Involvement of Protein Kinase C in γ-Aminobutyric Acid Release from Xenopus Oocytes Injected with Rat Brain mRNA

Abstract: Involvement of protein kinase C (PKC) in the release of γ-aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA-injected oocytes preloaded with [³H]GABA showed spontaneous release of [³H]GABA, ∼0.5% of GABA content per 1 min. Stimulation with either Ca²⁺ ionophore (A23187) or a high K⁺ concentration increased the release of [³H]GABA from slices of rat deep cerebellar nucleus and mRNA-injected oocytes but not from noninjected and water-injected oocytes. 12- O -Tetradecanoylphorbol 13-acetate (10–300 n M ) but not 4α-phorbol 12,13-didecanoate (300 n M ) potentiated the A23187-stimulated release of [³H]GABA from slices and from mRNA-injected oocytes, in a concentration-dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.     

The Dopamine Metabolite 3-Methoxytyramine Is Not a Suitable Indicator of Dopamine Release in the Rat Brain

It has been postulated that changes in the concentration of 3-methoxytyramine (3-MT) in the brain might reflect changes in the release of 3,4-dihydroxyphenylethylamine (DA, dopamine) and, therefore, might be used as an index of dopaminergic activity in the brain. 3-MT is known to accumulate rapidly after death. Killing by microwave irradiation (MWR) is considered to be the method of choice to obtain "undisturbed" 3-MT concentrations. We measured striatal 3-MT concentrations even lower than those following MWR when the brains were excised and frozen in dry ice very rapidly (typical time between decapitation and freezing of the brain 22 s). There was a linear increase in striatal 3-MT concentration when the time between decapitation and freezing was varied between 13 and 300 s. Extrapolation to time zero indicated negligible amounts of 3-MT at the time of decapitation. In addition, it was observed that DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid decompose during the cooling phase after heating the brain by microwave. It is concluded that MWR induces artifactual changes in the postmortem levels of DA and metabolites. Consequently 3-MT cannot be considered to be a reliable indicator of DA release in the rat brain.     

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.     

Expression of Native GABAA Receptors in Xenopus Oocytes Injected With Rat Brain Synaptosomes

Abstract: Oocytes from the frog Xenopus laevis were shown recently to express native nicotinic acetylcholine receptors after injection with purified Torpedo electroplaque membrane vesicles. Injection of Xenopus oocytes with rat cortical or nigral synaptosomes has now been shown to result in the expression of γ-aminobutyric acid type A (GABA_A) receptor-mediated Cl⁻ currents. Electrophysiological characterization of the responses of these receptors to GABA and other agents revealed that they were incorporated into the oocyte membrane and that they retained their original pharmacological properties, such as sensitivity to Cl⁻ channel blockers, benzodiazepines, and general anesthetics. These results suggest that this approach to the expression of heterologous proteins in Xenopus oocytes may facilitate the study of native synaptic proteins derived from brain tissue.     

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5′-(β,γ-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.     

Thyrotrophin-Releasing Hormone Analogues Increase Dopamine Release from Slices of Rat Brain

Abstract: Rat brain slices were incubated with a high concentration of K⁺, thyrotrophin-releasing hormone (TRH), or one of two biologically stable TRH analogues (CG 3509 or RX 77368). Basal release of endogenous dopamine, measured by electrochemical detection, was increased by K⁺ (30 m M ) from slices of hypothalamus, septum, nucleus accumbens, and striatum. CG 3509 (10⁵–10⁻³ M ) increased the release of dopamine from slices of nucleus accumbens, septum, and hypothalamus in a dose-dependent fashion, whereas RX 77368 (10⁻⁴ M ) increased the release of dopamine from the septum only. Neither analogue increased the release of striatal dopamine. The results provide further evidence for specific regional interactions between TRH and dopamine in rat brain.     

Unstimulated and Amphetamine-Stimulated Release of Endogenous Noradrenaline and Dopamine from Rat Brain In Vivo

Abstract: The in vivo release rates of endogenous noradrenaline from the hypothalamus and dopamine from the caudate nucleus of the rat have been determined. Artificial CSF perfusates collected from a push-pull cannula inserted into specific areas of the brain were assayed for the amines by a sensitive radioenzymatic procedure. The release rates of noradrenaline and dopamine into artificial CSF perfusates were 38 ± 6 and 46 ± 6 pg/h (225 ± 36 and 301 ± 39 fmol/h), respectively; when 0.5 mM amphetamine was added to the CSF, the release rates of noradrenaline and dopamine increased to 176 ± 50 and 1183 ± 453 pg/h (1041 ± 296 and 7732 ± 2961 fmol/h), respectively.     

In Vivo Release of Dopamine and Its Metabolites from Rat Striatum in Response to Domoic Acid

The microdialysis technique was used to examine the effect of the neurotoxin domoate, an analog of glutamic acid, on striatal dopamine activity. Our results show that the intracerebral administration of different concentrations of domoate (100 and 500 M) produced increases in the extracellular levels of dopamine associated to decreases in the extracellular levels of its metabolites dihydroxyphenylacetate and homovanillate from rat striatum. These changes seem to be related according to a time sequence, indicating a possible effect on the metabolism of dopamine. Changes were also observed in locomotor activity (cycling behavior, sniffing around and chewing) in rats during the domoate infusion. The physiological mechanism by which domoate increased dopamine release remains to be worked out.     

Xenopus Embryos as a Model to Study the Genetic Mechanisms of Brain Development

The review considers the advantages of Xenopus embryos as an experimental model to study the molecular-genetic mechanisms of embryo development. The results are described that were obtained with this model in studies on the early brain development within the framework of the Russian program Human Genome.     

Release of Immunoreactive Insulin from Rat Brain Synaptosomes Under Depolarizing Conditions

Synaptosome preparations were utilized to characterize the release and compartmentalization of immunoreactive insulin (IRI) in the adult rat brain. Depolarization of synaptosomes by elevation of the external potassium ion concentration elicited release of IRI from the synaptosomes into the incubation medium. This release was reduced or eliminated under three conditions known to prevent depolarization-induced Ca2+ flux: elevating the external MgCl2, adding CoCl2, and eliminating external Ca2+ with EGTA. Depolarization of synaptosomes by veratridine also elicited release of synaptosomal IRI. This release was inhibited by tetrodotoxin. The amount of IRI released under depolarizing conditions represented 3-7% of that contained in the synaptosomes. High levels of IRI release also were observed upon removal of external Na+ to allow depolarization-independent influx of external Ca2+ into the synaptosomal compartment. The Ca2+ dependency of synaptosomal IRI release suggests IRI is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association with neural activity.     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

Redox Modulation of N-Methyl-D-Aspartate-Stimulated Neurotransmitter Release from Rat Brain Slices

Rat brain cortical slices released tritiated norepinephrine ([3H]NA) during a 2-min stimulation with N-methyl-D-aspartate (NMDA). Dithiothreitol (DTT; 0.1-5 mM), present for 6 min prior to stimulation, dose-dependently increased the release of [3H]NA from cortical slices stimulated with a maximally effective concentration of NMDA (500 microM). Similar results were observed for [3H]NA release from hippocampal slices and tritiated and endogenous dopamine release from striatal slices. DTT treatment also markedly shifted the dose-response curve of NMDA to the left. Cortical slices released approximately the same amount of [3H]NA with 10 microM NMDA following DTT treatment (about 5%) as non-DTT-treated control slices did with 500 microM NMDA. The effects of DTT were fully reversed by subsequent treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.5 mM). DTT treatment did not significantly alter the ability of magnesium (1.3 mM) or the polyamine antagonist arcaine to block the NMDA-stimulated release of [3H]NA. In contrast, DTT treatment significantly attenuated the antagonist effects of the competitive glycine antagonist, 7-chlorokynurenic acid, and the competitive NMDA antagonist, 2-aminophosphonopentanoic acid. These results suggest that oxidation and reduction of disulfide bonds located within the NMDA receptor complex might regulate the activation of the NMDA receptor. This could have important consequences in vivo if endogenous oxidizing/reducing systems are found to have similar effects on NMDA-stimulated responses.     

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.     

Expression of tachykinin receptors in Xenopus oocytes injected with poly(A)+RNA from cat dorsal root ganglion

The expression of the types of tachykinin receptors in the dorsal root ganglion (DRG) neurons by means ofXenopus oocyte expressing system was studied. Poly(A)⁺ RNAs were extracted from cat cervical and lumbar DRG. Two days after injection of Poly (A)⁺ RNAs, the oocytes were recorded with the two-electrode voltage clamp technique. In the oocytes injected with DRG poly(A)⁺ RNA, [Sar⁹, Met(O₂)¹¹]-substance P(Sar -SP, 1 μmol/L), neurokinin A (NKA, 1 μmol/L) or [β-Ala⁸]-neurokinin A₍₄₋₁₀₎ (Ala-NKA, 1 μmol/L) produced an inward current comprising a rapid spike and a long sustained oscillatory component for several minutes. Sar-SP induced response was blocked by NK-1 antagonist L-668, 169 (1 μmol/L), but not by NK-2 antagonist L-659, 877(1μmol/L). In contrast, Ala-NKA and NKA responses were only blocked by L-659, 877. The oocytes injected with DH Poly(A)⁺RNA also responded to Sar-SP and NKA with similar inward currents, which were selectively blocked by L-668, 169 and L-659, 877, respectively. These tachykinins-induced responses had a potent desensitization. The present data indicate expression of NK-1 and NK-2 receptors in DRG neurons, suggesting that there may be tachykinin autoreceptors on the nociceptive primary afferent terminals. Project supported by the National Natural Science Foundation of China (Grant No. 39370249).     

Mechanism of 1-Methyl-4-Phenylpyridinium-Induced Dopamine Release from PC12 Cells

The molecular mechanism of 1-methyl-4-phenylpyridinium (MPP⁺), a Parkinsonism-inducing neurotoxin, has been studied in PC12 cells. The cells treated with MPP⁺ (100 μM) induced a rapid increase in phosphorylation of tyrosine residues of several proteins, including synaptophysin, a major 38 kDa synaptic vesicle protein implicated in exocytosis. An accelerated release of dopamine by MPP⁺ correlated with phosphorylation of synaptophysin. Exposing the cells to MPP⁺ triggered reactive oxygen species (ROS) generation within 60 min of treatment and the said effect was blocked by mazindol, a dopamine uptake blocker. In addition, pretreatment with 50–100 μM of selegiline, a selective MAO-B inhibitor, significantly suppressed MPP⁺-mediated ROS generation. These effects of MPP⁺ result in the generation of ROS, which may be involved in neuronal degeneration seen in Parkinson’s disease.     

Expression of an inwardly rectifying K channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Stimulation of Immunoreactive Insulin Release by Glucose in Rat Brain Synaptosomes

The effect of glucose on the release of immunoreactive insulin (IRI) in synaptosomes isolated from rat brain was studied. In the absence of glucose synaptosomes release about 4% (0.77 IU/mg protein) of total content. Glucose increases significantly the IRI released by synaptosomes. Addition of the glycolytic inhibitor iodoacetic acid (IAA), decreased the glucose-induced release of IRI by about 50%, suggesting that glucose metabolism is involved. The observation that glucose provides a concentration related signal for IRI release indicates that this synaptosomal preparation may be useful as a model for research on the mechanism of insulin release in brain.     

Identification of a Novel mRNA-Associated Protein in Oocytes ofPleurodeles waltlandXenopus laevis

Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the completePleurodelescDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS–PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)⁺RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein–protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinantPleurodelesRAP55 fragment recognize the protein fromPleurodelesandXenopus.The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.