Central nicotinic receptors in hypertension

继续项目期间的研究将主要基于一项关键发现，该发现是在去年对自发性高血压大鼠(SHR)和挪威布朗亲本的同源品系大鼠进行的。该毒株在8号染色体上含有SHR背景中的3个LCM替换，非常幸运的是，该区域包含一组烟碱型乙酰胆碱受体基因，编码在中枢神经系统表达的受体亚单位(α3、β4和α7)。与SHR对应的动物相比，这些动物的血压(收缩压和舒张压)都有所降低。更重要的是，鞘内注射尼古丁激动剂后，升压和伤害性反应明显减弱。一段时间以来，我们一直认为自发性高血压是一种增强的兴奋性，随着动物年龄的增长，血压成为这一点的表现之一。如果我们考虑更广泛的兴奋性反应，突触前控制神经递质释放的明显扩增位点将是一个合乎逻辑的起点。最近的研究还证明，大多数尼古丁受体似乎位于突触前，位于脊髓和棘上区域。我们选择研究脊髓系统，不是因为我们认为它是一个全球控制高血压的区域，而是因为可以在清醒的动物身上监测节段性脊髓区域注射尼古丁激动剂的升压反应。反过来，已经并将测量目标受体的数量、它们的亚型、细胞反应(即：在微透析液中检测到的神经递质的释放)，以及神经递质和受体亚型在脊髓不同板层和细胞类型中的细胞定位。因此，我们建议更全面地描述清醒SHR和SHR-LX大鼠的升压反应，然后通过划定烟碱受体基因簇周围的区域来定位负责不同升压反应的基因(S)。如果反应仍然与AChR基因替换有关，那么我们将试图通过编码和关键非编码区的特征来确定这三个基因中的哪一个参与其中。其他显示数量性状基因座的区域也将被调查。由于对脊髓尼古丁激动剂的反应窗口被扩大，我们将通过免疫细胞化学和共聚焦显微镜来表征该区域受体和递质的定位。此外，通过使用辣椒素和P-皂苷结合物，脊髓中相对特异的细胞消融是可能的，通过比较治疗和未治疗动物改变的升压反应，我们应该能够绘制涉及改变反应的脊髓通路和受体亚型。

Studies in the continuing project period will be based primarily on a critical finding, made over the last year with a congenic strain of rats derived from the Spontaneously Hypertensive Rat (SHR) and Brown Norway parentage. This strain contains a 3 lcM replacement in chromosome 8 into the SHR background, and quite fortuitously this region contains a cluster of nicotinic acetylcholine receptor genes that encode receptor subunits expressed in the CNS (alpha3, beta4 and alpha7). These animals show diminished blood pressure (systolic and diastolic) relative to their SHR counterparts. More importantly, intrathecal administration of nicotinic agonists shows markedly diminished pressor and nociceptive responses. For sometime we have felt that the SHR trait was one of enhanced excitability, and as the animal ages, blood pressure becomes one manifestation of this. If we consider more widespread excitability responses, loci of amplification evident in the presynaptic control of neurotransmitter release would be a logical starting point. Recent work has also documented that most nicotinic receptors appear to lie presynaptically in spinal and supraspinal regions. We have chosen to study the spinal system, not because we believe it to be an area that globally controls hypertension, but rather pressor responses to nicotinic agonists injected into segmental cord areas can be monitored in the conscious animal. In turn, measurements of target receptor number, their subtypes, cellular responses (ie: release of neurotransmitters detected in microdialysates), and the cellular localization of neurotransmitter and receptor subtype in various lamina and cell types in the cord have been and will be measured. Accordingly, we propose to more fully characterize the pressor response in conscious SHR and SHR-Lx rats, and then begin to localize the gene(s) responsible for the different pressor response by delimiting the region around the nicotinic receptor gene cluster. If the response remains associated with the AChR gene substitution, we then will attempt to identify which of the three genes are involved, through a characterization of the coding and critical noncoding regions. Other regions showing quantitative trait loci will also be investigated. Since the window of the response to spinal nicotinic agonists is augmented, we will characterize the localization of receptors and transmitters in this region through the use of immunocytochemistry and confocal microscopy. In addition, relatively specific cell ablation is possible in the spinal cord through the use of capsaicin and a Substance P-saporin conjuate, and by comparing altered pressor responses of treated and untreated animals, we should be able to map spinal pathways and receptor subtypes involved in the altered responses.