The action of v-src on gap junctional permeability is modulated by pH

The product of the viral src gene (v-src) is the protein tyrosine kinase pp60v-src. Among the known consequences of pp60v-src activity is the reduction in permeability of gap junctions, an effect that is counteracted by the calcium antagonist TMB-8 (8-N,N-[diethylamino]octyl-3,4,5-trimethoxybenzoate). We show here that a decrease in intracellular pH (pHi) also counteracts the v-src effect: junctional permeability of cells containing active v-src kinase rose with decreasing pHi in the range 7.15 to 6.75, whereas junctional permeability of cells containing inactive v-src kinase or no v-src at all was insensitive to pH in that range. Low pH also counteracted the known action of diacylglycerol on junction, but only when pp60v-src kinase was inactive. Immunoblots of whole-cell lysates using an antibody against phosphotyrosine show that phosphorylation on tyrosine of at least one cellular protein, specific for pp60v-src kinase activity, was reduced by low pH but not by TMB-8. These results suggest that TMB-8 does not inhibit v-src action on junctional permeability by interfering with tyrosine phosphorylation of a protein crucial for closure of gap junction channels, but that the inhibition by low pH may be via this mechanism.     

Regulation of cytoplasmic pH in resident and activated peritoneal macrophages

Cytoplasmic pH (pHi) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pHi and/or its regulation existed between activated and resident macrophages (RES MOs) which might explain the increased NADPH oxidase activity observed in the former. The pHi of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pHi did not differ between resident (RES) and elicited (ELI) MOs (7.16 +/- 0.05 and 7.20 +/- 0.05, respectively). pHi recovery after intracellular acid loading was partially dependent on the presence of Na+ in the extracellular medium, and was partially inhibited by the Na+/H+ antiport inhibitor, amiloride. At comparable pHi, the rate of acid extrusion during recovery was not different in RES and ELI MOs (1.48 +/- 0.12 and 1.53 +/- 0.06 mM/min, respectively). In both RES and ELI MOs, approx. 40% of total pHi recovery was insensitive to amiloride and independent of extracellular Na+. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pHi response: an initial acidification followed by a sustained alkalinization to a new steady-state pHi. This alkalinization was Na(+)-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na+/H+ antiport activity. The new steady-state pHi attained after TPA stimulation was equivalent in RES and ELI MOs (7.28 +/- 0.04 and 7.31 +/- 0.06, respectively), indicating comparable stimulated Na+/H+ antiport activity. However, the initial acidification induced by TPA was greater in ELI than in RES MOs (0.18 +/- 0.02 vs. 0.06 +/- 0.02 pH unit, respectively, P less than 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pHi reduction by only about 50%. This suggested that the TPA-induced pHi reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.     

Changes in pH in the cytoplasm of rat thymocytes after treatment with the mitogen concanavalin A and protein kinase C activator phorbol ester

The intracellular pH (pHi) of rat thymocytes has been measured with the fluorescent probe 2', 7'-bis(carboxyethyl)-5,6-carboxyfluorescein, both in the resting cells and under mitogenic stimulation. Concanavalin A (Con A) has been found to increase pHi from 7.16 +/- 0.02 to 7.30 +/- 0.02 during the first minutes after addition; the phorbol ester TPA raised pHi to 7.25 +/- 0.02. The Con A- and TPA-induced rise of pHi is due to activation of Na+/H+ exchange since it was abolished by amiloride, an inhibitor of Na+/H+ antiport, or in a low-Na+ medium. The elevation of intracellular cAMP level, decrease of cellular ATP, or the lowering of the temperature from 37 degrees down to 25 degrees C inhibited the pHi rise induced by Con A or TPA.     

An activator of protein kinase C inhibits gap junction communication between cultured bovine lens cells.

Currently little is known about the regulation of gap junction communication in the lens. We report here on the effects of the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on cultured bovine lens cells which appeared to be epithelial in nature. Dramatically reduced intercellular transfer of the fluorescent dye Lucifer yellow was observed when the cultured lens cells were treated with octanol, a known inhibitor of gap junction communication. TPA (4 beta isomer) was also shown to reduce intercellular permeability within these cultures. In contrast, an inactive form of TPA, 4 alpha-TPA, did not decrease dye transfer. Permeability was evaluated in terms of both the number of cells receiving dye and the rate of decrease in fluorescence intensity in the injected cell. The maximum decreases in dye transfer occurred at 2 h of TPA treatment and dye transfer gradually increased to control levels over a time course of many hours. Incubation of cultures with 32Pi and immunoprecipitation using antibodies to the N- and C-terminal regions of connexin43 demonstrated a gap junction phosphoprotein of 43,000 Da. Phosphorylation of connexin43 increased during the first 2 h of TPA treatment. These results suggest that protein kinase C has a direct or indirect effect on gap junction communication in cultured lens cells.     

Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level.     

Modulation of junctional permeability

Changes in intercellular coupling can be accomplished by continuous synthesis and destruction of intercellular channels and through a modulation of unit channel permeability. The increase in free [Ca2+]i caused by activation of sodium-calcium exchange or by metabolic inhibition leads to cell decoupling. In embryonic cells the conductance of the gap junction is strongly dependent on pHi. The exact role of Ca2+ and H+ in the physiological modulation of junctional conductance remains unknown. The cyclic AMP (cAMP)-calcium hypothesis is presented. According to this view, cAMP modulates the junctional permeability through specific kinases. A feedback mechanism between calcium and cAMP might be relevant in the physiological control of junctional conductance.     

The detergent resistance of Connexin43 is lost upon TPA or EGF treatment and is an early step in gap junction endocytosis

Gap junctions are plasma membrane domains containing channels that directly connect the cytosols of neighbouring cells. Gap junction channels are made of a family of transmembrane proteins called connexins, of which the best studied is Connexin43 (Cx43). MAP kinase-induced phosphorylation of Cx43 has previously been shown to cause inhibition of gap junction channel permeability and increased Cx43 endocytosis. As Cx43 assembles into gap junction plaques, Cx43 acquires detergent resistance. Here we report that the detergent resistance is lost after activation of MAP kinase. Treatment of IAR20 rat liver epithelial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or epidermal growth factor (EGF) caused a rapid increase in the solubility of Cx43 in Triton X-100. This process was mediated by MAP kinase and was initiated at the plasma membrane. The data suggest that loss of the detergent resistance of Cx43 is an early step in TPA- and EGF-induced endocytosis of gap junctions.     

Modulation of vincristine sensitivity of human kidney tumor cells by pharmacological agents interfering with intracellular signals. No apparent relationship to changes in cytoplasmic Ca2+ or pH

The effect of substances proposed to modulate intracellular signal systems on growth and sensitivity to vincristine in the human kidney tumor cell line ACHN was investigated and related to changes in cytoplasmic free Ca2+ concentration ([Ca2+]i) and cytoplasmic pH (pHi). Presence during culture of the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol 13-acetate (TPA) had no effect on cell growth but significantly increased the EC50 concentration for vincristine inhibited cell growth. There was no indication for endogenous PKC activity being responsible for basal vincristine insensitivity since it was not affected by the PKC inhibitor H-7. The Ca2+ ionophore ionomycin tended to increase cell growth and induced vincristine resistance, whereas the calmodulin inhibitor W-7 had opposite effects. Presence during culture of the adenylate cyclase activator forskolin did not affect basal cell growth but dose-dependently made the cells more sensitive to vincristine. The modulators of vincristine sensitivity had no immediate effect on pHi, whereas after 3 days of incubation ionomycin and forskolin tended to increase pHi. Ionomycin and forskolin induced an immediate increase in [Ca2+]i which remained after 3 days only for ionomycin, whereas TPA decreased [Ca2+]i, a change which tended to remain after 3 days of incubation. It is concluded that perturbation of the intracellular signal system may affect both cell growth and cytotoxic drug sensitivity. However, there is no apparent relationship between immediate or late changes in [Ca2+]i and pHi and vincristine sensitivity.     

Hypertonicity-induced intracellular pH changes in rat mast cells

In a non-isotonic environment, cells can shrink or swell and return to their normal shape by activating ion transport pathways. Changes in intracellular pH (pHi) after osmotic stress have been identified in several cells. In order to study the mechanisms that regulate cytosolic pH of rat mast cells in a hypertonic medium, we used the pH sensitive dye, BCECF. Under these hypertonic conditions, pHi undergoes an alkalinization following an initial acidification. The alkalinization is mediated by a Na+/H+ exchanger, since it is inhibited by amiloride and lack of extracellular sodium. Under these conditions, the alkalinization is increased with the PKC activators, TPA and OAG, and partially blocked with trifluoperazine, an unspecific protein kinase C (PKC) and Ca2+ calmodulin-dependent protein kinases (Ca2+/CaM K) inhibitor. There is also an anion exchanger, blocked with DIDS but not activated by PKC, that participates in the observed alkalinization. However, Na+/H+ exchanger is the main mechanism involved in the alkalinization of pHi of mast cells in a hyperosmotic environment.     

Cytoplasmic pH is differently regulated in the monoblastic U-937 and erythroleukemic K-562 cell lines

Regulation of cytoplasmic pH (pHi) of the human monoblastic U-937 and erythroleukemic K-562 cell lines was investigated. The apparent resting pHi, as assessed by the fluorescent pH probe quenel, were 6.61 and 6.75 for the U-937 and K-562 cells, respectively. When extracellular Na+ was substituted by equimolar choline+, pHi decreased by about 0.2 units. The protein kinase C activating beta-form of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10(-10) and 10(-7) M) induced a dose-dependent alkalinization in both cell types of 0.03-0.12 units, whereas the alpha-form was inactive. The response was detectable after about 2 min and reached steady-state 10-15 min later. In the K-562 cells the alkalinization was mediated by Na+/H+ exchange as it was accompanied by stimulation of H+ extrusion and abolished by Na+ removal. The TPA response in the U-937 cells, however, was unaffected by Na+ removal, not accompanied by H+-efflux, and thus unrelated to Na+/H+ exchange. Since electron microscopy indicated development of multivesicular bodies with an acidic interior, the alkalinization can probably be accounted for by an intracellular mechanism. Ionomycin (10(-5) M) induced a rapid increase in the cytoplasmic Ca2+ concentration of both cell types and this response was accompanied by acidification followed by a Na+-dependent recovery. In the U-937, but not in the K-562, cells this recovery was followed by a net alkalinization. It is concluded that both cell types possess a Na+/H+ exchange of importance for pHi but that this mechanism is regulated differently in the U-937 and K-562 cells.     

Early transitory rise in intracellular pH leads to Bax conformation change during ceramide-induced apoptosis

Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early (<30 min) increase in Bax labeling was observed after the addition of several ceramide species to several hemopoietic-related cell types. On U937, this increase was not due to antigens synthesis or processing, but rather an increased accessibility or reactivity of Bax antigens for antibodies. This increased immuno-reactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactivity was also observed after Fas ligation, but it occurred later (>2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (<10 min) transitory increase in pHi. Both Bax immuno-reactivity and pHi increases were dependent on the mitochondrial permeability transition pore (PTP) status. It was concluded from these results that C2-Cer induced a transitory increase in pHi in relation to the PTP. This rise in pHi led to conformational changes in Bax which could be responsible for further apoptosis in the C2-Cer pathway while it was a consequence of caspase activation in the Fas pathway.     

TPA induces cytoplasmic alkalinization in human monoblastic U-937 cells without activation of Na+/H+ exchange

The effect of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on cytoplasmic pH (pHi) and H+ extrusion was studied in the human monoblastic cell line U-937. About 2 min after addition of TPA, pHi started to increase and reached a steady state 10-15 min later. The resulting alkalinization corresponded to 0.03 and 0.09 pH units at 10(-10) and 10(-7) M TPA, respectively. The TPA-induced increase in pHi was independent of the presence of extracellular Na+. Moreover, TPA did not affect the H+ extrusion from the U-937 cells. Together these observations indicate the presence of a novel mechanism for TPA-induced cytoplasmic alkalinization. This mechanism is independent of Na+/H+ exchange across the plasma membrane, but may involve organelle sequestration of H+.     

Priming effect of hyperosmotic stress on TRH-induced activation of Na+/H+ exchange in GH4C1 rat pituitary cells

The effect of mild hyperosmotic stress on cytosolic pH (pHi) alone, and in combination with thyrotropin-releasing hormone (TRH) or the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was investigated in GH4C1 cells at resting pHi. Hyperosmotic stress induced by addition of 50 mM choline was without an effect on pHi. In cells stimulated with either TRH or TPA after choline, pHi increased 0.15 +/- 0.05 and 0.14 +/- 0.03 pH units, respectively (mean +/- SD). A similar response was obtained if TRH or TPA was added prior to choline. The effect was abolished by replacing extracellular Na+ with choline+, and by pretreatment of the cells with amiloride, indicating that the change in pHi probably was dependent on activation of Na+/H+ exchange. The results thus indicate that, in GH4C1 cells, hyperosmotic stress in combination with TRH or TPA can activate Na+/H+ exchange at resting pHi levels.     

Internal pH of Xenopus oocytes: a study of the mechanism and role of pH changes during meiotic maturation

The internal pH (pHi) of Xenopus laevis oocytes, as measured by the DMO method, covered a broad range of values from 7.06 +/- 0.01 to 7.93 +/- 0.01, with a mean value of 7.43 +/- 0.03. The pHi measured by DMO and microelectrodes was nearly identical in control and maturing oocytes from the same batch. The oocytes from most females elevated their pHi in response to progesterone, reaching a maximum elevation of 0.30 +/- 0.03 pH units above control values at 100% germinal vesicle breakdown (GVBD). However, some females were found to contain oocytes that already had an elevated pHi of 7.71 +/- 0.03 which did not significantly increase during maturation. Human chorionic gonadotrophin (hcG)-stimulated females had oocytes with slightly higher control pHi values than oocytes from nonstimulated females but still showed the same elevation in response to progesterone. Thus, the "stimulated" state of oocyte physiology as induced by hcG did not account for the variation in control pHi and responsiveness to progesterone. Other aspects of this variability are discussed. Elevating or lowering the external pH is shown to elevate and lower pHi, respectively, in a stable and predictable manner. Using this approach to change pHi we have found no effect of changes in pHi on the rate of protein synthesis in control and maturing oocytes. Similarly, pHi had only a slight facilitating effect on the rate of GVBD. A pH indicator gel was used to demonstrate that the pHi increase during oocyte maturation involved an acid efflux. We conclude that an elevated pHi is not necessary for oocyte maturation, yet the mechanism of the pHi elevation is discussed as a possible lead to events that are necessary.     

Oleic Acid Inhibits Gap Junction Permeability and Increases Glucose Uptake in Cultured Rat Astrocytes

Abstract: The role of oleic acid in the modulation of gap junction permeability was studied in cultured rat astrocytes by the scrape-loading/Lucifer yellow transfer technique. Incubation with oleic acid caused a dose-dependent inhibition of gap junction permeability by 79.5% at 50 µ M , and no further inhibition was observed by increasing the oleic acid concentration to 100 µ M . The oleic acid-mediated inhibition of gap junction permeability was reversible and was prevented by bovine serum albumin. The potency of oleic acid-related compounds in inhibiting gap junction permeability was arachidonic acid > oleic acid > oleyl alcohol > palmitoleic acid > stearic acid > octanol > caprylic acid > palmitic acid > methyloleyl ester. Oleic acid and arachidonic acid, but not methyloleyl ester, increased glucose uptake by astrocytes. Neither oleic acid nor arachidonic acid increased glucose uptake in the poorly coupled glioma C6 cells. These results support that the inhibition of gap junction permeability is associated with the increase in glucose uptake. We suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication.     

Cell and toxicant specific phosphorylation of conexin43: effects of lindane and TPA on rat myometrial and WB-F344 liver cell gap junctions

Previous studies showed that the pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells. The present study tested the hypothesis that lindane and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit gap junction communication in rat myometrial and liver WBr-F344 cells by the common mechanism of increasing phosphorylation of the gap junction protein connexin43. We evaluated changes of connexin43 phosphorylation using Western blot of standard SDS-PAGE gels and cell immunostaining, and we monitored gap junction communication using microinjection and transfer of Lucifer yellow dye. Exposure of rat myometrial cells to lindane or TPA nearly abolished dye transfer but did not alter the electrophoretic mobility of connexin43, and neither lindane nor TPA increased phosphorylation of connexin43 as assessed by immunoblot with anti-phospho-connexin43 (S368) antibody. However, TPA increased punctate immunofluorescence staining of phospho-connexin43 (S368) in myometrial cells whereas lindane had no such effect. In WBr-F344 cells, lindane and TPA inhibited dye transfer. Lindane increased immunostaining for phospho-connexin43 (S368) in WBr-F344 cells without altering the abundance, electrophoretic mobility or phosphorylation of connexin43 as detected in immunoblots. TPA intensified a slower migrating connexin43 band and increased phospho-connexin43 (S368) in immunoblots, and intensified phospho-connexin43 immunostaining at WBr-F344 cell interfaces and nuclear regions. These results show that phosphorylation of connexin43 at serine 368 occurred in cell and toxicant specific manners and was independent of changes in electrophoretic mobility in standard SDS-PAGE gels. Moreover, lindane inhibited gap junction communication in myometrial cells by a mechanism that was not be explained by changes in phosphorylation of connexin43.     

Independent elevation of cytosolic [Ca2+] and pH of mammalian sperm by voltage-dependent and pH-sensitive mechanisms

Previous work (Babcock, D. F., Rufo, G. A., and Lardy, H.A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1327-1331) established that increased cytosolic pH (pHi) promotes metabolic and swimming activity of bull sperm and that intracellular alkalinization results from elevated extracellular K+, presumably as a consequence of membrane depolarization. The present studies show that a persistent but reversible increase in [Ca2+]i accompanies the increase in pHi that similarly results from treatment of ram sperm with elevated [K+] in alkaline media. Because comparable increases in pHi occur in the presence or absence of external Ca2+ and because [Ca2+]i is unaltered by imposed changes in pHi alone, [Ca2+]i and pHi apparently are neither directly linked by transmembrane Ca2+/H+ exchange nor indirectly linked through Na+/H+ and Na+/Ca2+ exchange under these conditions. Instead, inhibition of K+-induced increases in [Ca2+]i (but not of increases in pHi) by prenylamine, diltiazem, nifedipine, or verapamil (C1/2 = 6, 20, 30, and 60 microM, respectively) indicates that voltage-dependent Ca2+ channels, distinct from previously described voltage-dependent effectors of pHi, operate in mammalian sperm to control [Ca2+]i. Treatment with Cs+ plus valinomycin (as an alternative method of membrane depolarization) increases pHi much more effectively than it increases [Ca2+]i, and thus also partially supports this contention. In contrast to an apparent insensitivity to pHi, K+-dependent increases in [Ca2+]i are promoted reversibly by elevation of pHo, probably reflecting local surface charge effects on channel activity (as suggested by patch-clamp studies in other systems). A selective increase in membrane permeability to Ca2+ that is induced by 12 mM NaF under nondepolarizing conditions is not a consequence of cellular aggregation, but is attenuated by the chelator deferoxamine, suggesting that GTP-binding protein additionally may couple sperm Ca2+ channels to surface receptors and promote channel opening during sperm capacitation, presumably in response to agonists produced within the mammalian female reproductive tract.     

Changes in intracellular pH affect calcium currents in Paramecium caudatum

The relation between intracellular pH and membrane excitability was studied in the holotrich ciliate Paramecium caudatum. Intracellular pH (pHi) was measured with recessed-tip ion-sensitive microelectrodes (Thomas 1974) and electrical properties were examined by current stimulation and conventional two-electrode voltage clamp. Under normal conditions the resting pHi of Paramecium was 6.80 +/- 0.05. Intracellular alkalinization enhanced the early Ca current, while internal acidification depressed the Ca current. Both effects occurred in a voltage-independent manner. The late outward current was relatively unaffected by these alterations. Results obtained with replacement of extracellular Ca2+ by Ba2+ also support a direct effect of pHi on current through the Ca channel. Intracellular alkalinization to pH 7.15 converted graded, quasi-regenerative Ca responses elicited by injected current pulses into all-or-none action potentials. This change to all-or-none behaviour is presumed to be due to the increase in Ca current and a consequent change in the balance of inward and outward currents. Extracellular pH changes had little effect on pHi, resting membrane potential or the current-voltage relations. The intracellular pH was also independent of shifts in membrane potential. The results are consistent with a model in which Ca channel permeability is blocked by intracellular protonation of a single titratable site having an apparent dissociation constant of 6.2.     

Intracellular pH and cell adhesion to solid substrate

It was shown that activation of the Na+/H+ antiporter resulting in an increase of intracellular pH (pHi) by 0.2-0.3 is a necessary stage of cell stimulation by soluble growth factors. Solid substrate can also be formally regarded as a growth factor since adhesion stimulates proliferation of various cell types. In the present study we have found that the attachment of mouse embryo fibroblasts to solid substrate is followed by an increase of pHi by approx. 0.3 units. pH shift occurs after the cell attaches to the substrate and is obligatory for cell spreading. The evidence for Na+/H+ antiporter involvement in the increase of pHi in substrate-attached cells is presented. It is suggested that signals for cell proliferation by chemical (soluble ligands) and physical (solid substrate) growth factors are transmitted similarly.     

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs

In the fertilization of sea urchin eggs, intracellular [Ca2+] (Cai) increases transiently and intracellular pH (pHi) elevates accordingly. Unlinking these two activating factors experimentally, the requirement of the increase in pHi for sperm aster formation in the sea urchin, Clypeaster japonicus, was investigated. When the eggs were injected with an EGTA or BAPTA solution, they incorporated sperm but did not organize the sperm aster. Using these sperm-incorporated eggs under the condition that an increase in Cai was blocked, pHi was regulated by two methods: (i) perfusing ammonium acetate-containing seawater; and (ii) injecting pH buffer solutions of various pH values. By either of the two methods, the sperm aster formed at pHi 7.0 or more and functioned in female pronuclear migration when the sperm aster reached the female pronucleus. Hence, the step of the transient increase in Cai at fertilization can be bypassed. In contrast, a pHi increase is indispensably required for sperm aster formation in sea urchin eggs. Moreover, under the condition that there was the transient increase in Cai, the threshold pHi value for sperm aster formation was pHi 7.0 or more. Consequently, whether a Cai increase on fertilization occurs or not, the threshold pHi value for sperm aster formation is constant in sea urchin eggs.