Combination of Ca2+ -activated K+ channel blockers inhibits acetylcholine-evoked nitric oxide release in rat superior mesenteric artery

Abstract

Background and purpose: The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation.

Experimental approach: Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured.

Key results: A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+ -activated K channels abolished acetylcholine (10 microM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 microM), an inhibitor of sarcoplasmic Ca2+ -ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA.

Conclusions and implications: Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.

Figure 1

(a) Representative recordings of NO microsensor calibration performed in PSS at 37°C with constant stirring. The vertical bars below the trace indicate injection of increasing concentrations of NO. The horizontal bar indicates time. (b) Linear regression analysis of the relationship between amount of NO added and current output of the microsensor for one electrode tip (ISONOP3020), r²=0.9878, 1 pA=0.49 nM.

Figure 2

Original recordings of endothelial cell membrane potential in two arterial segments showing that (a) ACh (10 μM)-induced hyperpolarization is abolished in the presence of apamin (500 nM) and charybdotoxin (100 nM), whereas (b) ACh induces hyperpolarization in the absence and the presence of BaCl₂ (30 μM) and ouabain (100 μM). (c) Average of ACh-evoked hyperpolarization in the absence and in the presence of the combination of BaCl₂ (30 μM) and ouabain (100 μM) or apamin (500 nM) and charybdotoxin (100 nM). The experiments were performed in the presence of 3 μM indomethacin. The points are means±s.e.m. of arteries from three to seven animals. Significant differences evaluated by one-way analysis of variance (ANOVA) followed by unpaired t-test: ^*P<0.05 versus control.

Figure 3

Oxyhaemoglobin reverses relaxations and increases in NO concentration persisting in the presence of an NOS inhibitor. Original trace showing recording of simultaneous changes in tension (upper traces) and NO concentration (lower traces) in a segment of rat superior mesenteric artery (a) with endothelium and (b) after mechanical removal of the endothelium. (c and d) Average increases in NO concentration and relaxations induced by ACh (10 μM) in NA (0.5 μM)-contracted preparations in the absence and the presence of indomethacin (3 μM), indomethacin and ADMA (300 μM), and indomethacin, ADMA plus oxyhaemoglobin (OxHb, 10 μM). The points are means±s.e.m. of arteries from four to five animals. Significant differences evaluated by one-way ANOVA followed by unpaired t-test: ^*P<0.05 versus control.

Figure 4

Raising the extracellular K⁺ concentration inhibits ACh-evoked increases in NO concentration. Average simultaneous increases in (a) NO concentration and (b) relaxations induced by ACh in rat superior mesenteric arterial segments contracted with either NA (6.7±0.8 Nm⁻¹, n=6), 50 mM K⁺ (K-50, 6.0±0.4 Nm ⁻¹, n=6) or 80 mM K⁺ (K-80, 8.1±0.7 Nm⁻¹, n=4). (c) Concentration–response curves for ACh in arterial segments contracted with 50 mM K⁺ in the absence and the presence of ADMA (300 μM), oxyhaemoglobin (OxHb, 10 μM) and ADMA plus OxHb. The experiments were performed in the presence of 3 μM indomethacin. The points are means±s.e.m. of arteries from five to seven animals. ^*P<0.05 versus control, ^†P<0.05 versus ADMA and ^#P<0.05 versus OxHb.

Figure 5

Effect of the combination of apamin and charybdotoxin on ACh-evoked relaxations and NO concentrations. Original trace recordings showing simultaneous changes in tension (a) and NO concentration (b) in a vascular segment of the rat superior mesenteric artery. Contraction was induced by NA and a first control relaxation and increase in NO concentration was obtained for ACh, and a second response in the presence of apamin (500 nM) and charybdotoxin (100 nM). Finally, the NO donor, SNAP, was added. Average increases in (d) NO concentration and (c) relaxations induced by ACh in the absence or the presence of both apamin and charybdotoxin. The experiments were performed in the presence of 3 μM indomethacin. The points are means±s.e.m. of arteries from six animals. ^*P<0.05 versus control.

Figure 6

Effect of the endogenous NOS inhibitor, ADMA, and the combination of apamin and charybdotoxin. Average increases in (a) NO concentration and (b) relaxations induced by ACh in the absence or the presence of ADMA (300 μM), ADMA, apamin (500 nM) and charybdotoxin (100 nM), and ADMA, BaCl₂ (30 μM) and ouabain (100 μM). (c and d) Effect of these treatments on NA-evoked NO concentration and contraction. The experiments were performed in the presence of 3 μM indomethacin. The points are means±s.e.m. of arteries from five to six animals. ^*P<0.05 versus control and ^†P<0.05 versus ADMA.

Figure 7

Role of extracellular calcium for the release of NO in rat superior mesenteric artery. Original recordings of four different vascular segments from two animals showing (a) increase in NO concentration to ACh in the presence and the absence of extracellular calcium, and (b) in the absence and the presence of the inhibitor of the store-operated calcium channels, SKF 96365. (c) Average of increases in NO concentration to ACh in the presence and the absence of Ca²⁺, and SKF 96365. The experiments were performed in the presence of 3 μM indomethacin. The points are means±s.e.m. of arteries from six animals. ^*P<0.05 versus control, Student's t-test.

Figure 8

Effect of apamin and charybdotoxin on CPA-induced increase in NO on the rat superior mesenteric artery. Original recordings of two different vascular segments showing increase in NO concentration, measured with the same microsensor, to CPA (a) in the presence and the absence of extracellular calcium, and (b) in the absence and the presence of apamin (500 nM) and charybdotoxin (100 nM). The ACh was added at the height of the CPA-induced increase in NO concentration. (c) Average of increases in NO concentration to CPA in the absence and the presence of extracellular calcium, and apamin plus charybdotoxin. The experiments were performed in the presence of 3 μM indomethacin. The points are means±s.e.m. of arteries from four to six animals. ^*P<0.05 versus control, Student's t-test.

Figure 9

Simultaneous measurements of endothelial cell calcium and tension in the rat superior mesenteric artery. NA-evoked increases (a) in endothelial cell calcium and (b) tension were, respectively, decreased and enhanced in the presence of the combination of apamin (500 nM) and charybdotoxin (100 nM). Addition of (c and d) ACh (10 μM) and (e and f) CPA (30 μM) increased endothelial cell calcium and caused relaxation. Apamin plus charybdotoxin did not change endothelial cell calcium (c and e), but decreased ACh and CPA-evoked relaxation (d and f). Changes in FURA-2 fluorescence is expressed as increases in ratio (Δ ratio) of emission intensities at the two excitation wavelengths (340 versus 380 nm). Basal fluorescence intensity was 0.828±0.010 and 0.832±0.013 (n=8), respectively, in the absence and the presence of apamin plus charybdotoxin. The results are means±s.e.m. of arteries from eight animals. The measurements were paired. Significant differences between curves were evaluated by comparison of the area under the curve followed by a paired t-test: ^*P<0.05 versus control.