Boursereau Raphael, Donadieu Arnaud, Dabertrand Fabrice, Dubayle David, Morel Jean-Luc
Univ. de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France; CNRS, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France.
University of Vermont, Department of Pharmacology, UVM College of Medicine, Burlington, VT, USA.
Eur J Pharmacol. 2015 Jan 15;747:141-9. doi: 10.1016/j.ejphar.2014.11.027. Epub 2014 Dec 13.
Alternative splicing of the ryanodine receptor subtype 3 (RyR3) produces a short isoform (RyR3S) able to negatively regulate the ryanodine receptor subtype 2 (RyR2), as shown in cultured smooth muscle cells from mice. The RyR2 subtype has a crucial role in the control of vascular reactivity via the fine tuning of Ca(2+) signaling to regulate cerebral vascular tone. In this study, we have shown that the inhibition of RyR3S expression by a specific antisense oligonucleotide (asRyR3S) was able to increase the Ca(2+) signals implicating RyR2 in cerebral arteries ex vivo. Moreover, we tried to inhibit the expression of RyR3S in vivo. The asRyR3S was complexed with JetPEI and injected intravenously coupled with several methods known to induce a blood brain barrier disruption. We tested solutions to induce osmotic choc (mannitol), inflammation (bacteria lipopolysaccharide and pertussis toxin), vasoconstriction or dilatation (sumatriptan, phenylephrine, histamine), CD73 activation (NECA) and lipid instability (Tween80). All tested technics failed to target asRyR3 in the cerebral arteries wall, whereas the molecule was included in hepatocytes or cardiomyocytes. Our results showed that the RyR3 alternative splicing could have a function in cerebral arteries ex vivo; however, the disruption of the blood brain barrier could not induce the internalization of antisense oligonucleotides in the cerebral arteries, in order to prove the function of RYR3 short isoform in vivo.
如在小鼠培养的平滑肌细胞中所示，兰尼碱受体亚型3（RyR3）的可变剪接产生一种短异构体（RyR3S），它能够对兰尼碱受体亚型2（RyR2）进行负调控。RyR2亚型通过对Ca(2+)信号进行微调以调节脑血管张力，在控制血管反应性方面起着关键作用。在本研究中，我们发现，用特异性反义寡核苷酸（asRyR3S）抑制RyR3S的表达能够增加离体脑动脉中涉及RyR2的Ca(2+)信号。此外，我们试图在体内抑制RyR3S的表达。将asRyR3S与JetPEI复合，并静脉注射，同时结合几种已知可诱导血脑屏障破坏的方法。我们测试了诱导渗透性休克（甘露醇）、炎症（细菌脂多糖和百日咳毒素）、血管收缩或扩张（舒马曲坦、去氧肾上腺素、组胺）、CD73激活（NECA）和脂质不稳定性（吐温80）的溶液。所有测试技术均未能将asRyR3靶向脑动脉壁，而该分子却存在于肝细胞或心肌细胞中。我们的结果表明，RyR3可变剪接在离体脑动脉中可能具有某种功能；然而，血脑屏障的破坏无法诱导反义寡核苷酸内化进入脑动脉，从而无法在体内证明RYR3短异构体的功能。
