The Physiologic Concentration of Inositol 1,4,5-Trisphosphate in the Oocytes of Xenopus laevis

Abstract

To measure the concentration of inositol 1,4,5-trisphosphate ([IP 3 ]) in small regions of single Xenopus oocytes, a biological detector cell was combined with capillary electrophoresis. This method is 10,000 times more sensitive than all existing assays enabling subcellular measurement of [IP 3 ] in Xenopus oocytes. Upon addition of lysophosphatidic acid to an oocyte, [IP 3 ] increased from 40 to 650 nM within 2 min. IP 3 concentrations as high as 1.8 M were measured after activation with lysophosphatidic acid, suggesting that the physiologic concentration of IP 3 ranges from the tens of nanomolar to a few micromolar in Xenopus oocytes. Since the IP 3 receptor in Xenopus oocytes is nearly identical to the type I receptor of mammalian cells, the range of [IP 3 ] in most mammalian cells is likely to be similar to that in the oocyte. By selecting or engineering the appropriate detector cell, this strategy should be applicable to cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate, and to the discovery of new Ca 2؉ -releasing second messengers.The transduction of many hormonal and sensory stimuli is mediated by an increase in the intracellular (IP 3 ) 1 (1, 2). This second messenger is produced by the action of phospholipase C on phosphatidylinositol 4,5-bisphosphate following the activation of G protein-and tyrosine kinase-linked receptors at the plasma membrane. IP 3 binds to and opens the IP 3 receptor/ channel on the endoplasmic reticulum (ER), releasing Ca 2ϩ into the cytosol. The interplay of [IP 3 ], the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ]), and the IP 3 receptor/channel is thought to lead to the Ca 2ϩ spikes and waves observed in many cell types (1-8). The measured dissociation constant for IP 3 and the IP 3 receptor/channel ranges from Ͻ1 to ϳ100 nM depending on the receptor isotype, tissue, and experimental conditions (9 -12). The wide range in affinities may also be due to the interconversion of the binding site between a high and low affinity state. Recently, another binding site with a dissociation constant of ϳ10 M has been reported (11). The binding sites or states involved in physiologic Ca 2ϩ signaling are not known since the intracellular [IP 3 ] has yet to be established. In addition to regulating the opening of the IP 3 receptor/channel, [IP 3 ] also controls the route of metabolism of IP 3 , and consequently the activation of downstream signal transduction events. At low [IP 3 ] nearly all of the IP 3 can be converted to inositol 1,3,4,5-trisphosphate which binds with high affinity to several key signal transduction proteins and activates a Ras-GTPaseactivating protein, exists for the maximal concentration attainable in cells (100 nM to 100 M) (22-35). The purpose of this investigation was to establish the range of [IP 3 ] in a cell, the Xenopus oocyte, before and after application of a physiologic stimulus. The Xenopus oocyte was chosen because of its large size and widespread use as a model cell in Ca 2ϩ signaling. EXPERIMENTAL PROCEDUR...

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