5-HT is a potent relaxant in rat superior mesenteric veins

Abstract

Serotonin (5-HT, 5-hydroxytryptamine) reduces blood pressure of the conscious rat when administered chronically (1 week). 5-HT does not directly relax isolated arteries, and microsphere experiments in 5-HT-infused rats suggested that 5-HT increased flow to the splanchnic bed. We hypothesized that 5-HT increased splanchnic flow because of direct venous relaxation; our focus was thus on the superior mesenteric vein (SMV) as an important vein in splanchnic circulation. Real-time RT-PCR, immunohistochemistry and Western analyses supported the predominant expression of the 5-HT2B and 5-HT7 receptor in the SMV. The SMV was mounted in tissue baths for measurement of isometric contraction. 5-HT caused a concentration-dependent relaxation of the endothelin-1 (ET-1)-contracted vein. The threshold of 5-HT-induced venous relaxation was significantly lower than for 5-HT-induced venous contraction (∼2 vs. 700 nmol/L, respectively). A series of serotonergic agonists established in their use of receptor characterization was tested, and the following rank order of potency found for agonist-induced relaxation (receptor selectivity): 5-CT (5-HT1/5-HT7)>5-HT = LP-44 (5-HT7)>PNU109291 (5-HT1D) = BW723C86 (5-HT2B). 8-OH-DPAT (5-HT1A/7), CP93129 (5-HT1B), mCPBG (5-HT3/4), AS19 (5-HT7) and TCB-2 (5-HT2A) did not relax the isolated vein. Consistent with these findings, two different 5-HT7 receptor antagonists SB 269970 and LY215840 but not the 5-HT2B receptor antagonist LY272015 nor the nitric oxide synthase inhibitor LNNA abolished 5-CT-induced relaxation of the isolated SMV. 5-CT (1 μg kg(-1) min(-1), sc) also reduced blood pressure over 7 days. These findings suggest that 5-HT directly relaxes the SMV primarily through activation of the 5-HT7 receptor.

Keywords: 5-HT; hypotension; venous circulation.

Figure 1

Real-time RT-PCR for 5-HT receptor mRNA in the superior mesenteric vein. The data are reported relative to β2-microglobulin (β2M; C_T = 21 ± 0.45 cycles). Bars represent means ± SEM for number of animals in parentheses.

Figure 2

Images from immunohistochemical experiments aimed to detect 5-HT_1B (top), 5-HT_2B (middle) and 5-HT₇ (bottom) receptor in the isolated superior mesenteric vein. Images are representative of a minimum of four (4) different animals; arrows point to regions of interest. The left column depicts images of sections incubated with the specific 5-HT primary antibody, and the right column sequential sections incubated without the primary antibody. P, pancreatic tissue; E, endothelium; M, media. Brain sections were used as a positive control for 5-HT₇ receptor, and these images are inset into the bottom row with experimental images.

Figure 3

Western analyses of 5-HT_2B (A) and 5-HT₇ (B) receptor in the isolated rat superior mesenteric vein. Each lane represents a different animal, and lanes on the far right hand side are lanes loaded with appropriate positive control (rat stomach fundus for 5-HT_2B, rat brain for 5-HT₇). Alpha actin was used as a loading control for the superior mesenteric vein. C depicts densitometric analyses of 5-HT_2B and 5-HT₇ receptor expression relative to alpha actin expression. Bars represent means ± SEM for number of animals in parentheses.

Figure 4

Effect of 5-HT in isolated superior mesenteric vein when 5-HT was added at baseline (squares; contraction) or after ET-1-(1 nmol/L) induced contraction was established (circles; relaxation). Contraction is reported as a percentage of initial contraction to NE (151 ± 22 mg) and relaxation is reported as the percentage of ET-1-induced contraction (146 ± 9.3 mg) remaining in the presence of 5-HT. Points represent means ± SEM for number of animals in parentheses.

Figure 5

Representative tracing of isolated rat superior mesenteric vein to 5-HT (A) or bolus of 5-CT (B). Response is reported in milligrams, and scale bars for force and time are in the corner of each tracing. ET was added at the first arrow, and then either 5-HT (A) or 5-CT (B) introduced. (C) depicts the comparative magnitude of relaxation of the endothelium-dependent agonist ACh to 5-CT-induced relaxation in ET-1 contraction superior mesenteric veins. Bars represent means ± SEM for the number of animals in parentheses.

Figure 6

Effects of efficacious serotonergic receptor agonists on ET-1 (1 nmol/L)-induced contraction in the isolated superior mesenteric vein. The Vehicle curve is repeated in each graph for comparison. Points represent means ± SEM for number of animals indicated in parentheses. (A) 5-HT, (B) 5-CT, (C) BW723C86, (D) LP-44, (E) PNU109291.

Figure 7

(A). Effect of 5-HT_2B (LY272015) and 5-HT₇ (SB269970 and LY215840) receptor antagonists on 5-CT relaxation stimulated in the ET-1 (1 nmol/L)-contracted isolated superior mesenteric vein. (B). Ability of 5-CT to lower blood pressure of the conscious rat. Data are graphed as a change from baseline mean arterial blood pressure (MABP) where vehicle MABP = 98.4 ± 2.2 mm Hg, 5-CT MABP = 99.4 ± 2.4 mmHg. Points represent means ± SEM for number of animals indicated in parentheses. *Signify significant differences from vehicle, †from baseline values.