Vasoactive intestinal polypeptide relaxes isolated rat pulmonary artery rings through two distinct mechanisms

Abstract

Vasoactive intestinal polypeptide (VIP), an endogenous neuropeptide normally present in lungs and other organs, relaxes pulmonary arteries (PAs) in different species, whereas the underlying mechanisms are still not fully understood. The aim of this study, therefore, is to investigate the signal transduction of VIP in the relaxation of isolated rat PA rings. The isometric tension of the rings was studied in vitro with force-electricity transducers. In endothelium-intact (EI) rings, VIP elicited concentration-dependent relaxation after the rings were pre-contracted by phenylephrine. A similar effect, though smaller, was observed in endothelium-denuded (ED) rings. Inhibition of the endothelial nitric oxide synthase (eNOS) by NG-nitro-L-arginine methyl ester diminished the VIP-induced vasodilatation of PA rings. The VIP-induced vasorelaxation was markedly reduced by the inhibition of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway with wortmannin and LY294002, respectively, which was seen in EI rings, but not in ED rings. Western blot analysis revealed that VIP increased the phosphorylation of eNOS at Ser 1177, but did not affect the overall expression of eNOS. In ED rings, the PKA inhibitor H-89 and K(ATP) channel inhibitor glibenclamide almost totally abolished the vasodilatation effect of VIP. The results suggested that the vasodilatation effect of VIP on rat PAs is mediated by both vascular endothelium and smooth muscle, involving respectively the PI3K/Akt-eNOS pathway and the PKA-K(ATP) channel pathway.

Fig. 1

a Concentration-dependent relaxation of EI and ED PA rings induced by VIP (10⁻⁹ to 10⁻⁵ M), which was pre-contracted with 10⁻⁶ M PE. b The relationship of VIP versus contractile force in EI (filled square, n = 6) and ED rings (open square, n = 6). The response is expressed as a percentage of the active tension originally generated by 10⁻⁶ M PE. Data are expressed as mean ± SE. *P < 0.05, **P < 0.01 at each point. c Based on the data in b, the VIP-force relationship of EI rings was described with the Hill equation (y = 1/(1 + (C/IC₅₀)^h), where y is normalized force, C is VIP concentration, IC₅₀ (10⁻⁸ M) is the VIP concentration at 50% force inhibition, and h (1) is the Hill coefficient. d Similar data fitting was done in ED rings. The IC₅₀ is 9 × 10⁻⁸ M and h is 1.2

Fig. 2

a Representative recording showing the effect of 10⁻⁶ M VIP on 10⁻⁶ M PE pre-contracted in the presence of l-NAME in EI rings (n = 6). b Representative recording showing the effect of 10⁻⁶ M VIP on 10⁻⁶ M PE pre-contracted in the presence of l-NAME in ED rings (n = 5). c In comparison to the EI control group, a treatment of the EI rings with 10⁻⁴ M l-NAME produced significantly smaller relaxation. d In comparison to the control ED group, similarly, treatment with 10⁻⁴ M l-NAME also diminished relaxation induced by VIP. Responses are expressed as percentage of the active tension originally generated by 10⁻⁶ M PE. Data were expressed as mean ± SE. *P < 0.05; **P < 0.01 versus control group

Fig. 3

a Representative recording showing the effects of 10⁻⁶ M VIP on 10⁻⁶ M PE pre-contracted in the presence of wortmannin (Wort) in EI rings (n = 6). b Representative recording showing the effect of 10⁻⁶ M VIP on 10⁻⁶ M PE pre-contracted in the presence of LY294002 (LY) in EI rings (n = 6). c In comparison to control, a treatment of the EI rings with either wortmannin or LY294002 produced significantly smaller relaxations. d On the other hand, treatment with wortmannin (n = 5) or LY294002 (n = 9) did not produce any significant relaxation in ED rings compared to control ED. Responses are expressed as percentage of the active tension originally generated by 10⁻⁶ M PE. Data were expressed as mean ± SE. *P < 0.05; **P < 0.01 versus control group

Fig. 4

a Bovine PAECs were pre-treated without (control) or with 10⁻⁶ M VIP for 5–7 min. Lysates were analyzed by Western blot with antibodies to p-eNOS-Ser¹¹⁷⁷ and eNOS. The panel included the same experiment twice on the left and on the right. b Phosphorylated Ser1177 eNOS was significantly increased in the VIP-treated group compared to the control group. Bovine PAECs were treated with 10⁻⁶ M VIP for 5–7 min. Data represent mean ± SE, where control groups were normalized as one from four experiments. *P < 0.05 versus control group (0 M VIP)

Fig. 5

a Representative recording showing the effect of 10⁻⁶ M VIP on 10⁻⁶ M PE pre-contracted in the presence of glibenclamide (Glib) in ED rings (n = 10). b Representative recording showing the effect of 10⁻⁶ M VIP on 10⁻⁶ M PE pre-contracted in the presence of H-89 in ED rings (n = 9). c The ED ring relaxation by 10⁻⁶ M VIP was significantly reduced by the 30-min pretreatment with 10⁻⁵ M glibenclamide or 5 × 10⁻⁷ M H-89, respectively. Responses are expressed as percentage of the active tension originally generated by 10⁻⁶ M PE. Data were expressed as mean ± SE. *P < 0.05; **P < 0.01 versus control group