5-HT7 receptors mediate dilation of rat cremaster muscle arterioles in vivo

Abstract

Objective: Serotonin (5-HT) infusion in vivo causes hypotension and a fall in total peripheral resistance. However, the vascular segment and the receptors that mediate this response remain in question. We hypothesized that 5-HT₇ receptors mediate arteriolar dilation to 5-HT in skeletal muscle microcirculation.

Methods: Cremaster muscles of isoflurane-anesthetized male Sprague-Dawley rats were prepared for in vivo microscopy of third- and fourth-order arterioles and superfused with physiological salt solution at 34°C. Quantitative real-time PCR (RT-PCR) was applied to pooled samples of first- to third-order cremaster arterioles (2-4 rats/sample) to evaluate 5-HT₇ receptor expression.

Results: Topical 5-HT (1-10 nmols) or the 5-HT_1/7 receptor agonist, 5-carboxamidotryptamine (10-30 nM), dilated third- and fourth-order arterioles, responses that were abolished by 1 μM SB269970, a selective 5-HT₇ receptor antagonist. In contrast, dilation induced by the muscarinic agonist, methacholine (100 nmols) was not inhibited by SB269970. Serotonin (10 nmols) failed to dilate cremaster arterioles in 5-HT₇ receptor knockout rats whereas arterioles in wild-type litter mates dilated to 1 nmol 5-HT, a response blocked by 1 μM SB269970. Quantitative RT-PCR revealed that cremaster arterioles expressed mRNA for 5-HT₇ receptors.

Conclusions: 5-HT₇ receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to 5-HT-induced hypotension, in vivo.

Keywords: 5-HT7 receptors; arterioles; serotonin; skeletal muscle; vascular smooth muscle; vasodilation.

Figure 1 –

Selective antagonism of 5-HT₇ receptors abolishes 5-HT-induced arteriolar dilation in SD rats. Panels A-D: Images of a branching arteriolar network in the cremaster of a SD rat before (panel A, Control) and at the peak of dilation induced by topical application of 10 nmols 5-HT (panel B) with internal diameters of the parent and daughter vessels shown. Panels C and D show the same network in the presence of 1 μM SB269970 (panel C), a selective 5-HT₇ antagonist, and at the same time after application of 10 nmols 5-HT as in Panel B, in the presence of SB269970 (panel D) showing substantial inhibition of the 5-HT dilation. Panel E shows the time course of dilation induced by topical application of 1 nmol 5-HT and the antagonism of this response by 1 μM SB269970. Panel F shows the time course of dilation induced by topical application of 100 nmols methacholine (MCh) in the same arteriole as in Panel E showing that the effects of 1 μM SB269970 were selective for 5-HT-induced dilation. Panel G shows summary data (mean diameters ± SE n= 14 arterioles in 5 rats) for experiments as in Panels A-E showing that topical 5-HT dilates arterioles and block of this response by 1 μM SB269970. Panel H shows summary data (mean diameters ± SE, n = 22 arterioles in 10 rats) for experiments as in Panel F demonstrating the lack of effect of 1 μM SB269970 on dilation induced by 100 nmols MCh.

Figure 2 –

Block of arteriolar dilation to the 5-HT_7/1 agonist, 5-carboxamidotryptamine (5-CT) by a selective 5-HT₇ antagonist. Shown are paired diameter responses before (baseline) and in the presence of 10-30 nM 5-CT, before (Control) and in the presence of 1 μM SB269970, a selective 5-HT₇ antagonist, along with means ± SE, for 12 arterioles in 5 SD rats with p-values for comparisons shown. 5-CT-induced dilation was abolished by SB269970.

Figure 3 -

Selective block of 5-HT₇ receptors abolishes 5-HT-induced arteriolar dilation in 5-HT₇ wild-type rats. Panel A shows paired diameter responses to 1-10 nmols topical 5-HT in the absence (Control) or presence (1 μM SB) of SB269970. Panel B shows lack of effect of SB269970 on dilation induced by topical application of 100 nmols methacholine. Data are means ± SE and p-values shown for 16 arterioles from 5 wild-type rats.

Figure 4 –

Lack of effect of topical 5-HT on arteriolar diameter in 5-HT₇ knockout rats. Shown are paired diameter responses for topical application of 5-HT (left) or methacholine (MCh, right) before and during application of the dilators, also shown are means ± SE and p-values for 16 arterioles from 5, 5-HT₇ knockout rats. 5-HT had no effect on arteriolar diameter in rats lacking the 5-HT₇ receptor, despite normal reactivity to MCh.

Figure 5 –

Expression of 5-HT receptors in SD rat cremaster arterioles. Data are mean ± SE Cycle Threshold (CT) values for 4 separate RT-PCR runs from samples pooled from 2-3 animals for each run. Shown are p-values for comparison of CT-values vs. a value of 40.5 (indicating expression below the limits of detection) by Dunnet’s test after one-way ANOVA.