Biomedical subjects

H D Lux

Publications and source records attributed to H D Lux.

At least 73 records · Page 4Linked to original sources

PubMed1987

Patch-clamp study of ion channels activated by GABA and glycine in cultured cerebellar neurons of the mouse.

gamma-Aminobutyric acid (GABA) and glycine receptor channels have been investigated using the patch-clamp technique. Six out of 12 membrane patches exposed to GABA as well as to glycine responded to both substances, 3 to only GABA, two to only glycine and one was insensitive to both transmitters which were applied in micromolar concentrations. Channel currents reversed at the potential predicted by the constant field equation (using Cl- activities), indicating that the charge was carried by Cl-. Although multiple-conductance states were induced by GABA as well as by glycine, the phenomenon was much more pronounced after glycine applications. GABA channel permeabilities ranged from 3.7 X 10(-14) to 6.0 X 10(-14) cm3 s-1. With Cl- distributed symmetrically at 145 mM, these values would correspond to conductances of 19 and 31 pS, respectively. Glycine channel permeabilities ranged from 3.3 X 10(-14) to 14.7 X 10(-14) cm3 s-1 (corresponding conductances 17 and 76 pS).

Animals↗

PubMed1987

Development of two types of calcium channels in cultured mammalian hippocampal neurons.

Calcium influx through voltage-gated membrane channels plays a crucial role in a variety of neuronal processes, including long-term potentiation and epileptogenesis in the mammalian cortex. Recent studies indicate that calcium channels in some cell types are heterogeneous. This heterogeneity has now been shown for calcium channels in mammalian cortical neurons. When dissociated embryonic hippocampal neurons from rat were grown in culture they first had only low voltage-activated, fully inactivating somatic calcium channels. These channels were metabolically stable and conducted calcium better than barium. Appearing later in conjunction with neurite outgrowth and eventually predominating in the dendrites, were high voltage-activated, slowly inactivating calcium channels. These were metabolically labile and more selective to barium than to calcium. Both types of calcium currents were reduced by classical calcium channel antagonists, but the low voltage-activated channels were more strongly blocked by the anticonvulsant drug phenytoin. These findings demonstrate the development and coexistence of two distinct types of calcium channels in mammalian cortical neurons.

Animals↗

PubMed1987

Voltage dependent calcium currents in PC12 growth cones and cells during NGF-induced cell growth.

The role of calcium currents in the regulation of neurite outgrowth is still rather speculative. As a contribution to this field, macroscopic voltage dependent calcium currents were investigated in relation to the nerve growth factor (NGF)-induced outgrowth of neurites in PC 12 cells. Calcium currents were recorded in isolated growth cones of PC 12 cells using the whole cell patch clamp method. The currents were activated at high voltages and only slightly inactivated with time. The currents were identical to those found in the cell soma of PC 12 cells and similar to the classical high-voltage-activated calcium current found in many neuronal cells. The peak current density in the growth cones was in the same range as in the cell somata. The calcium currents of the cell somata were not modified during the early phase of NGF application, despite the occurrence of NGF-induced soma growth and outgrowth of neurites. The current density at this time was therefore lower in NGF-treated cells than in untreated cells. In a later phase, maximal current amplitudes of NGF-treated cells were higher than in untreated cells indicating an increase in current density to values similar to that found in the untreated cells. In addition, the calcium current inactivation was found to be more pronounced in the NGF-treated cells by that time. The results are discussed with regard to a possible role of calcium currents in the regulation of NGF-induced neurite outgrowth in these cells.

Adrenal Gland Neoplasms↗

PubMed1987

Effect of menthol on two types of Ca currents in cultured sensory neurons of vertebrates.

The effect of menthol on voltage-dependent Ca currents was investigated in cultured dorsal root ganglion cells from chick and rat embryos. Bath application of menthol (0.1-1 mM) had different effects on the various Ca currents present in these neurons. Below -20 mV, the low threshold Ca currents were reduced in amplitude in a dose-dependent manner by menthol with little changes of their activation kinetics. In contrast to this, the time course of inactivation of the high-threshold Ca currents, activated above -20 mV from a holding potential of -80 mV, was drastically accelerated by external menthol. The action of menthol was unchanged with more positive holding potentials (-50 mV). Thus, a proposed third type of Ca current with transient activation and complete deactivation below -50 mV was either not present or not affected by menthol. Menthol exerted its action only when applied from the outside. Its effect was completely reversible within 15-20 min of wash-out. Our findings are consistent with the idea that menthol acts on two types of Ca channels coexisting on the membrane of cultured sensory neurons. Menthol blocks currents through the low voltage-activated Ca channel, and facilitates inactivation gating of the classical high voltage-activated Ca channel.

Animals↗

PubMed1987

Kinetics and selectivity of a low-voltage-activated calcium current in chick and rat sensory neurones.

1. Using the whole-cell recording mode of the patch-clamp technique, we have investigated kinetic and selectivity properties of a low-voltage-activated (l.v.a.) Ca2+ current in chick and rat dorsal root ganglion (d.r.g.) neurones. 2. L.v.a currents were activated at about -50 mV and reached maximum amplitudes between -30 and -20 mV with averages of -0.16 nA in chick and -0.3 nA in rat d.r.g. cells with 5 mM-extracellular Ca2+. Between -60 and -20 mV, the time to peak, tp, of this current decreased with increasing membrane depolarizations. An e-fold change of tp required a 14 mV potential change in chick and a 17 mV change in rat d.r.g. cells at 22 degrees C. 3. Between -50 and +20 mV inactivation of this current was fast, single exponential and voltage dependent. In rat, the time constant of inactivation, tau h, was smaller and less voltage dependent than in chick. 4. The amplitude of these currents increased by a factor of 5-10, when the extracellular Ca2+ concentration was changed from 1 to 95 mM. Amplitudes and kinetic parameters of the currents showed typical shifts along the voltage axis. No correlation between Ca2+ current amplitudes and activation-inactivation kinetics was found, suggesting that the reaction rates which control these processes are not dependent on Ca2+ entry. 5. Recovery from inactivation was voltage dependent and developed with a time constant, tau r, in the order of 1 s. tau r was nearly halved by changing the potential from -80 to -120 mV. 6. Tail currents associated with membrane repolarization were also voltage dependent and developed exponentially. Their time constant decreased by a factor of 3 when the potential was changed from -60 to -100 mV. 7. A second and more prominent Ca2+ current was activated at potentials positive to -20 mV (high-voltage-activated Ca2+ currents, h.v.a.), masking the time course of l.v.a. currents. Between -20 and 0 mV, time to peak of the entire current increased by a factor of 2 but decreased again at higher membrane potentials. Inactivation also became significantly slower in this potential range. 8. The contribution of the h.v.a. component to the total membrane current was markedly reduced using a high intracellular Ca2+ concentration, [Ca2+]i, or internal fluoride salts. This made it possible to study the kinetic parameters and the I-V characteristics of the l.v.a. current more precisely over a wider potential range (-50 to +30 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1987

Single low-voltage-activated calcium channels in chick and rat sensory neurones.

1. Single and multiple Ca2+ channel currents were recorded from outside-out and cell-attached patches of cultured chick and rat dorsal root ganglion cells, using the patch-clamp technique. 2. Outside-out patches containing a large number of Ca2+ channels revealed Ca2+ currents resembling those from the whole cell. A low-voltage-activated (l.v.a.) and a high-voltage-activated (h.v.a.) Ca2+ current similar to those described in the accompanying paper (Carbone & Lux, 1987 b) could be distinguished. The h.v.a. current component subsided within 10 min following the formation of the patch, while the l.v.a. component lasted much longer. 3. Unitary events related to the l.v.a. Ca2+ channel could be clearly resolved in outside-out patches formed in Na+- and K+-free media containing 5-50 mM-CaCl2. 4. The amplitudes of l.v.a. channel openings were bimodally distributed, indicating the presence of two conductive states. At -40 mV, mean amplitudes of the two events were -0.29 +/- 0.07 pA and -0.47 +/- 0.085 pA in 50 mM-CaCl2, with apparent slope conductances of about 3.6 and 5.2 pS, respectively. In 5 mM-CaCl2 both slope conductances were about 3 times smaller. The mean open times were similar for both states and were fitted by a simple exponential with a time constant of about 2.5 ms at -40 mV. The time constant decreased with more-negative membrane potentials and was 0.9 ms at -100 mV. Openings frequently occurred in bursts separated by longer-lasting closures. The mean closed time during bursts was 1.33 ms at -40 mV. 5. Time and amplitude distributions of elementary events were similar for chick and rat sensory neurones and with Ba2+ and Sr2+ replacing external Ca2+. 6. In the potential range examined (from -60 to -30 mV), the first-latency distribution function revealed a distinct rise to peak which occurred at considerably earlier times than peaks of macroscopic currents. The time course of macroscopic l.v.a. Ca2+ currents could be simulated in two ways: (a) by using a five-state Markov-chain model with rate constants estimated from the transition probabilities and dwell times of the channel states, and (b) by evaluating the convolution integral of the first-latency function and the burst open probability of the channel. Both approaches suggest that activation and inactivation are weakly coupled and that the l.v.a. channel of sensory neurones reopens several times before inactivating.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1987

Proton-induced transformation of calcium channel in chick dorsal root ganglion cells.

1. In dissociated and cultured 2-5-day-old chick dorsal root ganglion cells, a large transient inward current could be activated in response to a 'step' increase in [H+]o. 2. Using the single-electrode patch clamp technique in its whole-cell configuration, the proton-induced current was graded with [H+]o and relaxed in 1-2 s. 3. The pH dependence of the current was sigmoid with activation occurring at around pH 7.0 (at[Ca2+]o = 1 mM) and a maximum at pH 6.0-5.5. 4. Small increases of [H+]o, which by themselves failed to activate a significant amount of current, inactivated the proton-induced current. The half-maximum of inactivation occurred at pH 7.11 at [Ca2+]o = 5 mM, but this changed to pH 7.32 at [Ca2+]o = 1 mM. 5. The proton-induced inward current reversed direction at the Na+ equilibrium potential and was suppressed in the absence of [Na+]o. Measurement of the reversal potential at different [Na+]o and/or [Na+]i showed a linear relation with a slope of 58 mV/decade as predicted from the Nernst equation. Thus, proton-induced current was carried by Na+ and was abbreviated as INa(H). 6. The membrane conductance associated with INa(H) showed no voltage dependence, but did change in parallel with the activation of the current. The membrane conductance increased by a factor of 10-20-fold at the peak of the inward current. 7. INa(H) was blocked by organic and inorganic Ca2+ channel blockers (diltiazem, Cd2+ and Ni2+), but was unaffected by high concentrations of tetrodotoxin (TTX) or steady-state increases of the [Ca2+]i to 10(-4) M or the [H+]i to 10(-6) M. 8. In outside-out membrane patches, the single channel associated with the proton-induced current opened in bursts, with long pauses. The mean open time during the bursts was 1.26 ms and the channel had a conductance of 20-25 pS at -80 mV (120 mM [Na2+]o, 20 mM [Na2+]i). 9. Measurement of the voltage-gated Ca2+ current using short (30-50 ms) depolarizing pulses to zero showed that the Ca2+ current (ICa) but not the fast Na+ current (INa) was completely suppressed during the time course of activation of INa(H). 10. INa(H) was completely blocked by high (35-40 mM) [Ca2+]o. 11. Simultaneous elevation of [H+]o and [Ca2+]o failed to activate INa(H) but enhanced the voltage-gated Ca2+ channel. 12. Our data show that the proton-induced current is carried by Na+ flowing through a transformed Ca2+ channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1986

Calcium channel current inactivation is selectively modulated by menthol.

The effect of menthol on Ca channel current inactivation was studied in identified Helix neurons. External application of menthol accelerated the Ca-dependent rapid phase of inactivation. Menthol restored a fast inactivation phase after the Ca-dependent inactivation had been removed by strongly buffering changes in intracellular free Ca or by using Ba ions as current carriers. The menthol-induced inactivation was unchanged by variations in intracellular free Ca. A sensitizing effect of menthol on Ca-dependent inactivation appeared unlikely. Instead the results indicate a modulating action of menthol on Ca inactivation.

Animals↗

PubMed1986

The role of chloride transport in postsynaptic inhibition of hippocampal neurons.

Hippocampal inhibitory postsynaptic potentials are depolarizing in granule cells but hyperpolarizing in CA3 neurons because the reversal potentials and membrane potentials of these cells differ. Here the hippocampal slice preparation was used to investigate the role of chloride transport in these inhibitory responses. In both cell types, increasing the intracellular chloride concentration by injection shifted the reversal potential of these responses in a positive direction, and blocking the outward transport of chloride with furosemide slowed their recovery from the injection. In addition, hyperpolarizing and depolarizing inhibitory responses and the hyperpolarizing and depolarizing responses to the inhibitory neurotransmitter gamma-aminobutyric acid decreased in the presence of furosemide. These effects of furosemide suggest that the internal chloride activity of an individual hippocampal neuron is regulated by two transport processes, one that accumulates chloride and one that extrudes chloride.

Animals↗

PubMed1986

Effects of dopamine and noradrenaline on Ca channels of cultured sensory and sympathetic neurons of chick.

The effects of noradrenaline and dopamine on voltage-dependent Ca currents were investigated in cultured dorsal root and sympathetic ganglion neurons from chick embryos. At concentrations of 1 to 10 microM, bath application of the neurotransmitters caused a general depression of inward Ca currents. Above -20 mV the decrease of the current amplitude was reversible and accompanied by a 2-10-fold prolongation of the activation time course. Below -20 mV, where a low voltage-activated Ca component is turned on, the size of the currents was reduced by 40% with little effect on the time course. Despite extensive wash-out, little sign of reversibility was observed in this case. Single-channel current recording in outside-out membrane patches revealed that at low membrane potentials dopamine and noradrenaline reversibly reduced single Ca-channel activity. This finding supports the view that in sensory and sympathetic neurons, both neurotransmitters affect the membrane conductance by modulating Ca permeability and not by activating catecholamine-specific channels able to carry transient outward currents. The probability of Ca channel opening is strongly reduced by addition of 10 microM of either catecholamine to the bath. The possible involvement of a voltage-dependent block of Ca channels by the neurotransmitters is discussed.

Animals↗

PubMed1986

Step reductions in extracellular Ca2+ activate a transient inward current in chick dorsal root ganglion cells.

We investigated whether transient step reductions in divalent cations would produce detectable changes in neuronal excitability similar to those reported in the total absence of divalent cations. Using cultured chick dorsal root ganglion cells as a model system, our results indicate that a step reduction in divalent cations induces a transient inward current. This response is mediated by a tetrodotoxin-resistant, Na+-permeable, cation channel that is blocked by cadmium. This, and our observation that the response is abolished by verapamil, suggests that the current passes through calcium channels. This transient inward current was estimated to be activated by decreases in extracellular calcium ([Ca2+]o) as small as 0.5-0.8 mM and thus represents a different response from the one previously observed when steady-state [Ca2+]o levels were reduced to micromolar levels.

Animals↗

PubMed1986

Single calcium channels and their inactivation.

Inactivation of single Ca channels in snail neurons was examined to test the idea that entering Ca ions react directly with the channel to produce this effect. Simulations of specific models were used for comparison with the experimental data. The Ca-dependent model predicts time-dependent changes in the single-channel events which were not found experimentally. It is possible that Ca-dependent inactivation is mediated by a Ca-binding protein that is associated with but not part of the channel itself.

Animals↗

PubMed1986

Ionic changes and alterations in the size of the extracellular space during epileptic activity.

Experiments with ion-selective microelectrodes revealed that a considerable activity of K ions appears temporarily in the extracellular space (ES) during enhanced neuronal activity and is removed from the ES by diffusion, active uptake, and entry into glial cells. The glial uptake results from the preferential glial K permeability and spatial glial K buffering. The glia responds to the local extracellular accumulation by a depolarization of the exposed part of its membrane. This depolarization will spread along the glial syncytium or extended glial cells. At sites where the extracellular K concentration has not yet increased, the membrane potential will thus be depolarized with respect to the K diffusion potential. Here K will move from the intra- into the extracellular space, in order to restore the electrochemical equilibrium. This induces a current that carries K into glial cells at sites of maximal K accumulation and that transports K out of glial cells at remote areas. In this way K is spatially redistributed. The corresponding current loop in the ES is predominantly carried by Na and Cl, the majority ions. Thus, Na and Ca are transported to the site of K accumulation while Cl moves away. The Cl and K ions are only partially replaced by Na. Hence, a decrease of extracellular osmolarity results, which leads to a water flux from the ES into the cells, inducing a shrinkage of the ES at sites of maximal K accumulation. At remote sites, the opposite effect is expected due to K flow out of glia and Cl transport to these sites. Thus, remote from the area of maximal neuronal activity, an increase of the ES is expected. This mechanism can explain the measured depth profile of the changes in the ES. At sites of maximal neuronal activity, the extracellular space undergoes a reduction by more than 30%. The ionic changes are accompanied by slow negative potential shifts. An increase in intracellular osmolarity due to enhanced metabolic activity and possibly KCl uptake mechanisms contributes to the changes in volume and ionic concentration. Model calculations of the after-effects of the loss of positive charges from the extracellular space and the K-specific glial buffering could predict size and time course of these changes. Experimental tests of this view include observations during epileptiform activity in gliotic scar foci as well as in hippocampal slices with depressed synaptic transmission. The extra- and intracellular ionic changes influence the generation, spread, and termination of seizure activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1985

Action of organic antagonists on neuronal calcium currents.

The low-voltage activated, fast inactivating calcium (Ca) conductance recently found in dorsal root ganglion cells is largely inert to the Ca agonist and antagonists (BAY K 8644, nifedipine and verapamil). The ubiquitous, slowly inactivating Ca conductance is strongly depressed by verapamil (20 to 100 X 10(-6) M) and with [Ca]o greater than or equal to 2 mM also by BAY K 8644, while nifedipine was weakly agonistic or ineffective in a comparable range of drug concentrations (10(-9) to 5 X 10(-7 M). The difference in affinity to verapamil renders possible a pharmacological separation of the two channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1985

gamma-Aminobutyric acid-induced depression of calcium currents of chick sensory neurons.

The effects of gamma-aminobutyric acid (GABA) on calcium currents were investigated in avian dorsal root ganglion (DRG) cells. GABA was applied to the vicinity of the cells by ejection pipettes using constant-pressure pulses. GABA concentrations between 5 and 100 mu M reduced and slowed the calcium current in a dose-dependent manner. A contribution of K and Cl outward currents to the reduction of the inward current was minimized by using identical caesium chloride concentrations on both sides of the membrane. The onset of the effect was rapid and 80% of the effect was observed within 1 s. The attenuation of the Ca slope conductance by GABA was found to be independent of the membrane potential between -50 and +50 mV.

Animals↗

PubMed1985

Lithium slows neuronal calcium regulation in the snail Helix pomatia.

Steady-state and transient changes in intracellular calcium concentrations ([Ca2+]i) of snail neurons (Helix pomatia) were measured by the Ca2+ indicator Arsenazo(III) following manipulation of the extracellular concentration of lithium chloride (LiCl). Application of LiCl in concentrations equivalent to those used in the treatment of manic-depressive illness produces slowing in Ca2+ reequilibration after Ca2+-influx during depolarization, concomitantly with steady-state elevation of [Ca2+]i of about 100 nM, suggesting a change in Ca2+ reequilibration as a prominent action of LiCl. This mechanism may be relevant to the therapeutic effects of LiCl.

Animals↗

PubMed1985

Progenitor cells from embryonic chick dorsal root ganglia differentiate in vitro to neurons: biochemical and electrophysiological evidence.

We have analyzed the appearance of neurons and glial cells in chick dorsal root ganglia during development. Neurons were identified by the presence of polysialogangliosides recognized by tetanus toxin (GD1b, GT1) or by the monoclonal antibody Q211 directed against polysialogangliosides containing four, five and six sialic acid residues. Glial cells were identified by the presence of 04 antigen. A population of undifferentiated cells, i.e., cells which express neither neuronal nor glial cell surface antigens, present in dorsal root ganglia until embryonic day 7, was separated from the neuronal and glial population. This cell population contains neuronal progenitor cells which differentiate to neurons within 1 day in culture. This differentiation process is characterized by the appearance of neuronal morphology, of neuron-specific gangliosides and by the appearance of voltage-dependent sodium and calcium channels.

Animals↗

PubMed1985

Activation of a nonspecific cation conductance by intracellular Ca2+ elevation in bursting pacemaker neurons of Helix pomatia.

The pacemaker current of a bursting neuron of Helix pomatia was investigated using voltage-clamp and pressure-injection techniques. In the steady state the net membrane current was zero near threshold of the action potential at -45 mV. Negative to this potential the membrane current was inward and steady. During burst activity a long-lasting inward current instantaneously appeared with voltage steps to membrane potentials below -20 mV. This inward current was already present when the clamp step fell into the rising phase of the first spike and became larger during the depolarizing phase of the spike. The repolarization phase and the interspike interval did not add much current. As the spike duration became longer in the course of the burst discharge the inward current grew in amplitude, but its increase was not proportional to that of the spike duration. This was observed with clamp steps to the potassium equilibrium potential (EK = -70 mV). The inward current decayed during a hyperpolarizing step with a half time of approximately 400 ms, which was invariant to voltage as measured between -40 and -100 mV. It decreased linearly from -100 to -40 mV with an extrapolated zero potential of about -20 mV. The inward current was not generated by spikes if the Ca2+ conductance was blocked by Ni2+. At membrane potentials positive to EK the development of an outward current, probably carried by K+, could be observed during the burst. It overlasted the inward current and decayed with time constants of 6-7 s. This current grew successively in amplitude in the course of the burst discharge and finally nullified the inward-current component at potentials around spike threshold, thus terminating the burst. An inward current with properties similar to the spike-induced inward current was produced by pressure injecting CaCl2 into the neurons. This current was unselectively carried by cations as shown by both ion-substitution experiments and measurements with ion-selective microelectrodes. Large cations such as choline, TEA, and Tris passed through the channels nearly as well as Na+. Changes in the H+ or Cl- concentration were not seen to affect the inward current. Spike as well as the injection-induced currents were largest in bursting pacemaker cells compared with other cells of similar size. Both currents were found to be small or absent in nonbursting but regularly firing pacemaker cells, albeit these cells reveal a larger Ca2+ current density than the bursting pacemaker cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗