ABSTRACT
The elusive nature of endothelium-derived hyperpolarizing factor ( E D H F ) has hampered detailed study of the underlying currents in isolated smooth muscle cells with voltageclamp techniques. The aim of the present study was to characterize the components of current attributable to E D H F by recording from segments of intact submucosal arterioles (20-40 um outside diameter) of guinea-pigs. These arterioles consist of a single layer of smooth muscle cells which can be voltage-clamped with single intracellular microelec trodes coupled to a switching voltage clamp amplifier. For the voltage-clamp studies, arterioles were cut into segments (100-300 um in length), and voltage ramps were applied to determine current-voltage relationships. In the presence of nitric oxide and prosta glandin synthesis inhibitors, stimulation of the endothelium with acetylcholine evoked a hyperpolarization that was abolished by charybdotoxin plus apamin. Under voltageclamp, acetylcholine evoked an outward current. Charybdotoxin alone reduced the amplitude of the outward current, while the presence of apamin plus charybdotoxin abolished it. Subtraction of the currents revealed charybdotoxin-sensitive and apaminsensitive currents, and their separate current-voltage relationships were determined. Both currents reversed near the expected potassium equilibrium potential, were weakly out wardly rectifying, and displayed little, if any, time or voltage-dependent gating. The components have the biophysical and pharmacological characteristics of the intermediate conductance and small conductance calcium-activated potassium channels, respectively. Thus, the hyperpolarization attributed to endothelium-derived hyperpolarizing factor involves two current components, characteristics of which are consistent with activation of intermediate and of small conductance calcium-activated potassium channels. These channels are responsible for the prominent hyperpolarization evoked by endothelial stimulation in guinea-pig submucosal arterioles.
