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Identification of the potassium channels involved in EDHF-mediated responses. Characteristics of endothelium-dependent hyperpolarizations in the isolated carotid artery of the guinea-pig (in the presence of inhibitors of NO-synthase and cyclooxygenase). (A) Acetylcholine (ACh)-induced hyperpolarizations in the vascular smooth muscle of the guinea-pig carotid artery are endothelium-dependent. In the absence of the endothelium, acetylcholine no longer produces a hyperpolarization. This is not due to damage following removal of the endothelium since the K_ATP opener, cromakalim (10^-5 M), still hyperpolarizes the smooth muscle cells. In the presence of the endothelial cells, raising the extracellular potassium concentration from 5 to 35 mM depolarizes the smooth muscle cells and prevents the hyperpolarizing effect of acetylcholine, suggesting that the endothelium-dependent hyperpolarization involves the opening of a potassium conductance. (B) Glibenclamide, the K_ATP blocker, does not affect the endothelium-dependent hyperpolarization to acetylcholine but abolishes the endothelium-independent hyperpolarizations to the NO donor, S-nitroso-L-glutathione (SNOG) and that to the prostacyclin analogue, iloprost. (C) The combination of two blockers of calcium-activated potassium channels, apamin (a selective blocker of small conductance calcium-activated potassium channel, SK_Ca) and charybdotoxin (a non-selective blocker of large and intermediate conductance calcium-activated and some voltage-activated potassium channels: BK_Ca, IK_Ca, and K_V), inhibits the acetylcholine-induced endothelium-dependent hyperpolarization while the endothelium-independent hyperpolarizations to either S-nitroso-L-glutathione or to iloprost are unaffected by the combination of the two toxins. (D) The concentration- and endothelium-dependent hyperpolarizations produced by acetylcholine are not affected by the presence of charybdotoxin (ChTx), partially inhibited by that of apamin (Apa) and virtually abolished by the combination of the two toxins. Similarly, TRAM-34, a non-peptidic and selective blocker of IK_Ca, does not affect the hyperpolarization elicited by acetylcholine while UCL 1684, a selective blocker of SK_Ca, produces a partial inhibition. The combination of the two blockers prevents the hyperpolarizing effect of acetylcholine. TRAM-34, UCL 1684 and their combination mimic the effects of charybdotoxin, apamin, and the association of the two toxins, respectively. Taken into conjunction, these observations indicate that EDHF-mediated responses are selectively associated with the opening of the two potassium channels SK_Ca and IK_Ca.

Compensatory EDHF-mediated responses in eNOS knockout mice. (A) eNOS knock-out mice are hypertensive. (B) Acetylcholine (ACh) produces an endothelium-dependent relaxation in the isolated aortic rings of wild type mice [eNOS(+/+)] but no longer in that of eNOS knockout mice [eNOS(-/-)]. However, the NO-donor and endothelium-independent vasodilator sodium nitroprusside (SNP) produces a relaxation in the aorta from both strains. These results confirm the deletion of the eNOS gene and demonstrate that, in a large blood vessel such as the aorta, NO is the preponderant endothelium-derived relaxing factor released by acetylcholine. (C) Acetylcholine induces a vasodilatation in the hindlimb of both wild type and eNOS knockout mice. In the wild type mice, this vasodilatation is unaffected by a depolarizing solution (KCl) and partially inhibited by the combination of charybdotoxin (CTX) and apamin (apa) but not by charybdotoxin alone, indicating that an EDHF-mediated response may contribute to the mechanism of vasodilatation but is not the preponderant mechanism. In the eNOS knockout mice, the acetylcholine-induced vasodilatation is preserved and is blocked by KCl or the combination of charybdotoxin plus apamin, partially inhibited by charybdotoxin alone and unaffected by the combination of iberiotoxin (IbTX), apamin, glibenclamide (Glib) and 4-aminopyridine (4AP). These results indicate that acetylcholine induces an endothelium-dependent vasodilatation showing the hallmarks of EDHF-mediated responses. The EDHF-mediated response compensates the disruption of the eNOS gene. (D) Original recording of arterial blood pressure in an anesthetized eNOS knockout mouse treated with an inhibitor of cyclooxygenase, diclofenac, and an inhibitor of NO-synthase, L-arginine-methyl-ester (L-NAME). The intravenous administration of the endothelium-dependent vasodilator bradykinin induces a transient decrease in arterial blood pressure. The summary bar graphs show the concentration-dependent changes in mean arterial pressure (MAP) evoked by administration of bradykinin in wild type [eNOS(+/+)] and eNOS(-/-) mice under control conditions (top graphs) or after treatment with inhibitors of cyclooxygenases and NO-synthases (lower graphs). The amplitude of the hypotensive response induced by bradykinin is similar in eNOS knockout and wild-type animals. In both strains, the effect of bradykinin is unaffected by the presence of inhibitors of cyclooxygenases and NO-synthases. These data strongly suggest that EDHF-mediated responses play an essential role in the hypotensive effect of bradykinin [142]

Intermediate conductance calcium-activated potassium channels in endothelial cells. (A) The various configurations of the patch clamp technique are shown. The patch-clamp technique is an electrophysiological technique that allows the study of ionic channels expressed in the cell membrane or in a lipid bilayer. A polished glass pipette is brought into contact with a given cell. When the tip of the pipette touches the cell membrane, a tight seal is formed (giga-seal). Then, by applying various maneuvers, suction, excision, permeation of the membrane with toxins, the global electrophysiological properties of the ionic channel expressed by the cell and/or the properties of individual channels can be studied. (B) IK_Ca in freshly dissociated porcine coronary endothelial cells (outside-out configuration of the patch-clamp technique). Single-channel activity was recorded at the indicated holding potentials under asymmetrical K⁺ gradients with Ca²⁺ fixed at 250 nM in the pipette solution. Mean unitary currents were plotted against holding potential, fitted with a linear function and the slope conductance computed (17.5 pS). (C) The effects of iberiotoxin and the subsequent addition of charybdotoxin were recorded. Distributions of unitary conductance amplitude (recorded at 0 mV and using 250 nM free Ca²⁺ in the pipette solution) are shown, together with representative traces. A Gaussian fit was performed for every single channel experiment, and the amplitude of single channels was determined with the best fit. Then, the mean of the obtained amplitudes was calculated. These data show that the current is insensitive to iberiotoxin (IbTX) but blocked by charybdotoxin (ChTX).

SK_Ca in freshly dissociated porcine coronary endothelial cells (outside-out configuration of the patch-clamp technique). (A) A small-conductance channel, as observed in single-channel recordings (250 nM free Ca²⁺ pipette solution, in the presence of charybdotoxin, physiological K⁺ gradient). (B) The unitary conductance was determined by variance analysis of activity recorded at -20, 0, 20 and 40 mV (with the assumption of a single channel). Data were fitted to give slope conductance of a 6.8 pS channel. (C) Effect of Ca²⁺ on the open probability of SK_Ca. Distributions of unitary conductance amplitude recorded at 0 mV are shown, together with representative traces. A Gaussian fit was performed for every single channel experiment, and the amplitude of single channels was determined with the best fit. Then, the mean of the obtained amplitudes was calculated. The effect of Ca²⁺ was determined using pipette solutions with free Ca²⁺ fixed at 100 and 250 nM. (D) The inhibitory effects of apamin were recorded. Distributions of unitary conductance amplitude (recorded at 0 mV and using 250 nM free Ca²⁺ in the pipette solution) are shown, together with representative traces. A Gaussian fit was performed for every single channel experiment, and the amplitude of single channels was determined with the best fit. Then, the mean of the obtained amplitudes was calculated. (E) Endothelial localization of the SK3 α-subunit in porcine coronary arteries by immunofluorescence labeling. A cross-section labeled for SK3 and nuclei. The inset shows primary antibody pre-incubated with control peptide: sole the auto-fluorescence of the internal elastic lamina and nuclei are clearly visible). The scale bar represents 25 μm [161]. EC: endothelial cells; VSMC: vascular smooth muscle cells.