Bradykinin-induced relaxation of coronary microarteries: S-nitrosothiols as EDHF?

Abstract

1. To investigate whether S-nitrosothiols, in addition to NO, mediate bradykinin-induced vasorelaxation, porcine coronary microarteries (PCMAs) were mounted in myographs. 2. Following preconstriction, concentration-response curves (CRCs) were constructed to bradykinin, the NO donors S-nitroso-N-penicillamine (SNAP) and diethylamine NONOate (DEA-NONOate) and the S-nitrosothiols L-S-nitrosocysteine (L-SNC) and D-SNC. All agonists relaxed PCMAs. L-SNC was approximately 5-fold more potent than D-SNC. 3. The guanylyl cyclase inhibitor ODQ and the NO scavenger hydroxocobalamin induced a larger shift of the bradykinin CRC than the NO synthase inhibitor L-NAME, although all three inhibitors equally suppressed bradykinin-induced cGMP responses. 4. Complete blockade of bradykinin-induced relaxation was obtained with L-NAME in the presence of the large- and intermediate-conductance Ca(2+)-activated K(+)-channel (BK(Ca), IK(Ca)) blocker charybdotoxin and the small-conductance Ca(2+)-activated K(+)-channel (SK(Ca)) channel blocker apamin, but not in the presence of L-NAME, apamin and the BK(Ca) channel blocker iberiotoxin. 5. Inhibitors of cytochrome P450 epoxygenase, cyclooxygenase, voltage-dependent K(+) channels and ATP-sensitive K(+) channels did not affect bradykinin-induced relaxation. 6. SNAP-, DEA-NONOate- and D-SNC-induced relaxations were mediated entirely by the NO-guanylyl cyclase pathway. L-SNC-induced relaxations were partially blocked by charybdotoxin+apamin, but not by iberiotoxin+apamin, and this blockade was abolished following endothelium removal. ODQ, but not hydroxocobalamin, prevented L-SNC-induced increases in cGMP, and both drugs shifted the L-SNC CRC 5-10-fold to the right. 7. L-SNC hyperpolarized intact and endothelium-denuded coronary arteries. 8. Our results support the concept that bradykinin-induced relaxation is mediated via de novo synthesized NO and a non-NO, endothelium-derived hyperpolarizing factor (EDHF). S-nitrosothiols, via stereoselective activation of endothelial IK(Ca) and SK(Ca) channels, and through direct effects on smooth muscle cells, may function as an EDHF in porcine coronary microarteries.

Figure 1

Relaxations of PCMAs, preconstricted with U46619, to bradykinin in absence (control; a, b) or presence of 100 μM L-NAME (c, d) with one or more of the following inhibitors: 1 μM Hoe140, 10 μM ODQ, 10 μM indomethacin, 100 μM L-NAME, 200 μM hydroxocobalamin (HC), 0.5 mM ouabain, 30 μM BaCl₂, 1 μM glibenclamide, 5 mM 4-aminopyridine, 10 μM miconazole or 10 μM sulfaphenazole. Data (mean±s.e.mean; n=5–45) are expressed as a percentage of the contraction induced by U46619.

Figure 2

Relaxations of PCMAs, preconstricted with U46619, to bradykinin in absence (control; a, b) or presence of 100 μM L-NAME (c, d) with one or more of the following inhibitors: 100 nM charybdotoxin (char), 100 nM apamin (apa), or 100 nM iberiotoxin (iber). Data (mean±s.e.mean; n=5–45) are expressed as a percentage of the contraction induced by U46619.

Figure 3

Relaxations of PCMAs, preconstricted with U46619, to SNAP in the absence (control) or presence of one or more of the following inhibitors: 10 μM ODQ, 200 μM hydroxocobalamin (HC), 100 nM charybdotoxin (char) or 100 nM apamin (apa). Data (mean±s.e.mean; n=5–14) are expressed as a percentage of the contraction induced by U46619.

Figure 4

Relaxations of PCMAs, preconstricted with U46619, to DEA-NONOate in the absence (control) or presence of one or more of the following inhibitors: 10 μM ODQ, 200 μM hydroxocobalamin (HC), 100 nM charybdotoxin (char) or 100 nM apamin (apa). Data (mean±s.e.mean; n=4) are expressed as a percentage of the contraction induced by U46619.

Figure 5

Relaxations of PCMAs, preconstricted with U46619, to L-SNC or D-SNC in the absence (control) or presence of 10 μM ODQ and/or 200 μM hydroxocobalamin (HC). Data (mean±s.e.mean; n=4–18) are expressed as a percentage of the contraction induced by U46619.

Figure 6

Relaxations of PCMAs without (a) or with (b, c) endothelium, preconstricted with U46619, to L-SNC or D-SNC in the absence (control) or presence of one or more of the following inhibitors: 10 μM ODQ, 200 μM hydroxocobalamin (HC), 100 nM charybdotoxin (char) or 100 nM apamin (apa). Data (mean± s.e.mean; n=4-18) are expressed as a percentage of the contraction induced by U46619.

Figure 7

Relaxations of PCMAs, preconstricted with U46619, to L-SNC in the absence (control) or presence of one or more of the following inhibitors: 100 nM iberiotoxin (iber), 100 nM apamin (apa), 5 mM 4-aminopyridine (4-AP), 100 μM L-NAME, 1 μM glibenclamide, 0.5 mM ouabain or 30 μM BaCl₂. Data (mean±s.e.mean; n=4–9) are expressed as a percentage of the contraction induced by U46619.

Figure 8

Cyclic GMP levels (expressed as % of baseline) in PCMAs after 1 min exposure to (a) bradykinin (1 μM) or (b) L-SNC (10 or 100 μM) under control conditions (no blocker) and in the presence of 10 μM ODQ, 200 μM hydroxocobalamin (HC), 1 μM Hoe140 and/or 100 μM L-NAME. Data are mean±s.e.mean (n=3–10). ♯P<0.05 vs control, ^*P<0.05 vs no blocker.

Figure 9

Hyperpolarization of smooth muscle cells by 100 nM bradykinin and 50 μM L-SNC in porcine coronary arteries with or without endothelium. (a) resting membrane potential (RMP). (b) change in membrane potential. Experiments were performed in the presence of 300 μM L-NA, 10 μM diclofenac and 1 μM U46619. Data are mean±s.e.mean of five to six separate experiments; ^*P<0.01 vs control. U46619 did not significantly affect RMP (–44.5±1.2 mV vs –42.7±1.7 mV, n=6), and in parallel experiments, using arterial rings from the same pig and following preconstriction with the same U46619 concentration (1 μM), 100 nM bradykinin relaxed the arteries by 89±9% (n=6).