Despite earlier notions that intracellular pH (pHi) was invariant with time, recent studies have documented pHi changes of from 0.1 to 1.6 U during metabolic and developmental transitions in a variety of cells. Here we review the evidence for pHi-mediated regulation of gamete activation, cellular dormancy, the cell cycle, and stimulus-response coupling. Intracellular Ca2+ level changes also accompany many of these same transitions, and mounting evidence suggests that pHi and Ca2+ changes can be interdependent, both in their mechanisms and their effects. Although the significance of such interactions is still largely unclear, one example--the pronounced pH dependence of Ca2+ binding by calmodulin--suggests their potential importance in metabolic regulation. Similar evidence suggests that pHi changes also influence intracellular adenosine 3',5'-cyclic monophosphate levels, and vice versa. Finally we show that changes in adenylate energy charge can significantly alter pHi. In light of these interactions--and because pHi, unlike most other effectors, does not require specialized receptors--we suggest that pHi functions as a synergistic messenger, providing a metabolic context within and through which the actions of other effectors are integrated.

Calcium-induced calcium release (CICR) may function widely in calcium-mediated cell signaling, but has been most thoroughly characterized in muscle cells. In a homogenate of sea urchin eggs, which display transients in the intracellular free calcium concentration ([Ca2+]i) during fertilization and anaphase, addition of Ca2+ triggered CICR. Ca2+ release was also induced by the CICR modulators ryanodine and caffeine. Responses to both Ca2+ and CICR modulators (but not Ca2+ release mediated by inositol 1,4,5-trisphosphate) were inhibited by procaine and ruthenium red, inhibitors of CICR. Intact eggs also displayed transients of [Ca2+]i when microinjected with ryanodine. Cyclic ADP-ribose, a metabolite with potent Ca(2+)-releasing properties, appears to act by way of the CICR mechanism and may thus be an endogenous modulator of CICR. A CICR mechanism is present in these nonmuscle cells as is assumed in various models of intracellular Ca2+ wave propagation.

Wnt genes encode secreted proteins which are implicated in receptor-mediated cell-cell signaling events important in embryogenesis, but the second messenger systems modulated by Wnts have not been identified. We report that ectopic expression of Xwnt-5A in zebrafish embryos enhances the frequency of intracellular Ca2+ transients in the enveloping layer of the blastodisc, whereas Xwnt-8 does not. These transients are independent of extracellular Ca2+. Consistent with the observed Ca2+ transients playing a role in responses of embryos to Xwnt-5A, we report that the ligand-activated serotonin type 1C receptor, which stimulates PI cycle activity and Ca2+ signaling independent of Wnts, phenocopies embryonic responses to Xwnt-5A. These results suggest that intercellular signaling by a subset of vertebrate Wnts involves modulation of a intracellular Ca2+ signaling pathway, which may arise from phosphatidylinositol cycle activity.

Propagating Ca2+ waves are a characteristic feature of Ca(2+)-linked signal transduction pathways. Intracellular Ca2+ waves are formed by regenerative stimulation of Ca2+ release from intracellular stores by Ca2+ itself. Mechanisms that rely on either inositol trisphosphate or ryanodine receptor channels have been proposed to account for Ca2+ waves in various cell types. Both channel types contributed to the Ca2+ wave during fertilization of sea urchin eggs. Alternative mechanisms of Ca2+ release imply redundancy but may also allow for modulation and diversity in the generation of Ca2+ waves.

The eggs of most or all animals are thought to be activated after fertilization by a transient increase in free cytosolic Ca 2+ concentration ([Ca2+]i). We have applied Ca2+-selective microelectrodes to detect such an increase in fertilized eggs of the frog, Xenopus laevis. As observed with an electrode in the animal hemisphere, [Ca2+]~ increased from 0.4 to 1.2 pM over the course of 2 min after fertilization, and returned to its original value during the next 10 min. No further changes in [Ca2+]~ were detected through the first cleavage division. In eggs impaled with two Ca 2+ electrodes, the Ca 2+ pulse was observed to travel as a wave from the animal to the vegetal hemisphere, propagating at a rate of ~10 #m/s across the animal hemisphere. The apparent delay between the start of the fertilization potential and initiation of the Ca 2+ wave at the sperm entry site as ~1 min. Though these observations describe only the behavior of subcortical [Ca2+]i, we suggest that our data represent the subcortical extension of the cortical Ca 2+ wave thought to trigger cortical granule exocytosis, and we present evidence that both the timing and magnitude of the Ca 2+ pulse we observed are consistent with this identity. This first quantification of subcortical [Ca2+]~ during fertilization indicates that the Ca 2+ transient is available to regulate processes (e.g., protein synthesis) in the subcortical cytosol.The calcium theory of egg activation, which holds that an increase in free cytosolic Ca 2÷ concentration ([Ca2÷]i) 1 sets in motion the early events of the "program of fertilization" (e.g., see reference 34), is amply supported by a variety of studies (2, 5-7, 10, 13, 14, 23, 27, 28, 37, 38). Qualitative measurements of [Ca2÷]i changes after fertilization in eggs of the medaka fish (14, 23), sea urchin (7, 28), starfish (7a), and mouse (6) MATERIALS AND METHODS Procurement and Handling of Gametes:Mature Xenopus oocytes were squeezed from females induced to ovulate via subcutaneous injection of 800-1,000 IU of human chorionic gonadotropin (Sigma Chemical Co., St. Louis, MO) on the previous night. Sperm were prepared by mincing dissected testes in FI solution (see below). To prevent prick activation, eggs were impaled in FI that contained 10 mM chlorobutanol, which was replaced with regular FI just before insemination. FI solution was prepared as previously described (17). Experiments were conducted at room temperature, between 20.5 and 24"C. Fabrication of Microelectrodes:Our Ca 2+ electrodes were a modified version of those of Tsien and Rink (31,32). Chromic acid-cleaned borosilicate glass micropipettes without an inner fiber were broken to ~2-~m tip diameter and rendered hydrophobic by baking for 30 min at 200"C in a chamber that contained tri-N-chlorobutylsilane (Pfaltz & Bauer Inc., Stamford, CT) vapor in air. The pipettes were then backfilled with pCa 7 calibration buffer (see below) by applying gentle pressure from a syringe to the back end of the pipettes, then the tips were filled via suction with a 50-...