Openers of SKCa and IKCa channels enhance agonist-evoked endothelial nitric oxide synthesis and arteriolar vasodilation

Abstract

Recent data have led us to hypothesize that selective activation of endothelial small- and/or intermediate-conductance, calcium-activated potassium channels (SK(Ca) and IK(Ca) channels, respectively) by the opener compounds NS309 and DCEBIO would augment stimulated nitric oxide (NO) synthesis and vasodilation in resistance arteries. Experimentally, ATP-evoked changes in membrane potential, cytosolic Ca(2+), and NO synthesis were recorded by patch clamp and microfluorimetry in single human endothelial cells. Agonist-evoked inhibition of myogenic tone in isolated, pressurized arterioles from rat cremaster skeletal muscle was analyzed by video microscopy. NS309 and DCEBIO enhanced ATP-evoked membrane hyperpolarization and cytosolic Ca(2+) transients, along with acute NO synthesis in isolated endothelial cells. The acetylcholine-mediated inhibition of myogenic tone (IC(50)=237 nM) was left-shifted in the presence of NS309 and DCEBIO (10, 100, and 1000 nM) to IC(50) values of 101, 78, and 43 nM; endothelial denudation inhibited this drug effect. L-NAME attenuated the acetylcholine-induced inhibition of myogenic tone but did not interfere with NS309 and DCEBIO-evoked vasodilation. Collectively, our data demonstrate that drug-induced enhancement of endothelial SK(Ca) and IK(Ca) channel activities represents a novel cellular mechanism to increase vasodilation of small-resistance arterioles, thereby highlighting these channels as potential therapeutic targets in cardiovascular disease states associated with compromised NO signaling.

Figure 1.

K_Ca channel activators NS309 and DCEBIO enhance agonist-evoked increases in NO synthesis and intracellular Ca²⁺ transients in isolated human vascular endothelial cells. A) Microfluorimetric tracing of ATP-evoked increases in acute NO production recorded from a single human vascular endothelial cell in both the absence and presence of the K_Ca channel activators NS309 and DCEBIO (1 μM each). Horizontal bars above tracing indicate drug additions. B) Effects of NS309 and DCEBIO on ATP-evoked cytosolic Ca²⁺ transients in a single human endothelial cell. C, D) Histograms quantifying evoked peak fluorescence changes associated with de novo NO synthesis (C) and increases in cytosolic free Ca²⁺ (D). Results are from 6–8 single-cell measurements recorded under each experimental condition, expressed as means ± se.*P < 0.05 vs. control; Student’s t test.

Figure 2.

NS309 and DCEBIO induce membrane hyperpolarization and enhance hyperpolarizing responses evoked by ATP in single human endothelial cells. A) ATP-evoked changes in membrane potential were recorded using perforated patch-clamp methodology in absence and presence of NS309 and DCEBIO. Horizontal bars above tracing indicate exposures to ATP and/or K_Ca channel activators. B) Representative membrane potential recording of hyperpolarizing responses evoked by 1 μM NS309 and 1 μM DCEBIO, applied either alone or in combination. C) Quantification of evoked changes in membrane potential. Left: peak membrane hyperpolarizations in response to ATP in either absence (open bars) or presence of 1 μM NS309 + DCEBIO (solid bars). Right: hyperpolarizing responses to varying concentrations of NS309 alone, 1 μM DCEBIO alone, and 1 μM NS309 + DCEBIO in combination. Note that all individual mean values differed from 0 under all drug treatment conditions depicted in histogram; P < 0.05. Results represent means ± se calculated from 4–6 observations for each drug exposure condition; only one exposure to a particular drug was applied for a given cell. *P < 0.05 vs. ATP alone; Student’s t test. ^#P < 0.05 vs. 1 μM NS309 + DCEBIO.

Figure 3.

Membrane hyperpolarizations evoked by NS309 and ATP, either alone or in combination, are blocked in the presence of apamin and TRAM-34. A, B) Representative perforated patch-clamp recordings of membrane potential changes in a single human vascular endothelial cell in response to 1 μM NS309 and 10 μM ATP, applied either separately (A) or in an overlapping fashion (B). Horizontal bars above tracings indicate bath applications of NS309, ATP, and apamin + TRAM-34. C) Quantification of the membrane hyperpolarizations evoked by NS309 and ATP in either the absence or presence of 0.1 μM apamin + 1 μM TRAM-34. Data are means ± se from 4 separate single-cell recordings. *P < 0.05 vs. NS309 and/or ATP alone; Student’s t test.

Figure 4.

K_Ca channel openers NS309 and DCEBIO evoke relaxation of myogenic tone in pressurized rat cremaster arterioles and enhance vasodilation in response to acetylcholine. A) Pressurized vessels were briefly exposed (∼1 min) to increasing concentrations of NS309 and DCEBIO in combination, followed by washout; resulting changes in vessel diameter were monitored during each exposure. Following a 30-min recovery period, protocol was repeated using NS309/DCEBIO solutions containing a fixed concentration (0.1 μM) of acetylcholine. Results are expressed as drug-evoked change in vessel diameter, normalized to initial degree of myogenic tone developed by vessel; means ± se calculated from 4–9 individual arterioles. For reference, exposure to 0.1 μM acetylcholine alone produced a dilation of 45.0 ± 3.0% (mean±se, n=5). *P < 0.05 vs. 0.1 μM acetylcholine alone; Student’s t test. ^#P < 0.05 vs. NS309/DCEBIO alone. B) In a different series of pressurized arterioles, vessels were briefly exposed to increasing concentrations of acetylcholine either alone or in presence of a fixed concentration of NS309/DCEBIO that was present continuously in the bath solution. Results are expressed as acetylcholine-evoked change in vessel diameter, normalized to degree of myogenic tone observed under each separate experimental condition. Means ± se calculated from measurements taken from 5–8 separate arterioles.

Figure 5.

NS309- and DCEBIO-induced relaxation of myogenic tone in pressurized rat cremaster arterioles is unaffected by eNOS inhibitor l-NAME, but abolished by functional disruption of vascular endothelium. A) Vasodilatory responses of pressurized arterioles briefly exposed to concentrations of either acetylcholine or NS309/DCEBIO producing ∼80% dilation of the developed myogenic tone. Same arteriole was then treated with 0.1 mM l-NAME for 45–60 min and then reexposed to the same concentrations of acetylcholine and NS309/DCEBIO in the continued presence of l-NAME (bottom panel). B) Quantification of drug-induced dilations in both absence and presence of l-NAME. Results from 5 pressurized vessels, presented as means ± se. C) Quantification of evoked dilation observed in pressurized arterioles exposed to either acetylcholine, NS309/DCEBIO, or NO donor sodium nitroprusside (SNP) either prior to or following functional disruption of vascular endothelium. Endothelium was physically denuded by carefully passing an air bubble through arteriolar lumen following control responses to acetylcholine, K_Ca channel openers, or SNP. Reexposure to these agents was performed once the denuded arteriole regained steady-state myogenic tone. Results are means ± se from 4 separate vessels. *P < 0.05 vs. endothelium intact arterioles; Student’s t test.

Figure 6.

Schematic diagram of putative cellular mechanisms underlying agonist- and drug-induced vasodilation in arteriolar wall. In presence of a vasorelaxant hormone (i.e., acetylcholine), G protein-coupled receptors initiate release of internal Ca²⁺ store, triggering activation of SK_Ca and IK_Ca channels and store-operated Ca²⁺ entry (SOCE). Membrane hyperpolarization induced by K_Ca channel activity supports SOCE and subsequent production of NO by eNOS. Conduction of hyperpolarizing electrical signal through endothelial cells and transfer to vascular smooth muscle cells (VSMC) via gap junctions lowers activity of voltage-gated, L-type Ca²⁺ channels (LTCC), leading to decreased Ca²⁺ influx and vasorelaxation. When NS309 and DCEBIO (red circles) bind and stimulate K_Ca channels, several of these processes are augmented (highlighted in red). Increase in SK_Ca and IK_Ca currents enhances SOCE, leading to greater NO synthesis (red plus signs). Augmented membrane hyperpolarization (red minus signs) would conduct from endothelium to VSMCs (red arrows), causing a more pronounced decrease in LTCC activity. Our finding that K_Ca channel openers are effective in absence of agonist suggests that these agents augment basal SK_Ca and/or IK_Ca channel activity sufficiently to elicit membrane hyperpolarization and vasodilation. Diagram has been modified from refs. , .