The opening of vascular endothelial cell K⁺ channels is pivotal to endothelium-dependent hyperpolarization of arterial myocytes (Edwards et al., 1999; Busse et al., 2002). However, in porcine coronary arteries, such hyperpolarization results at least in part from bradykinin-induced synthesis of epoxyeicosatrienoic acids (EETs) within the endothelium (Fisslthaler et al., 1999). Using TRAM-39 and apamin (selective inhibitors of endothelial cell intermediate- and small-conductance calcium-sensitive K⁺ channels, IK_Ca and SK_Ca, respectively), t he aim of the present study was to determine whether the opening of endothelial cell K⁺ channels is essential for production of EETs.

Using sharp micro-electrodes, membrane potential was recorded in endothelial and smooth muscle cells in endothelium-intact segments of porcine coronary arteries at 37°C. Tissues were constantly superfused with a physiological salt solution containing 300µM L-nitroarginine and 10µM indomethacin. Bradykinin and substance P were added as bolus doses calculated to give, transiently, a concentration of 100nM.

The resting membrane potential of the endothelial cells was –48.9±0.2mV (mean + s.e.mean) (n=8) and substance P and bradykinin produced hyperpolarizations of 25.6±1.0mV and 27.0±0.9mV (each n=4) , respectively. Exposure to 10µM TRAM-39+ 100nM apamin (which depolarized the membrane by 2.5±0.4mV, n=4) prevented any effect of substance P but only partially reduced the hyperpolarization to bradykinin (7.1±1.2mV hyperpolarization in the presence of TRAM-39 + apamin, n=4) . In the continued presence of these inhibitors, the addition of iberiotoxin (100nM) abolished the residual endothelial cell hyperpolarization to bradykinin. Mean peak membrane potentials in the presence of 10µM levcromakalim (–59.1±0.7mV, n=4) were unaffected by TRAM-39 + apamin (–59.4±1.0mV, n=4).

Following gap junction inhibition (with 10 µM HEPES), although the endothelial cell hyperpolarisation to levcromakalim was lost, hyperpolarization to bradykinin was not affected (26.7±0.7mV, n=4). However, under these conditions, the combination of TRAM-39 + apamin abolished the endothelial cell response to bradykinin. Nevertheless, bradykinin still induced hyperpolarisation of the myocytes (12.9± 1.0 mV, n=4), a response which was iberiotoxin-sensitive.

It is concluded that bradykinin, but not substance P, stimulates the release from endothelial cells of a hyperpolarizing factor, most probably an EET, by a mechanism which is independent of the opening of endothelial cell K⁺ channels. The putative EET hyperpolarizes the smooth muscle by activating large-conductance calcium-sensitive K⁺ channels (BK_Ca) on the myocytes. In the presence of TRAM-39 + apamin the endothelial cell hyperpolarization to bradykinin which still occurs is concluded to result indirectly by gap junction transfer of hyperpolarization from the myocytes.

Busse, R. et al. (2002) Trends Pharmacol. Sci. 23, 374-380
Edwards, G. et al. (1998) Nature, 396, 269-272
Fisslthaler, B. et al. (1999) Nature 401, 493–497