Wibo M, Feron O, Zheng L, Maleki M, Kolar F, Godfraind T
Laboratoire de Pharmacologie, Université Catholique de Louvain, Brussels, Belgium.
Cardiovasc Res. 1998 Jan;37(1):151-9. doi: 10.1016/s0008-6363(97)00228-9.
The aim was to analyze the early postnatal changes in myocardial density, subsarcolemmal localization and isoform expression of dihydropyridine receptors in rat ventricle and the influence of thyroid status on these changes.
Newborn rats were treated from postnatal day 2 with L-triiodothyronine (T3) or 6-n-propyl-2-thiouracil )PTU) and ventricles were collected on day 1, 7 and 14. Radioligand binding and cell fractionation (density gradient centrifugation) techniques were used to determine the tissue density of various receptors and their subcellular localization. To analyze dihydropyridine receptor alpha 1 subunit isoform expression, cDNA fragments corresponding to a large portion of motif IV were amplified by reverse transcriptase-polymerase chain reaction and treated with appropriate restriction endonucleases to determine the frequency of splicing events at the level of motif IV.
The myocardial density of dihydropyridine receptors increased 3-fold from day 1 to day 14 in control rats, and this increase occurred predominantly in membrane entities equilibrating at high densities in sucrose gradient, that is, presumably, in junctional structures (dyadic couplings). This maturation was delayed after PTU-treatment, and somewhat accelerated by excess T3. The proportion of mRNA variants typical of foetal heart (IVS3A variant and 'deleted' variant, showing a 33-nucleotide deletion at the level of the extracellular loop between IVS3 and IVS4) decreased with age in control rats. This reduction was delayed after treatment with PTU but was not influenced by excess T3.
Hypothyroidism impaired the early postnatal maturation of dihydropyridine receptors as regards both their concentration into junctional structures and the decrease in the relative expression of alpha 1-subunit mRNA variants typical of foetal heart.
分析出生后大鼠心室中二氢吡啶受体的心肌密度、肌膜下定位及亚型表达的早期变化，以及甲状腺状态对这些变化的影响。
新生大鼠从出生后第2天开始用L-三碘甲状腺原氨酸（T3）或6-正丙基-2-硫氧嘧啶（PTU）进行处理，并在第1、7和14天收集心室。采用放射性配体结合和细胞分级分离（密度梯度离心）技术来确定各种受体的组织密度及其亚细胞定位。为了分析二氢吡啶受体α1亚基亚型的表达，通过逆转录-聚合酶链反应扩增对应于基序IV大部分的cDNA片段，并用适当的限制性内切酶处理，以确定基序IV水平上剪接事件的频率。
在对照大鼠中，二氢吡啶受体的心肌密度从第1天到第14天增加了3倍，这种增加主要发生在蔗糖梯度中高密度平衡的膜实体中，即可能在连接结构（二元偶联）中。PTU处理后这种成熟延迟，而过量T3则使其略有加速。对照大鼠中，胎儿心脏典型的mRNA变体（IVS3A变体和“缺失”变体，在IVS3和IVS4之间的细胞外环水平显示33个核苷酸缺失）的比例随年龄下降。PTU处理后这种下降延迟，但不受过量T3影响。
甲状腺功能减退在二氢吡啶受体出生后早期成熟方面，在其向连接结构的集中以及胎儿心脏典型的α1亚基mRNA变体相对表达的降低方面均造成损害。
