mRNA-injected Oocytes Research Articles

30] Measurement of erythroid band 3 protein-mediated anion transport in mRNA-injected oocytes of Xenopus laevis

Des mRNA de reticulocytes codant pour la bande 3, proteine membranaires, sont injectes dans des ovocytes de Xenopus laevis. Leur expression est mesuree, apres incubation. Une methode requerant un appareillage d'electrophysiologie est mise au point: elle permet de suivre parallelement a la synthese des transporteurs anionique, les flux de Cl - a travers la membrane plasmique de l'ovocyte

Characteristics of the Na +/K +-ATPase from Torpedo californica expressed in Xenopus oocytes: A combination of tracer flux measurements with electrophysiological measurements

The Na +/K +-ATPase from electroplax of Torpedo californica was incorporated into the plasma membrane of Xenopus oocytes by microinjection of mRNA coding for the α- and β-subunit of the enzyme; the mRNAs were obtained by in vitro translation of cloned cDNAs (Noguchi et al. (1988) FEBS Lett. 225, 27–32). (1) Measurements of ouabain-sensitive membrane current revealed that the Na +/K +-ATPase of Torpedo is less sensitive to ouabain than the endogenous enzyme. (2) The ouabain-sensitive membrane currents in mRNA-injected oocytes exhibit similar voltage dependence as the currents generated by the endogenous ATPase of Xenopus oocytes; in particular, the current-voltage relation exhibits a maximum and a negative slope at potentials more positive than + 20 mV. (3) A maximum can also be detected if the rate of 22Na + efflux is determined under different voltage-clamp conditions. If membrane current and rate of Na 2 + efflux are determined simultaneously, a voltage-independent ratio between current and flux is obtained suggesting voltage-independent Na +-K + stoichiometry. The data are compatible with a 3Na +–2K + stoichiometry.

Differences in receptor-evoked membrane electrical responses in native and mRNA-injected Xenopus oocytes.

Xenopus laevis oocytes are giant cells suitable for studies of plasma membrane receptors and signal transduction pathways because of their capacity to express receptors after injection of heterologous mRNA. We studied depolarizing chloride currents evoked by acetylcholine (AcCho) in native oocytes ("intrinsic AcCho response"), by thyrotropin-releasing hormone (TRH) in oocytes injected with pituitary (GH3) cell RNA ("acquired TRH response"), and by AcCho in oocytes injected with rat brain RNA ("acquired AcCho response"). We found differences in the latencies and patterns of these responses and in the responsiveness to these agonists when applied to the animal or vegetal hemisphere, even though all of the responses are mediated by the same signal transduction pathway. The common intrinsic response to AcCho is characterized by minimal latency (0.86 +/- 0.05 sec), a rapid, transient depolarization followed by a distinct prolonged depolarization, and larger responses obtained after AcCho application at the vegetal rather than the animal hemisphere. By contrast, the acquired responses to TRH and AcCho are characterized by much longer latencies, 9.3 +/- 1.0 and 5.5 +/- 0.8 sec, respectively, and large rapid depolarizations followed by less distinct prolonged depolarizations. The responsiveness on the two hemispheres to TRH and AcCho in mRNA-injected oocytes is opposite to that for the common intrinsic AcCho response in that there is a much greater response when agonist is applied at the animal rather than the vegetal hemisphere. We suggest that the differences in these responses are caused by differences in the intrinsic properties of these receptors. Because different receptors appear to be segregated in the same oocyte in distinct localizations, Xenopus oocytes may be an important model system in which to study receptor sorting in polarized cells.

Phorbol ester inhibition of current responses and simultaneous protein phosphorylation in Xenopus oocyte injected with brain mRNA.

The role of protein kinase C activation in a coupling of Ca2+-mobilizing receptors/GTP-binding protein/phospholipase C was examined using Xenopus oocytes before and after microinjection of mRNA purified from rat brains. Under voltage-clamp conditions, although the phorbol ester TPA per se never elicited any changes in ionic conductance, chloride current responses of mRNA-injected cells to 5-hydroxytryptamine and acetylcholine (ACh) were suppressed by an 8-min pretreatment of 12-O-tetradecanoyl-4 beta-phorbol-13-acetate (TPA), at nanomolar concentrations. Native ACh response in intact follicular oocytes was also inhibited by the TPA treatment. However, similar current responses triggered by the direct activation of their intracellular signalling pathway with guanosine-5'-O-(3-thio)triphosphate or Ca2+ were not affected by TPA. Biochemical analyses indicated that phosphorylation of 33,000- and 45,000-dalton proteins was markedly enhanced by TPA in vivo, and that stimulation of receptors with agonists as well as TPA treatment increased phosphoproteins in the membrane fraction of mRNA-injected oocytes. These observations suggest that protein kinase C may switch off the signal transduction from receptors to GTP-binding proteins and may participate in the negative feedback modulation of receptor-operated ion channel responses.

GTP-binding proteins G i and G o transplanted onto Xenopus oocyte by rat brain messenger RNA

After injection with messenger RNA (mRNA) isolated from rat brain, Xenopus laevis oocytes acquired electrophysiological responsiveness to externally perfused acetylcholine (ACh) or serotonin (5-HT), and elevated responsiveness to internally applied guanosine 5′-(3- O-thio)triphosphate (GTPγS). Compared with the membranes of native oocytes, those of mRNA-injected oocytes contained increased amounts of 39 and 41 kDa proteins, which could be [ 32P]ADP-ribosylated by pertussis toxin (PTX). The amplitude of the GTPγS response and the amounts of the 39 and 41 kDa proteins increased in a parallel manner for at least 3 days following mRNA injection. Current responses to internally applied GTPγS showed properties common to those of responses to ACh or 5-HT perfusion: both responses had reversal potentials close to the Cl − potential, were mimicked by intracellular injection of IP 3, desensitized by a large dose of IP 3, and inhibited by a simultaneous injection of neomycin or EGTA. Incubation of mRNA-injected cells with PTX inhibited both the 5-HT response and the [ 32P]ADP-ribosylation of the 39 and 41 kDa proteins in a parallel, dose-dependent manner. After pretreatment of oocytes with PTX followed by mRNA injection, the levels of the 39 and kDa proteins and the 5-HT response appeared to be similar to those of non-treated cells injected with mRNA, whereas no detectable amounts of these proteins were induced when PTX-pretreated cells were analyzed under the same conditions without mRNA injection. From these results, we concluded that (1) PTX substrate 39 and 41 kDa G proteins were expressed in Xenopus oocytes by translation of injected rat brain mRNA, although native oocytes have their own G proteins, and (ii) the newly synthesized G proteins could also participate in the transplanted Ca 2+-mobilizing receptor-mediated responses.

Expression of an omega-conotoxin-sensitive calcium channel in Xenopus oocytes injected with mRNA from Torpedo electric lobe.

Xenopus laevis oocytes were injected with poly(A)+ RNA isolated from the electric lobe of Torpedo californica. Six to nine days after mRNA injection of the oocytes a cadmium-sensitive inward current could be detected in oocytes bathed in a calcium- and chloride-free solution containing 40 mM barium. This inward current could be distinguished from the native barium current of control oocytes by its high sensitivity to blockade by cadmium ions and its inhibition by omega-conotoxin, a peptide neurotoxin from Conus geographicus. Neither the current of control cells nor that of injected cells was detectably affected by nisoldipine (1 microM) or nitrendipine (1 microM). However, the barium current of control oocytes showed appreciably more inactivation (in the barium solution used for recording) than the omega-conotoxin-sensitive current that develops in mRNA-injected oocytes. Culturing of mRNA-injected oocytes in medium containing actinomycin D failed to prevent the appearance of the omega-conotoxin-sensitive current. These results support the conclusion that mRNA from Torpedo electric lobe is translated to produce an additional calcium channel in Xenopus oocytes. The features of this channel suggest that it may be the same type of calcium channel that controls transmitter release at nerve endings in Torpedo electroplax.

Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTP-binding proteins in Xenopus oocyte after injection of rat brain messenger RNA.

The molecular mechanism underlying the signal transduction from muscarinic and serotonergic receptors to Cl- channels were investigated in Xenopus oocyte microinjected with rat brain poly(A)+ mRNA. Transient Cl- current responses of the mRNA-injected oocytes to acetylcholine (ACh) and serotonin (5-HT) were similar in amplitude and onset. Although pharmacological characterization indicated that distinct M1-like and S1-like receptors of rat brain are involved in the ACh and 5-HT responses, respectively, these responses cross-desensitized each other completely. A common involvement of the GTP-binding proteins coupled to phosphoinositide breakdown was suggested by the findings that intracellular application of guanosine 5'-O-(2-thio)bisphosphate (GDP beta S) or neomycin greatly suppressed both ACh and 5-HT responses. These responses were not affected by exposure of the mRNA-injected cells to cholera toxin, but they were inhibited by pertussis toxin. The increase in inositol trisphosphate (IP3) responsive both to ACh and 5-HT coincided with the expression of Cl- current responses. However, only 5-HT but not ACh slightly increased the cyclic AMP (cAMP) content of the mRNA-injected cells. Intracellular injection of either IP3 or Ca2+ produced a transient Cl- current in the mRNA-injected cells as well as in non-injected cells, while 1-oleoyl-2-acetylglycerol (OAG), cAMP or cyclic GMP (cGMP) never elicited chloride current responses. It was proposed that muscarinic and serotonergic receptors are commonly linked to phosphoinositide breakdown through the mediation of GTP-binding proteins Ni and/or No in mRNA-injected oocytes.

Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTP-binding proteins in Xenopus oocyte after injection of rat brain messenger RNA

The molecular mechanism underlying the signal transduction from muscarinic and serotonergic receptors to Cl − channels were investigated in Xenopus oocyte microinjected with rat brain poly(A) + mRNA. Transient Cl − current responses of the mRNA-injected oocytes to acetylcholine (ACh) and serotonin (5-HT) were similar in amplitude and onset. Although pharmacological characterization indicated that distinct M 1-like and S 1-like receptors of rat brain are involved in the ACh and 5-HT responses, respectively, these responses cross-desensitized each other completely. A common involvement of the GTP-binding proteins coupled to phosphoinositide breakdown was suggested by the findings that intracellular application of guanosine 5′-O-(2-thio)bisphosphate (GDPβS) or neomycin greatly suppressed both ACh and 5-HT responses. These responses were not affected by exposure of the mRNA-injected cells to cholera toxin, but they were inhibited by pertussis toxin. The increase in inositol trisphosphate (IP 3) responsive both to ACh and 5-HT coincided with the expression of Cl − current responses. However, only 5-HT but not ACh slightly increased the cyclic AMP (cAMP) content of the mRNA-injected cells. Intracellular injection of either IP 3 or Ca 2+ produced a transient Cl − current in the mRNA-injected cells as well as in non-injected cells, while 1-oleoyl-2-acetylglycerol (OAG), cAMP or cyclic GMP (cGMP) never elicited chloride current responses. It was proposed that muscarinic and serotonergic receptors are commonly linked to phosphoinositide breakdown through the mediation of GTP-binding proteins Ni and/or No in mRNA-injected oocytes.

Rat brain serotonin receptors in Xenopus oocytes are coupled by intracellular calcium to endogenous channels.

Serotonin activates chloride currents in Xenopus oocytes injected with a subfraction of rat brain poly(A)+ mRNA. Patch-clamp recordings from cell-attached patches showed that serotonin, applied locally outside the patch, caused the opening of channels of approximately equal to 3 pS conductance and an average lifetime of approximately equal to 100 msec. The extrapolated reversal potential indicated that the channels are chloride-selective. Single-channel currents with similar characteristics were observed in inside-out patches from native oocytes in response to elevated calcium concentrations on the cytoplasmic side. Measurements of intracellular calcium concentration ([Ca2+]i) by fura-2 fluorescence showed approximately equal to 10-fold increases in [Ca2+]i in response to serotonin application in both normal and calcium-free Ringer solution in mRNA-injected oocytes. Little or no response to serotonin was observed in native oocytes. These results suggest that serotonin activation of receptors that are inserted into the oocyte membrane following injection of rat brain poly(A)+ mRNA can induce calcium release from intracellular stores. The increase in [Ca2+]i subsequently activates calcium-dependent chloride channels. Because calcium-dependent chloride channels and a receptor-controlled mechanism of internal calcium release have been shown to exist in native oocytes, we conclude that the newly inserted serotonin receptors utilized the endogenous second-messenger-mediated calcium release to activate endogenous calcium-dependent chloride channels.

CDNA cloning of a serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes.

We describe a strategy for the cloning of neurotransmitter-receptor and ion-channel cDNAs that is based on electrophysiological assays of mRNA-injected Xenopus oocytes. This procedure circumvents the purification of these membrane proteins, which is hindered by their low abundance and their hydrophobic nature. It involves methods for RNA fractionation by high-resolution gel electrophoresis, directional cDNA cloning in a single-stranded vector, and screening of the cDNA library by voltage-clamp measurements of currents induced by serotonin in mRNA-injected oocytes. The applicability of our approach is demonstrated by the isolation of a serotonin receptor cDNA clone from a mouse choroid plexus papilloma. The clone was identified by hybrid-depletion and hybrid-selection procedures. The receptor expressed in oocytes injected with hybrid-selected RNA is fully functional, indicating that it is composed of a single subunit encoded by a 5-kilobase RNA. The pharmacology of the hybrid-selected receptor confirms that we have successfully cloned a serotonin 5-HT1C receptor cDNA.

Neurotensin and acetylcholine evoke common responses in frog oocytes injected with rat brain messenger ribonucleic acid.

1. The intracellular reaction mechanism underlying electrophysiological responses evoked by neurotensin (NT) was studied using Xenopus laevis oocytes injected with poly (A)+ messenger ribonucleic acid (mRNA) isolated from rat brains. 2. A few days after the injection of mRNA, oocytes were found to acquire sensitivity to NT and substance P. 3. Under voltage-clamp conditions (-60 mV), application of NT to mRNA-injected oocytes produced transient and oscillatory inward currents which began after a delay of several tens of seconds. These inward currents were accompanied by an increase in membrane conductance. 4. NT receptors on mRNA-injected oocytes showed essentially the same pharmacological properties as those of native NT receptors. 5. The NT response showed desensitization and was not readily recovered even after extensive washing of cells for more than 30 min. 6. NT response was suppressed when the muscarinic acetylcholine (ACh) response of the same cell, which was also induced by the same mRNA, was desensitized by a large dose of ACh. 7. NT response and ACh response showed many similarities: they were both inhibited by pertussis toxin and intracellular ethyleneglycol-bis-(beta-aminoethylether) N, N'-tetraacetic acid (EGTA), mimicked by intracellularly injected inositol 1, 4, 5-trisphosphate (InsP3), and suppressed when cell response to InsP3 was desensitized by a large dose of InsP3. Reversal-potential analyses indicated that both responses were mediated by an increase in membrane permeability to Cl-. 8. It is concluded that NT responses and muscarinic ACh responses of Xenopus oocytes induced by rat brain mRNA may most likely share a common reaction mechanism. The reaction sequence includes the activation of receptors, activation of inhibitory guanine nucleotide-binding regulatory protein, production of InsP3, intracellular Ca2+ mobilization, and increased membrane permeability to Cl-.

Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA

Injection of poly(A) + RNA from rat brain into Xenopus oocytes caused the appearance of Cl currents in response to serotonin (5-HT) and acetylcholine (ACh). Both neurotransmitters evoked two-component currents similar in their time course to the oocyte's endogenous cholinergic muscarinic response, which was shown in previous studies to be mediated by IP 3 synthesis leading to Ca release from intracellular stores. The responses to ACh and 5-HT exhibited self- and cross-desensitization, i.e., application of either ACh or 5-HT inhibited the subsequent response to either one of the two transmitters. Intracellular injection of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) mimicked the 5-HT and ACh response, and also completely suppressed the response to the subsequent application of either ACh or 5-HT. Treatment of the oocytes with pertussis toxin (PTX) caused a 50% attenuation of ACh and 5-HT responses. In the membranes of both control and mRNA-injected oocytes, PTX catalyzed the ADP-ribosylation of a single M r = ∼40,000 protein. Injection of the purified βγ-subunits of transducin enhanced the 5-HT response. The 5-HT and GTP-γ-S responses were inhibited by intracellular injection of the Ca 2+ chelator, EGTA, as previously shown for the ACh response. These data suggest that ACh and 5-HT receptors, synthesized in the oocytes on the template of brain mRNA, act through a common pathway that involves (a) a guanine nucleotide binding protein and (b) IP 3 production leading to Ca mobilization.

Induction of taurine responsiveness in Xenopus oocytes by messenger RNA from mouse brain

A taurine response was induced in the surface membrane of the oocytes of Xenopus laevis by injection of the mRNA from the neonatal mouse brain, and the response was studied electrophysiologically. The permeability of mRNA-injected oocytes to chloride ions was increased by the application of taurine in a dose-dependent manner. The same oocyte also responded to GABA, but bicuculline suppressed only the GABA response. These results suggest a possibility that taurine could be a neurotransmitter of certain neurons as yet unknown in the central nervous system.

Growing Xenopus oocytes have spare translational capacity.

Previous studies have shown that exogenous mRNAs injected into full-grown (stage 6) Xenopus oocytes are translated only at the expense of endogenous messages; translational capacity is limited. In this report, we demonstrate that injection of globin mRNA into small, stage 4 oocytes results in an increase in total protein synthesis without a concomitant decrease in the translation of endogenous mRNAs. The absence of competition with endogenous messages in stage 4 oocytes, injected with globin mRNA, compared with stage 6 oocytes, was not due to differential turnover of the injected mRNA. Hybridization of RNA from mRNA-injected oocytes at both stages revealed that similar amounts of globin mRNA were present. These results are interpreted to mean that protein synthesis in growing oocytes is limited by the availability of mRNA and not components of the translational machinery. This conclusion, however, does not apply to all mRNA classes. The capacity of stage 4 oocytes to translate a mRNA (zein) on membrane-bound polysomes is as restricted as reported previously for stage 6 oocytes. This result suggests that putative membrane binding sites or rough endoplasmic reticulum content are limited in oocytes at both stages. The possible role of association of injected mRNA with endogenous proteins that prevent translation is discussed.

Identification of a messenger ribonucleic acid fraction in human prostatic cancer cells coding for a novel osteoblast-stimulating factor.

Prostatic cancer is frequently associated with new bone formation although the tumor-derived factors responsible for changes in bone cell function have not been identified. We have examined the synthesis of osteoblast-stimulating factors in a cultured human prostatic cancer cell line (PC-3) and show that conditioned medium from PC-3 cells stimulate mitogenesis and alkaline phosphatase in cells with the osteoblast phenotype (cultured rat osteosarcoma cells) and collagen synthesis in fetal rat calvaria. In order to characterize tumor-derived gene products which stimulate cells of the osteoblast phenotype messenger RNA (mRNA) was isolated from PC-3 cells and microinjected into Xenopus laevis oocytes. mRNA-directed translation products which were secreted into the oocyte medium were collected and assayed for a number of osteoblast stimulating properties. Translation products from PC-3 mRNA-injected oocytes stimulated division of cultured osteosarcoma cells by 8-fold and increased DNA synthesis as measured by incorporation of [3H]thymidine into these cells. In addition, tumor-derived translation products stimulated the production of alkaline phosphatase activity, a marker enzyme for bone formation, in cultured osteosarcoma cells. Oocytes injected either with water or with mRNA from a tumor not associated with bone formation were devoid of these activities. Total mRNA from the human prostatic cancer cells was then denatured and fractionated by size by agarose gel electrophoresis. When individual fractions of mRNA were eluted from the gel, translated in Xenopus oocytes, and the secreted translation products were tested for alkaline phosphatase-stimulating activity on osteoblast-like cells, the majority of the activity could be recovered in a mRNA fraction which was approximately 1800 bases in length. These results indicate that the PC-3 prostatic cancer cell line synthesizes a mRNA of approximately 1800 bases which codes for a heretofore unrecognized osteoblast-stimulating factor.

The Biosynthesis of Biologically Active Proteins in mRNA-MicroinjectedXenopusOocyte

The basic properties of mRNA-injected Xenopus oocytes as a heterologous system for the production of biologically active proteins will be reviewed. The advantages and limitations involved in the use of this in ovo system will be discussed, as compared with in vitro cell-free translation systems and with in vivo microinjected mammalian cells in culture. The different assay systems that have been utilized for the identification of the biological properties of oocyte-produced proteins will be described. This section will review the determination of properties such as binding of natural ligands, like heme or alpha-bungarotoxin; immunological recognition by antibodies; subcellular compartmentalization and/or secretion; various enzymatic catalytic activities; and induction in ovo of biological activities that affect other living cells in culture, such as those of interferon and of the T-cell receptor. The limitations involved in interpretation of results obtained using mRNA-injected oocytes will be critically reviewed. Special attention will be given to the effect of oocyte proteases and of changes in the endogenous translation rate on quantitative measurements of oocyte-produced proteins. In addition, the validity of the various measurement techniques will be evaluated. The various uses of bioassays of proteins produced in mRNA-injected Xenopus oocytes throughout the last decade will be reviewed. Nuclear and cytoplasmic injections, mRNA and protein turnover measurements and abundance calculations, and the use of in ovo bioassays for molecular cloning experiments will be discussed in this section. Finally, potential future uses of the oocyte system in various fields of research, such as immunology, neurobiology, and cell biology will be suggested.

Choline acetyltransferase and acetylcholine in Xenopus oocytes injected with mRNA from the electric lobe of Torpedo.

Xenopus oocytes were injected with poly(A)+ mRNA obtained from the electric lobes of Torpedo marmorata and Torpedo ocellata, which contain the cell bodies of the neurons that innervate the electric organs. The electric lobe mRNA preparation induces the oocytes to synthesize a catalytically active form of the enzyme choline acetyltransferase (EC 2.3.1.6). Enzymatic activity is found almost exclusively in the cytoplasmic fraction of injected, but not control, oocytes. Evidence is presented that distinguishes between the induced choline acetyltransferase activity and an intrinsic carnitine acetyltransferase activity present in the oocytes. This latter enzyme is associated principally with particulate fractions of the oocyte. The level of acetylcholine, which accumulates in mRNA-injected oocytes, is relatively insensitive to pharmacological manipulations that alter the acetylcholine content of other cells. These results show that Xenopus oocytes may be used advantageously to study functional properties of polypeptides associated with presynaptic elements in the nervous system.

Messenger RNA from rat brain induces noradrenaline and dopamine receptors in Xenopus oocytes.

Xenopus oocytes were induced to acquire sensitivity to noradrenaline and dopamine, by injecting them with poly(A)+ mRNA isolated from rat brain. In mRNA-injected oocytes, both neurotransmitters elicited a smooth inward membrane current on which was superimposed an oscillatory inward current, which was carried mainly by chloride ions. This contrasts with the native responses that are sometimes seen in non-injected oocytes, where noradrenaline and dopamine both elicit smooth outward currents that are carried mainly by potassium ions. The serotonin antagonist methysergide blocked the induced responses to both noradrenaline and dopamine, and the noradrenaline response was blocked by propranolol.

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species.

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.