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Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

ABSTRACT: We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-? (PLC-?) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLC?, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLC?. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLC? as well as doubling the amount of PLC? in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLC? stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization.

SUBMITTER: Bates RC

PROVIDER: S-EPMC3922219 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

Bates Ryan C RC Fees Colby P CP Holland William L WL Winger Courtney C CC Batbayar Khulan K Ancar Rachel R Bergren Todd T Petcoff Douglas D Stith Bradley J BJ

Developmental biology 20131121 1

We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA productio ...[more]

PMID: 24269904

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Project description:During Xenopus fertilization, the initial intracellular calcium ((Ca2+)i) release at the sperm-egg binding site (hot spot) has not been described without the use of inhibitors, nor related to underlying ER structure. Without inhibitors, we now report that sperm induce an initial hot spot after sperm addition to Xenopus eggs that was ~25 µm. This area is consistent with the size of ER patches and clusters of IP3 receptors that have enhanced activity. Furthermore, we find a new mechanism for the fertilization (Ca2+)i wave; instead of outward diffusion of inositol 1,4,5-trisphosphate (IP3), we find that the wave was generated by an outward, clockwise rotation of a ~63 µm disk of elevated (Ca2+)i moving very rapidly at ~65 µm/s. We also suggest a new mechanism for the acceleration of the fertilization (Ca2+)i wave as the disk accelerated and was joined by other rotating disks (some rotating counterclockwise) at a time when the speed of the (Ca2+)i wave increases. To examine the role of phosphatidic acid (PA) in the release of (Ca2+)i during Xenopus fertilization, we find that two inhibitors of PA production delayed the appearance of fertilization hot spots by ~9-12 min but did not reduce the size of hot spots and actually accelerated the later (Ca2+)i wave. Surprisingly, global addition of PA to Xenopus eggs induced localized hot spots at a time and size that was similar to those induced after sperm addition. In contrast, sperm induce a rapid (Ca2+)i wave (~4 µm/s) within ~30 s after hot spot appearance, whereas hot spots induced by PA required an ~32 min to induce a very slow (~1 µm/s) (Ca2+)i wave with a lower peak of (Ca2+)i. Thus, PA may not be required for the initial release of (Ca2+)i at the sperm-egg binding site, but mimics sperm by inducing a similarly sized localized (Ca2+)i release. As compared with sperm, PA may induce a weak, slow (Ca2+)i wave by slowly increasing IP3 receptor clustering. Addition of PA to Xenopus oocytes, or Ca2+ ionophore to either Xenopus oocytes or eggs, did not induce hot spots but a global (Ca2+)i wave that rapidly moved at ~12 µm/s.

Project description:Hypochlorhydria during Helicobacter pylori infection inhibits gastric Sonic Hedgehog (Shh) expression. We investigated whether acid-secretory mechanisms regulate Shh gene expression through intracellular calcium (Ca2(+)(i))-dependent protein kinase C (PKC) or cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation.We blocked Hedgehog signaling by transgenically overexpressing a secreted form of the Hedgehog interacting protein-1, a natural inhibitor of hedgehog ligands, which induced hypochlorhydria. Gadolinium, ethylene glycol-bis(?-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) + 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), PKC-overexpressing adenoviruses, and PKC inhibitors were used to modulate Ca(2+)(i)-release, PKC activity, and Shh gene expression in primary gastric cell, organ, and AGS cell line cultures. PKA hyperactivity was induced in the H(+)/K(+)-?-cholera-toxin-overexpressing mice.Mice that expressed secreted hedgehog-interacting protein-1 had lower levels of gastric acid (hypochlorhydria), reduced production of somatostatin, and increased gastrin gene expression. Hypochlorhydria in these mice repressed Shh gene expression, similar to the levels obtained with omeprazole treatment of wild-type mice. However, Shh expression also was repressed in the hyperchlorhydric H(+)/K(+)-?-cholera-toxin model with increased cAMP, suggesting that the regulation of Shh was not solely acid-dependent, but pertained to specific acid-stimulatory signaling pathways. Based on previous reports that Ca(2+)(i) release also stimulates acid secretion in parietal cells, we showed that gadolinium-, thapsigargin-, and carbachol-mediated release of Ca(2+)(i) induced Shh expression. Ca(2+)-chelation with BAPTA + EGTA reduced Shh expression. Overexpression of PKC-?, -?, and -? (but not PKC-?) induced an Shh gene expression. In addition, phorbol esters induced a Shh-regulated reporter gene.Secretagogues that stimulate gastric acid secretion induce Shh gene expression through increased Ca(2+)(i)-release and PKC activation. Shh might be the ligand transducing changes in gastric acidity to the regulation of G-cell secretion of gastrin.

Dataset Information

Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

Publications

Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

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