Bradykinin-induced, endothelium-dependent responses in porcine coronary arteries: involvement of potassium channel activation and epoxyeicosatrienoic acids

Abstract

In coronary arteries, bradykinin opens endothelial intermediate- and small-conductance Ca2+-sensitive K+ channels (IK(Ca) and SK(Ca)) and, additionally, releases epoxyeicosatrienoic acids (EETs) from the endothelium. To clarify the involvement of these pathways in endothelium-dependent myocyte hyperpolarization, bradykinin-induced electrical changes in endothelial cells and myocytes of porcine coronary arteries (following nitric oxide (NO) synthase and cyclooxygenase inhibition) were measured using sharp microelectrodes. Hyperpolarization of endothelial cells by bradykinin (27.0 +/- 0.9 mV, n = 4) was partially inhibited (74%) by blockade of IK(Ca) and SK(Ca) channels using 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile) plus 100 nM apamin (leaving an iberiotoxin-sensitive component), whereas the response to substance P was abolished. After gap junction blockade with HEPES, (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulphonic acid)) hyperpolarization of the endothelium by 100 nM bradykinin was abolished by TRAM-39 plus apamin, whereas myocyte hyperpolarization still occurred (12.9 +/- 1.0 mV, n=4). The residual hyperpolarizations to 100 nM bradykinin were antagonized by the EET antagonist, 14,15-EEZE (14,15-epoxyeicosa-5(Z)-enoic acid) (10 microM), and abolished by iberiotoxin. Bradykinin-induced myocyte hyperpolarizations were also reduced by 14,15-EEZE-mSI (14,15-EEZE-methylsulfonylimide) (5,6- and 14,15-EET antagonist), whereas those to exogenous 11,12-EET were unaffected. These data show that bradykinin-induced hyperpolarization of endothelial cells (due to the opening of IK(Ca) and SK(Ca) channels) is electrotonically transferred to the myocytes via gap junctions. Bradykinin (but not substance P) also hyperpolarizes myocytes by a mechanism (independent of endothelial cell hyperpolarization) which involves endothelial cell production of EETs (most likely 14,15- and/or 11,12-EET). These open endothelial IK(Ca) and SK(Ca) channels and also activate large-conductance calcium-sensitive K+ channels (BK(Ca)) on the surrounding myocytes.

Figure 1

Effect of 10 μM TRAM-39 and 100 nM apamin on changes in endothelial cell membrane potential (m.p.) induced by substance P, bradykinin, 1-EBIO and levcromakalim in segments of endothelium-intact porcine coronary arteries in the presence of 300 μM nitro-L-arginine and 10 μM indomethacin. (a, c) Typical traces showing the responses before and in the presence of TRAM-39 and/or TRAM-39+apamin (a) and in the additional presence of 100 nM iberiotoxin (c). Graphical representation of data from four separate experiments of the types shown in (a) and (c) are shown in (b) and (d), respectively. Each column in (b) and (d) represents m.p.±s.e.m., before and during exposure to bolus doses of substance P, bradykinin, 1-EBIO or levcromakalim calculated to give, transiently, the final bath concentrations indicated.

Figure 2

Effect of TRAM-39 + apamin on endothelial cell and myocyte responses to bradykinin and 11,12-EET in endothelium-intact segments of porcine coronary arteries preincubated for 18 h in HEPES-buffered Tyrode solution to inhibit gap junctions. (a) Typical trace showing endothelial cell responses in the absence and presence of 10 μM TRAM-39+100 nM apamin. After recording from the endothelial cell, the electrode was withdrawn (^*) and a myocyte in the same artery segment was impaled. Myocyte responses to bradykinin, 11,12-EET and levcromakalim were then recorded in the continued presence of the TRAM-39+apamin. (b) Graphical representation of data from four separate experiments of the type shown in (a); each column represents the membrane potential (m.p.)±s.e.m., before and during exposure to bolus doses of levcromakalim, bradykinin and 11,12-EET calculated to give, transiently, the final bath concentrations indicated. Experiments were performed in the presence of 300 μM nitro-L-arginine and 10 μM indomethacin.

Figure 3

Effect of TRAM-39 + apamin and iberiotoxin on myocyte hyperpolarizations induced by bradykinin, 1-EBIO and 11,12-EET in endothelium-intact porcine coronary artery segments in the presence of 300 μM nitro-L-arginine and 10 μM indomethacin. (a) Typical trace showing responses in the absence and presence of 10 μM TRAM-39+100 nM apamin and in the additional presence of 100 nM iberiotoxin. Levcromakalim was used to demonstrate the integrity of the tissue. The slanting bar shows where the continuous trace was cut for publication purposes. (b) Graphical representation of data from four separate experiments of the type shown in (a); each column represents the membrane potential (m.p.)±s.e.m., before and during exposure to bolus doses of bradykinin, 1-EBIO, 11,12-EET and levcromakalim calculated to give, transiently, the final bath concentrations indicated.

Figure 4

(a) Typical trace showing effect of 10 μM 14,15-EEZE and 100 nM iberiotoxin on 10 μM TRAM-39+100 nM apamin-resistant myocyte hyperpolarizations induced by bradykinin and 11,12-EET in endothelium-intact porcine coronary artery segments in the presence of 300 μM nitro-L-arginine and 10 μM indomethacin. (b) Graphical representation of data from four separate experiments of the type shown in (a). Levcromakalim was used to demonstrate the integrity of the tissue. Each column represents the membrane potential (m.p.)±s.e.m. (n=4), before and during exposure to bolus doses of the agents indicated, calculated to give, transiently, the stated final bath concentrations.

Figure 5

Effect of 14,15-EEZE-mSI on myocyte hyperpolarizations induced by bradykinin, substance P and 11,12-EET in endothelium-intact segments of porcine coronary arteries in the presence of 300 μM nitro-L-arginine and 10 μM indomethacin. (a) Typical trace showing responses before and in the presence of 10 μM 14,15-EEZE-mSI. (b) Graphical representation of data from four separate experiments of the type shown in (a) in which each column represents the membrane potential (m.p.)±s.e.m. (n=4), before and during exposure to bolus doses of the agents indicated (calculated to give, transiently, the final bath concentrations indicated) in the absence and presence of 14,15-EEZE-mSI.

Figure 6

Effect of 10 μM 14,15-EEZE-mSI on myocyte hyperpolarizations induced by bradykinin, 11,12-EET, 14,15-EET and NS1619 in endothelium-denuded segments of porcine coronary arteries in the presence of 300 μM nitro-L-arginine and 10 μM indomethacin. (a) Typical trace from a single continuous impalement but with sections between responses removed for clarity. (b) Graphical representation of data from four experiments of the type shown in (a) in which each column represents the membrane potential (m.p.)±s.e.m. (n=4), before and after exposure to bolus doses of NS1619, 5,6-, 11,12- and 14,15-EET (calculated to give, transiently, the final bath concentrations indicated) in the absence and presence of 14,15-EEZE-mSI.

Figure 7

Typical traces comparing the effects of 14,15-EEZE (a) and iberiotoxin (b) on myocyte hyperpolarizations induced by transient exposure to bradykinin, substance P and 11,12-EET in endothelium-intact segments of porcine coronary arteries in the presence of 300 μM nitro-L-arginine and 10 μM indomethacin. (c, d) Graphical representation of data from experiments of the types shown in (a, b) in which each column represents the membrane potential (m.p.)±s.e.m. (n=4), before and during exposure to bolus doses of bradykinin, substance P and 11,12-EET calculated to give, transiently, the final bath concentrations indicated.

Figure 8

Bradykinin produces endothelium-dependent myocyte hyperpolarization in coronary arteries via two pathways. One of these (solid lines) involves the opening of endothelial SK_Ca and IK_Ca channels that can be blocked with apamin and TRAM-39, respectively. Substance P activates only this pathway. The other (dashed lines) involves the generation of epoxyeicosatrienoic acids (EETs) via a cytochrome P450 (CYP450)-dependent mechanism. EETs not only activate endothelial SK_Ca and IK_Ca channels but also open myocyte BK_Ca channels sensitive to iberiotoxin. This component is usually masked by the hyperpolarization resulting from the opening of endothelial SK_Ca and IK_Ca channels. Neither the generation of EETs nor their effect on BK_Ca requires endothelial cell hyperpolarization.