Potentiation and Inhibition of Ionotropic Neurotransmitter Receptors Expressed in Xenopus Oocyte by Linoleic Acid and Its Hydroperoxide

Abstract: To study the effects of lipid hydroperoxide on ionotropic neurotransmitter receptors, γ-aminobutyric acid (GABA), N -methyl- d -aspartate (NMDA), and non-NMDA receptors (GABARs, NMDARs, and non-NMDARs, respectively) were expressed in Xenopus oocytes that received an injection of mRNA prepared from rat whole brain. Linoleic acid (LA) and its hydroperoxide 13- l -hydroperoxylinoleic acid (LOOH) prepared with soybean lipoxygenase inhibited the response of GABARs in the presence of GABA at high concentrations. The inhibition was stronger when the inhibitors were perfused 1 min before a mixture of GABA and the inhibitors than when they were perfused simultaneously with GABA. On the other hand, only LOOH potentiated the response of GABARs in the presence of GABA at low concentrations, possibly increasing the affinity of GABA to the receptors. Both LA and LOOH accelerated the rate of desensitization of GABARs, but LOOH did not affect their equilibrium between the active and desensitized form of the receptors. They also inhibited the response of NMDARs in a noncompetitive manner but barely inhibited the response of non-NMDARs in the presence of kainate at various concentrations. These results suggest the possibility that production of lipid hydroperoxide modulates the neural transmission in the brain, especially through GABARs.     

Receptor-mediated regulation of IsK, a very slowly activating, voltage-dependent K+ channel in Xenopus oocytes.

Expression of IsK in Xenopus oocytes has been obtained in 2 ways: (i) by injection of cardiac polyA+ RNA from neonatal mouse heart; (ii) by injection of a cRNA synthesized in vitro. It was observed that polyA+ RNA not only directs the expression of the IsK channel but also contains purinergic P2 and endothelin receptors. Stimulation of these receptors, that produce intracellular Ca2+ increase together with diacylglycerol production activating protein kinase C, increases IsK activity. The same type of results and the same conclusions were obtained by co-injecting cRNA's corresponding to the 5-HT2 receptor and the IsK channel into oocytes. This stimulatory effect was shown to be due to Ca2+ via a calmodulin-dependent kinase process. Conversely, activation of protein kinase C pathway alone by phorbol esters leads to inhibition of IsK activity.     

Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.

Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.     

Li+ uptake into Xenopus and Cynops oocytes injected with exogenous mRNA, observed by flame emission spectroscopy

Li+ uptake into Xenopus oocytes was measured by flame emission spectroscopy. Li+ uptake into the oocytes increased proportionally with incubation time and was dependent on either pH or temperature. Maximum uptake of Li+ was observed around pH 7. Li+ uptake into Xenopus oocytes increased by a factor of roughly 7 over the range 4-30 degrees C. When mRNA prepared from electroplax of Electrophorus electricus was injected into Xenopus or Cynops oocytes, Li+ uptake into the injected oocytes increased by the addition of carbamylcholine (Carb), an agonist of the acetylcholine receptor (AChR). This increase of Li+ uptake by Carb was inhibited by d-tubocurarine, an antagonist of nicotinic AChR. Thus, a new method was established for detection of the activity of nicotinic AChR synthesized in oocytes injected with exogenous mRNA.     

On the orientation of foreign neurotransmitter receptors in Xenopus oocytes

Xenopus oocytes can be made to incorporate into their membrane foreign neurotransmitter receptors and voltage-activated sodium channels. In their original location the receptors are normally activated by the extracellular action of transmitter substances. Tests were made to see if some of the newly synthesized foreign receptors were inserted in the oocyte membrane with their active site facing inwards. Since intracellular injections of acetylcholine, gamma-aminobutyric acid, serotonin and kainic acid and tetrodotoxin into the oocyte failed to elicit a response, we conclude that very few, or none, of the receptor molecules expressed in the oocyte by the exogenous mRNA are inserted with the wrong orientation in the membrane.     

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Ca2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Ca2+o removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage-sensitive calcium channels. When oocytes were challenged with ACh in Ca2(+)-free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Ca2+o-dependent component of membrane electrical responses, the Ca2(+)-evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage-sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2(+)-evoked current, implying negative feedback of calcium channels by calcium. Injection of 10-100 fmol of inositol 1,4,5-trisphosphate (IP3) resulted in a two-component depolarizing current. IP3 injection promoted the appearance of Ca2+o-evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell-membrane muscarinic receptors causes opening of apparently voltage-insensitive and verapamil or diltiazem-resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor-operated channels described in other model systems.     

Progesterone treatment abolishes exogenously expressed ionic currents in Xenopus oocytes

Fully grown oocytes of Xenopus laevis undergo resumption of the meiotic cycle when treated with the steroid hormone progesterone. Previous studies have shown that meiotic maturation results in profound downregulation of specific endogenous membrane proteins in oocytes. To determine whether the maturation impacts the functional properties of exogenously expressed membrane proteins, we used cut-open recordings from Xenopus oocytes expressing several types of Na(+) and K(+) channels. Treatment of oocytes with progesterone resulted in a downregulation of heterologously expressed Na(+) and K(+) channels without a change in the kinetics of the currents. The time course of progesterone-induced ion channel inhibition was concentration dependent. Complete elimination of Na(+) currents temporally coincided with development of germinal vesicle breakdown, while elimination of K(+) currents was delayed by approximately 2 h. Coexpression of human beta(1)-subunit with rat skeletal muscle alpha-subunit in Xenopus oocytes did not prevent progesterone-induced downregulation of Na(+) channels. Addition of 8-bromo-cAMP to oocytes or injection of heparin before progesterone treatment prevented the loss of expressed currents. Pharmacological studies suggest that the inhibitory effects of progesterone on expressed Na(+) and K(+) channels occur downstream of the activation of cdc2 kinase. The loss of channels is correlated with a reduction in Na(+) channel immunofluorescence, pointing to a disappearance of the ion channel-forming proteins from the surface membrane.     

mRNA-induced expression of the cardiac Na+-Ca2+ exchanger in Xenopus oocytes

Xenopus oocytes were injected with total mRNA isolated from hearts of 1-day-old chicks. After 5 days of incubation the follicular cell layers were removed and the oocytes were loaded with Na+ by incubation in hypertonic EGTA solution at 37 degrees C. The Na+-loaded oocytes accumulated 45Ca2+ from a Na+-free medium at a 3-18-fold higher rate than noninjected oocytes or oocytes injected with control solution containing no mRNA. Oocytes not subjected to the Na+-loading procedure showed no mRNA-dependent 45Ca2+ uptake. Size fractionation of the mRNA using sucrose density gradient centrifugation under denaturing conditions led to the identification of a 25 S fraction competent for induction of the Na+-Ca2+ exchange system.     

Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes.

Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.     

Minimal model analyzing response of glycine receptors expressed in Xenopus oocyte: inhibition by a lipid hydroperoxide.

Glycine receptor (GlyR) was expressed in Xenopus oocytes by injecting rat brain mRNA. Glycine (Gly)-elicited responses in the oocyte were measured by the voltage-clamping method. The following measurements were made to establish the relationship between Gly concentration and the current: 1) Gly-induced membrane current before desensitization, 2) Gly-induced membrane current after desensitization equilibrium, 3) fraction of the active form of the receptor after desensitization equilibrium, 4) rate of recovery of the desensitized receptors upon removal of Gly. These results were analyzed on the basis of the minimal model proposed for nicotinic acetylcholine and gamma-aminobutyric acid A receptor. The equilibrium and rate constants of the model were evaluated for GlyR. The effects of procaine and 13-L-hydroperoxylinoleic acid (LOOH) on GlyR were examined electrophysiologically. LOOH noncompetitively inhibited the receptor with the inhibition constant of 27 microM, while 1 mM procaine, a local anesthetic, did not inhibit GlyR at all.     

Extracts from plants used in Mexican traditional medicine activate Ca(2+)-dependent chloride channels in Xenopus laevis oocytes.

The two-electrode voltage-clamp technique was employed to investigate the effects of chloroform-methanol (1:1) extracts derived from five medicinal plants on Xenopus laevis oocytes. When evaluated at concentrations of 1 to 500 microg/ml, the extracts prepared from the aerial parts of Baccharis heterophylla H.B.K (Asteraceae), Chenopodium murale L. (Chenopodiaceae), Desmodium grahami Gray (Leguminosae) and Solanum rostratum Dun (Solanaceae) produced concentration-dependent oscillatory inward currents in the oocytes, while the extract of Gentiana spathacea did not induce any response. The reversal potential of the currents elicited by the active extracts was -17 +/- 2 mV and was similar to the chloride equilibrium potential in oocytes. These ionic responses were independent of extracellular calcium. However, they were eliminated by overnight incubation with BAPTA-AM (10 microM), suggesting that the currents were dependent on intracellular Ca2+ increase. Thus the plant extracts activate the typical oscillatory Ca(2+)-dependent Cl- currents generated in the Xenopus oocyte membrane more probably via a mechanism that involves release of Ca2+ from intracellular reservoirs. These observations suggest that Xenopus oocyte electrophysiological recording constitutes a suitable assay for the study of the mechanisms of action of herbal medicines.     

Properties of human brain glycine receptors expressed in Xenopus oocytes

Glycine and gamma-aminobutyric acid (GABA) receptors from the foetal human brain were 'transplanted' into the Xenopus oocyte membrane by injecting the oocytes with poly(A)+-mRNA extracted from the cerebral cortex. Activation of both glycine and GABA receptors induced membrane currents carried largely by chloride ions. However, unlike the GABA-activated current, the glycine current was blocked by strychnine, and was not potentiated by barbiturate. At low doses, the glycine current increased with concentration following a 2.7th power relation, suggesting that binding of three molecules of glycine may be required to open a single membrane channel. The current induced by steady application of glycine decreased with hyperpolarization beyond about -60 mV.     

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.     

Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.     

A delayed rectifier potassium current in Xenopus oocytes.

A delayed voltage-dependent K+ current endogenous to Xenopus oocytes has been investigated by the voltage-clamp technique. Both activation and inactivation of the K+ current are voltage-dependent processes. The K+ currents were activated when membrane potential was depolarized from a holding potential of -90 to -50 mV. The peak current was reached within 150 ms at membrane potential of +30 mV. Voltage-dependent inactivation of the current was observed by depolarizing the membrane potential from -50 to 0 mV at 10-mV increments. Voltage-dependent inactivation was a slow process with a time constant of 16.5 s at -10 mV. Removal of Ca2+ from the bath has no effect on current amplitudes, which indicates that the current is Ca2+)-insensitive. Tail current analysis showed that reversal potentials were shifted by changing external K+ concentration, as would be expected for a K(+)-selective channel. The current was sensitive to quinine, a K+ channel blocker, with a Ki of 35 microM. The blockade of quinine is voltage-independent in the range of -20 to +60 mV. Whereas oocytes from the same animal have a relatively homogeneous current distribution, average amplitude of the K+ current varied among oocytes from different animals from 30 to 400 nA at membrane potential of +30 mV. Our results indicate the presence of the endogenous K+ current in Xenopus oocytes with characteristics of the delayed rectifier found in some nerve and muscle cells.     

Responses to GABA, glycine and beta-alanine induced in Xenopus oocytes by messenger RNA from chick and rat brain

Poly (A)+ messenger RNA (mRNA) was extracted from rat and chick brains, and injected into oocytes of Xenopus laevis. This led to the expression of receptors that evoked membrane currents in response to gamma-aminobutyric acid (GABA), glycine and beta-alanine. These currents all inverted at about the chloride equilibrium potential in the oocyte, and showed a marked rectification at negative potentials. Oocytes injected with mRNA from chick optic lobe gave large responses to GABA and beta-alanine, but small responses to glycine. In contrast, one fraction of mRNA from rat cerebral cortex (obtained by sucrose density gradient centrifugation) caused oocytes to develop sensitivity to GABA, glycine and beta-alanine, but very little to GABA. The pharmacological properties of the three amino acid responses also differed. Barbiturate and benzodiazepines potentiated the responses to GABA and beta-alanine, but not to glycine. Strychnine reduced the responses to glycine and beta-alanine, but not to GABA, whereas bicuculline reduced the responses to GABA and beta-alanine, but not to glycine. We conclude that different species of mRNA code for receptors to GABA and glycine, and possibly also for separate beta-alanine receptors.     

Functional reconstitution of fMet-Leu-Phe receptor in Xenopus laevis oocytes

fMet-Leu-Phe (fMLP) receptors were functionally reconstituted into Xenopus laevis oocytes by microinjection with RNA isolated from promyelocytic leukemia cells (HL-60) differentiated with 750 microM N6, O2-dibutyryl cyclic adenosine 3',5'-monophosphate. fMLP-induced Ca2+ mobilization was monitored by measurement of photon emission elicited by aequorin coinjected with RNA into albino X. laevis oocytes. Maximal expression of the fMLP receptor was achieved 48 h after microinjection of RNA. Dose-response experiments revealed a K0.5 of 9.5 nM fMLP which is in good agreement with the dissociation constants of the fMLP receptor complex in human neutrophils. Furthermore, the fMLP-induced Ca2+ mobilization in Xenopus oocytes was blocked by the fMLP receptor inhibitor t-butoxycarbonyl-Met-Leu-Phe. Size fractionation of the RNA and microinjection of the individual fractions indicated that messenger RNA for the fMLP receptor is between 1.5 and 2.0 kilobases. Reconstitution of the fMLP receptor into Xenopus oocytes can be employed to isolate the cDNA encoding the fMLP receptor as well as to study the regulation of the fMLP receptor in a functional system.     

Some properties of IgG against diphtheria toxin synthesized in Xenopus oocytes containing mRNA from hybridoma

When a mixture of mRNA from hybridoma producing IgG, anti-diphtheria toxin antibody, and mRNA from rat liver was injected into Xenopus oocytes, most of the IgG synthesized in the oocytes was not secreted into the medium and remained in the rough endoplasmic reticulum fraction. In contrast, rat serum albumin was rapidly secreted. The glycosylation of IgG in the oocytes was of a high-mannose type, while that of IgG secreted very slowly into the medium was of a complex type. The IgG in the membrane fraction and in the medium could both bind to diphtheria toxin.