Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.     

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.     

Zn2+对爪蟾卵母细胞表达鲫鱼脑GABA受体的调制作用

爪蟾卵母细胞注射鲫鱼脑ｍＲＮＡ后表达的ＧＡＢＡ受体中约８５％为ＧＡＢＡＡ受体。约１５％的成分为ＧＡＮＡＣ受体。本文利用双电极电压箝方法结合药物灌流研究了Ｚｎ６２＋对这两型受体的作用。我们观察到了Ｚｎ＾２＋对它们的调制都是可抑制性的,可逆的。     

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes

Opposite effects of Ni²⁺ on Xenopus and rat ENaCs expressed in Xenopus oocytes. Am J Physiol Cell Physiol 289: C946–C958, 2005. First published June 8, 2005; .—The epithelial Na⁺ channel (ENaC) is modulated by various extracellular factors, including Na⁺, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni²⁺ cation on ENaCs, we compared the Na⁺ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni²⁺, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC). The kinetics of amiloride binding were determined using noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp method, and Na⁺ conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni²⁺ on amiloride binding is not species dependent, because Ni²⁺ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na⁺. Importantly, using the TEVC method, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses. In WT xENaCs, the initial ohmic current response was stimulated by Ni²⁺, whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni²⁺ was obtained. To further study the origin of the xENaC stimulation by Ni²⁺, and based on the rationale of the well-known high affinity of Ni²⁺ for histidine residues, we designed -subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT - and -subunits. Changing His²¹⁵ to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na⁺ channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni²⁺, and neither did complete deletion of this segment. Next, we mutated His⁴¹⁶ flanked by His⁴¹¹ and Cys⁴¹⁷, a unique site for possible heavy metal ion chelation, and, with this quality, most proximal (100 amino acids upstream of the second putative amiloride binding site at the pore entrance), was found localized at M2. Replacing His⁴¹⁶ with arginine, aspartate, tyrosine, and alanine clearly affected amiloride binding in all cases, as well as Na⁺ conductance, as expressed in the xENaC current-voltage relationship, especially with regard to aspartate and tyrosine. However, similarly to those obtained with the WYRFHY stretch, none of these mutations could either abolish the stimulating effect of Ni²⁺ or reverse it to an inhibitory type. epithelia; divalent cations; amiloride; Na⁺; voltage clamp     

The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes

The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in XENOPUS: oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses.     

Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid.

To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 microM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.     

Single-channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes

The neuronal nicotinic acetylcholine receptor subunits alpha 2, alpha 3, and alpha 4 form functional receptors with the beta 2 subunit. Each of these subunit combinations shows two distinct open states (referred to as primary and secondary). The primary open states of alpha 2 beta 2, alpha 3 beta 2, and alpha 4 beta 2 receptors were 33.6 +/- 1.8 pS, 15.4 +/- 0.8 pS, and 13.3 +/- 1.5 pS, respectively. The open times of the alpha 3 beta 2 primary open state were significantly longer than the open times of the other primary conductance states. The secondary open states of alpha 2 beta 2 and alpha 3 beta 2 were 15.5 +/- 1.3 pS and 5.1 +/- 0.4 pS, respectively. Secondary open states were seen infrequently with alpha 4 beta 2. Oocytes injected with alpha 2 RNA and a 9-fold excess of beta 2 RNA showed an enhanced expression of the secondary open state.     

Differential effects of estrogen and progesterone on potassium channels expressed in Xenopus oocytes

HYPOTHESIS: Potassium (K(+)) channel activation contributes in part to estrogen-mediated vasorelaxation. However, the underlying mechanism is still unclear. We hypothesize that estrogen increases K(+) currents via membrane-associated, non-genomic interaction and that steroid hormones have differential effects on different types of K(+) channels. EXPERIMENTAL: Human large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) and human voltage-gated K(+) channels (K(V1.5)) were expressed in Xenopus oocytes, and K(+) currents elicited by voltage clamp were measured. RESULTS: Both 17beta-estradiol and BSA-conjugated 17beta-estradiol increased the BK(Ca) current in a concentration-dependent manner and this effect was abolished by tetraethylammonium ions and iberiotoxin (putative BK(Ca) channel blockers). 17beta-estradiol-stimulated increase in the BK(Ca) current was unaffected by treatment with ICI 182,780 (classic estrogen receptor antagonist), tamoxifen (estrogen receptor agonist/antagonist), actinomycin D (RNA synthesis inhibitor), or cycloheximide (protein synthesis inhibitor). In contrast, progesterone reduced the BK(Ca) current in the absence or presence of NS 1619 (BK(Ca) channel activator). Progesterone also inhibited 17beta-estradiol-stimulated increase in the BK(Ca) current. Finally, progesterone but not 17beta-estradiol reduced the K(V1.5) current. CONCLUSIONS: The present results show that 17beta-estradiol stimulates BK(Ca) channels without affecting K(V1.5) channels. This effect is ICI 182,780-insensitive and is likely mediated via a membrane-bound binding site. Progesterone inhibits both BK(Ca)- and K(V1.5)-encoded currents. The present results suggest that inhibition of K(+) channels may contribute in part to its reported antagonism against 17beta-estradiol-mediated vascular relaxation via BK(Ca) channels.     

NMDA receptors expressed in oligodendrocytes

Oligodendrocytes are known to express (Ca2+)-permeable glutamate receptors and to have low resistance to oxidative stress, two factors that make them potentially susceptible to injury. Oligodendrocyte injury is intrinsic to the loss of function experienced in conditions ranging from cerebral palsy to spinal cord injury, focal ischaemia and multiple sclerosis. NMDA receptors, a subtype of glutamate receptors, are vital to the remodeling of synaptic connections during postnatal development and associative learning abilities in adults and possibly in improvements in oligodendrocyte function. Previous studies had failed to detect NMDA receptor mRNA or current in oligodendrocytes but three new papers demonstrate NMDA receptor expression in oligodendrocytes and discuss its implications for ischaemia therapy.     

Protein kinase and phosphatase modulation of quail brain GABA(A) and non-NMDA receptors co-expressed in Xenopus oocytes

The GABA(A) receptor and the non-NMDA subtype of the ionotropic glutamate receptor were co-expressed in Xenopus oocytes by injection of quail brain mRNA. The oocytes were treated with various protein kinase (PK) and protein phosphatase (PP) activators and inhibitors and the effects on receptor functioning were monitored. Two phorbol esters, 4-beta-phorbol 12-myristate-13-acetate (PMA) and 4-beta-phorbol 12,13-dibutyrate (PDBu); the cGMP-dependent PK activators sodium nitroprusside (SNP) and S-nitrosoglutathione (SNOG); and the PP inhibitor okadaic acid (OA) reduced the amplitude of the GABA-induced currents, whilst the PK inhibitor staurosporine potentiated it. In addition, PMA, PDBu, SNP, and OA reduced the desensitization of the GABA-induced response. Identical treatments generally had similar but less pronounced effects on responses generated by kainate (KA) but the desensitization characteristic of the non-NMDA receptor was not affected. None of the treatments had any effect on the reversal potentials of the induced currents. Immunoblots revealed that the oocytes express endogenous PKG and guanylate cyclase. The results are discussed in terms of the molecular structures of GABA(A) and non-NMDA receptors and the potential functional consequences of phosphorylation/dephosphorylation.     

beta -Amyloid peptide activates alpha 7 nicotinic acetylcholine receptors expressed in Xenopus oocytes

The alpha7 nicotinic acetylcholine receptor is highly expressed in hippocampus and in cholinergic projection neurons from the basal forebrain, structures that are particularly vulnerable to the ravages of Alzheimer's disease. Previous work suggests that beta-amyloid peptide can interact with alpha7 nicotinic acetylcholine receptors, although the nature of this interaction has not been well characterized. To test whether beta-amyloid peptide can activate alpha7 nicotinic acetylcholine receptors, we expressed these receptors in Xenopus oocytes and performed two-electrode voltage clamp recordings, characterizing the response to beta-amyloid peptide 1-42 applied at concentrations ranging from 1 pm to 100 nm. In alpha7-expressing oocytes, beta-amyloid peptide 1-42 elicits inward currents at low concentrations (1-100 pm), whereas at higher concentrations (nm), less effective receptor activation is observed, indicative of receptor desensitization. Preincubation with the alpha7-selective agents, the antagonist methyllycaconatine, and the agonist 4-OH-GTS-21 blocked beta-amyloid peptide-induced receptor activation. beta-amyloid peptide 1-42 at low concentrations was able to activate the L250T mutant alpha7 receptor. The endogenous Ca(2+)-activated chloride current in Xenopus oocytes is recruited upon receptor activation since replacing Ca(2+) with Ba(2+) in the recording solution reduced current amplitude. Thus, when beta-amyloid peptide activation of alpha7 receptors occurs, these currents are comprised, at least in part, of Ca(2+).     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.     

Functional activity and regulation of human beta 2-adrenergic receptors expressed in Xenopus oocytes

The recently cloned human beta-adrenergic cDNA and several mutated forms have been expressed in Xenopus laevis oocytes by injection of RNA made from the cDNA under the control of the bacteriophage SP6 promoter. The cDNA and gene of the beta 2-adrenergic receptor possess the unusual feature of having a second upstream ATG (-101 base pairs) and a 19-codon open reading frame 5' to the initiator methionine codon of the receptor (Kobilka, B. K., Dixon, R. A. F., Frielle, T., Dohlman, H. G., Bolanowski, M., Sigal, I. S., Yang-Feng, T. L., Francke, U., Caron, M. G., and Lefkowitz, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 46-50). RNA lacking this upstream AUG and open reading frame was translated approximately 10-fold more efficiently both in an in vitro rabbit reticulocyte system and in oocytes. Injected oocytes but not water injected controls expressed typical beta 2-adrenergic receptors as assessed by ligand binding (450 fmol/mg membrane protein) and catecholamine-stimulated adenylate cyclase (approximately 20 fold). Moreover, these receptors displayed typical agonist-induced homologous desensitization when oocytes were incubated with isoproterenol at room temperature for 3-24 h. Among a series of mutations, truncations of the membrane-anchored core of the receptor eliminated receptor binding and cyclase stimulating activity. In contrast, disruption of one of the cAMP-dependent protein kinase phosphorylation sites or removal of the serine/threonine-rich carboxyl terminus had little or no effect on these functions or on the extent of agonist-induced desensitization relative to that observed with native receptor. These studies validate the beta 2-adrenergic nature of the cloned human beta-adrenergic cDNA, document the utility of the Xenopus oocyte system for studying functional and regulatory properties of receptors coupled to adenylate cyclase, and suggest the possibility that elements in the 5' untranslated region of the beta 2-adrenergic receptor RNA may regulate its translation in vivo.     

D1及α2受体激动剂对卵母细胞表达的GABAA受体的抑制

目的：研究SKF38393及Clonidine对表达的DRG神经元GABAA受体的调制作用,并与新鲜分离细胞相比较。方法：实施在注射鼠DRG神经元mRNA的卵母细胞上进行,以比电极电压钳技术进行研究。结果：①SKF38393及Clonidine对表达的DRG神经元GABAA受体有明显的抑制作用,其作用呈浓度依赖性。②SKF38393及Clonidine的诱导电流之间有相互抑制作用。③SKF38393及Clonidine对GABAA受体的抑制作用是非竞争性抑制,并且为非电压依赖性。结论：实验结果提示SKF38393及Clonidine可通过胞内转导,由第二信使介导GABAA受体的磷酸化而抑制GABAA诱导电流。     

Neurotensin and substance P receptors expressed in Xenopus oocytes by messenger RNA from rat brain

Xenopus oocytes were induced to acquire sensitivity to neurotensin and substance P, by injecting them with a fraction of poly(A)+ mRNA from rat brain. Non-injected oocytes, and oocytes injected with other brain mRNAs, failed to show responses, suggesting that receptors to these peptides were expressed by specific brain mRNAs. Responses to substance P and neurotensin comprised an oscillatory chloride current, and a smooth current having different ionic basis. These currents resembled those seen during activation of muscarinic and serotonergic receptors, but were not blocked by the corresponding antagonists atropine and methysergide. The responses to substance P, and to a lesser extent to neurotensin, showed a long-lasting desensitization. Similarities between the oscillatory currents evoked by the peptides acetylcholine and serotonin suggest that all these receptors may 'link in' to a common intracellular messenger pathway.     

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)     

Characteristics of P2X7 receptors from human B lymphocytes expressed in Xenopus oocytes

Human B lymphocytes express an ATP-gated ion channel (P2Z receptor), which shares similarities with the recently identified P2X7 receptor. Using gene specific primers, we have now isolated P2X7 cDNA from the total RNA of human B lymphocytes. This hP2X7 receptor subtype was expressed in Xenopus oocytes and electrophysiologically characterized. The hP2X7 receptor is similar to, but does not completely match, P2Z of human B cells. The hP2X7 receptors resemble the P2Z receptors with regard to the ATP concentration of half maximal activation, reproducibility, permeation characteristics and lack of desensitization of the ATP-evoked currents. However, in contrast to the native lymphocytic P2Z receptor, the time course of activation of hP2X7 displayed an additional linearly increasing current component. Furthermore, a second, small and slowly deactivating current component exists only in hP2X7 expressed in oocytes. The activation and deactivation kinetics as well as permeation characteristics of hP2X7 are different from rat P2X7 recently expressed in oocytes. Unlike in mammalian cells, hP2X7 expressed in Xenopus oocytes is not sufficient to induce large non-selective pores.     

NMDA and non-NMDA receptors mediate responses in the primary gustatory nucleus in goldfish

Primary gustatory afferents from the oropharynx of the goldfish, Carassius auratus, terminate in the vagal lobe, a laminated structure in the dorsal medulla comparable to the gustatory portion of the nucleus of the solitary tract in mammals. We utilized an in vitro brain slice preparation to test the role of different ionotropic glutamate receptor subtypes in synaptic transmission of gustatory information by recording changes in field potentials after application of various glutamate receptor antagonists. Electrical stimulation of the vagus nerve (NX) evokes two short-latency postsynaptic field potentials from sensory layers of the vagal lobe. 6,7-Dinitroquinoxaline-2,3-dione and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, two non-N-methyl-D-aspartate (NMDA) ionotropic receptor antagonists, blocked these short-latency potentials. Slower potentials that were revealed under Mg2+ -free conditions, were abolished by the NMDA receptor antagonist, D(-)-2-amino-5-phosphonovaleric acid (APV). Repetitive stimulation produced short-term facilitation, which was attenuated by application of APV. These results indicate that the synaptic responses in the vagal lobe produced by stimulation of the gustatory roots of the NX involve both NMDA and non-NMDA receptors. An NMDA receptor-mediated facilitation may serve to amplify incoming bursts of primary afferent activity.