Freinkel N, Younsi C E, Bonnar J, Dawson R M
J Clin Invest. 1974 Nov;54(5):1179-89. doi: 10.1172/JCI107861.
Anionic fluxes during the membrane realignments of stimulated insulin release have not been characterized previously although cations have been implicated in stimulus-secretion coupling. We have shown that a limited packet pulse of phosphate release ("phosphate flush") begins at the same time that the first phase of insulin secretion may occur. To demonstrate this phenomenon, we have prelabeled islets, obtained from rat pancreas by collagenase digestions, by incubation with [(32)P]orthophosphate. When such prelabeled islets are perifused with Krebs-Ringer bicarbonate containing 0.5 mg/ml D-glucose, a basal rate of efflux of radioactivity is established; transfer to perifusates containing 3.0 mg/ml D-glucose elicits an increased (32)P efflux within 1-2 min to peak values which are 7- to 21-fold greater than basal. The total duration of this "phosphate flush" approximates 10 min and exceeds the duration of the first phase of stimulated insulin secretion. With lesser concentrations of glucose, the flush exhibits dose-response relationships, and with 3 mg/ml glucose, a second flush can be elicited by restoring basal conditions and stimulating anew with 3 mg/ml glucose. The phenomenon is highly specific and can be reduplicated by other secretagogues (L-leucine) or sugars (D-mannose) which are also known to elicit insulin release but not by sugars which fail to affect insulin secretion (D-galactose, D-fructose, i-inositol, L-glucose). The efflux of radioactivity consists entirely of [(32)P]orthophosphate. Phosphate flush persists in phosphate-free media, Ca(++)-free media, and when insulin release is obtunded by adding Ni(++) (2 mM) to the perifusates. Thus, efflux of [(32)P]orthophosphate can be dissociated from insulin extrusion, and from net influx of ionic phosphate or calcium. Membrane stabilization with D(2)O or 1.0 mM tetracaine reversibly inhibits phosphate flush. Although the mechanism by which this effect occurs has not yet been established, the phosphate flush appears to constitute one of the earliest and hitherto unknown indices of the excitatory state in pancreatic islets.
尽管阳离子参与了刺激-分泌偶联过程，但刺激胰岛素释放过程中膜重排期间的阴离子通量此前尚未得到表征。我们已经表明，有限的磷酸盐释放脉冲（“磷酸盐冲洗”）在胰岛素分泌的第一阶段可能发生的同时开始。为了证明这一现象，我们通过用[（32）P]正磷酸盐孵育，对通过胶原酶消化从大鼠胰腺获得的胰岛进行了预标记。当用含有0.5mg/ml D-葡萄糖的 Krebs-Ringer 碳酸氢盐对这种预标记的胰岛进行灌流时，建立了放射性流出的基础速率；转移到含有3.0mg/ml D-葡萄糖的灌流液中会在1-2分钟内引起（32）P流出增加，达到峰值，该峰值比基础值高7至21倍。这种“磷酸盐冲洗”的总持续时间约为10分钟，超过了刺激胰岛素分泌第一阶段的持续时间。对于较低浓度的葡萄糖，冲洗呈现剂量反应关系，并且对于3mg/ml葡萄糖，通过恢复基础条件并用3mg/ml葡萄糖重新刺激可以引发第二次冲洗。该现象具有高度特异性，并且可以被其他已知能引发胰岛素释放的促分泌剂（L-亮氨酸）或糖类（D-甘露糖）重复，但不能被不影响胰岛素分泌的糖类（D-半乳糖、D-果糖、肌醇、L-葡萄糖）重复。放射性流出完全由[（32）P]正磷酸盐组成。磷酸盐冲洗在无磷酸盐培养基、无Ca（++）培养基中持续存在，并且当通过向灌流液中添加Ni（++）（2mM）使胰岛素释放受到抑制时也持续存在。因此，[（32）P]正磷酸盐的流出可以与胰岛素排出以及离子磷酸盐或钙的净流入解离。用D（2）O或1.0mM丁卡因进行膜稳定化可逆地抑制磷酸盐冲洗。尽管这种作用发生的机制尚未确定，但磷酸盐冲洗似乎构成了胰岛兴奋状态的最早且迄今未知的指标之一。
