Klenerova Vera, Chottova-Dvorakova Magdalena, Skopek Petr, Sida Pavel, Mistrova Eliska, Slavikova Jana, Hynie Sixtus
Laboratory of Biochemical Neuropharmacology, Institute of Medical Biochemistry, 1st Faculty of Medicine, Charles University in Prague, Czech Republic.
Neuro Endocrinol Lett. 2011;32(6):805-10.
Oxytocin (OT) is a neuropeptide acting both as a peripheral hormone and in the brain as neurotransmitter and neuromodulator. In addition to its well-known effects on milk-ejection and uterine contraction, OT was shown to exert neuroendocrine regulation of heart functions. The aim of this study was to investigate the expression of mRNA of OT receptors (OTR) in rat hearts by real-time quantitative PCR (qPCR). The study was performed in Sprague-Dawley (SD) and Lewis (LE) rat strains, the latter having lower activity of HPA axis.
We used adult male SD and LE rats. OTR mRNA expression was detected in all heart chambers by comparing their threshold cycle values (CT) to CT of reference gene β-actin. The relative expression ratios were calculated using the 2-ΔΔCT method. The specificity of reaction of primary antibody with OTRs was tested by Western Blot and localization of OTR in the heart compartments was performed by immunofluorescence with commercial OTR specific antibodies.
We found expression of OTR mRNA in all heart compartments. The expression of OTR mRNA in both atria (LA, RA) was much higher than in the ventricles (RV, LV). By using two-way ANOVA we found no statistical differences between corresponding compartments of SD and LE rats. Immunohistochemical studies showed that OTR staining is not related to neuronal tissue and findings from left atrium indicate that prevalent localization of OTR is on cell membranes of cardiomyocytes.
The finding of expression of OTR mRNA by real-time qPCR and proof of OTR staining by immunohistochemistry in all heart compartments indicate that OT and its receptors may have function as a cardiovascular hormone. The differences in the HPA axis activity, as is exemplified in Sprague-Dawley and Lewis rat strain, do not project in the expression of OTR mRNA under basal condition. The effect of activity of HPA on OTR expression should be studied under stimulated conditions as it was performed in the behavioral studies.
催产素（OT）是一种神经肽，既作为外周激素起作用，又在大脑中作为神经递质和神经调节剂发挥作用。除了其对射乳和子宫收缩的众所周知的作用外，OT还被证明对心脏功能具有神经内分泌调节作用。本研究的目的是通过实时定量PCR（qPCR）研究OT受体（OTR）的mRNA在大鼠心脏中的表达。该研究在斯普拉格-道利（SD）大鼠和刘易斯（LE）大鼠品系中进行，后者的下丘脑-垂体-肾上腺（HPA）轴活性较低。
我们使用成年雄性SD和LE大鼠。通过将所有心腔的阈值循环值（CT）与参考基因β-肌动蛋白的CT进行比较，检测OTR mRNA的表达。使用2-ΔΔCT方法计算相对表达率。通过蛋白质免疫印迹法检测一抗与OTR反应的特异性，并使用商业化的OTR特异性抗体通过免疫荧光法对OTR在心脏各腔室中的定位进行研究。
我们在所有心脏腔室中均发现了OTR mRNA的表达。在两个心房（左心房、右心房）中OTR mRNA的表达远高于心室（右心室、左心室）。通过双向方差分析，我们发现SD大鼠和LE大鼠相应腔室之间无统计学差异。免疫组织化学研究表明，OTR染色与神经组织无关，左心房的研究结果表明OTR主要定位于心肌细胞膜上。
通过实时qPCR发现OTR mRNA的表达，并通过免疫组织化学证明所有心脏腔室中均有OTR染色，这表明OT及其受体可能具有心血管激素的功能。如在SD大鼠和LE大鼠品系中所示，HPA轴活性的差异在基础条件下不会影响OTR mRNA的表达。HPA活性对OTR表达的影响应在刺激条件下进行研究，就像在行为学研究中那样。
