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Biomedical subjects

E Wanke

Publications and source records attributed to E Wanke.

At least 73 records · Page 4Linked to original sources
PubMed1987

Cholinergic receptors, ion channels, neurotransmitter synthesis, and neurite outgrowth are independently regulated during the in vitro differentiation of a human neuroblastoma cell line.

The differentiation of human nerve cells was investigated using a cell model comprising human neuroblastoma (IMR32) cells that were induced to differentiate by the addition of 5-bromo-2'-deoxyuridine (BrdU) or N6-O2-dibutyryl cyclic adenosine 3'-5' monophosphate (Bt2cAMP). As parameters of differentiation, we studied neurite outgrowth, cholinergic receptors, voltage-activated ion channels, tyrosine hydroxylase activity, and neurotransmitter content. BrdU induced marked morphological differentiation, as indicated by the number and length of neurites, as well as an increase in the number of alpha-bungarotoxin binding sites, muscarinic receptors, and voltage-dependent Na channels. In addition, BrdU induced an increase in tyrosine hydroxylase activity as well as in serotonin, dopamine, and noradrenaline content. Bt2cAMP had a less dramatic effect on the morphological appearance of the cells, induced the expression of alpha-bungarotoxin binding sites (but not of muscarinic receptors), and produced a marked increase in the serotonin and noradrenaline content. Not only the number but also the functional properties of nicotinic and muscarinic receptors were differently affected by the two drugs. We conclude that Bt2cAMP and BrdU induce a different pattern of differentiation in the same cells, and that the expression of specific neuronal markers can be modulated to yield functionally different neurons.

Axons↗
PubMed1986

Acetylcholine operated ion channel and alpha-bungarotoxin binding site in a human neuroblastoma cell line reside on different molecules.

In neurons, alpha-bungarotoxin is often associated with nicotinic receptor but does not always block the acetylcholine operated channel. In a human neuroblastoma cell line, IMR 32, we have demonstrated a large number of alpha-Bungarotoxin binding sites (2640 per cell in non differentiated cells and 4660 per cell in differentiated cells) in presence of 0 to 4 Acetylcholine activated-channels per cell. This neuronal cell line promises to be an useful model for the study of structure and function of the alpha-Bungarotoxin binding site not related to the nicotinic receptor.

Acetylcholine↗
PubMed1986

alpha Latrotoxin of the black widow spider venom opens a small, non-closing cation channel.

alpha Latrotoxin, a presynaptically acting polypeptide neurotoxin, induces massive neurotransmitter release from both synapses of vertebrates and the neurosecretory cells of the line PC12, derived from a rat pheochromocytoma. Single PC12 cells, differentiated by treatment with nerve growth factor, were used to investigate by the patch-clamp technique i) the alterations of the resting cell conditions (membrane potential and resistance) and ii) the microscopic mechanism of the permeability changes that underly the response to alpha LTx. The toxin was found to open a channel, 15 pS in conductance, that is permeable to various cations (Na+, K+ and probably Ca2+) and has little tendency to close. This channel is different from the classical voltage- and receptor-operated channels present in PC12 cells, as well as from the large conductances induced by the toxin in artificial lipid membranes.

Animals↗
PubMed1985

A fast transient outward current in the rat sympathetic neurone studied under voltage-clamp conditions.

Post-ganglionic neurones of the isolated rat superior cervical ganglion were voltage clamped at 37 degrees C using separate intracellular voltage and current micro-electrodes. Control experiments in current clamp suggested that the neurone is electrotonically compact, the soma and the proximal dendritic membranes being under good spatial voltage uniformity. Depolarizing voltage steps from membrane potentials near -50 mV evoked: (i) a voltage-dependent inward Na+ current, (ii) an inward Ca2+ current, (iii) a voltage-dependent outward K+ current, (iv) a Ca2+-activated K+ outward current. Depolarizations from holding potentials more negative than -60 mV elicited, besides the currents mentioned above, a fast transient outward current IA which peaked in 1-2.5 ms and then decayed to zero following an exponential time course. The IA current was shown to be primarily, if not exclusively, carried by K+. It was unaffected by removal of external Ca2+ or addition of Cd2+ and was weakly blocked by tetraethylammonium ions and partially by 4-aminopyridine. The IA current showed a linear instantaneous current-voltage relationship. Its activation ranged from -60 to 0 mV with a mid-point at -30 mV. The A conductance could be described in terms of a simple Boltzmann distribution for a single gating particle with a valency of +3. Both the development and removal of inactivation followed a single exponential time course with a voltage-dependent time constant which was large near the resting potential (42 ms at -70 mV) and small (11 ms) near -100 and -40 mV. Steady-state inactivation h infinity ranged from -100 to -50 mV, with a mid-point at -78 mV, suggesting that approximately 50% of the IA channels are available at the physiological resting potential. Action potentials elicited from various holding potentials showed maximal repolarization rates dependent on the holding potential itself. This voltage dependence was found to be in reasonably good agreement with that of h infinity curve. These data are consistent with the view that in the rat sympathetic neurone, under physiological conditions, it is the IA current rather than the delayed outward current that is responsible for the fast action potential repolarization.

4-Aminopyridine↗
PubMed1985

Identification of delayed potassium and calcium currents in the rat sympathetic neurone under voltage clamp.

Post-ganglionic neurones of the isolated rat superior cervical ganglion were studied at 37 degrees C under two-electrode voltage-clamp conditions. Membrane depolarization beyond -40 mV from holding levels between -50 and -100 mV produced a delayed outward current which exhibited no inactivation within this voltage range. The current is carried primarily by K+ ions and its instantaneous I-V relation is linear. The total outward current could be separated into two distinct components on the basis of ion-substitution experiments. A voltage-dependent component of the delayed current, termed IK(V), is activated by membrane depolarization beyond -40 mV when Ca2+ fluxes are selectively blocked by Cd2+ or in Ca2+-free solution. IK(V) develops following first-order kinetics and rises to a peak with a voltage-dependent delay (239 ms at -30 mV and 23 ms at +10 mV). GK(V) attains a saturating value of the order of 17 mS/cm2 at about +20 mV and can be described in terms of a simple Boltzmann distribution for a single gating particle with a valency equal to +2.5. A second component of the delayed outward current, termed IK(Ca), depends on Ca2+ entry for its activation and was isolated as difference current before and after block of Ca2+ movements across the membrane. IK(Ca) is larger and faster than IK(V): it is strictly related to Ca2+ influx and also depends on membrane potential depolarization. A distinct Ca2+ current, ICa, was recorded from the neurone exposed to Na+-free or tetrodotoxin solution. ICa was activated by membrane depolarization beyond -30 mV and reached a maximum value near 0 mV. Its activation agrees with fourth-order kinetics and becomes faster with increasing depolarization. The Ca2+ current developed with a voltage-dependent time to peak of 2.9-1.8 ms and thereafter completely inactivated. The relationship between ICa and IK(Ca) is discussed. The Ca2+-k+ repolarizing system is expected to be mainly associated with action potentials arising from a depolarized neurone, whereas the IA current (Belluzzi, Sacchi & Wanke, 1985) dominates the repolarization mechanism at the normal membrane potential. The effect of muscarine was examined. Muscarine (10-50 microM) produced a fall in conductance with a voltage dependence similar to that exhibited by GK(Ca) and was ineffective when removing extracellular Ca2+ or adding Cd2+. A partial suppression of ICa by muscarine is demonstrated. It is suggested that the decrease of the outward current magnitude in the presence of muscarine may be accounted for qualitatively by the reduction in ICa.

Action Potentials↗
PubMed1983

High intracellular pH reversibly prevents gating-charge immobilization in squid axons.

Squid giant axons were used to study the reversible effects of high intracellular pH (pHi) on gating currents. Under depolarization, when Na channels are activated, internal solutions buffered at high pHi (10.2) affect considerably the time course of gating charge associated with channel closing, QOFF, with almost no alteration of QON records. In particular, at pHi 10.2 the charge corresponding to the fast phase of IgOFF, measured after long depolarizing pulses (7.7 ms), was consistently larger than that recorded at physiological pHi (7.2). This suggests that high pH prevents immobilization of gating charges induced by Na inactivation. In this respect, the present data agree reasonably well with previous observations, which show that pHi greater than 7.2 reversibly removes the fast Na inactivation with little effects on activation kinetics (Carbone, E., P. L. Testa, and E. Wanke, 1981, Biophys. J., 35:393-413; Brodwick, M.S., and D. C. Eaton, 1978, Science [Wash. DC], 200:1494-1496). Unexpectedly, high pH increases the amount of charge associated with the slow phase of IgOFF. In our opinion, this might be the result of either an increment of the net charge produced by the exposure to high pHi or that gating charges that return to the closed state might experience a larger fraction of the potential drop across the membrane (Neumcke, B., W. Schwarz, and R. Stampfli, 1980, Biophys. J., 31:325-332).

Animals↗
PubMed1981

Intracellular pH and ionic channels in the Loligo vulgaris giant axon.

Squid giant axons were used to investigate the reversible effects of intracellular pH(pHi) on the kinetic properties of ionic channels. The pharmacologically separated K+ and Na+ currents were measured under: (a) internal perfusion, (b) enzymatic Pronase treatment, and (c) continuous estimate of periaxonal ion accumulation. Variation of internal pH from 4.8 to 11 resulted in: (a) a decrease of steady-state sodium inactivation at positive potentials similar to the effect of the proteolytic enzyme Pronase, (b) a shift of the h infinity (E) curve toward depolarizing voltages, and (c) a decrease of the time constant of inactivation for potentials below -20 mV (an increase above). A plot of the steady-state sodium conductance at E = +40 mV as a function of pHi suggests that two groups with pKa 10.4 and 5.6 affect respectively the inactivation gate and the rate constants for the transition from the inactivated to the second open state (h2) (Chandler and Meves, 1970b). The voltage shifts of the kinetic parameters predicted by the Gouy-Chapman-Stern theory are well satisfied at high pHi and less at low. Once corrected for voltage shifts, the forward rate constants for channel opening were found to be slowed with the acidity of the internal or external solution.

Animals↗
PubMed1980

The sodium channel and intracellular H+ blockage in squid axons.

Sodium channels in plasma membranes can be blocked by a large variety of toxins and local anaesthetics. This property, however, is not confined to relatively large molecules. For instance, extracellularly applied small ions like hydrogen may also prevent the passive transport of permeant cations across open Na+ channels. A typical feature of this phenomenon is that the blocking action of hydrogen is gradually relieved by increasing the voltage applied across the membrane. Although in the frog skeletal muscle there is no clear evidence for a similar intracellular action, we report here for the squid giant axon remarkable effects on the ionic permeability of Na+ channels when the internal perfusate contains an excess of protons. Analysing the action of low pH inside and outside the fibre in terms of a kinetic model, we could conclude that Na+ channels in squid axons are controlled by two independent groups: one with an apparent pKa of 4.6 and the other with pKa 5.8, the former feeling one-fifth of the applied membrane potential, the latter three-quarters. As with pharmacological agents, we also show that the voltage-dependence of the H+ blockage is not affected by the presence of the inactivation gate.

Animals↗
PubMed1979

K+ conductance modified by a titratable group accessible to protons from the intracellular side of the squid axon membrane.

In the range of pH examined (5.2-10), variations of internal pH from high to low values result in a reversible decrease of the conductance of the open K channels, without significantly affecting the kinetics parameters. A linear plot of the conductance versus internal pH suggests the existence of a titratable group that has an apparent pKa of about 6.9, and that is accessible to protons only from the intracellular side of the membrane.

Animals↗
PubMed1978

Action of extracellular pH on Na+ and K+ membrane currents in the giant axon of Loligo vulgaris.

Voltage-clamp currents and resting membrane potential of squid giant axons have been studied at extracellular pH varying between 4 and 10. The membrane currents, analyzed according to the Hodgkin-Huxley equations, showed that sodium permeability, PNa(E), and potassium conductance gK(E), curves were shifted toward positive voltages by different amounts and slightly depressed as the external pH was lowered. Under the same conditions, taum(E) and taun(E) were found to be enhanced and shifted to a larger extent in the same direction. The rate constants alpham and alphan were shifted substantially toward positive voltages, but betam and betan changed hardly at all. The shift of the alpham(E) curve was analyzed in terms of a fixed surface charge model; it indicates that unspecific negative groups with an approximate pKa of 4.5 are located in the vicinity of sodium active sites with an average charge separation of 8 A. A similar figure is obtained for the potassium system from the shift of the alphan(E) curve.

Animals↗
PubMed1975

Potassium and sodium ion current noise in the membrane of the squid giant axon.

1. The spectral density of current noise power from 20 mm segments of giant axons of the squid Loligo vulgaris has been measured under space-clamp and voltage-clamp conditions. From 4 to 1000 Hz the measured noise is larger by several orders of magnitude than the theoretical thermal noise. The amplifier's noise, which may yield appreciable contributions above 200 Hz, could be evaluated and subtracted from the total noise using direct measurements of the membrane impedance...

Action Potentials↗
PubMed1975

Potassium and sodium current noise from squid axon membranes.

Current noise under voltage clamp has been measured from the giant axon of Loligo vulgaris. Relatively large areas (up to 0.4 cm2) were used for these measurements, under standard space clamped conditions. Current noise spectral densities were studied in the range of 0 to 20 C, -100 to -40 mV and in normal, tetrodotoxin, and tetraethylammonium ion solutions. Three components of current noise were found, l/f-noise, and a K and a Na component.

Animals↗