Rager Christine, Klöpper Tobias, Tasch Sabine, Whittaker Michael Raymond, Exintaris Betty, Mietens Andrea, Middendorff Ralf
Institute of Anatomy & Cell Biology, Faculty of Medicine, Justus-Liebig-University, Aulweg 123, 35392 Giessen, Germany.
Drug Delivery, Disposition, and Dynamics (D4), Monash Institute of Pharmacy & Pharmaceutical Sciences (MIPS), Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.
Cells. 2025 Jan 4;14(1):51. doi: 10.3390/cells14010051.
Vascular smooth muscle cell (SMC) relaxation by guanylyl cyclases (GCs) and cGMP is mediated by NO and its receptor soluble GC (sGC) or natriuretic peptides (NPs) ANP/BNP and CNP with the receptors GC-A and GC-B, respectively. It is commonly accepted that cultured SMCs differ from those in intact vessels. Nevertheless, cell culture often remains the first step for signaling investigations and drug testing. Previously, we showed that even popular reference genes changed dramatically after SMC isolation from aorta. Regarding NP receptors, a substantial amount of data relies on cell culture. We hypothesize that the NP/cGMP system in intact aortic tunica media differs from isolated and cultured aortic SMCs. Therefore, we studied isolation and culturing effects on the expression of NP receptors GC-A, GC-B, and NP clearance receptor (NPRC) compared to sGC. We investigated intact tunica media and primary SMCs from the longitudinal halves of the same rat aorta. GC activity was monitored by cyclic guanosine monophosphate (cGMP). In addition, we hypothesize that there are sex-dependent differences in the NP/cGMP cascade in both intact tissue and cultured cells. We, therefore, analyzed a male and female cohort. Expression was quantified by RT-qPCR comparing aortic media and SMCs with our recently validated reference gene (RG) small nuclear ribonucleoprotein 2 (U2). Only GC-A was stably expressed. In intact media, GC-A exceeded GC-B and NPRC. However, GC-B, NPRC, and sGC were dramatically upregulated in cultured SMCs of the same aortae different from the stable GC-A. The expression was mirrored by NP-induced GC activity. In cultured cells, changes in GC activity were delayed compared to receptor expression. Minor differences between both sexes could also be revealed. Thus, isolation and culture fundamentally alter the cGMP system in vascular SMCs with potential impact on drug testing and scRNAseq. Especially, the dramatic increase in the clearance receptor NPRC in culture might distort all physiological ANP, BNP, and CNP effects.
鸟苷酸环化酶（GCs）和环磷酸鸟苷（cGMP）介导的血管平滑肌细胞（SMC）舒张是由一氧化氮（NO）及其受体可溶性GC（sGC）或利钠肽（NPs）介导的，其中，心房钠尿肽（ANP）/脑钠肽（BNP）和C型钠尿肽（CNP）分别与GC-A和GC-B受体结合。人们普遍认为，培养的SMC与完整血管中的SMC不同。然而，细胞培养通常仍然是信号研究和药物测试的第一步。此前，我们发现，即使是常用的参考基因，在从主动脉分离SMC后也会发生显著变化。关于NP受体，大量数据依赖于细胞培养。我们假设，完整主动脉中膜的NP/cGMP系统与分离和培养的主动脉SMC不同。因此，与sGC相比，我们研究了分离和培养对NP受体GC-A、GC-B和NP清除受体（NPRC）表达的影响。我们研究了来自同一只大鼠主动脉纵向两半的完整中膜和原代SMC。通过环磷酸鸟苷（cGMP）监测GC活性。此外，我们假设，在完整组织和培养细胞中，NP/cGMP级联反应存在性别差异。因此，我们分析了一个雄性和雌性队列。通过逆转录定量聚合酶链反应（RT-qPCR），使用我们最近验证的参考基因（RG）小核核糖核蛋白2（U2）比较主动脉中膜和SMC的表达量。只有GC-A稳定表达。在完整中膜中，GC-A超过GC-B和NPRC。然而，与稳定的GC-A不同，在来自同一主动脉的培养SMC中，GC-B、NPRC和sGC显著上调。NP诱导的GC活性反映了这种表达变化。在培养细胞中，与受体表达相比，GC活性的变化有所延迟。两性之间也存在细微差异。因此，分离和培养从根本上改变了血管SMC中的cGMP系统，可能对药物测试和单细胞RNA测序（scRNAseq）产生影响。特别是，培养中清除受体NPRC的显著增加可能会扭曲所有生理上的ANP、BNP和CNP效应。
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