Raymond T. Kado

Development of calcium release mechanisms during starfish oocyte maturation

In response to the maturation-inducing hormone 1-methyladenine, starfish oocytes acquire increased sensitivity to sperm and inositol trisphosphate (InsP3), stimuli that cause a release of calcium from intracellular stores and a rise in intracellular free calcium. In the immature oocyte, the calcium release in response to 10 sperm entries is less than that seen with a single sperm entry in the mature egg. Likewise, the sensitivity to injected InsP3 is less in the immature oocyte. Approximately 100 times as much InsP3 is required to obtain the same calcium release in an immature oocyte as in a mature egg. However, with saturating amounts of InsP3, immature oocytes and mature eggs release comparable amounts of calcium. These results indicate that although calcium stores are well-developed in the immature oocyte, mechanisms for releasing the calcium develop fully only during oocyte maturation.

The time course of cortical vesicle fusion in sea urchin eggs observed as membrane capacitance changes

Abstract The addition of cortical vesicle membrane to the plasma membrane during the first 2 min after insemination in Strongylocentrotus purpuratus eggs was monitored continuously by measuring the resulting capacitance increase with AC current (400 Hz), using intracellular electrodes. The capacitance increase was first detected at 30 to 45 sec after the rise of the activation potential, thus allowing a more precise determination of the “latent period.” The time interval from a 10 to 50% change in capacitance was always less than that from the 50 to 90% level, suggesting that the traveling wave of cortical vesicle fusion slows down as it propagates.

Ion channels and ATP-driven pumps involved in ion transport across the tonoplast of sugarbeet vacuoles

Abstract The electrical properties of the tonoplast of mature sugarbeet root vacuoles have been studied using the patch-clamp technique. In whole-vacuole recordings, the addition of 5 mM Mg-ATP to the external solution activated a proton-translocating ATPase which produced inward currents of up to 65 pA. Furthermore, we identified a voltage-dependent membrane conductance which activated at hyperpolarized (inside-negative) potentials and decreased at positive potentials. Outside-out membrane patches predominantly contained a channel which showed an increasing probability of opening at potentials more negative than about –20 mV. These channels can account for the macroscopic currents recorded in whole vacuoles. The permeability sequence of the channel for cations and anions was: PKstaggered+ = PNa+ >PAc− >PNO3− >PMal2− >PCl−. The unit conductance of this channel was about 70 pS in symmetrical 50 mM KCl and 180 pS in symmetrical 200 mM KCl solutions. Another channel type of smaller conductance (15 pS in 50 mM KCl) was also present, but its properties have not yet been studied. The permeability sequence of the nonselective channel corresponds to that found by tracer measurements in vacuole suspensions, implying that the channel studied may present the molecular pathway for the movement of ions across the tonoplast.

Aplysia Giant Cell: Soma-Axon Voltage Clamp Current Differences

Under voltage clamp, local membrane currents have been measured in several regions of the soma. The early inward current appears to pass largely through the membrane of the axon and of the soma near the axon in normal media. After 10-5 molar tetrodotoxin (TTX) is added to the bathing medium the larger inward current is found in the somatic membrane away from the axon. The late currents are larger at the soma in both normal and TTX media.

Propagating potassium and chloride conductances during activation and fertilization of the egg of the frog, Rana pipiens.

Fertilization or artificial activation of the frog egg (Rana pipiens) elicits a positive‐going shift in membrane potential which results from an opening of Cl‐ and K+ channels in the egg membrane. We examined the spatial localization of the currents produced by the opening of these channels, using large patch electrodes (tip diameters 3‐10 microns). We also used small patch electrodes (tip diameters about 1 micron) to study the single K+ channel currents. After activation, with the patch electrode at any position on the egg surface, we observed a transient current, with a main peak lasting several seconds. This activation current occurred after a variable delay of 0‐3 min following the rise of the activation potential. With 10% Ringer solution in the bath and pipette, the current was usually outward, although it sometimes had an inward component. With one patch electrode on the animal surface of the egg and another patch electrode on the vegetal surface, we observed that the activation current propagated over the egg surface. In experiments where the egg was activated by applying a hyperpolarizing pulse, the response in the animal half preceded that in the vegetal half by an average of about 1 min. The amplitude of the peak outward current was similar for animal and vegetal recordings (1‐2 mA/cm2). Tetraethylammonium (11 mM) in the patch pipette blocked most of the outward component of the activation current and revealed an underlying inward component. The inward component of the activation current was carried by Cl‐, since it could be reversed by raising the Cl‐ concentration in the pipette. The Cl‐ component of the activation current propagated over the egg surface, with timing similar to that of the total current. The average amplitude of the peak Cl‐ current was six or more times larger at the animal than the vegetal surface. Fertilization caused a current to propagate from the animal to the vegetal surface, like the current observed during activation. With a small patch electrode, single channel currents of the K+ component of the activation current could be seen. The probability that the channels were open increased at more positive potentials. The single channel conductance was estimated to be 25 pS, and the reversal potential to be ‐150 mV. Single Cl‐ channel currents have not yet been seen. Activation or fertilization of the frog egg resulted in a wave‐like opening of Cl‐ and K+ channels, which spread from the animal to the vegetal half of the egg.(ABSTRACT TRUNCATED AT 400 WORDS)

Divalent cations in cerebral impedance and cell membrane morphology.

Abstract Electrical impedance of cerebral cortex was measured at 1.0 kHz with low current levels (10 −13 A per square micron of electrode surface). Changes in impedance were observed after topical application of calcium and magnesium solutions. These modifications were further evaluated by asphyxiation, and by dehydration with intravenous urea. Asphyxiation of untreated cortex caused an initial fall in resistance and raised capacitance rapidly followed by raised resistance and reduced capacitance. Repeated topical application of small amounts of calcium solutions induced a progressive rise in resistance, typically between 20 and 30% of baseline values 10–20 KΩ. This increased impedance reached a plateau after 30–50 min, and further application of calcium was without effect. By contrast with untreated cortex, asphyxiation was followed by a rapid fall in resistance to values around baseline levels. This impedance curve was thus biphasic, with an inverse relationship between the size of the calcium-induced rise and the ensuing fall during asphyxiation. On the other hand, magnesium ions in equivalent amounts exerted only minor effects on cortical impedance, and subsequent asphyxiation produced a large impedance increase, as in untreated cortex. However, magnesium ions produced effects similar to calcium in normal and asphyxiated cortex, if the cortex was first dehydrated with intravenous urea, and even though urea alone was without major effects on impedance. Prior dehydration with urea had no effect on the course of impedance changes produced by calcium. These findings are interpreted in terms of conductance of impedance measuring currents mainly through an extracellular space containing a hydrated network of macromolecules that blend with similar external coats on cell membrane, and which bear numerous cationic binding sites. Evidence is presented in electron micrographs prepared with aldehyde fixation that excess calcium ions narrow intercellular spaces and that these changes reverse with asphyxiation. No such narrowing was induced by urea. On the other hand, magnesium ions were associated with much augmented intercellular spaces. It is proposed that conductance in the extracellular space is determined by the state of its structural elements, including interdependent factors such as hydration and divalent cation binding, as well as by changes in its cross-sectional area. The preeminent role of calcium ions in altering tissue impedance is discussed in relation to membrane excitation, with competitive binding of calcium and hydrogen ions to anionic macromolecular sites. Evidence relating these results to information storage at the neuronal surface is discussed.

Hyperpolarizing current of the Na/K ATPase contributes to the membrane polarization of the Purkinje cell in rat cerebellum

Abstract The contribution of the Na/K ATPase (pump) current to the polarization of the Purkinje cell has been studied using slices of the rat cerebellum by blocking the pump with dihydro-ouabain (DHO) while recording the membrane potential with microelectrodes in the somata. From our recordings, it appeared that blocking the pump depolarized the Purkinje cells more rapidly than might be expected from shifts in Na+ and K+ concentrations, suggesting the removal of a hyperpolarizing current. Application of DHO, in the presence of tetrodotoxin (TTX), led to calcium spike firing and plateau-like discharges suggesting activation of voltage-dependent calcium channels in the dendrites. Adding 2 mM Co2+ to the medium did not prevent the depolarizations. Removing calcium from the bathing medium containing 2 mM Co2+ blocked the spiking activity but DHO application still produced a depolarization. Experiments to measure the current inhibited by DHO indicated that the Na/K pump supplies a constant current of 240 pA. Substitution of the sodium with choline produced a hyperpolarization, during which DHO had no effect on the membrane potential. Substitution of the sodium with lithium produced only a slowly developing depolarization. It is concluded that in the cerebellar Purkinje cell, a continuous sodium ion influx activates the pumps which produce a current that directly contributes to the membrane polarization. Possible pathways for this sodium influx are discussed.

The Design and Use of an FM / AM Radiotelemetry System for Multichannel Recording of Biological Data

A multichannel telemetry system for EEG recording has been constructed for study of animal or human behavior correlates under natural, unrestrained conditions. To be useful in research of this kind, the transmitted radio signal must be independent of the environment, so that changes in antenna loading and in signal level cause no artifact. Standard IRIG proportional-bandwidth FM subcarrier channels are used. These subcarriers are generated by twin-T oscillators, modulated in turn by the amplified data signals. The FM subcarriers are then linearly summed and impressed upon a crystal-controlled AM transmitter. The system is also relatively insensitive to major shifts in supply voltage. Wherever possible, fabrication was by means of integrated circuits, thus reducing the bulk of the modules.

G-proteins and egg activation.

G-proteins are present in eggs, and experiments in which GTP-gamma-S, GDP-beta-S, cholera toxin and pertussis toxin have been injected into eggs have indicated the involvement of G-proteins in egg activation at fertilization and in oocyte maturation. Eggs into which serotonin or muscarinic acetylcholine receptors have been introduced by mRNA injection produce fertilization-like responses when exposed to serotonin or acetylcholine; since these neurotransmitter receptors act by way of G-proteins, this observation further supports the conclusion that a G-protein is involved in the fertilization process.

Receptors, G-Proteins, and Activation of the Amphibian Egg

We have investigated the role of guanine nucleotide-binding proteins (G-proteins) in activation of the egg of Xenopus laevis by injection of activators or inhibitors of G-proteins and by assaying for pertussis toxin or cholera toxin substrates. To examine the possibility that a receptor might be involved in the activation of a G-protein in the egg membrane, we introduced exogenous G-protein-related neurotransmitter receptors into the egg membrane, added the corresponding agonists, and looked for activation events. This paper reviews some of our published and unpublished experiments to test the hypothesis that activation of the amphibian egg is mediated by receptor activation of a G-protein in the egg membrane.