Wang Yu-Feng, Hatton Glenn I
Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, California 92521, USA.
J Neurosci. 2007 Feb 21;27(8):1902-12. doi: 10.1523/JNEUROSCI.5346-06.2007.
Pulsatile neuropeptide secretion is associated with burst firing patterns; however, intracellular signaling cascades leading to bursts remain unclear. We explored mechanisms underlying burst firing in oxytocin (OT) neurons in the supraoptic nucleus in brain slices from lactating rats. Application of 10 pm OT for 30 min or progressively rising OT concentrations from 1 to 100 pm induced burst firing in OT neurons in patch-clamp recordings. Burst generation was blocked by OT antagonist and ionotropic glutamate receptor blockers or tetanus toxin. Blocking G-protein activation with suramin or intracellular GDP-beta-S, but not intracellularly administered antibody against the OT-receptor (OTR) C terminus, blocked bursts. Moreover, pretreatment of slices with pertussis toxin, an inhibitor of G(i/o)-proteins, did not block OT-evoked bursts, suggesting that G(i)/G(o) activation is unnecessary for burst generation. Thus, we further examined G alpha(q/11)-associated signaling pathways in OT-evoked bursts. Inhibition of phospholipase C or RhoA/Rho kinase did not block bursts. Activation of G betagamma subunits using myristoylated G betagamma-binding peptide (mSIRK) caused bursts, whereas intracellularly loaded antibody against G beta subunit blocked OT-evoked bursts. Blocking Src family kinase, but not phosphatidylinositol 3-kinase, occluded OT-evoked bursts. Similar to the effects of OT on EPSCs, mSIRK inhibited tonic EPSCs and elicited EPSC clustering. Finally, suckling caused dissociation of OTRs and G beta subunits from G alpha(q/11) subunits shown by coimmunoprecipitation and immunocytochemistry, supporting crucial roles for OTRs and G betagamma subunits in the milk-ejection reflex. We conclude that G betagamma subunits play a dominant role in burst firing evoked by applied OT or by suckling.
脉冲式神经肽分泌与爆发式放电模式相关；然而，导致爆发式放电的细胞内信号级联反应仍不清楚。我们探究了泌乳大鼠脑片视上核中催产素（OT）神经元爆发式放电的潜在机制。在膜片钳记录中，施加10皮摩尔OT 30分钟或使OT浓度从1皮摩尔逐渐升至100皮摩尔可诱导OT神经元爆发式放电。OT拮抗剂、离子型谷氨酸受体阻滞剂或破伤风毒素可阻断爆发式放电的产生。用苏拉明或细胞内GDP-β-S阻断G蛋白激活，但细胞内注射抗OT受体（OTR）C末端的抗体则不能阻断爆发式放电。此外，用百日咳毒素（一种G(i/o)蛋白抑制剂）预处理脑片并不阻断OT诱发的爆发式放电，这表明G(i)/G(o)激活对于爆发式放电的产生并非必需。因此，我们进一步研究了OT诱发的爆发式放电中与Gα(q/11)相关的信号通路。抑制磷脂酶C或RhoA/Rho激酶并不阻断爆发式放电。使用肉豆蔻酰化的Gβγ结合肽（mSIRK）激活Gβγ亚基可引发爆发式放电，而细胞内加载抗Gβ亚基的抗体则可阻断OT诱发的爆发式放电。阻断Src家族激酶而非磷脂酰肌醇3激酶可消除OT诱发的爆发式放电。与OT对兴奋性突触后电流（EPSC）的作用类似，mSIRK可抑制持续性EPSC并引发EPSC簇集。最后，通过免疫共沉淀和免疫细胞化学显示，哺乳导致OTR和Gβ亚基与Gα(q/11)亚基解离，支持OTR和Gβγ亚基在射乳反射中起关键作用。我们得出结论，Gβγ亚基在应用OT或哺乳诱发的爆发式放电中起主导作用。
