Tonic GABA(A) receptor-mediated neurotransmission in the dorsal vagal complex regulates intestinal motility in rats

Tacrine and physostigmine were tested for direct nicotinic actions on Xenopus oocytes microinjected with Torpedo electroplaque membranes. In this preparation, responses to acetylcholine arise 6-8 h after microinjection, due to the incorporation of nicotinic receptors into the plasma membrane by a process not involving protein synthesis. Currents elicited by acetylcholine (100-1000 microM) were recorded by two-electrode voltage clamping. Tacrine (1-1000 microM) and physostigmine (1-100 microM) exerted a potent, reversible block of the nicotinic receptors. The concentration-dependence curves fitted simple hyperbolas, suggesting a stoichiometry of 1:1 in the drug-channel interactions. Currents elicited by the highest acetylcholine concentration were inhibited by tacrine with maximal affinity, indicating an action at a site other than the ligand-binding domain. Inhibition was reduced at depolarising potentials, which is consistent with a preferential interaction with the ligand-bound form of the receptor. Blockade by tacrine or physostigmine was accompanied by a concentration-dependent slowing of the desensitisation, resembling the action of local anaesthetics. These results could indicate a modulatory effect of these drugs on neurosecretion through nicotinic receptors.     

Human high affinity, Na(+)-dependent L-glutamate/L-aspartate transporter GLAST-1 (EAAT-1): gene structure and localization to chromosome 5p11-p12

Controversies remain over which ion channels are the most sensitive to ethanol. We have found that ethanol potently modulates the neuronal nicotinic acetylcholine receptor-channel at micromolar concentrations with an EC50 of 88.5 microM, which is significantly lower than most values previously reported for other ion channels. Prolonged application of ethanol accelerated the decay phase of acetylcholine-induced currents, caused single-channels to open in bursts, and shortened the mean open time, all of which reflect increased receptor desensitization. However, ethanol slowed the decay phase of the current induced by a brief application of acetylcholine, which may indicate that ethanol manifests its action by causing an increase in the affinity of the receptor for acetylcholine. These results suggest that neuronal nicotinic acetylcholine receptors may be important target sites of ethanol, particularly in the early stages of ethanol intoxication.     

Inhibition by anaesthetics of 14C-guanidinium flux through the voltage-gated sodium channel and the cation channel of the 5-HT3 receptor of N1E-115 neuroblastoma cells

The influence of local and general anaesthetics on cation influx through the fast, voltage-dependent sodium channel and the 5-HT3 receptor cation channel was studied in N1E-115 mouse neuroblastoma cells by measuring 2-min influx of the organic cation 14C-guanidinium induced by either veratridine (1 mmol/l) or 5-HT (100 mumol/l). The veratridine-induced influx of 14C-guanidinium was potentiated by scorpion toxin and inhibited by tetrodotoxin. The 5-HT-induced 14C-guanidinium influx was not affected by tetrodotoxin but it was inhibited by nanomolar concentrations of the selective 5-HT3 receptor antagonists ondansetron and ICS 205-930; at high micromolar concentrations these compounds also inhibited the veratridine-induced influx of 14C-guanidinium. The 14C-guanidinium influx through both channels was inhibited by local and general anaesthetics. The rank order of potency for inhibition of veratridine-induced influx by local anaesthetics was tetracaine > bupivacaine > cocaine > lidocaine > procaine and that for inhibition of the 5-HT3 receptor channel was tetracaine > bupivacaine > cocaine > procaine > lidocaine. With the exception of procaine and cocaine, which were equipotent at both channels, the local anaesthetics were 4.4-fold (lidocaine) to 25-fold (tetracaine) more potent at the fast sodium channel than at the 5-HT3 receptor channel. The rank order of potency for general anaesthetics was propofol > etomidate = alfaxalone = ketamine > thiopental = methohexital at the fast sodium channel, and propofol > or = etomidate > alfaxalone = methohexital > thiopental > ketamine at the 5-HT3 receptor channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of nicotinic receptor-mediated responses in bovine chromaffin cells by diltiazem

1. The effects of diltiazem on various functional parameters were studied in bovine cultured adrenal chromaffin cells stimulated with the nicotinic receptor agonist dimethylphenylpiperazinium (DMPP) or with depolarizing Krebs-HEPES solutions containing high K+ concentrations. 2. The release of [3H]-noradrenaline induced by DMPP (100 microM for 5 min) was gradually and fully inhibited by increasing concentrations of diltiazem (IC50 = 1.3 microM). In contrast, the highest concentration of diltiazem used (10 microM) inhibited the response to high K+ (59 mM for 5 min) by only 25%. 3. 45Ca2+ uptake into cells stimulated with DMPP (100 microM for 1 min) was also blocked by diltiazem in a concentration-dependent manner (IC50 = 0.4 microM). Again, diltiazem blocked the K(+)-evoked 45Ca2+ uptake (70 mM K+ for 1 min) only by 20%. In contrast, the N-P-Q-type Ca2+ channel blocker omega-conotoxin MVIIC depressed the K+ signal by 70%. In the presence of this toxin, diltiazem exhibited an additional small inhibitory effect, indicating that the compound was acting on L-type Ca2+ channels. 4. Whole-cell Ba2+ currents through Ca2+ channels in voltage-clamped chromaffin cells were inhibited by 3-10 microM diltiazem by 20-25%. The inhibition was readily reversed upon washout of the drug. 5. The whole-cell currents elicited by 100 microM DMPP (IDMPP) were inhibited in a concentration-dependent and reversible manner by diltiazem. Maximal effects were found at 10 microM, which reduced the peak IDMPP by 70%. The area of each curve represented by total current (QDMPP) was reduced more than the peak current. At 10 microM, the inhibition amounted to 80%; the IC50 for QDMPP inhibition was 0.73 microM, a figure close to the IC50 for 45Ca2+ uptake (0.4 microM) and [3H]-noradrenaline release (1.3 microM). The blocking effects of diltiazem developed very quickly and did not exhibit use-dependence; thus the drug blocked the channel in its closed state. The blocking effects of 1 microM diltiazem on IDMPP were similar at different holding potentials (inhibition by around 30% at -100, -80 or -50 mV). Diltiazem did not affect the current flow through voltage-dependent Na+ channels. 6. These data are compatible with the idea that diltiazem has little effect on Ca2+ entry through voltage-dependent Ca2+ channels in bovine chromaffin cells. Neither, does diltiazem affect INa. Rather, diltiazem acts directly on the neuronal nicotinic receptor ion channel and blocks ion fluxes, cell depolarization and the subsequent Ca2+ entry and catecholamine release. This novel effect of diltiazem might have clinical relevance since it might reduce the sympathoadrenal drive to the heart and blood vessels, thus contributing to the well established antihypertensive and cardioprotective effects of the drug.     

Open channel block and alteration of N-methyl-D-aspartic acid receptor gating by an analog of phencyclidine

We investigated inhibition of the N-methyl-D-aspartic acid (NMDA) receptor-channel complex by N-ethyl-1,4,9, 9alpha-tetrahydro-4alphaR-cis-4alphaH-fluoren-++ +4alpha-amine (NEFA), a structural analog of phencyclidine (PCP). Using the whole-cell recording technique, we demonstrated that NEFA inhibits NMDA responses with an IC50 of 0.51 microM at -66 mV. We determined that NEFA binds to the open channel, and subsequently the channel can close and trap the blocker. Once the channel has closed, NEFA is unable to dissociate until the channel reopens. Single-channel recordings revealed that NEFA reduces the mean open time of single NMDA-activated channels in a concentration-dependent manner with a forward blocking rate (k+) of 39.9 microM-1 s-1. A computational model of antagonism by NEFA was developed and constrained using kinetic measurements of single-channel data. By multiple criteria, only models in which blocker binding in the channel causes a change in receptor operation adequately fit or predicted whole-cell data. By comparing model predictions and experimental measurements of NEFA action at a high NMDA concentration, we determined that NEFA affects receptor operation through an influence on channel gating. We conclude that inhibition of NMDA receptors by PCP-like blockers involves a modification of channel gating as well as block of current flow through the open channel.     

Benzodiazepine modulation of recombinant alpha1beta3gamma2 GABA(A) receptor function efficacy determination using the Cytosensor microphysiometer

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC50 values of 1.2 +/- 0.03 microM (nH = 0.84 +/- 0.02) and 1.2 +/- 0.1 microM (nH = 1.1 +/- 0.1), respectively. Tamoxifen could also block native 5-HT3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC50 = 0.81 +/- 0.15 microM and nH of 1.3 +/- 0.3. The characteristics of block by tamoxifen at the 5-HT3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.     

The effects of propofol and enflurane on single calcium channel currents of guinea-pig isolated ventricular myocytes

1. The effects of the anaesthetics, propofol (100 microM) and enflurane (3%, 1.46 mM), on single L type calcium channel currents were investigated in single myocytes isolated from guinea-pig ventricles. Channel activity was recorded from membrane patches by use of the 'cell-attached' patch-clamp technique (pipette solution containing 110 mM BaCl2, 5 microM Bay K 8644, 5 microM HEPES, pH 7.4; temperature 36 degrees C). 2. Channel conductance was calculated from the slope of the relationship between single channel current and membrane potential during step depolarizations to activate the channel over a range of approximately -20 to +20 mV. Neither propofol (6 cells) nor enflurane (7 cells) caused any significant reduction in channel conductance. 3. Both propofol (7 cells) and enflurane (9 cells) decreased the probability of the channel being open during depolarizations to +10 mV (measured from histograms of the fraction of time spent by the channel at different current levels, taking areas under the Gaussian curves fitted to the open and closed components of the distributions to represent the proportion of time spent in the two states). 4. A fraction of the current traces showed no detectable channel openings in response to step depolarizations to +10 mV. Both propofol and enflurane significantly increased the fraction of silent traces. 5. Transitions across a threshold halfway between the open and closed levels were used to define periods spent in the open and closed states. Both propofol (7 cells) and enflurane (9 cells) reduced the mean open times and increased the mean closed times of the calcium channel. 6. Histograms were plotted showing the distributions of times spent by the channels in the open and closed states. Two exponentials were fitted to the open and closed time distributions. Both propofol (7 cells) and enflurane (9 cells) shortened both time constants fitted to the open times and lengthened both time constants fitted to the closed times.7. It is concluded that both propofol and enflurane appear to alter the kinetics of opening and closing of calcium channels to favour shut channels without altering channel conductance. This effect would be expected to result in a reduction of the macroscopic calcium current and thus contribute to the negative inotropic action of these anaesthetics.     

Effect of physical activity as a caddie on ultrasound measurements of the Os calcis: a cross-sectional comparison

Human epidermal keratinocytes synthesize, secrete, and degrade acetylcholine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetyl-choline. Because acetylcholine modulates transmembrane Ca2+ transport and intracellular metabolism in several types of cells, we hypothesized that cholinergic agents might have similar effects on keratinocytes. Nicotine increased in a concentration-dependent manner the amount of 45Ca2+ taken up by keratinocytes isolated from human neonatal fore-skins. This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinocyte nicotinic acetylcholine receptor(s). The sequences encoding the alpha 5 and alpha 7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes. These subunits, as well as the alpha 3, beta 2, and beta 4 subunits previously found in keratinocytes, can be components of Ca(2+)-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the rate of cell differentiation, we measured the nicotinic cholinergic effects on the expression of differentiation markers by cultured keratinocytes. Long-term incubations with micromolar concentrations of nicotine markedly increased the number of cells forming cornified envelopes and the number of cells staining with antibodies to suprabasal keratin 10, transglutaminase type I, involucrin, and filaggrin. The increased production of these differentiation-associated proteins was verified by Western blotting. Because nicotinic cholinergic stimulation causes transmembrane Ca2+ transport into keratinocytes, and because changes in concentrations of intracellular Ca2+ are known to alter various keratinocyte functions, including differentiation, the subcellular mechanisms mediating the autocrine and paracrine actions of epidermal acetylcholine on keratinocytes may involve Ca2+ as a second messenger.     

Two types of ACh receptors contribute to fast channel gating on mouse skeletal muscle

Single-channel recordings from mouse C2 myotubes indicate that maturation of skeletal muscle is accompanied by the appearance of two types of fast acetylcholine (ACh) receptor channels that are each functionally distinct from the embryonic receptor type present at early stages of differentiation. The embryonic receptor type has a low conductance (45 pS) and long channel open time, rendering slowly decaying synaptic currents. One fast channel type that appears during muscle maturation is distinguished from the embryonic receptor type on the basis of both higher conductance (65 pS) and shorter open time. However, single-channel recordings from differentiated mouse skeletal muscle cell line (C2) point to the existence of a second fast receptor type, which has a conductance similar to the embryonic receptor type (45 pS), yet significantly reduced mean channel open time. Analyses of individual channel function at high ACh concentrations directly demonstrate the coexistence of two kinetically distinct types of 45 pS ACh receptors. Openings by fast type and slow embryonic type of 45 pS receptors occurred in bursts, allowing distinction on the basis of both mean open time and open probability for individual receptors. The embryonic type of 45 pS receptor has an open time approximately twofold longer than the fast-receptor counterpart. Additional differences were reflected in the open probability distributions for fast and slow 45 pS receptor types. Both types of 45 pS receptor were kinetically distinguishable from the 65 pS receptor. We found no support for the idea that the slow and fast 45 pS receptor types result from the interconversion of dual gating modes involving the same receptor protein. Our results are consistent with the idea that the acquisition of fast synaptic current decay, required at mature neuromuscular synapses, is the result of the up-regulation of two distinct fast types of nicotinic ACh receptors during skeletal muscle development.     

Potentiation and inhibition of neuronal nicotinic receptors by atropine: competitive and noncompetitive effects

Atropine, the classic muscarinic receptor antagonist, inhibits ion currents mediated by neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. At the holding potential of -80 mV, 1 microM atropine inhibits 1 mM acetylcholine-induced inward currents mediated by rat alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7 nicotinic receptors by 12-56%. Inward currents induced with a low agonist concentration are equally inhibited (alpha3beta2, alpha3beta4), less inhibited (alpha2beta4, alpha7), or potentiated (alpha4beta2, alpha4beta4) by 1 microM atropine. Effects on the more sensitive alpha4beta4 nicotinic receptors were investigated in detail by systematic variation of acetylcholine and atropine concentrations and of membrane potential. At high agonist concentration, atropine inhibits alpha4beta4 nicotinic receptor-mediated ion current in a noncompetitive, voltage-dependent way with IC50 values of 655 nM at -80 mV and of 4.5 microM at -40 mV. At low agonist concentration, 1 microM atropine potentiates alpha4beta4 nicotinic receptor-mediated ion current. This potentiating effect is surmounted by high concentrations of acetylcholine, indicating a competitive interaction of atropine with the nicotinic receptor, and potentiation is also reversed at high atropine concentrations. Steady state effects of acetylcholine and atropine are accounted for by a model for combined receptor occupation and channel block, in which atropine acts on two distinct sites. The first site is associated with noncompetitive ion channel block. The second site is associated with competitive potentiation, which appears to occur when the agonist recognition sites of the receptor are occupied by acetylcholine and atropine. The apparent affinity of atropine for the agonist recognition sites of the alpha4beta4 nicotinic acetylcholine receptor is estimated to be 29.9 microM.     

Drug interactions with neuromuscular blockers

Drugs administered to patients undergoing anaesthesia may complicate the use of the neuromuscular blockers that are given to provide good surgical conditions. The various sites of interaction include actions on motor nerve conduction and spinal reflexes, acetylcholine (ACh) synthesis, mobilisation and release, sensitivity of the motor end plate to ACh and the ease of propagation of the motor action potential. In addition, many drugs affect the pharmacokinetics of neuromuscular blockers, especially as most drugs depend to a greater or lesser extent upon renal excretion. The clinically significant interaction between nondepolarisers and depolarisers may be due to blockade of the pre-synaptic nicotinic receptors by the depolarisers, leading to decreased ACh mobilisation and release. Synergism between nondepolarisers probably results from post-synaptic receptor mechanisms. Volatile anaesthetic agents affect the sensitivity of the motor end-plate (post-synaptic receptor blockade) in addition to having effects on pre-synaptic nicotinic function. The effects of nondepolarisers are likely to be potentiated and their action prolonged by large doses of local anaesthetics due to depression of nerve conduction, depression of ACh formation, mobilisation and release, decreases in post-synaptic receptor channel opening times and reductions in muscular contraction. Most antibacterials have effects on pre-synaptic mechanisms. Procainamide and quinidine principally block nicotinic receptor channels. Magnesium has a marked inhibitory effect on ACh release. Calcium antagonists could theoretically interfere with neurotransmitter release and muscle contractility. Phenytoin and lithium decrease ACh release, whilst corticosteroids and furosemide (frusemide) tend to increase the release of the transmitter. Ecothiopate, tacrine, organophosphates, propanidid, metoclopramide and bambuterol depress cholinesterase activity and prolong the duration of the neuromuscular block. The probability of clinically significant interactions increases in patients receiving several drugs with possible effects on neuromuscular transmission and muscle contraction.     

The effects of temperature on the interactions between volatile general anaesthetics and a neuronal nicotinic acetylcholine receptor

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were investigated under two-electrode voltage clamp. Neuronal nicotinic acetylcholine receptor (AChR) currents were studied at low acetylcholine concentrations (< or = 200 nM). 2. Inhibition of the ACh-induced currents by three volatile general anaesthetics (halothane, isoflurane and methoxyflurane) and the specific inhibitor (+)-tubocurarine was studied as a function of temperature (over the range 4-25 degrees C). 3. The inhibition by the volatile anaesthetics increased (inhibition constants decreased) with decreasing temperature while the inhibition by (+)-tubocurarine did not change significantly near room temperature, but decreased at lower temperatures. The (+)-tubocurarine inhibition appeared to be competitive in nature and showed no significant voltage-dependence. 4. The van't Hoff plots (logarithms of the dissociation constants against reciprocal absolute temperature) were linear for the anaesthetics, but markedly non-linear for (+)-tubocurarine. From these plots, values for the changes in the standard Gibbs free energy delta G degrees water-->AChR, enthalpy delta H degree water-->AChR, entropy delta S degree water-->AChR and heat capacity delta Cp degree water-->AChR were determined. Tubocurarine was found to bind very much tighter to the receptor than the volatile anaesthetics due, entirely, to a favourable increase in entropy on binding. 5. A comparison between the temperature-dependence of the anaesthetic inhibition of the ACh receptor and that of general anaesthetic potencies in animals indicates that the temperature-dependence of animal potencies might be simply accounted for in terms of changes in anaesthetic/receptor binding.     

Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique

Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or -closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned.     

Effects of Zn2+ on wild and mutant neuronal alpha7 nicotinic receptors

Zn2+ is a key structural/functional component of many proteins and is present at high concentrations in the brain and retina, where it modulates ligand-gated receptors. Therefore, a study was made of the effects of zinc on homomeric neuronal nicotinic receptors expressed in Xenopus oocytes after injection of cDNAs encoding the chicken wild or mutant alpha7 subunits. In oocytes expressing wild-type receptors, Zn2+ alone did not elicit appreciable membrane currents. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by Zn2+ in a reversible and dose-dependent manner, with an IC50 of 27 microM and a Hill coefficient of 0.4. The inhibition of IAcCho by Zn2+ was competitive and voltage-independent, a behavior incompatible with a channel blockade mechanism. In sharp contrast, in oocytes expressing a receptor mutant, with a threonine-for-leucine 247 substitution (L247Talpha7), subnanomolar concentrations of Zn2+ elicited membrane currents (IZn) that were reversibly inhibited by the nicotinic receptor blockers methyllycaconitine and alpha-bungarotoxin. Cell-attached single-channel recordings showed that Zn2+ opened channels that had a mean open time of 5 ms and a conductance of 48 pS. At millimolar concentrations Zn2+ reduced IAcCho and the block became stronger with cell hyperpolarization. Thus, Zn2+ is a reversible blocker of wild-type alpha7 receptors, but becomes an agonist, as well as an antagonist, following mutation of the highly conserved leucine residue 247 located in the M2 channel domain. We conclude that Zn2+ is a modulator as well as an activator of homomeric nicotinic alpha7 receptors.     

Dynamic properties of Na+ ions in models of ion channels: a molecular dynamics study

We present simulation results for the effective diffusion coefficients of a sodium ion in a series of model ion channels of different diameters and hydrophobicities, including models of alamethicin, a leucine-serine peptide, and the M2 helix bundle of the nicotinic acetylcholine receptor. The diffusion coefficient, which in the simulations has a value of 0.15(2) A2ps-1 in bulk water, is found to be reduced to as little as 0.02(1) A2ps-1 in the narrower channels, and to about 0.10(5) A2ps-1 in wider channels such as the nicotinic acetylcholine receptor. It is anticipated that this work will be useful in connection with calculations of channel conductivity using such techniques as the Poisson-Nernst-Planck equation, Eyring rate theory, or Brownian dynamics.     

The agonistic action of pentobarbital on GABAA beta-subunit homomeric receptors

Murine gamma-aminobutyric acid type A (GABAA) receptor beta 1, beta 2, and beta 3 subunits were expressed in Xenopus oocytes and studied using the two electrode voltage clamp technique. Although all three beta-subunits were unresponsive to GABA when expressed as homomers, the intravenous general anaesthetics pentobarbital, etomidate and propofol induced currents in beta 2 and beta 3 homomers. The pentobarbital-induced currents in beta 3 homomers showed a dose dependence with an ED50 of 89 +/- 8.9 microM and a Hill coefficient of 0.94 +/- 0.08. Zinc (50 microM) blocked (61.1 +/- 5.6% of control) and 200 microM lanthanum potentiated (139 +/- 8.6% of control) the pentobarbital-induced current. This current was also blocked by picrotoxin but was insensitive to the GABAA receptor antagonist bicuculline. These observations indicate that the full expression of the agonistic action of GABA requires the presence of an alpha-subunit, in contrast to the agonistic action of intravenous general anesthetics, where the presence of a beta2 or beta 3-subunit is sufficient. The difference in the agonistic action of intravenous anaesthetics among these highly homologous beta-subunits suggests that the beta-subunit homomeric receptors may be useful to further define the molecular sites of action of intravenous general anaesthetics and other functional domains on GABAA receptors.     

Differential effects of physostigmine and organophosphates on nicotinic receptors in neuronal cells of different species

The effects of the carbamate physostigmine and of the organophosphates (OPs) parathion, paraoxon and phenyl saligenin cyclic phosphate (PSP) were examined on different subtypes of neuronal nicotinic acetylcholine receptors (nAChR). Stimulation with 1 mM ACh induced transient nicotinic inward currents in mouse N1E-115 and human SH-SY5Y neuroblastoma and in locust thoracic ganglion cells. All four acetylcholinesterase (AChE) inhibitors reduced the nicotinic currents in a concentration-dependent manner. Parathion is about 50 times more potent in blocking nAChR, compared to its active AChE inhibiting metabolite paraoxon. The relative blocking potency of the different AChE inhibitors was the same in all cell types, and followed the order parathion > physostigmine > PSP > paraoxon. In N1E-115 cells the IC50 values of block amounted to 2 microM, 30 microM, 39 microM and 96 microM for parathion, physostigmine, PSP and paraoxon, respectively. In all cell types, the nicotinic currents were equally blocked by parathion. Human nAChR in SH-SY5Y cells appeared more sensitive to block by physostigmine, PSP and paraoxon, while these AChE inhibitors similarly inhibited nicotinic currents in insect cells and in mouse neuroblastoma cells. The observation that the concentration-dependence of block is different from that of AChE inhibition, indicates a distinct interaction of AChE inhibitors with nAChR. Only in locust cells physostigmine induced a non-desensitizing inward current, that appeared to originate from nAChR activation. Occasionally, the OPs were able to activate slow ionic currents in mouse, but not in human and locust cells. As the OP-induced agonistic activity in mouse cells was not associated with the blocking action, the target site appeared to be distinct from nAChR. These results show that AChE inhibitors block nAChR with different potencies, dependent on the compound and the receptor subtype, and may activate distinct ion currents in neuronal cells of different species origin.     

Block of the endplate acetylcholine receptor channel by the sympathomimetic agents ephedrine, pseudoephedrine, and albuterol

Recent observations suggest that some patients with congenital myasthenic syndromes respond favorably to ephedrine, pseudoephedrine, or albuterol. Conventional microelectrode studies, however, provide no clear explanation for a beneficial effect of ephedrine in endplate diseases. To gain further insight into how these drugs affect neuromuscular transmission, we investigated their effects on the kinetic properties of the acetylcholine (ACh) receptor. Single channel currents were recorded from rat lumbrical muscles endplates using low concentrations of ACh and 2.5-100 microM of drugs. Between 10-100 microM, each drug progressively increased the rate of channel closure in a concentration dependent manner, consistent with an open-channel block. Albuterol acted as a sequential fast-acting channel blocker, increasing the mean burst duration in a concentration dependent manner without altering the total open time per burst or the duration of intraburst blockages. Increasing concentrations of ephedrine and pseudoephedrine also increased the number of intraburst closures but decreased the total open time per burst. None of the drugs altered single channel conductance. The channel blocking effects of ephedrine and pseudoephedrine might reduce the synaptic overactivity that occurs in the slow-channel myasthenic syndromes or in endplate ACh esterase deficiency, but these effects occur at concentrations not attainable in clinical practice.     

Cyanotriphenylborate: subtype-specific blocker of glycine receptor chloride channels

The inhibitory glycine receptor is a ligand-gated ion-channel protein existing in different homo- and heterooligomeric isoforms. Here we show that the chloride channel of the recombinant alpha 1-subunit homooligomeric glycine receptor is efficiently blocked by cyanotriphenylborate (CTB) with a concentration effecting 50% inhibition (IC50) of 1.3 microM in the presence of 50 microM glycine. The antagonistic effect of CTB is noncompetitive, use dependent, and more pronounced at positive membrane potentials, suggesting open-channel block. In contrast to alpha 1-subunit receptors, alpha 2-subunit homooligomers are resistant to CTB (IC50 > 20 microM). By exchanging the channel-lining transmembrane segment M2 of the alpha 1 polypeptide by that of the alpha 2 polypeptide, we could transfer this resistance to alpha 1 channels, indicating that a single glycine residue at position 254 of the alpha 1 subunit is critical for CTB sensitivity. The blocker did not affect the cation-selective channel of the nicotinic acetylcholine receptor. Thus, CTB may prove useful as a tool to probe the subunit structure of native glycine receptors in mammalian neurons.     

Erosion and controlled release properties of semisolid vesicular phospholipid dispersions

Single adult guinea-pig and rat ventricular cardiac myocytes were used to study the effects of two members of the omega3 class of polyunsaturated fatty acids, docosahexaenoic acid and eicosapentaenoic acid, on the electrical and mechanical activity of cardiac muscle. Docosahexaenoic acid and eicosapentaenoic acid reduced the electrical excitability of both guinea-pig and rat cells in a dose-dependent manner. Both agents produced a dose-dependent negative inotropic response in guinea-pig cells but in the rat cells there was first a dose-dependent positive inotropic effect at low concentrations (< 10 microM) followed by a negative inotropic effect at higher concentrations (> 10 microM). Possible mechanisms by which these agents affect contraction were studied using conventional electrophysiological techniques. The polyunsaturated fatty acids reduced the action potential duration and the plateau potential of the guinea-pig cells in a simple, dose-dependent manner. In contrast, the effect on the rat action potential mirrored the inotropic effect. At low concentrations (< 10 microM) there was a concentration-dependent increase in action potential duration followed by a concentration-dependent decrease at higher concentrations (> 10 microM). Both polyunsaturated fatty acids decreased the fast Na+ current and the L-type Ca2+ current in a concentration-dependent but not use-dependent manner in cells from both species. In the rat cells these agents inhibited the transient outward current resulting in an increase in the duration of the rat action potential. The effects of polyunsaturated fatty acids on the Ca2+, Na+ and K+ currents underlie these changes in the action potentials in guinea-pig and rat heart cells. The effects on the L-type Ca2+ current and action potential duration can also explain both the simple negative inotropic effects of the agents on the guinea-pig cells and the more complex effects on the rat cells. These effects of polyunsaturated fatty acids on membrane currents may account for their anti-arrhythmic properties.