN^— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN^— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca²⁺.     

Few cultured rat primary sensory neurons express a tolbutamide-sensitive K+ current

The response of dorsal root ganglion (DRG) neurons to metabolic inhibition is known to involve calcium-activated K⁺ channels; in most neuronal types ATP-sensitive K⁺ channels (K_ATP) also contribute, but this is not yet established in the DRG. We have investigated the presence of a K_ATP current using whole-cell recordings from rat DRG neurons, classifying the neurons functionally by their "current signature" (Petruska et al, J Neurophysiol 84: 2365–2379, 2000). We clearly identified a K_ATP current in only 1 out of 62 neurons, probably a nociceptor. The current was activated by cyanide (2 mM NaCN) and was sensitive to 100 μM tolbutamide; the relation between reversal potential and external K⁺ concentration indicated it was a K⁺ current. In a further two neurons, cyanide activated a K⁺ current that was only partially blocked by tolbutamide, which may also be an atypical K_ATP current. We conclude that K_ATP channels are expressed in normal DRG, but in very few neurons and only in nociceptors.