Effects of Ghrelin on the Intracellular Calcium Concentration in Rat Aorta Vascular Smooth Muscle Cells

Huan Fang, Zhen Hong, Jun Zhang, Dai-Fei Shen, Fen-Fei Gao, Kenji Sugiyama, Hiroki Namba, Tetsuya Asakawa
2012 Cellular Physiology and Biochemistry  
Background/Aims: Ghrelin has been regarded as a cardioprotective factor with complicated mechanisms. Whether ghrelin is vasodilative or vasoconstrictive in nature is controversial, and the effects of ghrelin on intracellular calcium concentration are still unclear. To explore the mechanisms involved in the vasoactive regulation of ghrelin at the cellular level, we investigated the effects of ghrelin on calcium concentrations in rat aorta vascular smooth muscle cells (VSMCs). Methods: We
more » ... VSMCs via cell culture and stained the cells with Furo-2 AM. Western blotting was used to verify growth hormone secretagogue receptor (GHS-R1a) expression in VSMCs. The intracellular calcium variations affected by ghrelin and the interactions of ghrelin with angiotensin II (AngII), Sq22536, and potassium chloride (KCl) were observed using a calcium imaging and analysis system. Results: Western blotting revealed good GHS-R1a expression in VSMCs. The most prominent finding in the present study was that ghrelin inhibited the AngII-induced increase in the calcium concentration. This inhibition was reversed by the adenylate cyclase inhibitor Sq22536 and the GHS-R1a antagonist (D-Lys 3 )-GHRP-6. This finding revealed the potential vasodilative effects of ghrelin at the cellular level. We did not observe any effects of ghrelin on intracellular calcium concentrations in resting VSMCs or the increase of calcium concentration induced by KCl. Conclusion: Ghrelin inhibited the increase in the intracellular calcium concentration of rat aorta VSMCs induced by AngII, which may depend on the activation of the cAMP/PKA pathway. Fig. 4 . Effects of ghrelin on the calcium FI in VSMCs during the interactions of ghrelin with AngII, KCl, and Sq22536. (A) Calcium FI after AngII administration. AngII image nos. 4 and 5 are brighter than the control images, indicating that calcium FI was enhanced. In contrast, reduced luminance was observed in the no. 6 AngII image, indicating that calcium FI was attenuated. Such dynamic variation in the luminescence induced by AngII coincided with the FI ratio time curve shown in (B). (C) Calcium FI when VSMCs were preincubated with 5 × 10 −6 mol/L ghrelin. Image nos. 3 and 4 were not different from the control images, indicating that the administration of ghrelin did not directly affect calcium FI. Moreover, AngII image nos. 5 and 6 did not exhibit the same increasing effects as Fig. 3A , indicating that preincubation with ghrelin inhibited the enhancing effect of AngII. Then, when 4 × 10 −2 mol/L KCl was administered, as shown in image nos. 7, 8, and 9, the brightness of the images increased, indicating that calcium FI was increased by KCl. Such dynamic changes were also observed in the FI ratio time curve (D). (E) Calcium FI when VSMCs were preincubated with 1 × 10 −5 mol/L Sq22536 and 5×10 −6 mol/L ghrelin. AngII image nos. 4, 5, and 6 were brighter then the control image, indicating that Sq22536 inhibited the antagonizing effects of ghrelin on AngII. Such dynamic variations were also observed in the FI ratio time curve (F).
doi:10.1159/000343319 pmid:23160599 fatcat:n56bv4wrf5fpvg65vpi3dpbany