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-mu-M) and diphenylhydantoin (10-80-mu-M) 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 < 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 pH(i) and pH(o) revealed dramatic differences between amitriptyline and diphenylhydantoin. When pH(o) 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 < 0.0001) during exposure to amitriptyline, but no effect was seen on tau-r after exposure to diphenylhydantoin. Conversely, when pH(i) 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 < 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. Superfusion of cells with diphenylhydantoin did not change mean channel open time but increased the probability of failure of the channel to open. When diphenylhydantoin was placed in the micropipette, no effect on sodium channel kinetics could be demonstrated. Our experiments show that amitriptyline blocks sodium channels via a drug-receptor complex site sensitive to changes in pH(o), whereas diphenylhydantoin blocks from a pH(o)-insensitive site that is susceptible to changes in pH(i). The decrease in amitriptyline-induced tau-r by diphenylhydantoin may result from allosteric modulation and suggests the presence of a separate (intracellular) binding site for diphenylhydantoin action.
