INTRACELLULAR SIGNALING IN ENDOCRINE CELLS

本项目研究了内分泌和神经内分泌细胞中的细胞信号级联，以及质膜电事件与受体介导的钙信号之间的相互作用。目前的重点是表征atp门控受体通道（P2XR）在这些细胞。当P2XR亚型在可兴奋的GT1细胞中表达时，其钙反应的峰值振幅与受体表达水平无关，其顺序为：P2X1R < P2X3R < P2X4R < P2X2bR < P2X2aR < P2X7R。在长时间激动剂刺激下，钙信号以不同的速率脱敏：P2X3R > P2X1R > P2X2bR > P2X4R >> P2X2aR >> P2X7R。P2X2aR的缓慢脱敏模式被P2X3R部分模仿，而P2X4R突变体则完全模仿，其c端6个氨基酸（6-aa）序列被P2X2aR对应的精氨酸371至脯氨酸376序列取代。将P2X4R的6-aa序列的总净电荷向更正的方向改变也减缓了受体的脱敏。另一方面，将P2X2aR的精氨酸371 -脯氨酸376序列替换为P2X1R、P2X3R和P2X4R的相应序列增加了受体脱敏率。此外，野生型P2X2aR和突变型P2X3R的异源聚合导致P2X2aR的c -末端6-aa在其类似位置，导致一个功能通道，显着延迟脱敏。P2X1R、P2X3R和p2x4r衍生的钙信号主要依赖于电压敏感型钙内流的激活，而在表达P2X2aR-、P2X2bR-和p2x7r的细胞中，电压敏感和电压不敏感的进入途径同样有助于胞质钙反应。P2X7R、P2X3R和P2X4R的转录本和功能在乳营养细胞和GH3细胞中被发现，但在生长营养细胞和促性腺功能细胞中未被发现。在74%的乳养菌中鉴定出功能性P2X7R，而这些细胞中有50%表达P2X3R， 33%表达P2X4R。这些受体亚型在单个乳养菌中经常被观察到共同表达。纯化的生长激素表达P2X2aR和P2X2bR转录物，在生长激素和促性腺激素中发现了功能受体，但在乳养动物中没有。这些结果表明，在可兴奋细胞中表达的同源P2XRs钙信号是亚型特异性的。这提供了一个有效的机制，以产生可变的钙模式响应共同的激动剂。

This project addresses the cellular signaling cascade in endocrine and neuroendocrine cells and the interactions between plasma membrane electrical events and receptor-mediated calcium signaling. Current emphasis is on the characterization of ATP-gated receptor-channels (P2XR) in these cells. When expressed in excitable GT1 cells, P2XR subtypes differed in the peak amplitudes of their calcium responses independently of the level of receptor expression, with the order: P2X1R < P2X3R < P2X4R < P2X2bR < P2X2aR < P2X7R. During prolonged agonist stimulation, calcium signals desensitized with different rates: P2X3R > P2X1R > P2X2bR > P2X4R >> P2X2aR >> P2X7R. A slow desensitizing pattern of P2X2aR was mimicked partially by P2X3R and fully by P2X4R mutants in which six amino acid (6-aa) sequences in their C-termini were substituted with the corresponding arginine 371 to proline 376 sequence of P2X2aR. Changing the total net charge in the 6-aa sequence of P2X4R to a more positive direction also slowed receptor desensitization. On the other hand, substitution of the arginine 371 - proline 376 sequence of P2X2aR with the corresponding sequences of P2X1R, P2X3R, and P2X4R increased the rate of receptor desensitization. Furthermore, heterologous polymerization of wild-type P2X2aR and mutant P2X3R having the C-terminal 6-aa of P2X2aR at its analogous position resulted in a functional channel, with significantly delayed desensitization. The P2X1R, P2X3R, and P2X4R-derived calcium signals were predominantly dependent on activation of voltage-sensitive calcium influx, whereas voltage-sensitive and -insensitive entry pathways equally contributed to cytosolic calcium responses in P2X2aR-, P2X2bR- and P2X7R-expressing cells. The transcripts and functional P2X7R, P2X3R, and P2X4R were identified in lactotrophs and GH3 cells, but not in somatotrophs and gonadotrophs. Functional P2X7R were identified in 74% of lactotrophs, whereas 50% of these cells expressed P2X3R and 33% expressed P2X4R. Co-expression of these receptor subtypes in single lactotrophs was frequently observed. Purified somatotrophs expressed transcripts for P2X2aR and P2X2bR, and functional receptors were identified in somatotrophs and gonadotrophs, but not in lactotrophs. These results indicate that calcium signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific. This provides an effective mechanism for generating variable calcium patterns in response to a common agonist.