Characterisation of the nitric oxide synthase- and cyclooxygenase-independent, endothelium-dependent relaxation induced by A23187 in sheep coronary arteries RN Senaratna & G Boachie-Ansah. Sunderland Pharmacy School, University of Sunderland, Sunderland SR1 3SD.	Print Abstract Search PubMed for: Senaratna RN Boachie-Ansah G

In most arterial beds, a significant endothelium-dependent relaxant response to various stimuli persists even after inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX). This dilator response is usually associated with a hyperpolarisation of vascular smooth muscle cells, and has been attributed to a diffusible hyperpolarising factor released by the endothelium (EDHF), whose chemical identity and precise cellular mechanism of action remain as yet unclear (McGuire et al., 2001). We sought in this study to determine the characteristics of the 'EDHF-like' relaxant response induced by the Ca⁺⁺ ionophore, A23187, in sheep coronary arteries.

Paired sheep circumflex coronary artery rings were set up for isometric tension recording, as previously described (Grainger et al., 2001). In study series I, one of each pair of rings was left endothelium intact and untreated (control), while the other ring was endothelium denuded or pre-treated for 30 min with both the NO synthase inhibitor, L-NAME (100 µM) and cyclooxygenase inhibitor, indomethacin (3 µM). In study series II, rings were pre-treated with both L-NAME (100 µM) and indomethacin (3 µM) and then randomised to pre-treatment with either vehicle (control) or a cytochrome P450 (CYP) inhibitor, K⁺ channel blocker or the Na⁺-K⁺-ATPase inhibitor, ouabain (3 µM). Rings were precontracted to endothelin-1 (2-3 nM) or 25 mM KCl and subsequently relaxed by cumulative addition of A23817 (0.1-300 nM). Mean sensitivity (pEC₅₀ values) and % maximal relaxation (R_max) to A23817 in control and treated rings were compared using Student's t-test or ANOVA and Dunnett's post-hoc test, as appropriate.

A23817 (0.1-300 nM) induced concentration-dependent relaxation of endothelin-1-evoked contractions in rings with intact endothelium (mean pEC₅₀ of 8.0*0.8 and R_max of 102.1±2.1%), and this effect was completely abolished following endothelium denudation (n=6). In endothelium-intact rings pre-treated with L-NAME (100 µM) and indomethacin (3 µM), A23817 still induced concentration-dependent relaxation of endothelin-1-evoked contractions, but with reduced potency and efficacy (mean pEC₅₀ and R_max of 7.40±0.15 and 64.8±7.0%, compared with 8.12±0.19 and 97.3±5.6%, respectively, in control rings; P<0.03; n=6). This A23817-induced NOS- and COX-independent relaxant response was unaffected by pre-treatment with the CYP inhibitors, -naphthoflavone (30 µM), sulfaphenazole (30 µM), 1-aminobenzotriazole (3 mM), or 17-octadecynoic acid (20 µM), but was completely abolished in rings contracted to 25 mM KCl rather than to endothelin-1 (n=4).

Pre-treatment with the large conductance, Ca⁺⁺-activated K⁺ (BK_Ca) channel blockers, TEA (1 mM) and iberiotoxin (100 nM), the ATP-sensitive K⁺ (K_ATP) channel blocker, glibenclamide (3 µM), the delayed rectifier K⁺ (K_DR) channel blocker, 4-aminopyridine (1 mM), or the inward rectifier K⁺ (K_IR) channel blocker, BaCl₂ (100 µM; Nelson & Quayle, 1995), did not modify the A23817-induced, NOS- and COX-independent dilator response. By contrast, the small conductance, Ca⁺⁺-activated K⁺ (SK_Ca) channel blocker, apamin (100 nM), completely abolished the A23817-induced dilator response (n=6), whereas the Na⁺-K⁺-ATPase inhibitor, ouabain (3 µM), markedly attenuated the response, reducing the R_max of A23817 from 74.9±4.9% in controls to 33.9±9.1%.

These findings (i) indicate that A23817 induces a significant endothelium-dependent, NOS-, COX- and CYP-independent relaxation in sheep coronary arteries, and (ii) suggest that this dilator effect is mediated via the opening of small conductance, Ca⁺⁺-activated K⁺ (SK_Ca) channels and/or stimulation of the Na⁺-K⁺-ATPase.

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