Biomedical subjects

I Spector

Publications and source records attributed to I Spector.

At least 55 records · Page 3Linked to original sources

PubMed1983

Development of sodium channels during differentiation of chick skeletal muscle in culture. I. Binding studies.

[125I]Scorpion toxin and [3H]saxitoxin, two neurotoxins that bind specifically to different sites on sodium channels, were used to monitor the development of sodium channels during synchronous differentiation of chick skeletal muscle in culture. [3H]Saxitoxin-binding sites are not detectable in myoblasts. They appear only after myoblasts fuse to form myotubes and increase gradually to reach a maximal value of 58 fmol/mg of protein by 8 days in culture. In contrast, [125I]scorpion toxin-binding sites are first detected during the period of cell fusion and increase after fusion to reach a maximum by 4 days in culture, well before either the maximal value of the [3H]saxitoxin binding or the appearance of fully developed sodium action potentials. Neither [3H]saxitoxin binding nor [125I] scorpion toxin binding affinity changes during development; they have values of 1.4 and 62 nM, respectively. These data, and data described in the companion paper (Baumgold, J., J. B. Parent, and I. Spector (1983) J. Neurosci. 3: 1004-1013), suggest that sodium channel development involves two sequential steps: incorporation into the cell membrane of an immature form of the sodium channel capable of binding scorpion toxin alone, followed by the acquisition of the ability to bind saxitoxin and thus to become a mature and functional channel.

Amphibian Proteins↗

PubMed1981

Immobilization of concanavalin A receptors during differentiation of neuroblastoma cells.

Neuroblastoma cells serve as a useful model of neuronal development because compounds such as dimethyl sulphoxide (DMSO) and dibutyryl cyclic AMP cause them to undergo a process of controlled differentiation in tissue culture, during which they can extend long processes, develop characteristic excitability mechanisms, synthesize neurotransmitters and form synapses. We have used the technique of fluorescence photobleaching recovery to study the lateral mobility of cell-surface constituents during the differentiation of neuroblastoma clone N1E-115 cells. The concanavalin A (Con A) binding sites appear as discrete patches distributed over the entire cell surface and exhibit lateral mobility in undifferentiated cells comparable with that of surface glycoproteins of other cells. After induction of differentiation, however, the vast majority of Con A binding sites become immobilized, and we present data which suggest that the mechanism of this immobilization may involve linkage to the internal actin network.

Actins↗

PubMed1981

Potassium current suppression by quinidine reveals additional calcium currents in neuroblastoma cells.

Quinine and quinidine have been evaluated with regard to their effects on the electrical activity of neuroblastoma cells. Under voltage-clamp conditions, we have found that quinine and quinidine block both the voltage-dependent and Ca2+-dependent K+ conductances. Blockage of the voltage-dependent K+ channel is manifest as an increase in the amplitude and in the duration of the action potential. Blockage of the Ca2+-dependent K+ channel in Na+-free (replaced by Tris) solutions containing 6.8 mM Ca2+ and tetraethylammonium ion or 4-aminopyridine (to block the voltage-dependent K+ current) is seen as a further prolongation of the Ca2+ action potential and diminution of the after-hyperpolarization. A critical role of the Ca2+-dependent K+ conductance in modulation of the rate and duration of trains of Ca2+ action potentials is shown by the use of low concentrations (5-40 microM) of quinine or quinidine, which diminish the Ca2+-dependent K+ conductance in a graded manner. After complete blockade of K+ currents, the peak Ca2+ currents are enhanced at all voltages, especially at values more positive than -30 mV, where a steady-state inward current appears as well. In this same voltage range, the decay of the Ca2+ current exhibits two time constants--that of the transient inward current, which is about 20 msec, and a much slower (approximately 2000 msec) component. It is suggested that neuroblastoma cells have two types of calcium channels--one which generates the Ca2+ action potential and a second, distinguished by activation at more depolarized levels and by a slow rate of inactivation, which underlies the calcium entry necessary to activate the Ca2+-dependent K+ conductance.

Action Potentials↗

PubMed1979

The calcium action potential and a prolonged calcium dependent after-hyperpolarization in mouse neuroblastoma cells.

1. Action potentials elicited in solutions with elevated [Ca2+] (1.8-40 mM) have been studied in differentiated cells of mouse neuroblastoma clone N1E-115 in tissue culture. 2. The action potential in high [Ca2+] solutions containing eithr Na+ or Tris is followed by a prolonged after-hyperpolarization (a.h.p.) lasting 0.5-4 sec. The a.h.p. reverses sign between -75 and -85 mV. 3. Externally applied tetraethylammonium (TEA, 15 mM) increases the Ca2+ spike overshoot, prolongs the falling phase and enhances the a.h.p. duration. The a.h.p. is inhibited by Ca2+ antagonists such as La3+, Co2+ and Mn2+. 4. After replacement of Ca2+ by Ba+ or Sr2+ (20mM) action potentials can still be elicited in Na+-free solution, but no a.h.p. is observed. 5. Increasing [Ca2+] from 1.8 up to 20 mM results in an increased capability of neuroblastoma cells to fire repetitively and in a consistent reduction of the firing rate from about 4-10 sec-1 to 0.5-1.8 sec-1. 6. It is concluded that Ca2+ entry during the action potential activates a TEA-resistant K+ conductance which gives rise to the prolonged a.h.p. Data from repetitively firing cells are consistent with the view that the a.h.p. plays a role in the regulation of low-frequency firing.

Action Potentials↗

PubMed1979

The calcium current and the activation of a slow potassium conductance in voltage-clamped mouse neuroblastoma cells.

1. The Ca2+ inward current (ICa) and a slow outward current in differentiated cells of mouse neuroblastoma clone N1E-115 have been studied under voltage-clamp conditions. 2. ICa shows voltage- and time-dependent inactivation when evoked by step-wise depolarizations in Na+-free solution containing high [Ca2+] (20 nM) and tetraethylammonium (TEA, 25 mM). Ba2+ and Sr2+ can substitute for Ca2+. 3. Holding potentials below -70 mV maximal activate ICa. Half inactivation occurs at -56 mV and ICa is completely inactivated beyond holding levels of -30 mV. Maximum peak currents are of the order of 10(-4) A/cm2 and the reversal potential ranges from +40 to +60 mV. The ICa inactivation time course follows first-order kinetics with a voltage-depedent time constant ranging from 25 to 100 msec. 4. The striking resemblance between ICa and the Ca2+ current in the unfertilized mouse oocyte (Okamoto, Takahashi & Yamashita, 1977) is discussed. 5. A slow outward current with a rise time of several seconds is recorded on voltage steps beyond -20 mV in high [Ca2+] solutions. It is carried primarily by K+ on account of the value of the reversal potential and its dependence on [K]0. This K+ current is TEA-insensitive and is blocked by Ca2+ antagonists. 6. The slow K+ current (IK(Ca)) is suggested to be mediated by Ca2+ influx, but the voltage-dependence of the underlying conductance (GK(Ca)) differs significantly from the ICa voltage-dependence. 7. The results are consistent with the hypothesis that IK(Ca) depends both on ICa and on membrane potential. An alternative hypothesis is briefly discussed.

Animals↗

PubMed1978

Ionic currents in cultured mouse neuroblastoma cells under voltage-clamp conditions.

1. Ionic currents in differentiated cells of mouse neuroblastoma clone N1E-115 have been studied under voltage-clamp conditions. 2. Depolarizing voltage steps from a holding potential of -85 mV to levels more positive than -40 mV produced fast transient inward currents followed by delayed outward currents. 3. The fast inward current is carried by Na+: it is blocked by tetrodotoxin and is absent in Na+-free solutions. Its kinetic behaviour resembles that of the Na+ current in squid giant axon. A mean value of 85 mmho/cm2 was found for the maximum Na+ conductance (GNa).4. The delayed outward current is carried primarily by K+: it is blocked by externally applied tetraethylammonium (TEA, 15 mM) and has a reversal potential (mean -71 mV) close to the theoretical K+ equilibrium potential. Its instantaneous I--V curve is linear. By analogy with the formulation of Hodgkin & Huxley (1952c), the outward current can be described by IK = -GKn2(V--EK) where GK = 12 mmho/mc2. 5. During prolonged depolarizations the delayed outward current declines. This decline, which occurs in two phases, represents a partial inactivation of the K+ conductance. 6. A weak inward current with slow activation and inactivation kinetics appears in Na+-free solution containing 10 mM-Ca2+. It is activated at a membrane potential of -55 mV and reaches its maximum at -20 mV with a time to peak of about 10 msec. This current is tetrodotoxin-resistant, reversibly blocked by Co2+ (5mM) and is suggested to be carried by Ca2+. 7. An increase in the external divalent cation concentration results in a parallel shift of the steady-state I--V curve along the voltage axis in positive direction. The activation of delayed outward currents is suggested not to depend on Ca2+ influx. 8. It is concluded that separate voltage-dependent Na+, K+ and Ca2+ channels exist in the differentiated neuroblastoma membrane with kinetic and pharmacological properties similar to those observed in non-mammalian preparations.

Calcium↗

PubMed1978

Induction of differentiation in mouse neuroblastoma cells.

In the presence of 1--2% dimethyl sulfoxide (DMSO), mouse neuroblastoma cells are induced to differentiate morphologically as well as electrically. In addition, treatment of neurolbastoma cells with 2% DMSO results in a marked increase in the veratridine-activated K+ or Rb+ efflux. At 4% DMSO, neurite outgrowth is completely repressed and electrical activity is poorly developed. However, at this concentration, the cells have a relatively high resting potential which suggested that membrane components determining passive and active permeability properties are not necessarily under coordinated control. Induction of differentiation by 2% DMSO is also accompanied by an increase in a heavier molecular form of acetylcholinesterase sedimenting at 10.5S. The effect of other agents on the growth and differentiation of neuroblastoma cells is also presented.

Acetylcholinesterase↗

PubMed1977

Membrane currents examined under voltage clamp in cultured neuroblastoma cells.

Examination of ionic membrane currents in a voltage-clamped neuronal cell line derived from the mouse C1300 neuroblastoma disclosed four kinetically different components: sodium, potassium, calcium, and leakage current. The kinetics, voltage dependence, and pharmacological properties of the sodium and potassium currents qualitatively resemble those of the corresponding currents in squid giant axon and frog myelinated nerve fiber, suggesting that the molecular structures of the sodium and potassium channels in neuroblastoma are similar to those of the non-mammalian preparations.

Action Potentials↗

PubMed1977

Development of electrophysiological and biochemical membrane properties during differentiation of embryonic skeletal muscle in culture.

Newly fused chick myotubes undergo simultaneous and rapid changes in cell membrane properties during synchronous differentiation in culture. These changes are coordinately regulated and include increases in acetylcholine receptor, acetylcholinesterase, and resting potential, as well as the appearance of action potentials in discrete membrane areas upon stimulation. Subsequently, the acetylcholine receptor reaches maximal levels, whereas the development of electrical properties is marked by a further increase in resting potential, changes in the characteristics of the elicited action potential, and the recruitment of additional membrane areas for action potential generation. Maturation of electrical excitability, marked by the acquisition of the ability to fire repetitively and to conduct action potentials along the membrane, occurs well after resting potential has reached a maximum. During post-maturational development, myotubes exhibit spontaneous electrical and contractile activity, and levels of acetylcholine receptor accessible to externally applied 125I-labeled alpha-bungarotoxin decrease markedly. It is suggested that electrophysiological membrane maturation is autonomously regulated with no requirement for neuronal intervention and involves the coordinated biosynthesis of discrete membrane components and their subsequent organization in the myotube membrane.

Action Potentials↗

PubMed1976

Maturation of neuroblastoma cells in the presence of dimethylsulfoxide.

Addition of dimethylsulfoxide at concentrations of 1% and 2% (vol/vol) to cells of mouse neuroblastoma clone NIE-115 in the confluent phase of growth resulted in the production of morphologically differentiated cultures with extensive process formation. Cell maintained in 2% dimethylsulfoxide remained in a stable nondividing condition for periods of up to 4 weeks. A high degree of electrical excitability was found in these cells, but there was no clear correlation of this property with the level of induction of either acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) or tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2]. In addition, intracellular levels of cyclic 3':5'-AMP were not elevated in fully morphologically and electrically differentiated cells. While cell division was markedly inhibited by 2% or higher concentrations of dimethylsulfoxide, at 1% growth continued at a somewhat slowed rate and such cultures exhibited enhanced process formation and electrical activity for a relatively short period. High concentrations (3% or 4%) of dimethylsulfoxide totally suppressed process formation and did not result in increased excitability, but cells maintained high resting potentials. The results suggest that the development of the excitable membrane in neuroblastoma cells may be expressed independently of neurospecific enzyme induction, and does not require a sustained elevation of cyclic 3':5'-AMP levels.

Acetylcholinesterase↗

PubMed1975

Enhancement of the electrical excitability of neuroblastoma cells by valinomycin.

Mouse neuroblastoma cells in stationary phase of growth display partially developed electrical properties. Addition of the K+ selective carrier valinomycin to these cells causes rapid enhancement of electrical excitability. We suggest that the appearance of molecules with properties similar to valinomycin is essential for the full expression of electrical excitability in differentiating neuroblastoma.

Action Potentials↗

PubMed1975