Decreased Role for BKCa Channels in Endothelium‐Dependent Relaxation of Nitrate Tolerant Coronary Arteries

Abstract

Prolonged exposure to nitroglycerin (NTG) leads to tolerance and impaired endothelium‐dependent vasodilation. Causes of endothelial dysfunction in nitrate tolerant arteries are well studied, but little is known about mechanisms that underlie the responses to endothelium‐derived mediators in nitrate tolerant blood vessels. Here we evaluated the role of large conductance, calcium‐activated K channels (BKCa) in acetylcholine (ACh)‐induced relaxation of isolated coronary arteries. Rats (Sprague‐Dawley, 250–300g) were treated with or without transdermal NTG patches (0.6 mg/hr) for 3 days. Isolated coronary arterial rings (diameter: 150–300 μm) were mounted in a multi‐wire myograph for isometric tension recording. Relaxation responses were determined in rings contracted with 5‐HT (0.1–1μM). In some experiments, arterial segments were incubated with the selective BKCa channel inhibitor, iberiotoxin (IBTx, 100 nM), for 20 min prior to contraction. Expression of BKCa channel proteins was determined by Western blot. Nitrate tolerance was confirmed by the impaired relaxation response to NTG (1 nM – 100 μM) observed in rings from rats treated with NTG patches (pD2= 7.28 ± 0.1 vs 6.45 ± 0.2, control vs NTG patch‐treated, respectively; p<0.05). Relaxations to the endothelium‐dependent vasodilator, ACh (1 nM – 10 μM) were impaired in nitrate tolerant arteries (pD2 = 7.01 ± 0.1 vs 6.52 ± 0.1; control vs tolerant, respectively; n=8; p<0.05). Incubation with the eNOS inhibitor, nitro‐l‐arginine (30 μM), or the guanylyl cyclase inhibitor, ODQ (10 μM), markedly inhibited the response to ACh in control and tolerant arteries, consistent with the release of endothelium‐derived NO in ACh‐induced relaxation of both control and tolerant arteries. Similarly, IBTx markedly reduced the response to ACh in control and tolerant arteries; however, IBTx was a more effective inhibitor of ACh‐induced relaxation in control arteries. Relaxations to 8‐Br‐cGMP (100 nM – 300 μM), a stable, cell‐permeable cGMP analog, were inhibited by IBTx in control arteries (pD2 = 5.37 ± 0.2 vs 4.21 ± 0.2; in the absence and presence of IBTx, respectively; n=4; p<0.05), but not in tolerant arteries (pD2 = 4.45 ± 0.1 vs 4.03 ± 0.1; in the absence and presence of IBTx; n=3; p>0.05). Protein expression of BKCa α‐subunits and β‐subunits was decreased by more than 40% in nitrate tolerant arteries (n=4–5; p<0.05). Taken together, the data indicate that, under control conditions, BKCa channels play a pivotal role in endothelium‐dependent relaxation to ACh in rat coronary arteries. In nitrate tolerant coronary arteries, the response to ACh is impaired and the contribution of BKCa channels to ACh‐induced relaxation is diminished.Support or Funding InformationSupported in part by grant HL098896 from the National Institutes of Health