Biomedical subjects

D A Saint

Publications and source records attributed to D A Saint.

At least 37 records · Page 2Linked to original sources

PubMed1995

The temperature dependence of conductance of the sodium channel: implications for mechanisms of ion permeation.

Voltage-gated sodium channel currents were recorded in cell-attached and inside-out membrane patches from rat ventricular myocytes at temperatures ranging from 4 degrees C to 36 degrees C. The effects of changes in temperature on channel conductance were accurately determined using a digital signal processing technique based on hidden Markov models. We show that the cardiac sodium channel has multiple conductance levels, with the most frequently observed sublevel being either 11 pS or 22 pS at room temperature in 280 mM Na+, depending on the dissociation procedures adopted. The single channel current-voltage relationship is ohmic at all of the temperatures studied. The conductance increases steeply with temperature, with Q10 ranging from 1.4 to 1.5. The proportional change in channel conductance with increasing temperature was greater than the corresponding change in bulk conductivity of electrolyte solutions, suggesting that an ion traversing the channel needs to surmount a small additional energy barrier. An activation energy deduced from a plot of the logarithm of single channel conductance against the inverse of temperature is about 28 kJ mole-1. We provide one possible interpretation of the observed conductance-temperature relationship in terms of the details of the microscopic interactions operating between the protein wall, ions and water molecules.

Animals↗

PubMed1994

An analysis of cardiac sodium channel properties using digital signal processing techniques.

The properties of single sodium channels in membrane patches from isolated rat ventricular myocytes were analyzed. Both cell-attached and inside-out patch configurations were examined. A digital signal processing method was used which allowed accurate determination of single-channel conductance and mean open time, even in situations where many channels coexisted in the patch. The results show that the cardiac sodium channel has a conductance of 5.3 pS at room temperature, and shows no rectification or saturation in the physiological ranges of ion concentration and membrane potential. The active channels contained in the membrane patch opened and closed independently of each other. A transition probability matrix, which describes transitions between all current levels, can also be generated, analysis of which permits an estimate of individual mean channel open time as well as the degree of coupling between channels.

Animals↗

PubMed1994

An electrophysiological basis for the antiarrhythmic actions of the kappa-opioid receptor agonist U-50,488H.

This study examined the actions of the selective kappa-opioid receptor agonist, U-50,488H, on voltage activated Na+ and K+ currents in isolated rat cardiac myocytes. U-50,488H produced a concentration-dependent block of the transient Na+ current with an ED50 of about 15 microM, and, at higher concentrations (40-50 microM), a block of the plateau K+ current and an increase in the rate of decay of the transient K+ current. In addition U-50,488H produced a hyperpolarising shift in the inactivation curve for the transient Na+ current without altering the voltage dependence for activation and without an effect on the voltage dependence of inactivation or activation of K+ currents. The block of Na+ currents by U-50,488H showed pronounced use dependence. The kappa-opioid receptor antagonist MR2266 did not itself produce any change in the Na+ or K+ currents and did not change the channel blocking properties of U-50,488H. Thus, since the antiarrhythmic actions of U-50,488H are not blocked by MR2266 or naloxone, the effects of U-50,488H to block Na+ and K+ currents are the most likely reasons for its antiarrhythmic actions, rather than an action at kappa-opioid receptors.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1994

Inactivation-resistant channels underlying the persistent sodium current in rat ventricular myocytes.

Single-channel sodium currents that could be blocked with TTX were elicited by depolarizing voltage pulses in either cell-attached or inside-out patches from rat ventricular myocytes. A transient burst of channels was followed by late-opening (persistent) channels with low open probability. Conditioning depolarizing pre-pulses that inactivated transient channels and 'chattering' late-opening channels had no effect on persistent channels. The open probability of persistent channels reached a maximum at more negative potentials than transient channels. Between -70 mV and -40 mV, the average open time of persistent channels increased, whereas the average open time of transient channels did not change significantly, so the open times of the two channels diverged as the potential became more positive. The conductance of transient and persistent channels was similar, and the conductance of both kinds of channel increased at more depolarized potentials.

Animals↗

PubMed1993

Electrophysiological and antiarrhythmic actions of the kappa agonist PD 129290, and its R,R (+)-enantiomer, PD 129289.

1. The S,S (-)-enantiomer PD 129290, a kappa agonist, and its corresponding inactive R,R (+)-enantiomer (PD 129289) were studied in rat isolated hearts and in intact rats for cardiovascular and antiarrhythmic actions. The electrophysiological actions of PD 129290 were also studied in rat isolated cardiac myocytes using voltage clamp. 2. Ventricular pressure, heart rate and ECG were studied in isolated hearts while blood pressure, heart rate and ECG were studied in pentobarbitone-anaesthetized rats. In the latter, responses to electrical stimulation and coronary occlusion were also investigated. 3. In isolated hearts both enantiomers, over the concentration range 2-16 microM, dose-dependently reduced systolic ventricular pressure and heart rate while prolonging the P-R and QRS intervals of the ECG. 4. At doses of 1-32 mumol kg-1 both enantiomers reduced blood pressure and heart rate in anaesthetized rats. In addition, both enantiomers increased the size of the RSh and increased P-R, QRS, and Q-T intervals of the ECG. The thresholds for premature beats and ventricular fibrillation were dose-dependently increased by PD 129289. At lower doses PD 129290 decreased thresholds. These decreases were blocked by naloxone to reveal underlying increases similar to those seen with PD 129289. Both enantiomers increased refractory periods. 5. Naloxone (8 mumol kg-1) did not alter any of the actions of PD 129290, except to abolish the initial decreases in thresholds in intact rats seen with lower doses of PD 129290. 6. Both enantiomers (2 and 8 mumol kg-1) equally reduced arrhythmias in anaesthetized rats subject to occlusion of a coronary artery. 7. In rat isolated cardiac myocytes 20 microM PD 129290, in the presence and absence of naloxone decreased the amplitude of the transient sodium current by about 50% without affecting the voltage dependence of activation or inactivation of this current.8. The antiarrhythmic actions of both enantiomers appear to primarily result from their Class I(sodium channel blockade) properties which are independent of kappa agonism.

Animals↗

PubMed1992

The relation between transmitter release and Ca2+ entry at the mouse motor nerve terminal: role of stochastic factors causing heterogeneity.

The relation between quantal transmitter release and presynaptic Ca2+/Ba2+ entry at the mouse neuromuscular junction was studied, making use of the finding that in the presence of Ba2+ trains of nerve stimuli or brief nerve terminal depolarizations elicit "tails" of raised miniature end-plate potential frequency (fm) that reflect entry of Ba2+ per pulse, and hence effectiveness of pulses in opening Ca2+/Ba2+ channels; at the same time these pulses elicit end-plate potentials. With nerve stimulation in the presence of Ba2+ and Ca2+ and modulation of release by raised Mg2+ or bekanamycin, slopes of log quantal content (m) vs log apparent Ba2+ entry per pulse were close to 4, which is the same as the Hill coefficient for Ba2+ cooperativity derived from other data. With depolarizing pulses of varied intensity, however, similar plots gave slopes close to 2, with Ba2+ alone or in a mixture of Ca2+ and Ba2+. Thus, the relation between transmitter release and Ca2+ (or Ba2+) entry apparently depends upon how entry is varied; varying the numbers of channels opened is not the same as varying ion entry per channel. A mathematical model was developed to examine the consequences of heterogeneity of local Ca2+ (or Ba2+) between release sites, arising because of stochastic variation of number and time course of Ca2+ channels opened per site; the experimental results were consistent with this model. It was therefore concluded that release is normally governed by intracellular Ca2+ close to points of Ca2+ entry through channels; stochastic factors give rise to more release than if Ca2+ were homogeneously distributed. If Ca2+ channels are uniformly close to release sites the average number of channels opened per site per action potential may be as low as 4.

Animals↗

PubMed1992

Effects of lignocaine and quinidine on the persistent sodium current in rat ventricular myocytes.

1. The effects of the Class 1 antiarrhythmic agents lignocaine and quinidine on action potentials, and on sodium currents and potassium currents activated by depolarization, were examined in rat isolated ventricular myocytes by the whole cell, tight seal recording technique. 2. Tetrodotoxin and lignocaine shortened, whereas quinidine prolonged, the duration of the plateau phase of action potentials. 3. At low concentrations, lignocaine and quinidine blocked a persistent sodium current that was resistant to inactivation but they had only a small effect on the transient sodium current. At higher concentrations, they also blocked the transient sodium current. 4. Quinidine, but not tetrodotoxin or lignocaine, depressed potassium currents activated by depolarization and this could account for the prolongation of the plateau phase caused by quinidine. 5. It is suggested that block of the persistent sodium current may be responsible, at least in part, for the antiarrhythmic action of lignocaine and quinidine.

Action Potentials↗

PubMed1992

The effect of aliphatic alcohols on the transient potassium current in hippocampal neurones.

1. The transient potassium current was recorded in single hippocampal CA1 neurones from the rat by use of the whole-cell patch clamp technique. The effects on this current of a homologous series of aliphatic alcohols, ranging from butanol to octanol, were investigated. 2. The predominant effect of octanol (and the other alcohols) was to cause an increase in the initial rate of decay of the transient potassium current together with a slight decrease in the rate of decay of later phases of the current, such that the current decay became markedly non-monotonic. The alcohols also caused a decrease in peak current amplitude which could not be accounted for solely by the change in current decay kinetics. 3. The effect of the alcohols was concentration-dependent and readily reversible. Increasing chain length increased the potency of each alcohol by about 3 fold for each methylene group added. Other than a difference in potency, there appeared to be little difference in the action of aliphatic alcohols of different chain length on the transient current. 4. The alcohols did not appreciably change the voltage-dependence of steady state inactivation or activation of the transient potassium current. 5. The rate of inactivation of the transient current in these cells was only weakly voltage-dependent. This weak voltage-dependence was not changed by the presence of aliphatic alcohols, neither was the effect of the alcohols themselves voltage-dependent. 6. The potencies of each of the aliphatic alcohols were well correlated with their respective membrane/buffer partition coefficients, a finding which implies a hydrophobic locus of action.

Alcohols↗

PubMed1992

A persistent sodium current in rat ventricular myocytes.

1. The tight seal, whole-cell, voltage-clamp technique was used to record currents from single ventricular myocytes acutely dissociated from adult rat hearts. Subtraction of currents recorded in the presence and absence of tetrodotoxin (TTX, 50 microM) revealed a small, persistent, inward current following a much larger, transient, inward current. 2. Both currents were sodium currents because they reversed close to the sodium equilibrium potential and were depressed when choline was substituted for extracellular sodium. 3. The persistent sodium current could be recorded when the transient current had been inactivated with conditioning depolarization. Only slight inactivation of the persistent current occurred during depolarizing pulses lasting up to 900 ms. 4. A lower concentration of TTX (0.1 microM) blocked the persistent sodium current while having little effect on the transient sodium current. 5. The persistent sodium current was activated at more negative potentials than the transient sodium current. It cannot have been a window current because it was recorded at positive potentials when the transient current was completely inactivated. 6. Because the persistent and transient sodium currents had a different voltage dependence and sensitivity to TTX, it was concluded that different channels are responsible for the two currents.

Animals↗

PubMed1991

Modulation of the transient potassium current in rat hippocampal neurones by cholecystokinin.

Cholecystokinin (CCK) affects neuronal excitability in a variety of in vivo and in vitro preparations, apparently by modulating a resting potassium conductance. The data presented here show that CCK (applied as CCK8-S) also affects the transient potassium current in hippocampal neurones, by changing the voltage dependence of the inactivation and activation of the current. The way in which the voltage dependence is changed can lead to either an enhancement of the current or an attenuation, depending upon the voltage protocol used. This effect of CCK does not desensitise over a time period of minutes, and may therefore be important in controlling neuronal excitability in the CNS.

Animals↗

PubMed1990

GABAB agonists modulate a transient potassium current in cultured mammalian hippocampal neurons.

Depolarization of voltage-clamped cultured rat hippocampal neurons from holding potentials more negative than -60 mV produced a transient outward current with the characteristics of an A-current: it was 50% inactivated at a holding potential of -85 mV and blocked by 4-aminopyridine (1 mM). In the presence of GABA or baclofen (50-200 microM), with or without bicuculline, inactivation of this current was shifted to more positive potentials so that there was little inactivation at -70 mV. Activation of the A-current was also shifted to more positive potentials by these agonists, but the voltage dependence of activation of the sodium current was unaffected. If A-currents with similar properties can influence the time course of action potentials in presynaptic terminals. GABAB agonists could make action potentials briefer by potentiating the A-current and hence depress transmitter release.

4-Aminopyridine↗

PubMed1990

Effects of butanedione monoxime on neuromuscular transmission.

1. The amplitude of endplate potentials was increased by concentrations of butanedione monoxime (BDM, 5-20 mM) that typically caused muscle paralysis. 2. Although BDM slowed the decay of spontaneous miniature endplate currents, the effect was insufficient to explain most of the large increase in amplitude of endplate potentials. 3. The quantal content of endplate potentials was increased by BDM in a reversible, concentration-dependent manner. 4. The frequency of miniature endplate potentials was not changed by 10 mM BDM in the presence of normal or raised potassium concentrations, indicating that BDM does not change quantal content by a direct effect on calcium channels or on steady-state intracellular calcium concentration. 5. A change in the time course of the extracellularly recorded nerve terminal action potential caused by BDM was similar to the change produced by 4-aminopyridine (4-AP). 6. The increase in quantal content produced by BDM was only slightly reduced in the presence of 1 mM tetraethylammonium (TEA) but was significantly reduced in the presence of 0.5 to 1 mM 4-AP. 7. It was concluded that BDM blocks a 4-AP-sensitive potassium conductance in motor nerve terminals and, by increasing the duration of the action potential in this way, increases evoked transmitter release.

4-Aminopyridine↗

PubMed1989

Cholecystokinin modulates voltage dependent K+ currents in cultured rat hippocampal neurones.

Much interest has been focussed recently on neuroactive peptides originally found in the peripheral nervous system, but now, increasingly, being shown to be present in considerable amounts in the mammalian CNS. One of these peptides, cholecystokinin (CCK), is present in large amounts in higher brain areas. Immunoreactivity to CCK has been demonstrated in the mammalian hippocampus and dentate gyrus, localised in nerve terminals, and increasingly this peptide is being suggested as having a role as a transmitter in the CNS. Generally, CCK appears to produce depolarisations of neurones: e.g. mesenteric ganglion cells, and hippocampal neurones [5], although the mechanism by which it does so remains unclear, there being reports of either a decrease in input resistance, an increase, or both.

Animals↗

PubMed1989

The effects of 4-aminopyridine and tetraethylammonium on the kinetics of transmitter release at the mammalian neuromuscular synapse.

The effect of 4-aminopyridine and tetraethylammonium on the time course of neurotransmitter release was examined at the neuromuscular junction using a computer-aided method which directly measured the time of occurrence of individual quanta. It is apparent that the action of 4-aminopyridine, at concentrations of 0.1 to 1 mM, when examined in isolation from other experimental manipulations, is to cause a greatly enhanced probability of release at times subsequent to the time over which the initial phase is essentially unchanged, i.e., there is no evidence of an increased latency of release caused by 4-aminopyridine. Similar results were obtained with tetraethylammonium, although the prolongation of release was much less, even at a concentration of 1 mM. The results are consistent with the view that the predominant action of 4-aminopyridine is to block the potassium conductance responsible for repolarization of the action potential and hence cause a prolonged Ca2+ current. The action of tetraethylammonium is consistent with the block of a different K+ conductance, with consequent enhancement of action potential effectiveness, but with little prolongation of release. The observation of multiple peaks, or oscillations in the release probability function at high (ca. 1 mM) concentrations of 4-aminopyridine, may be related, as is suggested, to oscillations of presynaptic membrane potential, or perhaps to changes in the electrochemical gradient for Ca2+ influx.

4-Aminopyridine↗

PubMed1988

Single Ca2+ entry and transmitter release systems at the neuromuscular synapse.

The possibility that there exists more than one voltage-gated Ca2+ channel system subserving quantal release of neurotransmitter at nerve endings was examined by measuring the sensitivity of quantal release to agents that block Ca2+ or Ba2+ entry, namely Cd2+, Mg2+, neomycin, and bekanamycin. The results show equal effectiveness vs. release evoked by presynaptic action potentials, brief intense presynaptic depolarizations, or prolonged relatively mild depolarizations, from which it is concluded that the same channel system is involved in each case. In the presence of Ba2+ and no Ca2+, there occur essentially normal (but small) endplate potentials (epps), while in the presence of Ba2+ and Ca2+ epp amplitude and frequency of miniature epps co-modulate with stimulation frequency in a manner which corresponds to a residual Ba2+ model for augmentation. It is therefore also concluded that a single transmitter release system is responsible for normal phasic release and the asynchronous release that is mediated by Ba2+.

Action Potentials↗

PubMed1988

Transmitter release at mouse motor nerve terminals mediated by temporary accumulation of intracellular barium.

1. In isolated mouse diaphragm, tetanic nerve stimulation in the presence of Ba2+ causes an increase in frequency of MEPPs which continues as an after-discharge or 'tail' of raised MEPP frequency that subsides over a period of seconds, in addition to EPPs of low quantal content. 'Ba2+ tails' are also seen with focal depolarization of nerve terminals in the presence of tetrodotoxin. 2. The development of 'Ba2+ tails' could be inhibited or blocked by neomycin, raised Mg2+, or Cd2+ present at the time of stimulation; the presence of the blocking substance during the tail itself had no effect. 3. The time course of MEPP frequency changes during and after stimulation could be expressed as a simple exponential process, with the same time constant for both the rise and the fall, by taking as the time-dependent variable the nth root of MEPP frequency, n being 4 or 5. The time constant (tau) derived from the rate of fall of the 1/4 power of MEPP frequency during the tail was at most junctions between 3 and 6 s, and apparently unaffected by concentration of Ba2+, or of Ca2+, or by tonic depolarization of the nerve terminal. 4. The intensity of 'Ca2+ tails' was graded steeply with the number of stimuli applied, but was nearly independent of stimulus frequency, when train duration was kept brief compared to tau, i.e. about a second or less. The nth root of the number of MEPPs at a given time period in the tail was linearly related to the number of stimuli, when n was chosen to be 4 or 5. 5. The above data are consistent with a model in which (a) with each nerve impulse in a train there occurs the same entry of Ba2+ into the terminal, (b) transmitter release (MEPP frequency) is proportional to the fourth or fifth power of [Ba2+] at critical sites within the nerve terminal, (c) the Ba2+ leaves these sites as a first-order process with a time constant of a few seconds. Compared to Ca2+, Ba2+ persists longer but has lower potency. 6. With variation of external [Ba2+] over the range 50 microM to 6.4 mM, apparent Ba2+ entry per nerve impulse grew linearly with concentration. 7. Evidence is presented indicating that intraterminal Ba2+ can 'co-operate' with Ca2+ or La3+ in promoting transmitter release.

4-Aminopyridine↗

PubMed1987

Anion permeability of motor nerve terminals.

Motor nerve terminals in mouse and frog display behavior consistent with an appreciable permeability of the nerve terminal membrane to chloride. In mouse diaphragm, in the presence of 15 mM K+ and 2 mM or 8 mM Ca2+, replacement of Cl- by NO3-, Br- or acetate causes a transient increase in the quantal release of acetylcholine, measured as the frequency of spontaneously occurring miniature end plate potentials (FMEPP); a rapid rise in FMEPP is followed by a slow decline, with a half-time of about 4 min, to an equilibration level close to the control level. After equilibration in a solution in which the Cl- is replaced by another anion, return to Cl- -containing solution causes a transient decrease in FMEPP with a subsequent slow recovery. The data are consistent with transient nerve terminal depolarization or hyperpolarization, reflecting a nerve terminal permeability to anions in the sequence Cl- greater than Br- greater than NO3- greater than acetate. In 5 mM K+, changes in nerve terminal excitability, determined using focal stimulation, are also consistent with alteration of nerve terminal membrane potential as a consequence of anion substitution. The time course of relaxation of FMEPP after a change from Cl- to an anion of lower permeability, or vice versa, is considerably slower than that expected if Cl- permeability of nerve terminals is similar to that of skeletal muscle fibres, and if the nerve terminal behaves as a single compartment. In frog cutaneous pectoris, transient changes in FMEPP produced by substitution of anions in the bathing solution were similar to those produced in mouse diaphragm, but more rapid in time course.

Action Potentials↗

PubMed1987

Multiple potassium conductances at the mammalian motor nerve terminal.

The possibility that multiple K+ conductances are present in mammalian motor nerve terminals was investigated by measuring the differential effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on transmitter release and on nerve terminal excitability in mouse phrenic nerve-diaphram preparations. Neither 4-AP nor TEA alter the spontaneous frequency of miniature end-plate potentials (fmepp) and therefore evidently do not affect calcium channels directly. Both 4-AP and TEA increased the quantal content of end-plate potentials (epps) evoked by nerve stimulation but the effects were not mutually exclusive; TEA continued to act in the presence of a maximally effective concentration of 4-AP. The increase in transmitter release evoked by focal polarisation of the terminal was not affected by 4-AP, whereas TEA exerted an effect consistent with a reduction in membrane conductance. Similarly, threshold for nerve terminal action potential generation was not affected by 4-AP, but TEA caused a reduction in threshold and alteration of the 'accommodation curve' indicative of a reduction of membrane conductance. Under conditions where one would predict no contribution of calcium K+ conductance, i.e., when release was evoked by Ba2+ in the absence of Ca2+, the different and non-competing effects of TEA and 4-AP were still apparent. It is concluded, therefore, that the motor nerve terminal possesses at least two K+ conductances, not including calcium activated gK, which may be distinguished pharmacologically.

4-Aminopyridine↗