Electrophysiological analysis of the negative chronotropic effect of endothelin‐1 in rabbit sinoatrial node cells

Electrophysiological effects of endothelin-1 (ET-1) were studied in rabbit sinoatrial node (SAN) using conventional microelectrode and whole-cell voltage and current recordings. In rabbit SAN, RT-PCR detected ETA endothelin receptor mRNA. ET−1 (100 nm) increased the cycle length of action potentials (APs) from 305 ± 15 to 388 ± 25 ms; this effect was antagonised by the ETA receptor-selective antagonist BQ−123 (1 μm). ET-1 increased AP duration (APD50) by 22 %, depolarised the maximum diastolic potential (MDP) from −59 ± 1 to −53 ± 2 mV, shifted the take-off potential by +5 mV and decreased the pacemaker potential (PMP) slope by 15 %. Under exactly the same experimental conditions, ET-1 caused a positive chronotropic effect in guinea-pig SAN with a decrease of 13 % in APD50, a shift of −4 mV in the take-off potential and an increase of 8 % in the PMP slope. Rabbit SAN exhibited two major cell types, distinguished both by their appearances and by their electrophysiological responses to ET-1. Whereas the spontaneous pacing rate and the PMP slope were similarly decreased by ET-1 (10 nm) in both cell types, ET-1 depolarised MDP from −67 ± 1 to −62 ± 4 mV in spindle-shaped cells but hyperpolarised it from −73 ± 1 to −81 ± 3 mV in rod-shaped cells. ET-1 decreased APD50 by 8 and 52 % and shifted the take-off potential by +5 and −9 mV in spindle- and rod-shaped cells, respectively. ET-1 decreased the high-threshold calcium current (ICaL) by about 50 % in both cell types, without affecting its voltage dependence, and decreased the delayed rectifier K+ current (IK) with significant shifts (of +4.7 and +14.0 mV in spindle- and rod-shaped cells, respectively) in its voltage dependence. It was exclusively in rod-shaped cells that ET-1 activated a sizeable amount of time-independent inward-rectifying current. The hyperpolarisation-activated current (If), observed exclusively in spindle-shaped cells, was significantly increased by ET-1 at membrane potentials between −74.7 and −84.7 mV whereas it was significantly decreased at more negative potentials. ET−1 significantly decreased the slope of the current-voltage (I–V) relation of the If tail without changing its half-maximum voltage. The overall negative chronotropic influence of ET-1 on the whole rabbit SAN is interpreted as resulting from the integration of its different actions on spindle- and rod-shaped SAN cells through electrotonic interaction. The endothelins (ETs) are a family of potent endogenous peptides, consisting of 21 amino acid residues, termed endothelin-1, -2 and -3 (ET-1, -2 and -3) (Inoue et al. 1989). The first member of the family, ET-1, was initially described as a potent vasoconstrictor produced by vascular endothelial cells (Yanagisawa et al. 1988). ETs have a wide variety of biological actions that are mediated by specific cell-surface receptors belonging to the superfamily of heptahelical G-protein coupled receptors. To date, two subtypes of endothelin receptors, named ETA and ETB, have been identified (Arai et al. 1990; Sakurai et al. 1990; Hosoda et al. 1991; Lin et al. 1991; Sakamoto et al. 1991). The ETA receptor has an affinity rank order of ET-1 > ET-2 >> ET-3, whereas the ETB receptor exhibits similar affinities for all three isopeptides. It has been shown that ETA and ETB receptors couple with distinct signal transduction pathways through different populations of GTP-binding proteins and that they have distinct cell-type and/or tissue distributions. In the body, all of these combine to confer the wide variety of functions of the endothelin system (Masaki et al. 1992; Goto et al. 1996). In the heart, ET-1 was initially reported to exert strong positive inotropic (Ishikawa et al. 1988a) and chronotropic actions (Ishikawa et al. 1988b). These effects were first believed to be mediated by augmentation of the high-threshold, long lasting, voltage-dependent calcium channel current ICaL, since they were cancelled by blockers of ICaL. However, later electrophysiological studies showed that ET-1 failed to increase ICaL in guinea-pig ventricular myocytes (Tohse et al. 1990). Hence, there have so far been no satisfactory data which can explain in electrophysiological terms such strong cardiotonic actions of ET-1, although the effect of ET-1 in increasing ICaL (Lauer et al. 1992) and the T-type calcium current, ICaT (Furukawa et al. 1992), has been documented. In contrast, our electrophysiological studies have disclosed that ET-1 can exert a negative chronotropic action, mediated by the ETA receptor, under the stimulation of the β-adrenoceptor (Ono et al. 1994, 1995a, b). We have found that stimulation of the ETA receptor hyperpolarises the resting membrane potential and decreases the duration of AP in guinea-pig and rabbit atrial myocytes by activating the muscarinic potassium current (IK(ACh)) and by inhibiting the ICaL (Ono et al. 1994, 1995b). Recently, quite similar results have been reported indicating that ET-1 reduces the ICaL and the delayed rectifier potassium current (IK) in single rabbit SAN cells and decreases the rate of spontaneous APs in small clusters of SAN cells (Tanaka et al. 1997). More recently we have demonstrated the distinct roles of ETA and ETB receptors in negative and positive chronotropic responses, respectively, in guinea-pig and rat heart (Ono et al. 1998). The SAN cells play a central role in the initiation and hormonal regulation of heart pacing (DiFrancesco, 1993; Irisawa et al. 1993). During the last decade, intensive electrophysiological studies using isolated SAN cells have revealed the contribution of several distinct ionic currents to the pacemaker activity of the SAN (Irisawa et al. 1993). The most important ionic currents are ICaL (Hagiwara et al. 1988; Brown & Denyer, 1989), ICaT (Hagiwara et al. 1988), the hyperpolarisation-activated inward current, If (DiFrancesco, 1986, 1993; DiFrancesco et al. 1986; DiFrancesco & Tortora, 1991), IK (Anumonwo et al. 1992), IK(ACh) (Sakmann et al. 1983), the sustained inward current, Ist (Guo et al. 1995), and the background sodium current (Hagiwara et al. 1992). Modulation of these currents by plasma membrane receptors, such as β-adrenoceptors and muscarinic acetylcholine receptors, have also been well documented (DiFrancesco, 1993; Irisawa et al. 1993). However, very little is known about the ionic basis for the strong chronotropic actions of ETs in SAN. During the past decade, histological investigations on the architecture of the SAN have revealed that it is composed of several types of cells with quite divergent morphological appearances (Opthof et al. 1985; Verheijck et al. 1998). Correspondingly, regional variations within the SAN in the configuration of APs (Verheijck et al. 1998), in the contribution of ionic currents (Kodama et al. 1997) and in the responses to pharmacological agents (Nikmaram et al. 1997) have been reported. Therefore, it is important, in order to understand the activity and response of the whole SAN, to elucidate possible differences among cell types within the SAN in their responses to endogenous modulators, including ET-1. In the present study, we found that ET-1 causes a negative chronotropic response in rabbit SAN through stimulation of the ETA receptor. The underlying ionic mechanisms for this negative chronotropic action of ET-1 were further examined by analysing the effects of ET-1 on membrane currents, using the whole-cell voltage clamp technique. Striking differences were found between two major, morphologically distinct types of SAN cells in ET-1-induced changes in spontaneous activity and in the underlying membrane currents. These data provide insight into the mechanisms of negative chronotropy of ET-1 in each cell type and into their contribution to the overall response of the SAN to ET-1. Preliminary results have been reported previously (Ono et al. 1997).