Morinelli T A, Finley E L, Jaffa A A, Kurtz D T, Ullian M E
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, USA.
Biochem Pharmacol. 1997 Jun 15;53(12):1823-32. doi: 10.1016/s0006-2952(97)00005-1.
Thromboxane A2 (TXA2) interacts with its G-protein coupled receptor, the TP receptor, to produce contraction and proliferation of vascular smooth muscle cells. We have shown previously that proliferation of primary cultures of vascular smooth muscle cells initiated by [1S-(1alpha, 2beta(5Z), 3alpha(1E, 3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (I-BOP), a stable TXA2 mimetic, is mediated by activation of mitogen-activated protein (MAP) kinase. In the present study, we examined further the intracellular mediators involved in TXA2 activation of vascular smooth muscle cells. Transient transfection of the cDNA for the TP receptor into A7r5 vascular smooth muscle cells resulted in expression of TP receptors with a receptor density, Bmax, of 0.7 +/- 0.2 pmol/mg protein and a receptor affinity, Kd, of 0.6 +/- 0.1 nM (N = 7). Mock transfected cells lacked significant receptor expression. In TP receptor transfected cells, I-BOP increased the activation of MAP kinase 2-fold, stimulated tyrosine phosphorylation of cellular proteins of relative molecular mass (Mr) of 140, 85, 60, 56, and 45 kDa, and increased the message for c-jun, a nuclear transcription factor involved in mitogenesis, 2.6-fold. Immunoblot analysis indicated that the 85-kDa protein represented phosphoinositide 3-kinase (PI3-K), while the 60 kDa protein was the TP receptor. The activity of PI3-K was increased 3.5-fold by the addition of I-BOP (0.1 microM). In summary, the present study demonstrated that stimulation of the TP receptor results in tyrosine phosphorylation of the receptor and of PI3-K.
血栓素A2（TXA2）与其G蛋白偶联受体TP受体相互作用，从而引起血管平滑肌细胞收缩和增殖。我们先前已经表明，由稳定的TXA2模拟物[1S-(1α, 2β(5Z), 3α(1E, 3R), 4α]-7-[3-(3-羟基-4-(4'-碘苯氧基)-1-丁烯基)-7-氧杂双环-[2.2.1]庚烷-2-基]-5'-庚烯酸（I-BOP）引发的血管平滑肌细胞原代培养物增殖是由丝裂原活化蛋白（MAP）激酶的激活介导的。在本研究中，我们进一步研究了参与TXA2激活血管平滑肌细胞的细胞内介质。将TP受体的cDNA瞬时转染到A7r5血管平滑肌细胞中，导致TP受体表达，其受体密度Bmax为0.7±0.2 pmol/mg蛋白，受体亲和力Kd为0.6±0.1 nM（N = 7）。 mock转染的细胞缺乏明显的受体表达。在TP受体转染的细胞中，I-BOP使MAP激酶的激活增加了2倍，刺激了相对分子质量（Mr）为140、85、60、56和45 kDa的细胞蛋白的酪氨酸磷酸化，并使参与有丝分裂的核转录因子c-jun的信息增加了2.6倍。免疫印迹分析表明，8 kDa的蛋白代表磷脂酰肌醇3激酶（PI3-K），而60 kDa的蛋白是TP受体。加入I-BOP（0.1 microM）后，PI3-K的活性增加了3.5倍。总之，本研究表明，TP受体的刺激导致受体和PI3-K的酪氨酸磷酸化。
