Expression and function of melatonin receptors in canine isolated coronary arteries

Rachael Morris, Diane Ryan, James Root, Sarah Pullen, Sidath Katugampola, & Carolyn Napier, Candidate Research Group, Pfizer, Sandwich, Kent, CT13 9NJ, UK.

Melatonin receptors, coupled to Gi, mediate vascular effects in rat preparations (Doolen et al.,1998). Additionally, molecular biology techniques have identified mRNA for melatonin receptors in human coronary arteries (CA) (Ekmekcioglu et al., 2001). Therefore, the aim of this study was to determine whether mRNA encoding melatonin receptors was present in the canine CA and to subsequently investigate the functional role of melatonin in canine isolated endothelium intact CA.

Endothelium intact CA were obtained from beagle dogs (10-20 kg) of either sex. Animals were used in other pharmacological studies and Pfizer ethics committee ethically reviewed the protocols. To determine the presence of mRNA for melatonin receptors, RNA was extracted from the CA and converted into cDNA. PCR was performed using primers designed in-house to detect canine MT1 and MT2 receptors. For functional studies, the CA was dissected into 4 mm rings and mounted in 15 ml organ baths containing oxygenated 37˚C Krebs at isometric resting tension of 1g. Tissue and endothelial viability were assessed using KCl (25 mM) and acetylcholine (1 µM) respectively. The effects of melatonin (0.1 nM-100 µM) were investigated either on basal tension or pre-constricted tissues (30 mM KCl, 100 nM 5-HT or 10 µM phenylephrine, concentrations representing approximately EC60-EC80) . Further studies examined the role of melatonin (10 and 100 nM) on the relaxation produced by the nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) and the PDE3 inhibitor, milrinone on 30 mM KCl pre-constricted tissues. Data are mean ± s.e.mean, n-values refer to the number of dogs. The magnitude of response (Emax) was expressed as a percentage of the 30 mM KCl pre-constriction. Data were compared using Student’s t-test, with significance set at P<0.05.

Using PCR we have shown for the first time the presence of the mRNA encoding MT1 (but not MT2) melatonin receptor in dog CA. Within the concentration range tested, melatonin had no direct functional effect either on basal tension or pre-constricted canine isolated coronary arteries (n≥3). Additionally, m elatonin at 10 nM and 100 nM did not significantly (P>0.05) alter either the potency or efficacy of the relaxation produced by SNAP or milrinone (Table 1).

Table 1: Mean pEC50 and max values (± s.e.mean) for the different treatment groups.

In agreement with studies in pig CA (Yang et al., 2001), we saw no vasoconstrictor or vasodilator responses to melatonin in canine CA. We have also shown that the endothelium dependent vasodilator responses produced by nitric oxide donors are unaltered by supraphysiological concentrations of melatonin. Additionally, the vasodilator responses produced by milrinone, were unaffected by melatonin. In conclusion, these results suggest that melatonin may not play a major role in regulating vascular tone in canine coronary arteries and does not alter the vasodilator responses to either nitric oxide donors or agents that elevate intracellular cAMP in-vitro. Based on our findings, it is possible that a species difference may exist (rat compared to dog) for melatonin receptors. Additionally, a vascular role for melatonin in-vivo, via other mechanisms, cannot be ruled out and therefore warrant further investigation.

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