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Abstract: Our previous findings regarding the biological activities of small peptides revealed that a di-peptide, Trp-His (WH), could play a role in the prevention of vascular lesions, including cell proliferation and atherosclerosis. Its vasoprotective effects could be associated with suppression of the vasocontraction signaling cascade, but the underlying mechanism(s) remains obscure. In this study, we attempted to elucidate the vasoprotective mechanism of WH, in opposing the proliferation of rat vascular smooth muscle cells (VSMCs). In VSMCs from 8 week-old male Wistar rat thoracic aortae, WH evoked a significant dose-dependent anti-proliferation effect, without cytotoxicity. In mitogen-stimulated cell experiments, 300μM WH inhibited cytosolic Ca2+ elevation in VSMCs induced by 10μM angiotensin II (Ang II). Furthermore, WH suppressed extracellular Ca2+ entry into CaCl2-stimulated VSMCs. The biological capacity of WH as an intracellular Ca2+ ([Ca2+]i) suppressor was also proven when 50μM Bay K8644 was used to enhance Ca2+ entry via a voltage-dependent l-type Ca2+ channel (VDCC) and 300μM WH elicited a 23% reduction in [Ca2+]i. The absence of a reduction of the [Ca2+]i by the mixture of tryptophan and histidine revealed the importance of the peptide backbone in the [Ca2+]i reduction effect. Furthermore, the WH-induced [Ca2+]i reduction was abolished by verapamil, but not by nifedipine, indicating that WH likely binds to an extracellular site of the VDCC at a site similar to that of the dihydropyridine type-Ca2+ channel blockers. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, we primarily attempted to identify di- and tri-peptides showing potent vasodilation in 1.0μM phenylephrine-contracted thoracic aortas of Sprague-Dawley rats. Synthetic 15 Trp-His (WH) skeleton analogues were used for rat aorta ring''s force measurements, since WH was found to be a vasoactive di-peptide so far. Among the synthesized peptides consisted of both His and Trp amino acid residues, His-Arg-Trp (HRW) was found to evoke the most potent vasodilation with an EC50 value of 1.2±0.08mM in an endothelium-independent manner, while no effect was evoked by a mixture of individual amino acids. In addition to the structure of tri-peptides-activity relationship, chemically modified HRW analogues, i.e., 1- or 3-methyl-His-Arg-Trp and His-citrulline-Trp demonstrated the structural importance of tri-peptide to evoke the vasoactivity as following factors: (1) Neutral imidazole and indole groups from His and Trp residues at N- and C-terminals, respectively and (2) basic amino acids at the middle position. In mitogen (10μM angiotensin II or 50μM Bay K8644)-stimulated vascular smooth muscle cells, vasoactive HRW (100μM) caused significant [Ca2+] i reduction to an extent of >30%. Thus, our results suggest that HRW caused vasodilation action via an endothelium-independent mechanism which probably involves the suppression of extracellular Ca2+ influx through voltage-gated l-type Ca2+ channel. [Copyright &y& Elsevier]