K-Opioid Agonist Modulation of [3H]Thymidine Incorporation into DNA: Evidence for the Involvement of Pertussis Toxin-Sensitive G Protein-Coupled Phosphoinositide Turnover

Abstract: A body of evidence has indicated that μ-opioid agonists can inhibit DNA synthesis in developing brain. We now report that K -selective opioid agonists (U69593 and U50488) modulate [³H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose- and developmental stage-dependent manner. K agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the K -selective antagonist, norbinaltorphimine, whereas in 21-day brain cell aggregates a 3,5-fold increase was evident. Cell labeling by [³H]thymidine was also inhibited by the K -opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7-day cultures and elevated it in 21-day cultures. Control levels were restored by norbin-altorphimine. Pertussis toxin blocked U69593-mediated inhibition of DNA synthesis. The action of K agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7- and 21-day cultures. These results suggest that K agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin-sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1-fold, and this stimulation was reversed by the opioid antagonist naltrexone.     

Effect of an n-3 Fatty Acid-Deficient Diet on the Adenosine-Dependent Melatonin Release in Cultured Rat Pineal

Abstract: We studied the effect of a diet deficient in n-3 fatty acids on the adenosine-dependent melatonin release from cultured rat pineal gland after stimulation by 5'- N -ethylcarboxamidoadenosine (NECA), an A₂ adenosine agonist. Experiments were conducted with 2-month-old rats raised on semipurified diets containing either peanut oil (n-3 deficients) or peanut plus rapeseed oil (controls). The proportion of docosahexaenoic acid (22:6 n-3) in the pineal total lipid fraction and in phosphatidylcholine and phosphatidylethanolamine was significantly decreased in n-3-deficient rats. This was compensated for partially by an increase in 22:4 n-6 and 22:5 n-6 levels. The activity of the cultured rat pineal, in terms of cyclic AMP content and N -acetylserotonin and melatonin release in the medium, was lower after stimulation by 10^-5 mol/L NECA in the group fed peanut oil than in the group fed peanut plus rapeseed oil. The increased ratio of n-6/n-3 fatty acids in pineal total lipids and the major glycerophospholipids (phosphatidylcholine and phosphatidylethanolamine) may have an important influence on the rat pineal responses. The results are discussed in the context of changes in membrane-bound proteins, including enzymes and/or receptors involved in the rat pineal gland function.     

Developmentally Regulated Secreted Factors Control Expression of Muscarinic Receptor Subtypes in Embryonic Chick Retina

Abstract: Two molecular mass subtypes of muscarinic receptor are expressed by the chick retina (72 and 86 kDa). During development, the ratio of subtypes changes, with the 72-kDa form becoming predominant. We have found that subtype switch can occur in retina cell culture, and have investigated factors that influence this in vitro increase in the 72-kDa receptor. Increases similar to those in vivo occurred when cells were cultured at 10⁵ cells/cm², but not at 10-fold lower density. High-density cultures, maintained on coverslips, showed no receptor development when transferred to large volumes of fresh medium, indicating that cell-cell contact alone was not responsible for induction. However, replacement of fresh medium with conditioned medium (from high-density cultures) resulted in normal induction. There were no morphological differences between cultures with high and low levels of the 72-kDa receptor. Conditioned medium also induced 72-kDa receptors in low-density cultures, consistent with a minimal role for cell-cell contact. Efficacy of conditioned medium was markedly dependent on age. Media from cells cultured 1–4 days had no effect, but media from cells cultured 5–8 and 1–8 days elicited 1.6-fold and fourfold increases in the 72-kDa subtype, respectively. The data indicate that maturing retina cells secrete developmentally regulated factors that are necessary for abundant expression of the 72-kDa muscarinic receptor subtype.     

Nitric Oxide Synthase Activity Endogenously Modulates NMDA Receptors

Abstract: We tested the possibility that endogenous nitric oxide synthase activity regulated NMDA receptors in primary cultured striatal neurons. We monitored NMDA-induced increase in intra-cellular Ca²⁺ levels with fura-2 ratio imaging, while nitric oxide synthase activity was either increased with l -arginihe (the natural substrate of nitric oxide synthase) or inhibited using nitro- l -arginine (a specific inhibitor of nitric oxide synthase). We found that the NMDA receptor effect was slowly but strongly diminished after an l -arginine (1 m M , 15 min) treatment ( l -arginine preincubation reduced the 100 μM NMDA-induced maximal effect by 30–50%). The l -arginine blockade of NMDA receptors was long-lasting but could be partially reversed by hemoglobin (100 μM , 10 min), which binds nitric oxide. This was not observed when the neurons were treated with l -arginine together with nitro- l -arginine. Our data strongly suggest that physiological nitric oxide synthase activity could regulate NMDA receptors.     

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R_1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R_1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-t20 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R_1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R_1A and ANF-R_1C subtypes. A10 and NIH cells which express high density of ANF-R₂ receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R_1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R_1A and ANF-R_1C subtypes are modulated in the same manner. In the presence of Mn²⁺, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg²⁺ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R_1A, the ANF-R_1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R_1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R_1A subtype.Abbreviation ANF atrial natriuretic factor - BNP brain natriuretic peptide - CNP C-type natriuretic peptide - ATP adenosine-5-triphosphate - IBMX 3-isobutyl-1-methylxanthine - TPA 12-O-tetradecanoyl-phorbol-13-acetate - FKL forskolin - PKC calcium-phospholipid-dependent protein kinase - PKA cAMP-dependent protein kinase - PKG cGMP-dependent protein kinase - C-ANF [Cys¹¹⁶]-ANF-(102-116)-NH₂ - CC chromaffin cells     

Demonstration of melatonin-binding sites in cyclohexylamine-formaldehyde-fixed brain tissues

This study shows for the first time that perfusion of rat or hamster brain with a cyclohexylamine-paraformaldehyde mixture makes possible the observation by autoradiography of melatonin binding sites in structurally well-preserved fixed tissues. This result is a first step in the identification of melatonin-receptor-containing cell types by cytoautoradiography.     

卡介苗对枯否细胞生物特性影响的免疫细胞化学研究

卡介苗(BCG)是增强肝枯否细胞(KC)抗肝癌细胞作用的最佳免疫制剂之一。本实验是给大鼠BCG后,应用溶菌酶免疫细胞化学法显示KC,以图象分析系统测定KC的数量、分布和面积,以细胞分光光度计测定KC溶菌酶活性;分离KC体外受BCG作用后,用ELISA法测定RC的Fc受体表达。结果显示:BCG使KC数量增多一倍,尤以肝小叶周边带为显著;KC体积明显增大,细胞面积增大55.71％,周边带KC面积增大一倍上;细胞溶菌酶光密度增大64.35％;Fc受体表达增强64.82％。上述结果是BCG增强KC抗肝癌细胞作用有关细胞学机制。     

Effect of beta-N-oxalylamino-l-alanine on cerebellar cGMP level in vivo

Beta-N-oxalylamino-l-alanine (BOAA), a non-protein amino acid present in the seeds of Lathyrus Sativus (LS), is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal degeneration. In the present study, the in vivo acute effects of synthetic BOAA and LS seed extract were investigated on rat cerebellar cyclic GMP following intraperitoneal (10–100 mg/kg) or oral (100 mg/kg) administration of subconvulsive doses of toxin. Furthermore, the BOAA content in LS seeds and in the cerebellum of injected rats was determined by high performance liquid chromatograph analysis. A dose- and time-dependent increase of cerebellar cyclic guanosine monophosphate (cGMP) level was observed after intraperitoneal administration of synthetic BOAA or LS extract. The neurotoxin evoked a maximum stimulation 90 min after injection within the dose range of 50–75 mg/kg, elevating cGMP from basal levels of 5.3±0.5 pmol/mg protein to 15±1.3 pmol/mg protein. Similarly, the oral intake of LS-extracted neurotoxin resulted in the elevation of cGMP content. Kynurenic acid (300 mg/kg i.p.), a non specific excitatory amino acid antagonist, was effective in blocking LS BOAA-elicited cGMP enhancement. The data suggest that in the cerebellum acute administration of low concentrations of BOAA exert in vivo activation of glutamate receptors involved in the regulation of cGMP level.