Dopamine receptors expressed in the Xenopus oocytes injected with bovine striatal mRNA

The response to dopamine (DA) of Xenopus oocytes injected with bovine striatal mRNA was electrophysiologically and pharmacologically investigated under a voltage-clamped condition. In oocytes injected with bovine striatal mRNA and then treated with collagenase (denuded oocytes), DA applied by superfusion dose-dependently induced oscillatory inward currents with a long latency and an ED50 value of 12.0 microM. mRNA non-injected denuded oocytes did not respond to DA up to concentrations of 1 mM. The reversal potential of DA-induced currents was about -25 mV, indicating that DA increased a Cl(-)-conductance. DA-induced currents were easily desensitized by repeated applications of DA. Haloperidol and SCH-23390 abolished DA-induced currents, while propranolol and mianserin showed no effect. Furthermore, SK&F-38393, a specific agonist of the DA D1 receptor, induced similar oscillatory currents as DA did. These results suggest the possibility that functional D1 and D2 DA receptors were co-expressed in Xenopus oocytes by injection of bovine striatal mRNA.     

Actions of pentobarbital on rat brain receptors expressed in Xenopus oocytes

Functional receptor channels activated by GABA and other neurotransmitters were "transplanted" from rat brain to Xenopus oocytes by injecting the oocytes with total poly(A)+ mRNA isolated from rat or chick brain. Membrane currents elicited in the oocyte by GABA inverted polarity at about the chloride equilibrium potential (ca. -25 mV). Pentobarbital potentiated the GABA-activated currents, without appreciably changing the reversal potential or form of the current-voltage relationship. At low (less than 10(-5) M) concentrations of GABA, pentobarbital (100 microM) potentiated the responses by a factor of 10 or more, but responses to high (ca. 1 mM) concentrations of GABA were almost unchanged. Half-maximal activation of the response was obtained with about 3 X 10(-5) M GABA when applied alone and with about 4 X 10(-6) M GABA when applied together with 100 microM pentobarbital. At low doses of GABA, the size of the current increased as the 1.4th power of GABA concentration, but this relationship became nearly linear in the presence of pentobarbital. The potentiation of the GABA response increased linearly with concentrations of pentobarbital up to about 300 microM, reaching a maximum of about 50-fold. At higher concentrations of pentobarbital, the response to GABA declined. Relaxations of GABA-activated currents following voltage steps became slower in the presence of pentobarbital, suggesting that the open life-time of the channels was prolonged. In addition to actions on GABA-activated currents, pentobarbital itself elicited a small membrane current that inverted polarity at a potential (-10 mV) more positive than the GABA-activated current.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water transport by glucose transporter type 3 expressed in Xenopus oocytes

The hypothesis that the facilitative glucose transporter type 3 (GLUT3) in the brain also exhibits water channel properties similar to that of GLUT1 was tested in Xenopus oocytes expressing human GLUT3 or GLUT1. The volume of oocytes expressing GLUT3 exposed to hypotonic medium increased in an exponential manner. The osmotic water permeability (Pf) for GLUT3 or GLUT1 increased significantly compared with that for oocytes-injected water. The osmotic water transport by GLUT3 was completely inhibited by treatment with a selective GLUT inhibitor, cytochalasin B. The Pf values for GLUT3 significantly increased with increasing temperature of the extracellular medium and the activation energy for GLUT3 was almost the same as that for GLUT1. Thus, GLUT3 in the brain also exhibits water channel properties.     

Antibiotics cause changes in the desensitization of ACh receptors expressed in Xenopus oocytes.

ACh receptors (AChRs) synthesized in Xenopus oocytes which were cultured in medium containing gentamicin desensitized much more rapidly than those expressed in the absence of gentamicin. The effect caused by 24 h incubation in gentamicin could not be reversed by leaving oocytes in culture medium without gentamicin for 24 h. In addition, gentamicin exhibited a direct reversible blocking action on the function of AChRs. Our experiments indicate that antibiotics should be used cautiously in culturing oocytes when studying the function of induced neurotransmitter receptors and ion channels.     

Single-channel properties of mouse-Torpedo acetylcholine receptor hybrids expressed in Xenopus oocytes.

This report analyzes the contribution of individual nicotinic acetylcholine receptor (AChR) subunits to the single-channel properties of the AChR ion channel. By in vitro synthesis of mRNA from cDNA clones encoding each AChR subunit (alpha, beta, gamma, and delta) from mouse BC3H-1 cells and Torpedo electric organ and microinjection of appropriate mRNA combinations into Xenopus oocytes, we studied the single-channel properties of both 'homologous' (all subunits from the same species) and 'hybrid' (subunits from both species) AChRs as they were expressed in the oocyte membrane. AChR expression was determined by surface binding of 125I-labeled alpha-bungarotoxin to intact oocytes, and those with binding sites of 1 fmol/cell or more were chosen for patch-clamp studies. Our results indicate the following: (1) Species difference in single-channel conductance can be explained largely by the charge distribution flanking the M2 transmembrane domain. (2) The alpha and delta subunits from mouse AChR independently lengthen the channel open time, in some cases by 10-fold; the beta subunit from mouse shortens the channel open time; the mouse gamma subunit lengthens open time less dramatically. (3) Voltage sensitivity, as measured by the ratio of channel open times at -60 mV and +60 mV, is influenced by the beta and delta subunits, in agreement with our previous study by two-electrode voltage-clamp recording. We conclude that single-channel properties of the AChR are governed by multiple elements located on different AChR subunits.     

Stereoselective effects of AMOA on non-NMDA receptors expressed in Xenopus oocytes.

Pharmacological characterization of the action of the novel non-N-methyl-D-aspartate (non-NMDA) antagonist AMOA (2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate) on glutamate receptors was investigated in Xenopus oocytes injected with mouse brain mRNA. AMOA (150 microM) produced a nearly parallel shift to the right of the dose-response curve for kainate-induced currents. AMOA was found to have two different effects on AMPA receptors: 1) currents elicited by low concentrations of AMPA (6 microM) were inhibited by AMOA with an IC50 value of 160 +/- 19 microM and 2) currents elicited by high concentrations of AMPA (100 microM) were potentiated with an IC50 value of 88 +/- 22 microM. The maximal potentiating effect of AMOA on AMPA currents was around 170%. Furthermore, the two opposing effects of AMOA on AMPA responses are specific for the L-configuration of AMOA. This unusual antagonistic/agonistic property of AMOA may explain its unusual properties with regard to antagonism of non-NMDA receptor-mediated events previously described.     

Voltage-dependent blockade by bupivacaine of cardiac sodium channels expressed in Xenopus oocytes

Bupivacaine ranks as the most potent and efficient drug among class I local anesthetics, but its high potential for toxic reactions severely limits its clinical use. Although bupivacaine-induced toxicity is mainly caused by substantial blockade of voltage-gated sodium channels （VGSCs）, how these hydrophobic molecules interact with the receptor sites to which they bind remains unclear. Navl.5 is the dominant isoform of VGSCs expressed in cardiac myocytes, and its dysfunction may be the cause of bupivacaine- triggered arrhythmia. Here, we investigated the effect of bupivacaine on Navl.5 within the clinical concentration range. The electrophysiological measurements on Navl.5 expressed in Xenopus oocytes showed that bupivacaine induced a voltage- and concentration-dependent blockade on the peak of/Na and the half-maximal inhibitory dose was 4.51 pmol/L. Consistent with other local anesthetics, bupivacaine also induced a use-dependent blockade on Navl.5 currents. The underlying mechanisms of this blockade may contribute to the fact that bupivacaine not only dose-dependently affected the gating kinetics of Nay1.5 but also accelerated the development of its open-state slow inactivation. These results extend our knowledge of the action of bupivacaine on cardiac sodium channels, and therefore contribute to the safer and more efficient clinical use of bupivacaine.     

Inhibition of the glutamate transporter EAAC1 expressed in Xenopus oocytes by phorbol esters

Recent evidence indicates that second messengers and protein kinases regulate the activity and expression of glutamate transporters. The aim of the present study was to determine if direct activation of protein kinases C or A modulates the activity of the sodium-dependent glutamate transporter EAAC1. EAAC1 modulation was studied in cRNA-injected Xenopus oocytes by measuring [3H]L-glutamate uptake or glutamate-evoked uptake currents. We found that activation of PKA was ineffective, whereas treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) caused a significant decrease in EAAC1 transport activity (IC(50)=44.7+/-12 nM). PMA-induced EAAC1 inhibition was PKC-mediated because the inhibition could be blocked by specific PKC inhibitors and incubation with the inactive 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) did not affect EAAC1. Saturation studies of glutamate-evoked uptake currents showed that PMA-mediated inhibition was due to a decrease in I(max) with no change in K(m). PMA simultaneously decreased membrane capacitance (C(m)) and transport-associated current and increased cytosolic accumulation of EAAC1 protein, compared to control. These results suggest that PKC activation inhibits EAAC1 by promoting its retrieval from the plasma membrane. PMA also significantly decreased glutamate uptake in a Madin-Darby canine kidney (MDCK) cell line stably transfected with EAAC1 but enhanced EAAC1-mediated glutamate uptake in the rat C6 glioma cells, consistent with previous observations. Because activation of PKC by phorbol esters leads to opposite effects on EAAC1 activity in different culture models, we conclude that the PKC-mediated regulation of EAAC1 is cell-type specific.     

Glycine-insensitive NMDA-sensitive receptor expressed in Xenopus oocytes by guinea pig cerebellar mRNA

The electrophysiological and pharmacological properties of N-methyl-D-aspartate (NMDA)-sensitive receptors expressed in Xenopus oocytes by injection of total poly(A)+RNAs (mRNAs) from the cerebellum and cerebrum of guinea pigs were compared. The inward current induced by NMDA under voltage-clamp in cerebellar mRNA-injected oocytes was depressed in a voltage-dependent fashion by Mg2+ to show a negative slope conductance and selectively antagonized by D-2-amino-5-phosphonovalerate (D-APV) and phencyclidine (PCP). Glycine (0.01-10 microM) did not potentiate NMDA-induced currents in cerebellar mRNA-injected oocytes, while it potentiated NMDA-induced currents in cerebral mRNA-injected oocytes in a dose-dependent fashion. 6-Cyano-7-nitroquinoxaline-2,3-dione and 7-chlorokynure-nate suppressed the NMDA response but significantly less potently in cerebellar mRNA-injected oocytes than in cerebral mRNA-injected oocytes. These results suggest that the NMDA-sensitive receptor expressed in Xenopus oocytes by guinea pig cerebellar mRNA resembles the cerebral NMDA receptor in its high sensitivities to Mg2+, PCP, and D-APV, but it is distinct from the cerebral NMDA receptor in responsiveness to glycine.     

Distinct regulation of expressed calcium channels 2.3 in Xenopus oocytes by direct or indirect activation of protein kinase C

Protein kinase C (PKC)-dependent regulation of voltage-gated Ca (Ca(v); with alpha(1)beta1Balpha2/delta subunits) channel 2.3 was investigated using phorbol 12-myristate 13-acetate (PMA), or by M(1) muscarinic receptor activation in Xenopus oocytes. The inward Ca(2+)-current with Ba(2+) (I(Ba)) as the charge carrier was potentiated by PMA or acetyl-beta-methylcholine (MCh). The inactivating [I(inact)] and non-inactivating [I(noninact)] components of I(Ba) and the time constant of inactivation tau(inact) were all increased by MCh or PMA. This may be a PKC-dependent action since the effect of MCh and PMA was blocked by Ro-31-8425 or beta-pseudosubstrate. MCh effect was blocked by atropine, guanosine-5'-O-(2-thiodiphosphate) trilithium (GDPbetaS) or U-73122. The effect of MCh but not PMA was blocked by the inhibition of inositol-1,4,5-trisphosphate (IP3) receptors, intracellular Ca(2+) ([Ca(2+)](i)) or the translocation of conventional PKC (cPKC) with heparin, BAPTA and betaC2.4, respectively. While a lower concentration (25 nM) of Ro-31-8425 blocked MCh, a higher concentration (500 nM) of Ro-31-8425 was required to block PMA action. This differential susceptibility of MCh and PMA to heparin, BAPTA, betaC2.4 or Ro-31-8425 is suggestive of the involvement of Ca(2+)-dependent cPKC in MCh action, whereas cPKC and Ca(2+)-independent novel PKC (nPKC) in PMA action. PMA led to additional increase in I(Ba) that was already potentiated by preadministered MCh (1 or 10 microM), leading to the suggestion that differential phosphorylation sites for cPKC and nPKC may be present in the alpha(1)2.3 subunit of Ca(v) 2.3 channels.     

Electrochemical monitoring of transport by a vesicular monoamine transporter expressed in Xenopus oocytes

Xenopus laevis oocytes were injected with synthetic mRNA coding for a rat VMAT2 mutant (rVMAT2-I483A/L484A) shown previously to be retained on the plasma membrane as a result of a presumed reduction of endocytosis. Binding of the specific VMAT inhibitor [3H]dihydrotetrabenazine indicated that expression did occur at a level of approximately 3 fmol per oocyte. To determine if rVMAT2-I483A/L484A expressed in oocytes was capable of substrate transport, oocytes were placed in buffer at pH 6.0, dopamine substrate was injected into the cell, and egress of substrate was monitored by fast scan cyclic voltammetry using a carbon fiber microelectrode. Under these conditions, transport by oocytes injected with RNA coding for rVMAT2-I483A/L484A ranged from approximately 0.5 to more than 2.5 pmol/min. Water-injected and uninjected control oocytes did not exhibit appreciable transport activity. Transport by rVMAT2-I483A/L484A-injected oocytes was reduced to control levels by tetrabenazine, a known inhibitor of VMAT transport activity. Comparison of subtracted voltammograms obtained from transport assays with those for calibration experiments confirmed that the transported species was dopamine. These results suggest that expression of VMATs in oocytes may provide a useful model system for mechanistic and regulatory studies that would not be feasible using traditional methods.     

Desensitization of junctional and extrajunctional nicotinic ACh receptors expressed in Xenopus oocytes.

Desensitization of nicotinic acetylcholine receptors (AChRs) was studied using the Xenopus oocyte expression system. Mouse and cat extrajunctional AChRs expressed in oocytes desensitized more slowly than Torpedo AChRs. Substitution of the mouse gamma subunit into the Torpedo AChR reduced the rate of desensitization, making it similar to the mouse AChR. Likewise, substitution of the Torpedo gamma subunit into the mouse extrajunctional AChR increased the rate of desensitization, making it similar to the Torpedo AChR. Furthermore, mouse junctional AChRs in which the gamma subunit was replaced by the epsilon subunit desensitized more rapidly than either mouse or cat extrajunctional AChRs, and resembled Torpedo AChRs. Thus, in addition to other properties, junctional and extrajunctional AChRs differ with respect to desensitization, suggesting a possible role of desensitization in normal development of neuromuscular junction.     

Histamine effects on the 5-HT1c receptor expressed in Xenopus oocytes.

To analyze the cross-reactivity between serotonin (5-HT) and histamine, the in vitro transcribed RNA for the 5-HT1c receptor was functionally expressed in Xenopus oocytes. 5-HT significantly increased 45Ca2+ efflux in RNA-injected oocytes, but not in uninjected and water-injected control oocytes. Furthermore, histamine and the H1 receptor agonists, but not the H2 and H3 agonists, significantly induced 45Ca2+ efflux in 5-HT1c receptor RNA-injected oocytes, but not in uninjected and water-injected oocytes. However, the H1, H2, and H3 antagonists failed to inhibit histamine-induced 45Ca2+ efflux at 10(-6) M. This finding suggests that the 5-HT1c receptor can be activated by both 5-HT and histamine, although the action of histamine is different from classic histamine pharmacology.     

Depressive-like behaviour of mice lacking cellular prion protein

Cellular Prion Protein (PrP^C) is known to mediate a protective role in several neurological conditions such as ischemia and epilepsy. However, so far, little information is available concerning the role of PrP^C in psychiatric disorders such as depression. Here, we have used PrP^C null mice to examine a putative role of PrP^C in depressive-like states. Prion protein null mice exhibited depressive-like behaviour when compared to wild-type mice in both the Forced Swimming Test (FST) and Tail Suspension Test (TST). The clinical antidepressant drug imipramine and the NMDA receptor antagonist MK-801 reversed the depressive-like behaviour observed for knockout mice in the TST. The present data thus indicate that PrP^C exerts a critical role in modulating the depressive-like state in mice, reinforcing the notion that PrP^C might be associated with alterations in mood disorder states, and suggests a possible role of PrP^C as a potential drug target for treating depressive disorders.     

Allosteric modulation by benzodiazepine receptor ligands of the GABAA receptor channel expressed in Xenopus oocytes

Chick brain mRNA was isolated and injected into Xenopus oocytes. This led to the expression in the surface membrane of functional GABA-activated channels with properties reminiscent of vertebrate GABAA channels. The GABA-induced current was analyzed quantitatively under voltage-clamp conditions. Picrotoxin inhibited this current in a concentration-dependent manner with IC50 = 0.6 microM. The allosteric modulation of GABA currents by a number of drugs acting at the benzodiazepine binding site was characterized quantitatively. In the presence of the benzodiazepine receptor ligands diazepam and clorazepate, GABA responses were enhanced, and in the presence of the convulsant beta-carboline compound methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), they were depressed. Maximal stimulation of the response elicited by 10 microM GABA was 160% with diazepam and 90% with clorazepate, and maximal inhibition was 42% with DMCM, 30% with methyl beta-carboline-3-carboxylate (beta-CCM), 15% with ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4]benzodiazepine-3-carboxylate (Ro 15-1788), and 12% with ethyl beta-carboline-3-carboxylate (beta-CCE). Half-maximal stimulation was observed with 20 nM diazepam and 390 nM clorazepate, respectively, and half-maximal inhibition with 6 nM DMCM. beta-CCM had a similar effect to DMCM, whereas beta-CCE and Ro 15-1788 showed only small inhibition at low concentrations (less than 1 microM). All the tested carboline compounds and Ro 15-1788 showed a biphasic action and stimulated GABA current at concentrations higher than 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-gated influx ion channels expressed in Xenopus oocytes after the administration of brain mRNA]

Expression of "fast", TTX-sensitive sodium and high-threshold calcium channels in the membrane of Xenopus oocytes following mRNA injection from the rat brain has been detected using two microelectrode voltage clamp technique. Barium current through expressed calcium channels was blocked by 200 mumol/l Cd2+ and was insensitive to D-600 (20 mumol/l) and nitrendipine (50 mumol/l). Expressed barium current was inhibited within 20-40 min by omega-conotoxin, a peptide neurotoxin known to block high-threshold calcium channels of the neuronal membrane, in 1 mumol/l concentration. A steady-state inactivation curve for this current could be fitted by the Boltzmann relation with V1/2 = -50 mV and k = 14 mV. Voltage-dependent and pharmacological properties of calcium channels which appeared in the oocyte membrane following mRNA injection from the mammalian brain resembled most of all those of high-threshold inactivating (HTI- or N-type) calcium channels of neurons in spite they did not demonstrate prominent time-dependent inactivation. Evidences in favour of expressed calcium channels heterogeneity were not obtained.     

Properties of endogenous voltage-dependent sodium currents in Xenopus laevis oocytes.

Endogenous voltage-dependent sodium currents were recorded using standard 2-microelectrode techniques in Xenopus laevis oocytes. Maximal inward current occurred at -10 mV with an average amplitude of -279 +/- 17 nA and steady-state inactivation was half-maximal at a voltage of -38 +/- 0.5 mV. Currents were blocked by low concentrations of tetrodotoxin (TTX) with an IC50 value of 6 nM. These properties make the endogenous sodium current in Xenopus oocytes similar to sodium currents expressed following injection of mammalian brain RNA. While endogenous sodium channels have the potential to complicate analysis when using the oocyte expression system, they are only present at significant levels in rare batches of oocytes (less than 5%). Our results do stress the need, however, to reproduce results from exogenous expression studies across several batches of oocytes from different donors.     

Endogenous cellular prion protein regulates contractility of the mouse ileum

Background Cellular prion protein (PrP^C) is expressed in the enteric nervous system (ENS), however, its physiological role has not been identified. Studies suggest that PrP^C can function as a metal‐binding protein, as absence of the protein has been linked to altered copper metabolism and atypical synaptic activity. Because copper is known to modulate smooth muscle relaxation, we tested the hypothesis that PrP^C deficiency would alter intestinal contractility. Methods We examined electrically evoked ileal contractility in Prnp^?/? or wild type littermate mice and the effects of copper or copper chelation. PrP^C expression was studied in whole mount ileal preparations of mice and guinea pigs by immunohistochemistry. Key Results Relative to wild type mice, ileal tissues of Prnp^?/? mice exhibited reduced electrical field stimulation (EFS)‐evoked contractility. Furthermore, EFS‐induced relaxation, as a percentage of that induced by a nitric oxide donor, was enhanced. Addition of a copper donor to the organ bath increased, whereas the addition of a copper chelator inhibited, nitric oxide donor‐induced ileal relaxation in Prnp^?/? mice. PrP^C was expressed on nerve fibers or terminals, and some cell bodies in the myenteric and submucosal plexuses of wild type mice. PrP^C colocalized with a neuron‐specific ectonucleotidase, nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), but to only a limited extent with GFAP, a marker of enteric glia. Guinea pigs expressed PrP^C in nerve fibers or terminals and enteric glia in the myenteric and submucosal plexuses. Conclusions & Inferences Our findings suggest that PrP^C, which is abundant in the ENS, has a role in the regulation of ileal contractility.     

Hippocampal synaptic plasticity in mice devoid of cellular prion protein

The cellular prion protein plays a role in the etiology of transmissible and inherited spongiform encephalopathies. However, the physiological role of the cellular prion protein is still under debate. Results regarding the synaptic transmission using the same strain of animals where the cellular prion protein gene was ablated are controversial, and need further investigation. In this work, we have studied the hippocampal synaptic transmission in mice devoid of normal cellular prion protein, and have shown that these animals present an increased excitability in this area by the lower threshold (20 Hz) to generate long-term potentiation (LTP) in hippocampal dentate gyrus when compared to wild-type animals. The mice devoid of normal cellular prion protein are also more sensitive to the blocking effects of dizocilpine and 2-amino-5-phosphonopentanoic acid on the hippocampal long-term potentiation generation. In situ hydridization experiments demonstrated overexpression of the mRNAs for the N-methyl-D-aspartate (NMDA) receptor NR2A and NR2B subunits in mice devoid of normal cellular prion protein. Therefore, our results indicate that these animals have an increased hippocampal synaptic plasticity which can be explained by a facilitated glutamatergic transmission. The higher expression of specific N-methyl-d-aspartate receptor subunits may account for these effects.