Selective potentiation of N-methyl-D-aspartate-induced current by protein kinase C in Xenopus oocytes injected with rat brain RNA.

Glutamate receptors and protein kinase C (PKC) may play significant roles in long-term potentiation in hippocampus. To clarify the regulatory involvement of PKC in the functions of glutamate receptors, we examined the effects of PKC activation on current response induced by the activation of each subtype of glutamate receptor in Xenopus oocytes injected with rat brain RNA. Treatment with the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), potentiated N-methyl-D-aspartate (NMDA)-induced current by about 2.5-fold, although it did not affect kainate-induced current at all. Quisqualate-mediated oscillatory current was almost abolished by this treatment. The TPA-induced potentiation of NMDA current was suppressed by staurosporine, an inhibitor of protein kinases. Pretreatment with 4-O-methyl-TPA, an inactive phorbol ester, had no effect on NMDA current. Current response mediated by NMDA receptors would thus appear to be modulated by PKC.     

Roles of protein kinases in neurotransmitter responses in Xenopus oocytes injected with rat brain mRNA

Microinjection of rat brain mRNA in Xenopus oocytes induced acetylcholine, neurotensin, serotonin, and glutamate receptors in the cells. These receptors stimulate an intracellular reaction pathway, including G-protein activation, inositol trisphosphate (IP3) formation, and Ca2+-dependent Cl- channels. In the present study, we examined the roles of several protein kinases in these responses by means of inhibitors and activators of these kinases. Isoquinolinesulfonamides, inhibitors of protein kinases, caused no current responses and affected no receptor-mediated responses when injected into the oocytes at low doses (30-50 pmol), which inhibit cyclic nucleotide-dependent kinases or kinase C specifically, but abolished the receptor-mediated responses at a higher dose (300 pmol), which inhibit most protein kinases nonspecifically. Calmodulin inhibitors blocked the receptor-mediated responses strongly. Activation of cyclic nucleotide-dependent kinases or kinase C by injection of cAMP (or cGMP) or perfusion with phorbol esters caused no direct current responses but suppressed receptor-mediated responses. Current responses triggered by IP3 injection were not suppressed by these treatments. These results suggest that cAMP- (or cGMP-)dependent kinases or kinase C may not be involved in the pathway directly but may modulate it by inhibiting the initial part of the pathway (receptors, G-proteins, and/or phospholipase C), and they suggest that calmodulin may most likely be involved in the activation of Ca2+-dependent Cl- channels.     

Tetrabutylammonium induces a voltage-dependent block of kainate current in Xenopus oocytes injected with rat brain mRNA.

Following injection of rat striatal and cerebrellar mRNA, Xenopus oocytes were voltage clamped and current responses to the excitatory amino acid receptor agonist, kainate, were recorded. This nonspecific cationic current is carried principally by Na+ and K+ and reverses polarity at a membrane potential of approximately -5 mV. When the membrane potential was voltage clamped to -60 mV, bath-applied tetrabutylammonium (0.1-30 mM) produced a rapid, concentration dependent and reversible block of kainate-induced inward current with an IC50 of 1.3 mM. Tetraalkylammonium derivatives having shorter chains (methyl, ethyl, and propyl) were relatively ineffective blockers. Longer alkyl chain derivatives (pentyl, hexyl, and heptyl) were more potent than tetrabutylammonium but limited in their usefulness by their toxicity. The antagonism of kainate-induced current by tetrabutylammonium displayed apparently uncompetitive kinetics, in contrast with the competitive antagonism by gamma-D-glutamylaminomethylsulfonate. The block by tetrabutylammonium was strongly voltage dependent; an e-fold change in IC50 was observed for a 27 mV change in holding potential. Replacement of the Na+ in the medium with a more permeant cation (NH4+), a less permeant cation (tetramethylammonium), or an uncharged solute (mannitol) had little effect on the block of kainate-induced current by tetrabutylammonium. The rates of association and dissociation of tetrabutylammonium with the kainate receptor-channel are clearly rapid. These observations suggest that tetrabutylammonium enters and blocks the kainate receptor-associated cation selective channel. Tetrabutylammonium appears to traverse 80-90% of the membrane electrical field to reach a relatively low-affinity binding site that may simply be a narrowing of the channel.     

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3–1000 µ.M) induced a smooth inward current that was competitively inhibted by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 µM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (E_r) of -6.1 ± 0.5 mV In cerebellar mRNA-injected oocytes; E_r was nearly identical (-5.1 ± 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd⁺⁺ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.     

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3-1000 microM) induced a smooth inward current that was competitively inhibited by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 microM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (Er) of -6.1 +/- 0.5 mV. In cerebellar mRNA-injected oocytes; Er was nearly identical (-5.1 +/- 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd++ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.     

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.     

Characterization of protein kinase C in Xenopus oocytes.

Protein kinase C (PKC) was partially purified from Xenopus laevis oocytes by ammonium sulfate fractionation followed by DEAE-cellulose and hydroxyapatite column chromatography. In the latter chromatography, two distinct PKC activities were identified. Both PKC fractions contained an 80 kDa protein which was recognized by three antisera raised against the conserved regions of mammalian PKC. However, specific antisera against alpha, beta I, beta II, and gamma-subspecies of rat PKC did not recognize the protein. Kinetic properties of the Xenopus PKCs were very similar to those of the rat alpha PKC, and only a subtle difference was found in the mode of activation by arachidonic acid. When oocytes were treated with the tumor promoter, phorbol 12-myristate 13-acetate, one of the Xenopus PKCs was found to disappear very rapidly, while the other remained unchanged up to 2 hr.     

Molecular mechanism of protein kinase C modulation of sodium channel alpha-subunits expressed in Xenopus oocytes.

The mechanism of modulation of sodium channel alpha-subunits (Type IIA) by a protein kinase C (PKC) activator was studied on single channel level. It was found that: (i) time constants for channel activation were prolonged; (ii) inactivation remained virtually unchanged; (iii) peak sodium inward current was reduced as evidenced by calculation of average sodium currents; and (iv) time constants for current activation and decay were prolonged. (i), (iii) and (iv) were voltage dependent, being most prominent at threshold potentials. The data show that a voltage dependent action on the activation gate can account for the observed reduction of peak inward sodium current and prolongation of current decay in macroscopic experiments.     

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).     

Glutamate receptor subtypes may be classified into two major categories: a study on Xenopus oocytes injected with rat brain mRNA

Three major subtypes of glutamate receptors that are coupled to cation channels are known. Recently an additional subtype that is coupled to G proteins and stimulates inositol phospholipid metabolism (the metabotropic glutamate receptor) has been proposed. The pharmacological characteristics of this receptor have now been examined. Although it shares some agonists with N-methyl-D-aspartate- and quisqualate-subtype receptors, it shares virtually no antagonists with any of the three cation channel-coupled receptor subtypes. Thus the metabotropic glutamate receptor belongs to a receptor category that is completely different from that of the other three receptor subtypes, not only functionally, but also pharmacologically.     

注射热损伤大鼠纹状体mRNA的爪蟾卵母细胞对多巴胺的反应

将从正常大鼠和热损伤大鼠的中枢纹状体提取的poly(A) mRNA ,注入非洲爪蟾卵母细胞表达。用电生理方法检测多巴胺诱发的膜电位和电流的变化 ,分析热损伤对中枢多巴胺受体表达的影响。结果表明 ,注射大鼠纹状体mRNA后 ,卵母细胞的静息电位与注射前没有变化 ,但多巴胺能诱发膜电流。经验证 ,此受体电流的主要载流离子是Cl-。注射热损伤大鼠纹状体mRNA的卵母细胞对多巴胺反应的敏感性降低 ,与正常大鼠组相比有显著性差异。因此可以断定 ,热损伤对大鼠纹状体中多巴胺受体的基因表达产生了明显的影响 ,并可能有离子通道的参与。     

注射热损伤大鼠纹状mRNA的爪蟾卵母细胞对多巴胺的反应

将从下沉大鼠和热损伤大鼠的中枢纹状体提取的ｐｏｌｙ（Ａ）＾＋ｍＲＮＡ,注入非洲爪蟾卵母细胞表达。用电生理方法检测多巴胺诱发的膜电位和电流的变化,分析热损伤对中枢多巴胺受体表达的影响。结果表明,注射大鼠纹状体ｍＲＮＡ后,卵母细胞的静息电位与注射 前没有变化,但多巴胺能诱发膜电流。经验证,此受体电流的主要载流离子是Ｃ１＾－。注射热务大鼠纹状体ｍＲＮＡ的卵母细胞对多巴胺反应的敏感性降低,与正常大鼠组相比     

Confocal microscopic study of GABA(A) receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity

The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.     

Effect of lipid hydroperoxide on Xenopus oocytes and on neurotransmitter receptors synthesized in Xenopus oocytes injected with exogenous mRNA

The effect of 13-L-hydroperoxylinoleic acid (LOOH) on both Xenopus oocytes and neurotransmitter receptors synthesized in the oocytes was studied by electrophysiological and ion flux measurement. Addition of LOOH to the incubation mixture of the oocytes raised the membrane potential and decreased the membrane resistance of the oocytes. These effects of LOOH on the oocytes were reversed within a few hours by incubation with frog Ringer solution. Addition of LOOH also caused an increase of Li+ and 45Ca2+ uptake into the oocytes. However, production of alkoxy radicals by the addition of FeCl2 to the incubation mixture containing LOOH did not accelerate the damage to the oocytes by LOOH. So essential toxicity is caused possibly by an increase in the membrane permeability resulting from disturbance of the lipid bilayer arrangement, not from production of active alkoxy radicals during decomposition of LOOH. Nicotinic acetylcholine and gamma-aminobutyric acid receptors were synthesized in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and rat brain. LOOH noncompetitively inhibited the function of these receptors and also increased the rate of desensitization of the receptors.     

The pathway of MAP kinase mediation of CSF arrest in Xenopus oocytes.

A cytoplasmic activity in mature oocytes responsible for second meiotic metaphase arrest was identified over 30 years ago in amphibian oocytes. In Xenopus oocytes CSF activity is initiated by the progesterone-dependent synthesis of Mos, a MAPK kinase kinase, which activates the MAPK pathway. CSF arrest is mediated by a sole MAPK target, the protein kinase p90Rsk which leads to inhibition of cyclin B degradation by the anaphase-promoting complex. Rsk phosphorylates and activates the Bub1 protein kinase, which may cause metaphase arrest due to inhibition of the anaphase-promoting complex (APC) by a conserved mechanism defined genetically in yeast and mammalian cells. CSF arrest in vertebrate oocytes by p90Rsk provides a potential link between the MAPK pathway and the spindle assembly checkpoint in the cell cycle.     

A protein kinase C inhibitory activity is present in rat brain homogenate

The partial purification and characterization of (a) factor(s) from rat brain which inhibit(s) the activity of calcium and phospholipid-dependent protein kinase from the same tissue is described. This factor, present in 100 000 X g rat brain homogenate supernatant, is inactivated upon treatment by trypsin and pepsin and is therefore assumed to be a protein. It was partially purified by ion-exchange chromatography on DEAE-cellulose, ammonium sulfate precipitation and gel filtration. This inhibitor is not stable to heating at 70 degrees C for 10 min, however partial renaturation of the inhibitory activity can be observed after incubation of the denatured inhibitor for 24 h at 4 degrees C. It is precipitable by 10% trichloroacetic acid and by 2 M ammonium sulfate. It exhibits a Stokes radius of 20 A by gel exclusion chromatography, corresponding to a molecular mass of 20 kDa assuming a globular shape. Kinetic analysis of the inhibition of calcium-phospholipid-dependent histone kinase activity indicates that the inhibitor is competitive with respect to the protein substrate. No change was observed in the kinetic values of the kinase for ATP, Ca2+ and phospholipids.     

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na+ concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na+/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na+ concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.     

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.