Two components of use-dependent block of sodium current by lidocaine in voltage clamped cardiac Purkinje cells.

Lidocaine is an effective antiarrhythmic and local anesthetic agent, and it has also been a useful tool for characterizing drug interactions with the Na channel. We studied the block of sodium current (INa) caused by lidocaine (20 microM to 2 mM) in canine cardiac Purkinje cells at reduced Na (45 mM outside, 0 mM inside) and temperature (14-17 degrees C). Lidocaine block of INa developed in response to strains of depolarizations, was greater with increasing drug concentration, faster pulsing frequency, longer depolarization duration, and less negative holding potential. The time course of the fall in peak INa was best fitted by two exponentials. In contrast, block of peak INa in pulse trains by the permanently charged lidocaine analogue QX-314 was always best fitted by a single, slow exponential. When the pH was lowered from 7.4 to 6.8 to increase the proportion of the charged form of lidocaine, the contribution of the slow component increased both absolutely and relative to the fast component. The time course of the development of lidocaine block was characterized with a two pulse protocol, and it showed that block developed in a biexponential manner with initial rapid binding to a transiently available state (such as the open state) and with slower binding to a long-lived state (such as the inactivated state). We conclude that the complex binding and unbinding of lidocaine to different conformations of the cardiac Na channel is related to different affinities for the charged and uncharged forms of lidocaine.(ABSTRACT TRUNCATED AT 250 WORDS)

Is there a second external lidocaine binding site on mammalian cardiac cells?

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

Use-dependent block of sodium current by ethmozin in voltage-clamped internally perfused canine cardiac Purkinje cells.

Block of sodium current (INa) by ethmozin (moricizine), an antiarrhythmic drug, was investigated in isolated, voltage-clamped, canine cardiac Purkinje cells. Initial block of INa by ethmozin (2 microM) in noninactivated cells (held at -150 mV) was 9.3 +/- 1.2% (S.D.). Additional "use-dependent" block developed in response to repetitive depolarization. This block was both frequency-dependent and dose-dependent with the fall in peak INa greater at increasing depolarization frequencies (0.625 to 4 Hz) and with increasing dose (2 microM to 20 microM). Use-dependent block was modeled according to the guarded receptor hypothesis assuming ingress to the channel binding site during the open state of the channel, and egress from the channel independent of the kinetic state of the channel. The rate constants (on-rate = 2100 +/- 100 (S.D.)/M/ms and off-rate = 1.7 +/- 0.3 (S.D.) 10(-5)/ms) were used to predict the time course of INa block in response to repeated depolarizations and the dose-response relationship of steady-state used-dependent block measured in independent experiments. We conclude that ethmozin blocks INa in Purkinje cells in both a non-use-dependent and a use-dependent manner and that the guarded receptor model is useful in describing the use-dependent block.

Vitamin E attenuates the effects of FMLP on rabbit circulating granulocytes.

Exposure of circulating rabbit granulocytes to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) in vivo results in transient granulocytopenia, hypotension, and cardiorespiratory distress. The effectiveness of vitamin E in attenuating these responses was tested. Vitamin E accelerated the rate of return of granulocytes to the peripheral circulation after FMLP-induced granulocytopenia and mitigated the hypotension. The reversible adherence of FMLP-stimulated granulocytes to endothelium offers a plausible mechanism to explain the transient granulocytopenia. From in vitro studies it was found that FMLP-activated granulocytes from animals treated with vitamin E showed decreased adherence to cultivated aortic endothelial monolayers when compared with FMLP-activated granulocytes from control animals.

The effect of vitamin E on rabbit neutrophil activation.

Chemoattractants such as N-formylmethionyl leucyl phenylalanine (FMLP) cause neutropenia in vivo. The sequestered neutrophils may block the microvasculature and contribute to respiratory distress. Neutrophils from humans receiving 1600 units vitamin E per day have reduced oxidative activity. To test whether vitamin E attenuates the responses of neutrophils to FMLP in vivo we gave rabbits four daily intramuscular injections of 100 mg vitamin E. Serum levels of the vitamin were 2.34 +/- 0.15 mg% compared to 0.19 +/- 0.04 mg% in control rabbits receiving placebo injections. On the fifth day testing was done before and after injecting FMLP. Variables monitored were the absolute granulocyte count (AGC), systolic, diastolic and mean blood pressures (MBP), heart rate, PO2, PCO2, pH and respiratory rate. When 0.5 microgram FMLP was injected intravenously the AGC decreased (at 2.5 min the percentage change was -89.7 +/- 8.0 with vitamin E and -97.0 +/- 2.7 without vitamin E; P = 0.2). MBP decreased also (% change, -29.0 +/- 13.0 with vitamin E and -36.3 +/- 16.0, without vitamin E). By 15 min recovery was seen (AGC % change, -26.0 +/- 17 with vitamin E and -78.7 +/- 10.5, without vitamin E; P = 0.01; MBP % change, -9.3 +/- 3.8 with vitamin E and -52.3 +/- 10.1 without vitamin E). Chromatographic analysis of serum extracts revealed increases in 6-keto-PGF1 alpha after stimulation. Studies with [3h]thymidine-labelled neutrophils showed that the sequestered cells return to the circulation. Vitamin E might facilitate this return by altering the adherence of neutrophils to endothelium. This possibility was tested by measuring the adherence to cultivated rabbit aorta endothelial monolayers of FMLP-stimulated neutrophils from vitamin E-treated rabbits. The percentage of neutrophils adhering was 32.5 +/- 3.5 with vitamin E and 60.0 +/- 7.1, without vitamin E. Thus vitamin E promotes the return of neutrophils to the circulation after chemotactic challenge and may do so by reducing their adherence to endothelium.