Concentration Clamp Research Articles

P-345 - Two phases of sodium channel block by lidocaine during rapid concentration clamp in guinea-pig ventricular myocytes.

P-345 - Two phases of sodium channel block by lidocaine during rapid concentration clamp in guinea-pig ventricular myocytes.

A highly potent and selective [formula omitted] receptor antagonist from the venom of the Agelenopsis aperta spider

Agatoxin-489, extracted from the venom of the Agelenopsis aperta spider, was studied on acutely isolated perfused hippocampal neurons of rat using the concentration clamp technique. Agatoxin-489 proved to be a selective N- methyl- d-aspartate antagonist; responses to applications of N- methyl- d-aspartate or l-aspartate were blocked by concentrations of agatoxin-489 ranging between 0.1 nM and 1 μM, while responses to kainate were not affected by agatoxin-489 at concentrations up to 10μM. The actions of agatoxin-489 against responses to N- methyl- d-aspartate or l-aspartate were use- and voltage-dependent, being less pronounced with an increase in the holding potential from —100 to —30 mV. The action of agatoxin-489 could be completely or partially reversed only after washout in the presence of an N- methyl- d-aspartate agonist. The washout was more effective at positive membrane potentials ranging from 0 to +20mV. These results imply that the spider toxin agatoxin-489, like dizocilpine, is a potent and selective N- methyl- d-aspartate antagonist which preferentially interacts with activated N- methyl- d-aspartate receptors and/or open N- methyl- d-aspartate-activated ionic channels.

Effects of a high molecular weight toxin from the sea anemone Condylactis gigantea on cholinergic responses

1. The effects of a high molecular weight toxin isolated from the sea anemone Condylactis gigantea (Condytoxina 2) on the cholinergic responses were studied in two different preparations: identified cells of a land snail and enzymatically dissociated mice sensory neurons. These neurons were studied using intracellular recording and concentration clamp techniques respectively. 2. The toxin produces a concentration-dependent dual effect on the cholinergic responses in both preparations. Thus the application of the toxin at concentrations up to 25 nmol/1 produces a reversible block of the response whereas higher doses potentiates it. 3. These results suggest that Condytoxina 2 contains an active compound(s) with the capacity to bind to the nicotinic acetylcholine receptor of excitable cells in both snail and mice neurons. During this action complex allosteric interactions among the binding sites could occur.

Ouabain blocks some rapid concentration-induced clamp acetylcholine responses on Helix neurons.

1. The effects of ouabain, a potent inhibitor of Na(+)-K+ ATPase, were determined on the transmembrane responses of internally dialyzed Helix neurons to rapid acetylcholine (ACh) application using the "concentration clamp" technique. 2. Ouabain selectively depressed "A"-type responses to ACh, which are due to a selective increase in membrane permeability to chloride. In contrast, the "B"-type responses, due primarily to an increase in monovalent cation permeability, was unaffected. 3. The blockade of the Cl- responses was not associated with a change of the reversal potential of the response. Ouabain depressed the maximal response without shifting the dose-response curve. 4. Ouabain caused an increase in the time constant of decay of the ACh current, but the value in the presence of ouabain was not different from that of a lower concentration of ACh determined so as to give a response of the same peak amplitude. Therefore, the effect of ouabain is not on the process of receptor desensitization directly.

Potential- and acetylcholine-activated ionic currents of pheochromocytoma PC12 cells during incubation with nerve growth factor

Pheochromocytoma PC12 cells incubated with and without nerve growth factor were investigated using the patch-clamp technique in the whole-cell recording mode, and the concentration clamp method in the rat. On the fourth day of incubation with nerve growth factor, sodium potential-activated ionic currents appeared in the membranes of the most morphologically differentiated cells. At the same period a three-fold increase of acetylcholine-activated current density, compared with the cells incubated without nerve growth factor, was observed. Thus, the qualitative and quantitative changes in membrane properties can be a result of metabolic reorganization in PC12 cells induced by nerve growth factor and accompanied by morphological differentiation according to neuronal phenotype.

Primary receptor for inhibitory transmitters in lamprey spinal cord neurons

The action of glycine and GABA on isolated lamprey spinal cord neurons was investigated by means of intracellular perfusion and concentration clamp techniques. These amino acids activated desensitizing chloride ionic conductances. The concentrations of agonists evoking half-maximum effects ( ed 50) were equal to 16 μM and 1.5 mM for glycine- and GABA-activated currents, respectively. Increase in the transmitter concentration led to a decrease in the time constant of desensitization. Current-voltage relationships of glycine- and GABA-activated currents were strongly dose-dependent. At low agonist concentrations the time constant of activation decreased with membrane hyperpolarization. Glycine- and GABA-activated currents exhibited complete cross-desensitization. The specific glycine antagonist, strychnine, suppressed both glycine- and GABA-activated currents to the same degree. Selective antagonists of GABA receptors, bicuculline and picrotoxin, produced equal blocking effects on glycineas well as GABA-evoked responses. In the cells studied, taurine activated desensitizing ionic conductance. Responses evoked by taurine and glycine applications demonstrated complete cross-desensitization. Taurine-activated currents were sensitive to strychnine, bicuculline and picrotoxin. These results suggest the existence of one receptor-channel complex for the main inhibitory transmitters in lamprey spinal cord neurons. 3,4-Dioxy- l-β-phenylalanine evoked desensitizing strychnine-sensitive ionic responses which exhibited cross-desensitization with glycine-activated currents.

Non-competitive inhibition of GABAA responses by a new class of quinolones and non-steroidal anti-inflammatories in dissociated frog sensory neurones.

1. The interaction of a new class of quinolone antimicrobials (new quinolones) and non-steroidal anti-inflammatory agents (NSAIDs) with the GABAA receptor-Cl- channel complex was investigated in frog sensory neurones by use of the internal perfusion and 'concentration clamp' techniques. 2. The new quinolones and the NSAIDs (both 10(-6)-10(-5) M) had little effect on the GABA-induced chloride current (ICI) when applied separately. At a concentration of 10(-4) M the new quinolones, and to a lesser degree the NSAIDs, produced some suppression of the GABA response. 3. The co-administration of new quinolones and some NSAIDs (10(-6)-10(-14) M) resulted in a marked suppression of the GABA response. The size of this inhibition was dependent on the concentration of either the new quinolone or the NSAID tested. The inhibitory potency of new quinolones in combination with 4-biphenylacetic acid (BPAA) was in rank order norfloxacin (NFLX) much greater than enoxacin (ENX) greater than ciprofloxancin (CPFX) much greater than ofloxacin (OFLX), and that of NSAIDs in combination with ENX was BPAA much greater than indomethacin = ketoprofen greater than naproxen greater than ibuprofen greater than pranoprofen. Diclofenac, piroxicam and acetaminophen did not affect GABA responses in the presence of ENX. 4. In the presence of ENX or BPAA, there was a small shift to the right of the concentration-response curve for GABA without any effect on the maximum response. However, the co-administration of these drugs suppressed the maximum of the GABA concentration-response curve, indicating a non-competitive inhibition, for which no voltage-dependency was observed.5. Simultaneous administration of ENX and BPAA also suppressed pentobarbitone (PB)-gated Icl. On the other hand, both PB and phenobarbitone reversed the inhibition of GABA-induced Ic, by coadministration of ENX and BPAA.6. The effect on GABAA responses of co-administration of new quinolones and NSAIDs was not via an interaction with benzodiazepine receptors coupled to the GABAA receptor, since this effect was not reversed by Rol5-1788 or diazepam.7. It is concluded that the co-administration of new quinolones and some of the NSAIDs inhibit GABAergic transmission, and could result in convulsions.

Plasma concentration clamping in the rat using a computer-controlled infusion pump.

We have developed a computer-controlled infusion pump to achieve rapidly and then maintain stable plasma thiopental concentrations in rats. Initially we derived the parameters of a triexponential pharmacokinetic model for thiopental, administered as a brief infusion to 10 rats, using nonlinear regression and standard pharmacokinetic equations. These parameters were incorporated into the pharmacokinetic model of a computer-controlled infusion pump. In a second group of animals this device was used to maintain three consecutive target thiopental concentrations ranging from 5 to 100 micrograms/ml in a stepwise fashion. Arterial blood gases were kept normal through controlled ventilation when necessary. The plasma thiopental concentrations in this second group of animals were generally higher than the target concentrations. The bias in pump performance (median prediction error) was +25%, and the inaccuracy (median absolute prediction error) was 26%. We fit the parameters of a three-compartment model to the plasma thiopental concentrations observed in the second group of animals. This produced a second set of thiopental pharmacokinetic parameters with the unique characteristic of having been derived from a computer controlled infusion study. These parameters were tested prospectively with a computer-controlled infusion pump in a third group of animals. This second set of thiopental pharmacokinetic parameters performed better, with a median prediction error of 0% and a median absolute prediction error of 15%. This study shows that it is possible to achieve rapidly and maintain steady plasma thiopental concentrations in the rat. Our results suggest that it is feasible to derive robust pharmacokinetic parameters from unusual drug dosing approaches, such as employed by a computer-controlled infusion pump.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid interaction of tetraethylammonium with delayed K + channel in mammalian brain neuron

In order to study the rapidity of interaction between tetraethylammonium (TEA) and the delayed K + channel of the mammalian brain neuron, TEA was applied to isolated rat hippocampal pyramidal neurons in a time-controlled sequence with a voltage step using a concentration clamp technique. TEA at a concentration of 30 mM, externally applied, started to decrease the delayed K + current ( I KD) without any detectable delay after the onset of the drug application. The inhibitory action of TEA reached a steady state within 10 ms while the full recovery from the inhibition required 20–30 ms after removing TEA from the external solution. Ba 2+ at a concentration of 30 mM also reduced the I KD without any detectable delay. However, it took 40–60 s to reach the steady state of inhibition. Full recovery from the inhibition by Ba 2+ was obtained around 60 s after removal of Ba 2+ from the external solution. It is suggested that the interaction of TEA with the delayed K + channel is much faster than that of Ba 2+.

Action of precursors of noradrenaline synthesis on membrane receptors of chick embryo spinal neurons

Effects of noradrenaline precursors on glycine and N-methyl-D-aspartate (NMDA) receptors in spinal cord neurons recently isolated from chick embryo were investigated using whole cell patch-clamp and concentration clamp techniques. Both L-alanine and L-DOPA were found to be glycine agonists capable of potentiating NMDA response, while L-tyrosine does not activate glycine but can potentiate NMDA response. Lastly, L-phenylalanine and dopamine do not interact with either glycine or NMDA receptors.

A novel selective NMDA agonist, N-phthalamoyl-L-glutamic acid (PhGA)

Ionic currents elicited by N-phthalamoyl-L-glutamic acid (PhGA) were investigated on freshly isolated hippocampal neurons with the whole-cell voltage clamp and concentration clamp techniques. PhGA elicited desensitizing inward currents in Mg(2+)-free salines only in the presence of glycine. The dose-response relationship for PhGA was close to a Langmuir isotherm with Kd = 3.7 mM and saturating level 0.75 of that for L-aspartate (L-Asp). PhGA-activated currents were blocked by Mg2+, D-2-amino-5-phosphonovalerate and kynurenate, and had the same reversal potential as L-Asp-activated currents. Complete cross-desensitization was obtained between the responses to PhGA and L-Asp. We conclude that PhGA is a new selective 'superacidic' agonist of the N-methyl-D-aspartate type of glutamate receptor.

Voltage clamp analysis of the kinetics of piperidine-induced chloride current in isolated Aplysia neurons

The effect of piperidine (Pip) on isolated Aplysia neurons was investigated using the voltage clamp and concentration clamp techniques in which neurons were perfused internally and externally with Na+,K+ free solution. Pip induced a Cl-current (ICl) in a dose-dependent manner for doses ranging from 10(-4) M to 10(-2) M. The dose-response curve gave an apparent dissociation constant of 8.4 x 10(-4) M and a Hill coefficient of 1.7. The current-voltage relationship was linear in the voltage range examined (-70 to +30 mV). The equilibrium potential for Pip induced current was close to the calculated equilibrium potential for chloride ions (ECl), (-10.7 mV). The activation phase of the ICl was characterized by a single exponential at all concentrations. The time constant of this phase decreased with increasing concentrations of Pip but did not depend on the membrane potential. The deactivation phase of the ICl proceeded on a single exponential curve at concentrations of Pip less than 5 x 10(-4) M, but on a double exponential at concentrations of 5 x 10(-4) M and higher. The deactivation time constant also decreased with increasing concentrations of Pip, but showed no potential dependence. Pip- and ACh-induced IClS were not blocked by 10(-4) M atropine. However, Pip-induced ICl was abolished with 10(-4) M d-tubocurarine (dTC), and the ACh-induced ICl was depressed by the same dose of dTC. These results suggest that Pip acts on at least two components of the nicotinic receptor-Cl channel complex in Aplysia neurons to elicit the ICl.

Proteolytic enzymes do not destroy the [formula omitted] (NMDA) sensitivity of acutely isolated hippocampal CA1 and CA3 neurons from postnatal rats

Using whole cell recordings in combination with the concentration clamp technique it was shown that even enzymatically isolated hippocampal CA1 and CA3 neurons exhibit N- methyl- d-aspartate (NMDA) and l-aspartate currents. These currents were completely blocked by 0.5 mM Mg 2+ or partially blocked (72.7 ± 2.4%) by dl-2-amino-5-phosphonovalerate (50 μM) whereas 10 μM glycine (Gly) potentiated the NMDA responses (3.1 ± 1.6-fold). A half-maximum dose of 55 μM was calculated from the sigmoid NMDA dose-response curve in the presence of 10 μM Gly. The amplitudes of these currents did not depend on the type of the proteolytic enzyme used for cell isolation.

GABA affects the glutamate receptor-chloride channel complex in mechanically isolated and internally perfused Aplysia neurons

The effects of γ-aminobutyric acid (GABA) on the glutamate receptors chloride ion (Cl −) channel complex were examined in mechanically isolated and internally perfused Aplysia neurons using a concentration clamp technique. GABA at concentrations of 3 × 10 −6 M or more, concentration dependently delayed the recovery of the glutamate response from desensitization. This effect was independent of the GABA response and Cl − redistribution. Muscimol (10 −4 M) mimicked the effect of GABA. However, this was not the case for baclofen (10 −3 M). In some isolated neurons, GABA at concentrations of more than 10 −4 M clearly induced an additional Cl − current, the current kinetics of which were different from those induced by lower concentrations of GABA. Even in the continued presence of 10 −4 M GABA, which desensitized the fast GABA response, higher concentrations of GABA (3 × 10 −4 M to 10 −2 M) elicited the additional current in a concentration-dependent manner. The presence of 10 −4 M glutamate completely abolished this current, indicating cross-desensitization between the glutamate and slow GABA responses. High concentrations of GABA (3 × 10 −2 M) did not activate the glutamate receptor coupled to the large cation channel. The results suggest that, in Aplysia neurons, the glutamate receptor-Cl − channel complex has some similarities to the GABA receptor-Cl − channel complex.

Cross-desensitization Reveals Pharmacological Specificity of Excitatory Amino Acid Receptors in Isolated Hippocampal Neurons.

Ionic currents elicited by excitatory amino acids were studied, using the concentration clamp method, in enzymatically isolated rat hippocampal neurons. Cross-desensitization between the responses to various agonists was applied to separate the activity of two types of receptors, N-methyl-d-aspartate (NMDA) and non-NMDA. NMDA receptors were selectively activated by NMDA, l- and d-aspartate, d-glutamate and quinolinate. Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate appeared to be selective, and quisqualate relatively less selective non-NMDA agonists, acting on the same receptor type. l-Glutamate, l- and d-homocysteate activated both receptor types. It is supposed that two receptor sites, activation site and desensitization site, control the action of agonists at the non-NMDA receptor. When examined in the cross-desensitization experiments, NMDA and non-NMDA receptors appear to be represented by the two homogeneous and independent receptor populations operating different ionic channels.

Development of L‐glutamate‐ and glycine‐activated currents in spinal cord neurones during early chick embryogenesis.

1. The membrane currents elicited by L-glutamate and glycine applications in morphologically different neurone types were investigated in isolated spinal cord cells from the lumbar enlargement of 6 to 11-day-old chick embryos. The whole-cell patch-clamp technique and concentration clamp methods have been used. Isolated spinal cord neurones of four stages were investigated: 6, 7.5, 9 and 11 days of incubation (29th, 32nd, 35th and 37th stages of development, respectively). 2. The L-glutamate-activated conductance consisted of desensitizing and non-desensitizing components. The Hill coefficient for the first component was 1, and for the second was 2. The number of cells responding to L-glutamate application with only a desensitizing component decreased from 53.4% on the 6th day of incubation to 6.7% on the 11th day, whereas the number of cells responding with bicomponent responses increased during the same period from 13.3 to 87%. 3. From the 6th and 11th day of chick embryo development the characteristics of the desensitizing component of L-glutamate-activated conductance remained constant (half-maximal dose, (ED50 = 2.6 +/- 0.3 mM) whereas the ED50 for the non-desensitizing component decreased 10 times. 4. It was found that the density of the desensitizing L-glutamate-activated ionic current increased during morphological and age-dependent differentiation of spinal cord cells. 5. Of the investigated cells 88.7% were sensitive to glycine application. The smallest percentage of neurones responding to glycine application was observed at 7.5 days of incubation. Glycine-activated conductance did not change at the investigated stages (ED50, 71 +/- 2 microM; Hill coefficient, 2). 6. A significant decrease in the glycine-activated current density was observed on the 9th day of incubation in multipolar neurones with three neurites, against a tendency to an increase of glycine-activated ionic currents during morphological and age-dependent differentiation of cells. 7. Of the investigated cells 78% were sensitive to both glycine and L-glutamate application. A negative correlation between glycine- and L-glutamate-activated current densities (correlation coefficient, -0.71) was revealed by means of statistical analysis. 8. We conclude that these changes in chemosensitivity of the spinal cord neurones may underlie the increase in locomotor activity of the chick embryo observed on the 9th day of incubation.

Inhibition of nicotinic acetylcholine response by atropine in frog isolated sympathetic neurons

The effect of atropine on nicotinic acetylcholine (ACh) response was studied in frog isolated sympathetic ganglion cells using a ‘concentration clamp’ technique which combines intra-cellular perfusion with a rapid external solution change within 3 ms. When atropine (more than 6 × 10 −7 M) was simultaneously applied to neurons with ACh (6 × 10 −6 M), the current amplitude was instantaneously reduced in a dose-dependent fashion. Further decreases in the current amplitude were observed in a time-dependent manner during about 20 min after the start of drug-application. Thereafter the current amplitude gradually restored toward the control level in the following 90–120 min in spite of the continuous presence of atropine. However, because d-tubocurarine greatly inhibited the ‘restored’ current, the last-mentioned is suggested be also mediated by nicotinic ACh receptor-ionophore complex. Therefore, the inhibitory action of atropine on the peak amplitude of ACh response was examined at 15–20 min after adding the agent. It was dose-dependent but not voltage-dependent. Respective concentrations of atropine and d-tubocurarine causing half the maximum inhibition (IC 50) of the peak current evoked by ACh (6 × 10 −6 M) were 1.8 × 10 −5 M and 1.8 × 10 −6 M. Thus, the inhibitory potency was 10 times less tha of d-tubocurarine, an antagonist of nicotinic ACh receptors. The blockade of ACh response by d-tubocaririne was competitive while that by atropine was non-competitive. The current elicited by Ach consisted of two (fast and low) exponentiald components plus a steady0state one in the control period. Atropine (3 × 10 −6 M) greatly reduced the amplitude of exponential components but it exerted a small effect on the steady-state one. Therefore, the changes in current kinetics during the application of atropine may be due to a different sensitivity of each component to atropine.

Interaction between pentobarbital and GABA-activated ionic channels in rat cerebellar neurons

Using techniques of voltage clamping at the membrane, intracellular perfusion, and concentration clamping, GABA- and barbiturate-activated currents were investigated in single neurons isolated from the rat cerebellum. The dissociation constant for interaction between GABA and GABA receptors was measured at 3±0.8 × 10−5 M. The presence of pentobarbital in the bathing solution exerts a potentiating effect on GABA-induced conductance in isolated neurons, shifting the dose-response curve for GABA towards lower concentration values without increasing peak chloride conductance. The concentrations at which GABA effects are potentiated range between 10−6–10−4 M. High concentrations of pentobarbital inhibit GABA-activated conductance; at concentrations in excess of 5 × 10−4 M, it also brings about activation of chloride conductance, depressed by bicuculline and picrotoxin, in the absence of GABA. A short-term increase in membrane conductance is produced by rapid pentobarbital washout.

Desensitization of NMDA receptors does not proceed in the presence of kynurenate

The agonist-induced steady-state desensitization of the N- methyl- d-aspartate (NMDA) receptor was investigated by means of whole cell patch and concentration clamp in isolated pyramidal neurons from rat hippocampus. When administered against a background of previously applied agonist, glycine (Gly) produced a response that was smaller than the response elicited by simultaneous application of agonist and Gly. This feature could be explained by an agonist-induced suppression of NMDA receptor sensitivity to the facilitatory action of Gly. This type of ‘steady-state desensitization’ did not develop when the preincubating solutions contained kynurenate (250 μM). It is proposed that NMDA receptors at all concentrations of agonist may acquire two distinct and interconverting conformations that are sensitive or insensitive to the facilitatory action of Gly. Kynurenate shifts the equilibrium between these states keeping the receptor in a Gly-sensitive conformation.

Piperidine discriminates between the transient and the persistent components of the ACh-induced chloride current in Aplysia neurons

ACh-induced Cl −-current ( I Cl) is well known to desensitive with two components: an initial fast phase followed by a second, more slowly developing phase. In the present study, the influence of piperidine, a normal constituent in vertebrates and invertebrates, on ACh-induced I Cl in isolated neurons of Aplysia was investigated by using the concentration clamp in combination with the voltage clamp technique. Pretreatment with piperidine in doses greater than 2 × 10 −4M depressed the transient ACh-induced I Cl but had little effect on the persistent I Cl. Kinetic study of the desensitization phase of ACh-induced I Cl showed that the slow time constant of the desensitization phase of ACh-induced I Cl was not altered by pretreatment with piperidine. The present results indicate that piperidine can discriminate between the fast transient and slow persistent components of ACh-induced I Cl in Aplysia neurons, and also suggest that two components o the desensitization phase of ACh-induced I Cl function in an independent manner.