In freshly harvested aortic endothelial cells from rabbits, some cellular events associated with stimulation by acetylcholine (ACh) were analyzed. ACh (3 microM) induced a transient hyperpolarization of 8.3 +/- 2.5 mV, which peaked within 3-5 s and subsequently declined with a similar time course. Hyperpolarization was caused by a transient Ca2+-dependent outward current (IoACh), which was mainly carried by K+. ACh (3 and 10 microM) also evoked transient dose-dependent increases in the intracellular free Ca2+ concentration (Ca2+i). Pretreatment with atropine (1 and 3 microM) abolished both responses to ACh, the increase in Ca2+i as well as the transient outward current. It is concluded that IoACh and the rise in Ca2+i are two manifestations of muscarinic receptor stimulation. The rise in Ca2+i might be the primary event, leading to secondary membrane hyperpolarization.

Cell-attached patch-clamp experiments were performed in cultured cardiocytes of neonatal rats at 19 degrees C to analyze elementary currents through single Na+ channels under control conditions and in the presence of the class 1 antiarrhythmic drugs amiodarone, propafenone, and diprafenone. As observed in a cell-attached patch with only one functioning Na+ channel, repetitive stepping of the membrane at 0.4 Hz triggered periodically channel openings except during a silent period of about 1.5 min. The latter began and ceased abruptly and did not fit the monoexponential distribution of the run length of sweeps without activity (blank sweeps). Treating the cardiocytes with amiodarone, propafenone or diprafenone (10 to 20 mumol/liter) led rapidly to a blockage and reduced the likelihood that membrane depolarization triggers the opening of Na+ channels. The number of blank sweeps increased at the expense of the number of sweeps with activity. The fraction of activity sweeps with superpositions, indicating the simultaneous activation of two or more Na+ channels, also declined. As tested with amiodarone, the run length of blank sweeps is voltage- and time-dependent, analogous to the intensity of the block of macroscopic Na+ currents. Open time, open-time distribution, unitary current size and the tendency to reopen did not differ in unblocked cardiac Na+ channels (i.e. that channel fraction capable of opening in the presence of amiodarone or propafenone) from the respective control values obtained before superfusing the cardiocytes with these drugs. Apart from its blocking action, the propafenone derivative diprafenone exerted additionally a modifying effect and reduced mean open time by up to 45%. In contrast to the block, this reduction in conducting state proved insensitive to changes in holding potential, at least between -130 and -150 mV, the range tested. This means that block was attenuated on hyperpolarization whereas the reduction in open time persisted. It is concluded that, in the presence of these drugs, unblocked cardiac Na+ channels share a number of properties with normal Na+ channels in the absence of these drugs. Shortening of channel lifetime by diprafenone may be indicative of a channel modification brought about possibly by a receptor-mediated facilitation of the transition from the open to the inactivated state.

In cultured bovine aortic endothelial cells, elementary K+ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150 +/- 10 pS and a small channel with a mean unitary conductance of 12.5 +/- 1.1 pS. The 150-pS channel proved to be voltage- and Ca2+-activatable and seems to be a K+ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca2+ concentration at the cytoplasmic side of the membrane from 10(-7) to 10(-4) M. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced run-down nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K+ and Na+ whose open probability was minimal near -60 mV but increased on membrane hyperpolarization. An increase in internal Ca2+ from 10(-7) to 10(-4) M left the open probability unchanged. Although the K+ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca2+-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na+.

In patch-clamped membranes from neonatal rat cardiocytes, elementary Na + currents were recorded at 19° C for study of the inhibitory influence of several antiarrhythmic drugs including lidocaine, diprafenone, propafenone, and prajmalium on DPI-modified cardiac Na L ocal anesthetics and related antiarrhythmic drugs compose a structurally heterogeneous group of organic compounds capable of interacting with Na + channels in excitable membranes, including cardiac cells. Several lines of evidence strongly support the assumption that a channel-associated binding site exists 1 -3 whose interaction or binding with these drugs will finally block the channel and hinder sodium ions from passing through the pore. The resultant decrease in excitability may be an antiarrhythmic principle in heart From the Physiological Institute of the University Freiburg Freiburg/Br., Federal Republic of Germany.Supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-1), Bonn, Federal Republic of Germany.Address for correspondence: Professor Dr. M. Kohlhardt, Physiological Institute of the University, Hermann-Herder-Str. 7, D-78 Freiburg/Br., Federal Republic of Germany.Received January 20, 1988; accepted September 28, 1988. to suppress irregular impulses. The remarkably different manifestation of Na + current depression, which attracted considerable theoretical interest and was well documented in numerous biophysical studies during the past years, reflects the combined influence of such factors as voltage, driving rate, and drug hydrophobicity in determination of the strength of blockade of Na + currents. The rather complex block phenomenology finds elegant and conclusive explanations by the models of Hille 2 and Hondeghem and Katzung 3 on the one hand and Starmer et al< on the other hand. The HilleHondeghem-Katzung model postulates a modulated receptor whose drug affinity depends on the channel state, rested or activated/inactivated, while the Starmer model assumes a guarded receptor, that is, drug access is controlled by the channel state. Such modeling of block proved valuable and indispensable in the understanding of some elementary properties of voltage-dependent Na + channels.by guest on May 12, 2018

Elementary Na+ currents through single cardiac Na+ channels were recorded at 19 degrees C in patch clamp experiments with cultured neonatal rat cardiocytes. The metabolites of the glycolytic pathway, 2,3-diphosphoglycerate and glyceraldehyde phosphate, were identified as a novel class of modulators of Na+ channel activity. In micromolar concentrations (1-10 mumol/liter), their presence at the cytoplasmic membrane face increased the number of sequential openings during depolarization and prolonged the conductive channel state. As found after ensemble averaging, the decay kinetics of reconstructed macroscopic Na+ currents became retarded and slow Na+ inactivation may have been evoked. Both metabolites attenuated the rundown of channel activity that regularly develops after patch excision in the inside-out patch configuration. It is tempting to assume that interference with Na+ inactivation is the mode of action underlying the increase in single-channel activity.