Davies Suzy, Lujan Kiana S, Rappaport Ella J, Valenzuela Carlos F, Savage Daniel D
Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, NM, United States.
Front Neurosci. 2023 Jun 28;17:1192096. doi: 10.3389/fnins.2023.1192096. eCollection 2023.
We have reported that prenatal alcohol exposure (PAE) elevates histamine H receptor (H3R) agonist-mediated inhibition of glutamatergic neurotransmission in the dentate gyrus. Here, we hypothesized that PAE alters the expression of two prominent H3R isoforms namely, the rH and rH isoforms, which have differing intrinsic activities for H3R agonists, in a manner that may contribute to heightened H3R function in PAE rats. In contrast to our predictions, we found different effects of sex and PAE in various brain regions with significant interactions between sex and PAE in dentate gyrus and entorhinal cortex for both isoforms. Subsequently, to confirm the PAE-and sex-induced differences on H3R isoform mRNA expression, we developed a polyclonal antibody selective for the rH inform. Western blots of rH mRNA-transfected HEK-293 cells identified a ~ 48 kDa band of binding consistent with the molecular weight of rH, thus confirming antibody sensitivity for rH protein. In parallel, we also established a pan-H3R knockout mice line to confirm antibody specificity in rodent brain membranes. Both qRT-PCR and H3R agonist-stimulated [S]-GTPγS binding confirmed the absence of mH mRNA and H3 receptor-effector coupling in H3R knockout (KO) mice. Subsequent western blotting studies in both rat and mouse brain membranes were unable to detect rH antibody binding at ~48 kDa. Rather, the H3RA antibody bound to a ~ 55 kDa band in both rat and mouse membranes, including H3R KO mice, suggesting H3RA binding was not specific for H3Rs in rodent membranes. Subsequent LC/MS analysis of the ~55 kDa band in frontal cortical membranes identified the highly abundant beta subunit of ATPase in both WT and KO mice. Finally, LC/MS analysis of the ~48 kDa band from rH mRNA-transfected HEK-293 cell membranes was able to detect rH protein, but its presence was below the limits of quantitative reliability. We conclude that PAE alters rH and rH mRNA expression in some of the same brain regions where we have previously reported PAE-induced alterations in H3R-effector coupling. However, interpreting the functional consequences of altered H3R isoform expression was limited given the technical challenges of measuring the relatively low abundance of rH protein in native membrane preparations.
我们曾报道，产前酒精暴露（PAE）会增强组胺H受体（H3R）激动剂介导的对齿状回谷氨酸能神经传递的抑制作用。在此，我们推测PAE会改变两种主要的H3R亚型，即rH和rH亚型的表达，这两种亚型对H3R激动剂具有不同的内在活性，其改变方式可能导致PAE大鼠中H3R功能增强。与我们的预测相反，我们发现在不同脑区中，性别和PAE有不同的影响，在齿状回和内嗅皮质中，两种亚型的性别和PAE之间存在显著的相互作用。随后，为了证实PAE和性别对H3R亚型mRNA表达的影响，我们开发了一种对rH亚型具有选择性的多克隆抗体。对转染了rH mRNA的HEK - 293细胞进行的蛋白质免疫印迹分析，鉴定出一条约48 kDa的结合条带，与rH的分子量一致，从而证实了该抗体对rH蛋白的敏感性。同时，我们还建立了一个H3R全敲除小鼠品系，以确认该抗体在啮齿动物脑膜中的特异性。实时定量聚合酶链反应（qRT-PCR）和H3R激动剂刺激的[S]-GTPγS结合实验均证实，H3R敲除（KO）小鼠中不存在mH mRNA和H3受体效应偶联。随后在大鼠和小鼠脑膜中进行的蛋白质免疫印迹研究均未能检测到约48 kDa处的rH抗体结合。相反，H3RA抗体在大鼠和小鼠脑膜（包括H3R KO小鼠）中均与一条约55 kDa的条带结合，这表明H3RA结合在啮齿动物脑膜中对H3R不具有特异性。随后对额叶皮质膜中约55 kDa条带进行的液相色谱/质谱联用（LC/MS）分析，在野生型（WT）和KO小鼠中均鉴定出了高度丰富的ATP酶β亚基。最后，对转染了rH mRNA的HEK - 293细胞膜中约48 kDa条带进行的LC/MS分析能够检测到rH蛋白，但其含量低于定量可靠性的限度。我们得出结论，PAE会改变一些相同脑区中rH和rH mRNA的表达，我们之前曾报道在这些脑区中PAE会导致H3R效应偶联的改变。然而，鉴于在天然膜制剂中测量相对低丰度的rH蛋白存在技术挑战，对H3R亚型表达改变的功能后果的解读受到了限制。
