Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochromocytoma cells.

Serum contains a factor that co-purifies with albumin and causes neurite retraction in PC12 cells, inhibits the proliferation of tumor cells in vitro, and activates the phosphatidylinositol/Ca2+ second messenger system in Xenopus oocytes and other cells. The activity of serum albumin depends on several lysophospholipids bound to albumin. Thin layer chromatographic analysis of the lipids extracted by methanol from serum albumin revealed over a dozen components, several of which evoked oscillatory currents in oocytes. In contrast to serum albumin, most of these lipids were absent in plasma, which lacks the biological activity. The most abundant naturally occurring active component was identified as stearoyl-lysophosphatidic acid. Synthetically prepared lysophosphatidates reproduced the biological activities of the natural serum factor. Adding synthetic lysophosphatidates to inactive fatty acid-free albumin restored activity to the albumin, making the active factor nondialyzable against aqueous solvents and protecting against digestion by various lipases. Since the biologically active lysophosphatidates were produced during blood clotting, in the presence of platelets, and lysophosphatidates have been shown previously to activate platelets, we propose that lysophosphatidates may play an important role in linking platelet activation to receptor-mediated tissue regeneration.     

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.     

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.     

Sera from amyotrophic lateral sclerosis patients induce the non-canonical activation of NMDA receptors "in vitro"

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the selective loss of both upper and lower motoneurons (MNs). The familial form of the illness is associated with mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD-1) enzyme, but it accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form of ALS. Although many proposals have been suggested over the years, the mechanisms underlying the characteristic selective killing of MN in ALS remain unknown. In this study we tested the effect of sera from sporadic ALS patients on NMDA receptors (NMDAR). We hypothesize that an endogenous seric factor is implicated in neuronal death in ALS, mediated by the modulation of NMDAR.Sera from ALS patients and from healthy subjects were pretreated to inactivate complement pathways and dialyzed to remove glutamate and glycine. IgGs from ALS patients and healthy subjects were obtained by affinity chromatography and dialyzed against phosphate-buffered saline. Human NMDAR were expressed in Xenopus laevis oocytes, and ionic currents were recorded using the two-electrode voltage clamp technique.Sera from sporadic ALS patients induced transient oscillatory currents in oocytes expressing NMDAR with a significantly higher total electrical charge than that induced by sera from healthy subjects. Sera from patients with other neuromuscular diseases did not exert this effect. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. The PLC inhibitor, U-73122, and the IP₃ receptor antagonist, 2-APB, also inhibited the sera-induced currents. The oscillatory signal recorded was due to internal calcium mobilization. Isolated IgGs from ALS patients significantly affected the activity of oocytes injected with NMDAR, causing a 2-fold increase over the response recorded for IgGs from healthy subjects.Our data support the notion that ALS sera contain soluble factors that mobilize intracellular calcium, not opening directly the ionic conductance, but through the non-canonical activation of NMDAR.     

Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).

To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca(2+) dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the G(i)- or G(q)-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.     

Hypotonicity activates a native chloride current in Xenopus oocytes

Xenopus oocytes are frequently utilized for in vivo expression of cellular proteins, especially ion channel proteins. A thorough understanding of the endogenous conductances and their regulation is paramount for proper characterization of expressed channel proteins. Here we detail a novel chloride current (ICl.swell) responsive to hypotonicity in Xenopus oocytes using the two-electrode voltage clamp technique. Reducing the extracellular osmolarity by 50% elicited a calcium-independent chloride current having an anion conductivity sequence identical with swelling-induced chloride currents observed in epithelial cells. The hypotonicity-activated current was blocked by chloride channel blockers, trivalent lanthanides, and nucleotides. G- protein, cAMP-PKA, and arachidonic acid signaling cascades were not involved in ICl.swell activation. ICl.swell is distinct from both stretch-activated nonselective cation channels and the calcium- activated chloride current in oocytes and may play a critical role in volume regulation in Xenopus oocytes.     

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.     

Two novel Xenopus homologs of mammalian LP(A1)/EDG-2 function as lysophosphatidic acid receptors in Xenopus oocytes and mammalian cells

Lysophosphatidic acid (LPA) induces diverse biological responses in many types of cells and tissues by activating its specific G protein-coupled receptors (GPCRs). Previously, three cognate LPA GPCRs (LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7) were identified in mammals. By contrast, an unrelated GPCR, PSP24, was reported to be a high affinity LPA receptor in Xenopus laevis oocytes, raising the possibility that Xenopus uses a very different form of LPA signaling. Toward addressing this issue, we report two novel Xenopus genes, xlp(A1)-1 and xlp(A1)-2, encoding LP(A1) homologs (approximately 90% amino acid sequence identity with mammalian LP(A1)). Both xlp(A1)-1 and xlp(A1)-2 are expressed in oocytes and the nervous system. Overexpression of either gene in oocytes potentiated LPA-induced oscillatory chloride ion currents through a pertussis toxin-insensitive pathway. Injection of antisense oligonucleotides designed to inhibit xlp(A1)-1 and xlp(A1)-2 expression in oocytes eliminated their endogenous response to LPA. Furthermore, retrovirus-mediated heterologous expression of xlp(A1)-1 or xlp(A1)-2 in B103 rat neuroblastoma cells that are unresponsive to LPA conferred LPA-induced cell rounding and adenylyl cyclase inhibition. These results indicate that XLP(A1)-1 and XLP(A1)-2 are functional Xenopus LPA receptors and demonstrate the evolutionary conservation of LPA signaling over a range of vertebrate phylogeny.     

Induction of membrane chloride currents in Xenopus laevis oocytes by the sulfonyl amide sweeteners acesulfame K and saccharin

Sweet receptors have remained elusive. In Xenopus oocytes sulfonyl amide sweeteners but not sweet compounds belonging to other chemical classes dose dependently induced membrane chloride currents via the inositol trisphosphate/calcium pathway. Induction of membrane currents was exclusively observed following extracellular application of sulfonyl amides but not by intracellular pressure injection, suggesting the involvement of a plasma membrane receptor. The presence of this receptor in oocytes and the observed seasonal variation of the sweet response offers an opportunity for a molecular cloning approach.     

Activation of a common effector system by different brain neurotransmitter receptors in Xenopus oocytes

Xenopus oocytes possess 'native' muscarinic receptors, which give rise to oscillatory chloride currents; similar responses are elicited by activation of foreign receptors to serotonin, glutamate and noradrenaline, expressed in oocytes after injection of messenger RNA from rat brain. When low concentrations of two agonists are applied together, the combined response is greater than would be expected from the sum of the responses to each agonist applied alone. Potentiation of acetylcholine by serotonin is blocked by the serotonin antagonist methysergide; conversely, the potentiation of serotonin by acetylcholine is blocked by the muscarinic antagonist atropine. This indicates that each agonist acts on a distinct receptor. The interactions between serotonin, acetylcholine and other agonists provide further evidence that the different receptors may all 'link in' to a common receptor-channel coupling system, in which phosphoinositide metabolism and calcium liberation lead to the opening of chloride channels in the oocyte membrane.     

Changes in intracellular calcium and in membrane currents evoked by injection of inositol trisphosphate into Xenopus oocytes

Intracellular calcium was monitored by the use of aequorin in voltage-clamped oocytes of Xenopus laevis. Injection of inositol trisphosphate (IP3) into oocytes elicited slowly rising and decaying aequorin/calcium signals and produced oscillatory chloride membrane currents. These responses did not depend upon extracellular calcium, since they could be elicited in calcium-free solution and after addition of cobalt or lanthanum to block calcium channels in the surface membrane. We conclude that IP3 causes the release of calcium from intracellular stores in the oocyte. Injections of calcium gave aequorin and membrane current responses that were more transient than those seen with IP3.     

Expression of Olfactory Receptors in Xenopus Oocytes

The rat olfactory epithelium and the amino acid-sensitive catfish olfactory system have been used as models to study the molecular mechanisms of olfactory transduction. Here we report the functional expression of rat and catfish olfactory receptors in Xenopus oocytes injected with mRNA isolated from the respective tissues. Application of odor ligands to injected oocytes, monitored by two-electrode voltage clamp, activates stimulus-dependent transmembrane currents that reverse direction at about the chloride equilibrium potential. The currents show characteristic secondary oscillations that are presumed to reflect underlying Ca2+ oscillations. Similar ligand-activated membrane currents induced in oocytes after injection of other mRNAs have been shown to be due to activation of endogenous Ca(2+)-activated chloride channels. In summary, our results demonstrate the usefulness of the Xenopus oocyte expression system for cloning and characterization of olfactory receptors in both fish and mammalian species.     

Functional and Structural Effects of Amyloid-β Aggregate on Xenopus laevis Oocytes

Xenopus laevis oocytes exposed to amyloid-β aggregate generated oscillatory electric activity (blips) that was recorded by two-microelectrode voltage-clamp. The cells exhibited a series of “spontaneous” blips ranging in amplitude from 3.8 ± 0.9 nA at the beginning of the recordings to 6.8 ± 1.7 nA after 15 min of exposure to 1 μM aggregate. These blips were similar in amplitude to those induced by the channel-forming antimicrobial agents amphotericin B (7.8 ± 1.2 nA) and gramicidin (6.3 ± 1.1 nA). The amyloid aggregate-induced currents were abolished when extracellular Ca²⁺ was removed from the bathing solution, suggesting a central role for this cation in generating the spontaneous electric activity. The amyloid aggregate also affected the Ca²⁺-dependent Cl⁻ currents of oocytes, as shown by increased amplitude of the transient-outward chloride current (T_out) and the serum-activated, oscillatory Cl⁻ currents. Electron microcopy revealed that amyloid aggregate induced the dissociation of the follicular cells that surround the oocyte, thus leading to a failure in the electro-chemical communication between these cells. This was also evidenced by the suppression of the oscillatory Ca²⁺-dependent ATP-currents, which require proper coupling between oocytes and the follicular cell layer. These observations, made using the X. laevis oocytes as a versatile experimental model, may help to understand the effects of amyloid aggregate on cellular communication.     

非洲爪蟾血清白蛋白的分离纯化及胰蛋白酶抑制活性

通过凝胶过滤层析及两步阴离子交换层析,从非洲爪蟾(Xenopus laevis)的血清中获得了其68kDa的血清白蛋白。与大蹼铃蟾血清白蛋白相似,非洲爪蟾血清白蛋白也具有抑制胰蛋白酶的活性,但其抑制活力相对较低,180nmol/L的非洲爪蟾血清白蛋白能抑制84%的胰蛋白酶活性(30nmol/L)。经表面等离子共振法获得了其与胰蛋白酶的结合动力学常数,解离平衡常数KD=1.44×10-6mol/L。经Western blot分析发现,非洲爪蟾的皮肤中也分布有血清白蛋白。推测两栖类动物血清白蛋白具有的胰蛋白酶抑制活性可能是其抵御天敌捕食的一种防御措施。     

Effects of ethanol on three endogenous membrane conductances present in Xenopus laevis oocytes

The Xenopus oocyte expression and recording system has allowed a detailed analysis of the physiology and pharmacology of neuronal ion channels including their sensitivity to ethanol. It is important however, to ascertain the effects of a particular drug on the channels inherently expressed by oocytes to ensure that drug effects ascribed to the expressed recombinant receptors are manifested solely through those receptors. In this study, the effects of ethanol were determined on three endogenous currents that can be elicited in oocytes and other cells by various manipulations. The inward cation current, IC, was activated by perfusing naive oocytes with a divalent-free recording solution. Ethanol (25-100 mM) modestly inhibited IC with 100 mM ethanol producing a 7-8% inhibition of steady state currents. The store-operated or capacitative calcium current (I(SOC)) was activated in thapsigargin-treated oocytes by switching from a calcium-free solution to one containing 10 mM calcium. In thapsigargin-treated oocytes also injected with EGTA to block calcium-activated chloride currents, ethanol (100 mM) had no effect on the store-operated calcium current. In contrast, ethanol (10-100 mM) dose-dependently inhibited the calcium-dependent chloride current (I(Cl(Ca)) in thapsigargin-treated oocytes. A voltage-jump protocol was used to separate the two components of I(Cl(Ca)), I(Cl-1) and I(Cl-2). Under these conditions, ethanol (100 mM) inhibited I(Cl-1) currents to a greater extent (38%) than it did I(Cl-2) currents (14%). These results show that Xenopus oocytes express endogenous ion channels that are differentially sensitive to ethanol.     

Regulation of human cystic fibrosis transmembrane conductance regulator (CFTR) by serum- and glucocorticoid-inducible kinase (SGK1).

BACKGROUND: Serum- and glucocorticoid-inducible kinase-1 (SGK1) increases CFTR Cl currents in Xenopus oocytes by an unknown mechanism. Because SGK increases the plasma membrane expression of other ion channels, the goal of this paper was to test the hypothesis that SGK1 stimulates CFTR Cl currents by increasing the number of CFTR Cl channels in the plasma membrane. METHODS: CFTR Cl currents were measured in Xenopus oocytes by the two-electrode voltage clamp technique, and CFTR in the plasma membrane was determined by laser scanning confocal microscopy. RESULTS: wt-SGK1 stimulated CFTR Cl currents by 42% and increased the amount of CFTR in the plasma membrane by 35%. A kinase-dead SGK mutant (K127N) had a dominant-negative effect on CFTR, reducing CFTR Cl currents by 38%. In addition, deletion of the C-terminal PDZ-interacting motif (SGK1-DeltaSFL) increased CFTR Cl currents by 108%. Thus, SGK1-DeltaSFL was more effective than wt-SGK1 in stimulating CFTR Cl currents. Neither wt-SGK nor the K127N mutant had any effect on Cl currents in oocytes when expressed alone in the absence of CFTR. CONCLUSION: SGK1 stimulates CFTR Cl currents in Xenopus oocytes by increasing the number of channels in the plasma membrane. Moreover, the effect of SGK may be mediated by protein-protein interactions involving the PDZ interacting motif.     

Biosynthesis of the neurofilament heavy subunit in Xenopus oocytes microinjected with rat brain poly(A)+ RNA

In order to study the expression of the major subunit of neurofilaments (NFs), rat brain poly(A)⁺ RNA was purified by three different procedures and was injected in Xenopus laevis oocytes. This system was able to translate efficiently the 200 kDa NF subunit as shown by a dot-blot immunoassay and by immunoprecipitation of labeled NF polypeptides.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethanesulphonyl fluoride - PBS phosphate buffered saline - BSA bovine serum albumin