Effects of chronic in vivo administration of nitroglycerine on ACh-induced endothelium-dependent relaxation in rabbit cerebral arteries

Abstract

Background and purpose: In the setting of nitrate tolerance, endothelium-dependent relaxation is reduced in several types of peripheral vessels. However, it is unknown whether chronic in vivo administration of nitroglycerine modulates such relaxation in cerebral arteries.

Experimental approach: Isometric force and smooth muscle cell membrane potential were measured in endothelium-intact strips from rabbit middle cerebral artery (MCA) and posterior cerebral artery (PCA).

Key results: ACh (0.1-10 microM) concentration-dependently induced endothelium-dependent relaxation during the contraction induced by histamine in both MCA and PCA. Chronic (10 days) in vivo administration of nitroglycerine reduced the ACh-induced relaxation in PCA but not in MCA, in the presence of the cyclooxygenase inhibitor diclofenac (3 microM). In the presence of the NO-synthase inhibitor N (omega)-nitro-L-arginine (L-NNA, 0.1 mM) plus diclofenac, in MCA from both nitroglycerine-untreated control and -treated rabbits, ACh (0.1-10 microM) induced a smooth muscle cell hyperpolarization and relaxation, and these were blocked by the small-conductance Ca(2+)-activated K(+)-channel inhibitor apamin (0.1 microM), but not by the large- and intermediate-conductance Ca(2+)-activated K(+)-channel inhibitor charybdotoxin (0.1 microM). In contrast, in PCA, ACh (<3 microM) induced neither hyperpolarization nor relaxation under these conditions, suggesting that the endothelium-derived relaxing factor is NO in PCA, whereas endothelium-derived hyperpolarizing factor (EDHF) plays a significant role in MCA.

Conclusions and implications: It is suggested that in rabbit cerebral arteries, the function of the endothelium-derived relaxing factor NO and that of EDHF may be modulated differently by chronic in vivo administration of nitroglycerine.

Figure 1

Effects of ACh on membrane potential of smooth muscle cells in endothelium-intact strips of middle cerebral arteries. (a) and (b) Original recordings of membrane potential changes induced by ACh (3 μM) obtained from a nitroglycerine-untreated control rabbit. Applications of ACh, apamin (0.1 μM) and charybdotoxin (0.1 μM) are indicated by horizontal bars. (c) Concentration-dependent effects of ACh (0.1–10 μM) on membrane potentials in nitroglycerine-untreated control and nitroglycerine-treated rabbits. Data are shown as means (±s.e.mean) from five animals. Diclofenac (3 μM) and N^ω-nitro-L-arginine (0.1 mM) were present throughout the experiments. RMP, resting membrane potential.

Figure 2

ACh-induced relaxation during the contraction produced by histamine in endothelium-intact strips of middle cerebral artery (MCA, a) and posterior cerebral artery (PCA, b) in nitroglycerine-untreated control rabbits. L-NNA (−) and (+), in the absence and presence of N^ω-nitro-L-arginine (L-NNA), respectively. Apamin (−) and (+), in the absence and presence of apamin, respectively. L-NNA (0.1 mM) was given as pretreatment for 60 min and was present thereafter. Apamin (0.1 μM) was given as pretreatment for 3 min and was present during the cumulative application of ACh in the presence of L-NNA. Diclofenac (3 μM) was present throughout the experiments. The concentration of histamine used to produce contraction was 10 μM in the absence of L-NNA, but for amplitude matching this was reduced to 1–2 μM in the presence of L-NNA. The amplitude of contraction induced by histamine before application of ACh was normalized as a relative tension of 1.0 for each curve. Data are shown as means (±s.e.mean) from four to six animals. In MCA, the pD₂ values for ACh were: for ‘L-NNA (−), Apamin (−),' 5.80±0.07 and for ‘L-NNA (+), Apamin (−),' 5.72±0.12. For ‘L-NNA (−)' in PCA, the corresponding value was 5.85±0.08. ^**P<0.01 by a two-way repeated-measures ANOVA.

Figure 3

Effects of chronic in vivo administration of nitroglycerine on ACh-induced relaxation in endothelium-intact strips of middle cerebral artery (MCA, a) and posterior cerebral artery (PCA, b). L-NNA (−) and (+), in the absence and presence of N^ω-nitro-L-arginine (L-NNA), respectively. Apamin (−) and (+), in the absence and presence of apamin, respectively. Data for ‘Control rabbit' are replotted from Figure 1. L-NNA (0.1 mM) was given as pretreatment for 60 min and was present thereafter. Apamin (0.1 μM) was given as pretreatment for 3 min and was present during the cumulative application of ACh in the presence of L-NNA. Diclofenac (3 μM) was present throughout the experiments. The concentration of histamine used to produce contraction was 10 μM in the absence of L-NNA, but for amplitude matching this was reduced to 1–2 μM in the presence of L-NNA. The amplitude of contraction induced by histamine before application of ACh was normalized as a relative tension of 1.0 for each curve. Data are shown as means (±s.e.mean) from four to six animals. In MCA from nitroglycerine-treated rabbits, the pD₂ values for ACh were: for ‘L-NNA (−), Apamin (−),' 6.02±0.06 and for ‘L-NNA (+), Apamin (−),' 5.86±0.08. For ‘L-NNA (−)' in PCA from nitroglycerine-treated rabbits, the corresponding value was 5.96±0.01. ^**P<0.01 by a two-way repeated-measures ANOVA.

Figure 4

Effect of in vitro application of L-arginine (1 mM) on ACh-induced relaxation in posterior cerebral arteries from nitroglycerine-untreated control and nitroglycerine-treated rabbits. L-Arginine (−), in the absence of L-arginine; L-Arginine (+), in the presence of L-arginine, which was given as pretreatment for 60 min. The amplitude of contraction induced by histamine (10 μM) before application of ACh was normalized as a relative tension of 1.0 for each curve. Diclofenac (3 μM) was present throughout the experiments. Data are shown as means (±s.e.mean) from four animals. In control rabbits, the pD₂ values for ACh were: for ‘L-Arginine (−),' 6.10±0.17 and for ‘L-Arginine (+),' 6.00±0.14, with the corresponding values in nitroglycerine-treated rabbits being 6.05±0.10 and 6.01±0.11, respectively. ^*P<0.05 by two-way repeated-measures ANOVA.

Figure 5

Effect of the NO donor, NOC-7 (1–300 nM), on histamine-induced contraction in endothelium-intact strips of posterior cerebral artery from nitroglycerine-untreated control and nitroglycerine-treated rabbits. N^ω-nitro-L-arginine (0.1 mM) and diclofenac (3 μM) were present throughout the experiments. The amplitude of contraction induced by histamine (1 μM) before application of NOC-7 was normalized as a relative tension of 1.0 for each curve. Data are shown as means (±s.e.mean) from four animals.