Nitrogen-Fixing Nodules Are an Important Source of Reduced Sulfur, Which Triggers Global Changes in Sulfur Metabolism in Lotus japonicus.

We combined transcriptomic and biochemical approaches to study rhizobial and plant sulfur (S) metabolism in nitrogen (N) fixing nodules (Fix(+)) of Lotus japonicus, as well as the link of S-metabolism to symbiotic nitrogen fixation and the effect of nodules on whole-plant S-partitioning and metabolism. Our data reveal that N-fixing nodules are thiol-rich organs. Their high adenosine 5'-phosphosulfate reductase activity and strong (35)S-flux into cysteine and its metabolites, in combination with the transcriptional upregulation of several rhizobial and plant genes involved in S-assimilation, highlight the function of nodules as an important site of S-assimilation. The higher thiol content observed in nonsymbiotic organs of N-fixing plants in comparison to uninoculated plants could not be attributed to local biosynthesis, indicating that nodules are an important source of reduced S for the plant, which triggers whole-plant reprogramming of S-metabolism. Enhanced thiol biosynthesis in nodules and their impact on the whole-plant S-economy are dampened in plants nodulated by Fix(-) mutant rhizobia, which in most respects metabolically resemble uninoculated plants, indicating a strong interdependency between N-fixation and S-assimilation.

High dietary fat and the development of osteoarthritis in a rabbit model.

OBJECTIVE: Osteoarthritis (OA) is associated with obesity, although this relationship remains unclear. Proposed etiologies of OA in obesity include mechanical loading of malaligned joints and possible toxicity of dietary fat. The hypothesis tested in the present study was that increased dietary fat worsens OA in both malaligned and normal joints, detected by biochemical and histological cartilage markers. METHOD: 83 New Zealand white rabbits were divided among two conditions related to OA: bowing of the knee and a 14%kcal vs 47.8%kcal fat diet. Rabbit weights and knee angles were compared throughout the experiment. At 28 and 38 weeks, intra-articular forces were measured, animals sacrificed, and knee cartilage examined for histological changes, glycosaminoglycan content, 35S uptake, and aggrecanase-1 expression. RESULTS: There were no differences in animal weights or intra-articular forces between the two diets. Despite increased fat content in their diet, animals on the 47.8%kcal fat diet did not gain excess weight. Representative histology showed atypical shearing of articular cartilage among animals on the high fat diet. Animals on the 47.8%kcal fat diet had suppression of protein synthesis compared to the 14%kcal fat diet: lower glycosaminoglycan content and aggrecanase-1 expression in all knee compartments at both times, and lower 35S uptake at 38 weeks. CONCLUSION: These results suggest dietary fat, independent of animal weight, results in altered chondrocyte function. Increased dietary fat was associated with changes in rabbit cartilage in vivo and appears to be a risk factor for the development of OA.

Homocysteine is transported by the microvillous plasma membrane of human placenta.

Elevated maternal plasma concentrations of homocysteine (Hcy) are associated with pregnancy complications and adverse neonatal outcomes. The postulate that we wish to advance here is that placental transport of Hcy, by competing with endogenous amino acids for transporter activity, may account for some of the damaging impacts of Hcy on placental metabolism and function as well as fetal development. In this article, we provide an overview of some recent studies characterising the transport mechanisms for Hcy across the microvillous plasma membrane (MVM) of the syncytiotrophoblast, the transporting epithelium of human placenta. Three Hcy transport systems have been identified, systems L, A and y(+)L. This was accomplished using a strategy of competitive inhibition to investigate the effects of Hcy on the uptake of well-characterised radiolabelled substrates for each transport system into isolated MVM vesicles. The reverse experiments were also performed, examining the effects of model substrates on [³5S]L-Hcy uptake. This article describes the evidence for systems L, A and y(+)L involvement in placental Hcy transport and discusses the physiological implications of these findings with respect to placental function and fetal development.

Circulating prouroguanylin is processed to its active natriuretic form exclusively within the renal tubules.

The intestine and kidney are linked by a mechanism that increases salt excretion in response to salt intake. The peptide uroguanylin (UGn) is thought to mediate this signaling axis. Therefore, it was surprising to find (as reported in a companion publication) that UGn is stored in the intestine and circulates in the plasma almost exclusively in the form of its biologically inactive propeptide precursor, prouroguanylin (proUGn), and, furthermore, that infused proUGn leads to natriuretic activity. Here, we investigate the fate of circulating proUGn. Kinetic studies show rapid renal clearance of radiolabeled propeptide. Radiolabel accumulates at high specific activity in kidney (relative to other organs) and urine (relative to plasma). The principal metabolites found in kidney homogenates are free cysteine and methionine. In contrast, urine contains cysteine, methionine, and three other radioactive peaks, one comigrating with authentic rat UGn15. Interestingly, proUGn is not converted to these or other metabolites in plasma, indicating that circulating proUGn is not processed before entering the kidney. Therefore, our findings suggest that proUGn is the true endocrine agent released in response to salt intake and that the response of the kidney is dependent on conversion of the propeptide to an active form after it reaches the renal tubules. Furthermore, proUGn metabolites (other than small amounts of cysteine and methionine) are not returned to the circulation from the kidney or any other organ. Thus, to respond to proUGn released from the gut, any target organ must use a local mechanism for production of active peptide.

A novel radioligand for glycine transporter 1: characterization and use in autoradiographic and in vivo brain occupancy studies.

INTRODUCTION: In an effort to develop agents to test the NMDA hypofunction hypothesis of schizophrenia, benchmark compounds from a program to discover potent, selective, competitive glycine transporter 1 (GlyT1) inhibitors were radiolabeled in order to further study the detailed pharmacology of these inhibitors and the distribution of GlyT1 in brain. We here report the in vitro characterization of [35S](S)-2-amino-4-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide ([35S]ACPPB), a radiotracer developed from a potent and selective non-sarcosine-derived GlyT1 inhibitor, its use in autoradiographic studies to localize (S)-2-amino-6-chloro-N-(1-(4-phenyl-1-(propylsulfonyl)piperidin-4-yl)ethyl)benzamide (ACPPB) binding sites in rat and rhesus brain and for in vivo occupancy assays of competitive GlyT1 inhibitors. METHODS: Functional potencies of unlabeled compounds were characterized by [14C]glycine uptake into JAR (human placental choriocarcinoma) cells and synaptosomes. Radioligand binding studies were performed with tissue homogenates. Autoradiographic studies were performed on tissue slices. RESULTS: ACPPB is a potent (Kd=1.9 nM), selective, GlyT1 inhibitor that, when radiolabeled with [35S], is a well-behaved radioligand with low nondisplaceable binding. Autoradiographic studies of rat and rhesus brain slices with this ligand showed that specific binding sites were plentiful and nonhomogeneously distributed, with high levels of binding in the brainstem, cerebellar white matter, thalamus, cortical white matter and spinal cord gray matter. In vivo studies demonstrate displaceable binding of [35S]ACPPB in rat brain tissues following iv administration of this radioligand. CONCLUSIONS: This is the first report of detailed anatomical localization of GlyT1 using direct radioligand binding, and the first demonstration that an in vivo occupancy assay is feasible, suggesting that it may also be feasible to develop positron emission tomography tracers for GlyT1.

MUC16 is produced in tracheal surface epithelium and submucosal glands and is present in secretions from normal human airway and cultured bronchial epithelial cells.

The gel-forming MUC5AC and MUC5B mucins have been identified as major components of human airway mucus but it is not known whether additional mucin species, possibly with other functions, are also present. MUC16 mucin is a well-known serum marker for ovarian cancer, but the molecule has also been found on the ocular surface and in cervical secretions suggesting that it may play a role on the normal mucosal surface. In this investigation, the LUM16-2 antiserum (raised against a sequence in the N-terminal repeat domain) recognized MUC16 in goblet and submucosal gland mucous cells as well as on the epithelial surface of human tracheal tissue suggesting that the mucin originates from secretory cells. MUC16 mucin was present in 'normal' respiratory tract mucus as well as in secretions from normal human bronchial epithelial (NHBE) cells. MUC16 from NHBE cells was a high-molecular-mass, monomeric mucin which gave rise to large glycopeptides after proteolysis. N- and C-terminal fragments of the molecule were separated on gel electrophoresis showing that the MUC16 apoprotein undergoes a cleavage between these domains, possibly in the SEA domain as demonstrated for other transmembrane mucins; MUC1 and MUC3. After metabolic labeling of NHBE cells, most of the secreted monomeric, high-molecular-mass [(35)S]sulphate-labelled molecules were immunoprecipitated with the OC125 antibody indicating that MUC16 is the major [(35)S]sulphate-labelled mucin in NHBE cell secretions.

The transfer of different forms of 35S to goat milk.

Sulphur-35 is released during the routine operation of UK gas-cooled reactors. An experiment to determine the rates of transfer of different forms of (35)S to goat milk is described. Lactating goats received (35)S orally as single administrations of sulphate, L-methionine, or grass contaminated either through root uptake of (35)S as sulphate or through aerial deposition of (35)S as carbonyl sulphide onto the grass. Transfer was higher for (35)S administered as methionine compared with (35)S administered as sulphate. Changes in activity concentrations in milk for all sources of (35)S demonstrated two components of loss. The first component had a half-life of circa 1 d for all sources, the second was longer in goats administered carbonyl sulphide (44 d) than in all of the other treatments (circa 10 d). The rate of transfer of (35)S to milk of a further group of goats receiving (35)S-sulphate daily appeared to reach equilibrium within 30 d. Extrapolation of transfer parameters derived to other dairy ruminants is discussed.

Meniscal debridement with an arthroscopic radiofrequency wand versus an arthroscopic shaver: comparative effects on menisci and underlying articular cartilage.

PURPOSE: Meniscal debridement with an arthroscopic radiofrequency (RF) wand versus an arthroscopic shaver and their comparative effects on menisci and underlying articular cartilage were studied. METHODS: When repair is not feasible, degenerative or post-traumatic meniscal tears often need debridement. Six fresh bovine knees were harvested, the tibial plateau was dissected free from the femoral articulation and placed in a saline bath at 28 degrees C, with 10% to 15% of the posterior horn of menisci debrided arthroscopically, and the surfaces debrided using a basket punch plus shaver, punch plus RF wand, RF wand alone, and untreated control. Treatment time of each case was 24 seconds at wand power 7. We characterized an injury zone, as well as viability and metabolic activity of meniscal cells and tibial articular cartilage chondrocytes. RESULTS: Chondrocyte viability of the tibial articular surface was 96% to 98%. We saw no differences in viability or injury zone (0 to 150 microm) among debrided groups or versus the control for any experimental surface, with no significant difference in metabolic activity in menisci debrided surfaces versus control. Meniscal viability was variable with analyses showing substantial levels (150 to 500 microm) of cell death in debrided and control groups. Metabolic activity in treated meniscus was lower than in cartilage specimens. No significant differences were observed among treatment groups versus control. CONCLUSIONS: Focal areas of chondrocyte cell death were not seen. Meniscal samples showed cell death (150 to 500 mum) throughout the tissue. CLINICAL RELEVANCE: Debridement of menisci with a bipolar RF wand produces levels of cell injury and death similar to those of debridement with a basket punch mechanical shaver. The RF wand did not harm underlying articular surfaces and produced a precise cut to the meniscal surface.

Synthesis and binding characteristics of [(3)H]neuromedin N, a NTS2 receptor ligand.

Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [(3)H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454nM in rat brain and 425nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8±30.18nM, while the Bmax value corresponded to 3.8±0.2pmol/mg protein in rat brain membranes. The specific binding of [(3)H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na(+) ions was observed on the binding of [(3)H]NN, resulting in an IC50 of 150.6mM. In [(35)S]GTPγS binding experiments the Emax value was 112.3±1.4% in rat brain and 112.9±2.4% in rat spinal cord membranes and EC50 values of 0.7nM and 0.79nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [(3)H]NN labels NTS2 receptors in rat brain membranes.

Modelling the fate of sulphur-35 in crops. 1. Calibration data.

Gas-cooled nuclear power plants in the UK release sulphur-35 during their routine operation. The gas is in the form of COS which can be readily assimilated by vegetation. It is therefore necessary to be able to model the uptake of such releases in order to quantify any potential contamination of the food chain. To develop such models experimental data are required. A series of experiments was undertaken to determine the rate of deposition, the partition and subsequent loss of sulphur-35 in crops exposed to CO(35)S. The mass normalised deposition rate was similar for the range of crops tested, while the partition of the (35)S paralleled the growth of crop components. There was no significant loss of radioactivity other than that expected from radioactive decay.

Modelling the fate of sulphur-35 in crops. 2. Development and validation of the CROPS-35 model.

Gas-cooled nuclear power plants in the UK release sulphur-35 during their routine operation, which can be readily assimilated by vegetation. It is therefore necessary to be able to model the uptake of such releases in order to quantify any potential contamination of the food chain. A model is described which predicts the concentration of (35)S in crop components following an aerial gaseous release. Following deposition the allocation to crop components is determined by an export function from a labile pool, the leaves, to those components growing most actively post exposure. The growth rates are determined by crop growth data, which is also used to determine the concentration. The loss of activity is controlled by radioactive decay only. The paper describes the calibration and the validation of the model. To improve the model, further experimental work is required particularly on the export kinetics of (35)S. It may be possible to adapt such a modelling approach to the prediction of crop content for gaseous releases of (3)H and (14)C from nuclear facilities.

Transepidermal water loss does not correlate with skin barrier function in vitro.

The purpose of this study was to investigate the relationship between transepidermal water loss and skin permeability to tritiated water (3H2O) and the lipophilic penetrant sulfur mustard in vitro. No correlation was found between basal transepidermal water loss rates and the permeability of human epidermal membranes to 3H2O (p = 0.72) or sulfur mustard (p = 0.74). Similarly, there was no correlation between transepidermal water loss rates and the 3H2O permeability of full-thickness pig skin (p = 0.68). There was no correlation between transepidermal water loss rate and 3H2O permeability following up to 15 tape strips (p = 0.64) or up to four needle-stick punctures (p = 0.13). These data indicate that transepidermal water loss cannot be unconditionally ascribed to be a measure of skin barrier function. It is clear that further work should be conducted to interpret the significance of measuring transepidermal water loss by evaporimetry.

Functional and anatomical localization of mu opioid receptors in the striatum, amygdala, and extended amygdala of the nonhuman primate.

The subregional distribution of mu opioid receptors and corresponding G-protein activation were examined in the striatum, amygdala, and extended amygdala of cynomolgus monkeys. The topography of mu binding sites was defined using autoradiography with [(3)H]DAMGO, a selective mu ligand. In adjacent sections, the distribution of receptor-activated G proteins was identified with DAMGO-stimulated guanylyl 5'(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Within the striatum, the distribution of [(3)H]DAMGO binding sites was characterized by a distinct dorsal-ventral gradient with a higher concentration of binding sites at more rostral levels of the striatum. [(3)H]DAMGO binding was further distinguished by the presence of patch-like aggregations within the caudate, as well as smaller areas of very dense receptor binding sites, previously identified in human striatum as neurochemically unique domains of the accumbens and putamen (NUDAPs). The amygdala contained the highest concentration of [(3)H]DAMGO binding sites measured in this study, with the densest levels of binding noted within the basal, accessory basal, paralaminar, and medial nuclei. In the striatum and amygdala, the distribution of DAMGO-stimulated G-protein activation largely corresponded with the distribution of mu binding sites. The central and medial nuclei of the amygdala, however, were notable exceptions. Whereas the concentration of [(3)H]DAMGO binding sites in the central nucleus of the amygdala was very low, the concentration of DAMGO-stimulated G-protein activation in this nucleus, as measured with [(35)S]GTPgammaS binding, was relatively high compared to other portions of the amygdala containing much higher concentrations of [(3)H]DAMGO binding sites. The converse was true in the medial nucleus, where high concentrations of binding sites were associated with lower levels of DAMGO-stimulated G-protein activation. Finally, [(3)H]DAMGO and [(35)S]GTPgammaS binding within the amygdala, particularly the medial nucleus, formed a continuum with the substantia innominata and bed nucleus of the stria terminalis, supporting the concept of the extended amygdala in primates.

L-Quisqualic acid transport into hippocampal neurons by a cystine-sensitive carrier is required for the induction of quisqualate sensitization.

A brief exposure of hippocampal slices to L-quisqualic acid sensitizes CA1 pyramidal neurons 30-250-fold to depolarization by two classes of excitatory amino acid analogues: (1) those whose depolarizing effects are rapidly terminated following washout, e.g. L-2-amino-4-phosphonobutanoic acid (L-AP4) and L-2-amino-6-phosphonohexanoic acid (L-AP6) and (2) those whose depolarizing effects persist following washout, e.g. L-aspartate-beta-hydroxamate (L-AbetaH). This process has been termed quisqualate sensitization. In this study we directly examine the role of amino acid transport systems in the induction of quisqualate sensitization. We report that L-quisqualate is a low-affinity substrate (K(M)=0.54 mM) for a high capacity (V(max)=0.9 nmol (mg protein)(-1) min(-1)) Na(+)-dependent transport system(s) and a high-affinity substrate (K(M)=0.033 mM) for a low-capacity (V(max)=0.051 nmol (mg protein)(-1) min(-1)) transporter with properties similar to the cystine/glutamate exchange carrier, System x(c-). We present evidence that suggests that System x(c-) participates in quisqualate sensitization. First, simultaneous application of L-quisqualate and inhibitors of System x(c-), but not inhibitors of Na(+)-dependent glutamate transporters, prevents the subsequent sensitization of hippocampal neurons to phosphonates or L-AbetaH. Second, L-quisqualic acid only sensitizes hippocampal neurons to other substrates of System x(c-), including cystine. Third, immunocytochemical analysis of L-quisqualate uptake demonstrates that only inhibitors of System x(c-) inhibit the highly concentrative uptake of L-quisqualate into a widely dispersed group of GABAergic hippocampal interneurons. We conclude that quisqualate sensitization is a direct consequence of the unique interaction of various excitatory amino acids, namely L-quisqualate, cystine, and phosphonates, with the exchange carrier, System x(c-). Therefore, the results of this study have important implications for the mechanism by which L-quisqualate, and other substrates of this transporter which are also excitatory amino acid agonists (such as glutamate and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid, beta-L-ODAP) may trigger neurotoxicity.

Pseudoginsenoside-F11 attenuates morphine-induced signalling in Chinese hamster ovary-mu cells.

Pseudoginsenoside-F11 (PF11), an ocotillol type saponin isolated from Panax quinquefolium L., has been shown to antagonize the behavioral actions of morphine. Biochemical experiments revealed that PF11 could inhibit diprenorphine (DIP) binding with an IC50 of approximately 6.1 microM and reduced the binding potency of morphine in Chinese hamster ovary (CHO)-mu cells. Furthermore, PF11 significantly attenuated morphine-stimulated [35S]GTPgammaS binding in a dose dependent manner, and strongly decreased the efficacy of morphine to inhibit intracellular cAMP production. In addition, PF11 pretreatment could also significantly inhibit naloxone induced cAMP overshoot in the morphine-pretreated cells. However, PF11 per se had no effect on either [35S]GTPgammaS binding or intracellular cAMP accumulation. These data suggested that PF11 antagonized the morphine stimulated opioid receptor signalling directly at the cellular level.

The uptake of cysteine in cultured primary astrocytes and neurons.

One of the vitally important functions of glutathione (GSH) is to adequately protect cells against toxic chemicals, reactive oxygen metabolites and free radical species. The amino acid, cysteine, is the key rate-limiting substrate for the biosynthesis of GSH, and the maintenance of adequate intracellular GSH levels is dependent upon the extracellular availability and transport of cysteine into cells. In the present study, primary cultures of astrocytes and neurons were employed to characterize cysteine transport systems. Both astrocytes and neurons used Na(+)-dependent systems as the major route for cysteine uptake (80-90% of total), while Na(+)-independent uptake represented a minor component of total transport (10-20% of total). Among the Na(+)-dependent systems, X(AG(-)) was the major contributor (approx. 80-90%) for cysteine uptake in both neurons and astrocytes, with a minor contribution from the ASC transport system (Na(+)-dependent neutral amino acid transport system for alanine, serine, and cysteine). In the Na(+)-independent transport systems (10-20% of total cysteine transport), multifunctional ectoenzyme/amino acid transporter gamma-glutamyltranspeptidase (GGT), and the neutral amino acid L-system contributed approximately equally towards cysteine uptake, in both neurons and astrocytes. The present studies demonstrate that astrocytes and neurons accumulate cysteine by both Na(+)-dependent and Na(+)-independent uptake systems, with major uptake occurring through the X(AG(-)) system and minor uptake via the ASC, GGT and L-systems.

Dopamine receptor-modulated [35S]GTPgammaS binding in striatum of 6-hydroxydopamine-lesioned rats.

The role of dopamine receptor-G protein coupling in the development of striatal dopamine receptor supersensitivity was studied in rats with a 6-hydroxydopamine (6-OHDA)-induced unilateral lesion of the nigrostriatal pathway. This coupling was assessed by the measurement of dopamine agonist-induced guanosine 5'-O-(gamma[35S]thio)triphosphate ([35S]GTP-gammaS) binding in striatal membranes, at different periods of time (1-5 weeks) following the microinjection of the neurotoxin. From the first to the fifth week following the lesion, basal and dopamine-stimulated [35S]GTPgammaS-specific binding were found to be enhanced in the denervated striata as compared to their control counterpart. D2 dopamine receptors were clearly demonstrated to be involved in this supersensitivity, as assessed by measuring N-propylnorapomorphine (NPA)-, quinpirole- and bromocriptine-induced [35S]GTPgammaS-specific binding. The involvement of D1 dopamine receptors was indirectly studied by the combination of dopamine with a saturating concentration of the selective and potent D2 antagonist domperidone. In these conditions, the remaining response to dopamine was also found to be significantly increased following the lesion. These results are consistent with the hypothesis that, in addition to D2 dopamine receptor upregulation, modulation of dopamine receptor-G protein interaction is involved in the hypersensitivity accompanying striatal dopamine depletion.

Chronic intrathecal morphine administration produces homologous mu receptor/G-protein desensitization specifically in spinal cord.

Previous studies have shown that chronic i.v. treatment with morphine or heroin decreased mu opioid receptor activation of G-proteins in specific brain regions. The present study examined the effect of intrathecal (i.t.) morphine administration on receptor/G-protein coupling in the spinal cord. In spinal cord membranes, [35S]GTP gamma S binding was stimulated by agonists of several G-protein-coupled receptors, including mu opioid (DAMGO), delta opioid (DPDPE), GABA(B) (baclofen), cannabinoid CB(1) (WIN 55,212-2), muscarinic cholinergic (carbachol) and adenosine A(1) (PIA). [35S]GTP gamma S autoradiography revealed that most of this agonist activation of G-proteins was localized to laminae I and II of dorsal horn. To determine the effects of chronic morphine on these receptor activities, rats were treated for 7 days with 0.11 mg/kg/day i.t. morphine, and receptor activation of G-proteins was determined by [35S]GTP gamma S autoradiography of brain and spinal cord. In spinal cord sections, chronic morphine treatment decreased DAMGO-stimulated [35S]GTP gamma S binding in laminae I and II at all levels of spinal cord examined. There were no effects of morphine treatment on [35S]GTP gamma S stimulation in spinal cord by other receptor systems examined (Adenosine A(1) and GABA(B)), and no significant effects of chronic i.t. morphine treatment were observed in brain sections. These data show that homologous desensitization of mu receptor/G-protein coupling occurs specifically in spinal cord following chronic morphine administration.

Up-regulation of spinal mu-opioid receptor function to activate G-protein by chronic naloxone treatment.

The effects of repeated s.c. administrations of an mu-opioid receptor antagonist naloxone on the G-protein activation induced by mu-opioid receptor agonists [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO), endomorphin-1 and endomorphin-2 in the mouse spinal cord was studied, monitoring guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding. All mu-opioid receptor agonists concentration-dependently increased the [35S]GTPgammaS binding. The increases of [35S]GTPgammaS binding induced by agonists were significantly enhanced in mice pretreated with naloxone. Under the present condition, chronic treatment with naloxone significantly increased the density of [3H]DAMGO binding sites with an increase in K(d) values in spinal cord membranes, indicating an increase in mu-opioid receptors on the membrane surface. These findings suggest that chronic treatment with an mu-opioid receptor antagonist naloxone leads to the supersensitivity to activate G-protein by mu-opioid receptor agonists with an increase in mu-opioid receptors in membranes of the mouse spinal cord.

Isolation of a retinal pigment epithelial cell-derived fraction which promotes Müller cell proliferation.

Retinal pigment epithelial cells (RPE) secrete a factor(s) in vitro which promotes Müller cell proliferation. To begin the isolation of the active factor, medium conditioned by cultured RPE (RPE-CM) was fractionated by fast protein liquid chromatography. The fractions were tested in an assay for Müller cell proliferation and it was found that one fraction contained biological activity comparable to that of complete RPE-CM. Gel electrophoresis demonstrated that this fraction consisted of proteins with approximate molecular masses of 35-88 kDa. An affinity assay revealed that only an 88-kDa protein in the RPE-CM binds to Müller cells. This protein was also present in the active fraction; therefore, it is the most likely source of the mitogenic activity of RPE-CM.