Characteristics of the actions by which 5-hydroxytryptamine affects electrical and mechanical activities in rabbit jugular vein graft

Abstract

Background and purpose: The vasomodulating actions of 5-HT in vein grafts, and the underlying mechanisms, remain to be fully clarified. Here, we characterized the actions by which 5-HT affects electrical and mechanical activities in rabbit autologous jugular vein grafts.

Experimental approach: Smooth muscle cell (SMC) membrane potential and isometric tension were measured in vein grafts 4 weeks after implantation into carotid arteries. Changes in the expression of 5-HT receptor subtypes and in myosin heavy chain isoforms (SM1, SM2 and SMemb) were examined by immunohistochemistry and Western blot analysis.

Key results: The walls of grafted veins displayed massive increases in the number of SM1- and SM2-positive SMCs. 5-HT induced a large depolarization and contraction that were each reduced by both 5-HT(2A) - and 5-HT(1B/1D) -receptor antagonists. The 5-HT-induced contraction was not modified by a 5-HT₇ -receptor antagonist. The 5-HT₇ -receptor-selective agonist AS 19 did not induce relaxation during the contraction to prostaglandin F(2α) . Immunohistochemical and Western blot analyses revealed that immunoreactive responses against 5-HT(2A) and 5-HT(1B/1D) receptors were increased in the vein graft.

Conclusions and implications: 5-HT is able to induce a large contraction in rabbit autologous jugular vein grafts through (i) an increased number of differentiated contractile SMCs; (ii) an increased number of SMCs expressing contractile 5-HT(2A) - and 5-HT(1B/1D) receptors; and (iii) a down-regulation of the function of the relaxant SMC 5-HT₇ receptors. These changes in the vein graft may help it to resist the higher pressure present on the arterial side of the circulation.

Figure 1

5-HT-induced mechanical responses in endothelium-intact strips of normal vein and grafted vein. (A) Effects of l-NNA on the mechanical responses induced by high K⁺ (128 mM) and 5-HT (0.03–10 µM) in normal vein (a1) and grafted vein (a2). After recording control responses to high K⁺ and 5-HT (left column), l-NNA (0.1 mM) was applied for 60 min and high K⁺ and 5-HT were applied in the presence of l-NNA. Concentrations of 5-HT: (1) 0.03 µM; (2) 0.1 µM; (3) 0.3 µM; (4) 1 µM; (5) 10 µM. (B) Absolute maximum tension induced by 128 mM K⁺ or 10 µM 5-HT in the absence and presence of l-NNA in normal vein (‘Normal vein’) and vein graft (‘Grafted vein’) (n= 6 in each case). Data are shown as mean ± SEM. *P < 0.05 versus before l-NNA, ^††P < 0.01 versus ‘Normal vein’. (C) Effects of l-NNA on 5-HT-induced contractions in vein graft. Data are shown as mean ± SEM. *P < 0.05 after versus before l-NNA.

Figure 2

Haematoxylin-eosin staining in vascular wall of vein grafts. (A) Haematoxylin-eosin staining in ‘Normal vein’ (left panel) and ‘Vein-graft’ (right panel). (B) Number of nuclei in the media region of ‘Normal vein’ (n= 4) and in the intima/media region of ‘Vein-graft’ (n= 4). (C) Wall thickness of media region in ‘Normal vein’ (n= 4) and of intima/media region in ‘Vein-graft’ (n= 4). Data are shown as mean ± SEM. ***P < 0.001 versus ‘Normal vein’.

Figure 3

Immunohistochemical staining for α-smooth muscle actin and myosin heavy chain isoforms (SM1, SM2 and SMemb) in vascular wall of carotid artery (‘Carotid artery’), jugular vein from normal rabbit (‘Normal vein’) and jugular vein graft from vein-grafted rabbit (‘Vein-graft’). Carotid artery, a1–a4; Normal vein, b1–b4; Vein graft, c1–c4. Immunohistochemistry was performed using antibodies against α-smooth muscle actin (a1, b1, c1), SM1 (a2, b2, c2), SMemb (a3, b3, c3) and SM2 (a4, b4, c4). Note that strong green fluorescence indicates elastic lamina in carotid artery. Similar observations were made in three other preparations.

Figure 4

Expressions of SM1, SM2 and SMemb in ‘Control vein’ and ‘Vein-graft’. Protein expressions of SM1 (A), SM2 (B) and SMemb (C) were measured by Western blot analysis in ‘Control vein’ and ‘Vein-graft’. Each column represents the mean of data from five different preparations (each from a different animal) with SEM. **P < 0.01 versus ‘Control vein’.

Figure 5

Effects of the ATP-sensitive K⁺-channel opener Y-26763 on membrane potential in smooth muscle cells of non-grafted vein and grafted vein. (A) Actual traces of Y-26763 (10 µM)-induced membrane potential changes in normal vein (a1) and vein graft (a2). (B) Summary of the effects of Y-26763 on SMC membrane potential (n= 3–8). Data are shown as mean ± SEM.

Figure 6

5-HT-induced changes in membrane potential in smooth muscle cells of vein graft. (A) Actual traces of 5-HT (0.1 µM)-induced membrane potential changes. After recording the control 5-HT response (control), sarpogrelate (1 µM) was pretreated for 20 min and 5-HT was then applied in the presence of sarpogrelate. Next, GR55562 (1 µM) was added for 20 min in the presence of sarpogrelate. Finally, 5-HT was applied in the presence of sarpogrelate+GR55562. (B) b1, Effects of sarpogrelate and sarpogrelate+GR55562 on resting membrane potential in smooth muscle cells (n= 6). b2, Effect of sarpogrelate on the 5-HT-induced membrane depolarization in smooth muscle cells and effect of GR55562 on that response (n= 6). Data are shown as mean ± SEM. ***P < 0.001 versus control, ^†P < 0.05 versus sarpogrelate.

Figure 7

Effects of sarpogrelate and/or GR55562 on the contraction induced by 5-HT, together with the effects of TCB-2 and sumatriptan on mechanical activities in endothelium-denuded vein grafts. (A) Effects of sarpogrelate (1 µM) with or without GR55562 (1 µM) on 5-HT-induced contraction. Data are shown as mean ± SEM. ***P < 0.001 versus ‘Control’ (n= 6). ^†P < 0.05 versus sarpogrelate (n= 6). (B) Effects of GR-55562 alone. *P < 0.05 versus control (n= 6). (C) Effects of TCB-2 (n= 9) and sumatriptan (n= 7). The maximum tension induced by 10 µM 5-HT was normalized as a relative tension of 1.0 in any given strip. ***P < 0.001 for TCB-2 versus sumatriptan.

Figure 8

Effects of SB269970 on 5-HT-induced contraction in endothelium-denuded vein grafts. (A) SB269970 (1 µM) did not modify the contraction induced by 5-HT (0.01–10 µM; n= 4). (B) Effects of 5-HT (0.1–1 µM) and AS 19 (10 µM) during the contraction induced by 1 µM PGF_2α in the presence of sarpogrelate+GR55562. 5-HT was cumulatively applied in the presence of PGF_2α. Following a 20 min washout with Krebs solution, PGF_2α (1 µM) was again applied and AS 19 (10 µM) was added during the PGF_2α contraction.

Figure 9

Expressions of 5-HT_2A, 5-HT_1B/1D and 5-HT₇ receptors in vascular wall of normal vein and vein graft. (A) Normal vein: a1–a3. Vein graft: b1–b3. Immunofluorescence images showing localization of antibodies against the 5-HT_2A (a1, b1), 5-HT_1B/1D (a2, b2) or 5-HT₇ (a3, b3) receptor in cross-sections of jugular vein. Similar observations were made in three other preparations. (B) Western blot analysis of 5-HT_2A receptors (b1) and 5-HT_1B receptors (b2) in ‘Control vein’ and ‘Vein-graft’. Each column represents the mean of data from four to five different preparations (each from a different animal) with SEM. *P < 0.05 versus ‘Control vein’.

Figure 10

Effects of nifedipine, with or without Y-27632 or hydroxyfasudil, on contractions induced by high K⁺ and 5-HT in endothelium-denuded vein grafts. (A) Actual traces showing the effects of nifedipine (1 µM), in the absence or presence of Y-27632 (10 µM), on the contractions induced by 128 mM K⁺ (a1) and 10 µM 5-HT (a2). After the control response induced by either high K⁺ or 5-HT had been recorded, nifedipine was applied as a pretreatment for 30 min and the same stimulant was applied in the presence of nifedipine. Under these conditions, Y-27632 was applied as a pretreatment and the same stimulant was finally applied in the presence of nifedipine+Y-27632. (B) Summary of the results obtained for the effects of nifedipine with or without Y-27632 on the contractions induced by 128 mM K⁺ (b1) and 10 µM 5-HT (b2). The maximum tension induced by 10 µM 5-HT was normalized to 1.0 in each preparation. (C) Summary of the results obtained (using a similar protocol to that described above) for the effects of nifedipine with or without hydroxyfasudil on the contractions induced by 10 µM 5-HT. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001 versus ‘None’, ^††P < 0.01, ^†††P < 0.001 versus ‘Nifedipine’.