Bradykinin potentiation by ACE inhibitors: a matter of metabolism

Abstract

1. Studies in isolated cells overexpressing ACE and bradykinin type 2 (B(2)) receptors suggest that ACE inhibitors potentiate bradykinin by inhibiting B(2) receptor desensitization, via a mechanism involving protein kinase C (PKC) and phosphatases. Here we investigated, in intact porcine coronary arteries, endothelial ACE/B(2) receptor 'crosstalk' as well as bradykinin potentiation through neutral endopeptidase (NEP) inhibition. 2. NEP inhibition with phosphoramidon did not affect the bradykinin concentration-response curve (CRC), nor did combined NEP/ACE inhibition with omapatrilat exert a further leftward shift on top of the approximately 10 fold leftward shift of the bradykinin CRC observed with ACE inhibition alone. 3. In arteries that, following repeated exposure to 0.1 microM bradykinin, no longer responded to bradykinin ('desensitized' arteries), the ACE inhibitors quinaprilat and angiotensin-(1-7) both induced complete relaxation, without affecting the organ bath fluid levels of bradykinin. This phenomenon was unaffected by inhibition of PKC or phosphatases (with calphostin C and okadaic acid, respectively). 4. When using bradykinin analogues that were either completely or largely ACE-resistant ([Phe(8)psi(CH(2)-NH)Arg(9)]-bradykinin and [deltaPhe(5)]-bradykinin, respectively), the ACE inhibitor-induced shift of the bradykinin CRC was absent, and its ability to reverse desensitization was absent or significantly reduced, respectively. Caveolar disruption with filipin did not affect the quinaprilat-induced effects. Filipin did however reduce the bradykinin-induced relaxation by approximately 25-30%, thereby confirming that B(2) receptor-endothelial NO synthase (eNOS) interaction occurs in caveolae. 5. In conclusion, in porcine arteries, in contrast to transfected cells, bradykinin potentiation by ACE inhibitors is a metabolic process, that can only be explained on the basis of ACE-B(2) receptor co-localization on the endothelial cell membrane. NEP does not appear to affect the bradykinin levels in close proximity to B(2) receptors, and the ACE inhibitor-induced bradykinin potentiation precedes B(2) receptor coupling to eNOS in caveolae.

Figure 1

Relaxations of porcine coronary arteries, preconstricted with 1 μM U46619, to bradykinin in the absence (control) or presence of 10 μM quinaprilat, 10 μM omapatrilat or 1 μM phosphoramidon. Data (mean±s.e.mean of five experiments) are expressed as a percentage of the contraction induced by U46619.

Figure 2

Relaxations of porcine coronary arteries, following preconstriction with 1 μM U46619, to three consecutive bradykinin doses (0.1 μM; BK1, BK2, BK3), followed by 10 μM quinaprilat, 10 μM omapatrilat, or 1 μM phosphoramidon. Top, original tracing; bottom, mean±s.e.mean of five experiments (data are expressed as a percentage of the contraction induced by U46619).

Figure 3

Relaxations of porcine coronary arteries, following preconstriction with 1 μM U46619, to three consecutive bradykinin doses (0.1 μM; BK1, BK2, BK3), followed by 10 μM quinaprilat, in the absence (control) or presence of 1 μM calphostin C or 0.5 μM okadaic acid. Data (mean±s.e.mean of five experiments) are expressed as a percentage of the contraction induced by U46619.

Figure 4

Relaxations of porcine coronary arteries, preconstricted with 1 μM U46619, to bradykinin, PA-bradykinin or DP-bradykinin in the absence (control) or presence of 10 μM quinaprilat, 10 μM captopril or 10 μM angiotensin-(1–7). Data (mean±s.e.mean of 5–28 experiments) are expressed as a percentage of the contraction induced by U46619.

Figure 5

Relaxations of porcine coronary arteries, following preconstriction with 1 μM U46619, to three consecutive bradykinin, PA-bradykinin or DP-bradykinin doses (0.1, 0.1 and 0.03 μM, respectively; BK1, BK2, BK3), followed by 10 μM quinaprilat. Top, original tracings of experiments with PA-bradykinin and DP-bradykinin; bottom, mean±s.e.mean of 5–12 experiments (data are expressed as a percentage of the contraction induced by U46619). *P<0.01 vs bradykinin.

Figure 6

Metabolism of bradykinin by porcine coronary artery rings during incubation of 15 ml organ bath fluid at 37°C with 1 μM bradykinin in the absence (control) or presence of 10 μM quinaprilat. Data are mean±s.e.mean of five experiments and have been expressed as a percentage of the level at t=0 (0.70±0.04 and 0.82±0.04 μM without and with quinaprilat).

Figure 7

Relaxations of porcine coronary arteries, preconstricted with 1 μM U46619, to bradykinin (top panels) or PA-bradykinin (bottom panels) in the absence (control) or presence of 10 μM quinaprilat, following pretreatment with 4 μg ml⁻¹ filipin, 2% cyclodextrin or 20 μg ml⁻¹ nystatin. Data (mean±s.e.mean of 4–10 experiments) are expressed as a percentage of the contraction induced by U46619.

Figure 8

Relaxations of porcine coronary arteries, preconstricted with 1 μM U46619, to SNAP in the absence (control) or presence of 4 μg ml⁻¹ filipin, 2% cyclodextrin or 20 μg ml⁻¹ nystatin. Data (mean±s.e.mean of 5–21 experiments) are expressed as a percentage of the contraction induced by U46619.

Figure 9

Relaxations of porcine coronary arteries, following preconstriction with 1 μM U46619, to three consecutive bradykinin doses (0.1 μM; BK1, BK2, BK3), followed by 10 μM quinaprilat, in the absence (control) or presence of 4 μg ml⁻¹ filipin, 2% cyclodextrin or 20 μg ml⁻¹ nystatin. Data are mean±s.e.mean of 5–12 experiments and have been expressed as a percentage of the contraction induced by U46619.