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Regulation of intracellular pH by p90Rsk-dependent activation of an Na(+)/H(+) exchanger in starfish oocytes.

ABSTRACT: Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pH(i)) and release from arrest by cellular alkalization. To elucidate pH(i) regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na(+)/H(+) exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pH(i). This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pH(i) increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pH(i) increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pH(i) ( approximately 7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pH(i) increases fertilization success by delaying apoptosis initiation.

SUBMITTER: Harada K

PROVIDER: S-EPMC2911288 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Regulation of intracellular pH by p90Rsk-dependent activation of an Na(+)/H(+) exchanger in starfish oocytes.

Harada Kaori K Fukuda Eriko E Hirohashi Noritaka N Chiba Kazuyoshi K

The Journal of biological chemistry 20100527 31

Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pH(i)) and release from arrest by cellular alkalization. To elucidate pH(i) regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na(+)/H(+) exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphoryla ...[more]

PMID: 20507995

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Project description:The aim of the present study was to evaluate the regulatory relationship between the cytosolic free calcium concentration ([Ca2+]i and cytosolic pH (pHi). [Ca2+]i and pHi were measured using the fluorescent dyes fura-2 and BCECF [2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein] respectively. In a medium with 1 mmol/l extracellular calcium, thrombin (2.5 units/ml) induced an increment in [Ca2+]i of 638 +/- 31 nmol/l (n = 5) and an intracellular alkalinization of 0.14 +/- 0.01 pH units (n = 8). Both responses were dependent on the concentration of thrombin, displaying a sigmoidal dose-response pattern. The intracellular alkalinization was dependent upon extracellular Na+ and was amiloride-sensitive, indicating that it was mediated by activation of the Na+/H+ exchanger. When extracellular calcium was chelated with EGTA prior to the addition of thrombin, the intracellular alkalinization was not affected (0.15 +/- 0.02 at 2.5 units/ml thrombin, n = 8). Under these circumstances, the [Ca2+]i increment represents mobilization from internal stores, reaching 157 +/- 42 nmol/l at 2.5 units/ml thrombin. When platelets were preloaded with the intracellular calcium chelator MAPTAM (1,2-bis-5-methylaminophenoxylethane-NNN'-tetraacetoxymethyl acetate) to block the increase in [Ca2+]i induced by thrombin, no increment in pHi was observed. Moreover, MAPTAM-loaded calcium-depleted platelets had a basal pHi that was more acidic than in the presence of 1 mmol/l extracellular calcium (6.93 +/- 0.09 versus 7.14 +/- 0.01, n = 26, P < 0.001). Ionomycin induced an elevation of [Ca2+]i that was accompanied by a concomitant increase in pHi, which was Na(+)-dependent and amiloride-sensitive. [Ca2+]i and pHi increases induced by ionomycin were both dependent on the concentration of ionomycin. In conclusion, an increase in [Ca2+]i is necessary for the agonist-induced activation of the Na+/H+ exchanger in platelets. Non-agonist-induced increases in [Ca2+]i seems to prompt activation of the exchanger. In addition, Ca(2+)-depleted platelets have a more acidic basal pHi, indicating that the basal level of [Ca2+]i is also important for maintaining the basal pHi.

Dataset Information

Regulation of intracellular pH by p90Rsk-dependent activation of an Na(+)/H(+) exchanger in starfish oocytes.

Publications

Regulation of intracellular pH by p90Rsk-dependent activation of an Na(+)/H(+) exchanger in starfish oocytes.

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