INTRACELLULAR SIGNALING IN ENDOCRINE CELLS

本计画探讨内分泌及神经内分泌细胞的细胞信号级联反应，以及质膜电性事件与受体介导的钙信号之间的相互作用。 目前的重点是在这些细胞中的ATP门控受体通道（P2 XR）的表征。 当在可兴奋的GT 1细胞中表达时，P2 XR亚型在其钙反应的峰值幅度上不同，与受体表达水平无关，顺序为：P2 X1 R <P2 X3 R <P2 X4 R <P2 X2 bR <P2 X2 aR <P2 X7 R。 在长时间的激动剂刺激过程中，钙信号以不同的速率脱敏：P2 X3 R> P2 X1 R> P2 X2 bR> P2 X4 R>> P2 X2 aR>> P2 X7 R。 P2 X2 aR的慢脱敏模式部分由P2 X3 R和完全由P2 X4 R突变体模拟，其中在其C-末端的6个氨基酸（6-aa）序列被P2 X2 aR的相应的精氨酸371至脯氨酸376序列取代。将P2 X4 R的6-aa序列中的总净电荷改变为更正的方向也减缓了受体脱敏。 另一方面，用P2 X1 R、P2 X3 R和P2 X4 R的相应序列取代P2 X2 aR的精氨酸371 -脯氨酸376序列增加了受体脱敏的速率。此外，异源聚合的野生型P2 X2 aR和突变体P2 X3 R的C-末端6-aa的P2 X2 aR在其类似的位置导致在功能通道，具有显着延迟脱敏。 P2 X1 R，P2 X3 R和P2 X4 R衍生的钙信号主要依赖于电压敏感性钙内流的激活，而电压敏感性和不敏感的进入途径同样有助于P2 X2 aR，P2 X2 bR和P2 X7 R表达细胞的胞浆钙反应。 转录本和功能P2 X7 R，P2 X3 R和P2 X4 R在催乳素细胞和GH 3细胞中被鉴定，但在促生长素细胞和促性腺激素细胞中未被鉴定。 在74%的催乳素细胞中鉴定出功能性P2 X7 R，而这些细胞中的50%表达P2 X3 R，33%表达P2 X4 R。 在单个催乳素细胞中经常观察到这些受体亚型的共表达。 纯化的生长激素细胞表达转录P2 X2 aR和P2 X2 bR，和功能性受体被确定在生长激素细胞和促性腺激素细胞，但不是在催乳素。 这些结果表明，在可兴奋细胞中表达的同源P2 XRs的钙信号传导是亚型特异性的。 这提供了一种有效的机制，用于响应于共同的激动剂而产生可变的钙模式。

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.