Introduction

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Endothelin-1 is a peptide, released from endothelial cells, which causes profound vasoconstriction in both arterial and venous smooth muscle (Leach et al., 1990; Sudjarwo et al., 1993; Yanagisawa et al., 1988). As a consequence of the potent and long-lasting nature of this vasoconstriction, it has been suggested that ET-1 may act as a mediator of vasospasm and hypertension (Barnes, 1994; Rubanyi and Polokoff, 1994). In support of this, plasma levels of ET-1 have been shown to be elevated in patients suffering coronary vasospastic episodes (Matsuyama et al., 1991; Toyo-Oka et al., 1991; Toyo-Oka and Sugimoto, 1991), pulmonary hypertension (Stewart et al., 1991) and cerebral vasospasm after subarachnoid hemorrhage (Suzuki et al., 1990). It also has been reported that endothelin receptor antagonists can reduce blood pressure when it is raised to a pathological level (Shigeno et al., 1995; Clozel et al., 1993).

To date two ET receptor subtypes have been cloned in mammalian tissue (ET_A, Arai et al., 1990; and ET_B, Sakurai et al., 1990). The ET_B receptor coexists with the ET_A receptor on vascular smooth muscle cells (Fukuroda et al., 1994; Sumner et al., 1992; Panek et al., 1992) and is expressed on endothelial cells, where it was first described (Sakurai et al., 1990). Both ET_A and ET_B receptors mediate constriction of vascular smooth muscle (Warner et al., 1993; Douglas et al., 1994; Sudjarwo et al., 1993). Vasoconstriction mediated by ET-1 involves an increase in intracellular Ca⁺⁺ ([Ca⁺⁺]_i) either because of the mobilization of inositol triphosphate-sensitive Ca⁺⁺ stores (Sugiura et al., 1989; Van Renterghem et al., 1988) or an influx of extracellular Ca⁺⁺ via dihydropyridine-sensitive Ca⁺⁺ channels (Yanagisawa et al., 1988; Goto et al., 1989). This increase in [Ca⁺⁺]_i not only leads directly to a contractile response but may also activate Ca⁺⁺-dependent currents mediating depolarization and opening of further voltage-dependent Ca⁺⁺ channels. In contrast to its vasoconstricting effects the electrophysiological actions of ET-1 are still poorly understood. However, ET-1 is known to inhibit ATP-sensitive K⁺ channel currents in porcine coronary arteries (Miyoshi et al., 1992) and to activate a Cl current, leading to depolarization, in porcine coronary or human mesenteric arteries. ET-1 has also been reported to activate a nonselective cation current (I_NS) in vascular smooth muscle cells via a Ca⁺⁺-dependent mechanism (Chen and Wagoner, 1991; Nakajima et al., 1996; Van Renterghem et al., 1988) and to have a dual activating and inhibiting action on Ca⁺⁺-activated K⁺ (K_Ca) channels in kidney mesangial cells (Hu et al., 1991). We recently reported that ET-1 has three distinct electrophysiological effects on myocytes enzymatically isolated from the rat small pulmonary artery: activation of a Ca⁺⁺-activated K⁺ current (I_K(Ca)) and a Ca⁺⁺-activated Cl current (I_Cl(Ca)) and a gradual Ca⁺⁺-independent inhibition of the delayed-rectifier K⁺ current (I_K; Salter and Kozlowski, 1996). The present study was performed to determine whether these effects of ET-1 were specific to rat small pulmonary arterial myocytes, or whether they were common to other types of vascular smooth muscle cell in this species. We therefore have compared the electrophysiological actions of ET-1 in smooth muscle cells isolated from small pulmonary artery, aorta and basilar artery, and found significant differences between the vessel types.

	Methods

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Cell isolation. Male albino rats (200-250 g) were sacrificed by an overdose of Euthatol i.p.(pentobarbitone sodium B.P., Rhone Merieux, Ireland); and the small pulmonary artery, aorta and basilar artery were removed. Smooth muscle cells were isolated by an enzymatic dispersion method similar to that used for isolating small pulmonary arterial myocytes, which previously was described by us (Salter and Kozlowski, 1996). All cells were stored at 4°C before patch-clamp experiments and remained viable for up to 10 h.

Electrophysiology. Once isolated, myocytes were subjected to patch-clamp experiments. Most experiments used the perforated-patch configuration (Horn and Marty, 1988) of the whole-cell patch-clamp recording technique. This prevents dilution of the cell contents and subsequent current run-down. Both current- and voltage-clamp experiments were performed. On occasion it was necessary to use the conventional whole-cell configuration to control the composition of the solution dialyzing the cell interior. Single channel recordings from outside-out patches also were made from myocytes isolated from basilar artery. Patch pipettes were pulled from borosilicate glass capillaries (Clark Electromedical, Pangbourne, England) by a vertical puller (Narishige Ltd., Tokyo, Japan). To initiate voltage-activated outward currents, cells were voltage-clamped at 50 mV and the voltage stepped to 100 mV for 100 ms before application of ramp pulses from 100 mV to +50 mV (dv/dt = 1 V s¹) and back to 100 mV (dv/dt = 0.5 V s¹) at a frequency of 0.2Hz. This protocol also allowed the background current at a holding potential of 50 mV to be continually recorded if required. To record uninterrupted background current, cells were voltage-clamped continuously at a set potential in the absence of any other voltage protocols. For flash photolysis experiments (see below) cells were voltage-clamped at 50 mV, and a 50-mV hyperpolarizing square pulse (100 ms in duration) followed by a 130-mV depolarizing pulse (500 ms in duration) was applied at a frequency of 0.2Hz. Ionic currents were detected with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Series resistance and capacity compensation facilities were used as necessary. Data were filtered at 1 kHz, digitized at 2 kHz with a Digidata 1200 interface (Axon Instruments) and recorded, either on-line with a personal computer or off-line on a modified DAT recorder (Sony DTC-100ES). Data were analyzed with pClamp software (version 5.7 or 6.1; Axon Instruments).

Solutions. The composition of the solutions used throughout this study is given in table 1. For most electrophysiological recordings myocytes were bathed in a quasiphysiological solution (solution A). For perforated-patch recordings the pipette contained solution B to which 240 µg·ml¹ amphotericin B was added. In experiments designed to determine the involvement of K_Ca channels, IbTX (20 nM throughout) was added to solution C, which contained 2.5 mM EGTA to obviate the involvement of extracellular Ca⁺⁺. For single channel recording from outside-out patches, the intracellular surface of the patch was exposed to solution D. In these experiments the bath contained solution A. To verify the involvement of Cl ions in electrophysiological responses to extracellularly applied ET-1, cells were bathed in solution E while solution F (containing 240 µg·ml¹ amphotericin B) was held in the pipette. Experiments involving flash photolysis of Nitr-5 used the whole-cell configuration during which the cells were dialyzed with solution G. In these experiments the bath contained nominally Ca⁺⁺-free solution H. All experiments were performed at room temperature (20-24°C). Drugs were added to the bath solution as required. Perfusion of the bath was achieved by a gravity feed system, and complete solution exchange was achieved within 10s.

Flash photolysis. Photolysis of Nitr-5 was effected by a 1-ms flash of UV light from a Xenon flashlamp (Hi-Tech Scientific Ltd, Salisbury, UK) directed through the rear port of a Nikon Diaphot microscope (Telford, UK) onto the cell under test as described previously (Kozlowski et al., 1991; Clapp et al., 1996). To allow adequate dialysis of the Nitr-5-containing pipette solution, cells were held at 50 mV for at least 5 min before examining the effects of photoreleased Ca⁺⁺.

Data analysis. Data are presented as mean ± S.E.M. Statistical significance was assessed with a Students t-test. P values  .05 were considered significant. Changes in the magnitude of I_K (peak current at +50 mV) were assessed in each of the cell types by measuring the current in response to 12 ramps (before or after addition of drugs to the bath) and calculating the mean value. To estimate the magnitude of the inward current activated by ET-1, the current elicited 1 min after the onset of the first oscillation was digitized at 100 Hz and its area (nA·ms) determined by the integration facility provided on pClamp software (see Salter and Kozlowski, 1996). A similar method was used to determine the magnitude of the STOCs observed at a holding potential of 0 mV. Single channel data were analyzed with pClamp6 software, after digitization of the signal at 5 kHz, with methods previously used by us (Hartley and Kozlowski, 1996). Changes in channel activity are expressed as percentage changes in N·P_open, where N is the number of functional channels and P_open is the open-state probability of the channel.

	Results

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Introductory remarks. Throughout the course of this study ET-1 was used at a concentration of 16 nM unless otherwise stated. This concentration was chosen because it induces a submaximal response which is well characterized in both contractile (Leach et al., 1990) and electrophysiological studies on small pulmonary arterial smooth muscle cells (previously performed by us; Salter and Kozlowski, 1996).

Electrophysiological characterization of the outward K⁺ current in myocytes. Throughout most of the study, ramp pulses (see "Methods") were used to evaluate the electrophysiological effects of ET-1 on the myocytes maintained in the perforated-patch configuration (solution A in the bath and solution B containing 240 µg·ml¹ amphotericin B in the pipette throughout, unless otherwise stated). This protocol enabled the effect of ET-1 across a wide potential range to be studied during a relatively short time course. This is important because it allows responses with a short duration to be included. In all three smooth muscle cell types a voltage-activated outward current was evoked in response to the ramp pulses. Examples of this current and its mean amplitude at +50 mV for each of the cell types is shown in figure 1, a and b. The current evoked in response to these ramp pulses was sensitive to the K⁺ channel blockers TEA (10 mM) and 4-AP (1 mM) applied extracellularly in all three cell types (fig. 1c). These results, coupled with the classical activation profile of the outward current, suggest it is likely to be carried by K⁺ ions; it is therefore referred to as I_K. Interestingly I_K was differentially sensitive to 4-AP and TEA in the three different myocytes studied, which indicates that I_K itself consists of currents carried through the activity of a range of K⁺ channels.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   (a) Current-voltage relationships recorded under perforated-patch conditions. Records represent currents in response to depolarizing ramp pulses from 100 to +50 mV, in the three cell types, under control conditions. (b) Bar graph showing mean (± S.E.M.) peak current at +50 mV in the three types of myocyte, under perforated-patch recording conditions. (c) Bar graph showing percent inhibition (mean ± S.E.M.) of the voltage-dependent outward current by the K+ channel blockers TEA and 4-AP in the three cell types studied. Solid bars represent the magnitude of inhibition induced by 10 mM TEA during 2 min; hatched bars represent the inhibition induced by 1 mM 4-AP in 2 min. The number of cells is shown in parentheses in both panels b and c.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   (a) Current records obtained during perforated-patch recording from a basilar artery myocyte. The cell was voltage-clamped at 0, 20, 40 and 60 mV for 1 min periods. STOCs, seen as upward deflections in the trace, are clearly voltage dependent; increasing with depolarization. (b) Current records obtained from a basilar artery myocyte voltage-clamped at 0 mV, under perforated-patch conditions in the absence of extracellular Ca++ (solution C in the bath). Addition of IbTX (20 nM) to the bath caused a marked inhibition of the STOCs which was reversed partially on washout. (c) Current-voltage relationships recorded under the same conditions as panel b. Records represent currents in response to depolarizing ramp pulses from 100 to +50 mV in the absence (control) or presence of IbTX (20 nM) in aortic (i), basilar (ii) and pulmonary (iii) arterial myocytes. IbTX mediates a small inhibition in aortic and pulmonary arterial myocytes but markedly reduces the outward current in basilar myocytes. Note, because of the erratic nature of IK in basilar arterial myocytes it is not possible to include a trace showing the mean current (see panel d) in the absence and presence of iberiotoxin. (d) Bar graph showing inhibition (mean ± S.E.M.) of the outward current by IbTX during the 3 min course in the three cell types. Mean inhibition in each cell type was determined by averaging the peak current (at +50 mV) for 1 min before and after addition of IbTX to the bath. The number of cells is shown in parentheses.

ET-1 effects on membrane potential. In perforated-patch current-clamp experiments (solution A in the bath and solution B in the pipette) on cells isolated from the small pulmonary artery (n = 6), ET-1 (0.8 nM) induced a biphasic response. This consisted of a gradual depolarization onto which oscillations of membrane potential were superimposed. In cells isolated from the aorta (n = 4) similar responses were observed during a similar period. In basilar artery myocytes (n = 5), ET-1 induced a gradual membrane depolarization in the absence of depolarizing oscillations. Results of these experiments are illustrated in figure 3a and quantified in figure 3b.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   (a) Current-clamp recordings of cell membrane potential, obtained with the perforated-patch recording configuration. (i) Recording from a pulmonary arterial myocyte. Before application of ET-1 (0.8 nM) the resting potential of this cell was ~38 mV. After application of ET-1, a gradual depolarization was observed. Superimposed on this depolarization were oscillations of membrane potential. (ii) Current-clamp recording from an aortic myocyte. Before application of ET-1 (16 nM), the resting potential of this cell was ~59 mV. Application of ET-1 resulted in a gradual depolarization on which oscillations in membrane potential were superimposed. (iii) Current-clamp recording from a basilar artery myocyte. Before application of ET-1 (16 nM), the resting potential of this cell was ~45 mV. On applying ET-1, a gradual depolarization followed. Note, no oscillations in membrane potential were visible. (b) Bar graph showing ET-1-induced depolarization (mean ± S.E.M.) induced in the three cell types by ET-1 under the conditions described in panel a during the 3 min course. In each case the number of cells is shown in parentheses.

ET-1 effects on membrane current. Perforated-patch voltage-clamp experiments (solution A in the bath, solution B in the pipette), during which ramp pulses were applied, revealed that ET-1 had differential effects on cells from the three tissues. In small pulmonary arterial myocytes ET-1 induced oscillations in inward current, transient enhancements of I_K and a gradual inhibition of I_K (fig. 4a). These electrophysiological effects have been characterized previously in detail (Salter and Kozlowski, 1996) and are included here for comparative purposes only. In aortic myocytes the same three effects were observed in response to ET-1 (fig. 4b). In myocytes isolated from the basilar artery the only observed effect of ET-1 was an inhibition of I_K (fig. 4c). Because of the complex and differential electrophysiological effects of ET-1 on the arterial myocytes studied, each effect is considered independently.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   (a) Current record obtained under voltage-clamp during perforated-patch recording from a myocyte isolated from the pulmonary artery. Application of ET-1 has three electrophysiological effects: (i) activation of an oscillatory ICl(Ca), represented by downward deflections of the trace (illustrated on a faster time base in the inset); (ii) enhancement of IK; and (iii) a slowly developing inhibition of IK. (b) Current record obtained from an aortic myocyte under conditions identical with panel a. Addition of ET-1 to the bath induced a gradual inhibition of IK along with activation of an oscillatory inward current (illustrated on a faster time base in the inset) and transient enhancements of IK. (c) Current record obtained from a basilar artery myocyte under the conditions described in panel a. ET-1 causes a gradual inhibition of IK, but does not evoke oscillations in inward current (see inset).

ET-1-induced inhibition of I_K. The magnitude of the inhibition induced by ET-1 in all three cell types developed for 3 min and was not significantly different among the cell types (fig. 5a). Given the different nature of I_K among the three types of myocyte, it seemed prudent to determine whether this inhibition was caused by inhibition of I_KV or I_K(Ca). Consequently experiments were performed in the presence of IbTX, because if it was the delayed-rectifying component of I_K which was sensitive to ET-1, the degree of inhibition would be identical in the presence or absence of IbTX. Addition of ET-1 to the bath solution C in the presence of IbTX induced inhibition of I_K in both aortic and pulmonary arterial myocytes, the magnitude of which was not significantly different from that observed in the absence of IbTX. Addition of ET-1 to basilar artery myocytes in the presence of IbTX produced significantly less inhibition than when ET-1 was applied alone (fig. 5b). This result suggests that ET-1 induces inhibition of I_KV in the case of aorta and pulmonary artery but not basilar where it inhibits I_K(Ca). Consistent with this result, application of ET-1 to basilar artery myocytes continuously voltage-clamped at 0 mV, a potential where STOCs are highly active, revealed that they too were inhibited (fig. 6a). This effect of ET-1 on the STOCs was quantified in five cells by estimating their area, with the integration facility provided on pClamp software (see "Methods"), for 1 min before and for 3 min after application of ET-1 (fig. 6b). The STOCs were inhibited significantly after 3 min. To verify whether inhibition of I_K(Ca) was mediated through interference with an intracellular process or whether it involved a membrane-delimited, direct effect on K_Ca channels, we performed single-channel recordings on outside-out membrane patches excised from basilar artery myocytes (solution D in the pipette and solution A in the bath). Under these conditions the conductance of the K_Ca channels was ~95 pS. This was consistent with our earlier findings (Hartley and Kozlowski, 1996). Extracellular application of ET-1 to the outside-out membrane patches induced 71.4 ± 14.0% (n = 3) inhibition of K_Ca channel activity. A typical example of the inhibitory effect of ET-1 is illustrated in figure 6c.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   (a) Bar graph showing mean (± S.E.M.) inhibition of IK induced by ET-1 during the 3-min course under perforated-patch recording conditions. (b) Bar graph showing the effect of IbTX on the magnitude of inhibition (mean ± S.E.M.) induced by ET-1 in the three cell types investigated. Filled bars represent the size of IK (pA) inhibited by ET-1 alone. Hatched bars represent the magnitude of IK (pA) inhibited by ET-1 in the presence of IbTX. * P  .05 when compared with application of ET-1 alone. The number of cells is shown in parentheses.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   (a) Current record obtained during perforated-patch recording from a basilar artery myocyte voltage-clamped at 0 mV. In the absence of ET-1, STOCs were observed. These are illustrated more clearly in the inset which shows the highlighted section of trace on a faster time base. Addition of ET-1 to the bath caused a gradual, but marked, inhibition of the STOCs. (b) Bar graph showing 1 min integrals of STOCs (see "Methods"; mean ± S.E.M.) recorded from basilar artery myocytes under the conditions described in panel a. Bars represent the mean of five determinations under control conditions (filled bar) and in the presence of ET-1 (hatched bars). ET-1 significantly inhibited the magnitude of the STOCs after 3 min. * P  .05 when compared with control. (c) Single KCa channel currents recorded from an outside-out patch taken from a basilar artery myocyte at a holding potential of 0 mV. The upper trace represents the KCa channel activity under control conditions (N·Popen = 0.135), whereas the lower trace represents the channel activity after 3 min in the presence of ET-1 (N·Popen = 0.004).

ET-1 induced oscillations in inward current. Oscillations of inward current were observed in both aortic and pulmonary myocytes but not basilar myocytes. The magnitude of the inward current activated for 1 min after onset of the oscillations was determined (see "Methods") for each cell type. These results, together with the average time to onset of this effect after addition of ET-1 to the bath, are summarized quantitatively in figure 7a. This effect is well characterized in pulmonary arterial myocytes (Salter and Kozlowski, 1996) where the oscillations have been caused by activation of a I_Cl(Ca).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   (a) Bar graph showing an estimate of the magnitude of inward current (see "Methods") and time to onset of the current after addition of ET-1 to the bath. Hatched bars represent the magnitude (mean ± S.E.M.) of inward current activated by ET-1 (nA·ms). Filled bars represent the mean time to onset (± S.E.M.) of the oscillations in inward current. The number of cells is shown in parentheses. (b) Current record showing the effect of niflumic acid (50 µM) applied extracellularly to an aortic myocyte maintained under the perforated-patch recording configuration at a holding potential of 70 mV. The oscillatory inward current, previously activated by addition of ET-1 to the bath was inhibited by niflumic acid, an effect partially reversed on wash-out. (c) Current record obtained with the perforated-patch recording configuration in the absence of extracellular Ca++ (solution C in the bath) at a holding potential of 70 mV. Application of caffeine (10 mM) to the bath induced a transient inward current and prevented ET-1-induced periodic oscillations of inward current. (d) Current records obtained from a perforated-patch (experiment on an aortic myocyte under a Cl gradient of 155.2 mM [Cl]o:17 mM [Cl]i (solution E in the bath and solution F in the pipette). At a holding potential of 30 mV, ET-1 activated oscillations in both outward and inward current. When the holding potential was switched to 0 mV outward currents alone were observed. Finally, when the holding potential was changed to 60 mV the currents became unidirectional inward oscillations.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   (a) Current records from flash photolysis experiments on a pulmonary arterial myocyte (solution G, containing 2 mM Nitr-5 in the pipette and solution H in the bath). Cells were voltage-clamped at 50 mV, and a 50-mV hyperpolarizing square pulse (100 ms in duration) followed by a 130 mV depolarizing pulse (500 ms in duration) was applied at a frequency of 0.2 Hz. Current records shown were recorded before (trace 1), during (trace 2) and 50 s after the flash (trace 3). PR-Ca++ activated both inward current (at 100 mV; ) and outward current at +30 mV. (b) Current records from flash photolysis experiments on a basilar artery myocyte under the same conditions as in (a). PR-Ca++ only activated outward current (at +30 mV). (c) Bar graph showing the percentage change (mean ± S.E.M.) in outward current (at +30 mV) and inward current (at 100 mV) in myocytes isolated from basilar artery (filled bars) and pulmonary artery (hatched bars) under conditions described in panel a. The number of cells is shown in parentheses.

ET-1 induced enhancements of I_K. In myocytes isolated from the aorta and pulmonary artery, oscillations in inward current were accompanied by a transient enhancement in I_K. In both cell types, this enhancement was prevented by addition of IbTX to bath solution C (n = 4 and n = 5, respectively; data not shown). This suggests that in these cells ET-1 activates I_K(Ca) in addition to I_Cl(Ca).

Pharmacological characterization of ET-1 effects. Pharmacological experiments with 1 µM FR139317 (an ET_A receptor antagonist; Aramori et al., 1993) or 1 nM STXS6c (an ET_B receptor agonist; Williams et al., 1991) were performed to determine the receptor coupling underlying the electrophysiological effects of ET-1 described above. The ET receptor ligands were added to the bath (solution A) while the pipette contained solution B. Results of these experiments are summarized both qualitatively and quantitatively in table 2. FR139317 prevented activation of the oscillatory inward current and enhancement of I_K in both aortic and pulmonary myocytes as well as prevented inhibition of I_K in basilar myocytes (compare fig. 9a, i, ii and iii with fig. 4, a, b and c, respectively). These results suggest that ET_A receptor stimulation is responsible for these effects. Consistent with these findings STXS6c did not induce oscillatory inward currents or enhance I_K in aortic or pulmonary myocytes, nor did it mediate inhibition of I_K in basilar myocytes (compare fig. 9b, i, ii and iii with fig. 4, a, b and c, respectively). These data suggest that, unlike in pulmonary and aortic myocytes, stimulation of ET_A receptors in the basilar artery underlies the inhibition of I_K(Ca).

View larger version (38K): [in this window] [in a new window]   Fig. 9.   (a) Current records obtained under voltage-clamp during perforated-patch recording (solution A in the bath and solution B in the pipette) from a pulmonary (i), aortic (ii) and basilar (iii) arterial myocyte. FR139317 is present in the bath for the duration of the trace. FR139317 prevented activation of the oscillatory inward currents and enhancement of IK in both aortic and pulmonary myocytes, as well as preventing inhibition of IK in basilar myocytes. It did not inhibit ET-1-mediated inhibition of IK in aortic and pulmonary arterial myocytes. (b) Current records obtained under the conditions described in panel a from a pulmonary (i), aortic (ii) and basilar (iii) arterial myocyte. STXS6c did not induce oscillatory inward currents or enhance IK in aortic or pulmonary myocytes nor did it mediate inhibition of IK in basilar myocytes. It did mediate inhibition of IK in aortic and pulmonary arterial myocytes, however.

View larger version (41K): [in this window] [in a new window]   Fig. 10.   (a) Bar graph illustrating 1 min integrals (see "Methods") of STOCs (mean ± S.E.M.) recorded from basilar myocytes using the perforated-patch configuration of the patch-clamp. Bars represent control (no drug; filled bar), FR139317 alone (open bar) and FR139317 together with ET-1 (hatched bars). Each bar represents the mean of three determinations. Note, ET-1 does not cause inhibition of STOCs in the presence of FR139317 (compare with fig. 6b). (b) Bar graph illustrating 1 min integrals of STOCs (mean ± S.E.M.) in basilar artery myocytes under the conditions described in panel a), in the absence (filled bar) and presence (hatched bars) of STXS6c. Each bar represents the mean of six determinations. Note, STXS6c, unlike ET-1, has no inhibitory effect on the STOCs.

	Discussion

Top Abstract Introduction Methods Results Discussion References

We have previously characterized in detail the electrophysiological effects of ET-1 in pulmonary arterial smooth muscle (Salter and Kozlowski, 1996). The purpose of this, a follow-up study, was to compare the action of the peptide on three types of vascular smooth muscle. The results of our studies clearly show that ET-1 has significantly different effects in different types of arterial smooth muscle. These tissue-specific electrophysiological actions may lead to the identification of novel therapeutic strategies for vascular disease known to involve pathological effects of the peptide (e.g., cerebral vasospasm, Suzuki et al., 1990; and pulmonary hypertension, Stewart et al., 1991).

ET-1 causes a gradual depolarization in pulmonary, aortic and basilar myocytes. This process plays a significant role in smooth muscle contraction by promoting activation of voltage-gated Ca⁺⁺ channels, and is agrees with many contractile studies (e.g., Leach et al., 1990; Lembeck et al., 1989; Sakata et al., 1989; Sudjarwo et al., 1993). In aortic and pulmonary arterial myocytes, oscillations of membrane potential were superimposed onto the gradual depolarization induced by the peptide. These oscillations, which were not seen in basilar myocytes, probably also tend to promote constriction. However, it is presently difficult to envisage their specific role vis-a-vis contraction (which is slow and sustained; Yanagisawa et al., 1988) or why these oscillations are exhibited by thoracic and not by cerebral arterial myocytes.

In pulmonary arterial myocytes, ET-1 causes mobilization of intracellular Ca⁺⁺ stores leading to activation of oscillatory I_Cl(Ca) and I_K(Ca) (Bakhramov et al., 1996; Salter and Kozlowski, 1996). A similar process is likely to underlie the effect of ET-1 in aortic myocytes, because oscillations in these cells, like those in pulmonary arterial myocytes, are inhibited by caffeine. This notion is consistent with the findings of others (Kai et al., 1989). In both pulmonary (see Salter and Kozlowski 1996) and aortic myocytes, oscillatory inward currents are inhibited by niflumic acid which blocks Cl channels. Niflumic acid, however, like all known blockers of chloride channels, is poorly selective and elicits other actions (Greenwood and Large, 1995; Kirkup et al., 1996; Ottolia and Toro, 1994), which include inhibition of a nonselective cation current (Gogelein et al., 1990). We therefore performed ion-exchange experiments to verify the nature of the oscillatory inward current activated in response to ET-1 in aortic myocytes. These experiments revealed that oscillations in membrane current were at least partly caused by activation of I_NS (unlike pulmonary arteries; Salter and Kozlowski, 1996). I_NS has been identified in a variety of tissues including arterial smooth muscle (Chen and Wagoner, 1991; Nakajima et al., 1996, Van Renterghem et al., 1988). Thus it is not surprising that such a current, if present, will be activated in concert with other Ca⁺⁺-activated currents (including I_K(Ca) and I_Cl(Ca)) as the [Ca⁺⁺]_i fluctuates. In basilar myocytes ET-1 did not activate any oscillatory currents, whereas photorelease of caged Ca⁺⁺ did not result in activation of an inward current (that could have been carried either by Cl ions or cations). This strongly suggests that I_Cl(Ca) and I_NS are absent in basilar myocytes, but does not rule out the possibility that ET-1 is still inducing release of Ca⁺⁺ from intracellular stores. This is probably not the case, however, because ET-1 inhibits, rather than activates, I_K(Ca), STOCs and single K_Ca channel currents. Indeed, activation of I_K(Ca) is observed in both pulmonary and aortic myocytes after ET receptor stimulation and (caffeine-sensitive) Ca⁺⁺ release. Our work shows that in basilar artery myocytes K_Ca channels are coupled to ET-receptors (see below) via a membrane-delimited pathway (because ET-1 inhibits these channels in cell-free patches).

Pharmacological experiments suggest that ET_A receptor stimulation underlies activation of I_Cl(Ca) and I_K(Ca) in pulmonary, and also I_NS in aortic, myocytes, because these effects are blocked by FR139317 (a selective ET_A receptor antagonist) and are not mimicked by STXS6c (a selective ET_B agonist). This is consistent with the work of Enoki and co-workers (1995) who showed that the ET_A receptor can couple "functionally" to a nonselective cation channel. Both contractile and binding studies have shown that ET_A receptors predominate in rat aorta (Panek et al., 1992); hence it is possible that ET-1-mediated contraction of rat aorta is mainly the result of ET_A receptor stimulation causing release of intracellular Ca⁺⁺, with subsequent activation of I_NS and I_Cl(Ca) and consequently depolarization. The slow inhibition of I_K in pulmonary and aortic myocytes is the consequence of ET_B receptor stimulation because it is mimicked by STXS6c and is not inhibited by FR139317. In marked contrast, stimulation of ET_A receptors in basilar myocytes appears to inhibit I_K(Ca), because FR139317 prevents ET-1-induced inhibition of I_K(Ca) whereas STXS6c elicits no pharmacological effect.

In conclusion, ET-1 evokes differential electrophysiological effects in smooth muscle cells isolated from the rat aorta, basilar artery and small pulmonary artery. In aortic and pulmonary arterial myocytes ET-1 evokes the three distinct effects, after activation of both ET_A and ET_B receptors. ET_A receptor stimulation causes activation of an oscillatory I_Cl(Ca) and transient enhancements of I_K(Ca), whereas ET_B receptor stimulation induces a slowly developing inhibition of I_K. In addition, ET_A receptor stimulation also activates I_NS in aortic myocytes. In basilar artery myocytes, ET-1 stimulation of ET_A receptors results in an inhibition of K_Ca channel activity through a membrane-delimited pathway. These effects may underlie the potent vasoconstriction mediated by ET-1 in these tissues, and in the future, may allow for pharmacological exploitation and provide a novel approach for the therapy of specific, localized vascular disorders.