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A O Grant

Publications and source records attributed to A O Grant.

At least 19 recordsLinked to original sources
PubMed1992

On the mechanism of action of antiarrhythmic agents.

Cardiac arrhythmias arise from disturbances in the functioning of the specific ion channels that normally control excitation or from the functional expression of previously latent channels. Antiarrhythmic agents act by blocking the membrane sodium, potassium, and calcium channels, but no agent has exclusive action on a given type of channel. Arrhythmias resulting from reentry form the largest group of clinically significant arrhythmias. Most arrhythmias result from depressed sodium channel function. The local anesthetic class of sodium channel blockers (class I agents) acts by slowing conduction and converting regions of unidirectional block to bidirectional block. Class III agents act by prolonging the action potential duration. Because potassium currents are normally responsible for repolarization of the cardiac action potentials, these agents are generally assumed to be potassium channel blockers. Class IV antiarrhythmics--calcium channel blockers--are used when a group of reentrant arrhythmias arises in regions in which conduction is primarily sustained by increases in permeability to calcium ions. The mechanisms of action of antiarrhythmic agents are discussed with respect to the basic cellular mechanisms of cardiac arrhythmias.

Anti-Arrhythmia Agents↗
PubMed1992

Block and modulation of cardiac Na+ channels by antiarrhythmic drugs, neurotransmitters and hormones.

The Na+ channel is an important target for the action of antiarrhythmic drugs. Application of contemporary biophysical, biochemical and molecular biological techniques have added considerably to our knowledge of its structure, function, modulation and block by antiarrhythmic drugs. The increased mortality from the use of these drugs for prophylaxis of cardiac arrhythmias has forced a re-evaluation of their use and of the entire pharmacological strategy of arrhythmia management. Gus Grant and David Wendt review recent studies on the block and modulation of cardiac Na+ channels and the place of Na+ channel blockers in future antiarrhythmic drug development.

Animals↗
PubMed1992

Na channel kinetics remain stable during perforated-patch recordings.

The results of studies on modulation of Na channel function are often difficult to interpret due to time-dependent changes in channel kinetics. Although the "tight-seal" whole cell voltage-clamp technique has proved very useful in studying the properties of the cardiac Na current, the spontaneous shift of parameters of inactivation and activation gating to more negative potential is a serious limitation to the use of the technique. The shifts are believed to result from changes in the intracellular milieu effected by dialysis; moreover, use of a variety of different anions and cations in the internal micropipette solution has not obviated the problem. The perforated-patch technique permits low-resistance intracellular access without free dialysis between the intracellular solution and the recording micropipette. We have compared steady-state inactivation and peak current-voltage relationship of whole cell Na currents measured with the conventional whole cell and perforated-patch techniques in rabbit atrial myocytes at 17 degrees C. Although gating parameters shifted to more negative potentials when recorded with the conventional technique, stable kinetics could be observed for up to 150 min with the perforated-patch technique. The potential for one-half Na channel inactivation was -73 +/- 5.1 mV and is consistent with measurements made using indirect techniques such as upstroke velocity measurements. The fact that the intracellular milieu is left relatively intact makes the approach attractive for studying modulation of the Na current by neurotransmitters and hormones.

Animals↗
PubMed1992

Cardiac instability amplified by use-dependent Na channel blockade.

Drugs that exhibit use-dependent Na channel blockade, including antiarrhythmic agents, tricyclic antidepressants, opiate-like analgesics, and cocaine, are linked with an increased susceptibility to cardiac arrhythmias and sudden death. Computer simulations indicate that Na channel blockade retards recovery of excitability, thereby increasing the spatial dispersion of refractoriness, a precursor of many cardiac arrhythmias. In isolated rabbit left atria, stimuli timed to occur at increasing intervals following conditioning stimuli reveal an unstable interval (vulnerable period) during which single stimuli initiate trains of responses. The vulnerable period is extended by use-dependent Na channel blockade and provides a model for assaying proarrhythmic potential and probing cardiac instability.

Animals↗
PubMed1992

Kinetics of interaction of the lidocaine metabolite glycylxylidide with the cardiac sodium channel. Additive blockade with lidocaine.

The recovery of the sodium channel from blockade by local anesthetic antiarrhythmic drugs is voltage dependent. Recovery from lidocaine-induced blockade is accelerated by hyperpolarization, whereas that from glycylxylidide (GX) blockade has been reported to be slowed by hyperpolarization. This striking difference occurs despite similarities in chemical structure. The fast recovery from GX block at depolarized potentials may lead to a partial reversal of lidocaine blockade when the two drugs are combined. We have examined the kinetics of interaction of GX with the cardiac sodium channel over a range of membrane potentials by measuring whole-cell currents in isolated rabbit myocytes under voltage clamp at 15 degrees C. In the absence of drug, slow inactivation developed with a time constant of 10.7 +/- 5.1 seconds (n = 6). During exposure to 74 mumol/l GX, block developed with a time constant of 7.0 +/- 3 seconds (n = 6). Because of the similar time course of slow inactivation and block, we used a high concentration of GX to induce a level of block sufficient for analysis. The onset of block was slower than that induced by lidocaine and was unaffected by variation of external sodium from 20 to 75 mmol/l. Use-dependent blockade of sodium channels was greater when pulse trains were applied from a holding potential of -100 than -140 mV. This suggested that recovery from GX block might be slower at -100 than -140 mV. Direct measurements gave time constants of recovery of 10.3 +/- 4.2 seconds at -100 mV (n = 6) and 4.1 +/- 0.4 seconds at -140 mV (n = 4). The combination of GX with lidocaine produced only additive blocking effects when pulse trains were applied from both holding potentials. Computer simulations of the requirements for the competitive displacement of a sodium channel blocker with slow kinetics by one with fast kinetics suggest that the recovery time constant of the fast drug must be 10-100-fold smaller than that of the slow drug. Rapid association kinetics effected by a large binding rate constant or a higher concentration of the fast blocking drug is also important. The simulations suggest that, for the interaction of GX and lidocaine, only additive blocking action should be observed over the range of stimulus frequencies used in these experiments.

Animals↗
PubMed1992

Blockade of cardiac sodium channels. Competition between the permeant ion and antiarrhythmic drugs.

A number of basic and clinical studies suggest that elevation of external sodium concentrations, [Na]o, may reverse the cardiotoxic effect of local anesthetic-class drugs. The mechanisms of reversal are uncertain. The blocking action of lidocaine and disopyramide were studied over a range of [Na]o. Both whole-cell voltage clamp and single-channel recordings were performed on isolated rabbit myocytes at 17 and 22 degrees C, respectively. In the presence of lidocaine, an inactivated channel blocker, the level of steady-state block in response to pulse train stimulation was not affected by variations in [Na]o from 20 to 150 mM. Estimates of the rate of dissociation of drug from the channel also were unaffected. In contrast, steady-state block by disopyramide, a drug that blocks open channels, was decreased as [Na]o was increased. Single-channel measurements suggest that the influence of [Na]o on channel current amplitude was small, 12% for a 25 mM increase in [Na]o. This increase in single-channel current amplitude would affect drug-free channels only, in that our studies suggest that drug-associated channels do not conduct. The association rate constant of disopyramide with open single sodium channels was decreased from 10 x 10(6) to 5 x 10(6)/M per s by an increase in [Na]o from 120 to 180 mM. Elevation of [Na]o may reverse the blocking action of local anesthetic-class drugs by an increase in single-channel current amplitude or by a decrease in drug association rate with the sodium channel. The occurrence of the latter action depends on the mode of block of the specific agent.

Animals↗
PubMed1991

A source of bias in the analysis of single channel data: assessing the apparent interaction between channel proteins.

A recent study of single sodium channel currents in neuroblastoma cells suggested interaction between ion channels in close proximity to one another (T. Kiss and K. Nagy, Eur. Biophys. J. 12, 13, 1985). The opening of one channel appeared to affect the likelihood that neighboring channels might open. Some of the conclusions were based on the analysis of observed channel openings that were segregated depending on whether one channel or more than one channel was open at the same time. We hypothesized that the longer one channel remained open, the more likely another channel operating independently, would open, thereby creating the impression of an apparent coupling of channel behavior. We performed simulations and measurements of single sodium channel currents to determine whether the technique of event segregation could account for apparent channel interactions. The simulations showed that the segregation of overlapping (more than one channel open at the same time) and nonoverlapping events led to a bias in the estimated open time and the derived closing rate. To avoid the bias, we found that random pairing of opening and closing events provided an unbiased estimate of the mean closing rate. Using this random assignment approach, we showed that the mean closing rate of single sodium channels in neonatal rat myocytes decreased with depolarization over a limited range of membrane potential. This suggested that the underlying closure mechanism(s) was voltage dependent. From the analysis of open times, we found no evidence for channel interaction in the time scale of tens of milliseconds. Depolarizing steps without events occurred in runs suggesting the existence of long-lived shut state(s). Double pulse experiments with the prepulse and test pulse above threshold showed significant inactivation of channels that did not open. The rate of inactivation of shut channels was substantially slower than the closure rate of open channels. The rate of inactivation of cardiac sodium channels appeared to be strongly dependent on the initial channel state.

Animals↗
PubMed1991

Lidocaine blockade of continuously and transiently accessible sites in cardiac sodium channels.

Lidocaine binds to sodium channels in a voltage dependent manner where depolarization enhances block and hyperpolarization relieves block. Voltage--clamp studies demonstrate that there are two components of block: one involving interaction with a binding site that is accessible for the duration of a depolarizing clamp (continuous access or availability) and one involving interaction with a site that is transiently available or accessible during transitions between polarized and depolarized potentials. Here we report results demonstrating two distinct voltage dependencies of blockade. The voltage dependence of block of the transiently accessible site is similar to that of channel activation and exhibits a maximal binding rate of 1.37 x 10(6)/M/S and an unbinding rate of 39.5/s at -30 mV. Blockade of the sustained site exhibits a voltage dependence similar to inactivation with a maximal binding rate of 3.59 x 10(4)/M/S and an unbinding rate of 0.678/s at -30 mV. Recovery from blockade acquired by either process is voltage dependent and proportional to exp(-0.037 Vm). Drug induced shifts in channel availability and transient site block are accurately predicted from kinetic rates estimated from frequency dependent protocols.

Animals↗
PubMed1991

Proarrhythmic response to sodium channel blockade. Theoretical model and numerical experiments.

BACKGROUND: The use of flecainide and encainide was terminated in the Cardiac Arrhythmia Suppression Trial because of an excess of sudden cardiac deaths in the active treatment group. Such events might arise from reentrant rhythms initiated by premature stimulation in the presence of anisotropic sodium channel availability. Drugs that bind to sodium channels increase the functional dispersion of refractoriness by slowing (a result of the drug-unbinding process) the transition from an inexcitable state to an excitable state. It is interesting that encainide and flecainide unbind slowly (15-20 seconds), whereas lidocaine and moricizine unbind rapidly (0.2-1.3 seconds). METHODS AND RESULTS: With a computer representation of a cable with Beeler-Reuter membrane properties, we found a small (6 msec) vulnerable window that occurred 338 msec after the last drive stimulus. Premature stimuli falling within the vulnerable window resulted in unidirectional block and reentrant activation. In the presence of a slowly unbinding drug, the window was delayed an additional 341 msec, and its duration was extended to 38 msec. The delay (antiarrhythmic effect) before the onset of the vulnerable window and its duration (proarrhythmic effect) were both dependent on the sodium channel availability and the recovery process. Both effects were also prolonged when sodium channel availability was reduced by membrane depolarization. Defining the proarrhythmic potential as the duration of the vulnerable window, we found that hypothetical use-dependent class I drugs have a greater proarrhythmic potential than non-use-dependent drugs. CONCLUSIONS: The antiarrhythmic and proarrhythmic properties of pure sodium channel antagonists are both dependent on sodium channel availability. Consequently, the price for increased antiarrhythmic efficacy (suppressed premature ventricular contractions) is an increased proarrhythmic vulnerability to unsuppressed premature ventricular contractions.

Anti-Arrhythmia Agents↗
PubMed1991

Blockade of cardiac sodium channels by amitriptyline and diphenylhydantoin. Evidence for two use-dependent binding sites.

Cardiac toxicity is a frequent manifestation in amitriptyline overdose and is felt to be due, in part, to sodium channel blockade by the drug. Another agent with sodium channel blocking properties, diphenylhydantoin, has been used clinically to reverse cardiac conduction abnormalities induced by amitriptyline. This reversal of toxicity is believed to occur secondary to competition for the sodium channel binding site. We evaluated individually and in combination the effects of amitriptyline (0.4 microM) and diphenylhydantoin (10-80 microM) on the sodium current in isolated rabbit atrial and ventricular myocytes at 17 degrees C. Using the whole-cell variant of the patch-clamp technique, we found that both amitriptyline and diphenylhydantoin reduced the sodium current in a use-dependent fashion. The time constant of recovery (tau r) from block by amitriptyline at -130 mV was very slow (13.6 +/- 3.2 seconds), whereas tau r during diphenylhydantoin exposure was fast (0.71 +/- 0.21 seconds, p less than 0.0001 compared with amitriptyline). During exposure of cells to a mixture of the two drugs, tau r was found to be 6.6 +/- 1.8 seconds, but no evidence of direct competition between amitriptyline and diphenylhydantoin was seen. Attempts to fit the recovery data of the mixture to two exponentials resulted in no significant improvement in the fit when compared with that using a single exponential. Use of the sodium channel blocking agent lidocaine (similar kinetics to diphenylhydantoin) in competition with amitriptyline resulted in findings consistent with direct competition of these two drugs for a single binding site. These observations prompted us to evaluate the possibility that diphenylhydantoin was not acting at (and therefore not competing for) the same channel binding site as amitriptyline. Experiments altering pHi and pHo revealed dramatic differences between amitriptyline and diphenylhydantoin. When pHo was increased from 7.4 to 8.0, tau r was reduced approximately threefold (from 13.6 +/- 3.2 to 4.2 +/- 0.1 seconds, p less than 0.0001) during exposure to amitriptyline, but no effect was seen on tau r after exposure to diphenylhydantoin. Conversely, when pHi was increased from 7.3 to 8.0, tau r after amitriptyline was unaffected, but tau r after diphenylhydantoin markedly increased (from 0.71 +/- 0.21 to 2.60 +/- 1.30 seconds, p less than 0.001). Additionally, diphenylhydantoin block demonstrated profound voltage dependence across the range of -130 to -90 mV, whereas amitriptyline block appeared less voltage sensitive. Single-channel studies using patch-clamp techniques in isolated ventricular myocytes supported these data.(ABSTRACT TRUNCATED AT 400 WORDS)

Amitriptyline↗
PubMed1991

Models of drug interaction with the sodium channel.

The local anesthetic-class of anti-arrhythmic drugs block the inward sodium current in nerve and cardiac muscle. A number of models for the interaction of these drugs with the neuronal sodium channel have been extended to cardiac muscle. The models assume a single binding site for the entire class of agents. The kinetics of drug interaction with this site depend on the Na channel conformation, open and inactivated channels having greater affinity than resting channels. An alternative formulation considers drug-receptor affinity as fixed, but access to the binding site is controlled by channel gating. Several clinically relevant predictions, such as competitive displacement of multiple agents, can be made from these models.

Animals↗
PubMed1990

An object oriented user interface for analysis of biological data.

In a previous paper we described a self-documented file and a collection of general purpose programs or tools that facilitates the management and analysis of biological data. The tools can be specified in a pipeline to accomplish a specific analysis task. However, we found that it was difficult for investigators to learn the UNIX command language for specifying pipelines, specify selection tasks through a command language, and visualize the data as they were transformed and rearranged. To alleviate these problems we developed an object-oriented user interface for the pipeline programs. The system consists of four major programs for visualization: Vedit, Vgraf, Vscan, and V spread. Vedit is a simple text editor, Vgraf is a flexible graphics program, Vscan facilitates scanning graphically through large files, and Vspread provides spreadsheet-like capabilities. To demonstrate how the visualization programs are used together to accomplish the needed analysis we describe two case studies and then discuss how well the system accomplished the goals of visualization, short learning curve, and user adaptability.

Biology↗
PubMed1990

Use of ionic currents to identify and estimate parameters in models of channel blockade.

Models of ion channel blockade are frequently validated with observations of ionic currents resulting from electrical or chemical stimulation. Model parameters for some models (modulated receptor hypothesis) cannot be uniquely determined from ionic currents. The time course of ionic currents reflects the activation (fraction of available channels that conduct in the presence of excitation) and availability of channels (the ability of the protein to make a transition to a conducting conformation and where this conformation is not complexed with a drug). In the presence of a channel blocking agent, the voltage dependence of availability appears modified and has been interpreted as evidence that drug-complexed channels exhibit modified transition rates between channel protein conformations. Because blockade and availability both modify ionic currents, their individual contributions to macroscopic conductance cannot be resolved from ionic currents except when constant affinity binding to a bindable site is assumed. Experimental studies of nimodipine block of calcium channels and lidocaine block of sodium channels illustrate these concepts.

Animals↗
PubMed1990

Extracellular pH modulates block of both sodium and calcium channels by nicardipine.

Slowing of the recovery of the Na channel from local anesthetic block at low external pH has been well described. In contrast the Ca channel has been reported not to show such an effect. Because of the absence of this response, a two-site model for the interaction of Ca antagonists with the Ca channel has been proposed. We sought to determine whether these results were a consequence of utilizing the poorly lipid-soluble Ca antagonist diltiazem as the test drug. We have measured the time constants of recovery (tau r) of Na (INa) and Ca currents (ICa) recorded from rabbit atrial myocytes during control and exposure to the lipid-soluble Ca antagonist nicardipine as external pH was reduced from 7.8 to 6.9. In the absence of drug, tau r for INa decreased from 19 +/- 1.3 to 12.1 +/- 0.6 ms for the pH reduction. Similarly, tau r for ICa decreased from 53 +/- 7 to 33.6 +/- 7 ms. During exposure to nicardipine tau r for INa increased from 2.24 +/- 0.8 to 5.96 +/- 0.8 s as external pH was reduced. For ICa, tau r also increased from 1.43 +/- 0.55 to 2 +/- 0.32 s. For each channel type, the results are well explained by a single blocking site model with hydrophobic and hydrophilic pathways to a single membrane binding site as proposed by Hille (J. Gen. Physiol. 69: 497-517, 1977).

Animals↗
PubMed1990

Effects of amiloride on pH regulation in canine cardiac Purkinje fibers.

Myocardial cells utilize membrane transport systems for proton extrusion as well as internal buffers to preserve pH homeostasis. Our laboratory had shown previously that amiloride (0.01-1.0 mM) causes a time- and dose-dependent increase in action potential duration, early after depolarizations and enhanced automaticity. Ion-selective microelectrode technique was used to evaluate whether the observed electrophysiologic effects of amiloride are linked to inhibition of Na/H exchange and subsequent inability of the myocardial cell to maintain steady-state intracellular pH (pHi), either under normal physiological conditions or in the presence of an imposed acid load. We analyzed different components of intracellular pH transients that occur in response to NH4Cl exposure and washout, which allowed us to quantitatively describe the effects of Na/H exchange inhibition in a multicellular preparation. Amiloride (0.01-1.0 mM) did not change the steady-state pHi, but did cause a dose-dependent increase in both the time for the pHi to reach a minimum value (time-to-peak) during washout of NH4Cl as well as in the absolute minimum value of pHi (peak acid). The effects of amiloride on pHi transients are rapidly reversible and antagonized by physiologic values of extracellular sodium activity. We conclude that Na/H exchange inhibition by amiloride does not cause intracellular acidosis under normal physiologic conditions, despite the dramatic changes in action potential characteristics. However, amiloride affected the time-to-peak and the peak acid value of the pHi transient during NH4Cl washout at concentrations that had no discernible effect on the overall time course of pHi recovery.

Acid-Base Equilibrium↗
PubMed1989

Calcium channels reconstituted from the skeletal muscle dihydropyridine receptor protein complex and its alpha 1 peptide subunit in lipid bilayers.

In the first part of this study, we show that sDHPR and pDHPR preparations reconstituted into lipid bilayers formed on the tips of patch pipettes exhibit two divalent cation-selective conductance levels of 9 and 20 pS, similar in single-channel conductance to VSCC reported in a variety of intact preparations (see Pelzer et al. and Tsien et al. for review). The larger conductance level is similar to the VSCC identified in intact rat t-tubule membranes and described in sDHPR and pDHPR preparations, and shares many properties in common with activity from L-type VSCC. It is sensitive to augmentation by the DHP agonist (+/-)-BAY K 8644 and cAMP-dependent phosphorylation, and to block by the phenylalkylamine (+/-)-D600 and the inorganic blocker CoCl2. Its open-state probability and open times are increased upon depolarization as expected for a voltage-dependent activation process. Upon depolarization beyond the reversal potential, however, open-state probability and open times decline again. A reasonable way to explain the bell-shaped dependence of open times and open-state probability on membrane potential is to assume voltage-dependent ion-pore interactions that produce closing of the channel at strong negative and positive membrane potentials. By contrast, the smaller conductance level may be similar to the 10.6-pS t-tubule VSCC described by Rosenberg et al. and may best be compared with T-type VSCC. It is largely resistant to augmentation by (+/-)-BAY K 8644 and cAMP-dependent phosphorylation or block by (+/-)-D600, but is sensitive to block by CoCl2. Its open times and open-state probability show a sole dependence on membrane potential where depolarization increases both parameters sigmoidally from close to zero up to a saturating level. Both elementary conductance levels do not exhibit significant inactivation over a wide potential range, which may suggest that skeletal muscle VSCC inactivation is either poorly or not voltage-dependent at all. This possibility seems in agreement with bilayer recordings on reconstituted intact t-tubule membranes and voltage-clamp recordings on intact fibers. It supports the idea that the decline of Ca2+ current in intact skeletal muscle fibers may be due to Ca2+ depletion from the t-tubule system and/or to inactivation induced by Ca2+ release from the sarcoplasmic reticulum. We consistently observe two conductance levels of 9 and 20 pS, either singly, or together in the same bilayer from solubilized DHPR samples and even highly purified DHPR preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Blockade of rabbit atrial sodium channels by lidocaine. Characterization of continuous and frequency-dependent blocking.

Lidocaine block of the cardiac sodium channel is believed to be primarily a function of channel state. For subthreshold potentials, block is limited to the inactivated state, whereas above threshold, block results from the combination of open- and inactivated-state block. Since, in the absence of drug, inactivation develops with time constants that vary from several hundred milliseconds to a few milliseconds as potential is varied from subthreshold to strongly depolarized levels, we would predict a similar voltage dependence of at least a fraction of block. Prior theoretical analyses from our laboratory suggest that there should be a direct parallel between blockade determined with a single pulse and trains of pulses. We tested these predictions by measuring the blockade of sodium current in cultured atrial myocytes during exposure to 80 microM lidocaine. We selected two test potentials for most of our studies, -80 mV, which was clearly in the subthreshold range of potentials, and -20 mV, which was close to the peak of the current-voltage curve. With single pulses of increasing duration, block developed with a single exponential time course and with time constants that decreased from 694 +/- 117 msec at -80 mV to 373 +/- 54 msec at -20 mV. In the absence of drug, inactivation developed with a time constant 176 +/- 17 at -80 mV and 2.9 +/- .5 msec at -20 mV. Despite the much slower onset of inactivation at -80 mV, no second-order delay in block development was observed. This suggests that at -80 mV block is occurring to a channel conformation that is accessed without delay rather than the classical inactivated state. We compared the kinetics of block during a single continuous pulse with trains of pulses at -20 mV. The rate of block onset was faster during the pulse trains, suggesting an element of "activated state" block. We computed shifts in apparent inactivation from observed steady-state blockade. The computed shifts agree well with those observed, indicating that shifts in apparent inactivation result largely from voltage-sensitive equilibrium blockade. The classical states described in the Hodgkin-Huxley formalism may be too restrictive to fully describe the voltage- and time-dependent block of cardiac sodium channels.

Animals↗