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R Simantov

Publications and source records attributed to R Simantov.

At least 73 records · Page 4Linked to original sources
PubMed1988

pp60c-src expression in the developing rat brain.

We have studied pp60c-src expression in the striatum, hippocampus, and cerebellum of the developing rat brain. In the striatum, pp60c-src protein kinase activity peaks during embryonic development and then declines in the adult. The peak activity occurs in the striatum on embryonic day 20 (E20) when it is 18- to 20-fold higher than the activity in fibroblasts and 4- to 5-fold higher than the activity in the striatum at E15 or in the adult striatum. In the hippocampal region, pp60c-src activity reaches a maximum shortly after birth but remains high throughout life. On postnatal day 2 (P2) the activity in the hippocampus is 9- to 13-fold higher than the activity in fibroblasts and twice as high as the activity in the hippocampus at E18. In the cerebellum, the kinase activity remains constant from E20 onward and is 6- to 10-fold higher than that observed in fibroblasts. The increase in pp60c-src kinase activity observed during the development of the striatum and hippocampus is due to an increase in the amount of pp60c-src protein and to an increase in the specific activity of the kinase. The increase in specific activity in these regions coincides with the peak periods of neurogenesis and neuronal growth. In the striatum, we have found that the increase in pp60c-src activity also parallels the increase observed in culture as embryonic striatal neurons differentiate. Taken together, our results are consonant with the idea that pp60c-src is the product of a developmentally regulated gene that is important for the differentiation and/or the continuing function of neurons.

Aging↗
PubMed1987

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.

Animals↗
PubMed1986

Naloxonazine actions in vivo.

Naloxonazine is a relatively selective mu 1 affinity label in binding studies. Like naloxazone, naloxonazine has proven valuable in vivo in establishing a role for mu 1 sites in specific opiate actions. We now report a detailed characterization of naloxonazine's actions in mice and rats. Naloxonazine antagonized morphine analgesia for greater than 24 h without altering lethality. This prolonged action could not be easily explained by a slow rate of elimination since the terminal elimination half-life was estimated at less than 3 h. Furthermore, these actions were associated with a wash-resistant inhibition of binding lasting 24 h which was relatively selective for mu 1 sites. At a fixed morphine dose, increasing naloxonazine doses lowered peak tailflick latencies in a biphasic manner, suggesting that morphine analgesia consisted of both mu 1 (naloxonazine-sensitive) and a non-mu 1 (naloxonazine-insensitive) components. The ratio of mu 1 to non-mu 1 analgesia was greater at low morphine doses, implying that morphine activated mu 1 analgesic mechanisms with higher affinity. Our studies also emphasized that naloxonazine has reversible actions similar to those of naloxone; only its irreversible actions are relatively mu 1-selective. In addition, the selectivity of naloxonazine's irreversible actions is dose-dependent. High naloxonazine doses will irreversibly antagonize receptors other than mu 1.

Animals↗
PubMed1986

Plasticity in the phenotypic expression of brain opioid receptors: differential response of forebrain and hindbrain cultures to chemical depolarization.

Potassium chloride at a depolarizing concentration has a differential effect on the expression of opioid receptors in aggregating neuronal cultures prepared from the rat forebrain or hindbrain. The mu-selective enkephalin analogue DAGO was used to indicate that the effect of potassium chloride is not uniform on all subtypes of opioid receptors. These results suggest for the first time that chemical depolarization may modulate the phenotypic expression of opioid receptors.

Animals↗
PubMed1986

Calcitonin induced increase in ACTH, beta-endorphin and cortisol secretion.

The response of ACTH, beta-endorphin and cortisol to calcitonin administration was investigated in 8 subjects with recent fractures of the vertebrae due to postmenopausal or senile osteoporosis (Ost) and in seven normal healthy controls (NC). A significant increase of the three hormones was observed in 13 subjects. The maximum increase was observed between 15 and 60 min.: the cortisol level (microgram/100 ml) rose from 14.3 +/- 1.9 to 24.8 +/- 3.2 (P less than 0.05) in Ost and from 7.7 +/- 0.6 to 21.7 +/- 1.7 (P less than 0.001) in NC, the beta-endorphin (pmol/l) from 5.8 +/- 0.6 and to 21.2 +/- 1.3 in OST (P less than 0.001) and from 5.9 +/- 0.4 to 21.9 +/- 4.5 (P less than 0.01) in NC and the ACTH levels (pg/ml) from 21.3 +/- 5.7 to 61.7 +/- 3.6 (P less than 0.001) in OST and from 30.0 +/- 6.2 to 58.8 +/- 7.5 (P less than 0.05) in NC. The results indicate a possible role of calcitonin in modulating the anterior pituitary function. It also suggests that the analgesic effect of calcitonin might be mediated by the increase of beta-endorphin. The possibility that this analgesic effect of calcitonin is due to its direct binding to the opiate receptors was excluded in the present study by in vitro binding assay.

Adrenocorticotropic Hormone↗
PubMed1986

Subcellular compartmentation of opioid receptors: modulation by enkephalin and alkaloids.

A subclone of NG108-15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes beta-glucuronidase, galactosyltransferase, 5'-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [3H]D-Ala2-D-Leu5-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [3H]diprenorphine, chased with various unlabeled opiates, and the distribution of 3H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [3H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells.

Animals↗
PubMed1984

Up-regulation of opiate receptors by opiate antagonists in neuroblastoma-glioma cell culture: the possibility of interaction with guanosine triphosphate-binding proteins.

Neuroblastoma-glioma NG108-15 cells that were cultured for 48 h with the opiate antagonist, naloxone, respond to the guanosine 5'-triphosphate (GTP) analogue guanosine 5'-[beta, gamma-imido]-triphosphate (GMP-PNP) in the binding assay as the control, non-treated, cells. This was observed when the guanyl nucleotide was tested in the presence or absence of sodium chloride and also after subcellular fractionation of the membranes on a sucrose gradient which separated between two receptor-containing fractions. The findings suggest that the increase in delta type enkephalin receptors in naloxone-treated NG108-15 cells does not reflect an alteration in the interaction between the receptor and the adenylate cyclase-GTP-binding protein system.

Animals↗
PubMed1984

Characterization and down-regulation of opiate receptors in aggregating fetal rat brain cells.

Aggregating brain cells prepared from embryonic rats bind radioactive opiates in a stereospecific manner. The drug selectivity, receptor content during culturing and down-regulation of these apparent opiate receptors were studied in aggregates prepared from the embryonic hindbrain or forebrain. The receptor content in hindbrain but not forebrain aggregates was increased up to 3-fold after 21 days in culture. Differences between the receptors of the two types of aggregates were also observed in the affinity of opiate alkaloids and D-Ala2,D-Leu5-enkephalin (DADL). The potent opiate alkaloid etorphine induced down-regulation of opiate receptors in aggregates prepared from either brain region whereas DADL was a potent down-regulator in the forebrain but not in the hindbrain aggregates and morphine had no effect in both tissues. The implications of these results concerning the control of various types of opiate receptors in the whole animal are discussed.

Animals↗
PubMed1984

Selectivity in the control of opiate receptor density in the animal and in cultured fetal brain cells.

Two aspects of the mechanisms controlling down-regulation of opiate receptors were studied: 1. The possibility that morphine does not induce down-regulation of delta receptors is an observation confined to in vitro conditions was investigated by studying the regulation of receptors in neuroblastoma-glioma cells in diffusion chambers implanted in ICR mice peritonea. Injection of morphine for 9 days at a dose inducing opiate tolerance did not change the number of receptors in the chamber-implanted cells, whereas etorphine at a 1/100 dose had a profound effect. 2. Embryonic cells from rat forebrain or hindbrain were cultured with mu type opiate alkaloid (morphine) or peptide (morphiceptin) to further establish the selectivity of opiate action. A partial effect of morphiceptin but not morphine on the number of receptors in hindbrain aggregates was observed. Thus, conclusions derived from experiments with morphine may not be applicable to mu type peptides. The results suggest that the mammalian brain may contain sub-types of mu receptors. Alternatively, although interacting with a common mu receptor, morphine and mu opioid peptides may induce different regulatory mechanisms.

Animals↗
PubMed1983

Control of endogenous cell regulators by the second-stage tumor promoter phorbol-12-retinoate 13-acetate.

The phorbol esters phorbol 12-retinoate 13-acetate (RPA) and 12-O-tetradecanoyl phorbol 13-acetate (TPA) were used to investigate the role of tumor promoters in the control of hormone response in normal and leukemic myeloid cells. RPA and TPA inhibited the binding of [20-3H]phorbol 12,13-dibutyrate to the leukemic cells in a competitive manner with 50% inhibition values of 5.2 +/- 1.3 and 1.1 +/- 0.6 nM, respectively. RPA, like TPA, enhanced (1) prostaglandin E1-induced cyclic AMP synthesis, (2) the differentiation of leukemic cells induced by the normal myeloid differentiation-inducing protein, and (3) the formation of normal myeloid cells colonies induced by the normal myeloid growth-inducing protein. Both compounds can thus control endogenous cell regulators. Since RPA functions in the second stage of tumor promotion in mouse skin, it is suggested that the control of such endogenous regulators may involve biochemical pathways similar to those that are activated in the second stage of tumor promotion.

Animals↗
PubMed1983

Regulation of opiate receptors in mouse brain: arcuate nuclear lesion induces receptor up-regulation and supersensitivity to opiates.

Lesion of the hypothalamic arcuate nucleus of the mouse by neonatal application of monosodium glutamate (MSG) increased the binding of [3H]dihydromorphine to membranes prepared from the midbrain. A saturation curve of [3H]dihydromorphine binding indicated that MSG increased the number of the opiate receptors. The MSG-treated mice also exhibited an enhanced response to morphine and naltrexone regarding thermal pain sensitivity. The physiological implications of opiate receptors up-regulations upon arcuate nuclear lesion are discussed.

Animals↗
PubMed1983

Down-regulation of opiate receptors in serum-free cultures of aggregating fetal brain cells.

Brain cells from rat embryos that were cultured in serum-free medium form aggregates which possess stereospecific binding of radioactive opiates. The specificity of these opiate receptors and their regulation by opiate alkaloids and enkephalin was studied. D-ala2, D-leu5-enkephalin (DADL) induced down-regulation of opiate receptors in aggregates prepared from the forebrain but had no effect on hindbrain aggregates, whereas the potent alkaloid etorphine induced down-regulation of the receptors in both tissues. Interestingly, morphine did not induce down-regulation in both tissues. The significance of these findings and their relationship to the control of different types of opiate receptors are discussed.

Animals↗
PubMed1983

Enkephalins and opiate antagonists control calmodulin distribution in neuroblastoma-glioma cells.

The calcium binding protein calmodulin and the opiate receptor binding sites are unevenly distributed in various subcellular fractions of neuroblastoma-glioma NG108-15 cells. The crude mitochondrial-membrane fraction of these cells contains two membrane fractions that are separable by sucrose gradient centrifugation. These two differ in the content of both calmodulin and opiate receptors. Leucine enkephalin and D-Ala2-methionine enkephalinamide decrease the amount of membrane-bound calmodulin in the NC108-15 cells in a time- and dose-dependent manner, whereas the opiate antagonists naloxone and levallorphan have an opposite effect. Naloxone blocks the effect of leucine enkephalin and dextrallorphan has no significant effect. The opiate alkaloids entorphine and phenazocine induce changes similar to that of the enkephalins whereas morphine is inactive even at high concentrations. The alteration in the amount of membrane-bound calmodulin after a short incubation (15 min) with the enkephalins or with naloxone is reflected as an opposite change in the amount of calmodulin in the cell cytosol. Naloxone and levallorphan also increase the number of opiate receptors in NG108-15 cells but dextrallorphan has no such effect. Modulation of the intracellular distribution of calmodulin by opioid peptides and alkaloids may control the activity of various membrane-bound and cytosolic systems that are calmodulin- and/or calcium-dependent.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Down regulation of enkephalin (delta) receptors. Demonstration in membrane-bound and solubilized receptors.

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue D-Ala2-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to delta-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the alpha 2-adrenergic ligand, [3H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of delta-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.

Alkaloids↗
PubMed1982

A genetic approach to reveal the action of the opiate receptor in selected neuroblastoma-glioma cells. Interaction with alpha-adrenoceptors, calmodulin and Ca2+-ATPase.

Three clones of neuroblastoma-glioma cells that contain low amounts of calmodulin were selected from the NG108-15 cells after several treatments with high concentrations of chlorpromazine. Purified membranes of the three clones had decreased numbers of both alpha-adrenergic and opiate receptors, monitored with [3H]yohimbine and [3H,D-Ala2]methionine encephalinamide, respectively. No changes were observed in the affinity of these radioactive ligands to the receptors of the selected cells as compared to the parent cells. Addition of bovine brain calmodulin did not affect the binding of [3H,D-Ala2]methionine encephalinamide to the membranes of the selected cells and they had the same number of acetylcholine receptors, determined with 1-quinuclidinyl-[phenyl-4-3H]-benzilate, as the parent NG108-15 cells. The basal ATPase activity in the membranes of the selected cells was 35-50% of the parent cells, with a decreased V value and no significant change in the affinity constant Ka to ATP. Addition of Ca2+ to the purified membranes increased the V of the ATPase in the selected as well as the parent cells but the V of the selected cells remained lower than that of the parent cells. Ca2+ had no effect on the Ka to ATP in either cell type. The Ca2+-dependent ATPase activity of both the parent and the selected cells was also calmodulin-dependent dependent since it was blocked in vitro by chlorpromazine. The co-regulation of opiate and adrenergic receptors and their interaction with calmodulin and Ca2+-ATPase is discussed in view of recent observations indicating biochemical and physiological association between opiates, Ca2+ and adrenergic compounds.

Animals↗
PubMed1982

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells.

Phospholipase A2 activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A2 activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-3H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7-14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E1 did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A2 activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A2, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A2 activity was also related to previously found differences in the ability of the clones to develop desensitization to beta-adrenergic hormones or prostaglandin E1.

Animals↗