In vivo regulation of cyclic AMP phosphodiesterase in Xenopus oocytes. Stimulation by insulin and insulin-like growth factor 1

Cyclic AMP phosphodiesterase activity was measured in vivo after microinjection of [3H]cAMP into intact Xenopus oocytes. This activity was inhibited by extracellular application of methylxanthines, and the dose-dependent inhibition of phosphodiesterase activity correlated with the abilities of isobutylmethylxanthine and theophylline to inhibit oocyte maturation induced by progesterone, with IC50 values of approximately 0.3 and 1.5 mM, respectively. Insulin stimulated in vivo phosphodiesterase activity measured after microinjection of 200 microM [3H]cAMP in a time- and dose-dependent fashion without affecting phosphodiesterase activity measured after microinjection of 2 microM [3H]cAMP. Although progesterone alone had no effect on in vivo phosphodiesterase activity, low concentrations of progesterone (0.01 microM) accelerated the time course of insulin stimulation of both phosphodiesterase activity and oocyte maturation. The EC50 for stimulation of in vivo phosphodiesterase activity by insulin correlated with the IC50 for inhibition of oocyte membrane adenylate cyclase activity measured in vitro (2 and 4 nM, respectively). Twenty-fold higher concentrations of insulin were required to stimulate oocyte maturation. In contrast, insulin-like growth factor 1 stimulated in vivo phosphodiesterase, inhibited in vitro adenylate cyclase, and induced oocyte maturation at concentrations of 0.3-1.0 nM. These results demonstrate a dual regulation of oocyte phosphodiesterase and adenylate cyclase by insulin and insulin-like growth factor 1.     

Distinct, constitutively active MAPK phosphatases function in Xenopus oocytes: implications for p42 MAPK regulation In vivo

Xenopus oocyte maturation requires the phosphorylation and activation of p42 mitogen-activated protein kinase (MAPK). Likewise, the dephosphorylation and inactivation of p42 MAPK are critical for the progression of fertilized eggs out of meiosis and through the first mitotic cell cycle. Whereas the kinase responsible for p42 MAPK activation is well characterized, little is known concerning the phosphatases that inactivate p42 MAPK. We designed a microinjection-based assay to examine the mechanism of p42 MAPK dephosphorylation in intact oocytes. We found that p42 MAPK inactivation is mediated by at least two distinct phosphatases, an unidentified tyrosine phosphatase and a protein phosphatase 2A-like threonine phosphatase. The rates of tyrosine and threonine dephosphorylation were high and remained constant throughout meiosis, indicating that the dramatic changes in p42 MAPK activity seen during meiosis are primarily attributable to changes in MAPK kinase activity. The overall control of p42 MAPK dephosphorylation was shared among four partially rate-determining dephosphorylation reactions, with the initial tyrosine dephosphorylation of p42 MAPK being the most critical of the four. Our findings provide biochemical and kinetic insight into the physiological mechanism of p42 MAPK inactivation.     

Pendrin function and regulation in Xenopus oocytes

SLC26A4/PDS mutations cause Pendred Syndrome and non-syndromic deafness. but some aspects of function and regulation of the SLC26A4 polypeptide gene product, pendrin, remain controversial or incompletely understood. We have therefore extended the functional analysis of wildtype and mutant pendrin in Xenopus oocytes, with studies of isotopic flux, electrophysiology, and protein localization. Pendrin mediated electroneutral, pH-insensitive, DIDS-insensitive anion exchange, with extracellular K((1/2)) (in mM) of 1.9 (Cl(-)), 1.8 (I(-)), and 0.9 (Br(-)). The unusual phenotype of Pendred Syndrome mutation E303Q (loss-of-function with normal surface expression) prompted systematic mutagenesis at position 303. Only mutant E303K exhibited loss-of-function unrescued by forced overexpression. Mutant E303C was insensitive to charge modification by methanethiosulfonates. The corresponding mutants SLC26A2 E336Q, SLC26A3 E293Q, and SLC26A6 E298Q exhibited similar loss-of-function phenotypes, with wildtype surface expression also documented for SLC26A2 E336Q. The strong inhibition of wildtype SLC26A2, SLC26A3, and SLC26A6 by phorbol ester contrasts with its modest inhibition of pendrin. Phorbol ester inhibition of SLC26A2, SLC26A3, and SLC26A6 was blocked by coexpressed kinase-dead PKCδ but was without effect on pendrin. Mutation of SLC26A2 serine residues conserved in PKCδ -sensitive SLC26 proteins but absent from pendrin failed to reduce PKCδ sensitivity of SLC26A2 (190).     

Early stimulation of phospholipid methylation in Xenopus oocytes by progesterone

Progesterone induced a transient increase in the incorporation of [3H]methyl groups into phospholipids of Xenopus oocytes followed by a rise in 45Ca2+ uptake. Phospholipid methylation reached a maximum as early as 15 s after progesterone treatment and returned to basal level within 2 min. Steroids inactive in promoting oocyte maturation were less effective in affecting phospholipid methylation. Methyltransferase inhibitors, 3-deaza-SIBA, SIBA, and Sinefungin, inhibited progesterone-activated stimulation of phospholipid methylation, calcium uptake and meiotic maturation. Phospholipid methylation is the earliest detectable biochemical event occurring in oocytes after exposure to progesterone followed by calcium influx and leading to germinal vesicle dissolution.     

Modulation of the substrate specificity of the polycation-stimulated protein phosphatase from Xenopus laevis oocytes

A polycation-stimulated (PCS) protein phosphatase was isolated in high yield (280 micrograms/100 g ovaries) from Xenopus laevis oocytes through a procedure involving a tyrosine-agarose hydrophobic chromatography. The 220-kDa enzyme contains a 35-kDa and a 62-kDa subunit. It was identified as the low-Mr polycation-stimulated (PCSL) protein phosphatase. The labile p-nitrophenyl phosphatase activity, copurifying with the phosphorylase phosphatase activity, can be increased severalfold by preincubating the purified enzyme with ATP, its analogues or PPi. This activation is time-dependent and accompanied by a parallel decrease of the phosphorylase phosphatase activity. Although the stimulation was antagonized by metal ions during the preincubation, the basal and ATP-stimulated p-nitrophenyl phosphatase requires Mg2+ or Mn2+ in the assay, with pH optima of 8.5-9 and 7.5 respectively.     

Cyclic AMP synthesis in Xenopus laevis oocytes inhibition by progesterone

[α-³²P] ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.     

Functional activity and regulation of human beta 2-adrenergic receptors expressed in Xenopus oocytes

The recently cloned human beta-adrenergic cDNA and several mutated forms have been expressed in Xenopus laevis oocytes by injection of RNA made from the cDNA under the control of the bacteriophage SP6 promoter. The cDNA and gene of the beta 2-adrenergic receptor possess the unusual feature of having a second upstream ATG (-101 base pairs) and a 19-codon open reading frame 5' to the initiator methionine codon of the receptor (Kobilka, B. K., Dixon, R. A. F., Frielle, T., Dohlman, H. G., Bolanowski, M., Sigal, I. S., Yang-Feng, T. L., Francke, U., Caron, M. G., and Lefkowitz, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 46-50). RNA lacking this upstream AUG and open reading frame was translated approximately 10-fold more efficiently both in an in vitro rabbit reticulocyte system and in oocytes. Injected oocytes but not water injected controls expressed typical beta 2-adrenergic receptors as assessed by ligand binding (450 fmol/mg membrane protein) and catecholamine-stimulated adenylate cyclase (approximately 20 fold). Moreover, these receptors displayed typical agonist-induced homologous desensitization when oocytes were incubated with isoproterenol at room temperature for 3-24 h. Among a series of mutations, truncations of the membrane-anchored core of the receptor eliminated receptor binding and cyclase stimulating activity. In contrast, disruption of one of the cAMP-dependent protein kinase phosphorylation sites or removal of the serine/threonine-rich carboxyl terminus had little or no effect on these functions or on the extent of agonist-induced desensitization relative to that observed with native receptor. These studies validate the beta 2-adrenergic nature of the cloned human beta-adrenergic cDNA, document the utility of the Xenopus oocyte system for studying functional and regulatory properties of receptors coupled to adenylate cyclase, and suggest the possibility that elements in the 5' untranslated region of the beta 2-adrenergic receptor RNA may regulate its translation in vivo.     

Insulin and progesterone activate a common synthetic ribosomal protein S6 peptide kinase in Xenopus oocytes

A synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, the structure of which is based on that of a phosphorylated sequence in ribosomal protein S6, was employed as a probe for stimulated kinase activity in Xenopus laevis oocytes induced to mature with insulin or progesterone. Insulin elicited an early (20-30 min) 3-fold stimulation of S6 peptide phosphorylating activity that was not evident with progesterone. However, both hormones produced a delayed 7-12-fold stimulation of S6 peptide phosphorylating activity at the time of germinal vesicle breakdown. The results of DEAE-Sephacel, Sephacryl S-200, TSK-400, and heparin-Sepharose chromatographic fractionation experiments imply that a common S6 peptide kinase is activated as a consequence of short and long term insulin exposure, as well as in long term progesterone treatment of oocytes. Omission of potassium from the oocyte culture medium greatly facilitated insulin-induced meiotic maturation.     

cADPR stimulates SERCA activity in Xenopus oocytes

The intracellular second messenger cyclic ADP-ribose (cADPR) induces Ca²⁺ release through the activation of ryanodine receptors (RyRs). Moreover, it has been suggested that cADPR may serve an additional role to modulate sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump activity, but studies have been complicated by concurrent actions on RyR. Here, we explore the actions of cADPR in Xenopus oocytes, which lack RyRs. We examined the effects of cADPR on the sequestration of cytosolic Ca²⁺ following Ca²⁺ transients evoked by photoreleased inositol 1,4,5-trisphosphate (InsP₃), and by Ca²⁺ influx through expressed nicotinic acetylcholine receptors (nAChR) in the oocytes membrane. In both cases the decay of the Ca²⁺ transients was accelerated by intracellular injection of a non-metabolizable analogue of cADPR, 3-Deaza-cADPR, and photorelease of cADPR from a caged precursor demonstrated that this action is rapid (a few s). The acceleration was abolished by pre-treatment with thapsigargin to block SERCA activity, and was inhibited by two specific antagonists of cADPR, 8-NH₂-cADPR and 8-br-cADPR. We conclude that cADPR serves to modulate Ca²⁺ sequestration by enhancing SERCA pump activity, in addition to its well-established action on RyRs to liberate Ca²⁺.     

Antipain microinjection prevents progesterone to inhibit adenyl cyclase in Xenopus oocytes

Microinjection of antipain, an inhibitor of thiol and Ca2+-dependent proteases, in immature Xenopus oocytes inhibited meiotic maturation induced by progesterone, but not by transfer of cytoplasm taken from maturing oocytes. Oocytes could be released from antipain inhibition by increasing progesterone concentration. alpha-32P-ATP was microinjected to study adenylcyclase in ovo. As already reported, neosynthesis of cAMP was decreased following progesterone application. This decrease was not observed, or it was considerably reduced, in oocytes previously injected with antipain. In amphibian, full-grown ovarian oocytes are arrested at first meiotic prophase, and have a large nucleus known as the germinal vesicle. Progesterone induces the production of a cytoplasmic maturation-promoting factor (MPF), which itself triggers germinal vesicle breakdown (GVBD), and subsequent events of meiotic maturation (Masui and Markert, 1971; Gerhart et al., 1984). A considerable body of evidences support the view that release from prophase block is due to inactivation of a cAMP-dependent protein kinase (reviewed by Maller, 1983). On the other hand, progesterone has been shown to induce a transient decrease in cAMP level (Speaker and Butcher, 1977; Schorderet-Slatkine et al., 1982; Cicirelli et al., 1985), and this initial drop of cAMP, along with a number of studies indicating a decrease in adenylate cyclase activity (Mulner et al., 1979; Baltus et al., 1981; Sadler and Maller, 1981; Finidori-Lepicard et al., 1981; Jordana et al., 1981), provided key support to the theory that an early drop in cAMP led to the dephosphorylation of a hypothetical protein which initiates maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conventional protein kinase C isoforms regulate human dopamine transporter activity in Xenopus oocytes

The hypothesis that specific protein kinase C (PKC) isoforms regulate dopamine transporter (DAT) function was tested in Xenopus laevis oocytes expressing human (h)DAT. Activation of conventional PKCs (cPKCs) and novel PKCs (nPKCs) using 10 nM phorbol 12-myristate 13-acetate (PMA) significantly inhibited DAT-associated transport currents. This effect was reversed by isoform-non-selective PKC inhibitors, selective inhibitors of cPKCs and deltaPKC, and by Ca2+ chelation. By contrast, the epsilonPKC translocation inhibitor peptide had no effect on PMA-induced inhibition of hDAT transport-associated currents. Thus, the primary mechanism by which PMA regulates hDAT expressed in oocytes appears to be by activating cPKC(s).     

In vivo regulation of ovarian activity by alpha-fetoprotein

Adult female rats, each injected with 760 microgram alpha-fetoprotein (AFP) weekly, presented after the third week a decrease of ovarian activity evidenced by a decreased number of maturing follicles, corpora lutea, and a drop in ovarian weight. The level of progesterone was low and agreed with the decreased number of corpora lutea. Conversely, total and free estradiol-17 beta levels remained normal despite the known ability of AFP to bind estrogenic hormones. A regulatory role of AFP during the postnatal stage of sexual maturation is proposed.     

Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes

Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B.Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification.     

In vivo uptake of mitochondrial malate dehydrogenase from watermelon by mitochondria of Xenopus laevis oocytes

The heterologous in vivo translation system of Xenopus laevis oocytes was used to translate messenger RNA isolated from water-melon cotyledons. Immunocytochemistry was used to localize the translation products in situ within the oocyte. In addition, the translation products were immunoprecipitated from homogenized oocytes, separated on SDS-polyacrylamide electrophoresis and visualized by fluorography. A variety of watermelon proteins encoded in the injected mRNA were translated within the oocytes. Among them was the mitochondrial isoenzyme of malate dehydrogenase (mtMDH). The mtMDH was correctly imported into the mitochondria of the oocytes, as detected by immunocytochemistry.     

Dynamics of calcium regulation of chloride currents in Xenopus oocytes

Ca-activated Cl currents are widely expressed in many cell typesand play diverse and important physiological roles. TheXenopus oocyte is a good model systemfor studying the regulation of these currents. We previously showedthat inositol 1,4,5-trisphosphate (IP₃) injection intoXenopus oocytes rapidly elicits anoninactivating outward Cl current(I_Cl1-S)followed several minutes later by the development of slow inward(I_Cl2) andtransient outward(I_Cl1-T) Clcurrents. In this paper, we investigate whether these three currentsare mediated by the same or different Cl channels. Outward Cl currentswere more sensitive to Ca than inward Cl currents, as shown byinjection of different amounts of Ca or by Ca influx through aheterologously expressed ligand-gated Ca channel, the ionotropicglutamate receptor iGluR3. These data could be explained by twochannels with different Ca affinities or one channel with a higher Caaffinity at depolarized potentials. To distinguish between thesepossibilities, we determined the anion selectivity of the threecurrents. The anion selectivity sequences for the three currents werethe same (I > Br > Cl), butI_Cl1-Shad an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, afterconsideration of possible errors, the hypothesis that a single type ofCl channel underlies the complex waveforms of the three differentmacroscopic Ca-activated Cl currents inXenopus oocytes remains a viable alternative.

     

Potentiation of the effect of steroid hormones (progesterone and testosterone) inducing the reinitiation of meiosis in Xenopus oocytes in vitro, by inhibitors of phosphatase activity

The induction of meiosis reinitiation by steroid hormones (progesterone and testosterone) in Xenopus laevis oocytes was studied in vitro in presence of inhibitors of phosphatase activity such as beta-glycerophosphate, considered as a competitive inhibitor, and the two ions, Zn++ and MoO--4. Kinetics of the germinal vesicle breakdown indicating the reinitiation of meiosis, have shown that while these phosphatase inhibitors were not active by themselves under the present experimental conditions, they enhanced the process elicited by progesterone or testosterone.     

Cyclic AMP synthesis in Xenopus laevis oocytes: inhibition by progesterone.

[alpha-32P]ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.