Aerobic exercise enhanced endothelium-dependent vasorelaxation in mesenteric arteries in spontaneously hypertensive rats: the role of melatonin

Abstract

Melatonin, a neuroendocrine hormone synthesized primarily by the pineal gland, provides various cardiovascular benefits. Regular physical activity is an effective non-pharmacological therapy for the prevention and control of hypertension. In the present study, we hypothesized that melatonin plays an important role in the aerobic exercise-induced increase of endothelium-dependent vasorelaxation in the mesenteric arteries (MAs) of spontaneously hypertensive rats (SHRs) in a melatonergic receptor-dependent manner. To test this hypothesis, we evaluated the vascular mechanical and functional properties in normotensive Wistar Kyoto (WKY), SHRs, and SHRs that were trained on a treadmill (SHR-EX) for 8 weeks. Exercise training produced a significant reduction in blood pressure and heart rate in SHR, which was significantly attenuated by the intraperitoneal administration of luzindole, a non-selective melatonin receptor (MT1/MT2) antagonist. Serum melatonin levels in the SHR group were significantly lower than those in the WKY group at 8:00-9:00 and 21:00-22:00, while exercise training reduced this difference. Endothelium-dependent vessel relaxation induced by acetylcholine was significantly blunted in SHR compared with age-matched WKY. Both exercise training and luzindole ameliorated this endothelium-dependent impairment of relaxation in hypertension. Immunohistochemistry and Western blotting showed that the protein expression of the MT2 receptor and eNOS, as well as their colocalization in the endothelial cell layer in SHRs, was significantly decreased; as exercise training suppressed this reduction. These results provide evidence that regular exercise has a beneficial effect on improving endothelium-dependent vasorelaxation in MAs, in which melatonin plays a critical role by acting on MT2 receptors to increase NO production and/or NO bioavailability.

Fig. 1

Serum endogenous melatonin levels in rats. *P < 0.05; n = 6 in each group

Fig. 2

Effect of melatonin on NE-induced vessel contraction. A Example of real-time recording of vascular contractility in mesenteric arterial rings in control (a), with L-NAME pretreatment (b), and with Luz pretreatment (c). The dose–response curves of melatonin-induced mesenteric arterial relaxation. B Comparison of dose–response relations of melatonin-induced vasorelaxation in WKY, SHR-SED, and SHR-EX in control (without L-NAME and Luz pretreatment). *P < 0.05, vs. WKY; ^#P < 0.05, vs. SHR-SED. C–H Cumulative concentration–relaxation (%) curves for melatonin (10⁻⁷−10⁻³ M) in MAs against NE in control, with L-NAME pretreatment, and with Luz pretreatment in WKY (C and F), SHR-SED (D and G), and SHR-EX (E and H). *P < 0.05, vs. control. L-NAME, 10⁻⁴ M; Luz, 2 × 10⁻⁶ M, n = 6 in each group

Fig. 3

Effects of aerobic exercise on ACh-induced endothelium-dependent vasorelaxation in MAs. A Example of real-time recording of vascular contractility in mesenteric arterial rings in control (a), with Mel pretreatment (b), and with Luz + Mel pretreatment (c). B, C, and D Cumulative concentration–relaxation (%) curves for ACh (10⁻⁹–10−5 M) in MAs against NE in control, with Mel pretreatment, and with Luz + Mel pretreatment in WKY (B), SHR-SED (C), and SHR-EX (D). *P < 0.05, vs. control; ^#P < 0.05, vs. Mel. E, comparison of dose–response relations of ACh-induced vasorelaxation in WKY, SHR-SED, and SHR-EX in control (without Mel and Luz pretreatment). *P < 0.05, vs. WKY; ^#P < 0.05, vs. SHR-SED. Luz, luzindole (2 × 10⁻⁶ M); Mel, melatonin (10⁻⁴ M); NE, norepinephrine (10⁻⁵ M). Values were expressed as mean ± SEM, n = 6 in each group

Fig. 4

Effects of aerobic exercise on SNP-induced endothelium-independent vasorelaxation in MAs. A Example of real-time recording of vascular contractility in MAs in the presence of L-NAME in control (a) or with Mel pretreatment (b). B, C, and D Cumulative concentration–relaxation (%) curves for SNP (10⁻⁹–10⁻⁵ M) in MAs against NE in control and with Mel pretreatment in WKY (B), SHRSED (C), and SHR-EX (D). L-NAME N^ω-nitro-l-arginine methyl ester (10⁻⁴ M), SNP sodium nitroprusside; n = 6 in each group

Fig. 5

Protein expression of MT1, MT2 receptor, and eNOS in mesenteric artery homogenates. A Representative Western immunoblot and densitometric analysis of MT1 and MT2 receptor. B Representative Western blot and densitometric analysis of eNOS. *P < 0.05, **P < 0.01, relative to WKY; ^#P < 0.05, relative to SHR-SED; n = 6 in each group

Fig. 6

Immunofluorescent colocalization of eNOS and MT2 receptors in MAs. Colocalization of eNOS (red) and MT2 (green) staining is confirmed by the sharp merged image (yellow). Scale bar = 25 μm