Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery

Abstract

1. The effects of endothelium-derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12-EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. 2. Substance P (100 nM) and 11,12-EET (11,12-epoxyeicosatrienoic acid; 3 microM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100 nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100 nM). 3. Substance P (100 nM) and bradykinin (30 nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12-EET hyperpolarized de-endothelialized vessels by 12.6+/-0.3 mV, an effect abolished by 100 nM iberiotoxin. 4. 11,12-EET hyperpolarized intact arteries by 18.6+/-0.8 mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11, 12-EET and substance P were partially inhibited by 100 nM charybdotoxin and abolished by further addition of 100 nM apamin. 5. 30 microM barium plus 500 nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500 microM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. 6. Substance P-induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. 7. In pig coronary artery, 11,12-EET is not EDHF. Smooth muscle hyperpolarizations attributed to 'EDHF' are initiated by endothelial cell hyperpolarization involving charybdotoxin- (but not iberiotoxin) and apamin-sensitive K(+) channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded.

Figure 1

Typical traces comparing the smooth muscle hyperpolarizations in pig coronary artery induced by bolus application of 11,12-EET and its vehicle, ethanol (EtOH), with those induced by bolus application of substance P (SP), NS1619 and levcromakalim (LK). Microelectrode impalements were made through the adventitial surface. (a) In de-endothelialized vessels, substance P was without effect. Iberiotoxin (IbTX) abolished the response to NS1619 and reduced the magnitude of the hyperpolarization to 11,12-EET to that of its vehicle. (b,c) In intact vessels, iberiotoxin had little effect on the response to 11,12-EET or to substance P in the absence or presence of apamin. In contrast, charybdotoxin (ChTX) markedly reduced the hyperpolarization to 11,12-EET and slightly inhibited the response to substance P. Responses to 11,12-EET and substance P were abolished in the presence of both charybdotoxin and apamin. (a–c) Hyperpolarizations induced by levcromakalim were similar in the presence or absence of the endothelium and were not modified by the presence of the toxin K⁺ channel inhibitors.

Figure 2

Effect of iberiotoxin on smooth muscle hyperpolarizations induced by bolus exposure to 11,12-EET, its vehicle (ethanol, EtOH), NS1619 and levcromakalim (LK) in de-endothelialized segments of porcine coronary artery. The results are the mean of four experiments typified by the trace in Figure 1(a). In the absence of the endothelium, substance P (SP) was without effect. Each column indicates the membrane potential before and after exposure to the indicated substance, +or−s.e.mean.

Figure 3

Effects of iberiotoxin (a) and charybdotoxin (b) on smooth muscle hyperpolarizations induced by bolus application of 11,12-EET, its vehicle (ethanol, EtOH) and substance P (SP) in intact segments of porcine coronary artery. The results in each graph are the mean of four experiments typified by the traces in Figure 1(b) and (c). Each column indicates the membrane potential before and after exposure to the indicated substance, +or−s.e.mean.

Figure 4

Typical traces comparing the endothelial cell hyperpolarizations in pig coronary artery induced by bolus application of 11,12-EET and its vehicle, ethanol (EtOH), with those induced by bolus application of substance P (SP). (a) Iberiotoxin (IbTX) was without effect on the hyperpolarizations to substance P, to 11,12-EET or its vehicle, ethanol (EtOH). (b) Charybdotoxin (ChTX) markedly reduced the hyperpolarization to 11,12-EET but had little effect on the response to substance P. Responses to 11,12-EET and substance P were abolished in the presence of both charybdotoxin and apamin. (c and d) Graphical representation of data from four separate experiments of the types shown in (a) and (b), respectively in which each column represents the endothelial cell membrane potential (m.p.), +or−s.e.mean, before and after exposure to substance P, EtOH or 11,12-EET. Responses are shown before (control) or after exposure to (c) 100 nM iberiotoxin or (d) 100 nM charybdotoxin in the absence and presence of 100 nM apamin.

Figure 5

Effects of Ba²⁺ and ouabain and of pre-treatment with gap 27 on hyperpolarizations induced by bolus exposure to substance P (SP), acetylcholine (ACh) and levcromakalim (LK) in smooth muscle cells of pig intact coronary arteries. Microelectrode impalements were made through the adventitial surface. (a) The upper trace shows that substance P and levcromakalim each produced smooth muscle hyperpolarization whereas acetylcholine was without effect. Exposure to 30 μM Ba²⁺+500 nM ouabain produced a depolarization usually accompanied by rapid membrane potential oscillations (inset panel) and responses to substance P and levcromakalim were essentially unchanged. (b) After 60 min incubation of a different vessel in Krebs solution containing 500 μM gap 27, substance P generated only a small hyperpolarization whereas a substantial effect of levcromakalin was always observed. Exposure to Ba²⁺+ouabain produced a depolarization which was accompanied by fewer membrane potential oscillations. The effects of substance P were essentially absent while the effects of levcromakalim were unchanged or enhanced. Note that the rapid oscillations usually depolarized the membrane to approximately zero mV and that their indicated magnitude was limited by the speed of the A-D converter.

Figure 6

Graphical representation of data from four separate experiments of the types shown in Figure 5 in which the columns represent the membrane potential (m.p.)+or−s.e.mean in vessels exposed to 30 μM Ba²⁺+500 nM ouabain (Ba+ouab) and 500 μM gap 27. (a) Shows absolute values of membrane potential (m.p.) before and after exposure to the agonists to highlight the depolarizing effect of Ba²⁺+ouabain. (b) Hyperpolarizations (Δ m.p.) produced by substance P (SP), bradykinin (BK) and levcromakalim (LK) to emphasise the inhibitory effects of gap 27 and Ba²⁺+ouabain on responses to substance P and bradykinin while responses to levcromakalim were unaffected.

Figure 7

(a,b) Typical traces showing effects of Ba²⁺ and ouabain and of pre-treatment with gap 27 on hyperpolarizations induced by bolus application of substance P (SP) and levcromakalim (LK) in smooth muscle cells of pig intact coronary arteries impaled via the intimal surface. (a) Substance P and levcromakalim each produced smooth muscle hyperpolarization. Exposure to 30 μM Ba²⁺+500 nM ouabain produced an almost instantaneous sustained depolarization usually accompanied by a single, rapid, transient depolarization to 0 mV. The responses to substance P and levcromakalim were not reduced by the presence of barium and ouabain. (b) After 60 min incubation of a different vessel in Krebs solution containing 500 μM gap 27, responses to substance P and levcromakalim were similar to those before exposure to the gap junction inhibitor (see panel (a)). Subsequent exposure to barium+ouabain produced a sustained depolarization which developed more slowly than in the absence of gap 27 and which was never associated with a rapid, transient depolarization. The effects of substance P and levcromakalim were not reduced in the presence of barium and ouabain after exposure to gap 27. (c,d) Graphical representation of data from 4–5 separate experiments in which the columns represent the membrane potential (m.p.), +or−s.e.mean, before and after exposure to substance P or levcromakalim under control conditions and after exposure to 30 μM Ba²⁺+500 nM ouabain (Ba+ouab) and 500 μM gap 27 as indicated. (c) Representation showing absolute values of membrane potential (m.p.) to highlight the depolarizing effect of Ba²⁺+ouabain. (d) Hyperpolarizations (Δ m.p.) produced by 100 nM substance P and levcromakalim to emphasise the lack of inhibitory effects of gap 27 and Ba²⁺+ouabain.

Figure 8

(a,b) Typical traces showing effects of gap 27 plus Ba²⁺ and ouabain on smooth muscle hyperpolarization induced by continuous superfusion of substance P (SP) in pig intact coronary arteries impaled via the intimal (i) or adventitial (ii) surfaces. (a) The onset of the response to substance P was more rapid when myocytes were impaled from the intimal side. (b) Following 60 min exposure to gap 27, tissues were exposed to 30 μM barium (Ba²⁺)+500 nM ouabain (+inhibitors). Under these conditions, the magnitude of the hyperpolarization induced by substance P was not reduced in recordings made from the intimal side but was substantially diminished in recordings from the adventitial side. (c,d) Graphical representation of data from 7–14 separate experiments in which the columns represent the membrane potential (m.p.), +or−s.e.mean, when myocytes were impaled from either the intimal or adventitial sides under control conditions or after exposure to 30 μM Ba²⁺+500 nM ouabain and 500 μM gap 27 (+inhibitors). (c) Absolute values of membrane potential (m.p.) before and after exposure to 100 nM substance P to highlight the depolarizing effect of Ba²⁺+ouabain. (d) Hyperpolarizations (Δ m.p.) produced by substance P to emphasise the effects of the inhibitors.